Egr-1特异脱氧核酶下调cyclin D1、TGF-β1和MMPs抑制大鼠血管平滑肌细胞增殖和迁移
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摘要
前言
经皮腔内冠状动脉介入术(percutaneous coronary intervention,PCI)是目前广泛应用的治疗冠心病手段之一,但PCI术后半年内血管重建部位再狭窄(restenosis,RS)影响了它的远期疗效。RS的机制很复杂,包括早期的血管弹性回缩和后期的血管平滑肌细胞(vascular smooth muscle cell,VSMC)增生、迁移、细胞外基质的产生以及血管重塑等。寻找抑制PCI术后RS的方法仍是当前研究的热点。脱氧核酶(deoxyribozyme,DRz)是一种具有酶催化活性的DNA分子,作为一种基因抑制剂有着实际的治疗作用。早期生长反应因子-1(early growth responsefactor-1,Egr-1)是一种锌指结构转录因子,在动脉损伤等外界刺激下被激活,诱导VSMC的分裂、增殖和内膜增生。本研究应用合成的Egr-1特异脱氧核酶(DNAenzyme/10-23DRz,ED5)及杂码ED5(scrambled ED5,ED5SCR),转染至体外培养的大鼠胸主动脉平滑肌细胞中,观察其对大鼠VSMC增殖、凋亡和迁移的影响,探讨其抑制VSMC增殖和迁移的作用机制。
实验方法
1、寡脱氧核苷酸(oligodeoxynucleotide,ODN)ED5、ED5SCR的合成
ED5、ED5SCR由大连宝生物工程有限公司合成。ED5碱基序列为:5′-CCGCTGCCAGGCTAGCTACAACGACCCGGACGT-3′,ED5SCR碱基序列为:5′-GCCAGCCGCGGCTAGCTACAACGATGGCTCCAC-3′,两侧下划线所示部分为寡核苷酸识别序列,中间为保守的催化活性区,在3′和5′端各有两个磷酸二酯键进行硫代磷酸修饰,以增加其稳定性。部分5′端用FITC标记,以便于在荧光显微镜下观察转染后基因的分布。
2、大鼠VSMC培养及实验分组
采用组织块贴壁法原代培养:体重120～150g Wistar大鼠(中国医科大学实验动物中心提供),无菌条件下取出胸主动脉,中膜切块贴壁,滴加含20%胎牛血清(fetal bovine serum,FBS)、100U/ml青霉素、100μg/ml链霉素pH7.4的Dulbecco'smodified Eagle medium(DMEM),置于37℃、5%CO_2饱和湿度培养箱内培养。0.25%胰酶消化传代,传代以后使用含10%FBS、100U/ml青霉素、100μg/ml链霉素pH7.4的DMEM维持细胞生长。经形态学观察和采用α-平滑肌肌动蛋白(α-SM-actin)免疫细胞化学染色鉴定为VSMC。选取3～8代细胞进行实验。
实验分组为:对照组、ED5组、ED5SCR组。实验时各组细胞均使用含10%FBS的不含抗生素DMEM培养;ED5组转染0.1μmol/L ED5;ED5SCR组转染0.1μmol/LED5SCR。
3、VSMC ODN的转染
VSMC使用含10%FBS不含抗生素的DMEM培养,生长至70%后更换无血清无抗生素DMEM培养30h,进行第一次基因转染,18h后更换含10%FBS无抗生素的DMEM进行二次转染。FuGENE6/ODN转染复合物制备:在1.5ml无菌Eppendorf管中加入无血清无抗生素DMEM,再取适量的FuGENE6加入Eppendorf管中混匀并孵育5min,按FuGENE6与ODN 3:1(体积与质量之比)的比例分别加入ED5、ED5SCR并混匀,室温孵育15min。以0.1μmol/L浓度将转染复合物逐滴加入相应分组细胞中,转染结果用荧光显微镜和流式细胞仪(flowcytometry,FCM)检测。
4、实验检测方法
噻唑蓝[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide,MTT]比色法和5-溴脱氧尿嘧啶(5-bromodeoyuridine,BrdU)掺入法测定ODN转染对VSMC增殖的影响;FCM分析ODN转染对VSMC细胞周期分布的影响;用FCM和荧光显微镜检测ODN转染对VSMC凋亡的影响;用改良的Boyden小室法(Transwell)、划痕损伤模型测定ODN转染对VSMC迁移的影响;用RT-PCR检测Egr-1、PCNA、TGF-β1、p53、p21、Bax、MMP-14、MMP-2、TIMP-2mRNA表达的变化,用Westernblot检测细胞Egr-1、PCNA、TGF-β1、p53、p21、MMP-14、MMP-2、TIMP-2蛋白表达水平,免疫细胞化学法检测Egr-1、cyclin D1蛋白表达。
实验结果
1、组织块贴壁法培养5～6天后可见细胞从组织块边缘处长出,3周后长成单层,呈典型“峰-谷”样生长。VSMC特异性标志蛋白α-SM-actin免疫细胞化学染色阳性。贴壁法培养可获得高纯度的VSMC,可传代培养至24代,10代以后细胞的增殖能力逐渐降低。
2、倒置荧光显微镜检测ED5、ED5SCR转染VSMC阳性率分别为70.6±1.52%、72±2.73%,可以看到大多数为细胞浆染色,少数为细胞核染色,其机理为细胞内吞作用。流式细胞仪检测ED5、ED5SCR转染VSMC的摄取率分别为13.5%、15.2%。
