缺氧预处理对子宫内膜细胞缺氧的保护作用及其机制研究

发布时间：2018-05-09 02:44

本文选题：子宫内膜异位症 + 子宫内膜基质细胞　；参考：《苏州大学》2014年硕士论文

【摘要】：目的：子宫内膜异位症（endometriosis, EMs）是一种常见的妇科疾病，其严重影响着女性的生活和健康。本病常见于生育年龄女性，以25～45岁女性多见，发病率约为10%，且近年呈上升趋势。虽然其在形态学上呈良性表现，但在临床行为学上具有类似恶性肿瘤的特点，如种植、侵袭及远处转移等。本病的治疗方法有手术及药物，除根治性手术外，尚无一种理想的根治方法，无论是药物或保守性手术均有相当高的复发率。近年来，EMs与凋亡的关系日益受到重视，越来越多研究表明，异位子宫内膜细胞得以在宫腔外种植并继续存活，，与其凋亡特性改变有关。有研究表明内异症女性子宫收缩强度及频率显著高于正常女性，导致子宫轻度缺血缺氧（包括内膜）；而又有大量的研究证明组织器官经过缺氧预处理(hypoxia preconditioning, HPC)后，能够增强其对随后严重缺血缺氧所致凋亡的耐受性，HPC正成为组织损伤学和器官移植学研究的热点，而与之相似的缺氧预处理与凋亡的关系也成为研究的新方向。我们通过体外培养子宫内膜细胞，建立子宫内膜的HPC模型及其对照组，比较各组间凋亡有关基因及其蛋白的表达就可以进一步验证HPC对子宫内膜的保护作用。通过阻断缺氧预处理对凋亡的保护通路就可以有效抑制异位内膜的活性来预防其发生或降低复发，为EMS的临床治疗提供新的思路。方法：1.选择因子宫肌瘤行（次）全子宫切除手术的病例8名，术中刮取其子宫内膜,经酶解、过滤、贴壁纯化等技术体外分离培养子宫内膜基质细胞，并行免疫荧光细胞鉴定，通过CCK-8测定细胞生长曲线。2.对传代后的细胞进行处理，待细胞融合致70%时分为五组：HPC组、HPC+缺氧组，ly294002+HPC+缺氧组，缺氧组、对照组。1）HPC：细胞间断缺氧1h，复氧1h（共3个循环）后置于37℃、5%CO2的细胞培养箱培养；2）HPC+缺氧组：细胞间断缺氧1h，复氧1h（共3个循环）后置于缺氧培养箱培养；3）ly294002+HPC+缺氧组：ly294002以50um的浓度加入培养基，如2）处理。4）缺氧组：细胞置于缺氧培养箱中培养72小时；5）对照组：细胞置于37℃、5%CO2细胞培养箱培养。3.干预72小时后，撤离处理条件后同一时间点分别收集各种因素作用后的细胞，测定各组的凋亡指数及AKT、Bcl-2、HIF、VEGF的mRNA水平及AKT蛋白表达。结果：1.8例标本培养基质细胞，6例成功，1例细菌污染，另1例细胞数较少，培养失败。原代培养的细胞中，波形蛋白染色阳性。细胞在接种后5-8天接近铺满，生长至第6天进入指数生长期，传至2-3代后基质细胞纯度高达98%以上。2.HPC+缺氧组AKT、HIF、VEGF基因表达明显高于对照组（P＜0.05）， HPC+缺氧组AKT蛋白表达明显高于对照组。3. HPC+缺氧组细胞凋亡指数明显低于缺氧组（P＜0.05），ly294002+hpc+缺氧组凋亡指数明显高于hpc+缺氧组（P＜0.05）。结论：缺氧预处理可以保护缺氧对子宫内膜基质细胞的损伤，其作用的发挥跟AKT信号通路有密切关系。如果我们特异性控制AKT信号通路就可以有效调节子宫内膜异位症异位内膜对缺氧的耐受性，从而降低子宫内膜异位症的发病率和复发率。对子宫内膜的治疗开辟一种新的治疗方法。
[Abstract]:Objective: Endometriosis (EMs) is a common gynecologic disease, which seriously affects the life and health of women. This disease is common in women of childbearing age. It is common in women of childbearing age. The incidence of 25~45 year old women is more common, the incidence is about 10%, and it has an upward trend in recent years. Although it has a benign morphological appearance, it is in clinical behavior. Similar to malignant tumor, such as planting, invasion and distant metastasis. The treatment of this disease has surgery and drugs, and there is no ideal radical cure, no matter whether it is drug or conservative operation, there is a high recurrence rate. In recent years, the relationship between EMs and apoptosis has been paid more and more attention, more and more studies have shown that it is different. Studies have shown that the uterine contraction intensity and frequency of endometriosis women are significantly higher than those of normal women, leading to mild hypoxic hypoxia (including intima) in uterus, and a large number of studies have shown that the tissues and organs are pretreated by hypoxia preconditioning (hypoxia precon). After ditioning, HPC), it can enhance its tolerance to apoptosis induced by severe ischemia and hypoxia. HPC is becoming a hot spot in tissue injury and organ transplantation. The relationship between hypoxia preconditioning and apoptosis is a new direction of research. By culture of endometrium cells in vitro, we establish the HPC model of endometrium. And compared with the control group, the expression of apoptosis related genes and proteins in each group can further verify the protective effect of HPC on the endometrium. By blocking the protective pathway of hypoxia preconditioning, the activity of the ectopic endometrium can be effectively suppressed to prevent the occurrence of the endometrium and reduce the relapse, which provides a new way of thinking for the clinical treatment of EMS.
