小鼠卵巢生殖干细胞分离培养鉴定

Isolation,Culture and Identification of Ovarian Germline Stem Cells in Mus musculus

通过比较不同酶分离方法获得小鼠(Mus musculus)卵巢生殖干细胞的数量以及不同饲养层培养卵巢生殖干细胞的生长状态,筛选出最佳酶分离方法和饲养层,以期建立更优化的培养体系,并对优化培养体系长期培养的卵巢生殖干细胞进行功能鉴定。选取5日龄C57BL/6雌性乳鼠卵巢,机械法制备卵巢组织碎片后,分别采用传统两步酶法(胶原酶和胰蛋白酶)和改良两步酶法(胶原酶、DNA酶、透明质酸酶、分离酶Ⅱ和胰蛋白酶)分离卵巢生殖干细胞,差速贴壁法纯化后,观察不同酶消化方法对卵巢生殖干细胞数量的影响;将分离得到的卵巢生殖干细胞分别培养于小鼠胚胎成纤维细胞STO系[sandosinbred mice(SIM)embryo-derived thioguanine and ouabain-resistant]、多聚赖氨酸以及明胶3种不同的饲养层,观察卵巢生殖干细胞的生长增殖状态;将传至第6代的卵巢生殖干细胞用碱性磷酸酶检测法和免疫荧光法对卵巢生殖干细胞标志物MVH、Oct4、C-kit和增殖标记物Brdu进行鉴定。结果发现两种酶分离方法均可从卵巢中分离得到卵巢生殖干细胞,其中传统两步酶法获得的细胞经差速贴壁法纯化后含卵巢生殖干细胞数量较多;3种不同的饲养层均可使卵巢生殖干细胞生长,其中尤以STO细胞培养的干细胞状态最佳,可在其上生长增殖长达20 d,而多聚赖氨酸和明胶为饲养层培养的卵巢生殖干细胞在培养第5天时出现凋亡。卵巢生殖干细胞碱性磷酸酶染色呈阳性表达,且卵巢生殖干细胞标志物MVH/Oct4、Oct4/Brdu及C-kit蛋白均呈阳性表达。通过传统两步酶法分离得到的卵巢生殖干细胞悬液,经差速贴壁法纯化后,以STO细胞作为饲养层,更有利于卵巢生殖干细胞的生长增殖,且稳定培养后的卵巢生殖干细胞具有正常的生物学功能。

By comparing the number of ovarian germline stem cells obtained by different enzyme separation methods and the growth status of ovarian germline stem cells in Mus musculus cultured on different feeder layers,the best enzyme separation method and feeder layer were selected to establish an optimized culture system,and the ovarian germline stem cells cultured for a long time in the optimized system were identified.The ovaries of 5-day-old C57BL/6 female Mus musculus were mechanically treated into ovarian tissue fragments.The traditional two-step enzyme method(collagenase and trypsin)and improved two-step enzyme method(collagenase,DNase,hyaluronidase,isolate enzyme II,trypsin)were used to isolate ovarian germ stem cells,and the effects of different enzyme digestion methods on the number of ovarian germ stem cells were observed by differential adhesion method.The isolated ovarian germ stem cells were cultured on three different feeder layers including STO[sandosinbred mice(SIM)embryo-derived thioguanine and ouabain-resistant] line of mouse embryonic fibroblasts,polylysine and gelatin to observe the growth and proliferation of ovarian germ stem cells.Ovarian germ stem cells transmitted to the 6th generation were identified by alkaline phosphatase detection and immunofluorescence to identify ovarian germ stem cell markers MVH,Oct4,C-kit and proliferation marker Brdu.The results showed that both enzymatic separation methods could isolate ovarian germline stem cells from the ovaries.The number of ovarian germline stem cells purified by the traditional two-step enzymatic method was more than that obtained by the improved two-step enzymatic method.Three different feeder layers could promote the growth of ovarian germline stem cells.Ovarian germline stem cells cultured with STO cells that blocked mitosis were in good condition and could grow and proliferate for up to 20 days.Ovarian germline stem cells cultured with polylysine and gelatin as feeder layers showed apoptosis on the 5th day of culture.The results of alkaline phosphatase and the immunofluorescence assays for MVH/Oct4 protein,Oct4/Brdu protein,C-kit protein of ovarian germ stem cells were positive.The cell suspension isolated by the traditional two-step enzymatic method was purified by differential adhesion method,and the STO cells were used as the feeder layer,which was more conducive to the growth and proliferation of ovarian germ stem cells,and the stable cultured ovarian germ stem cells had normal biological functions.