慢病毒及脂质体法转染原代软骨细胞的实验研究

Experimental research of rat original chondrocytes transfected by lentiviral vector and lipidosome

目的利用慢病毒载体及Lipofectamine 2000脂质体转染大鼠原代软骨细胞,探讨转染软骨细胞的理想条件。方法采用机械—酶消化法获取原代软骨细胞,经Ⅱ型胶原(ColⅡ)鉴定后,使用慢病毒载体介导GFP及利用Lipofectamine 2000介导的FAM-siRNA分别转染原代软骨细胞,倒置荧光显微镜检测转染效率,MTT法测定原代软骨的增殖活力,流式细胞术检测细胞凋亡率。结果采用脂质法转染软骨细胞不理想,转染率不到10%。运用慢病毒载体后转染率明显高于脂质体法,且随感染复数(病毒滴度×病毒液体积/细胞数,MOI)增加而增加,MOI为100时,转染率可达92.85%。MTT及流式结果显示,低MOI时,软骨细胞活力及凋亡率与对照组相比差异无统计学意义;当高MOI(200)时,细胞活力[(81.32±10.72)%]明显低于对照组,而细胞凋亡率[(21.37±2.80)%]明显高于对照组。结论当MOI为100时可作为转染软骨细胞安全有效的条件,这为细胞转染技术在软骨细胞的研究领域提供实验基础。

Objective To explore the ideal conditions of transfection chondrocytes by lentiviral vector and Lipofectamine 2000.Method The primary chondrocytes were obtained by mechanical-enzyme digestion method,and be identified by immunohistochemical cells.Transfection chondrocytes by lentiviral vector-GFP and Lipofectamine 2000-FAM-siRNA.Transfection efficacy was evaluated by fluorescence microscopy.The cell viability was detected by MTT and the cell apoptosis was assessed by flow cytom etry.Result The transfection efficiency of Lipofectamine2000FAM-siRNA was under 10%.The transfection efficiency of lentiviral vector-GFP was higher than lipidosome,the efficiency was increased with increasing of infection plural（Virus drops degree by liquid deposition/cell number,MOI）,and the infection rate can be as high as 92.8 5% when MOI=100.Compared with control group,the cell viability and cell apoptosis there were no statistical significantly differences in low MOI groups.But when MOI=200the cell viability（81.32±10.72）% was decreaseed obviously and cell apoptosis（21.37±2.80）% was significantly increased（P〈0.05）.Conclusion The MOI= 100 is safe and effective conditions to transfection chondrocytes,it can be provided experimental basis for the research field of chondrocytes in transfection technique.