胃癌组织中胃癌相关成纤维细胞、胃正常成纤维细胞的分离鉴定及增殖情况观察

Isolation,identification,and proliferation of gastric cancer-associated fibroblasts and normal gastric fibroblasts

目的从胃癌组织中分离胃癌相关成纤维细胞(CAFs)及胃正常成纤维细胞(NFs),并鉴定其功能。方法3例胃癌患者,均行胃癌切除术,术中保留胃癌组织及胃正常黏膜组织。采用组织贴壁块法及胶原酶消化法分离胃癌组织中的胃CAFs及胃正常黏膜组织中的胃NFs。原代培养胃CAFs、胃NFs,差速贴壁传代法分离并纯化细胞,差速贴壁20 min。倒置显微镜下观察胃CAFs、胃NFs细胞形态,采用免疫细胞化学染色法检测细胞α-平滑肌肌动蛋白(α-SMA)、角蛋白18(CK18)、波形蛋白(Vimentin)。取3个来源分离的胃CAFs、胃NFs,分别于细胞贴壁24 h、2 d、3 d、4 d、5 d、6 d采用MTT法测算细胞光密度(OD)值,绘制细胞生长曲线。结果胃癌CAFs细胞大小不一,呈梭形或星形,排列不规则并呈重叠生长;胃NFs细胞均匀一致,呈长梭形,排列规则。胃CAFs细胞存在α-SMA、Vimentin阳性表达,CK18阴性表达,胃NFs细胞存在Vimentin阳性表达,α-SMA、CK18阴性表达。培养3~6天,各来源胃CAFs细胞OD值均高于胃NFs(P均<0.05)。结论成功分离胃CAFs、胃NFs。胃CAFs、胃NFs均存在Vimentin表达。胃CAFs增殖能力优于胃NFs。

Objective To isolate the gastric cancer-associated fibroblasts（CAFs） and normal fibroblasts（NFs） and identify their functions. Methods Three patients with gastric cancer underwent gastric resection,and gastric cancer tissues and normal gastric mucosa were preserved during operation. The tissue adhension method and collagenase digestion were used to isolate the gastric CAFs in gastric cancer tissues and gastric NFs in the normal gastric mucosa. Primary cultured gastric CAFs and gastric NFs were isolated and purified by differential adherence method,and the cells received differential adhension for 20 min. The morphology of gastric CAFs and gastric NFs cells was observed under inverted microscope,and α-smooth muscle actin（α-SMA）,keratin 18（CK18）,and Vimentin were detected by immunocytochemical staining. The isolated gastric CAFs and gastric NFs from 3 sources adhered to the wall for 24 h,2 d,3 d,4 d,5 d,and6 d,and the cell optical density（OD） was calculated by MTT. Results The morphology of CAFs was variable with spindle shape or star shape. They were irregularly arranged and in the overlapping growth. On the contrast,NFs were uniform,spindle-shaped,and arranged regularly. Gastric CAFs showed positive expression of α-SMA and Vimentin,negative expression of CK18; gastric NFs cells had positive expression of Vimentin,and negative expression of α-SMA and CK18. After culture for 3-6 days,the OD values of CAFs from all sources were higher than those of gastric NFs（all P〈0. 05）.Conclusions CAFs and NFs are isolated successfully. Vimentin is expressed in both CAFs and NFs. The proliferation of CAFs is stronger than that of NFs.