bFGF对牙周膜成纤维细胞与骨髓间充质干细胞共育影响的体外研究

Effect of bFGF on the Co-breeding of Periodontal Ligament Fibroblasts and Bone Marrow Mesenchymal Stem Cells in Vitro

目的探讨碱性成纤维细胞(重组人型)生长因子(basic fibroblast growth factor,bFGF)对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)与牙周膜成纤维细胞(periodontal ligament fibroblasts,PDLFs)共育条件下BMSCs向PDLFs分化的影响。方法体外培养大鼠BMSCs和PDLFs,并进行细胞鉴定。采用Millicell悬挂式细胞共育室建立BMSCs/PDLFs间接共培养模型。将实验分为5组:阴性对照组(单独BMSCs组);阳性对照组(单独PDLFs组);实验组:0 ng/mL bFGF+BMSCs/PDLFs组、1 ng/mL bFGF+BMSCs/PDLFs组和10 ng/mL bFGF+BMSCs/PDLFs组。在细胞共育的第3天、第5天和第7天进行ALP活性测定。培育至2 w后,停止共育,RT-PCR法监测各组细胞的骨钙素(osteocalcin,OCN)、Ⅲ型胶原(collagen typeⅢ,COLⅢ)、骨桥蛋白(osteopontin,OPN)和金属蛋白酶1(metalloproteinase 1,ADAMTS1)的mRNA含量。采用SPSS 17.0软件对数据进行统计学分析。结果ALP活性测定显示第3天、第5天和第7天共培养后,在各个时间点,各实验组与阴性对照组相比,差异有统计学意义(P<0.05);与阳性对照组相比,差异无统计学意义(P>0.05);各实验组之间差异无统计学意义(P>0.05)。RT-PCR检测结果显示阴性对照组与其余4组的OCN和OPN mRNA表达差异有统计学意义(P<0.05),后4组之间差异无统计学意义(P>0.05);单独BMSCs组与其余4组的COLⅢ和ADAMTS1 mRNA表达差异有统计学意义(P<0.05),其中10 ng/mL bFGF+BMSCs/PDLFs组的COLⅢ、ADAMTS1 mRNA表达量高于其余各组,差异有统计学意义(P<0.05)。结论bFGF能诱导BMSCs向PDLFs分化,高浓度组(10 ng/mL)bFGF的诱导效果优于低浓度组(1 ng/mL)。

Objective To investigate the effect of basic fibroblast growth factor(bFGF)on the differentiation of bone marrow mesenchymal stem cells(BMSCs)into periodontal ligament fibroblasts(PDLFs)in the co-breeding condition of BMSCs and PDLFs.Methods BMSCs and PDLFs were cultured in vitro and identified.BMSCs/PDLFs indirect co-culture model was established in Millicell hanging cell co-culture room.The experiment was divided into 5 groups:negative control group(single BMSCs group);positive control group(PDLFs group alone);experimental group:0 ng/mL bFGF+BMSCs/PDLFs group,1 ng/mL bFGF+BMSCs/PDLFs group and 10 ng/mL bFGF+BMSCs/PDLFs group.ALP activity was measured on days 3,5 and 7 of cell co-breeding.After cultured to 2 w,the co-breeding was stopped,and mRNA contents of osteocalcin(OCN),collagen typeⅢ(COLⅢ),osteopontin(OPN)and metalloproteinase 1(ADAMTS1)were monitored by RT-PCR.SPSS 17.0 software was used for statistical analysis of the data.Results The ALP activity test showed that there were statistically significant differences between the experimental group and the negative control group at each time point after 3 d,5 d and 7 d co-culture(P<0.05);compared with positive control group,the difference was not statistically significant(P>0.05);there was no statistical significance among all experimental groups(P>0.05).RT-PCR detection results showed that the mRNA expression of OCN and OPN between the negative control group and the other 4 groups had statistical significance(P<0.05),but there was no statistical significance between the latter 4 groups(P>0.05);the mRNA expressions of COLⅢand ADAMTS1 in the single BMSCs group were significantly different from those in the other 4 groups(P<0.05),among which the mRNA expressions of COLⅢand ADAMTS1 in the 10 ng/mL bFGF+BMSCs/PDLFs group were higher than those in the other 4 groups.The difference was statistically significant(P<0.05).Conclusion bFGF can induce BMSCs to differentiate into PDLFs,and the induction effect of bFGF in high concentration group(10 ng/mL)is better than that in low concentration group(1 ng/mL).