树突状细胞的分离培养及HIV-1假病毒感染实验研究

发布时间：2017-12-30 20:35

本文关键词：树突状细胞的分离培养及HIV-1假病毒感染实验研究　出处：《长春中医药大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：以人外周血来源的单个核细胞为前体细胞，建立体外快速分离和诱导培养未成熟树突状细胞（Immatural dendritic cell, iDC）的方法；构建携带增强型绿色荧光蛋白基因（Enhanced green fluorescent protein, EGFP）的HIV-1假病毒；通过HIV-1假病毒感染iDC实验探讨病毒与细胞的相互作用。方法：采用Ficoll密度梯度离心和MACS磁珠分选系统，收集高纯度的CD14~+单核细胞；用rhGM-CSF、rhIL-4联合诱导CD14~+单核细胞，获得未成熟树突状细胞（iDC），应用流式细胞术鉴定细胞表面标记和抗原吞噬能力，，用普通光学显微镜、扫描电镜和透射电镜观察细胞表面和内部结构特征；利用慢病毒包装系统构建携带EGFP基因的HIV-1假病毒，通过RT-PCR扩增HIV-1假病毒中的EGFP基因；最后利用HIV-1假病毒感染iDC，对HIV-1假病毒感染iDC后的基因组整合和转录水平进行检测，观察被感染iDC中EGFP基因的表达。结果：流式细胞仪检测结果表明，CD14免疫磁珠技术获得CD14~+单核细胞纯度达94％以上，诱导分化第4天的iDC具有吞噬能力，CD195的表达在95％以上，普通光学显微镜、扫描电镜和透射电镜观察到细胞出现典型DC特征；构建的HIV-1假病毒通过RT-PCR结果显示扩增得到EGFP基因；HIV-1假病毒感染iDC后，在基因组整合和转录水平检测中都扩增得到了EGFP基因，荧光显微镜观察被HIV-1假病毒感染的iDC，观察到iDC表达绿色荧光蛋白。结论：经过体外快速诱导培养获得大量具有典型特征的iDC，其可以应用于进一步实验研究；成功构建携带EGFP基因的HIV-1假病毒；HIV-1假病毒可以感染iDC，并将其遗传物质整合到iDC基因组中，并经过转录和翻译使iDC表达EGFP。
[Abstract]:Objective: to establish immatural dendritic cell from human peripheral blood mononuclear cells (PBMC) by rapid isolation and induction of immature dendritic cells in vitro. IDC-based method; Construction of HIV-1 pseudovirus carrying enhanced green fluorescent protein (EGFP) gene; The interaction between virus and cell was studied by iDC infection of HIV-1 pseudovirus. Methods: high purity CD14 ~ monocytes were collected by Ficoll density gradient centrifugation and MACS magnetic beads sorting system. CD14- monocytes were induced by rhGM-CSF-1 rhIL-4 and immature dendritic cells were obtained. Flow cytometry was used to identify the surface labeling and phagocytic ability of the cells. The surface and internal structure of the cells were observed by general optical microscope, scanning electron microscope and transmission electron microscope. HIV-1 pseudovirus carrying EGFP gene was constructed by using lentivirus packaging system and EGFP gene in HIV-1 pseudovirus was amplified by RT-PCR. Finally, the genomic integration and transcription level of HIV-1 pseudovirus infected with iDC were detected by using HIV-1 pseudovirus infection, and the expression of EGFP gene in infected iDC was observed. Results: the results of flow cytometry showed that the purity of CD14- monocytes obtained by CD14 immunomagnetic beads was more than 94%, and the iDC on the 4th day of differentiation had phagocytosis ability. The expression of CD195 was above 95%. Typical DC characteristics were observed by ordinary optical microscope, scanning electron microscope and transmission electron microscope. The EGFP gene was obtained by RT-PCR amplification of the constructed HIV-1 pseudovirus. The EGFP gene was amplified from HIV-1 pseudovirus infected with iDC in genomic integration and transcriptional level detection. The iDC infected by HIV-1 pseudovirus was observed by fluorescence microscope. IDC expression of green fluorescent protein was observed. Conclusion: a large number of typical iDCs can be obtained by rapid induction and culture in vitro, which can be used for further experimental research. HIV-1 pseudovirus carrying EGFP gene was successfully constructed. HIV-1 pseudovirus can infect iDCand integrate its genetic material into the iDC genome, and through transcription and translation, iDC can express EGFP.
【学位授予单位】：长春中医药大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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