Received
2001-04-27Accepted 2001-08-22
This work was
supported by the National Natural
Science Foundation of China (No.39670308)
*Corresponding
author. Tel: +86-10-63291921;
E-mail: anwei@cpums.edu.cn
生理学报, Feb. 2002,
54 (1): 12～16
Acta
Physiologica Sinica
研究论文
转染人血红素加氧酶-1基因对大鼠血管平滑肌细胞抗氧化损伤的作用
张敏1, 安威2, 杜海军2, 陈莉2, 张宝慧1
1北京大学第三医院运动医学研究所, 北京 100083; 2首都医科大学细胞生物系, 北京 100054
摘要: 本实验构建含人血红素加氧酶-1(hHO-1)基因的逆转录病毒载体XM-6/hHO-1,
将其导入离体培养的大鼠血管平滑肌细胞(vascular smooth muscle cells, VSMC), 观察外源性hHO-1基因在VSMC内的表达及其抗活性氧损伤作用。结果表明:
(1) hHO-1基因可在靶细胞中明显表达, 转染VSMC的HO-1蛋白表达和HO酶活性分别比非转染细胞高1.8倍和2.0倍; (2)转染hHO-1的VSMC可对抗大剂量H2O2对细胞的损伤作用,
表现为细胞存活率增加和乳酸脱氢酶(LDH)漏出减少，上述保护作用可被HO特异性抑制剂锌原卟啉IX (Zinc-protoporphyrin IX， ZnPP-IX)所阻断。研究结果提示, 外源性HO-1的过量表达可增加VSMC对抗氧化损伤的能力。
关键词: 血红素加氧酶; 逆转录病毒载体; 活性氧; 血管平滑肌
学科分类号: Q78; R363
Increased resistance against oxidant-induced injury in
the rat vascular
smooth muscle cells transfected with human heme
oxygenase-1 gene
ZHANG Min1, AN
Wei2, DU Hai-Jun2, CHEN Li2, ZHANG Bao-Hui1
1Institute of
Sports Medicine, The Third Hospital, Peking University, Beijing 100083;
2Department of Cell Biology, Capital University of Medical Sciences, Beijing
100054
Abstract: The heme oxygenase-1 (HO-1), a
rate-limiting enzyme in heme metabolism, has been recently defined as a novel
stress-stimulated protein, since the intracellular expression of HO-1 in
response to various stimuli as oxidation, ischemia and endotoxin injury has
been proved to be able to protect the cells from damage. In this study, a
retroviral vector containing human HO-1 gene was constructed and transfected to
rat vascular smooth muscle cells (VSMCs). Using Southern and Northern blot
analyses, the integration and mRNA expression of HO-1 gene in the transfected
cells were confirmed. The profound protein expression of HO-1 as well as HO
enzyme activity in the transfected cells increased by 1.8-fold and 2.0-fold
respectively as compared with the non-transfected cells. It was found that the
HO-1 transfected-VSMCs presented dominant resistance to toxicity produced by
exposure to H2O2, as a significant protective effect of HO-1 marked by cell
survival and LDH leakage was observed when 200, 400 and 600 μmol/L of H2O2 were used. The protection of HO-1 rapidly
declined after the transfected-VSMCs were pretreated 24 h with an HO-1 specific
inhibitor (ZnPP-IX). The results of this investigation suggest that the
functional expression of HO-1 gene within VSMCs raises an alternative ability
to protect the vascular cells against active oxygen injury.
Key words: heme oxygenase; retroviral vector; active oxygen; vascular smooth muscle cells
大量研究结果表明, 自由基损伤可导致血管疾病发生, 而抗氧化物具有保护血管细胞免于受损。动脉粥样硬化(atherosclerosis, AS)和血管功能障碍的发病机制主要与血浆中低密度脂蛋白及其氧化修饰产物的升高有关[1]。血红素加氧酶(heme
oxygenase, HO)是血红素分解代谢过程中的限速酶, 其产物胆绿(红)素和一氧化碳(carbon monoxide, CO)分别具有抗氧化损伤和血管调节等作用。近年来,
诱导型血红素加氧酶(HO-1)及其产物的抗氧化损伤作用日益受到人们的重视[2]。有资料显示, 在人的动脉粥样斑块内, 与谷胱苷肽有关的抗氧化酶类普遍减少, 相反HO-1却有增加[3],
推测此时血管细胞的抗氧化防御作用主要是HO-1所为。据此, 本实验构建含hHO-1基2虻哪孀疾《驹靥?
