| Oral squamous cell carcinoma (oral squamous cell carcinoma, OSSC) is the most common oral and maxillofacial malignant tumor, derived from stratified squamous epithelial cells of the skin and mucous membranes. Tongue squamous cell carcinoma is one of the most common types of oral squamous carcinoma. In recent years, the incidence of oral squamous cell carcinoma is rising; the age of onset tends to be younger. It becomes an urgent important task to search for an effective treatment way. The most main three kinds of treatment means of tongue squamous cell carcinoma are still surgical therapy, chemotherapy and radiation therapy, however, the injury and risk of surgical therapy as well as the great side effects of radiotherapy and chemotherapy have seriously effect on the quality life and survival rate of cancer patients. Therefore, it is imperative to research and develop a new treatment plan of with targeted, more secure, more efficient, low side effects, non-invasive or minimally invasive for tongue squamous cell carcinoma.Using the principle that tumor tissue compared with normal tissue was more sensitive to high temperature, tumor tissue was heated to above43℃, the tumor cells than normal cells could not tolerate continuous high temperature, which occurred different degrees of apoptosis or necrosis, this method of treatment of malignant tumors was called hyperthermia (Hyperthermia). In recent years, the rapid development of nanotechnology makes it a challenging innovation in the medical field. Magnetic fluid hyperthermia (MFH) was magnetic fluid of modified or added a specific antibody by arteriovenous or injected directly to the tumor, magnetic fluid was phagocytic by tumor cells or deposited between cells, in the alternating magnetic field, it heated up by magnetic vector rotation and physical rotation of magnetic nanoparticles, namely through the mechanism of Neil relaxation and Brown relaxation, so as to kill tumor cells, while the surrounding normal tissues temperature was not obvious, highly targeted, the presence of the magnetic fluid hyperthermia technology to solve the problems of the accurate location of lesions in hyperthermia, got better effect in tumor hyperthermia. New type of nanoparticles mediated magnetic fluid hyperthermia (magnetic fluid hyperthermia, MFH) is a new method of deep tissue thermal therapy, we can get hyperthermia temperature what we want in our experiment by adjusting magnetic fluid concentration or magnetic field strength, this experiment was realized by adjusting the magnetic fluid concentration. The local heat therapy can be divided into thermotherapy (42～46℃), hyperthermia(46～70℃), heat(above70℃)resection. Compared with the traditional heat treatment technologies such as infrared hyperthermia therapy, ultrasound hyperthermia, microwave hyperthermia and radio frequency electromagnetic wave hyperthermia, MFH technology has the features of treatment of deep tumors, the side effect is small, noninvasive or minimally invasive. Once the tumor location was single injection nano magnetic fluid, patients can be hyperthermia repeated several times.The immune function of mice was deficient, under certain conditions, not to the exclusion of tissue transplantation from xenogenetic animal, transplanted tumor remained original histological structure and biological characteristics, at the same time, and transplanted tumor metastasis was hardly seen. These conditions were the basis for successful establishment of human tongue cancer tumor modal, and also an important guarantee for the experiment; and axillary subcutaneous tumor modal in nude mice, tumor tissue grow superficially and could be palpated from the surface, rich blood supply to tumors, tumor growth was larger, it was beneficial to observe the therapeutic effect of magnetic fluid hyperthermia on tumor volume change. The application of magnetic fluid hyperthermia (MFH) with nanoparticles has been shown to inhibit tumor growth in several animal models. However, the feasibility of using MFH in vivo to treat tongue cancer is uncertain, and the mechanism is unclear. In the present study, this study aimed to establish tongue cancer animal model of axillary subcutaneous in nude mice, intratumoral injection of nanometer magnetic fluid and hyperthermia under alternating magnetic field, to observe the effect of magnetic fluid hyperthermia on tumor growth, and further verify the clinical application and the feasibility of nano-magnetic fluid hyperthermia treatment on malignant tumors.Objectives:To detect the potential heating performance of nanometer magnetic fluid under alternating magnetic field in vivo and in vitro, and to establish xenograft model of Tca8113of human tongue squamous cell carcinoma cells in nude mice. To evaluate the feasibility of local magnetic fluid hyperthermia on nude mice