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1、硕士学位论文凝血酶诱导人肺成纤维细胞表达单核细胞趋化蛋白-1及其信号通路的研究硕士研究生:袁超导师:傅玉才第二导师:邓小玲专业:遗传学研究方向:肺纤维化的细胞分子机制研究入学时间:2009年 9月中国汕头二零一二年五月IdentificationofSignalingPathwaysInvolvedinThrombin-induced CCL2/MCP-1 production by HumanLung Fibroblasts-1 CellYUAN ChaoSubmitted in Partial Fulfillment of the Requirements for theMaster of
2、 Science DegreeSupervisorProf. FU Yu-cai and Associate Prof. DENG Xiao-lingDepartment of Cell SenescenceMay, 2012学位论文原创性声明本论文是我个人在导师指导下进行的工作研究及取得的研究成果。论文中除了特别加以标注和致谢的地方外,不包含其他人或其它机构已经发表或撰写过的研究成果。对本文的研究做出贡献的个人和集体,均已在论文中以明确方式标明。本人完全意识到本声明的法律责任由本人承担。作者签名:日期:年月日学位论文使用授权声明本人授权汕头大学保存本学位论文的电子和纸质文档,允许论文被查阅和
3、借阅;学校可将本学位论文的全部或部分内容编入有关数据库进行检索,可以采用影印、缩印或其它复制手段保存和汇编论文;学校可以向国家有关部门或机构送交论文并授权其保存、借阅或上网公布本学位论文的全部或部分内容。对于保密的论文,按照保密的有关规定和程序处理。作者签名:导师签名:日期:年月日日期:年月日汕头大学医学院硕士学位论文摘要研究目的:本研究旨在验证凝血酶是否诱导人肺成纤维细胞(human lung fibroblast-1 , HLF-1)表达单核细胞趋化蛋白-1(monocyte chemoattractant protein-1 , MCP-1);并验证凝血酶是否通过蛋白激酶 C(prote
4、in kinase C , PKC)途径诱导人肺成纤维细胞表达 MCP-1和凝血酶能否增强 NF-B与 MCP-1基因启动子区的结合从而提高 MCP-1转录水平。方法:培养人肺成纤维细胞,分别用几种不同类型的蛋白激酶抑制剂预处理人肺成纤维细胞,再用凝血酶(10 nmol/L)刺激,利用 ELISA法检测细胞培养上清液中 MCP-1的蛋白表达;提取细胞裂解液中总 RNA并逆转录合成 cDNA,利用实时定量 PCR检测 MCP-1mRNA表达水平;采用用染色质免疫共沉淀(CHIPs)技术检测在凝血酶的诱导下 NF-B与 MCP-1启动子区的结合。结果:凝血酶诱导人肺成纤维细胞表达 MCP-1;广谱
5、型 PKC抑制剂 Bisindolylmaleimide I和RO-31-8220可抑制凝血酶诱导的人肺成纤维细胞 MCP-1蛋白释放及 mRNA表达,而 Ca2+依赖性 PKC抑制剂 G6976则没有抑制效果;IK抑制剂可抑制凝血酶诱导的人肺成纤维细胞 MCP-1蛋白释放及 mRNA表达;在凝血酶的诱导下 NF-B与 MCP-1启动子区的结合加强。结论:实验结果证实凝血酶可以呈剂量依赖和时间梯度诱导人肺成纤维细胞表达 MCP-1;结果提示凝血酶是通过非 Ca2+依赖性 PKC途径诱导人肺成纤维细胞表达 MCP-1;另外,凝I汕头大学医学院硕士学位论文血酶能够增强 NF-B与人肺成纤维细胞 M
6、CP-1基因启动子区的结合从而提高 MCP-1转录水平。本研究可能为肺成纤维化疾病提供新型的潜在的治疗靶点。关键词凝血酶;蛋白激酶抑制剂;单核细胞趋化蛋白-1;核因子-B;人肺成纤维细胞II汕头大学医学院硕士学位论文AbstractObjective:This study aims to prove thrombin can induce HLF-1 express MCP-1, and the mechanism itinduce HFL-1 express MCP-1 is through PKC pathway.In addition,thromin can enhance thecom
7、bination of NF-B and the MCP-1 gene promoter of HLF-1 to promote the transcription ofMCP-1.METHODS:HLF-1 were cultured in F12K. Cultured HLF-1 were preincubated by protein kinaseinhibitors with various concentrations followed by thrombin stimulation. MCP-l protein levels insupernatants were assessed
8、 by ELISA. Total RNA from cell Lysis Solution was isolated andretrovirus cDNA were synthesized.MCP-1 mRNA levels were measured by quantitative real timePCR. The effect of thrombin on NF-kB p65 subunit binding to the MCP-1 promoter was detectedby ChIP assays.RESULTS:Thrombin could induces HLF-1 cell
9、express MCP-1. Bisindolylmaleimide I andRO-31-8220 of Broad spectrum PKC inhibitors inhibited thrombin-induced HLF-1 MCP-lrelease and mRNA inhibited thrombin-induced HLF-1 MCP-l release and mRNA expressionThe ChIP assay results showed that NF-kB p65 subunit binding to the MCP-1 promoter wasenhanced
10、after thrombin stimulation.CONCLUSION:Our study indicates thrombin induces HLF-1 cell express MCP-1 in a dose dependent andtime course dependent manner. Through non calcium ion dependent PKC, thromin inducesHLF-1 cell express MCP-1 ,and it can enhance the combination of NF-B and the MCP-1 genepromot
11、er of HLF-1 to increase MCP-1 expression in transcription level. This study may identifypotential novel therapeutic intervention for pulmonary fibrosis.III汕头大学医学院硕士学位论文KEY WORDSThrombin; Protein Kinase inhibitor; Monocyte Chemoattractant Protein-l;NF-B; Human Lung Fibloblast-1IV汕头大学医学院硕士学位论文缩略语简表Acr
12、APAP-1BpBSADMSODNAEBEDTAEGTAERKF12KGHHRPIKMgMinMlMTTNF-BnmODAcrylamideAmmonium persulfateactivator protein- 1Base pairBovine Serum AlbuminDimethyl sulphoxideDeoxyribonucleic acidEthidium BromideEthylene Diamine Tetraacetic AcidEthylene glycol bis(2-aminoethyl) tetraacetic acidextracellular regulated protein kinasesNutrient Mixture F-1