家兔子宫内膜细胞和平滑肌细胞的分离培养及鉴定

Culture and identification of uterus endometrial cells and smooth muscle cells of rabbit

为了建立一种高效的子宫细胞分离培养方法,用酶消化、过滤、离心与差速贴壁纯化相结合的方法分离培养家兔子宫细胞,分别以上皮细胞角蛋白、波形蛋白、α-平滑肌肌动蛋白等为抗原的免疫荧光对分离培养的细胞进行鉴定,并采用流式细胞仪分析细胞纯度。结果表明,家兔子宫内膜上皮细胞培养24 h后大部分贴壁,培养3 d左右形成单层细胞集落,呈角蛋白阳性,细胞圆形或椭圆形,胞质呈红色,核呈蓝色,纯度达96%以上;子宫内膜基质细胞培养0.5 h后即有贴壁,培养2 d后呈单层细胞集落状生长,呈波形蛋白阳性,细胞多边形或梭形,胞质呈红色,核呈蓝色,纯度达95%以上;平滑肌细胞培养24 h后大部分贴壁,6-7 d融合成片,呈α-平滑肌肌动蛋白阳性,细胞大都长梭形,胞质呈红色,核呈蓝色,纯度达98%以上。说明该方法能够成功分离并得到高产量、高纯度、高增殖能力的子宫内膜细胞及平滑肌细胞。

The purpose of this study was to set up a method of cultivating highly purified rabbit uterus endometrial cells and smooth muscle cells. Rabbit uterus endometrial epithelial cells, stromal cells and smooth muscle cells were isolated and purified by digestion of trypsin, centrifugation and difference tempo adherence,and observed by light microscope and identified by immunocytochemical stain, and the purity was also analyzed by flow cytometry. The results showed that high purity epithelial cells （95%）,stromal cells （96%） and smooth muscle cells （98%） were obtained . the epithelial cells became adherent after 24 h of culture, formed monolayer cell colony after culturing 3 days and were positively stained by anti-cytokeratin antibody . the cells presented egg-shape,the cytoplasm showed red color,and the nuclus blue,the stromal cells became adherent after 0.5 h of culture, form monolayer cell colony after culturing 2 days and were positively stained by anti-vimentin antibody, the cells showed polygon or fusiform shape, the cytoplasm was red,and the nuclus blue;the smooth muscle cells became adherent after 24 h of culture,formed monolayer cell colony after culturing 6-7 days and were positive stained by smooth muscle actin antibody, the cells presented fusiform shape,the cytoplasm showed red, and the nuclus blue. It is evident that, rabbit uterus endometrial cells and smooth muscle cells of high outcome, high purity and high proliferation can be obtained by using this method.