自噬基因LC-3与子宫内膜异位症的相关性研究

发布时间：2018-08-08 21:20

【摘要】：研究背景：子宫内膜异位症（Endometriosis, EMs）是一种常见和多发的妇科内分泌疾病，其引起的不孕及慢性疼痛症状严重影响育龄妇女生殖健康和生活质量。目前EMs的发病机制不明。程序性细胞死亡(Programmed ce1l death，，PCD)由多种信号通路控制并维持细胞内环境的稳定性。PCD分为两种：细胞凋亡(属I型细胞死亡，由caspase介导)和自体吞噬(属II型细胞死亡，非caspase介导)。自噬与溶酶体相融合形成自噬溶酶体将吞噬物降解消化，吞噬物包括蛋白质及细胞器等。自噬在多种疾病的发生发展中发挥着作用。微管相关轻链蛋白-3（Microtubule associated protein light chain3，LC-3）是酵母细胞自噬相关基因ATG8的同源基因。LC-3在多种肿瘤疾病中的表达水平出现异常，而子宫内膜异位症与肿瘤又有相似的生物学行为，目前细胞自噬与子宫内膜异位症发病机制关系的研究报道较少，本实验对细胞自噬泡超微结构及自噬基因LC-3与子宫内膜异位症发病机制进行相关性研究。目的：探讨子宫内膜异位症患者在位内膜及异位内膜细胞自噬泡超微结构变化；LC-3mRNA和蛋白在子宫内膜异位症在位内膜和异位内膜细胞中的表达及意义。方法：本文收集31例内异症患者的异位内膜组织及该患者相应的在位内膜组织为实验组，31例正常的子宫内膜组织为对照组，应用透射电镜观察内异症在位及异位内膜中细胞自噬泡超微结构的改变；RT-PCR、Western-blotting方法检测LC-3mRNA和蛋白在内异症组在位内膜细胞、异位内膜细胞及正常子宫内膜细胞中的表达，利用SPSS19.0统计学软件分析实验结果。结果实验组和对照组患者的平均年龄分别为：(32.48±4.36)岁；(32.39±2.64)岁，两组间比较无统计学意义(P＞0.05);透射电镜结果分析显示：实验组的在位内膜细胞及异位内膜细胞中的自噬泡数目较对照组的正常子宫内膜细胞的自噬泡数目有所下调；Real time-PCR结果分析显示：LC-3mRNA在实验组的在位子宫内膜细胞及异位内膜细胞中表达均低于对照组内膜细胞(P=0.015)，实验组在位子宫内膜细胞的LC-3mRNA较对照组内膜细胞是下调的（0.435±0.227; P=0.013），实验组异位子宫内膜细胞的LC-3mRNA表达量比对照组降低(0.429±0.303; P=0.011)，但实验组在位内膜及异位内膜细胞的LC-3mRNA表达量无明显差异（1.282±0.534;P=0.949）；Western Blot结果分析显示：LC-3蛋白在实验组的在位子宫内膜细胞及异位内膜细胞中表达均低于对照组内膜(P=0.000)，实验组在位子宫内膜细胞的LC-3蛋白较对照组内膜细胞是下调的（0.242±0.08，0.440±0.08；P=0.000）,实验组异位子宫内膜细胞的LC-3蛋白表达量比对照组降低(0.309±0.09；P=0.000)，但同时实验组异位内膜细胞的LC-3蛋白表达量明显高于在位内膜组（P=0.003）。结论子宫内膜异位症患者的异位内膜细胞的自噬活性降低，同时在位内膜细胞的自噬活性也降低，自噬参与了子宫内膜异位症的发病。
[Abstract]:Research background:
Endometriosis (EMs) is a common and multiple gynecologic endocrine disease. The symptoms of infertility and chronic pain seriously affect the reproductive health and quality of life of women of childbearing age. The pathogenesis of EMs is unclear.
Programmed ce1l death (PCD) is controlled by a variety of signaling pathways and maintains the stability of the intracellular environment,.PCD is divided into two kinds: apoptosis (I cell death, caspase mediated) and autophagy (II cell death, non caspase mediating). Autophagy and lysosomes are fused to form autophagic lysosomes to reduce phagocytosis. Digestion and phagocytosis include proteins and organelles. Autophagy plays a role in the development of many diseases.
Microtubule related light chain protein -3 (Microtubule associated protein light chain3, LC-3) is the expression level of the homologous gene.LC-3 in yeast cell autophagy related gene ATG8 in a variety of tumor diseases, and endometriosis and tumor have similar biological behavior. At present, the pathogenesis of autophagy and endometriosis is the disease. There are few reports on the mechanism of endometriosis. In this study, the ultrastructure of autophagic vesicles and the correlation between autophagic gene LC-3 and the pathogenesis of endometriosis were studied.
Objective:
To investigate the ultrastructural changes of autophagic vesicles in eutopic and ectopic endometrium cells of endometriosis, and the expression and significance of LC-3mRNA and protein in eutopic and ectopic endometrium cells of endometriosis.
Method:
The ectopic endometrium of 31 patients with endometriosis and the corresponding eutopic endometrium in the experimental group were collected and 31 normal endometrium tissues were used as the control group. The ultrastructural changes of the autophagic vesicles in the eutopic and ectopic endometrium were observed by transmission electron microscopy, and the RT-PCR and Western-blotting methods were used to detect LC-3mRNA and protein. In endometriosis group, the expression of eutopic endometrial cells, ectopic endometrial cells and normal endometrial cells was analyzed by SPSS19.0 statistical software.
Result
The average age of the patients in the experimental group and the control group was (32.48 + 4.36) years and (32.39 + 2.64) years old, and there was no statistical significance between the two groups (P > 0.05). The number of autophagic vacuoles in the eutopic and ectopic endometrium cells of the experimental group was compared with the number of autophagic vesicles in the normal endometrium cells of the control group. The results of Real time-PCR analysis showed that the expression of LC-3mRNA in the eutopic endometrium and ectopic endometrium cells in the experimental group was lower than that of the control group (P=0.015). The LC-3mRNA of the eutopic endometrium cells in the experimental group was lower than that of the control group (0.435 + 0.227; P=0.013), and the endometrium endometrium was fine in the experimental group. The expression of LC-3mRNA in the cell was lower than that of the control group (0.429 + 0.303; P=0.011), but the expression of LC-3mRNA in the eutopic and ectopic endometrium cells of the experimental group was not significantly different (1.282 + 0.534; P=0.949). The analysis of Western Blot results showed that the expression of LC-3 protein in the eutopic and ectopic endometrium cells of the experimental group was lower than that of the control. The LC-3 protein of the eutopic endometrium cells in the experimental group was lower than that of the control group (0.242 + 0.08,0.440 + 0.08; P=0.000). The expression of LC-3 protein in the ectopic endometrium cells in the experimental group was lower than that of the control group (0.309 + 0.09; P=0.000), but the expression of LC-3 protein in the ectopic endometrium cells in the experimental group was obvious at the same time. Higher than the eutopic endometrium group (P=0.003).
conclusion
The autophagy activity of ectopic endometrium cells in patients with endometriosis is reduced and autophagy of the eutopic cells is also reduced, and autophagy is involved in the pathogenesis of endometriosis.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.71

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