慢病毒介导siRNA和细胞内抗病毒因子对HIV抑制作用研究
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摘要
随着人类免疫缺陷病毒（HIV）在全球蔓延，HIV感染已成为严重危害人类健康的疾病。高效抗逆转录病毒疗法能够显著地改善AIDS患者的症状，但需要终身用药，药物带来累积性毒性，一旦药物治疗失败就会产生耐药性病毒株。另一方面，现今还未找到一个完全有效的疫苗。随着对控制HIV机制的了解，很多研究者开始将注意力集中在基因治疗，将它作为单独的或者是辅助的治疗方式。
RNA干扰技术通过靶向于结构蛋白或者是调节蛋白，已经有效地应用于HIV的基因治疗。由于siRNA在体内不稳定，很快就被降解，体外合成和质粒介导的RNA干扰持续时间较短，不适用于长期抑制基因表达。通过慢病毒载体表达编码siRNA的shRNA在转录水平下调HIV病毒基因从而抑制病毒复制成为基因治疗HIV一种重要的方式。将抗病毒基因导入到干细胞的方式对基因治疗达到长期治疗效果提供了希望。
在筛选出针对HIV-1的tat、vpr和rev特异性siRNA的基础上，本研究分别构建了含有相应shRNA序列的重组慢病毒表达质粒，并对靶向基因的干扰效果进行了检测。在此基础上，在293T细胞中包装出含有相应shRNA序列的重组慢病毒并测定病毒滴度。将得到的重组慢病毒感染MT-4细胞，通过嘌呤霉素抗性筛选得到稳定表达siRNA的MT-4细胞系，进行HIV-1NL4-3毒株体外攻击试验，通过检测培养上清中的P24蛋白含量来比较siRNA体外抑制病毒效果。结果表明，靶向于vpr的shRNA可以在转录水平上有效地抑制vpr的表达，抑制率达到88.8%；靶向于tat的shRNA抑制率达到60.2%；稳定表达vprshRNA或tatshRNA的MT-4细胞系能够有效地抑制HIV的复制，短期内培养基中没有HIV的大量复制。
基于HARRT治疗的经验，将多个抗HIV基因联合应用同样可能起到更好的抗病毒效果，因此本研究还构建了同时含有靶向于vpr和tat基因的双shRNA的重组慢病毒表达质粒，分别由人U6启动子和H1启动子控制表达；体外与含有vpr及tat基因的质粒共转染表明该重组表达质粒对vpr和tat基因在转录水平上的干扰效果分别达到89.2%和62.0%；将重组慢病毒质粒与病毒包装质粒pNL4-3共转染后，P24定量检测结果显示对HIV病毒包装的抑制率可以达到99.05%，比单独表达靶向于vpr的shRNA或靶向tat的shRNA的抑制率都要高；包装出重组慢病毒后感染MT-4细胞，同样筛选稳定表达siRNA的MT-4细胞系，HIVNL4-3病毒攻击实验表明该细胞系可以有效地抑制病毒的复制。
此外，本研究还构建了同时含有基因改造的人源的Trim5a(R332P)和靶向于vpr基因的shRNA的重组慢病毒双表达质粒，分别由巨细胞病毒CMV启动子和U6启动子控制表达，两种抗HIV基因在HIV生命周期的不同阶段通过不同的机制去抑制病毒复制；共转染实验表明重组慢病毒表达质粒表达的vprshRNA可以在转录水平有效地抑制vpr基因的表达，可以达到84.05%，对HIV病毒包装的抑制率可以达到98.45%；Western blot结果表明改造的人源Trim5a(R332P)可以在细胞内表达，在TZM-BL细胞水平通过检测Luciferase表达水平表明人源的Trim5a(R332P)对HIV假病毒的抑制率可以达到63.30%；HIV NL4-3病毒攻击导入该重组慢病毒的阳性MT4细胞实验表明该阳性细胞系可以有效地抑制病毒的复制，证明含有Trim5a(R332P)和vprshRNA表达元件的联合应用具有明显地抗HIV病毒效果。
含有siRNA的微粒子是通过细胞胞吐的方式将细胞内表达的siRNA包裹着细胞膜分泌到细胞外。细胞膜成分不仅可以提高siRNA的体外稳定性，而且由于生物膜成分的相似性可提高siRNA导入靶细胞的效率。为降低其免疫原性和提高亲和性，本研究选用低免疫原性的脐带间充质干细胞作为微粒子的制备宿主细胞。首先，通过重组慢病毒感染的方式将人端粒酶催化亚基（hTERT）导入细胞，提高间充质干细胞的分化增殖能力，并对传代后细胞的干细胞性进行检测。其次，导入含有靶向HIV-1的shRNA的重组慢病毒建立了能够持续产生细胞微粒子的间充质干细胞系。结果表明，筛选到的阳性hucMSCs克隆在基因组DNA和mRNA水平检测了hTERT基因的表达水平；该细胞系在体外已经持续培养105代，远远超过人脐带间充质干细胞的分裂增值界限；TRAP-PCR证实该细胞系恢复了端粒酶活性；流式细胞仪结果显示建立的hucMSCs-htert细胞表面有人间充质干细胞特异性表面标记，如CD29，CD44和CD105，不表达CD106、CD45、CD19和HLA-DR；hucMSCs-htert细胞在刺激成骨培养基中培养后，碱性磷酸酶染色表明细胞能够向成骨细胞进行分化，证明该细胞系仍然保持体外分化的潜能；核型分析表明该细胞具有46XY二倍体核型，在SCID小鼠体内不能形成肿瘤，表明该细胞还没有致瘤倾向，是安全的。并对细胞微粒子的制备、对靶向基因的抑制效果进行了初步研究，该细胞微粒可以用于HIV的基因治疗。
