【作者】 叶顺传；

【导师】 曾秋棠；

【作者基本信息】 华中科技大学 ， 内科学， 2008， 博士

【摘要】 第一部分pEGFP-N1/TNFR 1载体的构建及鉴定目的:本研究旨在构建人肿瘤坏死因子受体(TNFR 1)绿色荧光蛋白表达载体pEGFP-N1/TNFR 1,为研究TNFR 1细胞内转位奠定实验基础。方法:培养U937细胞,提取RNA,RT-PCR扩增出TNFR 1 cDNA基因片段,将TNFR 1基因的RT-PCR纯化产物及质粒pEGFP-N1 DNA经Sac I和Kpn I双酶切、胶回收纯化酶切片段后,体外连接酶切片段,使其定向重组,再将重组DNA转化E. coli DH5α感受态细胞,经复苏后,在含Kanr的LB固体培养基上筛选出阳性克隆。结果:挑取的LB固体培养基上的单菌落经酶切及测序鉴定,证实均为阳性克隆,即TNFR 1与pEGFP-N1体外重组成功。结论:采用体外重组技术,成功地将人TNFR 1 cDNA插入了荧光蛋白表达载体pEGFP-N1中。第二部分重组载体pEGFP-N1/TNFR 1在人脐静脉血管内皮细胞中的表达目的:构建肿瘤坏死因子受体(TNFR 1)绿色荧光蛋白表达载体pEGFP-N1/TNFR 1,并在人脐静脉血管内皮细胞中表达。方法:分离并培养HUVECs,将已成功构建的融合表达载体pEGFP-N1/TNFR 1,通过阳离子脂质体Lipofectin介导的方法转染到HUVEC中,RT-PCR检测mRNA的表达,Western blot检测融合蛋白的瞬时表达,同时在在活细胞状态下用荧光显微镜直接观察。结果:成功分离人脐静脉血管内皮细胞,重组pEGFP-N1/TNFR 1融合表达载体转染HUVECs后,在细胞中检测到TNFR 1基因片段,Western blot可以检测到TNFR 1在HUVECs表达,用荧光显微镜观察到转染细胞中有绿色荧光蛋白表达。结论:重组pEGFP-N1/TNFR 1融合表达载体在HUVEC中获得了表达,融合蛋白具有TNFR 1和GFP的双重活性,为进一步研究TNFR转位的调控因素打下基础。第三部分肌球蛋白IIB调节人脐静脉血管内皮细胞肿瘤坏死因子受体1的转位目的:观察非肌肉肌球蛋白IIB(nonmuscle myosin heavy chain IIB,NMHC-IIB)对人脐静脉血管内皮细胞(Human Hmbilical Vein Endothelial Cells, HUVECs)中肿瘤坏死因子受体1(TNF receptor 1, TNFR 1)转位的影响。方法:将成功构建的重组pEGFP-N1/TNFR 1载体通过阳离子脂质体Lipofectin介导的方法转染入HUVECs,筛选获得稳定克隆株。设计并合成NMHC-IIB的小分子干扰RNA(small interference RNA,siRNA),用脂质体LipofectamineTM 2000转染进入该稳定克隆株,差速离心加蔗糖密度梯度离心收获细胞质膜,Western印迹法观察TNFα诱导前和诱导后细胞质膜上TNFR 1蛋白含量的变化。结果:与空白对照组比较,转染NMHC-IIB siRNA进入HUVECs,可以明显抑制细胞内NMHC-IIB mRNA的表达(0.4540±0.0411 vs. 0.6704±0.0242,P < 0.001),也可以明显减少TNFα诱导前和诱导后细胞质膜TNFR 1的含量(0.3248±0.0167 vs. 0.4306±0.0289,P < 0.01;0.4922±0.0217 vs. 0.6609±0.0265,P < 0.001)。然而,在转染NMHC-IIB siRNA的细胞,TNFα的诱导仍然可以提高细胞质膜TNFR 1的含量(0.4922±0.0217 vs. 0.3248±0.0167,P < 0.001)。结论:NMHC-IIB可能在TNFR 1从反式高尔基体到细胞质膜的转位或运输中起正向作用,但是可能并不是惟一的,或者决定性的因素。更多还原

