补肾活血方对hPTH（1-34）干预下成骨细胞的影响及其治疗肾性骨病患者的远期疗效观察
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摘要
肾性骨病(ROD)是由慢性肾脏疾病及终末期肾脏疾病引起的体内矿物质和骨代谢系统紊乱的一组临床症候症,它是慢性肾功能衰竭(CRF)常见、严重、治疗困难的并发症。当肾小球滤过率GFR<10ml/min时,几乎100%的CRF患者都会发生ROD。
高转运肾性骨病是三种肾性骨病中最常见的骨病类型。高转换型肾性骨病的主要原因是继发性甲旁亢,甲状旁腺激素(PTH)分泌亢进,过量的PTH使骨重建过程的吸收和生成两种功能均亢进,成骨、破骨细胞异常增生活跃,而当破骨细胞增生大于成骨细胞,最终骨吸收大于骨形成,形成骨病。而成骨细胞与破骨细胞之间信息交流的分子基础是成骨细胞表达的骨保护素(OPG)及其配体(RANKL)和破骨细胞表达的NF-kB的活化受体(RANK),即OPG/RANKL/RANK系统,PTH对其有调控作用。
目前西医对ROD主要采取对症治疗,包括饮食治疗、磷结合剂、活性维生素D类似物的应用、合理调整血钙、磷、iPTH水平、钙敏感受体激动剂的应用及透析方式的调整,虽能部分改善肾性骨病患者的临床症状,但临床远期疗效并不理想,可能导致软组织异位钙化,引发心脑血管并发症发生率增加,且存在药物价格昂贵等多个问题。中医从“肾主骨”理论出发,根据肾性骨病脾肾衰惫为本、浊瘀内阻为标的基本病机,以补肾活血为大法,自拟补肾活血方(主要组成：熟地、川断、狗脊、补骨脂、丹参、大黄、当归)治疗肾性骨病。以期通过调节骨代谢平衡,改善甲旁亢作用,发挥其治疗作用。
1、研究目的
本课题研究分为实验研究和临床研究两部分。
实验研究目的在于通过体外模拟肾性骨病继发性甲旁亢状态,以人类重组甲状旁腺激素(hPTH)干预的小鼠成骨细胞为研究对象,观察补肾活血含药血清对其增殖、分化的影响及其对OPG及RANKL基因表达的影响,以探讨补肾活血中药治疗肾性骨病的分子水平机制。
临床研究则以随访形式追踪观察曾经入组的服用补肾活血颗粒肾性骨病患者,从心血管并发症、终点事件、骨代谢水平、生存质量及营养状况方面评定补肾活血方的远期临床疗效,更为临床采用中医药治疗肾性骨病提供相对客观评价依据。
2、研究方法
2.1实验方法
①培养成骨细胞MC3T3-E1,配制终浓度为10ng/ml的hPTH(1-34)溶液作为体外模拟甲旁亢的干预条件,制备中药、西药及正常鼠含药血清,实验分组。
②用MTT法在细胞培养的24h、48h、72h分别检测不同浓度补肾活血方组对hPTH(1-34)干预下MC3T3-E1增殖的作用。
③用ELISA方法在细胞培养的24h、48h、72h分别检测不同浓度补肾活血含药血清组细胞在hPTH (1-34)干预下其上清液中PICP和ALP分泌水平。
④采用实时荧光定量PCR法在8h、12h、24h时,对PTH干预下的的小鼠成骨细胞的OPGmRNA、RANKLmRNA的表达进行绝对定量分析。
2.2临床研究方法
本研究以2008年10月-2009年3月期间曾经参与“补肾活血法改善肾性骨病病人生存质量及骨代谢异常临床研究”课题的维持性血液透析肾性骨病患者为研究对象,治疗组及对照组则根据当时是否接受中药干预治疗划分,其中对照组采取规律活血颗粒。采用病例对照研究的血透、常规补钙降磷等基本治疗,而治疗组则是在基本治疗基础上加以口服补肾方法,追踪观察两组患者3年。临床观察指标主要包含两部分,一为回顾性记录3年中骨化三醇冲击治疗次数、心脑血管发生率、一年及三年生存率、终点事件发生率；另一为评估在3年观察终位时间点时生存质量评分、营养状况评分,甲状旁腺激素水平、钙、磷等骨代谢指标、肾功能指标、钙、磷及钙磷乘积达标率,将两组观察指标进行比较。所得数据使用SPSS16.0软件进行统计学处理,正态分布的计量资料采用t检验,非正态分布计量资料采用秩和检验,描述性计数资料采用卡方检验。
3、结果
3.1实验研究部分
3.1.1各组细胞增殖水平的比较：随时间增加,中药各浓度组细胞增殖水平明显上升,而模型PTH组则从48h增殖率开始下降,当达到72h时,模型PTH组0D值明显低于正常组(P<0.05),而各浓度中药组细胞增殖水平均显著高于模型PTH组(P<0.01),且增殖水平呈剂量依赖性,即浓度越高增殖水平也越高。
3.1.2各组ALP、PICP分泌量的比较：①自48h始至72h,各浓度中药含药血清组ALP分泌量均显著高于模型PTH组(P<0.01)；②而中药高浓度组在72h时PICP分泌量显著高于模型PTH组(P<0.01)。
3.1.3各组OPGmRNA及RANKLmRNA表达水平的比较：①在经一定浓度(lOng/ml)的PTH持续刺激后,成骨细胞OPGmRNA表达先是出现略微增高趋势,之后开始出现表达抑制,至24h时OPGmRNA表达明显抑制,表达量与正常组相比显著减少,有统计学差异(P<0.01)；另一方面,随时间延长RANKL表达量则呈不断上升趋势,且各个时间点均较正常组RANKL水平明显升高,有显著性差异(P<0.01),表明PTH能明显上调RANKLmRNA的表达；②补肾活血各浓度组及西药骨化三醇组随时间延长,OPGmRNA水平逐渐升高,促进其表达,至24h时达到高峰；且各个时间点均较模型PTH组OPGmRNA水平增高,中药组呈剂量依赖性,即补肾活血含药血清浓度越高,OPGmRNA表达量也越高；③补肾活血各浓度组及西药骨化三醇组RANKL表达量随时间延长呈递增趋势,但从12h起,各组RANKL表达量均较模型组减低,且差异有显著性(P<0.01),至24h时,各组RANKL表达量仍较模型组低,且P<0.05,有显著性差异。中药各浓度组中,高浓度组RANKL表达量最低,中浓度组次之,低浓度组最高。西药骨化三醇在各个时间点RANKL表达量则与补肾活血中浓度组近似。
