The Regulation And Mechanism Of Survivin To Proliferation And Apoptosis Of Pulmonary Arterial Smooth Muscle Cell In Pulmonary Arterial Hypertension Induced By High Pulmonary Blood Flow In Rats

CHAPTER I The expression and significance of survivin in pulmonary arterial smooth muscle cell of pulmonary arterial hypertension induced by high pulmonary blood flow in ratsBackground Proliferation of pulmonary arterial smooth muscle cells (PASMC) and the remodeling of pulmonary vessels were the key pathological changes of pulmonary arterial hypertension (PAH) induced by high pulmonary blood flow of congenital left to right shunt heart diseases. However, the exact molecular mechanism remained unclear. Survivin was one of the most important of apoptosis protein inhibitor. The expression of survivin in PAH-PASMC induced by high pulmonary blood flow and its regulation to the proliferation and apoptosis of PASMC need to furtherly research.Objectives The aim of this study was to determine the expression of survivin in PAH-PASMC induced by high pulmonary blood flow and explored the possible regulation effect of survivin to PASMC.Methods Adult Sprague-Dawley (SD) rats for half of male and female were used in this study. These SD rats were divided into Control group, Sham group and Shunt group. The rats in the Control group were without any treatment, the rats in the Sham group received blocking-up for blood flow in abdominal aorta for ten minutes, and the rats in the Shunt group were created the shunt fistula from abdominal aorta to inferior vena cava so as to create the PAH model induced by high pulmonary blood flow. After 11 weeks from creation of the shunt fistula, ultrasonography parameters, right ventricular hypertrophy index (RVHI), pulmonary arterial pressure and lung biopsy HE staining were used to verify the success of the PAH model. The expression and distribution of survivin in lung tissues were measured by immunohistochemistry. The PASMC were primary cultured in each group. The mRNA and protein expression of survivin were measured by qRT-PCR and western blot, respectively. The proliferation of PASMC in each group was measured by cell proliferative assay (CCK8) and the apoptosis of PASMC was measured by cell apoptotic assay (flow cytometry)Results The PAH rat model induced by high pulmonary blood flow was verified as successful establishment. Survivin was high expression in the pulmonary arterial smooth muscle layer of PAH rats. The mRNA and protein expression of survivin were found in the PASMC of Shunt group while none expression of survivin in the PASMC of Control and Sham group by qRT-PCR and western blot analysis, respectively. The proliferation of PASMC significantly increased in the Shunt group compared with the Sham group, respectively. There was no difference of PASMC proliferation between the Control and Sham group. The apoptosis of PASMC significantly decreased in the Shunt group compared with the Sham group. There was no difference for the apoptosis of PASMC between the Control and Sham group.Conclusion (1) Survivin expression was found in the PAH-PASMC of rats induced by high pulmonary blood flow, while was not found in the PASMC of normal rats. (2) Survivin may be involved in the pathogenesis of PAH induced by high pulmonary blood flow through the regulation of proliferation and apoptosis to PAH-PASMC.CHAPTER II The regulation and its possible mechanism of survivin to proliferation and apoptosis of PASMC in pulmonary arterial hypertension induced by high pulmonary blood flow in ratsBackground The proliferation increased and apoptosis inhibited of PAH-PASMC were verified, and the survivin expression in PAH-PASMC were verified while none expression in PASMC of the Control group and Sham group according to the conclusion of chapter I. We speculated survivin might participate in the regulation of proliferation and apoptosis to PAH-PASMC.Objective Through the construction of siRNA-survivin lentivirus vector, the PAH-PASMC were infected by lentivirus vector so as to down-regulation of survivin expression, the aim of this chapter was to explore the effect of survivin involved in the regulation of the proliferation and apoptosis, and the possible regulation mechanism of survivin to PASMC in PAH induced by high pulmonary blood flow.Methods The primary cultural PAH-PASMC in the chapter I were used in this study. The PAH-PASMCs were divided into Blank Control