【作者】 谢小雪；

【导师】 唐劲天； 廖遇平；

【作者基本信息】 中南大学 ， 临床肿瘤学， 2010， 博士

【摘要】 研究背景恶性黑色素瘤是由黑色素细胞恶性转化形成的一种高度恶性肿瘤,发病率为全身恶性肿瘤的1%-2%,占皮肤恶性肿瘤的20%。其侵袭性强,易发生远处转移,最常见的转移部位包括肺、骨和肝脏,进展期黑色素瘤患者平均生存期仅6-9个月,对人民群众的生命带来极大危害。治疗恶性黑色素瘤的关键在于控制其远处转移,由于其对常规治疗手段如化疗、放疗等敏感性较低,目前对于进展期恶性黑色素瘤还无有效的预防治疗方法。肿瘤热疗(Hyperthermia)是一种以非电离辐射方式作用于肿瘤的物理治疗方法,与化疗和放疗相比其优势在于较少受限于其毒副作用,并能够多次重复使用,且可以增强放射治疗和化学药物治疗的效果,被国际医疗界称为“绿色疗法”。传统的肿瘤热疗方法常常不能将肿瘤加热到治疗温度,新近研发的磁感应加温治疗可以将肿瘤组织加热到50℃左右,而使正常组织保持不升温,有效杀灭靶区内的肿瘤组织。近年来的动物实验证明了热疗不仅可抑制肿瘤细胞的增殖,还可抑制其远处转移。也有研究报道了热疗对转移相关蛋白表达的影响。但对热疗抑制肿瘤侵袭转移能力的具体机制尚不十分清楚,有待进一步探索。基于以上观点,本研究将采用不同温度体内外加热小鼠黑色素瘤,系统的研究41℃-50℃范围内不同温度热疗对B16F10细胞的增殖、侵袭、运动能力的影响,并研究其相关分子机制,将有助于临床个体化的热疗方案,为热疗治疗恶性黑色素瘤的临床应用提供部分理论和实验依据。目的探讨不同温度热疗对黑色素瘤B16F10细胞增殖以及周期分布的影响。方法采用水浴对黑色素瘤B16F10细胞行41℃、43℃、45℃、47℃、50℃实验组和对照组(37℃)热疗,四甲基偶氮唑蓝(MTT)检测细胞增殖抑制率,流式细胞术仪测定细胞周期分布的变化。结果45℃,47℃,50℃组的细胞增殖抑制率在24h、48h、72h、96h明显高于41℃和43℃组(P<0.05)。47℃,50℃组的细胞增殖抑制率在24h、48h、72h、96h均明显高于45℃组(P<0.05)。在47℃、50℃两组之间,各时间段的差异无显著性(P>0.05)。与37℃对照组相比,41℃、43℃组的G0/G1期细胞明显增多,而S期细胞明显减少(P<0.05)。47℃和50℃组的G0/G1期细胞比37℃组明显减少(P<0.05),而S期细胞明显增多(P<0.05),在47℃和50℃组之间无明显差异。与37℃对照组相比,43℃组的G2/M期细胞明显增多(P<0.05)。结论45℃及以上温度热疗可明显抑制B16F10细胞的体外增殖能力；45℃热疗对B16F10细胞S期比例无明显影响,47℃及以上温度热疗使其发生S期阻滞。结果提示：45℃及以上温度热疗不仅可以有效抑制B16F10细胞的增殖活性,且可以减少热耐受的产生,而高于47℃的热疗并不产生更强的抑制作用。目的研究不同温度热疗对黑色素瘤B16F10细胞形态、运动能力及体外侵袭能力的影响。方法采用水浴对黑色素瘤B16F10细胞行41℃、43℃、45℃、47℃、50℃实验组和37℃对照组热疗,倒置显微镜下观察热疗后不同时间点细胞形态的变化；透射电镜观察热疗后24h细胞显微结构的改变。取43℃、45℃、47℃实验组和37℃对照组热疗后的细胞进行划痕实验检测细胞的运动能力；侵袭小室实验检测细胞体外侵袭能力；明胶酶谱法检测不同温度热疗后24h内B16F10细胞的MMP-2及MMP-9的分泌量及活性。结果(1)B16F10细胞在41℃、43℃加热后更趋于成熟,分化更好,恶性程度降低,而45℃及以上温度热疗后细胞结构破坏明显,细胞活力下降。(2)与37℃(对照组)相比,43℃、45℃、47℃组细胞运动能力均减弱,45℃、47℃组细胞运动能力下降尤其明显。(3)37℃(对照组)、43℃,45℃及47℃热疗后,平均每个视野中B16F1O细胞穿过Matrigel胶到达Transwell小室膜背面的细胞数随热疗温度升高而降低,各组间均存在明显差异(P<0.05)。(4)与37℃组相比,43℃、45℃、47℃热疗后24h内B16F10细胞MMP-2及MMP-9的分泌量及活性明显下降(P<0.05),并且与43℃组相比,45℃、47℃热疗组MMP-2的分泌量及活性进一步下降(P<0.05),而45℃、47℃热疗组之间无明显差异(P>0.05)。结论不同温度热疗后细胞形态及超微结构发生不同程度的改变,45℃及以上温度热疗的主要表现为细胞结构的破坏；43℃、45℃及47℃热疗均可抑制B16F10细胞的运动能力,且对黑素瘤细胞明胶酶活性均有抑制作用,45℃、47℃热疗组MMP-2的分泌量及活性较43℃下降更明显；43℃、45℃及47℃对肿瘤细胞的体外侵袭能力的抑制作用随热疗温度升高而增强。说明B16F10细胞侵袭能力的减弱主要与细胞运动能力及明胶酶活性受到抑制有关。目的研究小鼠黑色素瘤B16F10细胞经过不同温度热疗后TGF-β1等侵袭转移相关基因和蛋白的表达变化。方法采用水浴对黑色素瘤B16F10细胞行43℃、45℃、47℃实验组和37℃对照组热疗,免疫细胞化学检测不同温度热疗后24hTGF-β1、VEGF蛋白在细胞内的表达变化,rt-PCR检测不同温度热疗后继续培养2h、6h、12h、4h TGF-β1、Smad4、VEGF、MMP-2及MMP-9在mRNA水平的表达变化。Western Blotting检测不同温度热疗后继续培养2h、6h、12h、24h TGF-β1、Smad4在蛋白水平的表达变化。结果(1)不同温度热疗后24h细胞内TGF-β1蛋白表达降低,43℃、45℃、47℃组的平均光密度值(AOD)与37℃对照组相比,明显降低(P<0.05)；同样,VEGF蛋白表达也下降,43℃、45℃、47℃组的平均光密度值(AOD)与37℃对照组相比,也明显降低(P<0.05),二者均呈现一定程度的温度依赖性。(2)与37℃对照组相比,43℃热疗后各时间点MMP-9、VEGF mRNA的表达均降低(P<0.05),43℃热疗后6h-24h MMP-2、TGF-β1、mRNA的表达明显降低(P<0.05)；45℃及47℃热疗后各时间点TGF-β1、VEGF、MMP-2及MMP-9 mRNA的表达均降低；各时间点Smad4 mRNA的表达均无明显变化。(3)与37℃对照组相比,43℃热疗后12h-24h出现TGF-β1蛋白表达降低及Smad4蛋白表达升高(P<0.05)；45℃热疗后6h-24hTGF-β1蛋白表达明显下降,6h-12h Smad4蛋白表达升高(P<0.05)；47℃热疗后各个时间点TGF-β1蛋白的表达均降低(P<0.05),而Smad4蛋白表达的增加(P<0.05)只出现在热疗后2h-6h(P<0.05)。