抗Stat3多克隆抗体制备及其在乳腺癌诊断中应用.docVIP

抗Stat3多克隆抗体制备及其在乳腺癌诊断中应用 　 作者：李宏芬，单保恩，陈晶，董青，王晓玲，王永军，陈兴，董金琢，马洪 【关键词】 信号转导与转录激活因子3；抗体；乳腺肿瘤；组织学分级 　　Preparation of polyclonal antibody against Stat3 and its application in the diagnosis of breast cancer 　　【Abstract】 AIM: To prepare rabbit antibody against Stat3 and analyze its application in the diagnosis of breast cancer. METHODS: The male rabbits were immunized with recombinant mouse Stat3 protein to prepare antibody against Stat3. The purity and specificity of the antibody were analyzed by Western Blot. The expression of Stat3 protein in the 65 cases of breast cancer and 30 cases of normal breast tissue was detected by immunohistochemical staining. RESULTS: The titer of antiStat3 antibody in serum was gt;1∶256 000 (A450=0.40) at the 6th week after the immunization. Both SDSPAGE and Western Blot analysis showed that there was an obvious protein band with Mr 92 000, The intracytoplasmic expression levels of Stat3 protein in the different histological gradings of 65 cases of breast cancer showed significant difference (Plt;0.05), so did the intranuclear expression levels (Plt;0.05) . CONCLUSION: Polyclonal antibody against Stat3 with high titer and specificity can be prepared successfully, and it can be used to detect the expression of Stat3 protein in breast cancer tissue. 　　【Keywords】 Stat3; antibodies; breast neoplasms; histological grading 　　【摘要】 目的： 制备兔抗信号转导与转录激活因子3（Stat3）抗体，分析其在乳腺癌诊断中的价值. 方法： 应用人工重组小鼠Stat3蛋白，免疫雄性家兔制备抗Stat3抗体；用免疫印迹法分析检测抗Stat3抗体的纯度和特异性；应用免疫组化染色法检测65例乳腺癌和30例正常乳腺组织中Stat3蛋白的表达. 结果： 用Stat3蛋白免疫家兔被免疫6 wk后，其血清中抗Stat3抗体效价gt;1∶256,000（A450 nm=0.40）；SDSPAGE和免疫印迹分析均在分子质量（Mr）92 000处有明显的带；65例乳腺癌组织的细胞浆和细胞核中，Stat3蛋白表达量在不同的组织学分级之间有统计学差异(Plt;0.05). 结论： 高效价和特异性强的抗Stat3抗体被成功制备. 该抗体可用于乳腺癌组织细胞Stat3蛋白表达的检测. 　　【关键词】 信号转导与转录激活因子3；抗体；乳腺肿瘤；组织学分级 　　0引言 　　信号转导与转录激活因子3(signal transducer and activator of transcription; Stat3)是一类脱氧核糖核酸结合蛋白，于细胞浆内有少量表达，细胞核内无表达，是信号转导与转录激活因子家族的重要成员之一. 　　异常的信号转导使Stat3磷酸化而被持续激活，促进细胞的恶性转化［1］和阻断细胞凋亡［2］. Stat3也可以被多种细胞因子、生长因子、非受体酪氨酸激酶、G蛋白等分子激活，将细胞外信号转