NGX6蛋白在鼻咽癌中的表达及其与VEGF表达和LMVD的相关性研究
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摘要
第一部分鼻咽癌中NGX6蛋白的表达及其意义
目的:研究鼻咽癌中鼻咽癌相关基因6(nasopharyngeal carcinoma associatedgene 6,NGX6)在蛋白水平的表达,及其与年龄、性别、淋巴结转移以及T、N和临床分期等因素的关系。
方法:实验一采用EnVision二步法(Enhance Labelled Polymer System,ELPS法)进行免疫组化染色,检测20例鼻咽部非肿瘤性上皮组织(A组)、32例不伴颈部淋巴结转移的鼻咽癌组织(B组)及48例伴颈部淋巴结转移的鼻咽癌组织(C组)中NGX6蛋白的表达情况;结合患者年龄、性别、淋巴结转移、T、N分期和临床分期等多个因素,分析NGX6蛋白表达在鼻咽癌发生及发展进程中的意义。
实验二采用Western Blot法,检测4例鼻咽部非肿瘤性上皮组织(A组)、6例不伴颈部淋巴结转移的鼻咽癌组织(B组)及10例伴颈部淋巴结转移的鼻咽癌组织(C组)中NGX6蛋白的表达情况;分析NGX6蛋白表达与鼻咽癌淋巴结转移的关系。
结果:实验一中NGX6蛋白的免疫阳性产物为棕黄色颗粒,定位于鼻咽部上皮细胞和肿瘤细胞的细胞膜和细胞浆内。A、B、C三组中NGX6蛋白阳性表达率呈逐级下降趋势。非鼻咽癌组的NGX6蛋白阳性表达率为85%(17/20),明显高于鼻咽癌组的NGX6蛋白阳性表达率52.5%(42/80),差异有统计学意义(Χ2=6.9864,P<0.01);非转移鼻咽癌组的NGX6蛋白阳性表达率为68.75%(22/32),明显高于转移鼻咽癌组的NGX6蛋白阳性表达率41.67%(20/48),差异有统计学意义(Χ2=5.6475,P<0.025)。实验一鼻咽癌组织中T2期和T3/4期的NGX6蛋白阳性表达率(45.24%、18.18%)均较T1期(77.78%)明显降低(P<0.01,P<0.005);但是T2期和T3/4期鼻咽癌的NGX6蛋白阳性表达率无显著性差异(P>0.05)。N2/3期的NGX6蛋白阳性表达率(26.92%)较N0和N1期的NGX6蛋白阳性表达率(68.75%、59.09%)均明显降低(P<0.005,P<0.025);但是N0期和N1期鼻咽癌的NGX6蛋白阳性表达率无显著性差异(P>0.05)。Ⅲ/Ⅳ期的NGX6蛋白阳性表达率(36.37%)较Ⅰ/Ⅱ期(72.22%)明显降低(P<0.005)。此外,鼻咽癌中NGX6蛋白阳性表达率在不同性别及年龄间无显著差异(P>0.05)。
实验二A、B、C三组中NGX6蛋白的相对积分光密度值呈逐级下降趋势,三组间差异有显著性(F=38.02,Pr<0.0001);非鼻咽癌组的NGX6蛋白的相对积分光密度值(0.9098±0.2673)高于非转移鼻咽癌组(0.3218±0.2003)(P<0.05),而非转移鼻咽癌组的NGX6蛋白的相对积分光密度值(0.3218±0.2003)高于转移鼻咽癌组的NGX6蛋白的相对积分光密度值(0.0875±0.0432)(P<0.05)。
结论:鼻咽癌中NGX6在蛋白水平存在表达下调,并且其下调程度与淋巴结转移相关,鼻咽癌中NGX6蛋白表达越低,预示着淋巴结转移的发生率越高;鼻咽癌中NGX6蛋白的表达与淋巴结转移、T分期、N分期及临床分期均显著相关,但是与患者年龄和性别无关。因此,NGX6蛋白的检测有可能有助于鼻咽癌淋巴结转移风险的预测,并有利于预后的判断。
第二部分NGX6蛋白表达与LMVD的关系
目的:研究鼻咽组织中NGX6蛋白表达与微淋巴管密度(lymphatic microvessel density ,LMVD)的相关性,探讨NGX6对肿瘤淋巴管生成可能存在的调节作用。方法:材料来源同第一部分实验一。采用EnVision二步法进行D2-40的免疫组化染色,计算各样本的淋巴管密度;分析LMVD与NGX6蛋白表达的相关性。
结果: D2-40免疫阳性产物为棕黄色颗粒,定位于淋巴管内皮细胞的细胞浆中。A、B、C三组的LMVD值呈逐级上升趋势,非鼻咽癌组的LMVD值(3.45±1.94)低于非转移鼻咽癌组(6.02±2.96),非转移鼻咽癌组的LMVD值低于转移鼻咽癌组(10.99±5.10),三组间差异有显著性(F=29.98,Pr<0.0001)。NGX6蛋白表达与LMVD值存在明显的负相关(rs=-0.73221,P<0.0001),NGX6蛋白表达水平越高,LMVD值越小。
