极早产儿主要临床问题回顾性分析及人脐带血间充质干细胞制备研究
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摘要
第一部分极早产儿主要临床问题回顾性分析
目的：
极早产儿(very preterm infants, VPT,胎龄<32wk)由于其解剖生理特点,易发生各种并发症,病死率及后遗症的发生率较高,对极早产儿周密的全面管理,是新生儿监护病房(NICU)中体现水平的综合反映。国内在极早产儿NICU救治、预后等方面研究资料匮乏。本研究通过回顾性分析超大容量NICU极早产儿临床病例资料,旨在了解：(1)极早产儿围生期特征、NICU中主要疾病构成、治疗手段及疾病负担；(2)极早产儿存活率、主要并发症患病比例以及预后的高危因素；(3)新生儿转运以及超大容量NICU对极早产儿临床转归的影响；(4)国内极早产儿救治现状与发达国家救治情况比较。希望通过此文研究为当前极早产儿救治难点、重点提供可靠的临床研究资料,为更大规模的极早产儿流行病学研究提供重要的理论依据。
方法：
回顾性收集2010年10月1日至2011年09月30日连续12个月内,八一儿童医院极早产NICU中收治的极早产儿病例资料。病例纳入标准：出生胎龄(GA)小于32wk、生后24小时内入住极早产NICU者。
用SPSS软件进行统计分析。计量资料中连续变量呈正态分布者表示为均数±标准差,组间均数比较采用t检验或单因素方差分析；偏态分布者表示为中位数(范围或四分位数间距),组间均数比较采用秩和检验。计数资料以发生率或构成比表示,并对发生率作区间估计,发生率或构成比的比较采用卡方检验。因本研究为回顾性分析,结合研究人群特点,采用单因素Logistic回归模型对极早产儿死亡高危因素分析,进一步运用多因素Logistic回归模型进行多因素逐步回归分析以明确死亡的独立风险因素。变量间相关性采用Spearman等级相关分析。
结果：
1.人口学特征
本次研究共收集病例1262份,符合入选标准的病例729例。其中164例为超早产儿(胎龄,GA≤28wk),占总数的22.5%；478例为极低出生体重儿(VLBW<1500g),占总数的65.6%；68例为超低出生体重儿(ELBW<1000g),占总数的9.3%。平均GA为29.8±1.4(范围23.9-31.9)wk。平均出生体重(BW)为1399±285(范围610-2415)g。男婴430例,占总的例数的59.0%。76例(10.4%)为小于胎龄儿(SGA),199例(27.3%)为多胎。平均入NICU时龄为2.2±2.3(范围0.3-24)小时。
2.围产期特征
544例(74.6%)极早产儿接受了规律产前检查,141例(19.3%)产前检查大于1次或情况不详。199例(27.3%)应用产前激素,362例(49.7%)剖宫产。153例(21.0%)胎膜早破>24h,64例(8.8%)产前抗生素治疗。461例(63.3%)的极早产儿母亲存在明确妊娠期疾病,包括妊娠高血压疾病160例(21.9%)、胎膜早破245例(33.6%)、前置胎盘或胎盘早剥72例(9.9%)、糖尿病72例(9.9%)等。213例(29.2%)极早产儿中有的产房用氧复苏史,其中103例(14.1%)极早产儿接受过氧气或气囊加压给氧,110例(15.1%)极早产儿在产房接受了气管插管,25例(3.4%)极早产儿接受了产房心脏按压,14例(1.9%)极早产儿产房复苏药物。
3.极早产儿NICU主要疾病构成
极早产儿主要疾病构成与GA、BW成密切相关,胎龄、BW越小,患病比例越高。呼吸系统疾病是极早产儿最常见诊断疾病,主要包括：499例(68.4%)呼吸窘迫综合症(RDS),353例(48.4%)新生儿肺炎,61例(8.4%)肺出血,55例(7.5%)新生儿持续性肺动脉高压(PPHN),22例(3.0%,,n=606例)支气管肺发育不良(BPD),16例(2.2%)气胸等。其他常见疾病主要有动脉导管未闭(PDA)462例(63.4%),呼吸暂停287例(39.4%),败血症92例(12.6%),脑室内出血(IVH)157例(21.5%),脑室周围白质软化(PVL)11例(1.5%),坏死性小肠结肠炎(NEC)22例(3.0%),3期以上早产儿视网膜病(ROP)11例(1.7%)等。
4.呼吸系统疾病特征及临床治疗特点
499例RDS极早产儿中,447例(89.6%)接受了肺表面活性物质(PS)治疗,66例(13.2%)接受了超过1次的PS治疗。应用PS与未使用PS相比,存活率无差异,但PS患儿GA较低、并发症比例较高、MV和氧疗时间较长；产房使用PS与NICU使用PS相比,两者GA、存活率、并发症、MV和氧疗时间等均无显著差异(P>0.05)；与牛源性PS相比,猪源性PS患儿GA、BW更低,住院时间更长,存活率较低但无显著差异(x2=2.625,P=0.105),主要并发症、MV和氧疗时间等均无显著差异(P>0.05)。
RDS患儿499例(100%)接受机械通气(MV),其中79例(15.8%)只接受nCPAP,357例(71.5%)接受常频机械通气(CMV,不含HFOV),63例(12.6%)接受HFOV。三种通气方式中,HFOV存活率最低,MV时间最长,相关疾病或并发症比例最高(肺出血、气胸、重度IVH)；与仅nCPAP相比,CMV除肺出血比例较高外,存活率或主要并发症无差异。
358例新生儿肺炎患儿中,57例(16.1%)痰培养阳性,其中培养阳性率较高的有：28例(49.1%)鲍曼不动杆菌,18例(31.6%)肺炎克雷伯菌,6例(10.5%)大肠埃希菌和3例(7.0%)铜绿假单胞菌。
55例PPHN患儿中HFOV的比例为24例(43.6%),吸入NO治疗的比例为44例(80.0%)。PPHN与PDA密切相关,接受PDA结扎术患儿中患有PPHN的比例显著高于非PPHN患儿(16.4%vs1.9%,P<0.001)。48例吸入NO的患儿中,8例(16.7%)需要接受PDA结扎术,占所有PDA结扎术患儿(22例)的36.4%。
606例极早产儿纠正胎龄36wk治愈或好转出院无氧依赖,22例(3.6%)纠正胎龄36wk仍需氧疗诊断为BPD。其中7例(31.8%)在生后28天接受激素治疗,3例(13.6%)出科时需吸氧,19例(86.4%)治愈出院。
5.其他常见疾病特征及临床治疗特点
极早产儿入住NICU后根据病情需要至少进行1次床旁头颅超声。157例(21.5%)IVH是超声诊断比例最高的疾病,GA越小、BW越低,IVH比例越高。IVH在超早产儿比例为42.1%,VLBW比例为24.3%。
92例败血症极早产儿中,血培养阳性70例(76.1%)。其中,肺炎克雷伯菌培养阳性率最高37.1%(26例),其次为葡萄球菌30.0%(21例)、真菌12.9%(9例)、大肠埃希菌7.1%(5例)、鲍曼不动杆菌5.7%(4例)等。
22例NEC患儿中,只有7例(31.8%)接受手术治疗,病死率为36.4%(8例)。死亡患儿中3例(37.5%)接受了手术治疗,5例(62.5%)选择了放弃治疗。
638例极早产儿在出院或死亡之前至少接受了1次眼底筛查,106例(16.6%)极早产儿有不同程度ROP表现,其中127例受检超早产儿、410例受检VLBW患儿的检出比例分别为47.2%(60例)、23.4%(96例)。ROP患儿中11例(10.4%)接受了激光光凝手术治疗。与未检出ROP的存活极早产儿比较,ROP患儿的吸氧时间显著延长(中位数：26天vs7天,P<0.001)；与未接受手术治疗的ROP超早产儿相比,接受ROP光凝术的超早产儿氧疗时间显著较长(中位数：44天vs29天,P<0.001)。
极早产儿PDA比例按胎龄分层有显著差异,GA越小,患病比例越高。462例PDA患儿中,380例(82.3%)接受了布洛芬治疗,其中130例(34.2%)患儿接受布洛芬治疗超过1个疗程,GA越小,接受布洛芬治疗疗程越多。365例接受心脏超声复查,205例(56.2%)患儿首个疗程后PDA闭合,83例(22.7%)的患儿需接受多个疗程后PDA闭合；GA越小,PDA闭合的比例越低。22例(4.8%)PDA患儿在接受布洛芬治疗后选择PDA结扎术；GA越小,PDA结扎术的比例越高。82例(17.7%)PDA未接受布洛芬治疗,心脏超声复查PDA自发性闭合的比例较布洛芬治疗的比例高(90.2%vs78.9%,P<0.05),但布洛芬治疗者GA和BW均较低、合并肺出血的比例较高、接受MV、氧疗和住院时间较长以及住院费用较高,存活率无差异。接受多疗程布洛芬治疗的PDA患儿,GA较低,合并PPHN及吸入NO治疗的比例、PDA结扎术的比例均较高,接受MV、氧疗和住院时间较长以及住院费用较高,但存活率与1个疗程布洛芬PDA患儿相比无差异(92.8%vs94.6%,P=0.495)。
6.极早产儿转运特点以及对极早产儿临床转归的影响
入选研究对象中,73例(10%)为本院出生新生儿(inborn),656例(90%)为转运新生儿(outborn)。转运新生儿入院时龄为2.0小时(中位数),本院出生新生儿为0.5小时(中位数),两者有显著性差异；GA、BW无差异(P>0.05)。本院出生新生儿剖宫产、胎膜早破>24小时比例高于转运新生儿,而后者多胎比例高于前者(x2=4.173,P=0.041)。转运新生儿产房抢救措施比例较本院出生新生儿高,但无显著统计学差异(x2=3.588,P=0.058)。573例(78.6%)极早产儿入NICU前转运中接受氧疗。转运中氧疗模式主要为面罩319例(55.7%)和气管插管气囊加压人工呼吸240例(41.9%)；氧疗的比例与GA、BW密切相关,胎龄越小、BW越低,接受氧疗比例越高(x2=124.7,P<0.001)。
对极早产儿入院后呼吸系统主要疾病、治疗措施以及IVH比例比较,转运新生儿和本院出生新生儿无差异(P>0.05)。治愈或好转出院患儿中转运新生儿与本院出生新生儿的存活率无差异(91.3%vs94.5%,P=0.384),主要并发症比例、MV、氧疗和住院时间、住院费用均无统计学差异(P>0.05)。
对入院极早产儿按入院时龄2小时(中位数)分层比较,入院时龄≤2h组GA、 BW均无显著统计学差异。入院时龄<2h患儿母亲体外人工受精妊娠、妊娠高血压疾病、规律产检、产前激素治疗和选择剖宫产比例显著高于入院时龄>2h组,而后者生后窒息、产房有创性复苏措施比例较高。入院时龄≤2h组患儿存活率高于入院时龄>2h组(91.2%vs84.8%,P<0.05),且放弃治疗存活出院的比例显著低于后者(11.2%vs21.7%,P<0.01)；存活者中主要并发症比例、MV、氧疗时间和住院时间以及住院费用等均无统计学差异(P>0.05)。
7.死亡、存活者主要并发症高危因素分析
极早产儿NICU病死率为8.4%(61例),包括13例(1.8%)院内死亡和48例(6.6%)放弃治疗死亡。其中病死率超早产儿为21.3%(35例),VLBW为10.5%(50例),ELBW患儿为26.5%(18例)。GA、BW越低,病死率越高。57例(93.4%)患儿死于新生儿期。
多因素logistic回归分析表明低GA、有创MV、入院时龄>2h、合并气胸、合并肺出血、未使用PS治疗、严重先天畸形是极早产儿死亡的独立高危因素。
8.存活极早产儿住院时间和疾病负担
存活极早产儿住院时间为43天(中位数),住院费用为46409元(中位数),住院费用大约为同期北京市城市居民年收入的1.4倍。GA越小,住院时间越长、疾病负担越重。例如,住院时间(中位数)、住院费用(中位数)GA<26wk分别为92天、132771元,GA31wk分别为37天、38142元。
