人硫氧还蛋白基因修饰肝细胞移植治疗大鼠急性肝衰竭模型。

Transplantation of human thioredoxin gene-modified hepatocytes for treatment of acute liver failure in rat model.

机构信息

Liver Transplantation Center, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, China.

出版信息

Chin Med J (Engl). 2009 Nov 5;122(21):2631-5.

PMID:19951583

Abstract

BACKGROUND

Mostly because of the limited number and proliferative ability of the transplanted hepatocytes, hepatocyte transplantation offers only temporary support to the hepatic function with rather poor functional replacement of the damaged liver parenchyma. This study aimed to observe the therapeutic effect of human thioredoxin (hTrx) gene-modified hepatocytes on experimental acute liver failure in rats.

METHODS

hTrx cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from human osteosarcoma 143 (TK-) cells to construct the recombinant retrovirus vector pLEGFP/hTrx, which was packaged into PA317 cells to collect the recombinant retrovirus containing hTrx gene. After titration and characterization, the recombinant retrovirus was applied to primary cultured rat hepatocyte for infection to generate hTrx gene-modified rat hepatocytes, whose viability and antioxidative capacity were examined by immunohistochemistry and MTT assay, respectively. In a Sprague-Dawley (SD) rat model of acute liver failure, the modified hepatocytes were injected into the spleen, and the hepatic function and survival rate of the recipient rats were evaluated at different time points after the transplantation.

RESULTS

NIH3T3 cells infected by the recombinant retrovirus were capable of expressing bioactive hTrx in the form of fusion proteins. Immunohistochemistry demonstrated normal function of the hTrx gene-modified hepatocytes, which possessed strong antioxidative capacity as shown by MTT assay. Transplantation of the modified hepatocytes in rats with acute liver failure resulted in significantly lowered serum alanine aminotransferase (ALT) and total bilirubin (TBIL) levels (P < 0.05). The hepatocytes exhibited long-term survival and efficient proliferation after transplantation. Fourteen days after the operation, the rat models receiving hTrx gene-modified hepatocytes had significantly higher survival rate than those without the transplantation.

CONCLUSION

hTrx gene-modified hepatocyte transplantation can effectively alleviate acute liver failure in rats.

摘要

背景

由于移植肝细胞的数量有限和增殖能力，肝细胞移植只能为肝功能提供暂时的支持，对受损的肝实质的功能替代效果较差。本研究旨在观察人硫氧还蛋白（hTrx）基因修饰的肝细胞对大鼠实验性急性肝衰竭的治疗作用。

方法

采用反转录-聚合酶链反应（RT-PCR）从人骨肉瘤 143（TK-）细胞中获得 hTrx cDNA，构建重组逆转录病毒载体 pLEGFP/hTrx，包装入 PA317 细胞中收集含有 hTrx 基因的重组逆转录病毒。经滴定和鉴定后，将重组逆转录病毒用于原代培养的大鼠肝细胞感染，生成 hTrx 基因修饰的大鼠肝细胞，通过免疫组化和 MTT 检测分别检测其活力和抗氧化能力。在 Sprague-Dawley（SD）大鼠急性肝衰竭模型中，将修饰后的肝细胞注射到脾脏中，在移植后不同时间点评估受体大鼠的肝功能和存活率。

结果

感染重组逆转录病毒的 NIH3T3 细胞能够以融合蛋白的形式表达有生物活性的 hTrx。免疫组化显示 hTrx 基因修饰的肝细胞功能正常，MTT 检测显示其具有较强的抗氧化能力。在急性肝衰竭大鼠中移植修饰后的肝细胞可显著降低血清丙氨酸氨基转移酶（ALT）和总胆红素（TBIL）水平（P < 0.05）。肝细胞移植后长期存活并有效增殖。术后 14 天，接受 hTrx 基因修饰肝细胞移植的大鼠模型的存活率明显高于未移植的大鼠模型。