奶牛子宫内膜基质细胞原代培养方法的改进与细胞鉴定

Improvement of primary culture method and identification of bovine endometrial stromal cells

为了优化奶牛子宫内膜基质细胞的分离培养方法,以提高奶牛子宫内膜基质细胞的培养效率和细胞纯度,试验采用常规胰蛋白酶和胶原酶交替消化过筛法分离细胞,在样本选择、消化酶配方和筛网孔径方面进行了优化改进并传代纯化,通过细胞形态学观察、细胞生长曲线、免疫组化鉴定进行细胞鉴别。结果表明:处于发情期的子宫样本采用0.25%胰蛋白酶和0.25%Ⅱ型胶原酶及0.15 g/L DNaseⅠ的次序消化,经大孔径筛网过筛后获得基质细胞,纯度较高,且细胞活力更好;采用0.25%胰蛋白酶+0.02%EDTA传代消化可缩短传代时间;基质细胞贴壁后呈长梭形和多角形两种形态;经免疫组化鉴定该细胞具有波形蛋白免疫反应性,与子宫内膜切片中波形蛋白组织化学结果一致,表明其为来源于内膜基质层的基质细胞。奶牛子宫内膜基质细胞生长曲线呈'S'型,符合细胞生长的规律。说明优化培养方法后可更便捷、高效地分离培养较高纯度的奶牛子宫内膜基质细胞。

In order to optimize the cow cultivation method of endometrial stromal cells,to improve the efficiency of the cow cultivation of endometrial stromal cells and the cell purity,alternate digestion with conventional trypsin and collagenase and sifting methods were used to separate the cells.The sample selection,digestive enzyme formula and mesh aperture were optimized and purified.Cell identification was performed by morphological observation,cell growth curve and immunohistochemical identification.The results showed that uterine samples in heat stage were digested using 0.25%trypsin,0.25%Ⅱcollagenase and 0.15 g/L DNaseⅠin order and were screened by large aperture to obtain stromal cells,with high purity and better cell vitality.Passage time could be shortened by 0.25%trypsin+0.02%EDTA.After adherent to the wall,the stromal cells show two forms:long spindle and polygon.The vimentin immunoreactivity of the cells was identified by immunohistochemistry,which was consistent with the results of vimentin histochemistry in endometrial slices,indicating that the cells were derived from the stromal layer of the intima.The growth curve of cow endometrial stromal cells was S-shaped,which was in line with the law of cell growth.The results suggested that cow endometrial stromal cells with high purity could be isolated more easily and efficiently after optimized culture method.