【作者】 尚丹；

【导师】 郑启昌；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 研究背景自体静脉是血管重建术中最常用的移植材料。但自体静脉移植术后出现移植血管狭窄或再狭窄是限制其远期疗效的主要障碍。再狭窄是一个多因素、多环节、多阶段的复杂过程。移植后再狭窄的病理基础是内膜增生和远期动脉硬化。增生内膜的主要成分是血管平滑肌细胞和细胞外基质。而血管平滑肌细胞过度增殖与移行是导致其病理改变的关键,同时血管平滑肌细胞增殖和凋亡平衡的破坏为内膜增生提供了条件。抑制平滑肌细胞过度增殖及迁移是防治血管重建术后再狭窄的有效策略。arresten是Colorado等研究发现的一种新的强效血管生成抑制因子,是IV型胶原α1链的羧基末端NC1结构域多肽片段,其分子量约为26kD。arresten能有效抑制血管内皮细胞的增殖、迁移和管状形成,诱导内皮细胞凋亡;在体内可抑制新生血管形成及肿瘤的生长和转移。前期研究发现,arresten可有效抑制血管平滑肌细胞的体外增殖,我们推测血管生成抑制因子arresten通过抑制血管平滑肌细胞的增殖和迁移可能在内膜增生及血管重建术后再狭窄的发生发展中发挥抑制作用。因此,研究arresten在自体静脉移植术后内膜增生中的作用,对进一步阐明血管重建术后再狭窄的发病机制及提供有效的防治策略均具有重要的理论意义和应用前景。目的本实验中将构建arresten基因的真核表达载体,并在COS-7细胞中表达其分泌蛋白。组织块贴壁法建立体外培养的大鼠血管平滑肌细胞模型,研究真核表达的arresten蛋白对血管平滑肌细胞生物学行为的影响;建立大鼠自体静脉移植模型,局部定位转染arresten基因,探讨其对移植静脉内膜增生的作用。方法(1)根据重组质粒pGEMArr(前期构建)中的人arresten cDNA序列及其真核表达载体pSecTag2-B的多克隆位点编码序列,设计一对特异引物P1和P2。以重组质粒pGEMArr为模板进行PCR扩增,并对扩增产物用PCR纯化试剂盒进行回收和纯化,取纯化的PCR扩增产物经双酶切后与pSecTag2-B质粒载体大片段,在T4 DNA连接酶的作用下进行连接反应。采用内切酶BamHI和EcoRI进行双酶切鉴定重组质粒,命名为pSecTag2-AT。取纯化的重组质粒样品,进行核苷酸序列测定。用DNAssist及DNAMAN分析软件进行序列分析,并与前期克隆的人arresten cDNA序列进行序列比对分析,以确认目的基因序列及重组表达质粒的密码子阅读框是否正确。确定成功后,将重组质粒转染COS-7细胞。RT-PCR检测细胞中目的基因mRNA表达,Western blot分析检测上清液中目的蛋白的表达。(2)体外原代培养并鉴定大鼠血管平滑肌细胞,采用CCK-8法检测arresten蛋白对细胞增殖的影响;采用Transwell趋化小室法进行细胞迁移实验,检测arresten蛋白对血管平滑肌细胞体外迁移的影响;采用Annexin V/FITC法检测arresten蛋白对血管平滑肌细胞体外凋亡的影响。(3)以Sprague-Dawley大鼠为研究对象,建立自体静脉移植模型,局部转染重组质粒pSecTag2-AT。4周后取移植血管,RT-PCR检测血管中arresten基因mRNA的表达,常规HE及Verhoeff弹力纤维染色,利用计算机图象分析,检测移植静脉血管内膜及中膜厚度;内膜和中膜面积;免疫组化方法检测移植血管内膜平滑肌细胞肌动蛋白(α-SMA)及细胞增殖核抗原(PCNA)阳性表达情况;Western blot检测转化生长因子β1(TGF-β1)蛋白的表达。结果(1)重组质粒测序结果经DNAssist和DNAMAN分析软件分析显示,三个克隆的核苷酸序列完全相同;序列比对分析表明,测序结果与前期克隆的人arresten cDNA序列完全一致,即插入到表达载体中的目的基因正确。分析重组表达质粒的密码子开放阅读框,结果表明插入的arresten基因片段与表达载体的密码子阅读框相吻合;即arresten基因完整、准确地克隆入表达载体中,以保证了目的蛋白能正确表达。成功构建arresten基因的真核表达载体pSecTag2-AT。COS-7转染细胞RT-PCR结果显示,转染arresten基因的COS-7细胞中存在有目的基因特异性片段(449bp),表明基因转染成功;Western blot结果表明转染细胞上清液中有目的蛋白表达。(2)细胞体外增殖分析显示真核表达的arresten蛋白可显著抑制血管平滑肌细胞的增殖作用(F =40.154,P<0.01),且呈时间和剂量依赖性;Transwell小室法检测显示经对照细胞,空载体转染及重组质粒转染的COS-7细胞培养上清液处理的平滑肌细胞迁移数为(28.70±3.97)个,(26.10±4.53)个,(14.00±3.33)个,差异具有显著统计学意义(F =38.915,P<0.01);细胞凋亡分析表明arresten蛋白对平滑肌细胞的凋亡具有促进作用(F=29.32, P<0.01),不同作用时间的凋亡率无统计学差异(P>0.05)。(3)成功建立大鼠自体静脉移植模型,RT-PCR结果显示重组质粒pSecTag2-AT转染的血管组织中有目的基因mRNA的表达;病理图象分析结果显示,转染重组质粒组内膜、中膜面积小于空载体组及空白对照组;转染重组质粒组内膜增生程度轻于空载体组及空白对照组,差异有明显统计学意义(P<0.01)。而内膜面积/中膜面积无统计学差异(P>0.05);α-SMA染色表明增生内膜中的细胞是血管平滑细胞;PCNA阳性细胞数及表达指数,转染重组质粒组均少于空载体组及空白对照组,差异有统计学显著意义;转染重组质粒组TGF-β1蛋白的表达与空载体组及空白对照组相比明显下降。结论:在前期工作的基础上,成功构建了中国人arresten基因的真核表达载体,并在COS-7细胞中成功表达其分泌型蛋白。真核表达的人arresten蛋白能有效抑制体外培养的血管平滑肌细胞的增殖和迁移,并促进平滑肌细胞的凋亡。体内局部定位转染人arresten基因可显著抑制移植静脉内膜增生,改善其血管通畅程度。arresten在防治血管移植术后再狭窄方面显示出良好的临床应用前景。更多还原

【Abstract】 BACKGROUND:Autogenous vein is most commonly used for reconstruction vascular operation as the one of the best curative effect grafted materials. However, the long-term effectiveness of operation is limited by patency stenosis or restenosis rates after autogenous vein graft. Restenosis is a complicated course of multiple factor、multiple link and multiple stage. Intimal proliferation and long-term arteriosclerosis are the pathematology foundation of restenosis after transplantation. The essential component of hyperplasic intima are vascular smooth muscle cells and extracellular matrix, while excessive proliferation and migration to endomembrane are the important pathological base of neointima proliferation, and the crucial link of vascular restenosis resulted by diversified factors after reconstructive vascular operation.; meanwhile balance breakdown between the proliferation and apoptosis of vascular smooth muscle cells provide a chance for intima proliferation. It will be the