内源性干细胞对小鼠出血性脑损伤的修复作用

Repairing effect of endogenous stem cells on mouse brain after intracerebral hemorrhage

目的探究内源性CD133^+干细胞及脑损伤后获得干性的细胞对出血性脑损伤的修复作用。方法建立CD133^+细胞谱系示踪小鼠(CD133-Cre-ERT2;CAG-loxP-STOP-loxP-ZsGreen)。将24只CD133^+细胞示踪小鼠随机分为出血组与假手术组,每组12只,其中出血组再随机分为损伤后1、3、7、14 d组,每组3只。将30 μL自体尾静脉血立体定位注射至小鼠纹状体,在损伤后1、3、7、14 d分别进行神经功能损伤评分。采用免疫荧光法观察并统计损伤后1、3、7、14 d损伤侧及对侧相应位置PDGFRβ^+、GFAP^+细胞数量,以及损伤侧与对侧SVZ区DCX^+与DCX^+/ZsGreen^+细胞数量;采用PDGFRβ和Nestin抗体、Ki67和GFAP抗体组合免疫荧光法观察损伤侧纹状体周细胞干性获得以及新生星形胶质细胞的情况。结果损伤后1、3、7、14 d时损伤侧纹状体星形胶质细胞数量明显多于对照侧相应位置,差异具有统计学意义(P<0.05),且损伤侧纹状体星形胶质细胞数量随时间增加而进一步增加;而损伤后两侧周细胞的数量差异无统计学意义,且数量不随时间的增加而变化;损伤后7 d出血侧SVZ区有10.35%±3.01%DCX^+细胞与ZsGreen信号共定位,出血侧SVZ区的DCX^+细胞及DCX^+/Zsgreen^+细胞在损伤后7 d时数量最多且明显多于对照侧(P<0.05),而损伤后1、3、14 d时DCX^+与DCX^+/ZsGreen^+两种细胞数量较低。损伤灶周有少量GFAP^+/Ki67^+细胞与ZsGreen信号共定位,同时灶周一些PDGFRβ^+细胞与Nestin信号共定位。结论内源性CD133^+细胞在脑出血损伤后能向成神经细胞、星形胶质细胞方向分化,参与神经修复;且出血侧纹状体星形胶质细胞大量增殖,部分血管周细胞获得干性,对神经系统修复产生潜在作用。

Objective To investigate the repairing effect of endogenous CD133^+ stem cells on adult mouse brain after intracerebral hemorrhagic injury.Methods CD133^+ cell lineage-tagged mouse strain (CD133-Cre-ERT2;CAG-loxP-STOP-loxP-ZsGreen) was established.Twenty-four CD133^+ cell lineage tracing mice were randomly divided into hemorrhage group and sham group with 12 for each.Mice in hemorrhage group were randomly equally divided into four subgroups: 1,3,7 and 14 d.30 μL autologous blood was stereotactically injected into the striatum of the mice and neurological severity scores were measured at 1,3,7 and 14 d after surgery.Ki67^+,Ki67^+/GFAP+,DCX^+ and DCX/ZsGreen cells in two sides at different time were detected and counted by the immunofluorescence.The PDGFRβ and Nestin antibodies were used to observe the stemness of pericytes.Results The number of astrocytes in injured side on the 1st,3rd,7th and 14th day after injury was significantly higher than that on the control side (P<0.05),and the number of striatal astrocytes in the injured side increased with time.The number of pericytes on both sides after the injury was not significantly different,and the number did not change with time;at 7th day after injury,10.35%±3.01% DCX^+ cells were co-localized with ZsGreen signal in the SVZ region on the injury side and the number of DCX^+,DCX^+/ZsGreen^+ cells was significantly more than that in the control side(P<0.05).On the 1st,3rd and 14th day after injury,the number of DCX^+ and DCX^+/ZsGreen^+ cells was reduced.Conclusion Endogenous CD133^+ stem cells can differentiate into neuroblasts and astrocytes after cerebral hemorrhagic injury,while the astrocytes proliferate in a large amount,and the pericytes gain stemness,which may have potential effects on neural system repairing.