阿维A酸对小鼠黑色素瘤细胞株B16增殖和Livin、bFGF表达的影响
发布时间：2018-12-07 17:43
【摘要】： 目的:恶性黑色素瘤(malignant malanoma,MM)是一种主要发生于皮肤由黑色素细胞恶化形成的能够产生黑色素严重威胁人们生命的高度恶性肿瘤,其生物学行为高度恶性,具有侵袭组织和持续增殖能力,并可逃避免疫监视。目前为止还没有有效的预防治疗方法,外科手术仍然是现在治疗黑色素瘤的首选方法,但是即便外科手术切除,预后仍很不理想,进展期此肿瘤患者平均的生存时间仅为6~9个月。因此,初步研究阿维A酸治疗恶性黑色素瘤的作用及其机制,探讨作用机制是否与阿维A酸影响Livin、bFGF的表达有关,进而为临床诱导分化治疗黑色素瘤提供实验基础。 维甲酸类(retinoids)包括维生素A的天然和人工合成的衍生物。它对上皮细胞有抑制其异常增殖和分化的作用,并可促进上皮组织正常角化,溶解角质使其恢复正常的作用,在临床上已用来预防及治疗多种皮肤病。近来科学研究表明,维甲酸还具有很强的诱导分化抑制肿瘤细胞增殖的作用,具有明显的时间依赖性和浓度依赖性。 方法: 1细胞培养 在含10%胎牛血清的RPMI-1640培养基(含青霉素100U/ml,含链霉素100U/ml,PH7.2)中培养B16细胞,置于饱和湿度、37℃、5%CO2的培养箱内做常规传代培养。 2四甲基偶氮唑蓝(MTT)比色法 检测阿维A对B16细胞增殖的抑制作用。采用浓度不同的阿维A(0.1 umol/L、1umol/L、10umol/L、100umol/L)作用不同时间(24、48、72小时)后,检测对B16细胞增殖的抑制作用。 3倒置相差显微镜及HE染色法 观察经过不同浓度药物处理后B16细胞的分化及形态变化并同时把细胞HE染色后进行观察。 4免疫细胞化学s-p法 检测经不同浓度药物处理后的B16细胞Livin及碱性成纤维细胞生长因子(bFGF)蛋白的表达状况。 5流式细胞分析技术 检测细胞周期变化及凋亡的进展,将实验分为5组,分别为阴性对照组(不加任何药物),阿维A浓度分别为0.1 umol/L,1 umol/L, 10umol/L,100 umol/L各一组。各组不同浓度药物处理48小时之后,收集细胞,应用流式细胞仪,检测各组细胞周期分布、细胞凋亡情况及Livin、bFGF蛋白表达状况。 6统计软件SPSS13.0对数据进行统计分析。 结果: 1 MTT比色分析法检测 结果显示阿维A酸对B16细胞增殖有显著的抑制作用(相对抑制率),各个组间的差别有显著性,并且不同浓度阿维A酸作用于小鼠B16细胞后所产生的抑制作用表现出显著的时间依赖效应关系和剂量依赖效应关系。 2阿维A酸对B16细胞形态及分化的影响 阴性对照组:B16细胞呈梭形或多角形,贴壁生长良好,透明,折光性好,增殖旺盛;经阿维A酸处理B16细胞后,细胞变圆,贴壁差,生长不佳,折光性减弱,核固缩现象增多,皱缩,并且出现小部分无细胞生长区及细胞漂浮等现象增多,而且随着阿维A处理时间的延长及浓度的增加上述现象变得更加明显严重。 3免疫细胞化学s-p法检测B16细胞Livin、bFGF蛋白表达情况 Livin、bFGF蛋白主要表达于细胞浆内,DAB染色为深棕黄色。经不同浓度药物处理后的细胞蛋白阳性表达的程度不同,阴性对照组细胞表达较强,随着药物浓度的增加染色程度都有不同程度的减弱,并且差异有显著性(p0.05)。 4流式细胞术检测经不同浓度药物作用后,细胞的周期、凋亡变化 流式细胞术检测显示:G1期延滞,G1-S期转化抑制,G0/G1期的比例上升,S期细胞的比例减少。 5流式细胞术测定不同浓度药物作用后,B16细胞Livin、bFGF蛋白的表达 经不同浓度药作用作用后,与阴性对照组相比,表达Livin、bFGF的阳性细胞数逐渐降低,经阿维A酸作用细胞48小时后,各组Livin蛋白表达荧光指数(FI)分别为1.6500、1.4215、1.2100、1.1124,bFGF蛋白表达荧光指数(FI)分别为1.8421、1.6446、1.4300、1.2218。把数据分别进行两两比较,之间的差异均有显著性(p0.05)。 结论: 1维A酸可以抑制B16细胞增殖,而且在一定时间和浓度范围内,对B16细胞增殖的抑制具有时间依赖性和量效依赖性。 2四个浓度的阿维A酸均可以影响小鼠黑色素瘤细胞B16从G0/G1期进入S期,然而诱导B16细胞凋亡的作用并不十分得明显。 3阿维A酸对B16细胞增殖的抑制作用可能与下调凋亡抑制因子Livin和碱性成纤维因子bFGF的表达有关。这有可能是其抗肿瘤增殖和诱导癌细胞分化的机制之一。
[Abstract]:Objective: MALIGNANT MALIGNANT (MM) is a highly malignant tumor which is mainly caused by the deterioration of the skin caused by the deterioration of the melanocytes. The biological behavior of the malignant melanoma is highly malignant and has the ability to attack the tissue and the continuous proliferation. and immune surveillance can be avoided. So far, there is no effective method of prevention and treatment, and surgery is still the first choice for the treatment of melanoma, but even if the surgery is removed, the prognosis is still not ideal, and the average survival time of this tumor patient is only 6-9 months. Therefore, the role of AVIA in the treatment of malignant melanoma and its mechanism were studied, and whether the mechanism of action was related