摘要
目的肝癌衍生生长因子(HDGF)是近年来逐渐引起重视的一种细胞生长因子,在许多肿瘤细胞中高表达。本研究拟构建RNAi干扰慢病毒载体,沉默HDGF基因在大肠癌SW480细胞中的表达,观察细胞的增殖状态。方法RT-PCR方法检测HDGF基因在大肠癌中的表达。利用Invitrogen公司在线网站设计HDGF干扰序列,片段合成后构建慢病毒RNAi表达载体,随后利用293FT细胞将其包装成成熟慢病毒颗粒去感染SW480细胞,经Blasticidin抗性选择培养后,建立了稳定表达HDGFsiRNA的SW480细胞株。实时荧光定量PCR和Western Blot检测HDGF基因在mRNA和蛋白水平表达变化;生长曲线和MTT法观察细胞增值情况的改变。结果HDGF基因在大肠癌SW480细胞中表达较高。成功的构建了HDGF慢病毒RNAi表达载体,并建立了稳定表达靶向干扰HDGF基因表达的siRNASW480细胞株。沉默HDGF基因在表达后,能明显体外抑制细胞的增殖。结论针对HDGF基因的慢病毒RNAi载体能明显下调HDGF基因的表达,并在体外显著抑制大肠癌细胞增殖能力。
[Objective] HDGF （Hepatoma-derived growth factor）, a novel growth factor paid close attention in a recently few years, is shown to be highly expressed in many human tumor cells. It plays an important role in cell proliferation. This study will construct RNAi vector of Lentivirus, which is used to silence the expression of HDGF in colon cancer cells to observe the change of the ability of cell proliferation. [Methods] RT-PCR was used to detect the expression of HDGF in colon cancer. RNAi sequence of HDGF was designed by online internet of Invitrogen Inc. After synthesized of RNAi sequence, the sequence was constructed into Lentiviral siRNA vector. Subsequently,the vector was packaged into mature lentiviral particles by 293NF cell to infect SW480 cells. After accomplished by blasticidin selection culture,SW480 cells with constant expression of HDGF siRNA was established. Real-time fluorescence quantitation PCR and western blot were used to detect the alteration of the mRNA and protein of HDGF respeefively. The growth curve and MTT method were used to evaluate the ability of cell proliferation. [Results] Higher mRNA expression of HDGF gene was shown to be SW480 ceils. SiRNA of lentivirus targeting HDGF was successfully eonstructed and could inhibit the proliferation of SW480 cells of colon cancer. [Conclusion] Expression of HDGF increased obviously was shown in SW480 eells. Lenfiviral siRNA veetor targeting HDGF could markedly decrease the expression of HDGF, which led to conspicuously reduce the ability of the cell proliferation SW480 cells in vitro.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2009年第9期1322-1326,共5页
China Journal of Modern Medicine
