损伤反应性星形胶质细胞对成年大鼠室下区的再生作用

Effects on regeneration in subventricular zone about injuried reactive astrocyte following brain injury in adult rats

目的①观察液压性脑损伤后不同时期室下区Nestin和GFAP的表达及其细胞类型。②分离脑损伤室下区Nestin+/GFAP+共存细胞进行培养、诱导分化鉴定。方法①用液压冲击法建立SD大鼠动物模型,用免疫组化和免疫荧光双标法分析和显示不同时期(1、3、5 d,1、2、3、4、6、8周)Nestin和GFAP的表达变化以及细胞类型。②分离液压损伤SD大鼠室下区Nestin+/GFAP+共存细胞,制成单细胞悬液,进行培养、传代,以免疫荧光化学方法对原代和传代培养形成的神经球以及诱导分化的细胞进行鉴定。结果①室下区免疫荧光染色:正常组及空白对照组在前脑侧脑室SVZ区出现神经干细胞,主要集中在外侧壁上部和上角,其他部位及三脑室的SVZ区有少量神经干细胞。脑损伤模型组脑损伤24 h后,和正常组相比,室下区Nestin和GFAP的表达显著增加,阳性表达在3 d后逐渐达到高峰,持续1周。②在上述条件下培养及传代的细胞不断分裂增殖,形成悬浮生长的呈Nestin阳性的神经球;神经球贴壁后分化的细胞表现为少突胶质细胞、神经元和星形胶质细胞的形态,且分别呈GalC阳性、β-tublinⅢ阳性和GFAP阳性。结论①成年大鼠液压性脑损伤可诱导室下区表达Nestin和GFAP,Nestin的表达和反应性星形胶质细胞增生呈正相关,且表达多相互共存,并且具有星形胶质细胞的形态;室下区的星形胶质细胞和神经干细胞都对脑损伤都有反应,并可能参与中枢神经系统损伤修复。②成年大鼠液压性脑损伤后分离的室下区细胞具有自我更新能力和多分化潜能,可以分化为神经元、少突胶质细胞和星形胶质细胞,是中枢神经系统的干细胞。

Objective ① Using immunohistochemical method and immunofluorescence, the expressions of Nestin and glial fibrillary acidic protein （GFAP） in subventricular zone of rats with hydraulic injury, as well as its cell types were observed in different period. ② The coexisting cells of Nestin ^＋/GFAP ^＋ were cultured and induced to differentiate. Methods ① Animal madel was established by moderate lateral fluid percussion. Expression and cell types of Nestin and GFAP in different period （1 d, 3 d and 5 d; 1 w, 2 w, 3 w, 4 w, 6 w and 8 w） were measured by immunohistochemical method and immunofluorescence. ② Coexisting cells of Nestin^＋ /GFAP^＋ were separated from subventricular zone of SD rats to make single cell suspension. And then, the cells were cultured and subcultured. Next, primary and subculturing neuronosphere as well as primary and subculturing differentiated cells were measured with immunofluorescence. Results ; Immunofluorescence staining results： For normal controls, small nerve stem cells of round or fusiform shape appeared in SVZ of prosencephalon lateral ventricle, mainly in upper part of lateral wall and corn. Few nerve stem cells were located in SVZ of lateral ventricle and 3^rd brain ventricle. Brain injury： 24 hours after brain injury, expression of Nestin and GFAP increased obviously and reached the peak value 3 days later,and lasted for 1 week. ②Subcultured cells proliferated and positive-Nestin neuronosphere formed. Differentiated cells were oligodendrocytes （positive-galactocerebroside）, neurons （positive- fl-tublinIII） and orastrocytes （positive- GFAP）. Conclusion ① Hydraulic injury can induce the expression of Nestin and GFAP in brain ventricle sub-area of rats. The expression of Nestin has a positive correlation with reactive astrocyte regeneration and coexists with the form of astrocytes. Both astrocytes and nerve stem cells in brain subventricular zone can be responsive to injury and possibly take part in repair of CNS. ② Cells separated from brain subventricular zone have a great ability in differentiation and self-renewing. These cells can differentiate into neurons, oligodendrocytes or astrocytes. They are stem cells of CNS. So, injury reactive astrocyte can repair CNS through regenerating the subventricular zone in adult rats.