A1AT在多囊卵巢综合征中的作用及机制

发布时间：2018-04-01 07:57

  本文选题：多囊卵巢综合征　切入点：α1-抗胰蛋白酶　出处：《吉林大学》2014年博士论文

【摘要】：多囊卵巢综合征（polycystic ovary syndrome, PCOS）是一种女性生殖功能障碍与代谢异常并存的内分泌紊乱综合征。PCOS患者常伴有肥胖及慢性脂肪炎症等并发症。研究表明，PCOS患者血清中脂联素（Adiponectin）浓度低于正常人群、瘦素（Leptin）浓度高于正常人群。多种白介素类促炎性细胞因子及中性粒细胞分泌的蛋白酶类在PCOS患者血清中浓度增加，表明炎症有可能与PCOS患病机制相关。 α1-抗胰蛋白酶（Alpha1-antitrypsin，A1AT）是一种体内重要的丝氨酸蛋白酶抑制剂，它主要由肝细胞合成，广泛存在于动物血清中，能够抑制某些促炎性细胞因子和中性粒细胞分泌的蛋白酶。但是，蛋白酶类相关调控蛋白在PCOS发病中的作用还需要进一步研究。本实验前期通过酶联免疫技术比较临床数据，发现肥胖者及肥胖型PCOS患者体内血清中A1AT蛋白浓度明显低于正常人群，但是其作用与意义尚不十分清楚。另外，由于PCOS患者临床组织标本取材困难，因此建立理想的PCOS动物模型是探讨临床诊疗策略的关键所在。 本论文探索A1AT通过抑制促炎性细胞因子和蛋白酶类相关调控蛋白干预PCOS的发生，，主要开展了以下研究： 1.肥胖人群血清中A1AT含量低于正常人群：采用酶联免疫吸附法（ELISA）检测其血清A1AT、瘦素（Leptin）、脂联素（Adiponectin）、血糖（Glucose）及胰岛素（Insulin）水平。结果表明A1AT和Adiponectin随着身体质量指数（BodyMass Index，BMI）的增加而减少，差异显著（P0.05）。Leptin随着BMI的增加而增加，差异显著（P＜0.05）。A1AT在人血清中与BMI、Adiponectin、Leptin和Insulin的血清水平相关。肥胖者Leptin水平升高，A1AT与Leptin成负相关，Leptin水平升高影响A1AT表达，因此肥胖人群体内A1AT含量下降；与Adiponectin正相关。由于Adiponectin为一种抗炎性细胞因子，推测A1AT与Adiponectin作用机制类似，可能具有抑制肥胖人群促炎性细胞因子表达的作用。 2.多囊卵巢综合征患者血清中A1AT含量低于正常女性：采用酶联免疫吸附法（ELISA）检测血清中A1AT，发现在PCOS患者血清中，A1AT含量明显低于正常对照组，中性粒细胞弹性蛋白酶（NE）含量高于正常对照组，NE/A1AT比例提高，促炎性细胞因子IL-8、IL-1β含量显著升高，提示在PCOS患者体内A1AT减少导致NE增多、促炎性细胞因子IL-8、IL-1β增多，推测NE/A1AT增高导致了促炎性细胞因子IL-8及IL-1β堆积，A1AT可能通过抑制NE及IL-8及IL-1β参与了PCOS发病机制。 3.多囊卵巢综合征大鼠模型的构建：我们利用来曲唑灌胃法构建大鼠多囊卵巢综合征模型。结果显示：来曲唑灌胃23天，对实验造模组大鼠进行阴道涂片检查，大鼠动情周期失去规律性变化，无排卵。卵巢形态学观察，卵巢结构紊乱，卵泡呈囊状扩张状，显示有大量的闭锁卵泡和大的囊状卵泡出现，发育阶段卵泡及黄体数目明显减少，颗粒细胞层减少，卵泡的面积大于对照组，体重均高于对照组。经检测血清性激素验证，血清中LH、T显著增高，E2水平显著下降，符合PCOS模型标准。符合肥胖型PCOS患者疾病病理特征。 4. A1AT与促炎性细胞因子在多囊卵巢综合征中的作用机制研究： 人和鼠A1AT是同源蛋白，造模同时设置人A1AT（hA1AT）干预组，阴道脱落细胞涂片结果表明，没有和造模组一样失去周期性变化。卵巢形态学也表明干预组没有像造模组一样出现结构紊乱的迹象。进一步通过激素水平的检测发现治疗组与PCOS模型组差异显著，和空白组无明显差异。 利用ELISA方法分析检测血清中促炎性细胞因子IL-8、IL-1β含量、NE含量，发现A1AT干预组促炎性细胞因子含量，NE含量低于模型组，与对照组差异不明显。说明在A1AT干预下，PCOS大鼠病理状态减轻，NE含量降低，促炎性细胞因子IL-8和IL-1β含量降低，因此推测PCOS的发病机制可能与A1AT的缺乏有关：①A1AT缺乏导致NE含量的升高而导致促炎性细胞因子堆积。②A1AT直接与IL-8及IL-1β作用，降低细胞炎症反应。 利用insightII软件的ZDOCK模块模拟分子对接，表明A1AT与NE、IL-8和IL-1β有直接作用位点，符合上述A1AT与NE、IL-8和IL-1β作用的推断。 本研究结果表明，由于A1AT的缺乏而不能有效抑制促炎性细胞因子及NE，从而引起炎症并进一步引发PCOS；A1AT对PCOS具有潜在的治疗作用。
[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

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