综上所述，本研究采用慢病毒介导靶向于HIV-1调控基因的单个shRNA、双shRNA、经基因改造过的Trim5a(R332P)和vprshRNA组合的重组慢病毒载体，并且检测了它们对HIV的抑制效果。抗HIV重组慢病毒载体可以有效地感染人脐带造血干细胞，两种shRNA共同应用及不同抗HIV基因的联合应用为HIV的基因治疗提供了新的策略。另外成功地建立了永生化的人脐带间充质干细胞系，证明了外源表达人端粒酶催化亚基可以重构端粒酶的活性并能够增强细胞的增值活性；该细胞系维持了细胞的形态、细胞表面抗原和正常的二倍体细胞核型，在继代培养中仍保持了分化潜能，在SCID小鼠体内没有形成肿瘤，该细胞系为基因治疗、细胞治疗和组织工程提供了充足的细胞来源。该细胞系免疫原性非常低，被应用于制备含有靶向于HIV-1基因的成熟的siRNA的生物纳米微粒子，作为新型的HIV的基因治疗的一种方式。
With global spread of the human immunodeficiency virus, HIV infection hasbecome a serious hazard to human health. Although，highly active antiretroviralretroviral theray (HAART) have markedly changed the profile of progression to AIDSin HIV-infected individuals, they are not without significant problems and drawbacks,including the lifetime treatment, cumulative toxicities and viral resistance. So far, afully effective HIV vaccine was not available. With the increasing knowledge ofmechanisms that allow control of HIV infection, more and more investigators arefocusing their attention on gene therapy, either as a stand-alone approach or as anadjuvant to HAART.
RNA interference have been effectively used to inhibit the replication of HIVrecently, by targeting the structure gene gag-pol, env and the regulatory proteinsincluding tat, vpr, rev. Exogenous siRNA transfected is not stable and knocking-downeffect is transient as the siRNA is gradually degraded or diluted by cell growth andcell division. The siRNA could be produced by expressing short hairpin (shRNA)precursor, which is exported to the cytoplasm and processed by the RNAi machinery.Expressing shRNA encoding siRNAs targeting viral sequences from the backbone ofviral vectors has been used as a form of genetic therapy for HIV-1and associatedinfection, which could inhibit HIV-1replication by down-regulating the viral gene atthe HIV transcription level.
