纳米微泡搭载siRNA联合超声靶向破碎技术干扰胶质瘤细胞增殖的体外实验研究

doi:10.3877/cma.j.issn.1672-6448.2018.12.014

目的

以纳米微泡超声造影剂作为基因治疗中小干扰RNA（siRNA）的载体，探讨纳米微泡搭载siRNA联合超声靶向破碎技术对siRNA的转染及干扰效率以及对抑制胶质瘤肿瘤细胞生长、提高肿瘤细胞凋亡水平的作用。

方法

薄膜水化法制备粒径均一的纳米微泡，通过生物素-亲和素系统连接纳米微泡和siRNA，对胶质瘤细胞进行转染。将细胞分为纳米微泡-羧基荧光素（FAM）-乱序阴性对照siRNA（scrambled siRNA，SCR）-超声辐照组（NB-FAM-SCR-US），纳米微泡-FAM-SCR-无超声辐照组（NB-FAM-SCR）和正常细胞组，利用激光共聚焦显微镜检测各组细胞siRNA的转染效率；将细胞分为纳米微泡-siRNA-超声辐照组（NB-siRNA-US），纳米微泡-siRNA-无超声辐照组（NB-siRNA），纳米微泡-SCR-超声辐照组（NB-SCR-US），实时荧光定量PCR（Real-time PCR）及蛋白质免疫印迹（Western blot）检测各组细胞siRNA对目的基因的干扰效率；CCK-8及Annexin-V检测各组肿瘤细胞的活性及细胞凋亡率。

结果

通过薄膜水化法制备的纳米微泡平均粒径为（663.9±102.5）nm，纳米微泡-siRNA的平均粒径为（707.0±127.6）nm。激光共聚焦显微镜检测发现，与NB-FAM-SCR组相比，NB-FAM-SCR-US组可在细胞核（蓝色荧光）周边观测到更多的绿色荧光，正常细胞组仅可观测到细胞核的蓝色荧光，未见绿色荧光。Real-time PCR检测发现，与NB-SCR-US组相比，NB-siRNA-US组和NB-siRNA组IDH1基因的表达在mRNA水平均降低，差异具有统计学意义（t=-20.35、-4.27，P均<0.01）；而NB-siRNA-US组目的基因表达降低更为显著，与NB-siRNA组相比，差异具有统计学意义（t=-12.34，P<0.01）。Western blot检测发现，与NB-SCR-US组和NB-siRNA组相比，NB-siRNA-US组的IDH1基因表达水平明显减低。CCK-8及Annexin-V检测各组细胞活性及细胞凋亡率，结果显示与NB-siRNA组和NB-SCR-US组相比，NB-siRNA-US组的细胞活性明显减低，细胞凋亡率明显增高，差异有统计学意义（CCK-8组：t=-13.52，-31.55，P<0.01；Annexin-V组：t=8.30、9.79，P<0.01）。

结论

纳米微泡搭载siRNA联合超声靶向破碎技术，能有效提高siRNA的转染及干扰效率，可抑制肿瘤细胞生长，为胶质瘤的非侵入性治疗提供了新思路。

关键词: 纳米微泡, 造影剂, 超声, 干扰小RNA, 胶质瘤

Objective

To evaluate the ability of ultrasound contrast agent nanobubbles as carriers of siRNA combined with ultrasound targeted nanobubble destruction to improve the siRNA transfection and interference efficiency in glioma cells, so as to achieve the effect of inhibiting tumor cell growth.

Methods

Nanobubbles were fabricated using the thin-film hydration method. Nanobubbles carrying siRNA were fabricated using the biotin-avidin system. After siRNA was transfected into glioma cells, confocal laser scanning microscopy was applied to assess the transfection efficiency in three groups of cells: nanobubbles-carboxyfluorescein-scrambled siRNA-ultrasound (NB-FAM-SCR-US), NB-FAM-SCR, and normal control. NB-siRNA-US (containing siRNA specific the target gene), NB-siRNA, and NB-SCR-US were used for subsequent experiments. Real-time PCR and Western blot were used to assess interference efficiency. Cell viability and apoptosis were evaluated by CCK-8 and annexin-V analysis.

