通过电穿孔将裸肝细胞生长因子基因导入骨骼肌减轻小鼠急性肝损伤

Attenuated acute liver injury in mice by naked hepatocyte growth factor gene transfer into skeletal muscle with electroporation.

作者信息

Xue F, Takahara T, Yata Y, Minemura M, Morioka C Y, Takahara S, Yamato E, Dono K, Watanabe A

机构信息

Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan.

出版信息

Gut. 2002 Apr;50(4):558-62. doi: 10.1136/gut.50.4.558.

DOI:10.1136/gut.50.4.558

PMID:11889079

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1773169/

Abstract

BACKGROUND

Hepatocyte growth factor (HGF) plays an essential role in hepatic development and regeneration, and shows proliferative and antiapoptotic activity in hepatocytes.

AIMS

To establish an effective new method for HGF gene transfer in vivo and to investigate its effects in acute experimental liver injury.

ANIMALS

Eight week old female mice were used.

METHODS

Rat HGF gene in a modified pKSCX plasmid was transferred to the tibialis anterior muscle by electroporation using a pulse generator. Four days later, plasma HGF concentrations were determined by enzyme linked immunosorbent assay every two days for three weeks. To confirm the efficacy of electroporation, a plasmid bearing green fluorescence protein (GFP) was transferred similarly. Four days after electroporation, carbon tetrachloride (CCl(4)) was administered to mice to induce acute liver injury. Plasma alanine aminotransferase (ALT) activity was measured. Hepatic apoptosis was assessed by Hoechst 33258 staining and the TUNEL method.

RESULTS

Fluorescence microscopy showed strong green fluorescence where the GFP gene had been transferred into muscle. In mice given the HGF gene, HGF in plasma was increased up to fourfold from pretreatment amounts, peaking 6-9 days after electroporation and quickly decreasing within three weeks. Compared with the group without HGF transfer, the percentage of apoptotic hepatocytes after CCl(4) intoxication was significantly lower, as was ALT activity. In addition, ALT activity normalised more rapidly in the HGF gene transfer group.

CONCLUSIONS

Naked DNA injection and transfer by electroporation efficiently brings about HGF expression in vivo, which can attenuate acute liver injury.

摘要

背景

肝细胞生长因子（HGF）在肝脏发育和再生中起重要作用，并在肝细胞中显示出增殖和抗凋亡活性。

目的

建立一种有效的体内HGF基因转移新方法，并研究其在急性实验性肝损伤中的作用。

动物

使用8周龄雌性小鼠。

方法

使用脉冲发生器通过电穿孔将修饰的pKSCX质粒中的大鼠HGF基因转移到胫前肌中。4天后，每两天通过酶联免疫吸附测定法测定血浆HGF浓度，持续3周。为了确认电穿孔的效果，类似地转移携带绿色荧光蛋白（GFP）的质粒。电穿孔4天后，给小鼠注射四氯化碳（CCl₄）以诱导急性肝损伤。测量血浆丙氨酸氨基转移酶（ALT）活性。通过Hoechst 33258染色和TUNEL法评估肝细胞凋亡。

结果

荧光显微镜显示GFP基因已转移到肌肉中的部位有强烈的绿色荧光。在给予HGF基因的小鼠中，血浆HGF比预处理量增加了四倍，在电穿孔后6-9天达到峰值，并在三周内迅速下降。与未进行HGF转移的组相比，CCl₄中毒后凋亡肝细胞的百分比明显更低，ALT活性也是如此。此外，HGF基因转移组中ALT活性恢复正常的速度更快。