头颈恶性肿瘤和鼻息肉中标志物的检测及hNESPCs生长动力学研究
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摘要
研究背景：头颈鳞状细胞癌(HNSCC)是世界上第六大常见的恶性肿瘤,其中约1/4是喉鳞状细胞癌(LSCC).相比之下,下咽鳞状细胞癌(HSCC)的发病率要比LSCC低得多,但相当多的HSCC患者在诊断时已经是晚期,往往已发生临近部位侵犯和颈部淋巴结转移,这些患者的预后很差。目前虽然可以采取手术治疗辅以放射治疗或化学药物等治疗方法,近年来LSCC和HSCC患者的无瘤生存率仍然没有明显的改观。如果能找到可以监测肿瘤发生发展过程的标志物,将会提高LSCC和HSCC患者的生存率和生活质量。近年来,肿瘤标志物的检测已经成为热点。
SIRT1,又名Sirtuin1,是一种NAD+依赖的去乙酰化酶,参与多种生命过程,并且可能在肿瘤发生过程中发挥着重要作用。据报道,SIRTI在多种肿瘤中表达增高,如乳腺癌、卵巢癌、前列腺癌、胃癌、结肠癌、大B细胞淋巴瘤、急性髓性白血病和Bowen氏病等。然而,也有人报道SIRT1在乳腺癌、卵巢癌、膀胱癌、前列腺癌和恶性胶质瘤中的表达低于正常对照组。目前为止,SIRT1在LSCC和HSCC的表达情况尚无报道。DBC1(Deleted in Breast Cancer1)是SIRT1的负向调节剂,一些研究表明DBC1可以通过特异性抑制SIRT1的活性来促进p53介导的细胞凋亡,也是因为这个原因,DBC1被认为是肿瘤抑制剂。然而,另外一些研究表明,DBC1在乳腺癌和结直肠癌中呈现高表达,同时它还促进乳腺癌细胞的生存,从这个角度讲,DBC1可能在乳腺癌和结直肠癌中起着促癌基因的作用。因此DBC1在恶性肿瘤中的具体作用还存在争议,有待进一步研究确定。HIC1(Hypermethylated In Cancer1)是一种抑癌基因,它在乳腺癌、胃癌、肝癌、食管癌、前列腺癌、宫颈癌、结直肠癌、非小细胞肺癌、生殖细胞肿瘤、白血病、大B细胞淋巴瘤、儿童髓母细胞瘤和室管膜瘤等肿瘤中表现为表观遗传学上的沉默。SIRT1是HIC1的一个作用靶点,人类髓性白血病中HIC1的灭活可以导致SIRT1对p53的活性抑制,进而引起细胞生存和凋亡的改变,p53反过来又可以直接激活HIC1的转录过程,因此,SIRT1、DBC1和HIC1在恶性肿瘤的发生和发展中可能发挥着重要的作用,并可能存在着重要的联系。
实验目的：本课题的研究目的是了解这些新型标志物(SIRT1、DBCl和HIC1)在喉鳞状细胞癌和下咽鳞状细胞癌发生发展中的作用,这三个标志物之间的相关性以及它们与这两种恶性肿瘤的临床病理特征之间的关联。
实验方法：用实时定量PCR方法测定了54对肿瘤标本和癌旁黏膜中SIRT1, DBC1和HIC1mRNA的表达情况并用配对Wilcoxon符号秩和检验进行统计学分析；用免疫组织化学染色的方法测定了120例肿瘤和54例癌旁黏膜中SIRT1,DBC1和HIC1的蛋白表达并采用独立样本Mann-Whitney双尾检验进行分析；另外,用Spearman等级分析来研究mRNA水平各标志物之间的相关性,分析这些标志物和临床病理特征之间的关系时采用Pearson卡方检验。最后采用Log Rank test方法进行了生存分析。所有统计分析均为双侧,当P<0.05时,认为差异有统计学意义。
实验结果：在mRNA水平上,SIRT1和HIC1在肿瘤组织中的表达明显降低,而DBCl的mRNA在肿瘤组织中则比对照组显著增高；Spearman等级分析结果显示在肿瘤组织和癌旁黏膜组织(对照组)中SIRT1与DBC1、HIC1存在明显正相关关系,而DBC1和HIC1之间无明显相关性。
在蛋白水平上,SIRT1表达在细胞核和细胞质中，而DBC1和p53则主要在细胞核中表达；SIRT1在61%(34/54)的癌旁黏膜中高表达,而仅在31%(37/120)的肿瘤标本中高表达；有趣的是,DBC1在93%(50/54)的癌旁黏膜中高表达,而仅在43%(52/120)的肿瘤标本中高表达；统计分析结果显示,SIRT1和DBC1蛋白在肿瘤组织中的表达都比对照组明显降低；SIRT1蛋白表达与肿瘤的TNM分期(p=0.005)和淋巴结转移(p=0.021)有关系,DBCl蛋白表达与肿瘤的分化程度(p=0.026)、淋巴结转移(p=0.013)和p53的表达(p=0.009)有关系。
本课题中共包括120例恶性肿瘤患者(59例为喉鳞状细胞癌,61例为下咽鳞状细胞癌),其中9例失访,随访结束时,58例患者死亡,53例仍生存。我们采用Log Rank test方法进行了生存分析,发现在早期恶性肿瘤患者中，SIRT1和DBC1与患者的生存率相关。
实验结论：三种新型标志物(SIRT1、DBC1和HIC1)在喉和下咽鳞状细胞癌中(LSCC和HSCC)都发挥着抑癌基因的作用,SIRT1和DBC1与SCC和HSCC的淋巴结转移有关。在早期恶性肿瘤中,SIRT1和HDBC1与患者的生存率相关。此外,HIC1在mRNA水平与SIRT1关系密切。这三种新型标志物有可能成为喉和下咽鳞状细胞癌的筛选指标和评估患者预后的指标,并可能成为抗肿瘤药物治疗的新靶点。
研究背景：鼻息肉(NP)是上呼吸道常见的一种慢性炎症,常伴有炎症细胞,特别是中性粒细胞和嗜酸性粒细胞的浸润,以及异常组织重塑。尽管目前有很多关于鼻息肉基因表达的研究,其具体的发病机制尚不明确。我们之前的基因芯片研究发现,与对照组相比,NP中EMP1、EGF和NRG3等标志物表达明显降低。上皮膜蛋白1(EMP1)是一种完整的膜糖蛋白,最早是在胃肠道、皮肤、肺以及脑组织中检测到的,可以调节上皮细胞的生长和分化。EGF和NRG3是表皮生长因子受体(ERBB家族)的配体,ERBB家族包括EGFR, ERBB2,ERBB3和ERBB4,它们的配体除了EGF和NRG3外,还有AREG, HBEGF, TGF-α和其它NRGs等。EGF(表皮生长因子)是由Cohen等人发现的,它参与很多生命过程。NRGs(神经调节蛋白)是细胞间的信号蛋白,包括NRG1, NRG2, NRG3和NRG4,这些神经调节蛋白具有类似EGF的结构域。EMP1和EGF-ERBB家族在NP中的表达情况以及在上皮组织重塑中的作用尚不明确。
糖皮质激素是目前为止控制鼻息肉炎症的最有效的药物治疗措施,可以明显抑制嗜酸性粒细胞的浸润。也有人提出糖皮质激素在促进上皮修复和控制组织重塑中发挥着重要作用。它可以使与上皮修复有关的AP-1及相关基因的表达上调,同时可以使鼻息肉中与组织重塑有关的基因p63的表达下降,有研究发现糖皮质激素通过EGF/EGFR信号转导通路来影响鼻息肉和哮喘患者的上皮修复和组织重塑过程。
实验目的：本课题的目的是研究EMP1和EGF-ERBB家族在鼻息肉中的表达情况,它们在鼻息肉上皮组织重塑中的作用及对糖皮质激素治疗的反应。
实验方法：用实时定量PCR方法测定了55例未经激素治疗的鼻息肉组织(GC-Naive NP)和30例下鼻甲对照组织中EMP1和EGF-ERBB家族mRNA的表达情况并采用Mann-Whitney双尾检验进行统计分析；激素治疗前后鼻息肉组织(18对)中各标志物mRNA的比较采用配对Wilcoxon符号秩和检验进行统计学分析；另外用Spearman等级分析来研究mRNA水平各标志物之间的相关性。用免疫组织化学染色的方法测定了10例GC-Naive NP样本和10例对照组IT样本中EMP1蛋白的表达；所有统计分析均为双侧,当p<0.05时,认为差异有统计学意义。
实验结果：EMPl的mRNA在GC-Naive NP组中明显低于对照组；激素治疗后,EMPl的表达明显增高；伴有重度上皮增生的NP组中EMP1表达水平明显低于轻-中度上皮增生组,伴有鳞状上皮化生的NP组中EMP1表达明显低于不伴鳞状上皮化生组；EMP1与AP-1相关的基因呈正相关。另外在10例GC-Naive NP样本和10例对照组的IT样本中测定了EMP1蛋白的表达,GC-Naive NP组中的EMP1蛋白表达明显低于对照组,免疫细胞化学染色结果提示EMP1在基底细胞和分化细胞中都有表达。
