電壓門控鈉離子通道亞型Nav1.5的表達在口腔鱗癌細胞侵襲和轉移中作用的初步研究
發(fā)布時間：2018-12-13 05:04
【摘要】：目的口腔鱗狀細胞癌(oral squamous cell carcinoma,OSCC)是發(fā)生于人類口腔頜面部區(qū)域內,最多見、惡性程度較高、極易發(fā)生轉移的一種口腔癌。OSCC具有的易轉移特點是該病治療困難,致死率高的主要的原因。有針對性的分子靶向治療手段被越來越多學者在探尋遏制癌癥轉移方法中所重視。大量文獻報道,電壓門控鈉離子通道(voltage-gated sodium channels,VGSC)在上皮來源的轉移性癌細胞中出現(xiàn)異常的高表達且對癌細胞的擴散、侵襲、轉移等行為起促進作用,阻斷VGSC的通道活性可明顯降低癌細胞的轉移率,推測VGSC可能具有作為分子靶點用以遏制癌癥轉移的潛能。在對VGSC不同亞型與不同組織類型癌癥發(fā)生及轉移相關性的研究中,Nav1.5表現(xiàn)出較頻繁的功能作用,而在OSCC中Nav1.5是否也存在功能性表達未見報道。本研究通過檢測Nav1.5m RNA和蛋白在正�？谇火つそM織和有/無淋巴結轉移低分化OSCC組織中的表達情況,判斷Nav1.5的表達水平的強弱與OSCC細胞表現(xiàn)出的侵襲和轉移能力的高低是否有關,預測Nav1.5成為OSCC轉移的新型分子標記物和治療靶點的可能。方法收集安徽醫(yī)科大學第一附屬醫(yī)院口腔頜面外科26例患有原發(fā)性低分化OSCC的患者術中切除的腫瘤上皮組織作為試驗組,并按OSCC組織有無淋巴結轉移將試驗組分為無轉移組(16例)和有轉移組(10例)。另收集10例正�？谇火つそM織(如來源于頜面部外傷、拔牙等術中切除的上皮組織)作為對照組,采用免疫組化法、實時熒光定量PCR(Quantitative Real-time PCR,q PCR)、蛋白質印跡法、酶聯(lián)免疫吸附法(enzyme-linked immunosorbent assay,ELISA)分別檢測Nav1.5在對照組及試驗各組中m RNA或蛋白的表達水平。對試驗所測結果進行單因素方差分析,判斷差異是否有統(tǒng)計意義。結果在對照組、無轉移組和有轉移組中,q PCR法檢測Nav1.5m RNA的相對表達量分別為1.054±0.162、2.311±0.134、4.462±0.362,無轉移組和有轉移組均顯著高于與對照組(P=0.037;P=0.029),且有轉移組顯著高于無轉移組(P=0.031)。蛋白質印跡法檢測Nav1.5蛋白的相對表達量依次為0.080±0.010、0.143±0.005、0.253±0.015,無轉移組和有轉移組均顯著高于與對照組(P=0.034;P=0.026),且有轉移組較無轉移組表達更高(P=0.033)。免疫組化檢測結果顯示,Nav1.5蛋白的陽性表達率分別為對照組10%(1/10)、試驗組92.3%(24/26),差異有統(tǒng)計學意義(P=0.016)。有轉移組和無轉移組相比,Nav1.5蛋白的陽性表達差異亦有統(tǒng)計學意義(P=0.028)。ELISA法結果顯示:Nav1.5在正常、無轉移和有轉移三組中的表達結果分別為(0.834±0.103)μg/L、(1.578±0.167)μg/L和(3.882±0.422)μg/L。后兩組檢測結果均高于對照組(P=0.041;P=0.032),且兩組間亦有明顯的表達差異(P=0.030)。結論Nav1.5在低分化OSCC組織中高表達,且因OSCC組織是否伴淋巴結轉移其表達水平存在明顯差異。推測Nav1.5的表達可能促進OSCC細胞的侵襲和轉移,值得進一步探索。
[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 鹵0.162 鹵0.134 鹵4.462 鹵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 鹵0.010 ~ 0. 143 鹵0. 005 鹵0. 253 鹵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 鹵0.103) 渭 g / L, (1.578 鹵0.167) 渭 g / L and (3.882 鹵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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.8
本文編號：2375933
[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 鹵0.162 鹵0.134 鹵4.462 鹵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 鹵0.010 ~ 0. 143 鹵0. 005 鹵0. 253 鹵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 鹵0.103) 渭 g / L, (1.578 鹵0.167) 渭 g / L and (3.882 鹵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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.8
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