BMP4、IGF-I、TGF-β3和睾酮对鹿茸间充质细胞作用的研究

Effects of BMP4,IGF-I,TGF-β3 and Testosterone on Pilose Antler Reserve Mesenchymal Cells

目的 研究骨形态发生蛋白4(bone morphogenetic protein-4,BMP4)、胰岛素样生长因子-I(insulin-like growth factor-I,IGF-I)、转移生长因子-β3(transforming growth factorβ3,TGF-β3)、睾酮对鹿茸间充质细胞增殖和分化的作用,探讨鹿茸生长的机理。方法 体外分离培养鹿茸间充质细胞,采用MTT法测定BMP4、IGF-I对不同代次鹿茸间充质细胞的增殖作用,以及与不同浓度的睾酮组合对鹿茸间充质细胞增殖活性的影响;采用免疫荧光、阿尔新蓝染色、实时荧光PCR法检测分化诱导培养7、14、21 d的鹿茸间充质细胞中II、X、I型胶原蛋白(Collagen type II、Collagen type X、Collagen type I,COLII、COLX、COLI)和聚集蛋白聚糖的变化,考察BMP4、TGF-β3和睾酮对鹿茸间充质细胞向软骨分化的影响。结果 BMP4可提高鹿茸间充质细胞的增殖活性,且明显高于IGF-I,并与IGF-I有协同促进作用。早期、传代次数较少的幼嫩鹿茸间充质细胞对BMP4较敏感。不同浓度的睾酮,均显著抑制了BMP4的促增殖作用,尤其是生理浓度睾酮抑制作用最强。软骨诱导分化培养3 d鹿茸间充质细胞扁平膨大,形态发生明显变化,7 d COLII免疫荧光试验均阳性;14 d细胞集聚呈团,阿尔新蓝染色阳性;21 d BMP4提升COLII表达,具有一定的促进鹿茸间充质细胞成软骨分化的作用,并与睾酮、TGF-β3有协同促进作用,BMP4单独作用主要表现在提升COLX、COLI的表达量,促细胞肥大、成骨分化;生理浓度的睾酮不影响COLX表达,但降低了COLI的表达量;TGF-β3能诱导COLII表达,降低了COLX、COLI的表达量,显著减弱BMP4的促软骨细胞肥大和促成骨分化作用,而睾酮对TGF-β3的这个抑制作用无明显影响。结论 BMP4、IGF-I、TGF-β3和睾酮在鹿茸增殖、分化为软骨细胞、软骨内成骨的不同生长发育阶段中发挥着相互协同或抑制的作用。

Objective To investigate the mechanism of pilose antler growth by studying the effects of bone morphogenetic protein-4(BMP4),insulin-like growth factor-I(IGF-I),transforming growth factor beta 3(TGF-β3)and testosterone on the proliferation and differentiation of pilose antler reserve mesenchymal cells(RMCs).Methods RMCs was isolated and cultured in vitro.MTT assay was used to determine the effects of BMP4 and IGF-I on different generations of RMCs,and the effects of combination with different concentrations of testosterone on RMC proliferation.Immunofluorescence,Alcin blue staining and real-time PCR were used to detect the changes of collagen type II,collagen type X,collagen type I(COLII、COL X、COL I)and aggrencan in RMCs cultured for 7,14 and 21 days after differentiation induction.The effects of BMP4,TGF-β3 and testosterone on the chondrogenic differentiation of RMC were investigated.Results BMP4 could increase the proliferation activity of RMCs,which was significantly higher than IGF-I,and had synergistic promoting effect with IGF-I.The young RMCs in early stage with less passage times was more sensitive to BMP4.The proliferation promoting effect of BMP4 was significantly inhibited by different concentrations of testosterone,especially by physiological concentration of testosterone.After 3 days of chondrogenic differentiation culture,RMCs was flattened and expanded with significant morphological changes.On day 7,the immunofluorescence test of COLII was positive.On day 14,the cells were clustered and alcin blue staining was positive.BMP4 could promote the chondrogenic differentiation of antler mesenchymal cells by enhancing the expression of COL II on day 21,and it was also associated with testosterone and TGF-β3 had synergistic promoting effect.The effect of BMP4 alone was mainly manifested in increasing the expressions of COL X and COL I,promoting cell hypertrophy and osteogenic differentiation.The physiological concentration of testosterone did not affect the expression of COL X,but decreased the expression of COL I.TGF-β3 could induce the expression of COL II,reduce the expression levels of COL X and COL I,and significantly weaken the effect of BMP4 on promoting chondrocyte hypertrophy and promoting bone differentiation,while testosterone had no significant effect on the inhibitory effect of TGF-β3.Conclusion BMP4,IGF-I,TGF-β3 and testosterone play synergistic or inhibitory roles in different growth and development stages of pilose antler proliferation,differentiation into chondrocytes and endochondral ossification.