抑制素α亚基片段P33对大鼠离体培养黄体细胞凋亡的影响

我室先前的工作表明,抑制素α亚基片段Ｐ３３显著抑制离体培养大鼠黄体细胞的孕酮分泌,整体实验显示Ｐ３３促进黄体功能萎缩和细胞凋亡。本实验进一步在细胞水平探讨Ｐ３３促进黄体细胞凋亡的作用机制。应用ＤＮＡ电泳检测技术、ＤＮＡ荧光（ＡＯＥＢＰＩ）染色和流式细胞分析方法观察了Ｐ３３对ＰＭＳＧｈＣＧ假孕大鼠胶原酶ＤＮＡ酶分散的黄体细胞的自发凋亡的影响。结果三种方法一致显示,Ｐ３３（１μｇ／ｍｌ）促进黄体细胞的自发凋亡。阻断酪氨酸蛋白激酶活性（ｇｅｎｉｓｔｅｉｎ５０μｇ）则抑制Ｐ３３诱导的黄体凋亡;而阻断ＲＮＡ和蛋白质合成（Ｃｙｘ,５０μｇ／ｍｌ;ＡｃｔＤ,５０μｇ／ｍｌ）均不抑制Ｐ３３促进的黄体细胞凋亡。结果表明,Ｐ３３促进培养大鼠黄体细胞的自发凋亡,其作用机制可能与ＴＰＫ途径有关。本实验为抑制素α亚单位或其相关衍生物可能是卵巢局部调节因子之一的假说提供了又一证据。

Effect of human inhibin alpha-fragment 1-32-Tyr(P33) on apoptosis of cultured rat corpus luteal cells

Recently, increasing evidence suggests that alpha inhibin or related proteins may be a functional regulator in the ovary, which is independent of hetero-dimer inhibin. In our previous study, it was demonstrated that human inhibin alpha-N-terminal fragment Tyr-1-32 (P(33)) significantly inhibited progesterone production by rat corpus luteal cells in vitro, and stimulated luteal functional regression and apoptosis in vivo. In the present work, the action of P(33) on apoptosis was further studied in vitro in cultured rat CL cells. Gel electrophoretic analysis for detection of oligonucleosomal DNA fragmentation, AO-EB or PI assays and flow cytometry were used to observe the action of P(33) on the occurrence of spontaneous apoptosis by collagenase-DNase dispersed CL cells, obtained from PMSG-hCG induced pseudopregnant rats. The results showed that P(33) (1 microg/ml) stimulated spontaneous apoptosis of CL cells. The inhibitor of tyrosine protein kinase, genistein (50 microg/ml),inhibited P(33) enhanced spontaneous apoptosis. RNA and protein synthesis inhibitors cycloheximide (Cyx,50 microg/ml) and actinomycin D(Act D,50 microg/ml) did not protect the cells from apoptosis stimulated by P(33). The results suggest that P(33) stimulates spontaneous apoptosis in cultured rat CL cells with the involvement of tyrosine specific protein kinase system. This work provides further evidence for the hypothesis that alpha inhibin or related protein might be a functional regulator in the ovary.