9.放弃或姑息治疗
131例(18.0%)极早产儿家长放弃或姑息治疗,其中67例(51.1%)是由于不能支付继续住院治疗费用,31例(23.7%)与患儿治疗效果差有关,21例(16.0%)是由于伴有严重并发症,12例(9.2%)是由于严重先天性疾病。患儿放弃治疗后死于医院的比例为36.6%(48例)。
10.极早产儿临床特征与发达国家比较
本研究中极早产儿产科干预比例与发达国家相比明显较低,新生儿转运比例明显高于发达国家,后者以产前转运为主。极早产儿总体存活率与发达国家水平相当,但主要并发症比例较发达国家低,分析可能与本院超早产儿的比例低、存活率较低有关。
结论：
1、本研究首次回顾性全面分析了国内超大容量NICU极早产儿病例资料,开创性的总结了国内首个NICU亚专科监护病房极早产儿的救治经验。研究结果表明极早产儿的救治水平,如89.5%的VLBW患儿治愈或好转出院,与美国等发达国家同期NICU中VLBW患儿存活率90%相当。
2、本研究中极早产儿产科主动干预比例(如产前激素应用、剖腹产比例)明显低于发达国家水平,而后者大量文献研究表明极早产儿产前激素应用可以显著降低极早产儿的病死率和并发症比例,因此我们可能在一定程度上延缓了极早产儿抢救时机,影响了极早产儿,特别是超早产儿在NICU中的转归。如我院超早产儿存活率为78.7%,与美国(88%)等发达国家同期水平相比,仍然存在一定的差距。
3、本院已成功建立了目前国内最大的极早产儿转运中心,所收治极早产儿中,90%通过本院新生儿转运系统由分娩医院主动转入。众所周知,极早产儿转运可能增加死亡和颅内出血等并发症风险,但本研究结果表明转运新生儿和本院出生新生儿的存活率、主要并发症比例无显著差异,提示通过完善的转运网络,短距离、2小时内新生儿转运以及区域性大规模的Ⅲ级NICU救治可以将转运相关的不良事件降至最低。
4、极早产儿,特别是超早产儿的预后差风险高、疾病负担重,患儿家长救治意识(担心预后)以及社会、家庭经济水平严重影响这些患儿的预后。
第二部分人脐带血间充质干细胞制备研究
目的：
间充质干细胞(mesenchymal stem cells, MSCs)是干细胞家族的重要成员,具有高度增殖、自我更新能力,广泛分布于骨髓、脐带血、脐带、胎盘等各种组织中。由于脐带血来源丰富,采集方便,排斥小,无伦理争议等优点,脐带血来源MSCs作为新生儿严重疾病移植用种子细胞受到重视。然而,脐带血(Umbilical Cord Blood, UCB) MSCs分离培养成功率低,目前对于如何从脐带血中获取MSCs仍存在争议。本研究的目的：(1)研究UCB-MSCs其体外分离、纯化及培养条件,并对其进行生物学特性进行鉴定,建立了稳定的UCB-MSCs培养体系。(2)明确不同GA来源UCB-MSCs培养成功率是否有差异。
方法：
(1)无菌条件下收集新鲜健康新生儿UCB,采用密度梯度离心法结合直接贴壁法体外分离UCB-MSCs,加入含体积分数为10%人胎牛血清的DMEM/F12培养基,调整细胞密度为1×1011/L接种于胎牛血清包被过的塑料培养瓶中,置于37℃、体积分数为5%的C02饱和湿度环境下培养。当培养瓶中的细胞生长到80%融合时,胰酶-EDTA混合液消化传代。观察贴壁细胞的形态学特征,计算累积群体倍增水平,应用流式细胞技术分析细胞增殖活性、免疫表型,特异性染色方法鉴定细胞的多向分化潜能。(2)将UCB依GA分为3组：足月儿组(GA>36周；n=16)；早产儿组(GA32-36周；n=11)；极早产儿组(GA<32周；n=9)。采取相对适应的条件培养,观察能成功培养出MSCs的比率,探讨GA、UCB采集量、UCB单核细胞(monocuclear cells,MNCs)数量对MSCs培养成功率的影响。
结果：
(1)UCB-单核细胞分离接种4-5d后开始可见有少量细胞贴壁,后贴壁细胞逐渐增多,并出现少量梭形和多角形细胞,2周后可见细胞形成集落,培养4-5周呈现融合。第2代细胞接种后24h内开始贴壁,2周左右即可达80%～90%融合。流式细胞仪细胞表面分子标记检测,此类细胞稳定地表达相关MSCs抗原标记CD105、CD90、CD73和HLA-ABC,但不表达造血干细胞标志物CD14、CD34和CD45。经特殊培养基诱导细胞分化实验证实成功向软骨、脂肪分化。传代15代以内细胞数扩增能力逐渐降低,第3代至第7代扩增能力较明显。流式细胞仪细胞周期及细胞凋亡检测表明细胞增殖力和生长均良好。细胞反复冻存复苏后细胞生物特性稳定。
(2)GA、UCB采集量、MNCs数量对UCB-MSCs培养成功与否的有显著影响：①UCB-MSCs培养成功率为52.8%,成功者GA明显小于未成功者(34.0±4.2vs36.8±3.3wk;t=2.184,P=0.036),MSCs培养成功率随GA增加呈降低趋势,差异无显著统计学意义(x2=3.769,P=0.152)。②成功者采血量显著高于未成功者(43.2±13.6vs32.1±11.5ml;t=2.635,P=0.013)。成功者不同GA间UCB采血量有显著差异(F=4.003,P=0.039),GA越大,采血量越多。③成功培养MSCs的样本单位体积内MNCs数显著高于未培养出MSCs的样本(32.9±11.7vs23.7±6.5×106/ml；t=2.863,P=0.008)；成功者不同GA间UCB单位体积内MNCs数无显著差异(F=1.087,P=0.361)。④按GA分层对MSCs产率比较,结果发现三组间有显著性统计学差异(F=13.309,P=0.000):GA越小,MSCs产率越高。
结论：
(1)本实验通过对传统方法的优化,根据细胞生长形态、细胞表明标记分子以及体外分化实验结果表明我们成功分离UCB-MSCs,同时体外扩增可以获得丰量的UCB-MSCs,细胞经冻存后复苏培养、反复传代后检测细胞生物学特性稳定,表明我们已建立了稳定的UCB-MSCs培养体系。
(2)UCB培养成功率仍较低,其中GA.UCB采集量、MNCs数量对UCB-MSCs培养成功与否的有显著影响。小胎龄、UCB采集量越多、单位体积内MNCs数量越多等可能是UCB-MSCs相对容易培养成功的原因。小胎龄UCB-MSCs产率高于大胎龄,提示小胎龄UCB可能是更佳的MSCs体外分离培养来源。
PART Ⅰ A Retrospective Analysis of Clinical Characteristics of Very Preterm Infants
Objective:
Internationally, the neonatal outcomes of very preterm (VPT) infants (born at<32weeks of GA) has became the highest priority of clinical investigation. However, information regarding this topic is still scant in Chinese tertiary NICU. In the past two decades, the driving forces for major tertiary care centers with NICUs have emerged dramatically with an increase in the preterm birth rate in China. This change in the pattern of provided services has improved the morbidity and mortality of low gestational age (GA) high-risk infants, who are born at level Ⅰ and Ⅱ nurseries in community hospitals and transferred to a level Ⅲ medical center NICU for additional care. We have established a tertiary pediatric hospital (BaYi Children's Hospital, affiliated to the General Hospital of Beijing Military Region) with a specialized neonatal care center that serves as a referral centre for the city of Beijing in2007. The neonatal care center has developed with450infant incubators, more than8000admissions in2011and four subspecialty NICUs up to2012. As the largest tertiary care center, it serves a largely outborn population and cares for all regional neonates requiring complex medical and surgical subspecialty care in Beijing. Over the past years, active treatment of VPT infants has been a major topic of discussion in China, but information regarding clinical outcomes of VPT infants in tertiary NICU setting is scant. The aim of this study was to evaluate clinical practice, mortality and morbidity of VPT infants during hospitalization in this largest NICU from October2010to September2011. We paid special attention to the outcomes of outborn infants trasnported from lower level hospitals.
Methods:
Research personnel retrospectively analyzed clinical data collected by a standardized data collection sheet through medical record review. The study group consisted of infants born at<32weeks of GA and admitted within24hours of postnatal life to the VPT-NICU of BaYi Children's Hospital, between October1st,2010and September30th,2011. The admitted infants aged over24hours often with incomplete prenatal data or medical records were not included in the present study.
