Myosin IIB Regulate The Translocation Of Tumor Necrosis Factor 1 In Human Umbilical Vein Endothelial Cells

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.