不同浓度富血小板血浆（PRP）促进人骨髓间充质干细胞增殖和成脂成骨分化的实验研究

发布时间：2019-01-29 00:13

【摘要】：目的:富血小板血浆(platelet-rich plasma,PRP )是全血通过离心获得的富含血小板的血浆成分。因其来源于自体血,富含多种生长因子,生长因子具有自然配比,使其得以广泛应用于组织修复与再生的基础研究和临床治疗。骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells,BMSCs )是组织工程领域重要的“种子细胞”。其具有自身增殖能力强、分化范围广,能够修复损伤组织等功能特点,已经大量应用于组织工程研究。近年来,PRP被证明能有效促进BMSCs的增殖及成骨、成脂分化。但是,目前对PRP组织修复作用的认识还较为粗浅,临床应用也较为初步,尚缺乏对不同浓度PRP影响BMSCs增殖和分化的具体认识,也未探索出PRP促进BMSCs增殖和影响分化的最佳浓度,这些问题限制PRP的临床精准使用。方法:本研究阐述了不同浓度 PRP ( 200× 1 09/L, 500× 1 09/L, 800× 1 09/L, 1 000× 1 09/L,1200×109/L, 1500×109/L, 1800×109/L, 2000×109/L, 2500×109/L, 3000×109/L)的制备方法,通过流式细胞术检测了 PRP中血小板特异标记CD41a和CD42b的表达情况,CCK-8法检测了 PRP对BMSCs增殖的影响。借助电镜、油红O染色,ALP染色、Von Kossa染色、RT-PCR法在形态、细胞、分子层面分析了不同浓度PRP对于BMSCs增殖和成骨、成脂分化的影响,绘制了较为详细的作用曲线。结果:在BMSCs增殖方面,随着浓度的升高,在低于1500～1800×109/L时,PRP促进BMSCs增殖呈现浓度依赖的递增特性,在1800×109/L～3000×109/L区间达到平台期,组间无显著性差异。成骨分化方面,随浓度升高,PRP影响成骨分化呈现先升高后降低的特性,峰值出现在1500×109/L左右,1800×109/L以上浓度较1500×109/L下降明显,在成骨诱导剂协同下,浓度在1800×109/L～3000×109/L区间,促成骨作用逐渐回升,并在2500×109/L～3000×109/L再次达到峰值。成脂分化时,0～3000×109/L区间,PRP促进BMSCs向脂肪分化呈现浓度依赖的递增特性,且2000×109/L以下作用效果较弱,2000×109/L～3000×109/L之间作用效果较明显。成脂诱导剂联合作用时,不同浓度间差异更为明显。结论:PRP体外促BMSCs增殖的最优浓度为1800×109/L,促进成骨分化的最优浓度为1500×109/L,浓度低于2000×109/L时促进成脂肪分化作用较弱,大于2000×109/L作用较强。提示骨缺损、骨不连时可以选择促成骨、增殖最佳,促成脂作用较弱的PRP浓度,即1500×109/L;整容领域应用时可选用促增殖、成脂较佳,而促成骨作用较弱的PRP浓度,即3000×109/L。本研究探索了不同浓度PRP对BMSCs增殖和分化的影响,分别获得了促进BMSCs增殖、成骨、成脂分化的优化浓度,进而为临床上不同组织、不同疾病的PRP精准治疗提供理论依据和参考方法。
[Abstract]:Objective: platelet-rich plasma (platelet-rich plasma,PRP) is a platelet-rich plasma obtained from whole blood by centrifugation. Because it comes from autologous blood and is rich in many kinds of growth factors, it has a natural proportion, so it can be widely used in the basic research and clinical treatment of tissue repair and regeneration. Bone marrow mesenchymal stem cells (Bone marrow-derived mesenchymal stem cells,BMSCs) are important seed cells in tissue engineering. It has been widely used in tissue engineering because of its strong ability of proliferation, wide range of differentiation and ability to repair damaged tissue. In recent years, PRP has been proved to be effective in promoting the proliferation, osteogenesis and adipogenic differentiation of BMSCs. However, the current understanding of the role of PRP in tissue repair is relatively superficial, and its clinical application is relatively preliminary. There is a lack of specific understanding of the effects of different concentrations of PRP on the proliferation and differentiation of BMSCs, and the best concentration of PRP for promoting the proliferation and differentiation of BMSCs has not been explored. These problems limit the clinical accuracy of PRP. Methods: the preparation methods of PRP at different concentrations (200 脳 10 9 / L, 500 脳 10 9 / L, 800 脳 10 9 / L, 1 000 脳 10 9 / L, 1500 脳 10 9 / L, 1800 脳 10 9 / L, 2000 脳 10 9 / L, 2500 脳 10 9 / L, 3000 脳 10 9 / L) were described. The expression of platelet specific labeled CD41a and CD42b in PRP was detected by flow cytometry, and the effect of PRP on BMSCs proliferation was detected by CCK-8 assay. The effects of different concentrations of PRP on proliferation, osteogenesis and adipogenic differentiation of BMSCs were analyzed by means of electron microscope, oil red O staining, ALP staining, Von Kossa staining and RT-PCR method at the morphological, cellular and molecular levels. The effects of PRP on BMSCs proliferation, osteogenesis and adipogenic differentiation were plotted out in detail. Results: in the aspect of BMSCs proliferation, with the increase of concentration, PRP increased the proliferation of BMSCs in a concentration-dependent manner when the concentration was lower than 1500 脳 10 ~ (9) / L, and reached the plateau in the range of 1800 脳 109/L~3000 脳 10 ~ (9) / L, and there was no significant difference between the two groups. In osteogenic differentiation, PRP affected osteogenesis differentiation first and then decreased with the increase of concentration. The peak value was about 1500 脳 10 9 / L, and the concentration above 1800 脳 10 9 / L was significantly lower than that of 1500 脳 10 9 / L. In the range of 1800 脳 109/L~3000 脳 109 / L, the concentration of bone increased gradually and reached a peak at 2500 脳 109/L~3000 脳 109 / L. In adipogenic differentiation, PRP promoted the adipose differentiation of BMSCs in a concentration-dependent manner in the range of 3 000 脳 10 9 / L, and the effect below 2000 脳 10 9 / L was weak, and the effect between 2000 脳 109/L~3000 脳 10 9 / L was obvious. The difference between different concentrations was more obvious when combined with lipogenic inducer. Conclusion: the optimal concentration of PRP for BMSCs proliferation in vitro is 1800 脳 10 9 / L, the optimal concentration for promoting osteogenic differentiation is 1500 脳 10 9 / L, and when the concentration is below 2000 脳 10 9 / L, the effect of promoting adipogenic differentiation is weaker than 2000 脳 10 9 / L. The results suggest that PRP concentration, which is 1500 脳 109 / L, can be selected when bone defect and nonunion are caused by bone proliferation, and the concentration of PRP, which is 3000 脳 10 ~ 9 / L, which is weak in bone function, can be selected to promote proliferation and fat formation when applied in cosmetic surgery. In this study, the effects of different concentrations of PRP on the proliferation and differentiation of BMSCs were investigated, and the optimal concentrations of BMSCs proliferation, osteogenesis and adipogenic differentiation were obtained respectively. The accurate treatment of PRP for different diseases provides theoretical basis and reference method.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68

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