【作者】 胡守森；

【导师】 娄卫华；

【作者基本信息】 郑州大学 ， 耳鼻咽喉科学， 2010， 硕士

【摘要】 喉鳞状细胞癌(laryngeal squamous cell carcinoma, LSCC)是来源于喉粘膜上皮组织的恶性肿瘤,多见于中老年男性。其发生与吸烟、酗酒、长期吸入有害物质及乳头状瘤病毒感染等因素有关。在耳鼻喉科领域中仅次于鼻咽癌和鼻腔,鼻窦癌,居第三位,是耳鼻咽喉科常见的恶性肿瘤。发病率约占全身肿瘤的1-5%,好发年龄为45～75岁。男性发病率约是女性的10倍,城市明显高于农村。近年来其发病有逐渐升高及年轻化的趋势。对于单纯的喉癌病人,可以通过手术切除的方式取得较好的疗效,术后5年生存率可达到80%。而对于伴有颈部淋巴结转移的病人,即使通过颈部淋巴结清扫及术后放疗,5年生存率仅为46%,死亡率明显上升。肿瘤细胞的转移是一个非常复杂的过程。而趋化因子及受体与肿瘤细胞的恶性表现密切相关。趋化因子与肿瘤的关系具有两面性：一方面趋化因子能通过以自分泌或旁分泌的方式调节细胞因子的分泌,调节机体的免疫系统,逃避宿主抗肿瘤反应、趋化肿瘤细胞、促进肿瘤相关血管生成、消化细胞外基质、刺激肿瘤细胞生长等直接和间接作用促进肿瘤的增殖和转移；另一方面,趋化因子又可抑制血管生长,趋化免疫活性细胞浸润至肿瘤,激活宿主对肿瘤的特异性免疫应答来抑制肿瘤的增殖和转移,但其主要作用可能还是加速了肿瘤的生长和转移。尤其是CXCL12/CXCR4相互作用所构成的反应轴,在多种肿瘤的发生、生长增殖、浸润转移中发挥着重要的作用。趋化因子调控白细胞对肿瘤的浸润、引起机体免疫反应杀伤肿瘤细胞,促进肿瘤相关血管生成、以自分泌或旁分泌的方式刺激肿瘤细胞增殖、使肿瘤细胞突破基底膜发生侵袭,同时促使癌细胞的运动和远处转移,从而产生趋化运动和侵袭反应。肿瘤细胞高表达特异的趋化因子受体,而一些器官高表达其相应的趋化因子配体,肿瘤细胞就借助趋化因子与其受体的特异性结合力,向这些器官特异性的转移。在许多肿瘤中均发现CXCL12/CXCR4参与肿瘤的局部侵袭和器官特异性转移。目的通过观察趋化因子CXCL12及其受体CXCR4在喉鳞状细胞癌中的表达,探讨CXCL12-CXCR4生物轴与喉鳞癌的临床病理特征、肿瘤大小、侵袭转移行为和预后的关系,为临床治疗提供理论依据。材料与方法1、材料选取郑州大学第一附属医院耳鼻咽喉科2008年8月～2009年12月年手术切除标本77例,术后经病理检查确诊为喉鳞状细胞癌,病理分级：T分期：T1-T2期30人,T3-T4期47人；淋巴结：转移23人,无转移54人(TNM分类标准按国际抗癌协会UICC2002年方案)；分化程度：高分化41人,中、低分化36人(按病历资料病理报告分级)；其中另选取癌旁切缘5mm癌旁正常黏膜组织18例。所有病例术前均未行放疗、化疗及其他相关治疗,临床病理资料完整。2、方法采用免疫组化SP法分析CXCL12、CXCR4在喉癌组织及癌旁正常组织中的阳性表达。操作步骤按试剂盒上说明进行。评分标准参照文献,根据组织切片中阳性细胞百分数和着色强度进行半定量分级评分。阳性细胞百分数≤10%为0分,11%～25%为1分,26%～50%为2分,51%～75%为3分,≥76%为4分。着色强度：0分,不着色；1分,浅黄色；2分,黄色；3分,棕黄色。取上述两项分值的乘积作为总积分：0分为阴性(-),1-4分为弱阳性(+),6-8分为阳性(++),9～12分为强阳性(+++)。3、统计学处理采用SPSS13.0统计软件包进行数据分析,X2检验及Spearman等级相关分析。P<0.05说明有统计学意义。结果1、CXCR4在喉癌组织和正常组织中的表达77例喉癌组织中有49例CXCR4阳性表达,阳性表达率63.64%(49/77),CXCR4阳性显色为棕黄色至棕褐色颗粒,主要表达于喉癌组织细胞的胞质。正常对照组几乎无阳性表达。二者阳性率比较差异有统计学意义(X2=12.991, P<0.05),且两组阳性表达强度差异有统计学意义(X2=13.778,P<0.05)。喉鳞癌组织中的CXCR4阳性表达率与患者淋巴结转移有关,在喉鳞癌淋巴结转移阳性组中CXCR4阳性表达率为91.30%(21/23)明显高于淋巴结阴性组的51.85%(28/54),差异有统计学意义(P<0.05)；喉鳞癌组织中CXCR4阳性表达率与患者T分期有关,在T3、T4期患者组喉鳞癌组织中CXCR4阳性表达率为74.47%(35/47)明显高于TI、T：期患者组的46.67%(14/30),差异有统计学意义(P<0.05)。2、CXCL12在喉癌组织与正常组织中表达77例喉癌组织中有43例CXCL12阳性表达,34例阴性表达,阳性表达率55.84%(43/77),CXCL12阳性显色为棕黄色颗粒,表达于喉癌组织细胞的胞质和胞核,但以胞质表达为主。正常组织中CXCL12表达8例,阳性表达率44.44%(8/18)。两组阳性表达率没有统计学差异(X2=0.763,P>0.05),且两组阳性表达强度差异没有统计学意义(X2=5.861,P>0.05)。喉鳞癌组织中的CXCL12阳性表达率与患者淋巴结转移有关,在喉鳞癌淋巴结转移阳性组中CXCL12阳性表达率为73.91%(17/23)明显高于淋巴结阴性组的48.15%(26/54),差异有统计学意义(P<0.05)。3、喉鳞癌组织中CXCR4、CXCL12表达的相关性分析喉鳞癌组织中CXCR4表达与CXCL12表达经Spearman等级相关分析,二者呈正相关,即随CXCR4表达的增加,CXCL12高表达所占比例升高(r=0.252,p<0.05)。结论1、CXCR4可能与喉癌的发病相关,参与了肿瘤的发展和转移,能促进肿瘤细胞恶性分化和淋巴结转移。2、CXCL12可由组织的间质细胞分泌,CXCL12在癌旁淋巴结有高表达,是吸引表达CXCR4的肿瘤细胞逆浓度梯度,向特异淋巴结转移的重要因素。3、CXCL12/CXCR4在喉癌组织中的表达呈正相关(P<0.05),符合其配体-受体的特性。通过两者结合激活下游信号传导通路,促进细胞的生存和增殖,同时参与血管形成,促进肿瘤的侵袭和转移。更多还原

