【作者】 付鹏；

【导师】 孙英贤；

【作者基本信息】 中国医科大学 ， 内科学， 2007， 博士

【摘要】 富含半胱氨酸蛋白61RNA干扰重组质粒抑制大鼠血管平滑肌细胞增殖的实验研究前言动脉损伤后狭窄的形成是一个复杂的病理生理过程，已证实血管平滑肌细胞（vascular smooth muscle cells，VSMCs）的活化、增殖和迁移是导致动脉壁过度增生的中心环节，在内膜增生及血管重构中发挥重要作用。因而抑制VSMCs的过度增殖对预防和治疗动脉粥样硬化（atherosclerosis，As）和经皮腔内冠状动脉血管成形术（Percutaneous transluminal coronary angioplasty，PTCA）后再狭窄有重要意义。近年来的研究表明，一些小的双链RNA可以高效、特异地阻断体内特定基因表达，促使mRNA降解，诱发细胞表现出特定基因缺失现象，称为RNA干扰（RNAinterference，RNAi）。随后的研究中发现，RNAi现象广泛存在于线虫、果蝇、斑马鱼、真菌以及植物等生物体内，这些生物体利用RNAi来抵御病毒的感染，阻断转座子的作用。RNA能高效特异地阻断基因的表达，在线虫、果蝇体内，RNAi能达到基因敲除的效果。在小鼠和人的体外培养细胞中利用RNAi技术也成功地阻断了基因的表达，实现了细胞水平的基因敲除。RNAi是细胞本身固有的对抗外源基因侵害的一种自我保护现象，是双链RNA（double stranded RNA，dsRNA）分子在mRNA水平关闭相应序列及基因的表达使其沉默的过程，是一种转录后基因沉默（post transcriptional gene silencing，PTGS）。与其他方法相比，RNAi技术在基因功能研究上有其独特的优点：①简单易行，容易开展，干扰结果在短时间内产生，这是其他实验技术无法比拟的；②与基因敲除相比实验周期短，成本低；③与反义技术相比具有高度特异性和高效性；④可进行高通量基因功能分析。RNAi技术的诸多优点使它很快便被作为研究基因功能的主要方法。目前，RNAi技术的应用已经从基因组学研究逐步扩展到临床领域，并成为基因治疗手段。RNAi作为一种高效的序列特异性基因剔除技术在传染性疾病和恶性肿瘤基因治疗领域发展极为迅速。在利用RNAi技术对HIV-1、乙型肝炎、丙型肝炎等进行基因治疗研究中发现，选择病毒基因组中与人类基因组无同源性的序列作为抑制序列可在抑制病毒复制的同时避免对正常组织的毒副作用。同时将抑制序列选择在特定的位点，可对部分有明确基因突变的恶性肿瘤细胞如含有BCL／ABL或AML1／MTG8融合基因的白血病细胞产生凋亡诱导作用。富含半胱氨酸蛋白61（cysteine-ricn 61，cyr61）是CCN家族中第一个被克隆的基因，人的CYR61基因位于染色体1p22.3。它是由血清、纯化的血小板衍化生长因子（platelet-derived growch factor，PDGF）或转移生长因子β（transforming growmfactor-β，TGF-β）作用于成纤维细胞株产生的蛋白分子，属于CCN家族中的分泌性、细胞外基质交联的血管源性调节因子，它能够启动一些与创伤修复过程相关的细胞的生长，迁移和粘附，可以诱导血管平滑肌细胞的增殖、粘附、迁移。AndresHilfiker研究表明人正常动脉血管没有明显的Cyr61的染色，而在人颈动脉斑块旋切术和冠状动脉移植手术的动脉硬化血管中Cyr61mRNA及蛋白均呈高表达，在apo-E缺陷鼠动脉硬化模型上Cyr61mRNA及蛋白均高表达，Wu KJ等在对猴子PTCA术后的研究中发现，在损伤的内膜及增殖的VSMCs中，Cyr61呈现高表达，说明Cyr61参与动脉硬化的形成及血管的狭窄。此外，Cyr61具有明显的促有丝分裂活性和趋化性，可诱导成纤维细胞增殖和分泌细胞外基质（eXtracellularmatrix，ECM）参与调节细胞增生、分化、胚胎发育形成。利用RNAi技术建立Cyr61小干扰RNA（small interference RNA，siRNA）重组质粒抑制VSMCs增殖的研究目前在国内外尚未见报道。为此我们构建了大鼠Cyr61 siRNA重组质粒并将其转染VSMCs中，观察其对VSMCs增殖的抑制作用，以期为将来动脉粥样硬化及PTCA术后再狭窄等增生性疾病的基因治疗奠定基础。方法1．人工合成针对靶序列Cyr61基因的两条寡核苷酸链，寡核苷酸链两端加入酶切位点，便于和表达载体连接。取正反向寡核苷酸链，与退火缓冲液混匀，94℃水浴3min，退火自然冷却至室温。质粒37℃酶切1h，酶切产物电泳，凝胶回收试剂盒回收大片段。退火片段与线性化Pgenesil-1质粒表达载体连接，将连接产物按常规方法转化感受态大肠杆菌DH5a，含Kana^r抗性筛选，挑取阳性克隆，采用酶切和DNA序列分析进行鉴定。构建的质粒分别命名为pCyr61-shRA₁、pCyr61-shRNA₂和p-HK（空质粒对照）。2．应用构建的质粒pCyr61-shRNA转染大鼠VsMcs，24h后应用RT-PCR及Westem blot方法检测VSMCs中Cyr61的mRNA和蛋白表达的变化。3．应用构建的质粒pcyr61-shRNA转染加入AngⅡ刺激的大鼠VSMCs，观察转染后细胞形态学变化；台盼蓝染色法和MTT法检测VSMCs增殖的变化；³H标记胸腺嘧啶掺入法检测VSMCs DNA含量；流式细胞仪检测细胞周期；ELISA法检测分泌到细胞基质中的Cyr61蛋白。结果1．构建的质粒pCyr61-shRNA经酶切和DNA测序证实了表达载体构建成功。2．