GU Yang-lin,ZHU Wen-hui,WANG Yu-bin,et al.Phenotype transformation from canine skeletal myoblast to fibrochondrocyte in vitro[J].Journal of Tongji University(Medical Science),2012,33(2):1-6. [点击复制]
Objective To induce the phenotype transformation of canine skeletal myoblast into fibrochondrocyte in vitro.Methods The muscle from the hindlimb of adult Beagle canine was obtained,from which skeletal myoblasts were isolated and harvested by mechanical decomposition and two-step enzyme digestion.The cultured skeletal myoblasts of the fourth passage were digested and cultured in a medium containing human cartilage-derived morphogenic protein-2(hCDMP-2) in a 24-well plate at a density of 2.0×104/cm2 in the experimental group;while in the control group the DMEM-LG complete culture medium without hCDMP-2 was used.After 21days of incubation the cellular morphology,secretion function and expression of type Ⅱ collagen in cultured cells were examined by HE and toluidine blue staining,and immunohistochemistry,respectively.The expression of collagen Ⅰ,Ⅱ and Sox9 mRNA was detected by reverse transcription-polymerase chain reaction(RT-PCR).Results The skeletal myoblasts changed from a spindle-like appearance into a polygonal shape after induction.The extracellular matrix was positive stained by H-E and toluidine blue staining in experimental group,and the cells were positively stained by immunohistochemistry for type Ⅱ collagen.RT-PCR revealed that the expression of collagenⅠ,Ⅱ and Sox9 mRNA were significantly displayed in the experimental group.No positive expression was observed in the control group.Conclusion The canine skeletal myoblasts can be induced for phenotype transformation to chondrocy-tes by hCDMP-2 in vitro.
