内毒素相关受体基因共转染肠上皮细胞最佳转染条件的探索

Laboratory study on lipopolysaccharide related receptor genes cotransfection to human intestinal epithelial cells

目的探讨内毒素(LPS)相关受体CD14、Toll样受体4(TLR4)和MD-2基因共转染人小肠上皮细胞株(HIC)的最佳转染条件。方法用核糖核酸酶保护法(RPA)检测基因转染效率,分别研究质粒DNA的量、FuGENE 6转染试剂与质粒DNA的比例、转染试剂-质粒DNA复合物与靶细胞的作用时间及培养液中血清存在与否对转染效果的影响。结果FuGENE 6转染试剂转染HIC的最佳转染条件是:每种质粒DNA的量为2.0μg(使用6 cm培养皿)、转染试剂与质粒DNA的比例为4∶1、转染试剂-质粒DNA复合物与HIC的作用时间为5 h,而培养液中血清存在与否对转染效率无明显影响。结论建立了CD14、TLR4和MD-2三种表达质粒共转染HIC的优化转染方法。

Objective To investigate optimization of lipopolysaceharide- related reeeptor-CD14, TLR4 and MD-2 genes eotransfection to human intestinal epithelial cell line （HIC）.Methods Effects of DNA amount, ratio of the volume of Fu-GENE 6 transfection reagent（μl） and DNA amount （μg）,action time of transfecfion reagent： DNA complex on target cells and the presence or absence of serum on transfection efficiency were analyzed by detecting the expression of transfected genes using RNase protection assay （RPA） .Results The optimal genes transfection conditions of CD14, TLR4 and MD-2 genes to HIC using FuGENE 6 transfection reagent were that DNA amount was 2.0μg; the ratio of the volume of FuGENE 6 transfection reagent（μl） and DNA amount （μg） was 4：1;the action time of transfection reagent：DNA complex on HIC was 5h and there was no obvious effect in transfection with or without serum. Conclusion The optimal method of CD14, TLR4 and MD-2 genes cotransfection to HIC using FuGENE 6 transfection reagent was established.