二亚硝基哌嗪（DNP）上调HSP70-2在低转移潜能鼻咽癌6-10B细胞中的表达
发布时间：2018-12-16 08:30
【摘要】：目的：本文分析二亚硝基哌嗪(Dinitrosopiperazine, DNP)对鼻咽癌细胞热休克蛋白70-2 (Heat Shock Protein70-2,HSP70-2)表达的影响,探讨DNP参与鼻咽癌转移的分子机制。 方法： 1.选取来自同一母细胞的高转移潜能鼻咽癌5-8F细胞株和低转移潜能鼻咽癌6-10B细胞株作为研究材料,运用四甲基偶氮唑蓝(Methylthiazolyltertrazolium, MTT)实验分析DNP对6-10B细胞生长的影响,筛选出DNP对6-10B细胞的非毒性浓度(non-cytotoxic concentration, NCC)。 2.采用非毒性浓度的DNP处理6-10B细胞,并以未处理的6-10B细胞、5-8F细胞作为对照,进行细胞生物学和分子生物学实验。 3.采用细胞间接免疫荧光方法初步分析DNP对HSP70-2表达的影响,Western Blotting分析DNP诱导HSP70-2表达的量效关系。 4.采用Real-time RT PCR技术检测DNP对HSP70-2基因表达的影响。 5.利用SPSS18.0统计软件分析数据。结果： 1.MTT实验分析DNP对鼻咽癌6-10B细胞生长的影响,结果表明DNP在高浓度(100μmol/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).0～100μmol/L为DNP对6-10B细胞的非毒性浓度。 2.细胞间接免疫荧光实验观察DNP对鼻咽癌细胞HSP70-2表达的影响,结果显示DNP处理鼻咽癌6-10B细胞后,HSP70-2蛋白表达明显增强,细胞中的表达以胞浆为主,且DNP处理的6-10B细胞荧光强度同高转移潜能5-8F细胞一致。 3.Western Blotting分析DNP诱导HSP70-2表达的量效关系,结果显示不同浓度(50μmol/L、100μmol/L)DNP处理的6-10B细胞,HSP70-2蛋白水平明显高于未处理6-10B细胞,且随DNP量增加而HSP70-2逐步增高(P0.05) 4.Real-time PCR分析DNP对Hsp70-2基因表达的影响,结果显示,未处理6-10B细胞中Hsp70-2基因相对折合表达量(1.04±0.33)与不同浓度(50μmol/L、100μmol/L)DNP处理的6-10B细胞中Hsp70-2基因相对折合表达量(分别为0.81±0.14、0.81±0.26)无明显差异(P0.05),但5-8F细胞中的表达量(1.84±0.79)高于6-10B细胞。结论： 1.DNP处理6-10B细胞后,细胞中的HSP70-2蛋白水平明显增高,表明DNP能上调HSP70-2蛋白水平。 2.DNP处理6-10B细胞后,细胞中的Hsp70-2基因的相对折合表达量并不增高,DNP可能不是在基因水平调控HSP70-2,提示DNP可能通过转录后调控机制上调HSP70-2蛋白表达。 3.5-8F细胞中的HSP70-2蛋白水平高于6-10B细胞,提示HSP70-2可能与细胞的恶性程度(转移潜能)有关。
[Abstract]:Aim: to investigate the effect of dinitropiperazine (Dinitrosopiperazine, DNP) on the expression of heat shock protein 70 2 (Heat Shock Protein70-2,HSP70-2 (HSP70 2) in nasopharyngeal carcinoma (NPC) cells and to explore the molecular mechanism of DNP involved in NPC metastasis. Methods: 1. 5-8F cell lines with high metastatic potential and 6-10B cell lines with low metastatic potential from the same mother cell were selected as the research materials. The effects of DNP on the growth of 6-10B cells were analyzed by (Methylthiazolyltertrazolium, MTT) assay. The nontoxic concentration of DNP on 6-10B cells (non-cytotoxic concentration, NCC).) was screened out. 2. 6-10B cells were treated with non-toxic concentration of DNP. The untreated 6-10B cells and 5-8F cells were used as controls to carry out cell biology and molecular biology experiments. 3. The effect of DNP on HSP70-2 expression was preliminarily analyzed by indirect immunofluorescence assay., Western Blotting was used to analyze the dose-response relationship of HSP70-2 expression induced by DNP. 4. Real-time RT PCR technique was used to detect the effect of DNP on the expression of HSP70-2 gene. 5. SPSS18.0 statistical software is used to analyze the data. Results: 1.MTT assay was used to analyze the effect of DNP on the growth of nasopharyngeal carcinoma 6-10B cells. The results showed that DNP could inhibit the growth and proliferation of 6-10B cells at high concentration (more than 100 渭 mol/L) (p0.05). At 0 渭 mol/L, the growth of 6-10B cells was not significantly affected (p0.05). 0 渭 mol/L was the nontoxic concentration of DNP on 6-10B cells. 2. The effect of DNP on the expression of HSP70-2 in nasopharyngeal carcinoma (NPC) cells was observed by indirect immunofluorescence assay. The results showed that the expression of HSP70-2 protein was significantly increased in 6-10B cells treated with DNP, and the expression of HSP70-2 protein was mainly in cytoplasm. The fluorescence intensity of 6-10 B cells treated with DNP was the same as that of 5-8 F cells with high metastasis potential. 3.Western Blotting analysis showed that the level of HSP70-2 protein in 6-10B cells treated with DNP at different concentrations (50 渭 mol/L,100 渭 mol/L) was significantly higher than that in untreated 6-10B cells. With the increase of DNP, HSP70-2 increased gradually (P0.05). The effect of DNP on the expression of Hsp70-2 gene was analyzed by 4.Real-time PCR. The results showed that, Relative expression of Hsp70-2 gene (1.04 卤0.33) and different concentrations (50 渭 mol/L,) in untreated 6-10B cells There was no significant difference in the expression of Hsp70-2 gene in 6-10B cells treated with 100 渭 mol/L DNP (0.81 卤0.140.81 卤0.26, respectively), but the expression of Hsp70-2 gene in 5-8F cells (1.84 卤0.79) was higher than that in 6-10B cells. Conclusion: the level of HSP70-2 protein in 6-10B cells treated with 1.DNP was significantly increased, indicating that DNP could up-regulate the level of HSP70-2 protein. The relative folding expression of Hsp70-2 gene in 6-10B cells treated with 2.DNP did not increase. DNP may not regulate HSP70-2, at the gene level, suggesting that DNP may up-regulate HSP70-2 protein expression through the mechanism of posttranscriptional regulation. 