CAJ | 학술논문

目的体外培养脑白质胶质源性神经祖细胞并制备神经祖细胞的缺血模型,以期进一步研究脑白质缺血性损伤时脑白质的再生机制. 方法分离3日龄内SD新生大鼠的双侧脑室周围白质组织,原代培养脑白质神经祖细胞,每3～4天传代一次,并对神经祖细胞进行诱导分化培养.免疫细胞化学法分别鉴定原代和传代培养的神经球标志物神经胶质抗原2(NG2)和诱导分化后少突胶质细胞前体标志物O4的表达.应用自行研制的细胞缺氧箱,对传至第3～4代的神经祖细胞制备氧糖剥夺缺血性模型.建模后24 h应用Hoechst 33342/PI双染和CCK-8法分别评估氧糖剥夺30 min、45 min、60 min、2h及3h神经祖细胞存活率的变化. 结果脑白质神经祖细胞具有形成神经球和连续传代的能力,原代和传代培养的神经球均显示NG2阳性,并可被诱导分化为O4阳性的少突胶质细胞前体.正常培养的神经祖细胞无凋亡和坏死细胞,随着氧糖剥夺时间的逐步延长,凋亡细胞逐渐增多并出现坏死细胞.不同氧糖剥夺时间组细胞存活率均不同,而且随着氧糖剥夺时间的延长,细胞存活率下降,差异有统计学意义(P＜0.05). 结论成功培养了脑白质胶质源性神经祖细胞并建立了有效可靠的祖细胞氧糖剥夺模型,为进一步研究脑白质缺血性损伤时脑白质的神经再生机制奠定了基础.
목적 체외배양뇌백질효질원성신경조세포병제비신경조세포적결혈모형,이기진일보연구뇌백질결혈성손상시뇌백질적재생궤제. 방법 분리3일령내SD신생대서적쌍측뇌실주위백질조직,원대배양뇌백질신경조세포,매3～4천전대일차,병대신경조세포진행유도분화배양.면역세포화학법분별감정원대화전대배양적신경구표지물신경효질항원2(NG2)화유도분화후소돌효질세포전체표지물O4적표체.응용자행연제적세포결양상,대전지제3～4대적신경조세포제비양당박탈결혈성모형.건모후24 h응용Hoechst 33342/PI쌍염화CCK-8법분별평고양당박탈30 min、45 min、60 min、2h급3h신경조세포존활솔적변화. 결과 뇌백질신경조세포구유형성신경구화련속전대적능력,원대화전대배양적신경구균현시NG2양성,병가피유도분화위O4양성적소돌효질세포전체.정상배양적신경조세포무조망화배사세포,수착양당박탈시간적축보연장,조망세포축점증다병출현배사세포.불동양당박탈시간조세포존활솔균불동,이차수착양당박탈시간적연장,세포존활솔하강,차이유통계학의의(P＜0.05). 결론 성공배양료뇌백질효질원성신경조세포병건립료유효가고적조세포양당박탈모형,위진일보연구뇌백질결혈성손상시뇌백질적신경재생궤제전정료기출.
Objective To in vitro culture glial lineage progenitor cells derived from periventricular white matter in neonatal rats,and establish oxygen-glucose deprivation models of progenitor cells to further explore the mechanism of neurogenesis in the ischemic white matter injury.Methods The progenitor cells were isolated and cultured from the white matter of 3-d-old neonatal rats.Cells were passaged every 3 or 4 d,and were further induced differentiation.The primary and passaged neurospheres and their differentiated cells were respectively identified by immunocytochemical stanning of NG2 (Neurospheres marker) and O4 (oligodendrocyte precursor marker).The third-or fourth-generation progenitor cells were used to establish the ischemic model of oxygen-glucose deprivation (OGD).The influence of different OGD periods (30,45 and 60 min,2 and 3 h) in the survival rates of progenitor cells was assessed 24 h after success of model making by both CCK-8 and Hoechst 33342/PI stainning.Results The progenitor cells obtained from the white matter had the capacity of forming neurospheres and reproduction which showed NG2 immunoreactive positivity,and could be induced differentiation into O4-positive oligodendrocyte precursors.No apoptotic or necrotic cells were observed in normal cultured NG2-positive progenitor cells.The apoptotic and necrotic cells tended to increase significantly with the extension of OGD period gradually.The survival rates of progenitor cells in turn were (85.94±3.06) ％ for 30 min OGD,(62.68±2.66) ％ for 45 min OGD,(45.09±2.24) ％ for 60 min OGD,(36.70±2.84) ％ for 2 h OGD and (22.01±3.00) ％ for 3 h OGD,respectively,with significant differences between each two groups (P＜0.05).Conclusion The oxygen-glucose deprivation models of glial lineage progenitor cells derived from periventricular white matter of neonatal rats are successfully established,which may provide the foundation for further exploring the mechanism of neurogenesis in the ischemic white matter injury.