| 张磊 涂艳阳 张鹏幸 杨安钢 吴介恒 缪吉玉 温伟红 张永生.转录因子SOX9 基因对脑胶质瘤干细胞干性维持的相关性研究[J].,2015,15(18):3430-3434 |
| 转录因子SOX9 基因对脑胶质瘤干细胞干性维持的相关性研究 |
| Knock-down SOX9 Gene Regulated the Stemness Maintance of Glioma StemCell in Vitro |
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| DOI： |
| 中文关键词: SOX9 慢病毒 胶质瘤干细胞 |
| 英文关键词: SOX9 Lentiviral Glioma stemcell |
| 基金项目:国家自然科学基金项目（81272419） |
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| 中文摘要: |
| 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(shRNA)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(qRT-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(qRT-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。 |
| 英文摘要: |
| Objective:To construct lentiviral expression vectors for silencing SOX9 and observe its effect on the stemness
maintance of stem cell in vitro.Methods:2 types of transcription of the SOX9 short hairpin RNA (shRNA) sequences were designed and
two types of corresponding lentivirals were packaged, which can be used to infect U87 and U251 glioma cell line. 2 types of stably
transfected cells were screened by puromycin. The expression of SOX9 silenced at the level of transcription and protein level was
detected by Western blot and qRT-PCR . Stem cell associated markers molecules SOX2, Nestin was detected by real-time fluorescence
quantitative PCR (qRT-PCR) expression and immunofluorescence staining. The ability of the stemness maintance of glioma stem cells
was compared with the diameter of spheres, and the expression of stem cell related markers of SOX2, Nestin was detected by
immunofluorescence staining.Results:The lentiviral of SOX9 was successfully constructed, which was used to infect U87, U251 cells
efficiently. The inhibitory rates of expression levels of SOX9 were 83.74 % and 80.12 % by qRT-PCR. The expression of related stem
cell markers decreased obviously with knock-down the gene of SOX9 in the stably transfected cell lines. In the glioma stem cell,
Knock-down SOX9 could inhibit the stemness maintance of glioma stem cells obviously by sphere diameter (P<0.01),
immunofluorescence showed the expression of glioma stemcell related molecules SOX2, Nestin decreased obviously compared with that
in the control group.Conclusion:The slicencing of the endogenous expression of SOX9 can inhibit the stemness maintance of glioma
stemcell, which laid a foundation of potential therapeutic targets. |
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