结论:鼻咽癌中LMVD增高,并且其增高程度与淋巴结转移相关,鼻咽癌中LMVD越高,预示着淋巴结转移的发生率越高;鼻咽癌中LMVD与NGX6蛋白表达呈显著负相关,提示NGX6的表达缺失和高LMVD在鼻咽癌的发展中起协同作用,且NGX6可能具有抑制肿瘤淋巴管生成的作用。
第三部分NGX6蛋白表达与VEGF蛋白表达的关系
目的:研究鼻咽组织中NGX6蛋白与血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的相关性,探讨NGX6对鼻咽癌细胞中VEGF蛋白表达可能存在的调节作用。
方法:实验一材料来源同第一部分实验一。采用EnVision二步法进行免疫组化染色,检测并分析各组鼻咽组织中VEGF蛋白的表达情况;分析VEGF蛋白表达与LMVD的相关性;分析VEGF蛋白表达与NGX6蛋白表达的相关性。
实验二材料来源同第一部分实验二。采用Western Blot法,检测并分析各组鼻咽组织中VEGF蛋白的表达情况;分析VEGF蛋白表达与NGX6蛋白表达的相关性。
结果:实验一中VEGF蛋白免疫阳性产物为棕黄色颗粒,定位于鼻咽部上皮细胞和肿瘤细胞的细胞浆内。A、B、C三组中VEGF蛋白阳性表达率呈逐级上升趋势。非
非鼻咽癌组的VEGF蛋白阳性表达率为55%(11/20),明显低于鼻咽癌组的阳性表达率86.25%(69/80),差异有统计学意义(Χ2=9.7656,P<0.005);非转移鼻咽癌组的VEGF蛋白阳性表达率为78.13%(25/32),明显低于转移鼻咽癌组的阳性表达率91.67%(44/48),但是差异无统计学意义(Χ2=2.9688,P>0.05)。VEGF蛋白表达与LMVD值存在明显的正相关(rs=0.73332,P<0.0001),VEGF蛋白表达水平越高,LMVD值越大。VEGF蛋白表达与NGX6蛋白表达呈显著负相关(rs=-0.59988,P<0.0001),NGX6蛋白表达水平越低,VEGF蛋白表达水平越高。
实验二中A、B、C三组中VEGF蛋白的相对积分光密度值呈逐级增加趋势,三组间差异有显著性(F=20.13,Pr<0.0001);非鼻咽癌组的VEGF蛋白的相对积分光密度值(0.5419±0.1993)低于非转移鼻咽癌组的相对积分光密度值(1.3748±0.366(P<0.05),非转移鼻咽癌组的VEGF蛋白的相对积分光密度值(1.3748±0.3665)低于转移鼻咽癌组的相对积分光密度值(1.9939±0.4506)(P<0.05)。VEGF蛋白与NGX6蛋白的相对积分光密度值呈显著负相关(r5)s=-0.80902,P<0.0001),随着NGX6蛋白相对积分光密度值越低,VEGF蛋白相对积分光密度值越高。
结论:鼻咽癌中存在VEGF蛋白过度表达,并且其表达程度与淋巴结转移相关,鼻咽癌中VEGF蛋白表达越高,预示着淋巴结转移的发生率越高;鼻咽癌中VEGF蛋白表达与NGX6蛋白表达呈负相关,提示NGX6的表达缺失和VEGF的高表达在鼻咽癌的发展中起协同作用,VEGF表达可能受抑癌基因NGX6的负调控;鼻咽癌中LMVD值与VEGF蛋白表达呈正相关,进一步验证了VEGF对肿瘤淋巴管生成的促进作用。因此,联合检测NGX6蛋白、VEGF蛋白和LMVD可能更加有助于鼻咽癌淋巴结转移风险的预测,以及预后的判断。
Objectives: To study the expression of NGX6(a kind of tumor suppressor gene)at protein level, in human nasopharyngeal carcinoma. To investigate the relationship between the expression of NGX6 and the clinical characteristics, such as age, sex lymph node metastasis,clinical stage and T、N-stages, in nasopharyngeal carcinoma.
