摘要
目的
高血压是充血性心衰、中风、终末期肾病的重要危险因子。据WHO统计,全世界的成年人群约有15-37%患有高血压病,而60岁以上某些人群,高血压的发病率可达50%。预计到2025年,全世界高血压的发病人数将飚升至15亿6千万人。但令人遗憾的是,只有接近三分之一的患者降压治疗达标。主要的原因在于高血压病是一种终生性疾病,而目前所有降压药的半衰期相对较短,有效作用时间均不超过24小时,故必须每天服药,病人不易坚持。此外由于药物的非特异性和副作用,也使高血压的治疗难以达到预期目标。因此寻找一种长期有效且通过调节体内基因表达而控制高血压的方法即基因治疗一直是医学家们努力的方向和目标。
RNA干扰(RNA interference,RNAi)技术是近年来发展起来的基因阻断技术,该技术通过双链RNA(double-stranded RNA,dsRNA)的介导,被属于RNaseⅢ家族的Dicer内切酶切割产生大量的小干扰RNA(small interfering RNAs,siRNA),并特异性降解与之序列同源的mRNA,而导致转录后水平的基因沉默。研究表明当siRNA呈短发央状RNA(shorthairpin RNA,shRNA)时,其基因沉默的效应更强。本实验即利用先进的RNA干扰技术,选择与高血压发生发展密切相关的肾素一血管紧张素系统(Renin-angiotensin system,RAS)中的血管紧张素转换酶(Angiotensin-converting enzyme,ACE)和血管紧张素Ⅱ1型受体(AngiotensinⅡtype 1 receptor,AT_1R)为靶点,构建ACE和AT_1R串联的shRNA的表达载体,分别在体外和体内实验中运用该技术抑制ACE和AT_1R mRNA和蛋白质的表达,从而探索联合和单基因沉默在高血压治疗中的应用前景。方法
1.构建靶向抑制大鼠ACE和AT_1R基因的shRNA真核表达载体:首先在GeneBank上选取大鼠ACE和AT_1R mRNA序列,根据siRNA设计原则,各设计两条靶序列,并进行BLAST验证,我们课题组经大量前期试验筛选出基因沉默作用较强的针对大鼠ACEmRNA和AT_1RmRNA的两条siRNA序列;利用分子克隆技术,在体外合成编码ACE-shRNA和AT_1R-shRNA的寡核苷酸序列单链,退火形成双链之后,利用限制性内切酶技术将其分别与PEGFP6-1和Pgenesil-hH1质粒载体连接,成功的构建了质粒pACE-shRNA、pAT_1R-shRNA真核表达载体;再次利用限制性内切酶技术将目的基因片段ACE与目的基因片段AT_1R连接,成功的构建了携带hU6和hH1启动子的重组串联质粒pACE-AT_1R-shRNA真核表达载体。
2.质粒pACE-AT_1R-shRNA转染大鼠血管内皮细胞条件的优化:①植块法原代培养大鼠血管内皮细胞:将SD大鼠(体重120-150g)用颈椎脱臼处死,无菌状态下取出其胸、腹主动脉,剪成约1mm×1mm的血管片,内膜面壁贴于培养瓶内,在含有胎牛血清的DMEM培养液中进行大鼠动脉血管内皮细胞的原代和传代培养。倒置相差显微镜下观察内皮细胞的形态和生长特性,并进行血管内皮细胞中Ⅷ因子相关抗原(von Willebrand factor,vWF)的检测鉴定。②用不同比例的质粒pACE-AT_1R-shRNA与脂质体转染剂METAFECTENE2000的复合物转染大鼠血管内皮细胞。因质粒中含绿色荧光蛋白的报告基因,故转染后48小时可在荧光显微镜下观察绿色荧光表达并计算转染效率,同时用MTT法检测细胞存活率,寻求转染效率较高且细胞毒性较低的pACE-AT,R-shRNA与METAFECTENE2000的最佳配比。
3.质粒pACE-AT,R-shRNA抑制大鼠血管内皮细胞ACE和AT_1R表达的研究:用pACE-AT,R-shRNA与METAFECTENE2000的最佳配比复合物转染大鼠血管内皮细胞,分别于转染前及转染后的24h、48h、72h收集细胞,用RT-PCR及Western blot法检测ACE和AT_1RmRNA及相应功能蛋白的表达。大鼠血管内皮细胞分为五组:①空白对照组;②质粒对照组;③pACE-shRNA组:④pATiR-shPdqA组;⑤pACE-ATlR-shRNA组。
4.构建靶向抑制大鼠ACE和AT_1R基因的shRNA重组腺病毒载体:利用AdMax腺病毒包装系统,自先期构建的pACE-AT,R-shRNA真核表达载体中酶切出ACE-AT_1R-shRNA,克隆入穿梭质粒pDC316中,然后将穿梭质粒pDC316-EGFP-ACE-shRNA-U6和腺病毒骨架质粒pBHGlox-E1,3Cre共转染293细胞,包装成重组的腺病毒载体Ad5-EGFP-ACE-AT,R-shRNA并扩增、纯化、鉴定。
