缺氧條件下TGF-β1在子宮內(nèi)膜異位癥中的作用機(jī)制
發(fā)布時(shí)間：2018-08-15 14:06
【摘要】：背景子宮內(nèi)膜異位癥(Ems)是育齡婦女常見的疾病之一,在不孕女性中高達(dá)25%-40%。Ems作為一種良性病變,卻有與惡性腫瘤相似的侵襲、轉(zhuǎn)移、復(fù)發(fā)等特點(diǎn),但其病因及發(fā)病機(jī)制目前仍不清楚。經(jīng)血逆流學(xué)說被廣泛接受與支持,在位內(nèi)膜決定論是其補(bǔ)充和發(fā)展。Ems由于月經(jīng)前與月經(jīng)期間子宮平滑肌及螺旋動(dòng)脈不正常收縮,子宮內(nèi)膜在剝脫前后、逆流至腹腔過程中以及在異位粘附、侵襲、血管形成之前處于不同程度的缺氧狀態(tài)。轉(zhuǎn)化生長因子-β1 (TGF-β1)具有調(diào)節(jié)細(xì)胞增殖、分化、血管生成等多種功能。在腫瘤細(xì)胞中,缺氧狀態(tài)下TGF-β1表達(dá)上調(diào)能提高腫瘤細(xì)胞侵襲、血管新生、免疫抑制等能力。Ems患者的腹腔液、血清中以及在位內(nèi)膜細(xì)胞中TGF-β1高于正常婦女水平。而缺氧狀態(tài)下TGF-β1對(duì)于Ems的致病作用及機(jī)制目前并不清楚。因此,研究缺氧狀態(tài)下TGF-β1對(duì)Ems內(nèi)膜細(xì)胞的影響,可以作為Ems發(fā)生機(jī)制研究新的著眼點(diǎn),為該疾病的診斷、尋求新的治療思路等提供實(shí)驗(yàn)依據(jù)并具有一定的理論指導(dǎo)意義。目的研究缺氧條件下TGF-β1及相關(guān)因子在Ems發(fā)生發(fā)展中的作用及其相互關(guān)系,闡明缺氧選擇在Ems發(fā)病中的作用機(jī)制。方法檢測(cè)Ems患者在位內(nèi)膜、異位內(nèi)膜組織中TGF-β1、HIF-1α及VEGF的表達(dá);培養(yǎng)原代人子宮內(nèi)膜細(xì)胞,檢測(cè)經(jīng)缺氧干預(yù)、缺氧再灌注、缺氧與TGF-β1干預(yù)等不同組間細(xì)胞增殖、凋亡和TGF-β1、HIF-1α、VEGF及代謝相關(guān)因子的表達(dá);生物信息學(xué)軟件預(yù)測(cè)VEGF基因啟動(dòng)子區(qū)轉(zhuǎn)錄因子結(jié)合位點(diǎn),構(gòu)建啟動(dòng)子區(qū)系列截短熒光素酶報(bào)告基因表達(dá)載體,雙熒光素酶報(bào)告基因系統(tǒng)初步鑒定TGF-β1信號(hào)通路的SBE位點(diǎn),ChIP實(shí)驗(yàn)檢測(cè)HIF-1α和Smad與該區(qū)域的結(jié)合;建立NOD-SCID以及C57BL/6小鼠Ems模型,觀察經(jīng)缺氧及TGF-β1處理移植物后病灶的生長情況。應(yīng)用MTT、流式細(xì)胞儀分析、WesternBlot、定量RT-PCR、細(xì)胞免疫熒光、免疫組織化學(xué)技術(shù)等方法檢測(cè)細(xì)胞增殖、凋亡、血管生成和相關(guān)因子的表達(dá)。結(jié)果1、Ems在位內(nèi)膜及異位內(nèi)膜TGF-β1、HIF-1α及VEGF的mRNA與蛋白表達(dá)高于正常子宮內(nèi)膜,HIF-1α與VEGF、TGF-β1與VEGF的表達(dá)呈正相關(guān)。2、1%02缺氧16h內(nèi),細(xì)胞凋亡增多,增殖能力下降,16h后細(xì)胞凋亡數(shù)減少,增殖能力增強(qiáng),HIF-1α及VEGF的mRNA及蛋白表達(dá)升高,而TGF-β1表達(dá)無明顯變化。缺氧24-48h細(xì)胞增殖能力增強(qiáng)并穩(wěn)定,72h下降。