生理学报 Acta Physiologica Sinica, June 25, 2006, 58 (3): 281-286http://www.actaps.com.cn281低氧反应元件调控下h-VEGF165基因表达及其蛋白产物的延迟消失张宜乾1,*，张中明1，闫英群1，董红燕21徐州医学院附属医院胸心外科；2徐州医学院神经生物研究中心，徐州 221002摘 要：血管内皮生长因子(vascular endothelial growth factor, VEGF)转基因可促进心肌缺血区血管生成，改善心脏功能，然而长期高水平表达又会引起诸多副作用。为调控VEGF表达，在启动子区加入低氧反应元件(hypoxic response element,HRE)作为调控开关，研究氧环境对VEGF基因mRNA及蛋白产物表达的影响。重组腺相关病毒(recombinant adeno-associ-ated virus, rAAV)作为载体(rAAV-HRE-h-VEGF165)，转染离体培养大鼠心肌细胞，在常氧/缺氧(氧浓度1%)/缺氧复氧条件下进行培养，用酶联免疫特异性测定(enzyme linked immunosorbent assay, ELISA)方法检测培养液h-VEGF165蛋白浓度，细胞免疫荧光染色观测细胞内h-VEGF165蛋白表达，逆转录聚合酶链反应(reverse transcription polymerase chain reaction, RT-PCR)方法检测细胞h-VEGF165 mRNA表达。结果显示：未转染组、常氧培养组均无h-VEGF165 mRNA及其蛋白表达；缺氧培养组h-VEGF165 mRNA及蛋白均表达；缺氧复氧4 h组的h-VEGF165 mRNA消失，培养液中h-VEGF165蛋白减少，细胞内h-VEGF165蛋白存在；缺氧复氧8 h 及12 h组的h-VEGF165 mRNA消失，培养液和细胞内h-VEGF165蛋白均消失。研究表明，在HRE调控下，缺氧可促使h-VEGF165基因表达，复氧后，h-VEGF165 mRNA表达停止，蛋白产物延迟消失。关键词：血管内皮生长因子；低氧反应元件；心肌细胞中图分类号：R654.2Delayed disappearance of h-VEGF165 mRNA and protein under regulation ofhypoxic response elementZHANG Yi-Qian1,*, ZHANG Zhong-Ming1, YAN Ying-Qun1, DONG Hong-Yan21Department of Cardiothoracic Surgery, Affiliated Hospital; 2Neurobiological Research Center, Xuzhou Medical College, Xuzhou 221002,ChinaAbstract: Transfer of vascular endothelial growth factor (VEGF) gene to ischemic myocardium may provide a useful approach forangiogenesis and improve cardiac performance. However, uncontrolled expression of VEGF in vivo may result in certain side effects,such ashemangioma formation, retinopathy, and tumor development. We investigated the feasibility of using the nine copies of hypoxicresponse element (HRE) to control the expression of human VEGF165 (h-VEGF165) under anoxic condition at cell level and also observedthe synchron of h-VEGF165 mRNA and protein expressions. Recombinant adeno-associated viral (rAAV) vector was prepared by usingthe three-plasmid system and cotransfected to human embryo kidney 293T cells by the calcium phosphate precipitates method. TherAAV vector was purified by chloroform-PEG8000/NaCl-chloroform and added to cultured myocardiocytes. Myocardiocytes ofSprague-Dawley rat were cultured in serum-free medium and then randomly divided into eight groups. Group I: cultured