低氧反应元件(HRE)调控下h-VEGF165基因表达及其蛋白产物的延迟消失

https://www.360docs.net/doc/101706269.html,

研究论文

Received 2005-11-14 Accepted 2006-01-20

This work was supported by Scientific Foundation of Jiangsu Educational Commission (NO. 03JKA300140).

*

Corresponding author. Tel: +86-516-5802033; E-mail: jia007mi@https://www.360docs.net/doc/101706269.html,

低氧反应元件调控下h-VEGF 165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2

1

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-VEGF 165)，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-VEGF 165蛋白浓度，细胞免疫荧光染色观测细胞内h-VEGF 165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-VEGF 165 mRNA 表达。结果显示：未转染组、常氧培养组均无h-VEGF 165 mRNA 及其蛋白表达；缺氧培养组h-VEGF 165 mRNA 及蛋白均表达；缺氧复氧4 h 组的h-VEGF 165 mRNA 消失，培养液中h-VEGF 165蛋白减少，细胞内h-VEGF 165蛋白存在；缺氧复氧8 h 及12 h 组的h-VEGF 165 mRNA 消失，培养液和细胞内h-VEGF 165蛋白均消失。研究表明，在HRE 调控下，缺氧可促使h-VEGF 165基因表达，复氧后，h-VEGF 165 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：R654.2

Delayed disappearance of h-VEGF 165 mRNA and protein under regulation of hypoxic response element

ZHANG Yi-Qian 1,*, ZHANG Zhong-Ming 1, YAN Ying-Qun 1, DONG Hong-Yan 2

1

Department of Cardiothoracic Surgery, Affiliated Hospital; 2Neurobiological Research Center, Xuzhou Medical College, Xuzhou 221002,

China

Abstract: Transfer of vascular endothelial growth factor (VEGF) gene to ischemic myocardium may provide a useful approach for angiogenesis and improve cardiac performance. However, uncontrolled expression of VEGF in vivo may result in certain side effects,such as

hemangioma formation, retinopathy, and tumor development. We investigated the feasibility of using the nine copies of hypoxic response element (HRE) to control the expression of human VEGF 165 (h-VEGF 165) under anoxic condition at cell level and also observed the synchron of h-VEGF 165 mRNA and protein expressions. Recombinant adeno-associated viral (rAAV) vector was prepared by using the three-plasmid system and cotransfected to human embryo kidney 293T cells by the calcium phosphate precipitates method. The rAAV vector was purified by chloroform-PEG8000/NaCl-chloroform and added to cultured myocardiocytes. Myocardiocytes of Sprague-Dawley rat were cultured in serum-free medium and then randomly divided into eight groups. Group I: cultured under normoxic conditions (21% O 2) for 8 h as control; Group II: cultured under anoxic conditions (1% O 2) for 8 h; Group III: cultured under normoxic conditions (21% O 2) for 8 h with gene transfer; Group IV: cultured under anoxic conditions (1% O 2) for 8 h with gene transfer;Group V, VI, VII: cultured under anoxic conditions (1% O 2) for 8 h with gene transfer and then tured to normoxic conditions (21% O 2)for 4, 8 or 12 h, respectively; Group VIII: cultured under anoxic conditions (1% O 2) for 20 h with gene transfer. After completion of cell culture, the amount of h-VEGF 165 protein in

culture supernatant was quantified by using enzyme linked immunosorbent assay

(ELISA). Expression of h-VEGF 165 protein in cultured cardiacmyocytes was also evaluated by immunofluorescence. RT-PCR was employed to detect the expression of h-VEGF 165 mRNA. The results revealed that there were no expressions of h-VEGF 165 mRNA and protein in groups I, II, III, VI and VII. After gene transfer, the expressions of h-VEGF 165 protein and mRNA were significantly higher in 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 a promising regulator for h-VEGF 165 gene expression following the changes of oxygen environment. HRE can induce the expression of h-VEGF 165 gene after hypoxia, but in normal oxygen condition, the expression of h-VEGF 165 was inhibited. Although expression of h-VEGF 165 mRNA ceased in normal oxygen condition under the control of HRE, expression of h-VEGF 165 protein was hysteretic to h-VEGF 165 mRNA expression.

Key words: vascular endothelial growth factor; hypoxic response element; cardiacmyocyte

血管内皮生长因子(vascular endothelial growth factor, 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VEGF 165 (human VEGF 165, h-VEGF 165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 phosphate dehydrogenase, GAPDH)、一氧化氮合酶(nitric oxide synthase, 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-VEGF 165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-VEGF 165)，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-VEGF 165基因表达和蛋白产物的影响。

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

院实验动物中心提供。

1.2 实验材料 质粒rAAV-9HRE-h-VEGF 165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 2. 辅助质粒pAAV-RC 的结构图

Fig. 2. Structure of pAAV-RC.

