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
发布时间：2019-06-21 03:20
【摘要】：目的:通过动物实验,研究维生素E琥珀酸酯(vitamin E succinate,VES;α-tocopheryl succinate,α-TOS)对黑色素瘤细胞的增殖、分化、凋亡的影响。通过相关蛋白表达的变化,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:将40只雄性BALB/c小鼠随机分为5组,每组8只。将鼠黑色素瘤B16细胞悬液接种于各组小鼠右侧背部皮下,每只1×10~6个,建立小鼠黑色素瘤移植瘤模型。当多数小鼠荷瘤后开始给药,采用腹腔注射给药方式:实验组分别给予VES 12.5mg/kg、25mg/kg、50mg/kg腹腔注射,每天一次,连续五天,休两天,共两周;对照组给予芝麻油0.05ml/只,给药方法及时间同实验组;阳性对照组给予达卡巴嗪(Dacarbazine, DTIC)0.2ml (80mg/kg)腹腔注射,每天一次,连续五天。两周后,颈椎脱臼处死各组小鼠,剥瘤称重,计算抑瘤率;显微镜观察VES作用后的瘤细胞大体形态变化,透射电镜观察VES作用后的瘤细胞超微结构变化;流式细胞术检测各组瘤细胞周期分布及凋亡率;免疫组化法检测各组瘤细胞S-100,Survivin,Caspase-3的蛋白表达情况;运用SPSS 13.0统计学软件进行数据处理,统计分析。 结果: 1 VES可抑制小鼠肿瘤生长:VES(12.5mg/kg、25mg/kg、50mg/kg)各剂量组对小鼠B16移植瘤的抑制率分别为:13.72%、31.22%、45.58%,呈剂量依赖性。达卡巴嗪组抑瘤率为55.64%,高于VES各组。各组小鼠处死前的体重比较:VES各处理组与对照组相比,差异无统计学意义(p0.05);阳性对照组体重有较明显下降,与阴性对照组相比,差异有统计学意义(p 0.01)。 2 VES可阻滞瘤细胞周期、诱导分化和凋亡:流式细胞分析仪检测结果显示VES12.5mg/kg、25mg/kg、50mg/kg剂量组的细胞,S期比例逐渐增加,呈剂量相关性,与阴性对照组相比,差异有统计学意义(p0.05或p 0.01)。VES12.5mg/kg、25mg/kg剂量组瘤细胞的G0/G1期细胞比例逐渐增加,与阴性对照组相比,差异具有统计学意义(p 0.05或p 0.01)。VES各剂量组瘤细胞的凋亡率分别为:20.88±0.58%、22.71±0.55 %、27.22±0.59%,阴性对照组为6.73±0.97%,均低于阳性对照组凋亡率30.95±0.52%,VES各组与对照组、阳性对照组相比,差异均有统计学意义(p 0.01)。 3 VES对瘤组织细胞形态的影响: 3.1经苏木精-伊红(HE)染色后,电子显微镜下观察细胞形态:阴性对照组瘤组织边界不清,细胞密集排列,异型性明显,而VES治疗组(12.5mg/kg、25mg/kg、50mg/kg)瘤组织中心及边缘可见不同程度的片状或灶性坏死,黑色素颗粒释出,散布于细胞间。 3.2透射电镜观察:在未经VES处理过的阴性对照组移植瘤B16细胞中,胞膜完整,细胞核大,不规则形,核质比大,常染色质丰富,异染色质较少,胞质中细胞器少,未见典型的黑色素小体;然而,经VES(12.5mg/kg、25mg/kg)作用后,细胞表面微绒毛减少,细胞核变小,核内异染色质增多,部分浓缩,核质比变小,可见大量典型的黑色素小体; 50mg/kgVES作用后,细胞出现不同程度凋亡改变:胞质中出现空泡,线粒体脊和膜部分或大部分融合消失,核膜局部向外呈泡状突起,核染色质高度浓缩,电子密度增高,边集于核膜下,形成新月体状,即凋亡细胞的特征性形态改变,凋亡半月,但无凋亡小体。 4免疫组化检测瘤细胞S-100、Survivin、Caspase-3蛋白表达:按照IHS值法进行免疫组化评分。VES各组Survivin、S-100蛋白表达值(IHS值)随VES浓度增大而降低, Caspase-3蛋白表达值随VES浓度增大而升高。三种蛋白各自比较,VES处理组和阴性对照组,差异均有统计学意义(p 0.05或p 0.01)。且各治疗组Survivin的表达与Caspase-3、的表达呈负相关性(r_s=-0.705, p0.01);与S-100的表达呈正相关(r_s=0.797, p0.01)。 结论: 1 VES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。 2 VES有双重作用, 12.5mg/kg、25mg/kg VES可将细胞阻滞于G0/G1期,诱导部分恶性黑色素瘤细胞分化,透射电镜观察可见大量典型的黑色素小体。50mg/kgVES可以阻滞B16细胞周期于S期,诱导凋亡,抑制肿瘤增殖。 3 VES具有抑制黑色素瘤增殖,致S-100表达减低,降低其恶性程度的作用。其机制可能是一方面下调Survivin蛋白,调节瘤细胞分化;另一方面启动Caspase-3依赖的凋亡途径。 4 VES对鼠B16黑色素瘤移植瘤生长有较显著的抑制作用,低剂量诱导分化,高剂量诱导凋亡,为临床恶性黑色素瘤的治疗和化学药物预防提供了新的思路和理论依据。
[Abstract]:Objective: To study the effects of vitamin E succinate (VES) on the proliferation, differentiation and apoptosis of melanoma cells by animal experiments. The mechanism of VES to inhibit the growth of melanoma cells was further discussed by the change of related protein expression, thus providing a new method and corresponding theoretical basis for the treatment of melanoma. Methods:40 male BALB/ c mice were randomly divided into 5 groups. 8. The murine melanoma B16 cell suspension was inoculated subcutaneously in the right back of each group of mice, each of which was 10 to 6, and a mouse melanoma graft tumor was established. Model: After the tumor-bearing of most mice, the administration was started with intraperitoneal injection: the experimental group was given the intraperitoneal injection of VES 12.5 mg/ kg,25 mg/ kg and 50 mg/ kg. The control group received 0.05 ml of sesame oil per day for two days, and the control group received 0.05 ml/ kg of sesame oil, and the administration method and time were the same. The positive control group was given a dose of 0.2 ml (80 mg/ kg) of Dacrobazine (DTIC) and 0.2 ml (80 mg/ kg) for intraperitoneal injection. Five days. After two weeks, each group of mice and the tumor cells were removed and weighed, and the tumor inhibition rate