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

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

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