【作者】 宋燕；

【导师】 张水军；

【作者基本信息】 郑州大学 ， 外科学， 2010， 博士

【摘要】 自体静脉移植及血管腔内成形术是目前治疗血管闭塞性疾病的有效手段,但是术后血管早期再狭窄一直是困扰治疗效果的一大难题。血管损伤或血管吻合术后,血管平滑肌细胞(Vascular smooth muscle cell, VSMC)出现异常的增殖和迁移,这是导致血管早期再狭窄的主要原因。整合素是一类细胞膜表面糖蛋白受体家族分子,主要介导细胞与细胞外基质(Extracellular matrix, ECM)以及细胞与细胞之间的黏附,在调节细胞生长、分化、增殖等方面起着重要作用。整合素α5β1是胞外基质纤维连接蛋白(Fibronectin,FN)的主要受体,通过与配体结合,将细胞骨架与细胞连接起来,引起VSMC的移动,并参与VSMC的迁移、增殖以及血管损伤的修复过程。但是整合素α5β1与VSMC增殖及迁移的具体关系尚不明确,本研究通过转基因技术诱导整合素α5β1高表达和基因沉默诱导整合素α5β1低表达,观察其在VSMC增殖与迁移中的作用。目的本课题通过构建整合素(Integrin, ITG)α5、β1慢病毒表达载体及针对整合素α5、β1的慢病毒siRNA表达载体,采用双向调控的方式,分别诱导VSMC内ITGα5、ITGβ1基因高表达与抑制表达,建立整合素α5、β1诱导高表达与低表达VSMC细胞株。观察调控整合素α5、β1表达对VSMC增殖及迁移的影响,并检测在此过程中整合素信号传导通路中黏着斑激酶(Focal adhesion kinase, FAK)及整合素连接激酶(integrin linked kinase, ILK)的变化情况,探讨整合素介导的VSMC增殖迁移过程中可能的信号传导机制。为防治血管成形术后血管早期再狭窄提供实验依据与理论支持。方法1.构建载体：分别构建整合素α5β1的慢病毒表达载体及针对整合素α5、β1的慢病毒siRNA表达载体。1)亚克隆重组子：设计带有KpnⅠ和MluⅠ内切酶位点的上下游引物,以cDNA克隆pCMV-SPORT6-ITGα5和cDNA克隆pCMV-SPORT6-ITGβ1为模板,PCR扩增出ITGα5和ITGβ1 cDNA全长片段,分别与pGEM-T载体连接后,转化DH5α细胞,筛选得到白色阳性菌落,经PCR和DNA序列测定鉴定,获得阳性克隆重组子.pGEM-T-ITGα5、pGEM-T-ITGβ1。经KpnⅠ、MluⅠ双酶切,获得带有粘性末端的ITGα5和ITGβ1目的片段,与慢病毒表达载体pLenti连接,经KpnⅠ和MluⅠ双酶切鉴定得到亚克隆慢病毒重组子pLent-ITGα5、pLent-ITGβ1;2)克隆重组子：扫描整合素α5、整合素β1 cDNA序列,根据siRNA靶序列设计原则,分别设计出针对ITGα5和ITGβ1基因的发卡样靶序列,并在两端加上BamHⅠ和XhoⅠ酶切位点,复性和纯化发卡DNA, pRNAT-U6.2/Lenti质粒连接,转化DH5α细菌,经PCR扩增筛选和DNA测序,得到克隆重组子pRNAT-U6.2/Lenti-siITGα5-1、pRNAT-U6.2/Lenti-siITGα5-2、pRNAT-U6.2/ Lenti-siITGβ1-1和pRNAT-U6.2/Lenti-siITGβ1-2;3)慢病毒包装：分别提取上述pLent-ITGα5、pLent-ITGβ1、pRNAT-U6.2/Lenti-siITGα5-1、pRNAT-U6.2/Lenti-siITGα5-2、pRNAT-U6.2/ Lenti-siITGβ1-1和pRNAT-U6.2/Lenti-siITGβ1-2及pLentiGFP空载体、pRNAT-U6.2/Leni空载体质粒,与病毒包装系统共转染包装细胞293 FT,包装产生慢病毒,以293 FT细胞内GFP蛋白的表达水平测定病毒滴度;2.感染细胞株：重组慢病毒感染VSMC,建立调控整合素ITGα5、ITGβ1表达细胞株：上述8组慢病毒颗粒分别感染正常VSMC,经G418(新霉素)筛选培养,建立高表达ITGα5株(EX-ITGα5),高表达ITGβ1株(EX-ITGβ1);抑制表达ITGα5株1 (si-ITGα5-1),抑制表达ITGα5株2(si-ITGα5-2),抑制表达ITGβ1株1(si-ITGβ1-1),抑制ITGβ1株2(si-ITGβ1-2),空表达载体株(Con-Ex),空siRNA载体株(Con-si)。再次用高表达ITGα5的重组慢病毒感染EX-ITGβ1细胞,G418筛选培养,命名为D-EX；抑制ITGα5的重组慢病毒感染si-ITGβ1-2细胞,G418筛选培养,命名为D-si；荧光定量RT-PCR和Western blot法检测各组细胞ITGα5、ITGβ1基因mRNA及蛋白表达情况。观察细胞株生长情况；3.荧光定量RT-PCR检测调控ITGα5、TGβ1表达对VSMC细胞FAK、ILK mRNA的影响；4.Transwell法测定调控ITGα5、TGPβ1表达对VSMC细胞侵袭迁移的影响；5.MTT法测定调控ITGα5、ITGβ1表达对VSMC细胞生长的影响；6.流式细胞术分析调控ITGα5、ITGβ1表达对VSMC细胞周期的影响；结果1.载体的构建结果：1)通过PCR扩增出3150bp(ITGα5)和2397bp(ITGβ1)的基因片段,分别在3150bp+176bp和2397bp+176bp处出现扩增带,经同源性比较,插入序列与设计完全一致,证实得到阳性克隆重组子PGEM-ITGα5和pGEM-ITGβ1;KpnI和MluI双酶切及DNA测序,证实得到亚克隆慢病毒重组子pLent-ITGα5、pLent-ITGβ1;2)经BLAST同源性分析,ITGα5基因siRNA最终确定的靶序列是735-753(GGACCAGGAAGCTATTTCT)和970-988 (GCTATGTCACCATCCTTAA); ITGβ1基因siRNA最终确定的靶序列是600-618 (GAGCCACAGACATTTACAT)和1283-1301(GTCAGCAGTAGGAACATTA);发卡样DNA退火产物,位于100bp下,接近60bp处,与设计一致；PCR扩增,得到310bp的扩增片段的阳性克隆,DNA序列测定,与设计一致,证实得到pRNAT-U6.2/Lenti-siITGα5-1、pRNAT-U6.2/ Lenti-siITGα5-2、pRNAT-U6.2/Lenti-siITGβ1-1和pRNAT-U6.2/Lenti-siITGβ1-2；3)病毒包装情况：荧光下显微镜观察测定,各组重组慢病毒滴度均在7.7×105IU/mL以上；2.