富血小板血浆对体外培养条件下人真皮成纤维细胞增殖能力的影响

J4 ›› 2011, Vol. 37 ›› Issue (1): 84-88.

富血小板血浆对体外培养条件下人真皮成纤维细胞增殖能力的影响

王悦¹|马英智^2,3|朱喆³|苏学今³| 周延民¹

1．吉林大学口腔医院种植中心|吉林长春 130021；2．空军航空大学门诊部|吉林长春130022；3．吉林大学白求恩医学院病理生理学教育部重点实验室|吉林长春130021

收稿日期:2010-10-12 出版日期:2011-01-28 发布日期:2011-01-28
通讯作者: 周延民（Tel: 0431-88796025，E-mail:wangyue0431@gmail.com） E-mail:wangyue0431@gmail.com
作者简介:王悦（1971-）|女|江苏省武进市人|医学博士|主要从事富血小板血浆的基础及临床应用研究。
基金资助:
吉林省科技厅科研基金资助课题（200804423）

Effect of platelet-rich plasma on proliferation of human dermal fibroblasts in vitro

WANG Yue¹| MA Ying-zhi^2,3|ZHU Ze³|SU Xue-jing³|ZHOU Yan-min¹

1．Department of Implant Center|Stomatology Hospital|Jilin University|Changchun 130021|China；2．Clinic of Navigate Air Force University|Changchun 130022|China；3．Key Laboratory of Pathophysiology|Ministry of Education|Norman Bethune College of Medicine|Jilin University|Changchun 130021|China

Received:2010-10-12 Online:2011-01-28 Published:2011-01-28

摘要/Abstract

摘要：

目的：探讨富血小板血浆（PRP）对人真皮成纤维细胞（hDFbs）在体外培养条件下增殖能力的影响，探讨PRP促进皮肤、黏膜伤口愈合的机制。方法： PRP和hDFbs来源于健康成年人，两次离心法制备PRP，倒置相差显微镜观察0、12.5%、25.0%、50.0%和100.0%PRP浓度作用下 hDFbs的增殖；免疫细胞化学检测50.0%浓度不同剂量PRP作用下细胞血小板源性生长因子（PDGF）的表达；荧光染色技术观察PRP作用下hDFbs在纯钛材料表面的生长；流式细胞术检测PRP培养后不同时间hDFbs细胞周期；CCK-8法检测不同浓度PRP培养条件下细胞增殖活力。结果：倒置相差显微镜下见PRP各浓度组细胞数量均多于对照组，细胞数量增加、折光性增强；免疫细胞化学检测，30 μL PRP组PDGF表达量最高且细胞密度最大，但10 μL PRP组累积吸光度值(IOD)高于20 μL PRP组（836.27±21.15 vs 794.35±30.26，P<0.05）；荧光染色技术观察，PRP组材料表面hDFbs细胞密集，数量较对照组高；细胞周期检测，PRP促进细胞进入S期进行DNA复制，PRP作用后第2天PRP组S期细胞百分比高于空白组（34.41% vs 22.00% ，P<0.05），第8天PRP组G0/G1期细胞百分比高于空白组（95.07% vs 89.70% ，P<0.05）;CCK-8测定细胞增殖活性，100.0%PRP组吸光度A450值高于12.5%PRP组（34.41% vs 22.00%，P<0.05）。结论：高浓度的PRP虽然表现较强的促细胞增殖作用，但并不存在浓度、剂量依赖性，适宜浓度的PRP可促进hDFbs的增殖。

关键词: 人真皮成纤维细胞；富血小板血浆；细胞增殖；培养技术；细胞周期

Abstract:

Objective To evaluate the effects of platelet-rich plasma (PRP) on proliferation of human dermal fibroblasts (hDFbs) in vitro and investigate the mechanism of PRP promoting wound healing. Methods hDFbs and PRP were separated from a healthy adult donor. PRP was prepared by a double centrifugation technique. In the proliferation experiment ainverted phase contrast microscope was used to observe the growth of cells after treated with different concentrations PRP,such as 0,12.5%,25.0%,50.0% and 100.0%. Immunocytochemistry was used to evaluate the expressions of platelet-derived growth factor (PDGF) treated with 50.0% PRP in different volumes.Fluorescent coloration was used to observe the proliferation of the cells which adhered to the titanium material. Cell cycle of hDFbs was detected by flow cytometry. The cell proliferation was also evaluated by CCK-8 assay. Results The microscope investigation showed that the number of cells in 100%PRP group was higher than those in other groups. The significant increase in PDGF expression was observed when treated with 30 μL PRP，but the IOD in 10 μL PRP group was higher than that in 20 μL PRP group(836.27±21.15 vs 794.35±30.26，P<0.05). The fluorescent coloration showed that PRP improved the cell quantity on the material compared with control group. The flow cytometry showed that PRP stimulated the cells to enter the S phage to copy DNA. In the 2nd day after treated with PRP the percent of the cells in S phage in PRP group was higher than that in control group (34.41% vs 22.00%,P<0.05);In the 8th day the percent of the cells in G0G1 phage was higher than that in control group (95.07% vs 89.70%,P<0.05 ).The result of CCK-8 showed that the absorbency number in 100.0% PRP group was significantly higher than that in 12.5%group (1.13±0.05 vs 0.75±0.04,P<0.05) in the 4th day. Conclusion High concentration PRP has a positive influence on hDFbs proliferation, but it isn’t dose-dependent. An appropriate concentration PRP can promote the proliferation of hDFbs.

Key words: human dermal fibroblasts；platelet-rich plasma；proliferation;culture techniques;cell cycle

中图分类号:

R446.6

王悦, 马英智, 朱喆, 苏学今, 周延民. 富血小板血浆对体外培养条件下人真皮成纤维细胞增殖能力的影响[J]. J4, 2011, 37(1): 84-88.

WANG Yue, MA Ying-Zhi, ZHU Zhe, SU Xue-Jin, ZHOU Yan-Min. Effect of platelet-rich plasma on proliferation of human dermal fibroblasts in vitro[J]. J4, 2011, 37(1): 84-88.