血管内皮生长因子165基因对人胃癌细胞凋亡的影响及机制

目的: 本实验探讨血管内皮生长因子165(VEGF165)对体外培养的胃癌细胞株BGC-823凋亡的影响和机制.方法: 将BGC一823细胞分为对照组、感染复数(MOI=20)病毒Ad-GFP的Ad-GFP组,重组腺病毒Ad_VEGF165转染的Ad-VEGF165组,应用流式细胞仪检测细胞凋亡的百分率,R11-PCR方法检测凋亡抑制基因Bc1-2mRNA的表达,免疫细胞化学方法检测Bc1-2蛋白的表达情况.结果: 流式细胞仪测定显示Ad-VEGF165组的细胞凋亡率明显低于Ad-GFP组和对照组(4.6%±0.31% vs 8.37%±1.06%.7.73%±0.86%,P.<0.01):RT-PcR和细胞免疫化学结果显示VEGF165转染BGC.823细胞后促进了细胞Bc1-2mRNA和蛋白的表达,Ad.VEGF165组Bc1-2 mRNA和蛋白均高于对照组和Ad-GFP组(Bc1-2 mRNA:0.761±0.05 vs 0.363±0.12,0.356±0.08:Bc1-2蛋白:1.010±0.08 vs 0.865±0.07,0.901±0.05;P<0.01).结论: VEGF165通过上调凋亡抑制基因Bc1-2及其蛋白的表达,来抑制血浆饥饿诱导的胃癌细胞的凋亡.     

血管内皮生长因子165基因对人胃癌细胞体外侵袭转移的影响

目的:探讨血管内皮生长因子165基因(vascular endothelial growth factor 165,VEGF165)对胃癌侵袭转移的影响及可能的机制.方法:通过体外培养人胃癌细胞BGC-823,分别转染Ad-GFP及Ad-VEGF165*应用Millicell小室分析VEGF165转染前后胃癌细胞迁移能力的变化:逆转录聚合酶链反应(RT-PCR)研究VEGF懈转染前后BGC-823细胞MMP-2、u-PAmRNA表达变化.结果:迁移实验结果显示Ad-VEGF15组胃癌细胞迁移能力高于Ad-GFP组及对照组(217.53±15.77vs174.07±13.83,167.5±11.5,P＜0.05);RT-PCR结果显示VEGF懈转染后促进了BGC-823细胞MMP-2、u-PA的mRNA表达,Ad-VEGF165组MMP-2、u-PA的mRNA水平明显高于对照组及d-GFP组((MMP-2mRNA:0.6646±0.0486 vs 0.3803±0.0254.0.4096±0.0668;u-PA mRNA:0.8216±0.0798vs0.4043±0.0620,0.406±0.1158,均P＜0.05).结论:经VEGF165转染后,胃癌细胞迁移能力增强,MMP-2、u-PA的mRNA表达增加,MMP-2、u-PA表达的增加可能为VEGF促进胃癌侵袭转移机制.     

青蒿琥酯对人结肠癌HCT-8细胞诱导血管新生的影响及机制探讨

目的观察青蒿琥酯对人结肠癌HCT-8细胞诱导血管新生的影响,并探讨其作用机制。方法采用大鼠主动脉环体外培养实验和鸡胚绒毛尿囊膜体内血管生长实验观察青蒿琥酯对人结肠癌HCT-8细胞诱导血管新生的影响。采用Western blot法检测青蒿琥酯作用前后人结肠癌HCT-8细胞的血管内皮生长因子(VEGF)、血管生成素-2(Ang-2)蛋白。结果青蒿琥酯可明显减少HCT-8细胞诱导的血管生成。HCT-8细胞高表达VEGF、Ang-2蛋白,青蒿琥酯呈剂量依赖方式下调HCT-8细胞VEGF、Ang-2蛋白的表达(P均<0.05)。结论青蒿琥酯具有抗人结肠癌HCT-8细胞诱导血管新生的效应,与青蒿琥酯抑制HCT-8细胞的VEGF、Ang-2蛋白表达有关。     