EGF-ERBB家族8个成员在GC-Naive NP组织中的表达明显低于对照组并且差异都有统计学意义,其中EGF和NRG3在两组之间的差异最为显著,分别达26.32和37.91倍之多(p值均小于0.001)；经糖皮质激素治疗后EGF和NRG3表达明显增高,它们相关的4个受体(ERBB2、ERBB3、ERBB4和EGFR)表达也有不同程度的增加。Spearman等级分析显示EGF和NRG3之间存在密切正相关关系(r=0.621),AREG和HBEGF之间存在密切正相关关系(r=0.911),EGF与ERBB2.ERBB3和ERBB4存在关联(r值分别为0.484,0.422和0.435),NRG3与4个受体也都呈正相关；EGF-ERBB家族与AP-1家族中的一些标志物呈正相关；与嗜酸性粒细胞相关的些标志物呈负相关；与T辅助细胞相关的一些标志物呈正相关；EGF-ERBB家族中一些标志物还与CXCL12、CXCR4和CDKN1A呈正相关关系。
实验结论：EMP1可以作为判定慢性呼吸道炎症性疾病(鼻息肉等)中有无存在异常上皮组织重塑和鳞状上皮化生的特异性指标。EGF-ERBB家族可能参与了鼻息肉上皮损伤和修复的过程。EMPl和EGF-ERBB家族可能在鼻上皮细胞的生长和分化中发挥着重要的作用。
研究背景：健康人的鼻黏膜上皮包含四种细胞类型：基底细胞、纤毛细胞、柱状细胞和杯状细胞。通常认为基底细胞具有干细胞和祖细胞的特性,它具有自我更新和分化能力,可以分化为其它几种上皮细胞,如杯状细胞和纤毛细胞等。在此前的研究中,我们已成功分离并培养来自鼻息肉(NP)和下鼻甲(IT)组织的鼻上皮干/祖细胞(hNESPCs)
目前已经报道的关于鼻息肉粘膜上皮的研究大都是鼻息肉上皮的病理学改变,以及相应的分子标志物和基因调控等研究,而无人报道体外鼻上皮干/祖细胞(hNESPCs)的生物学特性。我们的研究是基于这样一个假设：鼻息肉和健康鼻黏膜上皮之间存在内在表型差异。这项研究通过体外细胞培养体系研究鼻上皮干/祖细胞的生长和增殖特性,进而比较鼻息肉和健康鼻黏膜上皮来源的鼻上皮干/祖细胞生长和增殖特性的差异。
实验目的：本课题的目的是体外研究鼻上皮干/祖细胞(hNESPCs)的生物学特性,并比较鼻息肉和对照组来源的hNESPCs克隆形成率和倍增时间的差异,以及体外培养的hNESPCs中p63及Ki67等标志物的表达等。
实验方法：鼻上皮干/祖细胞(hNESPCs)经过分离后,在培养过程中计算细胞倍增时间和克隆形成率；观察衰老相关标志物β-半乳糖苷酶在细胞克隆中的染色情况；通过免疫细胞化学染色观察相关标志物的表达情况；实时定量PCR测定p63和Ki67在鼻息肉和对照组中的表达情况；最后进行统计学分析：采用SPSS18.0软件进行统计分析,用Spearman等级分析来研究mRNA水平各标志物之间的相关性；Linear mixed models用来估计NP和对照组之间倍增时间和克隆形成率的差异；Mann-Whitney分析鼻息肉和对照组组织和细胞中Ki67+/p63+比例的差异。所有统计分析均为双侧,当p<0.05时,认为差异有统计学意义。
实验结果：鼻息肉和对照组来源的hNESPCs具有相似的生长模式；两组不同来源的细胞之间,在p0和p1两代时无明显形态学差异,而到了p2和p3时,NP来源的hNESPCs细胞出现了较多分化和衰老的细胞,显微镜下呈现“荷包蛋”样外观,这些细胞中β-半乳糖苷酶染色阳性,表明细胞出现了明显的衰老状态。对照组来源的细胞在传代过程中生长增殖速度明显比鼻息肉组来源的细胞快,表现为对照组来源的细胞克隆形成率(CFE)更高,倍增时间更短。通过统计学分析(Linear Mixed Models)得出两种不同来源的细胞在p1和p2两代中有显著性差异。通过标准差分析发现NP来源的细胞倍增时间和克隆形成率的变异也较大。
实时定量PCR结果显示：从p0到p2,鼻息肉和对照组来源的鼻上皮干/祖细胞(hNESPCs) p63mRNA的表达无统计学差异。在两种不同来源的细胞中,从p1到p2,Ki67的表达呈下降趋势。在pl和p2两代,鼻息肉来源的hNESPCs中Ki67mRNA的表达低于对照组。对照组来源的细胞不同代之间Ki67的表达较稳定,从p1到p3,Ki67+/p63+的比例在77%到93%之间。而NP来源的细胞随培养时间延长,Ki67的表达逐渐下降,从p1到p3, Ki67+/p63+的比例分别为84%,61%和36%。
在伴有上皮增生和化生的鼻息肉组织中Ki67的表达下降。免疫荧光染色结果显示：在伴上皮增生的鼻息肉组织中Ki67+/p63+的比例明显低于对照组(中位数分别为7.3%和13.1%,p<0.001)。此外,我们进一步在组织中证实了p63和KRT5可以同时在细胞中着色(双染)。在p63染色阳性的对照组组织中KRT14染色为阴性；KRT14在伴上皮组织重塑的鼻息肉组织中仅有弱表达。
实验结论：这项研究第一次揭示了鼻息肉来源的鼻上皮干/祖细胞(hNESPCs)生长增殖能力下降,这种生长增殖能力的变化可能是鼻息肉组织重塑的原因所在。
Background:Head and neck squamous cell carcinoma (HNSCC) is the sixth most malignancy in the world. Approximately, one-fourth of all HNSCCs are laryngeal squamous cell carcinoma (LSCC). In contrast, hypopharyngeal squamous cell carcinoma (HSCC) is not so common, but it is usually diagnosed in the advanced stage with poor prognosis. Although several treatment strategies, including surgery, radiotherapy, gene therapy and immunotherapy, have been developed for LSCC and HSCC, no treatment could achieve a satisfactory therapeutic outcome for them and the survival rate has not been improved significantly. Therefore, identification of prognostic markers will be important for prevention and therapy of LSCC and HSCC.
Sirtuin1(SIRT1), which is nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, belongs to the silent information regulator2family (Sir2) of sirtuin histone deacetylases (HDACs). It may play an important role in tumorigenesis. It is reported that SIRT1is increased in many human tumors, such as breast cancer, ovarian cancer, prostate cancer, gastric cancer, colon cancer, diffuse large B-cell lymphoma, acute myeloid leukemia and Bowen's disease. However, there is also some evidence to show that the expression of SIRT1in breast cancer, ovarian carcinoma, bladder carcinoma, prostate carcinoma, and glioblastoma, is lower than that in normal tissues. SIRT1can be activated by SRT, AROS and SUMO-1and can be inhibited by tenovins, DBC1and Tat.
