存活素促大鼠主动脉内皮细胞血管生成的实验研究

发布时间：2018-05-26 04:57

本文选题：存活素 + 大鼠主动脉内皮细胞　；参考：《重庆医科大学》2016年博士论文

【摘要】：周围动脉疾病(Periphery Artery Disease,PAD)是一组表现为外周动脉狭窄、闭塞的疾病,以下肢发病最为常见。目前对于PAD的治疗方法主要有药物治疗、外科手术治疗等。研究认为以上治疗方式在增加行走时间及距离,减少截肢率及降低死亡率等方面的效果并不理想。治疗性血管生成是治疗PAD一种手段,是以血管生长因子的基因、蛋白或内皮祖细胞等进行局部或系统干预以促进缺血组织血管生成,从而改善外周循环的方法。血管生成是指从已存在的微血管床上芽生出新的,以毛细血管为主的血管系统的过程,主要包括:细胞外基质的降解;血管内皮细胞的激活、增殖、迁移;管腔结构的形成及血管网的重塑。存活素(Survivin,SVV)基因是一种广泛表达于胚胎和肿瘤组织的多效性基因,SVV通过调节肿瘤细胞的凋亡、周期转换、侵袭性等信号通路促进肿瘤的生长、转移及血管生成。目前SVV基因促进肿瘤血管生成的研究已较深入,但关于SVV基因促进正常组织或细胞血管生成的研究甚少,实验采用腺病毒(Adenovirus,Ad)携带SVV基因转染大鼠主动脉内皮细胞(Rat Aortic Endothelial Cell,RAECs)的方式,观察SVV基因对RAECs增殖、迁移、抗凋亡及血管生成的影响,以期于将SVV基因做为新的PAD治疗的血管生成因子。第一部分腺病毒介导大鼠主动脉内皮细胞存活素基因的转染及鉴定目的:鉴定腺病毒介导SVV基因转染RAECs后细胞内SVV基因及蛋白的表达情况。方法:根据对RAECs不同的处理将其分为3组:SVV转染组(Ad-GFP/SVV):RAECs转染携带有SVV基因及GFP的腺病毒;阴性对照组(Ad-GFP):RAECs转染仅携带有GFP的腺病毒;空白对照组(RAEC):RAECs正常培养不做任何处理。用Ad-GFP/SVV组行最佳MOI的鉴定,分别用MOI等于0、10、20、50、100的携带有GFP及SVV基因的腺病毒与RAECs共培养,于12h、24h、48h、72h采用流式细胞仪计算感染率,确定最佳MOI和最佳感染时间后用q RT-PCR及Western blot检测各组RAECs内SVV的m RNA及蛋白的表达情况。结果:转染48h后MOI=50的感染率为(95.67±2.16)%,MOI=100的转染率为(94.53±1.76)%,组间差异无统计学意义(P0.05);q RT-PCR结果显示SVV转染组m RNA相对表达量显著上调,Western blot结果显示与q RT-PCR结果一致,SVV转染组与空白对照组及阴性对照组间差异具有统计学意义(P0.05)。结论:以MOI=50时携带SVV基因的腺病毒转染RAECs 48h可显著提高其SVV基因和蛋白的表达。第二部分存活素基因对大鼠主动脉内皮细胞增殖的影响目的:研究SVV基因对RAECs增殖的影响。方法:根据对RAECs不同的处理将其分为3组:SVV转染组(Ad-GFP/SVV):RAECs转染携带有SVV基因及GFP的腺病毒;阴性对照组(Ad-GFP):RAECs转染仅携带有GFP的腺病毒;空白对照组(RAEC):RAECs正常培养不做任何处理。按已确定的感染复数和感染时间用各组腺病毒转染RAECs,细胞免疫荧光法检测PCNA表达,MTT法检测RAECs的增殖活性,Western blot法检细胞周期蛋白的表达。结果:SVV转染组的PCNA阳性相对表达量为(85.35±4.93)%;阴性对照组为(51.04±6.86)%;空白对照组为(52.57±5.24)%。MTT法中转染后第1天SVV转染组的OD值为0.93±0.06;阴性对照组为0.77±0.09;空白对照组为0.71±0.14;转染后第2天SVV转染组的OD值为1.28±0.12;阴性对照组为0.91±0.05;空白对照组为0.87±0.06。SVV转染组的PCNA表达量及MTT的OD值显著高于阴性对照组及空白对照组,组间差异具有统计学意义(P0.05);SVV转染组的周期蛋白cyclin B1、cyclin D1、cyclin E、CDC2、CDK 4、CDK2表达明显上调,与空白对照组及阴性对照组间差异具有统计学意义(P0.05)。结论:SVV基因通过上调RAECs周期蛋白的表达促进RAECs的增殖。第三部分存活素基因对大鼠主动脉内皮细胞凋亡的影响目的:研究SVV基因对RAECs凋亡的影响。方法:根据对RAECs不同的处理将其分为4组:SVV转染组(Ad-GFP/SVV):RAECs转染携带有SVV基因及GFP的腺病毒后缺氧条件下培养12h;阴性对照组(Ad-GFP):RAECs转染仅携带有GFP的腺病毒缺氧条件下培养12h;缺氧对照组(Control):RAECs不行转染缺氧条件下培养12h;空白对照组(RAEC):RAECs不行转染常氧下培养12h。细胞转染后于缺氧条件下诱导细胞凋亡,采用鬼笔环肽荧光染色法标记微丝(F-actin)的表达情况,TUNEL绿色FITC标记荧光检测法及流式细胞仪检测细胞凋亡的数量,Western blot检RAECs内凋亡相关蛋白Cleaved caspase-3,8,9的表达。结果:各组细胞经低氧诱导凋亡后,光镜下SVV转染组与RAEC组的RAECs形态良好,细胞间联系紧密,荧光显微镜下阴性对照组、缺氧对照组出现核固缩、核碎裂等典型的凋亡变化;SVV转染组F-actin平行均匀分布于细胞中,与正常RAECs相似,在阴性对照组和缺氧对照组,F-actin降解增加,呈环形富集于细胞膜的周边;流式细胞仪分析结果示SVV转染组细胞凋亡率显著降低,与阴性对照组、缺氧对照组比较差异有统计学意义(P0.05);Western blot结果示SVV转染组RAECs的cleaved-Caspase-3,Caspase-8,Caspase-9的表达减少,与阴性对照组、缺氧对照组间差异具有统计学意义(P0.05)。结论:SVV基因通过下调Caspase-3、8、9的表达抑制低氧诱导的RAECs凋亡。第四部分存活素基因对大鼠主动脉内皮细胞迁移与侵袭的影响目的:研究SVV基因对RAECs迁移与侵袭的影响。方法:根据对RAECs不同的处理将其分为3组:SVV转染组(Ad-GFP/SVV):RAECs转染携带有SVV基因及GFP的腺病毒;阴性对照组(Ad-GFP):RAECs转染仅携带有GFP的腺病毒;空白对照组(RAEC):RAECs正常培养不做任何处理。细胞转染后采用划痕实验,Transwell实验、基质胶小室实验等评估RAECs的迁移能力,使用Western blot检测细胞内MMPs的表达水平。结果:SVV转染组在6,12,24h迁移距离均较阴性对照组和空白对照组迁移距离显著增加(P0.05);Transwell实验中,SVV转染组穿过聚碳酸酯膜的RAECs较阴性对照组和空白对照组显著增加(P0.05);Western blot结果示SVV转染组RAECs内MMP-2、7、8、9的表达水平均高于阴性转染组和空白对照组。结论:SVV通过上调MMP-2、7、8、9的表达增加RAECs迁移和侵袭能力。第五部分存活素基因对大鼠主动脉内皮细胞小管形成及血管生成的影响目的:观察SVV基因对RAECs在体外管腔形成及体内血管生成的影响。方法:根据对RAECs不同的处理将其分为3组:SVV转染组(Ad-GFP/SVV):RAECs转染携带有SVV基因及GFP的腺病毒;阴性对照组(Ad-GFP):RAECs转染仅携带有GFP的腺病毒;空白对照组(RAEC):RAECs正常培养不做任何处理。SVV基因转染后,将RAECs接种在Martigel基质胶预处理的48孔板中,观察管腔形成的情况,并用ELISA检测细胞培养基上清液中血管内皮生长因子(VEGF)的表达;将转染后的细胞与Martigel基质胶混合液接种至裸鼠皮下,7天后采用HE染色、Masson染色及CD31免疫组化染色评估裸鼠皮下血管生成的情况。结果:SVV转染组相对于正常对照组的小管与分支的比例显著高于阴性对照组(P0.05)。ELISA检测结果示SVV转染组VEGF表达量显著增加,组间差异具有统计学意义(P0.05)。在体实验中,SVV转染组可明显见到横行整齐排列的毛细血管,经量化统计分析SVV转染组被膜与胶栓内血管数均显著高于阴性对照组和正常对照组(P0.05)。结论:SVV基因使RAECs在基质胶中直接参与管腔形成。SVV基因提高RAECs移植周围组织中VEGF的表达水平,促进血管的生成。
