E1A激活基因阻遏子蛋白對病理性血管內(nèi)皮凋亡的調(diào)控作用及機制研究
發(fā)布時間：2018-07-17 15:27
【摘要】：目的:血管內(nèi)皮損傷和凋亡是高血壓、糖尿病、動脈粥樣硬化(atherosclerosis, AS)和冠狀動脈介入治療術(shù)后再狹窄等多種血管疾病的早發(fā)病理過程。大量研究證實,血流動力學改變、氧化應激、炎癥和免疫反應等諸多外源性病理因素刺激均可導致血管內(nèi)皮凋亡發(fā)生;這些外源性病理因素刺激血管內(nèi)皮細胞(Vascular endothelial cells, VECs),導致其自身穩(wěn)態(tài)調(diào)控失衡,引發(fā)細胞凋亡,則進一步加重了血管內(nèi)皮功能障礙,加速了血管疾病的演進。近年來,針對引起血管內(nèi)皮損傷和凋亡的外源性病理因素進行了積極的干預,在一定程度上抑制了血管內(nèi)皮損傷的發(fā)展,延緩了血管性疾病的進程,但都沒有真正增強或改善內(nèi)皮功能。迄今為止,從血管內(nèi)皮或者VEC的自穩(wěn)態(tài)調(diào)控角度進行血管內(nèi)皮凋亡機制的研究報道甚少。 人E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes, CREG)基因是新近報道的一個維持組織及細胞成熟分化穩(wěn)態(tài)的重要調(diào)控因子。我室前期研究證實,CREG基因過表達能夠抑制動脈平滑肌(vascular smooth muscle cells, VSMCs)和骨髓間充質(zhì)干細胞的凋亡。并且,CREG基因在成熟VECs中呈現(xiàn)高豐度表達;而在急性球囊損傷和高脂喂養(yǎng)AS動物模型等急慢性病理性損傷VECs中表達顯著降低。這些研究提示,CREG基因表達與VEC的病理性損傷及功能障礙的發(fā)生有關(guān)。但是,CREG在VEC凋亡中是否具有調(diào)控作用尚不清楚。因此,本實驗主要探討CREG基因表達對動脈內(nèi)皮凋亡的作用及其調(diào)控機制。 方法:(1)用體外培養(yǎng)的正常人動脈內(nèi)皮細胞株(VE)作為對照,脂質(zhì)體轉(zhuǎn)染法將CREG過表達的逆轉(zhuǎn)錄病毒表達載體pLNCX-CREG、CREG低表達的逆轉(zhuǎn)錄病毒表達載體pLXSN-shRNA-CREG及pLNCX空載體分別感染phoenix293細胞,包裝出完整的逆轉(zhuǎn)錄病毒后,感染VEC,經(jīng)G418和嘌呤霉素篩選獲得穩(wěn)定感染的細胞克隆。將細胞分為3組進行實驗:CREG表達正常的空載體組、過表達CREG組、CREG表達沉默組內(nèi)皮細胞株。(2)CD31免疫熒光染色進行感染前的內(nèi)皮細胞鑒定、Western blot檢測感染前后細胞中CREG蛋白表達(。3)應用TUNEL染色、流式細胞分析及caspase-3的活性檢測等方法分析CREG對于STS和VP-16兩種凋亡誘導劑對VECs凋亡的影響。(4)應用酶聯(lián)免疫吸附(ELISA)實驗檢測各組細胞中的VEGF的分泌變化。Western blot檢測信號轉(zhuǎn)導蛋白PI3K、p-Akt、t-Akt及VEGF表達;添加PI3K信號通路阻斷劑LY294002及VEGF的中和抗體,分別抑制PI3K/Akt和VEGF的作用后,觀察各組細胞凋亡的改變。(5)應用原位免疫熒光染色和Western blot檢測apoE-/-小鼠AS血管內(nèi)皮和正常小鼠血管內(nèi)皮中CREG的表達情況;應用的TUNEL染色和cleaved caspase-3免疫熒光染色評價STS誘導的apoE-/-小鼠AS血管內(nèi)皮和正常小鼠的血管內(nèi)皮的凋亡情況。應用腺病毒-CREG瞬時感染體外培養(yǎng)的血管組織,TUNEL染色和cleaved caspase-3免疫熒光染色評價CREG基因過表達對apoE-/-小鼠AS血管內(nèi)皮凋亡的影響。 結(jié)果:(1)將重組逆轉(zhuǎn)錄病毒表達載體pLNCX、pLNCX-CREG及pLXSN-shRNA-CREG用脂質(zhì)體法分別感染phoenix 293細胞,包裝生產(chǎn)病毒,裂解后以病毒感染VE,經(jīng)過G418及嘌呤霉素篩選、挑取單克隆,分別得到表達含空載體pLNCX的CREG表達正常(CREG-NR)、CREG過表達(CREG-UP)及表達沉默組(CREG-DW)VEC細胞。(2)細胞鑒定結(jié)果:VE細胞中CD31熒光染色均呈陽性;以正常未處理組(命名為untreated cells)及CREG-NR組為空白對照,Western blot結(jié)果表明感染CREG-UP組細胞中的CREG蛋白表達上調(diào)大約190%;而感染CREG-DW組細胞中CREG蛋白表達明顯下調(diào)至10%。(3)應用TUNEL和流式細胞學檢測三組細胞在給予凋亡誘導劑STS及VP-16后的細胞凋亡情況。