调控ITGα5、ITGβ1表达细胞株的建立三个对照组Con-si(ITGα5 mRNA 0.252±0.026,ITGβ1 mRNA 0.516±0.056)、Con-Ex(ITGα5 mRNA 0.251±0.021,ITGβ1 mRNA 0.505±0.062)及Con(0.238±0.021,0.471±0.051)之间ITGα5、ITGβ1 mRNA表达量相比较,差异无显著性(P>0.05)；与这三个对照组比较,高表达组EX-ITGα5(0.632±0.102,0.534±0.061)、EX-ITGβ1株(0.271±0.031,1.172±0.11)及D-EX(0.587±0.042,1.146±0.108)ITGα5、ITGβ1 mRNA表达量显著升高,差异具有显著性(P<0.05)；低表达组si-ITGα5-1(0.033±0.004,0.459±0.038), si-ITGα5-2(0.075±0.009,0.493±0.054)及si-ITGβ1-1(0.241±0.023, 0.182±0.021),si-ITGβ1-2(0.234±0.025,0.114±0.013)及D-si(0.036±0.005,0.124±0.017)ITGα5、ITGβ1 mRNA表达量显著降低,差异具有显著性(P<0.05)；与此相一致,各组在分子量150kDa处均有ITGα5免疫印迹出现,其中EX-ITGα5和D-EX免疫印迹最强,显著强于空白各组,EX-ITGα5,D-EX之间无明显差异；抑制组中si-ITGα5-1、si-ITGα5-2及D-si的ITGα5印迹显著弱于空白各组印迹；si-ITGα5-1与si-ITGα5-2比较,si-ITGα5-1的印迹更弱；各组在分子量138kDa处均有ITGβ1免疫印迹出现,其中EX-ITGβ1和D-EX的免疫印迹最强,显著强于空白各组的印迹,EX-ITGβ1和D-EX两组之间无明显差异；抑制组中si-ITGβ1-1、si-ITGβ1-2及D-si的印迹显著弱于空白各组的印迹；si-ITGβ1-1与si-ITGβ1-2比较,si-ITGβ1-2的印迹更弱；这表明调控ITGα5及ITGβ1表达细胞株构建成功。倒置显微镜下观察,EX-ITGα5、EX-ITGβ1、D-EX组细胞生长最快、细胞形态最好,成长条梭状；下调群3组细胞(si-ITGα5、si-ITGβ1、D-si)与对照群3组细胞(Con-Ex、Con-si和VSMC)基本一致,生长速度都略慢于上调群3组细胞,同时细胞形态条梭状没有上调群长,并且圆缩漂浮细胞多于上调群。3.调控ITGα5、ITGβ1表达对VSMC内FAK、ILK mRNA表达情况的影响三个对照组Con-Ex(0.142±0.011)、Con-si(0.129±0.012)、正常VSMC(0.137±0.012)FAK mRNA的表达量之间相比较,差异无显著性(P>0.05)与三个对照组相比较,EX-ITGα5(0.165±0.014)、EX-ITGβ1(0.357±0.0194). D-EX(0.419±0.033)、si-ITGα5(0.111±0.009)、si-ITGβ1(0.054±0.008)、D-si(0.034±0.004)差异均有显著性(P<0.05)；其中,EX-ITGβ1和D-EX组的表达量显著升高,差异有非常显著性(P<0.01),si-ITGβ1和D-si组表达量显著降低,差异有非常显著性(P<0.01)；三个对照组Con-Ex(0.211±0.019)、Con-si(0.194±0.017)及Con(0.203±0.016)ILK mRNA的相对表达量之间比较,差异无显著性(P>0.05)；与这三个对照组相比较,EX-ITGα5(0.216±0.021)、si-ITGα5(0.191±0.018)表达量无明显变化,差异无显著性(P>0.05)；EX-ITGβ1(0.247±0.024)、D-EX(0.256±0.023)表达量升高,差异有显著性(P<0.05)；si-ITGβ1(0.159±0.015)、D-si(0.153±0.014)的表达量降低,差异有显著性(P<0.05)。这表明,上调ITGα5基因表达对VSMC信号传导通路中FAK基因的表达只有少量的诱导提高作用,而对ILK则无提高作用；下调ITGα5基因表达对VAMC内FAK及ILK的基因表达均无作用；上调ITGβ1基因表达对VSMC内FAK、ILK基因表达都有诱导上调作用,并且其作用强于上调ITGα5表达所诱导的；下调ITGβ1基因表达对VSMC内FAK、ILK基因表达的诱导下调作用大于下调ITGα5所诱导的；FAK受到诱导时的变化明显强于ILK的变化。4.调控ITGα5、ITGβ1表达对VSMC细胞侵袭迁移的影响Transwell实验中,三个对照组Con-Ex(32.9±4.4)、Con-si(36.6±4.1)和Con(35.2±4.7)穿透细胞数之间相比较,差异无显著性(P>0.05)；与这三个对照组相比较,EX-ITGα5(41.5±5.6)和si-ITGα5(29.3±3.9)穿透细胞数差异无显著性(P>0.05)；EX-ITGβ1(62.3±6.8)、D-EX(65.7±7.2)穿透细胞数显著增加,差异有显著性(P<0.05);si-ITGβ1(16.2±2.1)、D-si(14.8±1.7)穿透细胞数显著减少,差异有显著性(P<0.05)。这表明,单独上调ITGβ1和同时上调ITGα5、ITGβ1基因表达可有效促进VSMC细胞侵袭和转移的能力；单独下调ITGβ1及同时下调ITGα5、ITGβ1基因表达可有效抑制VSMC细胞侵袭和转移的能力。单独调控ITGα5表达,对VSMC细胞侵袭和转移的能力无显著影响。5.调控ITGα5、ITGβ1表达对VSMC细胞生长的影响与对照组(Con-Ex、Con-si、Con之间P<0.05)比较,在接种3d、4d、5d时,EX-ITGβ1组和D-EX组MTT测定的吸光度A值显著升高(P<0.05),EX-ITGα5与si-ITGα5则无显著性差异(P>0.05)；si-ITGβ1和D-EX显著降低(P<0.05)。这表明,上调ITGβ1基因表达,对细胞生长有促进作用；下调ITGβ1基因表达,对细胞生长有抑制作用。单独调控ITGα5表达对细胞生长无明显作用。6.调控ITGα5、ITGβ1表达对VSMC细胞周期的影响情况经流式细胞学检测,3个对照组之间细胞G0-G1期、G2-M期、S期构成比例,差异无显著性(P>0.05),总的趋势为G0-G1期比例>S期,构成比例>G2-M期。EX-ITGα5、EX-ITGβ1、D-EX中,G0-G1期构成比例均显著下降,差异有显著性(P<0.05),G2-M期构成比例均显著上升,差异有显著性(P<0.05),EX-ITGβ1和D-EX两组S期构成比例显著上升(P<0.05)。