斑蝥酸钠维生素B6注射液诱导人骨肉瘤细胞MG-63凋亡及其机制的研究
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摘要
目的:以人成骨肉瘤细胞MG-63为研究对象,探讨斑蝥酸钠维生素B6注射液诱导人成骨肉瘤细胞株MG-63细胞凋亡的作用及其分子机制,为骨肉瘤的治疗提供实验基础。
方法:将人骨肉瘤细胞株MG-63细胞于37℃,5%CO2培养箱内按常规条件进行传代培养,待细胞进入对数生长期后用胰蛋白酶消化成单细胞悬液后进行操作。采用以下实验:
1采用四唑盐(MTT)比色法检测细胞增殖,将骨肉瘤细胞接种于96孔培养板上,贴壁后分别加入不同浓度的斑蝥酸钠维生素B6注射液,置于培养箱培养24、48、72小时后分别加入MTT10微升,孵育4小时,弃上清液加入DMSO振荡5min使结晶物充分溶解,在酶联免疫检测分析仪上测定OD492的值,根据公式计算抑制率。
2形态学观察细胞接种贴壁后分别加入不同浓度的药物后分别培养24、48、72小时后在倒置显微镜下观察细胞形态改变并照相。
3流式细胞术(FCM)检测细胞凋亡、细胞周期分布:收集不同浓度斑蝥酸钠维生素B6注射液处理24小时后培养细胞的MG-63及对照组细胞,冷PBS漂洗。预冷70%乙醇4℃,固定,上机检测前离心去固定夜,0.5%胃蛋白酶消化,碘化丙锭室温避光染色,流式细胞仪上机检测,应用软件进行分析。
4透射电子显微镜观察:取经不同浓度药物处理24小时的细胞,用体积分数为2.5%戊二醛4℃固定,用10 g/L锇酸后固定,丙酮系列脱水,最后用Epon812环氧树脂包埋,超薄切片机切片,醋酸铀与柠檬酸铅双重染色,电镜下观察细胞超微结构并拍照。
5反转录聚合酶链反应(RT-PCR)检测Survivin mRNA的表达:实验组和对照组细胞进行培养,收集细胞,提取细胞总RNA。采用一步法进行反转录和扩增。将RT-PCR产物用1.5%琼脂糖凝胶电泳后,应用凝胶成像系统及凝胶分析软件对电泳条带进行成像分析,用Survivin吸光度与β-actin吸光度的比值代表Survivin的相对含量。
结果: 1用不同浓度的斑蝥酸钠维生素B6注射液作用于MG-63细胞后结果显示斑蝥酸钠维生素B6注射液能明显抑制细胞的增殖,并呈剂量和时间依赖性。抑制率最高可达到(68.56±5.21)%。2倒置显微镜下观察对照组细胞呈贴壁生长,生长状态良好,细胞透明,呈长梭形,颗粒较少,细胞间界限清楚,细胞核隐约可见。经斑蝥酸钠维生素B6注射液处理组细胞,随斑蝥酸钠维生素B6注射液浓度的增加和作用时间的延长,细胞逐渐变圆,呈漂浮状态生长的细胞数量明显增加,细胞内部结构受到破坏,存活细胞显著减少,至5.0μg/ml时大部分细胞裂解,可见多量细胞碎片。透视电镜观察可见典型的细胞凋亡形态改变:细胞皱缩,胞膜结构保持完整,胞核固缩,核碎裂,染色质浓缩边集,形成新月形胞核,胞浆空泡化明显,细胞表面有较多浓缩的胞质突起。3经药物处理MG-63细胞24小时后,流式细胞仪分析结果显示,MG-63细胞G0/G1期细胞逐渐增多,S期和G2/M期的细胞逐渐减少。经2.5μg/ml斑蝥酸钠维生素B6注射液作用24h后MG-63细胞G0/G1期细胞由对照组的(51.08±2.42)%升高至(75.46±1.72)%,S期、G2/M期的细胞比例减少。FCM发现经斑蝥酸钠维生素B6注射液处理细胞24h后,凋亡率随药物浓度的增加逐渐增高。对照组和药物浓度为2.5μg/ml时凋亡率分别为(1.09±0.18)%和(9.60±0.65)%,两者有显著性差异(P<0.01)差异,经0.5μg/ml、1.0μg/ml、2.5μg/ml药物浓度处理MG-63细胞24小时后G0/G1期前出现凋亡峰。RT-PCR结果表明:经0.5μg/ml、1.0μg/ml、2.5μg/ml的斑蝥酸钠维生素B6注射液处理细胞24h后,随着药物浓度的增加, Survivin mRNA表达逐渐降低。
结论:斑蝥酸钠维生素B6注射液能明显抑制MG-63细胞增殖,使细胞周期阻滞在G0/G1期,并进一步诱导其凋亡。其机制可能与抑制Survivin的表达有关。确切机制有待于进一步研究。
Objective:this study was designed to investigate human osteosarcoma MG-63 cells apoptosis induced by Disodium Cantharidinate and Vitamin B6 Injection and its molecular mechanisms.It may provid experimental foundation for osteosarcoma.