5.腺病毒介导的ACE-AT_1R-shRNA对自发性高血压大鼠(spontaneous hypertensiverat,SHR)的治疗作用:自发性高血压大鼠随机分为五组:①空白对照组;②病毒对照组;③Ad5-EGFP-ACE-shRNA治疗组;④Ad5-EGFP-AT_1R-shRNA治疗组;⑤Ad5-EGFP-ACE-AT_1R-shRNA治疗组;⑥正常血压对照组。以上各组大鼠均注射两次,注射时间均为实验的第1天和第17天。实验期间做以下检测:①注射前后检测血压、心率的变化。②首次注射后第3天,取治疗组大鼠心、主动脉、肾脏、脑、肺组织,做冰冻切片,荧光显微镜下观察腺病毒载体吸收情况。③首次注射后第3天,心脏体外采血用ELISA法检测各组大鼠血浆中ACE和AngⅡ的含量,且用实时荧光定量PCR及Westernblot法检测心肌、脑、主动脉、肾脏组织中ACE和AT_1RmRNA及相应功能蛋白的差异表达。④实验结束后,取各组大鼠心、脑、主动脉、肾脏组织做光镜及电镜切片,并检测大鼠全心/体重、左心室/体重,及心肌羟脯氨酸、胶原蛋白的含量,以观察心肌重构的变化。同时采血检测肝肾功能。
6.腺病毒介导的ACE-AT_1R-shRNA对自发性高血压大鼠的治疗作用机制:自发性高血压大鼠分组和治疗同前,实验期间做以下检测:①注射前后检测血压、心率的变化。②首次注射后第3天,取治疗组大鼠心、脑、肾脏组织,用ELISA法检测各组大鼠组织中ACE和AngⅡ的含量,并且用实时荧光定量PCR及Western blot法检测心、脑、主动脉、肾脏组织中ACE2和AT_2R mRNA及相应功能蛋白的差异表达。③首次注射后第3天,每组随机取6只大鼠,心脏体外采血.同时取心、脑、肾组织按照NO测试盒说明操作检测NO。结果
1.经DNA测序和酶切鉴定,证明我们构建的靶向抑制大鼠ACE和AT_1R基因的shRNA真核表达载体一重组质粒pACE-AT_1R-shRNA无基因突变,符合实验要求。
2.①植块培养3天倒置相差显微镜观察即可发现有内皮细胞从组织块周围移出,大约10天细胞开始融合成片,呈“铺路石样”。传代培养的细胞和原代培养形态相似,生长较快,4-5天可以融合成单层,传至3-4代时,细胞开始变性变形、脱落,不再继续生长。用免疫荧光法鉴定可见胞浆内有大量绿色荧光。②根据转染效率以及细胞生存率测定结果,筛选出质粒与转染试剂复合物的最佳配比为pACE-AT_1R-shRNA 1.0μg、METAFECTENE2000 4μl,此时转染效率为57.7%,细胞生存率为91.6%,优于其他条件时的转染效率并能保证较低的细胞毒性,故作为我们进一步实验的最佳选择。
3.pACE-shRNA和pACE-AT_1R-shRNA组在转染后48小时细胞内ACEmRNA及相应蛋白表达均明显降低,pAT_1R-shRNA和pACE-AT_1R-shRNA组在转染后48小时AT_1RmRNA的表达明显降低,与空白对照组和质粒对照组相比均有统计学差异(P＜0.05),72小时表达更低(P＜0.01)。实验中发现pACE-AT_1R-shRNA的抑制作用更强一些,可能为ACE和AT_1R双基因联合沉默的协同效应所致,而空白对照组与质粒对照组转染前后各时间点ACE和AT_1R mRNA及相应蛋白的表达无明显变化。
4.经限制性内切酶、PCR检测和荧光显微镜观察,证实成功构建了能够表达ACE和AT_1R shRNA的重组腺病毒载体并制备出高滴度的重组病毒。
5.①干预前SHR各组之间尾动脉压差异无统计学意义(P＞0.05),而SHR各组均与正常血压对照组有显著性差异(P＜0.05)。SHR治疗组于首次注射后第3天,尾动脉压分别下降(25.2+5.3)mmHg,(23.5+4.8)mmHg,(27.1+6.4)mmHg,与治疗前比较有统计学差异(P＜0.05),降压作用可持续15天左右,最大降压幅度达24mmHg,22mmHg,26 mmHg,首次注射后约17天时血压开始回升;第2次注射后,尾动脉压再次明显下降(22+6)mmHg,(20+5)mmHg,(23+7)mmHg,降压作用可持续20天左右,最大降压幅度达23mmHg,22mmHg,23mmHg。而SHR空白对照组和病毒对照组尾动脉压持续升高,正常血压对照组尾动脉压无明显变化。②荧光显微镜下可见心肌、脑、主动脉、肾脏、肺组织的冰冻切片有大量绿色荧光表达,说明Ad5-EGFP-ACE-shRNA,Ad5-EGFP-AT_1R-shRNA,Ad5-EGFP-ACE-AT_1R-shRNA可被富含ACE和AT_1R的组织大量吸收。③SHR治疗组血浆ACE含量明显低于空白对照组与病毒对照组;SHR治疗组Ad5-EGFP-ACE-shRNA血浆AngⅡ显著低于SHR空白对照组和病毒对照组,差异有统计学意义(P＜0.05),SHR治疗组Ad5-EGFP-AT_1R-shRNA血浆AngⅡ明显升高与SHR空白对照组、SHR病毒对照组比较差异有统计学差异(P＜0.05),SHR治疗组Ad5-EGFP-ACE-AT_1R-shRNA血浆AngⅡ介于以上两个治疗组之间。SHR治疗组心、脑、主动脉、肾脏组织中AT_1R mRNA及相应蛋白表达较SHR空白对照和SHR病毒对照组明显降低(P＜0.05),以Ad5-EGFP-AT_1R-shRNA和Ad5-EGFP-ACE-AT_1R-shRNA治疗组明显低于Ad5-EGFP-ACE-shRNA治疗组和正常血压对照组;Ad5-EGFP-ACE-shRNA、Ad5-EGFP-ACE-AT_1R-shRNA治疗组和正常血压对照组ACE mRNA及相应蛋白表达较Ad5-EGFP-AT_1R-shRNA治疗组、SHR空白对照和SHR病毒对照组明显降低(P＜0.05)④光镜切片显示治疗组心肌细胞肥大明显减轻,脑、肾和主动脉细胞病理改变明显减轻;电镜切片显示治疗组心肌细胞和肾脏超微结构明显改善。左心室/体重之比与心肌胶原蛋白含量:治疗组显著低于空白对照组与病毒对照组,但还未降到正常血压对照组水平。⑤整个实验期间,各组心率及肝肾功能均无明显变化。
