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学科主题: 临床检验诊断学
题名:
一、Survivin特异性单克隆抗体检测膀胱癌和肾细胞癌应用基础研究 二、LAPTM4B基因多态性与肾细胞癌和膀胱癌易感性的关系
作者: 陈冬梅
答辩日期: 2016-05-19
导师: 陈冬梅
专业: 临床检验诊断学
授予单位: 北京大学
授予地点: 北京大学临床肿瘤学院
学位: 硕士
关键词: Survivin ; 单克隆抗体 ; 膀胱癌 ; 肾细胞癌 ; 肿瘤标志物 ; 酶联免疫吸附法(ELISA) ; LAPTM4B ; 多态性 ; 易感性
其他题名: 1.Detection of Survivin expression in bladder cancer and renal cell carcinoma with its specific monoclonal antibodies 2.Relationship between LAPTM4B gene polymorphism and susceptibility of renal cell carcinoma and bladder cancer
分类号: R730.43
摘要:

目的  Survivin高表达于大部分肿瘤,却在大部分成人的正常组织中不表达,已有研究表明Survivin作为肿瘤标志物在肿瘤的诊断、治疗和预后方面具有重要价值,但是需要进一步证实。本研究旨在利用Survivin特异性单克隆抗体建立检测膀胱癌和肾细胞癌的细胞系,血清标本,尿液标本和肿瘤组织中Survivin表达的方法,并进一步评价Survivin作为膀胱癌和肾细胞癌肿瘤标志物的价值。

方法  通过培养Survivin单克隆杂交瘤细胞株,测定培养上清中单抗的表达量、亚类、特异性等,选择单抗表达量较大、特异性好、滴度高的杂交瘤细胞株进行扩大培养,然后注射到Balb/c小鼠腹腔取腹水,利用盐析法和亲和层析柱法纯化得到单克隆抗体。将纯化的单克隆抗体分别用辣根过氧化物酶标记,并用葡聚糖凝胶纯化。用一株单抗包被酶标板,与另一株辣根过氧化物酶标记的单抗配对建立一步法夹心ELISA,MS2-Survivin融合蛋白作为标准蛋白。并对一步法夹心ELISA进行灵敏度、特异性、重复性评价。同时收集膀胱癌患者尿液标本105例和血清标本122例,以及肾细胞癌患者尿液标本124例和血清标本208例。健康对照组尿液标本131例和血清标本198例。用辣根酶标记的单抗通过Western blot以检测肿瘤细胞系5637(膀胱癌),A549(肺癌),EC109(食管癌)和HepG2(肝癌)中Survivin的表达情况。通过免疫组化的方法检测Survivin在膀胱癌组织中的表达。

结果  选择了5株杂交瘤细胞(D8,A6,C6,A9,E6)制备单抗,分别纯化并用辣根过氧化物酶标记。E6作为包被单抗和辣根过氧化物酶标记C6作为检测抗体配对,成功建立了一步法夹心ELISA,其板内变异系数为7.28%,而板间变异系数为9.58%。以MS2-Survivin融合蛋白作为标准蛋白,最小可检测的剂量0.98ng/ml。利用一步法夹心ELISA检测收集的尿液和血清标本,发现膀胱癌和肾细胞癌患者尿液和血清中Survivin表达与健康对照组之间均存在显著性差异(p<0.05)。而且Survivin表达与膀胱癌和肾细胞癌的临床病理特征未发现关联,除了在肾细胞癌患者血清中高血压因素(p<0.05)you有显著关联。膀胱癌尿标本、肾细胞癌尿标本、膀胱癌血清和肾细胞癌血清的ROC曲线下的面积(AUC)分别为0.800,0.812,0.691,和0.600。按照最佳约登指数,当膀胱癌(肾细胞癌)患者尿标本cutoff值设为8.2765(9.4985)ng/ml时,敏感度为76.20%(71.00%),特异性为88.60%(84.00%)。当膀胱癌(肾细胞癌)患者血清cutoff值设为1.2385(1.1625)ng/ml,灵敏度为71.3%(62.00%),特异性为56.10%(47.50%)。Survivin在尿液中的表达水平要高于血清。在Western blot中,辣根酶标记单抗能够识别16.5kD处肿瘤细胞系中Survivin的表达。免疫组化的结果也显示Survivin在膀胱癌患者的组织中有不同程度的表达。

结论  本研究成功标记了Survivin单抗并建立了灵敏度和特异性高的一步法夹心ELISA,检测了肿瘤细胞系(5637,A549,EC109,HepG2),肿瘤患者尿液和血清标本以及组织中Survivin的表达。从细胞系,血清,尿液和组织水平证明了Survivin在膀胱癌的表达,并进一步表明Survivin是监测膀胱癌以及肾细胞癌的高潜能肿瘤标志物。

目的 溶酶体相关四次跨膜蛋白B(Lysosomal Associated Protein Transmembrane 4B,LAPTM4B)基因具有两个等位基因,LAPTM4B* 1和LAPTM4B* 2。已有研究证明LAPTM4B* 2与许多癌症的易感性相关。本研究旨在探讨LAPTM4B基因多态性与肾细胞癌和膀胱癌易感性之间的关系。

方法 对180例肾细胞癌,91例膀胱癌和347健康对照组进行病例对照研究。利用聚合酶链反应和电泳分析LAPTM4B多态性。卡方检验用于分析癌症组和健康对照组之间基因型频率和其他癌症病理参数分布的关系。非线性逻辑回归分析模型用于计算95%置信区间(CI)比值(OR)。