Results

The particle size of nanobubbles fabricated by the thin film hydration method was (663.9±102.5) nm; the particle size of nanobubbles carrying siRNA was (707.0±127.6) nm. The results of laser confocal microscopy showed that the transfection efficiency of siRNA was successfully enhanced by ultrasound irradiating nanobubbles. Real-time PCR and Western blot showed that the expression of the target gene was significantly reduced in the NB-siRNA-US group (real-time PCR: t=-12.34, -20.35, P<0.01). In the CCK-8 and annexin-V experiments, the proliferation of cells in the NB-siRNA-US group decreased and apoptosis increased (CCK-8: t=-13.52, -31.55, P<0.01; annexin-V: t=8.30, 9.79, P<0.01).

Conclusion

SiRNA transfection and interference efficiency can be improved by ultrasound irradiating nanobubbles to achieve the aim of inhibiting the growth of glioma cells, which provides a new strategy for non-invasive treatment of gliomas.

Key words: Nanobubbles, Contrast agent, Ultrasound, siRNA, Glioma

表1 IDH1- siRNA和阴性对照siRNA的序列

类别	正义链序列（5′-3′）	反义链序列（5′-3′）
IDH1	GGAGATGAAATGACACGAATCT	AGATTCGTGTCATTTCATCTCC
SCR	UUCUUCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT

表1 IDH1- siRNA和阴性对照siRNA的序列

类别	正义链序列（5′-3′）	反义链序列（5′-3′）
IDH1	GGAGATGAAATGACACGAATCT	AGATTCGTGTCATTTCATCTCC
SCR	UUCUUCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT

表2 IDH1和GAPDH引物序列

类别	正向引物（5′-3′）	反向引物（5′-3′）
IDH1	TGT GGT AGA GAT GCA AGG AGA	TTG GTG ACT TGG TCG TTG GTG
GAPDH	ACC ACA GTC CAT GCC ATC AC	TCC ACC ACC CTG TTG CTG TA

表2 IDH1和GAPDH引物序列

类别	正向引物（5′-3′）	反向引物（5′-3′）
IDH1	TGT GGT AGA GAT GCA AGG AGA	TTG GTG ACT TGG TCG TTG GTG
GAPDH	ACC ACA GTC CAT GCC ATC AC	TCC ACC ACC CTG TTG CTG TA

图1~9 激光共聚焦显微镜检测细胞转染siRNA的效率。图1~3为纳米微泡-羧基荧光素（FAM）-乱序阴性对照siRNA（SCR）-超声辐照组（NB-FAM-SCR-US组）的细胞核染色图（DAPI染色，×200），图1见细胞核染色显示为蓝色荧光，图2见细胞转染了FAM-SCR显示为绿色荧光，图3为图1和图2的合并；图4~6为纳米微泡-FAM-SCR-无超声辐照组（NB-FAM-SCR组）的细胞核染色图（DAPI染色，×200），图4见细胞核染色显示为蓝色荧光，图5见少量细胞转染了FAM-SCR显示为微量绿色荧光，图6为图4和图5的合并；图7~9为正常细胞组的细胞核染色图（DAPI染色，×200），图7见细胞核染色显示为蓝色荧光，图8未见细胞转染FAM-SCR无绿色荧光，图9为图7和图8的合并

图10，11 蛋白免疫印迹（Western blot）在蛋白水平检测目的基因表达水平。图10从左至右依次为纳米微泡-siRNA-超声辐照组，纳米微泡-siRNA-无超声辐照组和纳米微泡-乱序阴性对照siRNA（SCR）-超声辐照组目的基因蛋白表达水平，从右至左各组基因蛋白表达水平依次降低，纳米微泡-siRNA-超声辐照组明显低于另外2组。图11为纳米微泡-siRNA-超声辐照组，纳米微泡-siRNA-无超声辐照组和纳米微泡-SCR-超声辐照组3组内参蛋白甘油醛-3-磷酸脱氢酶（GAPDH）表达水平，3组间未见明显差异