Methods: 1. a total of 8 cases of hysteromyoma underwent total hysterectomy, the endometrium was scraped in the operation, the endometrial stromal cells were isolated and cultured in vitro by enzyme hydrolysis, filtration, and adherent purification. Immunofluorescent cells were identified by immunofluorescent cells. The cell growth curve.2. was measured by CCK-8, and the cells were treated with the cell fusion. Combined 70% time was five groups: HPC group, HPC+ anoxic group, ly294002+HPC+ anoxic group, anoxic group, control group.1) HPC: cell intermittent hypoxia 1H, reoxygenation 1H (3 cycles) post at 37, 5%CO2 cell culture box culture; 2) HPC+ hypoxia group: cell intermittent hypoxia 1H, reoxygenation 1H (3 cycles) after hypoxia incubator culture; 3) ly294002+HPC + hypoxia group: LY294002 was added to the medium of 50um, for example 2) treatment of.4) anoxic group: cells were cultured in anoxic incubator for 72 hours; 5) control group: cells were placed at 37, 5%CO2 cell culture box was incubating.3. for 72 hours, and after evacuating treatment conditions, the cells after various factors were collected, and each group was determined. The apoptotic index and mRNA level of AKT, Bcl-2, HIF, VEGF and AKT protein expression.
Results: 1.8 specimens were cultured, 6 cases were successful, 1 cases were contaminated with bacteria, the other 1 cases were less, the culture failed. In the primary cultured cells, vimentin was stained positive. The cells were close to spread on the 5-8 day after inoculation and grew to sixth days to enter the exponential growth period, and the purity of the matrix cells reached more than 98%.2.HPC+ hypoxia group AKT, HI after the 2-3 generation. The expression of F, VEGF gene was significantly higher than that in the control group (P < 0.05). The expression of AKT protein in the HPC+ hypoxia group was significantly higher than that in the control group of.3. HPC+ hypoxia group (P < 0.05), and the apoptotic index in the ly294002+hpc+ hypoxia group was significantly higher than that of the hpc+ hypoxia group (P < 0.05).
Conclusion: hypoxia preconditioning can protect the damage to endometrial stromal cells of the endometrium, which is closely related to the AKT signaling pathway. If we specifically control the AKT signaling pathway, we can effectively regulate the tolerance of Endometriosis Endometrium to anoxia, thus reducing the incidence of endometriosis and the incidence of endometriosis. Recurrence rate. A new treatment for endometrium.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.71

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