转染VSMCs 24h后，用荧光显微镜观察，转染效率在60％左右，并能成功表达出绿色荧光蛋白；转染的pCyr61-shRNA₁（0.114±0.012）和pCyr61-shRNA₂（0.105±0.010）的mRNA表达均明显降低，与正常对照组（1.256±0.138）及转染p-HK组（p-HK组）（1.342±0.147）相比有显著性差异（P＜0.01），而转染的p-HK组与正常对照组相比没有明显改变，无统计学差异（p＞0.05）；Westem-Blot结果显示，转染的pCyr61-shRNA₁组（0.012±0.004）和pCyr61-shRNA₂组（0.009±0.001）的蛋白表达均明显降低，与正常对照组（0.984±0.082）及p-HK组（0.898±0.076）相比有显著性差异（p＜0.01），而转染的p-HK组与正常对照组相比没有明显改变，无统计学差异（p＞0.05）。3．加入AngⅡ刺激同时转染VSMCs48h后进行台盼蓝染色法计数细胞数，加入AngⅡ刺激同时转染pCyr61-shRNA₁组和pCyr61-shRNA₂组细胞数（2.031±0.096和2.431±0.765）明显减少，与正常对照组和p-HK组（8.234±0.428和9.642±0.465）相比均有显著性差异（p＜0.01）；而正常对照组和p-HK组相比均无显著性差异（p＞0.05）。MTT法VSMCs检测增殖结果显示，加入AngⅡ刺激同时转染pCyr61-shRNA₁（Ⅲ组）和pcyr61-shRNA₂（Ⅵ组）吸光光度值（0.145±0.008和0.175±0.013）明显降低，与正常对照组（Ⅰ组）和转染的p-HK组（Ⅱ组）（0.856±0.046和0.876±0.033）相比均有显著性差异（p＜0.01）；而Ⅰ组与Ⅱ组相比均无显著性差异（P＞0.05）。³H-TDR掺入法检测VSMCs DNA合成，结果表明，加入AngⅡ刺激同时转染pCyr61-shRNA₁（Ⅲ组）和pCyr61-shRNA₂（Ⅵ组）吸光光度值为（158.333±11.480和162.000±12.156）明显降低，与正常对照组（Ⅰ组）和p-HK（Ⅱ组）（543.000±27.622和536.667±36.866）相比均有显著性差异（P＜0.01）；而Ⅰ组与Ⅱ组相比均无显著性差异（P＞0.05）。4．ELISA检测结果表明，加入AngⅡ刺激同时转染pCyr61-shRNA₁（Ⅲ组）和pCyr61-shRNA₂（Ⅵ组）Cyr61蛋白明显降低，与正常对照组（Ⅰ组）和空质粒对照p-HK（Ⅱ组）相比均有显著性差异（P＜0.01）；而Ⅰ组与Ⅱ组相比均无显著性差异（P＞0.05）。5．流式细胞检测细胞周期结果表明，AngⅡ对照组，AngⅡ10^-6mol／l刺激24小时，不转染质粒（Ⅰ组）：G0／G1期52.7％、S期16.9％、G2／M期30.4％，细胞进入S期、G2／M期明显增加；AngⅡ10^-6mol／l刺激24小时，转染p-HK组（Ⅱ组）：G0／G1期53.8％、S期16.6％、G2／M期29.6％，细胞进入S期、G2／M期也明显增加，未被转染的p-HK抑制；AngⅡ10^-6mol／l刺激24小时，转染pCyr61-shRNA₁（Ⅲ组）：G0／G1期84.3％、S期7.69％、G2／M期8.1％，细胞进入S期、G2／M期明显减少，主要停留在G0／G1期；AngⅡ10^-6mol／l刺激24小时，转染pCyr61-shRNA₂（Ⅵ组）：G0／G1期82.5％、S期6.5％、G2／M期11％，细胞进入S期、G2／M期明显减少，主要停留在G0／G1期。结论1．成功构建的Cyr61RNA干扰重组质粒可用于Cyr61基因干扰的研究中。2．pCyr61-shRNA₁和pCyr61-shRNA₂均明显抑制平滑肌细胞中Cyr61的mRNA和蛋白的表达，具有RNAi作用。3．AngⅡ可刺激VSMCs分泌Cyr61蛋白，此作用通过AT-1型受体途径介导。4．pCyr61-shRNA₁和pCyr61-shRNA₂均抑制了AngⅡ刺激VSMCs分泌Cyr61蛋白，同时抑制VSMCs的增殖。更多还原

【Abstract】 PurposeThe formation of stenosis after artery injury is a complicated pathology and physiology process. It has already been confirmed that activation, proliferation and migration of vascular smooth muscle cells（VSMCs） were centro-link for hyperplasia. Inhibition of proliferation of VSMCs have significant for atherosclerosis and restenosis after percutaneous transluminal coronary angioplasty （PTCA）.1998, Fire and his colleagues found a phenomenon called RNA interference （RNAi）. This phenomenon refers to the gene target specificity post transcriptional gene silencing （PTGS） induced by the double-stranded RNA（dsRNA） which is injected into the Caenorhabditis