結(jié)果顯示,CREG表達抑制組凋亡的細胞明顯增多,兩者呈負性相關(guān)。應用Western blot檢測cleaved caspase-3活化情況也證實:CREG-DW組細胞中cleaved caspase-3表達明顯增多,而CREG-UP組則相反。這些研究結(jié)果提示,CREG過表達能夠?qū)筍TS或VP-16誘導的VE凋亡的發(fā)生。(4)應用ELISA檢測發(fā)現(xiàn),CREG-UP組細胞中VEGF分泌明顯增高;而CREG-DW組細胞中VEGF分泌減少。Western blot分析顯示,PI3K、pAkt在CREG-UP組細胞中表達明顯增多,而CREG-DW組中明顯下降,但t-Akt沒有顯著變化。添加了PI3K阻斷劑LY294002和VEGF的中和抗體后,STS和VP-16刺激的CREG-UP組細胞凋亡均較未阻斷前明顯增加。進一步應用Western blot分析證實,CREG-UP組中添加VEGF的中和抗體后,CREG-UP組細胞中PI3K、pAkt的蛋白表達明顯下調(diào);相反,添加LY294002不影響細胞中VEGF的表達。結(jié)果提示CREG通過介導VEGF/PI3K/AKT信號通路調(diào)控細胞凋亡。(5)通過體外組織學培養(yǎng)的方法,比較STS(600μmol/L)刺激前后正常小鼠血管內(nèi)皮和apoE-/-小鼠高脂喂養(yǎng)8周的AS血管內(nèi)皮中CREG表達及血管內(nèi)皮凋亡情況。結(jié)果發(fā)現(xiàn);apoE-/-小鼠AS血管內(nèi)皮中CREG表達比正常小鼠內(nèi)皮中明顯減少;給予STS(600μmol/L)刺激后,AS血管內(nèi)皮組織中凋亡陽性的細胞較正常小鼠血管內(nèi)皮組織相比明顯增加。應用AD-CREG預處理體外培養(yǎng)的apoE-/-小鼠AS血管,使其CREG表達增多,結(jié)果顯示,經(jīng)STS(600μmol/L)刺激誘導的血管內(nèi)皮凋亡現(xiàn)象明顯減少。 結(jié)論: CREG過表達能夠抑制血管組織內(nèi)皮和體外培養(yǎng)的血管內(nèi)皮細胞的凋亡,其對內(nèi)皮細胞凋亡的抑制作用可能是通過VEGF/PI3K/AKT信號轉(zhuǎn)導通路介導的。
[Abstract]:Objective: vascular endothelial injury and apoptosis are early pathological processes of various vascular diseases such as hypertension, diabetes, atherosclerosis, AS and restenosis after coronary intervention. Numerous studies have confirmed that many exogenous pathological factors such as hemodynamic changes, oxidative stress, inflammation and immune response can be stimulated. These exogenous pathological factors stimulate vascular endothelial cells (Vascular endothelial cells, VECs), which lead to the imbalance of their homeostasis and induce apoptosis, which further aggravate vascular endothelial dysfunction and accelerate the evolution of vascular diseases. In recent years, vascular endothelial damage and apoptosis have been caused. Exogenous pathological factors have been actively intervened, inhibiting the development of vascular endothelial damage to a certain extent and postponing the process of vascular diseases, but not truly enhancing or improving the endothelial function. So far, few reports have been reported on the mechanism of vascular endothelial apoptosis from the angle of vascular endothelium or VEC homeostasis.
