發(fā)布時間：2018-05-06 01:22

  本文選題：鼻咽癌 + SGK3��； 參考：《南方醫(yī)科大學》2017年碩士論文

【摘要】：目的:鼻咽癌(Nasopharyngeal Carcinoma,NPC)是一種具有區(qū)域性分布以及種族聚集性特征的頭頸部惡性腫瘤,好發(fā)于我國南方及東南亞地區(qū)。鼻咽癌發(fā)生于鼻咽部粘膜,其病理類型多為低分化鱗狀細胞癌,惡性程度較高,早期即可出現(xiàn)頸部淋巴結(jié)轉(zhuǎn)移。鼻咽癌大多對放療有中度敏感性,以適形調(diào)強放療為主的綜合治療極大地提高了鼻咽癌的局部控制率,卻未能顯著降低其遠處轉(zhuǎn)移率,治療失敗的主要原因是局部復發(fā)和遠處轉(zhuǎn)移。因此,深入研究鼻咽癌的發(fā)生與發(fā)展機制,對改善鼻咽癌治療效果、提高患者生存率有重大意義。血清和糖皮質(zhì)激素調(diào)節(jié)激酶(Serum and glucocorticoid-inducible kinase,SGK)是一種絲氨酸/蘇氨酸激酶,與蛋白激酶B(Protein kinase B,PKB or AKT)同屬于AGC蛋白激酶家族成員,與AKT高度同源并調(diào)節(jié)與其類似的細胞過程,參與調(diào)控細胞增殖凋亡、細胞周期以及離子通道等,是細胞磷酸化級聯(lián)反應和多種細胞信號通路的一個功能性交匯點。SGK3是SGK家族的一員,其Thr256位點可被PDK1磷酸化而激活,被認為是一種非AKT依賴性的腫瘤信號傳導方式。研究表明,SGK3在很多惡性腫瘤中高度表達,對腫瘤的發(fā)生、發(fā)展起著重要作用,如乳腺癌、肝癌、前列腺癌等,但SGK3與鼻咽癌的相關(guān)性研究目前國內(nèi)外尚未見報道。本文旨在研究SGK3在鼻咽癌組織和細胞中的表達情況,以及SGK3對鼻咽癌細胞生物學特性的影響,探討SGK3表達與鼻咽癌發(fā)生發(fā)展的關(guān)系。方法:1.免疫組化法檢測SGK3在42例鼻咽癌組織和9例慢性鼻咽炎癥組織中的蛋白表達,卡方檢驗比較鼻咽癌組織和慢性鼻咽炎組織中SGK3蛋白表達的組間差異,Logistic回歸分析研究SGK3蛋白表達水平與鼻咽癌患者臨床病理特征之間的相關(guān)性。2.Western blot檢測SGK3在鼻咽癌細胞株CNE-1、CNE-2、HNE-1、SUNE-1、HONE-1及永生化鼻咽上皮NP69細胞株中的蛋白表達,Image Pro Plus軟件對蛋白相對表達強度進行半定量分析,單因素方差分析比較不同細胞株中蛋白灰度值的組間差異。3.Lipofectamine 2000瞬時轉(zhuǎn)染鼻咽癌細胞,轉(zhuǎn)染24小時在熒光顯微鏡下確認轉(zhuǎn)染成功后,再培養(yǎng)24小時行Western blot檢測轉(zhuǎn)染后鼻咽癌細胞中SGK3的表達情況,選取SGK3敲低效果最顯著的最佳質(zhì)粒,構(gòu)建沉默SGK3的鼻咽癌細胞。4.細胞生物學功能試驗:MTT比色法檢測轉(zhuǎn)染最佳質(zhì)粒后鼻咽癌細胞的增殖能力,流式細胞術(shù)檢測其凋亡水平,劃痕試驗檢測其遷移能力,存活率、凋亡率采用單因素的方差分析進行組間比較,劃痕試驗采用析因設計的方差分析。結(jié)果:1.免疫組化結(jié)果表明:SGK3的蛋白表達主要見于細胞質(zhì),在42例鼻咽癌組織中,SGK3蛋白的陽性表達率為90.5%,在9例慢性鼻咽炎組織中,SGK3蛋白的陽性表達率為11.1%,兩組差異具有統(tǒng)計學意義(P0.01)。但SGK3的蛋白表達水平與患者的性別、年齡、TNM分期以及臨床分期并無明顯相關(guān)性(P0.05)。2.Western blot結(jié)果表明:SGK3在各鼻咽癌細胞株的蛋白表達水平高于永生化鼻咽上皮NP69細胞株,其中CNE-2、HNE-1的SGK3蛋白表達與其他各組相比具有明顯統(tǒng)計學差異(P0.01)。3.成功構(gòu)建了沉默SGK3的鼻咽癌細胞CNE-2/shSGK3,證實了SGK3表達下調(diào)可明顯抑制鼻咽癌細胞的體外增殖、存活、遷移能力,并促進其凋亡水平。結(jié)論:1.SGK3在鼻咽癌組織和細胞中呈高表達,但其在組織中的蛋白表達水平與鼻咽癌患者的性別、年齡、TMN分期、臨床分期等臨床病理特征無明顯相關(guān)性,提示SGK3參與了鼻咽癌的發(fā)生。2.通過沉默SGK3的鼻咽癌細胞體外試驗,證實了SGK3的表達失調(diào)可以影響鼻咽癌細胞增殖、凋亡、遷移的生物學特性,提示SGK3可能是促進鼻咽癌進展的癌基因。
[Abstract]:Objective: Nasopharyngeal Carcinoma (NPC) is a kind of head and neck malignant tumor with regional distribution and racial aggregation characteristics. It occurs in southern and Southeast Asia. Nasopharyngeal carcinoma occurs in nasopharyngeal mucosa. The pathological type is mostly low differentiated squamous cell carcinoma with high malignancy, and cervical lymph nodes can appear in the early stage. Nasopharyngeal carcinoma is mostly sensitive to radiotherapy, and the integrated therapy based on conformal intensity modulated radiotherapy greatly improves the local control rate of nasopharyngeal carcinoma, but does not significantly reduce its distant metastasis rate. The main reason for the failure of the treatment is local recurrence and distant metastasis. Therefore, the mechanism of the occurrence and development of nasopharyngeal carcinoma is studied, and the changes in the mechanism of nasopharyngeal carcinoma are studied. Serum and glucocorticoid-inducible kinase (SGK) is a serine / threonine kinase, which is a member of the protein kinase B (Protein kinase B, PKB or AKT), which is highly homologous and regulates its class. The cell process, which participates in the regulation of cell proliferation and apoptosis, cell cycle and ion channel, is a member of the SGK family, a functional intercourse of cell phosphorylation cascade reaction and multiple cell signaling pathways, and its Thr256 site can be activated by PDK1 phosphorylation and is recognized as a non AKT dependent tumor signal transduction pathway. The study shows that SGK3 is highly expressed in many malignant tumors and plays an important role in the development of tumor, such as breast cancer, liver cancer, prostate cancer and so on. But the relationship between SGK3 and nasopharyngeal carcinoma has not yet been reported at home and abroad. This paper aims to study the expression of SGK3 in nasopharyngeal carcinoma and cells and SGK3 to nasopharyngeal