这表明,单独上调ITGβ1或同时上调ITGα5、ITGβ1基因表达可促进细胞进入分裂周期。si-ITGα5、si-ITGβ1、D-si中G0-G1期构成比例均显著上升,差异有显著性(P<0.05),G2～M期构成比例均显著下降,差异有显著性(P<0.05),si-ITGβ1和D-si两组S期构成比例显著下降(P<0.05),这说明在VSMC细胞中,单独下调ITGβ1或同时下调ITGα5、ITGβ1基因表达可抑制进细胞进入分裂周期。结论1.构建出ITGα5、ITGβ1慢病毒表达载体和慢病毒siRNA表达载体,成功诱导了VSMC内ITGα5及ITGβ1基因高表达与基因沉默,并建立了ITGα5与ITGβ1高表达及低表达的VSMC细胞株(包括单独高、低表达及同时高、低表达ITGα5、ITGβ1细胞株),这为研究整合素ITGα5及ITGβ1两个亚基的不同功能奠定了实验基础。2.在整合素介导的VSMC增殖与迁移过程中,FAK、ILK参与了整合素介导的信号传导。在此信号的传导过程中,FAK的变化程度明显强于ILK, FAK可能是调控VSMC增殖与迁移的主要信号结构基础。3.单独或者同时调控VSMC中ITGβ1基因高表达,可以诱导VSMC重新进入细胞分裂周期,对VSMC细胞的生长有明显促进作用,VSMC细胞的增殖与迁移能力增强；单独或者同时调控VSMC中ITGβ1基因低表达,可以抑制VSMC细胞重新进入细胞分裂周期,对细胞的生长有明显抑制作用,细胞的增殖与迁移能力也受到明显抑制；单独调控ITGα5的基因表达,则对VSMC的生长、侵袭及转移能力无显著影响。4.整合素β1确实与VSMC的生长、增殖及迁移能力密切相关。通过RNAi同时或者单独抑制VSMC内整合素β1基因表达,可以抑制VSMC的异常增殖及迁移,这为临床防治血管成形术后血管早期再狭窄,提供了一个潜在的治疗靶点。更多还原

【Abstract】 Autogenous vein graft and transluminal angioplasty are the main effective treatment for vascular occlusive disease, but early restenosis after vascular treatment is a major problem troubling the effection. Vascular smooth muscle cells (VSMC) abnormal proliferation and migration, is the main reason leading to early vascular restenosis. Integrins are a family of glycoprotein receptor on cell surface and participate in cell adhesion with extracellular matrix (ECM), playing an important role in regulating cell growth, differentiation and proliferation. Integrinα5β1 is the main receptor of fibronectin (FN), linking the cell cytoskeleton by binding with ligand, which can cause the movement of VSMC, and also involves in vascular smooth muscle cell migration, proliferation and vascular injury repairing process. But the specific relationship between integrinα5β1 with proliferation and migration of VSMC, is still not clear. When integrinα5β1 is overexpressed by transgenic or induced gene silencing in VSMC, there has not been reported about whether it can induce VSMC proliferation and migration or not.ObjectiveTo explore the two-way control effects on proliferation and migration of VSMC, and investigate the changes of signaling pathway focal adhesion kinase (FAK) and integrin linked kinase (ILK), we constructed the lentiviral expression vector of integrinα5β1 and lentiviral vector of RNA silence interference (siRNA) of integrinα5β1, and induced integrinα5β1 overexpression and gene silencing in VSMC respectively. It would help us to understand the mechanisms of Integrinα5β1 regulating VSMC proliferation and migration, and provide experimental and theoretical evidence for precaution method for vascular early restenosis, as well as guide the clinical work.Methods1. Constructing the lentiviral expression vector of integrinα5,β1 and lentiviral vector of RNA silence interference(siRNA) of integrinα5,β11) Subcloning recombinant vector:The entire integrinα5 andβ1 cDNA were amplified by PCR from cDNA pCMV-SPORT6-ITGα5 and cDNA of