结论小鼠黑色素瘤B16F10细胞中TGF-β1、Smad4在mRNA和蛋白水平的表达变化与热疗温度和热疗后继续培养的时间有关。43℃及以上温度热疗使(MMP)-2/VEGF的基因表达明显抑制,同时有TGF-β1基因表达的下调,此三者之间可能存在相互影响。而TGF-β1蛋白水平的上调相对滞后于其基因水平的变化,Smad蛋白表达一过性增加。总之,实验结果说明了TGF-β1表达的下调可能为热疗抑制B16F10细胞增殖及体外侵袭能力的机制之一。目的探讨不同温度磁热疗对小鼠恶性黑色素瘤皮下移植瘤的作用。方法用小鼠黑色素瘤B16F10细胞株建C57BL6小鼠皮下移植瘤模型,将Fe3O4磁性纳米颗粒直接注入肿瘤组织内,在交变磁场中进行磁感应加热治疗,控温在45℃和50℃分别持续30min和10min,24h后重复加热,共加热3次,比较45℃组磁热疗组(H1)、50℃磁热疗组(H2)、单纯磁流体组(M组)、单纯磁场组(T组)和对照组的肿瘤体积的大小、生存期和TGF-β1的阳性指数。结果与对照组相比,45℃磁热疗组和50℃磁热疗组均能抑制肿瘤的生长(P<0.05)。与45℃磁热疗组比较,50℃磁热疗组抑瘤作用更显著,有57.1%的肿瘤完全消退率(P<0.05)。45℃MFH组和50℃MFH组的生存期分别为(40.286±8.592)d和(60.143±13.319)d比对照组的(17.429±3.054)d明显延长(P<0.05)。TGF-β1的阳性指数在50℃磁热疗组显著降低为16.4%,与其它组相比,差异具有显著性(P<0.05)。结论45℃磁热疗和50℃磁热疗均能抑制小鼠恶性黑色素皮下移植瘤的生长,延长荷瘤小鼠的生存期,抑制肿瘤组织中TGF-β1的表达。50℃磁热疗组的抑瘤作用较45℃磁热疗组更为显著,可以达到57.1%的肿瘤完全消退率。更多还原

【Abstract】 Background Melanoma is a cancer that arises from melanocytes, specialized pigmented cells that are found predominantly in the skin. The incidence of melanoma is rising steadily in China. Invation and metastasis is the most important biologic characteristics for malignant carcinoma, which also chiefly accounts for clinic death of cancer patients. Thus, inhibiting invation and metastasis is the key to succeed in curing cancer patient. In its early stages malignant melanoma can be cured by surgical resection, but once it has progressed to the metastatic stage it is extremely difficult to treat and does not respond to current therapies(such as radiation therapy and themotherapy).Hyperthermia is appealing because it is a physical treatment with fewer side effects than chemotherapy and radiotherapy, and repeated treatments should be feasible without concern for cumulative toxic side effects. Clinical experiments have shown that hyperthermia, as part of multimodal regimens, is a tolerable and clinically practical supplementary therapy for patients with advanced malignant tumor, recurrent tumor and metastatic disease. In recent years, magnetic targeted hyperthermia could heating at higher temperature(50℃) and avoiding nomal tissue to be damaged.Recently, it is reported that hyperthermia could delay tumor growth and inhibit lymph node metastasis in animal model. Furthermore, the expression of some genes associated with metastasis was shown to be down-regulated by heating, suggesting that hyperthermia may alter the character of tumor with high metastatic potential via regulating some metastasis related genes. However the concrete mechanism need futher research. B16F10 cells is a promising experimental model of invation for their metastasis biologic characteristics. Therefore, in order to find the appropriate temperature for melanoma treatment, we heat B16F10 cells at different temperature in vitro and in vivo then explore the effect on proliferation、invation and migration of cells.Objective To investigate the