血管内皮生长因子165基因对大鼠血管损伤后重塑的实验研究

目的探讨局部转染血管内皮生长因子165基因(VEGF165cDNA)对大鼠血管球囊拉伤术后血管重塑的影响。方法制备VEGF165cDNA基因,将30只SD大鼠随机分为基因组、手术对照组及正常对照组(各10只),基因组通过尾静脉输入VEGF165cDNA基因,手术对照组及正常对照组只注入生理盐水,应用免疫组织化学、电镜、光镜等方法观察球囊拉伤后的大鼠股动脉内膜的变化,检测拉伤局部内膜VEGF165基因作用效果。结果基因组球囊拉伤血管24 h后内膜处有少量中性粒细胞浸润,手术对照组损伤内膜有较多的中性粒细胞浸润,14 d后基因组球囊拉伤血管内膜厚度小于手术对照组(P<0.01),管腔内径大于手术对照组(P<0.01),内膜有大量VEGF蛋白表达。结论病理性重塑是血管损伤后再狭窄的主要原因,而VEGF165基因可促进内皮细胞增生,加速损伤内膜的修复,抑制血管内膜平滑肌细胞的增生进而防治再狭窄。     

“分子搭桥术”基因治疗闭塞性血管病的实验研究

目的 应用血管内皮生长因子（ＶＥＧＦ）基因治疗实验性闭塞性外周血管病。方法构建重组ＶＥＧＦ１６５基因的真核表达载体，其上游含巨细胞病毒（ＣＭＶ）启动子；通过基因缝线，向一侧髂外动脉结扎引起血管闭塞症大鼠的肌肉内转移ＶＥＧＦ基因（２００μｇ／只）；应用逆转录－聚合酶链反应，Ｗｅｓｔｅｒｎｂｌｏｔ以及免疫组织化学方法观察ＶＥＧＦ基因的表达，并通过血管造影技术观察ＶＥＧＦ基因导入大鼠体内的生物效应。结果转ｐｃＤＮＡ３／ＶＥＧＦ基因７天后，肌肉组织内ＶＥＧＦｍＲＮＡ及其表达产物明显高于对照组单纯转ｐｃＤＮＡ３组；血管造影可见大量新生血管和侧支循环的形成，在１４天和３０天时更明显，转ＶＥＧＦ基因可以明显促进闭塞性下肢血流的恢复和改善组织坏死的程度。结论肌肉内转移ＶＥＧＦ基因，通过促进血管新生和侧支循环建立，促进血流恢复，为闭塞性血管病的治疗，提供一种新的可能性──分子搭桥术。     

血管活性肽在大鼠血管再狭窄形成中的作用

目的 研究血管活性肽在血管再狭窄形成中的作用。方法 在大鼠主动脉内皮球囊拉伤模型上,采用放射免疫法测定大鼠血浆及主动脉组织内皮素（ＥＴ）、降钙素基因相关肽（ＣＧＲＰ）和肾上腺髓质素（Ａdm）含量以及组织中血管紧张素Ⅱ（ＡⅡ）含量,用∧３Ｈ－ＴdＲ掺入法和组织学分析观察血管平滑肌细胞（ＶＳＭＣ）增生以及血管内膜／中膜面积比值。并在培养的ＶＳＭＣ上,采用∧３Ｈ－ＴdＲ掺入法研研究血管活性肽对细胞增生的影响。采用ＲＴ－ＰＣＲ法观察血管活性肽对自发性高血压大鼠（ＳＨＲ）和ＷＫＹ大鼠的主动脉和ＶＳＭＣ高血压相关基因－１（ＨＲＧ－１）表达的影响。结果 术后１０d ,血浆及主动脉ＥＴ达高峰,分别较对照组升高６９％和1２４％（Ｐ＜０．０１）;主动态ＡⅡ含量升高８０％（Ｐ＜０．０１）。应用ＥＴ抗血清或卡托普利可明显抑制血管损伤诱导的ＶＳＭＣ增殖和内膜增厚。血浆和主动脉ＣＧＲＰ水平在术后３d升高６４％和８９％（Ｐ＜０．０１）, 术后１０d血浆和组织Ａdm分别升高１２９％和１０２％（Ｐ＜０．０１）。在体应用ＣＧＲＰ（２５μg/kg）可显著抑制管损伤诱导的ＶＳＭＣ增殖和内膜增厚（抑制率分别为６６％和７９％,Ｐ＜０．０１）。ＥＴ和ＡⅡ抑制血管ＨＲＧ－１表达,激活丝裂素活化蛋白素酶（ＭＡＰＫ）;ＣＧＲＰ和Ａdm诱导ＨＲＧ－１表达,并抑制ＭＡＰＫ活性。结论 ＥＴ和ＡⅡ可促进损伤血管内增殖,而ＣＧＲＰ和Ａdm具有代偿性抗内膜增殖作用。ＥＴ、ＡⅡ、ＣＧＲＰ和Ａdm等血管活性肽可通过调节ＨＲＧ－１表达和ＭＡＰＫ途径以调控ＶＳＭＣ增殖,影响损伤血管狭窄的形成。     