group, Lentivirus Negative Control group and RNA Interference group (received siRNA-survivin lentivirus vector infection). After successful infection to PAH-PASMC through lentivirus vector, survivin, Kvl.5, Kv2.1 mRNA and protein expression were measured in the PAH-PASMC of each group by qRT-PCR and western blot. The proliferation of PAH-PASMC in each group was measured by cell proliferative assay (CCK8) and the apoptosis of PAH-PASMC was measured by cell apoptotic assay (flow cytometry). The concentrations of caspase-3 and caspase-9 in PAH-PASMC supernate were measured by ELISA.Results Primary PAH-PASMCs were cultured, siRNA-survivin lentivirus vector was constructed with high transfection efficiency and transfered into PAH-PASMC successfully. The mRNA and protein expression of survivin obviously decreased in the RNA interference group compared with the Lentivirus Negative Congrol group, respectively. There was no significant difference for survivin mRNA and protein expression between the Blank Control group and the Lentivirus Negative Congrol group by qRT-PCR and western blot analysis. The mRNA and protein expression of Kv1.5 and Kv2.1 significantly increased in the RNA Interference group compared with the Lentivirus Negative Control group, while no significant difference for the mRNA and protein expression of Kv1.5 and Kv2.1 was found between the Blank Control group and the Lentivirus Negative Control group. The proliferation of PAH-PASMC significantly decreased in the RNA Interference group compared with the Lentivirus Negative Control group. No significant difference for the proliferation of PASMC was found between the Lentivirus Negative Control group and the Blank Control group. The apoptosis of PAH-PASMC significantly increased in the RNA Interference group compared with the Lentivirus Negative Control group. No significant difference for the apoptosis of PAH-PASMC was found between the Lentivirus Negative Control group and the Blank Control group. The concentrations of caspase-3 and caspase-9 in PAH-PASMC supernate increased in the RNA Interference group compared with the Lentivirus Negative Control group, while no significant difference was found between the Blank Control group and the Lentivirus Negative Control group.Conclusions (1) The siRNA-survivin lentivirus vector was constructed and PAH-PASMC were infected by the siRNA-survivin lentivirus vector successfully, which inhibited the expression of survivin, and furtherly inhibited the proliferation and induced the apoptosis of PASMC. (2) Survivin inhibited the expression of Kv1.5 and Kv2.1 and activated the apoptotic protein signal pathway of caspase-3 and caspase-9, which might be involved in the pathogenesis of pulmonary arterial hypertension induced by high pulmonary blood flow through promotion of proliferation and inhibition of apoptosis to PAH-PASMC.CHAPTER Ⅲ Regulation effect of YM155 as survivin inhibitor to proliferation and apoptosis of pulmonary arterial smooth muscle cell in vitro in pulmonary arterial hypertension induced by high pulmonary blood flow in ratsBackground The regulation of survivin to proliferation and apoptosis of PAH-PASMC were verified according to the conclusion of chapter I-II YM155 was a novel small molecular inhibitor of survivin, which inhibited the activity of survivin promotor. The inhibition effect of YM155 to PAH-PASMC need to furtherly research.Objective To investigate the effect of YM155 (inhibitor of survivin) to proliferation and apoptosis of PAH-PASMC in vitro.Methods The primary cultural PAH-PASMC in the chapter I were used in this study. The PAH-PASMC were divided into Control group and Treatment group. The PAH-PASMC of the Treatment group would receive the interference of YM155 in different concentration. The proliferation of PAH-PASMC was measured by cell proliferative assay (CCK8), and the apoptosis of PAH-PASMC was measured by cell apoptotic assay (flow cytometry).Results Multi-concentration gradient of YM155 in the Treatment group was used to interference for PAH-PASMC. The proliferation of PAH-PASMC significantly decreased in the Treatment group compared with the Control group, which was as the concentration dependence relation. The apoptosis of PAH-PASMC significantly increased in the Treatment group compared with the Control group.Conclusion YM155 promoted the apoptosis and inhibit the proliferation of PAH in vitro in rats induced by high pulmonary blood flow.