Survivin、TGFβ3和TIMP1基因重组慢病毒多表达载体构建及其表达活性检测

目的应用Gateway技术构建生存素(Survivin)、人转化生长因子β3(TGFβ3)和基质金属蛋白酶抑制剂1(TIMP1)慢病毒多表达质粒,并检测其表达活性。方法应用Gateway技术,以自剪切多肽2A串联Survivin、TGFβ3、TIMP1的3段外源基因,并克隆到慢病毒多表达质粒上,通过RT-PCR和Western-blot技术分别检测质粒转染293T细胞后目的基因mRNA和蛋白质表达水平。结果 RT-PCR和Western-blot检测结果显示,慢病毒多表达质粒成功表达了3种目的基因,且其mRNA和蛋白质表达水平较空载体对照组、PBS对照组明显升高,差异有显著性(F=17.87~69.11,q=6.94~15.47,P<0.05)。结论成功构建了慢病毒多表达质粒pLenti6.3-Survivin-P2A-TGFβ3-T2A-TIMP1,为下一步病毒的制备以及体内实验提供了基础。     

人基质金属蛋白酶组织抑制剂1腺相关病毒表达质粒的构建及检测

目的构建含人基质金属蛋白酶组织抑制剂1（tissue inhibitor of metalloproteinases1,TIMPl）的腺相关病毒（adenoassociatedvirus,AAV）表达质粒并观察其在人胚肾293（HEK293）细胞中的表达,为进一步逆转椎间盘退变提供依据。方法以含有人TIMP1基因序列的质粒pBLAST49-hTIMP1为模板,通过PCR的方法扩增出TIMP1基因,再采用分子克隆的方法,把TIMP1克隆到AAV表达质粒pSNAV2．0构建重组质粒pSNAV2-TIMP1。把重组质粒转染HEK293细胞,用免疫荧光和Westernblot的方法对重组质粒的表达进行研究,ELISA法检测细胞培养上清液中TIMP1蛋白的含量。结果PCR、酶切、DNA测序等方法均证实成功构建了含有完全正确的TIMP1基因序列的AAV表达质粒pSNAVz-TIMP1。体外成功转染入HEK293细胞,免疫荧光、Westernblot及ELISA的结果表明重组质粒能够高表达TIMP1蛋白。结论成功构建了表达TIMP1的重组质粒pSNAV2-TIMP1,并证实在蛋白质水平获得了表达,为进一步逆转椎间盘退变研究奠定了基础。     

腺相关病毒与人α1干扰素基因重组体的构建

目的 构建腺相关病毒载体(pWP8A)与人α1干扰素(hIFNα1)基因重组体。方法 用多聚酶链反应(PCR)方法扩增获得hIFNα1基因；以T-A克隆方法将PCR扩增hIFNα1基因亚克隆到PCR2，1质粒中，产生PCR2．1—hIFNα1；用EcoRI酶切PCR2．1—hIFNα1，后回收hIFNα1基因片断，将此片断连接到pWP8A的EcoRI位点上，构建重组体pWP8A—hIFNα1；pWP8A—hIFNα1转化JM109大肠杆菌扩增，hIFNα1基因经测序鉴定符合Genebank中hIFNα1序列，并用BanH I酶和Xbal I酶双酶切鉴定hIFNα1基因插入方向。结果 经MR扩增获得567bp hIFNα1基因；构建重组体pWP8A—hIFNα1，经测序证实pWP8A—hIFNα1中的hIFNα1与Genebank中hIFNα1序列相同，用BamH I酶和Xbal I酶双酶切鉴定hIFNα1基因插入方向正确的重组体pWP8A—hIFNα1。结论 成功构建腺相关病毒载体(pWP8A)与人α1干扰素(hIFNα1)基因的重组体。     

重组腺相关病毒介导的TIMP-1载体的构建及鉴定

目的：构建及鉴定载入金属蛋白酶组织抑制因子1(TIMP-1)的重组腺相关病毒载体。方法：应用基因重组的方法构建含全长TIMP-1的重组腺相关病毒骨架质粒(pAAV—TIMP-1)，利用磷酸钙沉淀法将腺相关病毒载体质粒共转染入HEK293细胞中，分别装配成rAAV—TIMP-1和rAAV—hrGFP，将rAAV-hrGFP转导HT1080细胞测定重组病毒的滴度，Westernblot方法检测rAAV—TIMP-1在HT1080细胞中的表达情况。结果：成功构建rAAV—TIMP-1，病毒滴度达到10^9 TU／ml，转导细胞后，转导组TIMP-1的表达水平高于对照组。结论：构建的rAAV—TIMP-1，为基因修饰胰岛移植提供了新的手段。     

