原代结直肠癌相关成纤维细胞培养及特征分析

Primary culture and characteristics of colorectal cancer-associated fibroblasts

目的分离培养结直肠癌相关成纤维细胞（CAF），分析其与正常成纤维细胞（NF）的特征差异。方法取新鲜结直肠癌及对应的正常结肠组织标本，采用组织块培养法分离培养CAF和NF，光镜、Westernblot和免疫荧光技术鉴定所分离的细胞，荧光定量聚合酶链反应（qPCR）、免疫荧光技术、电镜观察和流式细胞术分析成纤维细胞的活化和生长特征。结果组织块培养法成功分离CAF和NF，形态上NF为长梭形，呈束状、编织状、旋涡状排列生长，CAF呈胖梭形、星形或多边形，散在分布、排列无规则，胞质中可见较明显的肌动蛋白骨架；免疫表型上，二者均表达纤维连接蛋白，不表达上皮细胞钙黏蛋白、CD31和钙结合蛋白（caldesmon）。qPCR结果显示，CAF成纤维细胞活化蛋白mRNA表达高于NF，而成纤维细胞特异蛋白1mRNA表达则低于NF。Western blot结果显示NF和CAF平滑肌肌动蛋白α（α-SMA）表达量差异无统计学意义（P〉0．05），免疫荧光显微镜观察显示CAF胞质中α—SMA呈束状、平行、规则地沿细胞长轴排列，NF中α-SMA则无此特征。电镜显示CAF胞质中可见密集、排列规则的肌丝，而NF则无。流式细胞术检测显示CAF中S期和G2期细胞比例明显低于NF，qPCR结果显示CAF相比NF转化生长因子β1、基质源性因子和血小板源性生长因子（PDGF）DmRNA表达下降，PDGFCmRNA表达升高，表明CAF相比NF增殖下降，细胞因子分泌存在差异。结论CAF与NF在形态、活化特征及部分细胞因子分泌方面存在较大差异，CAF相比NF生长增殖能力下降。

Objective To compare the biological characteristics of colorectal cancer associated fibroblasts （CAFs） with normal fibroblasts （NFs）. Methods CAFs and NFs were isolated from fresh specimens of colorectal cancer and their paired normal colon tissue and cultured by tissue explant method. Light microscopy, quantitative polymerase chain reaction （qPCR）, Western blot, immunofluorescence microscopy, electron microscopy and flow cytometry were used to identify isolated fibroblasts and to explore their characteristics of activation and growth. Results Primary colorectal CAFs and NFs were isolated and cultured successfully. NFs showed spindled morphology and were arranged in interlacing or spiral bundles. CAFs were polygonal or spindle, but were fatter than NFs. They were distributed randomly and arranged irregularly, and had obvious actin expression. CAFs and NFs both expressed fibronectin, but not E,cadherin, CD31 and caldesmon, qPCR showed that CAFs expressed more fibroblast activation protein （FAP） and less fibroblast specific protein 1 （FSP1） than that of NFs. There was no difference in the expression of β-SMA between NFs and CAFs by Western blot. β-SMA was bundled in parallel to the long axis of the cell by immunofluorescence. By electron microscopy, CAFs but not NFs showed dense myofilament that was arranged regularly. Flow cytometry showed that the percentage of S- and G2-phase in CAFs were significantly lower than that in NFs. mRNA expression of transforming growth factor β1, stromal derived factor 1 （SDF-1） and platelet derived growth factor （PDGF）-D in CAFs were lower while that for PDGFC was higher than that in NFs. That indicated the proliferation of CAFs was inhibited and the secretion of some cytokines was different when compared with NFs. Conclusions CAFs show differences with NFs in morphology, characteristics of activation and secretion of some cytokines. The proliferation of CAFs is down regulated as compared with NFs.