Expression of human VEGF121 cDNA in mouse bone marrow stromal cells

目的构建人VEGF121逆转录病毒表达载体,探讨应用血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)基因治疗骨科缺血性疾患的可行性.方法采用分子克隆技术从pCDⅠ/VEGF121质粒获得hVEGF121cDNA, 并克隆到pLXSN逆转录病毒质粒.经酶切及测序鉴定正确后,包装成为pLXSN/VEGF121重组逆转录病毒, 并进行滴度测定.培养小鼠骨髓基质细胞,用重组病毒感染小鼠骨髓基质细胞.经筛选后,用PCR及RT-PCR方法测hVEGF121cDNA在基质细胞中的整合及转录;用免疫印迹及免疫组化检测VEGF121蛋白的表达;用MTT检测转基因细胞培养上清中VEGF的促人内皮细胞增殖活性.结果经酶切鉴定及基因测序证实重组逆转录病毒质粒正确,包装的病毒滴度为6×105.PCR证实基因组DNA有hVEGF121cDNA的整合;RT-PCR证实有hVEGF121mRNA的转录;Western blot及免疫组化检测有VEGF121蛋白的表达;MTT试验显示转基因细胞培养上清中VEGF121能促进人内皮细胞增殖.结论我们成功地构建了pLXSN/VEGF121重组逆转录病毒载体,该载体不仅能够有效地表达VEGF121蛋白,且表达的蛋白具有促进人内皮细胞增殖的生物学活性,这些均为进一步应用该载体研究VEGF基因治疗骨科缺血性疾患打下了基础.     

腺相关病毒介导bFGF基因转染促进人骨髓基质细胞成骨实验研究

目的:观察腺相关病毒介导人碱性成纤维细胞因子(bFGF)基因转染对人骨髓基质细胞(hMSCs)成骨能力的影响。方法:从健康志愿者全骨髓中分离培养人骨髓基质细胞,体外扩增纯化后随机分为4组。①腺相关病毒介导人碱性成纤维细胞因子(AAV-bFGF)组:培养液中加入人碱性成纤维细胞因子基因重组腺相关病毒1×1010OPU/ml,孵育24h后换普通培养液继续培养;②β半乳糖酐酶基因重组腺相关病毒(AAV-LacZ)组:用半乳糖酐酶基因重组腺相关病毒替换bFGF,其余处理与AAV-bFGF组相同;③阳性对照组:培养液中添加地塞米松(1nmol/L)、抗坏血酸(10mmol/L)和β-甘油磷酸钠(50mg/L);④空白对照组:不给予特殊处理。各组细胞处理后2wk,免疫组化观察bFGF表达,Von Kossa染色检测人骨髓基质细胞中骨胶原结节形成,全自动生化分析仪检测培养上清碱性磷酸酶(ALP)活性。结果:经多次换液传代,人骨髓基质细胞呈均一梭形形态。处理后AAV-bFGF组与阳性对照组细胞形态逐渐趋于扁平,突起减少。AAV-LacZ组和空白对照组形态无明显变化。免疫组化染色见bFGF主要在胞浆内表达,AAV-bFGF组bFGF表达明显强于其他3组。von Kossa染色AAV-bFGF组与阳性对照组可见大量红色骨胶原结节形成,显著多于AAV-LacZ组和空白对照组。培养上清液ALP活性检测AAV-bFGF组与阳性对照组显著高于AAV-LacZ组和空白对照组。结论:基因重组腺相关病毒人碱性成纤维细胞因子转染对人骨髓基质细胞成骨能力具有促进作用。     

