摘要
目的:构建表达产甲酸草酸杆菌(Ox.F)草酸分解基因的人肝细胞系,探索高草酸尿的治疗方法。方法:分离培养人肠道Ox.F并克隆其功能基因oxc和frc,构建同时表达oxc和frc的真核双表达载体质粒pIRES-oxc-frc,将pIRES-oxc-frc转染人正常肝细胞系L-02。用RT-PCR和Western blot技术了解转基因细胞的目的基因表达状况;用离子色谱法测定转基因后细胞培养液中草酸浓度,了解其草酸分解功能。结果:转基因后人肝细胞系L-02在mRNA和蛋白质水平均成功表达oxc和frc基因,其草酸分解功能较转染前明显增强(P<0.01)。结论:Ox.F分解草酸的两个重要功能基因oxc和frc能在体外转入人正常肝细胞(L-02)中,并使后者草酸分解能力明显增强。转草酸分解基因的人肝细胞系的构建,有望用于高草酸尿,尤其是PH的病因治疗,值得进一步研究。
Objective： To construct human hepatic cell line with oxalate-degradation genes from Oxalobacter formigenes, and explore the therapeutic method of hyperoxaluria. Methods：The Oxalobacter formigenes （Ox. F） from the human feces were isolated and cultured. The functional genes of the Ox. F-frc gene and oxc gene-were cloned. The dicistronic eukaryotic expression vector pIRES-oxc-frc was constructed. The normal human hepatic cell line（L-02 cells） were transfected with pIRES-oxc-frc. RT-PCR and Western blot were performed to detect the expression of the objective genes. The concentration of the oxalate in the culture medium which was used to culture the transgenic L-02 cells was determined by ion chromatography to explore the oxalate-degradation function of the transgenic L-02 cells. Results： After the gene transference, the oxc gene and frc gene could be expressed at mRNA and protein level in human hepatic cells （L-02 cells） successfully and the oxalate-degradation function of the cells increased（ P 〈0.01）. Conclusions：The two important functional genes of the Ox. F-frc gene and oxc gene-could be transfected into normal human hepatic cells（L-02 cells） in vitro successfully, and it increased the oxalate-degradation function of the latter. The successful construction of human hepatic cell line with oxalate-degradation genes might be used in the etilogical therapy of hyperoxaluria, especially primary hyperoxaluria , and is worth to be further studied.
出处
《临床泌尿外科杂志》
2007年第2期143-145,共3页
Journal of Clinical Urology
基金
国家自然科学基金资助课题(No.30371423)
关键词
高草酸尿
基因治疗
产甲酸草酸杆菌
肝细胞
Hyperoxaluria
Genetic therapy
Oxalobacter formigenes
Hepatic cell