DBC1(Deleted in Breast Cancer1) is well known as a negative regulator of SIRT1. Some studies reveal that DBC1promotes p53-mediated apoptosis through specific inhibition of SIRT1. Therefore, DBC1has been suggested as a tumor suppressor based on this evidence. However, some recent studies showed that DBC1is much more overexpressed in breast cancers and colorectal cancers than in normal tissues and it promotes the survival of breast cancer cells. Therefore, it is still difficult to determine whether DBC1is a tumor suppressor or a tumor promotor. HIC1is a tumor suppressor and SIRT1is one of its transcriptional targets. HIC1plays an important role in cell survival and apoptosis and it may also participate in tumorigenesis.
Objective:In the present study, we aim to elucidate whether SIRT1, DBC1and HIC1are involved in the development of laryngeal squamous cell carcinomas (LSCCs) and hypopharyngeal squamous cell carcinomas (HSCCs) and to identify the potential role of those markers in the prevention and therapy of the tumors.
Methods:The mRNA levels of SIRT1, DBC1and HIC1were measured in54paired LSCC or HSCC tumors and corresponding adjacent noncancerous mucosae using quantitative RT-PCR (qRT-PCR). The protein levels of SIRT1and DBC1were also evaluated in120cases of patients with LSCC or HSCC using immunohistochemical staining. SPSS18.0was used for statistical analysis. The correlation between the protein expression (SIRT1and DBC1) and clinical parameters was analyzed with Pearson chi-square test. Log Rank test was performed for the survival analysis.
Results:qRT-PCR assay showed that, compared with the paired adjacent noncancerous mucosae, SIRT1mRNA and HIC1mRNA were significantly decreased in tumors. In contrast, DBC1mRNA was significantly increased in tumors compared with noncancerous mucosae. The immunohistochemical results indicated that the SIRT1protein was downregulated in tumors compared with noncancerous mucosae and the DBC1protein was downregulated in tumors, which is inconsistent with the results obtained by qRT-PCR. Moreover, decreased SIRT1was significantly correlated with the tumor clinical stage and lymph node metastasis. The decreased DBC1protein was significantly correlated with tumor differentiation, lymph node metastasis, and p53expression. SIRT1was positively correlated with DBC1and HIC1both in tumors and in controls. Besides, we performed the survival analysis using Log Rank test and found that SIRT1(Fig.5A) and DBC1(Fig.5B) were correlated with patient survival.
Conclusion:SIRT1and DBC1play key roles in laryngeal and hypopharyngeal squamous cell carcinomas as tumor suppressors and are associated with lymph node metastasis in LSCCs and HSCCs. HIC1is also served as a tumor suppressor in LSCCs and HSCCs, which is closely correlated with SIRT1at mRNA level. Those novel markers (SIRT1, DBC1and HIC1) could be served as diagnostic indicators for LSCCs and HSCCs and they may become the new therapeutic targets of anticancer drugs.
