【摘要】： 目的:通過體外實(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)胞增殖、分化、細(xì)胞周期和相關(guān)蛋白表達(dá)以及黑色素小體改變的影響并進(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:體外培養(yǎng)小鼠黑色素瘤B16細(xì)胞,采用四甲基偶氮唑藍(lán)(MTT)比色法檢測不同濃度VES作用于B16細(xì)胞24h、48h、72h后對其增殖抑制情況及經(jīng)瑞-吉染色后光學(xué)顯微鏡觀察細(xì)胞形態(tài)變化并篩選出適合的藥物濃度進(jìn)行后續(xù)試驗(yàn)。采用流式細(xì)胞術(shù)(FCM)測定VES對小鼠黑色素瘤B16細(xì)胞作用48h后的細(xì)胞周期分布和凋亡率;應(yīng)用透射電鏡觀察黑色素小體;NaOH裂解法測定黑色素含量變化;采用流式細(xì)胞術(shù)檢測VES處理B16細(xì)胞48小時后細(xì)胞周期蛋白cyclinD1和P21蛋白的表達(dá)變化;免疫細(xì)胞化學(xué)法檢測各組腫瘤細(xì)胞cyclinD1和P21蛋白表達(dá)情況。從而初步探討VES對B16細(xì)胞的誘導(dǎo)分化作用及相應(yīng)機(jī)制。 結(jié)果: 1 VES抑制B16細(xì)胞增殖: MTT法結(jié)果顯示,5μg/ml、10μg/ml、20μg/ml VES處理細(xì)胞24～72h后,各處理組OD值均有不同程度下降,與對照組相比,有顯著性差異(P0.01),各實(shí)驗(yàn)組間比較,亦有顯著性差異(P0.01)。且隨藥物濃度的增大、作用時間的延長,VES各組OD值逐漸下降,抑制率升高,即VES對B16細(xì)胞的增殖抑制作用呈明顯的時間-劑量依賴效應(yīng)。 2 VES可誘導(dǎo)B16細(xì)胞周期阻滯和凋亡: 流式細(xì)胞儀檢測結(jié)果顯示,5、10、20μg/ml VES作用于B16細(xì)胞48h后,隨藥物濃度增加,G0/G1期細(xì)胞比例增加,S期比例下降,增殖指數(shù)PI明顯下降(Ρ0.01),證明VES可阻滯B16細(xì)胞周期進(jìn)程于G0/G1期,且該作用呈劑量依賴性。VES各劑量組凋亡率分別為0.42±0.14%、0.72±0.18%、1.06±0.72%。對照組凋亡率為0.25±0.04%。因凋亡率均低于5%,且無亞二倍體凋亡峰出現(xiàn),故在5-20μg/mlVES作用于B16細(xì)胞后,無凋亡出現(xiàn),證明在此范圍內(nèi)VES不能誘導(dǎo)鼠黑色素瘤B16細(xì)胞凋亡。 3 VES對B16細(xì)胞形態(tài)的影響及電鏡觀察黑色素小體: 經(jīng)瑞-吉染色后光鏡下觀察細(xì)胞形態(tài)發(fā)現(xiàn):正常的黑色素瘤B16細(xì)胞呈貼壁、多層生長,形態(tài)為圓形、橢圓形或多邊形,生長不規(guī)則。而VES作用于B16細(xì)胞后,細(xì)胞呈一定的極性生長,平行排列,不重疊,隨著時間延長,多數(shù)細(xì)胞體積變大,延長,細(xì)胞呈網(wǎng)狀排列,具有樹突狀結(jié)構(gòu),生長緩慢,細(xì)胞數(shù)明顯變少,此形態(tài)變化于作用48h后較為明顯。 電鏡結(jié)果顯示:在未經(jīng)VES處理過的B16細(xì)胞中,胞膜完整,表面有較多微絨毛,細(xì)胞核大,圓形,核漿比較大,常染色質(zhì)豐富,異染色質(zhì)較少,胞質(zhì)內(nèi)可見游離核糖體,其他細(xì)胞器少,未見典型的黑色素小體;然而,經(jīng)VES(10、20μg/ml)作用后,胞膜表面微絨毛較少,細(xì)胞核變小,核內(nèi)異染色質(zhì)增多,核漿比變小,細(xì)胞器豐富,可見大量典型的黑色素小體,未見凋亡形態(tài)的細(xì)胞,這與以上凋亡率的檢測結(jié)果一致。 