PSNAV2-CTGF重组质粒构建及其在人胚肾293细胞中的表达

目的 构建含人结缔组织生长因子（CTGF）的腺相关病毒（AAV）表达载体,并观察其在人胚肾293（HEK293）细胞中的表达。方法 以含有人CTGF基因序列的质粒pCMV-SPORT6为模板,应用PCR克隆出CTGF基因,采用分子克隆的方法把CTGF克隆到AAV表达载体pSNAV2.0上构建重组质粒pSNAV2-CTGF。把重组质粒转染HEK293细胞,用免疫荧光法对重组质粒目的蛋白的表达进行研究,MTT法检测细胞培养上清液中CTGF蛋白的生物学活性。结果 成功构建完全正确的CTGF基因序列的AAV表达载体pSNAV2-CTGF,将其转染HEK293细胞后,免疫荧光检测到目的蛋白的表达,转染后的细胞上清具有促使成纤维细胞增殖的生物学活性。结论 成功构建了真核表达载体pSNAV2-CTGF,转染HEK293细胞后能够表达具有生物学活性的CTGF蛋白。     

基质金属蛋白酶组织抑制剂1基因重组腺病毒载体的构建

目的 为探讨基因治疗逆转或延缓椎间盘退变的可行性,构建及制备人基质金属蛋白酶组织抑制剂1 (tissue inhibitor of metalloproteinase 1,TIMP1) cDNA重组腺病毒载体.方法 以含TIMP1 cDNA序列的质粒为模板,通过PCR方法扩增TIMP1cDNA片段,将TIMP1 cDNA全长定向克隆到Ad5MaxTM腺病毒系统的穿梭质粒pDC316上,构建pDC316-TIMP1穿梭质粒;使用Ad5MaxTM腺病毒包装系统,脂质体介导的穿梭质粒及骨架质粒pBHGlox-E1,3Cre共转染HEK293细胞,同源重组构建含TIMP1 cDNA的重组腺病毒Ad5-TIMP1,通过PCR方法鉴定重组腺病毒Ad5-TIM P1的正确性.通过阴离子柱层析方法纯化重组腺病毒Ad5-TIMP1并测定重组腺病毒感染滴度.结果 经PCR及酶切方法证实pDC316-TIMP1穿梭质粒中存在630 bp大小左右的插入片段,与目的基因TIMP1的cDNA大小一致;经PCR鉴定证实TIMP1 cDNA重组腺病毒载体Ad5-TIMP1构建成功,病毒感染性滴度为1×1010 IU/mL.结论 成功构建TIMP1 cDNA重组腺病毒载体,为应用基因治疗方法逆转或延缓椎间盘退变的研究奠定实验基础.     

人正常和退变腰椎间盘中TIMP1的表达及其意义

目的 探讨金属蛋白酶组织抑制因子1 (TIMP1)mRNA在正常与退变椎间盘中表达的差别及意义.方法 利用荧光定量PCR技术检测TIMP1 mRNA在正常椎间盘(9个)与退变椎间盘(30个)组织的含量,结合年龄、病程、病理类型进行分析.结果 TIMP1在正常椎间盘组织表达量为0.900±0.289,退变椎间盘中TIMP1 mRNA表达量为0.340±0.241,两者差异有显著性(t=5.841,P<0.05).退变椎间盘中,不同年龄、病程及病理类型组间TIMP1 mRNA的表达量差异无统计学意义.结论 TIMP1 mRNA 的含量下降在腰椎间盘退变过程中起着重要作用,但未见与年龄、病程及病理类型有明显的关系.     

介导人血红素加氧酶-1基因的重组腺相关病毒载体的构建

目的:构建含人血红素加氧酶-1(hHO-1)基因的重组腺相关病毒(AAV)载体并进行鉴定。方法:利用基因重组技术,采用限制性内切酶HindⅢ从质粒XM-6/hHO-1中将目的基因hHO-1基因片段切出,克隆到质粒PAAV-MCS的HindⅢ位点中,构建重组质粒pAAV-hHO-1,采用限制性内切酶酶切,PCR和DNA测序鉴定。结果:PCR扩增出1 317 bp大小的片段,酶切鉴定证实插入位点及方向与预期的完全一致;测序证实hHO-1与Gene Bank提供的原始序列完全一致。结论:成功构建了含hHO-1基因的重组腺相关病毒载体,为缺血性疾病基因治疗的研究奠定了基础。     