结果 健康对照组LAPTM4B* 2频率(24.06%)低于肾细胞癌组(31.70%,P = 0.008)和膀胱癌组(35.71%,P = 0.009),有显著性差异。携带LAPTM4B*2的个体肾细胞癌和膀胱癌易感性分别是LAPTM4B*1的1.467(95%CI1.104-1.950)和1.634(95%CI1.132-2.360)倍。 肾细胞癌患者携带LAPTM4B* 1/2和* 2/2是LAPTM4B*1/1的 1.476(95%CI1.008-2.161)和2.231(95%CI1.089-4.569)倍。膀胱癌患者携带LAPTM4B*1/2和LAPTM4B* 2/2是LAPTM4B*1/1的1.563倍(95%CI,0.932-2.622)和2.900-倍(95%CI,1.233-6.821)。 LAPTM4B基因型和肾细胞癌和膀胱癌患者的临床病理参数之间没有发现联系,除了与膀胱癌患者的吸烟状况有关联(p = 0.003)。

结论 研究结果表明,LAPTM4B*2是肾细胞癌和膀胱癌的危险因素。LAPTM4B是肾细胞癌和膀胱癌易感性的潜在生物标志物。

英文摘要:

1

Purpose Survivin, highly expressed in the majority of tumors, while not in most normal adult tissues, was significant in clinical application. In our study, with the Survivin-specific monoclonal antibodies (mAbs), we aimed to establish methods for detecting the expression of Survivin in cancer cell lines, serum samples, urine samples and cancer tissues from bladder cancer (BCA) and renal cell carcinoma (RCC) patients, and evaluate the efficacy of Survivin as tumor marker in surveillance of BCA and RCC.

Methods Firstly, Label mAbs with horseradish peroxidase (HRP), and develop the one step enzyme-linked immunosorbent assay (ELISA) with mAbs and HRP-conjugated mAbs to detect Survivin expression in serum and urine samples of BCA and RCC patients comparing with healthy controls. The one step ELISA system was evaluated according to intra-assay precision, inter-assay precision and minimum detectable dose (MDD). A total of 105 and 125 urine samples, and 122 and 208 correspondent serum samples of BC and RCC patients were collected. And healthy control groups included 131 urine samples and 198 serum samples. HRP-conjugated mAbs were also used to detect Survivin expression in cancer cell lines in western blot. Survivin expression in cancer tissues of BCA patients was evaluated by immunohistochemistry.

Results The one step ELISA was successfully established. The intra-assay CV (Coefficient of variation) of the assay was 7.28% and the inter-assay CV was 9.58%, indicating that the one-step sandwich ELISA was reproducible. The minimum detectable dose (MDD) of the assay was 0.98ng/ml. And significantly higher expression of Survivin was found in BCA and RCC patients as compared with healthy controls in both urinary and serum samples (p<0.05), which was more remarkable in urine. With optimal Youden’s index, the cut-off value 8.2765 (9.4985) ng/ml was proposed, corresponding to a sensitivity of 76.20% (71.00%) and specificity of 88.60% (84.00%) in BCA (RCC) urine. In BCA (RCC) serum, when cut-off value was 1.2385(1.1625) ng/ml, sensitivity of 71.3% (62.00%) and specificity of 56.10% (47.50%) was obtained. HRP-mAbs could recongnize Survivin in cancer cell lines. Western blot analysis and immunohistochemistry results confirmed Survivin expression in bladder cancer cell line (5637) and tissues. And the expression of Survivin in bladder cancer tissues, urine and serum were in consistant in our study.

Conclusion In conclusion, the one step ELISA successfully established in our study was of high sensitivity and specificity in detection of Survivin expression. Our study indicated Survivin as highly potential tumor marker in surveillance of BCA and RCC.

2

Purpose     Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) gene has two alleles, LAPTM4B*1 and LAPTM4B*2. Previous studies have demonstrated LAPTM4B*2 contributed to the risk of cancers. This study aimed to investigate the correlation between LAPTM4B polymorphism and the susceptibility to renal cell carcinoma (RCC) and bladder cancer (BC).

Methods    A case-control analysis was performed in 180 RCC and 91 BC cases and 347 controls. LAPTM4B polymorphism was analyzed by polymerase chain reaction and electrophoresis. Chi-square test was used to calculate genotype frequency and other parametric distributions between cancer cases and controls. Unconditional logistic regression analysis models were used to calculate odds ratio (OR) with 95% confidence interval (CI).

Results    Allelic frequency of LAPTM4B*2 in the controls (24.06%) was significantly lower than in RCC (31.70%, p=0.008) and BC (35.71%, p=0.009) cases. The risk for RCC and BC increased 1.467 (95% CI 1.104–1.950) and 1.634 (95% CI 1.132–2.360) times in individuals carrying LAPTM4B*2 compared with those carrying LAPTM4B*1. RCC cases with LAPTM4B*1/2 and *2/2 have 1.476 (95% CI 1.008–2.161) and 2.231 (95% CI 1.089–4.569) higher risk than those carrying LAPTM4B*1/1. BC risk in cases carrying LAPTM4B*1/2 and LAPTM4B*2/2 was 1.563-fold (95% CI, 0.932–2.622) and 2.900- fold (95%CI, 1.233–6.821) that of cases with LAPTM4B*1/1. No association between LAPTM4B genotypes and clinical parameters of RCC and BC was found, except for smoking status in BC.

Conclusions      These findings suggested that LAPTM4B*2 was a risk factor for RCC and BC. LAPTM4B is a potential biomarker for susceptibility of RCC and BC.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/125690
Appears in Collections:北京大学临床肿瘤学院_学位论文

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作者单位: 北京大学临床肿瘤学院

Recommended Citation:
陈冬梅. 一、Survivin特异性单克隆抗体检测膀胱癌和肾细胞癌应用基础研究 二、LAPTM4B基因多态性与肾细胞癌和膀胱癌易感性的关系[D]. 北京大学临床肿瘤学院. 北京大学. 2016.
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