摘要
目的：
1、检测并比较乳腺癌MDA-MB-231、MCF-7细胞葡萄糖转运蛋白1(Glut1)表达情况,揭示乳腺癌Glut1表达与恶性程度的关系,选择Glut1过表达最显著的细胞株作为实验模型。
2、评价荧光物质标记的2-脱氧-D-葡萄糖(2-DG)即2-NBDG被高表达Glut1乳腺癌细胞靶向摄取的可行性,以此证实2-DG分子能被Glut1高表达肿瘤细胞靶向摄取。
3、探索用2-DG标记USPIO构建靶向分子磁共振成像探针2-脱氧葡萄糖-超小超顺磁性氧化铁(2-DG-USPIO)可行性。
4、探索分子磁共振成像探针2-DG-USPIO靶向Glut1高表达乳腺癌细胞的效果。
材料和方法：
1、RT-PCR及免疫组化检测乳腺癌MDA-MB-231细胞Glut1 mRNA和蛋白表达,Westernblot比较乳腺癌MDA-MB-231及MCF-7细胞Glut1蛋白表达量。
2、2-NBDG孵育接种在六孔板里的人乳腺癌MDA-MB-231细胞,并用D葡萄糖进行竞争抑制对照,荧光显微镜及流式细胞仪观察、分析2-NBDG摄取结果。流式细胞仪检测比较MDA-MB-231及MCF-7细胞吸收2-NBDG量的差异。
3、采用化学交联法制备分子磁共振成像探针2-DG-USPIO,电镜观察形态、测量粒径及近红外光谱分析表征。
4、分别用2-DG-USPIO、单纯USPIO孵育人乳腺癌MDA-MB-231细胞10分钟至2小时后普鲁士蓝染色及体外磁共振成像检测人乳腺癌细胞(MDA-MB-231)吸收情况,电镜观察细胞内吸收铁的分布。
结果：
1、乳腺癌MDA-MB-231细胞和MCF-7细胞均有明显Glut1mRNA和蛋白过表达Westernblot测得MDA-MB-231细胞表达Glut1蛋白表达量为(0.946±0.007),高于MCF-7细胞Glut1蛋白表达量(0.833±0.010)。
2、荧光成像及流式细胞仪分析显示MDA-MB-231能快速摄取2-NBDG,且50 mM D型葡萄糖竞争抑制后,摄取2-NBDG的荧光强度降低46%。2-NBDG孵育乳腺癌细胞20分钟后流式细胞仪分析结果显示MDA-MB-231荧光强度(25.10±0.57)明显高于MCF-7(10.12±0.62)
3、电镜观察2-DG-USPIO纳米粒子成球形,平均粒径为10nm,近红外光谱分析证明2-DG-USPIO纳米粒子表面存在2-DG结构。
4、2-DG-USPIO孵育MDA-MB-231细胞10分钟后普鲁士蓝染色即显示胞浆内大量蓝色颗粒,单纯USPIO未见明显蓝色颗粒。临床1.5T磁共振T2加权成像显示2-DG-USPIO孵育MDA-MB-231组较单纯USPIO孵育MDA-MB-231组信号明显下降。
结论：
1、乳腺癌细胞Glut1表达程度与细胞的恶性程度密切相关,细胞恶性程度越高,Glut1蛋白表达量越大；高度恶性的乳腺癌MDA-MB-231细胞可作为高代谢的肿瘤细胞实验模型。
2、2-NBDG能迅速被高表达Glut1的乳腺癌细胞靶向吸收,可作为葡萄糖代谢的光学标记物来检测高代谢的恶性肿瘤,并说明2-DG分子能被过表达Glut1的肿瘤细胞靶向吸收。
3、2-DG成功标记到包覆有DMSA的USPIO表面,且2-DG-USPIO能被过表达Glut1的乳腺癌MDA-MB-231细胞靶向吸收。
Objective:
1、To compare Glucose transporter 1 (Glut1) mRNA and protein expressed in breast cancer MDA-MB-231 and MCF-7 cells for revealing the relationship between grade malignancy and Glutl expression in breast cancer,and select the most significant cell line of Glutl overexpression in breast cancer as an experimental model.
