Zhao Zhi-Ming, Cui Na, Xu Su-Xin, Gao Fu-Lu, Hao Gui-Min, Cao Jin-Feng
Department of Reproductive Medicine, the Second Hospital, Hebei Medical University, Shijiazhuang 050000, China.
Zhonghua Fu Chan Ke Za Zhi. 2008 Dec;43(12):918-22.
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.
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 co-treated 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 cells 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.
(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 A(405) 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).
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.
研究4-氨基吡啶（4-AP）通过抑制卵巢黄素化颗粒细胞电压门控钾通道（Kv）对细胞增殖、分泌及凋亡的影响。
从25例月经规律且接受体外受精程序的女性中获取卵巢黄素化颗粒细胞。将培养的颗粒细胞分为4组：空白组、4-AP处理组、人绒毛膜促性腺激素（hCG）诱导组和hCG + 4-AP联合处理组。hCG和4-AP的终浓度分别为1250 U/L和5 nmol/L。采用化学发光法检测孕酮分泌。通过RT-PCR检测人卵巢黄素化颗粒细胞中Kv mRNA的表达。采用流式细胞术观察4-AP对颗粒细胞早期凋亡的影响。采用比色法观察细胞半胱天冬酶-3活性，并用甲基噻唑基四氮唑（MTT）法研究对细胞增殖的抑制作用。
（1）颗粒细胞中表达Kv mRNA。（2）培养24小时后，空白组、4-AP处理组、hCG诱导组和hCG + 4-AP联合处理组的孕酮分泌量分别为（547±64）、（206±32）、（1991±172）和（763±79）nmol/L。颗粒细胞暴露于4-AP可降低空白组和hCG诱导组颗粒细胞的孕酮分泌。（3）流式细胞术分析和细胞半胱天冬酶-3 A（405）结果显示，4-AP增加了早期凋亡百分比（P<0.01）：4-AP处理组与空白组相比[（40±5）%和0.049±0.009]与[（17±4）%和0.029±0.008]，hCG + 4-AP联合处理组与hCG诱导组相比[（25±4）%和0.039±0.008]与[（15±3）%和0.022±0.007]。（4）用4-AP和hCG处理后24小时，4-AP处理组培养的颗粒细胞抑制率高于空白组（19.7%对0），hCG + 4-AP联合处理组明显高于hCG诱导组（34.6%对0，P<0.01）。
卵巢黄素化颗粒细胞表达的电压门控钾通道在细胞增殖、分泌及凋亡中起重要作用。4-AP可能通过抑制增殖和诱导凋亡来抑制颗粒细胞中孕酮的分化。
