富血小板纤维蛋白提取液对牙龈成纤维细胞增殖活性的影响

doi:10.12016/j.issn.2096-1456.2019.08.003

摘要/Abstract

摘要：

目的研究富血小板纤维蛋白提取液（platelet-rich fibrin extract, PRFe）及其释放的血小板衍生生长因子（platelet derived growth factor,PDGF)对牙龈成纤维细胞（human gingival fibroblasts, HGFs）增殖活性的影响,为其应用于辅助促进牙龈软组织增量提供实验依据。方法通过组织块培养法分离培养牙龈成纤维细胞,将收集的富血小板纤维蛋白（platelet-rich fibrin, PRF）转化为PRFe,电镜观察PRF三维结构并通过ELISA定量测定PRF中PDGF含量,并将PRFe配比为2.5%PRFe、5%PRFe、7.5%PRFe、10%PRFe、12.5%PRFe、15%PRFe,通过CCK8法检测不同浓度PRFe对牙龈成纤维细胞增殖活性的影响,确定最佳PRFe浓度后,流式细胞周期实验检测PRFe对细胞增殖周期的影响,并通过中和其释放的PDGF,观察PDGF对牙龈成纤维细胞增殖活性的影响。结果 PRF为三维网状结构,其间含有大量的生长因子,其中PDGF释放在第7天达到峰值;不同浓度PRFe对HGFs增殖活性结果显示,HGFs对PRFe呈浓度依赖性,但在5%PRFe浓度时效果最佳(P < 0.05),其后浓度增加对HGFs增殖影响差异无统计学差异（P > 0.05）;流式细胞周期实验结果表明,5%PRFe可以刺激牙龈成纤维细胞S期分裂增殖;而PDGF中和实验结果表明,中和部分PDGF对牙龈成纤维细胞的增殖呈抑制作用。结论 5%浓度的PRFe体外促进牙龈成纤维细胞的效果最佳,PRF所释放的PDGF在促进牙龈成纤维细胞的增殖中起着重要作用。

关键词: 富血小板纤维蛋白提取液, 血小板衍生生长因子, 牙龈成纤维细胞, 细胞增殖, 牙龈, 软组织增量

Abstract:

Objective To study the effects of platelet-rich fibrin extract (PRFe) and platelet-derived growth factor (PDGF) released from PRFe on the proliferation of human gingival fibroblasts (HGFs) and to provide an experimental basis for its application in promoting gingival soft tissue increment.Methods Platelet-rich fibrin (PRF) was transformed into PRFe by tissue culture. The three-dimensional structure of PRF was observed by electron microscopy, and the content of PDGF in PRF was quantitatively determined by ELISA. The ratios of PRFe examined were 2.5% PRFe, 5% PRFe, 7.5% PRFe, 10% PRFe, 12.5% PRFe and 15% PRFe. Gingival fibrosis was detected by the CCK-8 method. After determining the optimal concentration of PRFe, flow cytometry was used to detect the effect of PRFe on the proliferation cycle of human gingival fibroblasts, and the effect of PDGF on the proliferative activity of gingival fibroblasts was observed by neutralizing the release of PDGF.Results PRF is a three-dimensional reticular structure that contains a large number of growth factors. PDGF release peaked on the 7th day. The proliferative activity of HGFs cultured with different concentrations of PRFe was concentration-dependent, but the effect was optimal at 5% PRFe (P < 0.05). There were no significant differences in the effect of subsequent concentration increases on the proliferation of HGFs (P > 0.05). The flow cytometry results showed that 5% PRFe could significantly stimulate the S-phase division and proliferation of gingival fibroblasts, while the PDGF neutralization test showed that the proliferation of gingival fibroblasts was significantly inhibited by the neutralization of PDGF.Conclusion Overall 5% PRFe had the best effect on promoting gingival fibroblast proliferation in vitro. PDGF released from PRF plays an important role in promoting the proliferation of gingival fibroblasts.

Key words: platelet-rich Fibrin extract, PDGF growth factor, gingival fibroblasts, cell proliferation, gums, soft tissue increment

中图分类号:

R783.5

何家林, 徐燕, 谢贤哲, 王腾飞, 霍冬梅. 富血小板纤维蛋白提取液对牙龈成纤维细胞增殖活性的影响[J]. 口腔疾病防治, 2019, 27(8): 490-495.

HE Jialin, XU Yan, XIE Xianzhe, WANG Tengfei, HUO Dongmei. Effect of platelet-rich fibrin extract on the proliferation of gingival fibroblasts[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2019, 27(8): 490-495.

图/表 6

图1 牙龈成纤维细胞 × 200 a：第5天HGFs细胞呈梭形,排列疏松;b：第10天HGFs细胞密集,呈放射状排列

Figure 1 Gingival fibroblasts × 200

图2 抗Vimentin染色及抗Cytokeratin染色免疫荧光组化染色鉴定人牙龈成纤维细胞 × 400 a：抗波形蛋白抗体染色呈阳性,胞核为蓝色,胞浆为红棕色;b：抗角蛋白抗体染色呈阴性,胞核为蓝色,胞浆透明

Figure 2 Identification of human gingival fibroblasts by anti-vimentin staining and anti-cytokeratin immunohistochemical staining × 400

图3 富血小板纤维蛋白扫描电镜观察

Figure 3 Scanning electron microscopy of PRF

图4 富血小板纤维蛋白释放PDGF ELISA定量检测 PRF的PDGF水平在制备后第1个小时暴释,后逐渐降低,第1天后逐渐上升,第7天达到峰值

Figure 4 Quantitative detection of platelet-rich fibrin release by PDGF ELISA

表1 各时间点各组吸光度均值比较

Table 1 Comparison of mean absorbance values in each group at different time points $\bar{x}$ ± s

时间	对照组	2.5%PRFe	5%PRFe	7.5%PRFe	10%PRFe	12.5%PRFe	15%PRFe	F值	P值
1 d	0.417 ± 0.023	0.537 ± 0.008	0.574 ± 0.024	0.614 ± 0.025^1）	0.658 ± 0.069^1）	0.647 ± 0.01^1）	0.684 ± 0.014^1）	7.346	0.030
3 d	0.683 ± 0.022	1.301 ± 0.171^1）	1.461 ± 0.037^1）	1.457 ± 0.075^1）	1.41 ± 0.107^1）	1.478 ± 0.092^1）2)	1.502 ± 0.06^1)2）	52.553	0.028
5 d	0.932 ± 0.041	1.627 ± 0.051^1）	2.011 ± 0.024^1）2）	2.004 ± 0.024^1）2）	2.011 ± 0.031^1）2）	1.931 ± 0.019^1）2）	1.966 ± 0.048^1）2）	226.428	0.011

图5 中和PDGF对牙龈成纤维细胞增殖的影响注 *：P<0.05 vs 5%PRFe组

Figure 5 Effect of anti-PDGF on gingival fibroblast proliferation

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