| Background and Aim: Liver cancer is a severe disease which threatens human's life.It is of great importance to establish a fast and accurate experimental method to precisely diagnose liver cancer at an early stage, detect tiny satellite lesions during operation and evaluate the therapeutic effect of drugs. Benefiting from its fingerprint information of biological molecules, narrow bandwidth, less harm to biological samples and the ability to work under aqueous or CO2 environment, Raman spectroscopy is undoubtedly suitable for such investigation. In this paper, we aim to establish the standard to assess the therapeutic effect of different concentration of different drugs to different liver cancer lines as well as a method to distinguish liver cancer tissues from liver normal tissues by Raman spectroscopy.Methods: By Raman imaging of single cells, we compare single point spectra and whole cell image of different liver cancer cells(Huh7, 7402, 7721) after treatment of different concentration of different anticancer drugs(cisplatin, epirubicin, vincristine),to find the rules of change. In addition, in order to show the response of cells to drugs clearly and to fully uncover the mechanism of drugs, we do K-means clustering and multivariate curve resolution(MCR) analysis of Huh7 cells after treatment of cisplatin.Besides, in order to set the criterion for liver cancer diagnosis, we also do single point spectra analysis and Raman imaging of tissue samples followed by K-means clustering and comparison with HE staining.Results: After treatment of cisplatin, cytochrome c in all three kinds of liver cancer cells diffused in the cell and finally disappeared under high concentration. Cisplatin also induced the appearance and accumulation of lipid drops in 7402 and 7721 cells and made the boundary between Huh7 cells and background become vague. After treatment of epirubicin, the background of Raman spectra within Huh7 cells become higher, cytochrome c disappeared and the image of lipid and protein become vague under high concentration. After treatment of vincristine, cytochrome c in Huh7 cells diffused and disappeared, the boundary between cell membrane and background become vague and the cell nucleus expanded. By analyzing the Raman image of liver cancer tissue and liver normal tissue, we found that there is a high fluorescence background which is hard to be removed by pre-exposure in normal tissue. In addition,we found that compared to normal tissue, the peaks at 1175cm-1, 1300~1500cm-1,1650cm-1 decreased while the peak at 1750cm-1 increased and changed to a sharp peak in cancer tissue. Finally, the Raman image results corresponded to HE staining results well.Conclusions: Cisplatin can induce the release and disappearance of cytochrome c from the mitochondria of three kinds of liver cancer cells. It can also stimulate the loss of cellular compound of Huh7 cells and the accumulation of saturated lipid in 7402 and7721 cells, which proves that cisplatin can induce the production of reactive oxygen species within cells. Epirubicin can induce the disappearance of cytochrome c and break the structure of lipid and protein within Huh7 cells. The remaining of this drug in cells can also create a high fluorescence background. Vincristine can induce the release and disappearance of cytochrome c in Huh7 cells and make cell membrane loose. By experiment of liver tissue, we can diagnose liver cancer by fluorescence background as well as Raman peaks at 1175, 1300~1500, 1650 and 1750cm-1. The last but not least,compared with conventional data analysis method, K-means clustering and MCR analysis can give a much clearer contrast between different sets of data, and can provide a new insight into analysis of Raman data in medical diagnosis. |
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