维生素E琥珀酸酯诱导黑色素瘤B16细胞分化的体外实验研究
发布时间：2018-09-05 19:02
【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(P0.01),各实验组间比较,亦有显著性差异(P0.01)。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(Ρ0.01),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(P0.01),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(P0.01)。 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(P0.01)。 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。
[Abstract]:AIM: To investigate the effects of vitamin E succinate (VES) on the proliferation, differentiation, cell cycle and related protein expression of melanoma cells and the changes of melanosomes in vitro, and to further explore the mechanism of VES inhibiting the growth of melanoma cells so as to provide a basis for melanogenesis. Tumor therapy provides new methods and corresponding theoretical basis.
Methods: B16 melanoma cells were cultured in vitro. MTT colorimetric assay was used to detect the proliferation inhibition of B16 cells treated with different concentrations of VES for 24 hours, 48 hours and 72 hours. The morphological changes of B16 cells were observed by light microscope after Rui-Gill staining and the appropriate concentration of VES was selected for subsequent experiments. (FCM) The cell cycle distribution and apoptosis rate of B16 melanoma cells treated with VES for 48 hours were measured; the melanosomes were observed by transmission electron microscopy; the melanin content was determined by NaOH lysis method; the expression of cyclin D1 and P21 proteins was detected by flow cytometry after 48 hours of VES treatment. The expression of cyclin D1 and P21 protein in tumor cells was detected by ELISA, and the effect of VES on the differentiation of B16 cells was investigated.
Result:
1 VES inhibited B16 cell proliferation:
MTT assay showed that after treatment with 5, 10, 20 and 20 ug/ml VES for 24-72 hours, the OD value of each treatment group decreased to some extent, which was significantly different from that of the control group (P 0.01). There was also a significant difference among the experimental groups (P 0.01). With the increase of drug concentration and duration of action, the OD value of each VES group decreased gradually and the inhibition rate increased. High, that is, VES inhibited the proliferation of B16 cells in a time - to dose-dependent manner.
2 VES can induce B16 cell cycle arrest and apoptosis:
The results of flow cytometry showed that the proportion of G0/G1 phase cells increased, the proportion of S phase cells decreased, and the PI of proliferation index decreased significantly (0.01) with the increase of drug concentration 48 hours after treatment with 5,10,20 ug/ml VES. The results showed that VES could block the cell cycle progression of B16 in G0/G1 phase in a dose-dependent manner. The apoptotic rate of control group was 0.25 6550
3 the effect of VES on the morphology of B16 cells and electron microscopic observation of melanin bodies:
The normal melanoma B16 cells grew in a round, oval or polygonal shape and irregular shape. The cells grew in a certain polarity, arranged in parallel and not overlapping. With the prolongation of time, most of the cells became larger, longer and more irregular. Arranged in a network, with a dendritic structure, slow growth, the number of cells significantly reduced, this morphological change was more obvious after 48 hours.
Electron microscopic results showed that in B16 cells without VES treatment, the membrane was intact, with more microvilli on the surface, large nucleus, round nucleus, relatively large nucleoplasm, abundant euchromatin, less heterochromatin, free ribosomes in the cytoplasm, few other organelles, and no typical melanosomes were found; however, after VES (10,20 ug/ml) treatment, the surface of the membrane was large. There were fewer microvilli, smaller nuclei, more heterochromatins, smaller nucleoplasmic ratios and abundant organelles. There were a large number of typical melanosomes and no apoptotic cells.
4 Determination of melanin content:
Compared with normal melanoma cells, melanin synthesis ability of melanoma cells was lower. When induced by differentiation inducer, melanin production ability was significantly increased. Effective differentiation inducer should increase melanin content more than twice. The results showed that the melanin content of B16 cells treated with VES of 5, 10 and 20 ug/ml for 48 hours was black. Compared with the control group, the content of melanin was 1.006 (+ 0.2410) times, 1.813 (+ 0.4380) times and 3.654 (+ 0.7000) times of the control group, respectively.