3、ED5组VSMC的MTT吸光度值较ED5SCR、对照组显著降低,转染后24、48、72h,ED5对VSMC增殖抑制率分别为25%、20%、18%,ED5SCR组与对照组比较无差别。ED5可明显抑制10%FBS诱导的体外培养的大鼠胸主动脉平滑肌细胞增殖。
4、BrdU掺入实验显示:对照组、ED5组与ED5SCR组BrdU标记率分别为30.46±4.38%、15.37±2.32%、29.17±4.15%。与其它两组相比,ED5组BrdU标记率明显减少,差异具有显著性(P＜0.01);ED5SCR与对照组比较无差别。说明ED5转染显著抑制S期DNA的合成,抑制VSMC增殖。
5、细胞周期分析显示,ED5组G_0/G_1期细胞百分比高于对照组、ED5SCR组,而S期比例低于对照组、ED5SCR组,细胞增殖指数降低,表明ED5可阻止细胞周期由G_0/G_1期向S期推进,使细胞停留在G_0/G_1期,从而抑制VSMC增殖。
6、FCM AnnexinⅤ/FITC、PI双染及Hoechst33342/PI荧光双染测定ED5、ED5SCR对VSMC凋亡无影响。
7、划痕损伤模型测定转染72h后,对照组、ED5组与ED5SCR组VSMC迁移距离分别为159.62±9.57、65.35±5.04、162.84±8.43μm,结果发现同其它两组相比,ED5明显抑制VSMC迁移(P＜0.01),而ED5SCR对VSMC迁移无影响。
8、改良的Boyden小室法测定ED5转染后6h,VSMC迁移抑制率为32.18%(P＜0.01)。ED5SCR对VSMC迁移无影响。
9、RT-PCR检测到:与ED5SCR、对照组相比,ED5转染抑制VSMC Egr-1、PCNA、TGF-β1、MMP-14、MMP-2mRNA表达,对p53、p21、Bax、TIMP-2mRNA表达无影响。
10、Western blot检测到:与ED5SCR、对照组相比,ED5转染抑制VSMC Egr-1、PCNA、TGF-β1、MMP-14、MMP-2蛋白表达,对p53、p21、TIMP-2蛋白表达无影响。未检测到Bax蛋白表达。
11、免疫细胞化学检测到ED5转染抑制VSMC Egr-1、cyclin D1蛋白表达,ED5SCR对VSMC Egr-1、cyclin D1蛋白表达无影响。
结论
1、ED5可特异地抑制Egr-1及其相关基因的表达,抑制10%FBS诱导的体外培养的大鼠胸主动脉平滑肌细胞增殖,此作用与其阻止细胞周期由G_0/G_1期向S期推进,抑制S期细胞合成,抑制cyclin D1、TGF-β1表达有关。ED5SCR无上述作用。
2、ED5可特异地抑制Egr-1,抑制MMP-14表达,减少MMP-2的激活,抑制VSMC的迁移。ED5SCR无上述作用。
3、ED5、ED5SCR对VSMC凋亡无影响。
Objective
Percutaneous coronary intervention(PCI) is now one of widely used methods to treat coronary artery disease,but restenosis following PCI during 6 months limits its long-term benefits.The biological mechanisms are complex,involving elastic recoil in the first instance,followed by extracellular maxtrix production,smooth muscle cell proliferation and migration,and remodeling of the vessel.Therefore the hunt for efficient inhibitors of restenosis remains an ongoing challenge.Deoxyribozyme(DRz) is a DNA molecule with the activity of enzyme catalysis(DNAenzyme),which has practical therapeutic implications as a new category of gene-inhibitor.Early growth response factor-1(Egr-1)is a zinc finger structural factor that is activated under the outside stimulation of arterial injury leading to the splitting,migration and proliferation of vascular smooth muscle cell(VSMC)and neointimal hyperplasia.The aim of this study was to generate a novel DNAenzyme targeting Egr-1(10-23DRz,ED5)and a scrambled ED5(ED5SCR)as control to observe the effect of ED5 on proliferation, apoptosis and migration of rat vascular smooth muscle cells(VSMCs)and study its mechanism of inhibiting proliferation and migration.
