钙调蛋白依赖性激酶Ⅱγ RNA干扰载体构建及对破骨细胞分化和骨吸收的影响
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摘要
目的研究钙调蛋白依赖性激酶Ⅱγ(CaMKⅡγ)RNA干扰对破骨细胞分化和骨吸收的影响。方法构建3个CaMKⅡγ RNA慢病毒重组干扰载体;阴性载体转染RAW264.7细胞,确定最适转染滴度值(MOI)和转染效率。用重组载体转染细胞,确定干扰效果最佳的重组载体用于后续实验。细胞分为对照组、阴性载体组、干扰载体组;病毒转染5 d后通过TRAP染色及牙本质磨片骨吸收陷窝检测3组破骨细胞生成及骨吸收情况。结果成功构建了3个CaMKⅡγ重组干扰载体;最适MOI值为30,转染效率>80%。#3重组载体干扰效果最佳,干扰效率在mRNA及蛋白水平分别为78.16%和67.02%(P <0.01)。3组细胞中,干扰载体组多核破骨细胞数、牙本质吸收陷窝数和面积较对照组分别下降了59.99%、54.19%和57.94%(P <0.01),而阴性载体组和对照组比较差异无显著性(P> 0.05)。结论 CaMKⅡγ RNA干扰可显著抑制破骨细胞生成和骨吸收功能。
Objective To investigate the effects of calmodulin-dependent kinase IIγ(GaMKIIγ) RNA interference on osteoclast differentiation and bone resorption. Methods Three CaMKIIγ recomninant RNA interference vectors were constructed using lentivirus. Negative vector was used to transfect RAW264.7 cells and the multiplicity of infection(MOI) with the optimal transfection efficiency was determined. Recombinant vectors were also used to transfect cells to determine the one with the best interference effect for following experiments.Then, the cells were divided into control group, negative vector group and interference vector group. Five days after virus transfection, osteoclastogenesis and bone resorption function were determined by TRAP staining and dentin resorption lacunae detection. Results Three CaMKIIγ recombinant interference vectors were constructed,and the optimal MOI was 30, under which transfection efficiency was about 81%. The #3 recombinant vector showed the best interference effect and the interference efficiency was up to 78.16% at mRNA level and 67.02% at protein level. When compared with control group, the number of multi-nucleated osteoclasts, the number and area of dentin resorption lacunaes in interference vector group decreased 59.99%、54.19% and 57.94% respectively(P < 0.01). No significant difference were observed between negative vector group and control group(P > 0.05).Conclusion CaMKIIγ RNA interference significantly inhibits osteoclastogenesis and bone resorption.
Objective To investigate the effects of calmodulin-dependent kinase IIγ(GaMKIIγ) RNA interference on osteoclast differentiation and bone resorption. Methods Three CaMKIIγ recomninant RNA interference vectors were constructed using lentivirus. Negative vector was used to transfect RAW264.7 cells and the multiplicity of infection(MOI) with the optimal transfection efficiency was determined. Recombinant vectors were also used to transfect cells to determine the one with the best interference effect for following experiments.Then, the cells were divided into control group, negative vector group and interference vector group. Five days after virus transfection, osteoclastogenesis and bone resorption function were determined by TRAP staining and dentin resorption lacunae detection. Results Three CaMKIIγ recombinant interference vectors were constructed,and the optimal MOI was 30, under which transfection efficiency was about 81%. The #3 recombinant vector showed the best interference effect and the interference efficiency was up to 78.16% at mRNA level and 67.02% at protein level. When compared with control group, the number of multi-nucleated osteoclasts, the number and area of dentin resorption lacunaes in interference vector group decreased 59.99%、54.19% and 57.94% respectively(P < 0.01). No significant difference were observed between negative vector group and control group(P > 0.05).Conclusion CaMKIIγ RNA interference significantly inhibits osteoclastogenesis and bone resorption.