The human E1A activated gene repressor (cellular repressor of E1A-stimulated genes, CREG) gene is an important regulator in the newly reported homeostasis of tissue and cell maturation and differentiation. Previous studies in my room have confirmed that the overexpression of CREG gene can inhibit the arterial smooth muscle (vascular smooth muscle cells,) and bone marrow mesenchymal stem Apoptosis of cells. Moreover, the CREG gene is highly expressed in mature VECs, and a significant decrease in acute and chronic pathological injury VECs, such as acute balloon injury and high fat feeding AS animal model. These studies suggest that the expression of CREG gene is related to the pathological damage of VEC and the occurrence of functional obstacles. But, whether CREG is in VEC apoptosis or not. The regulatory role of CREG gene is not yet clear. Therefore, this experiment focused on the effect of the expression of the gene on apoptosis of the arterial endothelial cells and its regulatory mechanism.
Methods: (1) the normal human arterial endothelial cell line (VE) cultured in vitro was used as the control. The transfection of CREG, retrovirus expression vector pLNCX-CREG, CREG low expression retroviral vector pLXSN-shRNA-CREG and pLNCX empty vector were respectively infected with phoenix293 cells, and the complete retrovirus was packaged and infected with V. EC, a stable infection cell clone was screened by G418 and purinamycin. The cells were divided into 3 groups to perform the experiment: the normal CREG expression group, the overexpressed CREG group, the CREG expression silent group endothelial cell line. (2) CD31 immunofluorescence staining for the identification of the endothelial cells before infection, and the Western blot to detect the expression of CREG protein in the cells before and after infection. .3) the effects of CREG on VECs apoptosis were analyzed by TUNEL staining, flow cytometry and caspase-3 activity detection. (4) enzyme linked immunosorbent assay (ELISA) test was used to detect the secretion of VEGF in each cell, and.Western blot detection signal transduction protein PI3K. In addition to the neutralization antibodies of the PI3K signaling pathway blockers LY294002 and VEGF and the inhibition of the effect of PI3K/Akt and VEGF, the changes in apoptosis were observed in each group. (5) the expression of CREG in the AS vascular endothelium of apoE-/- mice and in the vascular endothelial cells of normal mice was detected by in situ immunofluorescence staining and Western blot; TUNEL staining and cleaved statements were used. Ase-3 immunofluorescence staining was used to evaluate the apoptosis of vascular endothelium in STS induced apoE-/- mice AS vascular endothelial cells and normal mice. The effects of TUNEL staining and cleaved caspase-3 immunofluorescence staining on the apoptosis of AS vascular endothelial cells in apoE-/- mice were evaluated by TUNEL staining and cleaved caspase-3 immunofluorescence staining with the transient infection of adenovirus -CREG in vitro.
Results: (1) the recombinant retroviral vector pLNCX, pLNCX-CREG and pLXSN-shRNA-CREG were infected with Phoenix 293 cells by liposome, and the virus was packaged and produced. After the lysis, the virus infected VE. After G418 and purinamycin screening, the monoclonal was selected and the CREG expression of the pLNCX containing empty carrier pLNCX was normal (CREG-NR) and CREG overexpressed (C). REG-UP) and the expression of the silent group (CREG-DW) VEC cells. (2) the results of cell identification: CD31 fluorescent staining in VE cells was positive; the normal untreated group (named untreated cells) and CREG-NR group were blank control. Western blot results showed that the expression of CREG protein expression in the infected CREG-UP group cells was up to 190%; The expression of protein expression was down to 10%. (3) using TUNEL and flow cytometry to detect the apoptosis of three groups of cells after giving apoptosis inducer STS and VP-16. The results showed that the apoptotic cells in the CREG expression inhibition group were significantly increased. The Western blot detection of cleaved caspase-3 activation also confirmed that CREG-DW group was fine. The expression of cleaved caspase-3 in the cell was significantly increased, while the CREG-UP group was the opposite. These results suggested that CREG