【摘要】：目的:富血小板血漿(platelet-rich plasma,PRP )是全血通過離心獲得的富含血小板的血漿成分。因其來源于自體血,富含多種生長因子,生長因子具有自然配比,使其得以廣泛應用于組織修復與再生的基礎研究和臨床治療。骨髓間充質(zhì)干細胞(Bone marrow-derived mesenchymal stem cells,BMSCs )是組織工程領域重要的“種子細胞”。其具有自身增殖能力強、分化范圍廣,能夠修復損傷組織等功能特點,已經(jīng)大量應用于組織工程研究。近年來,PRP被證明能有效促進BMSCs的增殖及成骨、成脂分化。但是,目前對PRP組織修復作用的認識還較為粗淺,臨床應用也較為初步,尚缺乏對不同濃度PRP影響B(tài)MSCs增殖和分化的具體認識,也未探索出PRP促進BMSCs增殖和影響分化的最佳濃度,這些問題限制PRP的臨床精準使用。方法:本研究闡述了不同濃度 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)的制備方法,通過流式細胞術檢測了 PRP中血小板特異標記CD41a和CD42b的表達情況,CCK-8法檢測了 PRP對BMSCs增殖的影響。借助電鏡、油紅O染色,ALP染色、Von Kossa染色、RT-PCR法在形態(tài)、細胞、分子層面分析了不同濃度PRP對于BMSCs增殖和成骨、成脂分化的影響,繪制了較為詳細的作用曲線。結果:在BMSCs增殖方面,隨著濃度的升高,在低于1500～1800×109/L時,PRP促進BMSCs增殖呈現(xiàn)濃度依賴的遞增特性,在1800×109/L～3000×109/L區(qū)間達到平臺期,組間無顯著性差異。成骨分化方面,隨濃度升高,PRP影響成骨分化呈現(xiàn)先升高后降低的特性,峰值出現(xiàn)在1500×109/L左右,1800×109/L以上濃度較1500×109/L下降明顯,在成骨誘導劑協(xié)同下,濃度在1800×109/L～3000×109/L區(qū)間,促成骨作用逐漸回升,并在2500×109/L～3000×109/L再次達到峰值。成脂分化時,0～3000×109/L區(qū)間,PRP促進BMSCs向脂肪分化呈現(xiàn)濃度依賴的遞增特性,且2000×109/L以下作用效果較弱,2000×109/L～3000×109/L之間作用效果較明顯。成脂誘導劑聯(lián)合作用時,不同濃度間差異更為明顯。結論:PRP體外促BMSCs增殖的最優(yōu)濃度為1800×109/L,促進成骨分化的最優(yōu)濃度為1500×109/L,濃度低于2000×109/L時促進成脂肪分化作用較弱,大于2000×109/L作用較強。提示骨缺損、骨不連時可以選擇促成骨、增殖最佳,促成脂作用較弱的PRP濃度,即1500×109/L;整容領域應用時可選用促增殖、成脂較佳,而促成骨作用較弱的PRP濃度,即3000×109/L。本研究探索了不同濃度PRP對BMSCs增殖和分化的影響,分別獲得了促進BMSCs增殖、成骨、成脂分化的優(yōu)化濃度,進而為臨床上不同組織、不同疾病的PRP精準治療提供理論依據(jù)和參考方法。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R68

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