白花蛇舌草注射液抑制HL-60细胞增殖及作用机理研究

Inhibition Effect of Hedyotic Diffusa Willd Injection on HL-60 Cells and Its Mechanism

本研究通过对白花蛇舌草注射液(hedyotic diffusa willdInjection,HDI)在体外抑制人白血病细胞株(HL-60)增殖作用的观察,以探讨HDI用于治疗白血病患者的作用机制。以白血病HL-60细胞株为靶细胞,采用MTT法观察不同浓度的HDI对HL-60细胞增殖的影响;用流式细胞术和DNA Ladder观察细胞凋亡、以及粒系细胞分化的表面标志(CD33、CD15)表达;RT-PCR检测HDI对survivin、bcl-2基因表达的影响。结果表明:低浓度的HDI(1.56ml/L)对HL-60细胞增殖无明显抑制作用(p>0.05),但当HDI浓度提高至3.12-12.5ml/L时,细胞则出现明显的增殖抑制,且随浓度增加抑制作用增强(p<0.05或p<0.01)。Annexin-V/PI双参数和DNA Ladder检测发现,HL-60细胞经不同浓度HDI诱导24、48、72小时后均未出现典型的凋亡现象,而粒系细胞表面标志CD15的表达,经不同浓度HDI诱导1周后均显著增高,CD33表达无显著变化。HL-60经HDI(6.25ml/L)处理2周后,survivin、bcl-2基因表达无明显变化;当处理3周后,survivin、bcl-2基因表达均明显低于未经处理过的细胞,分别低于60%和44%。结论:白花蛇舌草注射液在一定浓度下能够直接抑制白血病细胞增殖,其作用机制可能是通过诱导细胞分化、凋亡。

This study was aimed to explore the inhibition effect and mechanism of hedyotic diffusa willd injection （HDI） on leukemia cell line （HL-60） in vitro. The leukemia cell line HL-60 was used as target cells. The inhibitory effects of HDI on proliferation of HL-60 cells were observed by MTT assay. The positive rate of cell apoptosis and the surface marker of granulocytic differentiation （ CD33 and CD15 ） were measured by flow cytometry. The expressions of anti-apoptosis related gene （ survivin and bcl-2 ） were detected by RT-PCR. The results showed that the growth of HL-60 cells was inhibited by higher concentration of HDI （3. 12- 12.5 ml/L） and inhibited obviously in dose-dependent manner （p 〈0.05 or p 〈0.01 ）, but not suppressed by low concentration of HDI （ 1.56 ml/L） in liquid culture system （p 〉 0.05 ）. The FCM and DNA Ladder results showed that the phenomenon of typical apoptosis did not detected after HL-60 cells were treated with the different concentrations of HDI for 24, 48 and 72 hours respectively. After HL-60 cells were treated with HDI （ 1.56, 3.12, 6.25 and 12.5 ml/L） for one week, the expression level of CD15 surface marker was all enhanced obviously. When treated with HDI （6.25 ml/L） for 3 weeks, the expression levels of survivin and bcl-2 gene were also decreased obviously by 60% and 44% respectively. It is concluded that HDI can inhibit HL-60 cells in the presence of its higher concentrations. The mechanisms of HDI may induce HL-60 cells differentiation, and surppress the expression of anti-apoptosis related gene （ survivin or bcl-2） to inhibit the growth of HL-60 cells.