Tca8113tongue cancer model and investigate the effect of it on the growth of tumor volume.Methods:1. Cultivation of Tca8113cell:recovery tongue cancer cell line Tca8113, cultivated in RPMI1640medium in vitro, and observe the cell morphology and proliferation activity of Tca8113cell.2. Mn0.4Zn0.6Fe2O4nanometer magnetic fluid was tested by transmission electron microscope and hystersis loop, as well as heated under different magnetic field strength in vitro.3. The phagocytosis experiment of Tca8113human tongue cancer cells on Mno.4Zno.6Fe204nanometer magnetic particle:To take Tca8113cells in the logarithmic phase and Mn0.4Zn0.6Fe2O4nanoparticles coated silica co-cultured24h, prussian blue staining, to observe phagocytosis of cells on nanoparticles under inverted microscope.4. Tca8113cytotoxicity assay and cell morphology observation:To take RPMI1640 medium containing different concentrations of nanoparticles leaching solution co-cultured with Tca8113cells, observing the change of cell morphology, detecting the change of the cell proliferation activity, as well as to the cell toxicity test.5. Experiment in vivo:Totally27BALB/c-nu male mice were selected randomly, mouse models of tongue cancer was established by injecting Tca8113cells subcutaneously. Ten days after tumor implantation, the mice bearing Tca8113tongue tumor were randomly divided into five groups, including the blank control group (A), magnetic fluid control group (B), magnetic flied control group (C), magnetic fluid hyperthermia1group (D) and magnetic fluid hyperthermia2group (E). After the mice of magnetic fluid hyperthermia1group (D) and magnetic fluid hyperthermia2group (E) were given intratumoral direct injection of magnetic fluid24h and exposed to an alternating magnetic field, the temperature changes of tumor center, tumor edge and perianal in alternating magnetic field were measured by thermistor temperature sensors. When the temperature of the experimental tumor center stabilized at43℃, then continually hyperthermia for30minutes. Magnetic fluid hyperthermia2group (E) were repeated hyperthermia one time after5～7days. Heating time and heating conditions of magnetic flied control group identical with magnetic fluid hyperthermia group, but the blank control group without any treatment, the magnetic fluid control group only intratumoral injection of magnetic nano-magnetic fluid without heating. After treatment of24h, one mouse from each group was randomly selected and death by anesthesia overdose injection; tumor tissue, surrounding muscle tissue, heart, lung, liver, spleen and kidney were rapidly removed, pathological changes of tissues were generally observed, were cut and fixed immediately with formaldehyde, HE and Prussian blue staining, microscope was used to observe the histological changes of the tumors and with or without magnetic fluid residue in important organs. All the experimental nude mice were sacrificed after nine weeks, tumor volume was measured, the inhibition rate of tumor volume was calculated, and tumor specimens were observed generally and histopathologically.6. Statistical analysis:SPSS20.0software package was used for statistical analysis. Measurement date were presented as mean±tandard(x±s), absorbance value, tumor volume, tumor longest diameter and temperature of each group were used analysis of variance, between the two groups and group pairwise comparisons were used the LSD method. P<0.05was considered to be statistically significant.Results:1. Cultivation of Tca8113cell:Tongue cancer cells grew well. Tca8113cells grow stably and have normal morphology. They lived in groups, proliferate quickly, enter logarithmic growth phase at the second day, and flat phase at the fourth day.2. Observed under transmission electron microscope photos found that nanoparticles particles well dispersed in aqueous solution, particle size of about10nm, not gathered phenomenon; Mn0.4Zn0.6Fe2O4nanoparticles have the property of superparamagnetic through the hysteresis loop detection. The heating experiment testing of magnetic fluid found that nano magnetic fluid heating rate increases with the increase of magnetic fluid concentration in vitro, in the current of50A and the magnetic field frequency of237KHZ, magnetic fluid rapidly rose to80℃within20minutes slow last maintained at83℃.3. After Tca8113cells with Mn0.4Zn0.6Fe2O4particles co-cultured24h, inverted microscope found that Tca8113cells had iron oxide of blue change, whereas the blank control group no blue particles by Prussian blue staining.4. The leaching solution group of25%,50%and75%with Tca8113cells co-cultured, the number of cells increased with prolonged the incubation time, no significant changes in cell morphology, no cytotoxicity; The leaching solution group of100%grew slowly, showing a small amount of nuclear pyknosis, showed slight