本文關(guān)鍵詞：樹突狀細胞的分離培養(yǎng)及HIV-1假病毒感染實驗研究　出處：《長春中醫(yī)藥大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

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【摘要】：目的：以人外周血來源的單個核細胞為前體細胞，建立體外快速分離和誘導(dǎo)培養(yǎng)未成熟樹突狀細胞（Immatural dendritic cell, iDC）的方法；構(gòu)建攜帶增強型綠色熒光蛋白基因（Enhanced green fluorescent protein, EGFP）的HIV-1假病毒；通過HIV-1假病毒感染iDC實驗探討病毒與細胞的相互作用。 方法：采用Ficoll密度梯度離心和MACS磁珠分選系統(tǒng)，收集高純度的CD14~+單核細胞；用rhGM-CSF、rhIL-4聯(lián)合誘導(dǎo)CD14~+單核細胞，獲得未成熟樹突狀細胞（iDC），應(yīng)用流式細胞術(shù)鑒定細胞表面標記和抗原吞噬能力，，用普通光學(xué)顯微鏡、掃描電鏡和透射電鏡觀察細胞表面和內(nèi)部結(jié)構(gòu)特征；利用慢病毒包裝系統(tǒng)構(gòu)建攜帶EGFP基因的HIV-1假病毒，通過RT-PCR擴增HIV-1假病毒中的EGFP基因；最后利用HIV-1假病毒感染iDC，對HIV-1假病毒感染iDC后的基因組整合和轉(zhuǎn)錄水平進行檢測，觀察被感染iDC中EGFP基因的表達。 結(jié)果：流式細胞儀檢測結(jié)果表明，CD14免疫磁珠技術(shù)獲得CD14~+單核細胞純度達94％以上，誘導(dǎo)分化第4天的iDC具有吞噬能力，CD195的表達在95％以上，普通光學(xué)顯微鏡、掃描電鏡和透射電鏡觀察到細胞出現(xiàn)典型DC特征；構(gòu)建的HIV-1假病毒通過RT-PCR結(jié)果顯示擴增得到EGFP基因；HIV-1假病毒感染iDC后，在基因組整合和轉(zhuǎn)錄水平檢測中都擴增得到了EGFP基因，熒光顯微鏡觀察被HIV-1假病毒感染的iDC，觀察到iDC表達綠色熒光蛋白。 結(jié)論：經(jīng)過體外快速誘導(dǎo)培養(yǎng)獲得大量具有典型特征的iDC，其可以應(yīng)用于進一步實驗研究；成功構(gòu)建攜帶EGFP基因的HIV-1假病毒；HIV-1假病毒可以感染iDC，并將其遺傳物質(zhì)整合到iDC基因組中，并經(jīng)過轉(zhuǎn)錄和翻譯使iDC表達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.
【學(xué)位授予單位】：長春中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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