Methods: osteosarcoma MG-63 cells were cultured in the environment of 37℃5% CO2 with modified Eagle medium (MEM)medium.The logarithmically growing MG-63 cells were used.
1 The suppressive effects of Disodium Cantharidinate and Vitamin B6 Injection on the proliferaition of MG-63 cells were evaluated in vitro by MTT colorimetricassay. After cultured for several generations,the cells were digested and transferred into 96-well culture plates. after adherenced,add to Disodium Cantharidinate and Vitamin B6 Injection,culture 24,48,72 hours,add to MTT 10ul,incubation 4 hours,abandon to supernate fluid,add to DMSO, to swing for 5 min,determin OD492 with analysator,according to formula calculate inhibition ratio.
2 Using light microscope observe the change of morphology: MG-63 cells were treated with different concentrations of Disodium Cantharidinate and Vitamin B6 Injection for 24,48,72 hours,the change of morphology was observed by inverted phase contrast microscope in different times and photographed.
3 Flow cytometric analysis:MG-63 cells were treated with Disodium Cantharidinate and Vitamin B6 Injection for 24h.Experimental group and control group cells were harvested and washed twice with PBS.Cells were fixed with ice cold 70% ethanol at 4°C. Precipitates were digested with 0.5% pepsin after centrifugation.And then cells were resuspended in 0.5 mL propidium iodide(PI)/RNase A solution.Cells were incubated in the dark at room temperature for 15 min.The fluorescence emission of stained cells was measured with a flow cytometer.Data were analyzed with Multipcycle software.
4 Morphology analysis by transmission electron microscopy: MG-63 cells treated with different concentrations of Disodium Cantharidinate and Vitamin B6 Injection for 24 h, and pre-fixed with 2.5% glutaraldehyde at 4°C.The cells were postfixed for 1 h with 1% osmium tetroxide,then dehydrated through a graded ethanol series,and embedded in Epon 812.The ultrastructure of cells was analyzed in ultrathin sections in a transmission electron microscope after the sections were stained with uranylacetate and lead citrate.
5 Reverse transcription polymerase chain reaction (RT-PCR) were used to observed Survivin mRNA expression: The cells were collected after 24h of culture.Total RNA of MG-63 cells was extracted.Inverse transcription and amplification of Survivin mRNA were done using one-step method.The production was detected using electrophoresis method and then was analyzed using imaging system and analysis software.The relative amount of Survivin mRNA was accessed by the ratio of Survivin absorption factor andβ-actin absorption factor.
Results: 1.MG-63 cells were treated with at various Disodium Cantharidinate and Vitamin B6 Injection concentra- tions for 24h,48h,and 72h respectively,the growth of cells was inhibited significantly in a time and dose-dependent fashion. The biggest inhibitory rate was (68.56±5.21)%.
