【摘要】：目的：本文分析二亞硝基哌嗪(Dinitrosopiperazine, DNP)對(duì)鼻咽癌細(xì)胞熱休克蛋白70-2 (Heat Shock Protein70-2,HSP70-2)表達(dá)的影響,探討DNP參與鼻咽癌轉(zhuǎn)移的分子機(jī)制。 方法： 1.選取來自同一母細(xì)胞的高轉(zhuǎn)移潛能鼻咽癌5-8F細(xì)胞株和低轉(zhuǎn)移潛能鼻咽癌6-10B細(xì)胞株作為研究材料,運(yùn)用四甲基偶氮唑藍(lán)(Methylthiazolyltertrazolium, MTT)實(shí)驗(yàn)分析DNP對(duì)6-10B細(xì)胞生長(zhǎng)的影響,篩選出DNP對(duì)6-10B細(xì)胞的非毒性濃度(non-cytotoxic concentration, NCC)。 2.采用非毒性濃度的DNP處理6-10B細(xì)胞,并以未處理的6-10B細(xì)胞、5-8F細(xì)胞作為對(duì)照,進(jìn)行細(xì)胞生物學(xué)和分子生物學(xué)實(shí)驗(yàn)。 3.采用細(xì)胞間接免疫熒光方法初步分析DNP對(duì)HSP70-2表達(dá)的影響,Western Blotting分析DNP誘導(dǎo)HSP70-2表達(dá)的量效關(guān)系。 4.采用Real-time RT PCR技術(shù)檢測(cè)DNP對(duì)HSP70-2基因表達(dá)的影響。 5.利用SPSS18.0統(tǒng)計(jì)軟件分析數(shù)據(jù)。結(jié)果： 1.MTT實(shí)驗(yàn)分析DNP對(duì)鼻咽癌6-10B細(xì)胞生長(zhǎng)的影響,結(jié)果表明DNP在高濃度(100μmol/L以上)時(shí)對(duì)鼻咽癌6-10B細(xì)胞生長(zhǎng)增殖有明顯的抑制作用(p0.05),而在0～100μmol/L時(shí)對(duì)6-10B細(xì)胞的生長(zhǎng)沒有明顯影響(p0.05).0～100μmol/L為DNP對(duì)6-10B細(xì)胞的非毒性濃度。 2.細(xì)胞間接免疫熒光實(shí)驗(yàn)觀察DNP對(duì)鼻咽癌細(xì)胞HSP70-2表達(dá)的影響,結(jié)果顯示DNP處理鼻咽癌6-10B細(xì)胞后,HSP70-2蛋白表達(dá)明顯增強(qiáng),細(xì)胞中的表達(dá)以胞漿為主,且DNP處理的6-10B細(xì)胞熒光強(qiáng)度同高轉(zhuǎn)移潛能5-8F細(xì)胞一致。 3.Western Blotting分析DNP誘導(dǎo)HSP70-2表達(dá)的量效關(guān)系,結(jié)果顯示不同濃度(50μmol/L、100μmol/L)DNP處理的6-10B細(xì)胞,HSP70-2蛋白水平明顯高于未處理6-10B細(xì)胞,且隨DNP量增加而HSP70-2逐步增高(P0.05) 4.Real-time PCR分析DNP對(duì)Hsp70-2基因表達(dá)的影響,結(jié)果顯示,未處理6-10B細(xì)胞中Hsp70-2基因相對(duì)折合表達(dá)量(1.04±0.33)與不同濃度(50μmol/L、100μmol/L)DNP處理的6-10B細(xì)胞中Hsp70-2基因相對(duì)折合表達(dá)量(分別為0.81±0.14、0.81±0.26)無明顯差異(P0.05),但5-8F細(xì)胞中的表達(dá)量(1.84±0.79)高于6-10B細(xì)胞。結(jié)論： 1.DNP處理6-10B細(xì)胞后,細(xì)胞中的HSP70-2蛋白水平明顯增高,表明DNP能上調(diào)HSP70-2蛋白水平。 2.DNP處理6-10B細(xì)胞后,細(xì)胞中的Hsp70-2基因的相對(duì)折合表達(dá)量并不增高,DNP可能不是在基因水平調(diào)控HSP70-2,提示DNP可能通過轉(zhuǎn)錄后調(diào)控機(jī)制上調(diào)HSP70-2蛋白表達(dá)。 3.5-8F細(xì)胞中的HSP70-2蛋白水平高于6-10B細(xì)胞,提示HSP70-2可能與細(xì)胞的惡性程度(轉(zhuǎn)移潛能)有關(guān)。
[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 鹵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 鹵0.140.81 鹵0.26, respectively), but the expression of Hsp70-2 gene in 5-8F cells (1.84 鹵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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R739.63

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