effect of hyperthermia on the proliferation and cell cycle of B16F10 cells.Methods B16F10 cells were heated at 41℃,43℃,45℃, 47℃,50℃and 37℃in temperature-controlled water bath, MTT assay, flow cytometry were applied to analyze the growth inhibition and cell cycle.Results The inhibitive rates of cell growth after 24h,48h,72h,96h at 45℃、47℃and 50℃was remarkably higher than at 41℃and 43℃(P <0.05). The inhibitive rates of cell growth after 24h,48h,72h,96h at 47℃and 50℃was remarkably higher than that of at 45℃(P< 0.05).There is no significant difference in inhibitive effect of cell growth between 47℃and 50℃(P<0.05). Results from flow cytometry indicate G0/G1 phase cells after 24h at 41℃,43℃were remarkably higher than that of the control group (P<0.05). However, S phase cells was remarkably lower as compared with the control group (P<0.05).G0/G1 phase cells after 24h at 47℃,50℃were remarkably lower than that of the control group (P<0.05) and S phase cells was remarkably higher as compared with the control group (P<0.05).G2/M phase cells after 24h at 43℃were remarkably higher than that of control group.Conclusions The hyperthermia at 45℃or higher termperature could remarkably inhibit the proliferation of B16F10 cells. The hyperthermia at 47℃or higher termperature could arrest the cells cycle in S phase.Objective To investigate the effect of hyperthermia on migration and invasion ability in vitro of B16F10 cells.Methods To observe the B16F10 cells after heating under invert microscope and transmission electron microscope. B16F10 cells were harvested after heating treatment at 43℃,45℃,47℃and 37℃in temperature-controlled water bath. The migration ability was measured by the scratch wound assay. In vitro cells invasion ability was evaluated by matrigel invasion assay. The activity of matrix metalloproteinase (MMP)-2/9 was investigated by gelatin zymographic assays.Results (1) Cells morphous and ultra-structure changed to different extent after heating at different temperature and showed temperature dependence.(2) The migration ability of B16F10 cells was reduced after heated at 43℃, and remarkably reduced at 45℃and 47℃.(3) The number of invading cells were 29.6±6.0、18.2±2.4 and 1.6±1.1 at 43℃,45℃and 47℃respectively, which were remarkably lower than that of control group (P<0.05).(4) (MMP)-2/9 secretion and enzymatic activity were suppressed at 43℃,45℃and 47℃which showed temperature dependence.Conclusions Cells morphous and ultra-structure changed after heating; In vitro migration and invasion of B16F10 cells was inhibited by heating; (MMP)-2/9 secretion and enzymatic activity were suppressed after heating.All above showed temperature dependence. Objective To study the effect of temperatures for in vitro heating treatment of B16F10 cells on the protein and mRNA expression of TGF-β1/Smad4, mRNA expression of