3.2临床研究部分
3.2.1骨化三醇冲击治疗率：治疗组骨化三醇冲击治疗率明显低于对照组(P<0.05),但治疗组运用骨化三醇冲击治疗的频次与对照组总体无显著性差异。
3.2.2心血管并发症发生率：两组的心梗、脑梗、心绞痛及心律失常发生率虽无显著性差异(P>0.05),但治疗组急性心衰发生率明显低于对照组(P<0.01),且心绞痛发生频次也明显低于对照组,有显著性差异(P<0.05)
3.2.3终点事件：两组骨折发生率均较低,两组间比较无显著性差异(P>0.05)；两组间一年生存率相比无显著性差异,而三年生存率则有显著性差异,治疗组优于对照组(P<0.05)；两组患者的死亡原因主要为心脑血管疾病和感染。
3.2.4骨代谢指标：治疗组iPTH、血磷水平均显著低于对照组(P<0.05),血钙水平两组间比较无显著性差异(P>0.05)；治疗组钙磷乘积达标率明显高于对照组(P<0.05),结果有显著性差异。
3.2.5肾功能指标：两组血肌酐及尿素氮水平相比,P均>0.05,无显著性差异。
3.2.6生存质量：①八个维度比较：治疗组在生理机能(PF)和总体健康(GH)得分高于对照组,并有统计学差异(P<0.05),表明治疗组在生理机能(PF)和总体健康(GH)方面优于对照组；而生理职能(RP)、躯体疼痛(BP)得分与对照组相比则无显著性差异；治疗组一般精力(VT)和精神健康(MH)方面得分均高于对照组(P<0.01),有极显著性差异,表明治疗组患者一般精力及精神健康方面均明显优于对照组；而治疗组在社会功能(SF)及情感职能(RE)方面,评分也均高于对照组(P<0.05),有统计学差异,治疗组患者社会功能和情感职能也较对照组好。②在生理健康(PCS)总得分方面,治疗组评分高于对照组,P<0.01,有极显著性差异,说明治疗组生理健康优于对照组；而治疗组心理健康总得分(MCS)高于对照组,P<0.01,有极显著性差异,治疗组心理健康也优于对照组。③治疗组SF-36总分高于对照组,P<0.05,有显著性差异。
3.2.7营养状况：治疗组存在营养风险人数12人(25.5%),而对照组则有20人(47.6%),两组相比,治疗组存在营养风险人数比低于对照组,P<0.05,有统计学差异。说明治疗组患者营养状况较对照组好。
4、结论
4.1补肾活血方含药血清能改善甲旁亢作用下成骨细胞的骨代谢作用,在促进hPTH (1-34)干预下小鼠成骨细胞MC3T3-E1增殖的同时,也能很好地促进其进一步分化成熟,进而促进成骨细胞骨形成,且促进效应呈浓度依赖性。
4.2补肾活血方含药血清能上调PTH干预下OPGmRNA的表达,降低PTH干预下RANKLmRNA的高表达,提示补肾活血方可能通过很好地促进骨形成,抑制甲旁亢引起的过度骨吸收作用机制来改善骨代谢平衡。并且中药浓度越高,促进骨形成作用越明显,反之,中药浓度越高抑制骨吸收作用也最强。补肾活血方组在体外调整骨代谢作用与西药骨化三醇相近。
4.3补肾活血方能减少骨化三醇冲击治疗率,降低iPTH、高血磷水平,提高钙磷乘积达标率,并能改善ROD患者生存质量及营养状况,减少心血管并发症发生率,提高生存率,提示补肾活血方治疗肾性骨病患者远期疗效好。
5、创新点
5.1关于肾性骨病实验研究模型的造模方法多采用大鼠部分肾切除加高磷饮食,本课题实验研究则从微观入手,采用直接给予小鼠成骨细胞一定浓度PTH持续刺激的方法进行造模,在体外模拟甲旁亢状态,此造模方法国内外文献未见报道；
5.2观察补肾活血方能改善PTH干预下的OPG及RANKLmRNA的表达,从细胞分子生物学水平上评价补肾活血方促进骨形成,抑制过度骨吸收,改善骨代谢的作用；
5.3采用了目前最前沿的实验方法Realtime-PCR法检测小鼠成骨细胞中OPGmRNA及RANKLmRNA表达量,较以往普通PCR法更加灵敏、准确,而且使用了实时荧光定量中的Taq man探针法建立标准曲线对基因表达做到准确定量；
5.4首次对肾性骨病患者进行远期疗效观察,补充以往肾性骨病临床研究中没有的项目：包括心血管并发症、骨化三醇冲击治疗次数、实验室检查、生存质量评分、营养状况、终点事件等,并引入生存曲线概念,以评定补肾活血方的远期疗效,目前未见有中药方治疗肾性骨病远期疗效观察的相关临床报道。
Renal osteodystrophy (ROD) has a group of clinical symptoms caused, i.e. mineral and bone metabolism disorders in the body caused by chronic kidney disease and end stage renal disease. It is a common, serious and difficultly treated complication of chronic renal failure (CRF). In almost100%of patients with CRF, ROD occurs at GFR (glomerular filtration rate)<10ml/min.
