泰山磐石散对多囊卵巢综合征大鼠子宫内膜容受性及妊娠结局的影响

发布时间：2018-06-23 21:53

本文选题：多囊卵巢综合征 + 种植窗　；参考：《南京中医药大学》2016年博士论文

【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(P0.01)；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(P0.01)；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(P0.01,P0.05)。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(P0.01)；C组、D组明显低于A组,差异有显著(P0.01)。在围着床期：E2、P在d5组明显高于d3组,差异有显著性(P0.05)；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(P0.05)。PR (IOD)的表达为d4组d5组d3组,三组组间差异无显著性(P0.05)。HOXA10的mRNA表达d4组明显高于d5组、d3组,差异有显著性(P0.01,P0.05)。整合素αvβ3的mRNA表达d4组明显高于d5组、d3组,差异有显著性(P0.01);d5组明显高于d3组,差异有显著性(P0.05)。Emx2的mRNA表达d4组明显低于d5组,差异有显著性(P0.05)。LIF的mRNA表达d3组明显高于d4组,差异有显著性(P0.05)。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(P0.01,P0.05)。P在A组、C组、D组表达明显高于B组,差异有显著性(P0.01)。E2/P在A组、C组、D组表达明显低于B组,差异有显著性(P0.01)；A组明显低于C组、D组(P0.01,P0.05)。TNF-a在B组表达明显高于A组、C组(P0.01,P0.05);D组明显高于A组(P0.05)。PRL在各组表达A组C组DB组,但各组间差异无统计学意义(P0.05)。子宫内膜PR的表达A组明显高于B组,差异有显著性(P0.05);ICAM-1的表达B组明显低于A组、C组、D组,差异有显著性(P0.05)。ICAM-1与P呈正相关(r=8.223, P0.05); TNF-α与E2/P呈正相关(r=5.389,P0.05)。HOXA10的nRN A表达B组明显低于A组、D组,差异有显著性(P0.01,P0.05)；HOXA10的蛋白表达B组明显低于A组、C组、D组,差异有显著性(P0.01)；A组明显高于C组、D组,差异有显著性(P0.05,P0.01)。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(P0.01)；A组蛋白表达明显高于C组,差异有显著性(P0.01)。Emx2的mRNA表达A组明显低于B组、C组,差异有显著性(P0.01)；Emx2的蛋白表达A组、C组明显低于B组、D组,差异有显著性(P0.01)。LIF的mRNA表达B组显著低于A、C组,差异有显著性(P0.01)。LIF蛋白表达B组明显低于A组、C组、D组,差异有显著性(P0.01)；C组明显低于A组、D组,差异有显著性(P0.01,P0.05)。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。
[Abstract]:Objective: to establish a model of ovulation induction in polyeystic ovary syndrome (PCOS) rats. On the basis of this model, the changes of serum endocrine hormones, inflammatory factors and endometrial receptive factors in the peri planting window of PCOS pregnant rats and the early embryo implantation rate were studied. The effect and possible mechanism of Taishan Panshi powder on the outcome of early pregnancy were used in early luteal period. The mechanism of action and target of traditional Chinese medicine were expounded, so as to provide experimental basis and theoretical basis for the clinical practice of combining traditional Chinese and Western medicine to treat PCOS. Methods: 92 female rats of 21 days of age SD were divided into normal control group (21 rats) and PCOS model group (71 rats). After 23 days of age, the normal control group was injected with soybean oil subcutaneously on the back of the neck every day. The PCOS model group was subcutaneously injected with dehydroepiandrosterone (DHEA) + injection with soybean oil, and the normal control group was set up for 30 days, and the PCOS model group rats were established respectively. After the model end, the normal control group and the PCOS model group were taken one rat each, and the left ovary was taken with HE staining. Serum testosterone (T) was measured in the remaining rats. From 80 days old, normal saline (NS) was administered to the normal control group, and the PCOS model group was gavaged with clomiphene (CC) +NS daily. After 5 days of continuous modeling, the male rats were caged according to 2:1, and the second day was examined to find the vaginal suppository as the first day of pregnancy (D1), which were set to normal pregnancy and PCOS respectively. The two groups were randomly selected and 6 of the two rats were randomly selected by enzyme linked immunosorbent assay (ELISA) to determine serum estradiol (E2), progesterone (P), prolactin (PRL) and tumor necrosis factor (TNF-a). The normal pregnancy group was set as group A; PCOS pregnancy group was randomly divided into B, C, D three groups. A, B, C, D four groups of rats were sacrificed at D8 pregnancy to calculate the number of blastocysts in the uterus of bilateral pregnancy. The rats were randomly selected from group B to D3, D4, D5 (D4 group, D5 group), before death (D3, D4, D5). In the left uterus, the endometrium progesterone receptor (PR) and the expression of intercellular adhesion molecule -1 (ICAM-1) were measured by immunohistochemistry (Envision method); the right endometrium was scraped into the right endometrium to detect the homologous frame gene (HOXA10), integrin alpha v beta 3, homologous domain gene (Emx2) and leukemia inhibitory factor (LIF) in ml RNA expression. Four groups were randomly selected. In the middle part of the rats, the rats were killed at D5 pregnancy. Before death, the serum E2, P, PRL and TNF-a were measured by ELISA method. The endometrium PR of the uterus was measured by Envision method and the expression of ICAM-1 was measured by Envision method after death. The endometrium on the right side of the uterus was detected by RT-PCR and Western blot, respectively. The serum T of the type group