Methods: In Experiment one, immunohistochemical technique (EnVision) was used to detect the expression of NGX6 in 20 cases of non-tumor nasopharyngeal epithelial tissue(group A), 32 cases of NPC tissues without lymph node metastasis(group B) and 48 cases of NPC tissues with lymph node metastasis (group C). Analysis the difference of expreesion level among three groups. Analysis the relationship between expressive level of NGX6 and clinicopathological features of the patients, including age, sex, lymph node metastasis, clinical stage and T、N-stages, respectively.
In Experiment two, Western Blot technique was used to detect the expression of NGX6 in 4 cases of non-tumor nasopharyngeal epithelial tissue(group A), 6 cases of NPC tissues without lymph node metastasis(group B) and 10 cases of NPC tissues with lymph node metastasis (group C). Analysis the relationship between expression of NGX6 and lymph node metastasis in NPC.
Results: In Experiment one, the immunoreactive products of NGX6 protein were stained into Buffy granules, located in cytoplasm and cell membrane of nasopharyngeal epithelial cells and tumor cells. The positive expression of NGX6 protein showed a downward trend in turn A, B, C group. The pos-expressive rate of NGX6 protein in non-tumor tissue(85%, 17/20) was significantly higher than that in NPC tissues (52.5%, 42/80)(Χ2=6.9864,P<0.01). The pos-expressive rate of NGX6 protein in NPC tissue without lymph node metastasis(68.75%, 22/32) was significantly higher than that in NPC tissues with lymph node metastasis(41.67%, 20/48)(Χ2=5.6475,P<0.025).
The pos-expressive rate expression of NGX6 protein in NPC tissue of T2 stage (45.2%) and NPC tissue of T3/4 stage (18.18%) were all significantly lower than that in NPC tissues of T1 stage (77.78%) (P<0.01,P<0.005). No significant diference was found between the expression of NGX6 protein in NPC tissue of T2 stage and of T3/4 stage(P>0.05). The pos-expressive rate of NGX6 protein in NPC tissue of N2/3 stage (26.92%) was significantly lower than that in both NPC tissues of N0 stage (68.75%) and NPC tissue of N1 stage (59.09%) (P<0.005,P<0.025). No significant diference was found between the expression of NGX6 protein in NPC tissue of N0 stage and of N1 stage (P>0.05). The pos-expressive rate of NGX6 protein in NPC tissue ofⅢ/Ⅳstage (36.37%) was significantly lower than that in NPC tissues ofⅠ/Ⅱstage (72.22%) (P<0.005). Besides, no significant diference was found in the expression of NGX6 protein in different age and sex (P>0.05,respectively).
In Experiment two, the opposing integral optical density value (ODV) of NGX6 protein showed downward trend in turn A, B, C group and significant diference in each other(F=38.02,Pr<0.0001). The opposing integral ODV of NGX6 protein in non-tumor tissue(0.9098±0.2673) was significantly higher than that in NPC tissues without lymph node metastasis (0.3218±0.2003) (P<0.05). The opposing integral ODV of NGX6 protein in NPC tissue without lymph node metastasis(0.3218±0.2003) was significantly higher than that in NPC tissues with lymph node metastasis(0.0875±0.0432) (P<0.05).
Conclusions: The expression of NGX6 was down regulated at protein level in nasopharyngeal carcinoma. Furthermore, the down regulation of NGX6 in NPC significantly correlated with lymph node metastasis. Lower expression of NGX6 in NPC indicated a higher incidence of lymph node metastasis. In NPC, the expression of NGX6 protein was significantly correlated to the clinicopathological characteristics, such as lymph node metastasis, T stages, N stages and clinical stages. Therefore, detection of the NGX6 protein might be used to predict metastasis of nasopharyngeal carcinoma and assess prognosis. But no significant correlation was found in the expression of NGX6 protein to both age and sex of patients.
Part two Correlation of NGX6 protein expression to LMVD
Objectives: To study the correlation of NGX6 protein expression to LMVD in nasopharyngeal epithelial tissue,then presume the possible function of NGX6 in regulating the formation of lymphatic vessel in tumor.
Methods: Immunohistochemical technique (EnVision) was used to detect the expression of D2-40 in each group (same to Part one-Experiment one). Calculate the LMVD of each sample. Analysis the difference of LMVD among three groups. Analysis the correlation of NGX6 protein expression to LMVD.
