血管内皮细胞Deptor缺失mTOR信号通路活化促进血管增多

发布时间：2018-01-10 11:00

本文关键词：血管内皮细胞Deptor缺失mTOR信号通路活化促进血管增多　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：一、研究背景血管的生成具体指的是在原来存在的血管附近,生长出其他新生的血管。血管内皮生长因子(英文名字为VEGFs),是指可以促进血管生成的一种细胞分泌因子,可以很好的保持血管内膜的完整性。据报道,在体外实验中,DEPTOR在调节血管内皮细胞激活和在炎症及血管生成中起重要的作用。可是,关于DEPTOR在生物体内微血管的生成中的具体作用还不清楚。因此,我们想在体内首次证实DEPTOR与血管生成的关系。二、研究方法1、在Cre-loxP系统上,我们构建了在血管内皮细胞特异性进行DEPTOR敲除的小鼠。2、验证血管内皮细胞特异性DEPTOR敲除小鼠敲除效果。3、采用苏木素-伊红染色、免疫组织化学及western blot等方法,检测敲除小鼠中血管数量变化等表达情况。观察用雷帕霉素处理后血管数量的变化。4、检测KO小鼠VEGF和HIF-1α表达情况。观察用雷帕霉素处理后的变化。5、HUVECs转染DEPTORsiRNA,观察CD31以及促血管生成因子、缺氧诱导因子的变化情况。观察用雷帕霉素处理后的变化。设置对照组、转染DEPTOR siRNA组、转染DEPTOR siRNA +雷帕霉素组,观察各组HUVEC小管形成情况。三、统计学处理在此实验中,所有数据结果均重复3次或以上,结果用均数±标准误来表示,数组间用t检验及两因素方差分析进行分析。当P0.05代表结果有统计学意义。四、研究结果1、成功构建了在血管内皮细胞进行特异性DEPTOR敲除的小鼠。免疫荧光染色表明DEPTOR蛋白表达在CD31标记的KO小鼠的血管EC中降低。与WT小鼠相比,Western blot结果证明,在KO小鼠组织中的Deptor表达显著降低。2、血管内皮细胞特异性敲除Deptor激活mTOR信号。与WT小鼠相比,KO小鼠组织中pS6明显升高。3、血管内皮细胞敲除Deptor促进了组织血管生成,并且雷帕霉素可以逆转这一表型。与WT小鼠相比,KO小鼠组织,CD31组织化学染色数量显着增加。用雷帕霉素处理KO小鼠时,CD31组织化学染色减少。western blot结果与其一致。4、在KO小鼠中,VEGF和HIFM α的表达增加,雷帕霉素可以逆转这一表型。免疫组织化学染色显示,与WT小鼠相比,KO小鼠的组织中VEGF和HIF-1α的表达增加。用雷帕霉素处理后,与KO小鼠相比,KO + RAPA小鼠中VEGF的染色数量明显减少,Western blot结果一致。5、用 DEPTOR siRNA 转染 HUVEC,CD31,VEGF 和 HIF-1α 的表达也显著增加。用雷帕霉素处理,抑制了 pS6K,pS6的增加,且CD31,VEGF和HIF-1α的表达明显减少。与对照组相比,加入DEPTOR siRNA的HUVEC小管生成增加,而雷帕霉素处理组可逆转增加。五、结论1、在血管内皮细胞内敲除Deptor基因,可以促进mTOR信号通路的活化,进而促进组织血管的形成。2、血管内皮细胞内mTOR信号通路活化,促进VEGF、缺氧诱导因子的的释放,促进血管形成,加雷帕霉素可以减少这一表型。
[Abstract]:A generation of backgroundvascular specifically refers to the presence of the vessel in the vicinity of the original, the growth of new blood vessels. Other vascular endothelial growth factor (English name VEGFs), refers to a kind of cell factor secretion can promote angiogenesis, can maintain the integrity of intravascular membrane very well. According to reports, in vitro, DEPTOR in the regulation of vascular endothelial cell activation and inflammation in angiogenesis and plays an important role. However, the specific role on the formation of DEPTOR in vivo in microvascular remains unclear. Therefore, we want to in the body for the first time that the relationship between DEPTOR and angiogenesis. Methods 1, two. In Cre-loxP system, we construct the DEPTOR knockout mice.2 except in vascular endothelial cell specific verification, vascular endothelial cell specific DEPTOR knockout mice and.3 knockout effect, using hematoxylin eosin staining, immunohistochemistry Chemical methods such as blot and western, detection of knockout mice. The expression of vascular changes in the number of observed changes of.4 blood vessel number after treatment of rapamycin, detection of KO VEGF and HIF-1 expression in mice. Observe the changes of.5 after treatment with rapamycin, HUVECs transfected with DEPTORsiRNA, CD31 and to observe the angiogenesis, changes of hypoxia induced factors. Observe the changes after rapamycin treatment. The control group, DEPTOR transfection group siRNA transfection DEPTOR siRNA + rapamycin group, observed HUVEC tubule formation. Three, the statistical treatment in this experiment, all the data were repeated 3 times or more, the results were expressed as mean + standard error. Analysis by t test and two factor variance between the array. When P0.05 results were statistically significant. Results four, 1, the successful construction of the specificity of DEPT in vascular endothelial cells OR knockout mice. Immunofluorescence staining showed that DEPTOR protein expression decreased in CD31 labeled KO mice vascular EC. Compared with WT mice, Western blot results show that the expression of Deptor in tissue of KO mice significantly decreased.2, vascular endothelial cell specific knockdown of Deptor activated mTOR signal. Compared with WT mice. In the KO mice pS6 significantly increased.3, vascular endothelial cells knockdown of Deptor promotes angiogenesis, and rapamycin can reverse the phenotype. Compared with WT mice, KO mice, CD31 histochemical staining. A significant increase in the number of KO mice with hormone treatment by rapamycin, CD31 staining decreased.Western blot results consistent with.4, in KO mice, increased expression of VEGF and HIFM alpha, rapamycin can reverse the phenotype. The immunohistochemical staining showed that, compared with WT mice, VEGF mice and HIF-1 alpha KO in table Increased. After treatment with rapamycin, compared with KO mice, the number of VEGF KO + RAPA staining in mice was significantly reduced, Western blot.5 DEPTOR siRNA results, CD31, transfection of HUVEC, expression of VEGF and HIF-1 alpha has increased significantly. With rapamycin inhibited pS6K, pS6 and CD31, increased. The expression of VEGF and HIF-1 alpha was significantly reduced. Compared with the control group, HUVEC DEPTOR siRNA joined the tubular formation increases, and rapamycin treatment group can reverse the increase. Five, 1 in conclusion, vascular endothelial cells Deptor gene knockout can promote the activation of mTOR signaling pathway, and promote the formation of.2 tissue blood vessels, blood vessels activation of mTOR signaling pathway in endothelial cells, promoting VEGF, hypoxia inducible factor release, angiogenesis, and rapamycin can reduce this phenotype.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R363

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