缓激肽B2受体信号通路抑制氧化应激诱导的内皮祖细胞和心脏干细胞衰老的机制研究
发布时间：2019-01-17 19:54
【摘要】：第一部分 缓激肽抑制过氧化氢诱导的内皮祖细胞的衰老目的：探讨缓激肽抑制过氧化氢诱导的内皮祖细胞衰老的具体分子机制。方去：检测冠心病患者外周循环血中CD34+细胞表面B2受体的表达。ELISA法检测冠心病患者血浆髓过氧化物酶浓度。使用3000M过氧化氢处理人脐静脉血来源的内皮祖细胞,并使用不同浓度的缓激肽干预细胞,p半乳糖苷酶染色检测细胞衰老程度,DCFH-DA荧光探针检测细胞内氧自由基含量。衰老相关PCR芯片以及荧光定量PCR检测并验证相关信号通路分子的表达。分别使用B2受体小干扰RNA, B2受体拮抗剂,PI3K拮抗剂LY294002, EGFR拮抗剂AG1478阻断B2受体以及下游信号通路分子,检测信号通路阻断剂对BK保护作用的影响。使用流式细胞技术和荧光定量PCR检测过氧化氢干预前后B2受体的表达。Western blot技术检测各信号通路分子的表达。结果：冠心病患者外周循环血中CD34+细胞表面B2受体表达较健康对照显著下降,血浆髓过氧化物酶浓度显著升高,两者具有显著的相关性。β半乳糖苷酶染色和DCFH-DA荧光探针显示缓激肽可以抑制过氧化氢诱导的人脐静脉血来源的内皮祖细胞衰老并减少细胞内氧自由基形成。衰老相关PCR芯片和荧光定量PCR结果显示过氧化氢能够显著上调RB基因的表达,下调B2受体表达,而缓激肽可以显著下调RB基因的表达。Western blot结果显示缓激肽能够上调过氧化氢诱导下磷酸化RB,磷酸化AKT和cyclin D的表达。信号通路拮抗剂和小干扰RNA阻断缓激肽发挥抗衰老的保护作用。结论:缓激肽通过B2受体介导的P13K和EGFR信号通路抑制过氧化氢诱导的人内皮祖细胞的衰老。第二部分缓激肽抑制高糖诱导的心脏干细胞的衰老目的：探讨缓激肽抑制高糖诱导的心脏干细胞衰老的具体分子机制。方法：体外分离培养C57BL/6J小鼠c-kit阳性心脏干细胞。使用25mM D-葡萄糖处理心脏干细胞,并使用不同浓度的缓激肽干预细胞,p半乳糖苷酶染色检测细胞衰老程度,DCFH-DA荧光探针检测细胞内氧自由基含量。分别使用B2受体小干扰RNA,B2受体拮抗剂,P13K拮抗剂]LY294002,mTOR拮抗剂Rapamycin和P53拮抗剂PFT-a阻断B2受体以及下游信号通路分子,检测信号通路阻断剂对BK保护作用的影响,同时检测细胞内超氧化物浓度和ATP浓度,并使用流式细胞技术检测D-葡萄糖干预前后B2受体的表达。、Western blot技术检测各信号通路分子的表达。结果：β半乳糖苷酶染色和DCFH-DA荧光探针显示缓激肽可以抑制D-葡萄糖诱导的小鼠心脏干细胞衰老并减少细胞内氧自由基的形成,并能降低细胞内超氧化物浓度,促进ATP生成。D-葡萄糖显著下调B2受体表达。Western blot结果显示缓激肽能够上调高糖环境下磷酸化AKT,磷酸化nTOR的表达,下调P53和P16的表达。B2受体拮抗剂,P13K拮抗剂,mTOR拮抗剂和小干扰RNA阻断缓激肽发挥抗衰老的保护作用。P53拮抗剂同样抑制D-葡萄糖诱导的心脏干细胞衰老。结论：缓激肽通过B2R介导的PI3K/AKT/mTOR/P53信号通路抑制D-葡萄糖诱导的心脏干细胞的衰老。
[Abstract]:The first part of bradykinin inhibits the aging of endothelial progenitor cells induced by hydrogen peroxide: a specific molecular mechanism for the inhibition of the senescence of endothelial progenitor cells induced by bradykinin. Method: To detect the expression of CD34 + cell surface B2 receptor in peripheral circulating blood of patients with coronary heart disease. Enzyme-linked immunosorbent assay (ELISA) for detecting the plasma myeloperoxidase concentration in patients with coronary heart disease. Endothelial progenitor cells from human umbilical venous blood were treated with 3000M hydrogen peroxide, and different concentrations of bradykinin were used to detect the degree of cell senescence, and the content of oxygen free radicals in the cells was detected by the DCFH-DA fluorescence probe. The aging-related PCR chip and the fluorescence quantitative PCR detect and verify the expression of the related signal path molecules. The effect of the signal pathway blocking agent on BK protection was detected by using the B2 receptor small interfering RNA, the B2 receptor antagonist, the PI3K antagonist LY294002, the EGFR antagonist AG1478 to block the B2 receptor and the downstream signal pathway molecule, respectively. The expression of B2 receptor before and after the intervention of hydrogen peroxide was detected by flow cytometry and fluorescence quantitative PCR. Western blot was used to detect the expression of signal pathway. Results: The expression of B2 receptor in peripheral circulating blood of patients with coronary heart disease was significantly lower than that in healthy control group, and the concentration of plasma myeloperoxidase increased significantly. Dekallikrein and DCFH-DA fluorescence probe showed that bradykinin could inhibit the aging of endothelial progenitor cells from the human umbilical venous blood source induced by hydrogen peroxide and reduce the formation of oxygen free radicals in the cells. The results of aging-related PCR and quantitative PCR show that hydrogen peroxide can significantly increase the expression of RB gene and down-regulate the expression of B2 receptor, while bradykinin can significantly lower the expression of RB