【作者】 赵娜；

【导师】 蒋军广；

【作者基本信息】 郑州大学 ， 老年医学， 2010， 硕士

【摘要】 1目的肺癌是威胁人类生命的重大疾病,其发病率和死亡率正在逐年上升。由人γ-snyculien基因(SNCG)编码的蛋白-丫突触核蛋白(Y-synuclien)为天然无折叠蛋白,是公认的乳腺癌分子标记物。近来研究发现它在卵巢癌、消化系肿瘤中异常高表达,在晚期肿瘤中表达尤其显著。有望成为-个有效的肿瘤标志物。国内外文献对于γ-Synuclein在非小细胞肺癌中的表达及其临床意义报道较少。组织因子途径抑制物2(tissue factor pathway inhibitor2,TFPI-2)属于库尼(Kunitz)型丝氨酸蛋白酶抑制剂家族蛋白,是新近发现的-种丝氨酸蛋白酶抑制物,与多种肿瘤的发生,肿瘤细胞的侵袭、转移有关。其抑制肿瘤细胞生长、侵袭及转移的机制成为近年来研究的热点。关于TFPI-2在NSCLC中的表达如何国外有数篇报道,而国内尚无报道。鉴于以上研究现状,本实验用免疫组织化学的方法来探讨TFIP-2与γ-synuclien在NSCLC中的表达及NSCLC的发生、发展和转移的机制,以期对NSCLC的诊断、治疗及预后评估提供实验依据。2对象与方法2.1研究对象随机选取60例自2060年1月至2008年12月在郑州大学第一附属医院胸外科手术切除的NSCLC患者标本作为观察组,所有患者术前均未行任何抗肿瘤治疗,均经病理科证实,按美国联合癌症分类委员会(AJCC)和国际抗癌联盟(FIGO)2002年制定的分期标准:Ⅰ期16例、Ⅱ期12例、Ⅲ期21例、Ⅳ期11例。对照组为20例癌旁正常肺组织(均距癌组织3mc以上)。2.2实验方法采用免疫组织化学S-P方法。2.3结果判定2.3.γ-synuclein的阳性结果判定以免疫组化染色后细胞质或细胞核中出现棕黄色颗粒作为阳性判断标准。将染色程度分为3级:无着色(l级)评1分,浅黄色2(级)评2分,棕黄色3(级)评3分;高倍镜(400倍)下每个视野计数100个细胞,每张切片数5个视野。按照高倍镜下阳性染色细胞数占癌细胞总数的百分率判断,阳性细胞数≤25%为1级(分),26%~75%为2级(分),)≥76%为3级(分)。每张切片的最后得分为两次评分的乘积(1分-9分),最后得分大于7分的为阳性表达(+),小于7分的为阴性表达(-)。2.3.2TFPI-2的阳性结果判定阳性染色为胞质有棕黄色颗粒沉着,在高倍镜下(400倍)计数100个细胞,每张切片数5个视野,无阳性反应或阳性染色细胞数≤25%记作阴性(-),阳性染色细胞数25%-50%记作弱阳性(+),阳性染色细胞数>50%记作强阳性(++)。2.4统计学处理采用卡方检验及四格表资料的精确概率法,统计处理采用SPSS13.0软件包,a=0.05为检验水准。3结果3.1y-synuclein和TFPI-2在NSCLC组织中的表达免疫组化结果显示,γ-synuclein免疫组化阳性产物主要定位于细胞质,也可见定位于细胞核。60例NSCLC组织中y-synuclein表达的阳性率为46.7%(28/60),20例正常肺组织无一例阳性表达(0%),差异有统计学意义(P=0.000)。TFPI-2免疫组化阳性产物主要定位于胞质。60例NSCLC组织中TFPI-2表达的阳性率为65%(39/60),20例正常肺组织均为阳性表达(100%),差异有统计学意义(P=0.002)。3.2y-synuclein和TFPI-2的表达与NSCLC临床病理特征的关系高中分化的NSCLC患者中的y-synuclein的阳性表达率为34.8%,低分化为85.7%,差异有统计学意义(P=0.001)。TNM分期Ⅰ、Ⅱ期NSCLC患者中y-synuclein的阳性表达率为28.6%,Ⅲ、Ⅳ期患者y-synuclein的阳性表达率为62.5%,差异有统计学意义(P=0.009)。伴淋巴结转移的NSCLC患者中y-synuclein的阳性表达率为87.5%,不伴淋巴结转移的患者为19.4%,差异有统计学意义(P=0.000)。y-synuclein的表达与NSCLC患者的年龄、性别、肿瘤的大小、病理类型,临床分型、有无胸腔积液及是否吸烟无关。TNM分期Ⅰ、Ⅱ期NSCLC患者中TFPI-2的阳性表达率为78.6%,Ⅲ、Ⅳ期患者TFPI-2的阳性表达率为53.1%,差异有统计学意义(P=0.039)。伴淋巴结转移的NSCLC患者中TFPI-2的阳性表达率为45.8%,不伴淋巴结转移的患者为77.8%,差异有统计学意义(P=0.011)。TFPI-2的表达与NSCLC患者的年龄、性别、肿瘤的大小、分化程度、病理类型,临床分型、有无胸腔积液及是否吸烟无关。4结论(1)y-synuclein在NSCLC中的阳性表达率明显升高,提示其与NSCLC的发生密切相关,可能成为NSCLC个新的诊断标志。(2)y-synuclein的表达与NSCLC的分化程度、TNM分期及有无淋巴结转移密切相关,与患者的年龄、性别、肿瘤的大小、病理类型,临床分型、有无胸腔积液及是否吸烟无关。(3)TFPI-2在NSCLC中呈低表达,并随TNM分期的增高及出现淋巴结转移表达降低,与患者的年龄、性别、肿瘤的大小、病理类型,临床分型、分化程度、有无胸腔积液及是否吸烟无关。(4)y-synuclein和TFPI-2异常表达可能对NSCLC患者的早期诊断及预后判断提供依据,有望成为NSCLC治疗的新靶点。更多还原

【Abstract】 1 AimsLung cancer is the major diseases threatening human life, its incidence and mortality rates are increasing every year.y-synuclein is a natural non-folded protein encoded by the human y-synuclein gene(SNCG).It is a recognized molecular markers of breast cancer. Recently studies found that it expressed abnormally highly in ovarian cancer, digestive tumors, particularly in advanced tumors expressed higher. It is expected to become an effective tumor marker. Literature at home and abroad about the y-Synuclein in non-small cell lung carcinoma and its clinical significance are reported