血管内皮生长因子165基因对人胃癌细胞在体外生长及其受体表达的影响

目的:将已构建的重组腺病毒Ad-VEGF165进行扩增和纯化并观察其对人胃腺癌细胞(BGC-823)在体外生长及其受体表达的影响.方法:以人胚肾293细胞对重组腺病毒Ad-VEGF165和对照病毒Ad-GFP进行包装、扩增后感染BGC-823N胞,应用MTT比色法分析VEGF165对该种细胞体外生长的影响,并以免疫细胞化学方法检测该种细胞VEGFR-2的表达情况.结果:成功扩增及纯化了重组腺病毒Ad-VEGF165及对照病毒Ad-GFP,获得约3.2×1013pfu/L滴度的重组腺病毒和2.0×1013 pfu/L滴度的对照病毒.当感染复数(MOI)为20时转导率即接近100%而无明显细胞中毒现象.MTT结果显示Ad-VEGF165组吸光度明显高于Ad-GFP组(24 h:0.960±0.0 1 vs 0.737±O.01,P＜0.01:48 h:1.321±0.03 vs0.981±0.02,P＜0.01:72 h:1.663±0.03 vs 1.207±0.01,P＜0.01)及对照组(24 h:0.960±0.01 vs 0.724±0.03.P＜0.01:48 h:1.321±0.03 vs 0.968±0.01.P＜0.01:72 h:1.663±0.03 vs 1.185±0.02,P＜0.01).另外,在该细胞株上检测到了VEGFR-2的表达,且在Ad-VEGF165组其表达明显高于Ad-GFP组(62.5%vs37.6%,P＜0.01)和对照组(62.5%vs 34.1%,P＜0.01).结论:VEGF对胃癌细胞有直接的促生长作用,并可上调其主要受体VEGFR-2的表达水平,从而进一步证实了VEGF对胃癌细胞的自分泌作用.     

缺氧预适应通过蛋白激酶C途径上调心肌细胞促血管生成因子的表达

目的 :研究缺氧预适应 （HPC）对心肌细胞血管内皮生长因子 （VEGF）和碱性成纤维细胞生长因子表达的影响。方法 :建立体外培养的新生大鼠心肌细胞 HPC模型 ,免疫组织化学法结合计算机图像分析 ,研究 HPC对心肌细胞血管内皮生长因子 （VEGF）和碱性成纤维细胞生长因子 （b FGF）表达的影响以及蛋白激酶 C（PKC）在此过程中的作用。结果 :心肌细胞 HPC后 ,VEGF和 b FGF表达显著增多 ,阳性染色吸光度值明显升高。 PKC抑制剂 chel-erythrine chloride（Che）可拮抗 HPC促进心肌细胞表达 VEGF和 b FGF的作用。结论 :HPC可促进心肌细胞VEGF和 b FGF的表达 ,此作用是通过激活 PKC途径介导的     

心肌缺血的分子再血管化

应用促血管生成因子（AF）的蛋白多肽或表达基因治疗心肌缺血的分子再血管化技术成为人们关注的热点。AF包括一大类蛋白多肽,其中血管内皮细胞生长因子（VEGF）和成纤维细胞生长因子（FGF）的研究最为深入,心肌缺血时VEGF和FGF及其受体表达增加,应用外源VEGF或FGF可以促进侧支和新生血管生成、改善血液灌注及减少缺血面积。心肌缺血的基因治疗是将AF的表达基因转入心脏,在局部产生表达蛋白发挥作用,一系列动物实验证实了其可行性。尽管目前仍有许多问题有待解决,但分子再血管化为终末期冠心病治疗提供了新的希望。     

