摘要
目的探讨小干扰RNA(small interfering RNA,siRNA)慢病毒介导沉默P75神经生长因子受体(P75 neurotrophin receptor,P75NTR)基因对大鼠BMSCs成骨分化的影响。方法首先设计3条慢病毒介导P75NTR基因的siRNA序列(P75NTR-siRNA-1、2、3)以及阴性对照(negative control,NC)-siRNA,分别转染第3代SD大鼠BMSCs;倒置显微镜下观察细胞形态变化,实时荧光定量PCR及Western blot检测细胞P75NTR基因及蛋白表达情况,并筛选沉默效果最佳的P75NTR-siRNA进行后续成骨分化实验。取第3代SD大鼠BMSCs,随机分为实验组、阴性对照组及空白对照组;其中,阴性对照组及实验组分别采用NC-siRNA及筛选的P75NTR-siRNA慢病毒载体转染BMSCs,空白对照组为正常BMSCs。采用MTT法检测转染后细胞增殖情况;各组细胞经成骨诱导分化培养后,Western blot检测成骨相关蛋白[骨钙蛋白(osteocalcin,OCN)及Runx相关转录因子2(Runx related transcription factor 2,Runx2)]表达,免疫组织化学染色观察Ⅰ型胶原表达情况,ALP检测以及茜素红染色观察成骨情况。结果慢病毒介导P75NTR基因转染BMSCs后,其P75NTR mRNA及蛋白表达量均明显降低(P<0.05),其中P75NTR-siRNA-3沉默效果最佳。P75NTR基因沉默后,MTT检测示实验组细胞增殖较两对照组明显增快(P<0.05)。成骨诱导分化培养后,实验组OCN及Runx2蛋白相对表达量、Ⅰ型胶原蛋白表达、ALP活力均明显高于两对照组,差异有统计学意义(P<0.05);且随成骨诱导时间延长,矿化结节逐渐增多。结论siRNA慢病毒沉默P75NTR基因可以促进大鼠BMSCs成骨分化,为治疗骨缺损提供了新思路。
Objective To investigate the effect of small interfering RNA(siRNA)lentivirus-mediated silencing of P75 neurotrophin receptor(P75NTR)gene on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in rats.Methods Three lentivirus-mediated P75NTR gene siRNA sequences(P75NTR-siRNA-1,2,3)and negative control(NC)-siRNA were designed and transfected into the 3rd generation Sprague Dawley(SD)rat BMSCs.The cells morphological changes were observed under an inverted microscope,and the expressions of P75NTR gene and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot.Then the best silencing P75NTR-siRNA for subsequent osteogenic differentiation experiments was screened out.The 3rd generation SD rat BMSCs were randomly divided into experimental group,negative control group,and blank control group(normal BMSCs).The BMSCs of negative control group and experimental group were transfected with NC-siRNA and the selected P75NTR-siRNA lentiviral vector,respectively.The cells of each group were cultured by osteogenic induction.The expressions of osteogenic related proteins[osteocalcin(OCN)and Runx related transcription factor 2(Runx2)]were detected by Western blot;the collagen typeⅠexpression was observed by immunohistochemical staining;the osteogenesis of BMSCs was observed by alkaline phosphatase(ALP)detection and alizarin red staining.Results After lentivirus-mediated P75NTR transfected into BMSCs,the expressions of P75NTR mRNA and protein significantly reduced(P<0.05),and the best silencing P75NTR-siRNA was P75NTR-siRNA-3.After P75NTR gene was silenced,MTT test showed that the cell proliferation in the experimental group was significantly faster than those in the two control groups(P<0.05).After osteogenic induction,the relative expressions of OCN and Runx2 proteins,collagen typeⅠexpression,and ALP activity were significantly higher in the experimental group than in the two control groups,the differences were significant(P<0.05).With the prolongation of osteogenic induc
作者
王宁
陈俊毅
陈西淼
朱伦井
段江涛
王烨
贝朝涌
WANG Ning;CHEN Junyi;CHEN Ximiao;ZHU Lunjing;DUAN Jiangtao;WANG Ye;BEI Chaoyong(Department of Trauma and Limb Surgery,Affiliated Hospital of Guilin Medical College,Guilin Guangxi,541000,P.R.China;Guilin Medical College,Guilin Guangxi,541000,P.R.China)
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2020年第8期1052-1058,共7页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(81660366)。
关键词
P75神经生长因子受体
小干扰RNA
慢病毒
基因沉默
BMSCS
成骨分化
大鼠
P75 neurotrophin receptor
small interfering RNA
lentivirus
gene silencing
bone marrow mesenchymal stem cells
osteogenic differentiation
rat