undernormoxic conditions (21% O2) for 8 h as control; Group II: cultured under anoxic conditions (1% O2) for 8 h; Group III: cultured undernormoxic conditions (21% O2) for 8 h with gene transfer; Group IV: cultured under anoxic conditions (1% O2) for 8 h with gene transfer;Group V, VI, VII: cultured under anoxic conditions (1% O2) for 8 h with gene transfer and then tured to normoxic conditions (21% O2)for 4, 8 or 12 h, respectively; Group VIII: cultured under anoxic conditions (1% O2) for 20 h with gene transfer. After completion ofcell culture, the amount of h-VEGF165 protein inculture supernatant was quantified by using enzyme linked immunosorbent assayReceived 2005-11-14 Accepted 2006-01-20This work was supported by Scientific Foundation of Jiangsu Educational Commission (NO. 03JKA300140).* Corresponding author. Tel: +86-516-5802033; E-mail: jia007mi@163.com282 生理学报 Acta Physiologica Sinica, June 25, 2006, 58(3): 281-286(ELISA). Expression of h-VEGF165 protein in cultured cardiacmyocytes was also evaluated by immunofluorescence. RT-PCR wasemployed to detect the expression of h-VEGF165 mRNA. The results revealed that there were no expressions of h-VEGF165 mRNA andprotein in groups I, II, III, VI and VII. After gene transfer, the expressions of h-VEGF165 protein and mRNA were significantly higherin groups IV and VIII than those in other groups (P<0.01); Immunofluorescence positive cells were observed in groups IV, V and VIII.RT-PCR revealed that a 484-bp strip can be found in groups IV and VIII, but unavailable in other groups. We conclude that HRE is apromising regulator for h-VEGF165 gene expression following the changes of oxygen environment. HRE can induce the expression of h-VEGF165 gene after hypoxia, but in normal oxygen condition, the expression of h-VEGF165 was inhibited. Although expression of h-VEGF165 mRNA ceased in normal oxygen condition under the control of HRE, expression of h-VEGF165 protein was hysteretic to h-VEGF165 mRNA expression.Key words: vascular endothelial growth factor; hypoxic response element; cardiacmyocyte血管内皮生长因子(vascular endothelial growthfactor, VEGF)转基因治疗可促进心肌缺血区毛细血管生成，增加侧枝循环，减轻心肌缺血，改善心脏功能[1]。然而，人VEGF165 (human VEGF165, h-VEGF165基因的长期高水平表达又会引起诸多副作用，如：肿瘤、视网膜病、血管瘤、关节炎等[2-4]。