人血管内皮细胞生长因子165重组腺病毒的构建及其在真核细胞中的表达

目的:体外构建人血管内皮生长因子165(hVEGF165)的腺病毒表达载体,并检测其在HEK293细胞中的表达。方法:从重组质粒pcDNA3/hVEGF165中获得hVEGF165,经酶切及测序鉴定,将hVEGF165基因亚克隆到穿梭质粒pAdTrack-CMV,重组穿梭质粒经酶切线性化后,与pAEdasyl质粒在大肠杆菌BSJ183中进行同源重组,线性化后转染HEK293细胞进行包装扩增获取重组病毒上清,同时应用RT-PCR及ELISA法检测hVEGF165的表达。结果:目的基因的测序结果与人VEGF165序列(Genbank)相符。转染后经RT-PCR和ELISA检测证实转染重组质粒组hVEGF165 mRNA及蛋白表达,而转染空质粒组及未转染组没有检测到hVEGF165的表达。结论:本研究构建了人VEGF165重组腺病毒表达载体pAd-hVEGF165,并能成功地在HEK293细胞中表达。     

人骨髓间充质干细胞分离培养鉴定及VEGF基因的转染

目的：探讨人骨髓间充质干细胞（hMSC）分离培养、鉴定方法及人血管内皮生长因子（hVEGF）165基因转染hMSC后表达情况,拟构建血管化组织工程皮肤.方法：用密度梯度离心法分离骨髓间充质干细胞,筛选贴壁细胞培养并传代.观察细胞形态,生长情况.检验细胞CD44、CD29、CD14、CI45等表面标记.利用基因重组技术,构建pCDN3.1＋VEGF表达质粒,并将其转染第3代hMSC.经G418抗性筛选后扩大培养并用Western blot实验检测其蛋白产物.结果：骨髓密度梯度离心法分离hMSC,接种培养瓶后26 h呈对数生长,6d后达到高峰.hMSC表达CD29,CD44,不表达造血细胞标记物CD14和CD45.通过酶切鉴定验证成功构建pCDN3.1＋ VEGF表达质粒.Western blotting实验证实转染后hMSCs表达VEGF增加.结论：成功建立hMSC分离培养鉴定体系.利用基因重组技术可成功将hVEGF基因导入hMSC并产生预期效应.     

腺病毒介导的血管内皮生长因子体外靶向性转染心肌细胞

目的:构建以鼠心肌轻链蛋白基因(mlc-2v)为启动子、携带人血管内皮生长因子hVEGF165基因的重组腺病毒载体,检测该重组腺病毒载体对心肌细胞转染的靶向性.方法:酶切腺病毒载体PDC315自身启动子CMV,构建腺病毒载体PDC317,分别接入hVEGF165、mlc-2v基因,构建腺病毒载体PDC-mlc-hVEGF165.鉴定正确后,将PDC-mlc-hVEGF165与Lipofectamine共转染至293细胞,经同源重组获得以mlc-2v为启动子、携带hVEGF165基因的重组腺病毒Ad-mlc-hVEGF165,同步构建无靶向性的重组腺病毒Ad-hVEGF165.扩增并测定滴度后,Ad-hVEGF165、Ad-mlc-hVEGF165分别转染体外培养的心肌细胞、骨骼肌细胞及平滑肌细胞,利用ELISA、Western印迹分析等方法检测Ad-mlc-hVEGF165转染心肌细胞的靶向性.结果:构建的病毒正确,病毒滴度为2.8×109 pfu/ml.转染3 d后,Ad-hVEGF165组在各培养细胞的上清液及细胞内均检测到VEGF的表达,而Ad-mlc-hVEGF165组仅在心肌细胞中有VEGF的表达,且Ad-mlc-hVEGF165组心肌细胞分泌的VEGF要少于Ad-hVEGF165组.结论:成功构建了以mlc-2v为启动子、携带人VEGF基因的重组腺病毒Ad-mlc-hVEGF165;该重组载体能体外靶向性转染心肌细胞并使之表达VEGF.     

人VEGF 165基因转染大鼠内皮祖细胞及对其增殖的影响

目的探讨人血管内皮细胞生长因子165（hVEGF165）基因转染骨髓源性内皮祖细胞（EPCs）及对EPCs增殖的影响。方法体外分离、培养、鉴定EPCs,脂质体介导pcDNA3-hVEGF165质粒转染组、pcDNA3空质粒转染组、空白对照组。ELISA检测EPCs表达VEGF蛋白,MTT法检测hVEGF165基因修饰对EPCs增殖的影响。结果FITC-UEA-Ⅰ和DiI-ac-LDL荧光双染证实分化的EPCs,脂质体介导pcDNA3-hVEGF165质粒转染组EPCs培养基上清中表达VEGF,hVEGF165基因转染促进EPCs增殖。结论EPCs可以成功转染hVEGF165基因,并且可促进EPCs的增殖,为进一步研究hVEGF165基因修饰EPCs治疗血管内膜损伤性疾病提供实验依据。     

