本文選題：肺動脈高壓 + 肺動脈平滑肌細胞��； 參考：《廣西醫(yī)科大學》2016年博士論文

【摘要】：第一部分Survivin在高肺血流性肺動脈高壓大鼠肺動脈平滑肌細胞表達及意義背景：先天性左向右分流性心臟病致高肺血流性肺動脈高壓最重要的病理變化是肺動脈平滑肌細胞增殖,肺血管重構,確切的細胞及分子機制尚不明確。Survivin是重要的凋亡蛋白抑制劑,抑制細胞凋亡,延長細胞存活。Survivin是否在高肺血流肺動脈高壓肺動脈平滑肌細胞表達以及對肺動脈平滑肌細胞增殖和凋亡的調(diào)控有待進一步研究。目的：證實survivin在高肺血流性肺動脈高壓大鼠肺動脈平滑肌細胞上表達,探討survivin對肺動脈平滑肌細胞可能的調(diào)控作用。方法：將成年SD大鼠,雌雄各半,按隨機數(shù)字方法分為對照組、假手術組和分流組。對照組大鼠未作任何處理。假手術組夾閉腹主動脈十分鐘。分流組通過手術方法建立腹主動脈到下腔靜脈的分流瘺道,以建立高肺血流性肺動脈高壓大鼠模型。11周后,通過對分流瘺道B超檢查、右心室肥厚指數(shù)測定、肺動脈壓力測定、肺組織切片HE染色等確認建模是否成功。免疫組織化學法檢測survivin在肺組織的表達及分布。取各組肺動脈平滑肌組織進行原代肺動脈平滑肌細胞培養(yǎng)；用qRT-PCR法檢測肺動脈平滑肌細胞survivin mRNA表達水平；western blot檢測suvivin蛋白的表達水平；細胞增殖實驗(CCK8試劑盒法)檢測肺動脈平滑肌細胞增殖；細胞凋亡實驗(流式細胞學)檢測肺動脈平滑肌細胞凋亡。結果：通過分流組大鼠腹主動脈-下腔靜脈分流瘺道B超檢查、右心室肥厚指數(shù)、肺動脈壓力測定、肺組織進行病理切片HE染色,證實已成功建立高肺血流性肺動脈高壓大鼠模型。免疫組化顯示survivin在高肺血流性肺動脈高壓的肺動脈平滑肌上表達。通過qRT-PCR及western blot分析,survivin mRNA及蛋白在分流組肺動脈平滑肌細胞呈現(xiàn)陽性表達,未發(fā)現(xiàn)對照組與假手術組有survivin mRNA及蛋白表達。通過細胞增殖實驗(CCK8試劑盒法)檢測各組肺動脈平滑肌細胞增殖,分流組肺動脈平滑肌細胞增殖顯著增加,較假手術組有顯著差異(P0.05),而對照組與假手術組比較無顯著差異(P0.05)。通過細胞凋亡實驗(流式細胞學)檢測分析,分流組肺動脈平滑肌細胞凋亡較假手術組顯著下降(P0.05),而對照組與假手術組比較無顯著差異(P0.05)。結論：(1)Survivin在高肺血流性肺動脈高壓大鼠肺動脈平滑肌細胞表達,正常大鼠肺動脈平滑肌細胞未發(fā)現(xiàn)survivin表達。 (2)survivin可能通過調(diào)控肺動脈平滑肌細胞增殖與凋亡參與高肺血流性肺動脈高壓的發(fā)病機制。第二部分Survivin調(diào)控高肺血流性肺動脈高壓大鼠肺動脈平滑肌細胞增殖與凋亡及可能的調(diào)控通路背景：根據(jù)第一部分結論,證實高肺血流性肺動脈高壓大鼠的肺動脈平滑肌細胞增殖增加,凋亡受抑；并證實survivin在高肺血流性肺動脈高壓大鼠的肺動脈平滑肌細胞表達,而對照組或假手術組大鼠的肺動脈平滑肌細胞無survivin的表達。由此推測survivin可能參與高肺血流性肺動脈高壓大鼠肺動脈平滑肌細胞增殖與凋亡的調(diào)控。目的：通過構建siRNA-survivin慢病毒載體,對分流組肺動脈平滑肌細胞進行感染,干擾survivin表達,探索survivin是否調(diào)控高肺血流性肺動脈高壓大鼠肺動脈平滑肌細胞增殖與凋亡,以及可能的調(diào)控通路。方法：利用第一部分已證實為高肺血流性肺動脈高壓的分流組大鼠原代培養(yǎng)的肺動脈平滑肌細胞為研究對象,將其分為空白對照組(不作處理),空載病毒組(用陰性對照慢病毒感染)、慢病毒干擾組(siRNA-survivin慢病毒載體感染)。