4黑色素含量的測定: 黑色素瘤細(xì)胞與正常黑色素細(xì)胞相比,黑色素合成能力低下。當(dāng)被分化誘導(dǎo)劑誘導(dǎo)分化時,黑色素生成能力明顯增加,有效的分化誘導(dǎo)作用應(yīng)使黑色素含量增加2倍以上。本實(shí)驗(yàn)結(jié)果顯示,5μg/ml,10μg/ml,20μg/ml的VES作用于B16細(xì)胞48h后,黑色素含量與對照組比較,分別是對照組的1.006±0.2410倍,1.813±0.4380倍,3.654±0.7000倍。經(jīng)統(tǒng)計,10μg/ml,20μg/ml組較未加藥組有極顯著性差異(P0.01),其中20μg/ml濃度組是對照組黑色素含量的3.654±0.7000倍2倍,故20μg/ml VES有效地誘導(dǎo)了B16細(xì)胞的分化。 5流式細(xì)胞儀檢測B16細(xì)胞cyclinD1、P21蛋白表達(dá): 5、10、20μg/ml VES作用于B16細(xì)胞48h后,發(fā)現(xiàn)cyclinD1蛋白的熒光指數(shù)(FI)隨VES濃度增大而逐漸減少,P21蛋白的FI值則隨藥物濃度的增大而逐漸增大。對于每一種蛋白,其處理組和對照組FI值比較,均有極顯著性差異(P0.01)。 6免疫細(xì)胞化學(xué)法檢測腫瘤細(xì)胞cyclinD1、P21蛋白表達(dá)并進(jìn)行評分: 未加藥組,cyclinD1蛋白在鼠黑色素瘤B16細(xì)胞的細(xì)胞漿和核均有表達(dá),以胞漿為主,強(qiáng)度成強(qiáng)陽性;VES處理后,隨著VES濃度增加,蛋白在核、漿表達(dá)均逐漸減少,以核減少為主;評分統(tǒng)計后VES各組cyclinD1蛋白表達(dá)值隨VES濃度增加而逐漸減小。未加藥組,P21蛋白在鼠黑色素瘤B16細(xì)胞的細(xì)胞漿和核極少量表達(dá),強(qiáng)度較弱;VES處理后,隨著VES濃度的增加,蛋白在核漿表達(dá)逐漸增加,以核增加為主;評分統(tǒng)計后P21蛋白表達(dá)值隨VES濃度增加而逐漸增大。兩種蛋白各自比較,VES處理組和陰性對照組,均有顯著性差異(P0.01)。 結(jié)論: 1、在不表現(xiàn)明顯的細(xì)胞凋亡現(xiàn)象的劑量范圍內(nèi),VES可明顯抑制黑色素瘤B16細(xì)胞增殖,并呈劑量-時間依賴性。 2、VES可通過對黑色素瘤細(xì)胞周期阻滯誘導(dǎo)分化,將細(xì)胞阻滯于G0/G1期,該作用呈劑量依賴性,并以48小時作用最明顯。 3、VES對B16細(xì)胞有較強(qiáng)的分化誘導(dǎo)能力,形態(tài)學(xué)表現(xiàn)為生長緩慢,細(xì)胞連成網(wǎng)狀結(jié)構(gòu),電鏡可見VES處理過的B16細(xì)胞內(nèi)含大量典型黑色素小體。功能上表現(xiàn): VES作用于B16細(xì)胞后,黑色素含量明顯增加,尤其是20μg/ml VES。 4、VES具有誘導(dǎo)黑色素瘤細(xì)胞分化作用,其機(jī)制可能與其下調(diào)cyclinD1蛋白,上調(diào)P21蛋白的表達(dá)有關(guān)。 5、本實(shí)驗(yàn)結(jié)果顯示:VES具有抑制黑色素瘤細(xì)胞增殖、阻滯細(xì)胞周期、誘導(dǎo)分化的作用,為其用于黑色素瘤治療提供了新的思路和理論依據(jù)。
[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.
【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】：2010
【分類號】：R739.5

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 姬巧霞;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[D];河北醫(yī)科大學(xué);2011年

，

本文編號：2225181