Triton X-100对脂质体介导的BMP-2基因转染大鼠BMSCs的作用

EFFECT OF TRITON X-100 ON LIPOSOME MEDIATED BONE MORPHOGENETIC PROTEIN 2 BY TRANSFECTION OF RAT BONE MARROW MESENCHYMAL STEM CELLS

目的探讨Triton X-100对脂质体介导的BMP-2基因转染大鼠BMSCs的作用。方法 8周龄Wistar大鼠1只,雄性,体质量120 g。取Wistar大鼠股骨、胫骨骨髓,采用贴壁培养法分离培养BMSCs并鉴定。取第3代BMSCs,采用MTT法筛选Triton X-100适宜浓度。实验分成3组,实验组:BMP-2+脂质体+0.010%Triton X-100+BMSCs,常规转染组:BMP-2+脂质体+BMSCs,空白对照组:BMSCs+血清培养基。转染后48 h,倒置荧光显微镜观察各组绿色荧光蛋白表达情况;转染后72 h,采用实时荧光定量PCR检测BMP-2 m RNA表达。结果 0.010%Triton X-100既能使BMSCs保持一定的活力,又可达到一定的作用效果,故确定其为适宜浓度。转染后48 h,倒置荧光显微镜下见实验组和常规转染组均有绿色荧光蛋白表达,空白对照组无绿色荧光蛋白表达。转染后72 h,实验组BMP-2 m RNA相对表达量为5.94±0.12,常规转染组为4.99±0.08,差异有统计学意义(t=360.28,P=0.02)。实验组转染效率较常规转染组平均提高了19%。结论 0.010%Triton X-100可以促进脂质体介导的BMP-2基因转染大鼠BMSCs,并提高其转染效率。

Objective To study the effect of Triton X-100 promoting liposome-mediated bone morphogenetic protein 2（BMP-2） gene transfection of rat bone marrow mesenchymal stem cells（BMSCs）. Methods BMSCs were separated and cultured from the femur and tibia of healthy Wistar rats（8-week-old,male）. The 3rd passage BMSCs identified by detecting the surface antigen were used to transfect. The optimum concentration of Triton X-100 for liposome mediated gene transfection was determined with ELISA meter by the way of MTT. In optimum concentration of Triton X-100,liposome mediated BMP-2 gene was transfected to BMSCs. The experiment was divided into 3 groups according to types of trasfection agents： BMSCs were transfected with Triton X-100＋liposome＋BMP-2（experimental group）,with liposome＋ BMP-2（conventional transfection group）,and untransfected BMSCs served as blank control group. After 48 hours of transfecting,the green fluorescent protein（GFP） in cells was detected through inverted fluorescence microscope. After 72 hours of transfection,real-time fluorescence quantitative PCR was applied to measure the m RNA expression of BMP-2. Results 0.01% Triton X-100 was determined to be the optimum concentration for not only making the BMSCs maintain vitality,but also achieving a certain effect on BMSCs. After trasfecting for 48 hours,GFP was observed through inverted fluorescence microscope in the experimental group and conventional transfection group,but was not observed in the blank control group. After trasfecting for 72 hours,the relative BMP-2 m RNA expression level was 5.94±0.12 in the experimental group,and was 4.99±0.08 in the conventional transfection group,showing significant difference（t=360.28,P=0.02）. The transfection efficiency was increased by 19% in the experimentalgroup. Conclusion 0.010%Triton X-100 can promote the liposome mediated BMP-2 gene transfection of rat BMSCs,and can improve the transfection efficiency.