慢病毒介导siRNA和细胞内抗病毒因子对HIV抑制作用研究

发布时间：2018-07-12 18:59

本文选题：人类免疫缺陷病毒 + 基因治疗　；参考：《北京工业大学》2013年博士论文

【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。
[Abstract]:With the spread of human immunodeficiency virus (HIV) in the world, HIV infection has become a serious harm to human health. High performance antiretroviral therapy can significantly improve the symptoms of AIDS patients, but it needs life-long medication, the drug brings cumulative toxicity. Once the drug treatment fails, the drug resistant strain can be produced. On the other hand, it is present. A full - effective vaccine has not been found yet. With understanding of the control of the HIV mechanism, many researchers began to focus on gene therapy as a separate or auxiliary treatment.
RNA interfering technology has been effectively applied to HIV gene therapy by targeting structural proteins or regulating proteins. Because siRNA is unstable in the body, it is quickly degraded. The duration of RNA interference in vitro synthesis and plasmid mediated interference is short, and it is not suitable for long-term inhibition of gene expression. SiRNA shRNA is expressed by lentivirus vector. It is an important way to reduce the HIV virus gene at transcriptional level and thus inhibit virus replication as a gene therapy HIV. The way to introduce the antiviral gene into the stem cells provides hope for the long-term effect of gene therapy.
On the basis of screening HIV-1 specific tat, Vpr and rev specific siRNA, the recombinant lentivirus expressing plasmid containing corresponding shRNA sequence was constructed and the interference effect of the target gene was detected. On this basis, the recombinant lentivirus containing corresponding shRNA sequence was packed in 293T cells and the virus titer was measured. The recombinant lentivirus infected MT-4 cells were obtained by screening the MT-4 cell line that stably expressed siRNA through the resistance of the purinomycin resistance. The HIV-1NL4-3 strain in vitro attack test was carried out. The P24 protein content in the culture supernatant was detected to compare the effect of the siRNA in vitro inhibition of the virus. The results showed that the shRNA targeted to Vpr could be at the transcriptional level. The inhibition rate of Vpr was 88.8%, the inhibition rate of shRNA target to Tat reached 60.2%, and the MT-4 cell line expressing vprshRNA or tatshRNA could effectively inhibit the replication of HIV, and there was no large copy of HIV in the short term medium.
Based on the experience of HARRT therapy, combined application of multiple anti HIV genes may also be a better antiviral effect. Therefore, a recombinant lentivirus expression plasmid containing both Vpr and tat genes is also constructed, which is expressed by human U6 promoter and H1 promoter, respectively, and plasmids containing Vpr and tat genes in vitro. Co transfection showed that the recombinant expression plasmid interfered with Vpr and tat gene at the transcription level of 89.2% and 62%, respectively. After CO transfection of recombinant lentivirus plasmid and viral package plasmid pNL4-3, the P24 quantitative detection results showed that the inhibition rate of HIV virus package could reach 99.05%, compared to shRNA or target targeted to Vpr alone. The inhibition rate of shRNA in Tat is high; MT-4 cells are infected after the recombinant lentivirus is packaged and the MT-4 cell line that expresses siRNA is also screened. The HIVNL4-3 virus attack experiment shows that the cell line can effectively inhibit the replication of the virus.
In addition, the recombinant human Trim5a (R332P) and the recombinant lentivirus double expression plasmid targeting the shRNA of vpr gene were also constructed, which were controlled by cytomegalovirus CMV promoter and U6 promoter respectively. The two anti HIV genes inhibited the replication of the virus by different mechanisms at the different stages of HIV life cycle. The co transfection test showed that the vprshRNA expressed by recombinant Lentivirus Expression Plasmid could effectively inhibit the expression of vpr gene at the transcriptional level, reach 84.05%, and the inhibition rate of HIV virus package could reach 98.45%. Western blot results showed that the transformed human Trim5a (R332P) could be expressed in cells and detected at the level of TZM-BL cells. The expression level of Luciferase showed that the inhibitory rate of human Trim5a (R332P) to HIV pseudo virus could reach 63.30%, and the positive MT4 cell test of HIV NL4-3 virus attacking the recombinant lentivirus showed that the positive cell line could effectively inhibit the replication of the virus and proved the joint utensil containing Trim5a (R332P) and vprshRNA expression elements. There is an obvious anti HIV virus effect.
The particles containing siRNA are secreted by the cell membrane through cell exocytosis. The cell membrane composition not only improves the stability of the siRNA in vitro, but also improves the efficiency of the siRNA into the target cells because of the similarity of the biofilm components. In order to reduce the immunogenicity and improve the affinity, the cell membrane composition can reduce the immunogenicity and enhance the affinity of the cells. The study selected low immunogenic umbilical cord mesenchymal stem cells as microparticles to prepare host cells. First, human telomerase catalyzed subunit (hTERT) was introduced into cells by recombinant lentivirus infection, and the differentiation and proliferation ability of mesenchymal stem cells was improved, and the stem cell properties of the cells were detected. Secondly, the target was introduced into the target. The recombinant lentivirus of shRNA, HIV-1, was established to produce mesenchymal stem cells that could continue to produce cell particles. The results showed that the screened positive hucMSCs clones detected the expression level of the hTERT gene at the genomic DNA and mRNA levels; the cell line had been cultured for 105 generations in vitro, far more than the human umbilical cord mesenchymal stem cells. TRAP-PCR confirmed that the cell line resumed the telomerase activity, and the flow cytometry showed that the surface of the hucMSCs-htert cells had the specific surface markers of human MSCs, such as CD29, CD44 and CD105, and did not express CD106, CD45, CD19 and HLA-DR; hucMSCs-htert cells were cultured in the bone culture medium. Sex phosphatase staining shows that the cells can differentiate into osteoblasts and prove that the cell line still maintains the potential of differentiation in vitro. Karyotype analysis shows that the cell has 46XY diploid karyotype and can not form a tumor in SCID mice. It indicates that the cell has no tumorigenic tendency and is safe. The inhibitory effect of the gene has been preliminarily studied, which can be used for gene therapy of HIV.
To sum up, the present study uses lentivirus to mediate the single shRNA, double shRNA, recombinant lentivirus vector of the genetically modified Trim5a (R332P) and vprshRNA, and the inhibition effect on HIV. The anti HIV recombinant lentivirus vector can effectively infect human umbilical cord hematopoietic stem cells and two shRNA altogether. The combination of the same application and different anti HIV genes provides a new strategy for the gene therapy of HIV. In addition, the immortalized human umbilical cord mesenchymal stem cell line has been successfully established. It is proved that the exogenously expressed human telomerase catalyzed subunit can restructure the telomerase activity and enhance the cell value added activity; this cell line maintains the cell. Morphology, cell surface antigen and normal diploid cell karyotype still retain the differentiation potential in subculture, and there is no tumor in SCID mice. This cell line provides a sufficient cell source for gene therapy, cell therapy and tissue engineering. The cell line is very low immunogenicity and is used to prepare the target to HIV-1. The gene's mature siRNA nanoparticle is a new way of gene therapy for HIV.
【学位授予单位】：北京工业大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.91

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