In order to study the potential of Venus,lentiviral vector,applied to acute myeloid leukemia,the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calucium phosphate coprecipitation.All virus stocks were collected and transfected into HL-60,the GFP expression in HL-60 cells was measured by flow cytometry.The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry.The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed.The results indicated that the transfection efficacy of letivial vector on HL-60 cells was more than 95 ％,which meets the needs for further study.C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector.Before and after transfection,the proliferation and differentiation of cells were not changed much.It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene.Meanwhile,lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.%随着对急性髓系白血病(AML)发生、发展的基因机制深入研究,构建转染效率高且对细胞无毒性作用的载体变得十分重要.为了研究补体C3a的受体蛋白C3aR1(complement component 3a receptor 1)在AML中的作用,构建了携带C3aR1的第三代慢病毒载体Venus.本研究主要观察Venus慢病毒载体在急性髓系白血病中应用的可行性.采用磷酸钙沉淀法将重组载体Venus-C3aR1转染包装细胞293T细胞,产生慢病毒颗粒,感染HL-60细胞,通过检测绿色荧光蛋白(GFP)观察感染效率,用RT-PCR和流式细胞术鉴定感染后HL-60细胞C3aR1的表达情况.用CCK8法绘制细胞生长曲线、观察细胞诱导分化能力来判断慢病毒载体的细胞毒性作用.结果显示,流式细胞仪(FCM)检测慢病毒感染率＞95％,感染后HL-60细胞上C3aR1表达明显增加,病毒包装成功.慢病毒感染前后细胞生长曲线及诱导分化能力无明显变化,说明慢病毒载体不影响HL-60细胞的一般生物学特性,无明显的细胞毒性作用.结论:Venus慢病毒载体对AML细胞株HL-60的感染效率高,能够整合到细胞基因组中实现目的基因的稳定表达.慢病毒感染对细胞无明显的细胞毒性作用,不会干扰靶基因在细胞株中功能,因此慢病毒载体可以为白血病细胞的研究提供可靠的技术支持.
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