Background:Nasal polyposis (NP) is a common chronic inflammatory disease of the upper airways. The main characteristics of NP are the infiltration of various inflammatory cells and epithelial damage followed by aberrant tissue repair or remodeling. There are many studies about the molecular mechanisms of nasal polyposis. However, the pathogenesis of NP is still unknown. In our previous study, EMP1, EGF and NRG3were found to be downregulated in NP compared with controls by microarray analysis. Basal cells in nasal epithelium have sternness/progenitor characters and play essential roles in the epithelial remodeling in nasal polyposis (NP). We have successfully isolated and cultured human nasal epithelial stem/progenitor cells in vitro which will help us to study the mechanism of human airway inflammatory diseases including nasal polyposis.
Epithelial membrane protein1(EMP1) is an integral membrane glycoprotein. It was first identified as a peripheral myelin protein22(PMP22) related transcript and was detected in epithelial tissues from gastrointestinal tract, skin, lung, and brain. EMP1was found to regulate epithelial cell proliferation and differentiation. EGF and NRG3are ligands of type I growth factor receptor (ERBB family). EGF is an important growth factor and there are many studies of it in various tissues. NRGs are a group of cell-cell signaling proteins, including NRG1, NRG2, NRG3and NRG4, which have the EGF-like domain structure. Glucocorticosteroids (GCs) are the most effective anti-inflammatory therapy for NP and they can inhibit the infiltration of eosinophils. An important aspect of GCs is to promote tissue repair and remodeling in NP and asthma through transforming growth factor (TGF)-P and epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) signaling pathways. GCs can also upregulate AP-1related genes which are connected with epithelial repair and downregulate p63which relates to tissue remodeling.
Objective:We sought to determine the expression of EMP1and EGF-ERBB family in nasal polyposis and to find out if they play a role in the remodeling of chronically inflamed nasal epithelium in nasal polyposis.
Methods:NP tissues were obtained from55NP patients,18of whom were treated with nasal glucocorticosteroid (GC)(i.e., these18patients had2sets of NP biopsies taken before and after treatment). Biopsies of inferior turbinate mucosa from30healthy subjects were used as controls. Quantitative real-time PCR was performed to determine the mRNA expression levels of EMP1and EGF-ERBB family. Immunohistochemistry was performed to evaluate the protein level of EMP1, p63and Ki67in nasal polyps and controls. And we also investigated p63, CK18and EMP1expressions in epithelial cells by immunocytochemistry. SPSS18.0was used for the statistical analysis.
Results:The EMP1mRNA expression (2.77-fold) was significantly lower in tissues from NP patients before GC treatment than those from controls, and it was increased after GC treatment. EMP1was stained in nasal epithelium and was co-localized with both basal (p63+) and differentiated (CK18+) epithelial cells. Their immunoreactivity was significantly greater in controls than in NP patients, especially in those with severe hyperplastic (1.79-fold) or metaplastic (1.85-fold) epithelium. Positive correlations between EMP1and other epithelial cell related gene (e.g. JUN, PTGS2, and AREG, etc.) mRNAs were observed.
The mRNA expression of EGF-ERBB family was significantly lower in tissues from NP patients before GC treatment than those from controls, especially for EGF (26.32-fold) and NRG3(37.91-fold). After GC treatment, most of the family members (EGF, NRG3, EGFR, ERBB2, ERBB3and ERBB4) were increased. EGF was positively correlated with NRG3at mRNA level (r=0.621). Positive correlations between EGF-ERBB family and AP-1related genes (JUN, PTGS2, IL6and EGR1) mRNAs were observed. EGF-ERBB family was positively correlated with T help cell related markers (T-bet, RORC and GATA3) and other markers (CXCL12, CXCR4and CDKN1A). Besides, EGF-ERBB family was negatively correlated with eosinophil related markers (CCL13and CCL18).
Conclusion:EMP1could be a specific marker for aberrant epithelial remodeling and metaplasia in chronic inflammatory upper airway mucosa (e.g. NP). EGF-ERBB family may participate in the pathogenesis of nasal polyps. EMP1and EGF-ERBB family may serve as the drug treatment targets.
Background:There are four major cell types in healthy nasal epithelium, including basal cells, ciliated cells, non-ciliated columnar cells and goblet cells. Basal cells are considered to have stemness and progenitor properties, which can self-renew and differentiate into other epithelial cell types. In our recent study, we have successfully isolated and cultured human nasal epithelial stem/progenitor cells (hNESPCs) from human inferior turbinate tissues in a serum-free culture method. In this study, we investigated growth and proliferation properties of hNESPCs in an in vitro cell culture system and further confirmation was performed in nasal mucosal tissue obtained from healthy subjects and NP patients.
Objective:We investigate whether the human nasal epithelial stem/progenitor cells (hNESPCs) from patients with NP are inherently distinct from those obtained from healthy controls.
Methods:Epithelial basal cells were isolated and cultured for four passages from NP tissues and control nasal mucosa. The immunocytochemistry and qRT-PCR was performed to analyze the expression of markers.
Results:More than90%of the hNESPCs in the colonies were p63positive, and among these cells, approximately90%were co-localized with KRT5; while they did not express any differentiated nasal epithelial cell markers (e.g., betaⅣ-tubulin and MUC5AC). Another common stem cell marker KRT14was also stained in the colonies, but only a subset of p63or Ki67positive cells expressed KRT14. hNESPCs from both NP and control tissues showed a similar growth pattern throughout the4passages. More "fried-egg" shape phenotype cells were seen from the cell cultures of NP tissues in both P2and P3than those from controls. These cells were almost all β-galactosidase positive, showing a light blue staining mostly in perinuclear region, indicating more senescent cells in the colonies. Cells from control tissues proliferated more rapidly than those from NP tissues over subsequent passages, showing higher CFE values (difference from11.3%to8.5%through PI to P3) and shorter doubling times (difference from6.5hr to22hr through P1to P3). In addition, the variations (based on standard deviation) of CFE and doubling time values were larger in cell cultures derived from NP tissues.
The immunocytochemistry results showed that, in hNESPCs from NP samples, the ratio of Ki67+/p63+cells was significantly lower in P2and P3as compared to those cells from controls and the percentage of Ki67+over p63+cells decreased from P1to P3(median value,84%,61%, and36%respectively). The ki67expression level decreased from P1to P2in cells from NP and control samples. In addition, ki67mRNA level was lower in hNESPCs from NP as compared to controls in P1(1.5-fold, a borderline significant trend, p=0.05) and P2(2.1-fold, approaching borderline of significance, p=0.07).
We further analyzed expression levels of p63and Ki67in nasal mucosa from the same NP and control subjects. Ki67immunostaining decreased in the hyperplasia or metaplasia areas of NP epithelium as compared to control epithelium. Similarly, the percentage (median,25th-75th percentile) of Ki67+cells among p63+cells was significantly lower (p＜0.001) in the hyperplasic NP epithelium (7.3%,4.3%-9.9%) than in healthy controls (13.1%,11.4%-17.3%) by immuno fluorescence staining.