重组腺病毒介导血管内皮生长因子基因在骨髓间充质干细胞中的表达及其影响

目的观察重组腺病毒介导血管内皮生长因子基因(AdVEGF165)转染后大鼠骨髓间充质干细胞(BMSCs)中血管内皮生长因子的表达情况以及腺病毒载体对大鼠骨髓间充质干细胞的生长影响情况。方法体外培养大鼠骨髓间充质干细胞,用腺病毒载体转染BMSCs。采用ELISA法检测血管内皮生长子因子(VEGF)的表达和分泌。转染后用胰酶消化传代培养,MTT法绘制细胞生长曲线。结果AdVEGF165转染组上清液中VEGF蛋白表达量极显著高于空白对照组(P<0.01)。且AdVEGF165转染BMSCs后,生长曲线与正常培养细胞无显著差异(P>0.05)。结论重组腺病毒载体能够在BMSCs中介导VEGF的表达,且对BMSCs生长无影响。     

人骨髓间充质干细胞向血管内皮细胞诱导分化的实验研究

目的：探讨人骨髓间充质干细胞（hMSCs）向血管内皮细胞（ECs）诱导分化的可行性。方法：用贴壁法从成人骨髓中分离出间充质干细胞,培养扩增后,加入10ng／ml的碱性成纤维细胞生长因子（bFGF）和人血管内皮生长因子（hVEGF）向ECs诱导分化,分别于0、24d流式细胞术检测细胞表型及荧光染色鉴定细胞。结果：hMSCs向ECs诱导分化24d,细胞CD34、KDR转为阳性,CD54、CD106、vWF表达增加。可摄取ac—LDL,并可结合UEA。结论：hMSCs经bFGF和VEGF作用,分化为具有ECs的表型和部分功能的细胞。     

腺病毒介导的骨形态发生蛋白-2基因转移促进人间充质干细胞成骨化

目的:观察腺病毒介导的骨形态发生蛋白-2(BMP-2)基因转移对人间充质干细胞(hMSCs)成骨能力的影响。方法:从健康志愿者全骨髓中分离培养hMSCs,体外扩增纯化后随机分为4组。(1)Ad-BMP-2组:培养液中加入BMP-2基因重组腺病毒(Ad-BMP-2,1×1010OPU/mL)孵育24h;(2)Ad-LacZ组:培养液中加入半乳糖酐酶基因重组腺病毒(Ad-LacZ);(3)阳性对照组:培养液中添加地塞米松、抗坏血酸和β-甘油磷酸钠。(4)空白对照组。2w后行Von Kossa染色检测hMSCs中骨胶原结节形成;生化分析仪检测碱性磷酸酶(ALP)活性。结果:经多次换液传代,hMSCs呈均一梭形。Ad-BMP-2组与阳性对照组细胞形态逐渐趋于扁平,突起减少,VonKossa染可见大量红色骨胶原结节形成,ALP活性也显著增高。结论:Ad-BMP-2基因转染对hMSCs成骨能力具有促进作用。     