[Abstract]:Periphery Artery Disease (PAD) is a group of diseases characterized by peripheral artery stenosis and occlusion and the most common disease of the lower limbs. The main treatment methods for PAD are drug treatment and surgical treatment. The above treatment is considered to increase the time and distance of walking, reduce the rate of amputation and reduce the mortality. Therapeutic angiogenesis is a means to treat PAD, a means of local or systematic intervention by gene, protein or endothelial progenitor cells of vascular growth factor to promote angiogenesis in ischemic tissue and improve peripheral circulation. Angiogenesis refers to new buds from the existing microvascular beds, The process of vascular system based on capillaries mainly includes the degradation of extracellular matrix, activation, proliferation, migration of vascular endothelial cells, formation of the lumen structure and remodeling of vascular network. The Survivin (SVV) gene is a pleiotropic gene widely expressed in the embryo and tumor tissue, and SVV by regulating the apoptosis of tumor cells, Periodic transformation, invasive and invasive pathways promote tumor growth, metastasis and angiogenesis. At present, SVV gene promotes tumor angiogenesis, but the research on SVV gene promoting normal tissue or cell angiogenesis is very little. The experiment uses Adenovirus (Ad) to transfect the rat aortic endothelial cells with SVV gene. (Rat Aortic Endothelial Cell, RAECs), to observe the effect of SVV gene on RAECs proliferation, migration, anti apoptosis and angiogenesis, in order to make SVV gene as a new angiogenic factor in PAD therapy. The first part of adenovirus mediated gene transfection and identification of rat aortic endothelial cell survivin gene: identification of adenovirus mediated SVV Expression of SVV gene and protein in cells after RAECs gene transfection. Methods: according to the different treatment of RAECs, it was divided into 3 groups: SVV transfection group (Ad-GFP/SVV): RAECs transfected with SVV gene and GFP adenovirus; negative control group (Ad-GFP): RAECs transfection only carrying GFP adenovirus; blank control group (RAEC): normal culture do not do normal culture The best MOI was identified by the Ad-GFP/SVV group. The adenovirus carrying the GFP and SVV genes was co cultured with the GFP and SVV genes, respectively. The infection rate was calculated by the flow cytometry in 12h, 24h, 48h and 72h, and the best MOI and the best infection time were determined and the eggs were detected. Results: the infection rate of MOI=50 after transfection of 48h was (95.67 + 2.16)%, and the transfection rate of MOI=100 was (94.53 + 1.76)%, and there was no significant difference between the groups (P0.05). Q RT-PCR results showed that the RNA relative expression of m in SVV transfected group was significantly up. The Western blot results showed that the results of the Western blot were in accordance with the Q results, and the transfection group and blank control group and Yin were negative. The difference between the sex control group was statistically significant (P0.05). Conclusion: the expression of SVV gene and protein can be significantly improved by transfecting RAECs 48h with SVV gene with SVV gene at MOI=50. The effect of the second part of the survivin gene on the proliferation of rat aortic endothelial cells is to study the effect of SVV gene on the proliferation of RAECs. The same treatment was divided into 3 groups: SVV transfection group (Ad-GFP/SVV): RAECs transfected with SVV gene and GFP adenovirus; negative