The high transfer ROD is one of the three most common types of bone disease, and is mainly caused by secondary hyperparathyroidism. With excessive PTH, the two functions i.e. absorption and generation in the bone remodeling process is hyperactive, and there is abnormal proliferation in osteoblasts and osteoclasts. For the exchange of information between osteoblasts and osteoclasts, the molecular basis is OPG (expressed by osteoblast) and its ligand RANKL as well as receptor activator of NF-KB (RANK) expressed by osteoclast, that is, OPG/RANKL/RANK system, to which PTH has a regulatory role.
At present, for ROD, the western medicine mainly carries out symptomatic treatment, including diet therapy, phosphate binder, active vitamin D analogues, and calcium-sensing receptor agonists&dialysis. These techniques improve clinical symptoms partially in patients with ROD, but clinical long-term efficacy is not ideal in addition to ectopic calcification of soft tissue etc. With the theory of kidney governing bones, Chinese medicine considers a basic pathogenesis with spleen-kidney insufficiency as a root cause and branch excess with phlegm stagnation as a secondary cause, and adopts the method to invigorate kidney and promote blood circulation with self-prepared Bushenhuoxue Recipe. This recipe regulates bone metabolism and improves hyperparathyroidism so therapeutic.
1. Objectives
Observe the effect of Bushen Huoxue-drug serum on proliferation&differentiation and OPG/RANKL gene expression, and explore the molecular mechanism of ROD treatment via the said recipe in the study of hPTH-intervened mouse, in which in vitro simulation of ROD secondary hyperparathyroidism is made. In the follow-up of ROD patients in the group taking said recipe (particular), the assessment for its long-term clinical effect is made from cardiovascular complications, endpoint events, level of bone metabolism, quality of life and nutritional status, so as to provide a relatively objective assessment basis for clinical ROD treatment using Chinese medicine.
2. Methods
2.1Experimental method
①Culture of MC3T3-El-osteoblast, preparing liquid of hPTH(1-34) plus Chinese/Western medicine drug-containing serum, and experimental grouping;
②MTT is used to detect hPTH(1-34)-intervened proliferation of MC3T3-E1(respectively cultured in24h,48h, and72h) in Bushenhuoxue recipe group at different concentrations;
③ELISA is used to detect hPTH(1-34)-intervened PICP and ALP secretion levels in the supernatant of cell in Bushenhuoxue-containing serum group at different concentrations.
④QRT-PCR is used in absolute quantitative analysis for PTH-intervened OPGmRNA/RANKLmRNA expression of mouse osteoblasts at8h,12h and24h.
