Angiogenesis Effect Of Nanometer Magnetic Fluid Hyperthermia In Tumor Model Of Wistar Rat

Background:Disease incidence of malignant tumor is increasing gradually every year,and malignant tumor becomes the first cause of disease death.Although many works have been done to prevent and cure the malignant tumor in the world wide,improvement is slow.People are eagering for a method to break through this bottle neck so as to cut down disease incidence and elevate cure rate largely.Traditional hyperthermia for malignant tumor has a long history, which is another important tool for curing cancer after surgery, radiotherapy,chemotherapy and biotherapy.However,traditional hyperthermia is an auxiliary and palliative therapy because of its lower therapy temperature.In recent years,studies on nano-hitech have been fastly improved,magnetic nanoparticle can be used as drug vehicle and also produce heat energy under an alternating magnetic field with certain intensity and frequency.Magnetic nanoparticle can be implanted into the tumor tissues and accurately heat the tumor tissues,at the same time,the tissues beside tumor will not be heated,I.e,"comformal hyperthermia", which has been a "heat spot" and potential breach in biomedicine technology to oncology study.Tissue effect caused by focal hyperthermia application occurs in two distinct phases--direct effect and indirect effect.The first phase includes cell death and apoptosis.The second phase is an indirect effect that produces a progressive tissue damage,which may be more important in oncology hyperthermia.This progressive injury may involve a balance of several factors,such as ischemia-reperfusion injury,Kupffer cell activation,altered multi-cytokine expression,and modulation of the immune response,and so on.As we know,there are no reports about angiogeneosis of indirect injury after hyperthermia application. We make use of this magnetic nano-hitech to investigate angiogeneosis mechanism of malignant tumor after focal hyperthermia.If we know much better about the molecular and biological basis of hyperthermia to malignant tumor,we can do much better to prevent and cure malignant tumor.CHAPTER ONE:Establishment and biological characteristics of subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar ratsObjective:To study the feasibility of establishment of subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar rats and its tumor biological characteristics.Methods:Carcinomatous walker-256 cells(about 2×10⁶ cells with 0.6ml per rat) were injected into the right axilla to form subcutaneous tumor model in rats.Then the subcutaneous tumor model's growth was observed,and tumor node's pathology examination was made.Results:1) Malignant tumor node whose diameter was 1 centimeter or so would be found after carcinomatous walker-256 cells injected within 7 or 9 days,which was proved by pathology examination. 2) The successful rate of the subcutaneous tumor model was very high, about 97.78%(176/180 cases).3) The natural survival time of the subcutaneous tumor model in rats was very long,namely 36 days.4) Animal model was stable,and tumor node was never disappear as soon as its diameter was more than 0.5 centimeter.Conclusion:1) The tumor model of carcinomatous walker-256 cells in Wistar rats is an ideal model for studying the mechanism and basis of malignancy,just because of its high successful rate.2) Biologic character of tumor model is steady,and tumor nodes will not spontaneously disappear.The natural survival time of tumor model is enough long to do some experiments.CHAPTER TWO:Study on the therapeutic effect of Fe₃O₄ nanometer magnetic fluid hyperthermia on subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar ratsObjective:To study the therapeutic effect of Fe₃O₄ nanometer magnetic fluid hyperthermia under an alternating magnetic field on subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar rats in vivo.Methods:After subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar rats was established,Fe₃O₄ nanometer magnetic fluid was injected into tumor tissues.Then,these Wistar rats were placed under an alternating magnetic field for hyperthermia(1,2 or 3 times per group respectively) with temperature controlled at 50～55 centigrade.The ratio of volume inhibition and survival time to subcutaneous tumor model in Wistar rats with Fe₃O₄ magnetic fluid hyperthermia were measured in contrast to control group s.And then,the pathologic changes and calefactive effect of Fe₃O₄ nanometer magnetic fluid under alternating magnetic field were observed.Results:1) Temperature in tumor tissues could reached to 50～55℃within 6 or 10 minutes,however,temperature in normal tissues was unchanged.2) From the volume-time curve,we could see that Fe₃O₄ nanometer magnetic fluid hyperthermia inhibited the tumor proliferation within two weeks,which was remarkable in MFH3 and MFH2 group than that in MFH1 and MF group in contrast to NS group s.The ratio of volume inhibition in MFH2 and MFH3 was 