E1A激活基因阻遏子蛋白对病理性血管内皮凋亡的调控作用及机制研究
发布时间：2018-07-17 15:27
【摘要】：目的:血管内皮损伤和凋亡是高血压、糖尿病、动脉粥样硬化(atherosclerosis, AS)和冠状动脉介入治疗术后再狭窄等多种血管疾病的早发病理过程。大量研究证实,血流动力学改变、氧化应激、炎症和免疫反应等诸多外源性病理因素刺激均可导致血管内皮凋亡发生;这些外源性病理因素刺激血管内皮细胞(Vascular endothelial cells, VECs),导致其自身稳态调控失衡,引发细胞凋亡,则进一步加重了血管内皮功能障碍,加速了血管疾病的演进。近年来,针对引起血管内皮损伤和凋亡的外源性病理因素进行了积极的干预,在一定程度上抑制了血管内皮损伤的发展,延缓了血管性疾病的进程,但都没有真正增强或改善内皮功能。迄今为止,从血管内皮或者VEC的自稳态调控角度进行血管内皮凋亡机制的研究报道甚少。 人E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes, CREG)基因是新近报道的一个维持组织及细胞成熟分化稳态的重要调控因子。我室前期研究证实,CREG基因过表达能够抑制动脉平滑肌(vascular smooth muscle cells, VSMCs)和骨髓间充质干细胞的凋亡。并且,CREG基因在成熟VECs中呈现高丰度表达;而在急性球囊损伤和高脂喂养AS动物模型等急慢性病理性损伤VECs中表达显著降低。这些研究提示,CREG基因表达与VEC的病理性损伤及功能障碍的发生有关。但是,CREG在VEC凋亡中是否具有调控作用尚不清楚。因此,本实验主要探讨CREG基因表达对动脉内皮凋亡的作用及其调控机制。 方法:(1)用体外培养的正常人动脉内皮细胞株(VE)作为对照,脂质体转染法将CREG过表达的逆转录病毒表达载体pLNCX-CREG、CREG低表达的逆转录病毒表达载体pLXSN-shRNA-CREG及pLNCX空载体分别感染phoenix293细胞,包装出完整的逆转录病毒后,感染VEC,经G418和嘌呤霉素筛选获得稳定感染的细胞克隆。将细胞分为3组进行实验:CREG表达正常的空载体组、过表达CREG组、CREG表达沉默组内皮细胞株。(2)CD31免疫荧光染色进行感染前的内皮细胞鉴定、Western blot检测感染前后细胞中CREG蛋白表达(。3)应用TUNEL染色、流式细胞分析及caspase-3的活性检测等方法分析CREG对于STS和VP-16两种凋亡诱导剂对VECs凋亡的影响。(4)应用酶联免疫吸附(ELISA)实验检测各组细胞中的VEGF的分泌变化。Western blot检测信号转导蛋白PI3K、p-Akt、t-Akt及VEGF表达;添加PI3K信号通路阻断剂LY294002及VEGF的中和抗体,分别抑制PI3K/Akt和VEGF的作用后,观察各组细胞凋亡的改变。(5)应用原位免疫荧光染色和Western blot检测apoE-/-小鼠AS血管内皮和正常小鼠血管内皮中CREG的表达情况;应用的TUNEL染色和cleaved caspase-3免疫荧光染色评价STS诱导的apoE-/-小鼠AS血管内皮和正常小鼠的血管内皮的凋亡情况。应用腺病毒-CREG瞬时感染体外培养的血管组织,TUNEL染色和cleaved caspase-3免疫荧光染色评价CREG基因过表达对apoE-/-小鼠AS血管内皮凋亡的影响。 结果:(1)将重组逆转录病毒表达载体pLNCX、pLNCX-CREG及pLXSN-shRNA-CREG用脂质体法分别感染phoenix 293细胞,包装生产病毒,裂解后以病毒感染VE,经过G418及嘌呤霉素筛选、挑取单克隆,分别得到表达含空载体pLNCX的CREG表达正常(CREG-NR)、CREG过表达(CREG-UP)及表达沉默组(CREG-DW)VEC细胞。(2)细胞鉴定结果:VE细胞中CD31荧光染色均呈阳性;以正常未处理组(命名为untreated cells)及CREG-NR组为空白对照,Western blot结果表明感染CREG-UP组细胞中的CREG蛋白表达上调大约190%;而感染CREG-DW组细胞中CREG蛋白表达明显下调至10%。(3)应用TUNEL和流式细胞学检测三组细胞在给予凋亡诱导剂STS及VP-16后的细胞凋亡情况。结果显示,CREG表达抑制组凋亡的细胞明显增多,两者呈负性相关。应用Western blot检测cleaved caspase-3活化情况也证实:CREG-DW组细胞中cleaved caspase-3表达明显增多,而CREG-UP组则相反。这些研究结果提示,CREG过表达能够对抗STS或VP-16诱导的VE凋亡的发生。(4)应用ELISA检测发现,CREG-UP组细胞中VEGF分泌明显增高;而CREG-DW组细胞中VEGF分泌减少。Western blot分析显示,PI3K、pAkt在CREG-UP组细胞中表达明显增多,而CREG-DW组中明显下降,但t-Akt没有显著变化。添加了PI3K阻断剂LY294002和VEGF的中和抗体后,STS和VP-16刺激的CREG-UP组细胞凋亡均较未阻断前明显增加。进一步应用Western blot分析证实,CREG-UP组中添加VEGF的中和抗体后,CREG-UP组细胞中PI3K、pAkt的蛋白表达明显下调;相反,添加LY294002不影响细胞中VEGF的表达。结果提示CREG通过介导VEGF/PI3K/AKT信号通路调控细胞凋亡。(5)通过体外组织学培养的方法,比较STS(600μmol/L)刺激前后正常小鼠血管内皮和apoE-/-小鼠高脂喂养8周的AS血管内皮中CREG表达及血管内皮凋亡情况。结果发现;apoE-/-小鼠AS血管内皮中CREG表达比正常小鼠内皮中明显减少;给予STS(600μmol/L)刺激后,AS血管内皮组织中凋亡阳性的细胞较正常小鼠血管内皮组织相比明显增加。应用AD-CREG预处理体外培养的apoE-/-小鼠AS血管,使其CREG表达增多,结果显示,经STS(600μmol/L)刺激诱导的血管内皮凋亡现象明显减少。 结论: CREG过表达能够抑制血管组织内皮和体外培养的血管内皮细胞的凋亡,其对内皮细胞凋亡的抑制作用可能是通过VEGF/PI3K/AKT信号转导通路介导的。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
本文编号：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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
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