to the expression of Livin and bFGF in the AlveA acid, and further provided an experimental basis for the clinical-induced differentiation of melanoma. Retinoids include natural and synthetic vitamin A It has the effects of inhibiting the abnormal proliferation and differentiation of epithelial cells, promoting the normal keratinization of epithelial tissue, dissolving the cutin, and restoring normal function, and has been used in the prevention and treatment of various kinds of epithelial cells. Dermatology. Recent scientific research has shown that retinoic acid also has a strong induction. Differentiation-inhibiting the proliferation of tumor cells, with significant time-dependence and concentration dependence. Method: 1 Cell culture was cultured in RPMI-1640 medium containing 10% fetal bovine serum (including penicillin 100U/ ml, streptomycin containing 100U/ ml, pH 7.2), and then placed in saturated humidity, 37 鈩，
本文编号：2367587
[Abstract]:Objective: MALIGNANT MALIGNANT (MM) is a highly malignant tumor which is mainly caused by the deterioration of the skin caused by the deterioration of the melanocytes. The biological behavior of the malignant melanoma is highly malignant and has the ability to attack the tissue and the continuous proliferation. and immune surveillance can be avoided. So far, there is no effective method of prevention and treatment, and surgery is still the first choice for the treatment of melanoma, but even if the surgery is removed, the prognosis is still not ideal, and the average survival time of this tumor patient is only 6-9 months. Therefore, the role of AVIA in the treatment of malignant melanoma and its mechanism were studied, and whether the mechanism of action was related to the expression of Livin and bFGF in the AlveA acid, and further provided an experimental basis for the clinical-induced differentiation of melanoma. Retinoids include natural and synthetic vitamin A It has the effects of inhibiting the abnormal proliferation and differentiation of epithelial cells, promoting the normal keratinization of epithelial tissue, dissolving the cutin, and restoring normal function, and has been used in the prevention and treatment of various kinds of epithelial cells. Dermatology. Recent scientific research has shown that retinoic acid also has a strong induction. Differentiation-inhibiting the proliferation of tumor cells, with significant time-dependence and concentration dependence. Method: 1 Cell culture was cultured in RPMI-1640 medium containing 10% fetal bovine serum (including penicillin 100U/ ml, streptomycin containing 100U/ ml, pH 7.2), and then placed in saturated humidity, 37 鈩，
本文编号：2367587
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