【作者】 曹春晓；

【导师】 王玉华；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2010， 硕士

【摘要】 目的:通过体外实验研究维生素E琥珀酸酯(Vitamin E succinate,VES;α-tocopheryl succinate,α-TOS)对黑色素瘤细胞增殖、分化、细胞周期和相关蛋白表达以及黑色素小体改变的影响并进一步探讨VES抑制黑色素瘤细胞生长的作用机制,从而为黑色素瘤治疗提供新的方法及相应的理论依据。方法:体外培养小鼠黑色素瘤B16细胞,采用四甲基偶氮唑蓝(MTT)比色法检测不同浓度VES作用于B16细胞24h、48h、72h后对其增殖抑制情况及经瑞-吉染色后光学显微镜观察细胞形态变化并筛选出适合的药物浓度进行后续试验。采用流式细胞术(FCM)测定VES对小鼠黑色素瘤B16细胞作用48h后的细胞周期分布和凋亡率;应用透射电镜观察黑色素小体;NaOH裂解法测定黑色素含量变化;采用流式细胞术检测VES处理B16细胞48小时后细胞周期蛋白cyclinD1和P21蛋白的表达变化;免疫细胞化学法检测各组肿瘤细胞cyclinD1和P21蛋白表达情况。从而初步探讨VES对B16细胞的诱导分化作用及相应机制。结果:1 VES抑制B16细胞增殖:MTT法结果显示,5μg/ml、10μg/ml、20μg/ml VES处理细胞24～72h后,各处理组OD值均有不同程度下降,与对照组相比,有显著性差异(P<0.01),各实验组间比较,亦有显著性差异(P<0.01)。且随药物浓度的增大、作用时间的延长,VES各组OD值逐渐下降,抑制率升高,即VES对B16细胞的增殖抑制作用呈明显的时间-剂量依赖效应。2 VES可诱导B16细胞周期阻滞和凋亡:流式细胞仪检测结果显示,5、10、20μg/ml VES作用于B16细胞48h后,随药物浓度增加,G0/G1期细胞比例增加,S期比例下降,增殖指数PI明显下降(Ρ<0.01),证明VES可阻滞B16细胞周期进程于G0/G1期,且该作用呈剂量依赖性。VES各剂量组凋亡率分别为0.42±0.14%、0.72±0.18%、1.06±0.72%。对照组凋亡率为0.25±0.04%。因凋亡率均低于5%,且无亚二倍体凋亡峰出现,故在5-20μg/mlVES作用于B16细胞后,无凋亡出现,证明在此范围内VES不能诱导鼠黑色素瘤B16细胞凋亡。3 VES对B16细胞形态的影响及电镜观察黑色素小体:经瑞-吉染色后光镜下观察细胞形态发现:正常的黑色素瘤B16细胞呈贴壁、多层生长,形态为圆形、椭圆形或多边形,生长不规则。而VES作用于B16细胞后,细胞呈一定的极性生长,平行排列,不重叠,随着时间延长,多数细胞体积变大,延长,细胞呈网状排列,具有树突状结构,生长缓慢,细胞数明显变少,此形态变化于作用48h后较为明显。电镜结果显示:在未经VES处理过的B16细胞中,胞膜完整,表面有较多微绒毛,细胞核大,圆形,核浆比较大,常染色质丰富,异染色质较少,胞质内可见游离核糖体,其他细胞器少,未见典型的黑色素小体;然而,经VES(10、20μg/ml)作用后,胞膜表面微绒毛较少,细胞核变小,核内异染色质增多,核浆比变小,细胞器丰富,可见大量典型的黑色素小体,未见凋亡形态的细胞,这与以上凋亡率的检测结果一致。4黑色素含量的测定:黑色素瘤细胞与正常黑色素细胞相比,黑色素合成能力低下。当被分化诱导剂诱导分化时,黑色素生成能力明显增加,有效的分化诱导作用应使黑色素含量增加2倍以上。本实验结果显示,5μg/ml,10μg/ml,20μg/ml的VES作用于B16细胞48h后,黑色素含量与对照组比较,分别是对照组的1.006±0.2410倍,1.813±0.4380倍,3.654±0.7000倍。经统计,10μg/ml,20μg/ml组较未加药组有极显著性差异(P<0.01),其中20μg/ml浓度组是对照组黑色素含量的3.654±0.7000倍>2倍,故20μg/ml VES有效地诱导了B16细胞的分化。5流式细胞仪检测B16细胞cyclinD1、P21蛋白表达:5、10、20μg/ml VES作用于B16细胞48h后,发现cyclinD1蛋白的荧光指数(FI)随VES浓度增大而逐渐减少,P21蛋白的FI值则随药物浓度的增大而逐渐增大。对于每一种蛋白,其处理组和对照组FI值比较,均有极显著性差异(P<0.01)。6免疫细胞化学法检测肿瘤细胞cyclinD1、P21蛋白表达并进行评分:未加药组,cyclinD1蛋白在鼠黑色素瘤B16细胞的细胞浆和核均有表达,以胞浆为主,强度成强阳性;VES处理后,随着VES浓度增加,蛋白在核、浆表达均逐渐减少,以核减少为主;评分统计后VES各组cyclinD1蛋白表达值随VES浓度增加而逐渐减小。未加药组,P21蛋白在鼠黑色素瘤B16细胞的细胞浆和核极少量表达,强度较弱;VES处理后,随着VES浓度的增加,蛋白在核浆表达逐渐增加,以核增加为主;评分统计后P21蛋白表达值随VES浓度增加而逐渐增大。两种蛋白各自比较,VES处理组和阴性对照组,均有显著性差异(P<0.01)。结论:1、在不表现明显的细胞凋亡现象的剂量范围内,VES可明显抑制黑色素瘤B16细胞增殖,并呈剂量-时间依赖性。2、VES可通过对黑色素瘤细胞周期阻滞诱导分化,将细胞阻滞于G0/G1期,该作用呈剂量依赖性,并以48小时作用最明显。3、VES对B16细胞有较强的分化诱导能力,形态学表现为生长缓慢,细胞连成网状结构,电镜可见VES处理过的B16细胞内含大量典型黑色素小体。功能上表现: VES作用于B16细胞后,黑色素含量明显增加,尤其是20μg/ml VES。4、VES具有诱导黑色素瘤细胞分化作用,其机制可能与其下调cyclinD1蛋白,上调P21蛋白的表达有关。5、本实验结果显示:VES具有抑制黑色素瘤细胞增殖、阻滞细胞周期、诱导分化的作用,为其用于黑色素瘤治疗提供了新的思路和理论依据。更多还原

【Abstract】 Objective: This experiment aimed to study the effects of Vitamin E succinate on the proliferation, cell differentiation, distribution of cell cycle, expression of correlative protein and melanosome production of melanoma cells in vitro. Thus to provided new method and theoretical evidence for theatment of melanoma.Methods: Murine B16 cells were cultured in vitro. Effect of different dose of VES on the proliferation of B16 cells after 24h, 48h and 72h was measured by MTT colorimetric methods, and morphological changes of B16 cells was observed by light microscopy by Wright Giemsa stain. Distribution of cell cycle and apoptotic rate of B16 cells treated with VES at suitable drug concentration after 48h were examined by flow cytometry (FCM); melanosome was observed