【作者】 刘音吟；

【导师】 谈勇；

【作者基本信息】 南京中医药大学 ， 中医妇科学， 2016， 博士

【摘要】 目的：建立多囊卵巢综合征(polyeystic ovary syndrome, PCOS)大鼠促排卵妊娠模型,在此模型的基础上,研究PCOS促排妊娠大鼠围种植窗期血清内分泌激素、炎性因子及子宫内膜容受因子的变化及早期胚胎着床率。从子宫内膜容受性的角度探讨早黄体期运用泰山磐石散对早期妊娠结局的影响及可能作用机制,阐述中医药的作用途径及靶点,从而为临床开展中西医结合治疗PCOS提供实验基础及理论依据。方法：选用21日龄SD雌性大鼠92只,分为正常对照组(21只)和PCOS模型组(71只)。自23日龄始正常对照组每日颈背部皮下注射大豆油,PCOS模型组每日皮下注射脱氢表雄酮(DHEA)+注射用大豆油,连续造模30日分别建立正常对照组、PCOS模型组大鼠。造模结束后,正常对照组、PCOS模型组各取一只大鼠,取左侧卵巢行HE染色；剩余大鼠取血清行血清睾酮(T)测定。自80日龄起,正常对照组大鼠每日生理盐水(NS)灌胃,PCOS模型组每日以克罗米芬(CC)+NS灌胃,连续造模5天后分别与雄鼠按2：1合笼,次日检查以发现阴栓记为妊娠第一天(d1),分别设为正常妊娠组和PCOS促排妊娠组。两组分别随机选取其中的6只妊娠大鼠采用酶联免疫吸附测定(ELISA)法测定血清雌二醇(E2)、孕酮(P)、泌乳素(PRL)和肿瘤坏死因子(TNF-a)。将正常妊娠组设为A组；PCOS促排妊娠组随机分为B、C、D三组。从妊娠d1开始,A组、B组灌服NS,C组灌服泰山磐石散,组灌服地屈孕酮,连续灌胃直至处死前。随机选取A、B、C、D四组部分大鼠,于妊娠d8处死,取双侧子宫计算胚泡数量。随机选取部分B组大鼠于妊娠d3、d4, d5处死(设为d3组、d4组、d5组),处死前采用ELISA法测定血清E2、P. PRL和TNF-a;处死后取左侧子宫以免疫组化(Envision法)测定子宫内膜孕激素受体(PR)、细胞间粘附分子-1(ICAM-1)的表达；刮取右侧子宫内膜以RT-PCR检测同源框基因(HOXA10)、整合素αvβ3、同源结构域基因(Emx2)、白血病抑制因子(LIF)的ml RNA表达。随机选取A、B、C、D四组中部分大鼠,于妊娠d5处死,处死前采用ELISA法测定血清E2、P、PRL和TNF-a;处死后取左侧子宫以Envision法测定子宫内膜PR、ICAM-1的表达；刮取右侧子宫内膜分别以RT-PCR、 Western blot法检测HOXA10、整合素αvβ3、Emx2、LIF的mRNA及蛋白的表达。结果：PCOS模型组血清T明显高于正常对照组,差异有显著性(P<0.01)；PCOS模型组较正常对照组卵巢体积增大,颜色苍白、包膜增厚、包膜下隐见较多的囊状扩张卵泡,呈多囊样改变。正常对照组大鼠全部妊娠,PCOS模型组妊娠率71.43%,差异有显著性(P<0.01)；妊娠d1,正常妊娠组血清E2、PRL、TNF-α均明显低于PCOS促排妊娠组,而P明显高于PCOS促排妊娠组,差异有显著性(P<0.01,P<0.05)。妊娠d8,大鼠的着床率及胚泡着床数B组明显低于A、C、D组,差异有显著(P<0.01)；C组、D组明显低于A组,差异有显著(P<0.01)。在围着床期：E2、P在d5组明显高于d3组,差异有显著性(P<0.05)；E2/P、PRL、ICAM-1表达为d5组<d4组<d3组,但三组组间差异无显著性(P>0.05)。PR (IOD)的表达为d4组>d5组>d3组,三组组间差异无显著性(P>0.05)。HOXA10的mRNA表达d4组明显高于d5组、d3组,差异有显著性(P<0.01,P<0.05)。整合素αvβ3的mRNA表达d4组明显高于d5组、d3组,差异有显著性(P<0.01);d5组明显高于d3组,差异有显著性(P<0.05)。Emx2的mRNA表达d4组明显低于d5组,差异有显著性(P<0.05)。LIF的mRNA表达d3组明显高于d4组,差异有显著性(P<0.05)。在种植窗期：E2在B组、C组、D组表达明显高于A组,差异有显著性(P<0.01,P<0.05)。P在A组、C组、D组表达明显高于B组,差异有显著性(P<0.01)。E2/P在A组、C组、D组表达明显低于B组,差异有显著性(P<0.01)；A组明显低于C组、D组(P<0.01,P<0.05)。TNF-a在B组表达明显高于A组、C组(P<0.01,P<0.05);D组明显高于A组(P<0.05)。PRL在各组表达A组<C组<D<B组,但各组间差异无统计学意义(P>0.05)。子宫内膜PR的表达A组明显高于B组,差异有显著性(P<0.05);ICAM-1的表达B组明显低于A组、C组、D组,差异有显著性(P<0.05)。ICAM-1与P呈正相关(r=8.223, P<0.05); TNF-α与E2/P呈正相关(r=5.389,P<0.05)。HOXA10的nRN A表达B组明显低于A组、D组,差异有显著性(P<0.01,P<0.05)；HOXA10的蛋白表达B组明显低于A组、C组、D组,差异有显著性(P<0.01)；A组明显高于C组、D组,差异有显著性(P<0.05,P<0.01)。整合素αvβ3的mRNA及蛋白表达A组、C组、D组明显高于B组,差异有显著性(P<0.01)；A组蛋白表达明显高于C组,差异有显著性(P<0.01)。Emx2的mRNA表达A组明显低于B组、C组,差异有显著性(P<0.01)；Emx2的蛋白表达A组、C组明显低于B组、D组,差异有显著性(P<0.01)。LIF的mRNA表达B组显著低于A、C组,差异有显著性(P<0.01)。LIF蛋白表达B组明显低于A组、C组、D组,差异有显著性(P<0.01)；C组明显低于A组、D组,差异有显著性(P<0.01,P<0.05)。结论：PCOS模型大鼠表现为卵巢多囊样变、高雄激素及排卵障碍,采用CC能纠正排卵障碍,明显提高大鼠妊娠率。但是,PCOS妊娠大鼠内分泌的紊乱(高水平的E2、PRL.低水平的P)及炎性因子TNF-a增高,都有可能影响胚胎种植、发育,导致早期流产的发生。PCOS妊娠大鼠容易发生早期妊娠丢失,泰山磐石散或地屈孕酮均能有效降低早期流产率,提高胚胎着床率。PCOS促排妊娠大鼠在种植前期子宫内膜容受因子PR、ICAM-1、HOXA10、整合素avβ3、Emx2、LIF峰值出现的早晚,使子宫内膜开放时间与胚胎发育不同步,这是导致早期妊娠丢失的可能原因之一。种植窗期PCOS妊娠大鼠雌、孕激素及比值异常,高表达TNF-a,子宫内膜PR、ICAM-1、HOXA10、整合素αvβ3、LIF的低表达,Emx2的高表达,使子宫内膜容受性下降,内膜与胚胎发育不同步,最终导致低着床率的发生。泰山磐石散通过调整围种植期内分泌激素,改善卵巢功能；上调子宫内膜PR、ICAM-1、HOXA10、整合素(tvβ3、LIF表达,下调Emx2表达,改善子宫内膜容受性,这可能也是泰山磐石散改善妊娠结局的可能作用机制。更多还原

