【作者】 王峰；

【导师】 丁政云；

【作者基本信息】 河北医科大学 ， 皮肤病与性病学， 2012， 硕士

【摘要】 目的：恶性黑色素瘤（malignant melanoma, MM）是来源于黑色素细胞的恶性肿瘤，多发生于皮肤，占所有皮肤恶性肿瘤的第三位。其恶性程度高，早期易发生淋巴和血道转移，愈后差。手术切除、生物治疗、放射治疗及化学治疗是恶性黑色素瘤通常采用的治疗方法，但效果均不理想。随着早期诊断、外科手术及辅助治疗的发展，早期患者通过手术多可以治愈，5年生存率约95%，但进展期患者中生存期仅2-8个月，5年生存率不足5%。传统化疗对恶性黑色素瘤的疗效较差，因此，寻求疗效好、毒副作用小的新的治疗方法和药物是肿瘤学者研究的热点问题。维甲酸类（retinoid）是一组包括维生素A的天然及人工合成的衍生物，具有抗增殖及诱导分化等作用，对多种肿瘤细胞具有抑制作用，是一种有效预防皮肤肿瘤的药物。其抗皮肤黑色素瘤的机制尚未完全阐明。TNF-a具有广泛的生物学活性，具有强大的抗肿瘤作用，是迄今发现的抗肿瘤作用最强的细胞因子，但其副作用较大。二者联合应用，有可能提高肿瘤对诱导剂的敏感性，增强抗肿瘤作用，从而降低单药剂量，减少毒副作用，也可提高治疗效果。本研究旨以人黑色素瘤细胞株A375为研究对象，探讨维甲酸、TNF-a在不同浓度、不同作用时间对皮肤黑色素瘤细胞增殖抑制作用及形态分化的影响，筛选两药联合应用的最佳作用时间和剂量；探讨阿维A酸及两药联合应用对人A375细胞表达MMP-1、TIMP-1的影响。为TNF-a、维甲酸类药物诱导皮肤肿瘤细胞凋亡的基础研究及临床应用提供实验依据。方法：1细胞培养将A375恶性黑色素瘤细胞培养于含10%胎牛血清的DMEM培养基（含青霉素100U/ml，含链霉素100U/ml，PH7.2）中，放入37℃、5%CO2的培养箱内进行常规传代培养。2采用MTT比色法检测阿维A酸及阿维A酸联合TNF-a对A375细胞增殖的抑制作用。3倒置相差显微镜观察各组药物处理后A375细胞的分化及形态变化。4应用免疫细胞化学s-p法，检测不同浓度药物处理后A375细胞MMP-1、TIMP-1蛋白的表达状况。5采用流式细胞分析技术，检测A375细胞周期变化和凋亡。6采用统计软件SPSS13.0对数据进行统计分析。采用单因素方差分析，以ａ=0.05为显著性差异标准。结果：1MTT比色分析法检测各处理因素对人A375细胞增殖的影响（相对抑制率）。1.1阿维A酸组：浓度不同的阿维A酸组与对照组相比，阿维A酸在0.1umol/L至100umol/L范围内对人黑色素瘤A375细胞有明显的增值抑制作用，且以100umol/L浓度组作用48h抑制率最高。统计分析表明，在12、24及48三个时间段，阿维A酸对人黑色素瘤细胞A375的抑制作用呈现明显的剂量依赖效应关系和时间依赖效应关系，各组间差别具有显著性（p＜0.05）。而10、100μmol/L两组间比较,其抑制作用差异无统计学意义(P>0.05)1.2阿维A联合TNF-a组：将实验分为3组，分别为阴性对照组（不加任何药物）﹑TNF-a组(5u/L)及阿维A酸(10umol／L)联合TNF-a(5u/L)组。上述各组作用A375细胞48小时后，MTT法检测实验组抑制率TNF组为38.60%，联合组为58.45%，联合组明显高于对照组11.85%和阿维A酸组43.65%，可见阿维A酸和TNF-a联合应用明显抑制A375细胞增殖，与阿维A酸、TNF-a单独应用比较，差异具有显著性（p＜0.05）。2阿维A酸及阿维A联合TNF-a对A375细胞形态及分化的影响阴性对照组细胞贴壁生长良好,分布均匀，胞体胞浆饱满呈棱形、梭形或多角形，透明，折光性好，增殖旺盛；阿维A酸处理A375细胞24小时后，细胞变圆、皱缩、破裂，细胞透亮度和折光性减弱，贴壁差，并且出现细胞漂浮等现象，随着作用时间的延长上述现象更加明显；阿维A联合TNF-a作用细胞24小时后，细胞形态变化较单用阿维A更明显，细胞数目明显减少，显微镜下可见多量漂浮细胞。3免疫细胞化学法检测阿维A酸及阿维A酸联合TNF-a作用前后A375细胞MMP-1、TIMP-1蛋白表达情况MMP-1、TIMP-1蛋白主要表达于细胞浆内，DAB染色为棕黄或棕褐色。经不同浓度药物处理后的细胞蛋白阳性表达的程度不同，阴性对照组细胞MMP-1高表达，TIMP-1低表达，在阿维A组及联合用药组A375细胞中，MMP-1及TIMP-1蛋白表达均降低，且联合组MMP-1及TIMP-1蛋白表达降低更明显，差异具有显著性（p＜0.05）。4流式细胞术检测不同浓度药物处理后细胞的周期、凋亡变化流式细胞术检测显示：与对照组相比（28.29%），加药组（TNF组为38.35%、阿维A酸组为49.56%及联合组为58.62%）中处于G0／G1期的细胞百比例明显增加，而S期（对照组为54.8%、TNF组为42.71%、阿维A酸组为30.5%及联合组为22.03%）的细胞数量明显减少，各组药物均能使G1期延滞，G1-S期转化抑制，将细胞阻滞于G0／G1期，且联合组效果更明显。可见，阿维A联合TNF-a应用对细胞周期的影响具有协同作用。TNF-a及阿维A联合TNF-a均能诱导A375细胞凋亡，但阿维A联合TNF-a诱导A375细胞凋亡作用显著，差异具有显著性（p＜0.05）。结论：1阿维A酸和TNF-a都能抑制A375细胞的增殖，并且在一定的浓度和时间范围内，阿维A酸对A375细胞增殖的抑制率具有时间依赖性和量效依赖性。2阿维A联合TNF-a对A375细胞的增殖抑制率优于阿维A和TNF-a单用，二者联合应用具有协同作用。3TNF-a及阿维A酸联合TNF-a应用均可阻断人黑色素瘤细胞A375从G0/G1期进入S期，且联合用药诱导A375细胞凋亡的作用更明显4阿维A酸联合TNF-a对A375细胞的抑制作用可能与下调MMP-1、TIMP-1的表达致使MMPs/TIMPs平衡失调有关，这可能为其抗肿瘤机理有关。更多还原

