二亚硝基哌嗪（DNP）介导Ezrin磷酸化促进鼻咽癌转移的分子机制研究
发布时间：2018-08-30 07:56
【摘要】：目的：本文检测化学致癌物二亚硝基哌嗪(N,N'-Dinitrosopiperazine, DNP)处理鼻咽癌细胞(6-10B)后的运动侵袭能力,分析DNP对Ezrin和phos-Ezrin (phosphorylated ezrin, phos-Ezrin)表达的调控作用,探讨DNP参与鼻咽癌侵袭转移的分子机制。 方法： 1.鼻咽癌细胞6-10B为低转移、低成瘤鼻咽癌细胞系,来自鼻咽癌母细胞系SUNE-1,作为研究材料。来自同一母细胞系的5-8F,为高转移、高成瘤鼻咽癌细胞株,作为对照细胞。 2.连续稀释法制备不同浓度DNP,将不同浓度DNP处理6-10B细胞,四甲基偶氮唑蓝(Methylthiazolyltertrazolium, MTT)实验分析DNP处理6-10B细胞的非毒性浓度(non-cytotoxic concentration,NCC)。全自动生化仪检测DNP处理6-10B细胞培养液乳酸脱氢酶(Lactate dehydrogenas, LDH),进一步确证DNP非毒性浓度。 3.用DNP非毒性浓度处理6-10B细胞,Western Blotting分析DNP处理6-10B细胞后Ezrin和phos-Ezrin Thr567表达的影响。明确DNP是否诱导6-10B细胞Ezrin或phos-Ezrin Thr567表达。 4.用DNP非毒性浓度处理6-10B细胞,划痕实验和Transwell迁移及侵袭实验分析非毒性浓度的DNP对6-10B细胞迁移运动和侵袭能力的影响。明确DNP是否促进6-10B细胞运动迁移和侵袭。 5.通过尾静脉将6-10B细胞移植在BALB/c裸小鼠体内,皮下注射DNP,处理一定时间后,解剖裸鼠,观察6-10B细胞转移情况,常规病理学检测,确认6-10B细胞是否发生转移。分析DNP在体内对鼻咽癌细胞成瘤和转移的影响。 6.将6-10B转移癌组织病理切片,免疫组化SP方法检测Ezrin和phos-Ezrin Thr567在裸鼠组织中的表达,分析DNP在体内对6-10B细胞Ezrin和phos-Ezrin Thr567表达的影响。 7.利用SPSS17.0统计软件分析数据。 结果： 1.MTT实验和LDH检测结果显示,DNP在高浓度(100μ.mol/L以上)时对鼻咽癌6-10B细胞生长增殖有明显的抑制作用(P0.05),而在0～100μmol/L时对6-10B细胞的生长没有明显影响(P0.05)。DNP处理6-10B细胞的非毒性浓度为0～100μmol/L。 2. Western Blotting分析DNP诱导Ezrin和phos-Ezrin Thr567表达,结果显示不同浓度DNP处理6-10B细胞组和未处理组Ezrin的表达无明显差异(P0.05)；而DNP处理6-10B细胞phos-EzrinThr567表达水平明显高于未处理6-10B细胞(P0.05),且随剂量增加和处理时间延长phos-EzrinThr567逐步增高,呈现明显的处理剂量依赖性和时间依赖性。 3.划痕实验结果显示,DNP处理组迁移生长细胞数明显多未处理组；Transwell迁移和侵袭实验结果表明,DNP处理组穿过过滤膜细胞数显著多于未处理组(P0.05),这说明DNP能增强鼻咽癌6-10B细胞迁移和侵袭能力。 4.6-10B细胞移植在BALB/c裸小鼠体内,DNP处理一定时间后,在裸鼠肺组织观察到瘤组织,常规病理检查证实为鼻咽癌细胞肺转移瘤,表明DNP促进6-10B细胞肺转移。 5.转移癌组织免疫组化结果显示,DNP处理组裸鼠肺转移灶phos-EzrinThr567的表达明显高于对照组(P0.05),总Ezrin蛋白表达无明显差异。提示DNP通过上调phos-EzrinThr567表达促进鼻咽癌6-10B细胞转移。 结论： 1.DNP诱导鼻咽癌6-10B细胞phos-EzrinThr567表达,呈现明显的剂量和时间依赖性,但总Ezrin的表达无明显变化。 2.DNP促进鼻咽癌6-10B细胞迁移运动和侵袭能力,DNP可能参与了鼻咽癌侵袭转移。 3.DNP促进鼻咽癌6-10B细胞裸鼠肺转移,上调转移组织phos-EzrinThr567的表达,提示DNP可能通过上调phos-EzrinThr567表达促进鼻咽癌细胞侵袭与转移：
[Abstract]:Objective: To investigate the effect of DNP on the expression of Ezrin and phos-Ezrin (phos-Ezrin) in nasopharyngeal carcinoma cells (6-10B) treated with N, N'-Dinitrosopiperazine (DNP), and to explore the molecular mechanism of DNP in the invasion and metastasis of nasopharyngeal carcinoma.
