【作者】 韩伟；

【导师】 刘闺男；

【作者基本信息】 中国医科大学 ， 内科学， 2010， 博士

【摘要】 前言血管平滑肌细胞(vascular smooth muscle cell, VSMC)过度增殖在动脉粥样硬化、血管成形术后再狭窄等血管增殖性疾病的病理生理过程中起着重要作用,是引起血管内膜异常增生、血管重塑及血管再狭窄的主要细胞成分之一。从基因治疗角度调控VSMC的增殖、迁移及凋亡是当前心血管疾病防治领域的研究热点之一。早期生长反应因子-1(early growth response factor-1, Egr-1)是一种锌指结构转录因子(transcription factor,TF),调控着多种与VSMC增殖相关基因的表达,诱导VSMC的分裂、增殖和内膜增生等。在外源性刺激时Egr-1表达增多,促进VSMC增殖,导致血管再狭窄等病理性血管修复反应,抑制其表达能够有效防止VSMC的异常增生,从而达到治疗疾病的目的。诱骗寡脱氧核苷酸(decoy oligodeoxynucleotides, decoy ODNs)技术是将与TF靶相结合位点(顺式作用元件)序列相同的人工合成双链寡核苷酸导入细胞内,竞争性诱骗TF与其结合,使TF所调控的能促进细胞增殖的下游基因表达受抑,从而在转录水平上达到抑制细胞增殖的目的。VSMC增殖、凋亡及迁移是动脉粥样硬化、再狭窄发生发展的关键因素,细胞周期又是调控VSMC多种病理过程的最终共同通路。因此,在本研究我们人工设计合成了针对Egr-1mRNA的decoy ODNs,转染原代培养的大鼠VSMC,观察转染ODNs后对Egr-1、细胞周期、增殖、凋亡、迁移及其相关基因表达变化的影响,进一步探讨Egr-1 decoy ODNs调控VSMC增殖、凋亡及迁移的作用机制和途径,为基因治疗血管增殖性疾病提供一个新的思路。实验方法1、Egr-1诱骗及诱骗对照寡脱氧核苷酸(decoy ODNs和decoy ODNs SCR)的设计合成Egr-1的诱骗寡脱氧核苷酸(decoy ODNs)基因序列为：上游5’-TCG CCC TCG CCC CCG CTA AGGG-3’下游3’-AGC GGG GGC GGG GGC GAT TCCC-5’Egr-1的诱骗对照寡脱氧核苷酸(decoy ODNs SCR)基因序列为：同诱骗序列类似的错配双链寡脱氧核苷酸上游5’-AGC CGC ACC GGC CTG CCT CGTC-3’下游3’-TCG GCG TGG CCG GAC GGA GCAG-5’经聚丙烯酰胺凝胶电泳法纯化并冻干保存,在其3′和5′端进行硫代修饰,部分寡核苷酸的5′端用FITC标记,以便于在流式细胞仪(flow cytometry, FCM)和荧光显微镜下观察转染后基因的分布情况。寡核苷酸由大连宝生物试剂公司合成。2、原代大鼠VSMC培养及实验分组采用组织块贴壁法原代培养：体重100～120g Wistar大白鼠(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’s modified Eagle medium (DMEM),置于37℃、5%CO2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、诱骗组、SCR组。实验时各组细胞均使用含10%FBS的不含抗生素DMEM培养液培养；诱骗组转染0.1μmol/L Egr-1 decoy ODNs; SCR组转染0.1μmol/L Egr-1 decoy ODNs SCR。3、寡脱氧核苷酸(ODNs)的细胞内转染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-ODNs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Decoy ODNs、Decoy ODNs SCR并混匀,室温孵育15min。以0.1μmol/L终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FCM观察检测。4、电泳迁移率变动分析(Electrophoretic Mobility Shift Assay, EMSA)收集各组培养细胞提取核蛋白。4μg核蛋白提取物与30pmol生物素标记的探针(大连宝生物公司合成,序列略)进行杂交(按Roche说明书操作),取杂交反应液10μl经6%非变性聚丙烯酰胺凝胶电泳(100V,40分钟)后转膜(Amersham公司),加入1：20稀释的发光液(宁波唯奥)暗室曝光显影。为证明ODNs与Egr-1结合的特异性,加入突变探针进行对照。5、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ODNs转染对VSMC细胞周期分布的影响；用FCM、电镜和荧光显微镜检测ODNs转染对VSMC超微结构及凋亡的影响；用改良的Boyden小室法(Transwell)、划痕损伤法测定ODNs转染对VSMC迁移的影响：用RT-PCR检测Egr-1、PCNA、cyclin D1、CKD4、p53、p21、MMP-14、MMP-2、TIMP-2mRNA表达的变化,用Western blot检测细胞Egr-1、PCNA、cyclin D1、CKD4、p53、p21、MMP-14、MMP-2、TIMP-2蛋白表达水平。实验结果1、组织块贴壁法培养5-7天后可见细胞从组织块边缘爬出,3-4周后可长成单层融合细胞,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20余代,选取3-8代细胞用于实验,10代以后VSMC的增殖能力及性状逐渐改变。2、倒置荧光显微镜检测Decoy ODNs、Decoy ODNs SCR转染效率：VSMC阳性率分别为71±1.25%、69±2.47%,强绿色荧光大多数分布于细胞核内,少数可见于细胞浆,其机理为细胞的内吞噬作用。流式细胞仪检测Decoy ODNs、Decoy ODNs SCR转染VSMC的摄取率分别为13.9%、14.7%。3、EMSA结果显示转染的Egr-1 decoy ODNs能与Egr-1蛋白相结合,竞争性的占据了Egr-1与DNA的结合位点。因此,在提取的核蛋白中,Egr-1的量明显减少,即Egr-1 decoy ODNs有效地抑制Egr-1蛋白与DNA的结合活性。而Egr-1decoy ODNs SCR并无此作用。4、诱骗组VSMC的MTT吸光度值(A值)较对照组和SCR组显著降低,转染后24、48、72h, Egr-1 Decoy ODNs对VSMC增殖抑制率分别为22.8%、21.9%、20.7%；而与对照组相比,SCR组各时间点A值变化没有统计学意义。Egr-1Decoy ODNs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。5、BrdU掺入实验显示：转染Egr-1 Decoy ODNs能显著抑制VSMC的DNA合成。与对照组(39.30±4.51%)相比,诱骗组BrdU阳性率(20.38±2.87%)明显减少,且差异具有显著性(P<0.01)；SCR组与对照组无差别,阴性对照未见BrdU标记。说明Egr-1 Decoy ODNs转染能显著抑制S期DNA的合成,抑制VSMC增殖。6、FCM细胞周期分析显示：与对照组、SCR组比较,不同时间点(24、48、72h)的诱骗组G0／G1期细胞百分比高,而S期比例低,细胞增殖指数降低,表明Egr-1 Decoy ODNs可阻止细胞周期由G0／G1期向S期的转化进程,使细胞停留在G0／G1期,从而抑制VSMC增殖。7、FCM Annexin V/FITC、PI双染及Hoechst33342/PI荧光双染测定、电镜成像分析均显示：Egr-1 Decoy ODNs及Egr-1 Decoy ODNs SCR对VSMC凋亡无影响。8、划痕损伤法测定转染72h后,对照组、诱骗组与SCR组VSMC迁移距离分别为102.88±6.41、53.66±9.47、104.49±6.48μm,结果发现同其它两组相比,Egr-1Decoy ODNs能明显抑制VSMC迁移(P<0.01),而Egr-1 Decoy ODNs SCR对VSMC迁移无影响。