实时荧光定量聚合酶链反应筛选重组人骨形态发生蛋白-2诱导比格犬骨髓基质干细胞成骨分化差异表达的miRNAs  

Real-time quantitative polymerase chain reaction screening of the differential miRNAs during osteogenic differentiation of beagle bone marrow mesenchymal stem cells induced by recombinant human bone morphogenetic protein-2

作　　者：胡稷杰[1] 刘亚伟[2] 何敏毅[3] 金丹[1] 赵辉[1] 余斌[1] 

机构地区：[1]南方医科大学南方医院创伤骨科,510515 [2]南方医科大学基础医学院 [3]南方医科大学珠江医院器官移植科

出　　处：《中华创伤骨科杂志》2013年第3期256-260,共5页Chinese Journal of Orthopaedic Trauma

基　　金：广东省医学科学技术研究摹金（B2009147）;广东省自然科学基金（10451051501005738,S20120400070R1）：广东省教育厅育苗工程（LYM10050）;广东省科技计划项目（20118031300020）

摘　　要：目的利川实时荧光定镣聚合酶链反应（Q—PCR）疗法筛选骨形态发生蛋白．2（rhBMP．2）诱导比格犬骨髓基质干细胞（BMSCs）成骨分化的miRNAs，探讨过表达miRNAs对成骨分化的影响。方法选取4只比格犬分离BMSCs原代，取培养至第3代BMSCs分为2组：对照组（用基础培养基培养）和实验组（在基础培养基中打11人rhBMP-2培养7d，诱导细胞成骨分化）。采川碱性磷酸酶染色法验证细胞成骨分化情况，采用Q—PCR法筛选成骨分化细胞对照组细胞差异表达的miRNAs；随机选择其中-种低表达miRNA（miR-125b），采用Lipofe（qamine2000进行转染，其中实验组1转染miR-125bmimic·s以过表达miR—125b，其阴性对照实验组2转染mimics—NC，验证过表达miR-125b7cl后对rhBMP-2诱导的比格犬BMSCs成骨分化的影响。结果成功建立比格犬BMSCs培养方法．rhBMP-2诱导比格犬BMSCs成骨分化7d后，进行Q—PCR筛选，获得表达差异相对于埘照组达到2倍以上的miRNAs41个，其中表达上调的有13个，表达下调的有28个；实验组l相对于实验组2，细胞中miR-125b表达上调4．13倍（P〈0．05）。转染7d后，ALP染色结果湿示：实验组1细胞质中深蓝色柑大颗粒明显少于实验组2。结论采用Q—PCR方法筛选比格犬BMSCs成骨分化的miRNAs系统高效、准确。筛选扶得的miRNAs中，miR．125b过表达能够显著抑制比格犬BMSCs的成骨分化。Objective To screen Ihe differential miRNAs during osteogenic differentiation of beagle bone marrow mesenchymal stem cells （BMSCs） induced by recombinant human bone morphngenetie protein-2 （rhBMP-2） using real-time quantitative polymerase chain reaction （Q-PCR） and to determine the effect of river-expression of one of the miRNAs screened on the osteogenie differentiation. Methods Four beagles were used to cuhure and isolate primal＇ BMSCs. The third generation BMSCs were divided into a control group in which the cells were cultured in basal medium and an experimental group in which the cells were induced by rhBMP-2 and cultured for 7 days. Alkaline phosphatase （ALP） staining was used to detect nsteogenic diffe,- entiation, and the Q-PCR was used to screen the differential miRNAs. One of the miRNAs screened （miR-125b） was randomly selected for transfection with Lipofectamine2000 and divided into 2 groups. In group 1 miR-i25b was over-expressed after transfection with miR-125b mimics while in group 2 miR-125b was transfected with mimics-NC to determine the effect of over-expression of miR-125b for 7 days on the osteogenicdifferentiation of the beagle BMSCs induced by rhBMP-2. Results Beagle BMSCs were successfully cultured. Q-PCR screening resulted in 41 miRNAs whose expression differences were more than 2 folds com- pared with the controls. Of them, 13 were up-regulated and 28 down-regulated. In group 1, the expression of miR-125b in the cells was up-regulated by 4. 13 folds compared with group 2 （ P 〈 0. 05） . ALP staining revealed that group 1 had obviously fewer deep blue large particles than group 2. Conclusions The screening of differential miRNAs during osteogenic differentiation of beagle BMSCs by Q-PCR is an effective method. Of the miRNAs screened, miR-125b can remarkably inhibit the osteogenic differentiation of beagle BMSCs.

关 键 词：骨髓 间充质干细胞 聚合酶链反应 骨形态发生蛋白类 

分 类 号：R3[医药卫生—基础医学]