Results: The immunoreactive products of D2-40 were stained into Buffy granules, located in cytoplasm of lymphatic vessel endothelial cells. The LMVD showed a upward trend in turn A, B, C group and significant diference in each othe(rF=29.98,Pr<0.0001). The LMVD in non-tumor tissue(3.45±1.94) was significantly lower than that in NPC tissues without lymph node metastasis (6.02±2.96) (P<0.05). The LMVD in NPC tissue without lymph node metastasis(6.02±2.96) was significantly lower than that in NPC tissues with lymph node metastasis(10.99±5.10) (P<0.05).The LMVD showed a negative correlation to the expressive level of NGX6 (rs=-0.73221,P<0.0001). The more expression of NGX6, the less of LMVD.
Conclusions: The LMVD was high in nasopharyngeal carcinoma. Furthermore, the LMVD in NPC significantly correlated with lymph node metastasis. Higher LMVD in NPC indicated a higher incidence of lymph node metastasis. In NPC, the LMVD was significantly correlated to the expression of NGX6 protein. Then we suppose that, the lower expression of NGX6 and higher LMVD have a synergistic effect in advancing of NPC. The NGX6 protein might has depressant effect on the formation of lymphatic vessel.
Part three Correlation of NGX6 Expression to VEGF Expression
Objectives: To study the correlation of NGX6 protein expression to VEGF protein expression in nasopharyngeal epithelial tissue,then presume the possible function of NGX6 in regulating the expression of VEGF in NPC cell.
Methods: In Experiment one, immunohistochemical technique (EnVision) was used to detect the expression of VEGF in each group (same to Part one-Experiment one). Analysis the difference of expreesion level among three groups. Analysis the correlation of VEGF protein expression to LMVD. Analysis the correlation of NGX6 protein expression to VEGF protein expression.
In Experiment two, Western Blot technique was used to detect the expression of VEGF in each group (same to Part one-Experiment two). Analysis the difference of expreesion level among three groups. Analysis the correlation of NGX6 protein expression to VEGF protein expression.
Results: In Experiment one, the immunoreactive products of VEGF protein were stained into Buffy granules, located in cytoplasm of nasopharyngeal epithelial cells and tumor cells. The positive expression of VEGF protein showed a upward trend in turn A, B, C group. The pos-expressive rate of VEGF protein in non-tumor tissue(55%, 11/20) was significantly lower than that in NPC tissues (86.25%, 69/80)(Χ2=9.7656,P<0.005). The pos-expressive rate of VEGF protein in NPC tissue without lymph node metastasis(78.13%, 25/32) was significantly lower than that in NPC tissues with lymph node metastasis(91.67%, 44/48), but the difference was not significant(Χ2=2.9688,P>0.05). The LMVD showed a positive correlation to the expressive level of VEGF(rs=0.73332,P<0.0001). The more expression of VEGF, the more of LMVD. The expressive level of VEGF showed a negative correlation to the expressive level of NGX6(rs=-0.59988,P<0.0001). The less expression of NGX6, The more expression of VEGF.
In Experiment two, the opposing integral optical density value (ODV) of VEGF protein showed upward trend in turn A, B, C group and significant diference in each other(F=20.13,Pr<0.0001). The opposing integral ODV of VEGF protein in non-tumor tissue(0.5419±0.1993) was significantly lower than that in NPC tissues without lymph node metastasis (1.3748±0.3665) (P<0.05). The opposing integral ODV of VEGF protein in NPC tissue without lymph node metastasis(1.3748±0.3665) was significantly lower than that in NPC tissues with lymph node metastasis(1.9939±0.4506) (P<0.05). The expressive level of VEGF showed a negative correlation to the expressive level of NGX6(rs=-0.80902,P<0.0001). The less opposing integral ODV of NGX6, The more opposing integral ODV of VEGF.
Conclusions: The expression of VEGF protein was up regulated in nasopharyngeal carcinoma. Furthermore, the overexpression of VEGF protein in NPC significantly correlated with lymph node metastasis. Higher expression of VEGF in NPC indicated a higher incidence of lymph node metastasis. In NPC, the expression of VEGF protein was significantly negatively correlated to the expression of NGX6 protein. Then we suppose that, the lower expression of NGX6 and overexpression of VEGF have a synergistic effect in proceeding of NPC. VEGF expression might be negatively regulated by NGX6. The expression of VEGF protein was significantly correlated to the LMVD, which verify the promoting effect of VEGF on the formation of lymphatic vessel. Therefore, com-detection of the NGX6 protein , VEGF protein and LMVD might be more helpful to predict metastasis of nasopharyngeal carcinoma and assess prognosis.