9、Transwell小室法测定Egr-1 Decoy ODNs转染后24h,VSMC迁移抑制率为47.83%(P<0.01),而Egr-1 Decoy ODN对VSMC迁移无影响。10、电镜下原代VSMC胞质富含大量肌丝,细胞器较少,呈收缩型状态；多次传代的VSMC胞体肥大,胞浆内线粒体、粗面内质网等增多,肌丝含量减少,呈合成型状态；诱骗组VSMC胞体及核较小,胞浆内肌丝含量相对较多,细胞器较少,接近收缩型。而SCR组与多次传代的正常对照组VSMC形态相似,说明Egr-1 decoy ODNs可抑制VSMC的表型转化。11、RT-PCR结果提示：与对照组、SCR组相比,Egr-1 Decoy ODNs转染抑制VSMC的Egr-1、PCNA、cyclinD1、CDK4、MMP-14、MMP-2mRNA表达,而对p53、p21、TIMP-2mRNA表达无影响。12、Western blot结果提示：与对照组、SCR组相比,Egr-1 Decoy ODNs转染抑制VSMC Egr-1、PCNA、cyclinD1、CDK4、MMP-14、MMP-2蛋白表达,对p53、p21、TIMP-2蛋白表达无影响。结论1、Egr-1 Decoy ODNs可特异性地抑制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,此作用与其抑制cyclin D1、CDK4、PCNA表达,阻止细胞周期由G0／G1期向S期转换,抑制S期细胞合成有关。2、Egr-1 Decoy ODNs可特异性地与Egr-1蛋白相结合,竞争性的占据了Egr-1与DNA的结合位点,抑制Egr-1自身转录表达,从而抑制MMP-14表达,减少MMP-2的激活,进而能够抑制VSMC迁移。3、Egr-1 Decoy ODNs、Egr-1 Decoy ODNs SCR对VSMC凋亡无影响。更多还原

【Abstract】 ObjectiveThe excess proliferation of vascular smooth muscle cells (VSMC) plays an important role in the pathology of cardiovascular hyperplasia diseases such as arthrosclerosis (AS), coronary heart disease and post-angioplasty restenosis (RS). In fact, superfluous proliferation is one of the major cellular events resulting in neointimal hyperplasia, vascular wall remodeling and RS. In present studies of the prevention and treatment of cardiovascular disease, regulation of proliferation, migration and apoptosis of VSMCs via gene therapy is a promising new avenue.Early growth response factor-1 (EGR-1) is a zinc finger transcription factor (TF), which controls the expression of multiple genes related to cell proliferation. EGR-1 expression is activated by an exogenous stimulus, facilitating cell proliferation and neointimal hyperplasia, which can then result in a pathological vascular repair reaction such as RS. Inhibiting the expression of EGR-1 may effectively block excessive VSMC proliferation, and ultimately prevent disease. The decoy oligodeoxynucleotides (ODNs) strategy is involves transfection of double-stranded ODNs, the sequence of which corresponds to the binding site sequence of the TF in question. These ODNs can compete for TF binding with the native TF binding site, thus leading to the inhibition of downstream gene regulation by the targeted TF. To investigate the mechanism of this inhibition and its effect on VSMC proliferation and apoptosis, we designed and synthesized decoy ODNs targeting EGR-1 and transfected them into the primary cultures of VSMCs in rats. We then evaluated ODN binding to EGR-1 and the subsequent effect on downstream gene expression, cell cycle, apoptosis and migration.Methods1. EGR-1 decoy oligodeoxynucleotides (decoy ODNs) and scrambled decoy ODNs (SCR) design and synthesisDouble-stranded EGR-1 decoy ODNs were synthesized by TaKaRa Biotech Co. (Dalian, China) according to the GenBank published, cis-element sequence of the EGR-1 binding site. The sequence is as follows:5’-TCGCCCTCGCCCCCGCTAAGGG-3’,3’-AGCGGGGGCGGGGGCGATTCCC-5’;The sequence of the scrambled ODNs (SCR) was:5’-AGCCGCACCGGCCTGCCTCGTC-3’,3’-TCGGCGTGGCCGGACGGAGCAG-5’.3’and 5’phosphorothioate modifications were added to the ODNs to enhance their stability. The 5’terminus of a subset ODNs was labeled with fluorescein isothiocya-nate (FITC) to identify gene distribution under fluorescence microscopy.2. VSMC culture and experimental groupingAdherent culture was to be applied.Rat primary aortic smooth muscle cells were derived from the thoracic aorta medial explants of weighing 100-120g 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% CO2. 