Methods
1.Oligodeoxynucleotide(ODN) synthesis
ED5 and ED5SCR were synthesized by Takara and the base sequence of ED5 was:5'-CCGCTGCCAGGCTAGCTACAACGACCCGGACGT-3',ED5SCR:5'-GCCA GCCGCGGCTAGCTACAACGATGGCTCCAC-3',recognition sequence underlined; catalytic domain in the middle.ODN was modified by phosphorothioic acid at the 3' end and 5' end for increasing stability,and a small part was labelled with fluorescein isothiocyanate(FITC) at the 5' end for visualization of cellular uptake of ODN after transfection under fluorescence microscope.
2.VSMC culture and experimental grouping
Adherent culture was to be applied.Rat primary aortic smooth muscle cells were derived from the thoracic aorta medial explants of weighing 120-150g Wistar rats (Provided by China Medical University).The primary cells were cultured in Dulbecco's Modified Eagle Medium(DMEM),pH7.4,containing 20%fetal bovine serum(FBS), 100U/ml penicillin and 100μg/ml streptomycin at 37℃in a humidified atmosphere of 5%CO_2.Cells were passaged by washing once in phosphate buffered solution(PBS) followed by trypsinization.Subcultured strains were cultured in DMEM,pH7.4, containing 10%FBS,100U/ml penicillin and 100μg/ml streptomycin.Cultured cells were idenfied as VSMCs by morphology and immunocytochemistry forα-SM-actin.Subcultured strains were used between passages 3 and 8.
VSMCs were divided into three groups:the control group,the ED5-treated group and the ED5SCR-treated group according to ODN used.Each group of cells were cultured in DMEM containing 10%FBS without antibiotics,the ED5 group was transfected with 0.1μmol/L ED5;the ED5SCR group was transfected with 0.1μmol/L ED5SCR.
3.ODN transfection
VSMCs were cultured in DMEM containing 10%FBS without antibiotics, subconfluent VSMCs(70%) were growth-arrested in serum-free conditions for 30h before transfecting with ODN(0.1μmol/L) using FuGENE6.Cells were transfected a second time in the presence of 10%FBS 18h following the initial transfection.The transfected results were detected by fluorescence microscope and flow cytometry (FCM).
Preparation of FuGENE6 Reagent:ODN complex:add corresponding FuGENE6 Transfection Reagent to the serum-free medium in 1.5ml sterile Eppendorf tubes,mix and incubate for 5 minutes at room temperature.Add ODN to each tube respectively, using 3:1 ratio of FuGENE6 Transfection Reagent(μl) to ODN(μg),mix and incubate the transfection Reagent:ODN complex for 15 minutes at room temperature,then add the complex to the cells.
4.Experiment methods
The effect of ODN on proliferation of VSMC was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)metabolism measuring and 5-bromodeoxyuridine(BrdU) incorporation assay.FCM was performed to track cell cycle progression.Apoptosis in cultured VSMC was quantified by FCM and fluorescence microscope.The modified Boyden's chamber method (Transwell) and wound-healing assay were used to examine the abilities of VSMC migration.RT-PCR was done to detect the mRNA level of Egr-1,PCNA,TGF-β1,p53, p21,Bax,MMP-14,MMP-2 and TIMP-2.Western blot was performed to measure the protein expression of Egr-1,PCNA,TGF-β1,p53,p21,Bax,MMP-14,MMP-2 and TIMP-2.The protein expression of Egr-1,cyclin D1 was measured by immunocytochemistry.