SPSS (SPSS for Windows, version20.0, IBM-SPSS, Chicago, IL, USA) was used for statistical analysis. The primary goal of this study was to provide descriptive statistics of the patient population. Continuous variables were presented as means and SD or medians and range or interquartile range (IQR,25th to75th percentile), depending on whether their distributions were or were not highly skewed; categorical variables as counts or rates, and odds ratios (ORs) with95%confidence intervals (CI). Comparison between continuous variables was made by using a Mann-Whitney U test. Univariate analyses on categorical data were performed by using a2-tailed Pearson χ2or Fisher's exact test wherever appropriate. Logistic regression models were used to analyze the risk factors for death in VPT infants. Multivariate logistic regression analysis included GA, BW categories, gender, caesarean section, small for gestational age (SGA), premature rupture of membrane (PROM)>24h and admission age>2h as independent factors. Values of P<0.05were considered to be signifcant.
Results:
1. Study Population
A total of1262case records were retrospectively reviewed. Of these records,729infants born before32weeks'GA were subsequently admitted to the NICU within24hours of postnatal life; that is, they met the inclusion criteria for this study.
Of these VPT infants,22.5%were extremely preterm (EPT) infants (GA≤28wk, n=164),65.6%very low birth weight (VLBW,<1500g, n=478) infants and9.3%extremely low birth weight (ELBW,<1000g, n=68) infants.
The mean GA and BW of all infants was29.8±1.4(range23.9-31.9) weeks,1399±285(range610-2415) g, respectively. BW increased with every week of gestation (P<0.001). The male/female ratio was1.4:1. Boys weighed more than girls (1430±290vs1356±272g; P=0.001), whereas there was no significant difference in GA (29.7±1.4vs29.9±1.4weeks; P=0.054).
76(10.4%)VPT infants were small for gestational age infants,199(27.3%) multiple births. The mean admission age was2.2±2.3(range0.3-24) h.
2. Perinatal Characteristics and Delivery Room Interventions
Of all VPT infants(n=729),74.6%VPT infants received regular prenatal care,19.3%prenatal care more than once or no prenatal care.27.3%infants were treated with prenatal steroids. Cesarean sections were done in362(49.7%) deliveries and the cesarean section delivery rate increased with GA (OR1.607;95%CI1.425-1.812; P <0.001), with the steepest increase between GA of≤26and31weeks (17.9%at≤26weeks and70.1%at24weeks). There were more girls than boys (55.5%vs45.6%; P=.008) among infants born by cesarean section and also more SGA compared to those by vaginal route (17.7%vs3.3%; P<0.001).21.0%infants were born with PROM>24h,8.8%received antibiotics. Diagnosed gestational diseases were occurred in63.3%VPT infants, including21.9%gestational hypertension,33.6% PROM,9.9%both placenta praevia or abruption and gestational diabetes mellitus.
29.2%(n=213) VPT infants were treated with oxygen for resuscitation in the delivery room, and of whom48.3%received by nasal cannula or mask, the remaining51.7%received by tracheal intubation;3.4%received chest compression and1.9received resuscitation drugs.
3. Primary diagnosis in NICU
Infants at the lowest GA and BW were at the greatest risk for morbidities of prematurity. Diseases of respiratory system were the most common diagnosis in VPT infants. Overall,68.4%of infants experienced neonatal respiratory distress syndrome (RDS),48.4%pneumonia of newborn,8.4%pneumorrhagia,7.5%pulmonary hypertension,3.0%bronchopulmonary dysplasia (BPD),2.2%pneumothorax and so on. The other main diagnosis included68.4%patent ductus arteriosus (PDA),39.4%apnea,12.6%sepsis,21.5%intraventricular hemorrhage (IVH),1.5%periventricular leukomalacia (PVL),3.0%necrotizing enterocolitis (NEC),1.7%retinopathy of prematurity (ROP) more than stage3and so on.
4. Characteristics of Diseases of Respiratory System and Clinical Practices
Of all RDS infants (n=499),89.6%received pulmonary surfactant (PS), and13.2%received PS more than once. Campared with no PS treated infants with RDS, the survival rate of PS treated infants was insignificant, while lower mean GA, higher rates of morbidities, longer mean length of mechanical ventilation (MV) and oxygen supply was found in PS treated infants. The survival rate, mean GA, rates of morbidities, mean length of MV and oxygen supply was no significant difference between infants received PS in the delivery room and infants received PS in the NICU. In addition, the effect of different surfactants (poractant alpha, Curosurf and calfactant, Calfsurf) has not shown any significant difference in the survial rate (Curosurf vs Calfsurf:86.6%vs92.7%; P=0.105), rates of main severe morbidities, mean length of MV and oxygen supply. However, infants who received Curosurf were noted to have indications that they were more critically ill than Calfsurf infants, with lower mean GA and BW, longer length of hospital stay (P<0.05).
All of infants with RDS were treated with MV (n=499), and of whom15.8% only received nasal continuous positive airway pressure (nCPAP),71.5%received only conventional mechanical ventilation (CMV) and/or nCPAP, and12.6%received high frequency oscillatory ventilation (HFOV). The survival rate of infants who received HFOV was the lowest, the mean length of MV was the longest, the rates of main complications and severe morbidities (including pneumorrhagia, pneumothorax, IVH grade3or4) were the highest. For the infants treated with only CMV and/or nCPAP, the survival rate and rates of main complications and severe morbidities were insignificant except a higher rate of pneumorrhagia (11.2%vs0%; P<0.01) compared with infants received only nCPAP.
The positive rate of tracheal aspirates culture in pneumonia of newborns was16.1%(n=57). The main microorganisms recovered from tracheal aspirates culture were Acinetobacter baumannii (49.1%), Klebsiella pneumoniae (31.6%), Escherichia coli (10.5%) and Pseudomonas aeruginosa (7.0%).
Of all infants with pulmonary hypertension (n=55),43.6%were treated with HFOV,80.0%treated with iNO. Infants with pulmonary hypertension often diagnosed with PDA. The proportion of infants with PDA who were treated surgically were higher in infants with pulmonary hypertension compared with no pulmonary hypertension infants (16.4%vs1.9%; P<0.001). For the infants who were treated with iNO (n=48),16.7%received PDA ligation, which accounded for36.4%of infants with PDA treated surgiallly.
As one of the severe pulmonary morbidities,22(3.6%) of606infants needed oxygen therapy at36weeks and developed BPD. Postnatal steroids was given to7(31.8%) infants by intravenous injection after BPD diagnosed.3(13.6%) infants of BPD discharged home on oxygen and the remaining19(86.4%) infants were survived until discharge without oxygen.
5. Other Primary Diagnosis and Clinical Practices
All infants underwent≥1cranial ultrasound evaluation after admission;77.9%(n=572) of these results were normal. The lowest GA of infants were experienced the highest risk of brain injury. The proportion of EPT infants with FVH was42.1%, and24.3%in VLBW infants.
Of92infants diagnosed with sepsis,76.1%blood culture of them were positive.The main microorganisms recovered from blood culture were Klebsiella pneumoniae (37.1%), Staphylococcus (30.0%), Fungus(12.9%), Escherichia coli (7.1%) Acinetobacter baumannii (5.7%) and so on.
22(3.6%) infants developed for NEC and of whom36.4%died, which included62.5%withdrawn and only37.5%received surgical treatment.
Among729cases,87.5%(n=638) underwent at least an ophthalmologic examination before hospital discharge, death, or transfer.16.6%of these infants were diagnosed with ROP any stage, with47.2%in127EPT infants and23.4%in410VLBW infants.8.5%of122EPT infants with VLBW were performed retina photocoagulation for ROP stage≥3while infants born at higher GAs or BW≥1500g were not detected for development of severe ROP. The median length of oxygen supply was significantly longer in infants with ROP than those without ROP (26vs7days; P<0.001). It was also significantly longer in EPT infants treated with ROP photocoagulation than those ROP infants without surgery (44vs29days; P<0.001).
The rate of infants with PDA was inversely related to GAs.82.3%(n=380) of infants with PDA received oral ibuprofen administration, and of whom,34.2%received ibuprofen more than one course. The lower GAs infants had, with the more courses of ibuprofen they were treated, the lower proportion of PDA closed and the higher rate of PDA surgery after ibuprofen administration.56.2%of infants with PDA were closed after the first course of ibuprofen administered,22.7%closed with more courses of ibuprofen administered, and4.8%received PDA surgery. Only17.7%of infants with PDA were not treated with ibuprofen, and of whom,90.2%closed spontaneously, which was significantly higher than the rate (78.9%) in the infants treated with ibuprofen (P<0.05). Infants who received ibuprofen were found to have indications that they were more critically ill than no ibuprofen treated infants, with lower GA and BW, higher rate of pneumorrhagia, longer length of MV and oxygen supply, and more money spended in the NICU, while no significant difference in the survival rate (P=0.623). Similarly, infants who received more courses of ibuprofen often had lower GA, higher rate of pulmonary hypertension, iNO treated and PDA ligation, longer length of MV, oxygen supply and hospital stay, and more money spended in the NICU, whereas no significant difference in the survival rate (P=0.495) compared with infants treated with one course of ibuprofen.
6. Neonatal Transport and Outcome-based Comparison
An extremely high proportion (90.0%) of admissions were born at lower level centers and subsequently transported, the remaining10.0%were inborns. The median age of NICU admission was2.0(IQR:1.5-3) hours for the outborns, which was significantly longer than0.5(IQR:0.5-1) hours for the inborns (P<0.001). No difference was found in both the mean GA and BW for the inborn and outborn infants. The rates of cesarean section and PROM>24h were higher in inborns than the rates in outborns, while more multiple births in outborns than inborns (P<0.05). Outborns experienced higher but insignificant need for intensive delivery room and NICU interventions compared with inborns (P=0.084).566(78.6%) infants were treated with supplemental oxygen therapy in transport. The main types of oxygen supply were oxyhood (55.7%) and endotracheal intubation (41.9%) in the transportation. The rate of infants treated with oxygen supply was conversely related to GA and BW (P<0.001). However, rates of oxygen use and MV were similar for the inborn and outborn infants.