【Abstract】 Objective:To establish PCOS pregnant rats mode with Ovulation induction.On the basis of this model, the serum endocrine hormones、inflammatory factors、uterine receptivity factors and early embryo implantation rate in the peri implantation window phase of PCOS ovulation induction rats were studied. To investigate the impact of Taishan Panshi San in early luteal phase on the outcome of early pregnancy and the possible mechanism from the perspective of endometrial receptivity, expound the function way and target of Chinese medicine, so as to provide experimental basis and theoretical basis for the clinical development of the treatment for PCOS with traditional Chinese medicine and Western Medicine.Methods:Ninety-two SD female rats of 21 days age were divided into normal control group (21) and PCOS model group (71). Since the age of 23 days, the normal control group with daily subcutaneous injection of soybean oil, PCOS model group with subcutaneous injection of dehydroepiandrosterone (DHEA) and soybean oil for continuous 30 days were made separately into Normal control group and PCOS model group. After the models were finished, one ant in the normal control group and PCOS model group were selected from each group and the left ovary of selected rats were taken for HE staining.The Remaining rats were taken serum testosterone (T) determination.Since 80 days of age, the normal control group was irrigated with normal saline(NS), and PCOS model group was given with CC combined NS for 5 consecutive days. Then they were mated with male rats with 2:1 and were found the female plugs as the first day of pregnancy (dl).They were set to normal pregnancy group and PCOS pregnancy group. The 6 pregnant rats were randomly selected from two groups and were measured of Serum estradiol (E2), progesterone (P), prolactin (PRL) and tumor necrosis factor (TNF-a) by ELISA.The normal pregnancy group was set as A group, and PCOS pregnancy group was randomly divided into three groups(B, C, D). From the beginning of pregnancy (dl), group A^B^C and D were respectively administered with NS、NS、Taishan Panshi San、progesterone until death. At the 8th day of pregnancy, a part of rats from group A、B、C and D were killed to take bilateral uterine blastocyst calculation. At the 3th、4th、5th day of pregnancy, some rats of group B were selected as group d3、d4、d5, and determinated of E2、P、PRL、TNF-a by ELISA then be killed.The left uterus were obtained to be tested the expression of PR、 ICAM-1 by immunohistochemistry and right uterus and endometria to be detect the mRNA expression of HOXA10、integrinavP3、Emx2、LIF by RT-PCR. At the 5th day of pregnancy, some rats of group A、B、C、D were selected to be determinated of E2、P、PRL、TNF-a by ELISA then be killed.The left uterus were obtained to be detected the