6.①SHR各治疗组心、脑、肾脏组织的ACE,AngⅡ显著低于SHR空白对照组和病毒对照组,差异有统计学意义(P＜0.05),而与正常血压对照组无统计学差异(P＞0.05)。②SHR各治疗组血清和心、脑、肾脏组织NO显著高于SHR空白对照组和病毒对照组,差异有统计学意义(P＜0.05),而与正常血压对照组无统计学差异(P＞0.05)。③SHR治疗组心、脑、主动脉、肾脏组织的ACE2 mRNA及相应蛋白表达均较SHR空白对照组、SHR病毒对照组显著升高,有统计学意义(P＜0.05),而与正常血压对照组无统计学差异(P＞0.05)。SHR空白对照组、SHR病毒对照组和Ad5-EGFP-AT_1R-shRNA组心、脑、。肾、主动脉组织AT_2R mRNA及相应蛋白表达较WKY组、Ad5-EGFP-ACE-shRNA治疗组,Ad5-EGFP-ACE-AT_1R-shRNA治疗组明显升高,有统计学意义(P＜0.05)。其中单用Ad5-EGFP-AT_1R-shRNA组心、脑、肾、主动脉组织AT_2RmRNA升高最明显。结论:
1.成功构建了以大鼠ACE和AT_1R基因为靶位的真核表达载体pACE-AT_1R-shRNA。
2.通过血管植块方法实现了大鼠动脉血管内皮细胞的原代培养并传代,细胞纯度在90%以上。经过优化转染条件,脂质体转染剂METAFECTENE2000可以将重组质粒pACE-AT_1R-shRNA高效转染入大鼠血管内皮细胞,并保证较低的细胞毒性。
3.真核表达载体pACE-AT_1R-shRNA转染进大鼠血管内皮细胞后可以有效地实现对靶基因mRNA的降解,进而抑制相应功能蛋白的表达。
4.成功构建了能够表达ACE-AT_1R-shRNA的重组腺病毒载体。
5.腺病毒介导的Ad5-EGFP-ACE-shRNA,Ad5-EGFP-AT_1R-shRNA,Ad5-EGFP-ACE-AT_1R-shRNA注射入自发性高血压大鼠体内后,成功发挥了基因沉默作用,有效抑制了ACE和(或)AT_1RmRNA及相应功能蛋白的表达,起到了明显、持久的降压作用,并且显著改善了心肌重构,明显减轻了脑、肾脏、主动脉组织的病理改变,使心脏和肾脏超微结构明显改善,同时未发现明显的副作用。联合沉默组稍优于单基因沉默组,可能由ACE和AT_1R双基因联合沉默的协同效应所致。因此,运用RNA干扰技术进行降压治疗是一种非常具有潜力的新策略,本研究也为小剂量应用Ad5-ACE-AT_1R-shRNA联合基因沉默ACE和AT_1R治疗高血压提供了临床应用的前景。
6.腺病毒介导的Ad5-EGFP-ACE-shRNA,Ad5-EGFP-AT_1R-shRNA,Ad5-EGFP-ACE-AT_1R-shRNA注射入自发性高血压大鼠体内后,使心、脑、肾脏局部组织中ACE、AngⅡ水平降低和NO水平升高,同时使得AT_2RmRNA/AT_1RmRNA和ACE2 mRNA表达显著升高,进而使相应功能蛋白的表达升高,阻断了ACE和ACE2两种羧肽酶表达的失衡,防止了高血压的靶器官损害;由此产生明显持久的降压作用。
Objective
Hypertension is the importantly risk factor of congestive heart failure,stroke and renal failure.According to the statistics of World Health Organization,15-37%of all world adults are hypertensive patients.The incidence of hypertension even comes up to 50%in a group of over 60-year-olds.It is estimated that the number of hypertensive patients across the world will go up to 1,560,000,000 in 2025.However regrettably, only one third of patients has been treated successfully.The main cause is that hypertension is a kind of lifelong disease,but all drugs decreasing blood pressure have short half-life period