pCMV-SPORT6-ITGβ1, which the upstream and downstream primers have KpnI and MluI. endonuclease sites, and then ligated with pGEM-T vector. The ligation products were transformed into the E.Coli DH5a cells. The positive recombinant clones pGEM-T-ITGα5, pGEM-T-ITGβ1 were selected and identified byα-complementation, PCR, restriction endonuclease digestion and DNA sequencing. The cloning vector and the Lentivirus were cut by KpnI and MluI. Then they were ligated and transformed. The enzyme analysis and gene sequencing analysis were used to verify the accuracy of recombinant vector pLent-ITGα5 and pLent-ITGβ1.2) Recombinant vector:According to the nucleotide sequence of the integrinα5 and integrinβ1 gene in Genbank and the principles of siRNA design, each 2 segment sequences was chosen:735-753nt,970-988nt and 600-618nt,1283-1301nt. The effective sequence of siRNA targeting integrinα5 andβ1 were designed. Both ends of hairpin target sequences with BamHI and XhoI endonuclease sites. The complementary DNA containing both sense and antisense of the targeting sequence was designed synthesized and cloned into the pRNAT-U6.2/Lenti vector which contained H1 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing integrinα5 orβ1 shRNA were named pRNAT-U6.2/Lenti-si ITGα5-1、pRNAT-U6.2/Lenti-siITGα5-2、pRNAT-U6.2/Lenti-siITGβ1-1 and pRNAT-U6.2/Lenti-siITGβ1-2. Restriction endonuclease digestion and DNA sequencing to confirm the recombinant vector. PCR and gene sequencing analysis was used to verify the accuracy of recombinant vector.3) Lentiviruls packaging:Lentivirus packaging plasmids mixtures with Lentivirus-ITGα5 or Lentivirus-ITGβ1 or pRNAT-U6.2/Lenti-siITGα5-1 or pRNAT-U6.2/Lenti-siITGa5-2 or pRNAT-U6.2/Lenti-siITGβ1-1 or pRNAT-U6.2/ Lenti-silTGβ1-2 cotransfected 293FT cells. All virus stocks were produced by transfection Reagent Lipofectmaine 2000.The titer of virus was tested according to the expression level of GFP.2. Transfecting VSMC cells:Recombinate Lentivirus transfected into VSMC, ITGa5 and ITGβ1 gene up-regulated line and down-regulated line were established:Lentivirus-ITGa5,Lentivirus-ITGβ1,pRNAT-U6.2/Lenti-siITGα5-1, pRNAT-U6.2/Lenti-siITGα5-2, pRNAT-U6.2/Lenti-siITGβ1-1, pRNAT-U6.2/ Lenti-siITGβ1-2 and pLentiGFP empty vector、pRNAT-U6.2/Leni empty vector were transfected into different VSMC, G418 screening method was used to obtain the stable transfection VSMC cells. They were named ITGα5 up-regulated line (EX-ITGα5), ITGβ1 up-regulated line (EX-ITGβ1); ITGα5 down-regulated linel (si-ITGα5-1), ITGα5 down-regulated line2 (si-ITGα5-2), ITGβ1 down-regulated linel (si-ITGβ1-1), ITGβ1 down-regulated line2 (si-ITGβ1-2), pLentiGFP empty vector line (Con-Ex), and pRNAT-U6.2/Leni empty vector (Con-si), accordingly. After stable transfection, Lentivirus-ITGα5 was transfected into EX-ITGβ1 line, got the ITGα5 and ITGβ1 gene all