图12~14 Annexin V-FITC/PI双染流式细胞仪检测细胞凋亡率。图12为纳米微泡-siRNA-超声辐照组（NB-siRNA-US组）细胞凋亡结果；图13为纳米微泡-siRNA-无超声辐照组（NB-siRNA组）细胞凋亡结果；图14为纳米微泡-乱序阴性对照siRNA（SCR）-超声辐照组（NB-SCR-US组）细胞凋亡结果

表3 3组细胞活性及细胞凋亡率检测结果（±s，%）

组别	细胞活性	细胞凋亡率
NB-siRNA-US	44.29±3.14^a	31.5±5.2^a
NB-siRNA	78.71±4.75^b	5.5±1.0^b
NB-SCR-US	101.30±2.54	1.2±0.4
F值	318.16	81.39
P值	<0.01	<0.01

表3 3组细胞活性及细胞凋亡率检测结果（±s，%）

组别	细胞活性	细胞凋亡率
NB-siRNA-US	44.29±3.14^a	31.5±5.2^a
NB-siRNA	78.71±4.75^b	5.5±1.0^b
NB-SCR-US	101.30±2.54	1.2±0.4
F值	318.16	81.39
P值	<0.01	<0.01

1	Leng Q, Woodle MC, Mixson AJ. Targeted Delivery of siRNA Therapeutics to Malignant Tumors [J]. J Drug Deliv, 2017, 2017: 6971297.
2	Truong NP, Gu W, Prasadam I, et al. An influenza virus-inspired polymer system for the timed release of siRNA [J]. Nat Commun, 2013, 4: 1902.
3	汪朝霞, 王志刚. 超声微泡造影剂携基因或药物治疗研究[J]. 中国介入影像与治疗学, 2006, 3(4): 306-308.
4	Zhou Y, Yang K, Cui J, et al. Controlled permeation of cell membrane by single bubble acoustic cavitation [J]. J Control Release, 2012, 157(1): 103-111.
5	Cai WB, Yang HL, Zhang J, et al. The Optimized Fabrication of Nanobubbles as Ultrasound Contrast Agents for Tumor Imaging [J]. Sci Rep, 2015, 5: 13725.
6	Yang H, Cai W, Xu L, et al. Nanobubble-Affibody: Novel ultrasound contrast agents for targeted molecular ultrasound imaging of tumor [J]. Biomaterials, 2015, 37: 279-288.
7	杨恒丽, 蔡文斌, 张莉, 等. 纳米级分子靶向超声造影剂的制备及其体外肿瘤靶向性实验研究[J]. 中国超声医学杂志, 2016, 32(4): 367-370.
8	Aronovich EL, Mcivor RS, Hackett PB. The Sleeping Beauty transposon system: a non-viral vector for gene therapy [J]. Hum Mol Genet, 2011, 20(R1): R14-R 20.
9	Huang S, Kamihira M. Development of hybrid viral vectors for gene therapy [J]. Biotechnol Adv, 2013, 31(2): 208-223.
10	赵晓乐, 孔晓军, 李剑勇. 可递送siRNA的非病毒纳米载体的设计[J]. 国际药学研究杂志, 2016, 43(4): 677-681.
11	Li W, Hu ZF, Chen B, et al. Response of C2C12 myoblasts to hypoxia: the relative roles of glucose and oxygen in adaptive cellular metabolism [J]. Biomed Res Int, 2013, 2013: 326346.
12	Ballarin-Gonzalez B, Howard KA. Polycation-based nanoparticle delivery of RNAi therapeutics: adverse effects and solutions [J]. Adv Drug Deliv Rev, 2012, 64(15): 1717-1729.
13	陈中华, 朱德生, 李军, 等. 非病毒siRNA载体研究进展[J]. 中国药理学通报, 2015, 31 (7): 910-914.
14	Hobbs SK, Monsky WL, Yuan F, et al. Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment [J]. Proc Natl Acad Sci U S A, 1998, 95(8): 4607-4612.
15	Oeffinger BE, Wheatley MA. Development and characterization of a nano-scale contrast agent [J]. Ultrasonics, 2004, 42(1-9): 343-347.
16	Calvert AE, Chalastanis A, Wu Y, et al. Cancer-Associated IDH1 Promotes Growth and Resistance to Targeted Therapies in the Absence of Mutation [J]. Cell Rep, 2017, 19(9): 1858-1873.
17	Marmottant P, Hilgenfeldt S. Controlled vesicle deformation and lysis by single oscillating bubbles [J]. Nature, 2003, 423(6936): 153-156.
18	Ohl CD, Arora M, Ikink R, et al. Sonoporation from jetting cavitation bubbles [J]. Biophys J, 2006, 91(11): 4285-4295.
19	Wu G, Mikhailovsky A, Khant HA, et al. Remotely triggered liposome release by near-infrared light absorption via hollow gold nanoshells [J]. J Am Chem Soc, 2008, 130(26): 8175-8177.
20	Wu J. Shear stress in cells generated by ultrasound [J]. Prog Biophys Mol Biol, 2007, 93(1-3): 363-373.