转染CTGF基因对乳腺癌细胞MMP－2及TIMP－2表达的影响

目的: 通过研究结缔组织生长因子（CTGF）基因对人乳腺癌MCF-7细胞株基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶组织抑制因子-2（TIMP-2）表达的影响,深入探讨CTGF在乳腺癌侵袭和转移中的作用机制。方法: 采用脂质体介导法,将CTGF基因瞬时转染体外培养的人MCF-7细胞,并用免疫荧光、RT-PCR和蛋白质印迹法检测其转染成功与否;再采用蛋白质印迹法检测转基因MCF-7 MMP-2及TIMP-2表达改变。结果: 与对照组相比,转染CTGF基因的MCF-7,其CTGF mRNA和蛋白表达均显著增高,MMP-2蛋白表达水平显著增高,而TIMP-2蛋白表达水平显著降低。 结论: CTGF可能通过增加乳腺癌细胞MMP-2表达及抑制TIMP-2的生成而起到促进乳腺癌侵袭和转移的作用。     

CTGF对体外人晶状体上皮细胞表达MMP-2、TIMP-2的影响

目的 研究结缔组织生长因子(CTGF)对体外培养人晶状体上皮细胞(HLECs)基质金属蛋白酶-2(MMP-2)及基质金属蛋白酶抑制剂-2(TIMP-2)表达的影响.方法 采用免疫细胞化学染色法及反转录-聚合酶链反应(RT-PCR)法检测不同浓度CTGF对HLECs诱导MMP-2及TIMP-2的表达.结果 在无CTGF刺激的情况下,HLECs基本不表达MMP-2及TIMP-2;随着CTGF浓度增加,HLECs表达MMP-2增加,并且这种作用随浓度的增加而增强,每两组之间的比较差异均有统计学意义(P＜0.01或P＜0.05);对TIMP-2的表达无明显影响(P＞0.05).结论 CTGF能诱导HLECs表达MMP-2,对HLECs表达TIMP-2无影响;MMP-2/TIMP-2的比例失调,引起ECM的代谢紊乱,可能是后发性白内障的发病机制之一.     

人对氧磷酶1基因重组腺相关病毒载体的构建及表达

目的：构建带有人对氧磷酶1（hPONI）基因的重组腺相关病毒（rAAV）载体,并检测其转染大鼠骨髓内皮祖细胞（EPCs）后hPON1的表达。方法：提取流产胎儿肝脏的总RNA,RT-PCR获得cDNA,设计引物通过PCR获得PON!基因片段,将其与pGEM—TEasy载体连接,将PON1基因片段切下,插入到质粒pAAV—IRES—GFP的多克隆位点,获得质粒pAAV—PON1-IRES—GFP,经酶切和测序鉴定。包装的病毒rAAV—PON1用斑点杂交检测滴度,并用其转染EPCs检测hPON1在mRNA水平的表达。结果：pAAV—PON1-IRES-GFP酶切和测序结果完全正确。斑点杂交检测病毒的滴度为1．7×10^10g／mL,RT—PCR结果显示病毒转染EPCs后存在hPON1的表达。结论：成功构建了pAAV—PON1-IRES—GFP质粒,包装出较高滴度的rAAV—PON1,并且转染EPCs后可以检测到hPON1表达。     

HSV1-TK基因重组腺相关病毒载体的构建及表达

目的:构建含单纯疱疹病毒Ⅰ型胸苷激酶(HSV1-TK)基因的重组腺相关病毒载体质粒pSNAV2.0-TK,制备重组腺相关病毒rAAV2/HSV1-TK,并检测HSV1-TK基因在转染晶状体上皮细胞中的整合和表达,为后囊膜混浊的基因治疗奠定基础.方法:用基因重组技术构建含HSV1-TK基因的重组腺相关病毒载体质粒pSNAV2.0-TK,并通过PCR和酶切鉴定;将该质粒转染BHK-21细胞,经G418筛选出携带该质粒的载体细胞株BHK-21/TK,在辅助病毒的参与下包装成重组病毒rAAV2/HSV1-TK;用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)法和高效液相色谱(HPLC)法检测重组病毒rAAV2/HSV1-TK的纯度,用斑点杂交法检测重组病毒的滴度;重组病毒rAAV2/HSV1-TK转染兔晶状体上皮细胞N/N1003A,用PCR和RT-PCR检测HSV1-TK基因的整合和表达.结果:经PCR和酶切鉴定重组质粒pSNAV2.0-TK构建成功;成功制备了重组病毒rAAV2/HSV1-TK,病毒滴度达1×1012v.g./mL;重组病毒转染N/N1003A细胞后,PCR和RT-PCR检测显示HSV1-TK基因在细胞中整合并且有效表达.结论:成功构建了含HSV1-TK基因的重组腺相关病毒载体质粒,制备了高滴度重组病毒rAAV2/HSV1-TK,HSV1-TK基因在转染该病毒的晶状体上皮细胞中可有效表达.     