TGF-β1/Smad4、(MMP)-2 /9 and VEGF.Methods B16F10 cells were harvested at 2h,6h,12h and 24h after heating treatment at 43℃,45℃,47℃and 37℃for 30min in temperature-controlled water bath, rt-PCR, western blotting, as well as immunohistochemical technique were employed to determine the mRNA and protein expressions of TGF-β1/Smad4、MMP-2/9 and VEGF.Results (1) Immunohistochemical analysis demonstrates that the expression of TGF-β1 and VEGF decreased with the increase of the temperature for heating treatment. (2) Compared with the control group, the mRNA expression of MMP-2、VEGF at 43℃was lower within 24h (P <0.05);expression of MMP-9、TGF-β1 at 43℃was lower from 6h to 24h with statistical significance (P<0.05); expression of TGF-β1、VEGF、MMP-2 and MMP-9 at 45℃、47℃was lower within 24h(P<0.05). (3) When compared with the control group, the protein expression of TGF-β1 was remarkably decreased and Smad4 was increased(P<0.05) from 12 h to 24 h at 43℃; the protein expression of TGF-β1 was remarkably decreased from 6 h to 24 h and Smad4 was increased from 6 h to 12 h at 45℃(P<0.05); at 47℃the protein expression of TGF-P, was remarkably decreased within 24 h and Smad4 was increased from 2 h to 6 h(P<0.05).Conclusions Protein and mRNA expression of TGF-β1/Smad4 was dependent on the temperature and culture time. The upregulation of Smad4 protein lasts only a period of very short time.The hyperthermia at 43℃or higher termperature could inhibit the mRNA expression of MMP-2/9 and VEGF, which may be associated with down regulation of TGF-β1.Objective To study the temperature effect of magnetic fluid hyperthermia (MFH) at 45℃or 50℃in a murine xenograft model of mouse melanomaMethods Murine xenograft model of mouse melanoma was established by transplanting cultured B16F10 cells into the subcutaneous tissue of the back of nude mouse. The tumor-bearing mice then underwent radiation by an alternative magnetic filed (AMF) after the Fe3O4 magnetic fluid (MF) was locally injected in the tumor area. The parameters of the AMF were carefully adjusted until a local tumor temperature (45℃or 50℃) was maintained for 30 min and 10 min respectively. The MFH was performed three times with 24h interval. The tumour volume, mice survival and the index of TGF-β1 were examined.Results MFH at 45℃or 50℃could inhibit the tumor growth. Compared with 45℃MFH,50℃MFH had a greater inhibitive effect on tumor growth (P<0.05). The survival of 50℃MFH group and 47℃MFH group were more prolonged than that of control group (P<0.05). When compared with the other groups, the expression of TGF-β1 was remarkably decreased at 50℃MFH group (P<0.05).Conclusion MFH at 45℃or 50℃could inhibit the tumor growth, prolong survival as well as decreased the expression of TGF-β1 with more remarkable effect at 50℃MFH group, in which the rate of total tumour regression was 57.1%.更多还原