【作者】 杨阳；

【导师】 丁政云； 高顺强；

【作者基本信息】 河北医科大学 ， 皮肤病与性病学， 2008， 硕士

【摘要】 目的:从细胞实验和动物实验两个角度观察阿维A酸(Acitretin)及其联合抗癌药物顺铂(DDP)对鼠B16黑色素瘤细胞增殖、分化的影响,初步研究阿维A酸治疗恶性黑色素瘤的作用及其机制,探讨其作用机制是否与阿维A酸影响survivin、Fas及VEGF的表达有关,从而为临床诱导分化治疗恶性黑色素瘤提供实验基础。方法:1体外实验:1.1体外培养恶性黑色素瘤细胞株B16,采用四甲基偶氮唑蓝(tetrzolium-based colorimetric assay, MTT)比色法检测不同浓度阿维A酸作用不同时间后对该细胞增殖的影响;光学显微镜观察细胞形态学改变。1.2采用MTT比色法检测阿维A酸与抗癌药物顺铂联合应用对恶性黑色素瘤细胞株B16的增殖抑制作用,采用金氏公式分析相互作用指数(q),评价联合后的作用,如q>0.85表示相加作用,q<0.85为拮抗作用。2体内实验:C57BL/6J小鼠50只,于右前肢腋下皮下注射浓度为1×10~6个/ml的B16黑色素瘤细胞悬液0.2ml建立荷瘤小鼠模型。小鼠随机分为5组,每组10只,分别为阴性对照组,阿维A酸低剂量组,阿维A酸高剂量组,顺铂组及联合治疗组。于皮下接种瘤细胞第二天开始给药,用药共21天,每天观察小鼠的状态,每3天测量一次瘤体积,最后一次给药24小时后将小鼠处死,剥瘤称重,计算抑瘤率。将肿瘤组织沿最大面剖开,常规固定并进行HE染色及形态学观察。免疫组化染色观察药物对survivin、Fas及VEGF在瘤组织中表达的影响。运用统计软件SPSS12.0对数据进行统计分析。结果:1体外实验:1.1 MTT比色法显示:阿维A酸(0.1μmol/L、1.0μmol/L、10μmol/L和100μmol/L)对B16细胞增殖均具有抑制作用。不同浓度阿维A酸处理细胞24-72h后,与对照组相比,各处理组OD值均有不同程度下降,差异具有极显著性(P<0.01)。且随阿维A酸处理浓度的增大、作用时间的延长,OD值逐渐下降,阿维A酸对B16细胞的增殖抑制率也逐渐增大。1.2光镜下观察细胞形态结果显示:阴性对照组细胞贴壁生长良好,细胞呈梭形,透明,折光性好,细胞生长活跃。而用药组细胞皱缩,破裂,呈不规则形,有的细胞两端出现细长的伪足,细胞胞浆中出现空泡,脱落细胞增多。1.3 MTT比色法分析阿维A酸联合顺铂对B16细胞的增殖抑制作用,结果显示:金氏公式分析相互作用指数q值均大于0.85,即0.1、1.0、10、100μmol/L的阿维A酸可增强顺铂对B16细胞的增殖抑制作用,且随阿维A酸浓度的增大,其对顺铂的协同作用逐渐增强。2体内实验:2.1瘤体积-时间生长曲线:各治疗组小鼠肿瘤生长速度始终低于对照组,阿维A酸高剂量组、顺铂组和联合给药组抑制作用尤为显著。2.2瘤重、瘤体积及抑瘤率:阿维A酸低、高剂量组、顺铂组和联合给药组的平均瘤重均低于阴性对照组,抑瘤率分别为:26.94%、42.74%、60.64%和74.14%,与对照组比较差异有显著性(P<0.05)。各治疗组平均瘤体积和抑制率也明显低于对照组,与对照组比较差异有显著性(P<0.01)。2.3 HE染色观察瘤组织:对照组瘤组织边界不清细胞密集排列,异型性明显,而治疗组瘤组织中心及边缘可见不同程度的大片状或灶性坏死。2.4免疫组化结果显示:各治疗组的survivin,VEGF表达均低于对照组(P<0.05),Fas的表达均高于对照组(P<0.01),表明各治疗组能显著抑制凋亡基因survivin和VEGF的表达,上调促凋亡基因Fas的表达。且各治疗组Survivin的表达与Fas的表达呈负相关(rs=-0.766,P<0.01),与VEGF的表达成正相关(rs=0.724,P<0.01)。结论:1阿维A酸对鼠B16恶性黑色素瘤细胞具有增殖抑制及诱导分化的作用,为临床MM的治疗和化学预防提供了新的思路和理论依据。2阿维A酸在一定浓度和时间范围内对B16细胞增殖的抑制作用呈现量效相关性和时间依赖性。3阿维A酸可强化顺铂诱导B16细胞凋亡,推测阿维A酸可能增强B16细胞对化疗药的敏感性。4阿维A酸对鼠B16黑色素瘤移植瘤生长有显著的抑制作用,且呈一定的量效关系。5阿维A酸对鼠B16黑色素瘤的抑制作用与其下调抑凋亡基因survivin、上调促凋亡基因Fas的表达及抑制VEGF的表达有关。这可能是其抗肿瘤增殖和促癌细胞分化的机理之一。更多还原