5 flow cytometry was used to detect the expression of cyclinD1 and P21 protein in B16 cells.
The fluorescence index (FI) of cyclin D1 protein decreased gradually with the increase of VES concentration and the FI value of P21 protein increased gradually with the increase of VES concentration.
6 the expression of cyclinD1 and P21 protein in tumor cells was detected by immunocytochemistry.
Cyclin D1 protein was expressed in the cytoplasm and nucleus of murine melanoma B16 cells in the untreated group, mainly in the cytoplasm, and strongly positive in the intensity. After VES treatment, with the increase of VES concentration, the expression of cyclin D1 protein in the nucleus and plasma decreased gradually, mainly in the nucleus. After score statistics, the expression of cyclin D1 protein in each VES group decreased gradually with the increase of VES concentration. The expression of P21 protein in cytoplasm and nucleus of murine melanoma B16 cells was slightly weaker in VES treated group; the expression of P21 protein in nucleus and cytoplasm increased gradually with the increase of VES concentration, and the expression of P21 protein increased gradually with the increase of VES concentration. There was a significant difference between the two groups (P0.01).
Conclusion:
1. VES could inhibit the proliferation of melanoma B16 cells in a dose-time dependent manner without obvious apoptosis.
2. VES can induce the differentiation of melanoma cells through cell cycle arrest and block the cells in G0/G1 phase in a dose-dependent manner, and the effect is most obvious in 48 hours.
3. VES could induce the differentiation of B16 cells. The morphological features of VES were slow growth and reticular structure. Under electron microscope, a large number of typical melanosomes were found in B16 cells treated with VES.
4. VES can induce the differentiation of melanoma cells. The mechanism may be related to the down-regulation of cyclin D1 protein and up-regulation of P21 protein expression.
5. The results show that VES can inhibit the proliferation of melanoma cells, block cell cycle and induce differentiation, which provides a new idea and theoretical basis for the treatment of melanoma.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.5
本文编号：2225181
[Abstract]:AIM: To investigate the effects of vitamin E succinate (VES) on the proliferation, differentiation, cell cycle and related protein expression of melanoma cells and the changes of melanosomes in vitro, and to further explore the mechanism of VES inhibiting the growth of melanoma cells so as to provide a basis for melanogenesis. Tumor therapy provides new methods and corresponding theoretical basis.
Methods: B16 melanoma cells were cultured in vitro. MTT colorimetric assay was used to detect the proliferation inhibition of B16 cells treated with different concentrations of VES for 24 hours, 48 hours and 72 hours. The morphological changes of B16 cells were observed by light microscope after Rui-Gill staining and the appropriate concentration of VES was selected for subsequent experiments. (FCM) The cell cycle distribution and apoptosis rate of B16 melanoma cells treated with VES for 48 hours were measured; the melanosomes were observed by transmission electron microscopy; the melanin content was determined by NaOH lysis method; the expression of cyclin D1 and P21 proteins was detected by flow cytometry after 48 hours of VES treatment. The expression of cyclin D1 and P21 protein in tumor cells was detected by ELISA, and the effect of VES on the differentiation of B16 cells was investigated.
Result:
1 VES inhibited B16 cell proliferation:
MTT assay showed that after treatment with 5, 10, 20 and 20 ug/ml VES for 24-72 hours, the OD value of each treatment group decreased to some extent, which was significantly different from that of the control group (P 0.01). There was also a significant difference among the experimental groups (P 0.01). With the increase of drug concentration and duration of action, the OD value of each VES group decreased gradually and the inhibition rate increased. High, that is, VES inhibited the proliferation of B16 cells in a time - to dose-dependent manner.