血管内皮生长因子基因治疗兔下肢缺血的实验研究

目的：了解血管内皮生长因子（ＶＥＧＦ）基因治疗兔下肢缺血模型后新生血管和侧支循环形成状况。方法：构建ｈＶＥＧＦ１６５的真核表达载体,通过直接肌肉注射将ｈＶＥＧＦ１６５基因转染缺血部位肌细胞,经颈总动脉插管行下肢血管造影。结果：ＶＥＧＦ治疗组在质粒转染后２和４周动脉血管造影显示,新生血管数目和侧枝血管形成较对照组明显增加。结论：肌细胞能够吸收裸露的ＶＥＧＦ ｃＤＮＡ质粒并获得局部高效表达,产生刺激体     

血管内皮生长因子在脑星形细胞瘤中的表达及其临床意义

脑星形细胞瘤往往伴有血管增生,血管增生程度与其预后密切相关。血管内皮生长因子（ｖａｓｃｕｌａｒｅｎ－ｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）被认为是促内皮细胞有丝分裂和血管增生的重要血管生长因子之一。为探索ＶＥＧＦ蛋白表达与星形细胞瘤病理分级和临床预后的关系,采用免疫组织化学方法,结合临床随访,对１０７例脑星形细胞瘤瘤组织和２５例正常脑组织中ＶＥＧＦ蛋白表达水平进行定性和定量检测分析。结果显示：①正常脑组织ＶＥＧＦ表达极低,星形细胞瘤瘤组织ＶＥＧＦ表达明显增高（Ｐ＜０．００５）;②脑星形细胞瘤ＶＥＧＦ表达随其病理分级增加而增高（Ｐ＜０．０１）;③星形细胞瘤ＶＥＧＦ表达愈高的病人,其临床６月、１年、２年存活率愈低。因而,ＶＥＧＦ表达与脑星形细胞瘤病理分级和临床预后密切相关。ＶＥＧＦ表达水平的检测可能对判断脑星形细胞瘤病理分级及预后有重要的临床应用价值     

大鼠心肌缺血的血管内皮生长因子基因治疗

目的：应用血管内皮生长因子（ｖａｓｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈ ｆａｃｔｏｒ, ＶＥＧＦ） 基因治疗心肌缺血。方法：雄性Ｗｉｓｔａｒ 大鼠２０ 只,结扎冠状动脉前降支造成心肌梗死模型。心脏多点注射构建的ｐｃＤ２／ＶＥＧＦ真核表达质粒。应用逆转录聚合酶链反应,ＶＥＧＦ免疫组织化学染色,心肌碱性磷酸酶染色及血管计数等方法检测ＶＥＧＦ 基因在缺血心肌中的表达及生物学作用。结果：转移ＶＥＧＦ基因后在大鼠缺血心肌中有ＶＥＧＦｍＲＮＡ 高表达, ＶＥＧＦ 免疫组化染色可见ＶＥＧＦ蛋白表达水平升高。心肌碱性磷酸酶染色显示,转移ＶＥＧＦ基因能够促进缺血心肌新生血管的形成。结论：ＶＥＧＦ基因能够在大鼠缺血心肌中获得表达,并发挥其促进新血管形成的作用。为ＶＥＧＦ 基因治疗心肌缺血奠定基础。     