by the transmission electron microscopy. Melanin contents were determined by NaOH spallation;The expression of cyclinD1 and P21 proteins of B16 cells treated by VES after 48h were analyzed by flow cytometry and cell immunocytochemistry. Thus to initially investigated to support the mechanicsms of VES on induction differentiation of melanoma B16 cells.Results:1 VES inhibited the proliferation of B16 cells: after the administration of VES(5, 10, 20μg/ml) for 24h to 72h, the OD values of VES-treated groups decreased obviously. Compared with control group, there was statistically significant difference between control group and among every treatment group (P<0.01). Furthermore, with the increasing concentration of VES and prolong of treatment time, the OD values decreased gradually, the inhibitory rate increased, that was, VES inhibited the proliferation of B16 cells significantly in a dose and time dependent manner.2 VES could arrest cell cycle and induce apoptosis of B16 cells: After the administration of VES at the concentration of 5, 10, 20μg/ml for 48h, the number of cells in G0/G1 phase increased gradually and the number of cells in S phase decreased gradually, with the increasing concentration of VES, the proliferation index (PI) significantly decreased (P<0.01).It was demonstrated that, VES could induce an arrestment of cell cycle in G0/G1 phase in a dose-dependent manner. In addition, after the administration of VES, the apoptotic rate of every VES-treated group were 0.42±0.14%, 0.72±0.18%, 1.06±0.72%, respectively, and the control group was 0.25±0.04%; Because of the apoptotic rate of every VES-treated group all were smaller than 5% and without hypodiploid apoptotic peak, so 5-20μg/ml VES could not induce apoptosis to B16 cells.3 VES could cause morphological changes of B16 cells and observe melanosome by transmission electron microscope: After Wright Giemsa stain, the morphological changes of B16 cells were observed by light microscopy: The cells of normal B16 cells were adherence, multilayered and irregularly growth in vitro, round, oval and polygon. After treated with VES (10, 20μg/ml), cells show heteropolarity growth, parallel rank, without overlap; Following the time prolongation, the volume of the majority cells was gradually increasing and extent, and cellshad dendrite architecture, reticulate arrange, slow-growing, and cell population decreased obviously.The ultramicrostructure changes of B16 cells were observed by transmission electron microscope: the B16 cells without treatment with VES, cell membrane complete, more microvilli on the surface, cell nucleus bigger and round, karyoplasmic ratio larger, euchromatin more, heterochromatin less, intracytoplasm organelle junior except free ribosomes, without typical melanosome. However, after treatment with VES (10 and 20μg/ml), less microvilli on the surface of the cell membrane, cell nucleus smaller and irregular, heterochromatin more, karyoplasmic ratio smaller, more mitochondrion and rough endoplasmic reticulum and a great deal of melanosome in the cytoplasm, but without apoptosis cells could be seen, which were consisted with result of the flow cytometry above. 