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 identified 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 (normal culture cells), decoy group (transfection of EGR-1 decoy ODNs), SCR group (transfection of scrambled ODNs). Each group of cells were cultured in DMEM containing 10% FBS without antibiotics, the Decoy group was transfected with 0.1μmol/L Egr-1 decoy ODNs; the Decoy SCR group was transfected with 0.1μmol/L Egr-1 decoy ODNs SCR.3. ODNs transfectionVSMCs were cultured in DMEM containing 10% FBS without antibiotics, subconfluent VSMCs (70%) were growth-arrested in serum-free conditions for 30h before transfection with ODNs(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 ODNs to each tube respectively, using 3:1 ratio of FuGENE6 Transfection Reagent (μl) to ODN (μg), mix and incubate the transfection Reagent:ODNs complex for 15 minutes at room temperature, then add the complex to the cells.4. Electrophoretic Mobility Shift Assay (EMSA)Cells of every group were collected and the nucleoproteins were extracted according to the manufacturers instructions of Applygen Technologies Inc.(Beijing, China).4μg of nucleoprotein extraction were hybridized with 30 pmol biotin-labeled probe (TaKaRa Biotech Co. Dalian, China), according to the EMSA kit manufacturer’s instructions (Roche).10μl of each reaction was electrophoresed on 6% native polyacrylamide gels (100 V,40 min) and transferred onto nitrocellulose filters (Amersham). Gels were exposed to develop in dark room by adding illumination (1:20 dilution, Viagene Biotech, Ningbo Co.). As a control, a mutation probe was added to prove the specific binding of decoy ODNs to EGR-1.5. Experiment methodsThe 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, cyclinD1, CKD4, p53, p21, MMP-14, MMP-2 and TIMP-2. Western blot was performed to measure the protein expression of Egr-1, PCNA, TGF-β1, cyclinD1,CKD4, p53, p21, MMP-14, MMP-2 and TIMP-2.Results1. 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 more than 20 passages. After 10 passages, the cells lost the proliferation ability gradually.2. The ODNs were readily detectable apparently within the nucleus and, to a lesser extent, the cytoplasm by the fluorescence microscope as a result of endocytosis. The green fluorescent could be seen localized in the nucleus of 71±1.25%,69±2.47%. VSMCs respectively on the 24th hour after transfectiOn of FITC labeled ODNs. Uptaking rate of FITC-Decoy ODNs and FITC-Decoy ODNs SCR detected by FCM was 13.9%,14.7% respectively.3. EMSA results revealed that transfected EGR-1 decoy ODNs can bind to EGR-1 protein and competitively occupy the DNA binding site. Accordingly, the amount of EGR-1 was markedly diminished in decoy ODN-treated nuclear extracts. In a word, the EGR-1-binding activity was effectively inhibited by EGR-1 decoy ODNs but not by SCR ODNs.4. The optical density (OD) of MTT decreased significantly in VSMCs transfected with Egr-1 decoy ODNs compared with that in VSMCs non-transfected. The inhibition ratio of VSMC proliferation was 22.8%,21.9%,20.7%respectively after transfecting of Egr-1 decoy ODNs on the 24th,48th and 72th hour. There was no significant change between the Decoy SCR and Control group. Egr-1 decoy ODNs can significantly inhibit 10% FBS induced VSMC proliferation.5. The labeling ratio of BrdU of the group control, Decoy and Decoy SCR was 