【摘要】 目的:研究甜菜碱促进小鼠脾淋巴细胞的增殖作用与钙通道的关系。方法:采用清洁级BALB/c小鼠分离小鼠脾淋巴细胞后体外培养的方式获得小鼠脾淋巴细胞,分为阴性对照组、ConA组、甜菜碱0.04,0.4,4,20mmol.L-1组。分别采用MTT法观察甜菜碱对小鼠脾淋巴细胞增殖的作用,流式细胞术测定甜菜碱对小鼠脾淋巴细胞周期的变化,激光共聚焦扫描显微镜观察甜菜碱对小鼠脾淋巴细胞内钙浓度的变化及加入不同钙通道阻滞剂后细胞内钙浓度的变化。结果:4,20mmol.L-1甜菜碱体外作用于小鼠脾淋巴细胞12h促进小鼠脾淋巴细胞增殖,0.04,0.4,4,20mmol.L-1甜菜碱分别体外作用于小鼠脾淋巴细胞24,48h均促进小鼠脾淋巴细胞增殖,以4mmol.L-1甜菜碱作用24h效果最好;4mmol.L-1甜菜碱作用小鼠脾淋巴细胞4,6,18,24h能促使小鼠脾淋巴细胞由G0/G1期进入S期,并以18h效果最为明显;4mmol.L-1甜菜碱作用于淋巴细胞6,12,18h,脾淋巴细胞内Ca2+浓度明显升高(P<0.01),以6h效果最明显;钙通道阻滞剂硝苯地平、地尔硫卓、咪贝地尔、金雀异黄素对甜菜碱升高小鼠脾淋巴细胞内钙离子浓度没有影响,而维拉帕米、新霉素、肝素、普鲁卡因能阻断甜菜碱升高小鼠脾淋巴细胞内钙离子浓度。结论:甜菜碱通过升高小鼠脾淋巴细胞内钙离子浓度而促进小鼠脾淋巴细胞由G0/G1期进入S期,促小鼠脾淋巴细胞增殖的作用。细胞内钙离子浓度升高主要通过2个途径:影响G蛋白介导的L-型电压门控钙通道的α亚单位而引起外钙内流;影响胞内钙库的IPR钙通道和RyR钙通道而引起内钙释放。更多还原

【Abstract】 Objective:To study how the way in which betaine promotes the proliferation of mouse spleen lymphocytes is related to calcium channels.Method:BALB/c mice were used for this experiment.Mouse spleen lymphocytes were obtained through in vitro cultivation after they had been separated,and were divided into a negative control group,a ConA group,and 0.04,0.4,4,and 20 mmol·L-1 betaine groups.MTT was used to observe the effect of betaine on the proliferation of mouse spleen lymphocytes;flow cytometry was used to measure the changes in the cell cycle of mouse spleen lymphocytes;and laser confocal scanning microscopy was used to observe the changes in the intracellular [Ca2+]i of mouse spleen lymphocytes after betaine or different calcium channel blockers were applied.Result:Betaine was found to promote the proliferation of mouse spleen lymphocytes 12 h after it had been applied in vitro in concentrations of 4 and 20 mmol·L-1.It was also found to promote the proliferation of mouse spleen lymphocytes 24 h and 48 h after it had been applied in vitro in concentrations of 0.04,0.4,4,and 20 mmol·L-1,with the effect being most marked for the 4 mmol·L-1 group 24 h after its application.It was found to facilitate the entry of mouse spleen lymphocytes from the G0/G1 to the S phase 4,6,18,and 24 h after it had been applied to mouse spleen lymphocytes in a concentration of 4 mmol·L-1,with the effect being most marked at 18 h after its application.Intracellular [Ca2+]i in mouse spleen lymphocytes increased significantly(P<0.01) 6,12,18 h after 4 mmol·L-1 betaine had acted on the lymphocytes,with the effect being most marked at 6 h.The calcium channel blockers nifidipine,diltiazem,mibefradil,and genistein had no effect on the increase of the intracellular [Ca2+]i in mouse spleen lymphocytes due to the application of betaine,while verapamil,mycifradin,heparin,and procaine could block such increase.Conclusion:Betaine facilitates the entry of mouse spleen lymphocytes from the G0/G1 into the S phase by raising the intracellular [Ca2+]i in these cells,thus promoting their proliferation.Intracellular [Ca2+]i increases mainly in two ways:①By affecting the α1 subunit of the L-type voltage-gated calcium channel with mediation by G proteins and thus leading to an efflux of intracellular calcium;②By affecting the IP3R and RyR calcium channels of the intracellular calcium stores and thus leading to the release of intracellular calcium.更多还原