which is no longer than 24 hours.Hence,patients must take medicine every day,which is not easy to insist on.Moreover,the nonspecific effects and side effects of drugs are also not easy to accept for patients.Therefore,pursuing a kind of lasting and effective antihypertensive gene therapy by regulating gene expression is always scientists' aim and dream.
RNA interference(RNAi)is a new genetic interference technology,which specially degrades the corresponding sequence mRNA by the mediate of double-stranded RNA(dsRNA)which are diced up 21-23 nucleotide-long small interfering RNA(siRNA)by the enzyme Dicer to induce the post-transcriptional gene silencing.Short hairpin RNA(shRNA)is a kind of siRNA.The shRNA is more effective than the siRNA.In this study,we designed and constructed the expression vector of shRNA targeting ACE and AT_1R of RAS closely associated with hypertensive occurance and development in order to degrade ACE and AT_1R mRNA and decrease corresponding protein expression in vivo and vitro experiment with RNAi technology.By this way,we investigated the application prospective of RNAi in antihypertensive treatment.
Methods
1.Construction of eukaryotic expression vector for shRNA targeting ACE and AT_1R gene
Firstly,four RNAi sites targeting rat ACE and AT_1R gene in GenBank were selected according to the guidelines for the selection of highly effective siRNA sequences and then BLAST test was performed.We selected two siRNA sequences with stronger effects of gene silence aiming at rat ACE and AT1R by a lot of previous experiments.Secondly,using molecule cloning technique,two pairs of oligonuleotides fragments were synthesized and annealed to double strands.Thirdly,the double strands were cloned into plasmid vectors PEGFP6-1 and Pgenesil-hH1.Fourthly,The recombinant plasmids were identified by restriction enzyme digestion and assayed sequence of them.The two kinds of recombinant plasmids were named pACE-shRNA and pAT_1R-shRNA.Finally,The target gene fragments of ACE and AT1R were identified by restriction enzyme digestion and assayed sequence of them.The recombinant plasmid carrying the promoter of hU6 and hH1 was named pACE-AT_1R-shRNA.