目的:研究鼻咽癌中鼻咽癌相关基因6(nasopharyngeal carcinoma associatedgene 6,NGX6)在蛋白水平的表达,及其与年龄、性别、淋巴结转移以及T、N和临床分期等因素的关系。
方法:实验一采用EnVision二步法(Enhance Labelled Polymer System,ELPS法)进行免疫组化染色,检测20例鼻咽部非肿瘤性上皮组织(A组)、32例不伴颈部淋巴结转移的鼻咽癌组织(B组)及48例伴颈部淋巴结转移的鼻咽癌组织(C组)中NGX6蛋白的表达情况;结合患者年龄、性别、淋巴结转移、T、N分期和临床分期等多个因素,分析NGX6蛋白表达在鼻咽癌发生及发展进程中的意义。
实验二采用Western Blot法,检测4例鼻咽部非肿瘤性上皮组织(A组)、6例不伴颈部淋巴结转移的鼻咽癌组织(B组)及10例伴颈部淋巴结转移的鼻咽癌组织(C组)中NGX6蛋白的表达情况;分析NGX6蛋白表达与鼻咽癌淋巴结转移的关系。
结果:实验一中NGX6蛋白的免疫阳性产物为棕黄色颗粒,定位于鼻咽部上皮细胞和肿瘤细胞的细胞膜和细胞浆内。A、B、C三组中NGX6蛋白阳性表达率呈逐级下降趋势。非鼻咽癌组的NGX6蛋白阳性表达率为85%(17/20),明显高于鼻咽癌组的NGX6蛋白阳性表达率52.5%(42/80),差异有统计学意义(Χ2=6.9864,P<0.01);非转移鼻咽癌组的NGX6蛋白阳性表达率为68.75%(22/32),明显高于转移鼻咽癌组的NGX6蛋白阳性表达率41.67%(20/48),差异有统计学意义(Χ2=5.6475,P<0.025)。实验一鼻咽癌组织中T2期和T3/4期的NGX6蛋白阳性表达率(45.24%、18.18%)均较T1期(77.78%)明显降低(P<0.01,P<0.005);但是T2期和T3/4期鼻咽癌的NGX6蛋白阳性表达率无显著性差异(P>0.05)。N2/3期的NGX6蛋白阳性表达率(26.92%)较N0和N1期的NGX6蛋白阳性表达率(68.75%、59.09%)均明显降低(P<0.005,P<0.025);但是N0期和N1期鼻咽癌的NGX6蛋白阳性表达率无显著性差异(P>0.05)。Ⅲ/Ⅳ期的NGX6蛋白阳性表达率(36.37%)较Ⅰ/Ⅱ期(72.22%)明显降低(P<0.005)。此外,鼻咽癌中NGX6蛋白阳性表达率在不同性别及年龄间无显著差异(P>0.05)。
实验二A、B、C三组中NGX6蛋白的相对积分光密度值呈逐级下降趋势,三组间差异有显著性(F=38.02,Pr<0.0001);非鼻咽癌组的NGX6蛋白的相对积分光密度值(0.9098±0.2673)高于非转移鼻咽癌组(0.3218±0.2003)(P<0.05),而非转移鼻咽癌组的NGX6蛋白的相对积分光密度值(0.3218±0.2003)高于转移鼻咽癌组的NGX6蛋白的相对积分光密度值(0.0875±0.0432)(P<0.05)。
结论:鼻咽癌中NGX6在蛋白水平存在表达下调,并且其下调程度与淋巴结转移相关,鼻咽癌中NGX6蛋白表达越低,预示着淋巴结转移的发生率越高;鼻咽癌中NGX6蛋白的表达与淋巴结转移、T分期、N分期及临床分期均显著相关,但是与患者年龄和性别无关。因此,NGX6蛋白的检测有可能有助于鼻咽癌淋巴结转移风险的预测,并有利于预后的判断。
第二部分NGX6蛋白表达与LMVD的关系
目的:研究鼻咽组织中NGX6蛋白表达与微淋巴管密度(lymphatic microvessel density ,LMVD)的相关性,探讨NGX6对肿瘤淋巴管生成可能存在的调节作用。方法:材料来源同第一部分实验一。采用EnVision二步法进行D2-40的免疫组化染色,计算各样本的淋巴管密度;分析LMVD与NGX6蛋白表达的相关性。
结果: D2-40免疫阳性产物为棕黄色颗粒,定位于淋巴管内皮细胞的细胞浆中。A、B、C三组的LMVD值呈逐级上升趋势,非鼻咽癌组的LMVD值(3.45±1.94)低于非转移鼻咽癌组(6.02±2.96),非转移鼻咽癌组的LMVD值低于转移鼻咽癌组(10.99±5.10),三组间差异有显著性(F=29.98,Pr<0.0001)。NGX6蛋白表达与LMVD值存在明显的负相关(rs=-0.73221,P<0.0001),NGX6蛋白表达水平越高,LMVD值越小。
