c-myc基因慢病毒载体的构建及其意义

目的 构建携带c-myc基因野生型及T58A突变型的慢病毒载体.方法 应用聚合酶链反应(PCR)方法 扩增c-myc野生型及T58A突变型基因,利用Gateway技术构建携带绿色荧光蛋白(GFP)基因的慢病毒载体,经PCR及基因测序鉴定后,转染293FT包装细胞,产生相应慢病毒,测定其滴度.结果 PCR和测序证实,构建分别携带c-myc野生型及T58A突变型基因的慢病毒载体,并包装慢病毒,病毒滴度测定结果　分别为6.10 × 107、5.65×107 TU/ml.结论　成功构建含有c-myc野生型及T58A突变型基因的慢病毒载体并包装出具高效感染力的慢病毒颗粒.

Abstract：

Objective To construct a lentiviral vector with c-myc gene, including wild type and mutation type. Methods The lentiviral vector with green fluorescent protein (GFP) was constructed by polymerase chain reaction (PCR) and gateway technology and identified by PCR and gene sequencing,then transfecte dinto the package cells 293FT by lipofectin to produce mature lentivirus. The virus titer was measured. Results According to PCR and gene sequencing, the myc-lentiviral vectors were successfully constructed with the virus titer being 6. 10 × 107 ( wild type) and 5.65 × 107 ( mutation type) TU/ml, respectively. Conclusion The myc-lentiviral vector was successfully constructed and efficient lentivirus particles were packaged.     

人内皮型一氧化氮合成酶基因重组腺病毒载体构建及其在人血管平滑肌细胞的表达

目的 利用AdMax腺病毒载体系统构建人内皮型一氧化氮合成酶基因(heNOS)重组腺病毒并检测其在体外培养人血管平滑肌细胞中表达.方法 将质粒PMSCV-heNOS通过聚合酶链反应(PCR)法扩增出heNOS全长基因,插入PSUCMV中构建成腺病毒穿梭质粒PSUCMV-heNOS,转化DH5α大肠杆菌,挑选细菌单克隆进行DNA测序,酶切鉴定正确;通过PSUCMV-heNOS与质粒pBHGE3在293细胞中同源重组,得到携带heNOS基因的复制缺陷型重组腺病毒载体(AdCMV-he-NOS).转染体外培养的人血管平滑肌细胞,MTT检测细胞增殖,用免疫组织化学和Western blot等方法检测heNOS蛋白的表达.结果 (1)PCR产物电泳结果,酶切鉴定和测序鉴定结果证实构建人eNOS重组腺病毒载体成功,病毒滴度达3.5×l010PFU/ml;(2)根据体外培养的人血管平滑肌细胞(HVSMCs)形态学变化,免疫组织化学进行平滑肌细胞表型标志物α-actin染色,证实为HVSMCs;(3)转染120 h,AdCMV-heNOS病毒感染复数(MOI)分别为50、150、250、300、450、A570值分别为1.410±0.081、1.357±0.150、1.303±0.311、0.995±0.248、0.731±0.101,其中感染复数300明显稳定抑制血管平滑肌细胞的增殖,差异有统计学意义(P＜0.01).免疫组织化学和Western blot检测显示转染细胞heNOS蛋白明显表达.结论 heNOS重组腺病毒的构建及其表达为血管内膜增生等疾病的基因治疗提供可能.