引文
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[16]陆大壮.CaMKIIδ基因沉默对MAPKs、CREB信号及破骨细胞分化的影响[D].华北理工大学,2017.
[17]MA H,GROTH R D,COHEN S M,et al.γCaMKII shuttles Ca2+/CaM to the nucleus to trigger CREB phosphorylation and gene expression[J].Cell,2014,159(2):281-294.
[2]YAVROPOULOU M P,YOVOS J G.Osteoclastogenesis-current knowledge and future perspectives[J].J Musculoskelet Neuronal Interact,2008,8(3):204-216.
[3]NEGISHI-KOGA T,TAKAYANAGI H.Ca2+-NFATc1 signaling is an essential axis of osteoclast differentiation[J].Immunol Rev,2009,231(1):241-256.
[4]YAO C H,ZHANG P,ZHANG L.Differential protein and mRNA expression of CaMKs during osteoclastogenesis and its functional implications[J].Biochem Cell Biol,2012,90(4):532-536.
[5]ANG E S,ZHANG P,STEER J H,et al.Calcium/calmodulindependent kinase activity is required for efficient induction of osteoclast differentiation and bone resorption by receptor activator of nuclear factor kappa B ligand(RANKL)[J].J Cell Physiol,2010,212(3):787-795.
[6]SEALES E C,MICOLI K J,MCDONALD J M.Calmodulin is a critical regulator of osteoclastic differentiation,function,and survival[J].J Cell Biochem,2010,97(1):45-55.
[7]YE J,DAS S,ROY A,et al.Ischemic injury-induced CaMKIIδand CaMKIIγconfer neuroprotection through the NF-κB signaling pathway[J].Mol Neurobiol,2018:1-14.
[8]PARK-MIN K H,JI J D,ANTONIV T,et al.IL-10 suppresses calcium-mediated costimulation of receptor activator NF-kappa Bsignaling during human osteoclast differentiation by inhibiting TREM-2 expression[J].J Immunol,2009,183(4):2444-2455.
[9]WILLIAMS J P,MICOLI K,MCDONALD J M.Calmodulin-an often-ignored signal in osteoclasts[J].Ann N Y Acad Sci,2010,1192(1):358-364.
[10]HWAN P K,BORYUNG P,SUK Y D,et al.Zinc inhibits osteoclast differentiation by suppression of Ca2+-Calcineurin-NFATc1signaling pathway[J].Cell Commun Signal,2013,11(1):74-74.
[11]张艳波,戚孟春,董伟,等.CaMKⅡδ慢病毒过表达载体构建及其对破骨细胞分化的影响[J].实用医学杂志,2018,34(13):2123-2127.
[12]TIMMIN J M,OZCAN L,SEIMON T A,et al.Calcium/calmodulin-dependent protein kinase II links ER stress with Fas and mitochondrial apoptosis pathways[J].J Clin Invest,2009,119(10):2925-2941.
[13]MIYAZAKI T,KATAGIRI H,KANEGAE Y,et al.Reciprocal role of ERK and NF-κB Pathways in survival and activation of osteoclasts[J].J Cell Biol,2000,148(2):333-342.
[14]KIM H H,CHUNG W J,LEE S W,et al.Association of sustained ERK activity with integrin beta3 induction during receptor activator of nuclear factor kappaB ligand(RANKL)-directed osteoclast differentiation[J].Exp Cell Res,2003,289(2):368-377.
[15]刘娟娟,董伟,戚孟春,等.钙离子/钙调蛋白依赖性蛋白激酶ⅡδRNA干扰对下游基因表达及破骨细胞分化的影响[J].第三军医大学学报,2017(39):27.
[16]陆大壮.CaMKIIδ基因沉默对MAPKs、CREB信号及破骨细胞分化的影响[D].华北理工大学,2017.
[17]MA H,GROTH R D,COHEN S M,et al.γCaMKII shuttles Ca2+/CaM to the nucleus to trigger CREB phosphorylation and gene expression[J].Cell,2014,159(2):281-294.