control group (Ad-GFP): RAECs transfected only with GFP adenovirus; blank control group (RAEC): RAECs normal culture does not do any treatment. The expression of PCNA was detected by cell immunofluorescence, the proliferation activity of RAECs was detected by MTT, and the expression of cyclin was detected by Western blot. Results: the positive relative expression of PCNA in SVV transfected group was (85.35 + 4.93)%, and the negative control group was (51.04 + 6.86)%, and that of the blank control group was 0.93 + in the first day transfection group of (52.57 + 5.24)%.MTT. 0.06, the negative control group was 0.77 + 0.09 and the blank control group was 0.71 + 0.14, and the OD value of the SVV transfected group was 1.28 + 0.12 and the negative control group was 0.91 + 0.05 after the transfection. The PCNA expression in the blank control group and the MTT in the 0.87 + 0.06.SVV transfection group were significantly higher than those in the negative control group and the blank control group, and the difference between the groups was statistically significant (P0.05). The expression of cyclin cyclin B1, cyclin D1, cyclin E, CDC2, CDK 4, CDK2 expression was obviously up-regulated in the transfected group, and the difference between the blank control group and the negative control group was statistically significant (P0.05). Conclusion: the SVV gene promotes the proliferation of the SVV by up regulation of the expression of the periodic protein. The third part survivin gene is on the aortic endothelial cells in rat aorta. The effect of apoptosis: To study the effect of SVV gene on RAECs apoptosis. Methods: according to the different treatment of RAECs, it was divided into 4 groups: SVV transfection group (Ad-GFP/SVV): RAECs transfected with SVV gene and GFP of adenovirus to cultivate 12h; negative control group (Ad-GFP): RAECs transfection only carrying GFP adenovirus in hypoxia condition culture 12 H; hypoxia control group (Control): RAECs could not be transfected to 12h under hypoxia condition; blank control group (RAEC): RAECs was not transfected to induce apoptosis of 12h. cells under hypoxia and transfected in normal oxygen, and the expression of microfilament (F-actin) was marked by phofic cyclic peptide fluorescence staining, TUNEL green FITC labelled fluorescence detection and flow cytometry The number of apoptotic cells and the expression of apoptosis related protein Cleaved caspase-3,8,9 in RAECs were detected by Western blot. Results: after hypoxia induced apoptosis, the RAECs morphology of SVV transfected group and RAEC group under light microscope was good, the intercellular connection was close, the negative control group under the fluorescence microscope, the hypoxic control group had nuclear pyknosis and nuclear fragmentation. SVV transfected group F-actin was parallel and evenly distributed in cells, similar to normal RAECs, and in negative control group and hypoxia control group, the degradation of F-actin increased, and was enriched in the periphery of cell membrane. Flow cytometry showed that the mortality rate of SVV transfected group decreased significantly, compared with negative control group, the ratio of hypoxia control group was more than that of the negative control group. The difference was statistically significant (P0.05); Western blot results showed that the expression of cleaved-Caspase-3, Caspase-8, Caspase-9 in the SVV transfected group decreased, and the difference between the negative control group and the negative control group was statistically significant (P0.05). Conclusion: the SVV gene inhibits the