expression of PR、 ICAM-1 by immunohistochemistry and right uterus and endometria to be detect the mRNA and protein expression of HOXA10、integrinavβ3、Emx2、LIF by RT-PCR and Western blot.Results:PCOS model group serum T was significantly higher than the normal control group (P< 0.01).The ovary of PCOS model group compared with normal control group was pale, capsular thickening, subcapsular cystic dilatation of more hidden follicles and showed multiple follicular cysts.The pregnancy rate was 100% in normal control group, and that in PCOS model group was 71.43%, there was significant difference (P< 0.01).At the lth day of pregnancy, the serum levels of E2, PRL and TNF-a in normal pregnancy group were less than those in PCOS group, while P was more than that in PCOS group, there was significant difference between them (P< 0.01, P<0.05).At the 8th day of pregnancy, the rat implantation rate and implantation of group A were significantly higher than that of group A, C and D, there was significant difference (P< 0.01); there was significant difference betreen group C、D and group A (P< 0.01).In peri-implantation period:E2、P of d5 group was significantly higher than those in d3 group, the difference was significant (P< 0.05). E2/P, PRL and ICAM-1 were expressed as d5< d3< d4, but there was no difference among them (P> 0.05).PR (IOD) was expressed as d4>d5>d3, but there was no difference (P>0.05). The mRNA expression of HOXA10 and integrin avP3 in d4 group was more than that of d5 and d3 group, and there was Obvious difference (P< 0.01, P< 0.05). And d5 group was higher than d3 group in the mRNA expression of integrin avβ3, the difference was Obvious (P< 0.05). The mRNA expression of Emx2 in d4 group was significantly lower than that in d5, and the difference was obvious (P< 0.05). The mRNA expression of LIF was significantly higher than that in d4 group, and the difference was significant (P< 0.05).In implantation period:Group B、C、D about E2 much higher than Group A (P<0.01, P<0.05). Group A、C、D about P was significantly higher than group B (P<0.01).But group A、C、D about E2/P was lower than group B (P<0.01) and group A was much lower than group C、D, the difference was significant (P<0.01, P< 0.05). TNF-a in group B was higher than group A and C (P<0.01, P<0.05); TNF-a in group D was significantly higher than that in group A.The expression of PRL was group A<C<D<B, but there was no significant difference between four groups (P> 0.05). The expression of PR in group A was significantly higher than that in group B (P< 0.05). The expression of ICAM-1 in group B was significantly lower than that in group A、C、D, the difference was significant (P< 0.05). ICAM-1 and P were positively correlated (r=8.223, P<0.05); TNF-α and E2/P were positively correlated (r=5.389, P<0.05). The mRNA expression of HOXA10 in group B was significantly lower than that in group A、D, and