摘要
目的探究慢病毒介导神经生长因子(NGF)过表达转染促进骨髓间充质干细胞(BMSCs)向神经细胞转化的作用。方法体外分离,培养人BMSCs,利用基因过表达技术,构建NGF过表达慢病毒载体转染BMSCs。根据转染情况分成3组,空白对照组(未转染慢病毒载体的BMSCs)、空白载体病毒组(转染不含NGF过表达慢病毒载体的BMSCs)和过表达载体病毒组(转染过表达NGF慢病毒载体的BMSCs)。利用流式细胞仪检测BMSCs的表面标记物,利用实时荧光定量PCR(qRT-PCR)和Western blot分别检测神经细胞表面蛋白胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE) mRNA及蛋白的表达。结果 (1) BMSCs的表面蛋白标记物CD44和CD29在第2代BMSCs的表达为60. 2%和58. 3%,CD34和CD45在第2代BMSCs的表达为3. 4%和2. 6%。(2)慢病毒转染效率为90%以上。转染效率较高。(3)空白对照组、空白载体病毒组和过表达载体病毒组的GFAP和NSE mRNA的相对表达量三组比较有统计学差异(P <0. 05);其中空白载体病毒组和空白对照组的组间比较无统计学差异(P> 0. 05);过表达载体病毒组高于空白载体病毒组,组间比较有统计学差异(P <0. 05)。Western blot检测蛋白表达量也具有同样的表达特点。结论慢病毒介导NGF过表达转染促进BMSCs可以提高BMSCs向神经细胞转化的效率,可以为神经损伤和脊髓损伤提供种子细胞。
Objective To explore the effect of lentivirus-mediated neural growth factor( NGF) overexpression on promoting transformation of bone marrow mesenchymal stem cells( BMSCs) into neural cells. Methods Human BMSCs were isolated and cultured in vitro. Using gene overexpression technology,lentivirus NGF overexpression vector was constructed to transfect BMSCs. According to the transfection condition,the BMSCs were divided into three groups: blank control group( BMSCs without lentiviral transfection),lentiviral blank victor transfection group( BMSCs transferred by victor without NGF overexpression) and NGF overexpression lentiviral victor transfection group( BMSCs transferred by victor with NGF overexpression). Flow cytometry was used to detect the expressions of BMSCs surface markers. Real time fluorescence quantitative polymerase chain reaction( qRT-PCR) and Western blot were used to respectively detect the expressions of nerve cell surface proteins glial fibrillary acidic protein( GFAP) and neuron-specific enolase( NSE) mRNAs and proteins.Results( 1) The positive expression rates of BMSCs surface markers CD44 and CD29 in second generation BMSCs were60. 2% and 58. 3% respectively,and the positive expression rates of CD34 and CD45 in second generation BMSCs were3. 4% and 2. 6%,respectively,and this showed that these cells were derived from BMSCs.( 2) Transfection efficiency of lentiviruses was higher with transfection efficiency more than 90%.( 3) There were significant differences in the relative expression levels of GFAP and NSE mRNAs and proteins in blank control group,blank victor lentiviral transfection group and NGF overexpression lentiviral victor transfection group( all P < 0. 05),while there were no significant differences in them between blank control group and blank victor lentiviral transfection group( all P > 0. 05),and there were significant differences in them between NGF overexpression lentiviral victor transfection group and blank victor lentiviral transfection group( all P < 0. 05). Conclusion NGF overexpression lentivirus vector transfection to BMSCs can improve the efficiency of transformation to nerve cells and provide seed cells for nerve injury and spinal cord injury.
作者
邓明
谢萍
马永刚
明江华
周炎
陈庆
刘志勇
刘世清
DENG Ming;XIE Ping;MA Yong-gang;MING Jiang-hua;ZHOU Yan;CHEN Qing;LIU Zhi-yong;LIU Shi-qing(Department of Orthopaedics,Renmin Hospital of Wuhan University,Wuhan,Hubei 430060,China;不详)
出处
《中国临床研究》
CAS
2019年第2期145-149,共5页
Chinese Journal of Clinical Research
基金
国家自然科学基金(81802203)
中央高校基本科研业务费专项资金(2042017KF0139)~~
