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目的:应用慢病毒干扰SOX9的表达,观察其对脑胶质瘤干细胞干性维持的影响。方法:设计2条针对SOX9转录短发夹RNA(sh RNA)的DNA序列,构建慢病毒并感染胶质瘤细胞系U87、U251细胞,利用嘌呤霉素筛选稳转细胞。在转录水平及蛋白水平检测SOX9的沉默效果,利用荧光实时定量PCR(q RT-PCR)及免疫荧光染色检测相应干细胞相关标志分子SOX2、Nestin的表达差异。比较诱导形成的胶质瘤干细胞成球能力(肿瘤干细胞成球的直径),同时利用荧光实时定量PCR(q RT-PCR)及免疫荧光染色对干细胞相关标志物SOX2、Nestin的表达水平进行比较。结果:SOX9成功包装慢病毒并有效感染U87、U251细胞,实时荧光定量PCR检测其抑制率分别可降低83.74%和80.12%。并且在稳转细胞系水平相关干细胞标志分子表达含量有明显下降。在干细胞层面沉默SOX9可以明显抑制胶质瘤细胞诱导成球的直径(P<0.01),同时免疫荧光显示肿瘤干细胞相关干性分子SOX2、Nestin的表达水平较对照组明显降低。结论:慢病毒感染沉默SOX9基因可以抑制胶质瘤干细胞干性的维持,为胶质瘤的生物治疗提供了重要的靶点。
OBJECTIVE: To detect the effect of lentivirus on the expression of SOX9 and its effect on the maintenance of glioma stem cells. Methods: Two DNA sequences encoding short hairpin RNA (shRNA) of SOX9 were designed and constructed. Lentivirus was constructed and infected glioma cell lines U87 and U251. Puromycin was used to screen stable transfected cells. SOX9 silencing effect was detected at transcription and protein levels. The expression of SOX2 and Nestin were detected by qRT-PCR and immunofluorescence staining. The ability of glioma stem cells to form into spheres (the diameters of tumor stem cells into spheres) was compared. The expression levels of SOX2 and Nestin were compared by qRT-PCR and immunofluorescence staining. RESULTS: SOX9 successfully packaged lentivirus and effectively infected U87 and U251 cells. The inhibition rates of SOX9 were reduced by 83.74% and 80.12% respectively by real-time fluorescence quantitative PCR. And the expression level of related stem cell markers in stable cell lines decreased significantly. Silencing SOX9 at the stem cell level significantly inhibited the diameter induced by glioma cells (P <0.01). Meanwhile, the expression of SOX2 and Nestin in cancer stem cells correlated significantly lower than that in the control group by immunofluorescence. Conclusion: Silencing of SOX9 gene by lentivirus infection can inhibit the maintenance of glioma stem cells and provide an important target for the biological treatment of glioma.