血管内皮生长因子165及血管生成素-1减小大鼠心肌梗死面积的研究

目的 在体条件下探讨血管内皮生长因子165(VEGF165)和血管生成素-1(angiopoietin-1，Ang1)对心脏缺血损伤的保护作用及其机制。方法 构建编码人VEGF165和Ang-全长基因的复制缺陷型腺病毒载体Ad—VEGF165和Ad—Ang1。结扎SD大鼠冠状动脉前降支，在缺血区周围心肌内注射Ad—VEGF165和(或)Ad—Angl，术后72h检测心脏组织中三磷酸肌醇激酶活性和抗凋亡因子Bcl-2表达水平；术后1周收取心脏，分析结扎区内梗死心肌的面积变化。结果 Ang-1和VEGF165单用组及两者联用组心肌梗死面积显著减小。其中，Ang-1单用组和两者联用组效果要好于VEGF165单用组。VEGF165和(或)Ang-1转染后，心肌内三磷酸肌醇激酶活性和Bcl-2表达水平均显著增高。结论 VEGF165和(或)Ang-1可激活心脏中三磷酸肌醇激酶并促进Bcl-2表达，减小心肌梗死面积，有明确的心脏保护作用。     

芎芍胶囊及丹参对大鼠缺血心肌血管新生的作用

目的探讨中成药芎芍胶囊及丹参对心肌梗死大鼠缺血心肌血管新生的影响。方法采用开胸结扎冠状动脉左前降支建立心肌梗死模型,清洁级(或SPF级)SD大鼠24只(雌雄各半)分为3组,即模型组、芎芍胶囊组、丹参组,每组8只,分别用生理盐水、芎芍胶囊、丹参按中剂量进行灌胃给药,连续给药。6周后采集血,分离样本,并按规定保存,用免疫组化评分法、血管新生相关因子检测法;检测各组缺血心肌内皮细胞生长因子(VEGF)、成纤维细胞生长因子(FGF)、表皮细胞生长因子(EGF)、转化生长因子(TGF)、血小板源生长因子(PDGF)等促血管生成因子的表达。结果芎芍胶囊组对VEGF、EGF的促进作用较丹参组、对照组有统计学意义(P<0.05),其中芎芍胶囊组、丹参组对FGF的促进作用与对照组比较均有统计学意义(P<0.05),对照组对TGF、PDGF促进作用较芎芍胶囊组、丹参组有统计学意义(P<0.05)。结论中药具有促进心肌梗死大鼠缺血心肌血管新生、提高心肌梗死大鼠心肌细胞VEGF、FGF、EGF蛋白的表达、具有保护心肌缺血性损伤的作用。     

血管内皮生长因子基因转移对6-羟多巴胺诱导的多巴胺能细胞损伤的保护作用

目的探讨血管内皮生长因子(VEGF)基因转移对6-羟多巴胺(6-OHDA)诱导的多巴胺能细胞损伤的保护作用。方法用腺病毒载体携带VEGF165基因(Ad-VEGF165)感染PC12细胞,并设腺病毒携带的β-半乳糖苷酶基因(Ad-LacZ)感染组和磷酸盐缓冲液(PBS)处理组作对照,基因转移成功后,用6-OHDA处理细胞,另设正常对照(不加6-OHDA)。四甲基偶氮唑盐法(MTT)测定细胞活性,免疫荧光细胞化学法检测细胞酪氨酸羟化酶(TH)表达,高效液相色谱检测细胞分泌功能。结果Ad-VEGF165感染组细胞吸光度A_(570)(0.31±0.07),高于Ad-LacZ感染组(0.15±0.07)和PBS处理组(0.13±0.05),但低于正常对照组(0.55±0.10),分别与各组比较,差异均有统计学意义(均为P<0.01);免疫荧光染色显示Ad-VEGF组细胞胞浆平均荧光强度(86.75±21.62)高于Ad-LacZ组(51.53±1 7.49)及PBS组(54.19±15.82),但低于正常对照组(110.39±24.21),分别与各组比较,差异均有统计学意义(均为(P<0.05);Ad-VEGF感染组细胞培养液中多巴胺和去甲肾上腺素含量也高于Ad-LacZ感染组和PBS处理组。结论腺病毒介导的VEGF基因转移对6-OHDA诱导的多巴胺能细胞损伤具有保护作用。     