Results
1.Tissue explants adherent method was used to culture VSMCs,a few of cells erupted 5 to 6 days later and approached confluence approximately 3 weeks.VSMCs were characterized by specific hill-and-valley appearance and by positiveα-SM-actin immunocytochemical staining.Highly purified primary VSMCs were gained and can be propagated about 24 population doublings(PD).After PD 10,the cells lost the proliferation ability gradually.
2.The ODN was readily detectable apparently within the cytoplasm and,to a lesser extent,the nucleus by the fluorescence microscope as a result of endocytosis. The green fluorescent could be seen localized in the cytoplasm of 70.6±1.52%,72±2.73%VSMCs respectively on the 24th hour after transfecting of FITC labelled ED5 and ED5SCR.Uptaking rate of FITC-ED5 and FITC-ED5SCR detected by FCM was 13.5%、15.2%respectively.
3.The optical density(OD) of MTT decreased significantly in VSMCs transfected with ED5 compared with that in VSMCs non-transfected.The inhibition ratio of VSMC proliferation was 25%,20%,18%respectively after transfecting of ED5 on the 24th,48th and 72th hour.There was no significant change between the ED5SCR and control group.ED5 can significantly inhibite 10%FBS induced VSMC proliferation.
4.The labeling ratio of BrdU of the group control,ED5 and ED5SCR was 30.46±4.38%、15.37±2.32%、29.17±4.15%respectively.Many proliferous cells were labelled by BrdU in the control and ED5SCR group,while the proliferous cells lablled by BrdU were obviously decreased in the ED5 group(P＜0.01).There was no significant change between the ED5SCR and control group.The results of BrdU incorporation show that ED5 can significantly inhibite the DNA synthesis in S phase, and inhibite 10%FBS induced VSMC proliferation.
5.FCM analysis indicated that the G_0/G_1 phase fraction ratio of the ED5 group was higher than the control group and ED5SCR group,while its S-phase fraction ratio was lower than the control group and ED5SCR group,the proliferation index of the ED5 group decreased significantly.The result suggested that ED5 blocked VSMC cycle in G_0/G_1 phase,inhibited the proliferation of VSMC.
6.There was no significant difference about the ratio of apoptosis in VSMC among these groups by FCM analysis detected with Annexin V/FITC combined PI and fluorescence microscope detected with Hoechst33342/PI double labelled assay.
7.Wound-healing assay showed that the migration lenth of the group control,ED5 and ED5SCR was 159.62±9.57、65.35±5.04、162.84±8.43μm respectively.The results suggested that ED5 inhibited VSMC migration after mechanical injury(P＜0.01). ED5SCR had no effect to VSMC migration.
8.The modified Boyden's chamber method showed that the number of VSMC migration was reduced by 32.18%at 6 hour after transfecting of ED5.ED5SCR had no contribution to VSMC migration.
9.The results of RT-PCR showed that the mRNA transcription of Egr-1,PCNA, TGF-β1,MMP-14 and MMP-2 were decreased obviously in the ED5 group compared with the control group and ED5SCR group,while the mRNA transcription of p53,p21, Bax and TIMP-2 had no significant change.
10.The results of Western blot showed that protein translation of Egr-1,PCNA, TGF-β1,MMP-14 and MMP-2 were decreased obviously in the ED5 group compared with the control group and ED5SCR group,while the protein translation of p53,p21 and TIMP-2 had no significant change.The result of Bax was not shown.
11.The result of immunocytochemistry showed that protein translation of Egr-1, cyclin D1 was decreased obviously in the ED5 group compared with the control and ED5SCR group.ED5SCR had no effect to the protein translation of Egr-1,cyclin D1.
Conclusion
1.ED5 gene transfection may specially suppress the expression of Egr-1 and corresponding genes with Egr-1.ED5 can significantly inhibite 10%FBS induced VSMC proliferation.This effect may be related to its blocking VSMC cell cycle in G_0/G_1 phase,inhibiting the DNA synthesis in S phase,decreasing the expression of cyclin D1 and TGF-β1.ED5SCR gene transfection does not have the same effect as ED5.
2.ED5 gene transfection may specially suppress the expression of Egr-1,regulate the expression of MMP-14,decrease activation of MMP-2,and inhibit VSMC migration.ED5SCR gene transfection does not have the same effect as ED5.