【Abstract】 Objective: To observe the role of acitretin and its combinated with DDP inhibiting cell proliferation and inducing apoptosis in the mouse melanoma cell line B16 in vivo and in vitro.To study the inhibitory effect and mechanisms of acitretin on the treatment of melanoma. And to further study whether the mechanism is related with that acitretin affect the expression of surviving , Fas and VEGF. These results would be hoped to elicit a novel approach to the clinical treatment of melanoma.Methods:1 In vitro:1.1 The mouse melanoma cell line B16 were cultured in vitro,and depressant effect of different concentration of acitretin on the proliferation of B16 cells was measured by MTT colorimetric method at different time;obaervated the morphological changes in B16 cells.1.2 The depressant effect of acitretin combined with DDP on the proliferation of B16 cells was measured by MTT colorimetric method,and evaluated with iteraction index.If q<0.85,express additivity;If q>0.85,express antagonism. 2 In vivo:B16 melanoma cells were inoculated into the C57BL/6J mice to establish B16 melanoma model, in order to investigate the effect of acitretin on tumor growth in C57BL/6J mice with transplanting tumor of melanoma. Fifty C57BL/6J mice with melanoma tumor were randomized five groups: negative control group, acitretin high dose group, acitretin low dose group ,Cisplatin group and joint group. Observation on the status of mice every day;measuring tumor volume every 3 days. After 21 days’treatment, samples of tumor were collected,weighed and calculated the tumor inhibition rate. The morphological changes of tumor were observed by HE staining method.By immunohistochemical determinated the expression of Survivin、Fas and VEGF. The test data were analyzed with SPSS 12.0 staistic software.Results:1 In vitro:1.1 MTT assay showed that acitretin (0.1,1.0,10,100μmol/L) on B16 can inhibit cells proliferation. After cells dealed with different concentrations acitretin of 24-72 hours ,compared with the control group, the treatment group OD values have decreased to some extent, the difference was significant (P <0.01).With the increasing concentration of acitretin and the extension of time, OD value has been gradually declining, that Acitretin on B16 cells was significantly inhibited the proliferation of the time-a dose-dependent effect. 1.2 Cells morphology was observed under light microscope showed that: cells shrinkage after treatment, the breakdown was irregular in shape, some cells appear at both ends of the pseudo-foot slender, exfoliated cells increased significantly inhibited the B16 cells growth, cells apparent Morphological changes.1.3 MTT assay showed that acitretin (0.1,1.0,10,100μmol/L) can strengthen the inhibitory effect of DDP on B16 cells proliferation.With the increasing concentration of acitretin,the synergistic effect has been enhanced gradually.2 In vivo:2.1 The tumor growth velocity of negative control group was higher than that of other groups all along.2.2 The tumor weitht and volume of negative control group was significally higher than that of other four groups; difference was significant (P<0.05 and P<0.01). The tumor inhibition rate of each group (acitretin low, acitretin high,DDP and joint group) were 26.94%、42.74%、60.64% and 74.14% respectively.2.3 In the tumor tissue of negative control group,tumor cells were confertim and obvious heteromorphism ,but different degree of lamellar necrosis were showed in that of other groups.2.4 The expression of Survivin and VEGF on tumor tissue of negative control group was significally higher than that of other four groups; difference was significant (P<0.05). The expression of Fas on tumor tissue of negative control group was significally lower than that of other four groups; difference was significant (P<0.01).There was a close correlation between the expression of Survivin and the expression of Fas,VEGF in the mouse melanoma tissues.Conclusion:1 Acitretin can inhibit the proliferation of B16 cells for clinical treatment and chemical MM prevention provided a new way of thinking and theoretical basis.2 Acitretin inhibit B16 cells proliferation of a time - a dose-dependent relationship within the appropriate.3 Acitretin can enhance cisplatin on B16 cells proliferation inhibition.It had been presumed that acitrtin can strengthen the sensibility of B16 cells on conventional anticancer agents.4 Acitretin can inhibit obviously the growth of B16 transplantation tumor.5 Acitretin can reduce the expression of Survivin, VEGF and increase the expression of Fas to inhibit the proliferation, invasion and metastasis of B16 cells.更多还原