【作者】 崔晓峰；

【导师】 陈晓光；

【作者基本信息】 南方医科大学 ， 病原生物学， 2008， 博士

【摘要】 喉癌是我国北方地区头颈部最常见的恶性肿瘤，占全身恶性肿瘤的5．7％-7．6％，其病理类型以鳞状细胞癌为主，占全部喉癌的93％-99％。随着现代工业的发展和大气污染的加重，喉癌发病率一直呈明显增长趋势。喉癌发病率在世界各地差异很大，据报告意大利的瓦雷泽地区、巴西的圣保罗地区、印度的孟买地区属世界三大高发地区。我国的喉癌发病率地区差别也很大，东北地区发病率最高。喉癌的发病率城市高于农村，空气污染重的重工业城市高于污染轻的轻工业城市。发病年龄50-70岁为最多，35岁以下较为少见，男性喉癌发病率显著高于女性，吸烟为喉癌的主要患病因素之一。近年来随着诊断和治疗水平的不断进步，对喉癌患者术后的5年生存率、喉癌术后功能的修复和术后生存质量都有很大的提高，但是对于晚期喉癌患者的治疗效果仍不尽如人意。因此，对于喉癌发病相关因素的探讨，以期早期发现、早期治疗并提高临床疗效已成为临床研究的重点之一。细胞周期蛋白（Cyclin）是真核细胞细胞周期循环中主要的调节因子，控制细胞周期的进程，其表达水平具有细胞周期依赖性，是调节细胞活动的重要蛋白质。在人类肿瘤细胞中，已经发现多种细胞周期蛋白的异常表达。细胞周期蛋白（Cyclin G）是近年来新发现的一种周期蛋白，研究表明周期蛋白G的异常表达与多种肿瘤的发生密切相关。目前关于Cyclin G在喉癌中表达的报道甚少，与临床病理特征的关系尚无公认的结论。喉癌组织中cyclin G表达与微血管密度（MVD）关系的研究尚未见报道。为此，我们采用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶连结法（streptavidin-perosidase,SP）方法同步检测了81例喉鳞癌组织、20例癌旁正常喉粘膜组织和20例喉良性病变的Cyclin G1和Cyclin G2的表达，应用SPSS12．0统计软件，分别探讨两种蛋白的表达与喉鳞癌患者年龄、性别、临床分期、临床分型、肿瘤分化程度以及预后等的关系。采用逆转录聚合酶链反应（reversetranscription-polymerise chain reaction，RT-PCR）检测了40例喉鳞癌组织，10例癌旁正常喉粘膜和10例喉良性病变组织中的cyclin G1 mRNA和cyclin G2 mRNA的表达，通过RT-PCR半定量法研究cyclin G1 mRNA和cyclin G2 mRNA在喉鳞癌组织中的表达情况以及cyclin G1mRNA和cyclin G2 mRNA的表达强度与喉鳞癌患者的各种临床、病理指标之间的关系。探讨cyclin G1 mRNA和cyclin G2 mRNA在喉鳞癌中的表达及其临床意义。肿瘤血管的生成与恶性肿瘤的发生、发展、侵袭、转移及复发关系密切，在恶性肿瘤的诊断、预后及治疗中发挥重要作用。本文通过采用CD₃₄抗体标记血管内皮细胞，应用免疫组化SP法方法研究CD₃₄、Cyclin G1、Cyclin G2蛋白在喉鳞癌组织中的表达，计算CD₃₄标记的微血管密度（MVD），并与癌旁正常喉粘膜和喉良性病变组织中的表达作对比分析，评价微血管密度的临床意义，比较分析MVD与Cyclin G1、Cyclin G2蛋白之间的关系，探讨Cyclin G1、CyclinG2蛋白在喉鳞癌血管生成中的关系及临床病理意义，以期为Cyclin G1、CyclinG2在喉鳞癌的诊断、预后和治疗中的应用，为喉鳞癌综合治疗中的抗血管生成和基因治疗研究及应用提供一定的理论基础和实验依据。一、材料1病例选择1．1第一部分和第三部分收集2000年1月～12月间解放军463医院及解放军总医院根治性手术切除的喉鳞癌标本94例，其中13例因切片或免疫组化过程中出现缺失而排除除外，对81例进行研究，其中解放军463医院71例，解放军总医院10例，所有标本均经病理确诊为鳞状细胞癌。其中男71例，女10例；患者年龄36-82岁，平均年龄54．8岁；肿瘤发生部位：声门上型18例、声门型61例、声门下型2例；病理学分级：Ⅰ级42例，Ⅱ级30例，Ⅲ级9例；按国际抗癌联盟（UICC）1997年TNM分期标准进行临床分期：Ⅰ期15例，Ⅱ期24例，Ⅲ期29例，Ⅳ期13例；颈部淋巴结转移者13例，无颈淋巴结转移者68例；随访过程中复发21例；术前未行放疗、化疗，所有病例均获访，随访时间>5年。另收集癌旁正常喉粘膜组织块20例（取材部位距离癌组织2cm以上）及同期门诊手术病例，喉良性病变（10例声带息肉，10例声带乳头状瘤），均经病理确诊。1．2第二部分收集2000年1月～12月间解放军463医院根治性手术切除的喉鳞癌新鲜标本40例，所有标本均经病理活检确诊为鳞状细胞癌，其中男31例，女9例；患者年龄39-82岁，平均年龄60．8岁；肿瘤发生部位：声门上型10例、声门型28例、声门下型2例；病理学分级：Ⅰ级11例，Ⅱ级19例，Ⅲ级10例；按国际抗癌联盟（UICC）1997年TNM分期标准进行临床分期：Ⅰ期5例，Ⅱ期11例，Ⅲ期18例，Ⅳ期6例；颈部淋巴结转移者13例，无颈淋巴结转移者27例；术前未行放疗、化疗。另收集癌旁正常喉粘膜组织块10例（取材部位距离癌组织2cm以上）和同期门珍手术病例10例，其中喉良性病变（9例声带息肉，1例声带乳头状瘤），均经病理确诊。所有新鲜组织液氮速冻后置-70℃低温冰箱备用。二、方法2．1免疫组化法检测Cyclin G1和Cyclin G2蛋白表达及CD₃₄标记的血管内皮细胞。应用免疫组化SP法研究，鼠抗人Cyclin G1单克隆抗体、兔抗人Cyclin G2多克隆抗体，购自北京中杉金桥生物技术有限公司（Santa Cruz公司产品）、鼠抗人CD₃₄单克隆抗体及SP试剂盒均为北京中杉金桥生物技术有限公司产品。实验步骤按试剂盒说明书进行。以正常血清置换一抗作为空白对照，用已知阳性的乳腺癌组织切片作为阳性对照。Cyclin G1阳性物质分布在细胞核内，呈棕黄色颗粒。Cyclin G2阳性物质主要分布在细胞胞浆内，也有细胞浆、细胞核同时表达或单独存在细胞核中，呈棕黄色颗粒。CD₃₄染色，内皮细胞胞浆，呈棕黄色，参考Weidner的方法计算MVD值。2．2 RT-PCR检测cyclin G1 mRNA和cyclin G2 mRNA的表达取50-100mg组织粉末，加人1 ml Trizol溶液彻底匀浆。按Trizol试剂盒说明书用一步法提取RNA，用DNA／RNA测定仪测定RNA浓度和纯度。所用的cyclin G1的上、下游引物序列分别是：5’-GCCTTTCTCCACAATCCTGA-3’和5’-TGCATTTGTGAAATGGTGGT-3’，扩增片段长度为363bp。