2 VES can induce B16 cell cycle arrest and apoptosis:
The results of flow cytometry showed that the proportion of G0/G1 phase cells increased, the proportion of S phase cells decreased, and the PI of proliferation index decreased significantly (0.01) with the increase of drug concentration 48 hours after treatment with 5,10,20 ug/ml VES. The results showed that VES could block the cell cycle progression of B16 in G0/G1 phase in a dose-dependent manner. The apoptotic rate of control group was 0.25 6550
3 the effect of VES on the morphology of B16 cells and electron microscopic observation of melanin bodies:
The normal melanoma B16 cells grew in a round, oval or polygonal shape and irregular shape. The cells grew in a certain polarity, arranged in parallel and not overlapping. With the prolongation of time, most of the cells became larger, longer and more irregular. Arranged in a network, with a dendritic structure, slow growth, the number of cells significantly reduced, this morphological change was more obvious after 48 hours.
Electron microscopic results showed that in B16 cells without VES treatment, the membrane was intact, with more microvilli on the surface, large nucleus, round nucleus, relatively large nucleoplasm, abundant euchromatin, less heterochromatin, free ribosomes in the cytoplasm, few other organelles, and no typical melanosomes were found; however, after VES (10,20 ug/ml) treatment, the surface of the membrane was large. There were fewer microvilli, smaller nuclei, more heterochromatins, smaller nucleoplasmic ratios and abundant organelles. There were a large number of typical melanosomes and no apoptotic cells.
4 Determination of melanin content:
Compared with normal melanoma cells, melanin synthesis ability of melanoma cells was lower. When induced by differentiation inducer, melanin production ability was significantly increased. Effective differentiation inducer should increase melanin content more than twice. The results showed that the melanin content of B16 cells treated with VES of 5, 10 and 20 ug/ml for 48 hours was black. Compared with the control group, the content of melanin was 1.006 (+ 0.2410) times, 1.813 (+ 0.4380) times and 3.654 (+ 0.7000) times of the control group, respectively.
5 flow cytometry was used to detect the expression of cyclinD1 and P21 protein in B16 cells.
The fluorescence index (FI) of cyclin D1 protein decreased gradually with the increase of VES concentration and the FI value of P21 protein increased gradually with the increase of VES concentration.
6 the expression of cyclinD1 and P21 protein in tumor cells was detected by immunocytochemistry.
Cyclin D1 protein was expressed in the cytoplasm and nucleus of murine melanoma B16 cells in the untreated group, mainly in the cytoplasm, and strongly positive in the intensity. After VES treatment, with the increase of VES concentration, the expression of cyclin D1 protein in the nucleus and plasma decreased gradually, mainly in the nucleus. After score statistics, the expression of cyclin D1 protein in each VES group decreased gradually with the increase of VES concentration. The expression of P21 protein in cytoplasm and nucleus of murine melanoma B16 cells was slightly weaker in VES treated group; the expression of P21 protein in nucleus and cytoplasm increased gradually with the increase of VES concentration, and the expression of P21 protein increased gradually with the increase of VES concentration. There was a significant difference between the two groups (P0.01).
Conclusion:
1. VES could inhibit the proliferation of melanoma B16 cells in a dose-time dependent manner without obvious apoptosis.
2. VES can induce the differentiation of melanoma cells through cell cycle arrest and block the cells in G0/G1 phase in a dose-dependent manner, and the effect is most obvious in 48 hours.
3. VES could induce the differentiation of B16 cells. The morphological features of VES were slow growth and reticular structure. Under electron microscope, a large number of typical melanosomes were found in B16 cells treated with VES.
4. VES can induce the differentiation of melanoma cells. The mechanism may be related to the down-regulation of cyclin D1 protein and up-regulation of P21 protein expression.
5. The results show that VES can inhibit the proliferation of melanoma cells, block cell cycle and induce differentiation, which provides a new idea and theoretical basis for the treatment of melanoma.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.5
【引证文献】
相关硕士学位论文 前1条
1 姬巧霞;维生素E琥珀酸酯对黑色素瘤B16荷瘤小鼠抑瘤作用及其机制的实验研究[D];河北医科大学;2011年
，本文编号：2225181
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