【Abstract】 BackgroundLaryngeal squamous cell carcinoma (LSCC) is a malignant tumor derived from the laryngeal mucosa epithelium. It occurs with smoking, alcoholism, long-term inhalation of harmful substances and papilloma virus infection and other factors. It is a common malignancy otorhinolaryngology in the ENT field with nasopharyngeal and nasal sinus cancer. The incidence of LSCC accounts for approximately 1～5%of systemic cancer, and it is most common between the age of 45 and 75 years. At present,men are more frequently affected than women. The LSCC’incidence of people in the city was significantly higher than in rural areas. In recent years, the incidence has gradually increased especially in the young man. For patients of definitive LSCC, A good therapeutic efficacy can be get through laryngectomy. The overall 5 year survival rates are 80%after laryngectomy. For those Patients with cervical lymph node metastasis,the overall 5 year survival rates are 46.2%after laryngectomy,even though through radical neck disseetion and postoperation radiotherapy. Mortality rate increased significantly. Metastasis of tumor cells is a very complicated process. The chemokine and its receptor are closely related to the performance of malignant tumor cells,especically the CXCL12/CXCR4 interactions,which play an important role in a variety of tumor’s growth, proliferation, invasion and metastasis. Chemokine regulate infiltration of leukocyte to the tumor, causing the immune response to kill the tumor cells and promoting of tumor-associated angiogenesis, to autocrine or paracrine manner in order to stimulate tumor cell proliferation, for tumor cells invasion through breaking the basement membrane,to promote cancer movement and distant metastasis. With high expression of tumor cell-specific chemokine receptors and high expression of their corresponding chemokine ligands in some organs,tumor cells transfer to these specific-organ through the binding specificity of chemokine and its receptor. In lots of tumors, CXCL12/CXCR4 were found involving in the tumor local invasion and organ-specific metastasis.ObjectiveBy observing the expression of chemokine CXCL12 and its receptor CXCR4 in laryngeal squamous cell carcinoma,to explore the CXCL12-CXCR4 biological axis and clinical pathological features of laryngeal squamous cell carcinoma, tumor size, invasion and metastasis of the relationship between behavior and prognosis,to provide a theoretical basis for the clinical treatment.Materials and Methods1. MaterialsSpecimens of squamous cell carcinoma were obtained from 77 outpatients in the department of Otorhinolaryngology from August 2008 to December 2009,in the First Affiliated Hospital of Zhengzhou University. It is confirmed to be squamous cell carcinoma after postoperative pathological examination. Selecting 18 cases from adjacent normal mucosa. The clinical and pathological information of all cases is complete,without radiotherapy, chemotherapy and any other treatment before operation.2、MethodsSP immunohistochemistry method was used to measure the expression of CXCL12, CXCR4 in human laryngeal carcinoma and adjacent normal tissue. We can get half quantitation ranking scores according to stain intensity. Percentage of positive cells≤10%:0 point.11%to 25%:1 point,26%to 