小鼠睫状神经营养因子基因在视网膜色素上皮细胞系ARPE-19细胞中表达的研究

Cloning and eukaryotic expression of wild type and truncated mouse ciliary neurotrophic factor gene

目的探讨野生型和截短型小鼠睫状神经营养因子(CNTF)基因的真核表达及其在视网膜色素上皮细胞系ARPE-19细胞中目的蛋白的表达及意义。方法通过逆转录聚和酶链反应(RT-PCR)扩增小鼠CNTF野生型全长编码序列,体外定点突变获取截短型CNTF的互补DNA(cDNA)编码序列,构建上述两序列分别克隆至pTracer-CMV真核表达载体,转染ARPE-19细胞,免疫印迹检测两种CNTF的表达。通过噻唑蓝(MTT)和流式细胞仪定量凋亡检测观察两种CNTF基因在去血清培养的ARPE-19细胞中表达后产生的生物学效应。结果PCR成功扩增了野生型和截短型CNTF基因,DNA序列分析证实两种真核表达载体中的CNTF序列与GeneBank中目的序列一致。两种重组真核表达质粒转染ARPE-19细胞后,免疫印迹检测结果证实CNTF在ARPE-19细胞培养上清中有表达。MTT检测结果显示CNTF不能促进ARPE-19细胞的增殖,流式细胞仪凋亡分析提示CNTF在一定程度上可以抑制去血清培养引起的ARPE-19细胞凋亡。结论野生型和截短型CNTF基因在ARPE-19细胞系的真核表达为视网膜色素变性基因治疗研究奠定了基础。(中华眼科杂志,2006,421017-1022)

Objective To clone and eukaryotic express wild type and truncated mouse ciliary neurotruphic factor （CNTF） gene, and to observe the biological effect of two types of CNTF gene expressing in ARPE-19 cells. Methods RT-PCR was used to amplify the cDNA of CNTF gene, and truncated CNTF cDNA was obtained by site-directed mutagenesis. The two types of CNTF gene were cloned into plasmid pTracer-CMV and transfected to ARPE-19 cells. Dot blotting was used to detect the expression of CNTF. MTr and flow cytometry apoptosis assay were performed to observe the biological effect of CNTF expressing in ARPE-19 cells. Results Wild type and truncated CNTF gene were amplified by RT-PCR, and their eukaryotic expression plasmids were successfully constructed. After ARPE-19 cells transfected with two types of recombinant plasmids, the CNTF were detected in the supernatant of cells culture. MTr result shows that two types of CNTF have no proliferation promoting effect to ARPE-19 cells, and quantitive apoptosis assay implicated that CNTF could partially suppress the apoptosis that induced by the cells culturing with serum free culture. Conclusion Expression of two types of CNTF in ARPE-19 cells gets prepared for gene therapy research of retinitis pigmentosa.