人脐带间充质干细胞联合富含血小板血浆促进大鼠跟腱损伤的修复  

Synergistic effect of human umbilical cord mesenchymal stem cells and platelet-rich plasma on promoting healing of Achilles tendon injury in rats

作　　者：王位[1] 付宇翀[1] 周梅[1] 唐红[1] 张吉强[2] 唐康来[1] 

机构地区：[1]第三军医大学西南医院骨科全军矫形外科中心,重庆400038 [2]第三军医大学基础医学部神经生物学教研室,重庆400038

出　　处：《第三军医大学学报》2017年第2期185-191,共7页Journal of Third Military Medical University

基　　金：全军医学科研"十二五"计划(创新专项重点13CXZ008)~~

摘　　要：目的探讨不同浓度富含血小板血浆(platelet-rich plasma,PRP)在体外对人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,h UC-MSCs)增殖、分化的影响,评估h UC-MSCs联合PRP对大鼠跟腱损伤修复的影响。方法 PRP活化后成为PRP-clot releasate(PRCR)。取P3代h UC-MSCs于培养皿中培养24 h后,分别加入含0%(对照组)、10%、20%、40%PRCR的DMEM/F12培养基,于第1、2、3、4天测定h UC-MSCs的增殖速度。P3代h UC-MSCs按相同分组培养,于第1、3、5天收集细胞并提取蛋白。Western blot检测h UC-MSCs中成肌腱分化标志性基因肌腱蛋白C(tenascin c,TNC)、SCX(scleraxis)、Ⅰ型胶原的表达。选取48只8周龄雄性SD大鼠,Ⅰ型胶原酶溶液50μL注射于大鼠右侧跟腱制备跟腱损伤模型,3 d后于损伤部位再次分别注射50μL生理盐水(对照)、h UC-MSCs悬液、PRP或PRP+h UC-MSCs混悬液,术后2、4周安乐死大鼠,Western blot检测跟腱中TNC、SCX及Ⅰ型胶原的表达,跟腱切片HE染色行组织学评估。结果 PRCR在体外对h UC-MSCs增殖均有促进作用,20%PRCR组促增殖作用最显著(P<0.05);20%PRCR组较对照组在体外可以显著地促进h UCMSCs成肌腱分化标志性基因的表达(P<0.05);术后2、4周,PRP+h UC-MSCs组跟腱中成肌腱分化相关基因在蛋白水平表达更高,跟腱切片HE染色显示PRP+h UC-MSCs组纤维排列更规整,炎细胞更少。结论适宜浓度PRP在体外显著促进了h UC-MSCs的增殖、成肌腱分化;PRP联合h UC-MSCs能有效促进大鼠受损跟腱的修复。Objective To determine the effect of different concentrations of platelet-rich plasma （PRP） on the proliferation and differentiation of human umbilical cord mesenchymal stem cells （HUC-MSCs） in vitro, and evaluate the effect of hUC-MSCs combined with PRP in the treatment of collagenase-induced Achilles tendon injury in rats. Methods PRP was activated to prepare into PRP-clot releasate （PRCR）. hUC-MSCs at passage 3 were incubated in control medium or medium supplemented with 10% , 20% or 40% PRCR, and the proliferation of hUC-MSCs on days 1, 2, 3 and 4 was evaluated. Tenogenesis related genes （ scleraxis, tenascin-C, and collagen type Ⅰ） were assessed by Western blotting on days 1,3 and 5. In 2 and 4 weeks after hUC-MSCs, PRP, hUC-MSCs ＋ PRP or saline （ as control） injection into the injured Achilles tendon, the tendon tissue was harvested and evaluated by histology and Western blotting. Results PRCR promoted the proliferation of hUC-MSCs in vitro, and 20% PRCR prominently increased the number of hUC- MSCs （P 〈0. 05 ） as well as the protein levels of collagen type Ⅰ , tenascin-C and scleraxis （P 〈 0. 05 ）. Combination of hUC-MSCs and PRP resulted in better healing effect of the injured tendon by improving histological characteristics and increasing expression of tenogenesis related genes in 2 and 4 weeks after operation. Conclusion Appropriate concentration of PRP promotes the proliferation and tenogenic differentiation of hUC-MSCs. Combination of hUC-MSCs and PRP has synergistic effect on promoting healing of the injured Achilles tendon.

关 键 词：人脐带间充质干细胞 富含血小板血浆 大鼠模型 跟腱损伤 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R686.1[医药卫生—基础医学]