人sFlt-1基因转染骨髓基质细胞对K562细胞生长的影响

Effect of bone marrow stromal cells transfeted by human sFlt-1 gene on growth of K562 cells

目的:通过转基因的方法获得人可溶性血管内皮生长因子受体1(sFlt-1)蛋白的稳定表达,并检测其对K562细胞生长的影响。方法:构建真核表达载体pcDNA3-sFlt-1D4,用脂质体介导的方法将其转染骨髓基质细胞,采用RT-PCR、ELISA和MTT法检测转基因产物的表达及对K562细胞生长的影响。结果:流式细胞仪检测骨髓基质细胞的转染率为9.27%。ELISA法检测转染后24h、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)、(0.158±0.022)和(0.171±0.069)μg.L-1。ELISA法证实转基因组培养上清中VEGF含量降低。MTT法检测转染24h、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: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。

Objective To get the protein expression of sFh 1 by transfection and to investigate the effects of sFh-1 gene transfection on the growth of K562 cells. Methods The recombinant plasmid pcDNA3-sFh-1D4 was constructed. The recombinant plasmid pcDNA3-sFh-1D4 was transfected into the bone marrow stromal cells by Lipofectamine 2000, which was identified by RT-PCR, ELISA and MTT. Results The transfection efficiency identified by flow cytometry was 9.27%. The protein expression of sFh-1D4 was found in the culture supernatant 24 h, 48 h and 4 weeks after transfetion by ELISA and the expression concentrations were （0.104±0. 078 ）, （0. 158±0. 022） and （0. 171±0. 069）μg·L^-1, respectively. The content of VEGF secreted by K562 culturing with transfectant cells culture supernatant was reduced compared with control. The inhibitoy 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. Oonclusion The bone marrow stromal cells transfected with recombinant plasmid pcDNA3-sFh-1D4 could secrete sFh-1D4 and inhibit the proliferation of K562 cells.