半胱氨酸蛋白酶抑制素B在人上皮性卵巢肿瘤中的作用

发布时间：2018-05-21 20:18

本文选题：Cystatin + B　；参考：《复旦大学》2014年硕士论文

【摘要】：研究背景及目的：卵巢癌是导致女性死亡的首位生殖道恶性肿瘤,目前尚无有效的早期检测方法及治疗手段,因此寻找早期卵巢癌的生物标志物对卵巢癌的诊断及治疗具有重要意义。半胱氨酸蛋白酶抑制素B(cystatin B, stefin B, CSTB)是细胞内半胱氨酸蛋白超家族中的一员,在多种肿瘤中均有表达,而在卵巢癌中的报道尚且不多。转化生长因子-β (transforming growth factor-β, TGF-β)在卵巢癌的发生发展过程中起着重要作用。本课题主要研究CSTB的表达与卵巢肿瘤进展的相关性、TGF-p信号转导通路是否对它产生调控作用以及CSTB对卵巢癌细胞增殖的调控功能。方法：1.卵巢组织标本：收集自2005年1月至2012年12月以来复旦大学附属金山医院的27例卵巢肿瘤患者(包括浆液性、黏液性和透明细胞性卵巢肿瘤,分为良性肿瘤组、交界性肿瘤组和恶性肿瘤组)和6例卵巢无病变患者(对照组)的卵巢石蜡标本；2.免疫组织化学染色及分析：免疫组织化学染色法检测上述33例卵巢组织石蜡标本中CSTB是否表达,并对染色结果进行评分,分析其表达情况。收集患者的病史资料,对评分结果及临床病理参数(年龄、卵巢肿瘤组织学类型、肿瘤大小、淋巴结转移及临床分期等方面)之间的相关性做统计分析；3.卵巢癌细胞株培养,TGF-β与拮抗剂SB-431542干预：TGF-β不同浓度及时间干预上皮性卵巢癌OVCAR-3和SK-OV-3细胞株,观察TGF-β是否调控这两种细胞株内CSTB的表达。进而使用TGF-βI型受体拮抗剂SB-431542,观察细胞内CSTB的表达情况；4. Western blot和Real-time PCR:检测TGF-β与其拮抗剂SB-431542干预下卵巢癌细胞株内CSTB的表达情况；5.细胞转染及增殖检测：CSTB特异性的siRNA转染卵巢癌OVCAR-3细胸.实现CSTB基因敲减后进行细胞增殖检测。检测方法包括WST-1比色法及流式细胞仪分析细胞周期法；6.统计分析：所有数据应用SPSS19.0统计软件进行分析。结果：1.CSTB在人卵巢肿瘤中过表达：免疫组织化学染色发现,CSTB在良性、交界性和恶性卵巢肿瘤中有不同程度的表达,呈现依次增高的趋势,在三种组织学类型的卵巢癌(浆液性、粘液性和透明细胞性卵巢癌)中呈强阳性表达。卵巢肿瘤中CSTB的表达阳性率显著高于卵巢无病变组织(P0.01),而良性、交界性和恶性肿瘤之间的差异不显著(P0.05)。卵巢肿瘤中CSTB的免疫组化评分亦显著高于卵巢无病变组织(P0.01)。交界性和恶性肿瘤的CSTB免疫组化评分较良性卵巢肿瘤显著增高(P0.05),后两者间的差异不显著；2.CSTB与卵巢肿瘤临床病理参数的相关性无统计学意义：CSTB的表达与卵巢癌患者的临床病理特征(如年龄、组织学类型、肿瘤大小、淋巴结转移和临床分期等)无显著相关性；3. TGF-β1调控卵巢癌细胞CSTB的表达,且这种调控作用可被TGF-βI型受体阻断剂阻断：TGF-β1干预上皮性卵巢癌OVCAR-3和SK-OV-3细胞株后,CSTB表达下调,呈剂量依赖性。两种细胞中TGF-β1最佳抑制浓度分别为10ng/ml、1 ng/ml;最佳抑制时间是24 h(梯度设定为1 h、6 h和24 h)。使用TGF-βⅠ型受体抑制剂(SB-431542)后,出现CSTB mRNA和蛋白的表达上调；4.CSTB促进卵巢癌细胞的增殖：由WST-1比色法绘制细胞生长曲线知,CSTB特异性的siRNA干预组出现细胞增殖抑制；流式细胞仪分析,细胞周期阻滞于G2/M期,细胞增殖抑制。结论：1.CSTB是卵巢肿瘤的进展性标志物；2.CSTB的表达受TGF-p信号转导通路调控；3.CSTB可能促进卵巢癌细胞的增殖。
[Abstract]:Research background and objective: ovarian cancer is the first reproductive tract malignant tumor that causes female death. There is no effective early detection method and treatment. Therefore, it is of great significance to find the biomarkers of early ovarian cancer for the diagnosis and treatment of ovarian cancer. Cysteine egg white enzyme inhibin B (cystatin B, stefin B, CSTB) is fine. A member of the cysteine protein superfamily is expressed in a variety of tumors, but there are not many reports in ovarian cancer. Transforming growth factor- beta (TGF- beta) plays an important role in the development of ovarian cancer. This topic is to study the relationship between the expression of CSTB and the progress of ovarian tumors. Whether or not the TGF-p signal transduction pathway regulates it and regulates the proliferation of ovarian cancer cells by CSTB. Methods: 1. ovarian tissue specimens: 27 ovarian tumor patients (including serous, mucous and transparent cell ovarian tumors) were collected from January 2005 to December 2012 at Jinshan Hospital Affiliated to Fudan University. Ovarian paraffin specimens of benign tumor group, borderline tumor group and malignant tumor group) and 6 cases of ovarian non pathological patients (control group); 2. immunohistochemical staining and analysis: immunohistochemical staining method was used to detect the expression of CSTB in the paraffin specimens of the 33 cases of ovarian tissue, and the staining results were scored and the expression was analyzed. A statistical analysis of the correlation between scores and