低氧反应元件(hypoxic response element, HRE)是位于低氧相关基因3'或5'端的一段DNA序列，最先发现于促红细胞生成素(erythropoietin, EPO)基因的上游，核心序列为(A/G)CGT (G/C) C。HRE能与低氧诱导因子1α (hypoxia inducing factor, HIF-1α)特异结合，促使相关基因的表达上调，这些基因包括：VEGF、EPO、葡萄糖转运体1 (glucose transporter-1, Glut-1)、3-磷酸甘油醛脱氢酶(glyceraldehyde phosphatedehydrogenase, GAPDH)、一氧化氮合酶(nitric oxidesynthase, NOS)[5-9]，HIF-1α与HRE特异结合的拮抗剂可抑制这些基因的表达[10] 。表明HRE可以作为调控元件对转染的VEGF基因进行调控，而且这种调控和缺氧引起的HIF-1α密切相关，这也正是治疗缺血性心脏病所需要的调控元件。研究显示，9个拷贝的HRE作用最强[11-14]。本研究中，在h-VEGF165基因的启动子区加入9拷贝的HRE作为基因表达调控开关，重组腺相关病毒(recombinant adeno-associated virus, rAAV)作为载体(rAAV-9HRE-h-VEGF165)，转染离体培养的大鼠心肌细胞，利用常氧/缺氧(氧浓度1%)/缺氧复氧的细胞培养环境，监测对h-VEGF165基因表达和蛋白产物的影响。院实验动物中心提供。1.2 实验材料 质粒rAAV-9HRE-h-VEGF165由美国加利福尼亚大学的苏华教授惠赠(图1)，质粒helper、rep/cap由协和医院王任直教授提供(图2、3)。HEK 293T细胞购自协和医科大学细胞中心，大图 1. 重组腺相关病毒载体结构图Fig. 1. Structure of rAAV-9HRE-h-VEGF165. ITR, invertedterminal repeat. We inserted the nine copies of HRE consensussequence (CCGGGTAGCTGGCGTACGTGCTGCAG) to thehuman VEGF165-plasmid between Sma I and Hind III sites up-stream of the simian virus 40 (SV40) minimal promoter. We clonedthe expression cassette (nine copies of HRE, SV40 minimalpromoter, the human VEGF165 cDNA, and SV40 polyadenylationsignal) into an AAV vector between two inverted terminal repeatsto generate the rAAV-9HRE-h-VEGF165 vector.1 材料与方法1.1 实验动物 新生Sprague-Dawley (SD)大鼠，小于3 d，体重(50±4) g，雌雄不拘，由徐州医学图 2. 辅助质粒pAAV-RC的结构图Fig. 2. Structure of pAAV-RC. One helper plasmid has AAV repand cap genes.张宜乾等：低氧反应元件调控下h-VEGF基因表达及其蛋白产物的延迟消失283165图 3. 辅助质粒pHelper的结构图Fig. 3. Structure of pHelper. One helper plasmid has the adenoviralVA, E2A, and E4 regions that mediate AAV vector replication.肠杆菌Dh5α购自上海生工，h-VEGF165 ELISA试剂盒购自博士德，RT-PCR试剂盒购自北京鼎国，去内毒素质粒提纯试剂盒购自Qiagen公司。1.3 心肌细胞分离培养 新生SD大鼠，碘伏消毒后固定，麻醉，开胸、取心，置入D-hanks液洗涤2次，将心肌组织剪成糊状，加入0.08%胰蛋白酶溶液(0.5%胰蛋白酶和D-hanks液配置)，37 ℃水浴恒温、搅拌、分次消化，每次10 min，至心肌完全被消化。每次消化后将细胞悬液收集到离心管中，加适量含血清的M199培养液和胰蛋白酶。细胞悬液1 300 r/min离心7 min，弃上清，M199培养液重悬细胞，接种于50 ml螺口培养瓶中。培养2 h后将培养液转移(差速贴壁)至明胶处理过的24孔板中，加Brdu浓度至0.1 mmol/L，放入培养箱常规培养。1.4 rAAV-9HRE-h-VEGF165载体扩增、包装和纯化 取3份200 μl分装的感受态细菌(大肠杆菌Dh5α)，分别加入rAAV-9HRE-h-VEGF165、helper、rep/cap三种质粒各5 μl，培养过夜。挑取单个菌落接种培养，去内毒素质粒纯化试剂盒提纯质粒。HEK 293T接种，磷酸钙共转染法转染三种质粒，继续培养2~3 d，镜下见部分细胞脱壁，细胞毛玻璃样改变时，收集细胞沉淀，加入DNA酶Ⅰ和RNA酶，裂解，离心，抽提病毒上清。