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1.
血友病B是凝血因子Ⅸ(hFⅨ)缺乏所导致的严重凝血功能障碍、X-连锁隐性遗传性疾病,在男性中的发病率为三万分之一.目前没有理想的治疗措施,而基因治疗可能是根治该病的安全、有效方法.该实验构建了ubiquitin—c和ABP(肝特异性)启动子指导hFⅨ表达的载体FUXW、FAXW,利用磷酸钙法共转染三质粒制备高滴度的重组慢病毒.将不同剂量的重组FUXW、FAXW病毒,分别用尾静脉液压法和静脉缓注法注射入血友病B小鼠体内,各治疗组小鼠血浆均可持续检测到hFⅨ抗原,最高峰值为45ng/mL,表达持续超过60d.结果表明:hFⅨ蛋白的表达与病毒的剂量成正相关,重组FAXW病毒的hFⅨ表达量高于重组FUXW病毒,而尾静脉液压组和静脉缓注组在表达量、表达时间上没有显著差异.  相似文献   

2.
慢病毒是一种具有独特优点和巨大应用潜力的哺乳动物细胞基因转移载体,我们对慢病毒载体对不同哺乳动物细胞的基因转移及表达效率进行了平行比较研究.应用第三代重组慢病毒系统构建了携带CMV启动子-EGFP报告基因表达元件的重组慢病毒Lenti-EGFP,分别对多种不同哺乳动物细胞进行转导实验,在转导48 h后应用流式细胞仪检测报告基因在不同细胞株中的转移及表达效率.我们共使用了29种哺乳动物细胞株,包括14种人类组织细胞,5种猴组织细胞,9种鼠组织细胞,1种兔组织细胞.结果显示,重组慢病毒具有良好的基因转移能力,可有效进入多数哺乳动物细胞,对不同种属来源的细胞没有表现出特别的偏嗜性,但对贴壁培养细胞的基因转移效率明显高于对悬浮培养细胞.本研究为重组慢病毒系统的合理使用提供了基础.  相似文献   

3.
干细胞因子是一种多功能细胞因子,能在多级造血水平与其他细胞因子协同作用促进造血干/祖细胞及各种血细胞的存活、增殖和分化.重组人二连体干细胞因子具有比干细胞因子单体更高的比活,可以避免副反应.重组人二连体干细胞因子在Sf9细胞和大肠杆菌中表达时,发现有特异性降解.片段缺失实验证实切割位点位于重组人二连体干细胞因子的145位到165位氨基酸之间.丝氨酸蛋白酶抑制剂aprotinin和PMSF能抑制这种特异性降解.试验了不同浓度的aprotinin和PMSF对Sf9细胞存活和重组人二连体干细胞因子产量的影响,显示当aprotinin的浓度为1.0μg/mL时,重组人二连体干细胞因子的产量是没有加aprotinin时产量的2倍,而且aprotinin可以完全抑制这种特异性降解.  相似文献   

4.
目的:构建人血管紧张素Ⅱ2型受体(hAT2R)过表达稳定细胞系,探讨AT2R激动剂Compound 21等对前列腺癌细胞的作用及机制.方法:构建含hAT2R基因的慢病毒表达载体pLV-CMV-hAT2R-IRES-eGFP并包装成慢病毒.将重组慢病毒载体pLV-CMV-IRES-eGFP-hAT2R感染前列腺癌细胞,并利用流式细胞术筛选出单克隆细胞株,RT-PCR及Western blot方法检测重组细胞系中hAT2R表达水平,使用AT2R激动剂CGP42112检测受体功能.结果:重组慢病毒载体感染PC-3、DU145前列腺癌细胞24 h后均能观察到eGFP表达,用流式细胞术分别筛选出单克隆细胞株,RT-PCR及Western blot检测结果显示目的基因hAT2R在两株重组细胞系中表达显著升高,CGP42112处理24 h后,重组细胞系细胞活力较正常PC-3、DU145细胞显著降低.结论:成功构建hAT2R过表达稳定细胞系.  相似文献   

5.
目的构建SV40LT基因过表达慢病毒载体,并对其进行慢病毒包装,为建立永生化的uncv小鼠胚胎成纤维细胞奠定基础。方法从293T细胞中获得SV40LT基因,将其克隆到p Lenti-GFP质粒中,构建重组穿梭质粒p Lenti-GFP-SV40LT,测序鉴定后分别将鉴定的阳性p Lenti-GFP-SV40LT和包装质粒p MD2.0G和ps PAX2共转染293T细胞,包装产生慢病毒。结果 SV40LT基因过表达慢病毒载体的构建与包装成功。结论 SV40LT基因过表达慢病毒载体构建与包装的成功为uncv小鼠胚胎成纤维细胞的永生化提供了工具。  相似文献   