Abstract：

Objective To construct recombinant adenovirus vector carrying the human endothelial nitric oxide synthase gene (heNOS) and detect its expression in human vascular smooth muscle cells in vitro. Methods heNOS was cloned by polymerase chain reaction (PCR) using plasmid PMSCV-heNOS,heNOS cDNA was inserted into adenovirus shuttle plasmid-PSUCMV to generate a recombinant plasmid PSUCMV-heNOS, transfected into E. coli DH5α, and positive clones were correctly constructed and confirmed by DNA sequencing analysis and restriction endonucleas analysis. The plasmid PSUCMV-heNOS was co-transfected with pBHGE3 in 293 cells to generate replication-defective recombinant adenovirus vector carrying heNOS gene. Human vascular smooth muscle cells were transfected by Ad-heNOS. The effect on the proliferation of human vascular smooth muscle cells was investigated by MTT assay. The expression of heNOS was detected by immunohistochemistry staining and Western blotting. Results ( 1 ) PCR, restrictive digestion, and sequencing revealed the successful construction of the recombinant adenovirus vector carrying heNOS gene and its titer was 3.5 × 1010 PFU/ml; (2) It was confirmed that the human vascular smooth muscle cells cultivated in vitro were characterized by morphology and immunohistochemistry strain of α-actin; (3) At 120 h, the A570 values in multiplicity of infection (MOI) 50, 150,250,300,450 were respectively 1. 410 ±0. 081, 1. 357 ±0. 150, 1.303 ±0. 311,0. 995 ±0. 248 and 0. 731 ±0. 101. The proliferation of human vascular smooth muscle cells was significantly and stably inhibited with MOI 300 of AdCMVheNOS ( P ＜ 0. 01 ). Immunostaining and Western blotting with heNOS in engineered cells were positive.Conclusion The successful construction of AdCMV-heNOS and expression may provide an opportunity for the gene therapy of vascular intimal hyperplasia.     

RNA干扰下调p53抑制因子iASPP表达对膀胱癌细胞的影响

Objective To discuss the effects of silencing of iASPP gene on human bladder cancer cells. Methods RNAi silencing of iASPP gene in bladder cancer cell 5637 and T24 cells were used by lentiviral mediated interfering short hairpin RNAs. Cell proliferation was tested by MTT assay, and rate of colony was tested by colony formation assay. Cell cycles were tested by using fluorescence-activated cell sorting. Results Down-regulation of iASPP could inhibit the growth and proliferation of human bladder cancer cells (P＜0.05). iASPP know-down could decrease the colony formation of 5637 and T24 cells (P＜0, 05). Knocking down of iASPP in 5637 and T24 cells showed cell arrested at G1. Conclusions Silencing of iASPP gene could inhibit proliferation and colony formation of bladder cancer, iASPP might be an important target for gene therapy of bladder cancer.     

RNA干扰下调p53抑制因子iASPP表达对膀胱癌细胞的影响

Objective To discuss the effects of silencing of iASPP gene on human bladder cancer cells. Methods RNAi silencing of iASPP gene in bladder cancer cell 5637 and T24 cells were used by lentiviral mediated interfering short hairpin RNAs. Cell proliferation was tested by MTT assay, and rate of colony was tested by colony formation assay. Cell cycles were tested by using fluorescence-activated cell sorting. Results Down-regulation of iASPP could inhibit the growth and proliferation of human bladder cancer cells (P＜0.05). iASPP know-down could decrease the colony formation of 5637 and T24 cells (P＜0, 05). Knocking down of iASPP in 5637 and T24 cells showed cell arrested at G1. Conclusions Silencing of iASPP gene could inhibit proliferation and colony formation of bladder cancer, iASPP might be an important target for gene therapy of bladder cancer.     

