电脉冲介导基因转移效率的实验研究

Electric pulse mediated high efficient gene transfer

目的 研究电脉冲介导的基因转移效率及其最佳的基因转移的电脉冲参数。方法 用微量注射器将pcD2 /LacZ质粒 10 μg ,在昆明小鼠的股四头肌注射 ,1～ 2min内在注射部位给予不同参数的电脉冲刺激 (不同的电脉冲参数每组 10只小鼠 ) ,然后进行 β 半乳糖苷酶活性的测定或酶组织化学染色。同时设空白对照组及单纯注射组。结果 电脉冲可明显增加LacZ基因的表达 ,电脉冲组的β 半乳糖苷酶活性 (131.6U/mg± 86 .5U/mg蛋白 )是单纯注射组 (4 .9U/mg± 1.0U/mg蛋白 )的 30倍(P <0 .0 5 )。组织化学染色结果表明电脉冲组肌肉组织中 β 半乳糖苷酶蓝色颗粒的数目和染色的程度均明显高于单纯肌肉注射组。当电脉冲参数电压 2 0 0V/cm ,波宽 40ms,脉冲次数 6次和频率 1Hz时 ,可获得最高的基因表达效率。结论 在最佳的电脉冲参数条件下 ,电脉冲介导的基因转移可获得较高的基因表达。

Objective To study gene transfer mediated by electric pulse and optimize the parameters of electric pulse in vivo. Methods 10 μg plasmid pcD 2/LacZ was injected into the quadriceps of 220 Kunming mice. One to two minutes after the DNA injection electric pulse with different parameters was given to the inje ction site. Three days after, activity of β-galactosidase was measured, and t he expression of Laz2 gene in muscle was determined by histochemical staining. Results The activity of β-galactosidase in electric pulse group (131.6 U/mg±86 6 U/m g p rotein) was 30 fold higher than that in direct injection group (4.9 U/mg±1.0 U/ mg pr otein) ( P <0.05). Histochemical analysis of muscles injected with a LacZ expr essi on plasmid also showed that in vivo electric pulse increased both the number of positively stained muscle fibers and the density of staining. When the electric pulse was with the parameters of 200 V/cm, 40 ms, 6 pulses and 1HZ, maximal gene expression was achieved. Conclusion Electric pulse, with optimal parameters, inc reases gene expression. Electric pulse makes much more gene expression than mere intramuscular DNA injection.