【Abstract】 Part I Construction and Identification of mammalian cell expression vector for pEGFP-N1 and TNFR 1 fusion proteinAIM: The purpose of this study was to construct a eukaryotic fluorescent expression vector carrying human TNFR 1 gene.METHODS: TNFR 1 gene was cloned by RT-PCR. Both TNFR 1 gene and plasmid pEGFP-N1 DNA were digested with Sac I and Kpn I. After purification , the two fragments obtained were ligated by T4 DNA Ligase. This recombinant DNA was then transformed into E. coli Competent Cells DH5αand positive clones were selected on the LB agarose plate containing kanamycin (30μg/ml).RESULT: Single clones were identified by double digestion with Sac I and Kpn I,and two fragments with the size of 4.7 kb and 1.4 kb were produced as expected. Sequence analysis showed that expression vector PEGFP-N1/TNFR 1 had been constructed successfully.CONCLUSIONS: The TNFR 1 gene was successfully inserted into the eukaryote expression vector plasmid pEGFP-N1 by the recombination technique in vitro. Part II The Expression of recombinant vector for TNFR 1 and EGFP fusion protein in human umbilical vein endothelial cellsAIM: To construct eukaryotic fluorescent expression vector pEGFP-N1/TNFR 1 and induce the vector express in human umbilical vein endothelial cells.METHODS: Endothelial cells were acquired by filling collagen enzyme solution into the lumen of umbilical veins and then cultured in endothelial cell medium with 5% fetal bovine serum and endothelial cell growth supplement. They were identified by cell morphology and VIII factor immunostaining. TNFR 1 gene was cloned by RT-PCR and the gene was inserted into plasmid pEGFP-N1 to construct a vector for the fusion protein. Using lipofectin method, the recombinant expression plasmid pEGFP-N1/TNFR 1 was transfected into HUVECs. RT-PCR was used to detect TNFR 1 mRNA expression in transfected HUVECs, Western blot and fluorescence microscope to detect the expressed fusion protein as well.RESULT: A large number of high purified endothelial cells could be acquired by digestion of collagen enzyme. The restriction enzyme digestion and sequence analysis showed that expression vector pEGFP-N1/TNFR 1 had been constructed successfully. TNFR 1 gene and TNFR 1 protein were detected in the transfected HUVEC cell,meanwhile the expression GFP was observed under fluorescence microscope.CONCLUSIONS: The expression vector pEGFP-N1/TNFR 1 was constructed and expressed in HUVEC cells successfully. The expressed fusion protein showed double activity of TNFR 1 and GFP. It is helpful to research the regulation factor in relation to TNFR1 translocation. Part III Myosin IIB regulate the translocation of Tumor Necrosis Factor 1 in Human Umbilical Vein Endothelial CellsAIM:To observe the effects of nonmuscle myosin heavy chain IIB on the translocation of tumor necrosis factor 1 in human umbilical vein endothelial cells.METHODS : The expression vector pEGFP-N1/TNFR 1 was transfected into HUVECs by using lipofectin. After small interference RNA for nonmuscle myosin heavy chain IIB was designed and annealed, it was transfected into HUVECs by lipofectamine 2000. Plasma membranes were collected by means of differential centrifugation and sucrose density gradient centrifugation after 72 hours. The changes of TNFR 1 protein was characterized by western blot before and after the cells were induced by TNFα.RESULT:NMHC-IIB siRNA had been transfected into HUVECs successfully. Compared with blank control group, the mRNA expression of NMHC-IIB in NMHC-IIB siRNA group was significantly decreased (0.4540±0.0411 vs. 0.6704±0.0242,P < 0.001). The protein expression of TNFR 1 in plasma membranes was also reduced markedly after 72 hours, regardless of the existence of TNFαor not (0.3248±0.0167 vs. 0.4306±0.0289,P < 0.01;0.4922±0.0217 vs. 0.6609±0.0265,P < 0.001). However, TNFαincreased the expression of TNFR 1 protein in plasma membranes in NMHC-IIB siRNA groups (0.4922±0.0217 vs. 0.3248±0.0167,P < 0.001).CONCLUSIONS:NMHC-IIB may play a active role in the translocation of TNFR 1 from trans Golgi network. However, it may not be the only, or decisive factor.更多还原