effective prevention and cure approaches of preventing restenosis after reconstructive vascular operation that to inhibit the excessive proliferation and migration of VSMC. arresten, the NC1 domain of the alpha1 chain of type IV collagen, is a recently reported by Colorado angiogenesis inhibitor derived from vascular basement membrane. Its molecular weight is about 26kD. arresten has recently been shown effective in the inhibition of proliferation and migration of endothelial cells、tubiform formation and induction of endothelial apoptosis. It also can suppress neovascularization and tumor growth and metastases in vivo. We found that arresten may inhibit the proliferation of VSMC in vitro in prophase research, so we suppose that angiogenesis inhibitor arresten may produce a marked effect in the intimal hyperplasia and development of restenosis after reconstructive vascular operation. Hence, to research the effect of arresten on intimal hyperplasia after autogenous vein graft is of important significance and application perspective to elucidate the molecular mechanisms of intimal hyperplasia and vasotransplantation restennosis deeply, provide effective prevention and cure strategy.OBJECTIVE:To construct human arresten gene eukaryotic expression vector, express recombinant plasmid in COS-7 cells and excrete protein in our experiment. Rat vascular smooth muscle cells were cultured from thoracic aorta of male Sprague-Dawley rats using the tissue explants method and subcultured by trypsinization;To express human arresten gene in eukaryotic cell, and investigate its effect on the biological behavior off vascular smooth muscle cells in vitro. To construct the model of autogenous vein graft in rat, transfect recombinant plasmid pSecTag2-AT into vein grafts locally; to explore the effect of arresten on the intimal proliferation of venous autografts.METHODS: (1) We design a pair of specific primer P1 and P2 according to the cDNA sequence of human arresten in recombinant plasmid pGEMArr contrasted in prophase and multiclone encoding sequence in eukaryotic expression vector pSecTag2-B. arresten gene was amplified by PCR using recombinant plasmid pGEMArr as the template. The amplified target gene were purified and reclaimed by QIAquick PCR Purification Kits. The purified amplified conduction after coupled reaction was inserted into the pSecTag2-B vector by T4 DNA Ligase. The recombinant plasmid was identified using restriction analysis by incision enzyme BamHI and EcoRI, and it was named as pSecTag2-AT. The target gene was sequenced using the 377 DNA automatic sequencing meter. The target gene sequence and reading frame of codon was affirmed correctly or not through the analysis of the DNA sequence of the obtained gene using DNAssist and DNAMAN analysis software. The recombinant expression plasmid was transfected into COS-7 cells. RT-PCR was used to detect the expression of arresten mRNA in cells, while Western blot assay was applied to detect expressed arresten protein in concentrated supernatants. (2) Rat vascular smooth muscle cells (VSMC) were cultured and identified successfully in vitro. Its proliferation was detected using Cell Counting Kit-8 (CCK-8) in vitro. Migration of VSMC was assayed by a microchemotaxis chamber and a polycarbonate filter (Transwell’s chemotaxis chamber) with pores of 8μm in diameter. Annexin V/FITC assay was carried out to detect the effect of the arresten protein on apoptosis of VSMC in vitro. (3) The model of autogenous vein graft was prepared on Sprague-Dawley rat strains, and recombinant plasmid pSecTag2-AT was transfected into vein grafts locally. RT-PCR was used