脂质体方法转染人骨髓基质细胞的影响因素

目的探讨用脂质体介导的方法转染人骨髓基质细胞的影响因素。方法构建真核表达载体pcDNA3-sFlt-1D4，脂质体方法转染人骨髓基质细胞，流式细胞仪检测转染率，ELISA、MTT法检测转基因蛋白sFlt-1D4的浓度及功能。结果转染率为9．27％。ELISA法检测转染后24、48h及4周转基因骨髓基质细胞培养上清中sFlt-1D4蛋白的表达浓度分别为（0．104±0．078）μg／L、（0．158±0．022）μg／L和（0．171±0．069）μ5／L。ELISA法证实转基因组培养上清中血管内皮生长因子（VEGF）含量降低。MTr法证实转染24、48h及4周的转基因产物对K562细胞的抑瘤率分别为（9．41±4．71）％、（23．63±7．50）％和（33．13±6．93）％。结论血清、细胞铺板密度、转染后细胞筛选的方式等是影响转染质量的主要因素。转基因方法可以获得sFlt-1D4蛋白的表达，转基因蛋白具有抑制K562细胞生长的作用。

The influencing factor on human marrow stroma cell transfected by lipofectamine

Objective To investigate the influencing factor on human marrow stroma cell transfected by llpofectamine. Methods The recombinant plasmid pcDNA3 -sFlt-104 was constructed. The recombinant plasmld pcDNA3-sFlt-104 was transfected into the bone marrow stromal cells by lipofectamine 2000, and it was identified by ELISA and MTT. Results The transfecfion efficiency identified by flow cytometry was 9. 27%. The protein expression concentrations of sFlt-104 in the culture supematant 24 h ,48 h and 4 weeks after transfecfion by ELISA were （0. 104 ±0. 078 ）μg/L, （0. 158 ±0. 022） μg/L and（0. 171 ± 0. 069）μg/L,respectively. The content of vascular endothelial growth factor （VEGF） secreted by K562 culturing with transfectant cells culture supematant was reduced compared with control. The inhibitory rates on the proliferation of K562 cells via MTT assay were （9. 41 ± 4.71 ） %, （23.63 ± 7.50） % and （ 33. 13 ± 6. 93） % ,respectively. Condusions The main influencing factors on human marrow stroma cell transfected by lipofectamine are blood serum, planking density of human marrow stroma cell and bolting modality after being transfected. The hone marrow stromal cells transfected with recombinant plasmid pcDNA3 -sFlt-104 could secrete sFlt-104 and inhibit the proliferation of K562 cell.