Stathmin siRNA表达载体的构建及对LM8细胞生物学行为的影响

背景与目的:Stathmin在多种恶性肿瘤细胞中都有高水平表达,通过抑制其表达可以干扰恶性肿瘤细胞的分裂,影响肿瘤细胞的增殖与凋亡。本研究构建并观察stathmin基因RNA干扰表达载体转染LM8细胞系后对stathmin表达及细胞生物学行为的影响。方法:构建靶向stathmin干扰质粒pGenesil-1-Stathmin和通用对照质粒pGenesil-1-HK,以脂质体法将2个重组质粒分别转染骨肉瘤LM8细胞系。实时荧光定量PCR和Western印迹技术检测各组LM8细胞系stathmin在mRNA和蛋白水平的表达,MTT(噻唑蓝)法比色分析检测细胞体外生长抑制率,软琼脂集落形成实验观察单个细胞体外增殖活力,细胞HE染色观察细胞形态学的变化,Hoechst染色观察细胞凋亡特征并计算凋亡率,流式细胞术定量分析细胞周期变化,C3H小鼠移植瘤实验显示肿瘤细胞成瘤能力。结果:DNA测序证实成功构建pGenesil-1-Stathmin重组质粒。转染LM8细胞后,明显抑制stathmin基因表达;细胞体外生长抑制率显著增加;单个细胞体外增殖活力显著下降;细胞HE染色部分细胞呈典型的凋亡细胞形态学改变;Hoechst染色显示细胞凋亡特征,细胞凋亡率明显高于对照组;流式细胞术检测显示细胞周期阻滞于G2/M期,明显高于对照组;C3H小鼠移植瘤实验显示特异转染组致瘤率下降,肿瘤生长减缓。结论:成功构建的靶向干扰质粒pGenesil-1-Stathmin能有效抑制stathmin基因在骨肉瘤LM8细胞的表达并影响其相关生物学行为,其抑制骨肉瘤细胞的增殖与生长,为stathmin基因应用于骨肉瘤的基因治疗研究奠定了理论基础。

Construction of siRNA expression vectors targeting stathmin and its influcence on biological behaviour in LM8 cells

Background and purpose:Stathmin is expressed at high levels in many kinds of human cancers.Inhibition of stathmin expression in malignant cells results in reduced cellular proliferation and induces apoptosis. This study was to construct silence-small interfering RNA(siRNA) expressing vector and observe the expression of stathmin gene and its in? uence on biological behavior in LM8 cell line after the vector transfection. Methods:The designed siRNA expression vector targeting stathmin gene were constructed. The recombinant vectors of pGenesil-1-Stathmin and pGenesil-1-HK were transfected by liposome-mediated method into the LM8 osteosarcoma cell line. The expression of stathmin at the levels of mRNA and protein were detected by real-time quantitative PCR and Western blot. The cellular growth inhibiting rates in vitro were assayed by MTT colorimetry. The cellular growth activities were assayed by vitro clonogenic assays. The morphological assessment of apoptosis was studied by Hoechst staining and HE staining. The cell cycle was analysed by FCM. C3H mice-transplanted tumors were used to estimate the kinetics of cancer formation of tumours of the three kinds of cells. Results:The siRNA expression vectors of pGenesil-1-Stathmin was successfully constructed and confirmed by the DNA sequencing. Stathmin mRNA and protein after transfection were obviously down-regulated in the LM8 cells transfected by pGenesil-1-Stathmin plasmid. The cellular growth inhibiting rates increased obviously. The cellular growth activities decreased obviously in soft agar culture. Hoechst and HE staining showed that apoptosis was induced after transfection. The cells at G2/M phase were signifi cantly higher than those in the negative control group transfected with pGenesil-1-HK and the nontransfected group. The formation rate of C3H mice-transplanted tumor mice slowed down signifi cantly by transfection of pGenesil-1-Stathmin. Conclusion:pGenesil-1-Stathmin expression vectors targeting stathmin were successful constructed. The inhibitory effect of siRNA on the LM8 cells transfected by pGenesil-1-Stathmin expression vectors was confi rmed and the transfection also had impacted on biological behaviour of cells like tumor growth,this may served as a biological basis for the application in the gene therapy of osteosarcoma.