Conclusion:In conclusion, our study demonstrated significantly reduced growth and proliferation dynamics in hNESPCs from NP epithelium. These intrinsic differences in growth and proliferation properties could be the main cause of the persistence and aberrant remodeling in NPs.
SIRT1,又名Sirtuin1,是一种NAD+依赖的去乙酰化酶,参与多种生命过程,并且可能在肿瘤发生过程中发挥着重要作用。据报道,SIRTI在多种肿瘤中表达增高,如乳腺癌、卵巢癌、前列腺癌、胃癌、结肠癌、大B细胞淋巴瘤、急性髓性白血病和Bowen氏病等。然而,也有人报道SIRT1在乳腺癌、卵巢癌、膀胱癌、前列腺癌和恶性胶质瘤中的表达低于正常对照组。目前为止,SIRT1在LSCC和HSCC的表达情况尚无报道。DBC1(Deleted in Breast Cancer1)是SIRT1的负向调节剂,一些研究表明DBC1可以通过特异性抑制SIRT1的活性来促进p53介导的细胞凋亡,也是因为这个原因,DBC1被认为是肿瘤抑制剂。然而,另外一些研究表明,DBC1在乳腺癌和结直肠癌中呈现高表达,同时它还促进乳腺癌细胞的生存,从这个角度讲,DBC1可能在乳腺癌和结直肠癌中起着促癌基因的作用。因此DBC1在恶性肿瘤中的具体作用还存在争议,有待进一步研究确定。HIC1(Hypermethylated In Cancer1)是一种抑癌基因,它在乳腺癌、胃癌、肝癌、食管癌、前列腺癌、宫颈癌、结直肠癌、非小细胞肺癌、生殖细胞肿瘤、白血病、大B细胞淋巴瘤、儿童髓母细胞瘤和室管膜瘤等肿瘤中表现为表观遗传学上的沉默。SIRT1是HIC1的一个作用靶点,人类髓性白血病中HIC1的灭活可以导致SIRT1对p53的活性抑制,进而引起细胞生存和凋亡的改变,p53反过来又可以直接激活HIC1的转录过程,因此,SIRT1、DBC1和HIC1在恶性肿瘤的发生和发展中可能发挥着重要的作用,并可能存在着重要的联系。
实验目的：本课题的研究目的是了解这些新型标志物(SIRT1、DBCl和HIC1)在喉鳞状细胞癌和下咽鳞状细胞癌发生发展中的作用,这三个标志物之间的相关性以及它们与这两种恶性肿瘤的临床病理特征之间的关联。
实验方法：用实时定量PCR方法测定了54对肿瘤标本和癌旁黏膜中SIRT1, DBC1和HIC1mRNA的表达情况并用配对Wilcoxon符号秩和检验进行统计学分析；用免疫组织化学染色的方法测定了120例肿瘤和54例癌旁黏膜中SIRT1,DBC1和HIC1的蛋白表达并采用独立样本Mann-Whitney双尾检验进行分析；另外,用Spearman等级分析来研究mRNA水平各标志物之间的相关性,分析这些标志物和临床病理特征之间的关系时采用Pearson卡方检验。最后采用Log Rank test方法进行了生存分析。所有统计分析均为双侧,当P<0.05时,认为差异有统计学意义。
实验结果：在mRNA水平上,SIRT1和HIC1在肿瘤组织中的表达明显降低,而DBCl的mRNA在肿瘤组织中则比对照组显著增高；Spearman等级分析结果显示在肿瘤组织和癌旁黏膜组织(对照组)中SIRT1与DBC1、HIC1存在明显正相关关系,而DBC1和HIC1之间无明显相关性。
在蛋白水平上,SIRT1表达在细胞核和细胞质中，而DBC1和p53则主要在细胞核中表达；SIRT1在61%(34/54)的癌旁黏膜中高表达,而仅在31%(37/120)的肿瘤标本中高表达；有趣的是,DBC1在93%(50/54)的癌旁黏膜中高表达,而仅在43%(52/120)的肿瘤标本中高表达；统计分析结果显示,SIRT1和DBC1蛋白在肿瘤组织中的表达都比对照组明显降低；SIRT1蛋白表达与肿瘤的TNM分期(p=0.005)和淋巴结转移(p=0.021)有关系,DBCl蛋白表达与肿瘤的分化程度(p=0.026)、淋巴结转移(p=0.013)和p53的表达(p=0.009)有关系。
本课题中共包括120例恶性肿瘤患者(59例为喉鳞状细胞癌,61例为下咽鳞状细胞癌),其中9例失访,随访结束时,58例患者死亡,53例仍生存。我们采用Log Rank test方法进行了生存分析,发现在早期恶性肿瘤患者中，SIRT1和DBC1与患者的生存率相关。
实验结论：三种新型标志物(SIRT1、DBC1和HIC1)在喉和下咽鳞状细胞癌中(LSCC和HSCC)都发挥着抑癌基因的作用,SIRT1和DBC1与SCC和HSCC的淋巴结转移有关。在早期恶性肿瘤中,SIRT1和HDBC1与患者的生存率相关。此外,HIC1在mRNA水平与SIRT1关系密切。这三种新型标志物有可能成为喉和下咽鳞状细胞癌的筛选指标和评估患者预后的指标,并可能成为抗肿瘤药物治疗的新靶点。
研究背景：鼻息肉(NP)是上呼吸道常见的一种慢性炎症,常伴有炎症细胞,特别是中性粒细胞和嗜酸性粒细胞的浸润,以及异常组织重塑。尽管目前有很多关于鼻息肉基因表达的研究,其具体的发病机制尚不明确。我们之前的基因芯片研究发现,与对照组相比,NP中EMP1、EGF和NRG3等标志物表达明显降低。上皮膜蛋白1(EMP1)是一种完整的膜糖蛋白,最早是在胃肠道、皮肤、肺以及脑组织中检测到的,可以调节上皮细胞的生长和分化。EGF和NRG3是表皮生长因子受体(ERBB家族)的配体,ERBB家族包括EGFR, ERBB2,ERBB3和ERBB4,它们的配体除了EGF和NRG3外,还有AREG, HBEGF, TGF-α和其它NRGs等。EGF(表皮生长因子)是由Cohen等人发现的,它参与很多生命过程。NRGs(神经调节蛋白)是细胞间的信号蛋白,包括NRG1, NRG2, NRG3和NRG4,这些神经调节蛋白具有类似EGF的结构域。EMP1和EGF-ERBB家族在NP中的表达情况以及在上皮组织重塑中的作用尚不明确。
糖皮质激素是目前为止控制鼻息肉炎症的最有效的药物治疗措施,可以明显抑制嗜酸性粒细胞的浸润。也有人提出糖皮质激素在促进上皮修复和控制组织重塑中发挥着重要作用。它可以使与上皮修复有关的AP-1及相关基因的表达上调,同时可以使鼻息肉中与组织重塑有关的基因p63的表达下降,有研究发现糖皮质激素通过EGF/EGFR信号转导通路来影响鼻息肉和哮喘患者的上皮修复和组织重塑过程。
实验目的：本课题的目的是研究EMP1和EGF-ERBB家族在鼻息肉中的表达情况,它们在鼻息肉上皮组织重塑中的作用及对糖皮质激素治疗的反应。
实验方法：用实时定量PCR方法测定了55例未经激素治疗的鼻息肉组织(GC-Naive NP)和30例下鼻甲对照组织中EMP1和EGF-ERBB家族mRNA的表达情况并采用Mann-Whitney双尾检验进行统计分析；激素治疗前后鼻息肉组织(18对)中各标志物mRNA的比较采用配对Wilcoxon符号秩和检验进行统计学分析；另外用Spearman等级分析来研究mRNA水平各标志物之间的相关性。用免疫组织化学染色的方法测定了10例GC-Naive NP样本和10例对照组IT样本中EMP1蛋白的表达；所有统计分析均为双侧,当p<0.05时,认为差异有统计学意义。
实验结果：EMPl的mRNA在GC-Naive NP组中明显低于对照组；激素治疗后,EMPl的表达明显增高；伴有重度上皮增生的NP组中EMP1表达水平明显低于轻-中度上皮增生组,伴有鳞状上皮化生的NP组中EMP1表达明显低于不伴鳞状上皮化生组；EMP1与AP-1相关的基因呈正相关。另外在10例GC-Naive NP样本和10例对照组的IT样本中测定了EMP1蛋白的表达,GC-Naive NP组中的EMP1蛋白表达明显低于对照组,免疫细胞化学染色结果提示EMP1在基底细胞和分化细胞中都有表达。
EGF-ERBB家族8个成员在GC-Naive NP组织中的表达明显低于对照组并且差异都有统计学意义,其中EGF和NRG3在两组之间的差异最为显著,分别达26.32和37.91倍之多(p值均小于0.001)；经糖皮质激素治疗后EGF和NRG3表达明显增高,它们相关的4个受体(ERBB2、ERBB3、ERBB4和EGFR)表达也有不同程度的增加。Spearman等级分析显示EGF和NRG3之间存在密切正相关关系(r=0.621),AREG和HBEGF之间存在密切正相关关系(r=0.911),EGF与ERBB2.ERBB3和ERBB4存在关联(r值分别为0.484,0.422和0.435),NRG3与4个受体也都呈正相关；EGF-ERBB家族与AP-1家族中的一些标志物呈正相关；与嗜酸性粒细胞相关的些标志物呈负相关；与T辅助细胞相关的一些标志物呈正相关；EGF-ERBB家族中一些标志物还与CXCL12、CXCR4和CDKN1A呈正相关关系。
