緩激肽B2受體信號(hào)通路抑制氧化應(yīng)激誘導(dǎo)的內(nèi)皮祖細(xì)胞和心臟干細(xì)胞衰老的機(jī)制研究
發(fā)布時(shí)間：2019-01-17 19:54
【摘要】：第一部分 緩激肽抑制過氧化氫誘導(dǎo)的內(nèi)皮祖細(xì)胞的衰老目的：探討緩激肽抑制過氧化氫誘導(dǎo)的內(nèi)皮祖細(xì)胞衰老的具體分子機(jī)制。方去：檢測(cè)冠心病患者外周循環(huán)血中CD34+細(xì)胞表面B2受體的表達(dá)。ELISA法檢測(cè)冠心病患者血漿髓過氧化物酶濃度。使用3000M過氧化氫處理人臍靜脈血來源的內(nèi)皮祖細(xì)胞,并使用不同濃度的緩激肽干預(yù)細(xì)胞,p半乳糖苷酶染色檢測(cè)細(xì)胞衰老程度,DCFH-DA熒光探針檢測(cè)細(xì)胞內(nèi)氧自由基含量。衰老相關(guān)PCR芯片以及熒光定量PCR檢測(cè)并驗(yàn)證相關(guān)信號(hào)通路分子的表達(dá)。分別使用B2受體小干擾RNA, B2受體拮抗劑,PI3K拮抗劑LY294002, EGFR拮抗劑AG1478阻斷B2受體以及下游信號(hào)通路分子,檢測(cè)信號(hào)通路阻斷劑對(duì)BK保護(hù)作用的影響。使用流式細(xì)胞技術(shù)和熒光定量PCR檢測(cè)過氧化氫干預(yù)前后B2受體的表達(dá)。Western blot技術(shù)檢測(cè)各信號(hào)通路分子的表達(dá)。結(jié)果：冠心病患者外周循環(huán)血中CD34+細(xì)胞表面B2受體表達(dá)較健康對(duì)照顯著下降,血漿髓過氧化物酶濃度顯著升高,兩者具有顯著的相關(guān)性。β半乳糖苷酶染色和DCFH-DA熒光探針顯示緩激肽可以抑制過氧化氫誘導(dǎo)的人臍靜脈血來源的內(nèi)皮祖細(xì)胞衰老并減少細(xì)胞內(nèi)氧自由基形成。衰老相關(guān)PCR芯片和熒光定量PCR結(jié)果顯示過氧化氫能夠顯著上調(diào)RB基因的表達(dá),下調(diào)B2受體表達(dá),而緩激肽可以顯著下調(diào)RB基因的表達(dá)。Western blot結(jié)果顯示緩激肽能夠上調(diào)過氧化氫誘導(dǎo)下磷酸化RB,磷酸化AKT和cyclin D的表達(dá)。信號(hào)通路拮抗劑和小干擾RNA阻斷緩激肽發(fā)揮抗衰老的保護(hù)作用。結(jié)論:緩激肽通過B2受體介導(dǎo)的P13K和EGFR信號(hào)通路抑制過氧化氫誘導(dǎo)的人內(nèi)皮祖細(xì)胞的衰老。第二部分緩激肽抑制高糖誘導(dǎo)的心臟干細(xì)胞的衰老目的：探討緩激肽抑制高糖誘導(dǎo)的心臟干細(xì)胞衰老的具體分子機(jī)制。方法：體外分離培養(yǎng)C57BL/6J小鼠c-kit陽(yáng)性心臟干細(xì)胞。使用25mM D-葡萄糖處理心臟干細(xì)胞,并使用不同濃度的緩激肽干預(yù)細(xì)胞,p半乳糖苷酶染色檢測(cè)細(xì)胞衰老程度,DCFH-DA熒光探針檢測(cè)細(xì)胞內(nèi)氧自由基含量。分別使用B2受體小干擾RNA,B2受體拮抗劑,P13K拮抗劑]LY294002,mTOR拮抗劑Rapamycin和P53拮抗劑PFT-a阻斷B2受體以及下游信號(hào)通路分子,檢測(cè)信號(hào)通路阻斷劑對(duì)BK保護(hù)作用的影響,同時(shí)檢測(cè)細(xì)胞內(nèi)超氧化物濃度和ATP濃度,并使用流式細(xì)胞技術(shù)檢測(cè)D-葡萄糖干預(yù)前后B2受體的表達(dá)。、Western blot技術(shù)檢測(cè)各信號(hào)通路分子的表達(dá)。結(jié)果：β半乳糖苷酶染色和DCFH-DA熒光探針顯示緩激肽可以抑制D-葡萄糖誘導(dǎo)的小鼠心臟干細(xì)胞衰老并減少細(xì)胞內(nèi)氧自由基的形成,并能降低細(xì)胞內(nèi)超氧化物濃度,促進(jìn)ATP生成。D-葡萄糖顯著下調(diào)B2受體表達(dá)。Western blot結(jié)果顯示緩激肽能夠上調(diào)高糖環(huán)境下磷酸化AKT,磷酸化nTOR的表達(dá),下調(diào)P53和P16的表達(dá)。B2受體拮抗劑,P13K拮抗劑,mTOR拮抗劑和小干擾RNA阻斷緩激肽發(fā)揮抗衰老的保護(hù)作用。P53拮抗劑同樣抑制D-葡萄糖誘導(dǎo)的心臟干細(xì)胞衰老。結(jié)論：緩激肽通過B2R介導(dǎo)的PI3K/AKT/mTOR/P53信號(hào)通路抑制D-葡萄糖誘導(dǎo)的心臟干細(xì)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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R363
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