热化疗对小鼠移植性S_（180）肉瘤细胞凋亡、增殖及血管再生的影响及其机制研究
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摘要
目的:热疗(hyperthermia)可直接杀伤肿瘤细胞,并对肿瘤代谢、细胞周期分布、微循环产生作用,热疗还会诱导细胞凋亡、抑制肿瘤的侵袭转移,另外,热疗还可提高机体免疫功能。因此,热疗做为一种重要的肿瘤辅助性治疗手段,临床上常与其他常规治疗方法联合应用,以期提高肿瘤的治疗效果。但目前与肿瘤热疗相关的基础和临床资料均有待于更加深入和充分的研究,某些关键技术环节尚未取得突破,因此目前肿瘤热疗的巨大潜能尚未得到充分开发与应用。本研究拟通过S180荷瘤小鼠动物模型,经热疗、化疗单独或联合处理后,测量各组平均瘤重,计算抑瘤率,测定肿瘤细胞凋亡率和细胞周期以及S180移植瘤组织内的增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和血管内皮生长因子(vascular endothelial growth factor,VEGF)蛋白及基因表达水平,从而观察热、化疗单独及联合应用对小鼠移植性S180肉瘤的凋亡、增殖及血管再生的影响,并初步探讨其可能的作用机制。
方法:1、实验用昆明小鼠60只,右后肢股部皮下接种S180肿瘤细胞株,随机分为空白对照组、热疗组、化疗组、热化疗组。当肿瘤生长至1cm3时分组进行局部热疗、化疗和热化疗。给化疗组和热化疗组小鼠腹腔注射平阳霉素0. 2mg/只,热化疗组小鼠给药30分钟后立即对荷瘤的右后肢给予43±0. 2℃恒温水浴加热1小时。然后热疗组给予同样加热处理。各实验组在给予第一次治疗后每隔3天,重复治疗一次。于第三次治疗后24小时,将小鼠颈椎脱臼处死,于小鼠后肢同一水平面离断其荷瘤下肢,瘤体称重,计算抑瘤率。离体标本用生理盐水冲洗3次,迅速将肿瘤组织切成多个小块,部分置于70%乙醇4℃固定保存,以备流式细胞分析使用;部分置于10%中性甲醛固定室温保存,以备免疫组织化学染色使用;部分置于液氮中固定以备RT-PCR使用。2、用流式细胞分析技术(FCM)测定各组S180移植瘤组织细胞周期、凋亡率及PCNA蛋白表达水平。3、免疫组化方法检测VEGF蛋白表达水平,以微血管密度(MVD)作为定量检测指标。4、RT-PCR检测VEGF mRNA、PCNA mRNA。5、数据以均数±标准差(x±s)表示,采用SPSS13. 0统计学软件进行数据处理。多组间均数差异性比较采用单因素方差分析(one-way ANOVA),并用最小显著法(least significant difference,LSD)作两两比较。直线相关分析方法比较两指标的相关性。P<0. 05为差异有显著性,P<0. 01为差异有极显著性,P>0. 05为差异无显著性。
结果:1、瘤重及抑瘤率:空白对照组瘤重为(2.030±0.047)g;热疗组瘤重为(1.926±0.027) g ,抑瘤率为5.12% ;化疗组瘤重为(1.844±0.044)g,抑瘤率为9.16%;热化疗组瘤重为(1.702±0.028)g,抑瘤率为16.16%;按α=0.05水准,任意两组间总体均数差异均有统计学意义(P<0.05)。本文试用Burgi修正公式,对热疗与化疗的联合应用作疗效判别,q =1.17>1.15,因此热疗与化疗联合有协同作用。2、流式细胞分析技术检测结果:(1)、细胞周期及凋亡率:对照组、热疗组、化疗组、热化疗组G0/G1期细胞比例分别为43.94±2.06%、61.06±1.01%、58.02±2.62%和71.20±3.56%,S期细胞比例分别为33.92±1.36%、19.26±1.71%、25.58±1.89%和15.08±1.24%,其中各干预组与对照组相比,G0/G1期比例均上升、S期比例均下降,差异有统计学意义(P<0. 05),以热化疗组变化最为显著。热疗组与化疗组二者比较,热疗组S期细胞所占比例较少,化疗组G2/M期细胞所占比例较少,变化有统计学意义(P<0. 05)。