人角质形成细胞基因导入效率的影响因素研究

目的 了解不同大小质粒和不同基因转染法对人KC基因导入效率的影响.方法 采用脂质体转染法、阳离子多聚物转染法、电穿孔联合细胞核转染试剂转染法、慢病毒感染法,分别将不同大小的质粒[pSUPER-增强型绿色荧光蛋白(EGFP)、pEGFP-N2、pHSER-绿色荧光蛋白(GFP)、ploxP-EGFP]导入人永生化KC株HaCaT细胞和人胚肾细胞株293FT细胞(后者为对照).于倒置荧光显微镜下观察GFP的表达,计算转染率.结果 (1)采用脂质体转染法可将4种质粒导入HaCaT细胞(转染率1.0%～3.3%)及293FT细胞(转染率80.0%～84.7%).(2)阳离子多聚物转染法亦可将4种质粒导入HaCaT细胞(转染率为1.0%～3.7%)和293FT细胞(转染率81.3%～86.7%).(3)采用电穿孔联合细胞核转染试剂转染法,可以将2种较小片段质粒pSUPER-EGFP和pEGFP-N2导入HaCaT细胞,转染率分别为22.3%和19.0%;而2种较大片段质粒pHSER-GFP和ploxP-EGFP的转染率分别为4.0%和3.3%.(4)pHSER-GFP经慢病毒包装后,导入HaCaT细胞的转染率高达97.0%,明显优于前3种转染法.结论 脂质体转染法、阳离子多聚物转染法较难将外源性基因导入人KC,慢病毒感染法的转染率明显优于电穿孔联合细胞核转染试剂转染法;不同大小质粒对转染率有明显影响.

Study on influencing factors in efficiency of introducing gene into human keratinocyte(KC)

Objective To observe the effect of plasmids in different size and gene transfection pro-tocol on efficiency of introducing gene into human KC. Methods Four plasmids in different size, inclu-ding pSUPER-enhaneed green fluorescent protein (EGFP), pEGFP-N2, pHSER-green fluorescent protein (GFP) and ploxP-EGFP, were transfeeted into immortal human KC line (HaCaT) and human embryo kid-ney cell line (293FT) separately following transfection protocols of liposome (LTP), cation polymerizer (CPTP) , eleetroporation combined with nucleus transfeetion agent (ETP) and lentivirus. 293FT was used as control. GFP expression was observed under inverted fluorescence microscope. The transfection efficiency (TE) was calculated. Results ( 1 ) The four plasmids could be introduced into HaCaT ( TE, 1.0% -3.3% ) and 293FT (TE, 80. 0%-84.7% ) following LTP. (2) The four plasmids could also be introduced into HaCaT (TE, 1.0%-3.7% ) and 293FT (TE, 81.3%-86.7% ) following CPTP. (3) Two shorter plas-raids (pSUPER-EGFP and pEGFP-N2) could be introduced into HaCaT by ETP with higher TE than the oth-er two longer plasmids ( pHSER-GFP and ploxP-EGFP) , which were 22.3% and 19.0% vs. 4. 0% and 3.3% , respectively. (4) pHSER-GFP packaged by lentivirus could be introduced into HaCaT with the TE reaching 97.0% , which surpassed the above three protocols. Conclusions It is difficult to introduce ex-ogenous gene into human KC by LTP or CPTP; TE of lentivirus transfection protocol apparently surpasses ETP. Plasmid size can greatly affect TE.