| Tumor, especially the malignant tumor is becoming one of the deadly diseases for human life and health in the 21st century, It is the highest mortality rate compared with cardiovascular and cerebrovascular diseases, diabetes and other frequently-occurring disease, The current method of clinical treatment of cancer are surgery, radiation therapy, chemotherapy and traditional Chinese medicine treatment. These methods have great side effects and can cause great pain to patients, and they are not easy to relapse. Given the limitations of these traditional therapies, researchers have an urgent need to find a new cancer treatment that can effectively eradicate tumor. The establishment and development of Sonodynamic therapy is on the basis of Photodynamic therapy, it is a new anti-tumor therapy. Because the laser penetration of biological tissue is limited, thus limiting its treatment of deep tissue tumors. SDT is based on preferential and long time retention of sonosensitizers such as hematoporphyrin and its derivatives in tumor tissues and subsequent activation of the drugs by ultrasound and use the synergistic effects to induce tissue or cell destruction and produce significant anti-tumor effects. Compared with photodynamic therapy, SDT has a unique advantage in the treatment of deep tissue cancer, and thus has a better prospect for clinical application. Since 1989, scholars have carried out many studies around the different tumor models, ultrasonic and acoustic parameters, sonosensitizers and have achieved fruitful results. The current sonosensitizers that use in SDT is mainly acoustic sensitizer hematoporphyrin and its derivatives such as first-generation photosensitizer, their main drawback is that they are the mixture to form a complex and the effective components is not clear, and it led great difficulties to the scholars to study the mechanism of SDT. Therefore, scientists have been committed to the development of second generation photosensitizer. Chlorin e6 is the second generation of new photosensitizer and is a degradation product of chlorophyll. It has a clear chemical composition, a good retention in the tumor tissue and can rapidly excreted from the body. Many mechanisms of SDT have been advanced just like singlet oxygen and apoptosis, but its exact mechanism is still unclear because of its multiple factors. This paper is supported by the National Natural Science-Foundation of China. We use the spectrofluorophotometry, In vivo imaging system enzymatic chemical methods and immunohistochemistry to study the spectral characteristics of Ce6 and its changes in the distribution in different tissues. And we selected the optimum parameters and observed its anti-tumor effect, at last, we preliminary study the possible mechanism of SDT. The present experimental conclusions obtained are as follows: 1. We found the maximum excitation wavelength of Ce6 is 403nm, the maximum fluorescence emission wavelength of Ce6 is 669nm, which is slightly different to the reported in the literature. indicating Ce6 fluorescence spectra may be affected by the solvent micro-environment, the stability of the system and many other factors, suggesting that during the different studies, they should according to their own experimental conditions spectrometry, making the data more reliable.2. The results of Fluorescence Spectrophotometry showed that after intravenous injection, the ce6 in each tissue increased to a peak value within two hours, and the ce6 in the liver had the highest concentration, and then began to decrease with time delayed with liver excretion fastest. The ce6 in the tumor began to increased after injection and reached the highest level at 2 hours later, and then began to slowly decrease, its metabolic activity was slow between 2～10 h, showed the retention effect of the tumor. The concentration of ce6 in the tumor reduced to the minimum at 24 h, but still higher than other tissues.3. The results in vivo imaging system showed that after intravenous injection, the ce6 in the tumor increased to a peak value after two hours. The concentration of ce6 in the tumor is remained at a higher concentration between 2～11 h, this is basically consistent to the results of fluorescent spectrophotometry, which further confirmed the reliability of our results. On the background of our previous results, we choose 2h,4h,6h after intravenous injection as the ultrasonic treatment time.4. The findings of anti-tumor effect showed that in the Ce6 group there was no any anti-tumor effect, in the Ultrasound group there was a minor effect, and the anti-tumor effect has a increasing trend with the ultrasonic intensity increased and can last for longer time. The Ultrasound plus Ce6 group showed a better anti-tumor effect. When the Ce6 concentration is 10 mg/kg, it plus ultrasound showed a significant anti-tumor effect compared with the Control group. When the Ce6 concentration is 10 mg/kg and the ultrasonic intensity is 4 W/cm2, they demonstrate an ideal combination of synergistic anti-tumor effect. Therefore, we decide to choose this experiment parameters in our future experiments.5. The activities of antioxidant enzymes assay and the lipid peroxidation test showed that compared with the Control group, the enzymes of the Ce6 group and the Ultrasound group showed no significant change, but the Ce6 plus Ultrasound group showed significant change. Therefore, SDT may play its anti-tumor effect by increasing the level of tumor tissue lipid peroxidation and reducing antioxidant enzyme activity.6. We detected the expression of microvessel density and vascular endothelial growth factor by immunohistochemistry, and thus assess the tumor angiogenesis. Compared with the Control group, the expression of VEGF in the SDT group was significantly reduced, indicating that an important factor promote tumor angiogenesis was inhibited, while the intratumoral microvessel density was also significantly reduced, suggesting that SDT may inhibit tumor angiogenesis by blocking their supply of nutrients and thus to inhibit its growth.7. We detected the expression of apoptotic protein Bcl-2, Bax by immunohistochemistry. Compared with the Control group, the expression of Bax in the SDT group was significantly reduced, the expression of Bcl-2 decreased, and the ratio of Bcl-2/Bax decreased, suggesting that one factor that promote tumor cell apoptosis increased, while one factor that inhibit tumor cell apoptosis decreased, and SDT may promote tumor cell apoptosis and inhibit tumor growth in this way. We choice the new sonosensitizer-Ce6 to study the anti-tumor effect of EAT tumor-bearing, and we first introduced the vivo imaging system to monitor the metabolism of ce6 in rats in real time, and to make the experimental data more real and reliable; We filtered the parameters of ultrasonic intensity and the ce6 concentration; and then we discussed the possible mechanism of SDT, which may provide basis experimental data for SDT mediated clinical cancer therapy. |
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