2.2Clinical research method
The clinical observation objects are the continuous hemodiafiltration patients who were studied in "The clinical study of bushenhuoxue therapy improving the evaluation of quality of life and Bone Metabolism of renal osteodystrophy "form October2008to March2009. According to whether or not to accept Chinese medicine treatment, patients were divided into treatment group and control group. The control group was subject to basic techniques e.g. hemodialysis, regular calcium supplementation. The treatment group in addition to basic techniques was subject to oral Bushenhuoxue particles. Case-control study was conducted. The time of clinical followup observation is3years. There are two clinical Observe Gauge. On one hand, a retrospective record was made in the past3years for number of calcitriol pulse treatments, cardiovascular disease incidence, statistical survival rate, endpoint event rate in the two groups. On the other hand, in end time of Observation,carried out evaluation of quality of life and nutritional status scores in the two groups, obtained laboratory indicators of patients including bone metabolism and renal function the like,and the two groups were compared. Relevant statistical analysis was conducted by SPSS16.0.
3. Results
3.1Experimental result
3.1.1Cell proliferation:Over time, cell proliferation was significantly increased in the Chinese medicine group at different concentrations; model PTH group's48h proliferation rate began to decline, at72h, model PTH group OD value significantly was lower than the normal group (P<0.05). For the Chinese medicine group at various concentrations, the cell proliferation level was significantly higher than the model PTH group (P<0.01), and this level was dose-dependent.
3.1.2Secretion of ALP and PICP:①From48h to72h, ALP secreted in the Chinese medicine-containing serum group at various concentrations was significantly higher than that of the model PTH group (P<0.01);②PICP secreted in the Chinese medicine group at high concentration at72h was significantly higher than the model PTH group (P<0.01).
3.1.3Expression of OPGmRNA and RANKLmRNA:①In Bushenhuoxue group at each concentration and western medicine calcitriol group, OPGmRNA level gradually increased over time promoting its expression, and reached a peak in24h; OPGmRNA level increased at each time point compared with the model PTH group. The Chinese medicine group was dose-dependent.②In the Bushenhuoxue group at various concentrations and western medicine group, RANKL expression showed an increasing trend with time, but from12h to24h, RANKL expressed in every group was lower than the model group with a significant difference (P<0.01). In Chinese medicine group at various concentrations, RANKL expression in the high concentration group is lowest, and in the lower concentration group highest.
3.2Clinical result
3.2.1Calcitriol pulse therapy rate:Compared to the control group, the treatment group could significantly reduce the rate of calcitriol pulse therapies (P<0.05).
3.2.2Incidence of cardiovascular complications:the treatment group was significantly lower than the control group in incidence of acute heart failure (P<0.01) and angina frequency (P<0.05).
3.2.3Endpoint events:The two groups had a low incidence rate of fractures and showed no significant difference (P>0.05); the two groups had no significant difference in one-year survival rate, but significant in three-year survival rate (P<0.05) in which the treatment were better than the control group. For patients in the two groups, the death was mainly due to cerebrovascular disease and infection.
3.2.4Indicators of bone metabolism:Compared to the control group, the treatment group could reduce iPTH, serum phosphate levels and calcium-phosphate product (P<0.05), resulting in a significant difference.
3.2.5Indicators of renal function:Between the two groups, there was no significant difference in serum creatinine and blood urea nitrogen levels (P>0.05).
3.2.6Quality of life:①Comparison in8aspects:the treatment group was better than the control group in physiological function (PF) and general health (GH), and there was a significant difference (P<0.05); the treatment group had a higher score than the control group in vitality (VT),mental health (MH),function (SF) and emotional role (RE)with a very significant difference (P<0.05).②The treatment group was higher than the control group in total physical health score (PCS) and mental health score (MCS) with a very significant difference (P<0.01)③As to the total score of SF-36, the treatment group was higher than the control group with a significant difference (P <0.05).
3.2.7Nutritional condition:12patients (25.5%) had a nutritional risk in the treatment group, while20(47.6%) in the control group; the difference was statistically significant (P<0.05).
4. Conclusions
4.1Bushenhuoxue recipe can promote MC3T3-E1-osteoblast proliferation, differentiation and maturation under the intervention by hPTH (1-34) in mouse, thereby promotes bone formation improve bone metabolism of osteoblasts under hyperparathyroidism. The promotional effect is concentration dependent.
4.2Bushenhuoxue drug-containing serum can up-regulate the expression of OPGmRNA intervention by hPTH (1-34),and down-regulate the expression of RANKLmRNA intervention by hPTH (1-34).indicating that the Bushenhuoxue recipe promote bone formation, suppress excessive bone resorption caused by hyperparathyroidism, improve the balance of bone metabolism.