In the study, we constructed several recombinant lentiviral expression plasmidscontaining the shRNA targeting vpr、tat、 rev and vif genes and evaluated inhibitoneffects of the shRNA expressed on the targeted gene. Then the inhibitory effects onHIV-1production were detected by cotransfected with the packaging plasmidspNL4-3. The recombinant lentivirus containing the shRNA was packaged and titred.MT4cell was transduced with the recombinant virus and screened by FACs. Thetransduced MT4cells were further challenged with HIV-1strains. The result showedthat shRNA targeting vpr gene could efficiently inhibited the vpr expression onmRNA, about89.2%, whereas the tatshRNA could reach to60.2%agaist tat gene.The MT4transduced the vprshRNA and tatshRNA could inhibit the HIV-1replicationeffectively, but not the revshRNA.
To improve the inhibition effect on HIV, the study constructed the bispecificlentiviral vector harboring vprshRNA and tat shRNA expression cassettes from U6promotor and H1promotor respectively, which were cotransfected with recombinantplasmid expressing the vpr and tat gene. The result showed that the bispecificlentiviral vector could inhibit the vpr and tat effectively，with ratio of89.2%and62% respectively. Cotransfected with pNL4-3in293T cell, the bispecific plasmids showedhigher efficacy in down regulating the HIV NL4-3strains packaging production thanthe recombinant plasmids expressing the single shRNA. MT4cell clone transducedwith recombinant lentiviral vectors were screened and challenged with HIV NL4-3.P24ELISA test showed that MT4transduced with the combinational lentiviral vectorcould inhibit virus replication efficiently in vitro.
However, as HIV-1is prone to generating escape mutants, using a singleanti-HIV construct would not be adequate to afford long range viral protection.Combination of therapeutic genes targeting different viral products and steps in theviral life cycle was consider as an alternative effectively solution. Here, we reported anovel expression construct into the backbone of a replication defective lentiviralvector, encoding the Trim5ahumutant and short hairpin RNA targeting HIV-1vpr gene,which were expressed under control of the CMV promoter and U6promoter,respectively. Both of the two anti-HIV genes could block the HIV infection andinhibit replication by a distinct mechanism. Co-transfection assays in293T cellsshowed that the vprshRNA in the combination vector could knock down vprexpression with ratio of84.05%and inhibit the HIV-1NL4-3strain productioneffectively with ratio of98.45%. The western blotting showed that the TRIM5ahumutant could be expressed highly in transduced cells. Luciferase assay in Tzm-bl cellsstably expressing the TRIM5ahu showed that the ectopic protein could inhibitpseudotype HIV effectively with ratio of63.30%. MT4cell tranduced with thecombination lentiviral vector were challenged with HIV-1NL4-3. P24ELISA testshowed that MT-4transduced the combination vector could inhibit virus replicationefficiently for long time. These results suggested that combination of TRIM5ahumutant and shRNA targeting HIV-1vpr gene could lead to an effective gene therapyfor the HIV-AIDS control.