实验结论：EMP1可以作为判定慢性呼吸道炎症性疾病(鼻息肉等)中有无存在异常上皮组织重塑和鳞状上皮化生的特异性指标。EGF-ERBB家族可能参与了鼻息肉上皮损伤和修复的过程。EMPl和EGF-ERBB家族可能在鼻上皮细胞的生长和分化中发挥着重要的作用。
研究背景：健康人的鼻黏膜上皮包含四种细胞类型：基底细胞、纤毛细胞、柱状细胞和杯状细胞。通常认为基底细胞具有干细胞和祖细胞的特性,它具有自我更新和分化能力,可以分化为其它几种上皮细胞,如杯状细胞和纤毛细胞等。在此前的研究中,我们已成功分离并培养来自鼻息肉(NP)和下鼻甲(IT)组织的鼻上皮干/祖细胞(hNESPCs)
目前已经报道的关于鼻息肉粘膜上皮的研究大都是鼻息肉上皮的病理学改变,以及相应的分子标志物和基因调控等研究,而无人报道体外鼻上皮干/祖细胞(hNESPCs)的生物学特性。我们的研究是基于这样一个假设：鼻息肉和健康鼻黏膜上皮之间存在内在表型差异。这项研究通过体外细胞培养体系研究鼻上皮干/祖细胞的生长和增殖特性,进而比较鼻息肉和健康鼻黏膜上皮来源的鼻上皮干/祖细胞生长和增殖特性的差异。
实验目的：本课题的目的是体外研究鼻上皮干/祖细胞(hNESPCs)的生物学特性,并比较鼻息肉和对照组来源的hNESPCs克隆形成率和倍增时间的差异,以及体外培养的hNESPCs中p63及Ki67等标志物的表达等。
实验方法：鼻上皮干/祖细胞(hNESPCs)经过分离后,在培养过程中计算细胞倍增时间和克隆形成率；观察衰老相关标志物β-半乳糖苷酶在细胞克隆中的染色情况；通过免疫细胞化学染色观察相关标志物的表达情况；实时定量PCR测定p63和Ki67在鼻息肉和对照组中的表达情况；最后进行统计学分析：采用SPSS18.0软件进行统计分析,用Spearman等级分析来研究mRNA水平各标志物之间的相关性；Linear mixed models用来估计NP和对照组之间倍增时间和克隆形成率的差异；Mann-Whitney分析鼻息肉和对照组组织和细胞中Ki67+/p63+比例的差异。所有统计分析均为双侧,当p<0.05时,认为差异有统计学意义。
实验结果：鼻息肉和对照组来源的hNESPCs具有相似的生长模式；两组不同来源的细胞之间,在p0和p1两代时无明显形态学差异,而到了p2和p3时,NP来源的hNESPCs细胞出现了较多分化和衰老的细胞,显微镜下呈现“荷包蛋”样外观,这些细胞中β-半乳糖苷酶染色阳性,表明细胞出现了明显的衰老状态。对照组来源的细胞在传代过程中生长增殖速度明显比鼻息肉组来源的细胞快,表现为对照组来源的细胞克隆形成率(CFE)更高,倍增时间更短。通过统计学分析(Linear Mixed Models)得出两种不同来源的细胞在p1和p2两代中有显著性差异。通过标准差分析发现NP来源的细胞倍增时间和克隆形成率的变异也较大。
实时定量PCR结果显示：从p0到p2,鼻息肉和对照组来源的鼻上皮干/祖细胞(hNESPCs) p63mRNA的表达无统计学差异。在两种不同来源的细胞中,从p1到p2,Ki67的表达呈下降趋势。在pl和p2两代,鼻息肉来源的hNESPCs中Ki67mRNA的表达低于对照组。对照组来源的细胞不同代之间Ki67的表达较稳定,从p1到p3,Ki67+/p63+的比例在77%到93%之间。而NP来源的细胞随培养时间延长,Ki67的表达逐渐下降,从p1到p3, Ki67+/p63+的比例分别为84%,61%和36%。
在伴有上皮增生和化生的鼻息肉组织中Ki67的表达下降。免疫荧光染色结果显示：在伴上皮增生的鼻息肉组织中Ki67+/p63+的比例明显低于对照组(中位数分别为7.3%和13.1%,p<0.001)。此外,我们进一步在组织中证实了p63和KRT5可以同时在细胞中着色(双染)。在p63染色阳性的对照组组织中KRT14染色为阴性；KRT14在伴上皮组织重塑的鼻息肉组织中仅有弱表达。
实验结论：这项研究第一次揭示了鼻息肉来源的鼻上皮干/祖细胞(hNESPCs)生长增殖能力下降,这种生长增殖能力的变化可能是鼻息肉组织重塑的原因所在。
Background:Head and neck squamous cell carcinoma (HNSCC) is the sixth most malignancy in the world. Approximately, one-fourth of all HNSCCs are laryngeal squamous cell carcinoma (LSCC). In contrast, hypopharyngeal squamous cell carcinoma (HSCC) is not so common, but it is usually diagnosed in the advanced stage with poor prognosis. Although several treatment strategies, including surgery, radiotherapy, gene therapy and immunotherapy, have been developed for LSCC and HSCC, no treatment could achieve a satisfactory therapeutic outcome for them and the survival rate has not been improved significantly. Therefore, identification of prognostic markers will be important for prevention and therapy of LSCC and HSCC.
Sirtuin1(SIRT1), which is nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, belongs to the silent information regulator2family (Sir2) of sirtuin histone deacetylases (HDACs). It may play an important role in tumorigenesis. It is reported that SIRT1is increased in many human tumors, such as breast cancer, ovarian cancer, prostate cancer, gastric cancer, colon cancer, diffuse large B-cell lymphoma, acute myeloid leukemia and Bowen's disease. However, there is also some evidence to show that the expression of SIRT1in breast cancer, ovarian carcinoma, bladder carcinoma, prostate carcinoma, and glioblastoma, is lower than that in normal tissues. SIRT1can be activated by SRT, AROS and SUMO-1and can be inhibited by tenovins, DBC1and Tat.