所用的cyclin G2的上、下游引物序列分别是：5’-AACCGGACCACAAGAAACTG-3’和5’-ATGACAAGGAGGCCATTCAG-3’，扩增片段长度为324bp。内对照β-actin的上、下游引物序列分别是：5’-CTCTTCCAGCCTTCCTTCCT-3’和5’-CACCTTCACCGTTCCAGTTT-3’，扩增片段长度为511bp。PCR的反应体系为：3μl标本cDNA，10×Buffer 2．5μl，2．5 mmol／L dNTPs 2μl，cyclin G1或（cyclinG2）引物（终浓度50pmol／L）和β-actin引物（终浓度10 pmol／L）各4μl，Taq酶（5U／μl）0．2μl，灭菌蒸馏水补足25μl。PCR反应程序为：94℃预变性2分钟，94℃变性30秒，55℃退火1分钟，72℃延伸4分钟，30个循环结束。PCR产物用1．5％琼脂糖凝胶电泳，溴乙锭显色。使用Band Leader Ver．3．0图象分析软件对电泳条带进行灰度扫描，计算cycling与β-actin产物条带的灰度值比值，计算cyclinG表达的相对值。三、统计学处理选用SPSS12．0统计软件进行数据处理。计数资料用χ²检验、Fisher（Fisher’sexact test）检验进行统计分析。计量资料均采用（?）±SD表示，两组均数比较时采用独立t检验，多组均数比较采用单因素方差分析进行统计分析。生存分析采用Kaplan-Meier法。四、实验结果1)喉鳞癌组织，喉良性病变，癌旁正常喉粘膜组织中Cyclin G1阳性表达率分别为61．7％（50／81）、5％（1／20）、0％（0／20），Cyclin G1的表达与喉良性病变、癌旁正常喉粘膜组织相比，其差别具有显著性（P=0．000）。2)Cyclin G1在喉鳞癌组织中表达与肿瘤分化程度、颈淋巴结转移以及复发均有统计学意义（P值分别为0．007、0．013和0．035）。3)Cyclin G2在喉鳞癌，喉良性病变和癌旁正常喉粘膜组织表达阳性率分别为67．9％、90％、100％，差异具有显著性（P=0．001）。4)Cyclin G2在喉鳞癌中阳性表达率随分化程度的降低而降低，有、无淋巴结转移者阳性率分别为38．4％和73．5％，差异具有显著性（P值分别为0．033和0．013）。5)Cyclin G1mRNA的表达强度在喉鳞癌组织中明显高于癌旁正常喉粘膜及喉良性病变组（P=0．000）。中-低分化与高分化喉鳞癌比较，后者cyclin GlmRNA表达较低，差异具有统计学意义（P=0．001）。6)Cyclin G1mRNA在有淋巴结转移比无淋巴结转移组喉鳞癌的表达强度显著增高（P=0．000）。cyclin G1mRNA的表达与颈淋巴结转移相关，与患者年龄、性别、临床分期、临床分型等均无统计学意义。7)Cyclin G2mRNA在喉鳞癌组织中、癌旁正常喉粘膜组织、喉良性病变中表达强度分别为（0．65±0．13）、（0．94±0．11）、（0．91±0．14）。中-低分化与高分化喉鳞癌比较，后者cyclin G2表达较强，差异具有统计学意义（P=0．000）。8)Cyclin G2mRNA表达与患者年龄、性别、临床分期、临床分型等均无统计学意义，而cyclin G2基因在淋巴结阴性者表达强度为（0．69±0．11），阳性者为（0．57±0．12），两者差异有统计学意义（P=0．004）。9)喉鳞癌，喉良性病变和癌旁正常喉粘膜组织MVD分别为（44．88±15．11）mv，（5．30±1．03）mv和（4．70±1．30）mv，喉鳞癌与喉良性病变和癌旁正常喉粘膜组织MVD相比，差异具有显著性（P=0．000）；10)Cyclin G1蛋白表达阳性组MVD为（53．34±12．53）mv，阴性组为（30．74±3．49）mv，其差别具有显著性（P=0．000）。11)Cyclin G2蛋白表达阳性组MVD为（39．53±9．93）mv，阴性组为（55．62±17．82） mv，其差别具有显著性（P=0．000）。五、结论1)Cyclin G1蛋白在喉鳞癌中存在高表达，并与肿瘤分化程度、颈部淋巴结转移关系密切，Cyclin G1蛋白异常表达可能在喉鳞癌发生、发展及转移过程中发挥重要作用，并可能作为判断患者预后的指标之一。2)Cyclin G2蛋白表达强度与喉鳞癌的恶性程度及发展趋势明显负相关，并与MVD关系密切。3)Cyclin G1mRNA在喉鳞癌组织中有异常高表达，并与喉鳞癌发生发展有一定联系，其高表达可能是喉鳞癌预后不良的因素之一。4)Cyclin G2mRNA表达强度与喉鳞癌的恶性程度及发展趋势明显负相关，可作为预测喉鳞癌预后的生物学指标之一。5)免疫组化实验及RT-PCR实验结果具有一致性，由于免疫组化方法对实验设备要求不高，费用相对较低，可以通过这种方法对目的基因进行初步检测。更多还原

【Abstract】 The laryngeal carcinoma is one of the most common malignancies in North China, accounting for 5.7-7.6% of all kinds of human malignant tumors of whole body. Ninety-three to ninety-nine percent of the laryngeal carcinomas are squamous cell carcinoma （LSCC） in pathological type. The occurrence of the LSCC has been greatly increasing with the development of modern industry and aggravation of the air pollution. The distribution of LSCC cases is largely varied in different countries. It is reported that the first three areas with the highest incidence of LSCC are Varese of Italy, St Paulo of Brazil and Bombay of India. The occurrence rate of laryngeal carcinomas in China also has great variance regionally with the highest incidence in Northeast China. The incidence is higher in cities than that