低氧诱导因子1α联合Scleraxis修饰人羊膜间充质干细胞促进腱骨愈合的体外实验

An in vitro study of enhancing tendon-bone healing by human amniotic mesenchymal stem cells co-modified with hypoxia-inducible factor 1 alpha and Scleraxis gene

背景:由于肌腱与骨的愈合缓慢而复杂,往往影响临床治疗效果,提高腱骨接触面细胞活性是损伤治疗的新策略。Scleraxis是肌腱细胞的特异性标志分子,低氧诱导因子1α(hypoxia inducible factor 1α,HIF-1α)可通过多种途径诱导血管和骨组织的形成。但HIF-1α能否增强Scleraxis诱导人羊膜间充质干细胞分化并用于腱骨损伤的修复还尚未研究。目的:探讨HIF-1α联合Scleraxis修饰人羊膜间充质干细胞在体外促进腱骨愈合的能力并研究其分子机制。方法:取第3代人羊膜间充质干细胞,分别经AdHIF-1α、AdScx、AdGFP和AdHIF-1α+AdScx腺病毒载体感染第3天和第7天后检测肌腱、软骨和骨组织相关基因的表达。结果与结论:(1)倒置相差显微镜观察显示,第3代人羊膜间充质干细胞呈长梭形、漩涡状贴壁生长;(2)透射电镜观察显示,人羊膜间充质干细胞呈椭圆形,结构清晰,胞浆含丰富的内质网及线粒体;(3)荧光显微镜观察显示,腺病毒感染24 h后,AdHIF-1α组表达红色荧光,荧光表达量约为50%,AdScx组和Ad GFP组均表达绿色荧光,荧光表达量约为70%;(4)实时荧光定量PCR显示,感染第3天和第7天时,AdHIF-1α+Ad Scx组、AdH IF-1α组和Ad Scx组Ⅰ型胶原、Fibronectin、RUNX2、VEGF和ALP的mRNA表达量均高于AdGFP组(P<0.05),AdHIF-1α+AdScx组Ⅰ型胶原、Fibronectin、RUNX2、VEGF和ALP的mRNA表达量均显著性高于AdScx组(P<0.05);(5)荧光免疫组化分析显示,AdHIF-1α+AdScx组感染第7天时Ⅰ型胶原表达量高于感染第3天;(6)结果表明,体外HIF-1α和Scleraxis基因联合修饰后可促进人羊膜间充质干细胞表达肌腱细胞、软骨细胞和骨细胞的标志分子,具有促进腱骨愈合的能力。

BACKGROUND:Tendon-to-bone healing is a complex and slow process,which often hinders the therapeutic outcomes.To enhance the tendon-to-bone healing,increasing cell bioactivity on the contact surface is a new strategy.Hypoxia-inducible factor 1α(HIF-1α)can induce the formation of blood vessel and bone tissue via many ways.However,it is unclear whether HIF-1αcan strengthen differentiation of human amniotic mesenchymal stem cells(hAMSCs)induced by Scleraxis,a specific marker of tendon,and can be used in tendon and bone repair.OBJECTIVE:To investigate the ability of HIF-1αin enhancing Scleraxis to modify hAMSCs aiming to promote tendon-to-bone healing and its molecular mechanism.METHODS:Passage 3 hAMSCs were infected with AdHIF-1α,AdScx,AdGFP and AdHIF-1α+AdScx.The expressions of related genes of tendon,cartilage and bone tissue were detected at 3 and 7 days after infection.RESULTS AND CONCLUSION:Under the inverted phase contrast microscopy,the passage 3 hAMSCs were in spindle shape and presented with vortex-like adherent growth.Under the transmission electron microscopy,hAMSCs were in oval shape and had clear structure,with abundant endoplasmic reticulum and mitochondria.Fluorescence photographs were taken at 24 hours after adenovirus infection,showing about 50%red fluorescence expression in the AdHIF-1αgroup,while about 70%green fluorescence expression in the AdScx and AdGFP groups.Real time-PCR results showed that at 3 and 7 days after infection,the mRNA expressions of collagen type I,Fibronectin,RUNX2,VEGF and ALP in the AdHIF-1α+AdScx group,AdHIF-1αgroup and AdScx group were higher than those in the AdGFP group(P<0.05);the mRNA expressions of collagen type I,Fibronectin,RUNX2,VEGF and ALP in the AdHIF-1α+AdScx group were significantly higher than those in the AdScx group(P<0.05).Fluorescence immunohistochemistry results showed that the expression of collagen type I in the AdHIF-1α+AdScx group at 7 days after infection was higher than that at 3 days after infection.To conclude,HIF-1αcan enhance the ability of Scleraxis to modify hAMSCs to promote tendon-to-bone healing by upregulating the expressions of molecular markers of tenocytes,chondrocytes and osteocytes.