【作者】 姚辉；

【导师】 陈志南； 李郁；

【作者基本信息】 第四军医大学 ， 细胞生物学， 2009， 硕士

【摘要】 Cyclophilin A (CypA)是亲环素家族中的主要成员,分子量约18kDa,在人体内多种组织和细胞中广泛表达。CypA最早被发现的功能是作为免疫抑制药物环孢菌素A(Cyclosporine A,CsA)在细胞内的主要受体,二者形成的复合物可以通过抑制钙调神经素的磷酸酶活性,进而阻止活化T细胞核因子(NF-ATs)向核内的移位以及下游相关基因的转录。CypA另一个重要的功能是具有肽基脯氨酰顺反异构酶(peptidyl-prolyl cis-tranisomerase,PPIase)活性,可以催化蛋白折叠和构象变化时肽基脯氨酰键的顺反异构化。CypA的顺反异构酶活性影响了细胞内蛋白质的转运、线粒体功能的维持、mRNA前体的加工、多蛋白复合体结构完整性的维持等多种重要的细胞生理过程。人体内CypA表达和功能的异常,与艾滋病、类风湿性关节炎、动脉粥样硬化等疾病的发生和进展有着紧密的联系。近几年来的研究还发现,CypA在肺癌、胰腺癌、子宫内膜癌等癌组织中的表达显著升高,CypA表达增高可能与癌细胞的增殖能力和抗凋亡能力提高有关。但是,CypA在肝癌中的表达和功能目前尚不清楚。本课题的研究目的是:(1)通过杂交瘤技术制备抗人CypA蛋白的单克隆抗体,为后续的研究提供必备的抗体工具;(2)阐明CypA蛋白在人正常肝组织和肝癌组织中的表达情况,以及CypA在肝癌的发生和发展过程中的功能和作用机制。本课题包含两部分内容(:1)利用高纯度的重组CypA蛋白免疫BALB/c小鼠,通过间接ELISA和细胞免疫荧光染色法筛选出特异性分泌抗人CypA单抗的杂交瘤细胞株,制备腹水后采用蛋白A吸附层析法纯化抗体;采用间接ELISA、蛋白免疫印记、细胞荧光、免疫组化等方法对单抗的特异性进行鉴定;最后对抗体可变区的基因进行克隆。(2)通过对肝癌和正常肝组织进行免疫组化染色,分析CypA在二者中的表达差异;明确CypA在肝癌细胞中是否存在分泌表达的形式,探讨可能的分泌形式对肝癌细胞的功能和作用机制。我们取得了以下结果:(1)制备了两株稳定分泌抗人CypA抗体的杂交瘤细胞株,分别命名为C7B8F10和F6E4F8B2;两株杂交瘤制备的腹水中单抗的滴度均达到106,腹水中抗体纯化后纯度大于95.0%;两株抗体均为IgG1亚类,κ轻链;蛋白免疫印迹实验表明两个单抗均能与原核表达的CypA蛋白,以及肝癌细胞裂解液中的的CypA蛋白特异性地结合。(2)以C7B8F10进行免疫组化染色的结果表明,CypA在肝癌组织表达水平要显著高于正常肝组织;我们证实了培养的肝癌细胞可以分泌表达CypA,低氧的刺激会进一步增强这种分泌表达;重组CypA蛋白可以通过活化ERK1/2和p38MAPK信号途径促进肝癌细胞的增殖,这种促增殖作用不依赖于CypA的顺反异构酶活性。更多还原

【Abstract】 Cyclophilin A (CypA), the prototypical member of the cyclophilins family, is an 18 kDa protein which can be found in many kinds of human tissues and cells. It was first identified as the primary intracellular target of the immunosuppressant cyclosporine A. Through inhibition of the phosphatase calcineurin, CypA:CsA down-regulates the nuclear translocation of nuclear factors of activated T cells and down stream gene transcription. CypA is also a peptidyl-prolyl isomerase (PPIase) which can catalyzes cis-trans isomerization about peptidyl-prolyl bonds during protein folding or conformational changes. The PPIase activity of CypA has recently been shown to be involved in diverse cellular processes, including intracellular protein trafficking, mitochondrial function, pre-mRNA processing, and maintenance of multiprotein complex stability. Abnormal expression or dysfunction of CypA has a relationship with several kinds of human diseases, such as AIDS, rheumatoid arthritis, atherosclerosis, etc. Recent reports have shown that CypA is over expressed in many cancer cells, including human lung cancer, pancreatic cancer, endometrial carcinoma. Overexpression of CypA is involved in the increase of proliferation rate and anti-apoptosis ability of cancer cells. However, little is known about the expression and function of CypA in hepatocellular carcinoma (HCC).The first purpose of this study is to generate mAbs against CypA by hybridoma technology which can provide necessary antibodies for the future research. The second purpose is to analyze the expression level of CypA in normal human hepatic tissue and HCC tissue, and identify the functions of CypA in the initiation and progression of HCC.Our study included two parts. Firstly, we immunized BALB/c mice with highly purified recombinant CypA. Hybridomas secreting mAbs specific against CypA were screened by indirect ELISA and immunofluorescence cell staining. The mAbs in the ascites were purified by specific adsorbing chromatography, and the specificity of the mAbs were identified by indirect ELISA , western blot, immunofluorescence cell staining and immunohistochemistry (IHC). Genes encoding the variable region of mAbs were also cloned and sequenced. Secondly, we analyzed the different expression level of CypA between normal hepatic tissue and HCC tissue by IHC. We also tried to identify whether CypA could be released from HCC cells in vitro, and the main function of this secreting type of CypA.We generated two strains of hybridomas named C7B8F10 and F6E4F8B2. The titers of these mAbs in the ascites reached to 106 and the purity of these mAbs were over 90% after purification. They all belonged to IgG1 subclass,κ-light chain. Western blot showed that both of two mAbs could bind to recombinant CypA or CypA in HCC cell lysate. One of the mAbs, C7B8F10, could also be used as primary antibody for IHC, and the IHC showed that CypA was highly expressed in HCC tissues and much lower in normal hepatic tissues. Moreover, we found the phenomenon that HCC cells could secret CypA into the culture medium, which could be further enhanced under the hypoxia cultivation condition. The recombinant CypA could promote the proliferation of HCC cells by activating two important cell signal molecules ERK1/2 and p38MAPK, and this promotion is not dependt on the PPIase activity of CypA.更多还原