不同方法制备富血小板血浆对兔脂肪干细胞增殖和成骨分化能力的影响
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摘要
目的使用3种不同方法制备富血小板血浆(PRP),通过观察所得产物对脂肪干细胞(ADSCs)细胞增殖和成骨分化能力的影响,为PRP和ADSCs复合材料的应用提供理论基础。方法分别使用传统的二次离心法、三次离心法以及Tubex改良法制备PRP,运用血小板计数比较3种方法制备PRP的血小板采集率的大小;采用酶消化组织块法分离培养细胞并通过流式细胞术、成骨分化实验和成脂分化实验鉴定细胞;将5%浓度的PRP和ADSCs共培养,通过CCK-8法观察其对细胞增殖能力的影响;茜素红染色、碱性磷酸酶染色定量和RT-PCR方法观察其对细胞成骨分化能力的影响。结果血小板计数结果表明Tubex改良法制备的PRP血小板回收率明显高于其他两组;实验中获得的细胞表面特定抗原的表达具备成骨分化和成脂分化能力;ADSCs在培养21 d后,所有PRP处理组的ADSCs有矿化结节的形成,碱性磷酸酶活性也明显高于矿化诱导液处理组(P<0.05),RTPCR结果表明PRP处理细胞成骨分化标记基因的表达也明显高于矿化诱导组(P<0.05),Tubex法制备的PRP对ADSCs成骨分化的促进作用显著强于其他两种方法 (P<0.05)。结论 Tubex法制备PRP血小板回收率均要高于二次离心法和三次离心法,其对兔ADSCs的增殖、成骨分化的促进作用优于其他两种方法。通过Tubex密闭装置制备PRP,不仅操作简单,而且能够提高血小板的采集率,其内丰富的细胞因子有利于ADSCs的增殖和分化。
Objective Platelet-rich plasma( PRP) was prepared using three different methods. The effect of the obtained product on the cell proliferation and osteogenic differentiation of adipose stem cells( ADSCs) was observed,which provided a theoretical basis for the application of PRP and ADSCs composite materials. Methods PRP was prepared by using conventional secondary centrifugation,tertiary centrifugation,and Tubex modification,respectively. Platelet counts were evaluated to compare PRP platelet collection rates,and enzyme digestion was used to isolate cultured cells and identification of cells by flow cytometry,osteogenic differentiation assay,and adipogenic differentiation assay. The 5% concentration of PRP and ADSCs were co-cultured,and their effects on cell proliferation were observed by CCK-8 method;Alizarin red staining,alkaline phosphatase staining and RT-PCR methods were used to observe the effect of osteogenic differentiation. Results The results of platelet count showed that platelet recovery of PRP prepared by Tubex modified method was significantly higher than that of the other two groups. The expression of specific antigens on the cell surface obtained in the experiment had osteogenic differentiation and adipogenic differentiation ability. After ADSCs cultured for 21 days,ADSCs in all PRP treatment groups had the formation of mineralized nodules,and alkaline phosphatase activity was also significantly higher than that of mineralization inducer treatment group( P < 0. 05). RT-PCR results showed that the expression of osteogenic differentiation marker genes in PRP-treated cells was also significantly higher than that in the mineralization-induced group( P < 0. 05). The PRP prepared by Tubex method had a significantly stronger effect on osteogenic differentiation of ADSCs than the other two methods( P < 0. 05). Conclusion The platex recovery rate of platelets prepared from PRP is higher than that of secondary centrifugation and three centrifugation methods,and it is superior to the other two methods in promoting proliferation and osteogenic differentiation of rabbit ADSCs. The preparation of PRP by Tubex sealed device not only has simple operation,but also can increase the collection rate of platelets. The abundant cytokines in PRP facilitate the proliferation and differentiation of ADSCs.