1.5 心肌细胞转染，缺氧处理和分组 培养的心肌细胞共分为8组，每组12孔，滴加病毒液(不转基因组滴加等量PBS)，37℃，95%空气/5% CO2培养16 h，转基因后，更换培养液，按组别继续培养，缺氧培养环境为94% N2/5% CO2/1% O2；有氧培养环境为 95%空气/5% CO2，复氧环境同有氧培养环境。Ⅰ组：空白对照组，不转基因，细胞有氧培养8 h；Ⅱ组：缺氧对照组，不转基因，细胞缺氧培养8 h；Ⅲ组：转基因对照组，转基因，细胞有氧培养8 h；Ⅳ组：转基因缺氧一组，转基因，细胞缺氧培养8 h；Ⅴ组、Ⅵ组、Ⅶ组为转基因复氧组，转基因，细胞缺氧培养8 h后复氧，分别在有氧下继续培养4、8、12 h；Ⅷ组：转基因缺氧二组，转基因，细胞缺氧培养20 h。每组取6孔样品上清液做ELISA测h-VEGF165蛋白含量，六孔细胞做免疫荧光染色，6孔细胞做RT-PCR测h-VEGF165 mRNA含量。1.6 细胞培养液中h-VEGF蛋白含量测定 取h-VEGF165-ELISA试剂盒，按说明配置底物工作液及标准品液，建立标准曲线，取待测细胞培养液遵试剂盒说明书加样测定492 nm处吸光值。根据样品OD值在标准曲线上查出相应h-VEGF165蛋白浓度。1.7 免疫荧光染色 细胞弃培养液后，4%多聚甲醛室温固定20 min，PBS洗涤，加一抗抗h-VEGF165单克隆抗体(小鼠单抗体1:50)，用含0.1%Triton X-100的PBS稀释，FITC (马抗小鼠1:150)避光孵育2 h，PBS洗涤，液体石蜡封孔，荧光倒置相差显微镜观察。1.8 RT-PCR 测h-VEGF165 mRNA 弃孔内培养液，加 TRIzol 100 μl，离心取上清，加20 μl 氯仿，震荡，离心。吸上层水相至另一离心管中，加异丙醇，离心，弃上清，加100 μl 75%乙醇，振荡，离心，弃上清，加50 μl DEPC液60℃ 10min，260 nm的光吸收率测RNA含量。实验步骤按RT-PCR试剂盒提供，h-VEGF165上游引物为5'-GCGCCAGTACTTCATAGGC-3'，下游引物为5'-CGGTCTGAAAGGATGAGCC-3'，扩增目的片段为484 bp，GAPDH内参片断为579 bp。RT-PCR循环采用94℃ 2 min，94℃变性45 s，70℃ 复性45s，72℃延伸45 s，35个循环；末次延伸时间72℃ 5 min。最后用5 μl PCR产物在1%凝胶上电泳，56 V电泳60 min，EB 染色40 min，紫外光下摄像，用FR-980生物电泳图像分析系统(上海复日公司)对DNA条带进行光密度扫描，用Smartviewer 软件(上海复日公司)进行灰度分析，用h-VEGF165/GAPDH的IOD比值表示h-VEGF165 mRNA的相对含量。1.9 统计学分析 使用SPSS11.5统计软件对计量资料分析，计量资料以mean±SD表示，组间比较采用方差分析。284 生理学报 Acta Physiologica Sinica, June 25, 2006, 58(3): 281-2862 结果2.1 心肌细胞形态变化心肌细胞转染后，各组间细胞形态无显著差异，形态为多角形或梭形，见细胞搏动，缺氧组和复氧组细胞形态无显著不同，形态仍为多角形或梭形，搏动频率100次/min，组间无差异，大部分细胞相互聚集、连接，呈同步搏动(图4)2.2 细胞培养液中h-VEGF165蛋白含量测定Ⅳ组和Ⅷ组h-VEGF165蛋白含量明显高于其他组(P<0.01)，Ⅴ组h-VEGF165蛋白含量低于Ⅳ组和Ⅷ组，但高于Ⅰ组、Ⅱ组、Ⅲ组、Ⅵ组和Ⅶ组(P<0.01)(表1)。2.3 免疫荧光法检测细胞浆内h-VEGF165蛋白表达情况图 4. 倒置显微镜下培养的新生大鼠心肌细胞Fig. 4. Photos of neonatal cardiomyocytes detected under invertmicroscope.Ⅳ组、Ⅴ组和Ⅷ组荧光染色可观察到绿色荧光，其余组均为阴性(图5)。2.4 RT-PCR 结果各组均有内参片段，大小为579 bp，Ⅳ组、Ⅷ组可见484 bp大小片断(h-VEGF165 mRNA)，其他图 5. 心肌细胞h-VEGF165蛋白免疫荧光图Fig. 5. The immunofluorescence staining with an antibody to h-VEGF165 protein revealed intense expression in cardiocytes. Expressionof h-VEGF165 protein was present in groups IV, V and VIII, but absent in groups I, II, III, VI and VII. Scale bar, 10 μm.表1. 各组h-VEGF165蛋白含量Table 1. The amount of h-VEGF165 protein in each groupProtein (×10-6 g/ml) I h-VEGF1650.37±0.06 II0.38±0.03 III IV V VI VII VIII0.33±0.0630.64±3.60*#19.70±1.85*0.39±0.090.36±0.0631.00 ±3.33*#mean±SD, n = 6. *P<0.01 vs I, II, III, VI, VII groups; #P<0.01 vs V group.