4 The determination of the melanin contents:Comparing the melanoma cells with the normal melanocytes, Melanin synthesis capability became lower, which increased obviously when B16 cells were induced differentiation by inductor. However, when melanin content was more than Two-fold, the differentiation is effective.The experiment result show that: after 5μg/ml, 10μg/ml, 20μg/ml VES affected B16 cells, compared the melanin content of every VES treated groupto the control group, about 1.0057±0.241-fold, 1.813±0.438-fold, 3.654±0.7-fold respectively. To sum up, there was statistically significant differrence between the negative control group and the groups that 10μg/ml, 20μg/ml (P<0.01), and the formation rate of melanin that 20μg/ml VES was 3.654±0.7 times than the normal group. Consequently, VES (20μg/ml) induce B16 cells differentiation effectually.5 Analysis on expressions of cyclinD1 and P21 by FCM: after the administration of VES at the concentrations of 5, 10 and 20μg/ml for 48h, the FI values of cyclinD1 decreased with the increasing concentration of VES, and the FI values of P21 increased. There was statistically significant difference between control group and among VES-treated group (P<0.01).6 The expressions of cyclinD1 and P21 protein of B16 cells were analyzed by immunocytochemical method, and scored in IHS value manner.CyclinD1 protein expressed both in the cytoplasm and nucleus of B16 cells which untreated with VES, especially in the cytoplasm, masculine intension. When after treatment with VES, following the concentration of VES increasing, cyclinD1 protein decreased gradually in the both cytoplasm and nucleus, especially in the nucleus. After scoring and statistics, expressive values of cyclinD1 (IHS value) decreased with the increasing concentration of VES. P21 proteinum expressed both in cytoplasm and nucleus that was extreme manipulus and strength weaker of B16 cells which untreated with VES and increased gradually in the both cytoplasm and nucleus, When after treated with VES, following the concentrateon of VES increasing, P21 proteinum increased gradually both in the cytoplasm and nucleus, especially in the nucleus. After scoring and statistics, expressive values of P21(IHS value) increased with the increasei-ng concentration of VES. To the expressive values of both proteins, there was statistically significant difference between the negative controlgroup and every VES treated group (P<0.01).Conclusions:1 VES could inhibit proliferation of B16 cells in a dose and time-dependent manner within a certain concentration and could not induce apoptosis.2 VES could arrest cell cycle in G0/G1 phase and induce them differentiation in a dose-dependent manner and especially after 48h.3 VES induced B16 cells differentiation obviously, on morphous respect, cells grow slowly, cell join reticular formation and a great deal of melanin granule in the cytoplasm. In the function, melanin content generate obviously increased, especially treated with VES (20μg/ml).4 VES could induce differentiation of melanoma cells and the mechanisms might be related with down-regulation the expression levels of cyclinD1 and up-regulation the expression levels of P21.5 The experiment demonstrated that: VES had the effect of inhibiting proliferation, inducing differentiation and arresting cell cycle, which provided new mind and theoretical evidence for curing of melanoma.更多还原