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目的设计和构建特异性siRNA纳米载体SWCNT-siRNA,通过体外实验,研究其对脑胶质瘤的作用。方法设计合成特异性Bcl-2 siRNA,利用超声技术合成SWCNT-siRNA复合物,动态散射粒度分析仪进行粒径分析;体外培养U251胶质瘤细胞,SWCNT-siRNA复合物处理细胞后,激光共聚焦显微镜追踪siRNA进入细胞的分布。PCR检测靶基因Bcl-2沉默情况,Annexin-V FITC与PI联合标记细胞后,流式细胞仪检测细胞凋亡。结果通过粒径分析证明siRNA成功缠绕到SWCNT上形成稳定的复合物siRNA-SWCNT。利用激光共聚焦显微镜对标记红色荧光的siRNA进行追踪,发现siRNA成功投递到细胞内。PCR验证了被投递到胞浆的siRNA很好的沉默目标基因Bcl-2。通过流式细胞仪分析发现,与对照组比较发现其可以抑制肿瘤细胞生长,促进肿瘤细胞早期凋亡。结论 SWCNT作为纳米载体投递Bcl-2 siRNA进入U251胶质瘤细胞,通过降解Bcl-2 mRNA抑制Bcl-2的表达并促进胶质瘤细胞的凋亡。
Objective To design and construct a specific siRNA nanocarrier SWCNT-siRNA, and study its effect on glioma by in vitro experiments. Methods The specific Bcl-2 siRNA was synthesized, the SWCNT-siRNA complex was synthesized by ultrasound and the particle size was analyzed by dynamic scattering particle size analyzer. After U251 glioma cells and SWCNT-siRNA complex were treated in vitro, Microscopy tracks the distribution of siRNA into the cell. PCR was used to detect the silence of target gene Bcl-2. After Annexin-V FITC and PI were labeled, the apoptosis was detected by flow cytometry. Results The particle size analysis showed that the siRNA was successfully wound onto SWCNTs to form a stable complex siRNA-SWCNT. Using laser scanning confocal microscopy to trace the red fluorescent siRNA, it was found that the siRNA was successfully delivered into the cell. PCR validated that siRNA delivered to the cytoplasm was a good target for silencing Bcl-2. By flow cytometry analysis found that compared with the control group found that it can inhibit tumor cell growth and promote early apoptosis of tumor cells. CONCLUSIONS: SWCNTs delivered Bcl-2 siRNA into U251 glioma cells as nanocarriers and inhibited the expression of Bcl-2 gene and promoted the apoptosis of glioma cells by degrading Bcl-2 mRNA.