血管内皮生长因子受体介导的靶向性非病毒载体的改进与体内 …

目的 通过构建一种有相对靶向性的基因治疗载体,将外源基因导入新生血管内皮细胞,为开展靶向于肿瘤新生血管的基因治疗奠定基础。方法 根据血管内皮生长因子（ＶＥＧＦ）与ＶＥＧＦ受体结合位点,修改本实验室已有的酸体寡肽ＧＶ１、ＧＶ２氨基酸序列,并合成了基因转移载体系统。用载体包裹外源ＤＮＡ（β－ｇａｌ）,进行体内实验,观察外源基因在体内的分布和不同时间的表达情况。结果 报告基因在心、肺、肝和肾中无明显地表     

大鼠颌下腺血管内皮生长因子cDNA的转移及表达

目的：探讨以颌下腺为靶器官,进行血管内皮生长因子（ＶＥＧＦ）基因治疗后,颌下腺中ＶＥＧＦ含量变化。方法：构建了ＶＥＧＦ基因表达的真核细胞表达载体,以阳离子脂质体为介质,向大鼠颌下腺转染外源性的ＶＥＧＦ基因,应用ＲＴ－ＰＣＲ技术观察ＶＥＧＦ基因的表达,ＥＬＩＳＡ方法检测颌下腺中ＶＥＧＦ的含量变化。结果：转ｐＢＫＣＭＶ－ＶＥＧＦ１２１基因后第１天,大鼠颌下腺内ＶＥＧＦ的表达及涎液中ＶＥＧＦ的含量即开始     

大鼠脾源性内皮祖细胞移植在损伤血管内膜修复中的作用

目的研究血管内皮祖细胞（endothelial progenitor cells,EPCs）移植在损伤血管再内皮化及抑制新生内膜增生中的作用。方法大鼠脾源性单个核细胞贴壁培养法定向扩增EPCs,检测其内皮细胞特性。通过尾静脉将EPCs移植到颈动脉内皮损伤的大鼠体内。10只大鼠行偶氮蓝染色,观察内皮损伤血管段再内皮化情况;其余大鼠于术后14d处死,行病理学观察。结果脾源性单个核细胞体外可诱导出内皮祖细胞,表现为表达内皮细胞特异性标志。EPCs移植组损伤血管57％不被偶氮蓝着染,而球囊损伤组损伤血管几乎完全被偶氮蓝染成蓝色。球囊损伤＋EPCs移植组在球囊损伤2周后血管新生内膜增生明显减轻,血管腔狭窄程度显著减轻。球囊损伤＋EPCs移植组新生内膜与中膜比值显著低于单纯球囊损伤组及M199组。球囊损伤＋EPCs移植组PCNA阳性表达细胞较单纯球囊损伤组及M199组明显减少。结论EPCs参与损伤血管的再内皮化过程。增加循环EPCs数量可以促进损伤血管的再内皮化,抑制血管平滑肌细胞增殖,减轻新生内膜增生程度。     

血管内皮生长因子在人卵巢和子宫内膜上的表达

目的 探讨血管内皮生长因子（ＶＥＧＦ）在人卵巢、子宫内膜中的表达及其在月经周期中的变化规律。方法 收集人正常卵巢、子宫内膜组织标本各２０例，采用免疫组织化学方法检测ＶＥＧＦ的表达。结果 （１）卵巢ＶＥＧＦ表达定位于颗粒胞浆及胞膜，卵泡膜细胞也有少量分泌，卵泡液呈阳性表达；原始卵泡、初级卵泡、次级卵泡表达依次增强，三者之间表达有显著性差异（Ｐ〈０．００１），（２）子宫内膜ＶＥＧＦ表达定位于腔上皮及腺     

