Wistar大鼠卵巢颗粒细胞的分离、培养与鉴定

Isolation, culture and identification of granulosa cells from wistar rats’ ovary

目的探讨Wistar大鼠卵巢颗粒细胞的分离、培养与鉴定的方法。方法选用21～29天龄wistar大鼠,皮下注射PMSG,取出卵巢,采用机械分离方法释放卵巢颗粒细胞,于CO2培养箱中培养。用HE染色和免疫细胞化学法观察FSHR蛋白表达的方法来对卵巢颗粒细胞进行鉴定,并用MTT法测定细胞生长曲线,同时检测E2、P的分泌情况。结果分离培养出的Wistar大鼠卵巢颗粒细胞存活率>90%,细胞纯度达到90%以上;体外培养的颗粒细胞对数生长期为48～96小时;HE染色在镜下可见贴壁细胞形态完整,细胞浆和细胞核染色均匀,核染深蓝色,位于细胞中央,胞浆淡红色并含有许多颗粒;激素的分泌趋势与细胞的生长情况基本一致,于在96～120小时左右达到峰值,随后逐渐下降。结论采用机械分离法分离培养的卵巢颗粒细胞纯度高、活性好,用FSHR表达染色鉴定颗粒细胞是一种简便快速的方法。

Objective To obtain and identify cultured granulosa cells from the ovary of wistar rats. Methods Wistar rats were subcutaneously treated with PMSG. The ovaries were aseptically removed. Granulosa cells were then released by mechanical method. Granulosa cells were diluted and incubated at 37℃ in water-saturated environment of 5% CO2. Hematoxylin ＆ Eosin（HE） and immunohistochemical staining of FSHR were used for ovarian granulose cell identification. Additionally, the growth curves of granulose cells and hormone levels at different incubation times were evaluated as well. Results Over 90% of the cultured cells were o- varian granulosa cells. The exponentim phase of growth was between 48 and 96 hours of incubation. Using HE staining the adherent cells were morphologically intacted in the microscopically, the cytoplasm and nucleus were dyed uniformity, the nuclear was stained deep blue which was located in the cell cytoplasm central, the cytoplasm was reddish and contained many particles; the hormone secretion and cell growth trend were basically consistent, and in the 96 - 120hours reached a peak, then decreased gradually. Conclusion More than 90% of highly purified granulosa cells could be obtained by mechanical method, and identifications of granulosa cells by HE and FSHR staining were quick and convenient approaches.