Rates of diseases of respiratory system, main clinical practices and IVH were found no significant difference in the inborn and outborn infants (P>0.05). A little but not so high mortality was seen in the outborns when compared to the inborns (8.7%vs5.5%; P=0.348). Rates of main complications, length of MV, oxygen supply and hospital stay were also found insignificant difference among the inborn and outborn survivors (P>0.05).
Compared with different admission age, the mean GA and BW of infants with admission age≤2h were no significant difference with those of infants with admission age>2h. For the perinatal characteristics, tube baby, gestational hypertension diseases, regular prenatal care, prenatal steroids and cesarean section were prevalent in infants with admission age≤2h, while asphyxia and intensive delivery room interventions were more common in infants with admission age>2h. For the outcomes of VPT infants in the NICU, higher survival rate (91.2%vs84.8%; P<0.05) and lower proportion of withdrawal of care (11.2%vs21.7%; P<0.01) were found in infants with admission age<2h compared with infants with admission age>2h. There was no difference in occurrence of main severe morbidities, length of MV, oxygen use and hospital stay, and cost of hospital (P>0.05).
7. Mortality Rates and Risk Factors of Death
The overall mortality rate of VPT infants until discharge from the hospital was8.4%(n=61), which consisted of hospital deaths either in the process of intensive care (1.8%) or after the withdrawn/withhold of support (6.6%). The death rate of VLBW and ELBW infants was10.5%and26.5%, respectively, which was higher compared to4.4%of infants with BW≥1500g (P<0.001). In the lower ranges of GA and BW (both P<0.001), a striking stepwise reductions in mortality rate occur significantly with each additional week of gestation and250g increase in BW. Most of deaths occurred within neonatal period:44.3%occurred in the early neonatal period (0-6days postpartum),49.2%during the late neonatal period (7-28days postpartum), while only6.6%of deaths after neonatal period.
The results of the multivariate logistic regression analysis showed that lower GA, invasive MV, admission age>2h, air leak, pulmonary hemorrhage, no PS therapy and presence of a major anomaly were the independent risk factors for death.
8. Length and Costs of Hospitalization
The median LOS among survivors was43days, and decreased with increasing GA, from92days at26weeks to37days at31weeks (P<0.001). PMA at discharge decreased from39.2weeks for surviving infants born at GAs of26weeks to35.7weeks or36.3weeks for those born at30or31weeks, respectively. The median cost of surviving infants was1.4-fold to the entire annual income of a Beijing urban resident in2011(32903CNY). The median cost of hospital in infants with GA≤26wk and31wk was132771and38142CNY, respectively.
9. Withdrawal or Withholding of Care
18.0%(n=131) of VPT infants support were withdrawn from the treatment, including27.4%in EPT infants. The withdrawal rate of support was20.5%(n=98) among VLBW infants and25.0%among ELBW infants. Of all infants withdrawn from the treatment,51%were withdrawn due to the parents'inability to afford the high cost of continued medical treatment.36.6%of infants died after withdrawal of support in the care unit. No distinction of survival was detected in the gender discrepancy (males vs females:90.9%vs92.6%; P=0.412) and the treatment withdrawn happened almost equally to female and male infants (18.4%vs17.7%; P=0.803).
10. Outcome-based Comparison with Developed Countries
The rate of provide active obstetric care and initiate neonatal intensive care for the most-premature infats was obviously lower in Chinese NICU than the rate in developed countries. An extremely high rate of VPT infants transported was found in the present study, while a much lower rate was found in developed countries. More antenatal transferred VPT infants were found in developed NICU. The overall survival rate of the VPT infants was similar with the rate in developed countries; specifically, a relative lower proportion and survival rate of EPT was found in this study.
Conclusions:
1. This study is the first report to summarize the management, mortality and morbidity among very preterm infants born at less than32weeks of gestation at the very preterm-NICU of the largest neonatal intensive care center in China.89.5%of VLBW infants survived to discharge, this nearly achieves the rate of90%of infants born with VLBW in subspecialty perinatal centers in the United States in2010.
2. The rate of provide active obstetric care (including prenatal steriods, cesarean section) and initiate neonatal intensive care for the most-premature infants (timely fetus or infant transferred to a higher level hospital) was obviously lower in the present data than the rate in developed countries, which would result in delaying the time to rescue the VPT infants and partially explain the worse outcomes of EPT infants. As we know, prenatal steroids treatment has been applied in clinical practice decades and recent reports have affirmed that antenatal exposure to steroids was associated with a lower rate of severe morbidity, death or neurodevelopment impairment among VPT infants in developed countries.
3. This VPT-NICU has been the largest VPT infants referral centre in China.90%of infants were born at lower level centers and subsequently transported to the NICU. As many studies reported, outborn VPT infants may have poor outcomes with regards to survival and morbidity when compared to inborn infatns. An unexpected finding was that no significant difference was found in the rates of mortality and servere morbidity between the inborn and outborn infants; the results may suggest that both short-distance within about2hours neonatal ground transport by well-developed hospital referral/infant transport systems and high-volume NICU may minimize the adverse effects of transport.
4. Of concern, the cost of hospital stay, especially in EPT infants with severe morbidity, were very high in the present study. The direct costs incurred during neonatal intensive care unit hospitalization for such infants approximated the annual income of an urban resident family. In addition, it should be noted that death after withdrawal or withholding form intensive care, contributed to the high mortality which accounted for79%of the total death in this article. The high cost of continued medical treatment and the high risk of poor outcomes, often withdraw or withhold the parents'decisions to provide active intensive care for VPT infants in China. In fact, socioeconomic and culture status has been a substantial impact on the outcomes of VPT infants.
PART II A Lab Study on Isolation of Mesenchymal Stem Cell from Human Umbilical Cord Blood
Objective:
Mesenchymal stem cells (MSCs) consist of a rare population of multipotent progenitors having the capacity for self-renewal and differentiation into various lineages of mesenchymal tissues. This ability makes MSCs an attractive tool in the field of therapeutic use. These cells can be isolated from different tissues such as bone marrow, umbilical cord, adipose tissue, dental pulp, and umbilical cord blood (UCB). UCB is an interesting source of these cells because the collection process is painless and non-invasive, it causes no harm to the mother or infant, and it is a material usually discarded. Unfortunately, reliable procedures for efficient expansion and differentiation of UCB-MSCs remain to be established. The isolation, characterisation, in vitro expansion and differentiation of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were among the current aims of this study, and achieving these goals is a pre-requisite for extensive use of this novel approach for the treatment of a number of human diseases. Additionally, the factors that influence the rate of success of MSCs isolation culture and the yields of MSCs from UCB of different gestational age deliveries were investigated.
Methods:
(1) UCB units from full-term deliveries were collected with informed consent of the mothers. We separated cells on Ficoll density gradient, and the monocuclear cells (MNCs) fraction was collected and washed in PBS. MNCs were seeded in culture at a density of1×10/L into a25cm2culture flask, precoated with5%fetal bovine serum (FBS), in DMEM/F12+10%FBS medium in the presence of100units/ml penicillin and100U/ml streptomycin, at37℃, in5%CO2. Every3to4days, we changed the medium until subconfluency was obtained, and cells were harvested with a trypsin-EDTA solution and replated at at a density of1×108/L. The morphology of MSCs derived from the UCM was taken. Cumulative population doubling levels were calculated for each subcultivation. Immunophenotyping of MSCs was detected using flow cytometry. The adipogenic and chondrogenetic differentiation studies were induced.
(2) UCB was collected at birth in neonates of three different gestational groups, cultured with the same culture conditions. The relationship of the yields of MSCs derived from UCB with several factors such as GA, the collected volume of UCB and the MNCs count of UCB and the relationship among these factors were investigated.
Results:
(1) After plating the MNCs, a few cells attached to the plastic culture dishes and formed adherent cells within4to5d. Most of those cells were monocytes, which fused toform osteoclast-like cells. The onset of colony formation could be observed at first after2weeks. These appeared in80%to90%of the flask coated with FBS and reached a subconfluent condition within4to5weeks. The expression of cell-surface antigens by flow cytometry was evaluated at passage4. The isolated cells significantly expressed MSCs markers CD105、CD90、CD73and HLA-ABC, while they lacked expression of the hematopoietic markers CD14, CD34and CD45. Differentiation capacity of MSC derived from HUCB was cultured in differentiation medium to induce adipogenic and chondrogenetic differentiation respectively. The MSCs demonstrated a multilineage capacity of adipogenic and chondrogenetic differentiation. The cumulative population doubling levels increased sharply for the the passage3to passage7and apparent slowly after the passage9. The bioactivities of MSCs had no changes after cell culture from thawing of frozen cells.
(2) There were significant correlations between the success rate and such factors as the gestaional age (GA), the volume and the MNCs count of UCB.①The success rate of generating MSCs cells from UCB was up to52.8%. The GA of UCB deliveries with succesful isolating MSCs (the successful UCB) was significantly lower than that of UCB deliveries without isolating MSCs (the unsuccessful UCB;34.0±4.2vs36.8±3.3wk;t=2.184, P=0.036). The trend but insignificantly toward the success rate declined with the increasing GA (χ2=3.769, P=0.152).②The volume of the unsuccessful UCB was significantly more than that of the unsuccessful UCB (43.2±13.6vs32.1±11.5ml; t=2.635, P=0.013). There was a positive correlation between the volume and the GA in the successful UCB (F=4.003, P=0.039).③The density of MNCs in the unsuccessful UCB was significantly higher than that of the unsuccessful UCB (32.9±11.7vs23.7±6.5×106/ml;t=2.863, P=0.008). In average, the yield of the MNCs was comparable between the three GA groups (F=1.087, P=0.361).④The MSCs count as percentage of MNCs was in a significant inverse correlation with GA; that is, the density of MSCs and GA, which reduces as the maturity increased (F=13.309, P=0.000).
Conclusions:
(1) MSCs can be isolated and cultivated successfully in vitro from UCB with the optimal culture conditions,1×1011/L mononuclear cells seeded in DMEM/F12culture medium containing10%FBS and a culture flask precoated with FBS. The bioactivities of these MSCs were identical with that of the MSCs reported.
(2) The success rate of generating MSCs cells from UCB was low. Crucial points to isolate MSCs cells from UCB were the gestaional age (GA), the volume and the MNCs count of UCB. Lower GA, more volume and higher density of MNCs of UCB seems like with higher success rate isolating MSCs cells. The UCB from preterm infants is a better source of MSCs than that from full term neonates.
