miR?125b调控Runx2/Osx表达对骨髓间充质干细胞成骨机制的影响
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摘要
目的分析miR-125b调控成骨细胞特异性转录因子Runx2/Osx影响。方法 40只雌性大鼠随机分为对照组、骨折模型组、骨折模拟物组、骨折抑制剂组各10只,建立大鼠股骨干闭合骨折模型(对照组除外),造模后分离培养BMSCs,7 d后骨折模拟物组转染miRNA-125b模拟物(mimic),骨折抑制剂组转染miRNA-法检测成骨基因[骨钙素(OCN)骨桥蛋白(OPN)、Osx、Runx2蛋白]、成脂基因[过氧化物酶体增殖物激活受体(PPARγ)]的表达。结果 qPCR显示miR-125b在骨折模型BMSCs中表达上调[(1.48±0.18)vs(0.95±0.12),P<0.05];转染7 d时,骨折抑制剂组细胞质出现红染脂滴细胞,骨折模拟物组在第14天时出现一定脂肪细胞形态特征。骨折模拟物组Runx2、OCN、Osx分别为(0.20±0.03)、(0.45±0.05)、(0.34±0.04),均低于其他3组,而PPARγ(0.51±0.05)高于其他3组,骨折模拟物组OPN(0.52±0.06)低于骨折抑制剂组(0.88±0.02),差异有统计学意义(P<0.05),而与对照组、骨折模型组比较差异无统计学意义(P>0.05)。结论 miR-125b可通过调控Runx2/Osx而负性调节OCN表达,正性调节PPARγ、OPN的表达,从而调控BMSCs成骨能力。
ObjectiveTo analyze the effect of miR-125 b regulating the expression of Runx2/Osx on osteogenesis mechanism of bone mesenchymal stem cells(BMSCs).MethodsForty female rats were randomly divided into control group,fracture model group,fracture model with mimic group,fracture model with inhibitor group(10 rats per group). The rat models of femoral shaft closed fractures were established in the other three groups except control group. BMSCs were isolated and cultured after successful modeling. After 7 days,fracture model with mimic group was transfected with miR-125 b mimic. The fracture model with inhibitor group was transfected with miRNA-125 b inhibitor. The morphology,expression and proliferation of BMSCs were observed. The expressions of osteogenesis gene [osteocalcin(OCN) osteopontin(OPN),Osx,Runx2 protein]and lipogenic gene[peroxisome proliferator-activated receptor γ(PPARγ)] were detected by Western blot.ResultsqPCR showed that the expression of miR-125 b was up regulated in BMSCs of the fracture model[(1.48±0.18)vs(0.95±0.12)](P<0.05). Red stained lipid droplet cells appeared in the cytoplasm of fracture model with inhibitor group at 7 days after transfection. In fracture model with mimic group,morphological characteristics of adipocytes appeared on the 14 th day. Runx2,OCN and Osx in fracture model with mimic group[(0.20±0.03),(0.45±0.05),(0.34±0.04)]were lower than those in the other three groups,while PPARγ(0.51±0.05)was higher than that in the other three groups. OPN in fracture model with mimic group was lower than that in fracture model with inhibitor group[(0.52±0.06)vs(0.88±0.02)](P < 0.05). But there was no significant difference between control group and fracture model group(P>0.05).ConclusionThe miR-125 b may negatively regulate the expression of OCN by regulating Runx2/Osx,and positively regulate the expression of PPARγ and OPN,thus regulate the osteogenesis of BMSCs.