对照组、热疗组、化疗组、热化疗组细胞凋亡率分别为4.70±0.27%、6.18±0.28%、10.14±0.25%、21.06±0.30%,各干预组之间以及干预组与对照组之间差异均有统计学意义(P<0.05)。(2)、PCNA蛋白:对照组、热疗组、化疗组、热化疗组PCNA蛋白相对含量分别为319.24±1.34、313.50±1.18、310.74±0.77、304.40±1.45。各干预组之间以及干预组与对照组之间差异均有统计学意义(P<0.05),以热化疗组变化最显著。3、应用免疫组化法检测各组肿瘤组织内VEGF及MVD的表达水平:对照组、热疗组、化疗组、热化疗组VEGF蛋白表达值分别为45.8±1.64、34.4±1.67、29.2±2.17、20.2±1.48, MVD值分别为19.2±1.30、16.0±1.41、14.4±0.55、9.8±1.92,各干预组VEGF蛋白表达和MVD较对照组降低,除热疗组与化疗组MVD值比较差异无统计学意义(P>0.05)外,各干预组之间以及干预组与对照组之间差异均有统计学意义(P<0.05)。进行直线相关分析后,发现VEGF蛋白表达与MVD间存在正相关性(r=0.915,P<0.01)。4、采用半定量RT-PCR检测各组肿瘤组织内VEGF mRNA、PCNA mRNA的表达水平:对照组、热疗组、化疗组、热化疗组VEGF mRNA表达相对值分别为0.3880±0.0177、0.3392±0.0112、0.3178±0.0034、0.2952±0.0134,PCNA mRNA表达相对值分别为1.5522±0.0214、1.3348±0.0257、1.2776±0.0161、1.014±0.0255,各干预组VEGF mRNA、PCNA mRNA较对照组降低,各干预组之间以及干预组与对照组之间差异均有统计学意义(P<0.05)。
结论:1、热疗、化疗、热化疗均可减轻小鼠移植性S180肉瘤的重量,对小鼠移植性S180肉瘤的增殖具有抑制作用,热化疗作用最显著。2、热疗、化疗、热化疗均可提高小鼠移植性S180肉瘤的细胞凋亡率,并可影响移植瘤组织的细胞周期,高热使S期细胞比例下降,43℃热疗对S期细胞最敏感,促进了小鼠移植性S180肉瘤的凋亡,热化疗联合作用更强。3、热疗、化疗均可下调小鼠移植性S180肉瘤的PCNA mRNA及蛋白的表达,抑制小鼠移植性S180肉瘤的增殖,二者联合应用优于单独应用。4、热疗、化疗、热化疗均可下调小鼠移植性S180肉瘤中VEGF mRNA及蛋白的表达,以热化疗联合作用效果最佳,对小鼠移植性S180肉瘤血管生成具有抑制作用。为临床中热、化疗联合应用治疗恶性肿瘤提供了实验依据。
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.
方法:1、实验用昆明小鼠60只,右后肢股部皮下接种S180肿瘤细胞株,随机分为空白对照组、热疗组、化疗组、热化疗组。当肿瘤生长至1cm3时分组进行局部热疗、化疗和热化疗。给化疗组和热化疗组小鼠腹腔注射平阳霉素0. 2mg/只,热化疗组小鼠给药30分钟后立即对荷瘤的右后肢给予43±0. 2℃恒温水浴加热1小时。然后热疗组给予同样加热处理。各实验组在给予第一次治疗后每隔3天,重复治疗一次。于第三次治疗后24小时,将小鼠颈椎脱臼处死,于小鼠后肢同一水平面离断其荷瘤下肢,瘤体称重,计算抑瘤率。离体标本用生理盐水冲洗3次,迅速将肿瘤组织切成多个小块,部分置于70%乙醇4℃固定保存,以备流式细胞分析使用;部分置于10%中性甲醛固定室温保存,以备免疫组织化学染色使用;部分置于液氮中固定以备RT-PCR使用。2、用流式细胞分析技术(FCM)测定各组S180移植瘤组织细胞周期、凋亡率及PCNA蛋白表达水平。3、免疫组化方法检测VEGF蛋白表达水平,以微血管密度(MVD)作为定量检测指标。4、RT-PCR检测VEGF mRNA、PCNA mRNA。5、数据以均数±标准差(x±s)表示,采用SPSS13. 0统计学软件进行数据处理。多组间均数差异性比较采用单因素方差分析(one-way ANOVA),并用最小显著法(least significant difference,LSD)作两两比较。直线相关分析方法比较两指标的相关性。P<0. 05为差异有显著性,P<0. 01为差异有极显著性,P>0. 05为差异无显著性。