Tie2基因RNAi慢病毒载体的构建及其干预恶性黑色素瘤细胞的体外研究

目的 构建携带酪氨酸蛋白激酶受体2-小干扰RNA(Tie2-SiRNA)慢病毒载体,观察其对恶性黑色素瘤细胞的干扰作用.方法 将pSilencer 1.0-U6启动子-酪氨酸蛋白激酶受体2-小干扰RNA(pSilencer 1.0-U6-Tie2-siRNA)重组质粒经Xba Ⅰ酶切电泳鉴定后,与经Xba Ⅰ酶切电泳鉴定的带有加强绿色荧光蛋白的转移质粒(pNL-EGFP)载体连接,产生加强绿色荧光蛋白的转移质粒-u6启动子-酪氨酸蛋白激酶受体2-Ⅰ(pNL-EGFP-U6-Tie2-Ⅰ)、pNL-EGFP-U6-Tie2-Ⅱ慢病毒转移质粒,电泳筛选阳性克隆,测序鉴定.用连接成功的慢病毒转移质粒,分别与水疱性口炎病毒G蛋白(pVSVG)包膜质粒和pHelper包装质粒组成慢病毒三质粒转染系统,再共转染293T细胞,产生pNL-EGFP-U6-Tie2-Ⅰ、pNL-EGFP-U6-Tie2-Ⅱ慢病毒.收集病毒上清,测定病毒滴度.将收集的病毒上清感染恶性黑色素瘤细胞,通过实时荧光定量RT-PCR测定抑制Tie2基因表达的效率.结果 酶切电泳与测序鉴定证实成功构建了Tie2-SiRNA慢病毒载体,293T细胞测定病毒原液滴度为8.8×103/ml.实时荧光定量RT-PCR结果显示:Tie2-SiRNA慢病毒载体感染恶性黑色素瘤细胞,抑制了恶性黑色素瘤细胞中Tie2基因的表达,其中较高者可达68.55%,并且2种慢病毒载体之间差异无统计学意义(t=0.362,P＞0.05).结论 成功构建了Tie2-SiRNA慢病毒载体,体外研究显示其能抑制Tie2 mRNA的表达,为下一步进行抑制肿瘤生成的动物实验研究奠定了基础.

Abstract：

Objective To construct lentivector carrying Tie2-Small interfering RNA(SiRNA),so as to study its influence on malignant melanoma cells.Methods Recombinant plasmid pSilencer 1.0-U6-Tie2-siRNA and plasmid pNL-EGFP were digested with Xbal.ligated a target lentiviral transfer plasmid of pNL-EGFP-U6-Tie2-Ⅰ or pNL-EGFP-U6-Tie2-Ⅱ,and then the electrophoresis clones was sequenced.Plasmids of pNL-EGFP-U6-Tie2-1 and pNL-EGFP-U6-Tie2-Ⅱ were constructed and combined with pVSVG and pHelper,respcectively,to constitute lentiviral vector system of three plasmids.The Lentiviral vector svstem was transfected into 293T cell to produce pNL-EGFP-U6-Tie2-Ⅰ and pNL-EGFP-U6-Tie2-Ⅱ lentivirus.Then the supernatant was collected tO determine the titer.Malignant melanoma cells were infected by both lentiviruses and identified by Realtime RT-PCR to assess inhibitory efficiency. Results The recombinant lentiviral vectors of Tie2-RNAi were constructed successfully which were analyzed with restriction enzyme digestion and identified by sequencing.And the titer of lentiviral vector was 8.8×103/ml,which was determined by 293T cell.The results of Reahime RT-PCR demonstrated that the lentiviral vectors of Tie2-RNAi could infect malignant melanoma cells and inhibit the expression of Tie2 genes in malignant melanoma cells(P＜0.01).There was no significant difference in the expression level (P＞0.05)between the two lentiviral vectors of Tie2-RNAi.Conclusions Lentivector carrying Tie2-SiRNA can be constructed successfully and inhibit the expression of Tie2 gene in vitro significantly.The study will supply the theory basis for the further research on the inhibition of tumor growth in vivo.     

慢病毒介导SOX9基因转染骨髓间充质细胞中的基因表达

目的:构建携带小鼠SOX9基因的慢病毒载体,体外转染小鼠骨髓间充质细胞,观察小鼠SOX9基因在小鼠间充质细胞中的表达。方法:从含有小鼠SOX9基因的质粒提取总RNA,利用RT-PCR方法扩增目的基因。连接目的基因与经Age-Ⅰ酶切线性化的慢病毒载体,转化感受态的大肠杆菌对质粒进行扩增,筛出阳性转化子,经293T细胞包装,收集病毒后,通过基因测序和限制性核酸内切酶酶切的方法对质粒进行鉴定。Lenti-SOX9-EGFP体外转染小鼠骨髓间充质细胞,利用倒置荧光显微镜观察转染是否成功,并通过流式细胞仪测定转染效率。同时利用RT-PCR和Western Blot检测小鼠SOX9基因的表达。结果:成功构建了携带SOX9基因慢病毒载体,Lenti-SOX9-EGFP能高效转染小鼠骨髓间充质细胞。RT-PCR和Western Blot检测显示经SOX9基因转染的小鼠骨髓间充质细胞表达目的基因产物。结论:利用慢病毒介导SOX9基因成功地转染小鼠骨髓间充质细胞,而且SOX9基因在小鼠骨髓间充质细胞中得到表达,这为SOX9修复软骨损伤的进一步研究奠定了基础。     