目的：
极早产儿(very preterm infants, VPT,胎龄<32wk)由于其解剖生理特点,易发生各种并发症,病死率及后遗症的发生率较高,对极早产儿周密的全面管理,是新生儿监护病房(NICU)中体现水平的综合反映。国内在极早产儿NICU救治、预后等方面研究资料匮乏。本研究通过回顾性分析超大容量NICU极早产儿临床病例资料,旨在了解：(1)极早产儿围生期特征、NICU中主要疾病构成、治疗手段及疾病负担；(2)极早产儿存活率、主要并发症患病比例以及预后的高危因素；(3)新生儿转运以及超大容量NICU对极早产儿临床转归的影响；(4)国内极早产儿救治现状与发达国家救治情况比较。希望通过此文研究为当前极早产儿救治难点、重点提供可靠的临床研究资料,为更大规模的极早产儿流行病学研究提供重要的理论依据。
方法：
回顾性收集2010年10月1日至2011年09月30日连续12个月内,八一儿童医院极早产NICU中收治的极早产儿病例资料。病例纳入标准：出生胎龄(GA)小于32wk、生后24小时内入住极早产NICU者。
用SPSS软件进行统计分析。计量资料中连续变量呈正态分布者表示为均数±标准差,组间均数比较采用t检验或单因素方差分析；偏态分布者表示为中位数(范围或四分位数间距),组间均数比较采用秩和检验。计数资料以发生率或构成比表示,并对发生率作区间估计,发生率或构成比的比较采用卡方检验。因本研究为回顾性分析,结合研究人群特点,采用单因素Logistic回归模型对极早产儿死亡高危因素分析,进一步运用多因素Logistic回归模型进行多因素逐步回归分析以明确死亡的独立风险因素。变量间相关性采用Spearman等级相关分析。
结果：
1.人口学特征
本次研究共收集病例1262份,符合入选标准的病例729例。其中164例为超早产儿(胎龄,GA≤28wk),占总数的22.5%；478例为极低出生体重儿(VLBW<1500g),占总数的65.6%；68例为超低出生体重儿(ELBW<1000g),占总数的9.3%。平均GA为29.8±1.4(范围23.9-31.9)wk。平均出生体重(BW)为1399±285(范围610-2415)g。男婴430例,占总的例数的59.0%。76例(10.4%)为小于胎龄儿(SGA),199例(27.3%)为多胎。平均入NICU时龄为2.2±2.3(范围0.3-24)小时。
2.围产期特征
544例(74.6%)极早产儿接受了规律产前检查,141例(19.3%)产前检查大于1次或情况不详。199例(27.3%)应用产前激素,362例(49.7%)剖宫产。153例(21.0%)胎膜早破>24h,64例(8.8%)产前抗生素治疗。461例(63.3%)的极早产儿母亲存在明确妊娠期疾病,包括妊娠高血压疾病160例(21.9%)、胎膜早破245例(33.6%)、前置胎盘或胎盘早剥72例(9.9%)、糖尿病72例(9.9%)等。213例(29.2%)极早产儿中有的产房用氧复苏史,其中103例(14.1%)极早产儿接受过氧气或气囊加压给氧,110例(15.1%)极早产儿在产房接受了气管插管,25例(3.4%)极早产儿接受了产房心脏按压,14例(1.9%)极早产儿产房复苏药物。
3.极早产儿NICU主要疾病构成
极早产儿主要疾病构成与GA、BW成密切相关,胎龄、BW越小,患病比例越高。呼吸系统疾病是极早产儿最常见诊断疾病,主要包括：499例(68.4%)呼吸窘迫综合症(RDS),353例(48.4%)新生儿肺炎,61例(8.4%)肺出血,55例(7.5%)新生儿持续性肺动脉高压(PPHN),22例(3.0%,,n=606例)支气管肺发育不良(BPD),16例(2.2%)气胸等。其他常见疾病主要有动脉导管未闭(PDA)462例(63.4%),呼吸暂停287例(39.4%),败血症92例(12.6%),脑室内出血(IVH)157例(21.5%),脑室周围白质软化(PVL)11例(1.5%),坏死性小肠结肠炎(NEC)22例(3.0%),3期以上早产儿视网膜病(ROP)11例(1.7%)等。
4.呼吸系统疾病特征及临床治疗特点
499例RDS极早产儿中,447例(89.6%)接受了肺表面活性物质(PS)治疗,66例(13.2%)接受了超过1次的PS治疗。应用PS与未使用PS相比,存活率无差异,但PS患儿GA较低、并发症比例较高、MV和氧疗时间较长；产房使用PS与NICU使用PS相比,两者GA、存活率、并发症、MV和氧疗时间等均无显著差异(P>0.05)；与牛源性PS相比,猪源性PS患儿GA、BW更低,住院时间更长,存活率较低但无显著差异(x2=2.625,P=0.105),主要并发症、MV和氧疗时间等均无显著差异(P>0.05)。
RDS患儿499例(100%)接受机械通气(MV),其中79例(15.8%)只接受nCPAP,357例(71.5%)接受常频机械通气(CMV,不含HFOV),63例(12.6%)接受HFOV。三种通气方式中,HFOV存活率最低,MV时间最长,相关疾病或并发症比例最高(肺出血、气胸、重度IVH)；与仅nCPAP相比,CMV除肺出血比例较高外,存活率或主要并发症无差异。
358例新生儿肺炎患儿中,57例(16.1%)痰培养阳性,其中培养阳性率较高的有：28例(49.1%)鲍曼不动杆菌,18例(31.6%)肺炎克雷伯菌,6例(10.5%)大肠埃希菌和3例(7.0%)铜绿假单胞菌。
55例PPHN患儿中HFOV的比例为24例(43.6%),吸入NO治疗的比例为44例(80.0%)。PPHN与PDA密切相关,接受PDA结扎术患儿中患有PPHN的比例显著高于非PPHN患儿(16.4%vs1.9%,P<0.001)。48例吸入NO的患儿中,8例(16.7%)需要接受PDA结扎术,占所有PDA结扎术患儿(22例)的36.4%。
606例极早产儿纠正胎龄36wk治愈或好转出院无氧依赖,22例(3.6%)纠正胎龄36wk仍需氧疗诊断为BPD。其中7例(31.8%)在生后28天接受激素治疗,3例(13.6%)出科时需吸氧,19例(86.4%)治愈出院。
5.其他常见疾病特征及临床治疗特点
极早产儿入住NICU后根据病情需要至少进行1次床旁头颅超声。157例(21.5%)IVH是超声诊断比例最高的疾病,GA越小、BW越低,IVH比例越高。IVH在超早产儿比例为42.1%,VLBW比例为24.3%。
92例败血症极早产儿中,血培养阳性70例(76.1%)。其中,肺炎克雷伯菌培养阳性率最高37.1%(26例),其次为葡萄球菌30.0%(21例)、真菌12.9%(9例)、大肠埃希菌7.1%(5例)、鲍曼不动杆菌5.7%(4例)等。
22例NEC患儿中,只有7例(31.8%)接受手术治疗,病死率为36.4%(8例)。死亡患儿中3例(37.5%)接受了手术治疗,5例(62.5%)选择了放弃治疗。
638例极早产儿在出院或死亡之前至少接受了1次眼底筛查,106例(16.6%)极早产儿有不同程度ROP表现,其中127例受检超早产儿、410例受检VLBW患儿的检出比例分别为47.2%(60例)、23.4%(96例)。ROP患儿中11例(10.4%)接受了激光光凝手术治疗。与未检出ROP的存活极早产儿比较,ROP患儿的吸氧时间显著延长(中位数：26天vs7天,P<0.001)；与未接受手术治疗的ROP超早产儿相比,接受ROP光凝术的超早产儿氧疗时间显著较长(中位数：44天vs29天,P<0.001)。
极早产儿PDA比例按胎龄分层有显著差异,GA越小,患病比例越高。462例PDA患儿中,380例(82.3%)接受了布洛芬治疗,其中130例(34.2%)患儿接受布洛芬治疗超过1个疗程,GA越小,接受布洛芬治疗疗程越多。365例接受心脏超声复查,205例(56.2%)患儿首个疗程后PDA闭合,83例(22.7%)的患儿需接受多个疗程后PDA闭合；GA越小,PDA闭合的比例越低。22例(4.8%)PDA患儿在接受布洛芬治疗后选择PDA结扎术；GA越小,PDA结扎术的比例越高。82例(17.7%)PDA未接受布洛芬治疗,心脏超声复查PDA自发性闭合的比例较布洛芬治疗的比例高(90.2%vs78.9%,P<0.05),但布洛芬治疗者GA和BW均较低、合并肺出血的比例较高、接受MV、氧疗和住院时间较长以及住院费用较高,存活率无差异。接受多疗程布洛芬治疗的PDA患儿,GA较低,合并PPHN及吸入NO治疗的比例、PDA结扎术的比例均较高,接受MV、氧疗和住院时间较长以及住院费用较高,但存活率与1个疗程布洛芬PDA患儿相比无差异(92.8%vs94.6%,P=0.495)。
6.极早产儿转运特点以及对极早产儿临床转归的影响
入选研究对象中,73例(10%)为本院出生新生儿(inborn),656例(90%)为转运新生儿(outborn)。转运新生儿入院时龄为2.0小时(中位数),本院出生新生儿为0.5小时(中位数),两者有显著性差异；GA、BW无差异(P>0.05)。本院出生新生儿剖宫产、胎膜早破>24小时比例高于转运新生儿,而后者多胎比例高于前者(x2=4.173,P=0.041)。转运新生儿产房抢救措施比例较本院出生新生儿高,但无显著统计学差异(x2=3.588,P=0.058)。573例(78.6%)极早产儿入NICU前转运中接受氧疗。转运中氧疗模式主要为面罩319例(55.7%)和气管插管气囊加压人工呼吸240例(41.9%)；氧疗的比例与GA、BW密切相关,胎龄越小、BW越低,接受氧疗比例越高(x2=124.7,P<0.001)。
对极早产儿入院后呼吸系统主要疾病、治疗措施以及IVH比例比较,转运新生儿和本院出生新生儿无差异(P>0.05)。治愈或好转出院患儿中转运新生儿与本院出生新生儿的存活率无差异(91.3%vs94.5%,P=0.384),主要并发症比例、MV、氧疗和住院时间、住院费用均无统计学差异(P>0.05)。
对入院极早产儿按入院时龄2小时(中位数)分层比较,入院时龄≤2h组GA、 BW均无显著统计学差异。入院时龄<2h患儿母亲体外人工受精妊娠、妊娠高血压疾病、规律产检、产前激素治疗和选择剖宫产比例显著高于入院时龄>2h组,而后者生后窒息、产房有创性复苏措施比例较高。入院时龄≤2h组患儿存活率高于入院时龄>2h组(91.2%vs84.8%,P<0.05),且放弃治疗存活出院的比例显著低于后者(11.2%vs21.7%,P<0.01)；存活者中主要并发症比例、MV、氧疗时间和住院时间以及住院费用等均无统计学差异(P>0.05)。
7.死亡、存活者主要并发症高危因素分析
极早产儿NICU病死率为8.4%(61例),包括13例(1.8%)院内死亡和48例(6.6%)放弃治疗死亡。其中病死率超早产儿为21.3%(35例),VLBW为10.5%(50例),ELBW患儿为26.5%(18例)。GA、BW越低,病死率越高。57例(93.4%)患儿死于新生儿期。
多因素logistic回归分析表明低GA、有创MV、入院时龄>2h、合并气胸、合并肺出血、未使用PS治疗、严重先天畸形是极早产儿死亡的独立高危因素。
8.存活极早产儿住院时间和疾病负担
存活极早产儿住院时间为43天(中位数),住院费用为46409元(中位数),住院费用大约为同期北京市城市居民年收入的1.4倍。GA越小,住院时间越长、疾病负担越重。例如,住院时间(中位数)、住院费用(中位数)GA<26wk分别为92天、132771元,GA31wk分别为37天、38142元。
9.放弃或姑息治疗