Combined expression of CTGF and tissue inhibitor of metalloprotease-1 promotes synthesis of proteoglycan and collagen type II in rhesus monkey lumbar intervertebral disc cells in vitro

Background Low back pain has emerged as a widespread disease often caused by intervertebral disc degeneration.This study aimed to establish an in vitro cell culture model of rhesus monkey lumbar intervertebral discs and to investigate the effect of combined connective tissue growth factor （CTGF） and tissue inhibitor of metalloprotease-1（TIMP-1） expression mediated by adeno-associated virus （AAV） on collagen type Ⅱ and proteoglycan levels.The purpose of these investigations was to explore potential methods for relieving the degeneration of lumbar intervertebral disc cells.Methods Rhesus monkey lumbar intervertebral disc nucleus pulposus cells （NPCs） were isolated by enzyme digestion,cultured, and transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, or rAAV2-TIMP-1 at a multiplicity of infection （MOl） of 106.The expression of collagen type Ⅱ and proteoglycan was measured using RT-PCR and Western blotting.The synthetic rate of proteoglycan was measured using 35S incorporation.Results Rhesus monkey lumbar intervertebral disc NPCs were transduced with rAAV2-CTGF-IRES-TIMP-1,rAAV2-CTGF, and rAAV2-TIMP-1 and the transduced genes were expressed and detected.Compared to the control,CTGF promoted the synthesis of collagen type Ⅱ and proteoglycan.TIMP-1 showed an enhancing effect on the expression of proteoglycan but no effect on collagen type Ⅱ.Expression of both genes in rhesus monkey lumbar intervertebral disc NPCs significantly enhances the synthesis of proteoglycan and collagen type Ⅱ.Conclusions Single gene transduction of CTGF or TIMP-1 can enhanced synthesis of proteoglycan.CTGF expression can also enhance collagen type Ⅱ protein synthesis.Combined transduction of both CTGF and TIMP1 can significantly promote the expression of proteoglycan and collagen type Ⅱ to levels greater than transduction of a single gene alone.Our study provides a good basis for multi-gene therapy to treat lumbar intervertebral disc degeneration.     

Construction of recombinant adeno-associated virus vector co-expressing hVEGF_(165) and hBMP_7 gene

Objective: To construct recombinant adeno-associated virus co-expressing human vascular epithelial growth factor 165(hVEGF165) and bone morphogenetic protein 7(hBMP7),measure the virus titer and verify the recombination. Methods:The AAV helper-free system was used as basis to generate recombinant AAV. The IRES sequence of plasmid pIRES was cut down and subcloned into ITR/MCS containing vector pAAV-MCS to construct recombinant plasmid pAAV-MCSa-IRES-MCSb. The hVEGF165 and hBMP7 gene was amplified by PCR and inserted into upstream MCSa and downstream MCSb respectively. Then,recombinant plasmid pAAV-hVEGF165-IRES-hBMP7,pAAV-RC and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hBMP7 packaging. The GFP labeled rAAV-IRES-GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-hrGFP. The efficiency of AAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured by infecting AAV-HT1080 cells,and the recombinant AAV-hVEGF165-IRES-hBMP7 was verified by PCR of the exogenous interest genes. Results:Recombinant pAAV-hVEGF165-IRES-hBMP7 was verified by double digestion. GFP expression in AAV-293 could be observed under fluorescent microscope 72 h after transfection and the system provided a high packing ratio of 95%. The recombinant adeno-associated virus has a high titer of 5.5×1011vp/ml,and AAV-HT 1080 was infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP7 and hVEGF165 gene. Conclusion:Recombinant rAAV-hVEGF165-IRES-hBMP7 was successfully constructed with a high virus titer,which may offer foundation for in vitro and in vivo experiments of hVEGF165 and hBMP7 co-expression and provide a new method for gene therapy of bone regeneration.     