骨髓间充质干细胞的肌源性诱导分化及转染VEGF基因的表达

目的体外诱导兔骨髓间充质干细胞向肌源性细胞分化,并观察血管内皮生长因子AdTrackCMV-hVEGF165真核表达质粒转染骨髓间充质干细胞(mesenchymal stem cells, MSCs)后的表达。方法将20只兔随机分为对照组和实验组,从骨髓液中分离MSCs,对照组以低糖DMEM培养,实验组加用5-氮胞苷(10 mmol/L)诱导培养,第28天进行肌钙蛋白I免疫组化染色。另构建AdTrackCMV-hVEGF165真核表达质粒,通过脂质体转染对照组MSCs,用Northern blotting和Western blotting鉴定血管内皮生长因子(VEGF)的表达,并用ELISA法测定培养上清液中VEGF的浓度。结果成功分离培养出兔MSCs,实验组经5-氮胞苷诱导后MSCs向肌源性细胞分化,肌钙蛋白I免疫组化染色阳性。成功构建AdTrackCMV-hVEGF165真核表达质粒并通过脂质体转染MSCs,Northern blotting示转染的MSCs VEGF165表达信号明显强于未转染细胞,Western blotting 示转染VEGF165基因的MSC表达VEGF,ELISA法测定培养上清液中VEGF的浓度在转染后第3天(1 011 pg/ml)和第5天(1 027 pg/ml)达高峰,此后逐渐下降,但至第13天(349 pg/ml)仍显著高于空白对照(116 pg/ml)和pAdTrackCMV组(125 pg/ml),P<0.01。结论兔骨髓间充质干细胞可在体外向肌源性细胞分化,转染VEGF基因可表达VEGF,有望将干细胞移植和基因治     

An experiment study of osteogenesis of Ad-VEGF165 transfected human bone marrow mesenchymal stem cells in vitro

Objective:To evaluate the effect of osteogenic potential on human marrow mesenchymal stem cells (hMSCs) transferred with human vascular endothelial growth factor(VEGF) gene by adenovirus. Methods :hMSCs were isolated from human marrow, cultured in vitro and randomly divided into 3 groups:Ad-VEGF165 group: adding 1×1010OPU/ml Ad-VEGF in hMSCs culture fluid after incubating 24 hours, changing into ordinary complete culture and continuing culturing; Positive control group: Cultured hMSCs with 1 nmol/L dexamethasone, 10 mmol/L glycerophosphate and 50 mg/L vitamin C,exchanging this conditioned medium twice a week; blank control group :no special treatment but culturing hMSCs in DMEM.To evaluate osteogenesis competence, Von Kossa's staining and a quantitative alkaline phosphates (ALP) activity analysis were performed after 2 weeks treatment. Results :The calcified nodes formed after 2 weeks treatment in Ad-VEGF165 group and Positive control group but not in blank control group. ALP activities in Ad-VEGF165 group,Positive control group and blank control group were (7.91 ± 0.90)u/L, (8.18 ± 0.76 u/L) and (3.46 ± 0.49)u/L respectively. The differences were no statistical significance between Ad-VEGF165 group and positive control group (P ＞ 0.05), but Ad-VEGF165 group and Positive control group were significantly different with blank control group (P ＜ 0.05). Conclusion:Adenovirus mediated VEGF165 gene can transfect hMSCs and promote osteogenesis of hMSCs.     

VEGF逆转录病毒载体构建及在小鼠骨髓中的表达(英文)

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血管内皮细胞生长因子在哺乳动物细胞的表达

目的：本实验试图构建具有生物学活性的ＶＥＧＦ真核表达载体,为ＶＥＧＦ基因治疗提供载体。方法：将已获得的ｈＶＥＧＦ１６５ ｃＤＮＡ克隆到ＧＳＴ融合表达载体,构建原核表达质粒,转染大肠杆菌ＤＨ５α,ＩＰＴＧ诱导表达,Ｗｅｓｔｅｒｎ ｂｌｏｔ鉴定表达产物。在此基础上构建真核表达载体ｐｃＤＮＡ３ ／ｈＶＥＧＦ１６５,脂质体介导转染ＣＨＯ－Ｋ１细胞,Ｇ－４１８筛选,Ｎｏｒｔｈｅｒｎ ｂｌｏｔ,Ｗｅｓｔ－ｅｒｎ ｂｌｏｔ分别检测其在细胞内的转录和表达情况,３Ｈ－ＴｄＲ掺入法检测表达蛋白的活性。结果：实验组的Ｎｏｒｔｈｅｒｎ ｂｌｏｔ和Ｗｅｓｔｅｒｎ ｂｌｏｔ出现特异性条带,而对照组在相应部位未出现条带。实验组的大鼠心肌血管内皮细胞３Ｈ－ＴｄＲ掺入率明显高于对照组（Ｐ＜ ０．００１）。结论：本实验所构建的ＶＥＧＦ真核表达质粒能够在真核细胞内获得表达,其表达产物具有血管内皮细胞增殖刺激活性。     

