發(fā)布時(shí)間：2018-04-26 09:29

  本文選題：腫瘤 + 乳腺癌　； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景和目的對(duì)于人類表皮生長(zhǎng)因子受體家族中的成員HER2/P185/ErbB2來(lái)說(shuō),其過(guò)表達(dá)與臨床上多種腫瘤的發(fā)生發(fā)展密切相關(guān),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響腫瘤病人的化療效果及生存時(shí)間。目前臨床上通常用檢測(cè)腫瘤病人血清中HER2蛋白的表達(dá)水平用作是多種腫瘤的診斷以及治療方案制定的一個(gè)標(biāo)準(zhǔn),例如胃癌、乳腺癌等,其可認(rèn)為是判斷腫瘤預(yù)后的獨(dú)立危險(xiǎn)因素。在臨床上常常使用血清中HER2水平的變化趨勢(shì)用作評(píng)價(jià)腫瘤的治療效果以及作為腫瘤患者預(yù)后的評(píng)估指標(biāo),但是由于目前國(guó)內(nèi)靈敏度較高的血清HER2的檢測(cè)方法的技術(shù)尚缺,因此本實(shí)驗(yàn)一項(xiàng)人血清中HER2蛋白含量高靈敏檢測(cè)技術(shù),并進(jìn)行臨床標(biāo)本檢測(cè),為腫瘤治療及預(yù)后評(píng)估提供參考。材料與方法本實(shí)驗(yàn)室從以下幾方面來(lái)建立人血清HER2檢測(cè)方法:(1)抗HER2的單克隆抗體HuA21與HerA以及高純度的標(biāo)準(zhǔn)抗原HER2-ECD的制備:以適應(yīng)的濃度接種培養(yǎng)本實(shí)驗(yàn)室保存的CHO細(xì)胞,待細(xì)胞培養(yǎng)至90%密度時(shí)分別轉(zhuǎn)染含HuA21、HerA、HER2-ECD基因的表達(dá)載體,待24小時(shí)的繼續(xù)培養(yǎng)后用胰酶進(jìn)行消化并將其傳代培養(yǎng),并往培養(yǎng)基內(nèi)加入0.3mg/ml Zeocin混合成選擇性培養(yǎng)基用來(lái)篩選抗性克隆。將得到的高表達(dá)抗性克隆逐級(jí)擴(kuò)大培養(yǎng),收取培養(yǎng)的上清液,進(jìn)一步純化、濃縮,采用SDS-PAGE非還原電泳技術(shù)純化抗體HuA21、HerA,采用BCA法蛋白定量試劑盒測(cè)定HER2-ECD標(biāo)準(zhǔn)抗原的含量,并與美國(guó)批準(zhǔn)臨床使用的血清HER-2/neu ELISA測(cè)定試劑盒檢測(cè)結(jié)果進(jìn)行比對(duì)。(2)運(yùn)用雙抗體夾心酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)技術(shù),分別以抗HER2單克隆抗體HuA21為包被抗體,生物素標(biāo)記的HerA作為檢測(cè)抗體,將得到的抗原標(biāo)準(zhǔn)品HER2-ECD構(gòu)建檢測(cè)血清中HER2蛋白含量技術(shù),并以此作為配對(duì)抗體親和力、特異性、穩(wěn)定性的檢測(cè)以及配對(duì)抗體濃度、反應(yīng)所需試劑和反應(yīng)條件的優(yōu)化等。(3)收集合肥地區(qū)的乳腺癌患者的血清標(biāo)本,使用研制的試劑盒進(jìn)行檢測(cè),并與西門子醫(yī)學(xué)診斷產(chǎn)品有限公司的類似試劑盒對(duì)比實(shí)驗(yàn)進(jìn)行檢測(cè),運(yùn)用student’s t檢驗(yàn)等統(tǒng)計(jì)方法對(duì)實(shí)驗(yàn)結(jié)果進(jìn)行分析總結(jié)。結(jié)果純度較高的抗HER2單克隆抗體HuA21和HerA由本實(shí)驗(yàn)制備成功。對(duì)實(shí)驗(yàn)設(shè)計(jì)使用的試劑盒各項(xiàng)組分進(jìn)行優(yōu)化,從而確定了高靈敏人血清HER2檢測(cè)試劑盒的包被抗體和檢測(cè)抗體抗分別為HuA21和生物素標(biāo)記的HerA。由上述方法所建立的ELISA方法檢測(cè)靈敏度為7.8 pg/ml,檢測(cè)范圍為0-500 pg/ml,其批內(nèi)變異系數(shù)為0.2%~10.9%,批間變異系數(shù)為1.4%~12.4%,在人血清中的抗原回收率在86.84%~116.40%之間,與上海西門子公司的化學(xué)發(fā)光法試劑盒測(cè)定臨床血清HER2含量相比較,實(shí)驗(yàn)結(jié)果表明二者之間的差異無(wú)明顯的統(tǒng)計(jì)學(xué)意義(P0.05),且其兩者之間的相關(guān)性(R20.70)具有明顯的一致性。結(jié)論本實(shí)驗(yàn)成功研制了一項(xiàng)能夠檢測(cè)人血清中HER2含量的高靈敏試劑盒。通過(guò)檢測(cè)收集到的臨床血清樣品發(fā)現(xiàn),通過(guò)HER2陽(yáng)性的轉(zhuǎn)移性乳腺癌患者與早期乳腺癌患者及健康人的比較可知,其血清中HER2含量明顯高于早期患者及健康者的平均水平(P0.05),結(jié)果發(fā)現(xiàn)HER2在轉(zhuǎn)移性乳腺癌患者中的檢出陽(yáng)性率為37.5%,這基本符合國(guó)外報(bào)道的結(jié)果。通過(guò)使用本實(shí)驗(yàn)技術(shù)對(duì)臨床組織樣品的檢測(cè),檢測(cè)結(jié)果表明,血清HER2在HER2陽(yáng)性的轉(zhuǎn)移性乳腺癌患者中的含量明顯高于早期乳腺癌患者及健康人的平均水平(P0.05);通過(guò)針對(duì)使用曲妥珠單合并化療的乳腺癌患者和接受單純化療的乳腺腫瘤患者的血清HER2含量變化進(jìn)行跟蹤檢測(cè),結(jié)果發(fā)現(xiàn)對(duì)接受藥物治療的腫瘤患者的有效性與血清中HER2水平的降低之間有正相關(guān)的關(guān)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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 李梅;;HER-2陽(yáng)性乳腺癌靶向治療藥物研究進(jìn)展[J];實(shí)用醫(yī)藥雜志;2012年08期

2 楊玲;李連弟;陳育德;D．M．Parkin;;中國(guó)乳腺癌發(fā)病死亡趨勢(shì)的估計(jì)與預(yù)測(cè)[J];中華腫瘤雜志;2006年06期

，

本文編號(hào)：1805431