elegans. During the past a few years, substantial improvements about RNAi were gained worldwide. RNAi became a new gene inhib- iting technique, and can successfully interfere the express of the specific genes using RNAi in vitro-cultured mammalian cells such as from human or rat. This provides a new possibility for human geng therapy.Cysteine-rich61（Cyr61） is a heparin-binding, extracellular, matrix associated protein of the cysteine-rich 61/nephroblastoma family, which also includes connective tissue growth factor, nephroblastoma overexpressed, Wnt-induced secreted protein-1 （WISP-1）, WISP-2, and WISP-3. Cyr61 induces angiogenesis in vivo and supports cell adhesion, promotes cell migration, and enhances growth factor stimulated mitogenesis in fibroblasts and endothelial cells, s strongly expressed in smooth muscle cells of arterial walls during embryonic develooment. Immunoblot and histological analyses showed that Cyr61 accumulates at high levels in the vessel media and neointima from d7 to d14 after balloon angioplasty, corresponding to the period of migratory and proliferative activity of smooth muscle cells in restenosis. Cyr61 supported VSMCs migration, proliferation and adhesion, most likely because of saturation of the Cyr61 heparin-binding sites. In the present study we constructed plasmids with rat Cyr61 short haitpin RNA （shRNA） and transfect the plasmids into rat VSMCs to investigate the changes of the expression of Cyr61 mRNA and protein respectively and the effect on hyperplasia of VSMCs in rat.Methods1. Finding the sequence of rat Cyr61 mRNA in the GeneBank, putting into the corresponding software and designing the primer rank. The fragments amplificated by PCR were inserted to the pGenesil-1 vector, and then selected and identified.2. The plasmids containing the shRNA of Cyr61 were constructed, and the change of rnRNA and protein were detected by RT-PCR and Western blot after transfecting vascular smooth musle cells.3. The plasmids containing the shRNA of Cyr61 were constructed and transfe cted vascular smooth musle cells, observe the shape by the inverted phasecon trast microscope and fluorescence microscope, and detect the hyperplasia of VSMCs by trypan blues taining and MTT. Detected DNA content by incorporating ³H-TDR. Through FCM method, we detected VSMCs cycleResults1. The result of confirmation test is consistent with the prediction, and