GRO-α在宫颈肿瘤微环境中诱导间质细胞衰老的机制研究

发布时间：2018-05-09 06:05

本文选题：宫颈癌 + 肿瘤微环境　；参考：《复旦大学》2014年硕士论文

【摘要】：[背景与目的] 已有研究证实,在肿瘤发生的微环境中,间质成纤维细胞的衰老可能促进上皮性肿瘤的发生,但宫颈癌的发生是否与其组织中间质成纤维细胞的衰老相关有待深入研究。检测宫颈肿瘤微环境中间质成纤维细胞的GRO-α表达水平,分析GRO-α诱导宫颈间质成纤维细胞衰老研究的信号传导通路,可能为宫颈癌的早期诊断及治疗提供指导和帮助。[方法]宫颈癌组织和正常宫颈组织HE染色以及间质细胞和上皮细胞鉴定；分离纯化正常宫颈成纤维细胞(normal fibroblasts, NFs)和宫颈癌相关成纤维细胞(cancer-associated fibroblasts,CAFs),用ELISA的方法检测NFs和CAFs中GRO-α的表达水平,同时检测细胞因子白介素6(IL-6)和血管内皮生长因子(VEGF)的分泌水平；用衰老相关β-半乳糖甘酶染色(SA-β-gal)、细胞计数和蛋白检测法(Western Blot)分别鉴定细胞衰老、绘制细胞生长曲线和NFs与CAFs中VEGF和p16蛋白表达；用RT-PCR的方法检测GRO-α的受体CXCR2的表达情况；用人重组GRO-α细胞因子处理正常宫颈成纤维细胞后,再通过β-半乳糖苷酶染色法观察其衰老情况；用蛋白免疫印迹法探讨GRO-α对间质成纤维细胞作用的可能机理：用CAF-CM (conditioned medium), CAF-CM+ GRO-aAb,或GRO-α分别处理NFs,检测细胞中相关信号分子的表达变化。[结果]在宫颈癌相关间质成纤维细胞的条件培养基中GRO-α的分泌量较正常宫颈成纤维细胞中的明显增多；宫颈癌相关成纤维细胞分泌的细胞因子IL-6和VEGF也比正常宫颈成纤维细胞要高2倍以上(P0.05)；宫颈癌相关成纤维细胞中SA-β-gal的活性要比正常宫颈成纤维细胞高；宫颈正常成纤维细胞生长速度明显快于宫颈癌相关成纤维细胞生长速度；宫颈癌相关成纤维细胞中p16的表达水平比正常宫颈成纤维细胞中要高；宫颈癌相关成纤维细胞表面受体CXCR2较正常宫颈成纤维细胞中明显增强；用人重组GRO-α细胞因子处理正常宫颈成纤维细胞后,间质细胞呈现明显的衰老迹象,进一步的机理研究显不：GRO-α在肿瘤微环境中主要是通过PI3K/AKT、MAPK、NF-κB等信号传导通路来促进间质细胞的衰老。[结论]本实验证实宫颈癌的发生伴有间质成纤维细胞的衰老,细胞因子GRO-a可能通过PI3K/AKT、MAPK、NF-κB信号通路介导宫颈癌间质衰老,从而可能对宫颈癌的发生和发展具有促进作用。
[Abstract]:[background and objective] it has been demonstrated that in the microenvironment of tumorigenesis, the aging of mesenchymal fibroblasts may promote the development of epithelial tumors. However, whether the occurrence of cervical cancer is related to the senescence of fibroblasts in tissue needs further study. To detect the expression of GRO- 伪 in the mesenchymal fibroblasts of cervical neoplasms, and to analyze the signal transduction pathway of GRO- 伪 induced senescence of cervical stromal fibroblasts, which may provide guidance and help for the early diagnosis and treatment of cervical cancer. [methods] HE staining and identification of interstitial cells and epithelial cells in cervical carcinoma and normal cervical tissues; Normal fibroblasts (NFS) and cancer-associated fibroblasts (Cafs) were isolated and purified from normal cervical fibroblasts (NFS) and cancer-associated fibroblast cells (Cafs). The expression of GRO- 伪 in NFs and CAFs was detected by ELISA, and the secretion levels of interleukin 6 (IL 6) and vascular endothelial growth factor (VEGF) were detected. Senescence related 尾 -galactosidase staining, cell count and protein detection were used to identify cell senescence, cell growth curve and VEGF and p16 protein expression in NFs and CAFs, and CXCR2 expression of GRO- 伪 receptor was detected by RT-PCR method. After the normal cervical fibroblasts were treated with recombinant GRO- 伪 cytokines, the aging of normal cervical fibroblasts was observed by 尾 -galactosidase staining. The possible mechanism of effect of GRO- 伪 on mesenchymal fibroblasts was investigated by Western blotting. NFS were treated with CAF-CM conditioned mediums, CAF-CM GRO-aAbor GRO- 伪, respectively, and the expression of related signal molecules was detected. [results] the secretion of GRO- 伪 in the conditioned medium of cervical cancer associated mesenchymal fibroblasts was significantly higher than that in normal cervical fibroblasts. The cytokines IL-6 and VEGF secreted by cervical cancer associated fibroblasts were more than 2 times higher than those of normal cervical fibroblasts, and the activity of SA- 尾 -gal in cervical cancer associated fibroblasts was higher than that in normal cervical fibroblasts. The growth rate of normal cervical fibroblasts was significantly faster than that of cervical cancer related fibroblasts, and the expression level of p16 in cervical cancer associated fibroblasts was higher than that in normal cervical fibroblasts. The surface receptor CXCR2 of cervical cancer associated fibroblasts was significantly enhanced than that of normal cervical fibroblasts, and the interstitial cells showed obvious signs of senescence after treatment of normal cervical fibroblasts with human recombinant GRO- 伪 cytokines. The further study on the mechanism of GRO- 伪 in tumor microenvironment is to promote the senescence of interstitial cells through signal transduction pathways such as PI3K / AKTK MAPK- 魏 B and so on. [conclusion] this study confirmed that cervical cancer is accompanied by the senescence of interstitial fibroblasts. The cytokine GRO-a may mediate the interstitial senescence of cervical cancer through the signal pathway of PI3K / AKTK MAPK- 魏 B, which may promote the occurrence and development of cervical cancer.
【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】：R737.33

【共引文献】