DBC1(Deleted in Breast Cancer1) is well known as a negative regulator of SIRT1. Some studies reveal that DBC1promotes p53-mediated apoptosis through specific inhibition of SIRT1. Therefore, DBC1has been suggested as a tumor suppressor based on this evidence. However, some recent studies showed that DBC1is much more overexpressed in breast cancers and colorectal cancers than in normal tissues and it promotes the survival of breast cancer cells. Therefore, it is still difficult to determine whether DBC1is a tumor suppressor or a tumor promotor. HIC1is a tumor suppressor and SIRT1is one of its transcriptional targets. HIC1plays an important role in cell survival and apoptosis and it may also participate in tumorigenesis.
Objective:In the present study, we aim to elucidate whether SIRT1, DBC1and HIC1are involved in the development of laryngeal squamous cell carcinomas (LSCCs) and hypopharyngeal squamous cell carcinomas (HSCCs) and to identify the potential role of those markers in the prevention and therapy of the tumors.
Methods:The mRNA levels of SIRT1, DBC1and HIC1were measured in54paired LSCC or HSCC tumors and corresponding adjacent noncancerous mucosae using quantitative RT-PCR (qRT-PCR). The protein levels of SIRT1and DBC1were also evaluated in120cases of patients with LSCC or HSCC using immunohistochemical staining. SPSS18.0was used for statistical analysis. The correlation between the protein expression (SIRT1and DBC1) and clinical parameters was analyzed with Pearson chi-square test. Log Rank test was performed for the survival analysis.
Results:qRT-PCR assay showed that, compared with the paired adjacent noncancerous mucosae, SIRT1mRNA and HIC1mRNA were significantly decreased in tumors. In contrast, DBC1mRNA was significantly increased in tumors compared with noncancerous mucosae. The immunohistochemical results indicated that the SIRT1protein was downregulated in tumors compared with noncancerous mucosae and the DBC1protein was downregulated in tumors, which is inconsistent with the results obtained by qRT-PCR. Moreover, decreased SIRT1was significantly correlated with the tumor clinical stage and lymph node metastasis. The decreased DBC1protein was significantly correlated with tumor differentiation, lymph node metastasis, and p53expression. SIRT1was positively correlated with DBC1and HIC1both in tumors and in controls. Besides, we performed the survival analysis using Log Rank test and found that SIRT1(Fig.5A) and DBC1(Fig.5B) were correlated with patient survival.
Conclusion:SIRT1and DBC1play key roles in laryngeal and hypopharyngeal squamous cell carcinomas as tumor suppressors and are associated with lymph node metastasis in LSCCs and HSCCs. HIC1is also served as a tumor suppressor in LSCCs and HSCCs, which is closely correlated with SIRT1at mRNA level. Those novel markers (SIRT1, DBC1and HIC1) could be served as diagnostic indicators for LSCCs and HSCCs and they may become the new therapeutic targets of anticancer drugs.
Background:Nasal polyposis (NP) is a common chronic inflammatory disease of the upper airways. The main characteristics of NP are the infiltration of various inflammatory cells and epithelial damage followed by aberrant tissue repair or remodeling. There are many studies about the molecular mechanisms of nasal polyposis. However, the pathogenesis of NP is still unknown. In our previous study, EMP1, EGF and NRG3were found to be downregulated in NP compared with controls by microarray analysis. Basal cells in nasal epithelium have sternness/progenitor characters and play essential roles in the epithelial remodeling in nasal polyposis (NP). We have successfully isolated and cultured human nasal epithelial stem/progenitor cells in vitro which will help us to study the mechanism of human airway inflammatory diseases including nasal polyposis.
