miR-34a对脂肪干细胞移植治疗跟腱炎的影响

Effect of miR-34a expression on transplantation of adipose-derived stem cells for treatment of Achilles tendonitis

目的:探讨微小RNA-34a (miR-34a)的表达对人脂肪干细胞(hADSCs)移植治疗大鼠跟腱炎的影响。方法:CCK-8法检测h ADSCs被分别转染miR-34a mimic和miR-34a inhibitor 24 h、48 h、72 h和96 h后的细胞活力情况。注射胶原蛋白酶构建跟腱炎大鼠模型,建模后将大鼠随机分为5组:PBS注射对照组(PBS组)、h ADSCs治疗组(h ADSCs组)、转染阴性对照(NC)的h ADSCs治疗组(hADSCs+NC组)、转染miR-34a mimics的h ADSCs治疗组(h ADSCs+miR-34a组)和转染anti-sense miR-34a的h ADSCs治疗组(h ADSCs+anti-miR-34a组)。分离跟腱组织,检测跟腱组织的硬度、压力和最大负荷拉力等生物力学指标;利用RT-qPCR检测miR-34a及I型胶原蛋白(collagen I)、scleraxis (Scx)和生腱蛋白C(tenascin C,TNC) mRNA的表达水平; Western blot测定collagen I、Scx和TNC蛋白表达水平。结果:在体外过表达和敲减miR-34a可分别抑制和促进h ADSCs的活力; 5组大鼠跟腱组织生物力学测定结果显示,与PBS组相比,h ADSCs组大鼠跟腱组织硬度、压力和最大负荷拉力显著提高;与h ADSCs+NC组相比,h ADSCs+miR-34a组和h ADSCs+anti-miR-34a组分别显著降低和提高了上述生物力学指标。与PBS组相比,h ADSCs组大鼠collagen I、Scx和TNC的mRNA和蛋白表达水平上调;与h ADSCs+NC组相比,h ADSCs+miR-34a组miR-34a的表达水平升高,collagen I、Scx和TNC的mRNA和蛋白表达水平降低,而h ADSCs+anti-miR-34a逆转了上述趋势。结论:miR-34a通过调控脂肪干细胞活力和分化,影响跟腱炎的治疗效果。

AIM:To elucidate the role of microRNA-34a(miR-34a)in transplantation of human adipose-derived stem cells(hADSCs)for treatment of Achilles tendonitis.METHODS:The viability of hADSCs transfected with miR-34a mimic and miR-34a inhibitor for 24 h,48 h,72 h and 96 h was measured by CCK-8 assay.A rat model of collagenase-induced Achilles tendonitis was established.The rats with Achilles tendonitis were divided into 5 groups:PBS group,hADSCs group,hADSCs+NC group,hADSCs+miR-34a group and hADSCs+anti-miR-34a group.The tendon tissues were isolated to detect stiffness,stress and maximum loading tension by biomechanical evaluation and to assess miR-34a expression level as well as the expression of collagen I,scleraxis(Scx)and tenascin C(TNC)at mRNA and protein levels by RT-qPCR and Western blot.RESULTS:miR-34a over-expression and knockdown suppressed and enhanced the viability of hADSCs in a time-dependent manner,respectively.Moreover,stiffness,stress and maximum loading tension in hADSCs group were increased compared with PBS group,while these biomechanical indexes in hADSCs+miR-34a group and hADSCs+anti-miR-34a group were improved and reduced as compared with hADSCs+NC group,respectively.Furthermore,up-regulation of collagen I,Scx and TNC expression at mRNA and protein levels was observed in hADSCs group as compared with PBS group.Meanwhile,miR-34a expression was increased but the expression of collagen I,Scx and TNC at mRNA and protein levels declined in hADSCs+miR-34a group.In contrast,reversed effects on the trends mentioned above were observed in hADSCs+anti-miR-34a group.CONCLUSION:miR-34a affects therapeutic outcome for Achilles tendonitis by regulating the viability and differentiation of hADSCs.