131例(18.0%)极早产儿家长放弃或姑息治疗,其中67例(51.1%)是由于不能支付继续住院治疗费用,31例(23.7%)与患儿治疗效果差有关,21例(16.0%)是由于伴有严重并发症,12例(9.2%)是由于严重先天性疾病。患儿放弃治疗后死于医院的比例为36.6%(48例)。
10.极早产儿临床特征与发达国家比较
本研究中极早产儿产科干预比例与发达国家相比明显较低,新生儿转运比例明显高于发达国家,后者以产前转运为主。极早产儿总体存活率与发达国家水平相当,但主要并发症比例较发达国家低,分析可能与本院超早产儿的比例低、存活率较低有关。
结论：
1、本研究首次回顾性全面分析了国内超大容量NICU极早产儿病例资料,开创性的总结了国内首个NICU亚专科监护病房极早产儿的救治经验。研究结果表明极早产儿的救治水平,如89.5%的VLBW患儿治愈或好转出院,与美国等发达国家同期NICU中VLBW患儿存活率90%相当。
2、本研究中极早产儿产科主动干预比例(如产前激素应用、剖腹产比例)明显低于发达国家水平,而后者大量文献研究表明极早产儿产前激素应用可以显著降低极早产儿的病死率和并发症比例,因此我们可能在一定程度上延缓了极早产儿抢救时机,影响了极早产儿,特别是超早产儿在NICU中的转归。如我院超早产儿存活率为78.7%,与美国(88%)等发达国家同期水平相比,仍然存在一定的差距。
3、本院已成功建立了目前国内最大的极早产儿转运中心,所收治极早产儿中,90%通过本院新生儿转运系统由分娩医院主动转入。众所周知,极早产儿转运可能增加死亡和颅内出血等并发症风险,但本研究结果表明转运新生儿和本院出生新生儿的存活率、主要并发症比例无显著差异,提示通过完善的转运网络,短距离、2小时内新生儿转运以及区域性大规模的Ⅲ级NICU救治可以将转运相关的不良事件降至最低。
4、极早产儿,特别是超早产儿的预后差风险高、疾病负担重,患儿家长救治意识(担心预后)以及社会、家庭经济水平严重影响这些患儿的预后。
第二部分人脐带血间充质干细胞制备研究
目的：
间充质干细胞(mesenchymal stem cells, MSCs)是干细胞家族的重要成员,具有高度增殖、自我更新能力,广泛分布于骨髓、脐带血、脐带、胎盘等各种组织中。由于脐带血来源丰富,采集方便,排斥小,无伦理争议等优点,脐带血来源MSCs作为新生儿严重疾病移植用种子细胞受到重视。然而,脐带血(Umbilical Cord Blood, UCB) MSCs分离培养成功率低,目前对于如何从脐带血中获取MSCs仍存在争议。本研究的目的：(1)研究UCB-MSCs其体外分离、纯化及培养条件,并对其进行生物学特性进行鉴定,建立了稳定的UCB-MSCs培养体系。(2)明确不同GA来源UCB-MSCs培养成功率是否有差异。
方法：
(1)无菌条件下收集新鲜健康新生儿UCB,采用密度梯度离心法结合直接贴壁法体外分离UCB-MSCs,加入含体积分数为10%人胎牛血清的DMEM/F12培养基,调整细胞密度为1×1011/L接种于胎牛血清包被过的塑料培养瓶中,置于37℃、体积分数为5%的C02饱和湿度环境下培养。当培养瓶中的细胞生长到80%融合时,胰酶-EDTA混合液消化传代。观察贴壁细胞的形态学特征,计算累积群体倍增水平,应用流式细胞技术分析细胞增殖活性、免疫表型,特异性染色方法鉴定细胞的多向分化潜能。(2)将UCB依GA分为3组：足月儿组(GA>36周；n=16)；早产儿组(GA32-36周；n=11)；极早产儿组(GA<32周；n=9)。采取相对适应的条件培养,观察能成功培养出MSCs的比率,探讨GA、UCB采集量、UCB单核细胞(monocuclear cells,MNCs)数量对MSCs培养成功率的影响。
结果：
(1)UCB-单核细胞分离接种4-5d后开始可见有少量细胞贴壁,后贴壁细胞逐渐增多,并出现少量梭形和多角形细胞,2周后可见细胞形成集落,培养4-5周呈现融合。第2代细胞接种后24h内开始贴壁,2周左右即可达80%～90%融合。流式细胞仪细胞表面分子标记检测,此类细胞稳定地表达相关MSCs抗原标记CD105、CD90、CD73和HLA-ABC,但不表达造血干细胞标志物CD14、CD34和CD45。经特殊培养基诱导细胞分化实验证实成功向软骨、脂肪分化。传代15代以内细胞数扩增能力逐渐降低,第3代至第7代扩增能力较明显。流式细胞仪细胞周期及细胞凋亡检测表明细胞增殖力和生长均良好。细胞反复冻存复苏后细胞生物特性稳定。
(2)GA、UCB采集量、MNCs数量对UCB-MSCs培养成功与否的有显著影响：①UCB-MSCs培养成功率为52.8%,成功者GA明显小于未成功者(34.0±4.2vs36.8±3.3wk;t=2.184,P=0.036),MSCs培养成功率随GA增加呈降低趋势,差异无显著统计学意义(x2=3.769,P=0.152)。②成功者采血量显著高于未成功者(43.2±13.6vs32.1±11.5ml;t=2.635,P=0.013)。成功者不同GA间UCB采血量有显著差异(F=4.003,P=0.039),GA越大,采血量越多。③成功培养MSCs的样本单位体积内MNCs数显著高于未培养出MSCs的样本(32.9±11.7vs23.7±6.5×106/ml；t=2.863,P=0.008)；成功者不同GA间UCB单位体积内MNCs数无显著差异(F=1.087,P=0.361)。④按GA分层对MSCs产率比较,结果发现三组间有显著性统计学差异(F=13.309,P=0.000):GA越小,MSCs产率越高。
结论：
(1)本实验通过对传统方法的优化,根据细胞生长形态、细胞表明标记分子以及体外分化实验结果表明我们成功分离UCB-MSCs,同时体外扩增可以获得丰量的UCB-MSCs,细胞经冻存后复苏培养、反复传代后检测细胞生物学特性稳定,表明我们已建立了稳定的UCB-MSCs培养体系。
(2)UCB培养成功率仍较低,其中GA.UCB采集量、MNCs数量对UCB-MSCs培养成功与否的有显著影响。小胎龄、UCB采集量越多、单位体积内MNCs数量越多等可能是UCB-MSCs相对容易培养成功的原因。小胎龄UCB-MSCs产率高于大胎龄,提示小胎龄UCB可能是更佳的MSCs体外分离培养来源。
PART Ⅰ A Retrospective Analysis of Clinical Characteristics of Very Preterm Infants
Objective:
Internationally, the neonatal outcomes of very preterm (VPT) infants (born at<32weeks of GA) has became the highest priority of clinical investigation. However, information regarding this topic is still scant in Chinese tertiary NICU. In the past two decades, the driving forces for major tertiary care centers with NICUs have emerged dramatically with an increase in the preterm birth rate in China. This change in the pattern of provided services has improved the morbidity and mortality of low gestational age (GA) high-risk infants, who are born at level Ⅰ and Ⅱ nurseries in community hospitals and transferred to a level Ⅲ medical center NICU for additional care. We have established a tertiary pediatric hospital (BaYi Children's Hospital, affiliated to the General Hospital of Beijing Military Region) with a specialized neonatal care center that serves as a referral centre for the city of Beijing in2007. The neonatal care center has developed with450infant incubators, more than8000admissions in2011and four subspecialty NICUs up to2012. As the largest tertiary care center, it serves a largely outborn population and cares for all regional neonates requiring complex medical and surgical subspecialty care in Beijing. Over the past years, active treatment of VPT infants has been a major topic of discussion in China, but information regarding clinical outcomes of VPT infants in tertiary NICU setting is scant. The aim of this study was to evaluate clinical practice, mortality and morbidity of VPT infants during hospitalization in this largest NICU from October2010to September2011. We paid special attention to the outcomes of outborn infants trasnported from lower level hospitals.
Methods:
Research personnel retrospectively analyzed clinical data collected by a standardized data collection sheet through medical record review. The study group consisted of infants born at<32weeks of GA and admitted within24hours of postnatal life to the VPT-NICU of BaYi Children's Hospital, between October1st,2010and September30th,2011. The admitted infants aged over24hours often with incomplete prenatal data or medical records were not included in the present study.
