SGK3在鼻咽癌中的表达及其影响鼻咽癌细胞生物学特性的相关性研究

发布时间：2018-05-06 01:22

本文选题：鼻咽癌 + SGK3　；参考：《南方医科大学》2017年硕士论文

【摘要】：目的:鼻咽癌(Nasopharyngeal Carcinoma,NPC)是一种具有区域性分布以及种族聚集性特征的头颈部恶性肿瘤,好发于我国南方及东南亚地区。鼻咽癌发生于鼻咽部粘膜,其病理类型多为低分化鳞状细胞癌,恶性程度较高,早期即可出现颈部淋巴结转移。鼻咽癌大多对放疗有中度敏感性,以适形调强放疗为主的综合治疗极大地提高了鼻咽癌的局部控制率,却未能显著降低其远处转移率,治疗失败的主要原因是局部复发和远处转移。因此,深入研究鼻咽癌的发生与发展机制,对改善鼻咽癌治疗效果、提高患者生存率有重大意义。血清和糖皮质激素调节激酶(Serum and glucocorticoid-inducible kinase,SGK)是一种丝氨酸/苏氨酸激酶,与蛋白激酶B(Protein kinase B,PKB or AKT)同属于AGC蛋白激酶家族成员,与AKT高度同源并调节与其类似的细胞过程,参与调控细胞增殖凋亡、细胞周期以及离子通道等,是细胞磷酸化级联反应和多种细胞信号通路的一个功能性交汇点。SGK3是SGK家族的一员,其Thr256位点可被PDK1磷酸化而激活,被认为是一种非AKT依赖性的肿瘤信号传导方式。研究表明,SGK3在很多恶性肿瘤中高度表达,对肿瘤的发生、发展起着重要作用,如乳腺癌、肝癌、前列腺癌等,但SGK3与鼻咽癌的相关性研究目前国内外尚未见报道。本文旨在研究SGK3在鼻咽癌组织和细胞中的表达情况,以及SGK3对鼻咽癌细胞生物学特性的影响,探讨SGK3表达与鼻咽癌发生发展的关系。方法:1.免疫组化法检测SGK3在42例鼻咽癌组织和9例慢性鼻咽炎症组织中的蛋白表达,卡方检验比较鼻咽癌组织和慢性鼻咽炎组织中SGK3蛋白表达的组间差异,Logistic回归分析研究SGK3蛋白表达水平与鼻咽癌患者临床病理特征之间的相关性。2.Western blot检测SGK3在鼻咽癌细胞株CNE-1、CNE-2、HNE-1、SUNE-1、HONE-1及永生化鼻咽上皮NP69细胞株中的蛋白表达,Image Pro Plus软件对蛋白相对表达强度进行半定量分析,单因素方差分析比较不同细胞株中蛋白灰度值的组间差异。3.Lipofectamine 2000瞬时转染鼻咽癌细胞,转染24小时在荧光显微镜下确认转染成功后,再培养24小时行Western blot检测转染后鼻咽癌细胞中SGK3的表达情况,选取SGK3敲低效果最显著的最佳质粒,构建沉默SGK3的鼻咽癌细胞。4.细胞生物学功能试验:MTT比色法检测转染最佳质粒后鼻咽癌细胞的增殖能力,流式细胞术检测其凋亡水平,划痕试验检测其迁移能力,存活率、凋亡率采用单因素的方差分析进行组间比较,划痕试验采用析因设计的方差分析。结果:1.免疫组化结果表明:SGK3的蛋白表达主要见于细胞质,在42例鼻咽癌组织中,SGK3蛋白的阳性表达率为90.5%,在9例慢性鼻咽炎组织中,SGK3蛋白的阳性表达率为11.1%,两组差异具有统计学意义(P0.01)。但SGK3的蛋白表达水平与患者的性别、年龄、TNM分期以及临床分期并无明显相关性(P0.05)。2.Western blot结果表明:SGK3在各鼻咽癌细胞株的蛋白表达水平高于永生化鼻咽上皮NP69细胞株,其中CNE-2、HNE-1的SGK3蛋白表达与其他各组相比具有明显统计学差异(P0.01)。3.成功构建了沉默SGK3的鼻咽癌细胞CNE-2/shSGK3,证实了SGK3表达下调可明显抑制鼻咽癌细胞的体外增殖、存活、迁移能力,并促进其凋亡水平。结论:1.SGK3在鼻咽癌组织和细胞中呈高表达,但其在组织中的蛋白表达水平与鼻咽癌患者的性别、年龄、TMN分期、临床分期等临床病理特征无明显相关性,提示SGK3参与了鼻咽癌的发生。2.通过沉默SGK3的鼻咽癌细胞体外试验,证实了SGK3的表达失调可以影响鼻咽癌细胞增殖、凋亡、迁移的生物学特性,提示SGK3可能是促进鼻咽癌进展的癌基因。
[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.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.63

【参考文献】