overexpression could antagonize the occurrence of VE apoptosis induced by STS or VP-16. (4) ELISA detection showed that VEGF secreted significantly in the CREG-UP group cells, while the VEGF secretion decreased in the CREG-DW group cells. There was a significant increase in the expression of the cells in the REG-UP group, but the CREG-DW group decreased significantly, but there was no significant change in t-Akt. After adding the neutralization antibody of LY294002 and VEGF, the apoptosis of the CREG-UP group stimulated by STS and VP-16 was significantly higher than that before the blocking. Further application of Western blot analysis confirmed that the neutralization resistance of the CREG-UP group was added. After the body, the protein expression of PI3K and pAkt in the CREG-UP group was obviously downregulated. On the contrary, the addition of LY294002 did not affect the expression of VEGF in the cells. The results suggested that CREG mediated the apoptosis by mediating the VEGF/PI3K/AKT signaling pathway. (5) the vascular endothelial and apoE-/- of normal mice were compared before and after the stimulation of STS (600 u mol/L) by the method of tissue culture in vitro. The expression of CREG in AS vascular endothelium and vascular endothelial apoptosis in the AS vascular endothelium of rats were observed. The results showed that the expression of CREG in the AS vascular endothelium of apoE-/- mice was significantly lower than that in the normal mice. After the stimulation of STS (600 mol/L), the apoptotic cells in the vascular endothelial tissue of AS increased significantly compared with that of the normal mice. The AS blood vessels of apoE-/- mice cultured in vitro were pretreated with AD-CREG, and the expression of CREG was increased. The results showed that the apoptosis induced by STS (600 micron mol/L) induced vascular endothelial apoptosis was obviously reduced.
Conclusion: overexpression of CREG can inhibit the apoptosis of vascular endothelial cells and endothelial cells cultured in vitro. The inhibition of endothelial cell apoptosis may be mediated by VEGF/PI3K/AKT signal transduction pathway.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363
本文編號：2130096
[Abstract]:Objective: vascular endothelial injury and apoptosis are early pathological processes of various vascular diseases such as hypertension, diabetes, atherosclerosis, AS and restenosis after coronary intervention. Numerous studies have confirmed that many exogenous pathological factors such as hemodynamic changes, oxidative stress, inflammation and immune response can be stimulated. These exogenous pathological factors stimulate vascular endothelial cells (Vascular endothelial cells, VECs), which lead to the imbalance of their homeostasis and induce apoptosis, which further aggravate vascular endothelial dysfunction and accelerate the evolution of vascular diseases. In recent years, vascular endothelial damage and apoptosis have been caused. Exogenous pathological factors have been actively intervened, inhibiting the development of vascular endothelial damage to a certain extent and postponing the process of vascular diseases, but not truly enhancing or improving the endothelial function. So far, few reports have been reported on the mechanism of vascular endothelial apoptosis from the angle of vascular endothelium or VEC homeostasis.
The human E1A activated gene repressor (cellular repressor of E1A-stimulated genes, CREG) gene is an important regulator in the newly reported homeostasis of tissue and cell maturation and differentiation. Previous studies in my room have confirmed that the overexpression of CREG gene can inhibit the arterial smooth muscle (vascular smooth muscle cells,) and bone marrow mesenchymal stem Apoptosis of cells. Moreover, the CREG gene is highly expressed in mature VECs, and a significant decrease in acute and chronic pathological injury VECs, such as acute balloon injury and high fat feeding AS animal model. These studies suggest that the expression of CREG gene is related to the pathological damage of VEC and the occurrence of functional obstacles. But, whether CREG is in VEC apoptosis or not. The regulatory role of CREG gene is not yet clear. Therefore, this experiment focused on the effect of the expression of the gene on apoptosis of the arterial endothelial cells and its regulatory mechanism.
