【作者】 李静；

【导师】 康晓静；

【作者基本信息】 石河子大学 ， 病理学与病理生理学， 2013， 硕士

【摘要】 目的：检测恶性黑素瘤、色素痣及正常组织中BRAF基因表达，分析BRAF基因的表达与恶性黑素瘤临床表型的关系及其意义，探讨其与恶性黑素瘤形成及预后的关系，以期找到一个用于疾病早期诊断及判断预后的新指标。同时探讨从恶性黑素瘤石蜡包埋组织中提取RNA并进行实时荧光定量的可行性。方法：收集2007-2012年新疆维吾尔自治区人民医院病理科、皮肤科确诊的74例恶性黑素瘤石蜡包埋组织，并整理分析临床病理资料，并选取了35例色素痣及46例正常包皮的石蜡包埋组织。运用SYBR Green Real-time PCR（qRT-PCR）方法检测BRAF基因在恶性黑素瘤织、色素痣及正常皮肤组织中的相对表达量2-ΔCt,分析其mRNA表达与恶性黑素瘤临床表型关系。采用琼脂糖凝胶电泳、紫外分光光度仪检测恶性黑素瘤石蜡包埋组织提取的RNA质量，运用标准曲线及溶解曲线进行实时荧光定量方法可行性分析。统计学分析采用SPSS17.0软件。结果：1）74例恶性黑素瘤患者维吾尔族34例（45.9%），汉族40例（54.1%）；男性39例（52.7%），女性35例（47.3%），男女之比：1.11:1；平均年龄55.6±14.3岁，其中≦45岁11例（14.9%），46-59岁20例（27.0%），≥60岁43例（58.1%）；原发部位位于皮肤47例（63.5%），粘膜24例（32.4%），其它3例（4.1%）；其中原发于皮肤的47例患者中33例为肢端黑素瘤（70.2%）。2)维吾尔族、汉族恶性黑素瘤患者BRAF基因的表达水平（0.377±0.167）明显高于色素痣（0.159±0.167）、正常皮肤组织（0.134±0.050）(p<0.01)。3）新疆维吾尔族、汉族恶性黑素瘤患者性别、年龄、民族、发病部位、淋巴结转移等BRAF基因mRNA表达水平无统计学差异（P>0.05）。4）恶性黑素瘤石蜡包埋组织中提取的RNA可进行后续实时荧光定量PCR反应，实时荧光定量PCR检测结果显示溶解曲线呈单峰，目的基因与内参基因扩增效率基本一致。结论：1）新疆地区恶性黑素瘤发病年龄以45岁以上为主，无明显性别差异，好发部位为肢端。2）BRAF基因高表达可能参与了恶性黑素瘤的发生发展。更多还原

【Abstract】 Objective: To examed the difference in BRAF gene expression between malignant melanoma(MM),pigment nevi and normal skin. Analyzed the relationship among BRAF gene expression, malignantmelanoma clinical phenotype, so explored its relationship with the occurrence and development ofmalignant melanoma, in order to find a new indicator for disease prediction and prognosis. The method ofextraction of RNA from formalin fixed and paraffin embedded(FFPE) of MM tissue for reversetranscription and polymerase chain reaction(RT-PCR)were established. The feasibility of this method wereinvestigated.Methods：This study involved formalin-fixation and paraffin-embedding tissue samples from74casesmalignant melanoma patients,35cases pigment nevi and46cases Normal skin tissue in People’s Hospitalof Xinjiang Uygur Autonomous Region. Clinical data were collected and analyzed. The expression ofBRAF gene were detected by SYBR Green real time PCR in74cases of malignant melanoma and35casespigment nevi and46cases Normal skin tissue cases. Each sample were calculated and analysized on2-ΔCt.RNA were extracted from FFPE sections and examed by agarose gel electrophoresis and ultravioletspectrophotometry. Feasibility of RT-PCR is made of the establishment of solubility curves andamplification plot. The relationship between BRAF expression and clinical phenotype were analyzed by ttest or one-way ANOVA test according to the SPSS17.0Software.Results：34of74cases in malignant melanoma patients were Uygur races (45.9%),the Han nationalitywere40cases (54.1%); The males were39cases (52.7%), the females were35cases (47.3%), the ratiobetween male and female is1.11:1; The average age is55.6±14.3, the age of11cases is less than or equal45years (14.9%), the age of20cases is between46and59(27.0%), the age of43cases is more than orequal to60years (58.1%); the primary site was located in the skin which were47cases(63.5%),24cases in the mucosa(32.4%), other is3cases(4.1%);33cases of47cases were occurred in theextremities(70.2%).2)Comparison of the expression of BRAF gene among Uygur races and Hannationality malignant melanoma, pigment nevi and normal skin tissue: the expression of BRAF gene inmalignant melanoma was0.377±0.167, in pigment nevi was0.159±0.167and in normal skin tissue was0.134±0.050. The results showed that the expression of BRAF gene in malignant melanoma was highersignificantly than that in pigment nevi and skin normal tissue (p<0.01). The expression of BRAF inmelanoma patients were no significant correlation with Clinical pathology characteristics such as genderage, ethnicity, anatomical site, metastasis of lymph node, ulceration(P>0.05). The MM of extraction ofRNA from formalin fixed and paraffin embedded degrade at different degree. Concentration and purity ofRNA fit for the demand of experiment. The result of RT-PCR have shown that dissolved curve with asingle-peak. The target gene and reference gene to amplification efficiency are mostly identical.Conclusion:1）The incidence of melanoma mainly in the elderly, with small gender differences inXinjiang region, acral is predilection sites.2）The higher expression of BRAF gene may involve in theoccurrence and development of melanoma and play an important role in the melanoma.更多还原