沉默HIF-1α基因对大鼠BMMSCs在牵张力作用下表达BSP和Osterix的影响
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摘要
目的探究机械牵张力作用下沉默大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)的低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)基因对骨涎蛋白(bone sialoprotein,BSP)和Osterix表达的影响,为利用BMMSCs修复骨缺损提供新的思路。方法根据大鼠HIF-1α基因设计shRNA序列,PCR扩增后克隆入p GMLV-SC1RNAi慢病毒载体。转化感受态细胞筛选阳性克隆并测序鉴定后,由293T细胞进行包装和滴度测定。转染体外培养的大鼠BMMSCs,倒置荧光显微镜观察荧光,筛选稳定沉默HIF-1α的细胞株。RNA干扰回复实验分为空白对照组、HIF-1αshRNA基因沉默组、阴性对照组、回复组4组,Western blot检测4组HIF-1α蛋白表达情况,以验证靶基因的回复效果,排除脱靶效应;使用FlexcellFX-5000T细胞应力加载系统对细胞进行机械牵张力干预,分为空白对照组、HIF-1αshRNA基因沉默组、阴性对照组,qRTPCR和Western blot检测各组BSP和Osterix的mRNA和蛋白表达水平。结果 HIF-1αshRNA慢病毒干扰载体构建成功,RNA干扰回复实验结果显示,回复组与空白细胞组HIF-1α蛋白表达量差异无统计学意义(P>0.05),重组慢病毒能有效沉默BMMSCs中HIF-1α。机械牵张力作用BMMSCs后,相对于空白对照组和阴性对照组,HIF-1αshRNA基因沉默组成骨相关因子BSP和Osterix的mRNA、蛋白表达均增强,差异具有统计学意义(P <0.05);空白对照组与阴性对照组间BSP和Osterix的mRNA和蛋白表达差异无统计学意义(P>0.05)。结论机械牵张力作用下沉默BMMSCs中的HIF-1α可促进BSP和Osterix的表达。
Objective To explore the effect of hypoxia inducible factor 1α(HIF-1α) gene silencing in rat bone marrow mesenchymal stem cells(BMMSCs) under mechanical distraction on the expression of bone sialoprotein(BSP) and osterix and to provide a new idea for repairing bone defects with BMMSCs. Methods The shRNA sequence was de-signed according to the rat HIF-1α gene, and the pGMLV-SC1 RNAi lentiviral vector was cloned after PCR amplifica-tion. After screening positive clones and identifying competent transformed cells by sequencing, 293 T cells were pack-aged and titered, rat BMMSCs were transfected and cultured in vitro. Clones with stably silenced HIF-1α expression were screened by inverted fluorescence microscopy. The RNAi response experiment was divided into four groups: the blank control group, the HIF-1α shRNA group, the negative control group, and the response group. Western blot was used to detect the expression of HIF-1α protein in the four groups to verify the response of the target genes and exclude off-target effects. A Flexcell FX-5000 T cell stress loading system was used to intervene in the mechanical stretch of the cells. qRT-PCR and Western blot were used to detect the expression of BSP and osterix in the blank control group, HIF-1α shRNA group, and negative control group. Results The HIF-1α shRNA lentiviral vector was successfully con-structed. The results of the RNAi response showed no significant difference in the expression of HIF-1α between the re-sponse and the blank control group(P > 0.05). The recombinant lentivirus could effectively silence HIF-1α in BMMSCs. After mechanical distraction of the BMMSCs, compared with the blank and negative control groups, the HIF-1α shRNA group showed significantly increased mRNA and protein expression of the bone-related factors BSP and os-terix(P < 0.05); there was no significant difference in the mRNA and protein expression of BSP or osterix between the blank and negative control groups(P > 0.05). Conclusion Silencing HIF-1α in BMMSCs under mechanical distrac-tion can promote the expression of BSP and osterix.
Objective To explore the effect of hypoxia inducible factor 1α(HIF-1α) gene silencing in rat bone marrow mesenchymal stem cells(BMMSCs) under mechanical distraction on the expression of bone sialoprotein(BSP) and osterix and to provide a new idea for repairing bone defects with BMMSCs. Methods The shRNA sequence was de-signed according to the rat HIF-1α gene, and the pGMLV-SC1 RNAi lentiviral vector was cloned after PCR amplifica-tion. After screening positive clones and identifying competent transformed cells by sequencing, 293 T cells were pack-aged and titered, rat BMMSCs were transfected and cultured in vitro. Clones with stably silenced HIF-1α expression were screened by inverted fluorescence microscopy. The RNAi response experiment was divided into four groups: the blank control group, the HIF-1α shRNA group, the negative control group, and the response group. Western blot was used to detect the expression of HIF-1α protein in the four groups to verify the response of the target genes and exclude off-target effects. A Flexcell FX-5000 T cell stress loading system was used to intervene in the mechanical stretch of the cells. qRT-PCR and Western blot were used to detect the expression of BSP and osterix in the blank control group, HIF-1α shRNA group, and negative control group. Results The HIF-1α shRNA lentiviral vector was successfully con-structed. The results of the RNAi response showed no significant difference in the expression of HIF-1α between the re-sponse and the blank control group(P > 0.05). The recombinant lentivirus could effectively silence HIF-1α in BMMSCs. After mechanical distraction of the BMMSCs, compared with the blank and negative control groups, the HIF-1α shRNA group showed significantly increased mRNA and protein expression of the bone-related factors BSP and os-terix(P < 0.05); there was no significant difference in the mRNA and protein expression of BSP or osterix between the blank and negative control groups(P > 0.05). Conclusion Silencing HIF-1α in BMMSCs under mechanical distrac-tion can promote the expression of BSP and osterix.
引文
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[3]Heo JH,Choi JH,Kim IR,et al.Combined treatment with low-level laser and rhBMP-2 promotes differentiation and mineralization of osteoblastic cells under hypoxic stress[J].Tissue Eng Regen Med,2018,5(6):793-801.
[4]Chen XY,Liu Y,Ding WH,et al.Mechanical stretch-induced osteogenic differentiation of human jaw bone marrow mesenchymal stem cells(hJBMMSCs)via inhibition of the NF-kappa B pathway[J].Cell Death Dis,2018,9(2):207.
[5]Yao Q,Liu Y,Selvaratnam B,et al.Mesoporous silicate nanoparticles/3D nanofibrous scaffold-mediated dual-drug delivery for bone tissue engineering[J].J Control Release,2018,279:69-78.
[6]Will LA.Orthodontic tooth movement:a historic prospective[J].Front Oral Biol,2016,18:46-55.
[7]邓正炜,张亚东,赵世昌.组织工程骨激活低氧诱导因子HIF-1α促进骨修复[J].实用骨科杂志,2018,24(10):917-921.
[8]Wang Y,Wan C,Deng L,et al.The hypoxia-inducible factorαpathway couples angiogenesis to osteogenesis during skeletal development[J].J Clin Investig,2007,117(6):1616-1626.
[9]Yang DC,Yang MH,Tsai CC,et al.Hypoxia inhibits osteogenesis in human mesenchymal stem cells through direct regulation of RUNX2 by TWIST[J].PLoS One,2011,6(9):e23965.
[10]Carroll SF,Buckley CT,Kelly DJ.Cyclic tensile strain can play a role in directing both intramembranous and endochondral ossification of mesenchymal stem cells[J].Front Bioeng Biotechnol,2017,5:73.
[11]Boink MA,Van Den Broek LJ,Roffel SA,et al.Different wound healing properties of dermis,adipose,and gingiva mesenchymal stromal cells[J].Wound Repair Regen,2016,24(1):100-109.