3.ED5 and ED5SCR gene transfection does not influence the ratio of apoptosis in VSMC.
经皮腔内冠状动脉介入术(percutaneous coronary intervention,PCI)是目前广泛应用的治疗冠心病手段之一,但PCI术后半年内血管重建部位再狭窄(restenosis,RS)影响了它的远期疗效。RS的机制很复杂,包括早期的血管弹性回缩和后期的血管平滑肌细胞(vascular smooth muscle cell,VSMC)增生、迁移、细胞外基质的产生以及血管重塑等。寻找抑制PCI术后RS的方法仍是当前研究的热点。脱氧核酶(deoxyribozyme,DRz)是一种具有酶催化活性的DNA分子,作为一种基因抑制剂有着实际的治疗作用。早期生长反应因子-1(early growth responsefactor-1,Egr-1)是一种锌指结构转录因子,在动脉损伤等外界刺激下被激活,诱导VSMC的分裂、增殖和内膜增生。本研究应用合成的Egr-1特异脱氧核酶(DNAenzyme/10-23DRz,ED5)及杂码ED5(scrambled ED5,ED5SCR),转染至体外培养的大鼠胸主动脉平滑肌细胞中,观察其对大鼠VSMC增殖、凋亡和迁移的影响,探讨其抑制VSMC增殖和迁移的作用机制。
实验方法
1、寡脱氧核苷酸(oligodeoxynucleotide,ODN)ED5、ED5SCR的合成
ED5、ED5SCR由大连宝生物工程有限公司合成。ED5碱基序列为:5′-CCGCTGCCAGGCTAGCTACAACGACCCGGACGT-3′,ED5SCR碱基序列为:5′-GCCAGCCGCGGCTAGCTACAACGATGGCTCCAC-3′,两侧下划线所示部分为寡核苷酸识别序列,中间为保守的催化活性区,在3′和5′端各有两个磷酸二酯键进行硫代磷酸修饰,以增加其稳定性。部分5′端用FITC标记,以便于在荧光显微镜下观察转染后基因的分布。
2、大鼠VSMC培养及实验分组
采用组织块贴壁法原代培养:体重120～150g Wistar大鼠(中国医科大学实验动物中心提供),无菌条件下取出胸主动脉,中膜切块贴壁,滴加含20%胎牛血清(fetal bovine serum,FBS)、100U/ml青霉素、100μg/ml链霉素pH7.4的Dulbecco'smodified Eagle medium(DMEM),置于37℃、5%CO_2饱和湿度培养箱内培养。0.25%胰酶消化传代,传代以后使用含10%FBS、100U/ml青霉素、100μg/ml链霉素pH7.4的DMEM维持细胞生长。经形态学观察和采用α-平滑肌肌动蛋白(α-SM-actin)免疫细胞化学染色鉴定为VSMC。选取3～8代细胞进行实验。
实验分组为:对照组、ED5组、ED5SCR组。实验时各组细胞均使用含10%FBS的不含抗生素DMEM培养;ED5组转染0.1μmol/L ED5;ED5SCR组转染0.1μmol/LED5SCR。
3、VSMC ODN的转染
VSMC使用含10%FBS不含抗生素的DMEM培养,生长至70%后更换无血清无抗生素DMEM培养30h,进行第一次基因转染,18h后更换含10%FBS无抗生素的DMEM进行二次转染。FuGENE6/ODN转染复合物制备:在1.5ml无菌Eppendorf管中加入无血清无抗生素DMEM,再取适量的FuGENE6加入Eppendorf管中混匀并孵育5min,按FuGENE6与ODN 3:1(体积与质量之比)的比例分别加入ED5、ED5SCR并混匀,室温孵育15min。以0.1μmol/L浓度将转染复合物逐滴加入相应分组细胞中,转染结果用荧光显微镜和流式细胞仪(flowcytometry,FCM)检测。
4、实验检测方法
噻唑蓝[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide,MTT]比色法和5-溴脱氧尿嘧啶(5-bromodeoyuridine,BrdU)掺入法测定ODN转染对VSMC增殖的影响;FCM分析ODN转染对VSMC细胞周期分布的影响;用FCM和荧光显微镜检测ODN转染对VSMC凋亡的影响;用改良的Boyden小室法(Transwell)、划痕损伤模型测定ODN转染对VSMC迁移的影响;用RT-PCR检测Egr-1、PCNA、TGF-β1、p53、p21、Bax、MMP-14、MMP-2、TIMP-2mRNA表达的变化,用Westernblot检测细胞Egr-1、PCNA、TGF-β1、p53、p21、MMP-14、MMP-2、TIMP-2蛋白表达水平,免疫细胞化学法检测Egr-1、cyclin D1蛋白表达。
实验结果
1、组织块贴壁法培养5～6天后可见细胞从组织块边缘处长出,3周后长成单层,呈典型“峰-谷”样生长。VSMC特异性标志蛋白α-SM-actin免疫细胞化学染色阳性。贴壁法培养可获得高纯度的VSMC,可传代培养至24代,10代以后细胞的增殖能力逐渐降低。
2、倒置荧光显微镜检测ED5、ED5SCR转染VSMC阳性率分别为70.6±1.52%、72±2.73%,可以看到大多数为细胞浆染色,少数为细胞核染色,其机理为细胞内吞作用。流式细胞仪检测ED5、ED5SCR转染VSMC的摄取率分别为13.5%、15.2%。