clinicopathological parameters (age, ovarian tumor histology type, tumor size, lymph node metastasis and clinical staging) was collected, and 3. ovarian cancer cell lines were cultured, TGF- beta and antagonist SB-431542 intervention: TGF- beta concentration and time intervention in epithelial ovarian ovary OVCAR-3 and SK-OV-3 cell lines, observe whether TGF- beta regulates the expression of CSTB in these two cell lines, and then uses TGF- beta I receptor antagonist SB-431542 to observe the expression of CSTB in the cell, and 4. Western blot and Real-time PCR: to detect the expression of the ovarian cancer cell line under the interference of beta and its antagonist; 5. thin. Cell transfection and proliferation detection: CSTB specific siRNA transfected OVCAR-3 fine chest. Cell proliferation detection was performed after CSTB knockout. The detection methods included WST-1 colorimetric assay and flow cytometry analysis of cell cycle method; 6. statistical analysis: all data were analyzed by SPSS19.0 statistical soft parts. Results: 1.CSTB in human ovarian swelling. Over expression in the tumor: immunohistochemical staining showed that CSTB was expressed in a different degree in benign, borderline and malignant ovarian tumors, showing a trend in turn, strongly positive in three histological types of ovarian cancer (serous, mucinous and transparent cell ovarian cancer). The positive rate of CSTB in ovarian tumors was significant. The difference between borderline and malignant tumor was not significant (P0.05). The immunohistochemical score of CSTB in ovarian tumors was also significantly higher than that of ovarian nonpathological tissue (P0.01). The CSTB immunohistochemical score of borderline and malignant tumors was significantly higher than that of benign ovarian tumors (P0.05). The difference between the two groups was not significant (P0.05). There was no significant correlation between 2.CSTB and the clinicopathological parameters of ovarian tumors. There was no significant correlation between the expression of CSTB and the clinicopathological features of ovarian cancer patients (such as age, histological type, tumor size, lymph node metastasis and clinical staging, etc.); 3. TGF- beta 1 regulates the expression of CSTB in ovarian cancer cells, and this regulation can be used. Blocked by TGF- beta I receptor blocker: TGF- beta 1 interfered with OVCAR-3 and SK-OV-3 cell lines in epithelial ovarian cancer, CSTB expression was downregulated and dose-dependent. The optimal inhibitory concentration of TGF- beta 1 in two cells was 10ng/ml and 1 ng/ml, and the best inhibition time was 24 h (1 h, 6 h and 24). After that, the expression of CSTB mRNA and protein was up-regulated; 4.CSTB promoted the proliferation of ovarian cancer cells: the cell growth curve was plotted by WST-1 colorimetry, and the proliferation inhibition of CSTB specific siRNA intervention group appeared; flow cytometer analysis, cell cycle arrest in G2/M phase and cell proliferation inhibition. Conclusion: 1.CSTB is the progress of ovarian tumor. The expression of 2.CSTB is regulated by TGF-p signal transduction pathway; 3.CSTB may promote the proliferation of ovarian cancer cells.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【共引文献】