结论:鼻咽癌中LMVD增高,并且其增高程度与淋巴结转移相关,鼻咽癌中LMVD越高,预示着淋巴结转移的发生率越高;鼻咽癌中LMVD与NGX6蛋白表达呈显著负相关,提示NGX6的表达缺失和高LMVD在鼻咽癌的发展中起协同作用,且NGX6可能具有抑制肿瘤淋巴管生成的作用。
第三部分NGX6蛋白表达与VEGF蛋白表达的关系
目的:研究鼻咽组织中NGX6蛋白与血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的相关性,探讨NGX6对鼻咽癌细胞中VEGF蛋白表达可能存在的调节作用。
方法:实验一材料来源同第一部分实验一。采用EnVision二步法进行免疫组化染色,检测并分析各组鼻咽组织中VEGF蛋白的表达情况;分析VEGF蛋白表达与LMVD的相关性;分析VEGF蛋白表达与NGX6蛋白表达的相关性。
实验二材料来源同第一部分实验二。采用Western Blot法,检测并分析各组鼻咽组织中VEGF蛋白的表达情况;分析VEGF蛋白表达与NGX6蛋白表达的相关性。
结果:实验一中VEGF蛋白免疫阳性产物为棕黄色颗粒,定位于鼻咽部上皮细胞和肿瘤细胞的细胞浆内。A、B、C三组中VEGF蛋白阳性表达率呈逐级上升趋势。非
非鼻咽癌组的VEGF蛋白阳性表达率为55%(11/20),明显低于鼻咽癌组的阳性表达率86.25%(69/80),差异有统计学意义(Χ2=9.7656,P<0.005);非转移鼻咽癌组的VEGF蛋白阳性表达率为78.13%(25/32),明显低于转移鼻咽癌组的阳性表达率91.67%(44/48),但是差异无统计学意义(Χ2=2.9688,P>0.05)。VEGF蛋白表达与LMVD值存在明显的正相关(rs=0.73332,P<0.0001),VEGF蛋白表达水平越高,LMVD值越大。VEGF蛋白表达与NGX6蛋白表达呈显著负相关(rs=-0.59988,P<0.0001),NGX6蛋白表达水平越低,VEGF蛋白表达水平越高。
实验二中A、B、C三组中VEGF蛋白的相对积分光密度值呈逐级增加趋势,三组间差异有显著性(F=20.13,Pr<0.0001);非鼻咽癌组的VEGF蛋白的相对积分光密度值(0.5419±0.1993)低于非转移鼻咽癌组的相对积分光密度值(1.3748±0.366(P<0.05),非转移鼻咽癌组的VEGF蛋白的相对积分光密度值(1.3748±0.3665)低于转移鼻咽癌组的相对积分光密度值(1.9939±0.4506)(P<0.05)。VEGF蛋白与NGX6蛋白的相对积分光密度值呈显著负相关(r5)s=-0.80902,P<0.0001),随着NGX6蛋白相对积分光密度值越低,VEGF蛋白相对积分光密度值越高。
结论:鼻咽癌中存在VEGF蛋白过度表达,并且其表达程度与淋巴结转移相关,鼻咽癌中VEGF蛋白表达越高,预示着淋巴结转移的发生率越高;鼻咽癌中VEGF蛋白表达与NGX6蛋白表达呈负相关,提示NGX6的表达缺失和VEGF的高表达在鼻咽癌的发展中起协同作用,VEGF表达可能受抑癌基因NGX6的负调控;鼻咽癌中LMVD值与VEGF蛋白表达呈正相关,进一步验证了VEGF对肿瘤淋巴管生成的促进作用。因此,联合检测NGX6蛋白、VEGF蛋白和LMVD可能更加有助于鼻咽癌淋巴结转移风险的预测,以及预后的判断。
Objectives: To study the expression of NGX6(a kind of tumor suppressor gene)at protein level, in human nasopharyngeal carcinoma. To investigate the relationship between the expression of NGX6 and the clinical characteristics, such as age, sex lymph node metastasis,clinical stage and T、N-stages, in nasopharyngeal carcinoma.
