乳腺癌转移演进的关键分子ALDH2体外可视化研究

In vitro visualization of ALDH2,a key molecule in the evolution of breast cancer metastasis

目的:分析乙醛脱氢酶2(ALDH2)靶向脂质体纳米探针ALDH2-Cy7@LP-FA在细胞水平的成像能力和靶向性能。方法:研究制备荧光探针ALDH2-Cy7@LP-FA,通过粒径分析、紫外吸收和荧光发射光谱对其进行表征。利用Western印迹和免疫组化检测ALDH2在乳腺癌组织和细胞系的表达情况。以ALDH2高表达的乳腺癌细胞BT474、MDA-MB-231作为实验组,ALDH2低表达的MCF-10A作为对照组,通过流式细胞术及共聚焦荧光显微镜,观察探针的亚细胞器共定位及成像效果。采用CCK8法分析探针的细胞毒性和生物相容性,为体内成像提供实验依据。结果:脂质体探针ALDH2-Cy7@LP-FA粒径均一分布[(155±2)nm],脂质体的包载使得标记抗体的紫外吸收和荧光发射分别发生了4 nm和5 nm的蓝移,脂质体的包载实现了抗体纳米化。ALDH2蛋白在BT474细胞系中表达最高,在MCF-10A表达最低(t=29.123,P<0.001)。体外摄取及成像效果显示,ALDH2-Cy7@LP-FA可特异性靶向线粒体中的ALDH2,对BT474细胞的结合亲和力高于231细胞,对MCF-10A的结合最低,具有选择性结合ALDH2高表达癌细胞的能力(BT474:t=9.976,P<0.01;MDA-MB-231:t=12.026,P<0.05)。CCK8实验结果表明探针毒性极低,具有良好的生物相容性。结论:ALDH2在高转移乳腺癌细胞和组织中高表达,是识别转移细胞的关键分子,通过ALDH2-Cy7@LP-FA靶向ALDH2分子成像,可实现精准定位肿瘤边界。

Objective:To analyze the imaging ability and targeting performance of acetaldehyde dehydrogenase 2(ALDH2)-targeted liposomal nanoprobe ALDH2-Cy7@LP-FA in the cellular level.Methods:The fluorescent probe ALDH2-Cy7@LP-FA was prepared and characterized by particle size analysis,UV absorption and fluorescence emission spectroscopy.Western blotting and immunohistoche-mistry were used to detect the expression of ALDH2 in different breast cancer tissues and cell lines.Breast cancer cells BT474 and MDA-MB-231 with high ALDH2 expression were used as the experimental group,and MCF-10A with low ALDH2 expression was used as the control group and the subcellular organelle co-localization and imaging effect of the probes were observed by flow cytometry and confocal fluorescence microscopy.The cytotoxicity and biocompatibility of the probes were analyzed by CCK8 method to provide experimental basis for in vivo imaging.Results:The particle size of the liposomal probe ALDH2-Cy7@LP-FA was uniformly distributed[(155±2)nm],and the liposome encapsulation resulted in a blue shift of 4 nm and 5 nm in the UV absorption and fluorescence emission of the labeled antibody,respectively,and the liposome encapsulation realized the nanosizing of the antibody.The protein expression of ALDH2 was the highest in BT474 cell line,and the expression in MCF-10A was extremely low(t=29.123,P<0.001).In vitro uptake and imaging showed that ALDH2-Cy7@LP-FA could specifically target ALDH2 in mitochondria,with higher binding affinity to BT474 cells than to 231 cells,and the lowest binding to MCF-10A,with the ability to selectively bind to ALDH2 high-expressing cancer cells(BT474:t=9.976,P<0.01;MDA-MB-231:t=12.026,P<0.05).The results of CCK8 experiments demonstrated that the probe had extremely low toxicity and good biocompatibility.Conclusion:ALDH2 is highly expressed in highly metastatic breast cancer cells and tissues,and is a key molecule for identifying metastatic cells.Molecular imaging by targeting ALDH2 with ALDH2-Cy7@LP-FA can achieve precise localization of tumor boundaries.