less.Tissue factor pathway inhibitor 2 (TFPI-2) belongs to Kunitz-type serine protease inhibitor family of proteins, is a newly discovered serine protease inhibitor, with a variety of tumors, inhibit tumor cell invasion and metastasis.Its inhibition of tumor cell growth and the mechanism of invasion and metastasis become research hotspot in recent years. There are several papers about TFPI-2 expression in NSCLC in foreign countries, but in our country there is none.In view of the above study, the purpose of present study by immunohistochemical methods is exploring the expression of TFPI-2 and y-synuclein in NSCLC and the occurrence, development, and transfer mechanisms of NSCLC,providing experimental evidence for NSCLC diagnosis, treatment and prognosis evaluation.2 Study Object and Methods2.1 Study object Specimen (study group) were collected from NSCLC patients operated in the horacic surgery resection of the first affiliated hospital of zhengzhou university from January 2006 to December 2008,the total example is 60,all patients has not performed any anti-tumor therapy, and confirmed by pathologists.According to the U.S.Joint Committee on Cancer Classification (AJCC) and the International Union Against Cancer (FIGO) that was enacted in 2002,we divid the samples in: Ⅰstage 16 cases,Ⅱstage 12 cases,Ⅲstage 21 cases,Ⅳstage 11 cases.comparison group is 20 cases of adjacent normal lung tissue (from cancer were more than 3cm).2.2 Experimental |methods Methods is the SP Immunohistochemical method.2.3 Assessing result2.3.1 y-synuclein We require immunohistochemical products staining in the cytoplasm or the nucleus appears yellow particles as a positive criterion. The dye were divided into three stages:no color(1 stage) assess 0 point, light yellow (2 stage) assess the 2 points, brownish yellow (3 stage) assess 3 point; Counting 100 cells every visual field under high power lens(400 times),and counting 5 visual fields of every sample. High magnification of positively stained cells according to the number of accounts to judge the percentage of the total number of cancer cells, positive cells < 25% as one stage (point),26% to 75% as two stage (point),≥76% as three stage (point).The final score for each slice multiplied by two score (1 point to 9 points), final score greater than 7 points as the positive (+),less than 7 points for the negative expression(-).2.3.2 TFPI-2:We require immunohistochemical products staining in the cytoplasm or the nucleus appears yellow particles as a positive criterion. Counting 100 cells every visual field under high power lens(400 times),counting 5 visual fields.no stain or the percentage≤25% as negative(-),25%～50% as weakly positive (+),>50% as strong positive(++)..2.4 Treatment in statistics:Using chi-square test and exact probabilities in 2×2 table, using SPSS 13.0 package, a=0.05 for the test level.3 Result3.1 The expression of y-synuclein