摘要
背景及目的：肺癌中约75%-85%为非小细胞肺癌(NSCLC),由于发病隐袭,缺乏有效的早期筛查手段,导致大多数肺癌病人治疗难度大、预后差。现有的肿瘤标记物尚无法应用于肺癌的早期诊断。血清肿瘤标志物对于诊断、判断预后、疗效评价和随诊等方面具有重要意义。因此,寻找肺癌相关的血清标志物是目前研究的热点之一。基于双向凝胶电泳(2-DE)和基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)为核心的蛋白质组学研究技术具有高通量、灵敏、自动化的特点,是当今肿瘤标志物研究的重要技术手段之一。本课题拟应用蛋白质组学技术筛选和鉴定早期NSCLC血清的差异表达蛋白,联合传统实验方法,包括Western blot、荧光定量PCR、免疫组化和ELISA,旨在找到可用于NSCLC早期诊断的血清标志物。
方法：1.收集经手术治疗及病理确诊的Ⅰ期NSCLC患者术前血清作为实验组；收集经手术治疗及病理确诊的肺部良性病变患者血清作为良性对照组；收集同期健康体检者血清作为正常对照组。等质量混合各组血清标本,去除高丰度蛋白质后进行2-DE分离找到差异表达蛋白点,经MALDI-TOF-MS检测,览啊定早期NSCLC血清差异表达蛋白。
2.选取与肺癌/肿中瘤关系密切的差异表达蛋白作为候选目标。在3组血清混合样本中进行Western blot验证,以验证质谱鉴定的准确性,筛选具有统计学意义的差异表达蛋白。
3.将筛选出的差异表达蛋白通过荧光定量PCR方法在基因水平上进行验证,在蛋白质水平上进行免疫组化验证和ELISA验证,确定NSCLC早期诊断血清标志物。
结果：1.在3组所有凝胶上均得到了清晰的2-DE蛋白质表达谱,各组凝胶图像用PDquest软件进行点识别、背景消除、点匹配及差异蛋白分析。选取肺癌组与另2组比较均P<0.05的点为差异蛋白点。共有38个差异点符合以上标准。肺癌组上调的差异点24个,下调的差异点14个
2.共挖取38个差异蛋白点,经MALDI-TOF-MS分析,成功鉴定出12种蛋白质,其中6种在肺癌组表达上调,包括血红蛋白β链、冷凝集素、碳酸酐酶Ⅰ、羧肽酶N催化链、补体C3及补体C4A：6种在肺癌组表达下调,包括载脂蛋白J、触珠蛋白、转甲状腺素蛋白、补体C8β链、补体Cls亚成分及血色素结合蛋白。
3.选取与肺癌/肿瘤相关的上调蛋白血红蛋白β链、碳酸酐酶Ⅰ、补体C3及下调蛋白载脂蛋白J、转甲状腺素蛋白、血色素结合蛋白,进行血清Western blot筛选及验证,结果上述6种蛋白均出现了清晰的蛋白表达条带,证实了质谱鉴定的准确性。与正常对照组和良性对照组比较,肺癌组碳酸酐酶Ⅰ的表达明显上调,转甲状腺素蛋白的表达下调,为寻找肺癌血清标志物进一步明确了候选蛋白。而载脂蛋白J、补体C3、血红蛋白p链和血色素结合蛋白的表达未见统计学差异。
4.经荧光定量PCR、免疫组化和ELISA验证,发现碳酸酐酶Ⅰ和转甲状腺素蛋白是非小细胞肺癌早期诊断标志物。
结论：1.血清标本作为一种容易获取的实验材料,对其进行研究是有效的、可行的,其研究结果可直接应用于临床实践,具有重大的临床意义和实践价值。
2.应用蛋白质组学技术,联合传统实验方法,可以作为一种研发肺癌新型血清标志物的有效策略。
3.碳酸酐酶Ⅰ在Ⅰ期NSCLC血清中表达升高,转甲状腺素蛋白在Ⅰ期NSCLC血清中表达降低,是NSCLC早期诊断的血清标志物。
Background and objective:About75%-85%of lung cancer are non-small cell lung cancer (NSCLC). The existing tumor markers cannot be applied to early diagnosis of NSCLC yet. Therefore, looking for lung cancer related serum marker is currently one of research hot spots. Proteomics technique based on two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has the characteristics of high throughput, sensitiveness and automation. So it has been one of the important technical method to current research on tumor markers. This topic is plan to screening and identification of differentially expressed serum proteins of early stage NSCLC by Proteomics technique. Meanwhile, combined the traditional experiment methods, including Western blot, fluorescent quantitative PCR, immunohistochemistry and enzyme-linked immunosorbent assay, we aimed at finding serum biomarkers for early detection of NSCLC.
Methods:1.Sera from three groups, including lung cancer group (n=11), benign control group (n=12), and normal control group (n=30), were collected, balanced pooled intra-group and high abundance proteins were removed from the pooled samples.2DE and MALDI-MS were employed to separate, screen and identify the differentially expressed proteins related to stage I NSCLC.
2. Differentially expressed proteins closely related to lung or other cancer were selected as candidates. Western blot were conducted in the pooled samples of the three groups to confonn the accuracy of MS results and screen the significant differentially expressed proteins.
3.The screened proteins were tested via fluorescent quantitative PCR at the genetic level, and immunohistochemistry and enzyme-linked immunosorbent assay at the protein level, to determine the biomarkers for early diagnosis of NSCLC.
Results:1. Clear2DE protein expression profiling were obtained from the three groups. PDquest8.0image analysis software was used to scan and analyze images. Total38protein spots were considered as differential spots.
2.In contrast to the two control groups,12differentially expressed proteins were identified via PMF using MALDI-MS. Six proteins (hemoglobin beta, cold agglutinin chain A, carbonic anhydrase I, carboxypeptidase N catalytic chain precursor, complement C3precursor, and complement C4A) were up-regulated in the lung cancer group. The other six proteins (apolipoprotein J, haptoglobin, transthyretin precursor, complement C8beta propetide, complement C1s subcomponent precursor, and hemopexin precursor) were down-regulated in the lung cancer group.
3. Differentially expressed proteins closely related to lung or other cancer, including hemoglobin beta, carbonic anhydrase I, complement C3precursor, apolipoprotein J, transthyretin precursor and hemopexin precursor, were selected to be tested via Western blot. As a result, the6kinds of proteins appeared clear bands of protein expression, which confirmed the accuracy of the finding of MS. In contrast to the two control groups, the expression of carbonic anhydrase Ⅰ in the lung cancer group was significantly up-regulated, whereas, that of transthyretin precursor was down-regulated.
4. Carbonic anhydrase I and transthyretin precursor were effective biomarker for early detection of NSCLC via validation of fluorescent quantitative PCR, immunohistochemistry and enzyme-linked immunosorbent assay.
Conclusion:1.As an accessible experimental materials, serum specimen is effective and feasible research object. The results of study can be directly applied to clinical practice, which is of great clinical significance and practical value.
2. Application of proteomics technique, combined with traditional experiment method, can serve as a kind of effective strategies for developing new serum biomarkers of lung cancer.
3. Carbonic anhydrase I and transthyretin precursor are serum markers in early diagnosis of NSCLC.
引文
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