将其导入VSMCs使其高表达, 观察该转染细胞对抗活性氧损伤的作用。
1材料和方法
1.1 逆转录病毒载体XM-6/hHO-1的构建用HindIII酶切pGEM7/hHO-1质粒(美国纽约医学院Abraham NG教授惠赠), 得到987
bp的hHO-1全长cDNA片段。将该片段插入到XM-6的5′LTR启动子下游HindIII位点, 得到重组体XM-6/hHO-1, 以ApaI酶切鉴定插入方向正确与否(图1)。
图1.含人HO-1基因逆转录病毒重组体的构建及酶切鉴定
Fig.
1.Illustration of the construction of a retroviral vector containing hHO-1
gene.
1.2 逆转录病毒载体的包装及滴度测定[4]利用脂质体(lipofectin,
Boehringer Mannheim公司生产)介导的基因转移方法将XM-6及XM-6/hHO-1 DNA导入包装细胞系PA317。经G418 (GIBCO公司生产) 400 μg/ml筛选2周后可见阳性克隆, 选取10个克隆继续培养至汇合。提取细胞总RNA, 用α-32P-[dCTP]标记(标记试剂为Promega公司生产)的hHO-1
cDNA 为探针, 对G418抗性细胞RNA进行Northern blot分析。以NIH3T3为靶细胞, 加入经Northern杂交确认的转hHO-1基因的PA317细胞培养基上清,
测定阳性克隆细胞的病毒滴度。将滴度大于1×106 CFU/ml细胞克隆用于实验。
1.3 VSMCs细胞培养、 鉴定及转染用I型胶原酶(Sigma公司产品)消化分离正常血压WKY大鼠胸主动脉中层VSMCs细胞,
以含10%胎牛血清的DMEM培养基培养。应用小鼠抗SMC-α-actin的单克隆抗体 (Sigma公司生产) 进行VSMCs免疫荧光及免疫组化染色, 旨在鉴定细胞培养中是否混有成纤维细胞,
以NIH3T3细胞作阴性对照。接种2.5×105个VSMCs于50 ml培养瓶中培养, 待细胞长至汇合率达50%～70%时弃去培养基, 加入病毒上清液5 ml及8
μg/ml Polybrene, 感染6 h。之后更换成正常培养基继续培养24 h, 加入G418 (剂量同前)筛选, 2周后出现阳性克隆。将克隆继续扩大培养,
得到XM-6及XM-6/hHO-1的两种转化VSMCs细胞。
1.4 Southern和Northern杂交[5]以酚-氯仿法提取以上三种细胞基因组DNA,
取15 μg DNA, 以BamHI消化, 经0.8%琼脂糖凝胶电泳分离后将DNA转印至尼龙膜。用TRIzol (GIBCO公司生产)一步抽提法提取细胞总RNA。取30
μg RNA经甲醛变性凝胶电泳分离后转印至尼龙膜。α-32P-[dCTP]标记的hHO-1探针变性后与尼龙膜分别进行Southern 和Northern杂交,
洗膜后－70℃放射自显影。
1.5 细胞蛋白提取及Western杂交[6]以PBS洗涤细胞三次后,
用冰冷的裂解液(50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100,
0.1%脱氧胆酸钠, 0.1% SDS, 1 mmol/L PMSF, 0.1%抑蛋白酶肽)100 μl裂解细胞, 14000 g离心15 min (4℃),
收获上清液。用Bicinchoninic Acid (BCA)方法测定细胞蛋白含量。取80 μg蛋白, 进行12% SDS-PAGE分离。以湿性电转移法将胶中蛋白转印至硝酸纤维素膜上(tank-transfer,
Bio-Rad)。该膜经5%脱脂奶粉封闭过夜(4℃), 次日分别与anti-HO-1多克隆抗体(Affiniti公司产品, 英国)和鼠抗兔IgG抗体-辣根过氧化物酶(Jackson
Immuno Research Laboratories, 美国)各孵育2 h和1 h。采用增强化学发光法(Sante Cruz公司产品)检测信号。曝光于X光片信号的密度值经GS-700扫描仪(Bio-Rad)进行定量分析。
1.6 细胞HO活性的测定[7]5×106细胞加3
ml预冷的含蔗糖的Tris缓冲液, 匀浆后4℃离心(13500 g, 20 min), 上清再离心(105000 g, 90 min)分离微粒体, 沉淀用0.1
mol/L磷酸钾缓冲液(pH 7.4)溶解。利用硫酸铵沉淀法从大鼠肝脏中提取胆绿素还原酶。HO活性测定反应体系中含0.5 mg HO、 20 μmol/L
Hemin、 0.8 mmol/L NADP、 4 mg胆绿素还原酶、 4 mmol/L 6-磷酸葡萄糖及1U 6-磷酸葡萄糖脱氢酶, 反应体积1 ml。37℃水浴避光孵育15
min后置冰浴以终止反应。用分光光度仪(Lamda-Bio, 美国PE公司)测定OD463和OD530。HO活性用胆红素形成速率[nmol/(mg·h)]来表示。
1.7 细胞存活率检测细胞分种于24孔板(8×104个细胞/孔),
