本文選題：存活素 + 大鼠主動脈內(nèi)皮細(xì)胞　； 參考：《重慶醫(yī)科大學(xué)》2016年博士論文

【摘要】：周圍動脈疾病(Periphery Artery Disease,PAD)是一組表現(xiàn)為外周動脈狹窄、閉塞的疾病,以下肢發(fā)病最為常見。目前對于PAD的治療方法主要有藥物治療、外科手術(shù)治療等。研究認(rèn)為以上治療方式在增加行走時間及距離,減少截肢率及降低死亡率等方面的效果并不理想。治療性血管生成是治療PAD一種手段,是以血管生長因子的基因、蛋白或內(nèi)皮祖細(xì)胞等進(jìn)行局部或系統(tǒng)干預(yù)以促進(jìn)缺血組織血管生成,從而改善外周循環(huán)的方法。血管生成是指從已存在的微血管床上芽生出新的,以毛細(xì)血管為主的血管系統(tǒng)的過程,主要包括:細(xì)胞外基質(zhì)的降解;血管內(nèi)皮細(xì)胞的激活、增殖、遷移;管腔結(jié)構(gòu)的形成及血管網(wǎng)的重塑。存活素(Survivin,SVV)基因是一種廣泛表達(dá)于胚胎和腫瘤組織的多效性基因,SVV通過調(diào)節(jié)腫瘤細(xì)胞的凋亡、周期轉(zhuǎn)換、侵襲性等信號通路促進(jìn)腫瘤的生長、轉(zhuǎn)移及血管生成。目前SVV基因促進(jìn)腫瘤血管生成的研究已較深入,但關(guān)于SVV基因促進(jìn)正常組織或細(xì)胞血管生成的研究甚少,實驗采用腺病毒(Adenovirus,Ad)攜帶SVV基因轉(zhuǎn)染大鼠主動脈內(nèi)皮細(xì)胞(Rat Aortic Endothelial Cell,RAECs)的方式,觀察SVV基因?qū)AECs增殖、遷移、抗凋亡及血管生成的影響,以期于將SVV基因做為新的PAD治療的血管生成因子。第一部分腺病毒介導(dǎo)大鼠主動脈內(nèi)皮細(xì)胞存活素基因的轉(zhuǎn)染及鑒定目的:鑒定腺病毒介導(dǎo)SVV基因轉(zhuǎn)染RAECs后細(xì)胞內(nèi)SVV基因及蛋白的表達(dá)情況。方法:根據(jù)對RAECs不同的處理將其分為3組:SVV轉(zhuǎn)染組(Ad-GFP/SVV):RAECs轉(zhuǎn)染攜帶有SVV基因及GFP的腺病毒;陰性對照組(Ad-GFP):RAECs轉(zhuǎn)染僅攜帶有GFP的腺病毒;空白對照組(RAEC):RAECs正常培養(yǎng)不做任何處理。用Ad-GFP/SVV組行最佳MOI的鑒定,分別用MOI等于0、10、20、50、100的攜帶有GFP及SVV基因的腺病毒與RAECs共培養(yǎng),于12h、24h、48h、72h采用流式細(xì)胞儀計算感染率,確定最佳MOI和最佳感染時間后用q RT-PCR及Western blot檢測各組RAECs內(nèi)SVV的m RNA及蛋白的表達(dá)情況。結(jié)果:轉(zhuǎn)染48h后MOI=50的感染率為(95.67±2.16)%,MOI=100的轉(zhuǎn)染率為(94.53±1.76)%,組間差異無統(tǒng)計學(xué)意義(P0.05);q RT-PCR結(jié)果顯示SVV轉(zhuǎn)染組m RNA相對表達(dá)量顯著上調(diào),Western blot結(jié)果顯示與q RT-PCR結(jié)果一致,SVV轉(zhuǎn)染組與空白對照組及陰性對照組間差異具有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:以MOI=50時攜帶SVV基因的腺病毒轉(zhuǎn)染RAECs 48h可顯著提高其SVV基因和蛋白的表達(dá)。第二部分存活素基因?qū)Υ笫笾鲃用}內(nèi)皮細(xì)胞增殖的影響目的:研究SVV基因?qū)AECs增殖的影響。