Survivin对高肺血流性肺动脉高压大鼠肺动脉平滑肌细胞增殖与凋亡调控及其机制研究

发布时间：2018-05-03 20:12

本文选题：肺动脉高压 + 肺动脉平滑肌细胞　；参考：《广西医科大学》2016年博士论文

【摘要】：第一部分Survivin在高肺血流性肺动脉高压大鼠肺动脉平滑肌细胞表达及意义背景：先天性左向右分流性心脏病致高肺血流性肺动脉高压最重要的病理变化是肺动脉平滑肌细胞增殖,肺血管重构,确切的细胞及分子机制尚不明确。Survivin是重要的凋亡蛋白抑制剂,抑制细胞凋亡,延长细胞存活。Survivin是否在高肺血流肺动脉高压肺动脉平滑肌细胞表达以及对肺动脉平滑肌细胞增殖和凋亡的调控有待进一步研究。目的：证实survivin在高肺血流性肺动脉高压大鼠肺动脉平滑肌细胞上表达,探讨survivin对肺动脉平滑肌细胞可能的调控作用。方法：将成年SD大鼠,雌雄各半,按随机数字方法分为对照组、假手术组和分流组。对照组大鼠未作任何处理。假手术组夹闭腹主动脉十分钟。分流组通过手术方法建立腹主动脉到下腔静脉的分流瘘道,以建立高肺血流性肺动脉高压大鼠模型。11周后,通过对分流瘘道B超检查、右心室肥厚指数测定、肺动脉压力测定、肺组织切片HE染色等确认建模是否成功。免疫组织化学法检测survivin在肺组织的表达及分布。取各组肺动脉平滑肌组织进行原代肺动脉平滑肌细胞培养；用qRT-PCR法检测肺动脉平滑肌细胞survivin mRNA表达水平；western blot检测suvivin蛋白的表达水平；细胞增殖实验(CCK8试剂盒法)检测肺动脉平滑肌细胞增殖；细胞凋亡实验(流式细胞学)检测肺动脉平滑肌细胞凋亡。结果：通过分流组大鼠腹主动脉-下腔静脉分流瘘道B超检查、右心室肥厚指数、肺动脉压力测定、肺组织进行病理切片HE染色,证实已成功建立高肺血流性肺动脉高压大鼠模型。免疫组化显示survivin在高肺血流性肺动脉高压的肺动脉平滑肌上表达。通过qRT-PCR及western blot分析,survivin mRNA及蛋白在分流组肺动脉平滑肌细胞呈现阳性表达,未发现对照组与假手术组有survivin mRNA及蛋白表达。通过细胞增殖实验(CCK8试剂盒法)检测各组肺动脉平滑肌细胞增殖,分流组肺动脉平滑肌细胞增殖显著增加,较假手术组有显著差异(P0.05),而对照组与假手术组比较无显著差异(P0.05)。通过细胞凋亡实验(流式细胞学)检测分析,分流组肺动脉平滑肌细胞凋亡较假手术组显著下降(P0.05),而对照组与假手术组比较无显著差异(P0.05)。结论：(1)Survivin在高肺血流性肺动脉高压大鼠肺动脉平滑肌细胞表达,正常大鼠肺动脉平滑肌细胞未发现survivin表达。 (2)survivin可能通过调控肺动脉平滑肌细胞增殖与凋亡参与高肺血流性肺动脉高压的发病机制。第二部分Survivin调控高肺血流性肺动脉高压大鼠肺动脉平滑肌细胞增殖与凋亡及可能的调控通路背景：根据第一部分结论,证实高肺血流性肺动脉高压大鼠的肺动脉平滑肌细胞增殖增加,凋亡受抑；并证实survivin在高肺血流性肺动脉高压大鼠的肺动脉平滑肌细胞表达,而对照组或假手术组大鼠的肺动脉平滑肌细胞无survivin的表达。由此推测survivin可能参与高肺血流性肺动脉高压大鼠肺动脉平滑肌细胞增殖与凋亡的调控。目的：通过构建siRNA-survivin慢病毒载体,对分流组肺动脉平滑肌细胞进行感染,干扰survivin表达,探索survivin是否调控高肺血流性肺动脉高压大鼠肺动脉平滑肌细胞增殖与凋亡,以及可能的调控通路。方法：利用第一部分已证实为高肺血流性肺动脉高压的分流组大鼠原代培养的肺动脉平滑肌细胞为研究对象,将其分为空白对照组(不作处理),空载病毒组(用阴性对照慢病毒感染)、慢病毒干扰组(siRNA-survivin慢病毒载体感染)。感染成功后,通过qRT-PCR法检测各组肺动脉平滑肌细胞survivinN电压门控钾通道Kvl.5以及Kv2.1 mRNA的表达水平；Western blot法检测suvivin、Kv1.5以及Kv2.1蛋白的表达水平；细胞增殖实验(CCK8试剂盒法)检测肺动脉平滑肌细胞增殖情况；细胞凋亡实验(流式细胞学)检测肺动脉平滑肌细胞凋亡情况。ELISA法检测所培养的肺动脉平滑肌细胞上清液caspase-3以及caspase-9浓度。结果：成功培养了研究所需的高肺血流性肺动脉高压大鼠的肺动脉平滑肌细胞,成功构建并筛选出感染效率高的siRNA-survivin慢病毒载体,成功感染原代培养的肺动脉平滑肌细胞。比较mRNA表达,慢病毒干扰组survivin mRNA表达水平较空载病毒组显著下降(P0.05)。空载病毒组与空白对照组比较无显著性差异(P0.05)。慢病毒干扰组Kv1.5以及Kv2.1mRNA表达水平较空载病毒组明显增加(P0.05)；空载病毒组Kv1.5以及Kv2.1 mRNA表达水平与空白对照组无显著差异(P0.05)。比较蛋白表达,空载病毒组survivin蛋白表达水平与空白对照组比较无显著性差异(P0.05),慢病毒干扰组survivin表达较空载病毒组显著下降(P0.05)。慢病毒干扰组Kv1.5以及Kv2.1蛋白表达水平较空载病毒组明显增加(P0.05)；空载病毒组的Kv1.5以及Kv2.1蛋白表达水平较空白对照组无显著差异(P0.05)。通过CCK8检测肺动脉平滑肌细胞增殖活性,空载病毒组的肺动脉平滑肌细胞增殖活性与空白对照组比较无显著差异(P0.05),慢病毒干扰组肺动脉平滑肌细胞增殖活性较空载病毒组显著下降(P0.05)。通过流式细胞术检测肺动脉平滑肌细胞凋亡,空载病毒组肺动脉平滑肌细胞凋亡与空白对照组比较无显著差异(P0.05)；而慢病毒干扰组较空载病毒组细胞凋亡显著上升,与空载病毒组比较有显著性差异(P0.05)。所培养的肺动脉平滑肌细胞上清液的caspase-3以及caspase-9浓度,慢病毒干扰组caspase-3和caspase-9浓度较空载病毒组明显上升(P0.05)；空载病毒组的caspase-3和caspase-9浓度与空白对照组比较,无显著差异(P0.05)。结论： (1)成功将构建siRNA-survivin慢病毒载体并对高肺血流性肺动脉高压大鼠肺动脉平滑肌细胞进行体外干扰,能下调survivin表达,起到抑制肺动脉平滑肌细胞增殖、诱导凋亡的作用。(2)Survivin通过抑制电压门控性钾通道Kv1.5和Kv2.1表达,激活凋亡蛋白家族caspase-3及caspase-9信号通路,促进高肺血流性肺动脉高压大鼠肺动脉平滑肌细胞增殖、抑制凋亡,参与高肺血流性肺动脉高压的发病机制。第三部分Survivin抑制剂Y2M155体外对高肺血流性肺动脉高压大鼠肺动脉平滑肌细胞增殖与凋亡调控作用背景：前两个部分已证实survivin调控高肺血流性肺动脉高压的肺动脉平滑肌细胞的增殖与凋亡。YM155是一个新型的小分子survivin抑制剂,抑制survivin启动子的活性。YM155对高肺血流性肺动脉高压的肺动脉平滑肌细胞抑制作用有待进一步研究。目的：研究YM155在体外对高肺血流性肺动脉高压肺动脉平滑肌细胞增殖与凋亡的作用。方法：利用第一部分原代培养并经鉴定的高肺血流性肺动脉高压的肺动脉平滑肌细胞作为研究对象,将其分为对照组、药物组。药物组肺动脉平滑肌细胞给予不同浓度的YM155进行干预,用细胞增殖实验(CCK8试剂盒法)检测肺动脉平滑肌细胞增殖情况；细胞凋亡实验(流式细胞学)检测肺动脉平滑肌细胞凋亡情况。结果：药物组设计多个YM155浓度梯度对肺动脉平滑肌细胞进行干预,药物组较对照组细胞增殖活性显著下降,呈浓度依赖性关系。通过流式细胞术检测肺动脉平滑肌细胞凋亡,药物组细胞凋亡较对照组显著增加(P0.05)有显著统计学意义(P0.05)。结论：体外实验证实YM155能抑制高肺血流性肺动脉高压大鼠肺动脉平滑肌细胞增殖并促进其凋亡。