3、ED5组VSMC的MTT吸光度值较ED5SCR、对照组显著降低,转染后24、48、72h,ED5对VSMC增殖抑制率分别为25%、20%、18%,ED5SCR组与对照组比较无差别。ED5可明显抑制10%FBS诱导的体外培养的大鼠胸主动脉平滑肌细胞增殖。
4、BrdU掺入实验显示:对照组、ED5组与ED5SCR组BrdU标记率分别为30.46±4.38%、15.37±2.32%、29.17±4.15%。与其它两组相比,ED5组BrdU标记率明显减少,差异具有显著性(P＜0.01);ED5SCR与对照组比较无差别。说明ED5转染显著抑制S期DNA的合成,抑制VSMC增殖。
5、细胞周期分析显示,ED5组G_0/G_1期细胞百分比高于对照组、ED5SCR组,而S期比例低于对照组、ED5SCR组,细胞增殖指数降低,表明ED5可阻止细胞周期由G_0/G_1期向S期推进,使细胞停留在G_0/G_1期,从而抑制VSMC增殖。
6、FCM AnnexinⅤ/FITC、PI双染及Hoechst33342/PI荧光双染测定ED5、ED5SCR对VSMC凋亡无影响。
7、划痕损伤模型测定转染72h后,对照组、ED5组与ED5SCR组VSMC迁移距离分别为159.62±9.57、65.35±5.04、162.84±8.43μm,结果发现同其它两组相比,ED5明显抑制VSMC迁移(P＜0.01),而ED5SCR对VSMC迁移无影响。
8、改良的Boyden小室法测定ED5转染后6h,VSMC迁移抑制率为32.18%(P＜0.01)。ED5SCR对VSMC迁移无影响。
9、RT-PCR检测到:与ED5SCR、对照组相比,ED5转染抑制VSMC Egr-1、PCNA、TGF-β1、MMP-14、MMP-2mRNA表达,对p53、p21、Bax、TIMP-2mRNA表达无影响。
10、Western blot检测到:与ED5SCR、对照组相比,ED5转染抑制VSMC Egr-1、PCNA、TGF-β1、MMP-14、MMP-2蛋白表达,对p53、p21、TIMP-2蛋白表达无影响。未检测到Bax蛋白表达。
11、免疫细胞化学检测到ED5转染抑制VSMC Egr-1、cyclin D1蛋白表达,ED5SCR对VSMC Egr-1、cyclin D1蛋白表达无影响。
结论
1、ED5可特异地抑制Egr-1及其相关基因的表达,抑制10%FBS诱导的体外培养的大鼠胸主动脉平滑肌细胞增殖,此作用与其阻止细胞周期由G_0/G_1期向S期推进,抑制S期细胞合成,抑制cyclin D1、TGF-β1表达有关。ED5SCR无上述作用。
2、ED5可特异地抑制Egr-1,抑制MMP-14表达,减少MMP-2的激活,抑制VSMC的迁移。ED5SCR无上述作用。
3、ED5、ED5SCR对VSMC凋亡无影响。
Objective
Percutaneous coronary intervention(PCI) is now one of widely used methods to treat coronary artery disease,but restenosis following PCI during 6 months limits its long-term benefits.The biological mechanisms are complex,involving elastic recoil in the first instance,followed by extracellular maxtrix production,smooth muscle cell proliferation and migration,and remodeling of the vessel.Therefore the hunt for efficient inhibitors of restenosis remains an ongoing challenge.Deoxyribozyme(DRz) is a DNA molecule with the activity of enzyme catalysis(DNAenzyme),which has practical therapeutic implications as a new category of gene-inhibitor.Early growth response factor-1(Egr-1)is a zinc finger structural factor that is activated under the outside stimulation of arterial injury leading to the splitting,migration and proliferation of vascular smooth muscle cell(VSMC)and neointimal hyperplasia.The aim of this study was to generate a novel DNAenzyme targeting Egr-1(10-23DRz,ED5)and a scrambled ED5(ED5SCR)as control to observe the effect of ED5 on proliferation, apoptosis and migration of rat vascular smooth muscle cells(VSMCs)and study its mechanism of inhibiting proliferation and migration.