慢病毒载体介导的低氧诱导因子-1α在脂肪源性干细胞中的表达

目的 构建携带有增强绿色荧光蛋白(EGFP)和低氧诱导因子-1α(HIF-1α)的重组慢病毒载体,并感染人脂肪源性干细胞(hADSCs).方法 重组慢病毒载体Lenti-hHIF-1α-EGFP转染293T细胞,进行病毒包装并测定病毒滴度.按照MOI=20将Lenti-hHIF-1α-EGFP转染hADSCs,流式细胞仪分选EGFP标记的细胞.对转染后的hADSCs进行多向分化能力鉴定,并检测hADSCs内hHIF-1α的表达.结果病毒滴度为5×107 pfu/ml.Lenti-hHIF-1α-EGFP转染hADSCs后第3天,荧光镜下观察转染效率70％,经流式细胞仪分选后,98％以上的细胞均有EGFP的表达.转染后的hADSCs茜素红染色及油红O染色阳性,表明hADSCs仍具有多向分化的潜能.hADSCs可以高表达HIF-1α.结论成功构建带有报告基因EGFP和目的基因hHIF-1α的慢病毒表达系统,并以慢病毒为载体将hHIF-lα整合到hADSCs中实现其高效表达.     

携带人血管内皮细胞生长因子基因细菌内重组腺病毒转染兔骨髓基质干细胞的表达

目的应用高效细菌内同源重组系统pAd-Easy,制备含人血管内皮细胞生长因子121 (VEGF121)基因的重组腺病毒,感染兔骨髓基质干细胞(BMSCs),并检测外源基因的表达,为其进一步应用于血管化组织工程骨组织的构建打下基础。方法自pCDNA-VEGF121质粒中切取VEGF121基因,将VEGF基因克隆到穿梭质粒,在BJ5183细菌内重组,在293细胞中构建携带VEGF基因的重组腺病毒,将腺病毒感染BMSCs,利用ELISA、免疫组织化学的方法检测VEGF121在BMSCs中的表达。结果通过pAd-Easy系统成功构建高滴度的携带VEGF121基因重组腺病毒pAd-VEGF,ELISA检测显示经转染的BMSCs中VEGF121的表达均明显增强,随着感染病毒MOI值的增高,VEGF121的表达量相应增高,差异有统计学意义(P<0．01),免疫组化法显示经基因转染的BMSCs中有强阳性信号,未转染组出现阴性结果。结论Ad-VEGF转染BMSCs后能稳定提高VEGF蛋白的表达。     

猪TGF-β1基因重组慢病毒载体的构建及其在BMSCs中的表达

目的构建猪TGF-β1重组慢病毒表达载体,并转染BMSCs,为构建组织工程骨软骨提供TGF-β1修饰的BMSCs,作为持续、高效的种子细胞。方法将已获取的目的基因TGF-β1cDNA包装至慢病毒载体中,通过PCR及基因测序对阳性克隆进行鉴定,并测定病毒滴度。取2月龄巴马香猪(体重约15 kg)骨髓制备BMSCs,取第2～3代用于实验。用TGF-β1重组慢病毒载体以感染复数(multiplicity of infection,MOI)为10、50、70、100、150分别转染BMSCs,通过激光共聚焦显微镜观察,并以Western blot检测不同MOI值的转染效果,确定最佳MOI值。用TGF-β1重组慢病毒载体以最佳MOI值感染BMSCs作为实验组,以空载体转染的BMSCs(空载体组)及未转染的BMSCs(空白组)作为对照,通过RT-PCR、免疫细胞化学染色、ELISA等方法检测TGF-β1基因及蛋白在BMSCs中的表达情况,并检测Ⅱ型胶原表达情况。结果经PCR及基因测序鉴定TGF-β1重组慢病毒表达载体构建成功,并成功转染BMSCs,激光共聚焦显微镜下可观察到强绿色荧光;Western blot示MOI为70时转染效果最佳;RT-PCR示实验组TGF-β1基因的表达量明显高于空载体组及空白组,差异有统计学意义(P<0.05);免疫细胞化学染色示实验组TGF-β1蛋白及Ⅱ型胶原呈阳性表达,而空载体组及空白组呈弱阳性或阴性表达;ELISA示实验组TGF-β1蛋白至转染后21 d仍有较高表达。结论 TGF-β1重组慢病毒表达载体可成功转染BMSCs,TGF-β1蛋白可长期、稳定表达,促使BMSCs向成软骨细胞方向分化。     