Methods: In Experiment one, immunohistochemical technique (EnVision) was used to detect the expression of NGX6 in 20 cases of non-tumor nasopharyngeal epithelial tissue(group A), 32 cases of NPC tissues without lymph node metastasis(group B) and 48 cases of NPC tissues with lymph node metastasis (group C). Analysis the difference of expreesion level among three groups. Analysis the relationship between expressive level of NGX6 and clinicopathological features of the patients, including age, sex, lymph node metastasis, clinical stage and T、N-stages, respectively.
In Experiment two, Western Blot technique was used to detect the expression of NGX6 in 4 cases of non-tumor nasopharyngeal epithelial tissue(group A), 6 cases of NPC tissues without lymph node metastasis(group B) and 10 cases of NPC tissues with lymph node metastasis (group C). Analysis the relationship between expression of NGX6 and lymph node metastasis in NPC.
Results: In Experiment one, the immunoreactive products of NGX6 protein were stained into Buffy granules, located in cytoplasm and cell membrane of nasopharyngeal epithelial cells and tumor cells. The positive expression of NGX6 protein showed a downward trend in turn A, B, C group. The pos-expressive rate of NGX6 protein in non-tumor tissue(85%, 17/20) was significantly higher than that in NPC tissues (52.5%, 42/80)(Χ2=6.9864,P<0.01). The pos-expressive rate of NGX6 protein in NPC tissue without lymph node metastasis(68.75%, 22/32) was significantly higher than that in NPC tissues with lymph node metastasis(41.67%, 20/48)(Χ2=5.6475,P<0.025).
The pos-expressive rate expression of NGX6 protein in NPC tissue of T2 stage (45.2%) and NPC tissue of T3/4 stage (18.18%) were all significantly lower than that in NPC tissues of T1 stage (77.78%) (P<0.01,P<0.005). No significant diference was found between the expression of NGX6 protein in NPC tissue of T2 stage and of T3/4 stage(P>0.05). The pos-expressive rate of NGX6 protein in NPC tissue of N2/3 stage (26.92%) was significantly lower than that in both NPC tissues of N0 stage (68.75%) and NPC tissue of N1 stage (59.09%) (P<0.005,P<0.025). No significant diference was found between the expression of NGX6 protein in NPC tissue of N0 stage and of N1 stage (P>0.05). The pos-expressive rate of NGX6 protein in NPC tissue ofⅢ/Ⅳstage (36.37%) was significantly lower than that in NPC tissues ofⅠ/Ⅱstage (72.22%) (P<0.005). Besides, no significant diference was found in the expression of NGX6 protein in different age and sex (P>0.05,respectively).
In Experiment two, the opposing integral optical density value (ODV) of NGX6 protein showed downward trend in turn A, B, C group and significant diference in each other(F=38.02,Pr<0.0001). The opposing integral ODV of NGX6 protein in non-tumor tissue(0.9098±0.2673) was significantly higher than that in NPC tissues without lymph node metastasis (0.3218±0.2003) (P<0.05). The opposing integral ODV of NGX6 protein in NPC tissue without lymph node metastasis(0.3218±0.2003) was significantly higher than that in NPC tissues with lymph node metastasis(0.0875±0.0432) (P<0.05).
Conclusions: The expression of NGX6 was down regulated at protein level in nasopharyngeal carcinoma. Furthermore, the down regulation of NGX6 in NPC significantly correlated with lymph node metastasis. Lower expression of NGX6 in NPC indicated a higher incidence of lymph node metastasis. In NPC, the expression of NGX6 protein was significantly correlated to the clinicopathological characteristics, such as lymph node metastasis, T stages, N stages and clinical stages. Therefore, detection of the NGX6 protein might be used to predict metastasis of nasopharyngeal carcinoma and assess prognosis. But no significant correlation was found in the expression of NGX6 protein to both age and sex of patients.
Part two Correlation of NGX6 protein expression to LMVD
Objectives: To study the correlation of NGX6 protein expression to LMVD in nasopharyngeal epithelial tissue,then presume the possible function of NGX6 in regulating the formation of lymphatic vessel in tumor.
Methods: Immunohistochemical technique (EnVision) was used to detect the expression of D2-40 in each group (same to Part one-Experiment one). Calculate the LMVD of each sample. Analysis the difference of LMVD among three groups. Analysis the correlation of NGX6 protein expression to LMVD.
