电压门控钠离子通道亚型Nav1.5的表达在口腔鳞癌细胞侵袭和转移中作用的初步研究

发布时间：2018-12-13 05:04

【摘要】：目的口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)是发生于人类口腔颌面部区域内,最多见、恶性程度较高、极易发生转移的一种口腔癌。OSCC具有的易转移特点是该病治疗困难,致死率高的主要的原因。有针对性的分子靶向治疗手段被越来越多学者在探寻遏制癌症转移方法中所重视。大量文献报道,电压门控钠离子通道(voltage-gated sodium channels,VGSC)在上皮来源的转移性癌细胞中出现异常的高表达且对癌细胞的扩散、侵袭、转移等行为起促进作用,阻断VGSC的通道活性可明显降低癌细胞的转移率,推测VGSC可能具有作为分子靶点用以遏制癌症转移的潜能。在对VGSC不同亚型与不同组织类型癌症发生及转移相关性的研究中,Nav1.5表现出较频繁的功能作用,而在OSCC中Nav1.5是否也存在功能性表达未见报道。本研究通过检测Nav1.5m RNA和蛋白在正常口腔黏膜组织和有/无淋巴结转移低分化OSCC组织中的表达情况,判断Nav1.5的表达水平的强弱与OSCC细胞表现出的侵袭和转移能力的高低是否有关,预测Nav1.5成为OSCC转移的新型分子标记物和治疗靶点的可能。方法收集安徽医科大学第一附属医院口腔颌面外科26例患有原发性低分化OSCC的患者术中切除的肿瘤上皮组织作为试验组,并按OSCC组织有无淋巴结转移将试验组分为无转移组(16例)和有转移组(10例)。另收集10例正常口腔黏膜组织(如来源于颌面部外伤、拔牙等术中切除的上皮组织)作为对照组,采用免疫组化法、实时荧光定量PCR(Quantitative Real-time PCR,q PCR)、蛋白质印迹法、酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)分别检测Nav1.5在对照组及试验各组中m RNA或蛋白的表达水平。对试验所测结果进行单因素方差分析,判断差异是否有统计意义。结果在对照组、无转移组和有转移组中,q PCR法检测Nav1.5m RNA的相对表达量分别为1.054±0.162、2.311±0.134、4.462±0.362,无转移组和有转移组均显著高于与对照组(P=0.037;P=0.029),且有转移组显著高于无转移组(P=0.031)。蛋白质印迹法检测Nav1.5蛋白的相对表达量依次为0.080±0.010、0.143±0.005、0.253±0.015,无转移组和有转移组均显著高于与对照组(P=0.034;P=0.026),且有转移组较无转移组表达更高(P=0.033)。免疫组化检测结果显示,Nav1.5蛋白的阳性表达率分别为对照组10%(1/10)、试验组92.3%(24/26),差异有统计学意义(P=0.016)。有转移组和无转移组相比,Nav1.5蛋白的阳性表达差异亦有统计学意义(P=0.028)。ELISA法结果显示:Nav1.5在正常、无转移和有转移三组中的表达结果分别为(0.834±0.103)μg/L、(1.578±0.167)μg/L和(3.882±0.422)μg/L。后两组检测结果均高于对照组(P=0.041;P=0.032),且两组间亦有明显的表达差异(P=0.030)。结论Nav1.5在低分化OSCC组织中高表达,且因OSCC组织是否伴淋巴结转移其表达水平存在明显差异。推测Nav1.5的表达可能促进OSCC细胞的侵袭和转移,值得进一步探索。
[Abstract]:Objective oral squamous cell carcinoma (oral squamous cell carcinoma,OSCC) is a kind of oral squamous cell carcinoma, which occurs in the oral and maxillofacial region of human being. The most common, malignant and easily metastasized oral squamous cell carcinoma (OSCC) is characterized by its difficulty in treatment. The main cause of high mortality. Targeted molecular targeted therapy has been paid more and more attention by more and more scholars in seeking ways to curb cancer metastasis. It has been reported that voltage-gated sodium channel (voltage-gated sodium channels,VGSC) is highly expressed in epithelial metastatic cancer cells and promotes the proliferation, invasion and metastasis of cancer cells. Blocking the channel activity of VGSC can significantly reduce the metastasis rate of cancer cells. It is speculated that VGSC may have the potential as a molecular target to inhibit cancer metastasis. In the study of the relationship between different subtypes of VGSC and carcinogenesis and metastasis of different tissue types, Nav1.5 showed frequent functional effects, but whether there was functional expression of Nav1.5 in OSCC was not reported. In this study, we examined the expression of Nav1.5m RNA and protein in normal oral mucosa and poorly differentiated OSCC with and without lymph node metastasis. To determine whether the expression of Nav1.5 is related to the ability of invasion and metastasis of OSCC cells, and to predict the possibility of Nav1.5 becoming a new molecular marker and therapeutic target for OSCC metastasis. Methods 26 patients of oral and maxillofacial surgery in the first affiliated Hospital of Anhui Medical University who had primary poorly differentiated OSCC underwent intraoperative excision of tumor epithelium as experimental group. The experimental group was divided into no metastasis group (16 cases) and metastatic group (10 cases) according to whether OSCC tissue had lymph node metastasis or not. Another 10 cases of normal oral mucosal tissues (such as maxillofacial trauma, extraction of teeth, etc.) were collected as control group. Immunohistochemical method and real-time fluorescent quantitative PCR (Quantitative Real-time PCR,q PCR), Western blot were used. Enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay,ELISA) was used to detect the expression of m RNA or protein in control group and experimental group. The single factor ANOVA was used to determine whether the difference was statistically significant. Results the relative expression of Nav1.5m RNA detected by, q PCR method was 1.054 卤0.162 卤0.134 卤4.462 卤0.362 in the control group, no metastasis group and no metastasis group, respectively, and was significantly higher than that in the control group (P0. 037). The group with metastasis was significantly higher than the group without metastasis (P0. 031). The relative expression of Nav1.5 protein detected by Western blotting was 0.080 卤0.010 ~ 0. 143 卤0. 005 卤0. 253 卤0. 015, which was significantly higher in non metastasis group and without metastasis group than in control group (P0. 034). The expression of P0. 026 in the group with metastasis was higher than that in the group without metastasis (P0. 033). Immunohistochemical results showed that the positive expression rates of Nav1.5 protein were 10% (1 / 10) in control group and 92.3% (24 / 26) in test group respectively. The difference was statistically significant (P0. 016). The positive expression of Nav1.5 protein in the metastatic group was significantly different from that in the non-metastatic group (P < 0. 028). ELISA method). The results of expression were (0.834 卤0.103) 渭 g / L, (1.578 卤0.167) 渭 g / L and (3.882 卤0.422) 渭 g / L, respectively. The results of the latter two groups were higher than those of the control group (P < 0.041), and there was a significant difference between the two groups (P0. 030). Conclusion Nav1.5 is highly expressed in poorly differentiated OSCC tissues, and there is significant difference in the expression level of OSCC with lymph node metastasis. It is speculated that the expression of Nav1.5 may promote the invasion and metastasis of OSCC cells, which is worthy of further exploration.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8

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