2 Inverted phase contrast microscope observed,comparing with control group cells,the growth of drug-treated group cells were inhibited,cell granulations were increased and thicken,some cells became round and Suspended in the medium,internal structure of cells were demolished, the cells of survival notably decreased.When the concentration reached to 5.0μg/ml,most of the cells lysised and could observe numerous cell debris.Electron microscope revealed the characteristic apoptosis aletrations,shrinking cellular morphology,integrity cell membr- ane,karyopycnosis,fragment of nucleus,chromatin condensation and margination,crescent nucleus,cytoplasmiec vacuoles.
3 After cells were exposed to various Disodium Cantharidinate and Vitamin B6 Injection concentrations for 24h,The quantity of treated cells in G0/G1 phase increased but that in S、G2/M phase decreased. After cells were exposed to the concentration of 2.5μg/ml for 24h , MG-63 cells rates in G0/G1 phase were 75.46 ±1.72% whlie MG-63 cells rates in G0/G1 phase were 51.08±2.42% in control group. But that in S、G2/M phase decreased. The analysis of cellular DNA content by FCM showed that the apoptosis rates become higer with drug concentration increased.the apoptosis rates was 9.60±0.65%,which was significantly higher than control 1.09±0.18% (P<0.01).The analysis of cellular DNA content by FCM showed that there was a sub-G0/G1 peak in the graph of drug-treated groups(0.5μg/ml、1.0μg/ml、2.5μg/ml).That was a typical apoptotic peak,which was not shown in the graph of control groups. The result of RT-PCR showed that expression of Survivin mRNA was down-regulated.
Conclusion:Disodium Cantharidinate and Vitamin B6 Injection can inhibit proliferation and induce apoptosis of MG-63 cells.Its possible molecular mechanisms might be related to modulation the expression of Survivin.The Precise mechanisms should be investigated further.
方法:将人骨肉瘤细胞株MG-63细胞于37℃,5%CO2培养箱内按常规条件进行传代培养,待细胞进入对数生长期后用胰蛋白酶消化成单细胞悬液后进行操作。采用以下实验:
1采用四唑盐(MTT)比色法检测细胞增殖,将骨肉瘤细胞接种于96孔培养板上,贴壁后分别加入不同浓度的斑蝥酸钠维生素B6注射液,置于培养箱培养24、48、72小时后分别加入MTT10微升,孵育4小时,弃上清液加入DMSO振荡5min使结晶物充分溶解,在酶联免疫检测分析仪上测定OD492的值,根据公式计算抑制率。
2形态学观察细胞接种贴壁后分别加入不同浓度的药物后分别培养24、48、72小时后在倒置显微镜下观察细胞形态改变并照相。
3流式细胞术(FCM)检测细胞凋亡、细胞周期分布:收集不同浓度斑蝥酸钠维生素B6注射液处理24小时后培养细胞的MG-63及对照组细胞,冷PBS漂洗。预冷70%乙醇4℃,固定,上机检测前离心去固定夜,0.5%胃蛋白酶消化,碘化丙锭室温避光染色,流式细胞仪上机检测,应用软件进行分析。
4透射电子显微镜观察:取经不同浓度药物处理24小时的细胞,用体积分数为2.5%戊二醛4℃固定,用10 g/L锇酸后固定,丙酮系列脱水,最后用Epon812环氧树脂包埋,超薄切片机切片,醋酸铀与柠檬酸铅双重染色,电镜下观察细胞超微结构并拍照。
5反转录聚合酶链反应(RT-PCR)检测Survivin mRNA的表达:实验组和对照组细胞进行培养,收集细胞,提取细胞总RNA。采用一步法进行反转录和扩增。将RT-PCR产物用1.5%琼脂糖凝胶电泳后,应用凝胶成像系统及凝胶分析软件对电泳条带进行成像分析,用Survivin吸光度与β-actin吸光度的比值代表Survivin的相对含量。