the difference was significant (P<0.01, P <0.05). The protein expression of HOXA10 in group B was significantly lower than that in group A、C、D, and the difference was significant (P<0.01); that in group A was significantly higher than that in group C、D, and the difference was significant (P<0.05, P<0.01). Group A、C、D in the mRNA and protein expression of integrin avβ3 were higher than group B, and the difference was obvious (P<0.01); the protein expression of it in group A was significantly higher than that in group C, and the difference was so significant (P<0.01). Group A about the mRNA expression was significantly lower than group B、C, the difference was significant (P<0.01). The protein expression of Emx2 in group A、C was significantly lower than that in group B、D, and the difference was significant (P< 0.01).The mRNA expression of LIF in group B was significantly lower than that in group A、C, and the difference was significant (P <0.01). The protein expression of LIF in group B was significantly lower than that in group A、C、D, and the difference was significant (P<0.01); that in group C was significantly lower than that in group A、D, and the difference was significant (P<0.01, P<0.05).Conclusion:In PCOS model rats, it was characterized as the polycystic ovary, hyperandrogenism and Ovulation dysfunction. CC was used to correct ovulation disorders and this significantly improved the rate of pregnancy. However, the endocrine disorders (high levels of E2、PRL, low levels of P) and the inflammatory factor TNF-a in PCOS pregnancy rats may affect embryo implantation and development and lead to early abortion.PCOS pregnant rats proned to early pregnancy loss, Taishan Panshi San or dydrogesterone could effectively reduce the rate of early abortion, improve the embryo implantation rate.The peak expression of PCOS pregnant rats about uterine receptivity factor PR, ICAM-1、HOXA10、integrinavβ3、Emx2、LIF appeared sooner or later the endometrial opening time in peri-implantation, and it leaded to the time of the endometrium opening was not synchronized with the development of the embryo. This is one of the possible reasons for the loss of pregnancy.In the window of the implantation, estrogen and progesterone and the ratio of abnormal, high expression of TNF-a, low expression of endometrial PR, ICAM-1, HOXA10, integrin avβ3, LIF, the high expression of Emx2 of PCOS pregnancy rats, lowered endometrial receptivity, unsynchronized the endometrium and embryo development and resulted in low implantation rate at last.Taishan Panshi San improved endometrial receptivity by adjusting hormones, improving ovarian function; upregulation of the expression of endometrial PR, ICAM-1, HOXA10, integrinavβ3, LIF, down-regulation of Emx2 expression during the implantation window, and that may be the Possible mechanism of Taishan Panshi San to improve pregnancy outcome.更多还原