待细胞70%融合时加不同浓度H2O2 (200、 400与600 μmol/L)损伤细胞, 6 h后加0.1 ml 0.1%胰蛋白酶消化, 之后用0.3 ml培养基中和。细胞悬液(50
μl)加等体积的0.4%台盼蓝, 细胞活率以存活细胞(未染)占总细胞(未染+着染)百分率计算。参照Wroblewski[8]的方法测定乳酸脱氢酶
(lactate dehydrogenase, LDH), 作为细胞受损指标。
1.8
统计学处理 结果用mean±SD表示,组间差异用one way ANOVA分析,P<0.05为差异有显著性。
2结果
2.1 VSMCs的鉴定
大鼠VSMCs显微镜下呈梭形或长梭型, 可重叠生长, 高低起伏, 呈典型的“谷与峰”样。荧光染色可见胞浆中含有丰富的黄绿色丝状物, 免疫组化可见胞浆中黄棕色阳性颗粒, 且每个细胞均被染成黄棕色,
而NIH3T3细胞均未出现以上结果, 说明培养的VSMCs未混杂有成纤维细胞 (本组照片未示)。VSMCs长期培养稳定, 实验用2～25代细胞。
2.2 hHO-1基因在PA317和VSMCs中的表达
逆转录病毒载体XM-6/hHO-1和空载体XM-6转染包装细胞PA317后, 经G418筛选分别获得阳性克隆PA317/hHO-1和PA317/XM-6。病毒颗粒感染NIH3T3后,
其最大病毒滴度分别达到1.3×106和1.2×106 CFU/ml。Northern杂交结果显示(图2A): PA317/hHO-1和PA317/XM-6 均有neor基因表达,
但仅PA317/hHO-1细胞有hHO-1基因表达。以上两种病毒感染VSMCs后, 经G418筛选分别获得阳性细胞VSMCs/hHO-1和VSMCs/XM6。图2B显示:
外源性hHO-1基因mRNA可以在VSMCs中有效表达, 而正常VSMCs及转染XM-6空载体的VSMCs中未见hHO-1基因的杂交信号。另外,
Southern杂交结果表明, 逆转录病毒所携带的hHO-1基因已经整合到VSMCs基因组DNA中(照片未示)。
图2.Northern blot鉴定PA317细胞和VSMCs中hHO-1, neor和S26表达
Fig. 2.Detection of hHO-1, neor and S26 transcripts by Northern blot in
RNA extracted from PA317 cells (A)
and VSMCs (B). A: PA317 (lane 1), PA317/XM6
(lane 2), PA317/hHO-1 (lane 3). B:
VSMCs (lane 1), VSMCs/XM6 (lanes 3 and 5), VSMCs/hHO-1 (lanes 2,4 and 6).
图3.Western blot 检测VSMCs中HO-1蛋白表达结果
Fig. 3.Western
blot analysis of hHO-1 protein expression in the VSMCs nontransfected or transfected with XM6
or XM6/hHO-1. Values are expressed as means±SD of 3 experiments. Lane 1, VSMCs; lane 2, VSMCs/XM6; lane 3, VSMCs/hHO-1. *P<0.05 vs control
VSMCs.
2.3 转染VSMCs HO活性增强、 HO-1蛋白表达增加
比较正常VSMCs和转染空载体XM6的VSMCs之HO活性, 未见两者具有显著性差异[10.5±3.0 nmol/(mg·h) vs 8.7±3.1 nmol/(mg·h), P>0.05],
而转染XM-6/hHO-1的VSMCs
HO活性明显增加[21.1±6.7 nmol/(mg·h)], 比正常VSMCs高2.0倍(P<0.05)。为进一步验证HO活性增加与外源性hHO-1基因mRNA表达之间的关系,
提取三种细胞蛋白用anti-HO-1抗体进行Western blot。结果显示, 与正常细胞相比, VSMCs/hHO-1细胞HO-1蛋白表达增加1.8倍(图3)。表明表达hHO-1基因的VSMCs
HO活性增强是由于hHO-1蛋白表达增加所致。
2.4 转染VSMCs抗活性氧的损伤作用
用三种不同浓度的H2O2作用6 h后测定细胞存活率及LDH漏出量。结果显示, 随H2O2浓度的增加, 细胞损伤加重, 表现为死亡细胞数目增多和LDH漏出量增高,
并且呈剂量依赖关系。然而, 在相同浓度H2O2损伤下, VSMCs/hHO-1与对照VSMCs和VSMCs/XM6组相比, 细胞存活率增高(图4), LDH漏出量减少(图5)。而事先24
h向培养液中加入HO特异性抑制剂ZnPP-IX后(终浓度为20 μmol/L), HO-1的保护作用消失, 说明hHO-1在VSMCs内过量表达具有增强细胞抗活性氧损伤的能力。
图4.不同浓度H2O2损伤后细胞存活率的变化
Fig.