结果: 1用不同浓度的斑蝥酸钠维生素B6注射液作用于MG-63细胞后结果显示斑蝥酸钠维生素B6注射液能明显抑制细胞的增殖,并呈剂量和时间依赖性。抑制率最高可达到(68.56±5.21)%。2倒置显微镜下观察对照组细胞呈贴壁生长,生长状态良好,细胞透明,呈长梭形,颗粒较少,细胞间界限清楚,细胞核隐约可见。经斑蝥酸钠维生素B6注射液处理组细胞,随斑蝥酸钠维生素B6注射液浓度的增加和作用时间的延长,细胞逐渐变圆,呈漂浮状态生长的细胞数量明显增加,细胞内部结构受到破坏,存活细胞显著减少,至5.0μg/ml时大部分细胞裂解,可见多量细胞碎片。透视电镜观察可见典型的细胞凋亡形态改变:细胞皱缩,胞膜结构保持完整,胞核固缩,核碎裂,染色质浓缩边集,形成新月形胞核,胞浆空泡化明显,细胞表面有较多浓缩的胞质突起。3经药物处理MG-63细胞24小时后,流式细胞仪分析结果显示,MG-63细胞G0/G1期细胞逐渐增多,S期和G2/M期的细胞逐渐减少。经2.5μg/ml斑蝥酸钠维生素B6注射液作用24h后MG-63细胞G0/G1期细胞由对照组的(51.08±2.42)%升高至(75.46±1.72)%,S期、G2/M期的细胞比例减少。FCM发现经斑蝥酸钠维生素B6注射液处理细胞24h后,凋亡率随药物浓度的增加逐渐增高。对照组和药物浓度为2.5μg/ml时凋亡率分别为(1.09±0.18)%和(9.60±0.65)%,两者有显著性差异(P<0.01)差异,经0.5μg/ml、1.0μg/ml、2.5μg/ml药物浓度处理MG-63细胞24小时后G0/G1期前出现凋亡峰。RT-PCR结果表明:经0.5μg/ml、1.0μg/ml、2.5μg/ml的斑蝥酸钠维生素B6注射液处理细胞24h后,随着药物浓度的增加, Survivin mRNA表达逐渐降低。
结论:斑蝥酸钠维生素B6注射液能明显抑制MG-63细胞增殖,使细胞周期阻滞在G0/G1期,并进一步诱导其凋亡。其机制可能与抑制Survivin的表达有关。确切机制有待于进一步研究。
Objective:this study was designed to investigate human osteosarcoma MG-63 cells apoptosis induced by Disodium Cantharidinate and Vitamin B6 Injection and its molecular mechanisms.It may provid experimental foundation for osteosarcoma.
Methods: osteosarcoma MG-63 cells were cultured in the environment of 37℃5% CO2 with modified Eagle medium (MEM)medium.The logarithmically growing MG-63 cells were used.
1 The suppressive effects of Disodium Cantharidinate and Vitamin B6 Injection on the proliferaition of MG-63 cells were evaluated in vitro by MTT colorimetricassay. After cultured for several generations,the cells were digested and transferred into 96-well culture plates. after adherenced,add to Disodium Cantharidinate and Vitamin B6 Injection,culture 24,48,72 hours,add to MTT 10ul,incubation 4 hours,abandon to supernate fluid,add to DMSO, to swing for 5 min,determin OD492 with analysator,according to formula calculate inhibition ratio.
2 Using light microscope observe the change of morphology: MG-63 cells were treated with different concentrations of Disodium Cantharidinate and Vitamin B6 Injection for 24,48,72 hours,the change of morphology was observed by inverted phase contrast microscope in different times and photographed.
3 Flow cytometric analysis:MG-63 cells were treated with Disodium Cantharidinate and Vitamin B6 Injection for 24h.Experimental group and control group cells were harvested and washed twice with PBS.Cells were fixed with ice cold 70% ethanol at 4°C. Precipitates were digested with 0.5% pepsin after centrifugation.And then cells were resuspended in 0.5 mL propidium iodide(PI)/RNase A solution.Cells were incubated in the dark at room temperature for 15 min.The fluorescence emission of stained cells was measured with a flow cytometer.Data were analyzed with Multipcycle software.