50%:2 points,51%to 75%:3 points,≥76%as 4 points. Color intensity:no color:0 point, pale yellow,1 point; yellow,2 points; brown,3 points. Taking the product of these two scores as the total score:0 is divided into negative (-),1 to 4 were divided into weak positive (+),6 to 8 is divided into positive (++),9 to 12 are divided into strongly positive (+++).3、Statistical analysisSPSS 13.0 statistical package was used for data analysis and The data was analyzed by x2 test and Spearman rank correlation analysis. P<0.05 shows statistical significance.Results1、The expression of CXCR4 protein in carcinoma tissues and normal tissuesPositive expression rate of CXCR4 in 77 patients with laryngeal carcinoma was 63.64%(49/77). CXCR4 protein were located in the cytoplasm or/and cell membranes of laryngeal cancer cells, but not in the normal stromal cells. The positive expression rate of CXCR4 in laryngeal cancer cells was significant higher than that in normal controls (x2=12.991, P< 0.05). There was a significant difference in the positive rates of CXCR4 between the two groups (P<0.05). and the expression intensity between the two groups was statistically significant (x2=13.778,P 0.05). The expression of CXCR4 had significant relations with lymph node metastasis and T staging(P<0.05).2、The expression of CXCL12 protein in carcinoma tissues and normal tissuesPositive expression rate of CXCL12 in 77 patients with laryngeal carcinoma was 55.84%(43/77). CXCL12 protein expressed in the cytoplasm and nucleus of laryngeal squamous cell, but the mainly cytoplasm. Positive expression rate of CXCL12 in 10 normal patients was 44.44%(8/18). The positive expression rate of the two groups was not statistically significant (x2=0.763,P>0.05), and the intensity of positive expression between two groups was not statistically significant difference (x2=5.861,P>0.05). The expression of CXCL12 had significant relations with lymph node metastasis (P<0.05).3、The correlation analyse of expression of CXCR4, CXCL12 in LSCCPositive correlation was observed by Spearman rank correlation analysis between the expression of CXCR4, CXCL12 in Laryngeal squamous cell carcinoma.(r= 0.252, p<0.05).Conclusion1、The expression rate and intensity of positive expression of CXCR4 in laryngeal tissue aer significantly higher than the adjacent normal laryngeal tissue. It may be related to the growth and proliferation of laryngeal squamous cell carcinoma.2、The results show that CXCL12 protein is a evident in the expression of adjacent lymph nodes. The positive expression rate of CXCL12 between LSCC and the normal tissue is not statistically significant. Normal stromal cells can also secrete chemokines.3、There is a positive correlation between the expression of CXCL12 and CXCR4 in laryngeal carcinoma (P<0.05). Combination of CXCL12 and CXCR4 active the downstream signaling pathway, promotes cell survival and proliferation and tumor invasion and metastasis.更多还原