One helper plasmid has AAV rep

and cap genes.

图 1. 重组腺相关病毒载体结构图

Fig. 1. Structure of rAAV-9HRE-h-VEGF 165. ITR, inverted terminal repeat. We inserted the nine copies of HRE consensus sequence (CCGGGTAGCTGGCGTACGTGCTGCAG) to the human VEGF 165-plasmid between Sma I and Hin d III sites up-stream of the simian virus 40 (SV40) minimal promoter. We cloned the expression cassette (nine copies of HRE, SV40 minimal promoter, the human VEGF 165 cDNA, and SV40 polyadenylation signal) into an AAV vector between two inverted terminal repeats to generate the rAAV-9HRE-h-VEGF 165 vector.

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张宜乾等：低氧反应元件调控下h-VEGF 165基因表达及其蛋白产物的延迟消失

肠杆菌Dh5α购自上海生工，h-VEGF 165 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-ha nks 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-VEGF 165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-VEGF 165、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% CO 2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% N 2/5% CO 2/1% O 2；有氧培养环境为 95%空气/5% CO 2，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-VEGF 165蛋白含量，六孔细胞做免疫荧光染色，6孔细胞做RT-PCR 测h-VEGF 165 mRNA 含量。

1.6 细胞培养液中h-VEGF 蛋白含量测定 取h-VEGF 165-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-VEGF 165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-VEGF 165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-VEGF 165 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-VEGF 165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Smar t viewer 软件(上海复日公司)进行灰度分析，用h-VEGF 165/GAPDH 的IOD 比值表示h-VEGF 165 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。

图 3. 辅助质粒pHelper 的结构图

Fig. 3. Structure of pHelper. One helper plasmid has the adenoviral VA, E2A, and E4 regions that mediate AAV vector replication.

生理学报 Acta Physiologica Sinica , June 25, 2006, 58(3): 281-286

2842 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-VEGF 165蛋白含量测定

Ⅳ组和Ⅷ组h-VEGF 165蛋白含量明显高于其他组(P <0.01)，Ⅴ组h-VEGF 165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-VEGF 165蛋白表达情况

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-VEGF 165 mRNA)，其他

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 invert microscope.

表1. 各组h-VEGF 165蛋白含量

Table 1. The amount of h-VEGF 165 protein in each group

Protein (×10-6 g/ml) I II III

IV

V

VI

VII

VIII

h-VEGF 165

0.37±0.06

0.38±0.03

0.33±0.06

30.64±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.

图 5. 心肌细胞h-VEGF 165蛋白免疫荧光图

Fig. 5. The immunofluorescence staining with an antibody to h-VEGF 165 protein revealed intense expression in cardiocytes. Expression of h-VEGF 165 protein was present in groups IV, V and VIII, but absent in groups I, II, III, VI and VII. Scale bar, 10 μm.

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张宜乾等：低氧反应元件调控下h-VEGF 165基因表达及其蛋白产物的延迟消失

组几乎不能检测到目的片断(图6、7)。

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-VEGF 165基因转染，检测时特异性检测h-VEGF 165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 -VEGF 165 mRNA 表达，转染h-VEGF 165细胞在缺氧培养条件下有h-VEGF 165 mRNA 表达，说明缺氧条件下，转染的h-VEGF 165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-VEGF 165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-VEGF 165蛋白表达；复氧4 h ，h-VEGF 165 mRNA 已经消失，细胞浆内仍有h-VEGF 165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-VEGF 165蛋白分泌量已经明显减少；继续复氧8 h 和12 h 后，h-VEGF 165RNA 和细胞内外的h-VEGF 165蛋白均消失，而缺氧20 h 组仍有h-VEGF 165 mRNA 表达和h-VEGF 165蛋白，说明h-VEGF 165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-VEGF 165基因表达明显增加，而复氧4 h 后h-VEGF 165 mRNA 已经消失，虽h-VEGF 165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，

图 6. h-VEGF 165逆转录聚合酶链反应结果

Fig. 6. Expression of h-VEGF 165 mRNA. RT-PCR revealed that a 484-bp strip can be found in group IV and group VIII, but unavailable in other groups. M, marker; 579 bp, GAPDH band;484 bp, h-VEGF 165

mRNA.

图 7. h-VEGF 165逆转录聚合酶链反应检测半定量图Fig. 7. Semiquantitative results of h-VEGF 165 mRNA expression.After gene transfer, the expressions of h-VEGF 165 mRNA were higher in group IV and group VIII than those in other groups significantly. *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

生理学报Acta Physiologica Sinica, June 25, 2006, 58(3): 281-286 286

在治疗血管生成的安全性方面具有一定的临床实用价值。

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