【摘要】：研究目的：觀察補腎助孕方對米非司酮干預的大鼠黃體期子宮內膜細胞雌激素受體(ER)、基質金屬蛋白酶-9(MMP-9)、基質金屬蛋白酶抑制因子-1(TIMP-1)的調控及對大鼠黃體顆粒細胞分泌雌二醇(E2)、孕激素(P)水平的影響。探討補腎助孕方治療黃體功能不全性不孕的有效機理。研究方法：：1、含藥血清制備：選用普通雄兔5只,予補腎助孕方藥煎液40g/kg灌胃(相當于成人臨床劑量的10倍),2次/d,連續(xù)3d。第四天末次灌胃1h(灌胃前禁食不禁水12h),局部麻醉頸動脈采血,室溫靜置3h,3000rpm離心分離血清15min,收集血清,-20℃保存,56℃水浴滅活補體30min,0.22um微孔濾膜過濾除菌,使用前無血清DMEM稀釋至5%、20%濃度的含藥血清。2、子宮內膜細胞、卵巢黃體細胞的分離、培養(yǎng)：選擇體重50-60g未成年雌性大鼠5只,皮下注射PMSG 50U/只,60h卵泡發(fā)育成熟,在皮下注射HCG 35U/只,注射后第七天(HCG注射日為第1天),拉頸斷髓法處死,摘取卵巢、子宮,分離和培養(yǎng)子宮內膜細胞和卵巢黃體細胞。表型鑒定后將子宮內膜細胞、卵巢黃體細胞培養(yǎng)于含有100U/ml青霉素、100U/ml鏈霉素、10%空白血清、DMEM培養(yǎng)液中,置于37℃、5%CO2培養(yǎng)箱內培養(yǎng),每日觀察生長情況,貼壁3-5d傳代一次。傳代用0.25%胰酶+DMEM培養(yǎng)液。3、含藥血清干預：加用藥干預48h后,提取各組細胞蛋白、RNA及細胞上清液,觀察補腎助孕方含藥血清對子宮內膜細胞ERα、ERβ、MMP-9、TIMP-1及卵巢顆粒細胞分泌雌、孕激素水平的影響。Western blot方法檢測子宮內膜細胞ER α、ERβ蛋白表達,RT-PCR法檢測子宮內膜細胞ERα、ERβ、MMP-9、TIMP-1mRNA的表達量,放射免疫法檢測黃體顆粒細胞上清液雌、孕激素的水平。研究結果：(1)補腎助孕方含藥血清對子宮內膜細胞ERα、ERβ蛋白的表達有提高作用；同時明顯改善米非司酮所致的ERα、ERβ蛋白的低表達；(2)補腎助孕方含藥血清對子宮內膜細胞ERα、ERβ、MMP-9、TIMP-1mRNA的表達有提高作用；同時明顯改善米非司酮所致的ER、MMP-9、TIMP-1的低表達；(3)補腎助孕方含藥血清明顯提高卵巢黃體細胞分泌雌、孕激素水平。研究結論：(1)補腎助孕方通過提高子宮內膜細胞ER、MMP-9、TIMP-1的表達,改善子宮內膜的容受性,有利于胚胎著床妊娠,為孕卵著床奠定基礎；(2)補腎助孕方藥能顯著提高卵巢黃體細胞分泌E2、P水平,改善米非司酮所致的E2、P水平低表達,改善米非司酮所致的卵巢黃體功能下降,提高黃體功能,有利于胚胎著床和妊娠維持,提高臨床妊娠率。
[Abstract]:Objective: to observe the regulation of Bushen Zhuyun recipe on estrogen receptor (ER), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-1 (TIMP-1) and luteal granulosa cells in luteal phase of rats treated with mifepristone. Effects of estradiol (E 2) secretion and progesterone (P) levels. To explore the effective mechanism of tonifying kidney and helping pregnancy in the treatment of luteal insufficiency infertility. Methods: 5 male rabbits were treated with 40g/kg (equivalent to 10 times of adult clinical dose) twice a day for 3 days. At the end of the fourth day, blood was collected from carotid artery under local anesthesia for 1 hour (12 hours before fasting). Serum was centrifuged at room temperature for 3 h and 3000rpm. The serum was collected and stored at -20 鈩，

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