激活的富血小板血浆促进Pellet培养的脂肪间充质干细胞成软骨样分化

Effect of activated platelet-rich plasma on the chondrogenic differentiation of human adipose-derived stem cells

目的探讨激活的富血小板血浆(PRP)对Pellet培养的人脂肪来源间充质干细胞向软骨样细胞分化及相关信号通路的影响。方法添加不同比例(5%、10%和15%)的激活PRP,检测不同培养时间(1、3、5、7、9、11 d)脂肪干细胞的增殖能力。将采用Pellet培养的P3代的h ADSCs分为3组,对照组、激活PRP组和含TGF-β3的软骨诱导组,持续培养21 d后,阿利新蓝和苏木精-伊红染色进行软骨分化鉴定,real-time PCR测定Sox-9、Aggrecan和II型胶原的基因表达,DMMB法测定胞外基质中GAG含量,Western blot进一步检测对照组和激活PRP组中Sox-9、Gli-1和BMP-2蛋白的表达。结果 MTT实验结果显示,在10%激活PRP培养条件下,11 d的生长时间内hADSCs的增殖率最适宜。阿利新蓝和苏木精-伊红染色显示,激活PRP组和软骨诱导组中软骨表达呈阳性。Real-time PCR结果表明软骨诱导组其Sox-9、Aggrecan和II型胶原mRNA表达的能力高于激活PRP组(P<0.05)。GAG实验结果显示,软骨诱导组促GAG分泌的能力优于激活PRP组(P<0.05)。Western blot结果显示,相比对照组,激活PRP组中伴随Gli-1和BMP-2的蛋白表达升高,而Sox-9蛋白表达随之升高。结论激活PRP刺激hADSCs的最适增殖能力的浓度比例为10%。激活PRP和软骨诱导剂对脂肪干细胞成软骨分化中的诱导表达具有相似的能力,激活PRP对h ADSCs诱导成软骨作用与Hedgehog和BMP信号通路有关。

Objective To investigate the in vitro effect of activated platelet-rich plasma(PRP)on chondrogenic differentiation and related signaling pathway in human adipose-derived stem cells(hADSCs)in pellet culture system.Methods hADSCs were stimulated by different proportion of activated PRP(5%,10%and 15%)at different time points(1,3,5,7,9 and 11 d)to detect the proliferation ability.After the hADSCs were grown in pellet culture system to the 3rd generation,the cells were divided into 3 groups:the control group,the activated PRP group and the induction group containing TGF-β3 and cartilage induction agent.On the 21st day,the three groups were observed by Alcian blue and H&E staining.The expression level of glycosaminoglycan(GAG)in extracellular matrix was determined by dimethyl-methylene blue(DMMB)method.The expression of Sox-9,Aggrecan and type II collagen gene were assessed by RT-PCR.Sox-9,Gli-1 and BMP-2 from the control group and activated PRP group were detected by Western blot.Results Under the condition of 10%activated PRP,the proliferation rate of hADSCs was optimal within 11 days by MTT assay.By Alcian blue and H&E staining,positive expression of cartilage was observed in the activated PRP group and the chondrogenic induction group,and the ability to secrete GAG in the chondrogenic induction group was better than that of the activated PRP group(P<0.05).Gene expression of Sox-9,Aggrecan and type II collagen in the chondrogenic induction group was higher than that in the activated PRP group(P<0.05).Compared with the control group,the expression level of Gli-1 and BMP-2 in the activated PRP group was increased by Western blot,and the expression of Sox-9 was increased as well.Conclusion The optimal proportion for the proliferative capacity in hADSCs by activated PRP is 10%.The activated PRP and chondrogenic induction group have similar ability to induce chondrogenic differentiation of hADSCs,and the activated PRP could induce chondrogenesis,which may be related to the Hedgehog and BMP signaling pathway.