缺氧条件下TGF-β1在子宫内膜异位症中的作用机制

发布时间：2018-08-15 14:06

【摘要】：背景子宫内膜异位症(Ems)是育龄妇女常见的疾病之一,在不孕女性中高达25%-40%。Ems作为一种良性病变,却有与恶性肿瘤相似的侵袭、转移、复发等特点,但其病因及发病机制目前仍不清楚。经血逆流学说被广泛接受与支持,在位内膜决定论是其补充和发展。Ems由于月经前与月经期间子宫平滑肌及螺旋动脉不正常收缩,子宫内膜在剥脱前后、逆流至腹腔过程中以及在异位粘附、侵袭、血管形成之前处于不同程度的缺氧状态。转化生长因子-β1 (TGF-β1)具有调节细胞增殖、分化、血管生成等多种功能。在肿瘤细胞中,缺氧状态下TGF-β1表达上调能提高肿瘤细胞侵袭、血管新生、免疫抑制等能力。Ems患者的腹腔液、血清中以及在位内膜细胞中TGF-β1高于正常妇女水平。而缺氧状态下TGF-β1对于Ems的致病作用及机制目前并不清楚。因此,研究缺氧状态下TGF-β1对Ems内膜细胞的影响,可以作为Ems发生机制研究新的着眼点,为该疾病的诊断、寻求新的治疗思路等提供实验依据并具有一定的理论指导意义。目的研究缺氧条件下TGF-β1及相关因子在Ems发生发展中的作用及其相互关系,阐明缺氧选择在Ems发病中的作用机制。方法检测Ems患者在位内膜、异位内膜组织中TGF-β1、HIF-1α及VEGF的表达;培养原代人子宫内膜细胞,检测经缺氧干预、缺氧再灌注、缺氧与TGF-β1干预等不同组间细胞增殖、凋亡和TGF-β1、HIF-1α、VEGF及代谢相关因子的表达;生物信息学软件预测VEGF基因启动子区转录因子结合位点,构建启动子区系列截短荧光素酶报告基因表达载体,双荧光素酶报告基因系统初步鉴定TGF-β1信号通路的SBE位点,ChIP实验检测HIF-1α和Smad与该区域的结合;建立NOD-SCID以及C57BL/6小鼠Ems模型,观察经缺氧及TGF-β1处理移植物后病灶的生长情况。应用MTT、流式细胞仪分析、WesternBlot、定量RT-PCR、细胞免疫荧光、免疫组织化学技术等方法检测细胞增殖、凋亡、血管生成和相关因子的表达。结果1、Ems在位内膜及异位内膜TGF-β1、HIF-1α及VEGF的mRNA与蛋白表达高于正常子宫内膜,HIF-1α与VEGF、TGF-β1与VEGF的表达呈正相关。2、1%02缺氧16h内,细胞凋亡增多,增殖能力下降,16h后细胞凋亡数减少,增殖能力增强,HIF-1α及VEGF的mRNA及蛋白表达升高,而TGF-β1表达无明显变化。缺氧24-48h细胞增殖能力增强并稳定,72h下降。3、TGF-β1干预对细胞增殖、凋亡与HIF-1α、VEGF表达等无明显影响,而缺氧与TGF-β1共同干预下,细胞VEGF、HIF-1α的mRNA和蛋白表达显著升高。4、1%02缺氧24h后恢复常氧,细胞活力增强,凋亡相对减少,HIF-1α, VEGF表达明显升高。5、VEGF基因启动子上游SBE1与HRE分别是TGF-β1和HIF-1α的作用位点,VEGF基因表达受TGF-β1与HIF-1α调控。6、持续给予TGF-β1干预,NOD-SCID小鼠及C57BL/6小鼠Ems模型移植病灶周围新生血管形成,VEGF、HIF-1α的蛋白表达升高;缺氧与TGF-βp1共同预处理组织并在移植后持续给予TGF-β1,病灶生长明显,血管丰富,VEGF、HIF-1α、TGF-β1的蛋白表达明显升高。结论 1、Ems患者子宫内膜组织中TGF-β1、HIF-1α和VEGF的表达高于正常对照。2、缺氧、HPC、缺氧与TGF-β1共同干预能够刺激子宫内膜细胞增殖、HIF-1 α和VEGF表达增加,GLUT1表达降低,LDHA、PDK1表达增高。3、随着缺氧时间的延长,细胞凋亡增加,活力下降。缺氧16h后细胞活力增强,1%02缺氧24h细胞耐受缺氧能力最强。4、缺氧和TGF-β1对VG对F表达调控发生在转录水平,缺氧通过-975—-968区域的HRE发挥作用,Smad蛋白结合在-992—-986区域的SBE上,HIF-1α和Smad与HRE和SBE区域DNA序列相结合。5、TGF-β1对Ems内膜细胞的影响是在缺氧基础上显示的,TGF-β1促进缺氧对Ems的作用是通过VEGF实现的。对于VEGF基因表达缺氧作用强于TGF-β1。6、缺氧与TGF-β1可促进NOD-SCID及C57BL/6小鼠Ems模型移植病灶的生长及新生血管形成,二者具有协同作用。
[Abstract]:Background Endometriosis (Ems) is one of the most common diseases in women of childbearing age, with a high incidence of 25% - 40% in infertile women. As a benign lesion, Ems has the characteristics of invasion, metastasis and recurrence similar to malignant tumors, but its etiology and pathogenesis are still unclear. Because of the abnormal contraction of uterine smooth muscle and spiral artery before and during menstruation, the endometrium is in different degree of hypoxia during the process of reflux to abdominal cavity, ectopic adhesion, invasion, and angiogenesis. Transforming growth factor-beta 1 (TGF-beta 1) can regulate cell proliferation and differentiation. In tumor cells, the up-regulation of TGF-beta 1 expression under hypoxia can enhance the ability of tumor cell invasion, angiogenesis and immunosuppression. TGF-beta 1 in peritoneal fluid, serum and eutopic endometrial cells of patients with Ems is higher than that of normal women. The pathogenic effect and mechanism of TGF-beta 1 under hypoxia on Ems are present. It is not clear. Therefore, the study of the effect of TGF-beta 1 on the endometrial cells of Ems under hypoxia can serve as a new focus for the study of the pathogenesis of Ems, providing experimental basis for the diagnosis of the disease and seeking new treatment ideas, etc. It has certain theoretical significance. Objective To study the role of TGF-beta 1 and related factors in the occurrence and development of Ems under hypoxia. Methods The expression of TGF-beta 