[摘要] 目的:构建DEK基因的RNAi慢病毒表达载体,并对其生物学功能进行初步检测。方法:采用RNAi干扰技术,根据DEK基因的序列,确定其有效靶序列,合成DEK基因的Oligo DNA,退火形成双链DNA,将其克隆到经BamHI与EcoRI酶切后的siRNA表达载体PSIH-H1上,产生PSIH-H1-DEK慢病毒载体,筛选阳性克隆并测序鉴定。与3个包装质粒共转染293T细胞,包装成慢病毒后感染乳腺癌细胞ZR75-1,经Puro(嘌呤霉素)筛选2周后,收集部分细胞利用Realtime-PCR和Western blot分别检验DEK在mRNA和蛋白水平的敲低效果,并通过细胞生长实验来检测DEK对人乳腺癌细胞系ZR75-1细胞生长的影响。结果:PCR和DNA测序结果证实DEK siRNA慢病毒表达载体PSIH-H1-DEK构建成功。RT-PCR和Western blot结果显示,构建的DEK siRNA可有效抑制DEK基因的表达,并由此建立了敲低DEK的稳定克隆。生长曲线实验表明,DEK siRNA可抑制人乳腺癌细胞系ZR75-1细胞的生长。结论:成功构建了DEK基因的RNAi慢病毒表达载体,感染人乳腺癌细胞系ZR75-1细胞后,有效沉默了ZR75-1细胞中的DEK基因的表达,为进一步研究DEK基因在乳腺癌中的作用奠定基础。
Construction and functional characterization of a lentiviral vector mediating RNA interference of DEK gene
ZHU Jie1Δ,LIU Jie2Δ,DING Li-hua1,HE Ronghua2,ZHANG ya-nan2,LUO Xiao-Li2,YE Qi-Nong2*,LV Zhao-Hui 1*
1Department of endocrinology,Chinese PLA General hospital,Beijing 100853;
2Beijing Institute of Biotechnology, Beijing 100850;China Corresponding author: LV Zhao-Hui, E-mail:metabolism301@126.com;
YE Qi-Nong,E-mai:yeqn66@yahoo.com
[Abstract] Objective:To construct the lentiviral vector of RNA interference(RNAi) for DEK,and to detect its effect on breast cancer cell growth.Methods:The DEK siRNA was designed and constructed based on DEK sequence using a lentiviral vector.The lentivirul vector containing DEK siRNA was named PSIH-H1-DEK,which was confirmed by PCR and sequenceing.PSIH-H1-DEK was then packaged with accessory plasmids into lentivirus in 293T cells and selected for 2 weeks with puromycin and the mixed colonies stably expressing DEK siRNA were obtained and the DEK expression was detected by QPCR and Western blot.The effect of DEK siRNA on ZR75-1 cell growth was determined by cell counting kit.Results:Western blot and qPCR showed that PSIH-H1-DEK siRNA could suppress DEK gene expression.Suppression of DEK could markedly inhibited the growth of ZR75-1 cells.Conclusion:The lentivirus-mediated DEK siRNA were obtained,which lays the foundation for further research on DEK function in breast cancer development.
【摘要】 目的:急性肝衰竭是目前临床上导致死亡的重要病因之一,研究具有肝特异性、灵敏度高的肝损伤标志物对于严重肝病的早期诊断以及预后预测具有重要的意义。肝活素Hepassocin(HPS) 是一种具有高度组织特异性的促肝细胞增殖活性因子,在动物水平检测其在血清中的表达水平与肝损伤程度的相关性将为其开发成用于临床检测急性肝损伤的特异性标志物奠定基础。方法:利用腹腔注射不同剂量CCl4处理诱导小鼠急性肝损伤模型,检测小鼠的存活率、转氨酶水平、以及肝脏病理变化。通过ELISA检测小鼠血清的HPS水平;Real time PCR法检测HPS mRNA水平;Western blot 实验检测HPS的蛋白表达水平。结果:随着CCl4处理剂量的增加,小鼠死亡率显著升高,肝损伤程度显著增加,在此过程中,HPS在小鼠血清及肝组织中的表达水平也显著上调,并与肝损伤程度密切相关。结论:HPS可以做为一种新的小鼠肝损伤标志物。