| Part one Effects of ferromagnetic fluid hyperthermia induced by an alternative magnetic field on hepatoma cell HepG2 in vitroObjectiv To study effects of varying concentration nano-ferroferric oxide magnetic fluid hyperthermia induced by an alternating magnetic field on hepatoma cell HepG2 in vitro.Methods A human hepatoma cell HepG2 was cultured with virous concertration of ferroferric oxide(Fe3O4, 68nm) magnetic fluid (1.5～7.5mg/ml) and exposed to an alternative magnetic field(AMF) with 100 kHz and 15kA/m for 30 min, meanwhile temperatures were measured by optical fiber transducer thermometer. The optic density(OD) of viable cell, cytotoxity index, growth curve of cells, morphologic changes of cell, cell cycle and aposptosis were assayed by MTT method, cell count, light and fluorescence microscope and flow cytometry.Results With the increase of concentration of Fe3O4, the following outcomes occurred, including:①the temperature of the culture medium was 38.9℃～47.2℃; above 41℃was got with the concentration of Fe3O4≥4.5mg/ml;②the proliferation of HepG2 cell was increasingly inhitibited, the OD value of viable cell decreased and cytotoxity index(CI) increased; CI nearly exceeded 50％with the concentration of 4.5mg/ml Fe3O4; the effect was concentration-dependent(P＜0.001).③the apoptotic HepG2 cell were observed to have cell shrinkage, chromatin condensation, margination, unclear fragmentation, apoptotic bodies, and intact cell membrane. The typical cytomorpho logical features of apoptosis was more obvious when the concentration of Fe3O4 exceeded 4.5mg/ml.④the cycle of HepG2 cell was inhibiteded in the stage S, the percentage of cells in the stage S increase; the ratio of apoptosis of HepG2 cell increased and it was up to 27.06％～66.05％with the concentraton of Fe3O4 ranging from 4.5mg/ml to 6.0mg/ml; the apoptosis rate 14.2％with the 3.0mg/ml Fe3O4(P＜0.001).Conclusion Ferromagnetic fluid hyperthermia induced by AMF could inhibit the proliferation and promote apoptosis of human hepatoma HepG2 cells in a concentration-dependent manner.Part two Effects of galactosed human serum albumin adriamycin nanoparticles on human hepatoma cell HepG2 and human liver cell L-02 in vitro combined with an alternative magnetic fieldObjective To explore the effects of galactosed human serum albumin adriamycin nanoparticles (Gal-MADM-NP) on human hepatoma cell HepG2 and human liver cell L-02 in vitro combined with an alternative magnetic field and compare the effects between HepG2 and L-02 cell.Methods Human hepatoma cell HepG2 and human liver cell L-02 were cultured in vitro. HepG2 cell and L-02 cell were randomly divided into 5 groups: A1,A2(RPMI 1640 medium+AMF), B1,B2(ADM+AMF), C1,C2(Gal-MADM-NP), D1/D2 (ADM-NP+AMF), E1,E2(Gal-MADM-NP+AMF), respectively. Except group C1,C2, the other groups were exposed to an alternative magnetic field(AMF) with 100 kHz,15kA/m for 30 min, respectively, meanwhile, temperatures were measured by optical fiber transducer thermometer. The growth curve of cell, optic density(OD) of viable cell, cytotoxity index, cyctomorphologic changes, DNA fragmentation, cell cycle and aposptosis were assayed by cell count,MTT method,fluorescence microscope,agarose gel elctrophoresis and flow cytometry and comparision of effects among groups and between HepG2 cells and L-02 cells would be performed.Results①in experimental groups, the proliferation of hepG2 cell and L-02 cell were inhibited in varying degree and cytotoxity index(CI) of both cells increased with the concentration of ADM, the CI of both cells were higher in the group E1,E2 than the other groups(P＜0.001).②the typical DNA ladder apperanced in experienmenal groups, DNA ladders were markedly obvious in the group E1,E2 than the other groups.③in the group E1,E2, the apoptosis ratio of hepG2 cell and L-02 cell were 40.12±2.28％and 45.32±2.48％, respectively, and both of them were 4 times higher than the group B1,B2 and significantly higher than the other groups(P＜0.001); the percentage of both cells in phase S were 39.53％,53.46％, respectively and obviously higher than the other groups(P＜0.001).④the CI,apoptosis ratio and the percentage of cells in phase S of L-02 cells were higher(P＜0.05) and alternation of the apoptotic cytomorphology were more obvious than that of HepG2 cells in all experimental groups.Conclusion the proliferation inhabitation,cytotoxicity and apoptosis of HepG2 cells and L-02 cells were more effective and the effects on L-02 cell was more effective than HepG2 cell in the group E1,E2 than the other groups.Part three Effects of galactosed human serum albumin adriamycin nanoparticles on rabbit VX2 tumor combined with alternative magnetic fieldObjective To study effects of galactosed human serum albumin adriamycin nanoparticles on rabbit VX2 combined with an alternating magnetic field.Methods Rabbit's hepatic(left lobe) cancer implanted with VX2 carcinoma tissues were randomly divided into 6 groups, including Group A(namely control group, NS+AMF), B(ADM+AMF), C (Gal-MADM-NP), D(NP+AMF), E(ADM-NP+AMF), F (Gal-MADM-NP+AMF). The rabbits in groups were received a hepatic arterial infusion of NS, ADM, Gal-MADM-NP, NP, ADM-NP and Gal-MADM-NP, respectively. The other groups were exposed to an 15kA/m external alternative magnetic field at 100kHz except group C. During this period, the temperature of the center and margin of the tumors in the rabbits, normal liver parenchyma(right lobe) and rectum were monitored and recorded with optical fiber transducer thermometer. The volume and tumor growth rate of tumors were monitored and the pathology of tumor and normal liver tissue were observed after 14 days of treatment.Results The tumor temperatures of the centre(42.4℃, 42.6℃, 42.7℃) and margin(40.3℃, 40℃.6, 40.7℃) in group D,E,F were significantly higher than in their normal liver parenchyma(36.7℃, 36.8℃, 36.9℃) and rectum (36.4℃, 36.4℃, 36.2℃), respectively. The volume and growth rate of the tumor markedly decreased(P＜0.001) and there were massive necroses(exceeding 70％) in tumor of group F. The volume of tumor in other groups increased by 3.5～13 times and growth rate of tumor increased 127～568％and had only slight or middle necroses in tumor(0～70％).Conclusions above 42℃temperature of the centre of the group D,E,F could be got with AMF mediated by targeted liver arterial infusion of the rabbits. Hyperthermia could markedly enhance the anticancer effect of Gal-MADM-NP, which have unique targeting treatment effects for liver cancer. |
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