was calculated. The changes of the tumor cells after the action of the VES were observed by the microscope, and the ultrastructural changes of the tumor cells after the action of the VES were observed by the transmission electron microscope. The cell cycle distribution and the number of tumor cells in each group were examined by flow cytometry. The expression of S-100, Survivin and Caspase-3 in each group of tumor cells was detected by immunohistochemistry. Analysis. Results: 1VES inhibited the growth of mouse tumor: VES (12.5 mg/ kg,25 mg/ kg,50 mg/ kg). The inhibition rate of mice B16 transplanted tumor was 13.72%, 31.22%, 45.58, respectively. %, dose-dependent, and the tumor-inhibiting rate of the Dazapache group was 55.64%. The body weight of each group was higher than that of the control group (p0.05). The weight of the positive control group was significantly lower than that of the control group, and the difference was statistically significant compared with the negative control group. The results of flow cytometry showed that the percentage of cells in VES12.5 mg/ kg,25 mg/ kg,50 mg/ kg and 50 mg/ kg increased gradually, in dose-related, and the difference was statistically significant (p The proportion of cells in the G0/ G1 phase of the VES12.5 mg/ kg,25 mg/ kg group tumor cells increased gradually, with a statistically significant difference compared to the negative control group (p The apoptosis rate of each dose group of VES was 20.88% 0.58%, 22.71% 0.55%, 27.22% 0.59%, and the negative control group was 6.73% 0.97%, all of which were lower than that of the positive control group. Statistical significance (p.01).3 The effect of VES on the cell morphology of the tumor: 3.1 After the staining with hematoxylin-eosin (HE), the morphology of the cells was observed under the electron microscope: the tissue boundary of the negative control group was not clear, the cells were densely arranged, the heterotype was obvious, and the VES treatment group ( At 12.5 mg/ kg,25 mg/ kg,50 mg/ kg), the center and the edge of the tumor tissue were in different degrees of sheet or focal The cell membrane was intact, the nucleus was large, the irregular shape, the nuclear mass ratio was large, the euchromatin was rich, the heterochromatin was small, the organelles in the cytoplasm were few, and the typical melanin was not found. After the action of VES (12.5 mg/ kg,25 mg/ kg), the microvilli of the surface of the cell decreased, the nucleus became smaller, the heterochromatin in the nucleus increased, the concentration of the part was reduced, the nuclear mass ratio became smaller, and a large number of typical melanosomes were found; after the action of 50 mg/ kg of the VES, In the cytoplasm, vacuoles, mitochondrial ridges and membrane parts or most of the fusion disappear, and the nuclear membrane is locally expanded to form a bubble-like protrusion, the nuclear chromatin is highly concentrated, the electron density is increased, and the edge is collected under the nuclear membrane to form a crescent body, that is, the apoptotic cell The features of S-100, Survivin and Casas were detected by immunohistochemistry. e-3 protein expression: Immunohistochemistry was performed according to IHS method. The expression of Survivin and S-100 protein (IHS) in the VES group decreased with the increase of the VES concentration, Ca The expression of the spase-3 protein increased with the increase of the VES concentration. The three proteins were compared, the VES treatment group and the negative control