二亞硝基哌嗪（DNP）介導Ezrin磷酸化促進鼻咽癌轉(zhuǎn)移的分子機制研究
發(fā)布時間：2018-08-30 07:56
【摘要】：目的：本文檢測化學致癌物二亞硝基哌嗪(N,N'-Dinitrosopiperazine, DNP)處理鼻咽癌細胞(6-10B)后的運動侵襲能力,分析DNP對Ezrin和phos-Ezrin (phosphorylated ezrin, phos-Ezrin)表達的調(diào)控作用,探討DNP參與鼻咽癌侵襲轉(zhuǎn)移的分子機制。 方法： 1.鼻咽癌細胞6-10B為低轉(zhuǎn)移、低成瘤鼻咽癌細胞系,來自鼻咽癌母細胞系SUNE-1,作為研究材料。來自同一母細胞系的5-8F,為高轉(zhuǎn)移、高成瘤鼻咽癌細胞株,作為對照細胞。 2.連續(xù)稀釋法制備不同濃度DNP,將不同濃度DNP處理6-10B細胞,四甲基偶氮唑藍(Methylthiazolyltertrazolium, MTT)實驗分析DNP處理6-10B細胞的非毒性濃度(non-cytotoxic concentration,NCC)。全自動生化儀檢測DNP處理6-10B細胞培養(yǎng)液乳酸脫氫酶(Lactate dehydrogenas, LDH),進一步確證DNP非毒性濃度。 3.用DNP非毒性濃度處理6-10B細胞,Western Blotting分析DNP處理6-10B細胞后Ezrin和phos-Ezrin Thr567表達的影響。明確DNP是否誘導6-10B細胞Ezrin或phos-Ezrin Thr567表達。 4.用DNP非毒性濃度處理6-10B細胞,劃痕實驗和Transwell遷移及侵襲實驗分析非毒性濃度的DNP對6-10B細胞遷移運動和侵襲能力的影響。明確DNP是否促進6-10B細胞運動遷移和侵襲。 5.通過尾靜脈將6-10B細胞移植在BALB/c裸小鼠體內(nèi),皮下注射DNP,處理一定時間后,解剖裸鼠,觀察6-10B細胞轉(zhuǎn)移情況,常規(guī)病理學檢測,確認6-10B細胞是否發(fā)生轉(zhuǎn)移。分析DNP在體內(nèi)對鼻咽癌細胞成瘤和轉(zhuǎn)移的影響。 6.將6-10B轉(zhuǎn)移癌組織病理切片,免疫組化SP方法檢測Ezrin和phos-Ezrin Thr567在裸鼠組織中的表達,分析DNP在體內(nèi)對6-10B細胞Ezrin和phos-Ezrin Thr567表達的影響。 7.利用SPSS17.0統(tǒng)計軟件分析數(shù)據(jù)。 結(jié)果： 1.MTT實驗和LDH檢測結(jié)果顯示,DNP在高濃度(100μ.mol/L以上)時對鼻咽癌6-10B細胞生長增殖有明顯的抑制作用(P0.05),而在0～100μmol/L時對6-10B細胞的生長沒有明顯影響(P0.05)。DNP處理6-10B細胞的非毒性濃度為0～100μmol/L。 2. Western Blotting分析DNP誘導Ezrin和phos-Ezrin Thr567表達,結(jié)果顯示不同濃度DNP處理6-10B細胞組和未處理組Ezrin的表達無明顯差異(P0.05)；而DNP處理6-10B細胞phos-EzrinThr567表達水平明顯高于未處理6-10B細胞(P0.05),且隨劑量增加和處理時間延長phos-EzrinThr567逐步增高,呈現(xiàn)明顯的處理劑量依賴性和時間依賴性。 3.劃痕實驗結(jié)果顯示,DNP處理組遷移生長細胞數(shù)明顯多未處理組；Transwell遷移和侵襲實驗結(jié)果表明,DNP處理組穿過過濾膜細胞數(shù)顯著多于未處理組(P0.05),這說明DNP能增強鼻咽癌6-10B細胞遷移和侵襲能力。 4.6-10B細胞移植在BALB/c裸小鼠體內(nèi),DNP處理一定時間后,在裸鼠肺組織觀察到瘤組織,常規(guī)病理檢查證實為鼻咽癌細胞肺轉(zhuǎn)移瘤,表明DNP促進6-10B細胞肺轉(zhuǎn)移。 5.轉(zhuǎn)移癌組織免疫組化結(jié)果顯示,DNP處理組裸鼠肺轉(zhuǎn)移灶phos-EzrinThr567的表達明顯高于對照組(P0.05),總Ezrin蛋白表達無明顯差異。提示DNP通過上調(diào)phos-EzrinThr567表達促進鼻咽癌6-10B細胞轉(zhuǎn)移。 結(jié)論： 1.DNP誘導鼻咽癌6-10B細胞phos-EzrinThr567表達,呈現(xiàn)明顯的劑量和時間依賴性,但總Ezrin的表達無明顯變化。 2.DNP促進鼻咽癌6-10B細胞遷移運動和侵襲能力,DNP可能參與了鼻咽癌侵襲轉(zhuǎn)移。 3.DNP促進鼻咽癌6-10B細胞裸鼠肺轉(zhuǎn)移,上調(diào)轉(zhuǎn)移組織phos-EzrinThr567的表達,提示DNP可能通過上調(diào)phos-EzrinThr567表達促進鼻咽癌細胞侵襲與轉(zhuǎn)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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R739.63
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