目的:从人骨髓造血前体细胞体外培养扩增树突状细胞(dendritic cells,DCs),测定其表型及T细胞刺激活性.方法:采用Mini-MACS分离技术,从正常人骨髓、脐血分离CD34~ 造血干细胞,体外以重组hGM-CSF,hTNF-α,hIL-3诱导培养2周,流式细胞术检测扩增细胞的表面表型及细胞内IL-12的表达,体外同种混合淋巴细胞反应检测扩增DCs的T细胞刺激活性.结果:从正常人骨髓、脐血分离得到高纯度(>90%)的CD34~ 造血干细胞,经重组hGM-CSF,hTNF-α的共同诱导培养,扩增得到大量DCs,加人hIL-3可以进一步增加DCs产量;FACS检测表明,扩增的DCs表达HLA-DR,CD40,CD54,CD80,CD86分子,细胞内有hIL-12的P35,P40亚基的表达;与外周血单核细胞培养生成的DCs相比,由CD34~ 干细胞扩增的DCs具有更强的激发同种T细胞增殖的能力.结论:人CD34~ 干细胞体外经诱导培养,可以生成大量功能成熟的DCs,从而为进一步开展DCs的基础及临床研究打下了基础.

Objective: To amplify the tk gene of HSV- I strain Stocker and clone into a eukaryotic expressing plasmid. A high effective expressing vector containing HSV-I tk gene would be constructed. Methods: PCR amplification was performed using primers based on tk gene sequence of HSV-I strain CL101, nucleotide of strain Stocker as template. PCR product was cloned into plasmid pUCl 19 and the sequence of tk gene was analyzed. The tk gene was then cloned into a high effective eukaryotic expressing plasmid which contains CMV immediate early gene promoter. The recombinant pCR3-tk was further identified by restriction digest. Results: A 1427 bp DNA fragment was amplified. Sequence analysis and restriction digest demonstrated that the fragment cloned contained complete HSV-I tk and indicated a 98.5% identity between the cloned tk gene and that from HSV-I CL101. pCR3-tk vector was identified by restrition digest. Conclusion: The identity between the cloned tk gene and that from HSV-I CL101 is very high. The recombinant of pCR3-tk is a high effective expressing eukaryotic vector. It will play a fundamental role in further study of tumor gene therapy with HSV- I tk/GCV system expecially mediated by non-viral vectors such as cationic liposomes.

R730.5

国家863重大项目(Z20-01-03)资助