中医正骨
中醫正骨
중의정골
THE JOURNAL OF TRADITIONAL CHINESE ORTHOPEDICS AND TRAUMATOLOGY
2014年
7期
3-7
,共5页
丁永利%陈星%赵明明%蔡一强%姜勇
丁永利%陳星%趙明明%蔡一彊%薑勇
정영리%진성%조명명%채일강%강용
微RNAs%成骨细胞%骨形态发生蛋白质2%miR-133a%Runx-2%Osterix%碱性磷酸酶%动物实验
微RNAs%成骨細胞%骨形態髮生蛋白質2%miR-133a%Runx-2%Osterix%堿性燐痠酶%動物實驗
미RNAs%성골세포%골형태발생단백질2%miR-133a%Runx-2%Osterix%감성린산매%동물실험
MicroRNAs%Osteoblasts%Bone morphogenetic protein 2%miR-133a%Runx-2%Osterix%Alkaline phosphatase%Animal Experi-mentation
目的：探究miR-133a对骨形态发生蛋白2诱导的鼠前成骨细胞分化的影响及其作用机制。方法：检测骨形态发生蛋白2诱导的鼠前成骨细胞分化过程中miR-133a水平、碱性磷酸酶活性、Runx-2蛋白水平及Osterix蛋白水平，检测转染miR-133a后鼠前成骨细胞分化过程中碱性磷酸酶活性、Runx-2 mRNA水平、Runx-2蛋白水平、Osterix mRNA水平和Osterix蛋白水平。miR-133a测定采用qRT-PCR法，Runx-2和Osterix蛋白水平测定采用Western-blot法，Runx-2和Osterix mRNA水平测定采用RT-PCR法，碱性磷酸酶活性检测采用碱性磷酸酶检测试剂盒。采用HEK293细胞进行miR-133a靶基因验证实验，细胞中荧光值的检测采用荧光素酶报告基因检测系统。结果：miR-133a在骨形态发生蛋白2诱导的MC3T3-E1细胞分化过程中表达显著下调，碱性磷酸酶的活性则显著增高。转染miR-133a后，细胞中碱性磷酸酶活性受到明显抑制，Runx-2蛋白水平显著下调、Runx-2 mRNA水平未发生明显变化，Osterix的mRNA和蛋白水平均明显下调。荧光素酶报告基因检测结果显示，miR-133a可靶向作用于Runx-2 mRNA的3’-UTR区。结论：miR-133a通过抑制Runx-2的蛋白表达进而抑制骨形态发生蛋白2诱导的鼠前成骨细胞分化过程。
目的：探究miR-133a對骨形態髮生蛋白2誘導的鼠前成骨細胞分化的影響及其作用機製。方法：檢測骨形態髮生蛋白2誘導的鼠前成骨細胞分化過程中miR-133a水平、堿性燐痠酶活性、Runx-2蛋白水平及Osterix蛋白水平，檢測轉染miR-133a後鼠前成骨細胞分化過程中堿性燐痠酶活性、Runx-2 mRNA水平、Runx-2蛋白水平、Osterix mRNA水平和Osterix蛋白水平。miR-133a測定採用qRT-PCR法，Runx-2和Osterix蛋白水平測定採用Western-blot法，Runx-2和Osterix mRNA水平測定採用RT-PCR法，堿性燐痠酶活性檢測採用堿性燐痠酶檢測試劑盒。採用HEK293細胞進行miR-133a靶基因驗證實驗，細胞中熒光值的檢測採用熒光素酶報告基因檢測繫統。結果：miR-133a在骨形態髮生蛋白2誘導的MC3T3-E1細胞分化過程中錶達顯著下調，堿性燐痠酶的活性則顯著增高。轉染miR-133a後，細胞中堿性燐痠酶活性受到明顯抑製，Runx-2蛋白水平顯著下調、Runx-2 mRNA水平未髮生明顯變化，Osterix的mRNA和蛋白水平均明顯下調。熒光素酶報告基因檢測結果顯示，miR-133a可靶嚮作用于Runx-2 mRNA的3’-UTR區。結論：miR-133a通過抑製Runx-2的蛋白錶達進而抑製骨形態髮生蛋白2誘導的鼠前成骨細胞分化過程。
목적：탐구miR-133a대골형태발생단백2유도적서전성골세포분화적영향급기작용궤제。방법：검측골형태발생단백2유도적서전성골세포분화과정중miR-133a수평、감성린산매활성、Runx-2단백수평급Osterix단백수평，검측전염miR-133a후서전성골세포분화과정중감성린산매활성、Runx-2 mRNA수평、Runx-2단백수평、Osterix mRNA수평화Osterix단백수평。miR-133a측정채용qRT-PCR법，Runx-2화Osterix단백수평측정채용Western-blot법，Runx-2화Osterix mRNA수평측정채용RT-PCR법，감성린산매활성검측채용감성린산매검측시제합。채용HEK293세포진행miR-133a파기인험증실험，세포중형광치적검측채용형광소매보고기인검측계통。결과：miR-133a재골형태발생단백2유도적MC3T3-E1세포분화과정중표체현저하조，감성린산매적활성칙현저증고。전염miR-133a후，세포중감성린산매활성수도명현억제，Runx-2단백수평현저하조、Runx-2 mRNA수평미발생명현변화，Osterix적mRNA화단백수평균명현하조。형광소매보고기인검측결과현시，miR-133a가파향작용우Runx-2 mRNA적3’-UTR구。결론：miR-133a통과억제Runx-2적단백표체진이억제골형태발생단백2유도적서전성골세포분화과정。
Objective:To explore the effect of miR-133a on differentiation of murine preosteoblasts induced by bone morphogenetic protein 2(BMP-2)and the mechanism of action.Methods:The miR-133a levels,alkaline phosphatase(ALP)activities,Runx-2 protein levels and Osterix protein levels were detected in the process of murine preosteoblast differentiation induced by BMP 2.The ALP activities, Runx-2 mRNA levels,Runx-2 protein levels,Osterix mRNA levels and Osterix protein levels were detected in the process of murine preos-teoblast differentiation after miR-133a transfection.The qRT-PCR assays was used for miR-133a determination,Western-blot for Runx-2 and Osterix protein levels determination,RT-PCR for Runx-2 and Osterix mRNA levels determination and ALP Assay Kit for ALP activities determination.HEK293 cells were used to verify the target gene of miR-133a,and the luciferase reporter gene detection system was used for the determination of fluorescence value in cells.Results:A significant down-regulation of miR-133a expression was found in the process of MC3T3-E1 differentiation induced by BMP-2,while a significant increase was found in the ALP activities.After transfection of miR-133a, the ALP activities were inhibited significantly,and a significant down-regulation can be seen in the Runx-2 protein levels and the mRNA and protein levels of Osterix,while no evident changes was found in Runx-2 mRNA levels.The detection result of luciferase reporter gene showed that miR-133a can target the 3’-UTR region of Runx-2 mRNA.Conclusion:The miR-133a can suppress the murine preosteoblast differentiation induced by BMP-2 by suppressing the expression of Runx-2 protein.
