碱性成纤维细胞生长因子和血管内皮生长因子对人牙周膜干细胞体外增殖、迁移和黏附的影响

摘要

目的 研究碱性成纤维细胞生长因子(basic fibroblast growth factor,FGF-2)和血管内皮生长因子(vascular endothelial growth factor,VEGF)对体外培养的人牙周膜于细胞(periodontal ligament stem cells,PDLSC)增殖、迁移和黏附能力的影响,探讨两种生长因子促牙周再生的相关机制.方法 体外培养人PDLSC,分为A组:2％胎牛血清+α-最低必需培养基(α-minimum essential medium,α-MEM)(对照1组)；B组:20 μg/L FGF-2+A组；C组:10 μg/L VEGF+A组；D组:20 μg/L FGF-2+10 μg/L VEGF+A组；E组:10％胎牛血清+α-MEM(对照2组)；F组:20 μg/L FGF-2+E组；G组:10 μg/L VEGF+E组；H组:20 μg/L FGF-2+ 10 μg/L VEGF +E组,按分组要求施加刺激,第1、3、5、7天用可溶性噻唑盐法观察两种生长因子在不同体积分数的胎牛血清中对PDLSC增殖能力的影响；根据增殖实验结果重新分组:对照组:10％胎牛血清+ α-MEM;FGF-2组:20 μg/L FGF-2+对照组；VEGF组:10 μg/L VEGF+对照组；FGF-2+ VEGF组:20 μg/L FGF-2+ 10 μg/L VEGF+对照组,流式细胞仪、细胞黏附实验、划痕损伤愈合迁移实验观察细胞周期、迁移、黏附能力的变化.结果 在2％胎牛血清中,FGF-2和VEGF促增殖效果均不明显；在10％胎牛血清中,刺激第3、5、7天FGF-2组A值(分别为1.22±0.17、2.15±0.19、2.72 ±0.11)均显著高于对照组(分别为0.76 ±0.16、1.25±0.06、1.64 ±0.09) (P ＜0.01),但第5、7天A值均显著低于FGF-2+VEGF组(分别为2.46±0.17、3.18±0.27)(P＜0.05),第5、7天VEGF组A值(分别为1.66±0.05、2.13±0.13)显著高于对照组,但显著低于FGF-2组(P＜0.05)；流式细胞仪结果示VEGF组增殖指数[(34.3±2.0)％]显著低于FGF-2[(46.8±3.2)％]和FGF-2+VEGF组[(45.0±4.0)％],但显著高于对照组[(14.5±1.7)％](P＜0.01).细胞迁移实验结果示FGF-2组与对照组迁移至创伤区的细胞数比值差异无统计学意义(P＞0.05)；细胞黏附实验结果示FGF-2组黏附细胞数比值(79±4)显著高于VEGF组(62±4)(P＜0.05).光镜下观察示FGF-2组细胞在材料表面形态好于未加FGF-2组.结论 FGF-2和VEGF均可呈时间依赖性促进PDLSC增殖,二者联合应用有一定协同作用;FGF-2促PDLSC黏附能力较VEGF强；VEGF可促进细胞向创伤部位迁移.%Objective To evaluate the effects of basic fibroblast growth factor(FGF-2) and vascular endothelial growth factor (VEGF) on the proliferation,migration,and adhesion of human periodontal ligament stem cells(PDLSC) in vitro.Methods Human PDLSC were cultured in vitro using tissue culture method.The cells were cultured and incubated with various concentrations of FGF-2 and VEGF [A:α-MEM with 2％ fetal bovine serum(FBS) (control 1) ; B:A supplemented with 20 μg/L FGF-2 ; C:A supplemented with 10 μg/L VEGF;D:A supplemented with 20 μg/L FGF-2 and 10 μg/L VEGF; E:α-MEM with 10％ FBS(control 2) ; F:E supplemented with 20 μg/L FGF-2 ; G:E supplemented with 10 μg/L VEGF; H:E supplemented with 20 μg/L FGF-2 and 10 μg/L VEGF].Soluble tetrazolium salts assay was used to evaluate the proliferative capacity on the 1st,3rd,5th and 7th d.Then the groups were changed according to result of the proliferation assay (control:α-MEM with 2％ FBS; FGF-2 group:control supplemented with 20 μg/L FGF-2 ; VEGF:control supplemented with 10 μg/L VEGF ; Combination group:control supplemented with 20 μg/L FGF-2 and 10 μg/L VEGF).The cell cycle,migration and adhesion capacities were evaluated using flow cytometer,soluble tetrazolium salts assay,cell adhesion assay and scratch woundhealing motility assay.Results In 2％ volume fraction serum containing medium,FGF-2 and VEGF did not stimulate the cell proliferation.However,in 10％ serum condition,in groups treated with FGF-2 for 3,5 or 7 d,the A value was (1.22 ± 0.17,2.15 ± 0.19,2.72 ± 0.11) respectively,which were significantly higher than that in the control group (0.76 ± 0.16,1.25 ± 0.06,1.64 ±.0.09) (P ＜ 0.01) while lower than that in the group treated with FGF-2 and VEGF in combination on the 5 th and 7 th d (2.46 ±0.17,3.18 ±0.27) (P ＜0.05).The A value in the VEGF group on the 5 th and 7 th d is higher than the control group while lower than the FGF-2 group(1.66 ± 0.05,2.13 ± 0.13) (P ＜ 0.05).Flow cytometer showed that the proliferation index in VEGF group [(34.3 ±2.0)％] were significantly lower than those in FGF-2 [(46.8 ±3.2)％] group and (FGF-2 + VEGF) group[(45.0 ±4.0)％] but higher than in the control group [(14.5 ± 1.7)％] (P ＜ 0.01).The cell migration assay indicated that the group stimulated with FGF-2 showed no migration promoted effect.Cell adhesion assay showed that the ratio of the adhesive cells number to the original cells number is greater in the FGF-2 group(79 ±4) than in the VEGF group(62 ±4) (P ＜ 0.05).Light microscope identified a better cellular morphology on the adhesive surface in the group with FGF-2 than groups without FGF-2.Conclusions Both FGF-2 and VEGF could simulate the proliferation of PDLSC in a dose dependent manner,and showed an synergistic effect.FGF-2 was more effective to promote the adhesive capacity of PDLSC compared with VEGF.VEGF could facilitate the migration of PDLSC to the wound side.