65%±3.5%,71.6±4.2% after hyperthermia at 14th day.3) The mean survival time of NS group was 35.50±5.59 days,MFH3 group:41.88±6.48 days,MFH2 group: 42.13±4.98 days,MFH1 group:39.00±5.30 days,MF group:36.06±6.57 days.The mean survival time of MFH3 and MFH2 group was longer than that of NS group,and there were statistical significance,q=-6.375, -6.625;P=0.003,0.002,however,there were no statistical significance between MFH1 and MF group with NS group,q=-3.500,-0.563;P =0.093,0.785.4) The tumor surface of NS and MF group was smooth,a few haemorrhage and small necrotic areas.The cancer cells were thick and fast,big nucleus and obvious nucleolus,and nucleus split stage could be seen easily.In the hyperthermia groups,the tumor surface was accidented and eschar.There were obvious haemorrhage,big necrotic areas and red-dyed remnant without structure,or cavity,and some nucleus shrank,split and dissolved.These manifests in MFH3 and MFH2 were obvious than those in NS and MF group.Conclusion:1) Nanometer magnetic fluid hyperthermia can ideally heat target tissues,however,temperature in normal tissues is unchanged, which is so-called "comformal hyperthermia".2) Fe₃O₄ nanometer magnetic fluid hyperthermia can inhibit the proliferation,prolong survival and promote apoptosis of subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar rats.3) Animal experiments manifest that Fe₃O₄ nanometer magnetic fluid hyperthermia effect is relative to hyperthermia times and energy.4) From the above,we can draw a conclusion that Fe₃O₄ nanometer magnetic fluid hyperthermia may also act as a therapeutic tool for other solid tumors.CHAPTER THREE:Study on the angiogenesis effect of Fe₃O₄ nanometer magnetic fluid hyperthermia on subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar ratsObjective:To investigate the angiogenesis effect of Fe₃O₄ nanometer magnetic fluid hyperthermia under an alternating electromagnetic field on subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar rats in vivo.Methods:After subcutaneous tumor model of carcinomatous Walker-256 cells in Wistar rats was established,Fe₃O₄ nanometer magnetic fluid was injected into tumor tissues.Then,these Wistar rats were placed under an alternating magnetic field for hyperthermia(1,2 or 3 times per group respectively,I.e.MFH1,MFH2,MFH3) with temperature controlled at 50～55 centigrade.Reverse transcription-polymerase chain reaction and immunohistochemistry were used to analyze the vascular endothelial cell growth factor(VEGF) and receptor(Flk-1,Flt-1) mRNA,VEGF and microvessel density(MVD) among experiment groups and contrast group from specimens of subcutaneous tumor model in Wistar rats.Biological professional image analysis software(imagepro-plus 6.0) was used to analyze VEGF staining intensity(studied with the integrated optical density) and microvessel counting.At last,the correlation of VEGF and MVD was also analyzed.Results:1) The integrated optical density(IOD) values of VEGF dyed-cell and the numbers of MVD dyed-mirovessel in MFH3 and MFH2 groups with immunohistochemistry were lower than those in NS group, and the difference had statistical significance(p=0.000,0.001;0.000, 0.001).However,IOD values and the numbers of MVD in MFH1 and MF groups were not different from those in NS group,and statistical significance was not found(p=0.675,0.999;0.332,0.994).2) The correlation of VEGF and MVD was also analyzed,and statistical significance could be found,I.e.r=0.898,p=0.000.3) Fe₃O₄ nanometer magnetic fluid hyperthermia under an alternating electro-magnetic field could down-regulated VEGF mRNA and Flk-1 mRNA expression in MFH3 and MFH2 groups in contrast to those in NS group,and there were statistical significance,P=0.000,0.006;0.005,0.014.However,there were no statistical significance in MFH1 and MF groups,P=0.648,0.231; 0.994,0.158.4) Fe₃O₄ nanometer magnetic fluid hyperthermia could down-regulated Flt-1 mRNA expression in MFH3 groups in contrast to those in NS group,and there were statistical significance,P=0.009. However,there were no statistical significance in MFH1,MFH2 and MF groups,P=0.954,0.983,0.699.5) Flk-1 receptor was more sensitive to hyperthermia than Flt-1 receptor was at 50～55 centigrade,from the above,we could draw a conclusion that new-born vessel was more sensitive to hyperthermia than grown-up vessel was.Conclusion:1) Fe₃O₄ nanometer magnetic fluid hyperthermia under an alternating electro-magnetic field can down-regulate the expression of VEGF and VEGF receptors(Fit-1 and Flk-1).Down-regulation of these factors may give rise to reduce of vessel number,which is confirmed by microvessel density count.2) New-born vessel is easier damaged than grown-up vessel to magnetic fluid hyperthermia.It indicates that the reduce of vessel number in tumor tissues mainly attribute to reduce of new-born vessel number.In other words,magnetic fluid hyperthermia inhibits new-born vessels.Indirect effect of magnetic fluid hyperthermia may be a more significant mechanism in tumor therapy.