| Objective: Recent study has confirmed that CD133 is a surface marker of cancer stem cells in the Hep-2 human laryngeal carcinoma cell line (Hep-2 cells). The CD133 positive cancer cells possess the typical characteristics of cancer stem cell. In this study, the CD133+ cells ratio,the inhibition rate of cell proliferation and the cell cycle distribution following radiotherapy were measured in the Hep-2 cells to find the changing characteristics of detection index and the interrelationship among the various indicators. By doing so, we investigated the effect of radiotherapy on cancer stem cells (CSC), test and verify the radiation resistance of CSC and explore the role of CSC in tumor resistance to radiotherapy. Thus, the underlying mechanisms behind of the radiation resistance in head and neck cancer are discussed preliminarily at CSC level, which establish a favorable foundation for further study on the new experimental methods for eliminating the head and neck CSC to cure tumor.Methods: First the Hep-2 cells were cultured under the conditions of 37℃,5﹪CO2,20﹪O2 and 95﹪humidity. Cells at logarithmic growth phase were selected for experimental study after different doses of radiotherapy and proceeded to the following experiment.1. The cells were observed under the inverted fluorescence microscope after different doses of radiotherapy and the morphological changes of the cells were examined.2. The inhibition of cell proliferation in the Hep-2 cells that had received radiotherapy was measured by MTT assay. The growth pattern of Hep-2 cells underwent different doses of radiotherapy was observed.3. The rate of CD133+ cells and the difference among the various checkpoints were measured by FCM. The proper radiation dose and the time point at which the CD133+ cells ratio reached the maximal level was sereened as the best observation point.4. Cell cycle distribution of the Hep-2 cells after 20Gy of radiation was determined by flow cytometry to changing characteristics of the cell cycle distribution after radiotherapy.By analyzing the observed data in this experiment we explored the relationship between radiation resistance and the cancer stem cells in human laryngcal carcinoma.Results:1. By periodic observation under the inverted fluorescence microscope, we found the morphological changes of the Hep-2 cells after radiotherapy. These included reducing of cell volume, dasformation, shrinking of nucleus, cytoplasmic roughness, fewer cell division and poor cells adhesion, all of which are typical changes of apoptosis. The radiation promoted the number of apoptotic cells and the degree of cell morphological changes in a dose-dependent manner.2 The growth of the Hep-2 cells were inhibited after radiotherapy as measured by MTT, which indicates that the radiation dosage enhanced the cell proliferation inhibition rate in a dose-dependent manner.3. The ratio of CD133+ cells was increased in every group after different doses of radiotherapy as determined by FCM. There was a significant positive correlation between the ratio of CD133+ cells and the radiation dose in 0Gy to 20Gy dose range (r=0.22,p<0.05), and becameless prominentat 25Gy. The ratio of CD133+ cells in Hep-2 cells that has been given radiotherapy was initially increased and decreased subsequently. The highest point is at 48h, with the highest value of 2.70%±0.13%at 20Gy. This can be defined as the best observation condition for the future study.4. The cell cycle distribution of the Hep-2 cells changed after exposure to 20Gy radiotherapy as evaluated by FCM. The Hep-2 cells cell cycle arrest at G0/G1 phase was induced and the ratio of G0/G1 was increased at the beginning and decreased thereafter, with the highest ratio of 68.2%±4.16% at 48h. Conclusion:1. The Hep-2 cells showed significantly morphological changes after the radiation exposure, which are consistent with the morphological characteristics of apoptosis.2. The phenomenon that the ratio of CD133+cells in Hep-2 cells was increased after radiotherapy was the direct sign that Hep-2-CSC has the ability to resist radiotherapy.3. There was significant positive correlation between the ratio of CD133+ cells and the radiation dose in 0Gy to 20Gy dose range. The point (20Gy, 48h) could be regarded as the best observation point for the future study.4. The ratio of CD133+ cells of the Hep-2 cell line after radiotherapy were increased initially and decreased subsequently. Because the radiation resistance of the CSC was comparatively high at the beginning, then the CSC began to differentiate into non-cancer stem cell, and finally led to the ratio decreased.5. The ability of cell proliferation will be recovered gradually in the future. The ability of CSC such as radiation resistance, self-renewal and differentiation, can regain the proliferation of Hep-2 cells line.6. Above all, there is a really existence of the cell subsets which has the ability of radiation resistance in the Hep-2 cells, most likely the CSC. There maybe some connections among cell proliferation inhibition rate, cell cycle and CSC. For the definition of the related mechanisms from the molecular aspects further investigation is due to carry out.
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