化学合成P6对小鼠正常淋巴细胞损伤作用及其机制的观察

Effects of synthetic nucleoside P6 on normal lymphocytes of mice

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 investigate the effects of a syn thetic nucleoside 2＇-O-methylguanosine （P6） on normal immunocytes. METHODS：Balb/c mice were sacrificed and T lymphocytes isolated from spleens were divided into four groups： a control group （0 μg/mL P6 group）,50 μg/mL P6 group,100 μg/mL P6 group and 150 μg/mL P6 group. The proliferation inhibition effects of P6 were detected by means of the microscopical examination,MTT assay and flow cytometery analysis. The apoptosis inducing effects were evaluated by the flow eytometery analysis and RT-PCR analysis. RESULTS：The micoroscopy assay showed that after being treated with P6 for 72 hours, the activation and the cell density of the mouse lymphocytes were reduced accompanying with the P6 dose-increase. The MTT detection showed P6 inhibited the proliferation of the activated mouse lymphocytes in a dose-dependent manner compared with the control group, P〈0.05. The cell cycle detection by flow cytometry showed the G1- and G2-phase cells of the P6 treated groups were reduced,yet the S- and the sub G1 phse cells of the P6-treated groups were increased in a dose-dependent manner compared with the control group （P〈0. 05） ,which indicated P6 could suppress the cell proliferation by arresting them in S-phase and induce the cell apoptosis. Furthermore, P6 could up regulate the Bax gene expression and down-regulate the Bcl-2 gene expression assayed by RT PCR. CONCLUSIONS：The synthetic nueleotide P6 has the proliferation inhibition effects and the apoptosis-inducing effects on both tumor cells and normal immunocytes. For this reason,it is necessary to make more detailed studies on the action and the side effect of P6 before it undergoes clinical trails in the tumor treatment.