[Abstract]:The expression and significance of Survivin in pulmonary artery smooth muscle cells in high pulmonary blood flow pulmonary hypertension rats: the most important pathological changes in high pulmonary blood flow pulmonary hypertension caused by congenital left to right shunt heart disease are pulmonary artery smooth muscle cell proliferation, pulmonary vascular remodeling, the exact cell and molecular mechanism is not clear.Surviv In is an important inhibitor of apoptosis protein, which inhibits apoptosis, prolongs the expression of.Survivin in pulmonary artery smooth muscle cells with high pulmonary blood flow and pulmonary artery smooth muscle cells, and regulates the proliferation and apoptosis of pulmonary artery smooth muscle cells in high pulmonary blood flow. Objective: to confirm that survivin is in the pulmonary artery of high pulmonary blood flow pulmonary hypertension rats. Expression of smooth muscle cells to explore the possible regulatory effect of survivin on pulmonary artery smooth muscle cells. Methods: adult SD rats were divided into control group, sham operation group and shunting group according to random number method. The control group was not treated with any treatment. The sham operation group had closed the abdominal aorta for ten minutes. The shunt group was established by operation method. The shunt fistula of the abdominal aorta to the inferior vena cava was established for the establishment of a high pulmonary blood flow pulmonary hypertension rat model for.11 weeks. The success of the modeling was confirmed by the shunt fistula B ultrasound examination, the determination of the right ventricular hypertrophy index, the pulmonary artery pressure, and the HE staining in the lung tissue section. The expression of Survivin in the lung tissue was detected by immunohistochemistry. The primary pulmonary artery smooth muscle cells were cultured in each group. The expression level of Survivin mRNA in pulmonary artery smooth muscle cells was detected by qRT-PCR; Western blot was used to detect the expression of suvivin protein; cell proliferation test (CCK8 Kit) was used to detect the proliferation of pulmonary vein smooth muscle cells; apoptosis experiment (flow) The apoptosis of pulmonary artery smooth muscle cells was detected by cytology. Results: through the abdominal aorta and inferior vena cava shunt fistula B ultrasonic examination, the right ventricular hypertrophy index, the pulmonary artery pressure and the lung tissue were stained with HE, which confirmed the successful establishment of high pulmonary blood flow pulmonary hypertension rat model. Immunohistochemistry showed survivin Expression of the pulmonary artery smooth muscle in high pulmonary blood flow pulmonary hypertension. Through qRT-PCR and Western blot analysis, the