糖尿病鼠肾小管周围新生血管与促血管生长因子关系研究

目的探讨促血管生成素-2(Ang-2)和血管内皮生长因子(VEGF)在糖尿病肾脏血管新生中的作用及其意义。方法2004年3月至2005年3月在四川大学华西医院用SD大鼠建立链唑霉素(STZ)诱导的糖尿病肾病模型,定量分析肾脏VEGF和Ang-2的表达变化,用逆转录聚合酶链式反应技术(RT-PCR)检测肾脏VEGF、Ang-2的mRNA的表达。结果糖尿病组肾脏VEGFmRNA表达从2周开始持续上调,16～20周达高峰;免疫组化显示糖尿病组各时点肾小管的VEGF着染均强于对照组;糖尿病组仅于16周和20周时探及肾组织Ang-2mRNA表达,12～24周免疫组化显示皮质区管周Ang-2着染管周微血管,整个实验期间对照组未探及Ang-2mRNA和蛋白表达;VEGF与Ang-2的改变呈正相关。结论糖尿病肾脏中后期皮质区存在Ang-2着染的新生管周微血管,VEGF与Ang-2参与糖尿病肾脏新生血管生成。     

Vascular endothelial growth factor-165 gene therapy promotes cardiomyogenesis in reperfused myocardial infarction

BACKGROUND: Vascular endothelial growth factor (VEGF)-165 promotes cardiomyogenesis in chronic myocardial ischemia and nonreperfused myocardial infarction (MI). It is unknown whether this effect is present in reperfused MI. We sought to investigate the effect of VEGF-165 gene therapy on cardiomyogenesis after reperfused MI. METHODS AND RESULTS: Twenty-four Yucatan minipigs underwent thoracotomy and a vascular clamp was placed in the left circumflex artery. Reperfusion was reestablished after 90 minutes, and VEGF-165 gene therapy or placebo was administered. A replication-deficient recombinant human adenovirus serotype 5 was used for gene transfer (Ad5-VEGF165). The same viral vector devoid of VEGF gene (Ad5-beta-galactosidase) was used as placebo. Two administration routes were tested, intramyocardial (IM) injection and circumflex intracoronary (IC) infusion. The pigs were assigned to one of the following groups: IM Ad5-VEGF165 (n = 6), IM Ad5-betaGal (n = 6), IC Ad5-VEGF165 (n = 6), and IC Ad5-betaGal (n = 6). All pigs received 5-bromo-2'-deoxyuridine (BrdU) 250 mg IV twice a week to label cells undergoing DNA replication. The hearts were explanted at 4 weeks. BrdU-labeled cardiomyocytes in the peri-infarct area were counted by a pathologist blinded to group assignment. The number of BrdU-labeled cardiomyocytes per million cells was 4-fold higher in the group receiving IM VEGF-165 (64 +/- 11.4) vs. IM placebo (16 +/- 10.6), P = 0.034. No difference in infarct size or ventricular function was observed between the groups. CONCLUSIONS: IM VEGF-165 gene therapy promotes cardiomyogenesis in reperfused MI. However, no benefit in infarct size or cardiac function was observed at 4 weeks. The origin of these cells remains unknown and needs to be determined.     

Infection efficiency of type 5 adenoviral vectors in synovial tissue can be enhanced with a type 16 fiber

OBJECTIVE: To obtain an adenoviral vector with increased infection efficiency in the synovial tissue compared with conventional vectors based on adenovirus serotype 5 (Ad5), without compromising the specificity of infection. METHODS: Coxsackie adenovirus receptor (CAR) expression was assessed in cultured synoviocytes. Chimeric adenoviruses based on Ad5 but carrying the DNA encoding the fiber of adenovirus from subgroup B (Adll, 16, 35) or D (Ad24, 28, 33, 45, or 47) were constructed and produced on PER.C6 cells. The gene transfer efficiency of these chimera was tested on cultured synoviocytes and peripheral blood mononuclear cells (PBMC). RESULTS: No surface expression of CAR protein was observed on synoviocytes. CAR messenger RNA expression of synoviocytes was found to be low. Of all fiber chimeric vectors tested, vectors carrying the fiber of Ad16 (Ad5.fib16) were most potent, yielding approximately150 times increased transgene expression in cultured synoviocytes compared with those of Ad5. Flow cytometry showed that the increase in transgene expression was caused by the transduction of higher percentages of synoviocytes and higher gene expression per synoviocyte. Experiments with 500 virus particles/cell of Ad5.GFP or Ad5.fib16.GFP resulted in an infection efficiency of 0.6% and 1% in PBMC and 43% and 76% in synoviocytes, respectively. CONCLUSION: Synoviocytes hardly express CAR, which hampers Ad5-mediated gene transfer. Ad5.fib16 is superior to Ad5 vectors for transducing synoviocytes, without compromising the specificity of infection. Our data suggest that Ad5.fib16-mediated gene transfer to synovial tissue improves the therapeutic window.     

