| Objective:Hyperthermia can directly kill the tumor cells,and be effective to tumor metabolism,cell cycle,micro-circulation.Also,hyperthermia can induce cell apoptosis,inhibit tumor invasion and metastasis.In addition, hyperthermia can improve the body's immune function.As an important cancer supportive treatment in clinical practice,hyperthermia are often associated with other conventional treatment methods in combination with a view to enhance the therapeutic effect.But with tumor hyperthermia-related basic and clinical research were less in depth and full,some key technical aspects have not yet achieved a breakthrough,at present tremendous potential of hyperthermia have not yet been fully developed and applications.To study the apoptosis,proliferation and angiogenesis influence of thermochemotherapy, this experiment establish an S180 Tumor-bearing mice model and therapies with Blank control,Hyperthermia,Chemotherapy with Pingyangmycin and Thermochemotherapy,to observe tumor weight and the tumor necrosis rate of each group and to detect cell apoptosis,cell cycle and the expression of PCNA and VEGF protein and mRNA in the S180 transplant tumor tissue.The study is to reveal the influence of Hyperthermia and Chemotherapy individual or joint to the S180 transplanted tumor tissue as well as to explore it's mechanism.Methods:1. Sixty kunming mice were inoculated with S180 sarcoma cell by right hind leg to estabilish carcinoma model,which were randomly assigned into 4 groups:Blank control group,Hyperthermia group,Chemotherapy group and Thermochemotherapy group.When the tumors'volume reached 1cm3, Hyperthermia,Chemotherapy,Thermochemotherapy were given respectively. Each one of the Chemotherapy group and the Thermochemotherapy group animals were injected Pingyangmycin 0.2mg into abdominal,30 minutes later,the tumor-bearing parts in the right hind leg of the Thermochemotherapy group had a 43±0.2 degrees C water bath heating for 1 hour.Then the Hyperthermia group had the same heating treatment.Every 3 days,each group had the repeat treatment once again.On the 24th hour behind the third treatment,all mice were killed and the tumor tissues were taked and weighed.Then the inhibition rate of tumor growth were calculated.In vitro specimens were washed three times with saline,quickly cuted into several small pieces of tumor tissue,some were immersed in 70% ethanol,placed in 4℃fixed preservation for streaming cellanalysis;some were placed in 10% neutral at room temperature preservation for the use of immunohistochemical staining;some were placed in liquid nitrogen fixed in preparation for RT-PCR. 2. FCM was Used to analysis cell apoptosis,cell cycle and the expression of PCNA protein. 3.Immunohistochemistry(IHC) image analysis was performed for detecting VEGF and MVD.4.The expression levels of PCNA mRNA and VEGF mRNA were detected by reverse transcription-polymerase chain reaction(RT-PCR). All dates were showed by( x±s)and analysised with the SPSS 13.0 software, including one-way ANOVA analysis,least significant difference(LSD)analysis and linear correlation analysis.Results:1.The average weight of the tumors and inhibitory rates of the tumor:The average weight of the tumors in Blank Control group,Hyperthermia group,Chemotherapy group,Thermochemotherapy group were(2.030±0.047)g, (1.926±0.027)g,(1.844±0.044)g,(1.702±0.028)g,the inhibition rates of the tumors in Hyperthermia group,Chemotherapy group andThermochemotherapy group were 5.12%,9.16%,16.16%.According toα=0.05 level,the difference between any two groups were statistically significant(P<0.05).This trial Burgi correction formula to determine efficacy of theapplication of hyperthermia combined with chemotherapy,q=1.17>1.15,so hyperthermia combined with chemotherapy has significant effects. 2.The result of the FCM:(1).Cell cycle and apoptosis: the proportion of G0/G1phase cells of the Blank Control group,Hyperthermia group,Chemotherapy group,Thermochemotherapy group respectively were 43.94±2.06%,61.06±1.01%,58.02±2.62% and 71.20±3.56%, the proportion of S phase cells respectively were 33.92±1.36%, 19.26±1.71%, 25.58±1.89% and 15.08±1.24%.Compared with the control group,the G0/G1 phase fractions were raised and S phase fraction were descended in experimental groups,the differences were statistically significant(P<0.05), especially in the Thermochemotherapy group.The differences between Hyperthermia group and Chemotherapy group were that the proportion of S phase cells in Hyperthermia group and the proportion of G2/M phase cells in Chemotherapy group were less than the other,changes were statistically significant(P<0.05).The cell apoptotic rates in Blank Control group, Hyperthermia group,Chemotherapy group,Thermochemotherapy group were 4.70±0.27%,6.18±0.28%,10.14±0.25%,21.06±0.30%,the difference between each group was markable(P<0.05).(2).PCNA protein:the relative content of PCNA protein of Blank Control group,Hyperthermia group,Chemotherapy group,Thermochemotherapy group were 319.24±1.34,313.50±1.18,310.74±0.77,304.40±1.45.The difference between each group were statistically significant(P<0.05),especially in Thermochemotherapy group(P<0.01). 3.Immunohistochemistry staining:The expression of VEGF protein in Blank Control group,Hyperthermia group,Chemotherapy group,Thermochemo- therapy group were 45.8±1.64,34.4±1.67,29.2±2.17,20.2±1.48, MVD values were 19.2±1.30,16.0±1.41,14.4±0.55,9.8±1.92,each in the experimental groups were decreased,the difference was markedly(P<0.05),in addition,MVD between Hyperthermia group and Chemotherapy group was no significant (P>0.05).For linear correlation analysis revealed that the VEGF protein expression and MVD was positive correlated(r=0.915,P<0.01).4.RT-PCR:The relative values of VEGF mRNA expression in Blank Control group, Hyperthermia group,Chemotherapy group,Thermochemotherapy group were 0.3880±0.0177, 0.3392±0.0112, 0.3178±0.0034, 0.2952±0.0134, the relative values of PCNA mRNA expression were 1.5522±0.0214,1.3348±0.0257, 1.2776±0.0161,1.014±0.0255.Each in the experimental groups were lower than the control group.The difference between each group was markedly(P<0.05).Conclusion:1.Hyperthermia,Chemotherapy and Thermochemotherapy can lessen the average weight of the S180 transplanted tumor,which can inhibit the growth of the S180 transplanted tumor,the effect of Thermochemotherapy was strongest.2.Hyperthermia,Chemotherapy and Thermochemotherapy can improve the cell apoptosis rate in mice transplanted S180 sarcoma,the role of Thermochemotherapy was most significant.They can also affect the cell cycle of the transplanted tumor tissue,heat made the proportion of S phase cells decreased,S phase cells were most sensitive to 43 degrees C heat treatment, and heat promoted the apoptosisin mice transplanted S180 sarcoma.Thermo- chemotherapy can deserve much more better effection.3.Hyperthermia and Chemotherapy can reduce the expression of PCNA mRNA and protein,which can inhibit the growth of the S180 transplanted tumor.The effect of joint between hyperthermia and chemotherapy was better than individual apply.4.Hyperthermia,Chemotherapy,Thermochemotherapy can reduce the expression of VEGF mRNA and protein,which can inhibit tumor angiogenesis.The role of Thermochemotherapy was most effective.This text provide experiment evidence for the clinical application of Thermo- chemotherapy to treat cancer.
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