Methods
1.Oligodeoxynucleotide(ODN) synthesis
ED5 and ED5SCR were synthesized by Takara and the base sequence of ED5 was:5'-CCGCTGCCAGGCTAGCTACAACGACCCGGACGT-3',ED5SCR:5'-GCCA GCCGCGGCTAGCTACAACGATGGCTCCAC-3',recognition sequence underlined; catalytic domain in the middle.ODN was modified by phosphorothioic acid at the 3' end and 5' end for increasing stability,and a small part was labelled with fluorescein isothiocyanate(FITC) at the 5' end for visualization of cellular uptake of ODN after transfection under fluorescence microscope.
2.VSMC culture and experimental grouping
Adherent culture was to be applied.Rat primary aortic smooth muscle cells were derived from the thoracic aorta medial explants of weighing 120-150g Wistar rats (Provided by China Medical University).The primary cells were cultured in Dulbecco's Modified Eagle Medium(DMEM),pH7.4,containing 20%fetal bovine serum(FBS), 100U/ml penicillin and 100μg/ml streptomycin at 37℃in a humidified atmosphere of 5%CO_2.Cells were passaged by washing once in phosphate buffered solution(PBS) followed by trypsinization.Subcultured strains were cultured in DMEM,pH7.4, containing 10%FBS,100U/ml penicillin and 100μg/ml streptomycin.Cultured cells were idenfied as VSMCs by morphology and immunocytochemistry forα-SM-actin.Subcultured strains were used between passages 3 and 8.
VSMCs were divided into three groups:the control group,the ED5-treated group and the ED5SCR-treated group according to ODN used.Each group of cells were cultured in DMEM containing 10%FBS without antibiotics,the ED5 group was transfected with 0.1μmol/L ED5;the ED5SCR group was transfected with 0.1μmol/L ED5SCR.
3.ODN transfection
VSMCs were cultured in DMEM containing 10%FBS without antibiotics, subconfluent VSMCs(70%) were growth-arrested in serum-free conditions for 30h before transfecting with ODN(0.1μmol/L) using FuGENE6.Cells were transfected a second time in the presence of 10%FBS 18h following the initial transfection.The transfected results were detected by fluorescence microscope and flow cytometry (FCM).
Preparation of FuGENE6 Reagent:ODN complex:add corresponding FuGENE6 Transfection Reagent to the serum-free medium in 1.5ml sterile Eppendorf tubes,mix and incubate for 5 minutes at room temperature.Add ODN to each tube respectively, using 3:1 ratio of FuGENE6 Transfection Reagent(μl) to ODN(μg),mix and incubate the transfection Reagent:ODN complex for 15 minutes at room temperature,then add the complex to the cells.
4.Experiment methods
The effect of ODN on proliferation of VSMC was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)metabolism measuring and 5-bromodeoxyuridine(BrdU) incorporation assay.FCM was performed to track cell cycle progression.Apoptosis in cultured VSMC was quantified by FCM and fluorescence microscope.The modified Boyden's chamber method (Transwell) and wound-healing assay were used to examine the abilities of VSMC migration.RT-PCR was done to detect the mRNA level of Egr-1,PCNA,TGF-β1,p53, p21,Bax,MMP-14,MMP-2 and TIMP-2.Western blot was performed to measure the protein expression of Egr-1,PCNA,TGF-β1,p53,p21,Bax,MMP-14,MMP-2 and TIMP-2.The protein expression of Egr-1,cyclin D1 was measured by immunocytochemistry.