the pl asmid was certified to be in the right rank.2. After transfecting Cells, there was significant different（P＜0.01）in the expression of Cyr61 mRNA between the gene transfected group （pCyr61 -shRNA₁ 0.114±0.012; pCyr61-shRNA₂0.105±0.010）and the control group （Control group 1.256±0.138; p-Hk group 1.342±0.147）; and there was significant different （P＜0.01） in the expression of Cyr61 protein between the geng transfected group （pCyr 61-shRNA₁ 0.012±0.004; pCyr61-shRNA₂ 0.009±0.001）and the control group （Control group 0.984±0.082; p-Hk group 0.898±0.076）. It is shown that pCyr61- shRNA can decrease the expression of Cyr61 mRNA and protein. 3. After transfecting cells, there was significant different（P＜0.01）in the Cell number between the gene transfected group （pCyr61-shRNA₁2.031±0.096; pCyr 61-shRN A₂ 2.431±0.765）and the control group（Control group 8.234±0.428; p-Hk group 9.642±0.465）; there was significant different （P＜0.01） in the Ratio of light density by MTT between the gene transfected group （pCyr61 -shRNA₁0.145±0.008; pCyr61-shRNA₂ 0.175±0.013）and the control group （Control group 0.856±0.046; p-HKgroup0.876±0.033）; there was significant different （P＜0.01） in the DNA content by incorporating ³H-TDR between the gene transfected group （pCyr61-shRNA₁ 158.333±11.480; pCyr61- shRNA₂ 162.000±12.156）and the control group （Control group543.000±27.622; p-HK group 536.667±36.866）.4. Through ELISA method, we detected VSMCs substance Cyr61 protein, there was significant different（P＜0.01） between AngⅡgroup and Losartan group. （204.179±7.131 vs 115.423±5.782）.5. Through FCM method, we detected VSMCs cell cycle. We found there was significant different（P＜0.01）in the Cell cycle between the gene transfected group （pCyr61-shRNA₁ G0/Glphase 84.3%、S phase7.69%、G2/M phase8.1%; pCyr61shRNA₂ 0/G1 phase 82.5%、S phase6.5%、G2/M phase11%）and the control group （Control group G0/G1 phase52.7%、S phase16.9%、G2/M phase 30.4%; p-HK group G0/G1 phase53.8%、S phase16.6%、G2/M phase29.6%9.642±0.465）.Conclusion1. The successful construction of Cyr61 shRNA plamids have the RNA interference activity.2. The plasmids containing the shRNA of Cyr61 can inhibit the expression of Cyr61 mRNA and protein in VSMCs.3. VSMCs can excreted Cyr61 protein by stimuluing of AngⅡ.4. After transfecting cells, The plasmids containing the shRNA of Cyr61 inhibitted Cyr61 protein and inhibited VSMCs proliferation.更多还原