發(fā)布時(shí)間：2018-03-29 06:39

  本文選題：慢病毒載體　切入點(diǎn)：TNF-α基因　出處：《青島大學(xué)》2010年碩士論文

【摘要】： [目的]：構(gòu)建帶有人TNF-a基因的慢病毒載體,并觀察該基因在人臍血間質(zhì)干細(xì)胞中的表達(dá)。 [方法]：通過(guò)逆轉(zhuǎn)錄聚合酶鏈反應(yīng)從PCD DNA-TNF-a質(zhì)粒中獲得人TNF-α基因,利用Infusion技術(shù)重組構(gòu)建慢病毒載體穿梭質(zhì)粒pGC-FU-TNF-α,在脂質(zhì)體lipofectamine 2000介導(dǎo)下與結(jié)構(gòu)質(zhì)粒pHelperl.0和包膜質(zhì)粒pHelper2.0共轉(zhuǎn)染293T細(xì)胞包裝生產(chǎn)慢病毒。將人臍血間質(zhì)干細(xì)胞分為實(shí)驗(yàn)組(pGC-FU-TNF-a),空載體對(duì)照組(pGC-FU-EGFP)及空白組(UCBMSCs),分別用重組慢病毒,空載慢病毒,PBS感染后,采用RT-PCR以及Elisa檢測(cè)TNF-a表達(dá)情況。 [結(jié)果]：(1)所獲TNF-a基因測(cè)序證明與Gene Bank中序列一致：(2)重組慢病毒載體質(zhì)粒pGC-FU-TNF-a經(jīng)鑒定正確；(3)三質(zhì)粒共轉(zhuǎn)染293T細(xì)胞成功,收集、濃縮病毒后測(cè)定其滴度為2×107TU/L。(4)重組慢病毒感染人臍血間質(zhì)干細(xì)胞后行RT-PCR檢測(cè)三組細(xì)胞電泳結(jié)果顯示均有TNF-a mRNA表達(dá),其中實(shí)驗(yàn)組光密度值(2.71±1.57),與空載對(duì)照組(4.63±1.32),空白組(6.03±0.88)比較差異有統(tǒng)計(jì)學(xué)意義(F=11.677,P0.01)；ELISA檢測(cè)三組細(xì)胞TNF-a濃度結(jié)果顯示實(shí)驗(yàn)組(1.32±0.23ug/ml)與空載對(duì)照組(0.70±0.25ug/ml),空白組(0.76±0.11ng/ml)比較差異有統(tǒng)計(jì)學(xué)意義(F=21.321,P0.01)。 [結(jié)論]：(1)構(gòu)建帶有TNF-a基因的慢病毒載體,包裝生產(chǎn)高滴度重組慢病毒；(2)重組慢病毒成功感染臍血間質(zhì)干細(xì)胞并實(shí)現(xiàn)TNF-a在臍血間質(zhì)干細(xì)胞中的表達(dá)；(3)轉(zhuǎn)基因TNF-a臍血間充質(zhì)干細(xì)胞的構(gòu)建為治療胃癌的應(yīng)用奠定理論基礎(chǔ)。
[Abstract]:Objective: to construct a lentivirus vector containing human TNF-a gene and observe its expression in human umbilical cord blood mesenchymal stem cells. [methods] Human TNF- 偽 gene was obtained from PCD DNA-TNF-a plasmid by reverse transcription polymerase chain reaction (RT-PCR). Lentivirus shuttle plasmid pGC-FU-TNF- 偽 was constructed by Infusion technique and co-transfected with structural plasmid pHelperl.0 and encapsulated plasmid pHelper2.0 into 293T cells to produce lentivirus under liposome lipofectamine 2000. Human umbilical cord blood mesenchymal stem cells were divided into experimental group pGC-FU-TNF-a- 偽 and empty. The vector control group (pGC-FU-EGFP) and the blank group (UCB MSC) were treated with recombinant lentivirus, respectively. RT-PCR and Elisa were used to detect the expression of TNF-a. [results] the sequence of TNF-a gene obtained from Gene Bank confirmed that the recombinant lentivirus vector pGC-FU-TNF-a was cotransfected into 293T cells successfully, and the recombinant lentivirus vector pGC-FU-TNF-a was identified as correct. The recombinant lentivirus was infected with human umbilical cord blood mesenchymal stem cells. The results of RT-PCR analysis showed that there was TNF-a mRNA expression in all three groups. The optical density of the experimental group (2.71 鹵1.57) was significantly higher than that of the no-load control group (4.63 鹵1.32) and the blank group (6.03 鹵0.88). The TNF-a concentration of the three groups was detected by Elisa. The results showed that the concentration of TNF-a in the experimental group was 1.32 鹵0.23ugrmlml) and that in the blank group was 0.76 鹵0.11ng / ml. [conclusion] A lentivirus vector containing TNF-a gene was constructed. Packaging production of High titer Recombinant lentivirus (lentivirus 2) Recombinant lentivirus successfully infects umbilical cord blood mesenchymal stem cells and realizes the expression of TNF-a in umbilical cord blood mesenchymal stem cells. The construction of transgenic TNF-a umbilical cord blood mesenchymal stem cells lays a theoretical foundation for the treatment of gastric cancer.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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本文編號(hào)：1680001