CAJ | 학술논문

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.
구건선립체융합소기인2(Mfn2)단발잡RNA(Mfn2shRNA)표체질립,관찰류체역학주사법대록색형광단백기관파향성.근거Mfn2기인서렬,도선1조목표기인서렬화1조비특이성기인서렬,구건질립중조체병측서급감정.장24지BALB/c소서수궤분위정상대조조、음성대조조화전염조(n=8),음성대조조화전염조분별통과미정맥쾌속주입음성대조질립(HK)화Mfn2shRNA질립용액1.5 ml.24 h후채집간장、심장、기육、신장조직빙동절편,형광현미경하관찰,병취혈청측정곡병전안매(ALT)、곡초전안매(AST)농도.결과표명:질립HK화Mfn2shRNA구건성공;류체역학주사후,간장、심기급신장가견록색형광단백적표체;여정상대조조비교,음성대조조혈청ALT、AST유명현차이,전염조혈청ALT、AST교정상대조조급음성대조조명현승고.Mfn2shRNA질립재체적성공구건,류체역학간장파향성적기인전염방법,위활체내연구Mfn2공능제공료가고적재료화방법.
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 function.