【作者】 余贻汉；

【导师】 杨道锋；

【作者基本信息】 华中科技大学 ， 内科学， 2006， 硕士

【摘要】 肝纤维化在慢性肝病的病理生理过程中占重要地位,阻断其向肝硬化发展,促使其逆转具有重要意义,而转化生长因子β在该过程中起重要作用,本课题旨在利用现代分子生物学和基因工程技术构建一个没有胞内段的不能转导胞外信号的截断型人转化生长因子βII型受体的表达载体,为进一步研究其生物学效应及在肝硬化中的作用奠定基础。目的:构建无细胞内段基因的人转化生长因子βII型受体(ΔTβRII)的真核表达载体pEGFP/ΔTβRII,转染人胚肝细胞L02以表达融合蛋白EGFP/ΔTβRII,并检测其胞外段的生物学活性。方法:以质粒H2-3FF为模板,用PCR方法扩增得到人转化生长因子βII型受体(TβRII)胞外段和跨膜段的cDNA ,并在两端分别引入EcoRI和BamHI限制性内切酶位点,将该cDNA经EcoRI和BamHI双酶切、纯化回收后克隆到真核表达载体pEGFP-N2上,构建成pEGFP/ΔTβRII重组质粒。经双酶切和核酸测序鉴定,将该重组质粒由脂质体Lipofectamin2000介导转染人胚肝细胞L02,用倒置荧光显微镜观察融合蛋白EGFP的表达,加入G418筛选得到阳性克隆,并用四唑盐(MTT)比色法和流式细胞术(FCM)检测其生物学活性。结果:①重组质粒pEGFP/ΔTβRII经EcoRI和BamHI双酶切,得到两个阳性片断,其中小片段带与PCR产物条带在相同位置,大小分别约为619bp;大片段条带与空载体pEGFP-N2骨架在同一位置,大小约为4.7kb,符合预期结果,并经核酸测序鉴定序列正确,表明ΔTβRII已经克隆至pEGFP-N2载体中;②重组质粒pEGFP/ΔTβRII转染L02人胚肝细胞24h后在荧光显微镜下观察到融合蛋白EGFP的表达,并用G418筛选得到稳定表达的阳性克隆;③MTT比色结果:转染重组子的实验组OD值显著高于转染pEGFP-N2的对照组OD值(0.780±0.012 vs 0.335±0.018, p<0.05); FCM结果显示前者的G1%(63.58)明显低于后者的G1% (73.03),而增殖指数PrI值[ (S +M G2) %]前者比后者高(36.42 vs 26.98),证实融合蛋白EGFP/ΔTβRII减轻TGFβ1对L02细胞生长抑制作用。结论:成功构建了pEGFP/ΔTβRII重组质粒,并表达了融合蛋白EGFP/ΔTβRII,其胞外段有结合TGFβ1的活性,能显著减轻TGFβ1对L02细胞生长抑制作用,并能减轻TGFβ1对L02细胞G1~S的阻滞作用,为进一步探讨其生物学效应及在肝硬化中的作用奠定了基础。更多还原

【Abstract】 It is significant to reverse the progress of hepatic fibrosis which paly an important role in the physiopathologic process of chronic hepatic disease. In this paper, a eukaryotic expression vector containing truncated type II human transforming growth factor was established to set foundation for further research.Objective: To construct a eukaryotic expression vector pEGFP/ΔTβRII without the cytoplasmic domain of the cDNA of the human transforming growth factor-beta type II receptor (TβRII), to express the fusion protein EGFP/ΔTβRII in human embryonic hepatic cell line L02 and to determine the biological activity of its extracellular domain.Methods: The cDNA of type II truncated receptor(ΔTβRII) that lacks the cytoplasmic domain was amplified by PCR from the plasmid H2-3FF, and two restriction sites (EcoR I and BamHI ) were introduced into it at the same time. The PCR product was inserted into the vector pEGFP-N2 to construct the eukaryotic expression plasmid pEGFP/ΔTβRII, which was identified by double enzyme digestion and DNA sequencing. The recombinant plasmid was transfected into L02 cells by cation lipoid Lipofectamine2000, and the GFP fluorescence was detected under inverted fluorescence microscope after the transfection. To obtain stable cells line, G418 was added in RPMI-1640 culture medium to screen the positive cells. The biological activity of its extracellular domain was examined by MTT assay and flow cytometry (FCM).Results:①Recombinant plasmid, pEGFP/ΔTβRII, digested with EcoRI and BamHI, and two bands at 619bp and 4.7kb could be observed respectively by agarose gel electrophoresis. These results were consistent with expection and the result of DNA sequencing, which demonstrated the gene ofΔTβRII was cloned to the vector pEGFP-N2.②The recombinant plasmid was transfected into the human embryonic hepatic cell line L02 by lipoid, and the specific fusion protein, EGFP, was observed by inverted fluorescence microscope 24h later. The positive clone was obtained by G418 screening.③The optical density (OD) of the test group was significantly higher than that of the control group, however, decreased the G1-phase of cell cycle and improved the cell proliferation index(PrI),which demonstrated the fusion protein would attenuate the inhibitory effect of TGFβ1 on L02 cells significantly.Conclusion: The eukaryotic expression plasmid pEGFP/ΔTβRII has been constructed and the fusion protein EGFP/ΔTβRII was successfully expressed in cell L02. The extracellular domain of the fusion protein can bind cytokine TGFβ1 , attenuate the inhibitory effect of TGFβ1 on L02 cells significantly and these works will help to set up foundation for further research.更多还原