高灵敏人血清HER2蛋白检测方法的建立及临床应用

发布时间：2018-04-26 09:29

本文选题：肿瘤 + 乳腺癌　；参考：《安徽医科大学》2017年硕士论文

【摘要】：背景和目的对于人类表皮生长因子受体家族中的成员HER2/P185/ErbB2来说,其过表达与临床上多种肿瘤的发生发展密切相关,其中包括乳腺癌、胃癌、肺癌及卵巢癌等。血清中HER2蛋白的含量可直接影响肿瘤病人的化疗效果及生存时间。目前临床上通常用检测肿瘤病人血清中HER2蛋白的表达水平用作是多种肿瘤的诊断以及治疗方案制定的一个标准,例如胃癌、乳腺癌等,其可认为是判断肿瘤预后的独立危险因素。在临床上常常使用血清中HER2水平的变化趋势用作评价肿瘤的治疗效果以及作为肿瘤患者预后的评估指标,但是由于目前国内灵敏度较高的血清HER2的检测方法的技术尚缺,因此本实验一项人血清中HER2蛋白含量高灵敏检测技术,并进行临床标本检测,为肿瘤治疗及预后评估提供参考。材料与方法本实验室从以下几方面来建立人血清HER2检测方法:(1)抗HER2的单克隆抗体HuA21与HerA以及高纯度的标准抗原HER2-ECD的制备:以适应的浓度接种培养本实验室保存的CHO细胞,待细胞培养至90%密度时分别转染含HuA21、HerA、HER2-ECD基因的表达载体,待24小时的继续培养后用胰酶进行消化并将其传代培养,并往培养基内加入0.3mg/ml Zeocin混合成选择性培养基用来筛选抗性克隆。将得到的高表达抗性克隆逐级扩大培养,收取培养的上清液,进一步纯化、浓缩,采用SDS-PAGE非还原电泳技术纯化抗体HuA21、HerA,采用BCA法蛋白定量试剂盒测定HER2-ECD标准抗原的含量,并与美国批准临床使用的血清HER-2/neu ELISA测定试剂盒检测结果进行比对。(2)运用双抗体夹心酶联免疫吸附实验(ELISA)技术,分别以抗HER2单克隆抗体HuA21为包被抗体,生物素标记的HerA作为检测抗体,将得到的抗原标准品HER2-ECD构建检测血清中HER2蛋白含量技术,并以此作为配对抗体亲和力、特异性、稳定性的检测以及配对抗体浓度、反应所需试剂和反应条件的优化等。(3)收集合肥地区的乳腺癌患者的血清标本,使用研制的试剂盒进行检测,并与西门子医学诊断产品有限公司的类似试剂盒对比实验进行检测,运用student’s t检验等统计方法对实验结果进行分析总结。结果纯度较高的抗HER2单克隆抗体HuA21和HerA由本实验制备成功。对实验设计使用的试剂盒各项组分进行优化,从而确定了高灵敏人血清HER2检测试剂盒的包被抗体和检测抗体抗分别为HuA21和生物素标记的HerA。由上述方法所建立的ELISA方法检测灵敏度为7.8 pg/ml,检测范围为0-500 pg/ml,其批内变异系数为0.2%~10.9%,批间变异系数为1.4%~12.4%,在人血清中的抗原回收率在86.84%~116.40%之间,与上海西门子公司的化学发光法试剂盒测定临床血清HER2含量相比较,实验结果表明二者之间的差异无明显的统计学意义(P0.05),且其两者之间的相关性(R20.70)具有明显的一致性。结论本实验成功研制了一项能够检测人血清中HER2含量的高灵敏试剂盒。通过检测收集到的临床血清样品发现,通过HER2阳性的转移性乳腺癌患者与早期乳腺癌患者及健康人的比较可知,其血清中HER2含量明显高于早期患者及健康者的平均水平(P0.05),结果发现HER2在转移性乳腺癌患者中的检出阳性率为37.5%,这基本符合国外报道的结果。通过使用本实验技术对临床组织样品的检测,检测结果表明,血清HER2在HER2阳性的转移性乳腺癌患者中的含量明显高于早期乳腺癌患者及健康人的平均水平(P0.05);通过针对使用曲妥珠单合并化疗的乳腺癌患者和接受单纯化疗的乳腺肿瘤患者的血清HER2含量变化进行跟踪检测,结果发现对接受药物治疗的肿瘤患者的有效性与血清中HER2水平的降低之间有正相关的关系。
[Abstract]:Background and purpose for HER2/P185/ErbB2, a member of the human epidermal growth factor receptor family, its overexpression is closely related to the development of a variety of clinical tumors, including breast, gastric, lung, and ovarian cancer. The content of HER2 protein in the serum can directly affect the chemotherapy effect and survival time of the tumor patients. It is usually used to detect the expression level of HER2 protein in the serum of cancer patients as a criterion for the diagnosis of various tumors and the formulation of a therapeutic scheme, such as gastric cancer, breast cancer, etc., which can be considered as an independent risk factor for judging the prognosis of the tumor. The change trend of HER2 level in blood is often used to evaluate the tumor in clinical. The effect of the treatment and the evaluation index of the prognosis of the tumor patients, but because of the lack of the technique of detecting the serum HER2 with high sensitivity at home, a high sensitive detection technique of HER2 protein content in human serum, and the detection of clinical specimens, can be used as a reference for the treatment of tumor and the prognosis of the tumor. The method of human serum HER2 detection is established in the following aspects: (1) preparation of anti HER2 monoclonal antibody HuA21 and HerA and high purity standard antigen HER2-ECD: inoculating CHO cells stored in our laboratory at adaptive concentration, and transfecting HuA21, HerA, HER2-ECD gene expression to the cell culture to 90% density. After 24 hours of continuous culture, it was digested and cultured with trypsin, and then mixed with 0.3mg/ml Zeocin