不同浓度TGF-β_3对体外肌腱干细胞分化的影响研究

EFFECTS OF TRANSFORMING GROWTH FACTOR β_3 AT DIFFERENT CONCENTRATIONS ON DIFFERENTIATION OF TEENDON STEM CELLS IN VITRO

目的探讨不同浓度TGF-β3对体外大鼠跟腱来源肌腱干细胞(tendon stem cells,TSCs)分化的影响。方法取3周龄雄性SD大鼠跟腱组织,采用酶消化法分离、培养TSCs,取第3代TSCs以2×103个/cm2密度接种于细胞培养皿中,培养24 h观察细胞完全贴壁后,分别采用5.0、2.5、1.0、0 ng/m L TGF-β3培养,于1、3、5 d收集细胞进行实时荧光定量PCR检测,观察成肌腱分化标志性基因Ⅰ型胶原、肌腱蛋白C(tenascin C,TNC)、腱调蛋白(tenomoduli n,TNMD)和Scx(scleraxis),成骨分化标志性基因成骨特异性转录因子2(Runt related transcription factor 2,Runx2)、ALP,成软骨分化标志性基因Sox9、Ⅱ型胶原,成脂肪分化标志性基因AP2、过氧化物酶增殖物活化受体γ(peroxisome proliferator-activated receptorγ,PPARγ)表达情况。结果不同浓度TGF-β3处理不同时间可导致TSCs向不同方向分化。TGF-β3促进TSCs向肌腱方向分化,与TGF-β3浓度、处理时间均相关,且两者存在交互作用(P<0.05)。TGF-β3短时间、低浓度刺激会抑制TSCs向非肌腱方向分化,而高浓度、长时间刺激会同时导致TSCs向成骨、成软骨等方向分化。结论 TGF-β3对TSCs分化的作用呈多样性,随浓度和时间改变而不同,可能是TSCs正常分化和异常分化之间的关键因素。

Objective To investigate the effect of transforming growth factor β3（TGF-β3） at different concentrations on the differentiation of rat Achilles-derived tendon stem cells（TSCs） in vitro.Methods TSCs were isolated from the tendon tissue of male Sprague Dawley rats（aged 3 weeks） by enzymatic digestion method and cultured for 3 passages.The TSCs were stimulated with TGF-β3 at the concentrations of 5.0,2.5,1.0,and 0 ng/m L.At 1,3,and 5 days,the m RNA expressions of tendogenic differentiation related genes [collagen type I,tenascin C（TNC）,tenomodulin（TNMD）,scleraxis（Scx）],osteogenic differentiation related genes [Runt related transcription factor 2（Runx2） and alkaline phosphatase（ALP）],chondrogenic differentiation genes（Sox9 and collagen type II）,and adipogenic differentiation genes [AP2 and peroxisome proliferator-activated receptor γ（PPARγ）] were measured by realtime quantitative PCR（q RT-PCR）.Results TSCs could differentiated in different directions after treated with TGF-β3at different concentrations at different time points.TGF-β3 was able to induce TSCs differentiated into tenocytes,which was related to the concentration and time of duration,and the two factors have interaction.Stimulation of TGF-β3 at low concentration and for short time could inhibit non-tendogenic differentiation of TSCs,but at high concentration and for long time,TGF-β3 enhanced TSCs differented into osteocytes or chondrocytes.Conclusion Effects of TGF-β3 on TSCs differentiation are complicated and depend on the concentration and time of duration,which may be a key factor between tendogenic and non-tendogenic differentiations of TSCs.