大黄虫丸对基因转染慢性粒细胞白血病K562细胞趋化因子受体4表达的影响

Effects of Dahuang Shuchong Wan on the CXC Chemokine Receptor 4 Expression in Chronic Myeloid Leukemia K562 Cell with Gene Transfection

目的探讨大黄虫丸治疗慢性粒细胞白血病的可能作用机理。方法将CXCR4基因定向克隆到含有双启动子的真核表达载体p Bud CE4.1,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。16只裸鼠随机分为空白组和大黄虫丸大、中、小剂量组,每组4只。大黄虫丸大、中、小剂量组分别给予大黄虫丸270、135、70 mg/ml灌胃,每次3ml,空白组给予生理盐水3 ml灌胃,均每天1次,连续给药3天,第3天每隔2h给药1次,连续给药3次,末次给药1 h后,心脏采血,分离血清,制备大黄虫丸含药血清。应用MTT法检测不同浓度大黄虫丸含药血清对K562/CXCR4细胞增殖的影响,TUNEL法检测其对K562/CXCR4细胞凋亡的影响,Western blotting法检测各组细胞CXCR4表达的变化。结果与空白组比较,大黄虫丸大、中、小剂量组细胞在24、48、72、96 h时吸光度A490均明显降低(P<0.01);大黄虫丸大、中、小剂量组作用时间越长、药物浓度越高,细胞的抑制率越高。空白组细胞凋亡率为0.28%,大黄虫丸大、中、小剂量组细胞凋亡率分别为60.79%、28.91%和10.14%。大黄虫丸大、中、小剂量组细胞中CXCR4表达较空白组明显降低,并且随剂量增大CXCR4表达降低。结论大黄虫丸可能通过抑制K562/CXCR4细胞增殖,诱导其凋亡,并影响CXCR4的表达,进而干预慢性粒细胞白血病的发生发展过程。

Objective To explore the underlying possible mechanism of Dahuang Zhechong Wan （DZW 大黄[庶虫]虫丸 DZW） in treating chronic myeloid leukemia （CML）. Methods The CXCR4 gene was directional cloned to eukaryotic expression vector pBudCE4. 1 carrying with double promoters, and transfected into K562 cell. Screen the steady K562/CXCR4 cells with high expression of human CXCR4 protein. 16 nude mice were rar/domly divided to a control group and three DZW groups respectively with a large, medium and small dose, 4 mice in each group. The three DZW groups with a large, medium and small dose were respectively received DZW gavage in 270mg/ml, 135mg/ml and 70mg/ml, 3ml one time, once daily for 3 days. The control group was given normal saline gavage in 3ml, once daily for 3days. On the third day, give gavage once every 3hours for three times. The blood was collected from the heart one hour after the last administration, and the serum was separated for DZW serum preparation. Effects of DZW serum in different concentrations on K562/CXCR4 cells proliferation and apoptosis were respectively determined by MTY and TUNEL methods. Changes in the expression of different group CXCR4 cells were tested by Western blotting method. Results Compared with the control group, the A490 absorbance of three DZW group cells at large, medium and small doses were significantly lower at 24h, 48h, 72h and 96h. The longer the DZW action time with large, medium and small doses, the higher the drug concentration, and the higher was the cell inhibition rate. The cell apoptosis rate was 0. 28% in the control group, and in the large, medium and small doses DZW groups were 60. 79%, 28.91% and 10. 14%. CXCR4 expressions significantly decreased in the large, medium and small doses DZW groups than that in the control group. The increasing the dose, the lower was the CXCR4 expression. Conclusion DZW could intervene in the CML developing process by restraining K562/CXCR4 cells proliferation, inducing their apoptosis and decreasing CXCR4 expression.