| Backgrounds:Malignant carcinoma has become a serious threaten to human health nowadays. Traditional methods such as radiotherapy, chemotherapy and surgery have unbearable side effects, surgery can cause organ damage. Following surgery, radiotherapy, chemotherapy and immune therapy, hyperthermia has become another important method to treat cancer. The property of the thermotherapy is to directly kill the tumor cells; therefore it can prolong the survival time and improve the quality of life of patients. When it coupled with other treatment methods including radiotherapy and chemotherapy, it can significantly enhance the sensitivity and reduce the side effects of other treatment methods. Thus the thermotherapy is viewed as "green therapy" by the international medical community. According to traditional technology, the heating temperature is not high, uneven heating, which induce instability of therapeutic effect. The magnetic induction hyperthermia treatment can concentrate energy to the regional mass by magnetic medium, which is also called "comformal thermotherapy". As a result, there are many advantages including protecting normal tissue, shorter heating time, better heat disposition and less invasive operation. Thermoseeds has characteristic of automatic control of the temperature with Curie point, so it improves the safety of the thermetherapy. We firstly set the model of the mammary orthotopic transplantation tumors in rats by Walker-256 strain in the thermoseed hyperthermia treatment. We observe the effect conventional high-temperature in 45℃and 50℃-55℃thermal ablation therapy on rat anti-breast cancer and its influence on immune function. Magnetic nanoparticles media can carry anticancer drugs and radioactive substances and increase gene transfection efficiency, which were called thermo-chemotherapy, thermo-radiotherapy, and thermo-genetics respectively. Hyperthermia can increase the tumor cell membrane permeability to chemotherapeutic drugs and reduce drug resistance so that thermotherapy and chemotherapy can produce synergistic sensitizing effect. Methotrexate (MTX) is a folic acid analogue, whose anti tumor effects is obtained by interfering with the metabolism of tumor, but it is easy to tumor cell resistance. We use magnetic nanoparticles modified with polyethylene imine, and then covalent chemotherapy MTX, the magnetic nanoparticles in alternating magnetic field can generate heat on the tumor mass, while increase release MTX, promotes cell uptake of MTX to enhance efficacy of cancer treatment. MTX conjugated magnetic nano-particle size of about 10nm, showed superparamagnetic. Currently, MTX superparamagnetic nanoparticles cancer thermochemotherapy on animal experiments have not been reported, and we use the magnetic fluid on rat breast cancer treatment which aimed to research feasibility of magnetic induction to provide the experimental and theoretical basis. Objective To establish transplantable transplantable metastasis models of breast Cancer and observe their biological characteristics.Methods Two groups contains 40 rats in the inoculated group and 4 normal rats in the control group. The rats were inoculated by Walker-256 breast cancer cells lines in the rats'right mammary gland, then we observed the tumor growth, survival, pathological examination and the autopsy with or without visceral metastasis etc, which could help understand their biological characteristics. Two weeks and 3 weeks later, 2 rats were randomly killed, the tumor tissue was resected and were observed organ whether or not metastasis. Normal rats were sacrificed to the breast tissue.Results In normal rat, the mammary reveals the normal distribution of adipose tissue, mammary is epithelial monolayer without any change of the proliferation. The glands inoculated with cancer cells was found the solid tumor were round or oval-shaped protruding surface, and with epithelial hyperplasia, nuclear atypia change, and even carcinogenesis. Tumor formation rate is 80%, and the survival time is (37.10±8.54) days. No metastasis occurred in the 2nd week, and the metastasis happened in the 3th week,59.3%(19/32) of rats was occurred metastasis in the lung,12.5%(4/32) liver,6.25%(2/32) axillary lymph node,6.25%(2/32) subcutaneous transfer, bone metastasis was none. Conclusion The tumor grows relative rapid, and it was not similar to the common breast cancer