positive expression of Survivin mRNA and protein in the pulmonary artery smooth muscle cells in the shunt group was positive, and there was no survivin mRNA and protein expression in the control group and the sham operation group. The lungs were detected by the cell proliferation test (CCK8 Kit). The proliferation of arterial smooth muscle cells and the proliferation of pulmonary artery smooth muscle cells in the shunt group were significantly increased (P0.05), but there was no significant difference between the control group and the sham operation group (P0.05). The apoptosis of pulmonary artery smooth muscle cells in the shunt group was significantly lower than that in the sham operation group (P0.0 5) but there was no significant difference between the control group and the sham operation group (P0.05). Conclusion: (1) the expression of Survivin in the pulmonary artery smooth muscle cells of the high pulmonary blood flow pulmonary hypertension rats was not found. (2) survivin may be involved in the high pulmonary blood flow by regulating the proliferation and apoptosis of pulmonary artery smooth muscle cells. The pathogenesis of pulmonary hypertension. The second part Survivin regulates the proliferation and apoptosis of pulmonary artery smooth muscle cells in high pulmonary blood flow pulmonary hypertension rats and possible regulatory pathway background: according to the first conclusion, it is proved that the proliferation of pulmonary artery smooth muscle cells in the pulmonary hypertension rats with high pulmonary blood flow is increased and apoptosis is suppressed; and Sur is confirmed. The expression of vivin in pulmonary artery smooth muscle cells in high pulmonary blood flow pulmonary hypertension rats, and no survivin expression in the pulmonary artery smooth muscle cells in the control group or the sham operation group. Therefore, it is suggested that survivin may be involved in the regulation of proliferation and apoptosis of pulmonary artery smooth muscle cells in the high pulmonary blood flow pulmonary hypertension rats. Objective: to construct Si by constructing the pulmonary artery smooth muscle cells in the high pulmonary blood flow pulmonary hypertension rats. RNA-survivin lentivirus carrier, infection of pulmonary artery smooth muscle cells in shunt group, interfering survivin expression, and exploring whether survivin regulates the proliferation and apoptosis of pulmonary artery smooth muscle cells in high pulmonary blood flow pulmonary hypertension rats, and possible regulatory pathway. Method: the first part has been proved to be high pulmonary blood flow pulmonary hypertension The primary cultured pulmonary artery smooth muscle cells were divided into blank control group (no treatment), empty virus group (negative control lentivirus infection), lentivirus interference group (siRNA-survivin lentivirus carrier infection). After successful infection, qRT-PCR method was used to detect survivinN electricity of pulmonary artery smooth muscle cells in each group. The expression level of pressure gated potassium