2.Optimization of the transfection condition oftranfecting pACE-AT_1R-shRNA to rat aortic endothelial cells
(1)The aorta was removed from rat in bacteria free condition.The vessel was opened longitudinally and cut into lmmxlmm pieces.These pieces were pasted inside culture bottle. Aortic endothelial cells were cultured in Dulbecco's modified Eagle's medium(DMEM) containing 10%or 20%heat-inactivated fetal bovine serum(FBS).Cells were identified by morphological(growth characteristic)and immunological(cellular markers)criteria.(2)Rat aortic endothelial cells was transfected with complex of different amount of pACE-AT_1R -shRNA and different concentration of METAFECTENE 2000(polycationic liposome transfection reagent).Transfection efficiency was determined by using fluorescence microscope and cells viability was calculated by using MTT assay after 48h of transfection to select the optimal ratio of pACE-AT_1R-shRNA to METAFECTENE 2000.
3.Study pACE-AT_1R-shRNA on the inhibition of ACE and AT_1R mRNA and protein expression in rat aortic endothelial cells
Rat aortic endothelial cells were transfected with complex of the optimal ratio of pACE-shRNA,pAT_1R-shRNA and pACE-AT_1R-shRNA to METAFECTENE 2000.Total RNA and total protein were extracted from aortic endothelial cells before transfection and after 24h, 48h,72h of transfection.The expression level of ACE and AT1R mRNA was evaluated by real-time quantitative PCR and the expression level of ACE and AT1R protein was evaluated by Western blot.Rat aortic endothelial cells were divided into five groups:(1)blank control group, (2)plasmid control group,(3)pACE-shRNA group,(4)pAT_1R-shRNA group,(5) pACE-AT_1R-shRNA group.
4.Construction of recombinant adenoviral vectors expressing shRNA targeting rat ACE and AT_1R gene
The rat ACE-AT_1R-shRNA segments were obtained from plasmids PACE-AT_1R-shRNA which were constructed at an earlier date by restriction endonucleases digestion and then cloned into the shuttle plasmids pDC316 to form recombinant plasmids pDC316-hH1-AT_1RB-U6-ACEB-EGFP.The recombinant plasmids were cotransfected with genomic plasmids pBHGlox_E1,3Cre into 293 cells to package recombinant adenovirus which were named Ad5-hH1-AT_1RB-hU6-ACEB-EGFE Then the recombinant adenovirus were propagated,purified and identified.
5.Study on the therapeutic effects of shRNA targeting ACE and AT_1R gene by adenovirus-mediated in spontaneously hypertensive rats(SHR)
SHR were randomly divided into five groups:(1)blank control group(injected intravenously with normal saline),(2)adenovirus control group(injected intravenously with control adenovirus),(3)Ads-EGFP-ACE-shRNA treat group(injected intravenously with Ads-EGFP-ACE-shRNA),(4)Ad5-EGFP-AT_1R-shRNA treat group(injected intravenously with Ads-EGFP-AT_1R-shRNA),(5)Ad5-EGFP-ACE-AT_1R-shRNA treat group(injected intravenously with Ad5-EGFP-ACE-AT_1R-shRNA).At the same time,Wistar-Kyoto rats(WKY)were selected as normal control group which were injected intravenously with normal saline.All rats were injected two times at the first day and the seventeenth day in the experiment.The following items were measured during experiment.(1)Systolic blood pressure(SBP)of the caudal artery and heart rate were measured before and after injection.(2)Aorta,myocardium,cerebral,lung and kidney of rats in treat group were observed by using fluorescence microscope to identify the sites of Ads-EGFP-ACE-shRNA,Ad5-EGFP-AT_1R-shRNA and Ad5-EGFP-ACE-AT_1R-shRNA expression at the third day after first injection.(3)At the third day after first injection,serum concentration of ACE and AngⅡwere measured by ELISA.The expression levels of ACE and AT_1R mRNA in aorta,myocardium,cerebral and kidney were evaluated by real-time quantitative PCR and the expression levels of ACE and AT_1R protein in aorta,myocardium,cerebral and kidney were evaluated by Western blot.(4)When the experiment was finished,the ratio of total heart weight(HW)to body weight(BW)and left cardiac ventricle weight(LU)to body weight(BW)were calculated.Myocardial hydroxyproline and collagen levels were measured. Aorta,myocardium,cerebral and kidney pathological changes were observed by light microscope,myocardial and kidney ultrastructures were observed by transmission electron microscope.At the same time,the functions of liver and kidney were measured.
