神经生长因子对培养的小鼠卵巢腔前颗粒细胞增殖的影响

Effects of nerve growth factor on the proliferation of ovarian preantral granulosa cells in mice

目的:为了探讨神经生长因子(nerve growth factor,NGF)的促生殖作用及其机理,观察小鼠卵巢腔前颗粒细胞(granulosa cell,GC)增殖情况,同时建立便捷灵敏、毒性小、无污染的细胞增殖检测方法。方法:用NGF刺激培养的小鼠卵巢腔前GC,CCK-8实验分析,溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)掺入标记,免疫细胞化学染色法检测GC在体外培养中的增殖情况和BrdU标记指数(labeling index,LI);流式细胞技术分析细胞周期各时相的变化。结果:NGF明显促进GC增殖,并且具有一定的量效关系。光镜下可见BrdU阳性标记的GC,其细胞核内可见棕褐色阳性颗粒,提示处于细胞增殖期。NGF组的BrdU LI为40.62%,明显高于对照组23.57%(P=0.000)。NGF使增殖期的细胞显著增多及增殖指数(proliferation index,PI)升高,G0/G1期细胞相对比例减少(P=0.000)。结论:本研究证实NGF能够促进GC增殖,CCK-8分析和BrdU标记是高效、便捷的标记腔前GC增殖的方法,NGF促进GC增殖与促进GC DNA的合成有关系。

Objective：To observe the proliferation of mouse ovarian preantral granulosa cells in order to discuss the reproduction and mechanism of nerve growth factor（NGF）and to establish speedy,sensitive and pollution free detection of cell proliferation with low toxicity. Methods：The mouse ovarian preantral granulosa cells were treated with NGF in vitro and cell proliferation and labeling index（LI）were measured by CCK-8 assay,bromodeoxyuridine（BrdU）labeling and immunocytochemistry staining. Changes in cell cycle were analyzed with flow cytometry. Results：Data showed that NGF significantly promoted the proliferation of granulosa cells in a dose-dependent manner. Granulosa cells BrdU positively labeled were visible under light microscope after NGF treatment. Brown positive grains were visible in the nuclei,which indicated that cells were in proliferation period. BrdU LI（%）in the treatment was 40.62%,which was significantly more than that of control（23.57%,P=0.000）. Number of cells and the proliferation index were increased in proliferation period and the cell proportion was decreased in G0/G1 phase（P=0.000）after NGF treatment. Conclusions： CCK-8 assay and BrdU labeling are effective,speedy and sensitive in detecting preantral granulosa cell proliferation. The promotion effect of NGF on granulosa cell proliferation might be associated with promotion of DNA synthesis.