channel Kvl.5 and Kv2.1 mRNA; Western blot method to detect the expression level of suvivin, Kv1.5 and Kv2.1 protein; cell proliferation test (CCK8 Kit Method) detection of proliferation of pulmonary artery smooth muscle cells; apoptosis test (flow cytology) detection of pulmonary artery smooth muscle cell apoptosis by.ELISA assay The cultured pulmonary artery smooth muscle cell supernatant caspase-3 and caspase-9 concentration. Results: the pulmonary artery smooth muscle cells of the high pulmonary blood flow pulmonary hypertension rats were successfully cultured, and the siRNA-survivin lentivirus vector with high infection efficiency was successfully constructed and screened, and the pulmonary artery smooth muscle cells were successfully infected with the primary cultured pulmonary artery smooth muscle. Compared with mRNA, the expression of Survivin mRNA in the lentivirus group was significantly lower than that in the empty virus group (P0.05). There was no significant difference between the empty virus group and the blank control group (P0.05). The expression level of Kv1.5 and Kv2.1mRNA in the lentivirus group was significantly higher than that of the empty virus group (P0.05), and the Kv1.5 and Kv2.1 mRNA expression in the unloaded virus group was expressed. There was no significant difference between the level and the blank control group (P0.05). There was no significant difference in the expression of survivin protein in the empty virus group compared with the blank control group (P0.05). The expression of Survivin in the lentivirus interference group was significantly lower than that in the empty virus group (P0.05). The expression level of Kv1.5 and Kv2.1 protein in the lentivirus dry disturbance group was more than that of the empty virus group. The expression of Kv1.5 and Kv2.1 protein in the unloaded virus group had no significant difference compared with that of the blank control group (P0.05). The proliferation activity of pulmonary artery smooth muscle cells in the pulmonary artery was detected by CCK8. The proliferation activity of pulmonary artery smooth muscle cells in the unloaded virus group was not significantly different from that of the blank control group (P0.05), and the pulmonary artery of the lentivirus interference group was flat. The proliferation activity of smooth muscle cells was significantly lower than that in the empty virus group (P0.05). The apoptosis of pulmonary artery smooth muscle cells was detected by flow cytometry. The apoptosis of pulmonary artery smooth muscle cells in the empty virus group was not significantly different from that of the blank control group (P0.05), while the apoptosis of the lentivirus group was significantly higher than that of the empty virus group, and the ratio of the no-virus group to the empty virus group was significantly higher than that of the empty virus group. There was a significant difference (P0.05). The concentration of Caspase-3 and caspase-9 in the supernatant of the cultured pulmonary artery smooth muscle cells, the concentration of