摘要
【目的】制备出一种人硫氧还蛋白(hTrx)基因修饰的肝细胞。【方法】逆转录聚合酶链反应法扩增出hTrxcDNA,应用重组逆转录病毒制备hTrx基因修饰大鼠肝细胞。白蛋白免疫组化SABC法进行肝细胞活性鉴定,MTT比色试验进行抗氧化应激能力检测。【结果】克隆出hTrx开放阅读框cDNA并制备出重组逆转录病毒,感染真核细胞后可分泌融合表达的hTrx并具有生物学活性。制备出hTrx基因修饰大鼠肝细胞,免疫组化SABC法显示肝细胞功能正常,MTT比色法检测具有较强的抗氧化应激能力(P<0.05),并可有效增殖。【结论】成功制备出一种具有较强抗氧化应激的hTrx基因修饰肝细胞。
【Objective】 To prepare the human thioredoxin （hTrx） gene-modified hepatocytes. 【Methods】 hTrx cDNA was amplified by reverse transcription-polymerase chain reaction （RT-PCR）,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 with albumin-immunohistochemical staining and MTT assay,respectively. 【Results】 The hTrx open reading frames cDNA was cloned and assembled with recombinant retrovirus,then eukaryotic cells infected by this virus 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 and could be proliferated effectively. 【Conclusion】 The hTrx gene-modified hepatocyte with more stronger antioxidative capacity is prepared.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2010年第4期572-576,共5页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家重点基础研究发展计划(2009CB522404)
国家自然科学基金(30772135)
关键词
肝细胞
人硫氧还蛋白
基因修饰
hepatocyte
human thioredoxin
gene modification