apoptosis induced by hypoxia by downregulating the Caspase-3,8,9 expression. The fourth part of the gene is stored. The effect of the living factor gene on the migration and invasion of rat aortic endothelial cells: To study the effect of SVV gene on the migration and invasion of RAECs. Methods: according to the different treatment of RAECs, it was divided into 3 groups: SVV transfection group (Ad-GFP/SVV): RAECs transfected with SVV gene and GFP adenosis; negative control group (Ad-GFP): RAECs transfection only carried GFP. Adenovirus, blank control group (RAEC): RAECs normal culture did not do any treatment. After cell transfection, the migration ability of RAECs was evaluated by scratch test, Transwell experiment and matrix glue laboratory test. The expression level of MMPs in cell was detected by Western blot. Results: SVV transfer group was compared with negative control group and blank control group at 6,12,24h migration distance. The migration distance of the control group increased significantly (P0.05); in the Transwell experiment, the RAECs in the SVV transfected group was significantly higher than that in the negative control group and the blank control group (P0.05). The Western blot results showed that the expression level of MMP-2,7,8,9 in RAECs in SVV transfected group was higher than that in the negative transfected group and the blank control group. Conclusion: SVV by up regulation of MMP-2,7,8, 9 expression increased RAECs migration and invasiveness. Fifth the effect of the fifth part of the survivin gene on the formation and angiogenesis of rat aortic endothelial cells: To observe the effect of the SVV gene on the formation of RAECs in vitro and the angiogenesis in the body. Methods: according to the different location of RAECs, the gene was divided into 3 groups: SVV transfection group (Ad-GFP/SVV): RAE Cs transfected adenovirus carrying SVV gene and GFP; negative control group (Ad-GFP): RAECs transfection only carried GFP adenovirus; blank control group (RAEC): RAECs normal culture without any treatment of.SVV gene transfection, RAECs inoculated in the 48 pore plate pretreated by Martigel matrix gel, observe the formation of the lumen, and use ELISA to detect cell culture. The expression of vascular endothelial growth factor (VEGF) in the supernatant of the nutrient base, and inoculated the transfected cells with the mixture of Martigel matrix glue into the subcutaneous tissue of nude mice. 7 days later, HE staining, Masson staining and CD31 immunohistochemical staining were used to evaluate the angiogenesis in nude mice. Results: the ratio of SVV to the normal control group was compared with that of the normal control group. Significantly higher than the negative control group (P0.05).ELISA test results showed that the expression of VEGF in SVV transfected group increased significantly, and the difference between the groups was statistically significant (P0.05). In the body experiment, the SVV transfected group could obviously see the neatly arranged capillaries in the SVV transfection group. The quantitative statistical analysis was significantly higher than the negative control in the number of the membrane and the thrombus in the SVV transfected group. Group and normal control group (P0.05). Conclusion: SVV gene makes RAECs directly participate in the formation of.SVV gene in the matrix glue to improve the expression of VEGF in the surrounding tissue of RAECs transplantation and promote the formation of blood vessels.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543

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