电压门控钾离子通道对卵巢黄素化颗粒细胞增殖、分泌及凋亡的影响

Influence of 4-aminopyridine on human ovarian luteinized granulosa cell proliferation, production, and apoptosis through inhibiting voltage-gated K ^＋ channel

目的探讨电压门控钾离子通道（Kv）对人绒毛膜促性腺激素（hCG）诱导的卵巢黄素化颗粒细胞增殖、分泌及凋亡的影响。方法收集2007年11月至2008年2月行体外受精。胚胎移植患者的卵泡液共25份，密度梯度法获得卵巢黄素化颗粒细胞，采用RT—PCR技术检测颗粒细胞中Kv mRNA的表达。将培养贴壁后的颗粒细胞按干预方式分为空白组、4氨基吡啶（4-AP）组、hCG组和hCG＋4-AP组共4组，hCG和4-AP的终浓度分别为1250U／L、5nmol／L。培养24h后，采用化学发光法测定各组孕酮水平；采用流式细胞仪和分光光度法检测各组颗粒细胞的凋亡情况；采用四甲基偶氮唑蓝（MTT）比色法检测各组颗粒细胞的增殖、抑制情况。结果（1）培养24h后，空白组、4-AP组、hCG组和bCG＋4-AP组颗粒细胞中的孕酮水平分别为（547±64）、（206±32）、（1991±172）和（763±79）nmol／L。4-AP组、hCG组分别与空白组比较，差异均有统计学意义（P〈0．01）；hCG＋4-AP组与hCG组比较，差异也有统计学意义（P〈0．01）。（2）培养24h后，4-AP组颗粒细胞抑制率为19．7％，与空白组（0）比较，差异有统计学意义（P〈0．05）；hCG＋4-AP组为34．6％，与hCG组（0）比较，差异也有统计学意义（P〈0．01）。4-AP组、hCG＋4-AP组的颗粒细胞增殖率分别为80．3％、68．2％，分别与空白组（100％）比较，差异均有统计学意义（P〈0．05、P〈0．01）；hCG组（100．4％）高于空白组，但差异无统计学意义（P〉0．05）。（3）培养24h后颗粒细胞的凋亡率及半胱氨酸天冬氨酸蛋白酶（caspase-3）活性，4-AP组分别为（40±5）％和0．049±0．009，均高于空白组[（17±4）％和0．029±0．008]，hCG＋4-AP组[（25±4）％和0．039±0．008]也均高于hCG组[（15±3）％和0．022±0．007]，差异均有统计学意义（P〈0．01）。结论4-AP能够抑制卵巢黄素化颗粒细胞和经hCG诱导的颗粒细胞的孕酮分泌，可能与其抑制增殖、促进凋亡有关。Kv在卵巢黄素化颗粒细胞分泌、增殖及凋亡过程中起重要的作用。

Objective To study the influence of 4-aminopyridine （4-AP） on proliferation, production, and apoptosis through inhibiting voltage-gated K ^＋ channel（Kv） in ovarian luteinized granulosa cells. Methods Ovarian luteinized granulosa cells were recovered from 25 women with regular menses who underwent in vitro fertilization programme. The cultured granulosa cells were divided into 4 groups ： blank group, 4-AP treated group, human chorionic gonadotropin （hCG）-induced group and hCG ＋ 4-AP cotreated group. The final concentrations of hCG and 4-AP were 1250 U/L and 5 nmol/L respectively. The progesterone production was detected by the chemoluminescence method. The expression of Kv mRNA on human ovarian luteinized granulosa cell was detected by RT-PCR. The influence on the early apoptosis of granulosa ceils by 4-AP was observed by flow cytometry. Cellular caspase-3 activities were observed with colorimetric method and the inhibition of the cell proliferation was studied using methyl thiazolyl tetrazolium （MTT） method. Results （ 1 ） Kv mRNA was expressed in granulosa cell. （2） The progesterone production of the blank group, 4-AP treated group, hCG-induced group and hCG ＋ 4-AP co-treated group were （547 ± 64）, （206 ±32）, （1991 ± 172）and （763±79） nmol/L, respectively after 24 hours culture. Exposure of the granulosa cells to 4-AP reduced the production of progesterone in blank and hCG-induced granulosa cells. （3）The flow cytometry analysis and the cellular caspase-3 A405 showed that 4-AP increased the percentage of early phase apoptosis （P 〈0. 01 ） ： 4-AP treated group vs blank group [ （40 ±5）% and 0. 049 ±0. 009] vs [ （17 ±4）% and 0. 029 ±0. 008], hCG ＋4-AP co-treated group vs hCG-induced group [ （25 ±4）% and 0. 039 ± 0. 008 ] vs [ （ 15 ± 3） % and 0. 022 ± 0. 007 ]. （4） 24 hours after treated with 4-AP and hCG, the inhibitory rate of cultured granulosa cells of 4-AP treated group was higher than the blank group （ 19. 7% vs 0）, and that of hCG ＋4-AP co-treated group was obviously higher than hCG-induced group （34. 6% vs 0, P 〈 0. 01 ）. Conclusions The voltage-gated K^＋ channels expressed by ovarian luteinized granulosa cell play an important role in cell proliferation, production, and apoptosis. 4-AP may inhibit differentiation of progesterone in granulosa cells through the inhibition of proliferation and induction of apoptosis.