摘要
目的:对卵巢癌相关成纤维细胞(Carcinoma-associated fibroblasts,CAFs)及卵巢正常成纤维细胞(Normalfibroblasts,NFs)进行分离、培养、纯化,并分析两者多种基因表达的差异。方法:①用组织块贴壁培养法获得原代卵巢癌CAFs和卵巢NFs,通过胰酶消化法和反复传代法进行细胞的纯化;②观察细胞形态,多种基因的免疫细胞化学染色检测及相应mRNA的半定量分析,对CAFs和NFs进行初步鉴定;③应用RT-PCR的方法对CAFs和NFs中多种基因进行分析比较。结果:①获得了纯化的卵巢CAFs和NFs;②卵巢CAFs的细胞免疫化学染色α平滑肌肌动蛋白(α-SMA)染色呈阳性,角蛋白呈阴性,卵巢NFs波形蛋白(Vimentin)阳性,而α-SMA和细胞角蛋白阴性;③卵巢CAFs与NFs中Vimentin与α-SMA的mRNA发现均有表达,但CAFs中表达增加,差异有统计学意义(P<0.05);④与NFs相比,CAFs中多种mRNA的表达增加,其中包括生长因子,血管生成促进因子,细胞因子等。结论:与NFs相比,CAFs在mRNA、蛋白表达等方面均有统计学差异;卵巢癌中CAFs基因表达的检测表明,其基因表达有显著变化,提示这些成纤维细胞可能为卵巢癌细胞的生长提供了最适合的微环境。推测卵巢-宿主界面微环境在上皮性卵巢癌的发展中可能起重要作用。
Objective：To separate,culture and purify ovarian cancer-associated fibroblasts（CAFs） and normal fibroblasts（NFs）,analyze the differential gene expression.Methods：Attachment culture method was used to obtain primary ovarian CAFs and ovarian NFs,trypsin digestion method and repeated passage method were used to purify the cells;the cell morphology was observed,immunohistochemical staining and mRNA semi-quantitative analysis were used for primary definition of CAFs and NFs;RT-PCR was used to analyze and compare multiple genes in CAFs and NFs.Results：Purified ovarian CAFs and NFs were obtained;immunohistochemical staining of ovarian CAFs showed that α smooth muscle actin（α-SMA） was positive and cytokeratin was negative;immunohistochemical staining of ovarian NFs showed that vimentin was positive,α-SMA and cytokeratin were negative;vimentin mRNA and α-SMA mRNA expressed in ovarian CAFs and NFs,but their expressions increased in CAFs,there was significant difference（P〈0.05）;compared with ovarian NFs,the expressions of multiple mRNA in ovarian CAFs increased,including growth factors,angiogenesis factor and cytokines.Conclusion：Compared with ovarian NFs,there is significant difference in mRNA expression and protein expression of ovarian CAFs;the detection of gene expression in ovarian CAFs shows that the gene expression changes significantly,and indicates that the fibroblasts may provide optimum microenvironment for growth of ovarian cancer cells;microenvironment of ovarian tumor-host interface may play an important role in development of epithelial ovarian cancer
出处
《中国妇幼保健》
CAS
北大核心
2011年第6期909-912,共4页
Maternal and Child Health Care of China
基金
湖北省科技攻关计划资助项目〔2007AA301B19〕
