| 题名 | ERGIC3单克隆抗体制备 、鉴定及其初步应用研究 |
| 作者 | 林清海 |
| 学位类别 | 硕士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 曹毅 |
| 关键词 | 肺癌 肿瘤标志物 ERGIC3 单克隆抗体 miRNA |
| 其他题名 | Preparation and Identification of ERGIC3 Monoclonal Antibody and Preliminary Application Research |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 肺癌是一种常见肿瘤，其死亡率居恶性肿瘤之首，其患者的5 年生存率为10%到15%。世界卫生组织统计显示，2012年全球新增肺癌患者180万，死亡159万人。而中国肺癌的发病率和死亡率首当其冲，约占此类病例的1/3以上。肺癌病死率居高不下的一个重要原因是，肿瘤早期不易被及时检测出来，延误最佳治疗期。而现行的一些肿瘤检测往往存在假阳性高、敏感性差、操作复杂等缺点，于是寻找一种敏感性好，特异性高的肿瘤标记物（Tumor Marker），并开发关于其在肿瘤检测方面的简捷且可靠地方法对于提高肺癌的临床检出率至关重要。 本实验室在前期的工作中，通过SSH（抑制性消减杂交技术）建立肺腺癌的差异表达基因文库，通过进一步分析发现ERGIC3的mRNA水平和蛋白水平在肺癌组织和多个肺癌细胞系中异常高表达，而且ERGIC3的阳性率还与肺癌的分化程度成正相关， ERGIC3属于Ⅱ型跨膜蛋白，分子量43.2kDa，是内质网-高尔基体间的循环蛋白，其异常表达与内质网应激反应相关并且ERGIC3可以作为MiR-490-3p的靶基因参与肝癌细胞的生长和转移，对肝癌的EMT过程有重要作用。这些都提示ERGIC3 很可能是一个有潜力的的肺癌标记物。 本研究为进一步研究ERGIC3的病理生理功能及研发可能的肺癌早期诊断新方法，制备了ERGIC3鼠源单克隆抗体，通过数轮的亚克隆筛选，获得了ERGIC3特异性细胞株，并将其命名为6-C4，并对6-C4细胞株分泌抗体亚型亚类进行鉴定，结果为IgG2b，轻链为kappa链。通过进一步的抗体鉴定发现，该单克隆抗体可应用于ELISA、western-blot、细胞免疫荧光以及免疫组织化学等方面的检测，接着本实验应用6-C4分泌抗体对ERGIC3在正常人体组织和多种肿瘤组织的表达进行系统的研究，结果发现在人体23种正常组织中ERGIC3的表达仅在泌尿系统的肾组织的远球小管、近球小管细胞，消化系统的肝组织细胞和胰腺上皮细胞，以及消化道的胃腺上皮细胞和肠道绒毛、覆盖上皮细胞，及乳腺导管上皮细胞等均有ERGIC3的阳性表达；而在大多数人体其它组织中，免疫组化未见有明显染色。这种表达分布关系可能与ERGIC3参与细胞蛋白转运、分泌的功能有关。而在人类常见的15种恶性肿瘤组织中，在上皮来源的肿瘤组织中ERGIC3蛋白大都有不同程度的高表达，而在间叶组织肿瘤中，如恶性软骨肉瘤和恶性纤维肉瘤中无明显表达通过制备ERGIC3蛋白的单克隆抗体， 探究ERGIC3蛋白在人体正常组织以及常见肿瘤组织中的表达情况，为研究ERGIC3在人体中的生理及病理功能的提供依据，尤其是为肿瘤早期诊断研究的提供新的方法基础。 MicroRNAs（miRNAs）是一组约为18-22核苷酸的非编码RNA。人体有约3%的基因编码miRNA，而有约30%的人体基因受到miRNA的转录调节。人体的很多生物过程都受到microRNA的调控，包括细胞增殖、凋亡、分化及发育等过程。因此，microRNA表达异常也会导致人体一系列疾病，包括癌症等。 实验室前期工作，通过miRNA-seq的方法检测在肺癌细胞及肺永生化细胞系中差异表达的miRNAs。通过mirecord等生物分析软件发现了与ERGIC3相关的差异表达的miRNA，包括hsa-miR-140-3p、hsa-miR-203a等。有文献报道，miR-490-3p在肝癌中异常表达且参与ERGIC3的调控。经过对上述3个miRNA在肺癌细胞系及肺永生化细胞系表达情况的实时定量PCR验证，其结果基本与miRNA-的情况相同。接着对ERGIC3在肺癌细胞系中的mRNA及蛋白表达水平进行检测，其表达情况与异常表达的miRNA在细胞系中的表达情况存在一定的一致性。最后，对miRNA低表达的细胞系进行mimic处理，提取处理组以及control组细胞中的mRNA和蛋白，检测ERGIC3的变化情况。结果发现miR-140-3p、miR-129-5p及miR-490-3p在肺癌细胞系中进行mimic处理之后，ERGIC3的表达均出现了不同程度的下调，尤其是在XLA-07细胞系中经过miR-203a mimic处理后，实时定量检测ERGIC3的mRNA表达降低为原来的一半以下，Western-blot检测处理细胞蛋白的变化也有0.63左右的降低。表明miR-203a可能参与肺癌细胞中ERGIC3表达的调控。 |
| 英文摘要 | Lung cancer is a common cancer, the mortality rate ranks first in cancer, the 5-year survival rate of their patients was 10% to 15%. World Health Organization statistics show that in 2012 the world's 1.8 million new lung cancer patients, 159 people died. The Chinese lung cancer incidence and mortality bear the brunt of such cases account for about 1/3. An important reason for the high lung cancer mortality is early tumors can not easily be detected in time, delay optimal treatment period. And present some Tumor detection tend to have high false positive, poor sensitivity and complicated to operate, and other shortcomings, then find a good sensitivity, high specificity of Tumor markers, and the development of its simple and reliable method in Tumor detection is very important to improve the clinical detection of lung cancer. Our laboratory in the preliminary work, stablish lung adenocarcinoma differentially expressed gene library through SSH (suppression subtractive hybridization), further research indicated that mRNA and protein levels ERGIC3 abnormally high expression in lung cancer tissues and multiple lung cancer cell lines, And the positive rate of ERGIC3 is with lung cancer is closely relative to the degree of differentiation. ERGIC3 belongs to type Ⅱ transmembrane protein, molecular weight 43.2 kDa, is circulating protein between the endoplasmic reticulum and golgi apparatus. Studies have reported that disregulation of ERGIC3 would lead to endoplasmic reticulum stress resistant, while ERGIC3 as MiR-490-3p target genes involved in cell growth and metastasis of liver cancer and played important role in EMT process. All of these suggest ERGIC3 is likely to be a potential lung cancer markers. For further research on ERGIC3 pathological and physiological