up-regulated line, named D-EX. pRNAT-U6.2/Lenti-siITGα5-1 was transfected into si-ITGβ1-2, got the ITGα5 and ITGβ1 gene all down-regulated line, named D-si; Real time PCR and Western blot were used to detect the changes of integrinα5 andβ1 gene and protein in all the stable transfection cells. Observe cell growth by microscope.3. Real time PCR was used to detect the changes of FAK and ILK gene in the stable transfection cells.4. The changes of invasion and migration abilities were measured by Transwell chamber invasion assay in the stable transfection cells.5. MTT assay was used to detect the proliferative activity in the stable transfection cells. 6. The changes of cell cycle were detected by flow cytometry assay in the stable transfection cells.Results1. Constructing vector:1).The results of PCR, enzyme analysis and DNA sequencing analysis have confirmed the right ITGα5 and ITGβ1 gene were cloned(full length 3150bp+176bp and 2397bp+176bp), recombinant clones pGEM-T-ITGα5、pGEM-T-ITGβ1 confirmed by gene sequencing analysis. Enzyme analysis and gene sequencing, lentiviral expression vector Lentivirus-ITGα5 and ITGβ1 were successfully constructed.2). PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of integrinα5 andβ1 producing psiRNA-integrinα5 andβ1 were constructed successfully. They were named pRNAT-U6.2/Lenti-siITGα5-1、pRNAT-U6.2/ Lenti-siITGα5-2、pRNAT-U6.2/Lenti-siITGβ1-1 and pRNAT-U6.2/Lenti-siITGβ1-2;3). The titer of all the virus were above 7.7×105IU/mL according to testing the expression level of GFP.2. The established cell lines:Integrinα5 andβ1 mRNA expression level were measured by Real-time fluorescence quantitative PCR, the level of the control groups were as follows: Con-si (ITG a 5 mRNA 0.252±0.026, ITGβ1 mRNA 0.516±0.056)、Con-Ex (ITGα5 mRNA 0.251±0.021, ITGβ1 mRNA 0.505±0.062) and Con(ITGα5 0.238±0.021, ITGβ1 0.471±0.051), there were no differences between them (P>0.05);Compared with the above control groups, the ITGα5 mRNA of EX-ITGα5 (0.632±0.102,0.534±0.061) and D-EX (0.036±0.005,0.124±0.017) were significantly increased (P<0.05); and those of si-ITGα5-1 (0.033±0.004, 0.459±0.038) and D-si (0.036±0.005,0.124±0.017) were significantly lower (P <0.05);Compared with the control groups, the ITGβ1 mRNA of EX-ITGβ1 (0.271±0.031,1.172±0.11) and D-EX (0.036±0.005,0.124±0.017) were significantly increased (P<0.05), those of si-ITGβ1-1 (0.033±0.004,0.459±0.038), and D-si were significantly decreased (P<0.05);The same trend occurred in the changes of protein ITGα5 and ITGβ1 detected by Western blot.Observed under inverted microscope, cells of EX-ITGα5、EX-ITGβ1 and D-E growthed faster than the control groups cells,which having better cell morphology and spindle shape; cells of si-ITGα5、si-ITGβ1 and D-si were consistent with the control groups cells, but the growth rate was slightly slower than the control