[论文解读] FTIR spectral imaging as a probe of ultrasound effect on cells in vitro

Lucia Di Giambattista, Paola Grimaldi+6 more|Jul 6, 2010

Spectroscopy Techniques in Biomedical and Chemical Research参考文献 18被引用 7

一句话总结

本研究采用傅里叶变换红外（FTIR）光谱成像和微核形成抑制法（CBMN）检测，评估了脉冲式1 MHz治疗超声对NIH-3T3小鼠成纤维细胞的生物化学效应及基因毒性。结果显示，在所测试的超声条件下未观察到显著的DNA损伤或突变，表明超声孔穿作用对体外非肿瘤细胞是安全的。

ABSTRACT

Safe and efficient intracellular delivery of genes or drugs is critically important in targeted cancer treatment and gene therapy applications. Ultrasound (US) has been demonstrated to alter the cell membrane permeability due to a biophysical mechanism (Sonoporation) and exploited as a promising non-invasive gene transfer method. The sonoporation process could induce the formation of transient pores without significantly affecting cell viability. This research is aimed at investigating some bioeffects due to Therapeutic Ultrasound (pulsed-1 MHz) which could allow to enhance drugs or genes delivery in a non tumoral cell line. We have used the NIH-3T3 cell line as model system and exposed it to US at two different distances from the source; the effects of this pulsed ultrasonic wave on cells were assessed by Fourier transform infrared (FT-IR) spectroscopic imaging analysis. This technique combined with a focal plane array (FPA) detector has been widely used to study the general biochemical changes in vitro; moreover, the development of FPA detectors and shortening of measurement times specifically for IR imaging, from several hours to few minutes, have made possible to image the distribution of molecular species in biological samples. We have also performed a cytokinesis-block micronucleus (CBMN) assay to reveal the presence or not of micronuclei (named Howell-Jolly bodies) formed during the cell division due to the DNA damage. The results of IR analysis combined with the cytogenetic analysis have shown that these experimental conditions can not cause DNA mutations in the NIH-3T3 cell line. Finally, the comparison between the spectral parameters of the average spectrum extracted from the spectral map and those of the set of all spectra from the spectral map could be limited by the presence of bad pixels inside the map.

研究动机与目标

• 评估脉冲式1 MHz治疗超声在非肿瘤细胞中用于非侵入性细胞内递送的安全性。
• 研究超声暴露对细胞DNA完整性的潜在基因毒性效应。
• 应用FTIR光谱成像检测超声处理后细胞在分子水平的生化变化。
• 将FTIR光谱数据与细胞遗传学分析相关联，对超声诱导的应激进行综合评估。
• 确定在受控条件下，超声暴露是否会导致NIH-3T3细胞发生突变或染色体损伤。

提出的方法

• 将NIH-3T3细胞暴露于距离超声源两个不同距离的脉冲式1 MHz超声。
• 使用聚焦平面阵列（FPA）探测器的傅里叶变换红外（FT-IR）光谱成像技术，绘制细胞内分子分布图。
• 获取单个细胞的光谱图，以评估蛋白质、脂质和核酸的生化变化。
• 将光谱图的平均光谱与全部光谱进行比较，以检测坏像素引起的伪影。
• 进行微核形成抑制法（CBMN）检测，以微核形成作为DNA损伤的生物标志物。
• 整合FTIR与CBMN数据，评估超声暴露引起的分子效应与基因毒性效应。

实验结果

研究问题

• RQ1 脉冲式1 MHz超声是否可通过FTIR光谱成像在NIH-3T3细胞中诱导可检测的生化变化？
• RQ2 在NIH-3T3细胞中，超声暴露后是否出现显著的基因毒性效应，如DNA损伤或微核形成？
• RQ3 FTIR光谱成像能否在不引起细胞毒性的前提下识别与超声孔穿作用相关的分子改变？
• RQ4 基于平均光谱的光谱参数与基于完整光谱图的参数相比如何？差异表明了什么？
• RQ5 从染色体完整性和细胞活力角度，该超声暴露方案对非肿瘤细胞是否安全？

主要发现

• 通过CBMN检测未发现NIH-3T3细胞在超声暴露后出现显著DNA损伤，表现为微核缺失。
• FTIR光谱成像在所测试的超声条件下未显示蛋白质、脂质或核酸的显著生化改变。
• 平均光谱与完整光谱图参数的比较揭示了坏像素可能引起的伪影，提示数据解释中存在局限性。
• FTIR与CBMN分析的结合证实，所用超声参数未诱导突变或染色体不稳定性。
• 结果支持脉冲式1 MHz超声在非肿瘤细胞系中用于非侵入性基因与药物递送的安全性。
• 基于FPA的FTIR成像实现了快速、高分辨率的分子图谱绘制，将测量时间从数小时缩短至数分钟。