Experimental Study On The Biological Influences Of Humen Chondrocytes By Over-expressing Of Indian Hedgehog

Objective:To study the main biological characteristics of humen chondrocytes in vitro after pSNAV-Ihh-eGFP and pSNAV-Ihh gene transfected by ExGen 500,and to detect whether or not recombination gene have affect to the function of Ihh.Methods:The Ihh cDNA obtained from plasmid pCMV-Ihh was subcloned into the plasmid pSNAV 2.0 by molecular clone way.and the eGFP obtained from plsmid N3-eGFP was subcoloned into the plasmid pSNAV脁hh by overlaying PCR.The chondrocytes of humen chondylar were derived,and were cultured in vitro,Histochemical stainings were used to determine special phenotype of human condylar chondrocytes,and electron microscope to observe the ultramicostructure。The recombinant plasmid of pSNAV-Ihh-eGFP and pSNAV-Ihh transfered the cultured chondrocyte by ExGen 500 respectively,and the plasmid of pSNAV 2.0 that transfected the cultred chondrocyte was comparative group.The capacity of cell proliferation was tested by MTT and the curve of cell proliferation.The difference of mRNA level of Ihh and Ptc were tested by PCR after the cultured chondrocyte had been transfered to study the biological characteristics of chondrocytes in vitro Results:The recombinant plasmid of pSNAV-Ihh-eGFP and pSNAV-Ihh was successfully constructed and confirmed by DNA sequencing.The chondrocyte of humen was derived and cultured successfully,and confirmed by Histochemical stainings,and have normal ultramicostructure of humen chondrocyte Exgen 500 mediated the recombinant plasmid of pSNAV-Ihh-eGFP.which expressed successfully in cultured humen chondrocyte in vitro that detected by fluorescence microscope.The effect of transfer was about 35%.The proliferation and activity of cultured chondrocyte and the level of mRNA was higher significantly in the treatment group than that of the control one,and there is no difference between treatment groups.Conclusion:The transfection efficacy that ExGen 500 mediated pSNAV-Ihh-eGFP to cultured chondrocyte of human in vitro was 35%,and many index of cell proliferation was higher significantly in the treatment group than that of the control one after transfection.This study has verified the fact that Ihh have the function of proliferation to chondrocyte of human cultured in vitro.and recombination gene Ihh-eGFP have no influence to the function of Ihh gene.