Microvesicles as vesicles are released from cells with donor cells’ ligand andlipids which could serve as delivery vehicle transporting proteins, mRNA and miRNA.Use of microvesicles as siRNA carrier may be a key step toward clinical applicationof siRNA. Human umbilical cord is an alternative source of adult stem cells with lowimmunogenicity, which do not trigger an immense immune reaction in unrelateddonor transplantation. However, hMSCs have a limited life, gradually lose theirdifferentiation potential after several passages and will be senescent and die, whichrestricts the scientific and clinical application. Ectopic expression of humantelomerase reverse transcriptase (hTERT) extended life-span in certain cell types andpotentially immortalize them. In this study, we constructed a recombinant lentiviralvector including htert gene to infect the hucMSCs at PD2. Then hucMSCs transducedwith htert was screened by drug resistance and underwent a total of approximately105population doublings so far and continued to proliferate further. PCR amplified from genomic DNA and quantitative PCR manifested the ectopic hTERT has beenintegrated into the genomic DNA and expressed. TRAP PCR was used to determinewhether the exogenous hTERT gene affected the telomerase activity of the hucMSCs,which showed that the telomerase activity recovered in hucMSCs-hTERT cells.Moreover, our study showed that the established hucMSC-TERT were stronglypositive for the crucial markers of hMSCs, such as CD29, CD44and CD105andnegative for CD106, CD45, CD19and HLA-DR. When the hucMSCs-htert werecultured in osteogenic stimulatory medium, the cell could form the osteoblast,demonstrating the hucMSC-htert still maintain the differentiation potential. We alsoassessed the capacity of the immortalized hucMSCs-hTERT to produce heterologousproteins in vitro. The hucMSCs-htert maintained normal cell characteristics inmorphology, phenotype, karyotype and differentiation potential without malignanttransformation. The hucMSCs-htert cell line has the potential as a cell source for genetherapy as well as transplantation.The established hucMSCs-htert cell was transducedwith recombinant lentiviral vectors containing shRNA targeting the HIV-1and cellswere screened. The microvesicles were isolated and detected with quatitative PCR.The inhibition of the target gene with the microvesicles was studied. Themicrovesicles derived from hucMSCs containing the siRNA targeting HIV-1withoutimmunogenicity would be a novel therapy for AIDS-HIV.
In summary, we constructed recombinant lentiviral vetors containing singleshRNA targeting the regutory protein of HIV-1, bispecific lentiviral vector containingvpr and tatshRNA and combinational lentiviral vector containing the human TRIM5amutant and shRNA targeting vpr gene and evaluated their inhibitory effect on HIV-1.The recombinant lentivirus would be used to infect hemopoietic stem cell for genetherapy on HIV. Combination of therapeutic genes targeting different viral productsand steps was considered as an effective gene therapy for the HIV-AIDS control. Then,we have successfully immortalized human umbilical cord derived MSCs, whichdemonstrated ectopic expression of hTERT in hucMSCs could reconstitute theirtelomerase activity and promote their proliferative ability. The establishedhucMSCs-htert cell maintained stem cell markers, normal karyotype anddifferentiation potential without any tumorigenesis in SCID mice. The establishedhucMSCs-htert could be a potential cell source for transplantation and gene therapy.The cells will be used in producing the micorvesicles containing siRNA targeting theHIV-1for their low immunogenicity, which could be used in gene therapy on HIV.
