Mfn2基因shRNA质粒的构建及其流体力学转染技术的实验研究

Construction of the Mfn2 shRNA and Study of Systemic shRNA Delivery via Hydrodynamic Intravascular Injection

构建线粒体融合素基因2(Mfn2)短发卡RNA(Mfn2shRNA)表达质粒,观察流体力学注射法对绿色荧光蛋白器官靶向性。根据Mfn2基因序列,挑选1条目标基因序列和1条非特异性基因序列,构建质粒重组体并测序及鉴定。将24只BALB/c小鼠随机分为正常对照组、阴性对照组和转染组(n=8),阴性对照组和转染组分别通过尾静脉快速注入阴性对照质粒(HK)和Mfn2shRNA质粒溶液1.5 ml。24 h后采集肝脏、心脏、肌肉、肾脏组织冰冻切片,荧光显微镜下观察,并取血清测定谷丙转氨酶(ALT)、谷草转氨酶(AST)浓度。结果表明:质粒HK和Mfn2shRNA构建成功;流体力学注射后,肝脏、心肌及肾脏可见绿色荧光蛋白的表达;与正常对照组比较,阴性对照组血清ALT、AST有明显差异,转染组血清ALT、AST较正常对照组及阴性对照组明显升高。Mfn2shRNA质粒载体的成功构建,流体力学肝脏靶向性的基因转染方法,为活体内研究Mfn2功能提供了可靠的材料和方法。

To construct the expression plasmids of short hairpin RNA （shRNA） for Mfn2 gene, and to investigate the effects of target organ for gene transfection by injection of green fluorescent protein （GFP） - expressed plasmid using hydrodynamic technique. One target and one non - specificity gene orders were chosen according to Mfn2 gene order, pGensil - 1.2 vector were constructed, sequenced and identified. Twenty - four male BALB/c mice were randomly divided into normal control group, negative control group and transfection group according to random digits table. 1.5 ml GFP- expressed plasmid （negative control or Mfn2shRNA, 75ug for each mouse） was injected into the mice of negative control and transfection groups rapidly in 5s through vena caudalis. Twenty - four hours later, the tissue of live, heart, muscle, and kidney were collected and were determined under fluorescent microscope by frozen sections. The blood samples were collected to detect the contents of serum alanine aminotransferase （ALT） and aspartate transaminase （AST）. Results showed that the two plasmids expression vectors of synthesized shRNA were exactly identical with the design. The hydrodynamic intravascular injection resulted in GFP expression in liver, heart, and kidney. Compared with the normal control group, the levels of serum ALT and AST of negative control group had statistically increased （ P 〈 0.05）. The levels of serum ALT and AST were significantly elevated in the mice of transfection group, compared with two control groups （P 〈 0.001）. Successful construction of Mfn2 shRNA plasmid vevtors and hydrodynamic transfection methods to deliver plasmid to the livers of mice provide reliable materials and methods for the research of Mfn2 ftmction.