Experimental Study On BMSCs Induced To Tendon Cells By IGF-1and BFGF

Objective： To investigate the new method of inducing Bone marrowmesenchymal stem cells (BMSCs) to diffrenciate into tendon cells by basic fibroblastgrowth factor (bFGF) and insulin-like growth factor-1(IGF-1).Methods:1. Isolate and culture the BMSCs, and molecular biology identification.BMSCs isolated by Density gradient centrifugation in vitro,and then culturedunder stem cell phenotype identification and osteogenesis induction,lipogenesisinduction and chondrogenesis induction to confirm if the bone marrow stem cells withmulti-directional differentiation capability.2. Observe on BMSCs induced by IGF-1and bFGF to tendon cells.BMSCs will divided randomly into four groups,named the experimental group A(IGF-1+bFGF), the experimental group B (IGF-1), the experimental group C (bFGF)and the control group D, and then cultured in37℃,5%CO2bacteriologicalincubatorincubator. Observe the changes of the cells morphology underthe microscope. By the method of RT-PCR, detect the expression level of collagen I,collagen III and scleraxis mRNA in each group in the3rd days,7th days,14th days and21st days. In the14thday, detect the expression level of collagen I, collagen III in eachgroup, by Immunofluorescence stain.Results:1.In morphology, the isolated and cultured BMSCs are long spindle-shaped,growing swirly. The results from flow cytometry tests are that: CD29and CD90insurface cells express is positive, respectively99.24%,99.06%; CD45in the surface cellsexpress is negative,98.71%. Alizarin red, oil red O, Armour aniline blue and Collagen IIimmunohistochemical stain in induced BMSCs are all positive.2. Observe the induced BMSCs find that: as for the mRNA and protein levels in I, III and SCX, the experimental A、B、C groups compared to the control group havesignificant differences. gene and protein levels in experimental groups rise constantlyfrom the3rd day, while dropping in the14th day. And from the7th day to the14th day,the differences are obvious. The gene in I、III、SCX in the experimental group B expressthe most obviously. And in the immunofluorescence staining test, the expression levelof collagen I and collagen III in the experimental group B is highest.Conclusion:There are significant differences, in morphology and function, between the cellsfrom the experimental groups and the control.group, and the morphologicalcharacteristics and the expression of cytoplasmic matrixes are similar to tendon cells.Especially IGF-1group. Combined application of bFGF and IGF-1do not significantlysynergistic effect.