分泌型rIL-10基因在正常大鼠肝细胞系BRL中的稳定表达

Stable expression of secretary type rIL-10 in a normal murine liver cell line BRL

目的:建立稳定表达分泌型大鼠白细胞介素10(rIL-10)基因的正常大鼠肝细胞系(BRL细胞),为研究IL-10抗纤维化的基因治疗打下基础。方法:通过RT-巢式PCR技术从大鼠外周血单核细胞(PBMC)中扩增出rIL-10全长基因,克隆入pcDNA3真核表达载体构建重组表达载体pcDNA3-rIL-10。鉴定正确后,用受体介导的脂质体转染试剂将pcDNA3-rIL-10转染BRL细胞,高浓度G418筛选,RT-PCR及ELISA法证实rIL-10在BRL细胞中的稳定表达。结果:构建的真核表达载体pcDNA3-rIL-10经质粒双酶切及测序鉴定证实载体构建的正确性。RT-PCR及ELISA法检测出经G418筛选的BRL细胞稳定表达IL-10。结论:成功构建分泌型pcDNA3-rIL-10真核表达质粒及筛选出稳定表达分泌型rIL-10的BRL细胞株,为进一步研究IL-10抗纤维化的基因治疗打下基础。

Objective：To establish a gene-modified BRL cell line which can express rat interleukin 10（rIL-10） stably. Methods： rIL-10 gene was amplified by nest-RT-PCR from PBMCs, and then cloned into the eukaryotic expressing vector pcDNA3. After being confirmed by DNA sequencing, the pcDNA3-rIL-10 was transfected into BRL cells by asialoglycoprotein receptor mediated liposome PEIjet-gal. The expression of rIL-10 was detected by RT-PCR and ELISA. Results： DNA sequencing and restriction endonuclease digestion confirmed that eukaryotic expressing vector pcDNA3-rIL-10 was constructed successfully. After the cells were selected with G418, the stable expression of rIL-10 was showed in BRL cells by RT-PCR and ELISA analysis. Conclusion： The recombinant expression vector for rlL-10 has been constructed successfully and the gene-modified BRL cells are obtained, which can stably express target protein. This study lay a foundation for further studying of IL-10 gene therapy in liver fibrosis.