富血小板血浆对脂肪干细胞体外增殖及成脂分化的影响

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摘要目的：研究不同体积分数富血小板血浆（PRP）对脂肪干细胞（ASCs）体外增殖及成脂分化的影响。方法：采用酶消化组织块法分离培养ASCs，有限稀释法克隆纯化，检测细胞表面标志物并进行鉴定。改良两步离心法从人全血中制备PRP。利用细胞计数试剂盒（CCK-8）检测在培养基中分别添加不同体积分数（0、10%、20%、30%）PRP对ASCs增殖的影响。反转录聚合酶链反应（RT-PCR）检测在成脂诱导培养基中添加不同体积分数PRP对ASCs成脂分化相关基因过氧化物酶体增殖体激活受体γ（PPARγ）、脂蛋白脂肪酶（LPL）、脂肪分化相关蛋白（adipophilin）表达的影响。结果：CCK-8结果显示，培养基中添加PRP显著促进了ASCs的增殖（P＜0.01）；与10% PRP相比，20% PRP或30% PRP更好地促进了ASCs的增殖（P＜0.01），而20% PRP与30%
PRP相比，差异无统计学意义（P＞0.05）。RT-PCR结果显示，成脂诱导培养基中添加PRP时，PPARγ、LPL、ad-ipophilin表达均明显升高（P＜0.05），而加入PRP的体积分数为10%、20%、30%时，对PPARγ、adipophilin表达的影响差异无统计学意义（P＞0.05），以20% PRP对LPL表达的促进作用最为明显（P＜0.01）。结论：PRP可以促进ASCs体外增殖及成脂分化，对脂肪组织再生有重要研究价值。

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	李锋
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关键词 ：富血小板血浆, 脂肪干细胞, 细胞增殖, 成脂分化

Abstract：Objective: To evaluate the effects of platelet-rich plasma (PRP) on the proliferation and adipogenic differentiation of adipose-derived stem cells (ASCs) in vitro. Methods: ASCs were isolated and cultured from human free fat tissue. The surface molecular markers of ASCs were determined with flow cytometry. PRP was prepared from human whole blood. Cell count kit-8 (CCK-8) and reverse transcription polymerase chain reaction (RT-PCR) were used to evaluate the influence of PRP (0, 10%, 20%, and 30%) in medium on ASC proliferation and adipogenic differentiation, respectively. Results: The platelet count in PRP was over 5 times of that in whole blood. CCK-8 showed that medium supplemented with PRP promoted ASCs proliferation significantly and the stimulatory potency of 20% PRP was the greatest. RT-PCR detected upregulated adipogenic-related genes, such as peroxisome proliferator-activated receptor-γ (PPARγ), lipoprotein lipase (LPL) and adipophilin. Conclusion: These results indicates PRP facilitated the proliferation and adipogenic differentiation of ASCs, which is of great value for adipose tissue engineering.

Key words： platelet-rich plasma adipose-derived stem cells cell proliferation adipogenic differentiation

收稿日期: 2016-05-31

基金资助:国家自然科学基金资助项目（81500817）；浙江省自然科学基金资助项目（LY15H140008）；温州市公益技术研究医学项目（Y20140708）；浙江省医药卫生科技计划一般项目（2016KYB184）。

通讯作者: 邓辉，副教授，副主任医师，Email：dh0726@163.com。

作者简介: 李锋（1985-），男，山东泰安人，主治医师，博士。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2017/V47/I1/7

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