结果:1、瘤重及抑瘤率:空白对照组瘤重为(2.030±0.047)g;热疗组瘤重为(1.926±0.027) g ,抑瘤率为5.12% ;化疗组瘤重为(1.844±0.044)g,抑瘤率为9.16%;热化疗组瘤重为(1.702±0.028)g,抑瘤率为16.16%;按α=0.05水准,任意两组间总体均数差异均有统计学意义(P<0.05)。本文试用Burgi修正公式,对热疗与化疗的联合应用作疗效判别,q =1.17>1.15,因此热疗与化疗联合有协同作用。2、流式细胞分析技术检测结果:(1)、细胞周期及凋亡率:对照组、热疗组、化疗组、热化疗组G0/G1期细胞比例分别为43.94±2.06%、61.06±1.01%、58.02±2.62%和71.20±3.56%,S期细胞比例分别为33.92±1.36%、19.26±1.71%、25.58±1.89%和15.08±1.24%,其中各干预组与对照组相比,G0/G1期比例均上升、S期比例均下降,差异有统计学意义(P<0. 05),以热化疗组变化最为显著。热疗组与化疗组二者比较,热疗组S期细胞所占比例较少,化疗组G2/M期细胞所占比例较少,变化有统计学意义(P<0. 05)。对照组、热疗组、化疗组、热化疗组细胞凋亡率分别为4.70±0.27%、6.18±0.28%、10.14±0.25%、21.06±0.30%,各干预组之间以及干预组与对照组之间差异均有统计学意义(P<0.05)。(2)、PCNA蛋白:对照组、热疗组、化疗组、热化疗组PCNA蛋白相对含量分别为319.24±1.34、313.50±1.18、310.74±0.77、304.40±1.45。各干预组之间以及干预组与对照组之间差异均有统计学意义(P<0.05),以热化疗组变化最显著。3、应用免疫组化法检测各组肿瘤组织内VEGF及MVD的表达水平:对照组、热疗组、化疗组、热化疗组VEGF蛋白表达值分别为45.8±1.64、34.4±1.67、29.2±2.17、20.2±1.48, MVD值分别为19.2±1.30、16.0±1.41、14.4±0.55、9.8±1.92,各干预组VEGF蛋白表达和MVD较对照组降低,除热疗组与化疗组MVD值比较差异无统计学意义(P>0.05)外,各干预组之间以及干预组与对照组之间差异均有统计学意义(P<0.05)。进行直线相关分析后,发现VEGF蛋白表达与MVD间存在正相关性(r=0.915,P<0.01)。4、采用半定量RT-PCR检测各组肿瘤组织内VEGF mRNA、PCNA mRNA的表达水平:对照组、热疗组、化疗组、热化疗组VEGF mRNA表达相对值分别为0.3880±0.0177、0.3392±0.0112、0.3178±0.0034、0.2952±0.0134,PCNA mRNA表达相对值分别为1.5522±0.0214、1.3348±0.0257、1.2776±0.0161、1.014±0.0255,各干预组VEGF mRNA、PCNA mRNA较对照组降低,各干预组之间以及干预组与对照组之间差异均有统计学意义(P<0.05)。
结论:1、热疗、化疗、热化疗均可减轻小鼠移植性S180肉瘤的重量,对小鼠移植性S180肉瘤的增殖具有抑制作用,热化疗作用最显著。2、热疗、化疗、热化疗均可提高小鼠移植性S180肉瘤的细胞凋亡率,并可影响移植瘤组织的细胞周期,高热使S期细胞比例下降,43℃热疗对S期细胞最敏感,促进了小鼠移植性S180肉瘤的凋亡,热化疗联合作用更强。3、热疗、化疗均可下调小鼠移植性S180肉瘤的PCNA mRNA及蛋白的表达,抑制小鼠移植性S180肉瘤的增殖,二者联合应用优于单独应用。4、热疗、化疗、热化疗均可下调小鼠移植性S180肉瘤中VEGF mRNA及蛋白的表达,以热化疗联合作用效果最佳,对小鼠移植性S180肉瘤血管生成具有抑制作用。为临床中热、化疗联合应用治疗恶性肿瘤提供了实验依据。
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.
引文
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