国际口腔医学杂志 ›› 2021, Vol. 48 ›› Issue (5): 520-527.doi: 10.7518/gjkq.2021075

• 论著 • 上一篇    下一篇

富血小板血浆和浓缩生长因子对人牙周膜细胞增殖和成骨分化影响的研究

刘娟(),陈斌,闫福华()   

  1. 南京大学医学院附属口腔医院·南京市口腔医院牙周病科 南京 210008
  • 收稿日期:2021-02-03 修回日期:2021-06-02 出版日期:2021-09-01 发布日期:2021-09-10
  • 通讯作者: 闫福华
  • 作者简介:刘娟,主治医师,硕士,Email: 1044042989@qq.com
  • 基金资助:
    国家自然科学基金(81771078);南京口腔疾病临床医学研究中心项目(2019060009);江苏省临床医学科技专项(BL2013002)

Effects of platelet-rich plasma and concentrated growth factor on the proliferation and osteogenic differentiation of human periodontal cells

Liu Juan(),Chen Bin,Yan Fuhua()   

  1. Dept. of Periodontology, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing 210008, China
  • Received:2021-02-03 Revised:2021-06-02 Online:2021-09-01 Published:2021-09-10
  • Contact: Fuhua Yan
  • Supported by:
    National Natural Science Foundation of China(81771078);Project of Nanjing Clinical Research Center for Oral Diseases(2019060009);Key Project of Science and Technology Bureau of Jiangsu Province(BL2013002)

摘要:

目的 研究富血小板血浆(PRP)和浓缩生长因子(CGF)对人牙周膜细胞(hPDLCs)增殖及成骨分化的影响。方法 分离、培养并鉴定hPDLCs;制备人全血、PRP和CGF,分别进行血小板计数;分别将全血(对照组)、PRP(PRP组)和CGF(CGF组)与hPDLCs共培养,CCK-8法检测细胞增殖能力,细胞划痕实验检测细胞迁移能力;使用1%、5%、10%的PRP和CGF分别对hPDLCs处理24、48、72 h,进行成骨诱导,Western blot法检测成骨相关转录因子Runx2、Osx、Dlx5和Msx2的相对表达量。结果 PRP、CGF可促进hPDLCs的增殖和迁移(P<0.05),提高促进成骨相关因子Runx2、Osx、Dlx5的表达并降低抑制成骨因子Msx2的表达(P<0.05)。结论 PRP、CGF能促进hPDLCs的增殖和迁移,并促进成骨分化相关转录因子的表达。

关键词: 富血小板血浆, 浓缩生长因子, 人牙周膜细胞, 细胞增殖, 成骨分化

Abstract:

Objective The study aimed to analyze the effects of platelet-rich plasma (PRP) and concentrated growth factor (CGF) on the proliferation and osteogenic differentiation of human periodontal membrane cells (hPDLCs). Metho-ds The hPDLCs were cultured and isolated from periodontal ligament tissue. PRP and CGF were prepared in accordance with the standard protocols, and platelet count was performed separately. The hPDLCs were cultured to three groups: whole blood (control group), PRP (PRP group), and CGF (CGF group) at various time points. Cell proliferation was detected by the CCK-8 method, and cell migration was detected by the cell scratch assay. The hPDLCs were treated with 1%, 5%, and 10% of PRP and CGF for 24, 48, and 72 h, respectively, for osteogenic induction. Expression levels of osteogenic transcription factors Runx2, Osx, Dlx5, and Msx2 were detected by Western blot. Results Both PRP and CGF could promote the proliferation and migration of the hPDLCs (P<0.05). The expression levels of osteogenic factors Runx2, Osx, and Dlx5 increased significantly; the expression of Msx2, which inhibited osteogenesis, decreased significantly (P<0.05). Conclusion PRP and CGF could promote the proliferation and migration of hPDLCs and induce the expression of osteogenic differentiation-related transcription factors.

Key words: platelet-rich plasma, concentrated growth factor, human periodontal ligament cells, cell proliferation, osteogenic differentiation

中图分类号: 

  • Q25

图1

全血制备CGF 全血经CGF程序离心后分为3层,中间层为CGF层。"

图2

hPDLCs的分离培养及鉴定 a:培养4~5 d可见细胞游出;b:细胞形态呈长梭形;c:结晶紫染色显示细胞呈漩涡状排列;d:抗波形丝蛋白染色阳性;e:抗角蛋白染色阴性;f:CCK-8法检测细胞增殖能力,生长曲线基本呈S形;g:矿化诱导后茜素红染色,显示矿化结节形成;h:成脂诱导后油红O染色,显示脂滴形成。"

表 1

全血、PRP和CGF的血小板计数"

样本编号 志愿者基本资料 血小板计数/(109·L-1
性别 年龄/岁 全血 PRP CGF
1 22 278 2 459
2 22 190 1 305
3 23 193 2 558
4 26 263 2 553
5 25 210 3 127
6 24 269 1 953
7 23 237 2 044

图3

PRP、CGF对hPDLCs增殖能力的影响 *:与对照组相比,P<0.05。"

图4

PRP、CGF对hPDLCs迁移能力的影响 结晶紫染色 上:对照组;中:PRP组;下:CGF组。"

图5

Western blot 法检测PRP、CGF对成骨相关转录因子Msx2、Osx、Dlx5、Runx2表达的影响 左:培养24 h;中:培养48 h;右:培养72 h。Con:对照组。"

图6

PRP、CGF对成骨相关转录因子Runx2、Dlx5、Osx、Msx2表达的影响 a:Runx2;b:Dlx5;c:Osx;d:Msx2。*:与对照组相比,P<0.05;#:与PRP组相比,P<0.05。"

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