慢病毒介导兔脑红蛋白基因感染骨髓间充质干细胞的实验研究

[目的]构建携带兔脑红蛋白(neuroglobin,Ngb)基因的重组慢病毒,感染兔骨髓间充质干细胞(bonemarrow mesenchymal stem cells,BMSCs),建立稳定表达兔Ngb的BMSCs/Ngb细胞。[方法]构建携带Ngb基因的重组慢病毒载体,在脂质体Lipofectamine 2000作用下转染293 T细胞,Real-time PCR检测慢病毒滴度;以Ngb重组慢病毒感染BMSCs,通过荧光表达法判定感染复数(multiplicities of infection,MOI),实时荧光定量PCR(real-time PCR)、蛋白质印迹(Western blot)和酶联免疫吸附试验(ELISA)判定感染后的BMSCs中Ngb的表达情况。[结果]构建Ngb重组慢病毒载体,经酶切及测序鉴定完全正确,能够转染293 T细胞并表达,滴度为2×108 TU/ml,Ngb重组慢病毒感染BMSCs最佳MOI值为100,且Ngb重组慢病毒感染BMSCs能持续稳定高水平表达Ngb mRNA和蛋白。[结论]成功构建Ngb重组慢病毒,并将Ngb基因稳定感染至BMSCs中,实现Ngb的持续稳定高水平表...     

LMP-1基因慢病毒载体构建及其在大鼠骨髓间充质干细胞的表达

目的：构建人LMP-1重组慢病毒载体,体外转染大鼠骨髓间充质干细胞,检测LMP-1基因在大鼠骨髓间充质干细胞的表达。方法：利用PCR法从cDNA文库中钓取LMP-1基因,将其与经AgeI酶切线性化的慢病毒载体pGC-FU-EGFP相连接,转化感受态大肠杆菌,筛选出阳性克隆pGC-FU-LMP-1-EGFP,基因测序对其鉴定。经293T细胞包装后,收集富舍病毒颗粒LV-LMP-1-EGFP的细胞上清,浓缩并标定滴度,RT-PCR检测并鉴定。以最佳MOI值体外转染大鼠骨髓间充质干细胞,荧光显微镜观察转染是否成功,流式细胞仪检测转染效率,RT-PCR和Westernblot检测转染细胞LMP-1基因的表达。结果：①基因测序及RT-PCR检测证实携带人LMP-1基因的慢病毒载体构建成功,包装后获得滴度为2×10^8 TU／ml的LV-LMP-1-EGFP。②以MOI=100转染大鼠骨髓间充质干细胞,荧光显微镜下可见大量绿色荧光蛋白表达,转染效率可达93．5％,经RT-PCR与Westernblot检测,被转染细胞内有LMP-1基因表达。结论：成功构建携带人LMP-1基因的慢病毒载体,可高效转染大鼠骨髓间充质干细胞,被转染细胞可高效表达LMP-1基因。     

