本文選題：多囊卵巢綜合征　切入點：α1-抗胰蛋白酶　出處：《吉林大學》2014年博士論文

【摘要】：多囊卵巢綜合征（polycystic ovary syndrome, PCOS）是一種女性生殖功能障礙與代謝異常并存的內(nèi)分泌紊亂綜合征。PCOS患者常伴有肥胖及慢性脂肪炎癥等并發(fā)癥。研究表明，PCOS患者血清中脂聯(lián)素（Adiponectin）濃度低于正常人群、瘦素（Leptin）濃度高于正常人群。多種白介素類促炎性細胞因子及中性粒細胞分泌的蛋白酶類在PCOS患者血清中濃度增加，表明炎癥有可能與PCOS患病機制相關(guān)。 α1-抗胰蛋白酶（Alpha1-antitrypsin，A1AT）是一種體內(nèi)重要的絲氨酸蛋白酶抑制劑，它主要由肝細胞合成，廣泛存在于動物血清中，能夠抑制某些促炎性細胞因子和中性粒細胞分泌的蛋白酶。但是，蛋白酶類相關(guān)調(diào)控蛋白在PCOS發(fā)病中的作用還需要進一步研究。本實驗前期通過酶聯(lián)免疫技術(shù)比較臨床數(shù)據(jù)，發(fā)現(xiàn)肥胖者及肥胖型PCOS患者體內(nèi)血清中A1AT蛋白濃度明顯低于正常人群，但是其作用與意義尚不十分清楚。另外，由于PCOS患者臨床組織標本取材困難，因此建立理想的PCOS動物模型是探討臨床診療策略的關(guān)鍵所在。 本論文探索A1AT通過抑制促炎性細胞因子和蛋白酶類相關(guān)調(diào)控蛋白干預PCOS的發(fā)生，，主要開展了以下研究： 1.肥胖人群血清中A1AT含量低于正常人群：采用酶聯(lián)免疫吸附法（ELISA）檢測其血清A1AT、瘦素（Leptin）、脂聯(lián)素（Adiponectin）、血糖（Glucose）及胰島素（Insulin）水平。結(jié)果表明A1AT和Adiponectin隨著身體質(zhì)量指數(shù)（BodyMass Index，BMI）的增加而減少，差異顯著（P0.05）。Leptin隨著BMI的增加而增加，差異顯著（P＜0.05）。A1AT在人血清中與BMI、Adiponectin、Leptin和Insulin的血清水平相關(guān)。肥胖者Leptin水平升高，A1AT與Leptin成負相關(guān)，Leptin水平升高影響A1AT表達，因此肥胖人群體內(nèi)A1AT含量下降；與Adiponectin正相關(guān)。由于Adiponectin為一種抗炎性細胞因子，推測A1AT與Adiponectin作用機制類似，可能具有抑制肥胖人群促炎性細胞因子表達的作用。 2.多囊卵巢綜合征患者血清中A1AT含量低于正常女性：采用酶聯(lián)免疫吸附法（ELISA）檢測血清中A1AT，發(fā)現(xiàn)在PCOS患者血清中，A1AT含量明顯低于正常對照組，中性粒細胞彈性蛋白酶（NE）含量高于正常對照組，NE/A1AT比例提高，促炎性細胞因子IL-8、IL-1β含量顯著升高，提示在PCOS患者體內(nèi)A1AT減少導致NE增多、促炎性細胞因子IL-8、IL-1β增多，推測NE/A1AT增高導致了促炎性細胞因子IL-8及IL-1β堆積，A1AT可能通過抑制NE及IL-8及IL-1β參與了PCOS發(fā)病機制。 3.多囊卵巢綜合征大鼠模型的構(gòu)建：我們利用來曲唑灌胃法構(gòu)建大鼠多囊卵巢綜合征模型。結(jié)果顯示：來曲唑灌胃23天，對實驗造模組大鼠進行陰道涂片檢查，大鼠動情周期失去規(guī)律性變化，無排卵。卵巢形態(tài)學觀察，卵巢結(jié)構(gòu)紊亂，卵泡呈囊狀擴張狀，顯示有大量的閉鎖卵泡和大的囊狀卵泡出現(xiàn)，發(fā)育階段卵泡及黃體數(shù)目明顯減少，顆粒細胞層減少，卵泡的面積大于對照組，體重均高于對照組。經(jīng)檢測血清性激素驗證，血清中LH、T顯著增高，E2水平顯著下降，符合PCOS模型標準。符合肥胖型PCOS患者疾病病理特征。 4. A1AT與促炎性細胞因子在多囊卵巢綜合征中的作用機制研究： 人和鼠A1AT是同源蛋白，造模同時設(shè)置人A1AT（hA1AT）干預組，陰道脫落細胞涂片結(jié)果表明，沒有和造模組一樣失去周期性變化。卵巢形態(tài)學也表明干預組沒有像造模組一樣出現(xiàn)結(jié)構(gòu)紊亂的跡象。進一步通過激素水平的檢測發(fā)現(xiàn)治療組與PCOS模型組差異顯著，和空白組無明顯差異。 利用ELISA方法分析檢測血清中促炎性細胞因子IL-8、IL-1β含量、NE含量，發(fā)現(xiàn)A1AT干預組促炎性細胞因子含量，NE含量低于模型組，與對照組差異不明顯。說明在A1AT干預下，PCOS大鼠病理狀態(tài)減輕，NE含量降低，促炎性細胞因子IL-8和IL-1β含量降低，因此推測PCOS的發(fā)病機制可能與A1AT的缺乏有關(guān)：①A1AT缺乏導致NE含量的升高而導致促炎性細胞因子堆積。②A1AT直接與IL-8及IL-1β作用，降低細胞炎癥反應(yīng)。 利用insightII軟件的ZDOCK模塊模擬分子對接，表明A1AT與NE、IL-8和IL-1β有直接作用位點，符合上述A1AT與NE、IL-8和IL-1β作用的推斷。 本研究結(jié)果表明，由于A1AT的缺乏而不能有效抑制促炎性細胞因子及NE，從而引起炎癥并進一步引發(fā)PCOS；A1AT對PCOS具有潛在的治療作用。
[Abstract]:The polycystic ovary syndrome ( PCOS ) is a kind of endocrine disturbance syndrome of female reproductive dysfunction and abnormal metabolism . The study shows that the concentration of adiponectin in the serum of PCOS patients is lower than that in the normal population .

