富血小板纤维蛋白对人牙龈成纤维细胞增殖、迁移和分泌I型胶原的影响

Effects of Choukroun＇s platelet-rich fibrin on human gingival fibroblasts proliferation, migration and type I collagen secretion

目的体外研究富血小板纤维蛋白（platelet—richfibrin，PRF）对人牙龈成纤维细胞（humangingivalfibroblasts，HGF）增殖、迁移和I型胶原表达能力的影响。方法采用组织块培养法原代培养人牙龈成纤维细胞，采用Choukroun的方法制备PRF，将自体PRF与HGF共培养，分为PRFl组（含1片PRF膜）、PRF2组（含2片PRF膜）和空白对照组，每组设6个复孔，共培养1、3、5d后，甲基噻唑基四唑（methylthiazolyltetrazolium，MTＴ）法检测细胞毒性及增殖水平；酶联免疫吸附测定法检测培养上清液中I型胶原的分泌水平；制备PRF膜浸出液，分为PRFl组、PRF2组和空白对照组，每组设6个复孑L，Transwell系统检测PRF膜浸出液对HGF迁移的作用。结果细胞增殖实验显示，第1、3、5天PRFl组A值（0．615±0．036、0．686±0．006、0．693±0．004）和PRF2组A值（0．653±0．023、0．766±0．034、0．775±0．053）均显著高于空白对照组（0．514±0．020、0．544±0．006、0．545±0．009）（P〈0．01），PRFl组和PRF2组A值差异无统计学意义（P〉0．05）；各组内不同时间点问相比，随时间递增A值均显著增高（P〈0．01）。细胞迁移实验显示，PRFl组和PRF2组细胞迁移数[分别为（85．67±2．94）、（85．83±1．47）]均显著高于空白对照组（54．17±2．48）（P〈0．01）；PRFl组和PRF2组细胞迁移数差异无统计学意义（P〉0．05）；细胞分泌I型胶原实验显示，第l、3、5天PRFl组A值（0．184±0．004、0．200±0．004、0．204±0．009）和PRF2组A值（0．213±0．008、0．226±0．005、0．229±0．006）均显著高于空白对照组（0．174±0．002、0．184±0．002、0．186±0．003）（P〈0．01），PRFl组和PRF2组A值差异无统计学意义（P〉0．05）；各组内不同时间点间相比，随时间递增A值均显著增高（P〈0．01）。结论PRF对HGF的生物学行为具有显著促进作用，PRF与种子细胞HGF相结合，在治疗牙龈退缩及牙周组织工程中具有较大的临床应用潜能。

Objective To investigate the effect of platelet-rich fibrin （PRF）on human gingival fibroblasts （HGF） biological behavior such as proliferation, migration and collagen-1expression. Methods Human healthy gingival tissues were cultured according to the explant technique to obtain primary cultures. PRF was prepared by means of Choukroun＇ s. HGF were co-cultured with PRF membrane originating from the same donor as the explants, divided into three groups, PRF1 group, PRF2 group and blank control group. Methyl thiazolyl tetrazolium（MTI＇） assay was used for cytotoxicity and cell proliferation study, and enzyme-linked immunosorbent assay （ELISA） for collagen- I （ COL- I ） secretion study at the 1 st, 3rd, 5th day respectively. Eluates from PRF membrane was prepared, and divided into three groups, PRF1 group, PRF2 group and blank control group. Transwell chamber was utilized to determine the effect of PRF membrane eluate on cell migration. Results The A values of HGF in culture of the PRF1 （0. 615 ± 0. 036,0. 686 ± 0. 006,0. 693 ± 0. 004） and PRF2 groups （0. 653 ± 0. 023,0. 766 ± 0. 034,0. 775 ±_ 0. 053 ）were significantly higher than those of the control cultures （0. 514 ± 0. 020,0. 544 ± 0. 006,0. 545 ± 0. 009 ） （P 〈0. 01 ）, but the difference between PRFI and PRF2 group was not significant （P 〉 0. 05 ）. In each group at different time points, the HGF proliferation effect was significantly enhanced with time （P〈 0. 01）. Cell migration test showed that the migration numbers of HGF in PRF1 and PRF2 groups （ 85.67 ± 2. 94,85.83± 1.47） were significantly higher than those of the control group （54. 17± 2. 48） （ P 〈 0. 01 ）, but the difference between the two experimental groups was not significant （ P 〉 0. 05 ）. COL- I secretion test exhibited that the A values of COL- I in PRF1 （0. 184 ± 0. 004,0. 200 ± 0. 004,0. 204 ± 0. 009） and PRF2 group（0. 213 ±0. 008,0. 226 ±0. 005,0. 229 ±0. 006） were significantly higher than the A values of the control group （0. 174 ± 0. 002,0. 184 ± 0. 002, 0. 186 ± 0. 003 ） （P 〈 0. 01 ）, but the difference between the two experimental groups was not significant（ P 〉 0. 05 ）. In each group, the secretion level of COL- I increased significantly with time （ P 〈 0. 01 ）. Conclusions PRF could exert a positive effect on HGF biological behaviour and had clinical application potential in the treatment of gingival recession and in the periodontal tissue engineering when combined with seed cell HGF.