Methods: (1) the normal human arterial endothelial cell line (VE) cultured in vitro was used as the control. The transfection of CREG, retrovirus expression vector pLNCX-CREG, CREG low expression retroviral vector pLXSN-shRNA-CREG and pLNCX empty vector were respectively infected with phoenix293 cells, and the complete retrovirus was packaged and infected with V. EC, a stable infection cell clone was screened by G418 and purinamycin. The cells were divided into 3 groups to perform the experiment: the normal CREG expression group, the overexpressed CREG group, the CREG expression silent group endothelial cell line. (2) CD31 immunofluorescence staining for the identification of the endothelial cells before infection, and the Western blot to detect the expression of CREG protein in the cells before and after infection. .3) the effects of CREG on VECs apoptosis were analyzed by TUNEL staining, flow cytometry and caspase-3 activity detection. (4) enzyme linked immunosorbent assay (ELISA) test was used to detect the secretion of VEGF in each cell, and.Western blot detection signal transduction protein PI3K. In addition to the neutralization antibodies of the PI3K signaling pathway blockers LY294002 and VEGF and the inhibition of the effect of PI3K/Akt and VEGF, the changes in apoptosis were observed in each group. (5) the expression of CREG in the AS vascular endothelium of apoE-/- mice and in the vascular endothelial cells of normal mice was detected by in situ immunofluorescence staining and Western blot; TUNEL staining and cleaved statements were used. Ase-3 immunofluorescence staining was used to evaluate the apoptosis of vascular endothelium in STS induced apoE-/- mice AS vascular endothelial cells and normal mice. The effects of TUNEL staining and cleaved caspase-3 immunofluorescence staining on the apoptosis of AS vascular endothelial cells in apoE-/- mice were evaluated by TUNEL staining and cleaved caspase-3 immunofluorescence staining with the transient infection of adenovirus -CREG in vitro.
Results: (1) the recombinant retroviral vector pLNCX, pLNCX-CREG and pLXSN-shRNA-CREG were infected with Phoenix 293 cells by liposome, and the virus was packaged and produced. After the lysis, the virus infected VE. After G418 and purinamycin screening, the monoclonal was selected and the CREG expression of the pLNCX containing empty carrier pLNCX was normal (CREG-NR) and CREG overexpressed (C). REG-UP) and the expression of the silent group (CREG-DW) VEC cells. (2) the results of cell identification: CD31 fluorescent staining in VE cells was positive; the normal untreated group (named untreated cells) and CREG-NR group were blank control. Western blot results showed that the expression of CREG protein expression in the infected CREG-UP group cells was up to 190%; The expression of protein expression was down to 10%. (3) using TUNEL and flow cytometry to detect the apoptosis of three groups of cells after giving apoptosis inducer STS and VP-16. The results showed that the apoptotic cells in the CREG expression inhibition group were significantly increased. The Western blot detection of cleaved caspase-3 activation also confirmed that CREG-DW group was fine. The expression of cleaved caspase-3 in the cell was significantly increased, while the CREG-UP group was the opposite. These results suggested that CREG overexpression could antagonize the occurrence of VE apoptosis induced by STS or VP-16. (4) ELISA detection showed that VEGF secreted significantly in the CREG-UP group cells, while the VEGF secretion decreased in the CREG-DW group cells. There was a significant increase in the expression of the cells in the REG-UP group, but the CREG-DW group decreased significantly, but there was no significant change in t-Akt. After adding the neutralization antibody of LY294002 and VEGF, the apoptosis of the CREG-UP group stimulated by STS and VP-16 was significantly higher than that before the blocking. Further application of Western blot analysis confirmed that the neutralization resistance of the CREG-UP group was added. After the body, the protein expression of PI3K and pAkt in the CREG-UP group was obviously downregulated. On the contrary, the addition of LY294002 did not affect the expression of VEGF in the cells. The results suggested that CREG mediated the apoptosis by mediating the VEGF/PI3K/AKT signaling pathway. (5) the vascular endothelial and apoE-/- of normal mice were compared before and after the stimulation of STS (600 u mol/L) by the method of tissue culture in vitro. The expression of CREG in AS vascular endothelium and vascular endothelial apoptosis in the AS vascular endothelium of rats were observed. The results showed that the expression of CREG in the AS vascular endothelium of apoE-/- mice was significantly lower than that in the normal mice. After the stimulation of STS (600 mol/L), the apoptotic cells in the vascular endothelial tissue of AS increased significantly compared with that of the normal mice. The AS blood vessels of apoE-/- mice cultured in vitro were pretreated with AD-CREG, and the expression of CREG was increased. The results showed that the apoptosis induced by STS (600 micron mol/L) induced vascular endothelial apoptosis was obviously reduced.
Conclusion: overexpression of CREG can inhibit the apoptosis of vascular endothelial cells and endothelial cells cultured in vitro. The inhibition of endothelial cell apoptosis may be mediated by VEGF/PI3K/AKT signal transduction pathway.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363
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