Construction and Identification of Recombinant Adenovirus Vector Containing the hVEGF165 Gene

Thedevelopmentofgene therapieshas renderedits clinical application feasible.Vascular endothelialgrowth factor( VEGF) hasbeen approved to be usedfor clinical trials.VEGF plays an important role inangiogenesis and prevention of restenosis[1-8] ,butthelow efficiency of plasmid vector restricts its clinicalapplication.In this study,the h VEGF165c DNA wassubcloned into p ACCMV .p Lp A and subsequentlythis recombinant was co- transfected into 2 93cellswith p JM1 7to obtain the replication- …     

骨髓间充质干细胞的肌源性诱导分化及转染VEGF基因的表达

OBJECTIVE: To induce the differentiation of rabbit bone marrow mesenchymal stem cells (MSCs) into myogenic cells in vitro, and to investigate the expression of vascular endothelial growth factor (VEGF) gene in AdTrackCMV-hVEGF165- transfected MSCs. METHODS: Twenty rabbits were divided equally into control group and experimental group, and MSCs were isolated and purified from their bone marrow by Percoll (1.073 g/ml) followed by cell culture in low-glucose DMEM supplemented with 10% fetal bovine serum. 5-azacytidine (5-Aza) was added into the cell culture of the experimental group on the third day. The expression of troponin I in MSCs was assayed by immunohistochemistry on the 28th day. AdTrackCMV- hVEGF165 eukaryotic expression vector was constructed and transfected into the MSCs, and subsequent VEGF expression was detected by Northern blotting and Western blotting while enzyme-linked immunosorbent assay (ILISA) was employed to examine the VEGF concentration in the supernatant of the culture medium. RESULTS: Following successful isolation and culture of the MSCs from rabbit bone marrow, 5-Aza-induced differentiation of the cells into myogenic cells was demonstrated by their positive staining for cardiac troponin I (cTnI). Northern blotting showed that the expression of VEGF 165 mRNA was much higher in the VEGF165 gene-transfected cells than in the control cells. Western blotting showed VEGF expression in the transfected cells. The concentration of VEGF in the supernatant mounted to the peak level 3-5 d after VEGF165 gene transfection (1,011-1,027 pg/ml) and decreased gradually thereafter, but still maintaining higher levels than those in the control group and pAdTrackCMV group (349 pg/ml vs 116 pg/ml and 125pg/ml, respectively, P<0.01). CONCLUSION: MSCs can be induced to differentiate into myogenic cells in vitro and express VEGF after VEGF gene transfection, and this success may provided a basis for combining MSC transplantation with gene therapy for regeneration of the damaged myocardial cells.     

Experimental Study of Adenovirus Vector Mediated-hVEGF165 Gene on Prevention of Restenosis after Angioplasty

This study evaluated the effects of adenovirus vector mediated human vascular endotheli-al growth factor-165 (hVEGF165) gene on prevention of restenosis after angioplasty. Rabbit models of bilateral carotid artery injury were established by balloon denudation. The recombinant adenovi-ruses containing hVEGF165 cDNA was directly injected into left side of the injured carotid arteries.On day 3 and week 3 after transfection the expression of VEGF was observed by RT-PCR and im-munohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in ca-rotid arteries were evaluated by computerized image analysis system 3 weeks after gene transfer,The changes in the VEGF gene-treated side were compared with the control side. Our results showed that 3 days and 3 weeks after hVEGF165 gene transfer the VEGF mRNA and antigen ex-pression were detected in vivo. 3 weeks after the transfer, the carotid artery rET was markedly better in the VEGF gene-treated group compared with the control. The thrombokinesis, intima are-a/media area (I/M), maximal intimal and medial thicknesses (ITmax and MTmax) demonstrated a statistically significant decrease in arteries treated with VEGF gene as compared with the control group. It is concluded that VEGF gene transfer could be achieved by intra-arterial injection of re-combinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit throm-bokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty.