The Construction Of Eukaryotic Expression Vector Of Human Dickkopf-1 And The Initial Study In SHG₄₄

Nowadays,it is known that the hypothesis about the genesis of human glioblastoma is gene mutation in many stages. Normal cells gradually become malignant through a series of traveling transform。In the clonal evolutionary process, a series of gene mutation accumulate and the mutation refers to different genes of many chromosomes。So finding new related gene of cancer and interfering its main signaling pathway will take effective treatments to cancer.Dickkopf-l(DKK-1) was first discovered as an inducer of head formation in embryo and as a potent inhibitor of Wnt signaling pathway.DKK-1 protein plays many different effects in many cancers.Now the study of DKK-1 in the pathogenesis of glioma is very slight. So we study the function of DKK-1 in glioma by transfecting the recombinant vector into SHG44.This study contains two parts of work:the first, construct the recombinant plasmid of DKK-1 in order to lay the foundation for further study.The second,transfect the recombinant vector into SHG44 by Lipofectimine 2000 and study its function to SHG44.PartⅠ:Construction of recombinant plasmid pcDNA3.1-DKK-lObjective To construct the recombinant plasmid pcDNA3.1-DKK-l which is capable of expressing in mammalian cells for further study.Methods DKK-1 gene containing NHeⅠand EcoRⅠendoenzyme sites were obtained by RT-PCR.Double enzyme digestion was conducted for pcDNA3.1 and RT-PCR product of DKK-1 gene.Both fragments were connected by using T4 ligase and transferred to DH5a.Then the connected product was detected by PCR,NHeⅠand EcoRⅠdouble enzyme digestion and suquencing.Result One 816bp fragment was amplificated from human placenta by RT-PCR.The same size fragment was amplificated from colony by PCR .Two corresponding fragments were obtained when the recombinant plasmid was digested by NHeⅠ和EcoRⅠ.The result of sequencing was completely correct.Conclusion The recombinant plasmid pcDNA3.1-DKK-l was succesfully constructed,which may facilitate the study of the function of DKK-1 in carcinoma cell lines.PartⅡ:The study of effect of DKK-1 in SHG44 by transfection in vitro Objective To investigate the biologic activity of DKK-1 to human glioma cell line SHG44 by transfection in vitro.Methods The pcDNA3.1-DKK-1 were transfected into SHG44 cells by Lipofectamine 2000 and PCR,RT-PCR,Western blot were applied to identificate the stable transfection of SHG44.All cells dealed with by BCNU and dyed by AVF+PI were measured by flow cytometry .Result By PCR,RT-PCR,Western blot detection,the recombinant plasmid transfected cell showed visible fragments in DNA,mRNA and protein.The control group did not show any fragments in DNA and protein and showed undefined fragments in mRNA.The average apoptosis ratio of non-transfected cell,empty plasmid transfected cell,recombinant plasmid transfected cell which were dealed with BCNU is 1.0%,1.4%,8.0% respectively.Conclusion constructed the stable expressing cell named SHG44-DKK-1. DKK-1 can raise the sensitivity of SHG44 to BCNU initially.