外源性SOCS-3对异位子宫内膜间质细胞凋亡和增殖的影响研究
发布时间：2019-05-08 00:26
【摘要】：背景 子宫内膜异位症(内异症)是一种常见的妇产科疾病,在育龄女性中的发病率可达8%-10%,临床上常表现为痛经、慢性盆腔痛、性交不适、不孕等症状,严重影响女性的生活质量。内异症虽然是一种良性疾病,但却具有恶性肿瘤的特点,如增殖、复发、转移等。对于该病的发病有多种学说进行解释,例如体腔上皮化生学说、淋巴血管转移学说、干细胞学说、在位内膜决定论等等,其具体发病机制目前尚无定论,最广为接受的仍然是Sampson的经血逆流学说。但是异位子宫内膜细胞要在子宫腔及子宫肌层以外的部位生长增殖并形成内异病灶,则需要抵抗对机体内环境稳态具有维护作用的细胞凋亡的发生。我们可以试图对参与细胞增殖及凋亡的相关信号通路进行探索,从而为研究内异症的发病机制和治疗方案提供新的思路。 Janus激酶／信号转导与转录活化因子(Janus kinase/signal transducer and activator of transcription, JAK/STAT)是目前研究较多的一条信号通路,该通路广泛参与细胞的增殖、凋亡、转移、血管生成等过程。而细胞因子信号抑制因子(Suppressor of cytokine signaling, SOCS)是JAK/STAT言号通路的主要负反馈调节因子,其表达及活性对调节该通路发挥重要作用。研究发现在多种肿瘤细胞中均存在STAT-3的活性形式p-STAT-3的异常高表达及SOCS-3表达量的下调,且运用JAK-2特异性抑制剂AG490或外源性导入SOCS-3后可通过抑制p-STAT-3,导致肿瘤细胞增殖受阻,同时可促进细胞凋亡,最终达到抑制肿瘤细胞生长的作用。而内异症虽为良性疾病,但在许多生物学行为上具有与恶性肿瘤相似的特点,如过度增殖、凋亡抑制、转移、复发等。我们的前期研究也发现,异位子宫内膜组织与在位及正常子宫内膜组织相比较,存在p-STAT-3的高表达及SOCS-3的下调,进而我们推测异位子宫内膜中p-STAT-3和SOCS-3的异常表达及相互作用可能参与了子宫内膜异位症的发生发展过程。因此本研究利用慢病毒载体向异位子宫内膜间质细胞(Ectopic endometrial stromal cells, EESCs)导入外源性SOCS-3,观察SOCS-3上调后对EESCs中JAK/STAT-3信号通路的影响及其对细胞增殖和凋亡的调控。 方法 选取12例卵巢型内异症患者的异位子宫内膜囊壁进行细胞原代培养,获取EESCs,波形蛋白染色对EESCs进行纯度鉴定；利用重组构建的SOCS-3和GFP融合表达的慢病毒载体(LV-SOCS3-GFP)感染实验组EESCs,使其过表达SOCS-3,同时利用仅携带绿色荧光蛋白(GFP)基因序列的慢病毒载体(LV-GFP)感染同源EESCs,设为阴性对照组,荧光显微镜下观察细胞经慢病毒感染后GFP的表达,评估感染效率；Real-Time PCI检测各组细胞中SOCS-3、JAK-2、STAT-3mRNA表达水平；Western Blot检测(?)SOCS-3、JAK-2、STAT-3蛋白表达情况,以及JAK-2、STAT-3蛋白的磷酸化水平(p-JAK-2、p-STAT-3);流式细胞仪检测(?)SOCS-3过表达后对EESCs凋亡及周期的影响。 结果 慢病毒载体感染EESCs72h后,感染效率可达90%以上；实验组SOCS-3mRNA水平明显上调,与阴性对照组比较具有显著差异(P0.001), JAK-2、STAT-3mRNA表达与阴性对照组无显著差异；实验组SOCS-3蛋白表达明显上升,同时出现p-STAT-3蛋白表达的下调,而STAT-3. JAK-2及P-JAK-2蛋白水平与阴性对照组无差异；流式细胞仪检测结果显示,SOCS-3过表达可引起EESCs细胞凋亡比例的增加,且细胞周期中Go/G1期比例增高,与阴性对照组比较差异有统计学意义(P0.05)。 结论 外源性SOCS-3可抑制EESCs中STAT-3蛋白磷酸化,且该效应可进一步促进EESCs的凋亡并抑制细胞的周期转化,干扰EESCs细胞的生长。
[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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.71
本文编号：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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.71
【参考文献】
相关期刊论文 前1条
1 涂飞霞;阮菲;吴瑞瑾;詹宏;马俊彦;林俊;;STAT3和SOCS3与子宫内膜异位症的相关性研究[J];中国病理生理杂志;2012年01期
，本文编号：2471482
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