外源性SOCS-3對異位子宮內(nèi)膜間質(zhì)細胞凋亡和增殖的影響研究
發(fā)布時間：2019-05-08 00:26
【摘要】：背景 子宮內(nèi)膜異位癥(內(nèi)異癥)是一種常見的婦產(chǎn)科疾病,在育齡女性中的發(fā)病率可達8%-10%,臨床上常表現(xiàn)為痛經(jīng)、慢性盆腔痛、性交不適、不孕等癥狀,嚴重影響女性的生活質(zhì)量。內(nèi)異癥雖然是一種良性疾病,但卻具有惡性腫瘤的特點,如增殖、復(fù)發(fā)、轉(zhuǎn)移等。對于該病的發(fā)病有多種學說進行解釋,例如體腔上皮化生學說、淋巴血管轉(zhuǎn)移學說、干細胞學說、在位內(nèi)膜決定論等等,其具體發(fā)病機制目前尚無定論,最廣為接受的仍然是Sampson的經(jīng)血逆流學說。但是異位子宮內(nèi)膜細胞要在子宮腔及子宮肌層以外的部位生長增殖并形成內(nèi)異病灶,則需要抵抗對機體內(nèi)環(huán)境穩(wěn)態(tài)具有維護作用的細胞凋亡的發(fā)生。我們可以試圖對參與細胞增殖及凋亡的相關(guān)信號通路進行探索,從而為研究內(nèi)異癥的發(fā)病機制和治療方案提供新的思路。 Janus激酶／信號轉(zhuǎn)導與轉(zhuǎn)錄活化因子(Janus kinase/signal transducer and activator of transcription, JAK/STAT)是目前研究較多的一條信號通路,該通路廣泛參與細胞的增殖、凋亡、轉(zhuǎn)移、血管生成等過程。而細胞因子信號抑制因子(Suppressor of cytokine signaling, SOCS)是JAK/STAT言號通路的主要負反饋調(diào)節(jié)因子,其表達及活性對調(diào)節(jié)該通路發(fā)揮重要作用。研究發(fā)現(xiàn)在多種腫瘤細胞中均存在STAT-3的活性形式p-STAT-3的異常高表達及SOCS-3表達量的下調(diào),且運用JAK-2特異性抑制劑AG490或外源性導入SOCS-3后可通過抑制p-STAT-3,導致腫瘤細胞增殖受阻,同時可促進細胞凋亡,最終達到抑制腫瘤細胞生長的作用。而內(nèi)異癥雖為良性疾病,但在許多生物學行為上具有與惡性腫瘤相似的特點,如過度增殖、凋亡抑制、轉(zhuǎn)移、復(fù)發(fā)等。我們的前期研究也發(fā)現(xiàn),異位子宮內(nèi)膜組織與在位及正常子宮內(nèi)膜組織相比較,存在p-STAT-3的高表達及SOCS-3的下調(diào),進而我們推測異位子宮內(nèi)膜中p-STAT-3和SOCS-3的異常表達及相互作用可能參與了子宮內(nèi)膜異位癥的發(fā)生發(fā)展過程。因此本研究利用慢病毒載體向異位子宮內(nèi)膜間質(zhì)細胞(Ectopic endometrial stromal cells, EESCs)導入外源性SOCS-3,觀察SOCS-3上調(diào)后對EESCs中JAK/STAT-3信號通路的影響及其對細胞增殖和凋亡的調(diào)控。 方法 選取12例卵巢型內(nèi)異癥患者的異位子宮內(nèi)膜囊壁進行細胞原代培養(yǎng),獲取EESCs,波形蛋白染色對EESCs進行純度鑒定；利用重組構(gòu)建的SOCS-3和GFP融合表達的慢病毒載體(LV-SOCS3-GFP)感染實驗組EESCs,使其過表達SOCS-3,同時利用僅攜帶綠色熒光蛋白(GFP)基因序列的慢病毒載體(LV-GFP)感染同源EESCs,設(shè)為陰性對照組,熒光顯微鏡下觀察細胞經(jīng)慢病毒感染后GFP的表達,評估感染效率；Real-Time PCI檢測各組細胞中SOCS-3、JAK-2、STAT-3mRNA表達水平；Western Blot檢測(?)SOCS-3、JAK-2、STAT-3蛋白表達情況,以及JAK-2、STAT-3蛋白的磷酸化水平(p-JAK-2、p-STAT-3);流式細胞儀檢測(?)SOCS-3過表達后對EESCs凋亡及周期的影響。 結(jié)果 慢病毒載體感染EESCs72h后,感染效率可達90%以上；實驗組SOCS-3mRNA水平明顯上調(diào),與陰性對照組比較具有顯著差異(P0.001), JAK-2、STAT-3mRNA表達與陰性對照組無顯著差異；實驗組SOCS-3蛋白表達明顯上升,同時出現(xiàn)p-STAT-3蛋白表達的下調(diào),而STAT-3. JAK-2及P-JAK-2蛋白水平與陰性對照組無差異；流式細胞儀檢測結(jié)果顯示,SOCS-3過表達可引起EESCs細胞凋亡比例的增加,且細胞周期中Go/G1期比例增高,與陰性對照組比較差異有統(tǒng)計學意義(P0.05)。 結(jié)論 外源性SOCS-3可抑制EESCs中STAT-3蛋白磷酸化,且該效應(yīng)可進一步促進EESCs的凋亡并抑制細胞的周期轉(zhuǎn)化,干擾EESCs細胞的生長。
[Abstract]:background Endometriosis (endometriosis) is a common gynecological disease. The incidence of endometriosis can be up to 8% to 10% in the female of childbearing age. It is often manifested in the symptoms of dysmenorrhea, chronic pelvic pain, sexual discomforts, and infertility, which seriously affects the quality of life of women. A benign disease, but a characteristic of a malignant tumor, such as proliferation, recurrence, metastasis, For example, there are many theories on the pathogenesis of the disease, such as the theory of metaplasia on the body cavity, the theory of lymphatic vessel transfer, the stem cell theory, the in-place endodonism, and so on. The specific pathogenesis of the disease is not yet conclusive, and the most widely accepted is the blood-flow counter-current study of Sampson. It is suggested that, in the case of ectopic endometrial cells to grow and proliferate outside the uterine cavity and the myometrium, it is necessary to resist the development of the cell apoptosis which has the effect of maintaining the homeostasis of the environment in the body. We can try to explore the relevant signal pathways involved in cell proliferation and apoptosis, so as to provide new thinking for the pathogenesis and treatment of endometriosis. The pathway. Janus kinase/ signal transducer and activator of transcription (JAK/ STAT) is a signal pathway that is widely used in cell proliferation, apoptosis, metastasis and angiogenesis. The factor of cytokine signal suppression (SOCS) is the main negative feedback adjustment factor of the JAK/ STAT signal path, and its expression and activity play a role in regulating the pathway. It was found that the abnormal high expression of the activity form of STAT-3 and the down-regulation of the expression of SOCS-3 in the active form of STAT-3 in various tumor cells, and the inhibition