纳米载体SWCNT投递Bcl-2 siRNA促进U251细胞凋亡

doi:10.13418/j.issn.1001-165x.2017.05.009

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

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.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.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/Abstract

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

胡晓芳，邱仁杰，余磊，刘茂生，杨琳，王国保. 纳米载体SWCNT投递Bcl-2 siRNA促进U251细胞凋亡[J]. 中国临床解剖学杂志, 2017, 35(5): 521-525.

HU Xiao-fang1,2, QIU Ren-jie4, YU Lei2, LIU Mao-sheng1, YANG Lin1, WANG Guo-bao2,3. Synthesis nano-carrier SWCNT to deliver Bcl-2 siRNA and induction of U251 cell apoptosis[J]. Chinese Journal Of Clinical Anatomy, 2017, 35(5): 521-525.

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纳米载体SWCNT投递Bcl-2 siRNA促进U251细胞凋亡

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

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