【Abstract】 Objective：Malignant melanoma is derived from the melanin cell ofmalignant tumor, occurs more often in the skin, accounting for all skin ofmalignant tumor third.Its highiy malignant and prone to early lymphatic andhematogenous metastasis, the prognosis poor. Surgical resection, biologicaltreatment, radiotherapy and chemotherapy are commonly used treatment ofmalignant melanoma, but the results are not satisfactory. With early diagnosisand surgical intervention and the development of the auxiliary treatment,patients with early by more than surgery can be cured,5years of survival rateof about95%, but in patients with adanced surial only2to8months,5yearsof survival rate to less than5%. Traditional chemotherapy for malignantmelanoma curative effect is poorer, therefore, seeks good effect and sideeffects of small new treatment and drugs are hotspot oncolosts.Retinoids includes vitamin A, natural and synthetic derivatives. It has theantiproliferation and inducing differentiation effects on a variety of tumor cells.It is also a kind of effective prevention of skin cancer drugs, but the resistanceof skin melanoma mechanism has not been fully elucidated. TNF-a has a widerang of biological activity, with a strong anti-tumor effect, is found so fa, thestrongest anti-tumor effect of cytokines, but its side effects. They mayimprove the sensitivity of the tumor indecer, enhanced anti-tumor effect,which erduces the signle drug dose, and to erduce the toxic side effects, butalso can improve treatment. The aim of this study to the human melanoma cellline A375to investigate retinoid, TNF-a in different concentrations anddifferent time of skin melanoma cells inhibit the proliferation andmorphological differentiation of the screening of the two drugs combined bestthe role of time and dose.Explore the acitretin, and the combination of twodrug to the expression of MMP-1and TIMP-1in human A375cells.TNF-a, Retinoids for skin tumor cell apoptosis in basic research and clinicalapplication provide an experimental basis.Method:1Cell cultureThe human melanoma cell line A375were cultured in DMEM mediumsupplemented with10%heat inactivated fetal bovine serum,100u/mlpenicillin and100u/ml streptomycin and in a numidified atmosphere5%CO2at37℃.2Detection of cell growth inhibition with the MTT analysis method.3The morphological and differentiation changes of cells were detected by thereverse microscopy after the treatment of different drugs.4To detect the expression of MMP-1, TIMP-1of the human melanoma cellline A375in the different groups by immunohistochemical method.5Statistic analyses: Data were analyzed by statistical software Spss13.0forwindows. Based on the One-way ANOVA with a=0.05as significant differences standards.Results:1To detect growth inhibition rate of A375cells by MTT:1.1In group acitretin: The difference of inhibition rate among the three groupswas significant(p＜0.05).The results of MTT showed that acitretin inhibitedthe proliferation of A375cells ina dose-dependent and time-dependentmanner.1.2In group