function, also for development of potential new method for early diagnosis of lung cancer, this study firstly prepared ERGIC3 mouse monoclonal antibody.Through several rounds of screening subclones obtained ERGIC3 specific monoclonal cell lines, and named 6-C4, the subtype subclass of 6-C4 antibody-secreting cell lines were identified, the result is IgG2b, kappa light chain. Further antibody identification found that the monoclonal antibody can be used in ELISA and Western blot, Immunofluorescence and Immunohistochemistry detection, then the application of 6-C4 secreted ERGIC3 antibody were systematic studied in normal tissues and a variety of tumor tissue, the results found in the human body, 23 kinds of normal tissue ERGIC3 express only in urinary system far ball tubule of the kidney, juxtaglomerular tubule cells, liver cells of the digestive system and the epithelial cells of the pancreas, and the digestive tract of gastric and intestinal villi epithelium cells, epithelial cells, and ductal epithelial cells were positive expression of ERGIC3; In most other body tissues, no obvious on immunohistochemical staining.This express distribution may have relationship with ERGIC3’function of protein transport and secretion. 15 kinds of common malignant tumor in human organizations, in the tumor tissues of epithelium ERGIC3 proteins have different degrees of high expression, while in mesenchymal tissue tumors, such as malignant and malignant chondrosarcoma, fibrosarcoma no obvious expression were detected by our prepared ERGIC3 monoclonal antibody.The research of ERGIC3 protein in human normal tissues and the common expression in tumor tissue, provide the basis for the study of ERGIC3’ pathological and physiological features in humans, especially provides new methodological basis for the early diagnosis of cancer. MicroRNAs (miRNAs) are a group of approximately 18-22 nucleotides in non-coding RNA. About 3% of the human genes encoding miRNA, and about 30% of human genes have been miRNA regulated. Many biological processes are regulated by microRNA, including cell proliferation, apoptosis, differentiation and developmental processes. Thus, microRNA expression abnormalities can also cause a range of human diseases, including cancer. The early stage of the laboratory work has detected the differential expression of miRNAs in lung cancer cells and lung immortalized cell lines by the method of miRNA-seq. Through biological analysis software, such as mirecord, found ERGIC3 related differentially expressed microRNAs, including has-miR-140-3p and has-miR-203a. As previous literature reported, miR-490-3p was abnormal expression in liver cancer and participated in ERGIC3 regulation. The expression of above-mentioned 3 microRNAs were verified in lung cancer cell line and lung immortalized cell line by real-time quantitative PCR, the results were same as miRNA-seq. Then detected ERGIC3 mRNA and protein expression level in lung cancer cell line, its expression and the microRNAs abnormal expression exist a certain consistency. Finally, mimic the low expression miRNA cell line, and extract treatment group and control group of mRNA and protein in the cells, then detect the alteration of ERGIC3. After miR-140-3p, miR-490-3p and miR-203a mimic in lung cancer cell line, ERGIC3 expression had different degrees of decrease, especially in the XLA-07 cell lines after treating with the miR-203 a mimic, real-time quantitative detection of ERGIC3 decreased to less than half of the original mRNA expression, Western-blot detecting the change of cell protein also reduced to 0.67, demonstrate that miR-203a may participate in regulating ERGIC3 expression in lung cancer. |
| 语种 | 中文 |
| 公开日期 | 2014-07-01 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7919]  |
| 专题 | 昆明动物研究所_分子病理学 |
| 推荐引用方式 GB/T 7714 | 林清海. ERGIC3单克隆抗体制备 、鉴定及其初步应用研究[D]. 北京. 中国科学院研究生院. 2014. |
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