犬子宫内膜基质细胞的分离培养与鉴定

Isolation, culture and characterization of canine endometrial stromal cells

通过优化犬子宫内膜基质细胞原代及传代培养方法来提高分离培养细胞纯度和效率,以期为犬子宫内膜相关疾病研究建立模型。采用不同浓度胶原蛋白酶消化、二次网筛过滤及差时消化法,选择细胞消化最适酶浓度,改进纯化方法,观察犬子宫内膜基质细胞的形态学和生长特性,通过免疫荧光染色鉴定分离的细胞并作图像分析,MTT法检测不同时间点的细胞活率。结果:0.5%胶原酶Ⅰ消化1 h,经400目筛网过滤可获得大量活力较好的犬子宫内膜基质细胞,且处于发情期的子宫样本分离获得的基质细胞活力更好;细胞呈长梭形或多角形,平行或涡旋状生长,原代细胞培养2~3 d基本铺满培养瓶瓶底,可进行有限的传代培养,传代细胞生长迅速并维持基本的形态不变,P9代之前细胞增殖旺盛,冷冻前后均有较高的存活率;免疫荧光法鉴定表明,所获细胞波形蛋白抗体染色阳性,角蛋白抗体染色阴性,证明确为子宫内膜基质细胞,纯度可达95%。本试验为研究犬子宫内膜相关疾病提供了理想的细胞模型。

The purpose of this study was to improve the purity and efficiency of the cell culture in vitro by optimizing the primary culture and subculture of canine endometrial stromal cells, in order to establish an in vitro model for further research on canine endometrial-related diseases. Canine endometrial stromal cells were isolated and purified by collagenase enzymolysis, mesh filtration and the differential attachment technique, based on which the optimal enzyme concentration was determined, the cell purification methods were improved, and the morphological characteristics and growth characteristics of the canine endometrial stromal cells were observed. Finally, the phenotype of the stromal cells was identified by immunofluorescence, and the MTT method was used to detect cell activity at different time points. The results showed that the 0.5% collagenase I digested for 1 h, which was filtered through a 400 mesh screen to obtain a large number of viable canine endometrial stromal cells, and the cells isolated from the uterine samples in estrus had better viability. The canine endometrial stromal cells were fusiform or polygonal, in parallel or vortex growth. The primary cell culture almost fully spread over the bottom of the culture flask after 2~3 days of cultivation, and was ready for limited passage culture. And the passaged cells grew rapidly and remained almost morphologically unchanged. The stromal cells before the 9th passage proliferated vigorously, and their survival rate was high both before and after cryopreservation. Immunofluorescence identification showed the cells were stained positive for vimentin antibody, and the cytokeratin antibody was stained negative, indicating that the cells isolated in the experiment were endometrial stromal cells at a purity rate of up to 95%. This experiment provides an ideal cell model for the investigation of canine endometrial-related diseases.