富血小板血浆体外培养骨髓间充质干细胞的增殖与胶原产生

Proliferation and collagen production of bone marrow mesenchymal stem cells cultured in platelet-rich plasma in vitro

背景:间充质干细胞是可大量获取的肌腱组织工程种子细胞,但体外如何诱导其分化为功能化腱样细胞成为此项技术的关键。目的:观察富血小板血浆对体外培养骨髓间充质干细胞分裂增殖和胶原产生的影响。方法:分离培养兔骨髓间充质干细胞,并采用自体富血小板血浆对骨髓间充质干细胞进行干预(设立高、中、低剂量富血小板血浆组,并设空白对照组),每日计数细胞并绘制细胞生长曲线,用MTT比色法分析各组骨髓间充质干细胞的存活和增殖能力。以ELISA定量检测细胞的胶原产生,通过RT-PCR反应测定富血小板血浆干预前后细胞Ⅰ型胶原基因的表达。结果与结论:高、中、低浓度富血小板血浆组骨髓间充质干细胞均保持较高的增殖活性,呈快速生长,曲线上升幅度大,与空白对照组比较差异有显著性意义(P<0.05)。培养时间越长作用越明显,培养一定时间后其作用具有剂量依赖性,较高剂量的富血小板血浆对细胞的增殖作用较为明显。富血小板血浆能明显促进骨髓间充质干细胞的Ⅰ和Ⅲ型胶原合成,剂量越大,刺激胶原产生的作用越明显。说明骨髓间充质干细胞具备组织工程化肌腱种子细胞的基本条件,富血小板血浆具有促进间充质干细胞向肌腱细胞转化的作用。

BACKGROUND：Mesenchymal stem cells（MSCs） are seed cells that can be obtained greatly for tendon tissue engineering,but how to induce to differentiate into functional tendon-like cells in vitro is a key technology.OBJECTIVE：To explore the effect of platelet-rich plasma（PRP） on proliferation and collagen production of MSCs cultured in vitro.METHODS：Rabbit bone marrow mesenchymal stem cells（BMSCs） were isolated and cultured.Autologous PRP was used to intervene BMSCs.We set up PRP-high dose,PRP-moderate dose and PRP-low dose groups and a blank control group.Cells were counted and cell growth curve was drawn.Methyl thiazolyl tetrazolium assay was utilized to analyze BMSCs survival and proliferation ability in each group.Enzyme linked immunosorbent assay was employed to detect collagen.Reverse transcription-polymerase chain reaction was used to determine expression of type Ⅰ collagen gene prior to and following PRP intervention.RESULTS AND CONCLUSION：BMSCs had high proliferative activity,showing rapid growth and great curve upgrade range in the PRP-high dose,PRP-moderate dose and PRP-low dose groups,which showed significant difference as compared with blank control group（P 0.05）.Long culture time exhibited significant effect.Following culture for a while,its effect presented dose dependence.High dose of PRP had significant proliferative effect.PRP could obviously promote type Ⅰ and Ⅲ collagen synthesis in BMSCs.High dose showed significant effects on stimulating collagen production.These suggest that BMSCs had basic conditions of tissue engineered tendon seed cells.PRP can induce MSC to transform into tendon cells.