RNA干扰技术通过靶向于结构蛋白或者是调节蛋白，已经有效地应用于HIV的基因治疗。由于siRNA在体内不稳定，很快就被降解，体外合成和质粒介导的RNA干扰持续时间较短，不适用于长期抑制基因表达。通过慢病毒载体表达编码siRNA的shRNA在转录水平下调HIV病毒基因从而抑制病毒复制成为基因治疗HIV一种重要的方式。将抗病毒基因导入到干细胞的方式对基因治疗达到长期治疗效果提供了希望。
在筛选出针对HIV-1的tat、vpr和rev特异性siRNA的基础上，本研究分别构建了含有相应shRNA序列的重组慢病毒表达质粒，并对靶向基因的干扰效果进行了检测。在此基础上，在293T细胞中包装出含有相应shRNA序列的重组慢病毒并测定病毒滴度。将得到的重组慢病毒感染MT-4细胞，通过嘌呤霉素抗性筛选得到稳定表达siRNA的MT-4细胞系，进行HIV-1NL4-3毒株体外攻击试验，通过检测培养上清中的P24蛋白含量来比较siRNA体外抑制病毒效果。结果表明，靶向于vpr的shRNA可以在转录水平上有效地抑制vpr的表达，抑制率达到88.8%；靶向于tat的shRNA抑制率达到60.2%；稳定表达vprshRNA或tatshRNA的MT-4细胞系能够有效地抑制HIV的复制，短期内培养基中没有HIV的大量复制。
基于HARRT治疗的经验，将多个抗HIV基因联合应用同样可能起到更好的抗病毒效果，因此本研究还构建了同时含有靶向于vpr和tat基因的双shRNA的重组慢病毒表达质粒，分别由人U6启动子和H1启动子控制表达；体外与含有vpr及tat基因的质粒共转染表明该重组表达质粒对vpr和tat基因在转录水平上的干扰效果分别达到89.2%和62.0%；将重组慢病毒质粒与病毒包装质粒pNL4-3共转染后，P24定量检测结果显示对HIV病毒包装的抑制率可以达到99.05%，比单独表达靶向于vpr的shRNA或靶向tat的shRNA的抑制率都要高；包装出重组慢病毒后感染MT-4细胞，同样筛选稳定表达siRNA的MT-4细胞系，HIVNL4-3病毒攻击实验表明该细胞系可以有效地抑制病毒的复制。
此外，本研究还构建了同时含有基因改造的人源的Trim5a(R332P)和靶向于vpr基因的shRNA的重组慢病毒双表达质粒，分别由巨细胞病毒CMV启动子和U6启动子控制表达，两种抗HIV基因在HIV生命周期的不同阶段通过不同的机制去抑制病毒复制；共转染实验表明重组慢病毒表达质粒表达的vprshRNA可以在转录水平有效地抑制vpr基因的表达，可以达到84.05%，对HIV病毒包装的抑制率可以达到98.45%；Western blot结果表明改造的人源Trim5a(R332P)可以在细胞内表达，在TZM-BL细胞水平通过检测Luciferase表达水平表明人源的Trim5a(R332P)对HIV假病毒的抑制率可以达到63.30%；HIV NL4-3病毒攻击导入该重组慢病毒的阳性MT4细胞实验表明该阳性细胞系可以有效地抑制病毒的复制，证明含有Trim5a(R332P)和vprshRNA表达元件的联合应用具有明显地抗HIV病毒效果。
含有siRNA的微粒子是通过细胞胞吐的方式将细胞内表达的siRNA包裹着细胞膜分泌到细胞外。细胞膜成分不仅可以提高siRNA的体外稳定性，而且由于生物膜成分的相似性可提高siRNA导入靶细胞的效率。为降低其免疫原性和提高亲和性，本研究选用低免疫原性的脐带间充质干细胞作为微粒子的制备宿主细胞。首先，通过重组慢病毒感染的方式将人端粒酶催化亚基（hTERT）导入细胞，提高间充质干细胞的分化增殖能力，并对传代后细胞的干细胞性进行检测。其次，导入含有靶向HIV-1的shRNA的重组慢病毒建立了能够持续产生细胞微粒子的间充质干细胞系。结果表明，筛选到的阳性hucMSCs克隆在基因组DNA和mRNA水平检测了hTERT基因的表达水平；该细胞系在体外已经持续培养105代，远远超过人脐带间充质干细胞的分裂增值界限；TRAP-PCR证实该细胞系恢复了端粒酶活性；流式细胞仪结果显示建立的hucMSCs-htert细胞表面有人间充质干细胞特异性表面标记，如CD29，CD44和CD105，不表达CD106、CD45、CD19和HLA-DR；hucMSCs-htert细胞在刺激成骨培养基中培养后，碱性磷酸酶染色表明细胞能够向成骨细胞进行分化，证明该细胞系仍然保持体外分化的潜能；核型分析表明该细胞具有46XY二倍体核型，在SCID小鼠体内不能形成肿瘤，表明该细胞还没有致瘤倾向，是安全的。并对细胞微粒子的制备、对靶向基因的抑制效果进行了初步研究，该细胞微粒可以用于HIV的基因治疗。
综上所述，本研究采用慢病毒介导靶向于HIV-1调控基因的单个shRNA、双shRNA、经基因改造过的Trim5a(R332P)和vprshRNA组合的重组慢病毒载体，并且检测了它们对HIV的抑制效果。抗HIV重组慢病毒载体可以有效地感染人脐带造血干细胞，两种shRNA共同应用及不同抗HIV基因的联合应用为HIV的基因治疗提供了新的策略。另外成功地建立了永生化的人脐带间充质干细胞系，证明了外源表达人端粒酶催化亚基可以重构端粒酶的活性并能够增强细胞的增值活性；该细胞系维持了细胞的形态、细胞表面抗原和正常的二倍体细胞核型，在继代培养中仍保持了分化潜能，在SCID小鼠体内没有形成肿瘤，该细胞系为基因治疗、细胞治疗和组织工程提供了充足的细胞来源。该细胞系免疫原性非常低，被应用于制备含有靶向于HIV-1基因的成熟的siRNA的生物纳米微粒子，作为新型的HIV的基因治疗的一种方式。
With global spread of the human immunodeficiency virus, HIV infection hasbecome a serious hazard to human health. Although，highly active antiretroviralretroviral theray (HAART) have markedly changed the profile of progression to AIDSin HIV-infected individuals, they are not without significant problems and drawbacks,including the lifetime treatment, cumulative toxicities and viral resistance. So far, afully effective HIV vaccine was not available. With the increasing knowledge ofmechanisms that allow control of HIV infection, more and more investigators arefocusing their attention on gene therapy, either as a stand-alone approach or as anadjuvant to HAART.