of p-STAT-3 by using the JAK-2 specific inhibitor AG490 or the exogenous introduction of SOCS-3, could cause the proliferation of the tumor cells to be blocked, and also promote the fine Apoptosis, and finally, the growth of tumor cells is achieved. and has the characteristics of being similar to the malignant tumor in many biological behaviors, such as hyperproliferation, apoptosis inhibition and metastasis, Relapse, etc. Our previous studies have also found that ectopic endometrial tissue is compared with in-place and normal endometrial tissue, with high expression of p-STAT-3 and SOCS-3 The down-regulation of the abnormal expression and interaction of p-STAT-3 and SOCS-3 in the ectopic endometrium may be involved in the occurrence of endometriosis. The effect of the upregulation of SOCS-3 on JAK/ STAT-3 signaling pathway in ESCs and its effects on cell proliferation and apoptosis were observed by introducing exogenous SOCS-3 into ectopic endometrial stromal cells (EESCs) using lentiviral vectors. tone Methods A total of 12 patients with endometriosis were cultured by primary culture, EESCs were obtained, and the purity of EESCs was identified by the staining of ESCs. The lentiviral vector (LV-SOCS3-GFP), which was expressed by recombinant SOCS-3 and GFP fusion, was used to infect the EESCs of the experimental group. For SOCS-3, a lentiviral vector (LV-GFP) carrying a sequence of a green fluorescent protein (GFP) gene was used to infect homologous EESCs, which was set to a negative control group, and the expression of GFP after a slow viral infection was observed under a fluorescence microscope to assess the efficiency of infection; and the real-time PCI detected SOCS in each group of cells. 3. JAK-2, STAT-3 mRNA expression level; Western B lot detection (?) SOCS-3, JAK-2, STAT-3 protein expression, and phosphorylation of JAK-2, STAT-3 protein (p-JAK-2, p-STAT-3); flow type After the overexpression of SOCS-3, the cytometric device is responsible for EESCs. dead and Results The level of SOCS-3 mRNA in the experimental group was significantly higher than that of the negative control group (P 0.001), and the expression of JAK-2 and STAT-3 was not significantly different from that of the negative control group. The SOC of the experimental group was higher than that of the negative control group. The expression of S-3 protein was significantly increased, and the expression of p-STAT-3 protein was also observed. The results of flow cytometry showed that the overexpression of SOCS-3 could cause an increase in the percentage of apoptosis in the EESCs, and the ratio of the Go/ G1 phase in the cell cycle increased and the difference between the control group and the negative control group was higher. Ji Xue Conclusion Exogenous SOCS-3 can inhibit the phosphorylation of STAT-3 protein in EESCs, and the effect can further promote the apoptosis of EESCs and inhibit the cycle of the cells.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R711.71
本文編號：2471482
[Abstract]:background Endometriosis (endometriosis) is a common gynecological disease. The incidence of endometriosis can be up to 8% to 10% in the female of childbearing age. It is often manifested in the symptoms of dysmenorrhea, chronic pelvic pain, sexual discomforts, and infertility, which seriously affects the quality of life of women. A benign disease, but a characteristic of a malignant tumor, such as proliferation, recurrence, metastasis, For example, there are many theories on the pathogenesis of the disease, such as the theory of metaplasia on the body cavity, the theory of lymphatic vessel transfer, the stem cell theory, the in-place endodonism, and so on. The specific pathogenesis of the disease is not yet conclusive, and the most widely accepted is the blood-flow counter-current study of Sampson. It is suggested that, in the case of ectopic endometrial cells to grow and proliferate outside the uterine cavity and the myometrium, it is necessary to resist the development of the cell apoptosis