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  

Construction of CaMKⅡγ RNA interference vector with lentivirus and its effect on osteoclast differentiation and bone resorption function

作　　者：王艺睿 王会 戚孟春 董伟 冯晓洁 孙红 WANG Yirui;WANG Hui;QI Mengchun;DONG Wei;FENG Xiaojie;SUN Hong(Department of Oral and Maxillofacial Surgery,College of Stomatology,Tangshan 063000,China)

机构地区：[1]华北理工大学口腔医学院口腔颌面外科教研室,河北唐山063000 [2]华北理工大学基础医学院病理教研室,河北唐山063000

出　　处：《实用医学杂志》2019年第4期557-561,共5页The Journal of Practical Medicine

基　　金：国家自然科学基金项目(编号:81270965);河北省教育厅重点研究项目(编号:ZD2015005);河北省自然科学基金资助项目(编号:H2017209114)

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(CaMKⅡγ)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.

关 键 词：钙调蛋白依赖性激酶IIγ 破骨细胞 RNA干扰 慢病毒 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]