方法:根據(jù)對RAECs不同的處理將其分為3組:SVV轉(zhuǎn)染組(Ad-GFP/SVV):RAECs轉(zhuǎn)染攜帶有SVV基因及GFP的腺病毒;陰性對照組(Ad-GFP):RAECs轉(zhuǎn)染僅攜帶有GFP的腺病毒;空白對照組(RAEC):RAECs正常培養(yǎng)不做任何處理。按已確定的感染復(fù)數(shù)和感染時間用各組腺病毒轉(zhuǎn)染RAECs,細(xì)胞免疫熒光法檢測PCNA表達(dá),MTT法檢測RAECs的增殖活性,Western blot法檢細(xì)胞周期蛋白的表達(dá)。結(jié)果:SVV轉(zhuǎn)染組的PCNA陽性相對表達(dá)量為(85.35±4.93)%;陰性對照組為(51.04±6.86)%;空白對照組為(52.57±5.24)%。MTT法中轉(zhuǎn)染后第1天SVV轉(zhuǎn)染組的OD值為0.93±0.06;陰性對照組為0.77±0.09;空白對照組為0.71±0.14;轉(zhuǎn)染后第2天SVV轉(zhuǎn)染組的OD值為1.28±0.12;陰性對照組為0.91±0.05;空白對照組為0.87±0.06。SVV轉(zhuǎn)染組的PCNA表達(dá)量及MTT的OD值顯著高于陰性對照組及空白對照組,組間差異具有統(tǒng)計學(xué)意義(P0.05);SVV轉(zhuǎn)染組的周期蛋白cyclin B1、cyclin D1、cyclin E、CDC2、CDK 4、CDK2表達(dá)明顯上調(diào),與空白對照組及陰性對照組間差異具有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:SVV基因通過上調(diào)RAECs周期蛋白的表達(dá)促進(jìn)RAECs的增殖。第三部分存活素基因?qū)Υ笫笾鲃用}內(nèi)皮細(xì)胞凋亡的影響目的:研究SVV基因?qū)AECs凋亡的影響。方法:根據(jù)對RAECs不同的處理將其分為4組:SVV轉(zhuǎn)染組(Ad-GFP/SVV):RAECs轉(zhuǎn)染攜帶有SVV基因及GFP的腺病毒后缺氧條件下培養(yǎng)12h;陰性對照組(Ad-GFP):RAECs轉(zhuǎn)染僅攜帶有GFP的腺病毒缺氧條件下培養(yǎng)12h;缺氧對照組(Control):RAECs不行轉(zhuǎn)染缺氧條件下培養(yǎng)12h;空白對照組(RAEC):RAECs不行轉(zhuǎn)染常氧下培養(yǎng)12h。細(xì)胞轉(zhuǎn)染后于缺氧條件下誘導(dǎo)細(xì)胞凋亡,采用鬼筆環(huán)肽熒光染色法標(biāo)記微絲(F-actin)的表達(dá)情況,TUNEL綠色FITC標(biāo)記熒光檢測法及流式細(xì)胞儀檢測細(xì)胞凋亡的數(shù)量,Western blot檢RAECs內(nèi)凋亡相關(guān)蛋白Cleaved caspase-3,8,9的表達(dá)。結(jié)果:各組細(xì)胞經(jīng)低氧誘導(dǎo)凋亡后,光鏡下SVV轉(zhuǎn)染組與RAEC組的RAECs形態(tài)良好,細(xì)胞間聯(lián)系緊密,熒光顯微鏡下陰性對照組、缺氧對照組出現(xiàn)核固縮、核碎裂等典型的凋亡變化;SVV轉(zhuǎn)染組F-actin平行均勻分布于細(xì)胞中,與正常RAECs相似,在陰性對照組和缺氧對照組,F-actin降解增加,呈環(huán)形富集于細(xì)胞膜的周邊;流式細(xì)胞儀分析結(jié)果示SVV轉(zhuǎn)染組細(xì)胞凋亡率顯著降低,與陰性對照組、缺氧對照組比較差異有統(tǒng)計學(xué)意義(P0.05);Western blot結(jié)果示SVV轉(zhuǎn)染組RAECs的cleaved-Caspase-3,Caspase-8,Caspase-9的表達(dá)減少,與陰性對照組、缺氧對照組間差異具有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:SVV基因通過下調(diào)Caspase-3、8、9的表達(dá)抑制低氧誘導(dǎo)的RAECs凋亡。