携带靶向干扰VEGF基因shRNA重组腺相关病毒载体的构建和制备

目的：构建并制备携带靶向干扰VEGF165基因shRNA的重组腺相关病毒载体．方法：将PCR法扩增所得EGFP-U6-shRNA（VEGF165）片段插入载体质粒pSNAV2．0-lacz-α的EcoRI和SalI酶切位点,构建重组质粒pSNAV2．0-EGFP—U6-shRNA（VEGF165）．以HSV1-RapCap／△UL2为辅毒,包装rAAV2-EGFP-U6-shRNA（VEGF165）病毒．对所获病毒载体进行PCR,SDS-PAGE鉴定分析、滴度测定和EGFP的活性检测．结果：PCR,SDS-PAGE鉴定分析结果表明靶向干扰VEGF165基因的shRNA片段成功包装入重组AAV2载体,病毒纯度在98％以上．点杂交法测定重组病毒载体基因组滴度约为3×10^14v．g．（vector genomes）／L．EGFP活性检测显示重组AAV2感染细胞阳性率在30％以上．结论：成功制备了rAAV2-EGFP-U6-shRNA（VEGF165）病毒载体,为通过RNA干扰技术特异性抑制VEGF基因的表达来阐明VEGF在缺血性脑损伤后各种生物学事件中的作用奠定了实验基础．     

CTGF和TIMP1双基因体外联合转染恒河猴腰椎间盘细胞对II型胶原和蛋白多糖的影响

目的 建立恒河猴腰椎间盘细胞体外培养模型,研究腺相关病毒（adeno-associated virus,AAV）介导的结缔组织生长因子（connective tissue growth factor,CTGF）和基质金属蛋白酶组织抑制因子1(tissue inhibitor of metalloproteinases 1,TIMP1) 双基因体外联合转染恒河猴椎间盘细胞较单基因对II型胶原和蛋白多糖合成的影响的变化,以探讨体外延缓椎间盘细胞退变的方法。方法 应用酶消化法培养恒河猴腰椎间盘髓核细胞,以感染复数（MOI） 为106的rAAV2-CTGF-IRES-TIMP1及rAAV2-CTGF、rAAV2-TIMP1分别感染髓核细胞,应用Western blot鉴定和RT-PCR检测II型胶原及蛋白多糖mRNA的表达,35S整合法性行蛋白多糖合成率的测定,SP-ABC免疫组化法检测Ⅱ型胶原含量。结果 rAAV2-CTGF-IRES-TIMP1双基因及rAAV-CTGF、rAAV-TIMP1单基因能够转染恒河猴椎间盘髓核细胞并在其内表达细胞因子。与对照组相比,CTGF可以促进II型胶原及蛋白多糖的合成,TIMP1可以促进蛋白多糖的合成,对II型胶原的合成未见到明显作用;双基因联合感染可以显著促进髓核细胞蛋白多糖和II型胶原的合成。结论 CTGF和TIMP1 单基因转染均可以促进蛋白多糖的合成,CTGF 对 II型胶原的合成亦有促进作用;双基因联合感染可以明显促进蛋白多糖和II型胶原的合成,其效果优于单基因,为多基因治疗椎间盘退变奠定了良好的基础。     

Construction of adeno-associated virus coexpression system for human angiopoietin-1 and VEGF gene

Background Ischemic disease is one of the leading causes of death in the world. In order to further study gene therapy for ischemic disease, we constructed a recombinant plasmid for co-expression of human angiopoietin-1 and vascular endothelial growth factor 165 (VEGF165) gene in adeno-associated virus (AAV) gene delivery system.Methods Human angiopoietin 1 and VEGF165 gene were obtained using PCR. The upstream of angiopoietin 1 contained restriction enzyme site Hind Ⅲ,contained restriction enzyme site BamH Ⅰ. The upstreamand the downstream of angiopoietin 1 of VEGF165 contained restriction enzyme site Bgl Ⅱ, and the downstream of VEGF165 contained restriction enzyme site BamH Ⅰ, Using the multiple cloning sites (MCS) in plasmid pZero such as BamH Ⅰ , Bgl Ⅱ, Hind Ⅲ, Not Ⅰ , Xho Ⅰ ,Xba Ⅰ , Sal Ⅰ , BspH Ⅰ , Ksp Ⅰ and the corresponding MCS in plasmid pAAV-MCS, angiopoietin 1 and VEGF165 gene were subcloned into pAAV-MCS.Results DNA sequencing revealed that the PCR- amplified angiopoietin 1 and VEGF165 were consistent with NCBI Gene Bank. The recombinant plasmid was identified using PCR and digestion,which proved to be consistent with our hypothesis. In recombinant plasmid, angiopoietinl and VEGF possessed a CMV promoter and polyA terminator system respectively, thus assuring co-expression of the two genes.Conclusion Successful construction of AAV co-expression system for human angiopoietin 1 and VEGF165 gene will provide the foundation for gene therapy to cure severe ischemic disease.     

Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene

To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.