6.
为了研究肥胖基因FTO过表达对小鼠胰岛β细胞功能的影响以及基因表达谱的变化,构建小鼠FTO基因过表达慢病毒载体,包装慢病毒颗粒并感染小鼠胰岛MIN6细胞.利用QPCR和WesternBlot技术检测FTO基因在MIN6细胞中过表达情况,并检测葡萄糖刺激检测胰岛素的释放情况,进一步利用小鼠全基因芯片检测FTO过表达对小鼠胰岛MIN6细胞表达谱的影响.结果表明:慢病毒载体成功介导了FTO在MIN6细胞中过表达,FTO过表达可以显著抑制MIN6细胞的胰岛素释放.表达芯片的结果显示FTO改变MIN6细胞表达谱,发现多达922个的差异基因.FTO过表达改变了小鼠胰岛细胞的表达谱,差异基因通过一些重要信号通路影响小鼠胰岛β细胞的生物学功能.  相似文献   

7.
全红花 《科学技术与工程》2012,12(32):8637-8641
为了提高病毒产率并避免制备的重组腺病毒产物中残留的动物血清,将贴壁生长的Trex-293细胞用无动物血清的CD293培养基在搅拌式培养瓶中悬浮培养。分析比较其生长特征及不同条件下的病毒产率。结果表明悬浮培养的细胞生长为稳定状态的时间比贴壁培养时稍长,但每次以(4—6)×105cells/mL密度传代。(3—4)d后基本达到2×106cells/mL,21 d达3×106cells/mL以上,比贴壁细胞密度约高3倍。培养初期,悬浮细胞存活率稍低,培养14 d开始维持90%以上,细胞倍增时间约32 h。而贴壁细胞存活率是基本维持在95%以上。重组腺病毒以200,500和1 000 VP/cell的比例感染悬浮细胞所获产率均值各为18 000,14 000和990 VP/cell,而1 000 VP/cell的比例感染贴壁细胞所获产率均值为10 500 VP/cell。结论为Trex-293细胞用无动物血清的培养基在搅拌式培养瓶中可悬浮培养,当重组腺病毒感染比例为200 VP/cell,感染后48 h,细胞存活率50%时收获有利于提高病毒产率,且高于感染贴壁细胞所获产率。  相似文献   

8.
构建小鼠Sirt3基因过表达慢病毒载体,检测人神经母细胞瘤细胞(SK-N-SH细胞)感染Sirt3基因过表达慢病毒后Sirt3 mRNA和蛋白的表达,为在细胞和动物水平研究Sirt3的作用提供新的途径和工具。利用Genebank检索的小鼠Sirt3基因序列,人工合成法合成小鼠Sirt3基因的c DNA片段,将其克隆至慢病毒载体p CDH-CMV-MCS-EF1-cop GFP中,构建慢病毒表达质粒。进行酶切及测序验证后,将慢病毒表达质粒连同辅助包装原件载体质粒共同转染入293T细胞,收集上清,测定病毒滴度。用Sirt3基因过表达慢病毒感染SK-N-SH细胞,即为Sirt3过表达组(Sirt3);用空载体慢病毒感染的SK-N-SH细胞为空载体组(GFP);未感染病毒的SK-N-SH细胞为对照组(CON)。收集细胞后,用Real-time PCR法检测各组细胞Sirt3 mRNA的水平;用Western blotting法检测各组细胞Sirt3蛋白的表达。Sirt3基因过表达慢病毒载体构建成功,病毒滴度为1.0×10~9TU/m L。Sirt3组SK-N-SH细胞的Sirt3 mRNA和Sirt3蛋白水平均显著高于GFP组和CON组(P0.01)。成功构建的Sirt3基因过表达慢病毒载体具备高效感染力,能在SK-N-SH细胞高效过表达Sirt3,本研究结果为今后进一步在神经细胞和模型动物中研究Sirt3的作用提供了新的途径和工具。  相似文献   

9.
研究了悬浮培养中接种密度、换液和优化培养中所用细胞因子组合对造血细胞扩增的影响。结果表明,较高的接种密度(1.0×106cells/mL)比低接种密度(0.5×106cells/mL)更有利于总细胞、粒巨噬集落形成单位(CFU-GM)和巨核细胞集落形成单位(CFU-Mk)的扩增。换液能及时补充葡萄糖和移出培养中产生的乳酸,从而不对细胞生长造成影响,而且可以成功地控制pH不超出造血细胞正常生长所需pH范围。在SCF、IL-3和IL-6组合基础上添加Flt-3配体(FL)和促血小板生成素(TPO)能比原来的SCF、IL-3和IL-6组合更能促进总细胞、CFU-GM和CFU-Mk的扩增。  相似文献   