was significantly higher than that of the normal control group (P0.01). The ovarian volume of the PCOS model group was larger than that of the normal control group, the color was pale, the capsule thickened, and the cystic dilatation follicles under the envelope were more cystic dilatation follicles, and the normal control group was all pregnant, and the pregnancy rate of the PCOS model group was 71.43%, the difference was significant (P0.01). Pregnancy D1, normal pregnancy group E2, PRL, TNF- alpha were significantly lower than the PCOS group, and P significantly higher than the PCOS group, the difference was significant (P0.01, P0.05). Pregnancy D8, the rate of implantation and the number of blastocysts in the B group were significantly lower than the A, the group, the difference was significantly lower than that of the group. The difference was significant. Bed period: E2, P in group D5 was significantly higher than that in group D3, the difference was significant (P0.05); E2/P, PRL, ICAM-1 were expressed as D3 group in D5 group D4 group, but there was no significant difference between the three groups (P0.05). 5. The expression of mRNA in the D4 group of integrin alpha v beta 3 was significantly higher than that in group D5, and in group D3, the difference was significant (P0.01); the D5 group was significantly higher than that of the D3 group, and the difference was significant (P0.05).Emx2 mRNA expression D4 group was significantly lower than that of the group, the difference was significantly higher than that of the group, and the difference was significant. The expression of D group was significantly higher than that of A group, the difference was significant (P0.01, P0.05).P in group A, C group and D group was significantly higher than that of B group, and the difference was significant (P0.01).E2/P in A group. There was no significant difference between group A and C group in group A, but there was no significant difference between each group (P0.05). The expression of PR in endometrium was significantly higher than that of B group. The difference was significant (P0.05) in the expression of PR in endometrium than in B group (P0.05); the difference was significantly lower than that of the B group (P0.05). The nRN A expression in the related (r=5.389, P0.05).HOXA10 group was significantly lower than that of the A group, and the difference was significant (P0.01, P0.05) in the D group, and the HOXA10 protein expression in the B group was significantly lower than that of the group. The expression of protein expression in group A was significantly higher than that in group B (P0.01), and the expression of protein in A group was significantly higher than that in group C, and the difference was significant (P0.01).Emx2 in A group was significantly lower than that of B group, and C group was significant (P0.01). The expression of sex (P0.01).LIF protein in B group was significantly lower than that in group A, C group and D group (P0.01); C group was significantly lower than group A and D group was significant (P0.01, P0.05). Conclusion: the rat model was characterized by polycystic ovarian degeneration, hormone and ovulation disorder in Kaohsiung. Endocrine disorders in rats (high levels of E2, PRL. low P) and increased inflammatory factor TNF-a are all likely to affect embryo implantation and development, leading to early abortion in.PCOS pregnancy rats prone to early pregnancy loss, and Taishan Panshi powder or D progesterone can effectively reduce early abortion rate and improve embryo implantation rate.PCOS to promote pregnancy induced pregnancy. The early stage of endometrium receptive factors PR, ICAM-1, HOXA10, integrin AV beta 3, Emx2, LIF peak appeared in the early stage of pregnancy, which made the endometrial opening time unsynchronized with the embryonic development, which was one of the possible causes of early pregnancy loss. The female, progestin and ratio of PCOS pregnant rats during the window period were abnormal, high expression of TNF-a, and endometrial P. R, ICAM-1, HOXA10, integrin alpha v beta 3, low expression of LIF, high expression of Emx2, decreased endometrium receptivity, unsynchronized endometrium and embryo development, and eventually lead to low implantation rate. Taishan Panshi powder can improve the ovarian function by adjusting peri implant endocrine hormones, and up regulation of endometrium PR, ICAM-1, HOXA10, integrin (TV beta 3, LIF expression) Down regulating Emx2 expression and improving endometrial receptivity may also be a possible mechanism of Taishan Panshi San to improve pregnancy outcome.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R711.75

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