摘要
目前乳腺癌诊断主要通过影像学手段,存在一定的创伤性和危险性,不利于乳腺癌的早诊、早发现。应用血清学肿瘤标志物(Tumor Marker, TM)检测可以降低病人检查的创伤和痛苦。但现有的乳腺癌血清肿瘤标志物敏感性、特异性不高,因此,识别更多的敏感性和特异性高的乳腺癌血清肿瘤相关标志物进行联合检测,具有重要意义,已成为临床上的迫切需要。本试验希望筛选出一些较特异、敏感的乳腺癌肿瘤相关标志物,从而为开发出一种廉价的诊断用乳腺癌蛋白芯片打下基础。
试验目的：从乳腺癌T7噬菌体展示文库中筛选乳腺癌肿瘤相关标志物,用于乳腺癌的检测。试验方法：应用自身抗体原理结合蛋白芯片技术,用乳腺癌患者血清和正常人血清对文库进行大规模筛选,然后对所得数据进行标准化处理和t检验,以P<0.01为差异极显著。再结合线性回归分析,选择在病人中高表达的克隆做PCR鉴定,选取合适片段大小的克隆测序,序列结果做BLAST比对,选出和肿瘤相关的有意义蛋白克隆做进一步的验证,检测其对乳腺癌诊断的价值。试验结果：试验用15份乳腺癌病人血清和15份正常人血清对文库大规模筛选,共筛选出115个差异极显著的噬菌体克隆。结合线性回归分析,选取出其中的22个克隆其相应自身抗体在病人血清中高表达,对这22个克隆做PCR分析,最后选取12个克隆进行测序,经过与GeneBank数据库中的已知序列比对分析,发现了1个与肿瘤相关的蛋白为锌指蛋白143。
At present, the diagnosis of breast cancer is primarily focused on imaging study, which has a certain extent of misdiagnosis rate for early breast cancer, and also causes varies extent of dangers. The application of serological tumor markers (Tumor Marker, TM) testing can overcome these demerits. For the existing tumor markers in breast cancer are all with limited sensitivity and specificity, therefore, it is of great significance and timely to identify new breast cancer tumor-associated serological markers with higher sensitivity and specificity used in joint detection. I hope this experiment has picked out some more specific and sensitive markers, so as to develop a low-cost diagnostic protein chip for breast cancer detection.
Experimental Objective:Screening out breast cancer associated TMs from T7 phage cDNA libraries of breast cancer that can be used in breast cancer detection. Experimental Methods:Guided by the principle of auto-antibodies-binding protein-chip technology, carry out a large-scale library screening with 15 patients'and 15 normal plasma samples, then standardized the data and make t test. We define the difference is extremely significant if P<0.01. Combined with linear regression analysis, selected patient-based high-expression clones to do PCR, and clone fragments of appropriate size were sequenced and compared with the GenBank to identify meaningful tumor-associated protein clones, and evaluated the value of breast cancer diagnosis by further verification with ELISA. Experimental results: After the large-scale library screening,115 phage clones were screened out with significant differences; 22 clones in patients with high expression after linear regression analysis. These 22 clones were identified by PCR analysis, finally we got the sequences of 12 clones, after the BLAST with the already known sequences in GeneBank database, one tumor-associated protein was identified named zinc finger protein 143.
引文
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