张宜乾等：低氧反应元件调控下h-VEGF基因表达及其蛋白产物的延迟消失285165组几乎不能检测到目的片断(图6、7)。图 6. h-VEGF165逆转录聚合酶链反应结果Fig. 6. Expression of h-VEGF165 mRNA. RT-PCR revealed thata 484-bp strip can be found in group IV and group VIII, butunavailable in other groups. M, marker; 579 bp, GAPDH band;484 bp, h-VEGF165 mRNA.图 7. h-VEGF165逆转录聚合酶链反应检测半定量图Fig. 7. Semiquantitative results of h-VEGF165 mRNA expression.After gene transfer, the expressions of h-VEGF165 mRNA werehigher in group IV and group VIII than those in other groupssignificantly. *P<0.01 vs groups I、II、III、V、VI、VII.3 讨论将VEGF基因转移于靶器官使其长期高效表达是基因治疗的目的，若VEGF不能在心肌中长时间保持较高的浓度，仅引起短期反应，则不能有效地诱导血管生成或仅生成一些无功能的毛细血管[15]。Schwarz等[3]在大鼠缺血心肌边缘注射VEGF质粒，33 d后可见缺血边缘区新生血管，但并不成熟，对心肌供血无明显改善。腺相关病毒作为基因载体无免疫原性，可以使所携带基因长期表达(可达20个月以上) [16-18]，但是VEGF基因在体内长时间及高水平的表达若难以控制，循环中VEGF蛋白的持续升高可导致诸多副作用的发生，如：肿瘤、视网膜病、血管瘤、关节炎及软骨瘤的生成等，其中一些副作用所导致的并发症往往是十分严重的[2-4]。Wang等[19]最早发现缺氧可诱导一个与EPO3 增强子特异结合的DNA结合蛋白，当缺氧的细胞恢复到常氧环境后，此蛋白的DNA结合活性迅速丧失。而且在不缺氧或经热休克处理的细胞，其核抽提物中也无此蛋白DNA结合活性，并证实它是与EPO3增强子第一部分结合，缺氧诱导的此DNA结合蛋白(转录因子)即称HIF-1，此EPO3 增强子第一部分的DNA序列后来被称为HRE。在生理条件下，低氧是HIF-1的主要诱导因素[20]。这些诱导因素可以上调HIF-1的表达，活性增高，与HRE结合诱导HRE下游的基因(VEGF)表达。Birot 等[21]研究表明常氧大鼠心室即可有VEGF mRNA表达，缺氧时表达量增加，但2 d后又恢复至常氧水平。本实验采用的h-VEGF165基因转染，检测时特异性检测h-VEGF165的表达情况，而大鼠心肌细胞自身分泌的r-VEGF蛋白不在检测范围，这就排除了r-VEGF基因表达对本实验结果的干扰。实验中，未转染细胞、常氧培养细胞和缺氧/复氧培养细胞转染后均无h-VEGF165 mRNA表达，转染h-VEGF165细胞在缺氧培养条件下有h-VEGF165 mRNA表达，说明缺氧条件下，转染的h-VEGF165基因表达与大鼠自身心肌细胞的r-VEGF表达可能存在差异，因本实验未对r-VEGF进行检测，故无法进一步讨论其差异性。在缺氧、复氧的不同时间采用3种方法检测h-VEGF165基因的表达情况。实验结果表明，心肌细胞在转病毒并且缺氧后才有h-VEGF165蛋白表达；复氧4 h，h-VEGF165 mRNA已经消失，细胞浆内仍有h-VEGF165蛋白，但细胞外培养液中蛋白量低于单纯缺氧组，说明心肌细胞h-VEGF165蛋白分泌量已经明显减少；继续复氧8 h和12 h后，h-VEGF165RNA和细胞内外的h-VEGF165蛋白均消失，而缺氧20 h组仍有h-VEGF165 mRNA表达和h-VEGF165蛋白，说明h-VEGF165蛋白的消失并非基因转染时效引起，而是因为复氧后，缺氧环境消失，基因表达调控有效关闭HRE所致。本研究结果显示，细胞缺氧时，h-VEGF165基因表达明显增加，而复氧4 h后h-VEGF165 mRNA已经消失，虽h-VEGF165蛋白尚存，但其蛋白延迟消失时间并不超过8 h，蛋白的延迟消失可能与蛋白自身的代谢有关，而VEGF发生视网膜病等副作用常于20个月以后[11]，可知数小时的时间窗显然不足以引起VEGF的诸多副作用，进一步说明HRE对转导的VEGF基因表达调控是灵敏的，能够受细胞氧环境变化的影响，适时开启或闭合目的基因的表达，286 生理学报 Acta Physiologica Sinica, June 25, 2006, 58(3): 281-286在治疗血管生成的安全性方面具有一定的临床实用价值。参 考 文 献1Zhang DZ (张端珍), Gai LY, Chen YW, Fan RY, Wen YF,Dong W. 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