小鼠子宫内膜原代上皮细胞及间质细胞的优化分离和培养

Isolation and culture of primary mouse endometrial epithelial cells and stromal cells

目的:建立一种获得高纯度、高活性的子宫内膜上皮细胞和间质细胞的优化分离和体外培养的方法,提供研究子宫内膜相关疾病的理想体外模型。方法:双酶消化、过滤、离心法分离和培养小鼠子宫内膜上皮细胞和间质细胞。用光镜观察培养细胞;双重免疫荧光标记法检测上皮细胞角蛋白(CK)和间质细胞波形蛋白(Vim)的表达;MTT法测定间质细胞的生长曲线。结果:培养24h,小鼠子宫内膜上皮细胞呈多边形、铺路石样生长,CK免疫荧光阳性,纯度达95%以上。正常原代小鼠子宫内膜上皮细胞不能传代,培养5～6天后细胞逐渐走向衰老。间质细胞呈长梭形、漩涡状生长,Vim免疫荧光阳性,纯度达95%以上。原代间质细胞可有限传代,3代内细胞形态无改变。传代间质细胞第3～6天为细胞对数生长期,生长曲线呈"S"形,倍增时间约为56.97h。结论:双酶消化、过滤、离心法可获得高纯度、高活性的具有典型的生物学细胞特性的子宫内膜上皮细胞和间质细胞。

Objective：To furnish an ideal mouse model for the study of endometrial diseases, establishing a method of optimized separation and culture to obtain high purity and high active mouse uterine endometrial epithelial cells and stromal cells in vitro. Method：Sepa- rated cells were obtained using double enzymatic digestion, filtration and centrifugation, then i- dentified by light icroscopy and double immunofluorescence method with cytokeratin and vimen- tin as the antigen. MTT method was used to evaluate proliferative activity of stromal cells. Re- stilts：After 24hours culturing, epithelial cells had polygon and grew like pave road stone. They were stained positively by cytokeratin and negatively by vimentin （ 〉95% ）. Primary endometri- al epithelial cells failed to passage, and the cells aging after 5 - 6d. Stromal cells were long spindle and grew in swirl. They were stained positively by vimentin and negatively by cytokera- tin（ 〉95% ）. Primary stromal cells could passage, but it was limited. The cells retained their morphology within the first 3 passages. The passage cells were in logarithmic growth phase from day 3 to 6, the growth curve shape was ＂S＂, and doubling time was 56.97 hours. Conclusion：With double enzymatic digestion, filtration and centrifugation, we can obtain high purity and high active epithelial and stromal cells, and they had typical biological characteristic.