into the culture medium and mixed into a selective medium to screen the resistant clones. The high expression resistant clones were expanded to be cultured in order to collect the cultured supernatant, and to purify, concentrate and use the SDS-PAGE non reduction electrophoresis technique. The antibody HuA21, HerA, the content of HER2-ECD standard antigen was measured by BCA protein quantitative kit, and compared with the serum HER-2/neu ELISA test kit test results approved by the United States. (2) double antibody sandwich enzyme linked immunosorbent assay (ELISA) was used to resist HER2 monoclonal antibody HuA21 as a package, respectively. In vivo and biotin labeled HerA as a detection antibody, the obtained antigen standard HER2-ECD is constructed to detect HER2 protein content in serum, which is used as a matching antibody affinity, specificity, stability detection, paired antibody concentration, reaction requirements and optimization of reaction conditions. (3) collect breast cancer patients in Hefei area The serum samples were tested by the developed kit and tested with the similar kits of SIEMENS medical diagnosis Products Co., Ltd., and the results were analyzed by Student 's t test. The results showed that the high purity anti HER2 monclon antibody HuA21 and HerA were prepared successfully by this experiment. All the components of the kit used in the experimental design were optimized, and the sensitivity of the ELISA method established by the above method was 7.8 pg/ml, the detection range was 0-500 pg/ml, and the intra batch variation coefficient was 0.. The sensitivity of the high sensitive human serum HER2 detection kit was 7.8 pg/ml. 2%~10.9%, the coefficient of variation between the groups was 1.4%~12.4%, the recovery rate of antigen in human serum was between 86.84%~116.40%, and compared with the determination of serum HER2 content by the chemiluminescence reagent kit of SIEMENS company in Shanghai. The experimental results showed that there was no significant statistical significance between the two (P0.05), and the correlation between the two (R20.70). Conclusion this experiment has successfully developed a highly sensitive kit for detecting the content of HER2 in human serum. Through the detection of collected clinical serum samples, it is found that the serum HER2 content of the patients with HER2 positive metastatic breast cancer is obviously higher than that of early breast cancer patients and healthy people. The average level of patients and healthy persons (P0.05) showed that the positive rate of HER2 in patients with metastatic breast cancer was 37.5%, which was basically in line with the results of foreign reports. The results of detection of clinical tissue samples using this technique showed that serum HER2 was significant in patients with HER2 positive metastatic breast cancer. Compared to the average level of early breast cancer patients and healthy people (P0.05), the changes in serum HER2 levels of breast cancer patients and breast cancer patients receiving chemotherapy with trastuzuma and chemotherapy were tracked, and the results were found to be effective and the level of HER2 in the serum was reduced. There is a positive correlation between them.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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