感染成功后,通過qRT-PCR法檢測各組肺動脈平滑肌細胞survivinN電壓門控鉀通道Kvl.5以及Kv2.1 mRNA的表達水平；Western blot法檢測suvivin、Kv1.5以及Kv2.1蛋白的表達水平；細胞增殖實驗(CCK8試劑盒法)檢測肺動脈平滑肌細胞增殖情況；細胞凋亡實驗(流式細胞學)檢測肺動脈平滑肌細胞凋亡情況。ELISA法檢測所培養(yǎng)的肺動脈平滑肌細胞上清液caspase-3以及caspase-9濃度。結果：成功培養(yǎng)了研究所需的高肺血流性肺動脈高壓大鼠的肺動脈平滑肌細胞,成功構建并篩選出感染效率高的siRNA-survivin慢病毒載體,成功感染原代培養(yǎng)的肺動脈平滑肌細胞。比較mRNA表達,慢病毒干擾組survivin mRNA表達水平較空載病毒組顯著下降(P0.05)�？蛰d病毒組與空白對照組比較無顯著性差異(P0.05)。慢病毒干擾組Kv1.5以及Kv2.1mRNA表達水平較空載病毒組明顯增加(P0.05)；空載病毒組Kv1.5以及Kv2.1 mRNA表達水平與空白對照組無顯著差異(P0.05)。比較蛋白表達,空載病毒組survivin蛋白表達水平與空白對照組比較無顯著性差異(P0.05),慢病毒干擾組survivin表達較空載病毒組顯著下降(P0.05)。慢病毒干擾組Kv1.5以及Kv2.1蛋白表達水平較空載病毒組明顯增加(P0.05)；空載病毒組的Kv1.5以及Kv2.1蛋白表達水平較空白對照組無顯著差異(P0.05)。通過CCK8檢測肺動脈平滑肌細胞增殖活性,空載病毒組的肺動脈平滑肌細胞增殖活性與空白對照組比較無顯著差異(P0.05),慢病毒干擾組肺動脈平滑肌細胞增殖活性較空載病毒組顯著下降(P0.05)。通過流式細胞術檢測肺動脈平滑肌細胞凋亡,空載病毒組肺動脈平滑肌細胞凋亡與空白對照組比較無顯著差異(P0.05)；而慢病毒干擾組較空載病毒組細胞凋亡顯著上升,與空載病毒組比較有顯著性差異(P0.05)。所培養(yǎng)的肺動脈平滑肌細胞上清液的caspase-3以及caspase-9濃度,慢病毒干擾組caspase-3和caspase-9濃度較空載病毒組明顯上升(P0.05)；空載病毒組的caspase-3和caspase-9濃度與空白對照組比較,無顯著差異(P0.05)。結論： (1)成功將構建siRNA-survivin慢病毒載體并對高肺血流性肺動脈高壓大鼠肺動脈平滑肌細胞進行體外干擾,能下調(diào)survivin表達,起到抑制肺動脈平滑肌細胞增殖、誘導凋亡的作用。(2)Survivin通過抑制電壓門控性鉀通道Kv1.5和Kv2.1表達,激活凋亡蛋白家族caspase-3及caspase-9信號通路,促進高肺血流性肺動脈高壓大鼠肺動脈平滑肌細胞增殖、抑制凋亡,參與高肺血流性肺動脈高壓的發(fā)病機制。第三部分Survivin抑制劑Y2M155體外對高肺血流性肺動脈高壓大鼠肺動脈平滑肌細胞增殖與凋亡調(diào)控作用背景：前兩個部分已證實survivin調(diào)控高肺血流性肺動脈高壓的肺動脈平滑肌細胞的增殖與凋亡。YM155是一個新型的小分子survivin抑制劑,抑制survivin啟動子的活性。YM155對高肺血流性肺動脈高壓的肺動脈平滑肌細胞抑制作用有待進一步研究。目的：研究YM155在體外對高肺血流性肺動脈高壓肺動脈平滑肌細胞增殖與凋亡的作用。方法：利用第一部分原代培養(yǎng)并經(jīng)鑒定的高肺血流性肺動脈高壓的肺動脈平滑肌細胞作為研究對象,將其分為對照組、藥物組。藥物組肺動脈平滑肌細胞給予不同濃度的YM155進行干預,用細胞增殖實驗(CCK8試劑盒法)檢測肺動脈平滑肌細胞增殖情況；細胞凋亡實驗(流式細胞學)檢測肺動脈平滑肌細胞凋亡情況。結果：藥物組設計多個YM155濃度梯度對肺動脈平滑肌細胞進行干預,藥物組較對照組細胞增殖活性顯著下降,呈濃度依賴性關系。通過流式細胞術檢測肺動脈平滑肌細胞凋亡,藥物組細胞凋亡較對照組顯著增加(P0.05)有顯著統(tǒng)計學意義(P0.05)。結論：體外實驗證實YM155能抑制高肺血流性肺動脈高壓大鼠肺動脈平滑肌細胞增殖并促進其凋亡。
[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.