group cells, and spindle cell morphology was less than cells of EX-ITGα5, EX-ITGβ1 and D-EX, but the floating shrinking cells were more.3. FAK mRNA expression level was measured by Real-time fluorescence quantitative PCR, the level of the control groups were as follows:Con-Ex (0.142±0.011)、Con-si (0.129±0.012) and Con (0.137±0.012),there were no difference between them (P>0.05). Compared with the control groups, the FAK mRNA expression level of EX-ITGα5 (0.165±0.014), those of EX-ITGβ1 (0.357±0.0194) and D-EX (0.419±0.033) were higher, the difference were significant (P<0.05); but those of EX-ITGβ1 (0.357±0.0194) and D-EX (0.419±0.033)were markedly higher (P<0.01). Compared with the control groups, the FAK mRNA expression level of si-ITGα5 (0.111±0.009)、si-ITGβ1 (0.054±0.008) and D-si (0.034±0.004) were gone down (P<0.05), but si-ITGβ1 (0.054±0.008) and D-si (0.034±0.004) were significantly lower (P<0.01);ILK mRNA expression level measured by Real-time fluorescence quantitative PCR, the level of the control groups were as follows:Con-Ex (0.211±0.019)、Con-si (0.194±0.017) and Con (0.203±0.016),there were no difference between them (P >0.05). Compared with the control groups, the ILK mRNA expression level of EX-ITGα5 (0.216±0.021) and si-ITGα5 (0.191±0.018) had no differences (P>0.05); the ILK mRNA expression level of EX-ITGβ1 (0.247±0.024) and D-EX (0.256±0.023) were higher, the difference were significant (P<0.05); Compared with the control groups, the ILK mRNA expression level of si-ITGβ1 (0.159±0.015),D-si (0.153±0.014) were lower, the difference were significant (P<0.05)From those data, we could know, up-regulation expression of ITG a 5 have only a small increase role on FAK, but no effect on ILK; Up-regulation expression of ITGβ-1 had stronger role on FAK and ILK than that of ITG a 5.4. Transwell chamber invasion assay:the number of passed through the artificial basement membrane of the control groups were as follows:Con-Ex (32.9±4.4)、Con-si (36.6±4.1) and Con (35.2±4.7),there were no difference between them (P<0.05); Compared with the control groups,the number of EX-ITGα5 (41.5±5.6) and si-ITGα5 (29.3±3.9) had no difference between them (P>0.05); the number of EX-ITGβ1 (62.3±6.8) and D-EX (65.7±7.2) were higher (P<0.05), the number of si-ITGβ1 (16.2±2.1) and D-si (14.8±1.7) were lower (P<0.05).From those data, we could know, there was no effect on VSMC cell invasion and metastasis by regulating ITG a 5 expression only.5. MTT assay showed the proliferative activity of the cells, there were no differences between the control groups (P>0.05); and the speed of cell proliferation among all the groups had no significant difference on the second day (P> 0.05); from the third day, the absorbance values A of MTT were significantly higher among EX-ITGβ1 and D-EX (P<0.05); and si-ITGβ1 and D-EX were significantly lower (P<0.05); but there had no significant difference among EX-ITGα5 and si-ITGα5 (P> 0.05). From those data, we could know, up-regulation ITGβ1 expression could induce cell proliferation, in the other, down-regulation ITGβ1 expression could hinder cell proliferation, regulating ITGα5 gene expression had no effect on cells growth.6. Flow cytometry results showed that, among the three control groups, the distribution of the proportion of cells in G0～G1,G2～M,and S phase were basically similar(P>0.05). G0～G1 phase were more than S phase, which were the general trend among the three control groups; Among EX-ITGα5、EX-ITGβ1 and D-EX, cells in G0～G1 phase were much lower, and G2～M phase were much higher (P<0.05); EX-ITGβ1 and D-EX had more S phase(P>0.05); there were more G0～G1 phase in si- ITGα5±si-ITGβ1 and D-si, and less G2～M phase(P>0.05); among si-ITGβ1 and D-si, cells in S phase were much lower(P>0.05); from those data, we could know, up-regulation ITGβ1 expression could induce cell division, on the other hand, down-regulation ITGβ1 expression could hinder cell proliferation.Conclusion1. Lentiviral expression vector Lentivirus-ITGα5 and ITGβ1 were successfully constructed. Lentivirus RNAi vector of integrinα5 andβ1 producing psiRNA-integrinα5 andβ1 were constructed successfully, also. ITGα5 and ITGβ1 up-regulation expression and down-regulation expression VSMC cell lines were established (Including separate high and low expression of ITGα5, ITGβ1 cell lines,all high and low expression of ITGα5, ITGβ1 cell lines). Form this, different functions of the ITGα5 and ITGβ1 subunits of the foundation could be explored deeply further.2. During integrin-mediated proliferation and migration of VSMC, focal adhesion kinase (FAK) and integrin linked kinase (ILK) were involved in the signaling pathway. In the process of signal transduction, the change of FAK was stronger than that of ILK. FAK may be the main structural basis for the signal conduction of regulating VSMC proliferation and migration.3. Up-regulation expression of ITGβ1, VSMC could be induced to re-enter the cell division cycle, and the ability of invasion, proliferation, and migration could be induced stronger. On the other hand, down-regulation expression of ITGβ1, VSMC could be hindered to re-enter the cell division cycle, and the ability of growth, proliferation, and migration could be induced inferior. There were no effect on the ability of growth, proliferation, and migration by only regulating expression of VSMC gene ITGα5.4. This indicates that integrinβ1 was really involved in the progress of proliferation and migration in VSMC. Down-regulation expression of integrinβ1 gene in VSMC by RNAi, the abnormal proliferation and migration of VSMC could be inhibited. This might provide a possible therapeutic target of prevention and treatment for early vascular restenosis.更多还原