本文選題：HOXB4 + 人臍帶間充質(zhì)干細胞��； 參考：《青島大學》2012年碩士論文

【摘要】：目的本實驗旨在構(gòu)建人同源盒基因(Homeobox Gene) HOXB4的慢病毒載體,探討慢病毒介導的HOXB4感染人臍帶間充質(zhì)干細胞后的變化。 方法利用PCR擴增獲得HOXB4,并克隆入慢病毒穿梭載體Lenti-shuttle。運用四質(zhì)粒慢病毒包裝系統(tǒng)轉(zhuǎn)染HEK293T細胞,48h收獲慢病毒Lenti-HOXB4,并測定慢病毒滴度。將Lenti-HOXB4惑染人臍帶間充質(zhì)干細胞,倒置熒光顯微鏡觀察細胞感染效果,確定最佳的病毒感染復數(shù)(MOI)。同時,利用RT-PCR、免疫熒光染色、流式細胞術(shù)檢測HOXB4的表達情況,CCK-8檢測HOXB4對細胞生長的影響。 結(jié)果利用四質(zhì)粒共轉(zhuǎn)染293T,成功獲得Lenti-HOXB4,測定的病毒滴度為3×108TU/ml；當MOI為20時,Lenti-HOXB4對人臍帶間充質(zhì)干細胞具有較高的轉(zhuǎn)染效率,達80%以上。感染Lenti-HOXB4的人臍帶間充質(zhì)干細胞,在nRNA、蛋白水平上均檢測到了目的基因的表達,其能促進臍帶間充質(zhì)干細胞增殖一倍,流式結(jié)果表明：CD340.79%CD451.48%CD10592.68%CD4497.41%與轉(zhuǎn)染前比較CD340.13%CD450.14%.CD10599.95%CD9099.88%,造血細胞表面標志略有增高,間充質(zhì)干細胞的表面標志無明顯改變。 結(jié)論1.成功構(gòu)建了HOXB4慢病毒載體并獲得了HUCMSCs-HOXB4基因工程細胞。2.HOXB4基因能促進臍帶間充質(zhì)干細胞的增殖3.HOXB4基因轉(zhuǎn)染并不影響臍帶間充質(zhì)干細胞表面標志的表達。
[Abstract]:Objective to construct the lentivirus vector of Homeobox Gene (Homeobox Gene) HOXB4 and to investigate the changes of Homeobox Gene HOXB4 infected with human umbilical cord mesenchymal stem cells. Methods HOXB4 was amplified by PCR and cloned into lentivirus shuttle vector Lenti-shuttle. Lentivirus Lenti-HOXB4 was harvested from HEK293T cells transfected with four plasmids lentivirus packaging system for 48 h and the titer of lentivirus was measured. Lenti-HOXB4 was used to infect human umbilical cord mesenchymal stem cells, and the effect of cell infection was observed by inverted fluorescence microscope, and the optimal number of virus infection (moi) was determined. At the same time, RT-PCR, immunofluorescence staining and flow cytometry were used to detect the expression of HOXB4. CCK-8 was used to detect the effect of HOXB4 on cell growth. Results Lenti-HOXB4 was successfully transfected into 293T by four plasmids, and the titer of the virus was 3 脳 108TU / ml. When moi was 20:00, Lenti-HOXB4 had a higher transfection efficiency to human umbilical cord mesenchymal stem cells (more than 80%). Human umbilical cord mesenchymal stem cells infected with Lenti-HOXB4 detected the expression of the target gene at the level of nRNA and protein, which could double the proliferation of umbilical cord mesenchymal stem cells. The flow rate showed that CD340.79451.4810592.684497.41% was higher than that before transfection. CD340.13450.14.CD10599.959099.88, the surface markers of hematopoietic cells increased slightly, but the surface markers of mesenchymal stem cells did not change significantly. Conclusion 1. HOXB4 lentivirus vector was successfully constructed and HUCMSCs-HOXB4 gene was obtained. 2. HOXB4 gene could promote the proliferation of umbilical cord mesenchymal stem cells 3.HOXB4 gene transfection did not affect the expression of surface markers of umbilical cord mesenchymal stem cells.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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本文編號：2075186