摘要
目的探究miR-9是否通过调控β-微管蛋白-Ⅲ(β-tubulinⅢ)、胶质纤维酸性蛋白(GFAP)影响神经干细胞(NSCs)的增殖。方法对健康孕鼠进行NSCs分离培养并将分离出的细胞均分为NO组(无转染NSCs细胞株)、NN组(NSCs转染miR-9-NC)、NM组(NSCs转染miR-9 mimics)、NI组(转染miR-9 inhibitor)。通过免疫荧光鉴定巢蛋白(Nestin),qRT-PCR法检测miR-9、β-tubulinⅢ、GFAP含量,MTT法检测NSCs增殖情况,流式细胞术检测NSCs凋亡情况,Western blot检测miR-9、β-tubulinⅢ、GFAP蛋白表达。结果Nestin在90%的NSCs中呈现阳性,且与细胞核共定位,提示NSCs分离成功。NM组NSCs中miR-9 mRNA表达量在四组中最高,说明转染成功。四组中NM组β-tubulinⅢmRNA、蛋白表达量最高,GFAP mRNA和蛋白表达量最低,NI组与之相反(P<0.05)。24 h后四组中NM组NSCs活性最强且呈现随时间延长而逐渐增强的趋势(P<0.05),NI组的细胞活性较其他三组减弱(P<0.05)。四组中NM组细胞凋亡率少于其他三组(P<0.05),NI组的细胞凋亡率较其他三组升高(P<0.05),NO组与NN组细胞上述指标差异无统计学意义。结论过表达miR-9通过促进β-tubulinⅢ表达、抑制GFAP表达,从而促进NSCs增殖。
Objective To explore whether miR-9 affects the proliferation of neural stem cells(NSCs)by regulatingβ-tubulinⅢand glial fibrillary acidic protein(GFAP).Methods NSCs cells were isolated and cultured on the purchased healthy pregnant mice and the isolated cells were divided into NO group(non-transfected NSCs cell line),NN group(NSCs transfected with miR-9-NC),NM group(NSCs transfected)transfected with miR-9 mimics,NI group(transfected with miR-9 inhibitor).Nestin was identified by immunofluorescence,the contents of Mir-9,β-tubulinⅢand GFAP were measured by QRT-PCR,the proliferation of NSCs was measured by MTT method,the apoptosis of NSCs was measured by flow cytometry,and the protein expressions of Mir-9,β-tubulinⅢand GFAP were detected by Western blot.Results The detection index Nestin of NSCs for 24 h after cell passage was positive in 90%of the NSCs,and co-localized with the nucleus,indicating the successful isolation of NSCs.The expression of miR-9 mRNA in NSCs of the NM group was the highest among the four groups,indicating that the transfection was successful.Among the four groups,β-tubulinⅢmRNA and protein expression was the highest in the NM group,and the GFAP mRNA and protein expression was the lowest.The NI group was the opposite(P<0.05).In the four groups after 24 h,the cell activity of NSCs in the NM group was the strongest and showed a trend of gradually increasing with time(P<0.05).The cell activity of NI was significantly weaker than that of the other three groups(P<0.05).Among the four groups,the apoptosis rate of the NM group was lower than that of the other three groups(P<0.05),and the apoptosis rate of NI was higher than that of the other three groups(P<0.05).The difference in the above indicators between the NO group and the NN group was smaller.Conclusion Overexpressed miR-9 promotes the proliferation of NSCs by promoting the expression ofβ-tubulinⅢand inhibiting the expression of GFAP.
作者
王煜
赵岚
阚伯红
史慧妍
Wang Yu;Zhao Lan;Kan Bohong;Shi Huiyan(Dept of Acupuncture,The First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin 300193)
出处
《安徽医科大学学报》
CAS
北大核心
2022年第1期10-14,共5页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81704148)
天津市教委科研计划项目(自然科学)(编号:2017KJ149)。
关键词
神经干细胞
神经退化性疾病
胶质细胞
分化
neural stem cells
neurodegenerative diseases
glial cells
differentiation