1, HIF-1a and VEGF in eutopic endometrium and ectopic endometrium of patients with Ems was detected, and the primary human endometrial cells were cultured, and the proliferation, apoptosis and TGF-beta 1 apoptosis were detected after hypoxia intervention, hypoxia-reperfusion, hypoxia and TGF-beta 1 intervention. Bioinformatics software predicted transcription factor binding sites in the promoter region of VEGF gene, constructed a series of truncated luciferase reporter gene expression vectors, identified the SBE sites of TGF-beta 1 signaling pathway by dual luciferase reporter gene system, and detected HIF-1a and Smad by ChIP assay. The NOD-SCID and C57BL/6 mice Ems models were established to observe the growth of the grafts after hypoxia and TGF-beta 1 treatment.MTT, flow cytometry, Western Blot, quantitative RT-PCR, immunofluorescence and immunohistochemistry were used to detect cell proliferation, apoptosis, angiogenesis and expression of related factors. Results 1. The mRNA and protein expression of TGF-beta 1, HIF-1a and VEGF in eutopic endometrium and ectopic endometrium of Ems were higher than that in normal endometrium. HIF-1a and VEGF, TGF-beta 1 were positively correlated with the expression of VEGF. Hypoxia 24-48 hours increased and stabilized the proliferation of cells, 72 hours decreased. 3. TGF-beta 1 intervention on cell proliferation, apoptosis and HIF-1 alpha, VEGF expression were not significantly affected, and hypoxia and TGF-beta 1 co-intervention, the expression of VEGF, HIF-1 alpha mRNA and protein increased significantly. 4, 1% 02 hypoxia 24 hours after recovery to normal oxygen, cells. The expression of HIF-1a and VEGF was significantly increased. 5. SBE1 and HRE were the sites of TGF-beta 1 and HIF-1a, respectively. The expression of VEGF gene was regulated by TGF-beta 1 and HIF-1a. 6. TGF-beta 1 was continuously given to intervene, neovascularization was observed in NOD-SCID mice and C57BL/6 mice. Hypoxia and TGF-beta-p1 preconditioned tissues and continued to give TGF-beta-1 after transplantation. The expression of VEGF, HIF-1a and TGF-beta-1 in the lesions was significantly increased. Conclusion 1. The expression of TGF-beta-1, HIF-1a and VEGF in the endometrium of Ems patients was higher than that of normal controls.2. Hypoxia, HPC, hypoxia and TGF-beta-1 co-existed. The same intervention could stimulate the proliferation of endometrial cells, increase the expression of HIF-1a and VEGF, decrease the expression of GLUT1, increase the expression of LDHA and PDK1. 3. With the prolongation of hypoxia time, apoptosis increased and activity decreased. The cell viability increased after 16 hours of hypoxia, and 1% 02 hypoxia 24 hours was the strongest. 4. Hypoxia and TGF-beta 1 regulated the expression of VG on F. At transcriptional level, hypoxia is mediated by HRE in the - 975 -- 968 region, Smad protein binds to SBE in the - 992 -- 986 region, HIF - 1A and Smad bind to DNA sequences in the HRE and SBE regions. 5. TGF - beta 1 affects the endometrial cells of Ems on the basis of hypoxia, and TGF - beta 1 promotes hypoxia through VEGF. The expression of hypoxia was stronger than that of TGF-beta 1.6. Hypoxia and TGF-beta 1 could promote the growth and angiogenesis of NOD-SCID and C57BL/6 mice Ems model.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R711.71

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