中国临床解剖学杂志 ›› 2017, Vol. 35 ›› Issue (5): 521-525.doi: 10.13418/j.issn.1001-165x.2017.05.009

• 实验研究 • 上一篇    下一篇

纳米载体SWCNT投递Bcl-2 siRNA促进U251细胞凋亡

胡晓芳1,2, 邱仁杰4, 余磊2, 刘茂生1, 杨琳1, 王国保2,3   

  1. 1.遵义医学院珠海校区人体解剖与组织胚胎学教研室,  广东   珠海    519041; 2.南方医科大学 广东省组织构建与检测重点实验室,  广州   510515; 3.南方医科大学南方医院肾内科,  广州   510515; 4.江西中医药大学科技学院,  江西   南昌 330004
  • 收稿日期:2017-06-28 出版日期:2017-09-25 发布日期:2017-10-30
  • 通讯作者: 王国保,主任医师,教授,E-mail:wgbandyl@medmail.com.cn
  • 作者简介:胡晓芳(1986-), 女,安徽安庆人,讲师,硕士,研究方向:肿瘤的基因治疗,E-mail:xiaofang4231@163.com
  • 基金资助:

     贵州省科学技术基金(黔科合J字[2014]2179号)

Synthesis nano-carrier SWCNT to deliver Bcl-2 siRNA and induction of U251 cell apoptosis

HU Xiao-fang 1,2, QIU Ren-jie 4, YU Lei 2, LIU Mao-sheng 1, YANG Lin1,  WANG Guo-bao 2,3   

  1. 1.Department of Anatomy, Zhuhai Campus of Zunyi Medical College, Guangdong Zhuhai 519041,China; 2. Department of Key Laboratory of Construction and Detection of Guangdong Province, Southern Medical University, Guangzhou 510515,China; 3. Department of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515,China; 4.College of Jiangxi University of Traditional Chinese Medicine, Jiangxi Nanchang 330004, China
  • Received:2017-06-28 Online:2017-09-25 Published:2017-10-30

摘要:

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。

关键词: 脑胶质瘤,  siRNA,  碳纳米管,  Bcl-2,  U251

Abstract:

Objective Design and synthesis Nano-Carrier of SWCNT-siRNA, and study the effect on glioma in vitro. Methods Design and synthesis Bcl-2 siRNA, and assemble them into nano-carrier. Characterization of the SWCNT-siRNA delivery system was measured by dynamic light scattering (DSL). U251 cell were cultured in vitro, and then treated with SWCNT-siRNA. The intracellular distribution of siRNA was detected by confocal laser scanning microscopy. The expression of Bcl-2 was detected by qPCR. Cells were stained with annexin V-FITC and propidium iodide (PI). The percentages of apoptotic and necrosis cells were quantified using a flow cytometer. Results Through particle size analysis, we proved that siRNA strongly adsorbed onto the surface of pristine SWCNTs to form the steady complex SWCNT-siRNA. siRNA labeled red fluorescence(cy3-siRNA)was traced by Laser Scanning Confocal Microscope and we found that siRNA was successful to be delivered into intracellular. The result of PCR showed that the expression of Bcl-2 was reduced in the group of SWCNT-siRNA. Flow Cytometer revealed SWCNT-siRNA could inhibit the growth of cells and induce early apoptosis and death. Conclusion SWCNT as nano-carrier could deliver Bcl-2 siRNA into intracellular, significantly cleaved the mRNA of Bcl-2, inducing lower levels of Bcl-2 expression and markedly triggering cell apoptosis.

Key words:  , Glioma; siRNA; SWCNT; Bcl-2; U251