人结肠癌相关成纤维细胞的原代培养及其生物学特性

Primary culture of human colon carcinoma-associated fibroblasts and biological characteristics

目的探讨人结肠癌相关成纤维细胞（CAFs）及结肠正常成纤维细胞（NFs）的培养和鉴定方法，分析两者在生物学特性和蛋白表达方面的差异。方法采用组织块法和胶原酶消化法进行人结肠CAFs和NFs的原代培养；选择第3代人结肠CAFs及NFs，通过形态学观察和免疫细胞化学染色进行鉴定；细胞计数试剂盒（CCK-8）法检测人结肠CAFs和NFs的增殖活性，并绘制7d生长曲线；酶联免疫吸附试验（ELISA）法测定结肠CAFs和NFs细胞上清液中骨桥蛋白（OPN）、转化生长因子-β（TGF-β）、血管内皮生长因子（VEGF）及基质金属蛋白酶（MMP）-2含量。结果结肠CAFs胞质突减少，细胞不规则，排列紊乱，可见双核及多核细胞，除细胞角蛋白（CK）外，波形蛋白（vimentin）与α平滑肌肌动蛋白（α-SMA）均为阳性表达；与结肠NFs比较，结肠CAFs增殖活性明显增强；ELISA法测定结肠CAFs和NFs细胞上清液中蛋白含量，其中OPN分别为（2．106±0．137）μg／L和（1．499±0．151）μg／L（P〈0．01），TGF-β分另0为（331．203±12．203）ng／L和（315．391±12．998）ng／L（P〈0．01），VEGF分别为（17．216±0．632）ng／L和（14．887±0．661）ng／L（P〈0．01），MMP-2分别为（1．830±0．192）坤g／L和（1．436±0．102）μg／L（P〈0．01）。结论通过组织块法和胶原酶消化法原代培养皆可获得高纯度的人结肠CAFs和NFs；两者在形态结构、增殖活性和蛋白表达等方面差异有统计学意义，这些差异可能是人结肠CAFs发挥其促结肠癌发生发展作用的生物学基础。

Objective To investigate the primary culture and identification of human colon carci- noma-associated fibroblasts （CAFs） and normal fibroblasts （NFs）, and analyze their differences in biological eharacteristics and protein expression patterns. Methods The primary human colon CAFs and NFs were obtained by tissue culture and digestion methods. The CAFs and NFs at third generation were identi- fied by morphological observation and immunocytochemistry （ICC） staining. Cell counting kit-8 （CCK-8） assay was used to detect the proliferative activity and a growth curve during seven days was drawn. Osteopon- tin （OPN）, transforming growth factor-β （TGF-β）, vascular endothelial growth factor （VEGF） and matrix metalloproteinase （MMP）-2 were measured by using enzyme linked immunosorbent assay （ELISA）. Results The cytoplasmic processes of CAFs were decreased, CAFs were arranged haphazardly and their shape was-ir- regular, with conjugate nuclei or more nuclei. ICC staining revealed negative cytokeratin （CK）, and positive vimentin and a-smooth muscle actin （α-SMA） in CAFs. The proliferative activity of CAFs was significantly enhaneed as compared with NFs. The expression levels of OPN in the culture supernatants of CAFs and NFs were （2. 106±0. 137） and （1.499 ±0. 151） μg/L （P〈0. 01）, those of TGF-β （331. 203 ±12. 203） and （315. 391± 12. 998） ng/L （P 〈0. 01 ）, those of VEGF （ 17. 216 ±0. 632） and （ 14. 887 ±0. 661 ） ng/L （ P 〈 0. 01 ）, and those of MMP-2 （ 1. 830 ± 0. 192 ） and （ 1. 436 ± 0. 102） μg/L （ P 〈 0.01 ）, respectively. Conclusion The purified human colon CAFs and NFs can be obtained successfully by both tissue culture and digestion methods. There are significant differences in morphological characteristics, proliferative activity and expression perterns of certain proteins between the CAFs and NFs, which may be a biological basis forhuman colon CAFs to play their role in promoting the development of colon cancer.