gene. Western blot showed that bradykinin could increase the expression of phosphorylated RB, phosphorylated AKT and cyclin D under the induction of hydrogen peroxide. Signal pathway antagonists and small interfering RNA blocking bradykinin play an anti-aging protective role. Conclusion: Bradykinin inhibits the senescence of human endothelial progenitor cells induced by hydrogen peroxide via a B2 receptor-mediated P13K and EGFR signaling pathway. The second part of bradykinin inhibits the aging of high-sugar-induced cardiac stem cells: a specific molecular mechanism for bradykinin inhibition of high-sugar-induced cardiac stem cell senescence. Methods: c-kit positive cardiac stem cells were isolated and cultured in vitro. The cardiac stem cells were treated with 25mM D-glucose and the cell senescence was detected using a different concentration of bradykinin-treated cells, p-half-lactase staining, and the DCFH-DA fluorescence probe was used to detect the oxygen free-radical content in the cells. The effect of the signal pathway blocking agent on BK protection was detected by using the B2 receptor small interfering RNA, the B2 receptor antagonist, the P13K antagonist, the LY294002, the mTOR antagonist Rapamycin and the P53 antagonist PFT-a, respectively, and the intracellular superoxide concentration and the ATP concentration were detected. The expression of B2 receptor before and after D-glucose was detected by flow cytometry. Western blot was used to detect the expression of signal pathway. Results: The DCFH-DA fluorescent probe showed that bradykinin could inhibit the aging of mouse cardiac stem cells induced by D-glucose and reduce the formation of oxygen free radicals in the cells, and decrease the intracellular superoxide concentration and promote the production of ATP. D-glucose significantly reduced the expression of the B2 receptor. Western blot showed that bradykinin could increase the expression of phosphorylated AKT and phosphorylated nTOR in high sugar environment and down-regulate the expression of P53 and P16. The B2 receptor antagonist, the P13K antagonist, the mTOR antagonist and the small interfering RNA block bradykinin as an anti-aging protective effect. The P53 antagonist also inhibits the aging of D-glucose-induced cardiac stem cells. Conclusion: Bradykinin inhibits the aging of D-glucose-induced cardiac stem cells through the B2R-mediated PI3K/ AKT/ mTOR/ P53 signaling pathway.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R363
[Abstract]:The first part of bradykinin inhibits the aging of endothelial progenitor cells induced by hydrogen peroxide: a specific molecular mechanism for the inhibition of the senescence of endothelial progenitor cells induced by bradykinin. Method: To detect the expression of CD34 + cell surface B2 receptor in peripheral circulating blood of patients with coronary heart disease. Enzyme-linked immunosorbent assay (ELISA) for detecting the plasma myeloperoxidase concentration in patients with coronary heart disease. Endothelial progenitor cells from human umbilical venous blood were treated with 3000M hydrogen peroxide, and different concentrations of bradykinin were used to detect the degree of cell senescence, and the content of oxygen free radicals in the cells was detected by the DCFH-DA fluorescence probe. The aging-related PCR chip and the fluorescence quantitative PCR detect and verify the expression of the related signal path molecules. The effect of the signal pathway blocking agent on BK protection was detected by using the B2 receptor small interfering RNA, the B2 receptor antagonist, the PI3K antagonist