ObjectiveTo analyze the effect of miR-125 b regulating the expression of Runx2/Osx on osteogenesis mechanism of bone mesenchymal stem cells(BMSCs).MethodsForty female rats were randomly divided into control group,fracture model group,fracture model with mimic group,fracture model with inhibitor group(10 rats per group). The rat models of femoral shaft closed fractures were established in the other three groups except control group. BMSCs were isolated and cultured after successful modeling. After 7 days,fracture model with mimic group was transfected with miR-125 b mimic. The fracture model with inhibitor group was transfected with miRNA-125 b inhibitor. The morphology,expression and proliferation of BMSCs were observed. The expressions of osteogenesis gene [osteocalcin(OCN) osteopontin(OPN),Osx,Runx2 protein]and lipogenic gene[peroxisome proliferator-activated receptor γ(PPARγ)] were detected by Western blot.ResultsqPCR showed that the expression of miR-125 b was up regulated in BMSCs of the fracture model[(1.48±0.18)vs(0.95±0.12)](P<0.05). Red stained lipid droplet cells appeared in the cytoplasm of fracture model with inhibitor group at 7 days after transfection. In fracture model with mimic group,morphological characteristics of adipocytes appeared on the 14 th day. Runx2,OCN and Osx in fracture model with mimic group[(0.20±0.03),(0.45±0.05),(0.34±0.04)]were lower than those in the other three groups,while PPARγ(0.51±0.05)was higher than that in the other three groups. OPN in fracture model with mimic group was lower than that in fracture model with inhibitor group[(0.52±0.06)vs(0.88±0.02)](P < 0.05). But there was no significant difference between control group and fracture model group(P>0.05).ConclusionThe miR-125 b may negatively regulate the expression of OCN by regulating Runx2/Osx,and positively regulate the expression of PPARγ and OPN,thus regulate the osteogenesis of BMSCs.
引文
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[2]Chen J,Qiu M,Dou C,et al.MicroRNAs in bone balance and os?teoporosis[J].Drug Dev Res,2015,76(5):235-245.
[3]Du X,Li X,Chen L,et al. Hepatic miR-125b inhibits insulin sig?naling pathway by targeting PIK3CD[J].J Cell Physiol,2018,233(8):6052-6066.
[4]黄科.miR-125b通过Cbfβ调节间充质干细胞成骨分化的分子机制研究[D].第三军医大学,2014.
[5]张苗苗,包翠芬,王艳,等.ZHX3基因沉默对BMSCs中成骨相关因子表达的影响[J].天津医药,2015,43(12):1356-1360.
[6]刘凯,李宗杰,吴海卫,等.重组人促红细胞生成素联合骨髓间充质干细胞移植对大鼠体外循环肾炎症反应的影响[J].医学研究生学报,2016,29(4):364-368.
[7]Grottkau BE,Yang X,Zhang L,et al.Comparison of Effects of Mechanical Stretching on Osteogenic Potential of ASCs and BM?SCs[J].Bone Res,2013,1(3):282-290.
[8]高璐,郑洪新,陈谊敬,等.补肾中药成分配伍调控Runx2、OSX对大鼠BMSCs成骨分化的影响[J].世界中西医结合杂志,2014,9(4):425-429.
[9]Hu HL,Nie ZQ,Lu Y,et al.Circulating miR-125b but not miR-125a correlates with acute exacerbations of chronic obstructive pulmonary disease and the expressions of inflammatory cytokines[J].Med,2017,96(51):e9059.
[10]李钧,童哲,魏勇,等.miR-125b靶向Smad4调控非创伤性股骨头坏死骨髓基质干细胞成骨分化与增殖的相关性[J].中国老年学杂志,2017,37(1):306-307.
[11]汪华清.miR-125b调控人骨髓间充质干细胞成骨机制的研究[D].第三军医大学,2015.
[12]陆细红,邓敏,贺洪辉,等.miR-125b通过靶向抑制Smad4调控骨髓间充质干细胞成骨分化[J].中南大学学报(医学版),2013,38(4):341-346.
[13]Wu R,Wang N,Li M,et al.Experimental study on the facilita?tive effects of miR-125b on the differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells[J].Cell Biology International,2013,37(8):812-819.
[14]吕本浩,李劲峰,马源,等.双基因重组载体转染乙醇诱导的兔干细胞对其成脂与成骨基因表达的影响[J].中华实验外科杂志,2017,34(12):2187-2190.
[15]安敏.小鼠成骨样细胞和骨样细胞在体外成脂分化的实验研究[D].遵义医学院,2015.
[16]代光明,任磊,陈虹,等.下调骨细胞TGF-β/Smad4信号可抑制小鼠BMSCs成骨及破骨分化[J].基础医学与临床,2017,37(6):786-791.
[17]Fu X,Li Y,Huang T,et al.Runx2/Osterix and Zinc Uptake Syn?ergize to Orchestrate Osteogenic Differentiation and CitrateCon?taining Bone Apatite Formation[J].Adv Sci,2018,28,5(4):1700755.