in countryside, and higher in cities with severe air pollution than those with lightly polluted cities. The majority of LSCC present during the sixth to eighth decades in age, and less occur younger than 35 years old. It is more common in males than in females. Several factors have been related to LSCC. There is emerging evidence that the lifestyle, in particular, smoking, is associated with an increased risk.Along with the continuous development in diagnosis and treatment technic, there is obviously progress in the reservation of laryngeal function, the post-treatment living time of the patient has been longer than before. But the therapeutic efficacy of advanced stage cancer is still not as good as what we hoped. In order to improve the therapeutic efficiency, the research about the correlation factors of this disease has become one of the major tasks of early discovery and early treatment.Cyclin is the major modulators in cell cycle of eucaryotic cell, it control the course of cell cycle, and is one of the most important proteins in cell activities. It has been found there are aberrant expressions of many kinds of Cyclin in human neoplasms. Recent researches show that the abnormal expression of Cyclin G is closely related to various tumors.At present, the reports about Cyclin G expression in laryngeal squamous cell carcinoma were absence and the correlation between its expression and clinical pathology was not clear. There were no reports about the relationship between Cyclin G expression and angiogenesis of ISCC. The expression of Cyclin G1 and Cyclin G2 protein in 81 cases of laryngeal squamous cell carcinoma, 20 cases of benign tumors and 20 cases of laryngeal normal tissues was detected by the immunohistochemistry method. The relationship between clinical data including age, gender, tumor stage, pathological type, and prognosis were analyzed using software SPSS 12.0. Forty cases of fresh LSCC tissue samples. 10 cases each of normal laryngeal mucosa tissue and polyp of vocal cord were collected, respectively, and the mRNA expression level of cyclin Gl and cyclin G2 were examined by using reverse transcription polymerase chain reaction （RT-PCR）. The relationship was statistically analyzed between expression levels of cyclin Gl, cyclin G2 and clinicopathological characteristics of the patients. To explore the mRNA expression of cyclin G1 and cyclin G2 in laryngeal squamous cell carcinoma （LSCC） and its clinical significance. Angiogenesis is essential for growth, development, invasion, metastasis and recurrence of malignant tumours.The process of angiogenesis play an important role in the aspect of diagnosis, treatment and prognosis in malignant tumours. The vascular endothelial cells were marked by CD34 to evaluate the clinical significance of micro vessel density（MVD）and investigate the relationship of CD34, Cyclin