不同代数永生化支气管上皮细胞分泌蛋白的分布特征

发布时间：2018-03-11 10:14

本文选题：M细胞　切入点：高效液相色谱　出处：《大连医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的: 肺癌作为严重威胁人类健康的重大疾病,其发病率和死亡率均居恶性肿瘤之首,其主要原因在于传统的影像学和细胞学诊断还不能有效地检测出早期肺癌,多数患者一经发现已经转移。因此,寻求新的、有效的早期诊断技术对于降低肺癌死亡率具有非常重要的意义。本论文的研究工作期望通过血浆检测发展肺癌早期诊断的有效方法,为进一步探讨肺组织恶变的机制,寻找可能的癌症早期诊断标记物提供基础的研究资讯。方法: M细胞系是SV40-Large T抗原转化的人支气管上皮永生化细胞系,为研究细胞从正常到癌变过程中的遗传学的改变提供了良好的模型。我们利用M细胞系作为模型,在无血清培养集中培养后,收集分泌蛋白,经浓缩、除盐处理,进一步采用凝胶分离(SDS-PAGE)、高效液相色谱(HPLC)、二维高效液相色谱(2D-HPLC)与质谱(MS)联用技术对不同代数永生化支气管上皮细胞的分泌蛋白加以研究,说明肺癌发生过程中分泌蛋白分布的变化特征。结果: 1.SDS-PAGE的实验结果表明,在MP49中,分泌蛋白的分子量分布较广,几乎在10-100kD范围内都有蛋白分布。相反,在MP188中,分泌蛋白主要集中在高分子量和低分子量范围内(60kD,10kD),而在10-50kD范围内蛋白含量较小。 2.两个样品得到的酶解产物的疏水性质相似,但是在MP49的分泌蛋白中含有一些疏水性较强的肽段,而MP188的分泌蛋白中亲水性肽段较多。 3.2D-HPLC/MS分析肽段的分布。在MP49的分泌蛋白中含有更多的肽段,肽段的疏水性质与被检测的离子的质荷比分布较为均匀。而MP188分泌蛋白的酶解产物则数量较少,主要得到一些质荷比相对较小的肽段。 4.强阳离子交换色谱对酶解产物肽段分布加以评价,MP49中显酸性的肽段较多。 5.采用质谱结合数据库对两个样品中的蛋白质加以鉴定。对MP49,可以确定120个蛋白;对MP188;可以确定85个蛋白。结论: 1.采用SDS-PAGE和HPLC、2D-HPLC分离结合MS检测技术对不同代数的永生化支气管上皮细胞MP49和MP188的分泌蛋白分布加以研究。结果表明:随着代数越大,其蛋白质的分子量分布将逐渐向高分子量范围和低分子量范围过渡,而相对中等分子量的蛋白质逐渐减少。 2.MP49对比MP188分泌蛋白的酶解产物,MP188中的亲水性肽段明显多于MP49,质谱鉴定也得到同样的结果。 3.与SDS-PAGE相比,2D-HPLC/MS系统有助于发现一些低丰度的功能性蛋白质。 4.在支气管上皮的恶性演变过程中寻找可能的癌症早期诊断蛋白质标记物,从亲水性较强的高分子量或低分子量蛋白质入手可能会得到更好的结果。
[Abstract]:Objective:. Lung cancer, as a serious disease threatening human health, has the highest morbidity and mortality in malignant tumors. The main reason is that traditional imaging and cytological diagnosis can not detect early lung cancer effectively. Most patients are found to have metastasized. Effective early diagnosis technique is of great significance in reducing lung cancer mortality. The present study hopes to develop an effective method for early diagnosis of lung cancer by plasma detection, in order to further explore the mechanism of lung cancer malignancy. Search for possible markers for early diagnosis of cancer to provide basic research information. Methods:. M cell line is an immortalized human bronchial epithelial cell line transformed by SV40-Large T antigen, which provides a good model for studying the genetic changes of cells from normal to cancerous. After concentrated culture in serum-free culture, secretory proteins were collected, concentrated and desalted. The secreted proteins of different algebraic immortalized bronchial epithelial cells were further studied by gel separation, high performance liquid chromatography (HPLC) HPLCX, two-dimensional high performance liquid chromatography (HPLC) 2D-HPLC and mass spectrometry (MS). The change of secretory protein distribution during the development of lung cancer. Results:. 1. The results of SDS-PAGE showed that the molecular weight distribution of secretory protein in MP49 was relatively wide, almost in the range of 10-100kD, but in MP188, The secretory protein was mainly concentrated in the range of high molecular weight and low molecular weight, and the protein content was small in the range of 10-50 KD. 2. The hydrophobic properties of the two samples were similar, but there were some hydrophobic peptides in the secretory proteins of MP49 and more hydrophilic peptides in the secreted proteins of MP188. 3.2D-HPLC / MS analysis showed that there were more peptides in the secretory protein of MP49, the hydrophobic property of the peptide was more uniform than the mass charge ratio of the detected ions, while the number of hydrolyzed products of MP188 secreted protein was relatively small. Some peptides with relatively small mass charge ratio were obtained. 4. The distribution of peptide segments in enzymatic hydrolysis products was evaluated by strong cationic exchange chromatography. 5. The proteins in two samples were identified by mass spectrometric binding database. For MP49, 120 proteins could be identified; for MP1888; 85 proteins could be identified. Conclusion:. 1. The secretory protein distribution of MP49 and MP188 in immortalized bronchial epithelial cells of different algebras was studied by SDS-PAGE and HPLCX 2D-HPLC and MS detection technique. The molecular weight distribution of the protein will gradually transition to the high molecular weight range and the low molecular weight range, while the protein with relatively medium molecular weight will gradually decrease. 2. Compared with MP188 secreting protein, MP49 had more hydrophilic peptides than MP49, and the same result was obtained by mass spectrometry. 3.Compared with SDS-PAGE, the 2D-HPLC / MS system can help to find some low abundance functional proteins. 4. In the process of malignant evolution of bronchial epithelium, a better result may be obtained by looking for possible protein markers for early diagnosis of cancer, starting with high molecular weight or low molecular weight proteins with strong hydrophilicity.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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