本文選題：多囊卵巢綜合征 + 種植窗��； 參考：《南京中醫(yī)藥大學(xué)》2016年博士論文

【摘要】：目的：建立多囊卵巢綜合征(polyeystic ovary syndrome, PCOS)大鼠促排卵妊娠模型,在此模型的基礎(chǔ)上,研究PCOS促排妊娠大鼠圍種植窗期血清內(nèi)分泌激素、炎性因子及子宮內(nèi)膜容受因子的變化及早期胚胎著床率。從子宮內(nèi)膜容受性的角度探討早黃體期運用泰山磐石散對早期妊娠結(jié)局的影響及可能作用機制,闡述中醫(yī)藥的作用途徑及靶點,從而為臨床開展中西醫(yī)結(jié)合治療PCOS提供實驗基礎(chǔ)及理論依據(jù)。方法：選用21日齡SD雌性大鼠92只,分為正常對照組(21只)和PCOS模型組(71只)。自23日齡始正常對照組每日頸背部皮下注射大豆油,PCOS模型組每日皮下注射脫氫表雄酮(DHEA)+注射用大豆油,連續(xù)造模30日分別建立正常對照組、PCOS模型組大鼠。造模結(jié)束后,正常對照組、PCOS模型組各取一只大鼠,取左側(cè)卵巢行HE染色；剩余大鼠取血清行血清睪酮(T)測定。自80日齡起,正常對照組大鼠每日生理鹽水(NS)灌胃,PCOS模型組每日以克羅米芬(CC)+NS灌胃,連續(xù)造模5天后分別與雄鼠按2：1合籠,次日檢查以發(fā)現(xiàn)陰栓記為妊娠第一天(d1),分別設(shè)為正常妊娠組和PCOS促排妊娠組。兩組分別隨機選取其中的6只妊娠大鼠采用酶聯(lián)免疫吸附測定(ELISA)法測定血清雌二醇(E2)、孕酮(P)、泌乳素(PRL)和腫瘤壞死因子(TNF-a)。將正常妊娠組設(shè)為A組；PCOS促排妊娠組隨機分為B、C、D三組。從妊娠d1開始,A組、B組灌服NS,C組灌服泰山磐石散,組灌服地屈孕酮,連續(xù)灌胃直至處死前。隨機選取A、B、C、D四組部分大鼠,于妊娠d8處死,取雙側(cè)子宮計算胚泡數(shù)量。隨機選取部分B組大鼠于妊娠d3、d4, d5處死(設(shè)為d3組、d4組、d5組),處死前采用ELISA法測定血清E2、P. PRL和TNF-a;處死后取左側(cè)子宮以免疫組化(Envision法)測定子宮內(nèi)膜孕激素受體(PR)、細胞間粘附分子-1(ICAM-1)的表達；刮取右側(cè)子宮內(nèi)膜以RT-PCR檢測同源框基因(HOXA10)、整合素αvβ3、同源結(jié)構(gòu)域基因(Emx2)、白血病抑制因子(LIF)的ml RNA表達。隨機選取A、B、C、D四組中部分大鼠,于妊娠d5處死,處死前采用ELISA法測定血清E2、P、PRL和TNF-a;處死后取左側(cè)子宮以Envision法測定子宮內(nèi)膜PR、ICAM-1的表達；刮取右側(cè)子宮內(nèi)膜分別以RT-PCR、 Western blot法檢測HOXA10、整合素αvβ3、Emx2、LIF的mRNA及蛋白的表達。結(jié)果：PCOS模型組血清T明顯高于正常對照組,差異有顯著性(P0.01)；PCOS模型組較正常對照組卵巢體積增大,顏色蒼白、包膜增厚、包膜下隱見較多的囊狀擴張卵泡,呈多囊樣改變。正常對照組大鼠全部妊娠,PCOS模型組妊娠率71.43%,差異有顯著性(P0.01)；妊娠d1,正常妊娠組血清E2、PRL、TNF-α均明顯低于PCOS促排妊娠組,而P明顯高于PCOS促排妊娠組,差異有顯著性(P0.01,P0.05)。妊娠d8,大鼠的著床率及胚泡著床數(shù)B組明顯低于A、C、D組,差異有顯著(P0.01)；C組、D組明顯低于A組,差異有顯著(P0.01)。在圍著床期：E2、P在d5組明顯高于d3組,差異有顯著性(P0.05)；E2/P、PRL、ICAM-1表達為d5組d4組d3組,但三組組間差異無顯著性(P0.05)。PR (IOD)的表達為d4組d5組d3組,三組組間差異無顯著性(P0.05)。HOXA10的mRNA表達d4組明顯高于d5組、d3組,差異有顯著性(P0.01,P0.05)。