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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 investigate the eukaryotic expression of ciliary neurotrophic factor (CNTF) gene in wild-type and truncated mice and its expression in human retinal pigment epithelial cell line ARPE-19. Methods The full length coding sequence of mouse CNTF was amplified by reverse transcription polymerase chain reaction (RT-PCR), and the complementary DNA (cDNA) coding sequence of truncated CNTF was obtained by site-directed mutagenesis in vitro. The two sequences were cloned into The pTracer-CMV eukaryotic expression vector was transfected into ARPE-19 cells to detect the expression of two kinds of CNTF. The biological effects of two kinds of CNTF gene expressed in serum-deprived ARPE-19 cells were observed by quantitative MTT assay and flow cytometry. Results The wild-type and truncated CNTF genes were successfully amplified by PCR. DNA sequence analysis confirmed that the CNTF sequences in the two eukaryotic expression vectors were consistent with the target sequences in GeneBank. After transfection of ARPE-19 cells with the two recombinant eukaryotic expression plasmids, the results of immunoblotting confirmed that CNTF was expressed in the culture supernatant of ARPE-19 cells. The results of MTT showed that CNTF could not promote the proliferation of ARPE-19 cells. Flow cytometry analysis indicated that CNTF could inhibit ARPE-19 cell apoptosis induced by serum-depleted culture to a certain extent. Conclusion The eukaryotic expression of wild-type and truncated CNTF genes in ARPE-19 cell line lays the foundation for gene therapy of retinitis pigmentosa. (Chinese Journal of Ophthalmology, 2006,421017-1022)