to detect the expression of arresten mRNA in blood vessel; the intimal and medial areas and thickness were measured by computerized planimetry under a light microscope to compare the degree of intimal hyperplasia (IH) by the calculated ratio between intima (I) and media (M) after staining with hematoxylin-eosin (HE) and Verhoeff (elastic fibers). Immunohistochemical labeling and morphologic analysis of vein graft sections were used to identify proliferating cell nuclear antigen (PCNA )positive cells and smooth muscle alpha-actin(α-SMA) positive cells; Western blot were used to detect the protein of transforming growth factor--β1 (TGF-β1).RESULTS: (1) arresten gene eukaryotic expression vector was identified correctly using restriction analysis, and the sequence was identified and confirmed by DNAssist and DNAMAN analytical software. Three clonged nucleotide sequence were in the same manner; analysis of sequence comparison showed that sequenced result was as the same as the sequence of human arresten cDNA clonged in prostage. Target gene inserted into expression vector was correct. There was coincidence between arresten gene fragment inserted into vector and reading frame of codon of expression vector. It suggested that arresten gene was clonged into expression vector completely and exactly to assure the correct expression of target protein. Eukaryotic expression plasmid pSecTag2-AT was constructed successfully. RT-PCR revealed that arresten-transferred cells contained a 449bp specific fragment of arresten gene. Successful protein expression in supernatants was confirmed by Western blot. (2) CCK-8 assay showed that the proliferation of VSMC were inhibited significantly by arresten protein as compared with control group (F=40.154, P<0.01). It was certain time and concentration dependence. Transwell’s chamber showed that the number of control group, pSecTag2 transfected group and pSecTag2-AT transfected group were 28.70±3.97,26.10±4.53,14.00±3.33, and the differences were statistically significant (F =38.915,P<0.01);apoptosis assay showed that arresten protein promoted the apoptosis of VSMC(F=29.32,P<0.01), and the rate of apoptosis in different time had no statistical significance (P>0.05).(3)RT-PCR revealed that the genome of arresten-transferred tissue contained a 449bp specific fragment of arresten gene; the intimal and medial areas of pSecTag2-AT transfected group was less than that of control group, and the difference were statistically significant (P<0.01), while I/M had no statistics difference. A less intimal thickness of pSecTag2-AT transfected group was seen compared with the control group and pSecTag2 transfected group.α-SMA staining suggested that VSMC were in the hyperplasic intima; the number of PCNA-positive-stained cells and expression index was lower as compared with that of the control group, and the differences were also statistically significant (P<0.01). Western blot revealed that protein level of TGF-β1 decreased obviously compared with the control group and pSecTag2 transfected group.CONCLUSIONS: We constructed eukaryotic expression vector of human arresten gene successfully, expressed recombinant plasmid in COS-7 cells and excreted protein. arresten protein expressed in eukaryotic cells can inhibit proliferation and migration of VSMC effectively and promote the apoptosis of VSMC in vitro. Local transfection of human arresten gene can inhibit the intimal hyperplasia of venous autografts and improve patency level of blood vessel. These showed the good clinical application perspective for prevention and cure of restenosis after transplantation of blood vessel.更多还原