4.3This recipe can reduce the rate of calcitriol pulse therapies, iPTH level, high phosphorus and calcium-phosphate product, improve the quality of life of ROD patients and their nutritional condition, reduce the incidence of cardiovascular complications and improve survival rates. The clinical long-term efficacy is better in the treatment group than the control group.
高转运肾性骨病是三种肾性骨病中最常见的骨病类型。高转换型肾性骨病的主要原因是继发性甲旁亢,甲状旁腺激素(PTH)分泌亢进,过量的PTH使骨重建过程的吸收和生成两种功能均亢进,成骨、破骨细胞异常增生活跃,而当破骨细胞增生大于成骨细胞,最终骨吸收大于骨形成,形成骨病。而成骨细胞与破骨细胞之间信息交流的分子基础是成骨细胞表达的骨保护素(OPG)及其配体(RANKL)和破骨细胞表达的NF-kB的活化受体(RANK),即OPG/RANKL/RANK系统,PTH对其有调控作用。
目前西医对ROD主要采取对症治疗,包括饮食治疗、磷结合剂、活性维生素D类似物的应用、合理调整血钙、磷、iPTH水平、钙敏感受体激动剂的应用及透析方式的调整,虽能部分改善肾性骨病患者的临床症状,但临床远期疗效并不理想,可能导致软组织异位钙化,引发心脑血管并发症发生率增加,且存在药物价格昂贵等多个问题。中医从“肾主骨”理论出发,根据肾性骨病脾肾衰惫为本、浊瘀内阻为标的基本病机,以补肾活血为大法,自拟补肾活血方(主要组成：熟地、川断、狗脊、补骨脂、丹参、大黄、当归)治疗肾性骨病。以期通过调节骨代谢平衡,改善甲旁亢作用,发挥其治疗作用。
1、研究目的
本课题研究分为实验研究和临床研究两部分。
实验研究目的在于通过体外模拟肾性骨病继发性甲旁亢状态,以人类重组甲状旁腺激素(hPTH)干预的小鼠成骨细胞为研究对象,观察补肾活血含药血清对其增殖、分化的影响及其对OPG及RANKL基因表达的影响,以探讨补肾活血中药治疗肾性骨病的分子水平机制。
临床研究则以随访形式追踪观察曾经入组的服用补肾活血颗粒肾性骨病患者,从心血管并发症、终点事件、骨代谢水平、生存质量及营养状况方面评定补肾活血方的远期临床疗效,更为临床采用中医药治疗肾性骨病提供相对客观评价依据。
2、研究方法
2.1实验方法
①培养成骨细胞MC3T3-E1,配制终浓度为10ng/ml的hPTH(1-34)溶液作为体外模拟甲旁亢的干预条件,制备中药、西药及正常鼠含药血清,实验分组。
②用MTT法在细胞培养的24h、48h、72h分别检测不同浓度补肾活血方组对hPTH(1-34)干预下MC3T3-E1增殖的作用。
③用ELISA方法在细胞培养的24h、48h、72h分别检测不同浓度补肾活血含药血清组细胞在hPTH (1-34)干预下其上清液中PICP和ALP分泌水平。
④采用实时荧光定量PCR法在8h、12h、24h时,对PTH干预下的的小鼠成骨细胞的OPGmRNA、RANKLmRNA的表达进行绝对定量分析。
2.2临床研究方法
本研究以2008年10月-2009年3月期间曾经参与“补肾活血法改善肾性骨病病人生存质量及骨代谢异常临床研究”课题的维持性血液透析肾性骨病患者为研究对象,治疗组及对照组则根据当时是否接受中药干预治疗划分,其中对照组采取规律活血颗粒。采用病例对照研究的血透、常规补钙降磷等基本治疗,而治疗组则是在基本治疗基础上加以口服补肾方法,追踪观察两组患者3年。临床观察指标主要包含两部分,一为回顾性记录3年中骨化三醇冲击治疗次数、心脑血管发生率、一年及三年生存率、终点事件发生率；另一为评估在3年观察终位时间点时生存质量评分、营养状况评分,甲状旁腺激素水平、钙、磷等骨代谢指标、肾功能指标、钙、磷及钙磷乘积达标率,将两组观察指标进行比较。所得数据使用SPSS16.0软件进行统计学处理,正态分布的计量资料采用t检验,非正态分布计量资料采用秩和检验,描述性计数资料采用卡方检验。
3、结果
3.1实验研究部分
3.1.1各组细胞增殖水平的比较：随时间增加,中药各浓度组细胞增殖水平明显上升,而模型PTH组则从48h增殖率开始下降,当达到72h时,模型PTH组0D值明显低于正常组(P<0.05),而各浓度中药组细胞增殖水平均显著高于模型PTH组(P<0.01),且增殖水平呈剂量依赖性,即浓度越高增殖水平也越高。
3.1.2各组ALP、PICP分泌量的比较：①自48h始至72h,各浓度中药含药血清组ALP分泌量均显著高于模型PTH组(P<0.01)；②而中药高浓度组在72h时PICP分泌量显著高于模型PTH组(P<0.01)。
3.1.3各组OPGmRNA及RANKLmRNA表达水平的比较：①在经一定浓度(lOng/ml)的PTH持续刺激后,成骨细胞OPGmRNA表达先是出现略微增高趋势,之后开始出现表达抑制,至24h时OPGmRNA表达明显抑制,表达量与正常组相比显著减少,有统计学差异(P<0.01)；另一方面,随时间延长RANKL表达量则呈不断上升趋势,且各个时间点均较正常组RANKL水平明显升高,有显著性差异(P<0.01),表明PTH能明显上调RANKLmRNA的表达；②补肾活血各浓度组及西药骨化三醇组随时间延长,OPGmRNA水平逐渐升高,促进其表达,至24h时达到高峰；且各个时间点均较模型PTH组OPGmRNA水平增高,中药组呈剂量依赖性,即补肾活血含药血清浓度越高,OPGmRNA表达量也越高；③补肾活血各浓度组及西药骨化三醇组RANKL表达量随时间延长呈递增趋势,但从12h起,各组RANKL表达量均较模型组减低,且差异有显著性(P<0.01),至24h时,各组RANKL表达量仍较模型组低,且P<0.05,有显著性差异。中药各浓度组中,高浓度组RANKL表达量最低,中浓度组次之,低浓度组最高。西药骨化三醇在各个时间点RANKL表达量则与补肾活血中浓度组近似。