人血管内皮生长因子165与嵌合水蛭肽融合基因防治兔颈动脉损伤后再狭窄的实验研究

目的观察人血管内皮生长因子165（hVEGF165）与嵌合水蛭肽融合基因（fused hirudin,FH）对兔颈动脉损伤后再狭窄的防治作用,为再狭窄的基因治疗提供新的思路。方法通过基因重组构建hVEGF。与FH的融合基因（hVEGF165-FH）。制备免颈总动脉血管成形术损伤后再狭窄模型,损伤局部转染该融合基因作为治疗组。同时局部转染pcDNA3．0空质粒作为对照组,通过血管造影、病理分析等观察融合基因是否抑制兔颈总动脉血管成形术后再狭窄。结果局部转染融合基因后1周和3周,造影显示治疗组血管狭窄程度与对照组比较显著减轻;病理分析显示。治疗组较对照组管腔狭窄程度显著减轻（1周：11．50％vs33．25％;3周：19．75％vs52．25％,P〈0．05）;治疗组较对照组内膜增生程度（内膜／中膜,I／M）显著减轻（1周：0．12vs0．50;3周：0．35vs1．07,P〈0．05）。同时融合基因对血管损伤处的血栓形成有抑制作用,但对系统凝血指标无影响,无全身出血并发症。结论融合基因hVEGF165-FH可有效地预防兔颈动脉成形术后再狭窄。以内皮修复为基础,构建双靶点甚至多靶点的融合基因可能是未来再狭窄防治的一个有效策略。     

VEGF165真核表达载体的构建及其在鼠膀胱平滑肌细胞的表达

目的 构建血管内皮生长因子(VEGF165)真核表达载体pcDNA3.1(-)/VEGF165,转染鼠膀胱平滑肌细胞并检验其生物学活性.方法 将VEGF165 cDNA克隆于真核表达载体pcDNA3.1(-),构建真核表达载体pcDNA3.1(-)/VEGF165;并转染入鼠膀胱平滑肌细胞,采用RT-PCR和免疫荧光染色检测VEGF165基因在鼠膀胱平滑肌细胞中的表达,并用MTT法检测转染后细胞上清液中VEGF165的生物学活性.结果 和结论将构建的真核表达载体pcDNA3.1(-)/VEGF165转染入鼠膀胱平滑肌细胞后,VEGF的表达增高,转染后细胞上清液具有促使内皮细胞增殖的生物学活性.     

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.     

VEGF真核表达载体构建及其转染骨髓间充质干细胞的表达

目的 构建大鼠血管内皮细胞生长因子164（vascular endothelial growth factor,VEGF164）真核表达载体pcDNA3.1(-)/VEGF164,观察大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)转染pcDNA3.1(-)/VEGF164基因后,外源性基因VEGF在蛋白水平的表达,为其在治疗肺组织损伤中的应用奠定基础。方法 应用DNA重组技术构建VEGF164真核表达载体并鉴定。采用脂质体介导的转染方法将pcDNA3.1(-)/VEGF164转染大鼠MSCs,并设立空质粒对照组（转染pcDNA3.1空载体）、脂质体对照组,用细胞免疫组化和Western blot检测大鼠MSCs 中VEGF164蛋白的表达情况。结果 重组质粒pcDNA3.1(-)/VEGF164测序结果与基因库(GENEBANK)中序列一致。细胞免疫组化检测显示pcDNA3.1(-)/VEGF164转染组MSCs中可见散在黄色颗粒,而空质粒对照组和脂质体对照组均未见明显黄色颗粒出现。Western blot检测pcDNA3.1(-)/VEGF164转染MSCs 72h后, 细胞裂解产物中VEGF含量明显高于空质粒对照组和脂质体对照组。结论 成功构建了大鼠VEGF164真核表达载体,实现VEGF164基因在大鼠MSCs的成功转染,并有外源性基因及蛋白的表达,具有促进肺组织损伤修复的应用前景。     