Results: The immunoreactive products of D2-40 were stained into Buffy granules, located in cytoplasm of lymphatic vessel endothelial cells. The LMVD showed a upward trend in turn A, B, C group and significant diference in each othe(rF=29.98,Pr<0.0001). The LMVD in non-tumor tissue(3.45±1.94) was significantly lower than that in NPC tissues without lymph node metastasis (6.02±2.96) (P<0.05). The LMVD in NPC tissue without lymph node metastasis(6.02±2.96) was significantly lower than that in NPC tissues with lymph node metastasis(10.99±5.10) (P<0.05).The LMVD showed a negative correlation to the expressive level of NGX6 (rs=-0.73221,P<0.0001). The more expression of NGX6, the less of LMVD.
Conclusions: The LMVD was high in nasopharyngeal carcinoma. Furthermore, the LMVD in NPC significantly correlated with lymph node metastasis. Higher LMVD in NPC indicated a higher incidence of lymph node metastasis. In NPC, the LMVD was significantly correlated to the expression of NGX6 protein. Then we suppose that, the lower expression of NGX6 and higher LMVD have a synergistic effect in advancing of NPC. The NGX6 protein might has depressant effect on the formation of lymphatic vessel.
Part three Correlation of NGX6 Expression to VEGF Expression
Objectives: To study the correlation of NGX6 protein expression to VEGF protein expression in nasopharyngeal epithelial tissue,then presume the possible function of NGX6 in regulating the expression of VEGF in NPC cell.
Methods: In Experiment one, immunohistochemical technique (EnVision) was used to detect the expression of VEGF in each group (same to Part one-Experiment one). Analysis the difference of expreesion level among three groups. Analysis the correlation of VEGF protein expression to LMVD. Analysis the correlation of NGX6 protein expression to VEGF protein expression.
In Experiment two, Western Blot technique was used to detect the expression of VEGF in each group (same to Part one-Experiment two). Analysis the difference of expreesion level among three groups. Analysis the correlation of NGX6 protein expression to VEGF protein expression.
Results: In Experiment one, the immunoreactive products of VEGF protein were stained into Buffy granules, located in cytoplasm of nasopharyngeal epithelial cells and tumor cells. The positive expression of VEGF protein showed a upward trend in turn A, B, C group. The pos-expressive rate of VEGF protein in non-tumor tissue(55%, 11/20) was significantly lower than that in NPC tissues (86.25%, 69/80)(Χ2=9.7656,P<0.005). The pos-expressive rate of VEGF protein in NPC tissue without lymph node metastasis(78.13%, 25/32) was significantly lower than that in NPC tissues with lymph node metastasis(91.67%, 44/48), but the difference was not significant(Χ2=2.9688,P>0.05). The LMVD showed a positive correlation to the expressive level of VEGF(rs=0.73332,P<0.0001). The more expression of VEGF, the more of LMVD. The expressive level of VEGF showed a negative correlation to the expressive level of NGX6(rs=-0.59988,P<0.0001). The less expression of NGX6, The more expression of VEGF.
In Experiment two, the opposing integral optical density value (ODV) of VEGF protein showed upward trend in turn A, B, C group and significant diference in each other(F=20.13,Pr<0.0001). The opposing integral ODV of VEGF protein in non-tumor tissue(0.5419±0.1993) was significantly lower than that in NPC tissues without lymph node metastasis (1.3748±0.3665) (P<0.05). The opposing integral ODV of VEGF protein in NPC tissue without lymph node metastasis(1.3748±0.3665) was significantly lower than that in NPC tissues with lymph node metastasis(1.9939±0.4506) (P<0.05). The expressive level of VEGF showed a negative correlation to the expressive level of NGX6(rs=-0.80902,P<0.0001). The less opposing integral ODV of NGX6, The more opposing integral ODV of VEGF.
Conclusions: The expression of VEGF protein was up regulated in nasopharyngeal carcinoma. Furthermore, the overexpression of VEGF protein in NPC significantly correlated with lymph node metastasis. Higher expression of VEGF in NPC indicated a higher incidence of lymph node metastasis. In NPC, the expression of VEGF protein was significantly negatively correlated to the expression of NGX6 protein. Then we suppose that, the lower expression of NGX6 and overexpression of VEGF have a synergistic effect in proceeding of NPC. VEGF expression might be negatively regulated by NGX6. The expression of VEGF protein was significantly correlated to the LMVD, which verify the promoting effect of VEGF on the formation of lymphatic vessel. Therefore, com-detection of the NGX6 protein , VEGF protein and LMVD might be more helpful to predict metastasis of nasopharyngeal carcinoma and assess prognosis.
引文
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