3.2临床研究部分
3.2.1骨化三醇冲击治疗率：治疗组骨化三醇冲击治疗率明显低于对照组(P<0.05),但治疗组运用骨化三醇冲击治疗的频次与对照组总体无显著性差异。
3.2.2心血管并发症发生率：两组的心梗、脑梗、心绞痛及心律失常发生率虽无显著性差异(P>0.05),但治疗组急性心衰发生率明显低于对照组(P<0.01),且心绞痛发生频次也明显低于对照组,有显著性差异(P<0.05)
3.2.3终点事件：两组骨折发生率均较低,两组间比较无显著性差异(P>0.05)；两组间一年生存率相比无显著性差异,而三年生存率则有显著性差异,治疗组优于对照组(P<0.05)；两组患者的死亡原因主要为心脑血管疾病和感染。
3.2.4骨代谢指标：治疗组iPTH、血磷水平均显著低于对照组(P<0.05),血钙水平两组间比较无显著性差异(P>0.05)；治疗组钙磷乘积达标率明显高于对照组(P<0.05),结果有显著性差异。
3.2.5肾功能指标：两组血肌酐及尿素氮水平相比,P均>0.05,无显著性差异。
3.2.6生存质量：①八个维度比较：治疗组在生理机能(PF)和总体健康(GH)得分高于对照组,并有统计学差异(P<0.05),表明治疗组在生理机能(PF)和总体健康(GH)方面优于对照组；而生理职能(RP)、躯体疼痛(BP)得分与对照组相比则无显著性差异；治疗组一般精力(VT)和精神健康(MH)方面得分均高于对照组(P<0.01),有极显著性差异,表明治疗组患者一般精力及精神健康方面均明显优于对照组；而治疗组在社会功能(SF)及情感职能(RE)方面,评分也均高于对照组(P<0.05),有统计学差异,治疗组患者社会功能和情感职能也较对照组好。②在生理健康(PCS)总得分方面,治疗组评分高于对照组,P<0.01,有极显著性差异,说明治疗组生理健康优于对照组；而治疗组心理健康总得分(MCS)高于对照组,P<0.01,有极显著性差异,治疗组心理健康也优于对照组。③治疗组SF-36总分高于对照组,P<0.05,有显著性差异。
3.2.7营养状况：治疗组存在营养风险人数12人(25.5%),而对照组则有20人(47.6%),两组相比,治疗组存在营养风险人数比低于对照组,P<0.05,有统计学差异。说明治疗组患者营养状况较对照组好。
4、结论
4.1补肾活血方含药血清能改善甲旁亢作用下成骨细胞的骨代谢作用,在促进hPTH (1-34)干预下小鼠成骨细胞MC3T3-E1增殖的同时,也能很好地促进其进一步分化成熟,进而促进成骨细胞骨形成,且促进效应呈浓度依赖性。
4.2补肾活血方含药血清能上调PTH干预下OPGmRNA的表达,降低PTH干预下RANKLmRNA的高表达,提示补肾活血方可能通过很好地促进骨形成,抑制甲旁亢引起的过度骨吸收作用机制来改善骨代谢平衡。并且中药浓度越高,促进骨形成作用越明显,反之,中药浓度越高抑制骨吸收作用也最强。补肾活血方组在体外调整骨代谢作用与西药骨化三醇相近。
4.3补肾活血方能减少骨化三醇冲击治疗率,降低iPTH、高血磷水平,提高钙磷乘积达标率,并能改善ROD患者生存质量及营养状况,减少心血管并发症发生率,提高生存率,提示补肾活血方治疗肾性骨病患者远期疗效好。
5、创新点
5.1关于肾性骨病实验研究模型的造模方法多采用大鼠部分肾切除加高磷饮食,本课题实验研究则从微观入手,采用直接给予小鼠成骨细胞一定浓度PTH持续刺激的方法进行造模,在体外模拟甲旁亢状态,此造模方法国内外文献未见报道；
5.2观察补肾活血方能改善PTH干预下的OPG及RANKLmRNA的表达,从细胞分子生物学水平上评价补肾活血方促进骨形成,抑制过度骨吸收,改善骨代谢的作用；
5.3采用了目前最前沿的实验方法Realtime-PCR法检测小鼠成骨细胞中OPGmRNA及RANKLmRNA表达量,较以往普通PCR法更加灵敏、准确,而且使用了实时荧光定量中的Taq man探针法建立标准曲线对基因表达做到准确定量；
5.4首次对肾性骨病患者进行远期疗效观察,补充以往肾性骨病临床研究中没有的项目：包括心血管并发症、骨化三醇冲击治疗次数、实验室检查、生存质量评分、营养状况、终点事件等,并引入生存曲线概念,以评定补肾活血方的远期疗效,目前未见有中药方治疗肾性骨病远期疗效观察的相关临床报道。
Renal osteodystrophy (ROD) has a group of clinical symptoms caused, i.e. mineral and bone metabolism disorders in the body caused by chronic kidney disease and end stage renal disease. It is a common, serious and difficultly treated complication of chronic renal failure (CRF). In almost100%of patients with CRF, ROD occurs at GFR (glomerular filtration rate)<10ml/min.