VEGF真核表达载体构建及其转染骨髓间充质干细胞并鉴定

目的 构建大鼠血管内皮细胞生长因子164（vascular endothelial growth factor,VEGF164）真核表达载体pcDNA3.1(-)/VEGF164,观察大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)转染pcDNA3.1(-)/VEGF164基因后,外源性基因VEGF在蛋白水平的表达,为其在治疗肺组织损伤中的应用奠定基础。方法 应用DNA重组技术构建VEGF164真核表达载体并鉴定。采用脂质体介导的转染方法将pcDNA3.1(-)/VEGF164转染大鼠MSCs,并设立空质粒对照组（转染pcDNA3.1空载体）、脂质体对照组,用细胞免疫组化和Western blot检测大鼠MSCs 中VEGF164蛋白的表达情况。结果 重组质粒pcDNA3.1(-)/VEGF164测序结果与基因库(GENEBANK)中序列一致。细胞免疫组化检测显示pcDNA3.1(-)/VEGF164转染组MSCs中可见散在黄色颗粒,而空质粒对照组和脂质体对照组均未见明显黄色颗粒出现。Western blot检测pcDNA3.1(-)/VEGF164转染MSCs 72h后, 细胞裂解产物中VEGF含量明显高于空质粒对照组和脂质体对照组。结论 成功构建了大鼠VEGF164真核表达载体,实现VEGF164基因在大鼠MSCs的成功转染,并有外源性基因及蛋白的表达,具有促进肺组织损伤修复的应用前景。     

Treatment of hemangioma by transfection of antisense VEGF gene

The effect of transfection of antisense vascular endothelial growth factor （VEGF） gene on the growth of hemangioma was studied. A total of 49 cases of capillary hemangiomas of the skin were collected. Immunohistochemical method was used to detect the expression of PCNA in hemangioma tissues. According to the finding, 49 cases of hemangiomas fell into proliferating phase （27 cases） and involuting phase （22 cases） respectively. Another 5 cases of normal skin tissues adjacent to the tumor tissues served as control. Immunohistochemical staining was performed to detect the expression of VEGF in the tumor tissues and the normal tissues. The average absorbance （A） values and the average positive area rate of VEGF were measured by image analysis system （HPIAS-2000）. Endothelial cells from the tumor tissues in proliferating phase were cultured. Eukaryotic expression vector was constructed by sub-cloning, and transfected into human hemangioma endothelial cells by using cation liposome as vector. The expression of VEGF mRNA and protein was detected by RT-PCR and indirect immunofluorescence assay （IFA）, respectively, and the biological characteristics of the transfected endothelial cells were examined by MTT assay and flow cytometry （FCM） after transfection. Immunohistochemical results showed that the expression of VEGF in proliferating endothelial cells was remarkably higher than those in involuting endothelial cells and normal endothelial cells （P〈0.01）, but there was no significant difference in the expression of VEGF between involuting endothelial cells and normal ones （P〉0.01）. Electrophoresis and sequencing indicated that the eukaryotic expression vector containing antisense VEGF gene, i.e. pcDNA3.1-VEGF, was success- fully constructed. After VEGF antisense RNA recombinant was transfected into hemangioma endothelial cells, RT-PCR revealed that the expression of VEGF mRNA in pcDNA-VEGF （V） group and blank group was obviously higher than that in pcDNA-VEGF （A） group, and that the expression of endogenous VEGF mRNA in pcDNA-VEGF （A） group was significantly inhibited. Immunohistochemical result demonstrated that, compared with blank group, there was statistically significant difference between pcDNA-VEGF （A） and pcDNA-VEGF （V） groups （P〈0.01）, but there was no significant difference between pcDNA-VEGF （V） group and blank group （P〉0.05）. The activity of endothelial cell proliferation was reduced significantly after transfection, and obvious apoptosis occurred in hemangioma endothelial cells after transfection of antisense VEGF. It was suggested that VEGF plays an important role in the pathological change of hemangiomas by promoting endothelial cell proliferation and angiogenesis. Antisense VEGF gene transfection could effectively inhibit the growth of hemanioma endothelial cells.     