and TFPI-2 in NSCLC The result of immunohistochemistry displayed that y-synuclein mainly located in the cytoplasm, nucleus occasionally,in 60 cases of NSCLC tissues the expression of y-synuclein-positive rate was 46.7% (28/60), in 20 cases of normal lung tissues there was none of the positive expression (0%), the difference was statistically significant (P=0.000). TFPI-2 immunohistochemistry positive products were mainly located in the cytoplasm.In 60 cases of NSCLC tissues the expression of TFPI-2 positive rate was 65% (39/60),20 cases of normal lung tissue were positive expression(100%), the difference was statistically significant (P=0.002).3.2 The relation of y-synuclein and TFPI-2 with the clinical path-character High differentiation of NSCLC patients in the y-synuclein positive expression rate was 34.8%, poorly differentiated was 85.7%,the difference was statistically significant (P=0.001).TNM stageⅠ,Ⅱperiod of NSCLC patients with y-synuclein positive expression rate was 28.6%,Ⅲ,Ⅳpatients with y-synuclein positive expression rate was 62.5%,the difference was statistically significant (P=0.009).With lymph node metastasis in NSCLC patients with y-synuclein positive expression rate was 87.5%, non-patients with lymph node metastasis was 19.4%, the difference was statistically significant (P=0.000). The expression rate of y-synuclein was not related to the patient’s age, sex, tumor size, pathologic type, clinical type, whether pleural effusion or smoking.TNM stageⅠ,Ⅱperiod of TFPI-2 in NSCLC patients with positive expression rate was 78.6%,Ⅲ,Ⅳpatients with TFPI-2 positive expression rate was 53.1%,the difference was statistically significant (P=0.039).With lymph node metastasis in NSCLC patients with TFPI-2 positive expression rate was45.8%, without lymph node metastasis was 77.8%, the difference was statistically significant (P=0.011).The expression rate of TFPI-2 was not related to the patient’s age、sex、tumor size、degree of differentiation、pathologic type、clinical type、whether pleural effusion or smoking.4 Conclision(1)The expression rate of y-synucleinin in NSCLC increased significantly than nomal tissues, suggesting that they were close correlation.γ-synucleinin may become a new diagnostic marker for NSCLC.(2) The espression rate of y-synuclein was close related to the differentiation、TNM stage、lymph node metastasis and has nothing to the patient’s age, sex, tumor size, pathologic type, clinical type, whether pleural effusion or smoking.(3)The expression of TFPI-2 in NSCLC was low and appears lower with the TNM staging increased and lymph node metastasis.and was not related to the patient’s age、sex、tumor size、pathologic type、differentiation、clinical type、whether pleural effusion or smoking. (4) The abnormal expression ofγ-synuclein and TFPI-2 in NSCLC patients’ early diagnosis and prognosis provide a reliable basis and provide a new treatment target.更多还原