第四部分存活素基因?qū)Υ笫笾鲃用}內(nèi)皮細(xì)胞遷移與侵襲的影響目的:研究SVV基因?qū)AECs遷移與侵襲的影響。方法:根據(jù)對RAECs不同的處理將其分為3組:SVV轉(zhuǎn)染組(Ad-GFP/SVV):RAECs轉(zhuǎn)染攜帶有SVV基因及GFP的腺病毒;陰性對照組(Ad-GFP):RAECs轉(zhuǎn)染僅攜帶有GFP的腺病毒;空白對照組(RAEC):RAECs正常培養(yǎng)不做任何處理。細(xì)胞轉(zhuǎn)染后采用劃痕實驗,Transwell實驗、基質(zhì)膠小室實驗等評估RAECs的遷移能力,使用Western blot檢測細(xì)胞內(nèi)MMPs的表達(dá)水平。結(jié)果:SVV轉(zhuǎn)染組在6,12,24h遷移距離均較陰性對照組和空白對照組遷移距離顯著增加(P0.05);Transwell實驗中,SVV轉(zhuǎn)染組穿過聚碳酸酯膜的RAECs較陰性對照組和空白對照組顯著增加(P0.05);Western blot結(jié)果示SVV轉(zhuǎn)染組RAECs內(nèi)MMP-2、7、8、9的表達(dá)水平均高于陰性轉(zhuǎn)染組和空白對照組。結(jié)論:SVV通過上調(diào)MMP-2、7、8、9的表達(dá)增加RAECs遷移和侵襲能力。第五部分存活素基因?qū)Υ笫笾鲃用}內(nèi)皮細(xì)胞小管形成及血管生成的影響目的:觀察SVV基因?qū)AECs在體外管腔形成及體內(nèi)血管生成的影響。方法:根據(jù)對RAECs不同的處理將其分為3組:SVV轉(zhuǎn)染組(Ad-GFP/SVV):RAECs轉(zhuǎn)染攜帶有SVV基因及GFP的腺病毒;陰性對照組(Ad-GFP):RAECs轉(zhuǎn)染僅攜帶有GFP的腺病毒;空白對照組(RAEC):RAECs正常培養(yǎng)不做任何處理。SVV基因轉(zhuǎn)染后,將RAECs接種在Martigel基質(zhì)膠預(yù)處理的48孔板中,觀察管腔形成的情況,并用ELISA檢測細(xì)胞培養(yǎng)基上清液中血管內(nèi)皮生長因子(VEGF)的表達(dá);將轉(zhuǎn)染后的細(xì)胞與Martigel基質(zhì)膠混合液接種至裸鼠皮下,7天后采用HE染色、Masson染色及CD31免疫組化染色評估裸鼠皮下血管生成的情況。結(jié)果:SVV轉(zhuǎn)染組相對于正常對照組的小管與分支的比例顯著高于陰性對照組(P0.05)。ELISA檢測結(jié)果示SVV轉(zhuǎn)染組VEGF表達(dá)量顯著增加,組間差異具有統(tǒng)計學(xué)意義(P0.05)。在體實驗中,SVV轉(zhuǎn)染組可明顯見到橫行整齊排列的毛細(xì)血管,經(jīng)量化統(tǒng)計分析SVV轉(zhuǎn)染組被膜與膠栓內(nèi)血管數(shù)均顯著高于陰性對照組和正常對照組(P0.05)。結(jié)論:SVV基因使RAECs在基質(zhì)膠中直接參與管腔形成。SVV基因提高RAECs移植周圍組織中VEGF的表達(dá)水平,促進(jìn)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R543

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1 王福O，

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