[1]	王亚红, 蔡胜, 葛志通, 杨筱, 李建初. 颅骨骨膜窦的超声表现一例[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(11): 1089-1091.
[2]	李晓妮, 卫青, 孟庆龙, 牛丽莉, 田月, 吴伟春, 朱振辉, 王浩. 超声心动图在孤立性左心室心尖发育不良疾病中的应用价值[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(10): 937-942.
[3]	陈慧, 姚静, 张宁, 刘磊, 马秀玲, 王小贤, 方爱娟, 管静静. 超声心动图在多发性骨髓瘤心脏淀粉样变中的诊断价值[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(10): 943-949.
[4]	戴飞, 赵博文, 潘美, 彭晓慧, 陈冉, 田园诗, 狄敏. 胎儿心脏超声定量多参数对主动脉缩窄胎儿心脏结构及功能的诊断价值[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(10): 950-958.
[5]	章建全, 程杰, 陈红琼, 闫磊. 采用ACR-TIRADS评估甲状腺消融区的调查研究[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(10): 966-971.
[6]	罗辉, 方晔. 品管圈在提高甲状腺结节细针穿刺检出率中的应用[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(10): 972-977.
[7]	杜祖升, 赵博文, 张帧, 潘美, 彭晓慧, 陈冉, 毛彦恺. 应用二维斑点追踪成像技术评估孕周及心尖方向对中晚孕期正常胎儿左心房应变的影响[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(09): 843-851.
[8]	杨忠, 时敬业, 邓学东, 姜纬, 殷林亮, 潘琦, 梁泓, 马建芳, 王珍奇, 张俊, 董姗姗. 产前超声在胎儿22q11.2 微缺失综合征中的应用价值[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(09): 852-858.
[9]	包艳娟, 杨小红, 张涛, 赵胜, 张莉. 阴道斜隔综合征的超声诊断与临床分析[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(09): 859-864.
[10]	汪洪斌, 张红霞, 何文, 杜丽娟, 程令刚, 张雨康, 张萌. 低级别阑尾黏液性肿瘤与阑尾黏液腺癌超声及超声造影特征分析[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(09): 865-871.
[11]	农云洁, 黄小桂, 黄裕兰, 农恒荣. 超声在多重肺部感染诊断中的临床应用价值[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(09): 872-876.
[12]	刘思锐, 赵辰阳, 张睿, 张一休, 杨萌. 多普勒超声对孕鼠子宫动脉不同节段血流动力学参数的评估[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(09): 877-883.
[13]	孙佳丽, 金琳, 沈崔琴, 陈晴晴, 林艳萍, 李朝军, 徐栋. 机器人辅助超声引导下经皮穿刺的体外实验研究[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(09): 884-889.
[14]	宋勇, 李东炫, 王翔, 李锐. 基于数据挖掘法分析3 种超声造影剂不良反应信号[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(09): 890-898.
[15]	常小伟, 蔡瑜, 赵志勇, 张伟. 高强度聚焦超声消融术联合肝动脉化疗栓塞术治疗原发性肝细胞癌的效果及安全性分析[J/OL]. 中华普外科手术学杂志(电子版), 2025, 19(01): 56-59.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

In vitro effect of nanobubbles carrying siRNA combined with ultrasound targeted destruction on glioma cell proliferation

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

In vitro effect of nanobubbles carrying siRNA combined with ultrasound targeted destruction on glioma cell proliferation