39.30±4.51%,20.38±2.87%,38.47±3.55%respectively. Many proliferous cells were labeled by BrdU in the Control and Decoy SCR group, while the proliferous cells labeled by BrdU were obviously decreased in the Decoy group (P<0.01). There was no significant change between the Decoy SCR and Control group. The results of BrdU incorporation show that Egr-1 decoy ODNs can significantly inhibit the DNA synthesis in S phase, and inhibit 10% FBS induced VSMC proliferation.6. FCM analysis indicated that the G0/G1 phase fraction ratio of the Decoy group was higher than the Control group and Decoy SCR group, while its S-phase fraction ratio was lower than the Control group and Decoy SCR group, the proliferation index of the Decoy group decreased significantly. The result suggested that Egr-1 decoy ODNs blocked VSMC cycle in G0/G1 phase, inhibited the proliferation of VSMC.7. 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 labeled assay. 8. Wound-healing assay showed that the migration lenth of the group control, Decoy and Decoy SCR was 102.88±6.41,53.66±9.47,104.49±6.48μm respectively. The results suggested that EGR-1 decoy ODNs inhibited VSMC migration after mechanical injury (P<0.01). EGR-1 decoy ODNs SCR had no effect to VSMC migration.9. The modified Boyden’s chamber method showed that the number of VSMC migration was reduced by 47.83%(P<0.01) at 24 hours after transfection of EGR-1 decoy ODNs. EGR-1 decoy ODNs SCR had no contribution to VSMC migration.10. Under TEM, the primary VSMCs of contractile phenotype were rich in cytoplasmic myofilaments, and little rough endoplasmic reticulum was observed. High passage VSMCs exhibited hypertrophy, increased mitochondia and rough endoplasmic reticulum, and a decrease in cytoplasmic myofilaments, indicating a synthetic phenotype. In the decoy ODN-transfected group, VSMCs had a contractile phenotype, with small nuclei, decreased organelles and a relatively normal amount of myofilaments. This indicates that EGR-1 decoy ODNs inhibited the transformation of VSMCs. SCR-transfected cells were similar to controls.11. The results of RT-PCR showed that the mRNA transcription of Egr-1, PCNA, cyclinDl, CDK4, MMP-14 and MMP-2 were decreased obviously in the Decoy group compared with the Control group and SCR group, while the mRNA transcription of p53, p21 and TIMP-2 had no significant change.12. The results of Western blot showed that protein translation of Egr-1, PCNA, cyclinD1, CDK4, MMP-14 and MMP-2 were decreased obviously in the Decoy group compared with the Control group and SCR group, while the protein translation of p53, p21 and TIMP-2 had no significant change.Conclusion1. Egr-1 Decoy ODNs gene transfection may specially suppress the expression of Egr-1 and corresponding genes with Egr-1. Egr-1 Decoy ODNs can significantly inhibit 10%FBS induced VSMC proliferation. This effect may be related to its blocking VSMC cell cycle in G0/G1 phase, inhibiting the DNA synthesis in S phase, decreasing the expression of cyclinDl, CDK4 and TGF-β1.2. Egr-1 Decoy ODNs can bind to EGR-1 protein and competitively occupy the DNA binding site. Accordingly, the amount of EGR-1 was markedly diminished in decoy ODN-treated nuclear extracts, then specially suppress the expression of Egr-1, regulate the expression of MMP-14, decrease activation of MMP-2, and inhibit VSMC migration.3. Egr-1 Decoy ODNs and Egr-1 Decoy ODNs SCR gene transfection does not influence the ratio of apoptosis in VSMC.更多还原