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。     

Experimental study of adenovirus vector mediated-h VEGF<Subscript>165</Subscript> gene on prevention of restenosis after angioplasty

Summary This study evaluated the effects of adenovirus vector mediated human vascular endothelial 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 adenoviruses 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 immunohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in carotid 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 expression were detectedin 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 area/media area (I/M), maximal intimal and medial thicknesses (IT_max and MT_max) 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 recombinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit thrombokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty. Liu Qigong, male, born in 1968, Associate Professor This project was supported by a grant from Wuhan Chenguang Program (No. 20015005048)     

RI基因真核表达载体的构建及其对人脐静脉内皮细胞的影响

目的构建融合表达载体pcDNA3.1-RI,并检测核糖核酸酶抑制因子(ribonuclease inhibitor,RI)基因与血管生成素(angiogenin,ANG)的关系及对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖及迁移能力的影响。方法用RT-PCR方法扩增RI基因,酶切后将其插入pcDNA3.1,构建融合表达载体pcDNA3.1-RI,在脂质体介导下转染HUVECs,RT-PCR检测RI、ANG基因的mRNA表达水平;Western blot检测RI、ANG、MMP-2、MMP-9的表达水平;CO-IP法检测ANG和RI的相互作用,MTT法检测细胞的增殖活力,流式细胞仪检测细胞周期分布。结果真核表达质粒构建成功;转染pcDNA3.1-RI组细胞RI基因的mRNA及蛋白的表达较2个对照组(转染pcDNA3.1空载体组和未转染质粒组)均呈显著性增加(P<0.05),而ANG基因的mRNA及蛋白的表达均降低(P<0.05),MMP-2、MMP-9蛋白表达水平亦降低(P<0.05);CO-IP法检测到ANG和RI在细胞内能结合;转染pcDNA3.1-RI质粒到HUVECs细胞后细胞的增殖活力明显降低(P<0.05),G0～G1期比例明显增加,S期减少。结论成功构建的真核表达质粒能显著增加RI基因及其蛋白水平的表达,RI可以直接在转录水平上降低ANG的表达,在细胞内与ANG结合,从而影响内皮细胞的增殖、迁移能力。     

人VEGF基因杆状病毒表达载体的构建及生物学活性鉴定

目的构建含人血管内皮细胞生长因子(VEGF)基因的杆状病毒表达载体,并对其表达产物的生物学活性进行测定。方法用HindⅢ对pcDNA3.1/VEGF进行酶切,回收575 bp的VEGF片段;pFast a载体用HindⅢ酶切回收,与VEGF片段连接。NcoI酶切鉴定方向,得到正向连接重组载体pFast/VEGF;将其转化DH10BAC感受态细胞,经卡那霉素、四环霉素、庆大霉素及蓝白斑筛选得到重组杆状病毒载体pBacmid-Fa-VEGF,PCR扩增鉴定得到单一VEGF条带。将该病毒载体转染sf9细胞,用噬斑筛选及SDS-PAGE和Western blot检测VEGF表达,并通过MTT法检测VEGF促内皮细胞生长活性。结果病毒液在稀释到10-7时出现噬斑,Western blot检测出现单一条带,MTT检测促内皮细胞生长率为19.44%。结论成功构建了含VEGF基因的杆状病毒表达载体,且表达产物具有显著的促内皮细胞生长作用。