of human, but to the inflammatory breast cancer, which would be used as a cancer model for basic research.Objective The study was to evaluate the effect of thermoseed inductive heating on mammary orthotopic transplantation tumors and immunologic function in rats.Methods Walker-256 tumor cells were inoculated subcutaneously into the mammary glands of Wistar rats. Rats were allocated to five groups:(I) C group (control group,37 rats); (II) M group (magnetic field group,31 rats); (III) T group (thermoseed control group,31 rats); (IV) H1 group (Hyperthermia treatment:45℃for 30 min,43 rats); (V) H2 group (Hyperthermia treatment:50℃-55℃for 10 min,43 rats). Immediately, 12 h and 24 h after hyperthermia, two rats were sacrificed in each group for pathological examination. Immunohistochemical method was used to examine the expression of PCNA, HSP70, Bax, and Bcl-2 and detect apoptosis. T lymphocyte subgroups, IL-2 and IFN-γlevels were measured in C, H1 and H2 groups after two weeks hyperthermia. Tumor volume was measured and long-term survival was observed.Results Breast tissue was infiltrated by the tumor, with the epithelial hyperplasia, marked nuclear atypia and even carcinomatous. After the hyperthermia, the Bax protein expression increased (P<0.05) amd Bcl-2 expression decreased (P<0.05) in H1 group. And the apoptosis was statistically different compared with H2 group (P<0.05). The expression of PCNA began to decrease immediately after the heating in group H1 and H2 and few residual tumor cells expressed PCNA after treatment of 12h or 24h, which was significant different from the C, M and T group (P<0.05). The expression of HSP70 began to increase immediately after the heating in group H1 and H2, few residual tumor cells expressed HSP70 after 12h and 24h's treatment, which was significantly different from the C, M and T group(P<0.05). And the difference of the tumor volume between the H1, H2 and the other 3 controlled groups was significant (P<0.05). The survival time of the H1 Group and H2 group was longer than the other groups (P<0.01), which of H2 group was longer than H1 (P<0.05). CD4+T lymphocyte subsets of peripheral blood in the H1 group, H2 group was statistically higher than in the C group (P<0.05), which in H1 group was statistically higher than the H2 group (P<0.05). CD4+/CD8+ ratio in H1 group, H2 group was higher than in the C group (P<0.05), which in H1 group was higher than in H2 group (P<0.05). Serum levels of IL-2 of H1 group, H2 group was higher than in C group (P<0.05). Serum levels of IFN-y of H1 group, H2 was higher than in C group (P<0.05), which in H2 group was higher than in H, group (P<0.05).Conclusion Both general hyperthermia in 45℃and thermal ablation therapy in 50℃-55℃on breast cancer orthotopic transplantation inhibited the growth, thermal ablation treatment in 50℃55℃is of the high survival rate of rats, and immune function was enhanced in both groups.Objective Magnetic nanoparticles has been widely applied in the field of the biomedical engineering, such as local tumor magnetic hyperthermia, drug delivery, magnetic resonance imaging and gene transfer, etc. Methotrexate (MTX) is a folic acid analogue, which has antitumor effect by interfering with the metabolism.But MTX was own seriously toxic side effects, and it is easy to tumor cell resistance. To reduce its side effects and avoid drug resistance, researchers often use their carrier with a number of polymer coupling, but this approach can only produce a single effect of chemotherapy. In this study, MTX covalent coupled with polyethylenimine (PEI) which was the magnetic induction thermochemotherapy achieves the dual therapeutic effect. We observe the effect to tumor-bearing rats and impact on immune function through the magnetic induction thermochemotherapy and explore feasibility of experiments and theoretical basis.Methods The Fe3O4 magnetic nanoparticles modified by PEI were superparamagnetic, with the size of about lOnm. The magnetic particles covalent modified by MTX was also superparamagnetic and with the size of 10nm. We inoculated the walker-256 breast cancer cells to the right mammary of the rats, the ones whose tumor size grew to 1.5-2cm after the inoculation were selected in to the experiment. The tumor was injected with the 300mg/ml magnetic fluid with the PEI or MTX, which was a half volume of the tumor.24h later, 1ml magnetic