Method:
1. Nasopharyngeal carcinoma cell line 6-10B is low metastasis, low tumorigenesis nasopharyngeal carcinoma cell line, from nasopharyngeal carcinoma mother cell line SUNE-1, as research materials. 5-8F from the same mother cell line, is high metastasis, high tumorigenesis nasopharyngeal carcinoma cell line, as control cells.
2. Different concentrations of DNP were prepared by continuous dilution method. 6-10B cells were treated with different concentrations of DNP. The non-cytotoxic concentration (NCC) of 6-10B cells treated with DNP was analyzed by MTT assay. Lactate dehydrogenase (LDH) in 6-10B cells treated with DNP was detected by automatic biochemical analyzer. ENAs, LDH) further confirmed the non toxic concentration of DNP.
3. Non-toxic concentration of DNP was used to treat 6-10B cells. Western Blotting was used to analyze the effect of DNP on the expression of Ezrin and phos-Ezrin Thr567 in 6-10B cells.
4. Non-toxic concentration of DNP was used to treat 6-10B cells. Scratch test and Transwell migration and invasion test were used to analyze the effect of non-toxic concentration of DNP on migration and invasion of 6-10B cells.
5. 6-10B cells were transplanted into BALB/c nude mice by tail vein. DNP was injected subcutaneously. After treatment for a certain period of time, the metastasis of 6-10B cells was observed. The metastasis of 6-10B cells was confirmed by routine pathological examination.
6. Immunohistochemical SP method was used to detect the expression of Ezrin and phos-Ezrin Thr567 in the tissues of 6-10B metastatic carcinoma. The effect of DNP on the expression of Ezrin and phos-Ezrin Thr567 in 6-10B cells was analyzed.
7. use SPSS17.0 statistical software to analyze data.
Result:
1. MTT assay and LDH assay showed that DNP significantly inhibited the growth and proliferation of nasopharyngeal carcinoma 6-10B cells at high concentrations (above 100 micromol/L) (P 0.05), but had no significant effect on the growth of 6-10B cells at 0-100 micromol/L (P 0.05). The non-toxic concentration of 6-10B cells treated with DNP was 0-100 micromol/L.
2. Western Blotting analysis of DNP-induced Ezrin and phos-Ezrin Thr567 expression showed that there was no significant difference in the expression of Ezrin between 6-10B cells treated with different concentrations of DNP and untreated cells (P 0.05), but the expression level of phos-Ezrin Thr567 in 6-10B cells treated with DNP was significantly higher than that in untreated 6-10B cells (P 0.05), and pH increased with the increase of dosage and treatment time. Os-EzrinThr567 progressively increased, showing a dose-dependent and time-dependent manner.
3. Scratch test showed that the number of migrating and growing cells in DNP treatment group was significantly higher than that in untreated group; Transwell migration and invasion test showed that the number of cells passing through filter membrane in DNP treatment group was significantly higher than that in untreated group (P 0.05), which indicated that DNP could enhance the migration and invasion ability of nasopharyngeal carcinoma 6-10B cells.
4.6-10B cells were transplanted into BALB/c nude mice. After treatment with DNP for a certain time, tumor tissue was observed in the lung tissue of nude mice. Routine pathological examination confirmed that nasopharyngeal carcinoma cell lung metastases, indicating that DNP promotes lung metastasis of 6-10B cells.
5. Immunohistochemical results showed that the expression of phos-EzrinThr567 in the lung metastases of nude mice treated with DNP was significantly higher than that of the control group (P 0.05), and the expression of total Ezrin protein was not significantly different.
Conclusion:
1. DNP induced phos-EzrinThr567 expression in nasopharyngeal carcinoma 6-10B cells in a dose-and time-dependent manner, but the total Ezrin expression did not change significantly.
2.DNP promotes the migration and invasion of nasopharyngeal carcinoma 6-10B cells, and DNP may be involved in invasion and metastasis of nasopharyngeal carcinoma.
