The Construction Of Eukaryotic Expression Vector Of Human Decorin And Study On Its Anti-tumor Effect In Vivo And Vitro

DCN belongs to a member of the proteoglycans and to be a pleiotropia molecule that affect matrix assembly,cellular proliferation,collagen fibrillation and TGF-βactivity adjusting,so it can antitissue fibrosis and inhibit tumor growth.The cDNA of DCN extron,was composed of 1080 bp of nucleotides and encoded a core protein of 395 amino acids.Objectives:To establish the eukaryotic(pcDNA3.1(+))expression vector of DCN,and transfect the recombinant to tumor cell through lipidosome,then detect apoptosis indexes of tumor cell by FCM,and do tumor depression experimentation in mice.Methods:1.Full length DCN gene was amplified from cDNA of DCN extron was obtained through PCR by using surivin specific primers.Then,DCN cDNA was inserted into pcDNA3.1(+) and verified by sequencing.2.Verified cDNA gene was subcloned into pcDNA3.1(+).The recombinant plasmid was introduced into S180 cells by lipidosome transfection.The transformants were contained different concentrations of G418 for selecting the positive clones. Get out RNA of the positive clones,then confirmed by RT- PCR.3.To detect apoptosis indexes and analyze cell cycles of S180 with transfected the recombinant plasmid,transfected void plasmid and not transfected.4.Inject pcDNA3.1(+)-DCN/S180,pcDNA3.1(+)/S180 and S180 to mice with randomization,and observe tumors growth of the three groups of mice.5.To execute mice and dissect tumor for pathologic section identification.Results:1.The result of sequencing human cDNA compared with the DCN sequence in the GenBank is the same.2.We have made up an eukaryotic(pcDNA3.1(+))expressive vector(pcDNA3.1(+)-DCN).After the recombinant vector was introduced into S180 cells by lipidosome transfection,we have selected positice clones against G418(900μg/ml)and confirmed by RT-PCR.3.FCM detected S180 transfected recombinant plasmid apoptosis index higher than transfected void plasmid and not transfected,and cell percentage of G₀/G₁ higher than the other two groups,cell percentage of G₂/M and S lower than the others.Differences between the three groups exist statistical significance(P＜0.05).4.Cultivante the positive clones multiplicitily,inject them into mice,and select S180 and S180 transfected void vector(pcDNA3.1(+)/S180)to control groups,results show mice injected S180 transfected recombinant vector (pcDNA3.1(+)-DCN/S180),tumor grew slower than control groups.There was a statistically significant difference in the growth rates among the three groups with different cells(P＜0.05).5. Pathologic sections show the three groups of tumor cells proliferated,and pcDNA3.1(+)-DCN/S180 group proliferates not as highly as pcDNA3.1(+)/S180 and S180 group,but its inflammation cells infiltration significantly more than the other two groups. Conclusions:To establish the eukaryotic(pcDNA3.1(+))expression vector of DCN,transfect the recombinant to S180 tumor cell,can make tumor cell apoptosis,vivo experiment showed mice injected pcDNA3.1(+)-DCN/S180,tumor grew depressed than the other two groups,mice injected pcDNA3.1(+)/S180 and S180.