The influence of synovial fluid on adenovirus-mediated gene transfer to the synovial tissue

OBJECTIVE: To determine the effect of synovial fluid (SF) from rheumatoid arthritis (RA) patients on adenovirus type 5 (Ad5)-mediated gene transfer to synoviocytes, and to explore new strategies for vector development based on the neutralization data obtained. METHODS: SF was derived from 63 randomly selected R4 patients. Ten samples were used to study the effect of SF on Ad5-mediated gene transfer in synoviocytes. IgG and <100-kd fractions were purified from these 10 SF, and their effect on gene transfer was determined. Neutralizing activity against wild-type Ad5 (wt-Ad5), wt-Ad26, wt-Ad34, wt-Ad35, and wt-Ad48 was tested in the SF from the remaining 53 patients. RESULTS: Seven of 10 SF samples inhibited Ad5-mediated gene transfer. Purified antibodies exhibited inhibition patterns similar to those seen with unfractionated SF. In 5 of 10 SF samples, low molecular weight fractions inhibited gene transfer at low dilutions. Neutralization of wt-Ad35 by SF from RA patients was less frequent than neutralization of other wt-Ad tested (4% versus 42-72%; n = 53). CONCLUSION: SF from 70% of the RA patients contained neutralizing antibodies that hamper Ad5-mediated gene transfer to synoviocytes. The activity of neutralizing antibodies may be circumvented in the majority of RA patients when vectors based on an Ad35 backbone are used.     

The influence of synovial fluid on adenovirus‐mediated gene transfer to the synovial tissue

Objective

To determine the effect of synovial fluid (SF) from rheumatoid arthritis (RA) patients on adenovirus type 5 (Ad5)–mediated gene transfer to synoviocytes, and to explore new strategies for vector development based on the neutralization data obtained.

Methods

SF was derived from 63 randomly selected RA patients. Ten samples were used to study the effect of SF on Ad5‐mediated gene transfer in synoviocytes. IgG and <100‐kd fractions were purified from these 10 SF, and their effect on gene transfer was determined. Neutralizing activity against wild‐type Ad5 (wt‐Ad5), wt‐Ad26, wt‐Ad34, wt‐Ad35, and wt‐Ad48 was tested in the SF from the remaining 53 patients.

Results

Seven of 10 SF samples inhibited Ad5‐mediated gene transfer. Purified antibodies exhibited inhibition patterns similar to those seen with unfractionated SF. In 5 of 10 SF samples, low molecular weight fractions inhibited gene transfer at low dilutions. Neutralization of wt‐Ad35 by SF from RA patients was less frequent than neutralization of other wt‐Ad tested (4% versus 42–72%; n = 53).

Conclusion

SF from 70% of the RA patients contained neutralizing antibodies that hamper Ad5‐mediated gene transfer to synoviocytes. The activity of neutralizing antibodies may be circumvented in the majority of RA patients when vectors based on an Ad35 backbone are used.

     

中缅树鼩自然感染五种高致病性人腺病毒的血清流行病学分析

目的 分析中缅树鼩(Tupaiabelangeri, Tree Shrew)自然感染5种高致病性人腺病毒(Human Adenovirus, HAdv)的血清流行病学,以探讨人腺病毒感染树鼩的可能性。方法 采用固定病毒稀释血清法测定34份树鼩血清对Ad3、Ad4、Ad7、Ad14和Ad55的中和抗体水平。结果 4种B组腺病毒的中和抗体阳性率及中和效价较E组4型的大,阳性率由大到小依次为:Ad14(55.88%)、Ad3(47.06%)、Ad55(29.71%)、Ad7(14.71%)、Ad4(8.82%),树鼩血清主要表现为Ad3、Ad14和Ad55混合型多价抗血清。结论 中缅树鼩可自然感染5种人腺病毒,有望以树鼩为实验动物,建立一种成熟的人腺病毒感染模型。     