G1 and Cyclin G2 protein and their significance of clinical pathology, we studied the expression of CD34, Cyclin G1 and Cyclin G2 protein in laryngeal carcinoma and compared the relevance among them in order to provide theoretic basis and experimental evidence for application of Cyclin Gl and Cyclin G2 in the aspect of diagnosis, treatment and prognosis and research about anti-angiogenic and genic therapy in laryngeal carcinoma.1 .Materials1)Cases of partⅠ81 cases of laryngeal squamous cell carcinoma （LSCC） treated at Shenyang 463 Hospital and the General Hospital of Chinese PLA in between Jan., 2000 to Dec, 2000 was collected. 71 cases were male and 10 cases were female. The patient age ranged from 36 to 82 years old with the average of 54.8. According to the tumor position, 61 cases of glottic carcinoma , 18 cases of supraglottic carcinoma and 2 cases of infraglottic carcinoma; According to the differentiating degree, the cases were classified as high differentiated （42 cases）, moderately differentiated （30 cases） and poorly differentiated （9 cases）; According to 1997 UICC stating criteria, 15 cases were classified as stageⅠ, 24 casesⅡ, 29 casesⅢand 13 cases IV; The cervical lymph node metastasis napped in 13 cases and 68 cases were free. None of the patients received radiotherapy or chemiotherapy before operation. 20 cases of normal laryngeal mucosa tissues adjacent to the malignant tumors and 20 cases of laryngeal benign diseases （10 case of laryngeal papilloma and 10 cases of polyp of vocal cord） were obtained in the same time.2)Cases of partⅡ 40 cases of fresh laryngeal squamous cell carcinoma （LSCC） treated at Shenyang 463 Hospital between Jan., 2000 to Dec, 2000 was collected. The patient age ranged from 39 to 82 years old with the average of 60.8. As to the tumor position, 28 cases were of glottic, 10 of supraglottic and 2 of infraglottic carcinoma; According to the differentiating degree, the cases were classified as high differentiated （11 cases）, moderately differentiated（19 cases） and poorly differentiated（10 cases）; According to 1997 UICC stating criteria, 5 cases were classified as stageⅠ,11 casesⅡ, 18 casesⅢand 6 casesⅣ; The cervical lymph node metastasis napped in 13 cases and 27 cases were free. None of the patients received radiotherapy or chemiotherapy before operation. 10 cases of normal tissues adjacent to the carcinomas and 10 cases of laryngeal benign lesions （1 cases of laryngeal papilloma and 9 cases of polyp of vocal cord） were obtained in the same time. All fresh tissues were snap frozen in liquid nitrogen and stored at -70℃.2 methods1) Immunohistochemistry for Cyclin G1 and Cyclin G2 protein and microvascular density labled by CD₃₄Immunohistochemical stainings were performed by SP method. The SP Kit was produced by BeiJing ZhongShan Biological Corp China. The primary antibodies were mouse