The high transfer ROD is one of the three most common types of bone disease, and is mainly caused by secondary hyperparathyroidism. With excessive PTH, the two functions i.e. absorption and generation in the bone remodeling process is hyperactive, and there is abnormal proliferation in osteoblasts and osteoclasts. For the exchange of information between osteoblasts and osteoclasts, the molecular basis is OPG (expressed by osteoblast) and its ligand RANKL as well as receptor activator of NF-KB (RANK) expressed by osteoclast, that is, OPG/RANKL/RANK system, to which PTH has a regulatory role.
At present, for ROD, the western medicine mainly carries out symptomatic treatment, including diet therapy, phosphate binder, active vitamin D analogues, and calcium-sensing receptor agonists&dialysis. These techniques improve clinical symptoms partially in patients with ROD, but clinical long-term efficacy is not ideal in addition to ectopic calcification of soft tissue etc. With the theory of kidney governing bones, Chinese medicine considers a basic pathogenesis with spleen-kidney insufficiency as a root cause and branch excess with phlegm stagnation as a secondary cause, and adopts the method to invigorate kidney and promote blood circulation with self-prepared Bushenhuoxue Recipe. This recipe regulates bone metabolism and improves hyperparathyroidism so therapeutic.
1. Objectives
Observe the effect of Bushen Huoxue-drug serum on proliferation&differentiation and OPG/RANKL gene expression, and explore the molecular mechanism of ROD treatment via the said recipe in the study of hPTH-intervened mouse, in which in vitro simulation of ROD secondary hyperparathyroidism is made. In the follow-up of ROD patients in the group taking said recipe (particular), the assessment for its long-term clinical effect is made from cardiovascular complications, endpoint events, level of bone metabolism, quality of life and nutritional status, so as to provide a relatively objective assessment basis for clinical ROD treatment using Chinese medicine.
2. Methods
2.1Experimental method
①Culture of MC3T3-El-osteoblast, preparing liquid of hPTH(1-34) plus Chinese/Western medicine drug-containing serum, and experimental grouping;
②MTT is used to detect hPTH(1-34)-intervened proliferation of MC3T3-E1(respectively cultured in24h,48h, and72h) in Bushenhuoxue recipe group at different concentrations;
③ELISA is used to detect hPTH(1-34)-intervened PICP and ALP secretion levels in the supernatant of cell in Bushenhuoxue-containing serum group at different concentrations.
④QRT-PCR is used in absolute quantitative analysis for PTH-intervened OPGmRNA/RANKLmRNA expression of mouse osteoblasts at8h,12h and24h.
