壳聚糖/小干扰RNA溶液对大鼠原代心肌细胞转染方法学的研究

Methodology research on chitosan/small interfering RNA solution transfection towards primary myocardial cells in rat

目的探讨壳聚糖(chitosan,CS)/小干扰RNA(small interfering RNA,siRNA)溶液对大鼠原代心肌细胞转染的优化条件。方法应用粒度仪检测氮磷比70∶1 CS/NC-siRNA的粒径分布和Zeta电位;实验分为12 h孵育组和24 h孵育组,各组分别与7个不同浓度的CS/NC-siRNA孵育(分别为0、4×10^(-3)、8×10^(-3)、12×10^(-3)、16×10^(-3)、20×10^(-3)、24×10^(-3) mg/mL),应用CCK8法检测孵育后各组心肌细胞的活性;选择12 h孵育组,分别应用活细胞成像系统和激光共聚焦显微镜检测CS/cy5-NC-siRNA转染效率。结果氮磷比70∶1 CS/NC-siRNA纳米颗粒粒径为370.50 nm,Zeta电位为28.17 mV。12 h孵育组中0 mg/mL组细胞活性与其他浓度组相比,差异无明显统计学意义(P>0.05)。24 h孵育组中0 mg/mL组细胞活性高于其他浓度组,差异有统计学意义(P<0.05)。12 h孵育组中各个浓度组(除外0 mg/mL组)的细胞活性明显比24 h孵育组中对应浓度组的细胞活性高,差异有统计学意义(P<0.01)。活细胞成像系统检测表明,各浓度组于转染24 h后转染效率均高于对应浓度组于转染12 h的转染效率,差异有统计学意义(P<0.0001);20×10^(-3) mg/mL组于转染24 h后转染效率与其他浓度组比较,差异无明显统计学意义(P>0.05);24×10^(-3) mg/mL组于转染48 h后转染效率与20×10^(-3) mg/mL组比较,差异无统计学意义(P>0.05),但明显高于其他浓度组(P<0.0001)。激光共聚焦显微镜检测表明,20×10^(-3) mg/mL组和24×10^(-3) mg/mL组于转染24 h后能充分转染心肌细胞,且两者转染效率比较,差异无统计学意义(P>0.05)。结论氮磷比为70∶1的CS/siRNA适合于转染原代心肌细胞,CS/siRNA溶液与原代心肌细胞12 h的孵育可以提高转染效率同时可以减少细胞毒性,浓度为20×10^(-3) mg/mL和24×10^(-3) mg/mL的CS/siRNA溶液转染效率优于本实验其他浓度CS/siRNA溶液。

Objectives To explore the optimal transfection protocol of chitosan(CS)/small interfering RNA(siRNA)solution towards primary myocardial cells in rat.Methods The distribution of particle size and Zeta potential in CS/NC-siRNA with N/P ratios 70:1 were detected by laser particle size analyzer;the experiment was classified into 12 h incubation group and 24 h incubation group,and each group was incubated with 7 kinds of CS/NC-siRNA with distinct concentrations respectively including 0,4×10^(-3),8×10^(-3),12×10^(-3),16×10^(-3),20×10^(-3),24×10^(-3)mg/mL,whose myocardial cells viability was measured by CCK8 after incubation;12 h incubation group was chosen for detection of the transfection efficiency of CS/cy5-NC-siRNA by live cell imaging system and laser scanning confocal microscope.Results The particle size of CS/NC-siRNA nanoparticles with nitrogen(N)/phosphorus(P)ratios 70∶1 was 370.50 nm,and its Zeta potential was 28.17 mV.There were no statistically significant differences between 0 mg/mL group and other groups on the cell viability in 12 h incubation group(P>0.05),while the cell viability of 0 mg/mL group was higher than those of the other groups in 24 h incubation group(P<0.05),and meanwhile,the cell viabilities of each groups in 12 h incuba⁃tion group were distinctly superior to those of 24 h incubation group′s corresponding groups,except 0 mg/mL group(P<0.01).According to live cell imaging system,each groups possessed more efficient transfections in 24 h after the admin⁃istration of CS/cy5-NC-siRNA compared with corresponding groups in 12 h(P<0.0001),yet there were no signifi⁃cant differences between 20×10^(-3) mg/mL group and other groups on transfection efficiency in 24 h after the adminis⁃tration of CS/cy5-NC-siRNA(P>0.05),and after the administration of CS/cy5-NC-siRNA in 48 h,the transfection ef⁃ficiency of 20×10^(-3) mg/mL group was not preferable to 24×10^(-3) mg/mL group(P>0.05)which possesses more efficient transfection than other groups(P<0.0001).The detection of laser scanning confocal microscope manifested that both 20×10^(-3) mg/mL group and 24×10^(-3) mg/mL group can be transfected by CS/cy5-NC-siRNA perfectly after administration in 24 h,and also the transfection efficiency was similar between them without statistically significant difference(P>0.05).Conclusions Besides being suitable for the transfection of primary myocardial cells,CS/siRNA solution with N/P ratios 70∶1 possesses more efficient transfection and less toxicity towards primary myocardial cells after incubation in 12 h,and furthermore,both 20×10^(-3) mg/mL group and 24×10^(-3) mg/mL group show more efficient transfection of CS/siR⁃NA solution than other groups in this study.