monoclonal anti-human antigen Cyclin G1, rabbit polyclonal anti-human antigen Cyclin G2, mouse monoclonal anti-human antigen CD₃₄ and all the procedures were followed by the instruction. The negative control was prepared by replacing first antibodies with PBS, the positive control for staining was obtained from the company. The positive were defined as brown to yellow staining in cell nuclear （Cyclin G1） , nuclear and cytoplasm （Cyclin G2） or cytoplasm （CD34）. The MVD values were calculated by Weidner’s method.2) RT-PCR for cyclin G1 mRNA and cyclin G2 mRNA Total RNA from each sample （contain 50-100mg tissue） were isolated by using Trizol Solution, then the concentration and purity of RNA were detected by DNA/RNA detector.RT-PCR was done for cyclin G1, cyclin G2 and 6-actin （the primers and PCR product are listed in the following table）.The RT solution contained 25mmol /L MgCl₂ 4μl, 10×RNA PCR Buffer 2μl, RNase Free dH20 3.75μl, 10 mmol/L dNTP 2μl, RNase inhibitor 1μL AMV reverse tanscriptaselμl, Oligo dT-Adaptor Primer1μl, RNA 10μg and add RNase Free ddH₂O to 20μl. Followed by the condition: 60min at 42℃,5min at 99℃, 5 min at 4℃.PCR amplification was performed on a PCR thermal cycler. 3μl of cDNA mixture was subject to amplification in 25ul solution containing the following: 10×PCR buffer 2.5μl, 2.5 mmol/L dNTPs 2μl, Taq DNA polymerase （5U/μl） 0.2μl, each of 5’and 3’primers 2μl. Add sterile purified water to25μl. PCR conditions were initial denaturation for 3 min at 94℃, followed by 30 cycles of denaturation of 94℃for 30sec and annealed at 55℃for1 min, then extended at 72℃for 4min. PCR productswere resolved on a 1.5% agarose gel and the bands were visualized by ethidiumbromide staining. Leader Ver 3.0 software was used to analysis electrophoresisstrip.the relative values were obtained by calculating the values between cyclinG andβ-actin.3. Statistical analysisThe SPSS software 12.0 analyzed the data. Chi-squared test and Fisher （Fisher’s exact test） test was used in data of quantity. Independent-Samples T and One-way ANOVA test was used in data of quality. Kaplan-Meier method was used in cumulative survival rate of patients with CyclinG 1 or CyclinG2 in laryngeal squamous cell carcinoma.4.Results1) The positive rates of Cyclin G1, laryngeal benign diseases and the normal laryngeal mucosa were 61.7%（50/81）, 5%（1/20）, 0%（0/20） respectively. The expression of Cyclin Gl was higher in LSCC than that in laryngeal benign diseases and normal tissues（P=0.000）.2) The overexpression of Cyclin G1 in LSCC was related to the histological grade, cervical lymph node metastases and recurrence of tumor （P value is 0.007, 0.013 and 0.035, respectively）.3) The expression of Cyclin G2 protein: 67.9% （55/81） in LSCC, 90% （18/20） in the laryngeal benign diseases and 100% （20/20） in the normal tissues. The difference is significant （P=0.001）.4) The expression of Cyclin G2 in LSCC was related to the histoiogical grade and cervical lymph node metastases （P value is 0.033 and 0.013）.5) The mRNA expression of cyclin Gl was higher in LSCC than that in normal tissues and laryngeal benign disease （P=0.000）. The middle-poor differentiation LSCCS had significantly higher cyclin G1 level than that in well differentiation ones（P=0.001）.6) The mRNA expression of cyclin Gl in the LSCC with lymph node metastasis was higher than that without lymph node metastasis （P=0.013）. cyclin G1 mRNA expression in LSCC not correlated with age, gender, clinical stage, and clinical type.7) The expression of cyclin G2 mRNA was （0.65±0.13） in LSCC, （0.94±0.11） in normal tissue and （0.91±0.14） in laryngeal benign disease. The well differentiation LSCCS had significantly higher cyclin G2 level than that in middle-poor differentiation ones （P=0.000）.8) The expression of cyclin G2 mRNA was （0.69±0.11）in those without lymph node metastasis and（0.57±0.12） in those with lymph node metastasis （P=0.004）. Cyclin G2 mRNA expression in LSCC not correlated with age, gender, clinical stage, and clinical type.9) The expression of MVD was （44.88±15.11） mv in LSCC, （5.30±1.03）mv in the laryngeal benign diseases and （4.70±1.30） mv in the normal tissues. The difference is statistically significant （P=0.000）.10) The mean value of MVD was （53.34±12.53）mv in Cyclin G1 positive group, and （30.74±3.49） mv in negative ones. The difference is significant （P=0.000）.11) The mean value of MVD was （39.53±9.93） mv in Cyclin G2 positive group, and （55.62±17.82） mv in negative ones. The difference is statistically significant （P=0.000）.5 Conclusions1) Cyclin Gl is highly expressed in laryngeal squamous cell carcinoma, and is associated with differentiation degree and tumor angiogenesis. It may play an important role in carcinogenesis and metastasis in LSCC, and may be one of prognostic index for LSCC patients.2) Cyclin G2 protein highly expressed in LSCC and had correlation with the cervical lymph node metastasis and MVD.The expression level of Cyclin G2 negatively correlates with the malignant degree and development tendency of LSCC. Cyclin G2 may thus further restrain tumor angiogenesis, invasion and metastasis of the laryngeal carcinoma.3) The high expression of cyclin G1mRNA is correlated with the pathogenesis of laryngeal carcinoma and may be one of the indexes for the poor prognosis of LSCC patients.4) The expression level of cyclin G2mRNA negatively correlates with the malignant degree and development tendency of LSCC. cyclin G2mRNA is an important biological factor affecting the prognosis.5) There was concordance between the methods of immunohistochemistry and RT-PCR, the test rigs for immunohistochemistry technic was simpler and the cost was lower, it can be applied in initial detection of gene target.更多还原