RNA interference have been effectively used to inhibit the replication of HIVrecently, by targeting the structure gene gag-pol, env and the regulatory proteinsincluding tat, vpr, rev. Exogenous siRNA transfected is not stable and knocking-downeffect is transient as the siRNA is gradually degraded or diluted by cell growth andcell division. The siRNA could be produced by expressing short hairpin (shRNA)precursor, which is exported to the cytoplasm and processed by the RNAi machinery.Expressing shRNA encoding siRNAs targeting viral sequences from the backbone ofviral vectors has been used as a form of genetic therapy for HIV-1and associatedinfection, which could inhibit HIV-1replication by down-regulating the viral gene atthe HIV transcription level.
In the study, we constructed several recombinant lentiviral expression plasmidscontaining the shRNA targeting vpr、tat、 rev and vif genes and evaluated inhibitoneffects of the shRNA expressed on the targeted gene. Then the inhibitory effects onHIV-1production were detected by cotransfected with the packaging plasmidspNL4-3. The recombinant lentivirus containing the shRNA was packaged and titred.MT4cell was transduced with the recombinant virus and screened by FACs. Thetransduced MT4cells were further challenged with HIV-1strains. The result showedthat shRNA targeting vpr gene could efficiently inhibited the vpr expression onmRNA, about89.2%, whereas the tatshRNA could reach to60.2%agaist tat gene.The MT4transduced the vprshRNA and tatshRNA could inhibit the HIV-1replicationeffectively, but not the revshRNA.
To improve the inhibition effect on HIV, the study constructed the bispecificlentiviral vector harboring vprshRNA and tat shRNA expression cassettes from U6promotor and H1promotor respectively, which were cotransfected with recombinantplasmid expressing the vpr and tat gene. The result showed that the bispecificlentiviral vector could inhibit the vpr and tat effectively，with ratio of89.2%and62% respectively. Cotransfected with pNL4-3in293T cell, the bispecific plasmids showedhigher efficacy in down regulating the HIV NL4-3strains packaging production thanthe recombinant plasmids expressing the single shRNA. MT4cell clone transducedwith recombinant lentiviral vectors were screened and challenged with HIV NL4-3.P24ELISA test showed that MT4transduced with the combinational lentiviral vectorcould inhibit virus replication efficiently in vitro.
However, as HIV-1is prone to generating escape mutants, using a singleanti-HIV construct would not be adequate to afford long range viral protection.Combination of therapeutic genes targeting different viral products and steps in theviral life cycle was consider as an alternative effectively solution. Here, we reported anovel expression construct into the backbone of a replication defective lentiviralvector, encoding the Trim5ahumutant and short hairpin RNA targeting HIV-1vpr gene,which were expressed under control of the CMV promoter and U6promoter,respectively. Both of the two anti-HIV genes could block the HIV infection andinhibit replication by a distinct mechanism. Co-transfection assays in293T cellsshowed that the vprshRNA in the combination vector could knock down vprexpression with ratio of84.05%and inhibit the HIV-1NL4-3strain productioneffectively with ratio of98.45%. The western blotting showed that the TRIM5ahumutant could be expressed highly in transduced cells. Luciferase assay in Tzm-bl cellsstably expressing the TRIM5ahu showed that the ectopic protein could inhibitpseudotype HIV effectively with ratio of63.30%. MT4cell tranduced with thecombination lentiviral vector were challenged with HIV-1NL4-3. P24ELISA testshowed that MT-4transduced the combination vector could inhibit virus replicationefficiently for long time. These results suggested that combination of TRIM5ahumutant and shRNA targeting HIV-1vpr gene could lead to an effective gene therapyfor the HIV-AIDS control.