which has the effect of maintaining the homeostasis of the environment in the body. We can try to explore the relevant signal pathways involved in cell proliferation and apoptosis, so as to provide new thinking for the pathogenesis and treatment of endometriosis. The pathway. Janus kinase/ signal transducer and activator of transcription (JAK/ STAT) is a signal pathway that is widely used in cell proliferation, apoptosis, metastasis and angiogenesis. The factor of cytokine signal suppression (SOCS) is the main negative feedback adjustment factor of the JAK/ STAT signal path, and its expression and activity play a role in regulating the pathway. It was found that the abnormal high expression of the activity form of STAT-3 and the down-regulation of the expression of SOCS-3 in the active form of STAT-3 in various tumor cells, and the inhibition of p-STAT-3 by using the JAK-2 specific inhibitor AG490 or the exogenous introduction of SOCS-3, could cause the proliferation of the tumor cells to be blocked, and also promote the fine Apoptosis, and finally, the growth of tumor cells is achieved. and has the characteristics of being similar to the malignant tumor in many biological behaviors, such as hyperproliferation, apoptosis inhibition and metastasis, Relapse, etc. Our previous studies have also found that ectopic endometrial tissue is compared with in-place and normal endometrial tissue, with high expression of p-STAT-3 and SOCS-3 The down-regulation of the abnormal expression and interaction of p-STAT-3 and SOCS-3 in the ectopic endometrium may be involved in the occurrence of endometriosis. The effect of the upregulation of SOCS-3 on JAK/ STAT-3 signaling pathway in ESCs and its effects on cell proliferation and apoptosis were observed by introducing exogenous SOCS-3 into ectopic endometrial stromal cells (EESCs) using lentiviral vectors. tone Methods A total of 12 patients with endometriosis were cultured by primary culture, EESCs were obtained, and the purity of EESCs was identified by the staining of ESCs. The lentiviral vector (LV-SOCS3-GFP), which was expressed by recombinant SOCS-3 and GFP fusion, was used to infect the EESCs of the experimental group. For SOCS-3, a lentiviral vector (LV-GFP) carrying a sequence of a green fluorescent protein (GFP) gene was used to infect homologous EESCs, which was set to a negative control group, and the expression of GFP after a slow viral infection was observed under a fluorescence microscope to assess the efficiency of infection; and the real-time PCI detected SOCS in each group of cells. 3. JAK-2, STAT-3 mRNA expression level; Western B lot detection (?) SOCS-3, JAK-2, STAT-3 protein expression, and phosphorylation of JAK-2, STAT-3 protein (p-JAK-2, p-STAT-3); flow type After the overexpression of SOCS-3, the cytometric device is responsible for EESCs. dead and Results The level of SOCS-3 mRNA in the experimental group was significantly higher than that of the negative control group (P 0.001), and the expression of JAK-2 and STAT-3 was not significantly different from that of the negative control group. The SOC of the experimental group was higher than that of the negative control group. The expression of S-3 protein was significantly increased, and the expression of p-STAT-3 protein was also observed. The results of flow cytometry showed that the overexpression of SOCS-3 could cause an increase in the percentage of apoptosis in the EESCs, and the ratio of the Go/ G1 phase in the cell cycle increased and the difference between the control group and the negative control group was higher. Ji Xue Conclusion Exogenous SOCS-3 can inhibit the phosphorylation of STAT-3 protein in EESCs, and the effect can further promote the apoptosis of EESCs and inhibit the cycle of the cells.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R711.71
【參考文獻】
相關(guān)期刊論文 前1條
1 涂飛霞;阮菲;吳瑞瑾;詹宏;馬俊彥;林俊;;STAT3和SOCS3與子宮內(nèi)膜異位癥的相關(guān)性研究[J];中國病理生理雜志;2012年01期
，本文編號：2471482
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