group. The expression of Survivin in each treatment group was negatively correlated with the expression of Caspase-3 (r _ s =-0.705, p0.01); and S-100. expression Positive correlation (r _ s = 0.797, p0.01). Conclusion: 1VES versus rat B The growth of 16 melanoma xenografts has a significant inhibitory effect and has a certain dose-effect relationship. The VES has a double effect, 12.5 mg/ kg and 25 mg/ kg of VES can block the cells in G0/ G1 phase, induce partial malignant melanoma cell differentiation, and a large number of typical melanosomes can be observed by transmission electron microscopy. / kgVES can block B16 cell cycle in S phase, induce apoptosis, and inhibit tumor proliferation. VES has the effect of inhibiting the proliferation of melanoma, reducing the expression of S-100, and reducing the degree of malignancy. On the other hand, caspase-3-dependent apoptosis was initiated.4-VES had a significant inhibition on the growth of murine B16 melanoma, and the low-dose induction of differentiation was high.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.5
本文编号：2503746
[Abstract]:Objective: To study the effects of vitamin E succinate (VES) on the proliferation, differentiation and apoptosis of melanoma cells by animal experiments. The mechanism of VES to inhibit the growth of melanoma cells was further discussed by the change of related protein expression, thus providing a new method and corresponding theoretical basis for the treatment of melanoma. Methods:40 male BALB/ c mice were randomly divided into 5 groups. 8. The murine melanoma B16 cell suspension was inoculated subcutaneously in the right back of each group of mice, each of which was 10 to 6, and a mouse melanoma graft tumor was established. Model: After the tumor-bearing of most mice, the administration was started with intraperitoneal injection: the experimental group was given the intraperitoneal injection of VES 12.5 mg/ kg,25 mg/ kg and 50 mg/ kg. The control group received 0.05 ml of sesame oil per day for two days, and the control group received 0.05 ml/ kg of sesame oil, and the administration method and time were the same. The positive control group was given a dose of 0.2 ml (80 mg/ kg) of Dacrobazine (DTIC) and 0.2 ml (80 mg/ kg) for intraperitoneal injection. Five days. After two weeks, each group of mice and the tumor cells were removed and weighed, and the tumor inhibition rate was calculated. The changes of the tumor cells after the action of the VES were observed by the microscope, and the ultrastructural changes of the tumor cells after the action of the VES were observed by the transmission electron microscope. The cell cycle distribution and the number of tumor cells in each group were examined by flow cytometry. The expression of S-100, Survivin and Caspase-3 in each group of tumor cells was detected by immunohistochemistry. Analysis. Results: 1VES inhibited the growth of mouse tumor: VES (12.5 mg/ kg,25 mg/ kg,50 mg/ kg). The inhibition rate of mice B16 transplanted tumor was 13.72%, 31.22%, 45.58, respectively. %, dose-dependent, and the tumor-inhibiting rate of the Dazapache group was 55.64%. The body weight of each group was higher than that of the control group (p0.05). The weight of the positive control group was significantly lower than that of the control group, and the difference was statistically significant compared with the negative control group. The results of flow cytometry showed that the percentage of cells in VES12.5 mg/ kg,25 mg/ kg,50 mg/ kg and 50 mg/ kg increased