carcinoma cells. The relationship between the expression of SGK3 and the development of nasopharyngeal carcinoma. Methods: 1. immunohistochemistry was used to detect the protein expression of SGK3 in 42 cases of nasopharyngeal carcinoma and 9 cases of chronic nasopharyngitis. The chi square test compared the differences in the expression of SGK3 egg white in the nasopharyngeal and chronic nasopharyngeal tissues, and the Logistic regression analysis The correlation between the expression level of SGK3 protein and the clinicopathological features of nasopharyngeal carcinoma patients.2.Western blot detection of SGK3 in nasopharyngeal carcinoma cell lines CNE-1, CNE-2, HNE-1, SUNE-1, HONE-1, and immortalized nasopharyngeal epithelial NP69 cell lines, and Image Pro software has a semi quantitative analysis of the expression intensity of protein phase and single factor variance. Analysis and comparison of the difference of the protein gray value in different cell lines.3.Lipofectamine 2000 transient transfection of nasopharyngeal carcinoma cells. After transfection for 24 hours to confirm the successful transfection under the fluorescence microscope, the expression of SGK3 in the transfected nasopharyngeal carcinoma cells was detected by Western blot for 24 hours, and the best plasmid with the most significant effect of SGK3 knockout was selected. The.4. cell biological function test of nasopharyngeal carcinoma cells with silent SGK3 was built. MTT colorimetric assay was used to detect the proliferation ability of the nasopharyngeal carcinoma cells after transfection of the best plasmid. Flow cytometry was used to detect the apoptosis level. The scratch test was used to detect the migration ability, survival rate and apoptosis rate by single factor difference analysis. The scratch test adopted factorial analysis. Results of variance analysis. Results: 1. immunohistochemical results showed that the protein expression of SGK3 was mainly found in the cytoplasm. The positive expression rate of SGK3 protein was 90.5% in 42 nasopharyngeal carcinoma tissues. The positive expression rate of SGK3 protein was 11.1% in 9 cases of chronic nasopharyngitis, and the difference between the two groups was statistically significant (P0.01). But the protein expression level of SGK3 was compared with that of SGK3. There was no significant correlation between the sex, age, TNM staging and clinical staging of the patients (P0.05).2.Western blot results showed that the protein expression level of SGK3 in each nasopharyngeal carcinoma cell line was higher than that of the immortalized nasopharyngeal epithelial NP69 cell line, and the SGK3 protein expression of CNE-2, HNE-1 was significantly different from that of the other groups (P0.01).3.. The silenced SGK3 nasopharyngeal carcinoma cell CNE-2/shSGK3 confirmed that the downregulation of SGK3 could significantly inhibit the proliferation, survival, migration and apoptosis of nasopharyngeal carcinoma cells in vitro. Conclusion: the expression of 1.SGK3 in nasopharyngeal carcinoma tissues and cells is highly expressed, but the expression level of egg white in the tissues is related to the sex, age, and TMN of nasopharyngeal cancer patients. There is no significant correlation between clinical and pathological features, such as clinical stage, suggesting that SGK3 participates in the occurrence of nasopharyngeal carcinoma in.2. through in vitro test of nasopharyngeal carcinoma cells with silent SGK3, which confirms that the dysregulation of SGK3 can affect the proliferation, apoptosis and migration of nasopharyngeal carcinoma cells, suggesting that SGK3 may be the oncogene that promotes the progression of nasopharyngeal carcinoma.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.63

【參考文獻】

相關(guān)期刊論文 前1條

1 李官成 ,謝鷺 ,周國華 ,孫去病 ,符紅普 ,周建華;用抗獨特型疫苗主動免疫治療鼻咽癌病人的臨床研究(英文)[J];Chinese Medical Journal;2002年04期

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本文編號：1850154