羧甲基葡聚糖磁性纳米颗粒与脂质体介导人可溶性fms-样酪氨酸激酶受体-1的转染比较

Comparison of the transfection effects on soluble fins-like tyrosine kinase receptor-1 gene mediated by carboxymethylated dextran coated nanoparticle and lipofectamineTM 2000

目的 比较羧甲基化葡聚糖（CMD）磁性纳米颗粒与脂质体LipofectamineTM2000对人可溶性fms-样酪氨酸激酶受体-1（sFlt-1）第2～4区基因片段的转染率.方法 构建真核表达质粒编码增强型绿色荧光蛋白质粒（pcDNA3.1-EGFP）/sFlt-1（2～4）,采用酶切、电泳及基因测序鉴定.将实验分为磁颗粒组、脂质体组和未转染对照组进行.转染后24、48 h,倒置相差荧光显微镜下观察细胞绿色荧光分布;流式细胞仪检测细胞绿色荧光表达率;逆转录聚合酶链反应（RT-PCR）法和免疫蛋白印迹（Western blot）法检测sFlt-1（2-4）mRNA和蛋白表达;噻唑蓝（MTT）比色法观测细胞生长情况,计算各组细胞相对增长率（RGR）;Hoeehst细胞核染色法观察各组细胞凋亡情况.结果 重组质粒pcDNA3.1/sFlt-1（2～4）酶切产物在琼脂糖凝胶电泳时出现大小为915碱基对的条带.流式细胞仪检测发现,磁颗粒组平均转染率为45%,脂质体组平均转染率21%;二者比较,差异有统计学意义（t=2.541,P〈0.05）.RT-PCR和Western blot观察发现,转染后48 h磁颗粒组细胞sFlt-1（2～4）mRNA和蛋白表达均明显高于脂质体组（t=2.454,2.398;P值均〈0.05）.转染后24、48 h,磁颗粒组RGR与未转染对照组间差异无统计学意义（t=1.436,P〉0.05）,脂质体组RGR与未转染对照组及磁颗粒组间差异均有统计学意义（t=2.412,2.545,P值均〈0.05）;磁颗粒组细胞凋亡率与未转染对照组间差异无统计学意义（t=1.436,P〉0.05）,与脂质体组间差异有统计学意义（t=2.236,P〈0.05）.结论 CMD磁性颗粒较脂质体LipofeetamineTM2000可获得更高的sFlt-1（2～4）基因片段转染率.

Objective To compare the transfection effects on soluble fins-like tyrosine kinase receptor1 （sFlt-1） gene （2-4 transcellular region） mediated by carboxymethylated dextran coated nanoparticle and lipofectamineTM 2000. Methods The plasmid pcDNA3. 1-EGFP/sFlt-1（2-4） was constructed and assessed by enzyme cut, electrophoresis, and genetic sequencing. Three groups were divided： nanoparticle group,lipofectamine group, and non-transfeeted group. Twenty-four and 48 hours after the transfection, the distribution of cellular green fluorescence was oberved under the inverted phase contrast fluorescence microseope; the expression rate of green fluorescence was measured by flow cytometry; the expression of sFlt-1（2-4） mRNA and the protein was detected by reverse transcription-polymerase chain reaction （RTPCR） and Western blot; the growth of the cells was observed by methyl thiazolyl tetrazolium （MTT）colorimetry and the relative growth rate （RGR） of the cells in each group was ealeulated; the cellular apoptosis in each group was detected by Hoechst staining. Results The sequence of sFlt-1（2-4） gene was equal to 915 base pair （bp）. The transfection rate was 45 % in nanoparticle group and 21% in lipofectamine group; the difference between the two groups was significant （t = 2. 541, P〈0.05）. Forty-eight hours after the transfection, the expression of sFlt-1 （2-4）mRNA and protein was obviously higher in nanopartiele group than that in lipofeetamine group （t= 2.454, 2.398; P〈0.05） . Twenty-four and 48 hours after the transfection, the difference of RGR of the cells between nanoparticle and non-transfected group was not significant （t=1. 436, P〉0. 05）; the RGR in lipofectamine group differed much from that in nontransfected and nanoparticle group （t= 2. 412,2. 545; P〈0. 05） ; the difference of cellular apoptosis was not significant between nanoparticle and non-transfected group （t = 1. 436, P 〉 0. 05）, but significant between nanoparticle and lipofectamine group （t= 2. 236, P〈0.05）. Conclusion The transfection rate of sFlt-1（2-4） mediated by carboxymethylated dextran coated nanoparticle was higher than that mediated by lipofectamineTM 2000.