整合素αvβ3的mRNA表達d4組明顯高于d5組、d3組,差異有顯著性(P0.01);d5組明顯高于d3組,差異有顯著性(P0.05)。Emx2的mRNA表達d4組明顯低于d5組,差異有顯著性(P0.05)。LIF的mRNA表達d3組明顯高于d4組,差異有顯著性(P0.05)。在種植窗期：E2在B組、C組、D組表達明顯高于A組,差異有顯著性(P0.01,P0.05)。P在A組、C組、D組表達明顯高于B組,差異有顯著性(P0.01)。E2/P在A組、C組、D組表達明顯低于B組,差異有顯著性(P0.01)；A組明顯低于C組、D組(P0.01,P0.05)。TNF-a在B組表達明顯高于A組、C組(P0.01,P0.05);D組明顯高于A組(P0.05)。PRL在各組表達A組C組DB組,但各組間差異無統(tǒng)計學(xué)意義(P0.05)。子宮內(nèi)膜PR的表達A組明顯高于B組,差異有顯著性(P0.05);ICAM-1的表達B組明顯低于A組、C組、D組,差異有顯著性(P0.05)。ICAM-1與P呈正相關(guān)(r=8.223, P0.05); TNF-α與E2/P呈正相關(guān)(r=5.389,P0.05)。HOXA10的nRN A表達B組明顯低于A組、D組,差異有顯著性(P0.01,P0.05)；HOXA10的蛋白表達B組明顯低于A組、C組、D組,差異有顯著性(P0.01)；A組明顯高于C組、D組,差異有顯著性(P0.05,P0.01)。整合素αvβ3的mRNA及蛋白表達A組、C組、D組明顯高于B組,差異有顯著性(P0.01)；A組蛋白表達明顯高于C組,差異有顯著性(P0.01)。Emx2的mRNA表達A組明顯低于B組、C組,差異有顯著性(P0.01)；Emx2的蛋白表達A組、C組明顯低于B組、D組,差異有顯著性(P0.01)。LIF的mRNA表達B組顯著低于A、C組,差異有顯著性(P0.01)。LIF蛋白表達B組明顯低于A組、C組、D組,差異有顯著性(P0.01)；C組明顯低于A組、D組,差異有顯著性(P0.01,P0.05)。結(jié)論：PCOS模型大鼠表現(xiàn)為卵巢多囊樣變、高雄激素及排卵障礙,采用CC能糾正排卵障礙,明顯提高大鼠妊娠率。但是,PCOS妊娠大鼠內(nèi)分泌的紊亂(高水平的E2、PRL.低水平的P)及炎性因子TNF-a增高,都有可能影響胚胎種植、發(fā)育,導(dǎo)致早期流產(chǎn)的發(fā)生。PCOS妊娠大鼠容易發(fā)生早期妊娠丟失,泰山磐石散或地屈孕酮均能有效降低早期流產(chǎn)率,提高胚胎著床率。PCOS促排妊娠大鼠在種植前期子宮內(nèi)膜容受因子PR、ICAM-1、HOXA10、整合素avβ3、Emx2、LIF峰值出現(xiàn)的早晚,使子宮內(nèi)膜開放時間與胚胎發(fā)育不同步,這是導(dǎo)致早期妊娠丟失的可能原因之一。種植窗期PCOS妊娠大鼠雌、孕激素及比值異常,高表達TNF-a,子宮內(nèi)膜PR、ICAM-1、HOXA10、整合素αvβ3、LIF的低表達,Emx2的高表達,使子宮內(nèi)膜容受性下降,內(nèi)膜與胚胎發(fā)育不同步,最終導(dǎo)致低著床率的發(fā)生。泰山磐石散通過調(diào)整圍種植期內(nèi)分泌激素,改善卵巢功能；上調(diào)子宮內(nèi)膜PR、ICAM-1、HOXA10、整合素(tvβ3、LIF表達,下調(diào)Emx2表達,改善子宮內(nèi)膜容受性,這可能也是泰山磐石散改善妊娠結(jié)局的可能作用機制。
[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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R711.75

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