3、TGF-β1干預(yù)對(duì)細(xì)胞增殖、凋亡與HIF-1α、VEGF表達(dá)等無明顯影響,而缺氧與TGF-β1共同干預(yù)下,細(xì)胞VEGF、HIF-1α的mRNA和蛋白表達(dá)顯著升高。4、1%02缺氧24h后恢復(fù)常氧,細(xì)胞活力增強(qiáng),凋亡相對(duì)減少,HIF-1α, VEGF表達(dá)明顯升高。5、VEGF基因啟動(dòng)子上游SBE1與HRE分別是TGF-β1和HIF-1α的作用位點(diǎn),VEGF基因表達(dá)受TGF-β1與HIF-1α調(diào)控。6、持續(xù)給予TGF-β1干預(yù),NOD-SCID小鼠及C57BL/6小鼠Ems模型移植病灶周圍新生血管形成,VEGF、HIF-1α的蛋白表達(dá)升高;缺氧與TGF-βp1共同預(yù)處理組織并在移植后持續(xù)給予TGF-β1,病灶生長明顯,血管豐富,VEGF、HIF-1α、TGF-β1的蛋白表達(dá)明顯升高。結(jié)論 1、Ems患者子宮內(nèi)膜組織中TGF-β1、HIF-1α和VEGF的表達(dá)高于正常對(duì)照。2、缺氧、HPC、缺氧與TGF-β1共同干預(yù)能夠刺激子宮內(nèi)膜細(xì)胞增殖、HIF-1 α和VEGF表達(dá)增加,GLUT1表達(dá)降低,LDHA、PDK1表達(dá)增高。3、隨著缺氧時(shí)間的延長,細(xì)胞凋亡增加,活力下降。缺氧16h后細(xì)胞活力增強(qiáng),1%02缺氧24h細(xì)胞耐受缺氧能力最強(qiáng)。4、缺氧和TGF-β1對(duì)VG對(duì)F表達(dá)調(diào)控發(fā)生在轉(zhuǎn)錄水平,缺氧通過-975—-968區(qū)域的HRE發(fā)揮作用,Smad蛋白結(jié)合在-992—-986區(qū)域的SBE上,HIF-1α和Smad與HRE和SBE區(qū)域DNA序列相結(jié)合。5、TGF-β1對(duì)Ems內(nèi)膜細(xì)胞的影響是在缺氧基礎(chǔ)上顯示的,TGF-β1促進(jìn)缺氧對(duì)Ems的作用是通過VEGF實(shí)現(xiàn)的。對(duì)于VEGF基因表達(dá)缺氧作用強(qiáng)于TGF-β1。6、缺氧與TGF-β1可促進(jìn)NOD-SCID及C57BL/6小鼠Ems模型移植病灶的生長及新生血管形成,二者具有協(xié)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.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R711.71
本文編號(hào)：2184434
[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.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R711.71
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 趙軍;劉肖;李亞里;譚明華;;低氧預(yù)處理對(duì)大鼠子宮內(nèi)膜異位病灶形成的影響[J];南方醫(yī)科大學(xué)學(xué)報(bào);2016年10期
2 Hong-Tao Yan;Guan-Fang Su;;Expression and significance of HIF-1α and VEGF in rats with diabetic retinopathy[J];Asian Pacific Journal of Tropical Medicine;2014年03期
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