腺病毒转染的肝细胞生长因子对冠心病患者外周造血干细胞的动员作用

目的 探讨经冠状动脉内注射腺病毒（adenovirus,Ad,）转染人肝细胞生长因子（HGF）对严重冠状动脉狭窄患者外周造血干细胞的动员作用。方法 本研究人选18例冠心病患者,分为两组：给药组9例,经冠状动脉注入Ad5-HGF;对照组9例,经冠状动脉注入同等量的生理盐水。所有患者均于冠状动脉造影术前、术后6h、24h及6天抽取外周血,并应用流式细胞仪单克隆荧光抗体标记法检测其外周造血干细胞表面抗原CD34^＋、CD38^＋及CD117^＋。结果 给药组术后6h的CD34^＋明显高于对照组（0,104±0．082比0．022±0．012,P=0．021）,0h、24h及6天的CD34^＋两组之间差异无统计学意义;给药组术后6天的CD117^＋明显高于对照组（0．058±0．058比0．012±0．009,P=0,034）,差异有统计学意义,其余时间点两组间差异均无统计学意义;CD38^＋在各时间点两组间差异均无统计学意义。结论 经冠状动脉内注射转染A5-HGF可能在短时间内引起外周造血干细胞的动员。动员外周造血干细胞可能是HGF参与血管新生和器官组织损伤修复及抗凋亡、改善心功能的一个重要机制。     

Antigen detection with monoclonal antibodies for the diagnosis of adenovirus gastroenteritis

A monoclonal antibody-based enzyme immunoassay (EIA) was developed for direct detection of enteric adenoviruses in stool specimens from individuals with gastroenteritis. Tests specific for each of the enteric adenoviruses, adenovirus type 40 (Ad 40) and type 41 (Ad 41), were designed. The sensitivity of the assay was determined by comparing the results of the EIA with isolation of virus in Graham 293 cells from stools that contained particles having adenovirus morphology. The standard for specificity was analysis of adenovirus genome profiles after digestion with SmaI endonuclease. The sensitivity was 95.8% (23 of 24) for Ad 40 and 97.1% (34 of 35) for Ad 41. The specificity was 95.7% (45 of 47) and 97.2% (35 of 36), respectively. The two type-specific monoclonal antibodies could be mixed in an EIA for identification of enteric adenoviruses in stools without loss of reactivity in either type. The EIA permits rapid diagnosis and type-specific identification of enteric adenoviruses in gastroenteritis.     

Epidemiology of enteric adenoviruses 40 and 41 in acute gastroenteritis in infants and young children in the Tokyo area.

82/2,223 stool specimens, collected 1982-1988, from children with enteritis (3.7%) were found to contain adenoviruses; 17 adenovirus-positive samples were provided from other institutes. 89 adenoviruses were isolated in Graham 293 cells from these 99 specimens and were typed by DNA restriction enzyme analysis with Sma I. 37 strains were typed as adenovirus 40 (AD40), and 37 strains as adenovirus 41 (Ad41). Although most strains had the same DNA profiles, a few strains had 3 kinds of different electropherotypes generated by Sma I. Five strains were identified as adenovirus 31. The remaining 10 strains were adenovirus 1 (2 strains), adenovirus 2 (3 strains), adenovirus 3 (1 strain), adenovirus 5 (1 strain), and a non-classified adenovirus (3 strains). Ad40 and Ad41 infections were found throughout the year, but peaked between September and November. 80% of the children with adenovirus infections were less than or equal to 2 years of age. The highest incidence of diarrhea caused by Ad40 or Ad41 was in 6-11 months old children. 1982-1984, the rate of Ad40 infection was 91.7%, while the rate of Ad41 infection was only 8.3%. The prevalence of Ad40 infection gradually diminished from 1985. During 1987 and 1988 the reverse ratios, 20.6% and 79.4%, respectively, of Ad40 and Ad41 infections were observed. Thereafter, Ad41 infection became predominant.