富血小板血浆对人成骨样细胞MG63增殖能力的影响

doi:10.3969/j.issn.1000-1182.2012.02.006

华西口腔医学杂志

富血小板血浆对人成骨样细胞MG63增殖能力的影响

王悦1 刘春丽2 王静3 杨旭芳4 周延民1 马英智5

1.吉林大学口腔医院种植中心； 2.口腔颌面外科，长春130021；3.长春市南关区中医院口腔科，长春130041； 4.牡丹江医学院病理生理学教研室，牡丹江157011；5.空军航空大学飞行训练基地门诊部，长春130000

收稿日期:2012-04-25 修回日期:2012-04-25 出版日期:2012-04-01 发布日期:2012-04-01
通讯作者: 周延民，Tel：0431-88796025
作者简介:王悦（1971—），女，江苏人，博士
基金资助:
吉林省科技厅科研基金资助项目（200804423）

Effect of platelet rich plasma on the proliferation behavior of human MG63 osteoblast-like cells in vitro

Wang Yue1, Liu Chunli2, Wang Jing3, Yang Xufang4, Zhou Yanmin1, Ma Yingzhi5

1. Dept. of Implant Center, School of Stomatology, Jilin University, Changchun 130021, China; 2. Dept. of Oral and Maxillofacial Surgery, School of Stomatology, Jilin University, Changchun 130021, China; 3. Dept. of Stomatology, Nanguan Traditional Chinese Medicial Hospital of Changchun City, Changchun 130041, China; 4. Dept. of Pathophysiology, Mudanjiang Medical College, Mudanjiang 157011, China; 5. Clinic of Flight Training Base, Aeronautical University of Air Force, Changchun 130000, China

Received:2012-04-25 Revised:2012-04-25 Online:2012-04-01 Published:2012-04-01
Contact: Zhou Yanmin，Tel：0431-88796025
About author:Wang Yue（1971—），女，江苏人，博士

摘要/Abstract

摘要：

目的研究富血小板血浆（PRP）对人成骨样细胞MG63增殖能力的影响，探讨PRP的生物学功能。方法采集健康志愿者的静脉血，两次离心法制备PRP，氯化钙加人凝血酶激活后离心提取PRP萃取液。碱性磷酸酶（ALP）染色检测不同体积分数PRP（0、1%、2%、3%）对MG63分化活性的影响，碘化丙啶（PI）荧光染色观察PRP作用下MG63细胞形态的变化，免疫细胞化学检测PRP对MG63表达转化生长因子-β（TGF-β）的影响，扫描电子显微镜（SEM）观察PRP对MG63在人工骨材料表面生长情况的影响，CCK-8法检测PRP对MG63增殖活性的影响，流式细胞术（FCM）检测经PRP培养后不同时间MG63的细胞周期，逆转录聚合酶链反应（RT-PCR）检测PRP作用下MG63中Ⅰ型胶原（Col-Ⅰ）mRNA的表达量。结果ALP检测见3%PRP组ALP阳性细胞染色最深，并随体积分数的增加染色强度增加，且PRP组细胞均出现不同程度的脱片现象。PI荧光染色见PRP组细胞核荧光染色较对照组增强。免疫细胞化学检测见3%PRP组TGF-β表达量最高（P＜0.05）。SEM观察：PRP组材料表面MG63密集，生长状态良好。CCK-8法测定细胞增殖活性，显示4.8%PRP组吸光度A450 nm值高于对照组（P＜0.05）。FCM检测表明：PRP组第2天S期细胞百分比高于对照组（P＜0.05）；第10天PRP组G0/G1期细胞百分比高于对照组（P＜0.05）；除第6天外，PRP组G2/M期细胞百分比均高于对照组。RT-PCR结果表明：PRP组MG63的Col-ⅠmRNA表达量较对照组明显上调。结论适宜体积分数的PRP对MG63的增殖、迁移和相关蛋白、基因的表达有促进作用，PRP具有统一、协调细胞生物学行为的作用。

关键词: 富血小板血浆, 人成骨样细胞MG63, 细胞增殖

Abstract:

Objective To assess the effect of platelet rich plasma（PRP） on proliferation and differentiation of human MG63 osteoblast-like cells and the biological function of PRP in vitro. Methods PRP was obtained from venous blood of a health volunteer by two step centrifugation. CaCl2 and thrombin were used to activate PRP. The differentiation of MG63 cells, which were exposed to various concentrations of PRP（0, 1%, 2%, 3%） was detected by alkaline phosphatase（ALP） activity. Propidium iodide（PI） fluorescent coloration staining was used to observe the morphology of cells. Immunocytochemistry was used to evaluate the expression level of transforming growth factor-β（TGF-β） in MG63 cells in different concentration of PRP. The cells adhered to calcium phosphate material was observed by scanning electron microscopy（SEM）. The proliferation was evaluated by cell counting kit-8（CCK-8） proliferation assay. The cell cycle assay was performed by flow cytometry（FCM） to detect the effect of PRP on MG63 cells in different time points. The mRNA level of Col-Ⅰ in MG63 cells cultured under different concentration PRP was checked by reverse transcription polymerase chain reaction（RT-PCR）.Results ALP activity experiment demonstrated that the maximum effect was got in 3% PRP group. PRP had a positive effect on the proliferation of MG63 cells but cells also presented disengage phenomena from the glass slides. The PI staining showed that PRP improved fluorescent intensity of cell nucleus. Immunocytochemistry showed that TGF-β expression level was significantly enhanced on 3% PRP group（P＜0.05）. SEM showed that cells grew well on material in PRP group. The results of CCK-8 showed that the mean absorbency number A450 nm of 4.8% PRP was significantly higher than that of control group（P＜0.05）. FCM showed that S period cells percentage of PRP group was higher than that of control group in the 2nd day（P＜0.05）; G0/G1 period cells percentage of PRP group was significant increased than that of control group in the 10th day（P＜0.05）; G2/M period cells percentage of PRP group was higher than that of control group except the 6th day. PRP promoted the expression of Col-Ⅰ in MG63 cells by RT-PCR. Conclusion These data suggest that PRP has a positive influence on MG63 proliferation, transference and the expression of relative protein and gene in an appropriated concentration. The findings of this study also demonstrated that PRP may play a beneficial role of unifying and modulating the biological behavior of MG63 cells.

Key words: platelet rich plasma, human MG63 osteoblast-like cell, cell proliferation

王悦刘春丽王静杨旭芳周延民马英智. 富血小板血浆对人成骨样细胞MG63增殖能力的影响[J]. 华西口腔医学杂志, doi: 10.3969/j.issn.1000-1182.2012.02.006.

Wang Yue1, Liu Chunli2, Wang Jing3, Yang Xufang4, Zhou Yanmin1, Ma Yingzhi5. Effect of platelet rich plasma on the proliferation behavior of human MG63 osteoblast-like cells in vitro[J]. West China Journal of Stomatology, doi: 10.3969/j.issn.1000-1182.2012.02.006.

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富血小板血浆对人成骨样细胞MG63增殖能力的影响

Effect of platelet rich plasma on the proliferation behavior of human MG63 osteoblast-like cells in vitro

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