6.Study on the therapeutic mechanism of shRNA targeting ACE and AT_1R gene by adenovirus-mediated in spontaneously hypertensive rats(SHR)
SHR group and therapy were same as above.The following items were measured during experiment.(1)Systolic blood pressure(SBP)of the caudal artery and heart rate were measured before and after injection.(2)At the third day after first injection,tissue content of ACE and AngⅡwas measured by ELISA in myocardium,cerebral and kidney.The expression levels of ACE2 and AT_2R mRNA in aorta, myoeardium,cerebral and kidney were evaluated by real-time quantitative PCR and the expression levels of ACE2 and AT_2R protein in aorta,myocardium,cerebral and kidney were evaluated by Western blot.(3)At the third day after first injection,serum concentration of NO was measured, tissue content of NO was measured in myocardium,cerebral and kidney.
Results
1.Restriction enzyme digestion analysis showed that eukaryotic expression vectors of shRNA targeting ACE and AT_1R gene had been constructed successfully.Sequencing results revealed that the sequences of the vectors were all right.
2.(1)After three days of explant culture,endothelial cells migrating out of the aortic intima could be seen at the edge by a inverted phase-contrast microscope and formed confluent monolayers which showed a cobblestone shape 10 days later.Passaged cells grew faster than primary cells and could form confluent monolayers 4-5 days later.After passaged four generations,cells became deformed,detached from the culture bottle.Characteristic green fluorescence was observed by fluorescence microscope after immunofluorescence labeling for von Willebrand factor.(2)The optimal ratio of pACE-AT_1R-shRNA to METAFECTENE 2000 was“1ug:4ul”according to transfection efficiency and cells viability.At this transfection condition,transfection efficiency was 57.7%and cells viability was 91.6%.
3.Compared with control groups,the expression level of ACE mRNA and protein of pACE-shRNA group and pACE-AT_1R-shRNA group were significantly suppressed after 48h of transfection(P＜0.05),the expression level of AT_1R mRNA and protein of pAT_1R-shRNA group and pACE-AT_1R-shRNA group were significantly suppressed after 48h of transfection (P＜0.05)and both were nearly undetectable 72h later(P＜0.01).The suppression effect of pACE-AT_1R-shRNA group was stronger,the result was caused by cooperative effects of double genes combined silencing.There were no significant difference about expression level of ACE and AT_1R mRNA and protein in blank control group and plasmid control group before and after transfection.
4.Recombinant adenoviral vectors Ad5-EGFP-ACE-AT_1R-shRNA were constructed successfully,which was confirmed by restriction enzyme digestion and PCR and EGFP expression.
5.(1)There was no significant difference in the SBP of the caudal artery among SHR groups before intervention(P＞0.05),but SBP of the caudal artery of all SHR groups was much higher than that of normal control group(P＜0.01).After two days of single injection,SBP of the caudal artery of treat group was effectively reduced by(25.2±5.3)mmHg,(23.5±4.8)mmHg and (27.1±6.4)mmHg respectively.The biggest range reached 24mmHg,22mmHg and 26 mmHg. This antihypertensive effect could last up to 15 days.After two days of the second injection,SBP of the caudal artery of treat group was effectively reduced by(22±6)mmHg,(20±5)mmHg and (23±7)mmHg.The biggest range reached 23mmHg,22mmHg and 23mmHg.This antihypertensive effect could last up to 20 days.However,rats of blank control group and adenovirus control group produced a continued increase in blood pressure and without significant difference between these two groups.(2)Under the fluorescence microscope,marked expression could be observed in heart,cerebral,lung,aorta and kidney.(3)Serum concentration of ACE in each treat group was significantly lower than that of blank control group and adenovirus control group respectively(P＜0.05),similar to that of normal control group(P＞0.05). Serum concentration of AngⅡin Ad5-EGFP-ACE-shRNA group was significantly lower than that of blank control group and adenovirus control group(P＜0.05).Serum concentration of AngⅡin Ad5-EGFP-AT_1R-shRNA group was significantly higher than that of blank control group and adenovirus control group(P＜0.05).Serum concentration of AngⅡin AdS-EGFP-ACE-AT_1R-shRNA was between above two treatment groups.The expression levels of ACE and(or)AT1R mRNA and protein in heart,cerebral,aorta and kidney of each treat group were significantly decreased compared with blank control group and adenovirus control group(all P＜0.05),The expression levels of ACE mRNA and protein in AdS-EGFP-AT_1R-shRNA group were similar to that of blank control group and adenovirus control group(P＞0.05).(4)Compared to blank control group and adenovirus control group,myocardial,cerebral,aorta and kidney hypertrophy and ultrastrueture of myocardial and kidney in each treat group were significantly improved.The LV/BW and the level of myocardial collagen of treat group were all significantly lower than those of blank control group(both P＜0.05)and adenovirus control group(both P＜0.05),hut still higher than those of normal control group (P＞0.05).(5)The heart rate and the function of kidney and liver were not affected during the experiment.
