IGF-1和bFGF对藻酸盐微球中兔耳弹性软骨细胞增殖活性和基质合成的影响

Effects of IGF-1and bFGF on the ECM synthesization and cell proliferation by cultured rabbits auricular elastic chondrocytes in alginate beads

目的利用藻酸盐微球体外培养兔耳弹性软骨细胞,观察IGF-1和bFGF对细胞增殖和基质合成的影响及协同作用效果。方法酶消化法从兔耳获取软骨细胞,与1.2%藻酸钠混合,细胞浓度107/mL,经注射器滴入102mM的CaCl2溶液,形成软骨细胞/藻酸钙凝胶微球,用含10%FBS的DMEM培养液体外培养,对照组不加生长因子,实验A、B和C组分别加入25ng/mLIGF-1、50ng/mLbFGF和25ng/mLIGF-1+50ng/mLbFGF,14d后终止培养,采用生物化学方法分析微球中的细胞DNA和GAG总量。结果对照组、IGF-1组、bFGF组和IGF-1+bFGF组单个微球的DNA总量分别为3.5μg、3.6μg、6.6μg和6.2μg,统计分析结果显示IGF-1组的DNA含量与对照组之间、bFGF组和IGF-1+bFGF组之间没有明显差异,而IGF-1+bFGF组与对照组、IGF-1组之间均有明显差异,DNA含量明显增加。GAG含量为15.5μg、23.8μg、29.0μg和38.9μg,各实验组与对照组及各组之间均有显著差异。结论IGF-1和bFGF对软骨细胞的影响体现在不同方面,IGF-1可以提高细胞的GAG合成总量,但并不促进软骨细胞的增殖,而bFGF的作用主要体现在细胞的增殖方面,IGF-1和bFGF联合应用的协同作用效果明显好于因子的单独应用,细胞增殖明显增加,GAG的含量也明显增加,表明不同生长因子的联合应用可能会产生更为理想的软骨形成条件。

Objective To investigate effects of insulin-like growth factor and basic fibrobla st growth factor on the cell proliferation and synthesized extr acellular matrix of cultured chondr ocytes.Methods Isolated chondrocytes from rabbit auricular cartilage by enzyme digestion were encapsulated in alginate i n a density of 10 7 cells /ml.The alginate beads of chondrocytes were prepared by dropping the cells /alginate into 125mM Ca Cl2solution through a 22gauge needl e and were allowed to polymerized for 10min.The beads were cultured for 14days in vitro with DMEM added with 10%FBS for bio-chemical analysis.In groups A,B and C,25ng /ml IGF-1,50ng /ml bFGF,25ng/ml IGF-1and 50ng /ml bFGF were added respectively in the same culture med ium.Results14days later,each beads contained a n average of 3.5μg,3.6μg,6.2μg and 6.6μg DNA.There was no statistical signi ficance between group A and control beads,and the same result was in group B and C.However,the amount of D NA demonstrated a significant incre ase in group C compared to group A and c ontrol.The amount of GAG were 15.5μg,23.8μg,29.0μg and 38.9μg.It was statistically significant in all groups.Conclusions This study demonstrates that IGF-1a nd bFGF influence differently on the ECM synthesization and cell prolife ration of elastic chondrocytes.IGF-1increases the a mount of GAG,but has no significant i nfluence on the cell proliferation.In reverse,the effect of bFGF is on the cell proliferation primarily.When incubated in c ulture medium with both 2growth factors,the chon-drocytes show increasingly prolife ration ability and GAG synthesizati on.It suggests that the combination of different growth factors has an advantageous effect o n the chondrogenesis.[