4 Morphology analysis by transmission electron microscopy: MG-63 cells treated with different concentrations of Disodium Cantharidinate and Vitamin B6 Injection for 24 h, and pre-fixed with 2.5% glutaraldehyde at 4°C.The cells were postfixed for 1 h with 1% osmium tetroxide,then dehydrated through a graded ethanol series,and embedded in Epon 812.The ultrastructure of cells was analyzed in ultrathin sections in a transmission electron microscope after the sections were stained with uranylacetate and lead citrate.
5 Reverse transcription polymerase chain reaction (RT-PCR) were used to observed Survivin mRNA expression: The cells were collected after 24h of culture.Total RNA of MG-63 cells was extracted.Inverse transcription and amplification of Survivin mRNA were done using one-step method.The production was detected using electrophoresis method and then was analyzed using imaging system and analysis software.The relative amount of Survivin mRNA was accessed by the ratio of Survivin absorption factor andβ-actin absorption factor.
Results: 1.MG-63 cells were treated with at various Disodium Cantharidinate and Vitamin B6 Injection concentra- tions for 24h,48h,and 72h respectively,the growth of cells was inhibited significantly in a time and dose-dependent fashion. The biggest inhibitory rate was (68.56±5.21)%.
2 Inverted phase contrast microscope observed,comparing with control group cells,the growth of drug-treated group cells were inhibited,cell granulations were increased and thicken,some cells became round and Suspended in the medium,internal structure of cells were demolished, the cells of survival notably decreased.When the concentration reached to 5.0μg/ml,most of the cells lysised and could observe numerous cell debris.Electron microscope revealed the characteristic apoptosis aletrations,shrinking cellular morphology,integrity cell membr- ane,karyopycnosis,fragment of nucleus,chromatin condensation and margination,crescent nucleus,cytoplasmiec vacuoles.
3 After cells were exposed to various Disodium Cantharidinate and Vitamin B6 Injection concentrations for 24h,The quantity of treated cells in G0/G1 phase increased but that in S、G2/M phase decreased. After cells were exposed to the concentration of 2.5μg/ml for 24h , MG-63 cells rates in G0/G1 phase were 75.46 ±1.72% whlie MG-63 cells rates in G0/G1 phase were 51.08±2.42% in control group. But that in S、G2/M phase decreased. The analysis of cellular DNA content by FCM showed that the apoptosis rates become higer with drug concentration increased.the apoptosis rates was 9.60±0.65%,which was significantly higher than control 1.09±0.18% (P<0.01).The analysis of cellular DNA content by FCM showed that there was a sub-G0/G1 peak in the graph of drug-treated groups(0.5μg/ml、1.0μg/ml、2.5μg/ml).That was a typical apoptotic peak,which was not shown in the graph of control groups. The result of RT-PCR showed that expression of Survivin mRNA was down-regulated.
Conclusion:Disodium Cantharidinate and Vitamin B6 Injection can inhibit proliferation and induce apoptosis of MG-63 cells.Its possible molecular mechanisms might be related to modulation the expression of Survivin.The Precise mechanisms should be investigated further.