3. DNP promotes lung metastasis of nasopharyngeal carcinoma 6-10B cells in nude mice and up-regulates the expression of phos-EzrinThr567 in metastatic tissues, suggesting that DNP may promote invasion and metastasis of nasopharyngeal carcinoma cells by up-regulating the expression of phos-EzrinThr567.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63
[Abstract]:Objective: To investigate the effect of DNP on the expression of Ezrin and phos-Ezrin (phos-Ezrin) in nasopharyngeal carcinoma cells (6-10B) treated with N, N'-Dinitrosopiperazine (DNP), and to explore the molecular mechanism of DNP in the invasion and metastasis of nasopharyngeal carcinoma.
Method:
1. Nasopharyngeal carcinoma cell line 6-10B is low metastasis, low tumorigenesis nasopharyngeal carcinoma cell line, from nasopharyngeal carcinoma mother cell line SUNE-1, as research materials. 5-8F from the same mother cell line, is high metastasis, high tumorigenesis nasopharyngeal carcinoma cell line, as control cells.
2. Different concentrations of DNP were prepared by continuous dilution method. 6-10B cells were treated with different concentrations of DNP. The non-cytotoxic concentration (NCC) of 6-10B cells treated with DNP was analyzed by MTT assay. Lactate dehydrogenase (LDH) in 6-10B cells treated with DNP was detected by automatic biochemical analyzer. ENAs, LDH) further confirmed the non toxic concentration of DNP.
3. Non-toxic concentration of DNP was used to treat 6-10B cells. Western Blotting was used to analyze the effect of DNP on the expression of Ezrin and phos-Ezrin Thr567 in 6-10B cells.
4. Non-toxic concentration of DNP was used to treat 6-10B cells. Scratch test and Transwell migration and invasion test were used to analyze the effect of non-toxic concentration of DNP on migration and invasion of 6-10B cells.
5. 6-10B cells were transplanted into BALB/c nude mice by tail vein. DNP was injected subcutaneously. After treatment for a certain period of time, the metastasis of 6-10B cells was observed. The metastasis of 6-10B cells was confirmed by routine pathological examination.
6. Immunohistochemical SP method was used to detect the expression of Ezrin and phos-Ezrin Thr567 in the tissues of 6-10B metastatic carcinoma. The effect of DNP on the expression of Ezrin and phos-Ezrin Thr567 in 6-10B cells was analyzed.
7. use SPSS17.0 statistical software to analyze data.
Result:
1. MTT assay and LDH assay showed that DNP significantly inhibited the growth and proliferation of nasopharyngeal carcinoma 6-10B cells at high concentrations (above 100 micromol/L) (P 0.05), but had no significant effect on the growth of 6-10B cells at 0-100 micromol/L (P 0.05). The non-toxic concentration of 6-10B cells treated with DNP was 0-100 micromol/L.
2. Western Blotting analysis of DNP-induced Ezrin and phos-Ezrin Thr567 expression showed that there was no significant difference in the expression of Ezrin between 6-10B cells treated with different concentrations of DNP and untreated cells (P 0.05), but the expression level of phos-Ezrin Thr567 in 6-10B cells treated with DNP was significantly higher than that in untreated 6-10B cells (P 0.05), and pH increased with the increase of dosage and treatment time. Os-EzrinThr567 progressively increased, showing a dose-dependent and time-dependent manner.
3. Scratch test showed that the number of migrating and growing cells in DNP treatment group was significantly higher than that in untreated group; Transwell migration and invasion test showed that the number of cells passing through filter membrane in DNP treatment group was significantly higher than that in untreated group (P 0.05), which indicated that DNP could enhance the migration and invasion ability of nasopharyngeal carcinoma 6-10B cells.
4.6-10B cells were transplanted into BALB/c nude mice. After treatment with DNP for a certain time, tumor tissue was observed in the lung tissue of nude mice. Routine pathological examination confirmed that nasopharyngeal carcinoma cell lung metastases, indicating that DNP promotes lung metastasis of 6-10B cells.
5. Immunohistochemical results showed that the expression of phos-EzrinThr567 in the lung metastases of nude mice treated with DNP was significantly higher than that of the control group (P 0.05), and the expression of total Ezrin protein was not significantly different.
Conclusion:
1. DNP induced phos-EzrinThr567 expression in nasopharyngeal carcinoma 6-10B cells in a dose-and time-dependent manner, but the total Ezrin expression did not change significantly.
2.DNP promotes the migration and invasion of nasopharyngeal carcinoma 6-10B cells, and DNP may be involved in invasion and metastasis of nasopharyngeal carcinoma.
3. DNP promotes lung metastasis of nasopharyngeal carcinoma 6-10B cells in nude mice and up-regulates the expression of phos-EzrinThr567 in metastatic tissues, suggesting that DNP may promote invasion and metastasis of nasopharyngeal carcinoma cells by up-regulating the expression of phos-EzrinThr567.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63
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