【學位授予單位】：廣西醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R725.4

【參考文獻】

相關期刊論文 前10條

1 徐剛;高文祥;陳德偉;李曉栩;劉福玉;黃tJ;高鈺琪;;小鼠PE右心室導管的制作及應用研究[J];重慶醫(yī)學;2014年19期

2 鄒麗珍;陳馬云;黃曉穎;王良興;;改良右心導管法測量大鼠肺動脈壓力的實驗方法研究[J];中國病理生理雜志;2014年04期

3 Ying Yang;Feng Sun;Chen Zhang;Hao Wang;Guoyao Wu;Zhenlong Wu;;Hypoxia promotes cell proliferation by modulating E2F1 in chicken pulmonary arterial smooth muscle cells[J];Journal of Animal Science and Biotechnology;2013年03期

4 陳果;何建國;柳志紅;顧晴;倪新海;熊長明;;不同類型肺動脈高壓患者臨床特征和血流動力學的比較分析[J];中國循環(huán)雜志;2013年04期

5 姚健;侯明曉;尚長青;趙科研;徐林;;Tocilizumab對肺動脈高壓大鼠IL-6及survivin的影響[J];中國實驗診斷學;2013年05期

6 李廣;戴愛國;;大鼠肺動脈平滑肌細胞原代培養(yǎng)及低氧對缺氧誘導因子-1α的影響[J];腫瘤藥學;2011年02期

7 楊杰章;黃石安;陳燦;李波;高漢華;龐玲品;;大鼠經(jīng)頸外靜脈導絲引導插管測肺動脈壓與傳統(tǒng)方法的比較[J];中國比較醫(yī)學雜志;2010年09期

8 曾曉春;伍偉鋒;黃凱;;先天性心臟病肺動脈高壓與外周血存活素的關系[J];實用醫(yī)學雜志;2010年17期

9 錢國清;王良興;陳嬋;黃曉穎;葉舒婷;;大鼠細小肺動脈平滑肌細胞原代培養(yǎng)和鑒定方法的研究[J];中國應用生理學雜志;2010年01期

10 唐旭東;肖穎彬;周建國;郝嘉;;Survivin在先天性心臟病肺動脈高壓發(fā)生機制中的作用[J];第三軍醫(yī)大學學報;2008年01期

，

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