CXCR4基因表达增强K562细胞的趋化作用

CXCR4 gene expression promotes K562 cells chemotaxis

目的构建高表达人CXCR4蛋白的白血病细胞系并测定CXCR4基因对其侵袭转移能力的影响。方法从外周血淋巴细胞中提取基因组RNA,用RT-PCR扩展编码CXCR4基因片段,将CXCR4基因定向克隆到含有双启动子的真核表达载体PBudCE4.1,采用酶切和序列测定方法鉴定。将所构建的重组质粒用脂质体2000转染K562细胞,Zeocin筛选阳性克隆。流式细胞术和RT-PCR对阳性克隆进行鉴定;用Transwell板检测转染与未转染CXCR4的K562细胞对趋化因子SDF-1趋化活性。结果成功构建编码CXCR4基因的真核表达质粒PBudCE4.1/CXCR4,经转染入K562细胞,筛选获得能稳定高表达人CXCR4蛋白的K562/CXCR4细胞;K562/CXCR4细胞对SDF-1的趋化能力较K562细胞明显增强。结论成功构建了转染人CXCR4基因的白血病细胞系,为进一步研究白血病细胞髓外浸润的分子机制奠定基础。

Objective To construct the tranfected cell expressing the human CXCR4 gene and to identify the effect on its immigration. Methods Total RNA was isolated from peripheral blood monouclear cell （PBMC）, the fulllength CXCR4 gene was amplified by RT-PCR and was inserted into plasmids PBudCF4. 1 which have two promtors, after the identification by digestion and sequencing, the recombinant was transfected into K562 cell by lipofectamineTM2000. After screening culture by zeocin, stable transfected K562 cell line was established, and transcription and exression of CXCR4 were checked by flow cytometry ; the chemotactic activity of K562 cell transfected and untrandfected CXCR4 was analyzed by Transell plate. Results The eukaryotic expression plasmid PBudCF4. 1/CXCR4 was constructed successfully. The stable trasfected K562/CXCR4 cell lines which highly express CXCR4 was established, the chemotactic activity of K562/CXCR4 was increased significiantly than K562. Conclusion CXCR4 transfected K562 cell line was successfully established, and it can make the basis for the further research on mechanism of extramedullary infiltration in leukemia.