6.(1)Serum concentration of ACE and AngⅡin myocardial,cerebral and kidney of each treat group was significantly lower than that of blank control group and adenovirus control group respectively(P＜0.05),similar to that of normal control group(P＞0.05).(2)Serum and tissue of myocardial,cerebral and kidney concentration of NO in each treat group was significantly higher than that of blank control group and adenovirus control group(P＜0.05),similar to that of normal control group(P＞0.05).(3)The expression levels of ACE2 mRNA and protein in heart, cerebral,aorta and kidney of each treat group were significantly higher compared with blank control group and adenovirus control group(all P＜0.05),similar to that of normal control group(P＞0.05).The expression levels of AT_2R mRNA and protein in heart,cerebral,aorta and kidney of blank control group,adenovirus control group and Ad5-EGFP-AT_1R-shRNA group were significantly higher compared with normal control group,Ad5-EGFP-ACE-shRNA group, Ad5-EGFP-ACE-AT_1R-shRNA group(all P＜0.05),especially Ad5-EGFP-AT_1R-shRNA group was higher.
Conclusions
1.Construct successfully eukaryotic expression vector pACE-AT_1R-shRNA.
2.By explant technique,we successfully accomplished the isolation,primary culture and passaged culture of rat aorta vascular endothelial cells.The cells purity was above 90%.By optimizing the parameters,METAFECTENE 2000 could transfect pACE-AT_1R-shRNA to rat aorta vascular endothelial cells with high effect,at the same time,with high cells viability.
3.Transfection of pACE-AT_1R-shRNA to rat aorta vascular endothelial cells could initiate sequence-specific post-transcriptional gene silencing and inhibit the expression of ACE and AT_1R mRNA,subsequently inhibit the protein expression.
4.Recombinant adenovirus vectors expressing ACE-AT_1R-shRNA were constructed successfully,which laid the foundation of application ofgene therapy to antihypertension.
5.Introduction of shRNA targeting ACE and AT_1R gene by adenovirus-mediated to SHR led to successful inhibition of ACE and(or)AT_1R mRNA and protein,which subsequently produced significance and lasting decrease in blood pressure.Moreover,myocardial remodeling was significantly improved.Myocardial,cerebral,aorta and kidney hypertrophy and ultrastructure of Myocardial and kidney in each treat group were significantly improved.At the same time,there were no obvious side-effects.Combined gene silence group was superior to single gene group(P＞0.05),probably by the cooperative effects of double genes combined silencing.The RNAi technology may become a new strategy of gene therapy for hypertension.The experiment may offer clinical application prospect for combined silencing therapy to antihypertension with low dosage of Ad5-ACE-AT_1R-shRNA.
6.Introduction of shRNA targeting ACE and AT_1R gene by adenovirus-mediated to SHR led to successful expression of AT_2RmRNA/AT_1RmRNA and ACE2 mRNA and protein,block the imbalance expression of two types carboxypeptidase,which subsequently produced significance and lasting decrease in blood pressure and avoided damage of target organs in hypertension;meanwhile,ACE and AngⅡin myocardial,cerebral and kidney of each treat group was significantly lower,NO in each treat group was significantly higher than that of blank control group and adenovirus control group(P＜0.05).
引文
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