不同浓度的A-PRF膜萃取液对人牙龈成纤维细胞增殖及OPG表达的影响

Effects of different concentrations of A-PRF membrane extracts on proliferation and expression of OPG in human gingival fibroblasts

目的比较不同浓度的富血小板纤维蛋白(A-PRF)膜萃取液对人牙龈成纤维细胞(HGF)增殖能力及骨保护素(OPG)表达的影响。方法将健康成人的牙龈依照组织块培养法进行传代,对其来源进行免疫荧光染色鉴定,取浓度0、10%、50%、100%的A-PRF提取液100μl分别加入到人牙龈成纤维细胞培养基内,标记为A组、B组、C组、D组,观察人牙龈成纤维细胞的生长密度与形态;用酶联免疫吸附剂测定(ELISA)检测OPG表达水平,并进行组间比较。结果利用组织块贴壁的培养方法培养HGF。在倒置相差显微镜下进行观察,可见HGF呈现梭形或者星形,细胞排列十分规则,呈束状或旋涡状。用免疫荧光染色法鉴别HGF组织的来源,抗角蛋白抗体染色结果无染色呈阴性,抗波形丝蛋白抗体染色呈现阳性,胞浆呈现荧光绿色,HGF的细胞有明显细长的伪足呈长梭形,胞核多呈椭圆形位于细胞的中央,照相记录HGF的细胞形态,证实本实验所培养的细胞为来源于中胚层的纤维母细胞,具有HGF的形态特点。随着A-PRF膜萃取液浓度的增高牙龈成纤维生长密度增加,细胞数量明显增多。HGF在低浓度的生长因子萃取液刺激的条件下,OPG表达水平随着萃取液浓度的增高而不断增加。经不同浓度A-PRF膜萃取液刺激后,A组、B组、C组、D组的OPG表达分别为(2490.69±255.94)、(3336.86±245.41)、(5434.33±256.56)、(6931.46±132.72)pg/ml,四组的OPG表达水平比较差异均具有统计学意义(P<0.05)。结论A-PRF膜萃取液能够很好的促进牙龈成纤维细胞的增殖,并且OPG的含量也相应有所增加,更加证实了A-PRF膜在促进牙龈组织愈合及骨组织愈合具有很好的贡献。

Objective To compare the effects of different concentrations of advanced platelet-rich fibrin(A-PRF)membrane extracts on proliferation and expression of osteoprotegerin(OPG)in human gingival fibroblasts(HGF).Methods The gingiva of healthy adults was passaged according to the tissue block culture method,and its source was identified by immunofluorescence staining.100μl of A-PRF extracts at concentrations of 0,10%,50%,and 100%were added to human gingival fibroblast culture medium and labeled as group A,B,C,and D,respectively.The growth density and morphology of human gingival fibroblasts were observed;the expression level of OPG was detected by enzyme-linked immunosorbent assay(ELISA)and compared between groups.Results HGF was cultured by tissue block adherent culture method.Observation under an inverted phase contrast microscope showed that HGF was in the shape of a fusiform or a star,and the cells were arranged very regularly,in the shape of a bundle or a vortex.Immunofluorescence staining was used to identify the origin of HGF tissue.The results of anti-keratin antibody staining were negative,the anti-vimentin antibody staining was positive,the cytoplasm was fluorescent green,the cells of HGF had obvious elongated pseudopods in long shuttle shape,the nucleus was mostly oval in the center of the cells,and the cell morphology of HGF was photographed and recorded.It was confirmed that the cells cultured in this experiment were fibroblasts from mesoderm and had the morphological characteristics of HGF.With the increase of the concentration of A-PRF membrane extract,the growth density of gingival fibroblasts increased,and the number of cells increased significantly.The OPG expression level of HGF stimulated by low concentration of growth factor extracts increased continuously with the increase of extract concentration.After stimulation with different concentrations of A-PRF membrane extract,the OPG expression in group A,group B,group C and group D were(2490.69±255.94),(3336.86±245.41),(5434.33±256.56),(6931.46±132.72)pg/ml,and the difference was statistically significant(P<0.05).Conclusion The A-PRF membrane extract can well promote the proliferation of gingival fibroblasts,and the content of OPG also increases accordingly,which further confirms that A-PRF membrane has a good contribution to the promotion of gingival tissue healing and bone tissue healing.