白花蛇舌草注射液抑制HL-60细胞增殖及作用机理研究

本研究通过对白花蛇舌草注射液（hedyotic diffusa willd Injection，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。

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

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.