Caspase-3 and caspase-9 in the lentivirus group was significantly higher than that of the empty virus group (P0.05), and the concentration of Caspase-3 and caspase-9 in the unloaded virus group was not significantly different from that of the empty white control group (P0.05). (1) Successful construction of siRNA-survivin lentivirus vector and in vitro interference with pulmonary arterial smooth muscle cells of high pulmonary blood flow pulmonary hypertension rats, can reduce the expression of survivin, inhibit the proliferation of pulmonary artery smooth muscle cells and induce apoptosis. (2) Survivin activates apoptosis by inhibiting the expression of Kv1.5 and Kv2.1 of electrical pressure gated potassium channels. Protein family caspase-3 and caspase-9 signaling pathway, promote the proliferation of pulmonary artery smooth muscle cells in high pulmonary blood flow pulmonary hypertension rats, inhibit apoptosis and participate in the pathogenesis of high pulmonary blood flow pulmonary hypertension. The third part of Survivin inhibitor Y2M155 in vitro proliferation and proliferation of pulmonary artery smooth muscle cells in high pulmonary blood flow pulmonary vein hypertension rats in vitro Background of apoptosis regulation: the first two parts have confirmed that survivin regulates the proliferation and apoptosis of pulmonary artery smooth muscle cells in high pulmonary blood flow hypertension..YM155 is a new small molecule survivin inhibitor. Inhibition of survivin promoter activity.YM155 inhibits pulmonary artery smooth muscle cells with high pulmonary hemodynamic pulse high pressure The purpose of this study is to study the effect of YM155 on the proliferation and apoptosis of pulmonary arterial smooth muscle cells of high pulmonary blood flow in vitro. Methods: the pulmonary artery smooth muscle cells in the first part of the primary culture and identified high pulmonary blood flow pulmonary arterial hypertension were used as the control group, and the drug group was divided into the control group and the drug group. The pulmonary artery smooth muscle cells in the drug group were given different concentrations of YM155, and the proliferation of pulmonary artery smooth muscle cells was detected by the cell proliferation test (CCK8 Kit). Apoptosis test (flow cytology) was used to detect the apoptosis of pulmonary artery smooth muscle cells. Results: the drug group designed multiple YM155 concentration gradient to smooth the pulmonary artery. The proliferation activity of the drug group was significantly lower than that of the control group. The apoptosis of pulmonary artery smooth muscle cells was detected by flow cytometry. The apoptosis in the drug group was significantly higher than that of the control group (P0.05). (P0.05). Conclusion: real YM155 can inhibit the high pulmonary blood flow in vitro. Pulmonary artery smooth muscle cells proliferate and promote apoptosis in rats with pulmonary hypertension.

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R725.4

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