Objective Platelet-rich plasma( PRP) was prepared using three different methods. The effect of the obtained product on the cell proliferation and osteogenic differentiation of adipose stem cells( ADSCs) was observed,which provided a theoretical basis for the application of PRP and ADSCs composite materials. Methods PRP was prepared by using conventional secondary centrifugation,tertiary centrifugation,and Tubex modification,respectively. Platelet counts were evaluated to compare PRP platelet collection rates,and enzyme digestion was used to isolate cultured cells and identification of cells by flow cytometry,osteogenic differentiation assay,and adipogenic differentiation assay. The 5% concentration of PRP and ADSCs were co-cultured,and their effects on cell proliferation were observed by CCK-8 method;Alizarin red staining,alkaline phosphatase staining and RT-PCR methods were used to observe the effect of osteogenic differentiation. Results The results of platelet count showed that platelet recovery of PRP prepared by Tubex modified method was significantly higher than that of the other two groups. The expression of specific antigens on the cell surface obtained in the experiment had osteogenic differentiation and adipogenic differentiation ability. After ADSCs cultured for 21 days,ADSCs in all PRP treatment groups had the formation of mineralized nodules,and alkaline phosphatase activity was also significantly higher than that of mineralization inducer treatment group( P < 0. 05). RT-PCR results showed that the expression of osteogenic differentiation marker genes in PRP-treated cells was also significantly higher than that in the mineralization-induced group( P < 0. 05). The PRP prepared by Tubex method had a significantly stronger effect on osteogenic differentiation of ADSCs than the other two methods( P < 0. 05). Conclusion The platex recovery rate of platelets prepared from PRP is higher than that of secondary centrifugation and three centrifugation methods,and it is superior to the other two methods in promoting proliferation and osteogenic differentiation of rabbit ADSCs. The preparation of PRP by Tubex sealed device not only has simple operation,but also can increase the collection rate of platelets. The abundant cytokines in PRP facilitate the proliferation and differentiation of ADSCs.
引文
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[9]陈博.血小板裂解液(platelet lysate,PL)对牙源性间充质干细胞的生物学影响[J].第四军医大学学报,2012,5(3):22-6.
[10]Shen J,Li S,Chen D.TGF-beta signaling and the development of osteoarthritis[J].Vasc Surg,2014,6(1):19-20.
[11]Marx R E.Platelet-rich plasma(PRP):what is PRP and what is not PRP[J].Implant Dent,2001,10(4):225-8.
[12]Harrison S,Vavken P,Kevy S,et al.Platelet activation by collagen provides sustained release of anabolic cytokines[J].Am J Sports Med,2011,39(4):729.
[2]Park A,Hogan M C,Kesturu G S,et al.Adipose-derived mesenchymal stem cells treated with growth differentiation factor-5 express tendon-specific markers[J].Tissue Eng Part A,2010,16(9):2941-51.
[3]Desai V D,Hsia H C,Schwarzbauer J E.Reversible modulation of myofibroblast differentiation in adipose-derived mesenchymal stem cells[J].PLo S One,2014,9(1):e86865.
[4]Neo P Y,See E Y,Toh S L,et al.Temporal profiling of the growth and multi-lineage potentiality of adipose tissue-derived mesenchymal stem cells cell-sheets[J].J Tissue Eng Regen Med,2016,10(7):564-79.
[5]Monaco E,Bionaz M,Hollister S J,et al.Strategies for regeneration of the bone using porcine adult adipose-derived mesenchymal stem cells[J].Theriogenology,2011,75(8):1381-99.
[6]Yu F,Dong Y,Yang Y W,et al.Effect of an experimental direct pulp-capping material on the properties and osteogenic differentiation of human dental pulp stem cells[J].Sci Rep,2016,6:34713.
[7]樊明山.富血小板血浆在皮肤抗衰老中作用机制的研究进展[J].中国美容医学杂志,2012,21(13):1874-6.
[9]陈博.血小板裂解液(platelet lysate,PL)对牙源性间充质干细胞的生物学影响[J].第四军医大学学报,2012,5(3):22-6.
[10]Shen J,Li S,Chen D.TGF-beta signaling and the development of osteoarthritis[J].Vasc Surg,2014,6(1):19-20.
[11]Marx R E.Platelet-rich plasma(PRP):what is PRP and what is not PRP[J].Implant Dent,2001,10(4):225-8.
[12]Harrison S,Vavken P,Kevy S,et al.Platelet activation by collagen provides sustained release of anabolic cytokines[J].Am J Sports Med,2011,39(4):729.
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