慢病毒载体Venus在HL-60细胞应用的可行性研究

Transfection of HL-60 Cells by Venus Lentiviral Vector

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。

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 lenfiviral 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 lefivial 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, lenfiviral vector can not affect HL-60 cell ability to proliferate and differentiate.