cytotoxicity. With the increase of concentration of leaching solution, cell proliferation activity decreased obviously by MTT test.5. Tumors on axillary subcutaneous in nude mice grow to an average of about0.8cm～1cm in diameter after inoculation of10days, the tumorigenicity in nude mice was100%. All nude mice were randomly divided into five groups, and there were no significant differences between the tumor volume of nude mice of each group before hyperthermia(.P>0.05). The tumor center temperature of magnetic fluid hyperthermia groups could be relatively stable43℃, tumor marginal zone temperature lower2～3℃than the central area of the tumor was about40℃, rectum almost no heated. Magnetic field control group without warming, tumor center temperature was the same as body temperature. The tumor center temperature of blank control group and magnetic fluid control group were stable at30.8℃, also without warming. Tumor center temperature and tumor marginal temperature of magnetic fluid hyperthermia1group and magnetic fluid hyperthermia2group were compared with the blank control group, the difference was statistically significant (P<0.05). Magnetic fluid hyperthermia1group were compared with magnetic fluid hyperthermia2group, the difference was no statistically significant (P>0.05). There was significant difference between tumor center temperature and tumor marginal temperature of magnetic fluid hyperthermia group (P<0.05). All experimental mice could tolerate hyperthermia. The tumor volume of nude mice in each group showed a certain degree of increasing trend after hyperthermia treatment. Blank control group and magnetic field group increased obviously, the growth trend of magnetic fluid group was slightly lower. The tumor volumes of three control groups namely magnetic field control group, magnetic fluid control group and blank control group were compared, the difference was no statistically significant (P>0.05). The tumor volumes within6week namely after inoculation the second, third, fourth, fifth week of magnetic fluid hyperthermia group were compared with control group, and the difference was statistically significant (P<0.05), especially the tumor volume of magnetic fluid hyperthermia2group was compared with each control group, and the difference was more statistically significant (P<0.01);The tumor volume after inoculation the third, fourth, fifth week of the magnetic fluid hyperthermia groups were compared, and the difference was statistically significant (P<0.05), but after6weeks due to residual tumor cells arounding hyperthermia tumor tissue continue to grow, there was no significant difference between each group after6weeks (P>0.05). After magnetic fluid hyperthermia1group and magnetic fluid hyperthermia2group were injected of magnetic fluid and hyperthermia treatment, tumor growth was inhibited, the inhibition of magnetic fluid hyperthermia2group was more pronounced, the inhibition rate of tumor volume after inoculation between2and5weeks of magnetic fluid hyperthermia1group and magnetic fluid hyperthermia2group were23.80%～41.81%and34.92%～56.76%respectively. There was no metastasis of tumor cells in heart, lung, liver, kidney and spleen in each group, but also no nano-magnetic fluid residue, no obvious abnormalities.Conclusions:1. Water solubility of Mn0.4Zn0.6Fe2O4nano magnetic fluid was good, had the characteristic of magnetic; Mn0.4Zn0.6Fe2O4nano magnetic fluid had a good characteristic of temperature rising both in vitro and in vivo, tumor area could obtained43℃hyperthermia treatment temperature of relatively homogeneous distribution, while the remaining normal tissue without heating up in vitro.2. Human tongue Tca8113cell could phagocytic Mn0.4Zn0.6Fe2O4nanoparticles.3. Mn0.4Zn0.6Fe2O4had no obvious cytotoxicity to cells, cytotoxicity was closely to the concentration of the magnetic fluid, and the magnetic fluid had no significant effect on cell morphology of tongue Tca8113cells.4. Tongue animal modal which was identified as Tca8113squamous cell carcinoma by histopathological observation was successfully constructed. Magnetic fluid hyperthermia of43℃could lead to tumor tissue necrosis and significantly inhibited the growth of tumor; Similarly, the magnetic fluid hyperthermia could promote the distribution of intratumoral magnetic fluid, to achieve surrounding effect of hyperthermia. The killing effect of magnetic fluid hyperthermia on tumor tissue was related to hyperthermia frequency. Nano magnetic fluid was injected of disposable intratumoral, magnetic fluid mediated43℃hyperthermia treatment of nude mice subcutaneously transplanted tumor of Tca8113was security, effective, feasible, low side effect, better targeting, and could be repeated hyperthermia, had potential clinical applications valuable to treat tumors. |
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