CAJ | 학술논문

目的通过体内基因转染方法在肝细胞中建立持续、高效表达外源性肝细胞生长因子(HGF)的体系,探讨其在肝细胞凋亡中的作用.方法通过尾部静脉快速注射大量pCMV-HGF质粒,ELISA检测外周血和肝组织中HGF表达的高峰和持续时间.实验动物分为模型组、pcDNA3空质粒保护组、pCMV-HGF质粒保护组和0.9%氯化钠溶液对照组,每组10只,Western印迹检测Caspase-3,tBid、Bax、细胞色素C的表达.所有数据组间均数比较采用单因素方差分析,均数间的两两比较用q检验.结果在小鼠尾静脉快速大量注射pCMV-HGF 4 h后就有外源性HGF表达,外周血和肝组织中分别于12 h和8 h达高峰,于注射后第6天仍可检测到HGF表达.D-氨基半乳糖胺/脂多糖可诱导明显的细胞凋亡,并导致tBid、Bax、Caspase-3及细胞色素C的表达明显增加.与模型组和pcDNA3空质粒保护组相比,pCMV-HGF保护组tBid、Bax、Caspase-3及细胞色素C的表达明显减少.结论外源性HGF能抑制小鼠肝细胞凋亡,通过抑制Bid的活性片段tBid的表达,减少下游凋亡蛋白的激活.
목적 통과체내기인전염방법재간세포중건립지속、고효표체외원성간세포생장인자(HGF)적체계,탐토기재간세포조망중적작용.방법 통과미부정맥쾌속주사대량pCMV-HGF질립,ELISA검측외주혈화간조직중HGF표체적고봉화지속시간.실험동물분위모형조、pcDNA3공질립보호조、pCMV-HGF질립보호조화0.9%록화납용액대조조,매조10지,Western인적검측Caspase-3,tBid、Bax、세포색소C적표체.소유수거조간균수비교채용단인소방차분석,균수간적량량비교용q검험.결과 재소서미정맥쾌속대량주사pCMV-HGF 4 h후취유외원성HGF표체,외주혈화간조직중분별우12 h화8 h체고봉,우주사후제6천잉가검측도HGF표체.D-안기반유당알/지다당가유도명현적세포조망,병도치tBid、Bax、Caspase-3급세포색소C적표체명현증가.여모형조화pcDNA3공질립보호조상비,pCMV-HGF보호조tBid、Bax、Caspase-3급세포색소C적표체명현감소.결론 외원성HGF능억제소서간세포조망,통과억제Bid적활성편단tBid적표체,감소하유조망단백적격활.
Objective To establish high level expression system of exogenous hepatocyte growth factor(HGF) protein in mouse livers by in vivo gene transfection and to observe the inhibition effect of exogenous HGF on hepatocyte apoptosis in mice. Methods Mice were divided into four groups, with 10 mice in each arm, which were injected with control solution, empty pcDNA3 plasmids, pCMV-HGF plasmid or 0.9% sodium chloride solution by tail vein. Enzyme-linked immunosorbent assay(ELISA) was used to determine the peak level and the expression duration of HGF protein in the peripheral blood and liver tissue. Western blotting was performed to measure the Caspase-3, tBid, Bax and Cytochrom C in the hepatocyte homogenatea and mitochondrion. Results HGF protein was detected in the mice blood as early as 4 hours after single injection of pCMV-HGF plasmid. The peak level of HGF protein in liver and plasma was respectively achieved by 8 hours and 12 hours after first injection while HGF protein was still detectable in the blood 6 days after the initial injection. D-Galactosamine/lipopolysaeeharide (LPS) led to obvious hepatocyte apoptnsis and induced an increased concentration of tBid, Bax, Caspase-3 and Cytochrom C in the hepatocyte homogenates and mitochondrion. Compared to sodium chloride control group and empty pcDNA3 protected group, the expression of tBid, Bax, Caspase-3 and Cytochrom C decreased in pCMV-HGF plasmid protecting group. Conclusions Hepatocyte apoptosis can be inhibited by exogenous HGF protein expression in mouse livers, which is induced by in vivo gene transfection. Moreover, it may inhibit the activation of downstream apoptotic proteins by blocking the expression of tBid.