LY294002, the EGFR antagonist AG1478 to block the B2 receptor and the downstream signal pathway molecule, respectively. The expression of B2 receptor before and after the intervention of hydrogen peroxide was detected by flow cytometry and fluorescence quantitative PCR. Western blot was used to detect the expression of signal pathway. Results: The expression of B2 receptor in peripheral circulating blood of patients with coronary heart disease was significantly lower than that in healthy control group, and the concentration of plasma myeloperoxidase increased significantly. Dekallikrein and DCFH-DA fluorescence probe showed that bradykinin could inhibit the aging of endothelial progenitor cells from the human umbilical venous blood source induced by hydrogen peroxide and reduce the formation of oxygen free radicals in the cells. The results of aging-related PCR and quantitative PCR show that hydrogen peroxide can significantly increase the expression of RB gene and down-regulate the expression of B2 receptor, while bradykinin can significantly lower the expression of RB gene. Western blot showed that bradykinin could increase the expression of phosphorylated RB, phosphorylated AKT and cyclin D under the induction of hydrogen peroxide. Signal pathway antagonists and small interfering RNA blocking bradykinin play an anti-aging protective role. Conclusion: Bradykinin inhibits the senescence of human endothelial progenitor cells induced by hydrogen peroxide via a B2 receptor-mediated P13K and EGFR signaling pathway. The second part of bradykinin inhibits the aging of high-sugar-induced cardiac stem cells: a specific molecular mechanism for bradykinin inhibition of high-sugar-induced cardiac stem cell senescence. Methods: c-kit positive cardiac stem cells were isolated and cultured in vitro. The cardiac stem cells were treated with 25mM D-glucose and the cell senescence was detected using a different concentration of bradykinin-treated cells, p-half-lactase staining, and the DCFH-DA fluorescence probe was used to detect the oxygen free-radical content in the cells. The effect of the signal pathway blocking agent on BK protection was detected by using the B2 receptor small interfering RNA, the B2 receptor antagonist, the P13K antagonist, the LY294002, the mTOR antagonist Rapamycin and the P53 antagonist PFT-a, respectively, and the intracellular superoxide concentration and the ATP concentration were detected. The expression of B2 receptor before and after D-glucose was detected by flow cytometry. Western blot was used to detect the expression of signal pathway. Results: The DCFH-DA fluorescent probe showed that bradykinin could inhibit the aging of mouse cardiac stem cells induced by D-glucose and reduce the formation of oxygen free radicals in the cells, and decrease the intracellular superoxide concentration and promote the production of ATP. D-glucose significantly reduced the expression of the B2 receptor. Western blot showed that bradykinin could increase the expression of phosphorylated AKT and phosphorylated nTOR in high sugar environment and down-regulate the expression of P53 and P16. The B2 receptor antagonist, the P13K antagonist, the mTOR antagonist and the small interfering RNA block bradykinin as an anti-aging protective effect. The P53 antagonist also inhibits the aging of D-glucose-induced cardiac stem cells. Conclusion: Bradykinin inhibits the aging of D-glucose-induced cardiac stem cells through the B2R-mediated PI3K/ AKT/ mTOR/ P53 signaling pathway.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R363
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