SPSS (SPSS for Windows, version20.0, IBM-SPSS, Chicago, IL, USA) was used for statistical analysis. The primary goal of this study was to provide descriptive statistics of the patient population. Continuous variables were presented as means and SD or medians and range or interquartile range (IQR,25th to75th percentile), depending on whether their distributions were or were not highly skewed; categorical variables as counts or rates, and odds ratios (ORs) with95%confidence intervals (CI). Comparison between continuous variables was made by using a Mann-Whitney U test. Univariate analyses on categorical data were performed by using a2-tailed Pearson χ2or Fisher's exact test wherever appropriate. Logistic regression models were used to analyze the risk factors for death in VPT infants. Multivariate logistic regression analysis included GA, BW categories, gender, caesarean section, small for gestational age (SGA), premature rupture of membrane (PROM)>24h and admission age>2h as independent factors. Values of P<0.05were considered to be signifcant.
Results:
1. Study Population
A total of1262case records were retrospectively reviewed. Of these records,729infants born before32weeks'GA were subsequently admitted to the NICU within24hours of postnatal life; that is, they met the inclusion criteria for this study.
Of these VPT infants,22.5%were extremely preterm (EPT) infants (GA≤28wk, n=164),65.6%very low birth weight (VLBW,<1500g, n=478) infants and9.3%extremely low birth weight (ELBW,<1000g, n=68) infants.
The mean GA and BW of all infants was29.8±1.4(range23.9-31.9) weeks,1399±285(range610-2415) g, respectively. BW increased with every week of gestation (P<0.001). The male/female ratio was1.4:1. Boys weighed more than girls (1430±290vs1356±272g; P=0.001), whereas there was no significant difference in GA (29.7±1.4vs29.9±1.4weeks; P=0.054).
76(10.4%)VPT infants were small for gestational age infants,199(27.3%) multiple births. The mean admission age was2.2±2.3(range0.3-24) h.
2. Perinatal Characteristics and Delivery Room Interventions
Of all VPT infants(n=729),74.6%VPT infants received regular prenatal care,19.3%prenatal care more than once or no prenatal care.27.3%infants were treated with prenatal steroids. Cesarean sections were done in362(49.7%) deliveries and the cesarean section delivery rate increased with GA (OR1.607;95%CI1.425-1.812; P <0.001), with the steepest increase between GA of≤26and31weeks (17.9%at≤26weeks and70.1%at24weeks). There were more girls than boys (55.5%vs45.6%; P=.008) among infants born by cesarean section and also more SGA compared to those by vaginal route (17.7%vs3.3%; P<0.001).21.0%infants were born with PROM>24h,8.8%received antibiotics. Diagnosed gestational diseases were occurred in63.3%VPT infants, including21.9%gestational hypertension,33.6% PROM,9.9%both placenta praevia or abruption and gestational diabetes mellitus.
29.2%(n=213) VPT infants were treated with oxygen for resuscitation in the delivery room, and of whom48.3%received by nasal cannula or mask, the remaining51.7%received by tracheal intubation;3.4%received chest compression and1.9received resuscitation drugs.
3. Primary diagnosis in NICU
Infants at the lowest GA and BW were at the greatest risk for morbidities of prematurity. Diseases of respiratory system were the most common diagnosis in VPT infants. Overall,68.4%of infants experienced neonatal respiratory distress syndrome (RDS),48.4%pneumonia of newborn,8.4%pneumorrhagia,7.5%pulmonary hypertension,3.0%bronchopulmonary dysplasia (BPD),2.2%pneumothorax and so on. The other main diagnosis included68.4%patent ductus arteriosus (PDA),39.4%apnea,12.6%sepsis,21.5%intraventricular hemorrhage (IVH),1.5%periventricular leukomalacia (PVL),3.0%necrotizing enterocolitis (NEC),1.7%retinopathy of prematurity (ROP) more than stage3and so on.
4. Characteristics of Diseases of Respiratory System and Clinical Practices
Of all RDS infants (n=499),89.6%received pulmonary surfactant (PS), and13.2%received PS more than once. Campared with no PS treated infants with RDS, the survival rate of PS treated infants was insignificant, while lower mean GA, higher rates of morbidities, longer mean length of mechanical ventilation (MV) and oxygen supply was found in PS treated infants. The survival rate, mean GA, rates of morbidities, mean length of MV and oxygen supply was no significant difference between infants received PS in the delivery room and infants received PS in the NICU. In addition, the effect of different surfactants (poractant alpha, Curosurf and calfactant, Calfsurf) has not shown any significant difference in the survial rate (Curosurf vs Calfsurf:86.6%vs92.7%; P=0.105), rates of main severe morbidities, mean length of MV and oxygen supply. However, infants who received Curosurf were noted to have indications that they were more critically ill than Calfsurf infants, with lower mean GA and BW, longer length of hospital stay (P<0.05).
All of infants with RDS were treated with MV (n=499), and of whom15.8% only received nasal continuous positive airway pressure (nCPAP),71.5%received only conventional mechanical ventilation (CMV) and/or nCPAP, and12.6%received high frequency oscillatory ventilation (HFOV). The survival rate of infants who received HFOV was the lowest, the mean length of MV was the longest, the rates of main complications and severe morbidities (including pneumorrhagia, pneumothorax, IVH grade3or4) were the highest. For the infants treated with only CMV and/or nCPAP, the survival rate and rates of main complications and severe morbidities were insignificant except a higher rate of pneumorrhagia (11.2%vs0%; P<0.01) compared with infants received only nCPAP.
The positive rate of tracheal aspirates culture in pneumonia of newborns was16.1%(n=57). The main microorganisms recovered from tracheal aspirates culture were Acinetobacter baumannii (49.1%), Klebsiella pneumoniae (31.6%), Escherichia coli (10.5%) and Pseudomonas aeruginosa (7.0%).
Of all infants with pulmonary hypertension (n=55),43.6%were treated with HFOV,80.0%treated with iNO. Infants with pulmonary hypertension often diagnosed with PDA. The proportion of infants with PDA who were treated surgically were higher in infants with pulmonary hypertension compared with no pulmonary hypertension infants (16.4%vs1.9%; P<0.001). For the infants who were treated with iNO (n=48),16.7%received PDA ligation, which accounded for36.4%of infants with PDA treated surgiallly.
As one of the severe pulmonary morbidities,22(3.6%) of606infants needed oxygen therapy at36weeks and developed BPD. Postnatal steroids was given to7(31.8%) infants by intravenous injection after BPD diagnosed.3(13.6%) infants of BPD discharged home on oxygen and the remaining19(86.4%) infants were survived until discharge without oxygen.
5. Other Primary Diagnosis and Clinical Practices
All infants underwent≥1cranial ultrasound evaluation after admission;77.9%(n=572) of these results were normal. The lowest GA of infants were experienced the highest risk of brain injury. The proportion of EPT infants with FVH was42.1%, and24.3%in VLBW infants.
Of92infants diagnosed with sepsis,76.1%blood culture of them were positive.The main microorganisms recovered from blood culture were Klebsiella pneumoniae (37.1%), Staphylococcus (30.0%), Fungus(12.9%), Escherichia coli (7.1%) Acinetobacter baumannii (5.7%) and so on.
22(3.6%) infants developed for NEC and of whom36.4%died, which included62.5%withdrawn and only37.5%received surgical treatment.
Among729cases,87.5%(n=638) underwent at least an ophthalmologic examination before hospital discharge, death, or transfer.16.6%of these infants were diagnosed with ROP any stage, with47.2%in127EPT infants and23.4%in410VLBW infants.8.5%of122EPT infants with VLBW were performed retina photocoagulation for ROP stage≥3while infants born at higher GAs or BW≥1500g were not detected for development of severe ROP. The median length of oxygen supply was significantly longer in infants with ROP than those without ROP (26vs7days; P<0.001). It was also significantly longer in EPT infants treated with ROP photocoagulation than those ROP infants without surgery (44vs29days; P<0.001).
The rate of infants with PDA was inversely related to GAs.82.3%(n=380) of infants with PDA received oral ibuprofen administration, and of whom,34.2%received ibuprofen more than one course. The lower GAs infants had, with the more courses of ibuprofen they were treated, the lower proportion of PDA closed and the higher rate of PDA surgery after ibuprofen administration.56.2%of infants with PDA were closed after the first course of ibuprofen administered,22.7%closed with more courses of ibuprofen administered, and4.8%received PDA surgery. Only17.7%of infants with PDA were not treated with ibuprofen, and of whom,90.2%closed spontaneously, which was significantly higher than the rate (78.9%) in the infants treated with ibuprofen (P<0.05). Infants who received ibuprofen were found to have indications that they were more critically ill than no ibuprofen treated infants, with lower GA and BW, higher rate of pneumorrhagia, longer length of MV and oxygen supply, and more money spended in the NICU, while no significant difference in the survival rate (P=0.623). Similarly, infants who received more courses of ibuprofen often had lower GA, higher rate of pulmonary hypertension, iNO treated and PDA ligation, longer length of MV, oxygen supply and hospital stay, and more money spended in the NICU, whereas no significant difference in the survival rate (P=0.495) compared with infants treated with one course of ibuprofen.
6. Neonatal Transport and Outcome-based Comparison
An extremely high proportion (90.0%) of admissions were born at lower level centers and subsequently transported, the remaining10.0%were inborns. The median age of NICU admission was2.0(IQR:1.5-3) hours for the outborns, which was significantly longer than0.5(IQR:0.5-1) hours for the inborns (P<0.001). No difference was found in both the mean GA and BW for the inborn and outborn infants. The rates of cesarean section and PROM>24h were higher in inborns than the rates in outborns, while more multiple births in outborns than inborns (P<0.05). Outborns experienced higher but insignificant need for intensive delivery room and NICU interventions compared with inborns (P=0.084).566(78.6%) infants were treated with supplemental oxygen therapy in transport. The main types of oxygen supply were oxyhood (55.7%) and endotracheal intubation (41.9%) in the transportation. The rate of infants treated with oxygen supply was conversely related to GA and BW (P<0.001). However, rates of oxygen use and MV were similar for the inborn and outborn infants.