Lentivirus-based gene delivery in mouse embryonic stem cells

BACKGROUND: Embryonic stem (ES) cells are widely used in therapeutic research as an unlimited source of cell therapy. Therefore, it is of great value to find a way to efficiently manipulate ES cells. HIV-1-derived lentiviral vectors are now considered to be an efficient vehicle for delivering genes into a variety of cells. In this study, we examined the efficacy of lentivirus-based gene delivery into mouse ES (mES) cells. MATERIALS AND METHODS: Recombinant HIV-I-based lentiviral vectors Lt-GFP, expressing green fluorescent protein (GFP), and Lt-LacZ, expressing E. coli LacZ gene in conjunction with neomycin resistance gene, were generated using a FuGENE 6 transduction method and used for transducing ES cells derived from 129Sv mice. Lentiviral transduction efficacy was evaluated by GFP expression assay using flow cytometry and by X-gal staining. The in vivo potential of developing teratoma of such transduced mES cells was examined in severe combined immunodeficiency (SCID) mice. RESULTS: FuGENE 6 showed no considerable transduction-associated cytotoxicity. The expression rate of GFP and LacZ of mES cells increased on a multiplicity of infection (MOI)-dependent manner with the amount of Lt-GFP and Lt-LacZ used. Approximately 42% of mES cells were positive for GFP after infection of Lt-GFP at an MOI of 30. Notably, after G418 selection, nearly 100% of Lt-LacZ-transduced mES cells were positive for LacZ and formed teratomas in SCID mice. CONCLUSIONS: This work demonstrates that HIV-I-based lentiviral vectors are capable of transducing mES cells. Lentiviral vectors may facilitate an advance in the field of gene transfer and expression in various types of ES cells, including human ES cells.     

人VEGF基因慢病毒介导RNA干扰有效靶点的设计及筛选

目的:设计并筛选人血管内皮生长因子(VEGF)有效RNA干扰片段,构建VEGF慢病毒表达载体。方法:对人VEGF基因编码区分析,筛选序列3条,阴性对照序列l条,通过连接线性化的plenti6.3-MIR载体,构建miRNA慢病毒载体质粒,并转化至感受态细胞DH5α,进行测序验证。在脂质体介导下转染293T细胞,包装生产慢病毒,测定其滴度。慢病毒载体转染人肝癌细胞MHCC97L,用Real-time PCR检测干扰效果。结果:测序证实3个VEGF基因RNAi慢病毒载体质粒构建成功。慢病毒载体经293T细胞包装成功,测定病毒的滴度分别为3.23×109、3.30×109、3.73×109TU/mL。3个慢病毒载体转染人肝癌细胞MHCC97L后,VEGF基因在mRNA水平受到抑制,其中miR-200序列效果最佳,对VEGF基因表达的干扰效率可达72%。结论:成功构建并筛选了人VEGF基因RNAi慢病毒载体及有效靶点,为进一步深入研究VEGF基因与抗肿瘤药物药效关系提供实验基础。     

RNA干扰阻断OX40/OX40L共刺激通路对CD4+CD25+T调节细胞的影响

目的 探讨慢病毒介导的RNA干扰技术诱导小鼠树突状细胞OX40L基因沉默对调节性T细胞的影响. 方法 设计针对小鼠OX40L基因的RNA于扰序列,通过外源筛靶筛选出干扰效果最佳的序列.以293T为包装细胞,制备含OX40L的siRNA序列的慢病毒载体OX40L-RNAi-LV和阴性对照载体NC-GFP-LV.采用磁式分选器分离培养骨髓来源的小鼠树突状细胞(DCs),以MOI为25进行转染,分别将转染OX40L-RNAi-LV(实验组)、转染NC-GFP-LV(阴性对照组)和未转染(空白对照组)的DCs与磁式分选器分选得到的CD4+CD25+T调节细胞共培养,6 d后通过流式细胞仪检测T调节细胞的增殖和凋亡情况. 结果外源筛靶筛选出干扰效果最佳的RNA干扰序列(靶序列为GCTCATACAAGAATGAGTA),OX40L蛋白表达的抑制率为73.1%.感染复数为25时,慢病毒载体感染DCs的效率为86.4%.DCs与CD4+CD25+T调节细胞共培养后,实验组的凋亡细胞比例为8.7%,显著低于阴性对照组(20.1%)和空白对照组(19.8%),F=244.22,P=0.000;而增殖细胞前体频率为38.3%,明显高于阴性对照组(24.5%)和空白对照组(22.9%),F=95.40,P=0.000.结论 小鼠OX40L的siRNA慢病毒载体可以有效降低树突状细胞OX40L的表达,对体外培养的CD4+CD25+T调节细胞具有显著地促进增殖、减少凋亡的作用.