gradually, in dose-related, and the difference was statistically significant (p The proportion of cells in the G0/ G1 phase of the VES12.5 mg/ kg,25 mg/ kg group tumor cells increased gradually, with a statistically significant difference compared to the negative control group (p The apoptosis rate of each dose group of VES was 20.88% 0.58%, 22.71% 0.55%, 27.22% 0.59%, and the negative control group was 6.73% 0.97%, all of which were lower than that of the positive control group. Statistical significance (p.01).3 The effect of VES on the cell morphology of the tumor: 3.1 After the staining with hematoxylin-eosin (HE), the morphology of the cells was observed under the electron microscope: the tissue boundary of the negative control group was not clear, the cells were densely arranged, the heterotype was obvious, and the VES treatment group ( At 12.5 mg/ kg,25 mg/ kg,50 mg/ kg), the center and the edge of the tumor tissue were in different degrees of sheet or focal The cell membrane was intact, the nucleus was large, the irregular shape, the nuclear mass ratio was large, the euchromatin was rich, the heterochromatin was small, the organelles in the cytoplasm were few, and the typical melanin was not found. After the action of VES (12.5 mg/ kg,25 mg/ kg), the microvilli of the surface of the cell decreased, the nucleus became smaller, the heterochromatin in the nucleus increased, the concentration of the part was reduced, the nuclear mass ratio became smaller, and a large number of typical melanosomes were found; after the action of 50 mg/ kg of the VES, In the cytoplasm, vacuoles, mitochondrial ridges and membrane parts or most of the fusion disappear, and the nuclear membrane is locally expanded to form a bubble-like protrusion, the nuclear chromatin is highly concentrated, the electron density is increased, and the edge is collected under the nuclear membrane to form a crescent body, that is, the apoptotic cell The features of S-100, Survivin and Casas were detected by immunohistochemistry. e-3 protein expression: Immunohistochemistry was performed according to IHS method. The expression of Survivin and S-100 protein (IHS) in the VES group decreased with the increase of the VES concentration, Ca The expression of the spase-3 protein increased with the increase of the VES concentration. The three proteins were compared, the VES treatment group and the negative control group. The expression of Survivin in each treatment group was negatively correlated with the expression of Caspase-3 (r _ s =-0.705, p0.01); and S-100. expression Positive correlation (r _ s = 0.797, p0.01). Conclusion: 1VES versus rat B The growth of 16 melanoma xenografts has a significant inhibitory effect and has a certain dose-effect relationship. The VES has a double effect, 12.5 mg/ kg and 25 mg/ kg of VES can block the cells in G0/ G1 phase, induce partial malignant melanoma cell differentiation, and a large number of typical melanosomes can be observed by transmission electron microscopy. / kgVES can block B16 cell cycle in S phase, induce apoptosis, and inhibit tumor proliferation. VES has the effect of inhibiting the proliferation of melanoma, reducing the expression of S-100, and reducing the degree of malignancy. On the other hand, caspase-3-dependent apoptosis was initiated.4-VES had a significant inhibition on the growth of murine B16 melanoma, and the low-dose induction of differentiation was high.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.5
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