3.5-8F cells had higher levels of HSP70-2 protein than 6-10B cells, suggesting that HSP70-2 might be related to the degree of malignancy (metastasis potential) of the cells.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63
[Abstract]:Aim: to investigate the effect of dinitropiperazine (Dinitrosopiperazine, DNP) on the expression of heat shock protein 70 2 (Heat Shock Protein70-2,HSP70-2 (HSP70 2) in nasopharyngeal carcinoma (NPC) cells and to explore the molecular mechanism of DNP involved in NPC metastasis. Methods: 1. 5-8F cell lines with high metastatic potential and 6-10B cell lines with low metastatic potential from the same mother cell were selected as the research materials. The effects of DNP on the growth of 6-10B cells were analyzed by (Methylthiazolyltertrazolium, MTT) assay. The nontoxic concentration of DNP on 6-10B cells (non-cytotoxic concentration, NCC).) was screened out. 2. 6-10B cells were treated with non-toxic concentration of DNP. The untreated 6-10B cells and 5-8F cells were used as controls to carry out cell biology and molecular biology experiments. 3. The effect of DNP on HSP70-2 expression was preliminarily analyzed by indirect immunofluorescence assay., Western Blotting was used to analyze the dose-response relationship of HSP70-2 expression induced by DNP. 4. Real-time RT PCR technique was used to detect the effect of DNP on the expression of HSP70-2 gene. 5. SPSS18.0 statistical software is used to analyze the data. Results: 1.MTT assay was used to analyze the effect of DNP on the growth of nasopharyngeal carcinoma 6-10B cells. The results showed that DNP could inhibit the growth and proliferation of 6-10B cells at high concentration (more than 100 渭 mol/L) (p0.05). At 0 渭 mol/L, the growth of 6-10B cells was not significantly affected (p0.05). 0 渭 mol/L was the nontoxic concentration of DNP on 6-10B cells. 2. The effect of DNP on the expression of HSP70-2 in nasopharyngeal carcinoma (NPC) cells was observed by indirect immunofluorescence assay. The results showed that the expression of HSP70-2 protein was significantly increased in 6-10B cells treated with DNP, and the expression of HSP70-2 protein was mainly in cytoplasm. The fluorescence intensity of 6-10 B cells treated with DNP was the same as that of 5-8 F cells with high metastasis potential. 3.Western Blotting analysis showed that the level of HSP70-2 protein in 6-10B cells treated with DNP at different concentrations (50 渭 mol/L,100 渭 mol/L) was significantly higher than that in untreated 6-10B cells. With the increase of DNP, HSP70-2 increased gradually (P0.05). The effect of DNP on the expression of Hsp70-2 gene was analyzed by 4.Real-time PCR. The results showed that, Relative expression of Hsp70-2 gene (1.04 卤0.33) and different concentrations (50 渭 mol/L,) in untreated 6-10B cells There was no significant difference in the expression of Hsp70-2 gene in 6-10B cells treated with 100 渭 mol/L DNP (0.81 卤0.140.81 卤0.26, respectively), but the expression of Hsp70-2 gene in 5-8F cells (1.84 卤0.79) was higher than that in 6-10B cells. Conclusion: the level of HSP70-2 protein in 6-10B cells treated with 1.DNP was significantly increased, indicating that DNP could up-regulate the level of HSP70-2 protein. The relative folding expression of Hsp70-2 gene in 6-10B cells treated with 2.DNP did not increase. DNP may not regulate HSP70-2, at the gene level, suggesting that DNP may up-regulate HSP70-2 protein expression through the mechanism of posttranscriptional regulation. 3.5-8F cells had higher levels of HSP70-2 protein than 6-10B cells, suggesting that HSP70-2 might be related to the degree of malignancy (metastasis potential) of the cells.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63
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