Rates of diseases of respiratory system, main clinical practices and IVH were found no significant difference in the inborn and outborn infants (P>0.05). A little but not so high mortality was seen in the outborns when compared to the inborns (8.7%vs5.5%; P=0.348). Rates of main complications, length of MV, oxygen supply and hospital stay were also found insignificant difference among the inborn and outborn survivors (P>0.05).
Compared with different admission age, the mean GA and BW of infants with admission age≤2h were no significant difference with those of infants with admission age>2h. For the perinatal characteristics, tube baby, gestational hypertension diseases, regular prenatal care, prenatal steroids and cesarean section were prevalent in infants with admission age≤2h, while asphyxia and intensive delivery room interventions were more common in infants with admission age>2h. For the outcomes of VPT infants in the NICU, higher survival rate (91.2%vs84.8%; P<0.05) and lower proportion of withdrawal of care (11.2%vs21.7%; P<0.01) were found in infants with admission age<2h compared with infants with admission age>2h. There was no difference in occurrence of main severe morbidities, length of MV, oxygen use and hospital stay, and cost of hospital (P>0.05).
7. Mortality Rates and Risk Factors of Death
The overall mortality rate of VPT infants until discharge from the hospital was8.4%(n=61), which consisted of hospital deaths either in the process of intensive care (1.8%) or after the withdrawn/withhold of support (6.6%). The death rate of VLBW and ELBW infants was10.5%and26.5%, respectively, which was higher compared to4.4%of infants with BW≥1500g (P<0.001). In the lower ranges of GA and BW (both P<0.001), a striking stepwise reductions in mortality rate occur significantly with each additional week of gestation and250g increase in BW. Most of deaths occurred within neonatal period:44.3%occurred in the early neonatal period (0-6days postpartum),49.2%during the late neonatal period (7-28days postpartum), while only6.6%of deaths after neonatal period.
The results of the multivariate logistic regression analysis showed that lower GA, invasive MV, admission age>2h, air leak, pulmonary hemorrhage, no PS therapy and presence of a major anomaly were the independent risk factors for death.
8. Length and Costs of Hospitalization
The median LOS among survivors was43days, and decreased with increasing GA, from92days at26weeks to37days at31weeks (P<0.001). PMA at discharge decreased from39.2weeks for surviving infants born at GAs of26weeks to35.7weeks or36.3weeks for those born at30or31weeks, respectively. The median cost of surviving infants was1.4-fold to the entire annual income of a Beijing urban resident in2011(32903CNY). The median cost of hospital in infants with GA≤26wk and31wk was132771and38142CNY, respectively.
9. Withdrawal or Withholding of Care
18.0%(n=131) of VPT infants support were withdrawn from the treatment, including27.4%in EPT infants. The withdrawal rate of support was20.5%(n=98) among VLBW infants and25.0%among ELBW infants. Of all infants withdrawn from the treatment,51%were withdrawn due to the parents'inability to afford the high cost of continued medical treatment.36.6%of infants died after withdrawal of support in the care unit. No distinction of survival was detected in the gender discrepancy (males vs females:90.9%vs92.6%; P=0.412) and the treatment withdrawn happened almost equally to female and male infants (18.4%vs17.7%; P=0.803).
10. Outcome-based Comparison with Developed Countries
The rate of provide active obstetric care and initiate neonatal intensive care for the most-premature infats was obviously lower in Chinese NICU than the rate in developed countries. An extremely high rate of VPT infants transported was found in the present study, while a much lower rate was found in developed countries. More antenatal transferred VPT infants were found in developed NICU. The overall survival rate of the VPT infants was similar with the rate in developed countries; specifically, a relative lower proportion and survival rate of EPT was found in this study.
Conclusions:
1. This study is the first report to summarize the management, mortality and morbidity among very preterm infants born at less than32weeks of gestation at the very preterm-NICU of the largest neonatal intensive care center in China.89.5%of VLBW infants survived to discharge, this nearly achieves the rate of90%of infants born with VLBW in subspecialty perinatal centers in the United States in2010.
2. The rate of provide active obstetric care (including prenatal steriods, cesarean section) and initiate neonatal intensive care for the most-premature infants (timely fetus or infant transferred to a higher level hospital) was obviously lower in the present data than the rate in developed countries, which would result in delaying the time to rescue the VPT infants and partially explain the worse outcomes of EPT infants. As we know, prenatal steroids treatment has been applied in clinical practice decades and recent reports have affirmed that antenatal exposure to steroids was associated with a lower rate of severe morbidity, death or neurodevelopment impairment among VPT infants in developed countries.
3. This VPT-NICU has been the largest VPT infants referral centre in China.90%of infants were born at lower level centers and subsequently transported to the NICU. As many studies reported, outborn VPT infants may have poor outcomes with regards to survival and morbidity when compared to inborn infatns. An unexpected finding was that no significant difference was found in the rates of mortality and servere morbidity between the inborn and outborn infants; the results may suggest that both short-distance within about2hours neonatal ground transport by well-developed hospital referral/infant transport systems and high-volume NICU may minimize the adverse effects of transport.
4. Of concern, the cost of hospital stay, especially in EPT infants with severe morbidity, were very high in the present study. The direct costs incurred during neonatal intensive care unit hospitalization for such infants approximated the annual income of an urban resident family. In addition, it should be noted that death after withdrawal or withholding form intensive care, contributed to the high mortality which accounted for79%of the total death in this article. The high cost of continued medical treatment and the high risk of poor outcomes, often withdraw or withhold the parents'decisions to provide active intensive care for VPT infants in China. In fact, socioeconomic and culture status has been a substantial impact on the outcomes of VPT infants.
PART II A Lab Study on Isolation of Mesenchymal Stem Cell from Human Umbilical Cord Blood
Objective:
Mesenchymal stem cells (MSCs) consist of a rare population of multipotent progenitors having the capacity for self-renewal and differentiation into various lineages of mesenchymal tissues. This ability makes MSCs an attractive tool in the field of therapeutic use. These cells can be isolated from different tissues such as bone marrow, umbilical cord, adipose tissue, dental pulp, and umbilical cord blood (UCB). UCB is an interesting source of these cells because the collection process is painless and non-invasive, it causes no harm to the mother or infant, and it is a material usually discarded. Unfortunately, reliable procedures for efficient expansion and differentiation of UCB-MSCs remain to be established. The isolation, characterisation, in vitro expansion and differentiation of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were among the current aims of this study, and achieving these goals is a pre-requisite for extensive use of this novel approach for the treatment of a number of human diseases. Additionally, the factors that influence the rate of success of MSCs isolation culture and the yields of MSCs from UCB of different gestational age deliveries were investigated.
Methods:
(1) UCB units from full-term deliveries were collected with informed consent of the mothers. We separated cells on Ficoll density gradient, and the monocuclear cells (MNCs) fraction was collected and washed in PBS. MNCs were seeded in culture at a density of1×10/L into a25cm2culture flask, precoated with5%fetal bovine serum (FBS), in DMEM/F12+10%FBS medium in the presence of100units/ml penicillin and100U/ml streptomycin, at37℃, in5%CO2. Every3to4days, we changed the medium until subconfluency was obtained, and cells were harvested with a trypsin-EDTA solution and replated at at a density of1×108/L. The morphology of MSCs derived from the UCM was taken. Cumulative population doubling levels were calculated for each subcultivation. Immunophenotyping of MSCs was detected using flow cytometry. The adipogenic and chondrogenetic differentiation studies were induced.
(2) UCB was collected at birth in neonates of three different gestational groups, cultured with the same culture conditions. The relationship of the yields of MSCs derived from UCB with several factors such as GA, the collected volume of UCB and the MNCs count of UCB and the relationship among these factors were investigated.
Results:
(1) After plating the MNCs, a few cells attached to the plastic culture dishes and formed adherent cells within4to5d. Most of those cells were monocytes, which fused toform osteoclast-like cells. The onset of colony formation could be observed at first after2weeks. These appeared in80%to90%of the flask coated with FBS and reached a subconfluent condition within4to5weeks. The expression of cell-surface antigens by flow cytometry was evaluated at passage4. The isolated cells significantly expressed MSCs markers CD105、CD90、CD73and HLA-ABC, while they lacked expression of the hematopoietic markers CD14, CD34and CD45. Differentiation capacity of MSC derived from HUCB was cultured in differentiation medium to induce adipogenic and chondrogenetic differentiation respectively. The MSCs demonstrated a multilineage capacity of adipogenic and chondrogenetic differentiation. The cumulative population doubling levels increased sharply for the the passage3to passage7and apparent slowly after the passage9. The bioactivities of MSCs had no changes after cell culture from thawing of frozen cells.
(2) There were significant correlations between the success rate and such factors as the gestaional age (GA), the volume and the MNCs count of UCB.①The success rate of generating MSCs cells from UCB was up to52.8%. The GA of UCB deliveries with succesful isolating MSCs (the successful UCB) was significantly lower than that of UCB deliveries without isolating MSCs (the unsuccessful UCB;34.0±4.2vs36.8±3.3wk;t=2.184, P=0.036). The trend but insignificantly toward the success rate declined with the increasing GA (χ2=3.769, P=0.152).②The volume of the unsuccessful UCB was significantly more than that of the unsuccessful UCB (43.2±13.6vs32.1±11.5ml; t=2.635, P=0.013). There was a positive correlation between the volume and the GA in the successful UCB (F=4.003, P=0.039).③The density of MNCs in the unsuccessful UCB was significantly higher than that of the unsuccessful UCB (32.9±11.7vs23.7±6.5×106/ml;t=2.863, P=0.008). In average, the yield of the MNCs was comparable between the three GA groups (F=1.087, P=0.361).④The MSCs count as percentage of MNCs was in a significant inverse correlation with GA; that is, the density of MSCs and GA, which reduces as the maturity increased (F=13.309, P=0.000).
Conclusions:
(1) MSCs can be isolated and cultivated successfully in vitro from UCB with the optimal culture conditions,1×1011/L mononuclear cells seeded in DMEM/F12culture medium containing10%FBS and a culture flask precoated with FBS. The bioactivities of these MSCs were identical with that of the MSCs reported.
(2) The success rate of generating MSCs cells from UCB was low. Crucial points to isolate MSCs cells from UCB were the gestaional age (GA), the volume and the MNCs count of UCB. Lower GA, more volume and higher density of MNCs of UCB seems like with higher success rate isolating MSCs cells. The UCB from preterm infants is a better source of MSCs than that from full term neonates.
引文
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