10.
李腾 《科学技术与工程》2013,13(22):6389-6393
构建并鉴定UBXN1-shRNA慢病毒表达载体,以便应用RNAi技术以及慢病毒感染系统建立稳定干涉细胞系并进一步研究UBXN1的功能。将携带不同特异性干涉序列的DNA片段插入PLKO.1载体中构建慢病毒表达载体,并制备慢病毒颗粒。将慢病毒颗粒感染U2OS细胞,建立稳定干涉细胞系,应用real-time PCR和western blot技术分别检测U2OS细胞中UBXN1 mRNA和蛋白质水平的表达差异。重组克隆经酶切证实shRNA正确插入慢病毒载体,DNA测序证实插入的序列正确,western blot检测证实设计的五条shRNA干扰序列有效的敲低U2OS细胞中内源性UBXN1的表达。成功制备UBXN1的慢病毒干涉颗粒,并建立UBXN1稳定下调的U2OS细胞系。  相似文献   

11.
To explore the expression of human clotting factor Ⅸ (hFⅨ) cDNA in vitro and the feasibility of gene therapy for hemophilia B mice mediated by recombinant lentiviral vector, a recombinant hFⅨ lentiviral vector driven by ubiquitin-C promoter, FUXW, and by ABP liver specific promoter, FAXW, was constructed respectively. Recombinant lentivirus was harvested from 293T cells by calcium phosphate-mediated transient cotransfection of three plasmids (transgene vector, CMV腞8.2, VSV-G). hFⅨ expression was detected in supernatant of 293T, BHK and L-02 cells infected with FUXW virus, whereas higher expression of hFⅨ levels (630 ng/106 cells/48 h) was detected only in L-02 cells infected with FAXW virus. Serum hFⅨ antigen was detected in all hemophilia B mice treated with FAXW virus by tail vein injection, an efficiency level of hFⅨ was observed (45 ng/mL, approximately 1% of normal human levels), the expression lasted for more than 60 d. The results indicated that HIV-based lentiviral vectors offer a promising approach to the gene therapy of hemophilia B.  相似文献   

12.
研究旨在构建Npas4基因过表达慢病毒,为进一步深入探索Npas4基因的功能奠定基础。用人工合成大鼠Npas4基因c DNA片段,将其插入p CDH-CMV-MCS-EF1-cop GFP构建慢病毒表达质粒p CDH-Npas4。酶切、测序验证质粒后,将p CDHNpas4和辅助质粒共转染包装细胞293T,浓缩上清得病毒颗粒并测定病毒滴度。取病毒颗粒感染SK-N-SH细胞48 h,收集细胞采用Western blotting法检测Npas4蛋白的表达。p CDH-Npas4携载正确Npas4基因,将其包装293T细胞后能产生病毒。病毒滴度为1.05×109TU/m L。相比于转染GFP病毒对照组(GFP)和未转染对照组(control),Npas4重组慢病毒组(Npas4)的细胞Npas4蛋白表达显著增高。成功构建Npas4基因过表达的重组慢病毒载体p CDH-Npas4,并获得高效的重组慢病毒,能将外源Npas4基因导入SK-N-SH细胞,为进一步研究Npas4基因的相关功能奠定了基础。  相似文献   

13.
Human FⅨ expression vector pCMVⅨ was packaged by effectene^TM reagent and injected into mice seminiferous tubules with glass pipettes.The expressional frame of pCMVⅨ was examined by PCR and Southern blotting among 41 progenies.There were 2(4%) mice being integrated with hFⅨ gene into chromosomes.4.6ng/mL of hFⅨ protein was expressed in plasma of one mouse,which was tested by ELISA.We demonstrated that building of transgenic animals by spermatogonial stem cells is an efficient method.Meanwhile,it has also been proved to be an alternative choice for mammary gland bioreactor.  相似文献   

14.
《科学通报(英文版)》1998,43(15):1294-1294
The DNA of human factor Ⅸ (hFⅨ) gene vector pMCⅨm, which had been proven to be able to express in in vitro and living cells, was introduced into 586 zygotes of Kunming White Mice by positive pressure microinjection technique with manual operation. The 499 survival embryos after microinjection were then transferred into pseudopregnant recipient mice and 216 F 0 pups were born. The analysis of PCR and Southern blot hybridization showed that, of the 216, 6 (2 females and 4 males) were integrated with foreign DNA in their genomes, giving an integration frequency of 3% (6/216). Two F\-0 female transgenic mice could express hFⅨ protein in their milk and the content was over 100 ng/mL as measured with ELISA. The biological activities of hFⅨ in the milk of two F\-0 mice were 44 67% and 79 43%, respectively.  相似文献   

