神经生长因子及抑制剂K252α和UO126对颗粒细胞增殖及PCNA蛋白表达的影响

Effect of nerve growth factor and K252α and UO126 on proliferation of granulosa cells and expression of proliferating cell nuclear antigen

目的:观察神经生长因子(nerve growth factor,NGF)及抑制剂K252α、UO126对颗粒细胞增殖情况以及增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白表达的影响,同时探索其作用机理。方法:用NGF及K252α、UO126作用培养的小鼠卵巢腔前颗粒细胞,采用2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑单钠盐(cell counting Kit-8,CCK-8)法实验分析颗粒细胞在体外培养中的增殖情况,应用Western blot方法检测PCNA表达的改变。结果:NGF组(A值1.58)明显促进颗粒细胞增殖,与对照组(A值1.31)比较差异有统计学意义(PNGF=0.000),而应用K252α、UO126之后,颗粒细胞的增殖下降,K252α组(A值1.21),NGF+K252α组(A值1.19)、NGF+UO126组(A值1.14)比对照组明显减少,差异具有统计学意义(PK252α=0.001,PNGF+K252α=0.001,PNGF+UO126=0.000)。同时NGF组PCNA相对表达量为5.21,与对照组(表达量1.65)比较差异有统计学意义(PNGF=0.000);而K252α组、NGF+K252α组、NGF+UO126组PCNA相对表达量分别为1.48、0.84、0.41,与对照组比较有统计学意义(PK252α=0.02,PNGF+K252α=0.000,PNGF+UO126=0.000);同时与NGF组比较,差异均有统计学意义(PK252α=0.000,PNGF+K252α=0.000,PNGF+UO126=0.000)。结论:NGF促进颗粒细胞增殖的同时也刺激PCNA表达增高;抑制剂能够抑制细胞的增殖,其机制可能与抑制PCNA的表达有关。

Objective：To observe the mouse ovarian preantral granulose cells proliferation and expression of proliferating cell nuclear antigen（PCNA）in order to investigate the mechanism of nerve growth factor（NGF）and K252α and UO126. Methods：The mouse ovarian preantral granulose cells were treated with NGF and inhibitors K252α and UO126 in vitro. Cell proliferation was measured by cell counting Kit-8（CCK-8）assay and change in the expression of PCNA was detected by Western blot. Results：The data showed that the proliferation of granulose cells was significantly increased in NGF group（A value=1.58）than in control group（A value =1.31）（PNGF=0.000）. The proliferation of granulose cells was significantly decreased in K252α group（A value=1.21）,NGF＋K252α group（A value=1.19）,NGF＋UO126 group（A value=1.14）than in control group（PK252α=0.001,PNGF＋K252α=0.001,PNGF＋UO126=0.000） after applying K252α and UO126. Relative expression of PCNA was higher in NGF group（5.21）than in control group（1.65）（PNGF=0.000）. Relative expression of PCNA was respectively 1.48,0.84,0.41 in K252α group,NGF ＋K252α group and NGF ＋UO126 group,with statistical differences compared with that in control group（PK252α=0.02,PNGF＋K252α=0.000,PNGF＋UO126=0.000） and that in NGF group（PK252α=0.000,PNGF＋K252α=0.000,PNGF＋UO126=0.000）. Conclusion：NGF could significantly promote the proliferation of granulose cell and at the same time induce the expression of PCNA. However,the inhibitors suppress the proliferation of granulose cell,which might be associated with the declining expression of PCNA.