骨髓造血干祖细胞改善慢性约束应激诱导的小鼠脾细胞数减少

Bone marrow hematopoietic stem-progenitor cells improve chronic restraint stress-induced spleen cells depletion in mice

目的观察CD34+骨髓造血干祖细胞(CD34+HSPC)对慢性约束应激小鼠脾细胞数的影响。方法将10只8周龄雄性BALB/c小鼠分为2组(n=5):①慢性应激组:小鼠放入约束应激管中约束12 h/d(早9:00-晚9:00),解除约束12 h/d(晚9:00-早9:00),连续处理3 d以建立慢性约束应激的小鼠模型;②对照组:正常饲养不施加任何处理。慢性应激第3天结束时即取每组小鼠脾并计数脾细胞。HSPC体内干预实验中,20只雄性BALB/c小鼠分为4组(n=5):HSPC干预+慢性应激组,溶剂对照+慢性应激组,HSPC干预组和溶剂对照组。HSPC干预+慢性应激组和溶剂对照+慢性应激组慢性应激第3天结束时即取每组小鼠脾并计数脾细胞,流式细胞术检测其中活细胞、凋亡细胞及坏死细胞比例。绿色荧光标记HSPC做体内示踪试验,检测迁移至脾的HSPC。HSPC体外干预实验中,细胞培养分为4组:HSPC干预+地塞米松组,脾细胞+地塞米松组,HSPC干预组和脾细胞单独培养组,培养18 h,流式细胞术检测其中活细胞、凋亡细胞及坏死细胞的比例。结果脾细胞计数结果表明,与对照组相比,慢性应激组小鼠的脾细胞总数显著减少(P<0.05)。HSPC体内干预实验中,与溶剂对照+慢性应激组相比,HSPC干预+慢性应激组小鼠脾细胞计数增加(P<0.05)。流式细胞术结果表明,与溶剂对照组相比,溶剂对照+慢性应激组小鼠脾细胞凋亡及坏死比例增加(P<0.05);与溶剂对照+慢性应激组相比,HSPC干预+慢性应激组小鼠脾细胞凋亡比例减少,活细胞比例增加(P<0.05)。体内示踪试验结果显示,慢性应激过程中HSPC可迁移至脾中。体外实验结果表明,与脾细胞单独培养组相比,脾细胞+地塞米松组脾细胞凋亡及坏死比例增加(P<0.05);与脾细胞+地塞米松组相比,HSPC干预+地塞米松组脾细胞坏死比例减少,活细胞比例增加(P<0.05)。结论CD34+HSPC可改善慢性约束应激诱导的小鼠脾细胞数的减少。

Objective To investigate the effect of CD34+bone marrow hematopoietic stem-progenitor cells(CD34+HSPCs)on the number of splenic cells in mice suffering from chronic constraint stress.Methods Ten 8-week-old male BALB/c mice were divided into two groups(n=5 in each group):chronic stress group and control group.In chronic stress group,the mice were placed into the constraint stress tube for 12 h/d(9:00a.m.-9:00p.m.)and released for 12 h/d(9:00p.m.-9:00a.m.)for continuous 3 d to establish a mouse model of chronic constraint stress.In control group,the mice were fed normally without any treatment.At the third day of chronic stress,the spleens of mice were obtained to detect the number of spleen cells.In the in-vivo HSPC intervention experiment,20 male BALB/c mice were divided into 4 groups(n=5 in each group):HSPC intervention+chronic stress group,solvent control+chronic stress group,HSPC intervention group and solvent control group.At the third day of chronic stress,the spleens of mice were obtained to detect the number of spleen cells,and the percentages of living,apoptotic and necrotic cells were determined using flow cytometry.HSPCs were labeled with green fluorescence for in-vivo tracing experiment to detect the homing of HSPCs to the spleen.In the in-vitro HSPC intervention experiment,the cell cultures were divided into 4 groups:HSPC intervention+dexamethasone group,spleen cells+dexamethasone group,HSPC intervention group and blank control group.After the cells were cultured for 18 h,the percentages of living,apoptotic and necrotic cells were detected by flow cytometry.Results The results of cell counts showed that compared with control group,the total number of spleen cells in mice was significantly reduced in chronic stress group(P<0.05).In the in-vivo HSPC intervention experiment,compared with solvent control+chronic stress group,the number of spleen cells of mice in HSPC intervention+chronic stress group was increased(P<0.05).Flow cytometry results showed that compared with solvent control group,the apoptosis rate and necrosis rate of spleen cells of mice in solvent control+chronic stress group were increased(P<0.05).Compared with solvent control+chronic stress group,the apoptosis rate of spleen cells of mice in HSPC intervention+chronic stress group was decreased,while the percentage of living cells was increased(P<0.05).The results of in-vivo tracing experiment displayed that HSPCs homed to the spleen during chronic constraint stress.The results of in-vitro experiments showed that compared with blank control group,the apoptosis rate and the necrosis rate were increased in spleen cells+dexamethasone group(P<0.05).Compared with spleen cells+dexamethasone group,the necrosis rate of spleen cells in HSPC intervention+dexamethasone group was decreased,while the percentage of living cells was increased(P<0.05).Conclusion CD34+HSPCs can improve the chronic restraint stress-induced spleen cells depletion in mice.