牛成纤维细胞体外分离培养方法比较及细胞特征鉴定

Comparison of isolation and culture methods and identification of cell characteristics of bovine fibroblasts in vitro

为了获得牛成纤维细胞最佳体外分离培养方法并建立成纤维细胞特征鉴定方法,试验采集牛耳皮肤组织块,利用眼科剪修剪成大小为0.5~2 mm^(3)的组织块,分别采用组织块贴壁法、胶原酶Ⅱ+组织块黏附法、双酶消化法(胰蛋白酶+胶原酶Ⅱ)+组织块黏附法进行原代牛耳成纤维细胞的分离培养,观察各方法培养的细胞生长情况,利用成纤维细胞特异性标志蛋白(波形蛋白)免疫荧光方法对分离获得细胞的纯度进行鉴定,采用CCK-8试剂盒对传代细胞增殖情况进行检测,绘制增殖曲线。结果表明:组织块贴壁法在培养至第7天,开始有成纤维细胞缓慢游出,并向组织块周围迁移;培养至第17天,细胞大约汇合至培养皿的80%。胶原酶Ⅱ+组织块黏附法培养至第3天时,在镜下可观察到呈絮状团块、圆形的细胞;培养至第5天时,细胞迅速生长,并且呈梭形、胞质伸展、胞核清晰、单层融合;培养至第11天时,絮状团块和培养皿边缘的细胞出现放射样和旋涡样生长晕,汇合至培养皿的70%~80%。双酶消化法+组织块黏附法培养至第3天时,在镜下观察到细胞大多呈梭形;培养至第5天时,细胞开始逐渐增加,同时细胞迁移数量、聚集程度都明显优于胶原酶Ⅱ+组织块黏附法;培养至第9天时,细胞出现旋涡样生长晕,大约汇合至培养皿的80%,可进行传代纯化。细胞染色呈阳性,且纯度高达98%。增殖曲线呈典型的S形,且2~5 d是细胞的最佳增殖期。说明通过双酶消化法+组织块黏附法可以快速分离、获得牛耳成纤维细胞,是最佳的牛成纤维细胞分离培养方法。

In order to obtain the best method for isolation and culture of bovine fibroblasts in vitro and establish a method for cell characterization, in the experiment, the tissue blocks of auricle skin were collected and trimmed to 0.5-2 mm^(3) by ophthalmic scissors, the primary bovine ear fibroblasts were isolated and cultured by tissue adherence method, collagenase Ⅱ + tissue block adhesion method, double enzyme digestion method(trypsin + collagenase Ⅱ) + tissue adhesion method, respectively. The growth of cultured cells was observed. The purity of the isolated cells was identified by immunofluorescence method of fibroblast specific marker protein(vimentin). CCK-8 kit was used to detect the proliferation of the sub-culture and draw the proliferation curve. The results showed that fibroblasts began to swim out slowly and migrate around the tissue block on the 7 th day of culture with tissue adherent method. On the 17 th day, the cells converged to about 80% of the dish. On the third day of culture with collagenase Ⅱ + tissue adhesion method, round cells were observed at the edge of flocculent mass under microscope. On the 5 th day, the cells grew rapidly and showed fusiform shape, extended cytoplasm, clear nucleus and monolayer fusion. On the 11 th day of culture, the flocculent mass and the cells on the edge of the dish showed radial and vortex like growth halos, and confluenced to 70%-80% of the dish. On the third day of culture by double enzyme digestion and tissue adhesion, most of the cells were spindle shaped. On the 5 th day, the number of cells began to increase gradually, and the number of cell migration and the degree of cell aggregation were significantly higher than those of collagenase Ⅱ + tissue block adhesion method. On the 9 th day of culture, the cells appeared vortex like growth halo, which confluent to about 80% of the dish and could be purified by subculture. Cell staining was positive and the purity was up to 98%. The proliferation curve showed a typical S-shape, and day 2 to 5 was the best proliferation period. The results indicated that the method of double enzyme digestion and tissue block adhesion was the best method for the isolation and culture of bovine ear fibroblasts.