4.Survival of VSMCs transfected with or without retroviral vectors (XM6/hHO-1
or XM6) after exposure to different concentrations of H2O2. Data are shown as
means±SD of 4 independent experiments. A:
Treatment with H2O2 for 6 h. B: Pretreatment with 20 μmol/L ZnPP-IX for 24 h followed by exposure to H2O2 for 6
hours. *P<0.05 vs control
VSMCs.
图5.不同浓度H2O2损伤后细胞LDH的漏出量
Fig. 5.LDH
leakage of VSMCs transfected with or without retroviral vectors (XM6/hHO-1 or
XM6 ) after exposure to different concentration of H2O2. Data are shown as
means±SD of 6 independent experiments.
A: Treatment with H2O2 for
6 h. B: Pretreatment with 20 μmol/L ZnPP-IX for 24 h followed by exposure
to H2O2 for 6 hours. *P<0.05 vs control VSMCs.
3讨论
逆转录病毒载体具有感染效率高和稳定表达等优点[9]。虽然其病毒滴度低于腺病毒载体, 但由于腺病毒不能在靶细胞长期表达以及可引起机体严重的免疫反应甚至导致患者死亡[10],
因此在基因治疗中选择逆转录病毒作为载体构建重组基因更受青睐。本实验所用逆转录病毒载体XM6属N2系列, 其5′LTR可对插入的外源基因进行有效表达[11]。我们将含hHO-1的重组病毒经包装后导入大鼠VSMCs,
结果表明, 病毒滴度可达1.3×106 CFU/ml, 且目的基因hHO-1与靶细胞基因组DNA整合后可有效转录和翻译, 导入hHO-1基因后其蛋白表达量较正常细胞升高1.8倍,
HO酶活性增加2倍。经检测hHO-1基因在VSMCs传代25次后仍可表达, 说明本载体构建策略正确并实现了在VSMCs中的长期有效表达。
HO可分三型, 为不同基因所编码。各型HO在组织分布、 表达调节及分子性质方面差异较大[12]。血管系统只表达HO-1和HO-2, 后者属结构型,
一般认为在生理状态下起调节作用。而HO-1是诱导型, 细胞在缺氧、 氧化应激、 氧化型低密度脂蛋白、 重金属、 内毒素等应激条件下其表达增高[13]。据此认为HO-1可能为一种热休克蛋白[14],
是细胞的一种内源性保护蛋白质。有资料显示, HO-1表达升高可增强肺内皮细胞、 皮肤成纤维细胞及肾上皮细胞等的抗氧化能力。本实验证明, 将人HO-1基因导入大鼠VSMCs使其过量表达可增强细胞对抗大剂量H2O2攻击的能力。而一旦抑制HO-1的活性,
则转染HO-1细胞抗损伤的特性即可消失。该结果充分显示HO-1基因在抗活性氧损伤中的作用。虽然目前HO-1表达升高的细胞保护机制尚不甚明了, 但可能与以下因素有关:
(1) HO分解血红素生成胆绿(红)素, 不仅减少了血红素本身的氧化作用, 而且胆绿(红)素被证实是一类很重要的生理性氧自由基清除剂, 其抗脂质过氧化的能力优于维生素E[15]; (2)在血红素降解过程中产生的亚铁, 能够诱导转铁蛋白的表达, 后者可减少活性氧产生而发挥抗氧化作用;
(3)所产生的内源性CO具有舒张血管和抑制血小板聚集等作用。
许多疾病如AS、 高血压、 血管再狭窄等均与活性氧关系密切。在AS形成过程中, 随着血管管壁细胞内一些关键性抗氧化酶的普遍降低和血管内皮细胞NO产生减少[16],
其抗活性氧损伤能力大大降低。此时如果刺激HO-1表达, 启动HO/CO信号通路, 则为血管管壁细胞抵御AS形成提供了另一道重要防线。另外我们的研究发现,
hHO-1在VSMCs表达可抑制细胞增殖(资料另文发表), 因此, HO/CO通路的激活可能通过重新建立血管壁抗氧化和促氧化之间的平衡、 抗细胞增殖、 维持血流等作用,
在防治AS的形成和发展中起重要作用。
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