不同浓度PRP对骨骼肌干细胞成骨分化的影响

EFFECT OF VARIOUS CONCENTRATIONS OF PLATELET-RICH PLASMA ON OSTEOGENIC DIFFERENTIATION OF SKELETAL MUSCLE-DERIVED STEM CELLS

目的探讨不同浓度的PRP对体外培养的兔骨骼肌干细胞(skeletal muscle stem cells,SMSCs)诱导成骨的影响。方法1岁龄新西兰大白兔9只,体重2.5～3.0kg。从兔耳中央动脉取血参照Landesberg法制备PRP,并行全血和PRP中PLT计数。取兔右后肢比目鱼肌,体外培养兔SMSCs,取传至第3代的SMSCs,随机分为实验组与对照组。实验组加入含不同浓度(6.25%、12.50%、25.00%和50.00%)自体PRP的条件培养液进行干预,对照组不加PRP。于干预不同时间点行ALP染色观察、ALP活性测定、钙结节茜素红染色、骨钙素免疫荧光染色检测各组细胞成骨活性,RT-PCR检测成骨启动基因核心结合因子α1(core binding factor alpha1,Cbfa1)的表达。结果全血PRP及PLT计数为(3.06±0.46)×105个/μL和(18.08±2.10)×105个/μL;ALP染色观察示,培养7d各实验组细胞染色呈阳性,胞浆内可见大量黑色颗粒沉积;对照组细胞染色呈阴性。各实验组ALP活性均高于对照组(P<0.05),其中12.50%浓度组各时间点均高于其他浓度组(P<0.05)。培养14d各实验组经茜素红染色可见桔红色钙结节形成;对照组未见钙结节形成,矿化率为0。各实验组矿化率与对照组比较差异均有统计学意义(P<0.05),12.50%浓度组矿化率均高于其余浓度组(P<0.05)。培养7d各实验组骨钙素免疫荧光染色呈阳性,胞浆出现黄色荧光;对照组细胞染色呈阴性。RT-PCR检测示,培养4、12和24h对照组基因表达不同时间点无明显变化,实验组基因表达显著强于对照组,于12h时作用最明显,其中12.50%浓度组强于其他各浓度组。结论PRP能明显促进体外培养的兔SMSCs向成骨方向分化,其作用效果与PRP浓度有关,12.50%PRP作用效果较好。

Objective To investigate the effect of various concentration of platelet-rich plasma （PRP） on osteogenic differentiation of rabbit skeletal muscle-derived stem cells （SMSCs） cultured in vitro. Methods Blood drawn from the central ear arteries of 9 one-year-old New Zealand white rabbits weighing 2.5-3.0 kg （male and female） was used to prepare PRP （Landesberg method）. Full blood count and ptatelet count in PRP were tested. Soleus muscle of right hindlimb in rabbit was obtained and used to culture SMSCs in vitro. The cells at passage 3 were randomly divided into different groups： the experimental groups in which the cells were treated by conditioned culture media with various concentrations of autologous PRP （6.25%, 12.50%, 25.00%, 50.00%）, and the control group in which the cells were treated with the media without PRP. At different time points after intervention, osteogenetic activity of the cells was detected by ALP staining observation, ALP activity detection was conducted, alizarin red staining for calcium nodules and immunofluorescence staining for osteocalcin were performed, and core binding factor α1 （Cbfα1） of osteogenic gene expression was tested by RT-PCR. Results The full blood PRP count and the platelet count in PRP was （3.06 ± 0.46） × 10^5/μL and （18.08 ± 2.10） × 10^5/μL, respectively. ALP staining： the cells in all the experimental groups were positive for the staining with many black sediment particles in cytoplasm; the cells in the control group were negative staining. ALP activity： all the experimental groups were higher than the control group （P 〈 0.05）, the experimental group at 12.50% was superior to other experimental groups at each time point （P 〈 0.05）. Alizarin red staining： at 14 days after culture, orange-red calcium nodules were evident in all the experimental groups; no orange-red calcium nodules were observed in the control group with a mineralization rate of zero; there were significant difference between the experimental groups and the control group in terms of mineralization rate （P 〈 0.05）, the experimental group at 12.50% had a higher mineralization rate than other experimental groups （P 〈 0.05）. Immunofluorescence staining for osteocalcin： at 7 days after culture, the experimental groups were positive for the staining with yellow fluorescence in cytoplasm, and the result of the control group was negative. RT-PCR detection： no obvious changes of the gene expression were noted at 4, 12, and 24 hours after culture in the control group; the gene expression in all the experimental groups was significant superior to that of control group, especially at 12 hours, and the expression in the experimental group at 12.50% was the highest. Conclusion PRP can obviously promote the osteogenic differentiation of SMSCs cultured in vitro in a concentration-dependent manner, and the 12.50% is proved to be the ideal concentration.