电穿孔辅助肌内注射肝细胞生长因子质粒DNA对博来霉素诱导的大鼠肺纤维化的影响

Effect of intramuscular injection of hepatocyte growth factor plasmid DNA with electroporation on bleomycin-induced lung fibrosis in rats.

作者信息

Long Xiang, Xiong Sheng-dao, Xiong Wei-ning, Xu Yong-jian

机构信息

Institute of Pulmonary Disease, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430030, China.

出版信息

Chin Med J (Engl). 2007 Aug 20;120(16):1432-7.

PMID:17825173

Abstract

BACKGROUND

So far, there is no efficient treatment for pulmonary fibrosis. The objective of this study was to determine whether intramuscular injection of the hepatocyte growth factor (HGF) plasmid DNA by in vivo electroporation could prevent bleomycin-induced pulmonary fibrosis in rats, and to investigate the possible mechanisms.

METHODS

Twenty male Wistar rats were randomly divided into four groups: control group (group C), model group (group M), early intervention group (group I) and late intervention group (group II). Groups M, I and II were intratracheally infused with bleomycin, then injected the plasmid pcDNA3.1-hHGF to group I on day 7, 14 and 21. Group II received the same treatment like Group I on day 14 and 21. All the rats were killed on day 28 after bleomycin injection. We detected Homo HGF expression in the rats with ELISA method and estimated the pathological fibrosis score of lung tissue using hematoxylin eosin (HE) and Massion staining. The mRNA expression of transforming growth factor-beta1 (TGF-beta1), cycloxygenase-2 (COX-2), and rat HGF in rat pulmonary parenchyma were evaluated by RT-PCR. Immunohistochemistry and Western blotting were performed to determine the protein expression of transforming TGF-beta1 and COX-2 in lung parenchyma.

RESULTS

The plasmid pcDNA3.1-hHGF could express hHGF in NIH3T3 cells and the hHGF protein is secreted into the culture medium. The expression of hHGF protein could be monitored in quadriceps muscle, plasma and lung in Groups I and II. Pulmonary fibrosis levels of Groups I and II were obviously lower than that of group M (P < 0.05). Expression of TGF-beta1 protein and mRNA in lung tissue was markedly decreased in Groups I and II compared with Group M (P < 0.05). The level of expression of HGF and COX-2 mRNA was higher in Groups I and II than in Group M (P < 0.05).

CONCLUSIONS

Injection of the plasmid pcDNA3.1-hHGF into skeletal muscle with electroporation has a potential role in the treatment of bleomycin-induced lung fibrosis. Exogenous HGF may inhibit the expression of TGF-beta1 and regulate the crosstalk between AECs and mesenchymal fibroblasts.

摘要

背景

迄今为止，尚无针对肺纤维化的有效治疗方法。本研究的目的是确定通过体内电穿孔肌内注射肝细胞生长因子（HGF）质粒DNA是否能预防博来霉素诱导的大鼠肺纤维化，并探讨其可能的机制。

方法

将20只雄性Wistar大鼠随机分为四组：对照组（C组）、模型组（M组）、早期干预组（I组）和晚期干预组（II组）。M组、I组和II组经气管内注入博来霉素，然后I组在第7、14和21天注射质粒pcDNA3.1-hHGF。II组在第14和21天接受与I组相同的治疗。所有大鼠在博来霉素注射后第28天处死。我们用ELISA法检测大鼠体内HGF的表达，并用苏木精-伊红（HE）和Masson染色评估肺组织的病理纤维化评分。通过RT-PCR评估大鼠肺实质中转化生长因子-β1（TGF-β1）、环氧化酶-2（COX-2）和大鼠HGF的mRNA表达。进行免疫组织化学和蛋白质印迹法以确定肺实质中TGF-β1和COX-2的蛋白质表达。

结果

质粒pcDNA3.1-hHGF可在NIH3T3细胞中表达hHGF，且hHGF蛋白分泌到培养基中。I组和II组的股四头肌、血浆和肺中均可监测到hHGF蛋白的表达。I组和II组的肺纤维化水平明显低于M组（P<0.05）。与M组相比，I组和II组肺组织中TGF-β1蛋白和mRNA的表达明显降低（P<0.05）。I组和II组中HGF和COX-2 mRNA的表达水平高于M组（P<0.05）。