慢病毒介导酪氨酸激酶受体A过表达载体促进神经干细胞增殖和分化的实验研究  

Experimental study on the lentivirus⁃mediated overexpression vector of tyrosine kinase receptor A promotes proliferation and differentiation of neural stem cells

作　　者：邓明[1] 谢萍 马永刚[1] 周炎[1] 贺斌[1] 陈庆[1] 吴飞[1] 陈忠辉 明江华[1] DENG Ming;XIE Ping;MA Yonggang;ZHOU Yan;HE Bin;CHEN Qing;WU Fei;CHEN Zhonghui;MING Jianghua(Department of Orthopedics,Renmin Hospital of Wuhan University,Wuhan,Hubei 430060,China;Department of Chinese Traditional Medicine,Tongren Hospital of Wuhan University(Wuhan Third Hospital),Wuhan,Hubei 430060,China)

机构地区：[1]武汉大学人民医院骨科,湖北武汉430060 [2]武汉市第三医院中医科,湖北武汉430060

出　　处：《安徽医药》2020年第12期2410-2414,I0003,共6页Anhui Medical and Pharmaceutical Journal

基　　金：湖北省自然科学基金面上项目(2019CFB457);中央高校基本科研业务费专项资金(武汉大学青年教师资助项目)(2042017kf0139);武汉大学医学部创新种子基金培育项目(TFZZ2018027)。

摘　　要：目的探究酪氨酸激酶受体A(Trk A)过表达载体对神经干细胞增殖和分化能力的影响。方法取SD大鼠海马组织进行提取神经干细胞,采用免疫荧光的方法对神经球进行鉴定,利用siRNA技术构建Trk A过表达载体,将培养的神经干细胞分成三组,空白对照组为未做任何处理的细胞组,阴性对照组是经空白载体的慢病毒转染的细胞组,实验组是由Trk A的过表达载体构建的慢病毒转染细胞组。逆转录PCR(RT⁃PCR)检测Trk A和神经干细胞分化标志基因Tuj1,GFAP和CNPase的表达,CCK⁃8试剂盒检测各组细胞的增殖率。结果免疫荧光染色显示神经干细胞细胞球Nestin(红色荧光),Brud染色(绿色荧光)阳性,且占细胞量的90%以上;荧光双标染色(棕色荧光)显示双染细胞量占总细胞量的90%以上,说明培养的细胞是神经干细胞。慢病毒滴度的检测结果:Trk A过表达慢病毒载体转染滴度为3×10^8 TU/mL。RT⁃PCR结果表明,空白对照组(1.09±0.09)和阴性对照组(1.11±0.05)的Trk A的mRNA相对表达量比较差异无统计学意义(P>0.05),实验组的Trk A的mRNA相对表达量(7.12±1.32)要明显高于空白对照组和阴性对照组(P<0.05);神经干细胞分化标志基因Tuj1,GFAP和CNPase的表达与Trk A相同。CCK⁃8试剂盒检测结果表明,空白对照组、阴性对照组及实验组的细胞增殖率分别为(87.92±9.21)%、(88.22±9.37)%、(176.01±11.33)%(F=18.082,P<0.05),其中空白对照组和阴性对照组的细胞增殖率比较差异无统计学意义(P>0.05),实验组的细胞增殖率要明显高于空白对照组和阴性对照组(P<0.05)。结论Trk A过表达载体可以促进神经元干细胞向神经元细胞,星型胶质细胞和少突胶质细胞的分化,并且可以促进神经元干细胞的增殖。Objective To investigate the effect of tyrosine kinase receptor A(Trk A)overexpression vector on the proliferation and differentiation of neural stem cells.Methods Neural stem cells were extracted from the hippocampus of SD rats and identified by immunofluorescence.Trk A overexpression vector was constructed by siRNA technology.The cultured neural stem cells were as⁃signed into three groups.The blank control group was a cell group without any treatment.The negative control group was a cell group transfected by lentivirus with blank vector.The experimental group was overexpressed by Trk A.Lentivirus transfection cell group constructed by vector.RT⁃PCR was used to detect the expression of Trk A and Tuj1,GFAP and CNPase.CCK⁃8 kit was used to detect the proliferation rate of cells in each group.Results The result of the immunofluorescence staining showed that the Nes⁃tin(red fluorescence)and Brud staining(green fluorescence)of neural stem cell spheroids were positive,and accounted for more than 90%of the cell volume;the double staining(brown fluorescence)showed that the amount of double staining cells accounted for more than 90%of the total cell volume,indicating that the cultured cells were neural stem cells.The results showed that the ti⁃ter of Trk A overexpression vector was 3×10^8 TU/mL.The results of the RT⁃PCR showed that there was no significant difference in the mRNA expression of Trk A between the blank control group(1.09±0.09)and the negative control group(1.11±0.05)(P>0.05),and the expression of Trk A mRNA in the experimental group(7.12±1.32)was significantly higher than that in the blank control group and the negative control group(P<0.05);the expression of TuJ1,GFAP and CNPase was the same as that of Trk A.The results of CCK⁃8 kit showed that the cell proliferation rate of blank control group was(87.92±9.21)%,that of negative control group was(88.22±9.37)%,and that of experimental group was(176.01±11.33)%.The cell proliferation rate of the three groups was statistically different(F=1

关 键 词：受体 Eph家族 神经干细胞 慢病毒属 转染 增殖 分化 大鼠 Sprague⁃Dawley 

分 类 号：R651.2[医药卫生—外科学]