Alpha 1 - antitrypsin ( A1AT ) is an important serine protease inhibitor in vivo , which is mainly synthesized by hepatocytes . It is widely used in animal serum to inhibit certain pro - inflammatory cytokines and neutrophils . However , the role and significance of the protease - related regulatory protein in the pathogenesis of PCOS are not very clear . In addition , the establishment of an ideal animal model of PCOS is the key to the clinical diagnosis and treatment strategy .

In this paper , we explored the occurrence of PCOS by inhibiting pro - inflammatory cytokines and protease - related regulatory proteins , and the following studies were carried out :

The serum levels of A1AT , leptin , Adipose , Glucose and Insulin were detected by enzyme - linked immunosorbent assay ( ELISA ) . The results showed that A1AT and Adipose decreased with the increase of body mass index ( BMI ) , and the difference was significant ( P < 0.05 ) .
Because Adipose is an anti - inflammatory cytokine , it is speculated that the mechanism of A1AT is similar to that of Adipose , which may have the effect of inhibiting the expression of pro - inflammatory cytokines in obese people .

2 . The content of A1AT in serum of patients with polycystic ovary syndrome was lower than that of normal control group . In the serum of PCOS patients , the content of A1AT was significantly lower than that of normal control group , the content of NE / A1AT was higher than that of normal control group , and the content of IL - 8 and IL - 1尾 in the serum of PCOS patients was higher . It was suggested that the increase of IL - 8 and IL - 1尾 in patients with PCOS could increase the accumulation of pro - inflammatory cytokines IL - 8 and IL - 1尾 , and A1AT might participate in the pathogenesis of PCOS by inhibiting NE and IL - 8 and IL - 1尾 .

3 . Construction of rat model of polycystic ovary syndrome : A model of polycystic ovary syndrome in rats was constructed by using letrozole gavage . The results showed that the rats underwent vaginal smear examination for 23 days .

4 . Effects of A1AT and pro - inflammatory cytokines in polycystic ovary syndrome : a study of mechanism :

Human and murine A1AT were homologous proteins , and human A1AT ( hA1AT ) intervention group was set up at the same time . The results of vaginal exfoliated cell smear showed that there were no signs of structural disorder in the intervention group , but there were no signs of structural disorder in the intervention group . The difference between treatment group and PCOS model group was significantly different from the detection of hormone level , and there was no significant difference between treatment group and blank group .

The levels of pro - inflammatory cytokines IL - 8 , IL - 1尾 and NE in serum were analyzed by ELISA . It was found that the levels of pro - inflammatory cytokines , NE content and the content of IL - 1尾 in patients with PCOS were lower than those in the model group . The results suggested that the pathogenesis of PCOS might be related to the lack of A1AT : 鈶

本文編號：1694636