- 學位論文
RU486對人類卵巢顆粒細胞第二型環氧酶表達 之傳導路徑之調控
Signaling Transduction Pathway Involves in Mifepristone (RU486) Regulating hCG-induced Cyclooxygenase-2 Expression in Human Granulosa-Luteal Cells
摘要
黃體激素(luteinizing hormone；LH)在哺乳類動物體內控制排卵機轉上扮演著重要角色。許多證據指出，促性腺激素(gonadotropins)，如黃體激素或人類絨毛膜促性腺激素(human chorionic gonadotropin；hCG)，經由活化cAMP/第一型蛋白激酶A (PKA) 及下游絲裂原活化蛋白激酶(mitogen-activated protein kinase；MAPK)蛋白家族如ERK1/2、p38等蛋白，進而刺激第二型環氧酶(cyclooxygenase-2；COX-2)蛋白生成。第二型環氧酶基因缺失或前列腺素 (prostaglandins；PGs)的缺乏，皆會造成生殖障礙；而前列腺素的合成更受到第二型環氧酶及前列腺素合成酶(prostaglandin synthase)等酵素調控。RU486，一種固醇類黃體素拮抗劑，在靈長類動物活體(in vivo)實驗中，證明除了能抑制黃體素(progesterone；P)作用，也能抑制第二型環氧酶的催化作用及前列腺素E2的合成，影響排卵及著床，達到避孕效果。但目前關於RU486對人類生殖的影響報告，仍著重在黃體素合成及著床上；因此，本論文將著重探討人類絨毛膜促性腺激素對人類卵巢顆粒細胞的作用，及RU486是否會經由絲裂原活化蛋白激酶級聯途徑(MAPK cascade)影響第二型環氧酶、前列腺素E合成酶(prostaglandin E synthase；PGEsyn) mRNA表現及前列腺素E2分泌的影響，阻礙排卵。我們分別以接受人工生殖計畫的婦女之卵巢濾泡中所分離出的人類卵巢顆粒細胞(human granulosa-luteal cell；hGLC)及人類卵巢顆粒細胞HO23細胞株為實驗材料，利用RT-PCR、西方點墨法及細胞免疫化學染色等方法，發現人類卵巢顆粒細胞在人類絨毛膜促性腺激素(hCG)刺激下，前列腺素E合成酶mRNA都呈時間和劑量相關的上升趨勢；而第二型環氧酶 mRNA及蛋白表現和ERK1/2被磷酸化的情形，雖也隨人類絨毛膜促性腺激素劑量及作用時間增加而上升，但卻都在人類絨毛膜促性腺激素作用12小時後下降。我們也在人類卵巢顆粒細胞培養中投予(MEK1/2)抑制劑 PD98059，證實人類絨毛膜促性腺激素刺激ERK1/2磷酸化程度與第二型環氧酶mRNA及蛋白表現有關；而RU48對人類絨毛膜促性腺素所誘發之ERK1/2磷酸化及第二型環氧酶mRNA及蛋白表現有相似的抑制效果。以酵素免疫分析法(enzyme immunoassay；EIA)分析細胞培養液，發現在人類絨毛膜促性腺激素刺激下前列腺素E2 (prostaglandin E2；PGE2)分泌呈劑量相關增加，而隨著RU486濃度增加，前列腺素E2分泌也逐漸下降；但RU486對PGEsyn mRNA表現沒有明顯抑制。 綜合以上結果，我們認為在人類卵巢顆粒細胞中，RU486抑制ERK1/2磷酸化，降低人類絨毛膜促性腺激素誘發之第二型環氧酶合成而非前列腺素E合成酶，影響人類卵巢顆粒細胞前列腺素E2分泌，抑制排卵。
並列摘要
In the past decade, a critical role for the luteinizing hormone (LH) surge in initiating periovulatory events in mammals is well established. There are also several evidence indicate that gonadotropins, i.e., LH and human chorionic gonadotropin (hCG), treatments were able to increase cyclooxygenase-2 (COX-2) protein expression through the cAMP/type I protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) family activation signaling pathway. COX-2, a rate-limiting enzyme of prostaglandins (PGs) synthesis, and PGs-deficient mice will lead to infertility. RU486, a synthetic steroid, can provide a significant degree of pregnancy protection, preventing implantation or ovulation by inhibiting progesterone (P), COX-2 and prostaglandin E2 (PGE2) productions in primates. However, most of researches about RU486 in human reproduction realm still focus on progesterone synthesis and its effect on implantation. In the present study, we examine the effects of RU486 on COX-2, prostaglandin E synthase (PGEsyn) mRNA and PGE2 secretion levels in human granulosa-luteal cells (hGLC), and whether RU486 can affect COX-2 expression by altering phosphorylation level of mitogen-activated protein kinase (MAPK) family proteins. Thus, we have used primary cultures of hGLC collected from follicular fluid of women participated in in vitro fertilization (IVF) program and hGLC cell line, HO23, as our target, by using reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunocytochemistry, we found hCG stimulation could increase PGEsyn mRNA expression in time- and does-dependent manner; COX-2 protein and ERK1/2 phosphorylation level were also increased in a does-dependent character. However, in time-course experiment, both COX-2 protein and phosphorylated ERK1/2 protein expressions decreased after 12 hours hCG administration. By using MEK1/2 inhibitor, PD98059, hCG increased COX-2 mRNA and protein expression via activating ERK1/2 protein. Meanwhile, in HO23 cells co-cultured with hCG and RU486, we found that hCG-induced ERK1/2 phosphorylation level and COX-2 expressions were profoundly suppressed by RU486 in a similar pattern. However, RU486 has no significant influence on PGEsyn mRNA expression level. However, an increasing level of prostaglandin E2 secretion in the HO23 cell culture medium after giving hCG have been discovered, and RU486 administration will inhibit hCG from increasing PGE2 secretion level in a does-dependent manner. In summery, we conclude that RU486 might inhibit hCG-induced COX-2 expression by direct or indirect suppression of ERK1/2 protein phosphorylation, leading to a decrease in PGE2 secretion, and as a result inhibiting ovulation and luteinization in women.