15.
目的 为小鼠白细胞减少症模型的建立提供基础资料。方法 选用ICR小鼠和C57BL/6J小鼠以5Gy的剂量60Co-γ射线一次性全身照射小鼠,测定小鼠外周血白细胞总数。结果 在本实验条件下,ICR小鼠的正常白细胞数量极显著低于C57BL/6J小鼠;雄性C57BL/6J在照射后第3 d白细胞数量显著高于其雌性小鼠。结论 小鼠的品系、性别对60Co-γ射线照射建立的白细胞减少症模型具有一定的影响。  相似文献   

16.
The DNA of human factor IX (hF IX) gene vector pMC IX m, which had been proven to be able to express inin vitro and living cells, was introduced into 586 zygotes of Kunming Whlte Mice by positive pressure microinjection technique with manual operation. The 499 survival embryos after microinjection were then transferred into pseudopregnant recipient mice and 216 F, pups were born. The analysis of PCR and Southern blot hybridization showed that, of the 216, 6 (2 females and 4 males) were integrated with foreign DNA in their genornes, giving an integration frequency of 3% (6/216). Two F0 female transgenic mice could express hF IX protein in their milk and the content was over 100 ng/mL as measured with ELISA. The biological activities of hF IV in the milk of two F0 mike were 44.67 % and 79.43 %, respectively.  相似文献   

17.
摘要:构建人mir-218-2, pre-miRNA慢病毒表达载体,为研究miR-218在人体的功能及作用机制打下基础。以人hsa-mir-218-2前体序列,设计部分互补的正反向引物,进行引物退火,形成引物二聚体,PCR扩增引物二聚体,酶切后插入到线性化pGCSIL-GFP慢病毒表达载体中,对重组质粒进行双酶切鉴定,并进行慢病毒的包装与滴度检测。用构建好的慢病毒表达载体感染人胃癌细胞MKN-28,qPCR检测细胞内miR-218表达。结果显示,重组质粒经双酶切分析及转化菌液测序,插入序列正确,慢病毒表达载体感染人胃癌细胞后qPCR检测显示能显著增高miR-218的表达。说明本实验成功构建了hsa-mir-218-2慢病毒表达载体,感染人胃癌细胞后能有效提高miR-218的表达。为进一步研究miR-218在人体的功能及作用机制建立了实验基础。  相似文献   

18.
慢病毒载体感染小鼠曲细精管的研究   总被引:1,自引:0,他引:1  
目的探讨基于慢病毒载体曲细精管注射方法建立转基因动物的可行性。方法将8只4w~5w龄的雄性昆明小鼠分为高剂量(2只)、低剂量(6只)2个实验组,曲细精管注射滴度分别为1×109、2×107TU/mL的绿色荧光蛋白慢病毒载体(LV-GFP),注射量均为20μL/testis。注射后第4w、8w分别处死高剂量组小鼠各1只,于第5w、13w、17w各处死2只低剂量组小鼠,取睾丸,通过PCR、荧光显微镜和免疫组化等方法检测睾丸组织中GFP基因及表达。结果3只低剂量和2只高剂量小鼠睾丸组织中均可检测到GFP基因;但GFP表达仅见于高剂量组小鼠睾丸,其分布范围主要集中于曲细精管基膜及管间隙。结论慢病毒载体可通过曲细精管注射感染小鼠睾丸组织,但其感染效率与病毒滴度有关。  相似文献   

19.
A series of adeno-associated viral vectors containing a mutation of human factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25×1012 particle/mL, and then, a mammalian cell line, C2C12 and the factor Ⅸ knock-out mice were transfected with the rAAV-hFⅨR338A in vitro and in vivo. The results show that the high-level expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(106 cells)-1·(24 h)-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt (2565.76±64.36) ng·(106 cells)-1·(24 h)-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFⅨ.  相似文献   

20.
目的比较三种常用麻醉药阿佛丁、水合氯醛、乌拉坦的麻醉效果,探索最适合于C57BL/6J品系小鼠的麻醉方法。方法三种麻醉药物分别以剂量梯度对C57BL/6J小鼠进行腹腔注射,比较不同剂量下的麻醉起效时间、麻醉深度、麻醉维持时间及恢复时间。结果阿佛丁(1.2%)以0.60~0.65mL/20g剂量效果最好,水合氯醛(4%)以0.20mL/20g剂量效果较好,乌拉坦不适用于C57BL/6J小鼠的麻醉实验。结论腹腔注射阿佛丁(0.60mL/20g)和水合氯醛(0.20mL/20g)是适合于C57BL/6J品系小鼠全身麻醉的起效时间最短,维持时间和麻醉深度适度,死亡率为0,麻醉效果较佳的麻醉方法。  相似文献   

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