引文
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5 Chen YN, Chen JC, Yin SC, et al. Effector mechanisms of norcantharidin-induced mitotic arrest and apoptosis in human hepatoma cells. Int J Cancer , 2002, 100(2):158-165
6 Cao JH, Xu B, Li M, et al.Effect of norcantharidin's derivative Nd3 on proliferation of human ovarian cancer cell line SKOV3 and its possible mechanisms. Ai Zheng, 2007,26(4): 361-366
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8 Fan YZ, Fu JY, Zhao ZM, et al. Effect of norcantharidin on proliferation and invasion of human gallbladder carcinoma GBC-SD cells. World J Gast roenterol, 2005, 11(16): 2431-2437
9 Zhang JP, Ying K, Xiao ZY, et a1.Analysis of gene expression profiles in human HL-60 cell exposed to cantharidin using cDNA microarray.Int J Cancer, 2004, 108(2):212-218
10刘秀芳,王炳胜,丁瑞亮,等.奇宁注射液联合放疗化疗治疗中晚期非小细胞肺癌50例.中医杂志, 2003 , 44 (1): 50-50
11张斌.艾易舒注射液联合动脉栓塞化疗治疗肝细胞性肝癌的临床观察.中国医药导报,2007,4(26):48-49
12刘思海,冯雯,朱瑞霞,等.斑蝥酸钠维生素B6注射液联合草酸铂与替加氟持续泵人治疗晚期胃癌的比较研究.实用中西医结合临床.2008, 8(4):3-4,17
13周文丽,考军,于长青,等.吡柔比星+泰素帝联合奇宁注射液治疗晚期乳腺癌临床观察.肿瘤研究与临床, 2005,
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16 Hill TA, Stewart SG, Gordon CP, et al. Norcantharidin analogues: synthesis, anticancer activity and protein phosphatase 1 and 2A inhibition. ChemMedChem, 2008, 3(12):1878-1892
17 Bonness K, Aragon IV, Rutland B, et al. Cantharidin- induced mitotic arrest is associated with the formation of aberrant mitotic spindles and lagging chromosomes resulting, in part, from the suppression of PP2Aalpha. Mol Cancer Ther, 2006, 5(11):2727-2736
18梁枫,王明艳,许冬青.斑蝥酸钠诱导人胃癌BGC823细胞凋亡的实验研究.南京中医药大学学报, 2006,22(3):171- 172
19刘展华,曹洋,陈锐深,等.斑蝥酸钠对人肺癌LAC细胞增殖的抑制作用.中药新药与临床药理, 2008, 19(1):32-33
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27 Tsaia WC, Chub CH, Yua CP, et al.Matriptase and survivin expression associated with tumor progression and malignant potential in breast cancer of Chinese women:Tissue microarray analysis of immunostaining scores with clinicopathological parameters. Disease Markers, 2008, 24 (2): 89-99
28 Kleinberg L, Florenes VA, Silins I, et a1.Nuclear expression of survivin is associated with improved survival in metastatic ovarian carcinoma.Cancer, 2007, 109(2):228-238
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34 Trieb K, Lehner R, Stulnig T, et al.Survivin expression in human osteocarcoma is a marker for survival.Eur J Surg Oncol, 2003, 29:379-382
35高嵩,罗建平,郑稼,等.人骨肉瘤组织中survivin蛋白表达及其临床意义.实用诊断与治疗杂志, 2007, 21(12): 893-894, 898
36 Wang W, Luo H, Wang A Expression of survivin and correlation with PCNA in osteosarcoma. J Surg Oncol, 2006, 93(7):578-584
37 Zhang H Z,W ang Y,Gao P,et a1.Silencing Stathmin Gene Expression by Survivin Promoter-Driven siRNA Vector to Reverse Malignant Phenotype of Tumor Cells.Cancer Biol Ther, 2006, 5(11):1457-1461
1 Chen YJ, Shieh CJ, Tsai TH, et al. Inhibitory effect of norcantharidin,a derivative compound from blister beetles, on tumor invasion and metastasis in CT26 colorectal adenocarcinoma cells. Anticancer Drugs, 2005, 16(3): 293-299
2 Hill TA, Stewart SG, Sauer B, et al.Heterocyclic substituted cantharidin and norcantharidin analogues-synthesis, protein phosphatase (1 and 2A) inhibition, and anti-cancer activity. Bioorg Med Chem Lett, 2007, 17(12): 3392-3397
3 Rauh R, Kahl S, Boechzelt H, et al.Molecular biology of cantharidin in cancer cells. Chin Med, 2007, 2: 8.
4 Fan YZ, Fu JY, Zhao ZM, et al. Inhibitory effect of norcantharidin on the growth of human gallbladder carcinoma GBC-SD cells in vitro.Hepatobiliary Pancreat Dis Int, 2007, 6(1): 72-80
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