Results
1.Tissue explants adherent method was used to culture VSMCs,a few of cells erupted 5 to 6 days later and approached confluence approximately 3 weeks.VSMCs were characterized by specific hill-and-valley appearance and by positiveα-SM-actin immunocytochemical staining.Highly purified primary VSMCs were gained and can be propagated about 24 population doublings(PD).After PD 10,the cells lost the proliferation ability gradually.
2.The ODN was readily detectable apparently within the cytoplasm and,to a lesser extent,the nucleus by the fluorescence microscope as a result of endocytosis. The green fluorescent could be seen localized in the cytoplasm of 70.6±1.52%,72±2.73%VSMCs respectively on the 24th hour after transfecting of FITC labelled ED5 and ED5SCR.Uptaking rate of FITC-ED5 and FITC-ED5SCR detected by FCM was 13.5%、15.2%respectively.
3.The optical density(OD) of MTT decreased significantly in VSMCs transfected with ED5 compared with that in VSMCs non-transfected.The inhibition ratio of VSMC proliferation was 25%,20%,18%respectively after transfecting of ED5 on the 24th,48th and 72th hour.There was no significant change between the ED5SCR and control group.ED5 can significantly inhibite 10%FBS induced VSMC proliferation.
4.The labeling ratio of BrdU of the group control,ED5 and ED5SCR was 30.46±4.38%、15.37±2.32%、29.17±4.15%respectively.Many proliferous cells were labelled by BrdU in the control and ED5SCR group,while the proliferous cells lablled by BrdU were obviously decreased in the ED5 group(P＜0.01).There was no significant change between the ED5SCR and control group.The results of BrdU incorporation show that ED5 can significantly inhibite the DNA synthesis in S phase, and inhibite 10%FBS induced VSMC proliferation.
5.FCM analysis indicated that the G_0/G_1 phase fraction ratio of the ED5 group was higher than the control group and ED5SCR group,while its S-phase fraction ratio was lower than the control group and ED5SCR group,the proliferation index of the ED5 group decreased significantly.The result suggested that ED5 blocked VSMC cycle in G_0/G_1 phase,inhibited the proliferation of VSMC.
6.There was no significant difference about the ratio of apoptosis in VSMC among these groups by FCM analysis detected with Annexin V/FITC combined PI and fluorescence microscope detected with Hoechst33342/PI double labelled assay.
7.Wound-healing assay showed that the migration lenth of the group control,ED5 and ED5SCR was 159.62±9.57、65.35±5.04、162.84±8.43μm respectively.The results suggested that ED5 inhibited VSMC migration after mechanical injury(P＜0.01). ED5SCR had no effect to VSMC migration.
8.The modified Boyden's chamber method showed that the number of VSMC migration was reduced by 32.18%at 6 hour after transfecting of ED5.ED5SCR had no contribution to VSMC migration.
9.The results of RT-PCR showed that the mRNA transcription of Egr-1,PCNA, TGF-β1,MMP-14 and MMP-2 were decreased obviously in the ED5 group compared with the control group and ED5SCR group,while the mRNA transcription of p53,p21, Bax and TIMP-2 had no significant change.
10.The results of Western blot showed that protein translation of Egr-1,PCNA, TGF-β1,MMP-14 and MMP-2 were decreased obviously in the ED5 group compared with the control group and ED5SCR group,while the protein translation of p53,p21 and TIMP-2 had no significant change.The result of Bax was not shown.
11.The result of immunocytochemistry showed that protein translation of Egr-1, cyclin D1 was decreased obviously in the ED5 group compared with the control and ED5SCR group.ED5SCR had no effect to the protein translation of Egr-1,cyclin D1.
Conclusion
1.ED5 gene transfection may specially suppress the expression of Egr-1 and corresponding genes with Egr-1.ED5 can significantly inhibite 10%FBS induced VSMC proliferation.This effect may be related to its blocking VSMC cell cycle in G_0/G_1 phase,inhibiting the DNA synthesis in S phase,decreasing the expression of cyclin D1 and TGF-β1.ED5SCR gene transfection does not have the same effect as ED5.
2.ED5 gene transfection may specially suppress the expression of Egr-1,regulate the expression of MMP-14,decrease activation of MMP-2,and inhibit VSMC migration.ED5SCR gene transfection does not have the same effect as ED5.
3.ED5 and ED5SCR gene transfection does not influence the ratio of apoptosis in VSMC.
引文
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