Dickopff-1的真核表达载体构建及其对SHG44的作用

Construction of eukaryotic expression vector of human Dickopff-1 and the initial study in human glioma cell line SHG44

目的构建Dickopff-1（DKK-1）的真核表达质粒，研究SHG44转染DKK-1并经BCNU处理后其生物学特性的变化。方法将纯化扩增的人DKK-1与真核表达载体pcDNA3．1连接后转化人大肠杆菌DH5α感受态扩增重组，以Lipofectamine^TM2000介导将重组质粒转染SHG44细胞，经G418筛选后进行鉴定。BCNU处理转染DKK-1基因后的SHG44细胞，流式细胞仪检测细胞特性变化。结果扩增获得的816bp特异片段，重组质粒经鉴定及测序分析结果完全一致。重组载体转染SHG44细胞经G418筛选后，转染细胞在DNA、mRNA、蛋白水平均有DKK-1的表达。BCNU处理后的未转染、空质粒转染、重组质粒转染的三组SHG44细胞，平均凋亡率分别为1．0％、1．4％、8．0％。结论本实验成功构建了pcDNA3．1-DKK-1真核表达质粒，并建立稳定表达DKK-1蛋白的胶质瘤细胞株（SHG44-DKK-1）。转染DKK-1能增强SHG44对BCNU的敏感性，促进肿瘤细胞的凋亡。

Objective To construct the eukaryotic expression vector of human Dickopff - 1 （ DKK - 1 ） and to investigate its biologic activity to SHG44 transfection of DKK - 1 gene. Method Total RNA was prepared from normal human placenta tissue. Then RNA was reverse transcripted and cDNA was subjected to PCR amplification. PCR product was cloned into pcDNA3.1 and then the recombinant was transformed into DH5α E. coll. Constructed pcDNA3.1 - DKK - 1 was identified by PCR,restriction enzyme digestion analysis （ NHe I and EcoR Ⅰ ） and DNA sequencing. The pcDNA3.1 - DKK - 1 were transfected into SHG44 cells by Lipofectamine^TM2000 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. Results RT - PCR product of DKK - 1 coding sequence was 816 bp specific segment. By PCR,restriction enzyme digestion（ NHe I and EcoR I ） and DNA sequencing,the pcDNA3. 1 - DKK - 1 was amplified and digested into corresponding fragments respectively and DNA sequence was fight completely. By PCR, RT- PCR ,Western blot detection, the recombinant plasmid transfeeted cell showed visible fragments in DNA and protein and showed undefined fragments in mRNA. The control group did not show any fragments. 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. Conclusions The enkaryotic expression vector of human DKK - 1 is constructed successfully and constructed the stable expressing cell named SHG44 - DKK - 1. DKK - 1 can raise the sensitivity of SHG44 to BCNU initially and possess good biologic activity.