夜香树多糖诱导人骨肉瘤细胞MG-63凋亡作用的探讨

目的:探讨夜香树多糖对人骨肉瘤细胞MG-63凋亡诱导作用并研究其可能的作用机制。方法:从夜香树叶中提取夜香树多糖,分别用终质量浓度为2.5,5,10 mg·L^-1的夜香树多糖处理处于对数生长期的MG-63细胞,另设MG-63细胞为空白组;不同质量浓度夜香树多糖作用72 h后,采用MTT法检测MG-63细胞增殖抑制作用;不同质量浓度夜香树多糖作用46 h后,采用透射电镜观察经处理过的细胞超微结构的改变,采用流式细胞术检测夜香树多糖对MG-63细胞凋亡率的影响,Western blot法分析半胱氨酸天冬氨酸蛋白酶3(Caspase-3),Caspase-8,Caspase-9和B细胞淋巴瘤-白血病2(Bcl-2)的蛋白表达水平。结果:夜香树多糖显著抑制人骨肉瘤细胞MG-63增生,在5~10 mg·L^-1质量浓度显著抑制细胞增殖,并呈浓度和时间的依赖性;电镜下可发现夜香树多糖能诱导细胞凋亡,胞质内出现大量空泡,细胞核染色质固缩,细胞表面绒毛减少;流式细胞仪检测表明,5 mg·L^-1夜香树多糖可使MG-63细胞阻滞在G₀/G₁;夜香树多糖能剂量依赖诱导MG-63凋亡,浓度依赖性的降低MG-63细胞的Bcl-2蛋白表达和升高Caspase-8的蛋白表达(P<0.05,P<0.01)。结论:夜香树多糖能抑制MG-63细胞增殖,诱导人骨肉瘤细胞MG-63凋亡,与作用剂量和作用时间呈正相关,这一过程可能与夜香树多糖下调Bcl-2和激活Caspase-8有关。

Experiment on Apoptosis of Human Osteosarcoma Cell Line MG-63 Induced by Polysaccharide Extracted from Cestrum nocturnum

Objective: To investigate the effect of polysaccharide extract obtained from the Cestrum nocturnum(PCN)on the apoptosis of human osteosarcoma cell line MG-63 as well as it possible mechanism. Method: PCN was separated, and MG-63 cells in logarithmic phase were processed using PCN with the final concentration of 2.5, 5, 10 mg·L^-1. Blank group was established with MG-63 cells. After 72 h of PCN (different mass concentration) action, MTT assay was used to determine the inhibition of cell proliferation. After 46 h of PCN (different mass concentration) action, transmission electron microscope was used to observe the morphological changes of processed cells. Its effect on apoptosis of MG-63 cells was detected by flow cytometry, and the protein expression of Caspase-3, Caspase-8, Caspase-9 and Bcl-2 were detected by Western blot. Result: PCN significantly inhibited the proliferation of MG-63 cells in a time and dose-dependent manner. It could significantly inhibit cells proliferation within the concentration range of 5-10 mg·L^-1. Under electron microscopy, it could be found that PCN can induce cell apoptosis, with a large number of vacuoles in cytoplasm, and chromatin pyknosis was present for cell nucleus, and villus on cell nucleus surface was reduced. Flow cytometry indicated that 5 mg·L^-1 can make MG-63 cells blocked in G₀/G₁. PCN induced apoptosis of MG-63 cells in a dose dependent manner, reduced Bcl-2 expression and increased Caspase-8 expression in a concentration dependent manner (P<0.05, P<0.01). Conclusion: PCN could effectively inhibit cell proliferation and induce MG-63 cells apoptosis, with a direct relation to the dose and time. This process may be associated with down-regulating protein expressions of Bcl-2 and activating the expressions of Caspases-8.