Microvesicles as vesicles are released from cells with donor cells’ ligand andlipids which could serve as delivery vehicle transporting proteins, mRNA and miRNA.Use of microvesicles as siRNA carrier may be a key step toward clinical applicationof siRNA. Human umbilical cord is an alternative source of adult stem cells with lowimmunogenicity, which do not trigger an immense immune reaction in unrelateddonor transplantation. However, hMSCs have a limited life, gradually lose theirdifferentiation potential after several passages and will be senescent and die, whichrestricts the scientific and clinical application. Ectopic expression of humantelomerase reverse transcriptase (hTERT) extended life-span in certain cell types andpotentially immortalize them. In this study, we constructed a recombinant lentiviralvector including htert gene to infect the hucMSCs at PD2. Then hucMSCs transducedwith htert was screened by drug resistance and underwent a total of approximately105population doublings so far and continued to proliferate further. PCR amplified from genomic DNA and quantitative PCR manifested the ectopic hTERT has beenintegrated into the genomic DNA and expressed. TRAP PCR was used to determinewhether the exogenous hTERT gene affected the telomerase activity of the hucMSCs,which showed that the telomerase activity recovered in hucMSCs-hTERT cells.Moreover, our study showed that the established hucMSC-TERT were stronglypositive for the crucial markers of hMSCs, such as CD29, CD44and CD105andnegative for CD106, CD45, CD19and HLA-DR. When the hucMSCs-htert werecultured in osteogenic stimulatory medium, the cell could form the osteoblast,demonstrating the hucMSC-htert still maintain the differentiation potential. We alsoassessed the capacity of the immortalized hucMSCs-hTERT to produce heterologousproteins in vitro. The hucMSCs-htert maintained normal cell characteristics inmorphology, phenotype, karyotype and differentiation potential without malignanttransformation. The hucMSCs-htert cell line has the potential as a cell source for genetherapy as well as transplantation.The established hucMSCs-htert cell was transducedwith recombinant lentiviral vectors containing shRNA targeting the HIV-1and cellswere screened. The microvesicles were isolated and detected with quatitative PCR.The inhibition of the target gene with the microvesicles was studied. Themicrovesicles derived from hucMSCs containing the siRNA targeting HIV-1withoutimmunogenicity would be a novel therapy for AIDS-HIV.
In summary, we constructed recombinant lentiviral vetors containing singleshRNA targeting the regutory protein of HIV-1, bispecific lentiviral vector containingvpr and tatshRNA and combinational lentiviral vector containing the human TRIM5amutant and shRNA targeting vpr gene and evaluated their inhibitory effect on HIV-1.The recombinant lentivirus would be used to infect hemopoietic stem cell for genetherapy on HIV. Combination of therapeutic genes targeting different viral productsand steps was considered as an effective gene therapy for the HIV-AIDS control. Then,we have successfully immortalized human umbilical cord derived MSCs, whichdemonstrated ectopic expression of hTERT in hucMSCs could reconstitute theirtelomerase activity and promote their proliferative ability. The establishedhucMSCs-htert cell maintained stem cell markers, normal karyotype anddifferentiation potential without any tumorigenesis in SCID mice. The establishedhucMSCs-htert could be a potential cell source for transplantation and gene therapy.The cells will be used in producing the micorvesicles containing siRNA targeting theHIV-1for their low immunogenicity, which could be used in gene therapy on HIV.
引文
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