裸质粒与脂质体介导转染方法对大鼠平滑肌细胞CYP2J3基因表达的影响

Influence of Liposome-mediated and Naked Plasmid DNA Methods on Expression of CYP2J3 Gene in Rat Smooth Muscle Cells

目的比较脂质体介导法和裸质粒直接转染法导入CYP2J3基因表达的效率。方法将CYP2J3真核表达质粒导入原代培养大鼠平滑肌细胞,用半定量RT-PCR方法检测CYP2J3 mRNA表达,Western blotting方法测定CYP2J3蛋白表达,高效液相色谱法测定培养液11,12-EET含量。实验分为裸质粒转染组(2J3)、脂质体介导转染组(L2J3)和对照组,转染后24h和48h收集细胞。结果转染后24h,2个转染组CYP2J3 mRNA表达均较对照组明显增高,差异有统计学意义(P<0.01);L2J3组蛋白含量较对照组低,差异有统计学意义(P<0.01),2J3组蛋白含量较L2J3组高,差异有统计学意义(P<0.05)。培养液11,12-EET含量在3组间差异无统计学意义。转染后48h2个转染组CYP2J3 mRNA表达较24h时弱,但仍较对照组高,脂质体转染组较裸质粒转染组CYP2J3 mRNA表达增强;2J3组及L2J3组蛋白含量均较对照组低,差异有统计学意义(P<0.01);2个转染组培养液中11,12-EET浓度较对照组高,脂质体转染组表达高于裸质粒转染组,差异有统计学意义(P<0.05)。结论裸质粒直接转染能成功导入CYP2J3基因并表达产物。

Objective To study the expression efficiency of CYP2J3 gene in rat smooth muscle cells（SMCs） by liposome mediated and naked plasmid transfection. Methods The expression of CYP2J3 mRNA was tested by RT-PCR method, expression of CYP2J3 protein was determined by Western blotting method, and content of 11,12-EET was examined by HPLC system in SMCs. Cells were divided into 3 groups ： pcDNA3.1-CYP2J3 group, liposome mediated group and control group ; cells were collected after 24 h and 48 h. Results At 24 h after transfection, expression of CYP2J3 mRNA in 2 transfected groups was significantly higher than that of control group（ P 〈 0. 01 ）. Content of CYP2J3 protein in L2J3 group was decreased compared with control group （ P 〈 0.01 ）, and there was no significant difference among the 3 groups in content of CYP2J3 protein and 11,12-EET. At 48 h after transfection, expression of CYP2J3 mRNA in 2 transfected groups was decreased compared with 24 h after transfection （ P 〈 0.01 ）, but was still higher than that of control group （ P 〈 0.01 ）. Expression of CYP2J3 protein in control group was higher than those of the 2 transfected groups （P 〈 0.01 ）. And content of 11,12-EET in 2 transfected groups was significantly higher than that of control group, and that of L2J3 group was also higher as compared with 2J3 group （P 〈 0.05 ）. Conclusion The methods of naked plasmid direct transfection can introduce CYP2J3 gene and express its product.