成年小鼠触液神经元体外分离培养及自我更新能力的鉴定  

Isolation and culture of adult mouse cerebrospinal fluid-contacting neurons in vitro and characterization of self-renewal capacity

作　　者：上官泽宇 陈婵娟 李琦哲 谭伟 颜海健 王春庆 豆晓伟 李青 Shangguan Zeyu;Chen Chanjuan;Li Qizhe;Tan Wei;Yan Haijian;Wang Chunqing;Dou Xiaowei;Li Qing(Department of Traumatic Orthopedics,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou Province,China;School of Clinical Medicine,Guizhou Medical University,Guiyang 550025,Guizhou Province,China;Clinical Research Center,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou Province,China)

机构地区：[1]贵州医科大学附属医院创伤骨科,贵州省贵阳市550004 [2]贵州医科大学临床医学院,贵州省贵阳市550025 [3]贵州医科大学附属医院临床研究中心,贵州省贵阳市550004

出　　处：《中国组织工程研究》2025年第13期2728-2735,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(82160249,81960234),项目负责人:李青。

摘　　要：背景:课题组前期成功在体外分离培养乳鼠触液神经元,尚无研究报道有效分离培养高纯度成年小鼠触液神经元的方法,且触液神经元的自我更新能力是否随着年龄发生变化尚无研究。目的:建立一种高纯度成年小鼠触液神经元体外分离培养的方法,并鉴定成年小鼠触液神经元与乳鼠触液神经元在体外的自我更新能力。方法:从成年3月龄小鼠颈髓分离含有触液神经元的原代细胞贴壁培养并利用融合多模态成像基因的慢病毒转染细胞,通过嘌呤霉素筛选得到高纯度成年小鼠触液神经元细胞,在完全培养基中悬浮培养。通过免疫荧光检测成年小鼠触液神经元表达神经干细胞标记物巢蛋白(Nestin)及SOX2情况,观察成年小鼠触液神经元体外成球与传代能力;将同等数量(5×10^(3))的第3代成年小鼠及乳鼠触液神经元在同等条件下分为2组,分别接种在含有完全培养基的超低黏附培养板中,在体积分数5%CO_(2),37℃恒温箱悬浮培养,通过体外成球、CCK8实验、qPCR和Western blot鉴定成年小鼠及乳鼠触液神经元的自我更新能力。结果与结论:①实验成功在成年小鼠体内分离出高纯度触液神经元,在体外表达Nestin及SOX2,能形成神经球并连续传代。②成年小鼠触液神经元体外自我更新能力较乳鼠相比明显减弱,细胞培养到第4天时乳鼠触液神经元已经形成直径约为150μm的神经球,而成年小鼠触液神经元所形成的神经球直径仅为40μm(P<0.0001)。③CCK8增殖实验结果表明,成年小鼠触液神经元的增殖活性在培养后各时间点显著弱于乳鼠(P<0.0001)。④qPCR和Western blot检测发现成年小鼠触液神经元Nestin及SOX2的mRNA(P<0.0001)和蛋白表达量(P<0.01)较乳鼠显著下降。⑤上述结果证实,成年小鼠触液神经元的体外自我更新能力显著弱于乳鼠。BACKGROUND:We have successfully isolated and cultured neonatal mouse cerebrospinal fluid-contacting neurons in vitro,but there is no study that reports an effective method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons.There is no study on whether the self-renewal ability of cerebrospinal fluid-contacting neurons changes with age.OBJECTIVE:To establish a method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons in vitro,and to characterize the self-renewal ability of adult mouse cerebrospinal fluid-contacting neurons and neonatal mouse cerebrospinal fluid-contacting neurons in vitro.METHODS:Primary cells containing cerebrospinal fluid-contacting neurons were isolated from the cervical medulla of adult mouse(3 months of age)in adherent culture and transfected with lentivirus fused with multimodal imaging genes.High-purity adult mouse cerebrospinal fluid-contacting neurons were obtained by puromycin screening in suspension culture in complete medium.The expression of neural stem cell markers Nestin and SOX2 was detected by immunofluorescence in adult mouse cerebrospinal fluid-contacting neurons,and the ability of adult mouse cerebrospinal fluid-contacting neurons to form spheres and pass on in vitro was observed.An equal number(5×10^(3)/mL)of passage 3 adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons were divided into two groups under the same conditions and inoculated into ultra-low adhesion plates containing complete medium in suspension culture at 5%CO_(2),37℃thermostat,respectively.The self-renewal capacity of adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons was characterized by in vitro spheroid formation,CCK8 assay,qPCR,and western blot assay.RESULTS AND CONCLUSION:(1)High-purity cerebrospinal fluid-contacting neurons were successfully isolated from adult mouse,which expressed Nestin and SOX2 in vitro,and were able to form neurospheres and pass on continuously.(2)The in vitro self-r

关 键 词：脊髓损伤 成年小鼠触液神经元 内源性神经干细胞 自我更新能力 体外培养 细胞提纯 融合多模态成像基因的慢病毒 干细胞潜能鉴定 

分 类 号：R459.9[医药卫生—治疗学] R363[医药卫生—临床医学] R364