fluids were inoculated at the peripheral area of tumor. After 30 minutes, rats were placed in the alternating magnetic fields (300 kHz) to receive hyperthermia. And then we monitor the temperature in vitro and in vivo. The rats were divided into 12 groups:47℃thermochemotherapy group (47TC):24 rats were treated with MTX modified magnetic fluid in an alternating magnetic field in the temperature to 47℃, the treatment time is 30 min; 47℃hyperthermia group (47T):24 rats were with PEI modified magnetic fluid in the same temperature to 47℃, the treatment time is 30 min; single 42℃thermochemotherapy (single 42TC):21 rats with MTX modified magnetic fluid in magnetic field heating to 42℃, the treatment time is 60 min; Single 42℃hyperthermia group (single 42T): 21 rats, PEI modified magnetic fluid in magnetic field heating to 42℃, treatment time is 60min; repeatedly at 42℃chemotherapy group (repeated 42TC):12, with MTX modified magnetic fluid in magnetic field heating to 42℃, the treatment time is 60 min, interval of 72h,2-5 times; Repeated 42℃hyperthermia group (repeated 42T):12 rats, PEI modified magnetic fluid in magnetic field heating to 42℃, the treatment time is 60 min, and interval of 72h,2-5 times; magnetic fluid chemotherapy group (MFC):16 rats injected with MTX modified magnetic fluid did not enter the magnetic field, After 24 h two rats were killed; blank control group (C):21, tumor growth in rats without any treatment; magnetic control group (M):12, rats were exposured 30min in the alternating magnetic field; magnetic fluid control group (MF):12 rats injected with PEI modified magnetic fluid did not enter the magnetic field; MTX chemotherapy group (MTX):16 rats were intratumoral injected with 20mg/kg of MTX; normal group (Normal):10 rats, observe the survival without tumor inoculation. Immediately after treatment in each group except normal group, MFC group, MTX group were killed for the pathological examination. After 12h,47TC,47T, single 42TC, single 42T, C group were sacrificed 2 rats each. After 24h,3 rats were killed for pathological examination, electron microscopy, PCNA, HSP70 immunohistochemistry and routine blood and biochemical examination.2 weeks later,4 rats were killed in 47TC,47T, single 42TC, single 42T, MFC, MTX, C group and the normal group for detect the IL-2, IFN-γ, IL-4 and routine blood and biochemical examination, the remaining rats were observed survival time.Results Magnetic fluid in vivo, in vitro temperature well. After treatment, tumor tissues revealed a large number of magnetic nano-particle distribution. Thermochemotherapy and hyperthermia induced apoptosis and necrosis, the 47℃treatment group was of the most distinction.24 h later, HSP70, PCNA expression in 47TC and 47T were significantly lower than that of single 42TC, single 42T, MFC, MTX, C groups (P<0.05). Two weeks after treatment, tumor volume in 47TC,47T, and repeated 42T group was significantly small than that in single 42TC, single 42T, MFC, M, MF, MTX, C group (P<0.05), and repeated 42TC group was also small than that in single 42T, MFC, M, MF, MTX, C group (P<0.05). The survival time of the 47TC,47T, repeated 42TC, and repeated 42T were longer than M, MF, MTX, C group (P <0.05).47TC group survival rate was 84.6%(11/13). The serum levels of IL-2 with 47TC and 47T were significantly higher than of single 42T and C (P<0.05), and there were no difference between 47TC,47T group and normal groups. IFN-γlevels in 47TC,47T, and single 42TC were significantly higher than of C group (P<0.05). IL-4 levels in C group were significantly higher than of other groups (P<0.05). Methotrexate modified Magnetic fluid reduced the number of white blood cells in 24h after thermochemotherapy, and transaminase of some rats were elevated. Two weeks after treatment, routine blood, liver and renal function were normal.Conclusion Thermochemotherapy is more effective than thermotherapy or chemotherapy single, and 47℃thermochemotherapy is the group of the highest survival rates.47℃thermochemotherapy/ hyperthermia and repeated 42℃thermochemotherapy/hyperthermia can inhibite tumor growth to a certain extent, and 47℃thermochemotherapy /hyperthermia can activate immune function than single 42℃thermochemotherapy/hyperthermia. MTX modified magnetic fluid has not serious toxicity, which is worth to further study.
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