2.2Clinical research method
The clinical observation objects are the continuous hemodiafiltration patients who were studied in "The clinical study of bushenhuoxue therapy improving the evaluation of quality of life and Bone Metabolism of renal osteodystrophy "form October2008to March2009. According to whether or not to accept Chinese medicine treatment, patients were divided into treatment group and control group. The control group was subject to basic techniques e.g. hemodialysis, regular calcium supplementation. The treatment group in addition to basic techniques was subject to oral Bushenhuoxue particles. Case-control study was conducted. The time of clinical followup observation is3years. There are two clinical Observe Gauge. On one hand, a retrospective record was made in the past3years for number of calcitriol pulse treatments, cardiovascular disease incidence, statistical survival rate, endpoint event rate in the two groups. On the other hand, in end time of Observation,carried out evaluation of quality of life and nutritional status scores in the two groups, obtained laboratory indicators of patients including bone metabolism and renal function the like,and the two groups were compared. Relevant statistical analysis was conducted by SPSS16.0.
3. Results
3.1Experimental result
3.1.1Cell proliferation:Over time, cell proliferation was significantly increased in the Chinese medicine group at different concentrations; model PTH group's48h proliferation rate began to decline, at72h, model PTH group OD value significantly was lower than the normal group (P<0.05). For the Chinese medicine group at various concentrations, the cell proliferation level was significantly higher than the model PTH group (P<0.01), and this level was dose-dependent.
3.1.2Secretion of ALP and PICP:①From48h to72h, ALP secreted in the Chinese medicine-containing serum group at various concentrations was significantly higher than that of the model PTH group (P<0.01);②PICP secreted in the Chinese medicine group at high concentration at72h was significantly higher than the model PTH group (P<0.01).
3.1.3Expression of OPGmRNA and RANKLmRNA:①In Bushenhuoxue group at each concentration and western medicine calcitriol group, OPGmRNA level gradually increased over time promoting its expression, and reached a peak in24h; OPGmRNA level increased at each time point compared with the model PTH group. The Chinese medicine group was dose-dependent.②In the Bushenhuoxue group at various concentrations and western medicine group, RANKL expression showed an increasing trend with time, but from12h to24h, RANKL expressed in every group was lower than the model group with a significant difference (P<0.01). In Chinese medicine group at various concentrations, RANKL expression in the high concentration group is lowest, and in the lower concentration group highest.
3.2Clinical result
3.2.1Calcitriol pulse therapy rate:Compared to the control group, the treatment group could significantly reduce the rate of calcitriol pulse therapies (P<0.05).
3.2.2Incidence of cardiovascular complications:the treatment group was significantly lower than the control group in incidence of acute heart failure (P<0.01) and angina frequency (P<0.05).
3.2.3Endpoint events:The two groups had a low incidence rate of fractures and showed no significant difference (P>0.05); the two groups had no significant difference in one-year survival rate, but significant in three-year survival rate (P<0.05) in which the treatment were better than the control group. For patients in the two groups, the death was mainly due to cerebrovascular disease and infection.
3.2.4Indicators of bone metabolism:Compared to the control group, the treatment group could reduce iPTH, serum phosphate levels and calcium-phosphate product (P<0.05), resulting in a significant difference.
3.2.5Indicators of renal function:Between the two groups, there was no significant difference in serum creatinine and blood urea nitrogen levels (P>0.05).
3.2.6Quality of life:①Comparison in8aspects:the treatment group was better than the control group in physiological function (PF) and general health (GH), and there was a significant difference (P<0.05); the treatment group had a higher score than the control group in vitality (VT),mental health (MH),function (SF) and emotional role (RE)with a very significant difference (P<0.05).②The treatment group was higher than the control group in total physical health score (PCS) and mental health score (MCS) with a very significant difference (P<0.01)③As to the total score of SF-36, the treatment group was higher than the control group with a significant difference (P <0.05).
3.2.7Nutritional condition:12patients (25.5%) had a nutritional risk in the treatment group, while20(47.6%) in the control group; the difference was statistically significant (P<0.05).
4. Conclusions
4.1Bushenhuoxue recipe can promote MC3T3-E1-osteoblast proliferation, differentiation and maturation under the intervention by hPTH (1-34) in mouse, thereby promotes bone formation improve bone metabolism of osteoblasts under hyperparathyroidism. The promotional effect is concentration dependent.
4.2Bushenhuoxue drug-containing serum can up-regulate the expression of OPGmRNA intervention by hPTH (1-34),and down-regulate the expression of RANKLmRNA intervention by hPTH (1-34).indicating that the Bushenhuoxue recipe promote bone formation, suppress excessive bone resorption caused by hyperparathyroidism, improve the balance of bone metabolism.
4.3This recipe can reduce the rate of calcitriol pulse therapies, iPTH level, high phosphorus and calcium-phosphate product, improve the quality of life of ROD patients and their nutritional condition, reduce the incidence of cardiovascular complications and improve survival rates. The clinical long-term efficacy is better in the treatment group than the control group.
引文
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