combination of acitretin and TNF-a: The A375cells were dividedin there groups and treated with acitretin in combination or not with TNF-a invitro. The results showed that the combination of acitretin with TNF-ainhibited growth of A375cells, inhibitory rate of combination has remarkabledifference compared with acitretin and control group. The effect of acitretinand TNF-a were synergistic.2To observe the differentiation and morphology of A375cell by differentdrugs.Cells in the negative control group grow well stick wall, uniform distribution, moderately sized, cytoplasm full, most in shuttle or polygon shap,transparent, refractive index of proliferation exuberant. Cells go round, shrinks,rupture after treatment by acitretin after24hours. Refraction weakened andfloating cells appeared, along with the role the extension of time of the abovephenpmenon more apparent. The cells treated by acitretin and TNF-a werepresent in the supernatant. The density of adherence cells was diminishedwhen compared with the control.3To detect the exepression of MMP-1, TIMP-1protein by cellimmunochemistry.MMP-1, TIMP-1protein was expressed in the intracytoplasm of A375cell. We can detect the expression of MMP-1, TIMP-1protein diminished inarsenic acitretin-treated cells compared with the untreated controls. MMP-1,TIMP-1protein in A375cells treated with acitretin/TNF-a was diminishedsignificantly compared with control and acitretin, TNF-a alone. There hasdifference between the control and the exeperiment groups (p<0.05).4To detect the cell apoptosis rate and the cell cycle distribution by flowcytometry machine (FCM):Compared with the control group, medication in each group couldincreased G1-phase population and reduced the S-phase population in A375cells. Application of acitretin and TNF-a combined has synergistic effect;However, TNF-a and combination therapy could induce the apoptosis of A375cells. The combination therapy-induced apoptosis of A375cells moresignificant.Conclusion:1Acitretin and TNF-a could significantly inhibit the proliferation of A375cells. All these effects were in dose-dependent and time-dependent mannerwithin the appropriate extent.2The synergistic effects of a combination of acitretin with TNF-a on inducingdifferentiation, apoptosis and growth inhibition of A375cells which is moreoutstanding than used alone significantly.3The experiment groups all could increase the proportion of G0/G1period and decrease the proportion of S period, and the apoptosis of A375cells wasmore observed. But combination therapy-induced apoptosis of A375cells ismore obvious.4The combination of acitretin and TNF-a can inhibit the proliferation of A375cell, which may relate to decreasing the expression of MMP-1, TIMP-1.Thismay be related to its anti-tumor mechanism.更多还原