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目的:在证实化学合成2′-氧-甲基鸟嘌呤核苷(P6)具有抑制肿瘤细胞增殖及诱导肿瘤细胞凋亡作用的基础上,观察其对正常免疫细胞的损伤作用。方法:Balb/c小鼠断颈处死后,制备脾脏T淋巴细胞并分为对照组、50μg/mL P6组、100μg/mL P6组及150μg/mL P6组。采用细胞形态学观察、MTT实验、流式细胞术等方法检测P6对细胞的增殖抑制作用;流式细胞术、RT-PCR技术检测P6对细胞的凋亡诱导作用。结果:形态学观察表明,P6作用72 h后,小鼠淋巴细胞的活化程度和密度随P6浓度的增高逐渐降低;MTT检测表明,与空白对照组相比,P6各浓度组均可抑制活化小鼠淋巴细胞的增殖(P<0.05),该抑制作用具有剂量依赖性;流式细胞术检测细胞周期发现,与空白对照组相比,P6各浓度组G1、G2期细胞减少,S、sub-G1期(凋亡峰)细胞增多(P<0.05),呈剂量依赖性。表明P6可抑制淋巴细胞增殖,使其阻滞于细胞周期中的S期,并促进其凋亡。RT-PCR法检测淋巴细胞促凋亡基因Bax和抗凋亡基因Bcl-2的表达发现,P6可促进Bax mRNA的表达,并抑制Bcl-2 mR-NA的表达。结论:化学合成P6除抑制肿瘤细胞增殖并诱导其凋亡以外,同样具有抑制正常免疫细胞增殖及促进凋亡的作用。P6作为新的抗肿瘤药其药效及毒副反应尚需进一步研究。
OBJECTIVE: To observe the effect of 2’-oxo-methylguanosine (P6) on the proliferation of tumor cells and the induction of apoptosis of tumor cells on the basis of the observation of its effect on normal immune cells. Methods: The splenic T lymphocytes were prepared from Balb / c mice and divided into control group, 50μg / mL P6 group, 100μg / mL P6 group and 150μg / mL P6 group. Cell proliferation was measured by MTT assay and flow cytometry. Cell apoptosis was detected by flow cytometry and RT-PCR. Results: Morphological observation showed that the activation and density of mouse lymphocytes decreased gradually with the increase of P6 concentration after 72 hours of P6 treatment. The MTT assay showed that the activation of lymphocytes in P6 group was significantly inhibited compared with the blank control group (P <0.05), and the inhibition was dose-dependent. The cell cycle was detected by flow cytometry. Compared with the blank control group, the cells in G1 and G2 phases of P6 concentration decreased, S, G1 phase (apoptotic peak) cells increased (P <0.05), in a dose-dependent manner. P6 inhibits lymphocyte proliferation, arrests S phase in the cell cycle, and promotes apoptosis. The expression of Bax and anti-apoptotic gene Bcl-2 in lymphocytes were detected by RT-PCR. P6 could promote the expression of Bax mRNA and inhibit the expression of Bcl-2 mR-NA. Conclusion: Chemically synthesized P6 can inhibit the proliferation and induce the apoptosis of tumor cells in vitro. P6 as a new antitumor drug efficacy and toxicity need further study.