本文選題：子宮內(nèi)膜異位癥 + 子宮內(nèi)膜基質(zhì)細(xì)胞��； 參考：《蘇州大學(xué)》2014年碩士論文

【摘要】：目的：子宮內(nèi)膜異位癥（endometriosis, EMs）是一種常見的婦科疾病，其嚴(yán)重影響著女性的生活和健康。本病常見于生育年齡女性，以25～45歲女性多見，發(fā)病率約為10%，且近年呈上升趨勢。雖然其在形態(tài)學(xué)上呈良性表現(xiàn)，但在臨床行為學(xué)上具有類似惡性腫瘤的特點，如種植、侵襲及遠(yuǎn)處轉(zhuǎn)移等。本病的治療方法有手術(shù)及藥物，除根治性手術(shù)外，尚無一種理想的根治方法，無論是藥物或保守性手術(shù)均有相當(dāng)高的復(fù)發(fā)率。近年來，EMs與凋亡的關(guān)系日益受到重視，越來越多研究表明，異位子宮內(nèi)膜細(xì)胞得以在宮腔外種植并繼續(xù)存活，，與其凋亡特性改變有關(guān)。有研究表明內(nèi)異癥女性子宮收縮強度及頻率顯著高于正常女性，導(dǎo)致子宮輕度缺血缺氧（包括內(nèi)膜）；而又有大量的研究證明組織器官經(jīng)過缺氧預(yù)處理(hypoxia preconditioning, HPC)后，能夠增強其對隨后嚴(yán)重缺血缺氧所致凋亡的耐受性，HPC正成為組織損傷學(xué)和器官移植學(xué)研究的熱點，而與之相似的缺氧預(yù)處理與凋亡的關(guān)系也成為研究的新方向。我們通過體外培養(yǎng)子宮內(nèi)膜細(xì)胞，建立子宮內(nèi)膜的HPC模型及其對照組，比較各組間凋亡有關(guān)基因及其蛋白的表達(dá)就可以進(jìn)一步驗證HPC對子宮內(nèi)膜的保護(hù)作用。通過阻斷缺氧預(yù)處理對凋亡的保護(hù)通路就可以有效抑制異位內(nèi)膜的活性來預(yù)防其發(fā)生或降低復(fù)發(fā)，為EMS的臨床治療提供新的思路。 方法：1.選擇因子宮肌瘤行（次）全子宮切除手術(shù)的病例8名，術(shù)中刮取其子宮內(nèi)膜,經(jīng)酶解、過濾、貼壁純化等技術(shù)體外分離培養(yǎng)子宮內(nèi)膜基質(zhì)細(xì)胞，并行免疫熒光細(xì)胞鑒定，通過CCK-8測定細(xì)胞生長曲線。2.對傳代后的細(xì)胞進(jìn)行處理，待細(xì)胞融合致70%時分為五組：HPC組、HPC+缺氧組，ly294002+HPC+缺氧組，缺氧組、對照組。1）HPC：細(xì)胞間斷缺氧1h，復(fù)氧1h（共3個循環(huán)）后置于37℃、5%CO2的細(xì)胞培養(yǎng)箱培養(yǎng)；2）HPC+缺氧組：細(xì)胞間斷缺氧1h，復(fù)氧1h（共3個循環(huán)）后置于缺氧培養(yǎng)箱培養(yǎng)；3）ly294002+HPC+缺氧組：ly294002以50um的濃度加入培養(yǎng)基，如2）處理。4）缺氧組：細(xì)胞置于缺氧培養(yǎng)箱中培養(yǎng)72小時；5）對照組：細(xì)胞置于37℃、5%CO2細(xì)胞培養(yǎng)箱培養(yǎng)。3.干預(yù)72小時后，撤離處理條件后同一時間點分別收集各種因素作用后的細(xì)胞，測定各組的凋亡指數(shù)及AKT、Bcl-2、HIF、VEGF的mRNA水平及AKT蛋白表達(dá)。 結(jié)果：1.8例標(biāo)本培養(yǎng)基質(zhì)細(xì)胞，6例成功，1例細(xì)菌污染，另1例細(xì)胞數(shù)較少，培養(yǎng)失敗。原代培養(yǎng)的細(xì)胞中，波形蛋白染色陽性。細(xì)胞在接種后5-8天接近鋪滿，生長至第6天進(jìn)入指數(shù)生長期，傳至2-3代后基質(zhì)細(xì)胞純度高達(dá)98%以上。2.HPC+缺氧組AKT、HIF、VEGF基因表達(dá)明顯高于對照組（P＜0.05）， HPC+缺氧組AKT蛋白表達(dá)明顯高于對照組。3. HPC+缺氧組細(xì)胞凋亡指數(shù)明顯低于缺氧組（P＜0.05），ly294002+hpc+缺氧組凋亡指數(shù)明顯高于hpc+缺氧組（P＜0.05）。 結(jié)論：缺氧預(yù)處理可以保護(hù)缺氧對子宮內(nèi)膜基質(zhì)細(xì)胞的損傷，其作用的發(fā)揮跟AKT信號通路有密切關(guān)系。如果我們特異性控制AKT信號通路就可以有效調(diào)節(jié)子宮內(nèi)膜異位癥異位內(nèi)膜對缺氧的耐受性，從而降低子宮內(nèi)膜異位癥的發(fā)病率和復(fù)發(fā)率。對子宮內(nèi)膜的治療開辟一種新的治療方法。
[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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R711.71

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