白及有效部位诱导HL60细胞凋亡及其作用机制的研究

Studies on Active Fraction in Rhizoma Bletillae Striatae Inducing HL60Cells Apoptosis and Its Related Mechanisms

目的:研究白及有效部位诱导HL60细胞凋亡及其作用机制。方法:采用乙醇回流提取白及,系统溶剂萃取法萃取后得到白及氯仿层,采用folin-酚比色法测定氯仿层的总酚含量。将白及氯仿层配成50、100、150μg/mL后,作用于HL60细胞,应用Hoechst33342染色和透射电子显微镜观察细胞的形态学变化,应用流式细胞术分析白及氯仿层对细胞凋亡的影响,并用RT-PCR法检测Bcl-2和Bax mRNA的表达。结果:白及氯仿层总酚含量为25.35%,药物对HL60细胞作用24 h后,Hoechst33342染色后荧光显微镜下可见明显的胞核固缩、深染及碎裂等多种形态改变,透射电子显微镜下可见典型的凋亡细胞,流式细胞仪分析结果显示,白及氯仿层可显著诱导HL60细胞凋亡,凋亡率为24.9%,36.0%,51.3%,RT-PCR结果显示,Bcl-2 mRNA的表达随着药物浓度增加而下降,Bax mRNA表达增加。结论:白及能有效诱导HL60细胞凋亡,抑制HL60细胞增殖,其机制可能与Bax基因表达增加及Bcl-2基因表达降低有关。

Objective：To study the active fraction in Rhizoma Bletillae Striatae inducing HL60 cells apoptosis and its related mechanisms. Methods： 95% alcohol as well as the distilled water was used to extract Bletilla striata by backflow method. The system resolver extraction method was applied to divide the 95% alcohol extraction and then the chloroform level was obtained, determining the total phenol content by Folin colorimetry method. The chloroform level was dispensing into 50,100,150 Ixg/mL and then added to the cells for 24 h, and the morphology of the cells was observed by Hoechst33342 stain and transmission electron microscope. The cells apoptosis was analyzed by flow cytometer. The expressions of Bcl - 2 and Bax mRNA were measured by RT - PCR. Results ： The total phenol content in the chloroform level was 25.35%. Hoechst33342 stain showed that after giving drug 24 h later, we could find karyopyknosis, chromatin margination and anachromnsis in the cells, and we also could see cells apoptosis in the transmission electron microscope. The apoptosis rates of the different drug concentrations （50,100,150 μg/mL） were 24.9% ,36.0% ,51.3% respectively. The RT - RCR showed that B. striata can down -regulate the Bcl -2 mRNA expression and up -regulate the Bax expression. Conclusion：Bletillae striata can induce HL60 cells apoptosis and refrain its multiplication, the probably mechanism is regulation of the Bcl - 2 mRNA expression and up - regulation of the Bax expression.