維生素E琥珀酸酯對黑色素瘤B16荷瘤小鼠抑瘤作用及其機(jī)制的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2019-06-21 03:20
【摘要】：目的:通過動(dòng)物實(shí)驗(yàn),研究維生素E琥珀酸酯(vitamin E succinate,VES;α-tocopheryl succinate,α-TOS)對黑色素瘤細(xì)胞的增殖、分化、凋亡的影響。通過相關(guān)蛋白表達(dá)的變化,進(jìn)一步探討VES抑制黑色素瘤細(xì)胞生長的作用機(jī)制,從而為黑色素瘤治療提供新的方法及相應(yīng)的理論依據(jù)。 方法:將40只雄性BALB/c小鼠隨機(jī)分為5組,每組8只。將鼠黑色素瘤B16細(xì)胞懸液接種于各組小鼠右側(cè)背部皮下,每只1×10~6個(gè),建立小鼠黑色素瘤移植瘤模型。當(dāng)多數(shù)小鼠荷瘤后開始給藥,采用腹腔注射給藥方式:實(shí)驗(yàn)組分別給予VES 12.5mg/kg、25mg/kg、50mg/kg腹腔注射,每天一次,連續(xù)五天,休兩天,共兩周;對照組給予芝麻油0.05ml/只,給藥方法及時(shí)間同實(shí)驗(yàn)組;陽性對照組給予達(dá)卡巴嗪(Dacarbazine, DTIC)0.2ml (80mg/kg)腹腔注射,每天一次,連續(xù)五天。兩周后,頸椎脫臼處死各組小鼠,剝瘤稱重,計(jì)算抑瘤率;顯微鏡觀察VES作用后的瘤細(xì)胞大體形態(tài)變化,透射電鏡觀察VES作用后的瘤細(xì)胞超微結(jié)構(gòu)變化;流式細(xì)胞術(shù)檢測各組瘤細(xì)胞周期分布及凋亡率;免疫組化法檢測各組瘤細(xì)胞S-100,Survivin,Caspase-3的蛋白表達(dá)情況;運(yùn)用SPSS 13.0統(tǒng)計(jì)學(xué)軟件進(jìn)行數(shù)據(jù)處理,統(tǒng)計(jì)分析。 結(jié)果: 1 VES可抑制小鼠腫瘤生長:VES(12.5mg/kg、25mg/kg、50mg/kg)各劑量組對小鼠B16移植瘤的抑制率分別為:13.72%、31.22%、45.58%,呈劑量依賴性。達(dá)卡巴嗪組抑瘤率為55.64%,高于VES各組。各組小鼠處死前的體重比較:VES各處理組與對照組相比,差異無統(tǒng)計(jì)學(xué)意義(p0.05);陽性對照組體重有較明顯下降,與陰性對照組相比,差異有統(tǒng)計(jì)學(xué)意義(p 0.01)。 2 VES可阻滯瘤細(xì)胞周期、誘導(dǎo)分化和凋亡:流式細(xì)胞分析儀檢測結(jié)果顯示VES12.5mg/kg、25mg/kg、50mg/kg劑量組的細(xì)胞,S期比例逐漸增加,呈劑量相關(guān)性,與陰性對照組相比,差異有統(tǒng)計(jì)學(xué)意義(p0.05或p 0.01)。VES12.5mg/kg、25mg/kg劑量組瘤細(xì)胞的G0/G1期細(xì)胞比例逐漸增加,與陰性對照組相比,差異具有統(tǒng)計(jì)學(xué)意義(p 0.05或p 0.01)。VES各劑量組瘤細(xì)胞的凋亡率分別為:20.88±0.58%、22.71±0.55 %、27.22±0.59%,陰性對照組為6.73±0.97%,均低于陽性對照組凋亡率30.95±0.52%,VES各組與對照組、陽性對照組相比,差異均有統(tǒng)計(jì)學(xué)意義(p 0.01)。 3 VES對瘤組織細(xì)胞形態(tài)的影響: 3.1經(jīng)蘇木精-伊紅(HE)染色后,電子顯微鏡下觀察細(xì)胞形態(tài):陰性對照組瘤組織邊界不清,細(xì)胞密集排列,異型性明顯,而VES治療組(12.5mg/kg、25mg/kg、50mg/kg)瘤組織中心及邊緣可見不同程度的片狀或灶性壞死,黑色素顆粒釋出,散布于細(xì)胞間。 3.2透射電鏡觀察:在未經(jīng)VES處理過的陰性對照組移植瘤B16細(xì)胞中,胞膜完整,細(xì)胞核大,不規(guī)則形,核質(zhì)比大,常染色質(zhì)豐富,異染色質(zhì)較少,胞質(zhì)中細(xì)胞器少,未見典型的黑色素小體;然而,經(jīng)VES(12.5mg/kg、25mg/kg)作用后,細(xì)胞表面微絨毛減少,細(xì)胞核變小,核內(nèi)異染色質(zhì)增多,部分濃縮,核質(zhì)比變小,可見大量典型的黑色素小體; 50mg/kgVES作用后,細(xì)胞出現(xiàn)不同程度凋亡改變:胞質(zhì)中出現(xiàn)空泡,線粒體脊和膜部分或大部分融合消失,核膜局部向外呈泡狀突起,核染色質(zhì)高度濃縮,電子密度增高,邊集于核膜下,形成新月體狀,即凋亡細(xì)胞的特征性形態(tài)改變,凋亡半月,但無凋亡小體。 4免疫組化檢測瘤細(xì)胞S-100、Survivin、Caspase-3蛋白表達(dá):按照IHS值法進(jìn)行免疫組化評分。VES各組Survivin、S-100蛋白表達(dá)值(IHS值)隨VES濃度增大而降低, Caspase-3蛋白表達(dá)值隨VES濃度增大而升高。三種蛋白各自比較,VES處理組和陰性對照組,差異均有統(tǒng)計(jì)學(xué)意義(p 0.05或p 0.01)。且各治療組Survivin的表達(dá)與Caspase-3、的表達(dá)呈負(fù)相關(guān)性(r_s=-0.705, p0.01);與S-100的表達(dá)呈正相關(guān)(r_s=0.797, p0.01)。 結(jié)論: 1 VES對鼠B16黑色素瘤移植瘤生長有顯著的抑制作用,且呈一定的量效關(guān)系。 2 VES有雙重作用, 12.5mg/kg、25mg/kg VES可將細(xì)胞阻滯于G0/G1期,誘導(dǎo)部分惡性黑色素瘤細(xì)胞分化,透射電鏡觀察可見大量典型的黑色素小體。50mg/kgVES可以阻滯B16細(xì)胞周期于S期,誘導(dǎo)凋亡,抑制腫瘤增殖。 3 VES具有抑制黑色素瘤增殖,致S-100表達(dá)減低,降低其惡性程度的作用。其機(jī)制可能是一方面下調(diào)Survivin蛋白,調(diào)節(jié)瘤細(xì)胞分化;另一方面啟動(dòng)Caspase-3依賴的凋亡途徑。 4 VES對鼠B16黑色素瘤移植瘤生長有較顯著的抑制作用,低劑量誘導(dǎo)分化,高劑量誘導(dǎo)凋亡,為臨床惡性黑色素瘤的治療和化學(xué)藥物預(yù)防提供了新的思路和理論依據(jù)。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R739.5
本文編號：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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R739.5
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