2、To assess the feasibility of fluorescent 2-deoxyglucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl) amino]-2-deoxyglucose (2-NBDG) was targetedly taken up by breast cancer cells that overexpress Glutl, which confirmed that 2-DG molecule could target tumor cell with Glut1 expression.
3、To explore the feasibility of USPIO labeled with 2-DG construction for preparation tumor targeted molecular magnetic resonance imaging probe 2-deoxyglucose-ultra-small super-paramagnetic iron oxide (2-DG-USPIO).
4、To explore the effect of the molecular magnetic resonance imaging probe 2-DG-USPIO targeting breast cancer cells with Glut1 overexpression.
Methods:
1、Firstly, mRNA and protein expression of Glutl in breast cancer MDA-MB-231 cells were detected via RT-PCR and immunohistochemistry respectively,then protein expression of Glutl between breast cancer MDA-MB-231 cells and MCF-7 cells were compared via Western blot.
2、MDA-MB-231 cells were grown in 6-well plates for analysis of 2-NBDG uptake via fluorescence microscopy imaging and flow cytometer. And the control was addion D glucose in the medium of 2-NBDG for competitive inhibition.The amount differences of absorption of 2-NBDG between MDA-MB-231 and MCF-7 cells were compared by flow cytometry.
3、The molecular magnetic resonance imaging probe was prepared through 2-DG conjugated to USPIO using chemical method. The agent were tested by transmission electron microscope and infra-red spectrum.
4、Human breast cancer cells (MDA-MB-231) were incubated with 2-DG-USPIO or USPIO for 10 minute to 2 hour respectively. Accumulation in the cells was evaluated using Prussian blue staining and magnetic resonance (MR) imaging, further intracellular iron absorption observed by electron microscopy.
Results:
1、MDA-MB-231 cells and MCF-7 cells overexpress Glut1 mRNA and protein.MDA-MB-231 cells express Glutl protein(0.946±0.007) higher than MCF-7 cells express (0.833±0.010).
2、Fluorescence microscopy imaging and flow cytometer analysis displayed MDA-MB-231 cells could uptake 2-NBDG. Addition of 50 mM D-glucose to the media reduced 2-NBDG uptake by 46%. Furthermore, fluorescence intensity of MDA-MB-231 cells (25.10±0.57) is higher than MCF-7 cells'(10.12±0.62) when incubated by 2-NBDG for 20 minute.
3、The result of transmission electron microscope displayed 2-DG-USPIO nanoparticles were spherical. The diameters of particles were 10 nm. The result of infra-red spectrum proved that 2-DG was conjugated to the surfaces of USPIO.
4、After 10 mins, a large number of blue granules was observed in the cytoplasm of MDA-MB-231 incubated by 2-DG-USPIO, whereas there was no significant blue granules in cells incubated by USPIO.The signal of MDA-MB-231 cells after 10-mins incubation with 2-DG-USPIO was clearly lower than the signal of MDA-MB-231 cells incubated with plain USPIO in clinical 1.5-T MR T2-weighted image.
Conclusion:
1、Glutl expression is closely related to malignant grade in breast cancer cells, the higher cells malignant grade was,the higher Glutl protein expressed,so highly malignant breast cancer MDA-MB-231 cells was chosed as the tumor cells with high metabolic experimental model.
2、The preliminary data clearly demonstrate 2-NBDG was taken up and accumulated in breast cancer cells with overexpressing Glutl,and may as a optic probe for glucose uptake in hypermetabolism malignant cells, And indicate the 2-DG molecule can be targetedly uptaken by tumor cells with Glutl overexpression.
3、2-DG was labelled to the surface of USPIO coated with DMSA successfully. The molecular probe 2-DG-USPIO could be targetedly uptaken by breast cancer MDA-MB-231 cells with Glutl overexpression.
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