摘要
目的探讨有效靶向人表皮生长因子受体(EGFR)基因siRNA质粒表达载体的构建及其慢病毒包装。方法针对人EGFR基因的3个亚型同源区设计siRNA序列并进行siRNA表达载体的构建;然后转染HT1080细胞,荧光实时定量PCR筛选出最有效的siRNA序列,再进行慢病毒颗粒的包装和生产,同时检测体内抑瘤效率。结果针对人EGFR基因成功设计了siRNA序列并构建了siRNA表达载体。2号EGFR siRNA序列对目的基因EGFR的抑制最为明显,抑制率达92.2%,显著高于1号的69.75%和3号的31.80%,差异有高度统计学意义(P<0.01)。72 h后2号EGFR siRNA细胞感染率稳定在75%以上,病毒滴度为1.3412×105 ifu/μl,体内对前列腺癌细胞系PC-3移植瘤生长抑制率达34.83%,明显高于阴性对照组,差异有统计学意义(P<0.05)。结论针对人EGFR基因最有效的siRNA表达载体质粒的构建、鉴定和慢病毒包装,为其研究肿瘤靶向基因治疗奠定了基础。
Objective To screen and enclose an effective recombinant lentivirus carrying small interfering RNA（siRNA） against epithelial growth factor receptor （EGFR） gene. Methods Three pieces of siRNA sequences on the human EGFR gene were designed and then a recombinant plamid carrying siRNA was constructed, which was used to transfer HT1080 cells in order to screen the most effective sequence among the three siRNA sequences against EGFR with fluorescent realtime quantified PCR. Finally, the most effective EGFR siRNA sequence was enclosed and produced with Lentivirus which was used to determine the inhibitory effects on tumor growth in vivo. Results The siRNA sequences targeted to EGFR gene were designed and their expression vectors were cloned and verified successfully. Its transfeet efficiency was about 70%. Real-time PCR analysis revealed that siRNA expression vectors can inhibit the expression of EGFR. The No. 2 vector with EGFR siRNA showed a higher inhibition rate on the the expression of EGFR gene （92.20%） than No.1 vector （69.75%） or No. 3 vector （31.80%）（P〈0.01）. The infected rate on HT1080 cells of Lentivirus carrying No. 2 vector with EGFR siRNA was over 75% after 72h＇s infection and virus titre was measured 1.3412×10^5 ifu/ul, and the growth of transplanted tumors was reduced to 34.83% which was higher than that in the control group （P〈0.05）. Conclusion Construction, sreening and enclosing with lentivirus the most effective human EGFR siRNA are successful, and then the recombinant might be very useful for targeted tumor gene therapy in the next step.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2009年第2期216-219,393,共5页
Suzhou University Journal of Medical Science
关键词
表皮生长因子受体
基因
慢病毒
小干扰
构建
epithelial growth factor receptor
gene
lentivirus
small interfering construction