Epithelial membrane protein1(EMP1) is an integral membrane glycoprotein. It was first identified as a peripheral myelin protein22(PMP22) related transcript and was detected in epithelial tissues from gastrointestinal tract, skin, lung, and brain. EMP1was found to regulate epithelial cell proliferation and differentiation. EGF and NRG3are ligands of type I growth factor receptor (ERBB family). EGF is an important growth factor and there are many studies of it in various tissues. NRGs are a group of cell-cell signaling proteins, including NRG1, NRG2, NRG3and NRG4, which have the EGF-like domain structure. Glucocorticosteroids (GCs) are the most effective anti-inflammatory therapy for NP and they can inhibit the infiltration of eosinophils. An important aspect of GCs is to promote tissue repair and remodeling in NP and asthma through transforming growth factor (TGF)-P and epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) signaling pathways. GCs can also upregulate AP-1related genes which are connected with epithelial repair and downregulate p63which relates to tissue remodeling.
Objective:We sought to determine the expression of EMP1and EGF-ERBB family in nasal polyposis and to find out if they play a role in the remodeling of chronically inflamed nasal epithelium in nasal polyposis.
Methods:NP tissues were obtained from55NP patients,18of whom were treated with nasal glucocorticosteroid (GC)(i.e., these18patients had2sets of NP biopsies taken before and after treatment). Biopsies of inferior turbinate mucosa from30healthy subjects were used as controls. Quantitative real-time PCR was performed to determine the mRNA expression levels of EMP1and EGF-ERBB family. Immunohistochemistry was performed to evaluate the protein level of EMP1, p63and Ki67in nasal polyps and controls. And we also investigated p63, CK18and EMP1expressions in epithelial cells by immunocytochemistry. SPSS18.0was used for the statistical analysis.
Results:The EMP1mRNA expression (2.77-fold) was significantly lower in tissues from NP patients before GC treatment than those from controls, and it was increased after GC treatment. EMP1was stained in nasal epithelium and was co-localized with both basal (p63+) and differentiated (CK18+) epithelial cells. Their immunoreactivity was significantly greater in controls than in NP patients, especially in those with severe hyperplastic (1.79-fold) or metaplastic (1.85-fold) epithelium. Positive correlations between EMP1and other epithelial cell related gene (e.g. JUN, PTGS2, and AREG, etc.) mRNAs were observed.
The mRNA expression of EGF-ERBB family was significantly lower in tissues from NP patients before GC treatment than those from controls, especially for EGF (26.32-fold) and NRG3(37.91-fold). After GC treatment, most of the family members (EGF, NRG3, EGFR, ERBB2, ERBB3and ERBB4) were increased. EGF was positively correlated with NRG3at mRNA level (r=0.621). Positive correlations between EGF-ERBB family and AP-1related genes (JUN, PTGS2, IL6and EGR1) mRNAs were observed. EGF-ERBB family was positively correlated with T help cell related markers (T-bet, RORC and GATA3) and other markers (CXCL12, CXCR4and CDKN1A). Besides, EGF-ERBB family was negatively correlated with eosinophil related markers (CCL13and CCL18).
Conclusion:EMP1could be a specific marker for aberrant epithelial remodeling and metaplasia in chronic inflammatory upper airway mucosa (e.g. NP). EGF-ERBB family may participate in the pathogenesis of nasal polyps. EMP1and EGF-ERBB family may serve as the drug treatment targets.
Background:There are four major cell types in healthy nasal epithelium, including basal cells, ciliated cells, non-ciliated columnar cells and goblet cells. Basal cells are considered to have stemness and progenitor properties, which can self-renew and differentiate into other epithelial cell types. In our recent study, we have successfully isolated and cultured human nasal epithelial stem/progenitor cells (hNESPCs) from human inferior turbinate tissues in a serum-free culture method. In this study, we investigated growth and proliferation properties of hNESPCs in an in vitro cell culture system and further confirmation was performed in nasal mucosal tissue obtained from healthy subjects and NP patients.
Objective:We investigate whether the human nasal epithelial stem/progenitor cells (hNESPCs) from patients with NP are inherently distinct from those obtained from healthy controls.
Methods:Epithelial basal cells were isolated and cultured for four passages from NP tissues and control nasal mucosa. The immunocytochemistry and qRT-PCR was performed to analyze the expression of markers.
Results:More than90%of the hNESPCs in the colonies were p63positive, and among these cells, approximately90%were co-localized with KRT5; while they did not express any differentiated nasal epithelial cell markers (e.g., betaⅣ-tubulin and MUC5AC). Another common stem cell marker KRT14was also stained in the colonies, but only a subset of p63or Ki67positive cells expressed KRT14. hNESPCs from both NP and control tissues showed a similar growth pattern throughout the4passages. More "fried-egg" shape phenotype cells were seen from the cell cultures of NP tissues in both P2and P3than those from controls. These cells were almost all β-galactosidase positive, showing a light blue staining mostly in perinuclear region, indicating more senescent cells in the colonies. Cells from control tissues proliferated more rapidly than those from NP tissues over subsequent passages, showing higher CFE values (difference from11.3%to8.5%through PI to P3) and shorter doubling times (difference from6.5hr to22hr through P1to P3). In addition, the variations (based on standard deviation) of CFE and doubling time values were larger in cell cultures derived from NP tissues.
The immunocytochemistry results showed that, in hNESPCs from NP samples, the ratio of Ki67+/p63+cells was significantly lower in P2and P3as compared to those cells from controls and the percentage of Ki67+over p63+cells decreased from P1to P3(median value,84%,61%, and36%respectively). The ki67expression level decreased from P1to P2in cells from NP and control samples. In addition, ki67mRNA level was lower in hNESPCs from NP as compared to controls in P1(1.5-fold, a borderline significant trend, p=0.05) and P2(2.1-fold, approaching borderline of significance, p=0.07).
We further analyzed expression levels of p63and Ki67in nasal mucosa from the same NP and control subjects. Ki67immunostaining decreased in the hyperplasia or metaplasia areas of NP epithelium as compared to control epithelium. Similarly, the percentage (median,25th-75th percentile) of Ki67+cells among p63+cells was significantly lower (p＜0.001) in the hyperplasic NP epithelium (7.3%,4.3%-9.9%) than in healthy controls (13.1%,11.4%-17.3%) by immuno fluorescence staining.
Conclusion:In conclusion, our study demonstrated significantly reduced growth and proliferation dynamics in hNESPCs from NP epithelium. These intrinsic differences in growth and proliferation properties could be the main cause of the persistence and aberrant remodeling in NPs.
引文
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