本文關(guān)鍵詞：血管內(nèi)皮細(xì)胞Deptor缺失mTOR信號(hào)通路活化促進(jìn)血管增多　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 血管形成 血管內(nèi)皮細(xì)胞 哺乳動(dòng)物雷帕霉素靶蛋白 促血管生成因子 缺氧誘導(dǎo)因子

【摘要】：一、研究背景血管的生成具體指的是在原來存在的血管附近,生長出其他新生的血管。血管內(nèi)皮生長因子(英文名字為VEGFs),是指可以促進(jìn)血管生成的一種細(xì)胞分泌因子,可以很好的保持血管內(nèi)膜的完整性。據(jù)報(bào)道,在體外實(shí)驗(yàn)中,DEPTOR在調(diào)節(jié)血管內(nèi)皮細(xì)胞激活和在炎癥及血管生成中起重要的作用�？墒�,關(guān)于DEPTOR在生物體內(nèi)微血管的生成中的具體作用還不清楚。因此,我們想在體內(nèi)首次證實(shí)DEPTOR與血管生成的關(guān)系。二、研究方法1、在Cre-loxP系統(tǒng)上,我們構(gòu)建了在血管內(nèi)皮細(xì)胞特異性進(jìn)行DEPTOR敲除的小鼠。2、驗(yàn)證血管內(nèi)皮細(xì)胞特異性DEPTOR敲除小鼠敲除效果。3、采用蘇木素-伊紅染色、免疫組織化學(xué)及western blot等方法,檢測敲除小鼠中血管數(shù)量變化等表達(dá)情況。觀察用雷帕霉素處理后血管數(shù)量的變化。4、檢測KO小鼠VEGF和HIF-1α表達(dá)情況。觀察用雷帕霉素處理后的變化。5、HUVECs轉(zhuǎn)染DEPTORsiRNA,觀察CD31以及促血管生成因子、缺氧誘導(dǎo)因子的變化情況。觀察用雷帕霉素處理后的變化。設(shè)置對照組、轉(zhuǎn)染DEPTOR siRNA組、轉(zhuǎn)染DEPTOR siRNA +雷帕霉素組,觀察各組HUVEC小管形成情況。三、統(tǒng)計(jì)學(xué)處理在此實(shí)驗(yàn)中,所有數(shù)據(jù)結(jié)果均重復(fù)3次或以上,結(jié)果用均數(shù)±標(biāo)準(zhǔn)誤來表示,數(shù)組間用t檢驗(yàn)及兩因素方差分析進(jìn)行分析。當(dāng)P0.05代表結(jié)果有統(tǒng)計(jì)學(xué)意義。四、研究結(jié)果1、成功構(gòu)建了在血管內(nèi)皮細(xì)胞進(jìn)行特異性DEPTOR敲除的小鼠。免疫熒光染色表明DEPTOR蛋白表達(dá)在CD31標(biāo)記的KO小鼠的血管EC中降低。與WT小鼠相比,Western blot結(jié)果證明,在KO小鼠組織中的Deptor表達(dá)顯著降低。2、血管內(nèi)皮細(xì)胞特異性敲除Deptor激活mTOR信號(hào)。與WT小鼠相比,KO小鼠組織中pS6明顯升高。3、血管內(nèi)皮細(xì)胞敲除Deptor促進(jìn)了組織血管生成,并且雷帕霉素可以逆轉(zhuǎn)這一表型。與WT小鼠相比,KO小鼠組織,CD31組織化學(xué)染色數(shù)量顯著增加。用雷帕霉素處理KO小鼠時(shí),CD31組織化學(xué)染色減少。western blot結(jié)果與其一致。4、在KO小鼠中,VEGF和HIFM α的表達(dá)增加,雷帕霉素可以逆轉(zhuǎn)這一表型。免疫組織化學(xué)染色顯示,與WT小鼠相比,KO小鼠的組織中VEGF和HIF-1α的表達(dá)增加。用雷帕霉素處理后,與KO小鼠相比,KO + RAPA小鼠中VEGF的染色數(shù)量明顯減少,Western blot結(jié)果一致。5、用 DEPTOR siRNA 轉(zhuǎn)染 HUVEC,CD31,VEGF 和 HIF-1α 的表達(dá)也顯著增加。用雷帕霉素處理,抑制了 pS6K,pS6的增加,且CD31,VEGF和HIF-1α的表達(dá)明顯減少。與對照組相比,加入DEPTOR siRNA的HUVEC小管生成增加,而雷帕霉素處理組可逆轉(zhuǎn)增加。五、結(jié)論1、在血管內(nèi)皮細(xì)胞內(nèi)敲除Deptor基因,可以促進(jìn)mTOR信號(hào)通路的活化,進(jìn)而促進(jìn)組織血管的形成。2、血管內(nèi)皮細(xì)胞內(nèi)mTOR信號(hào)通路活化,促進(jìn)VEGF、缺氧誘導(dǎo)因子的的釋放,促進(jìn)血管形成,加雷帕霉素可以減少這一表型。
[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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Nan Wang;Ruijuan Wu;Xiaoheng Cheng;Jin Jin;Zongchao Jia;Jimin Zheng;;New insights into mTOR structure and regulation[J];Chinese Science Bulletin;2014年24期

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本文編號(hào)：1404981