Roles of TGF-β Signaling Pathway in Endoplasmic Reticulum Stress in Endothelial Cells Stimulated with Cigarette Smoke Extract / 华中科技大学学报（医学）（英德文版）

To investigate the role of signaling pathway in the effect of endoplasmic reticulum stress (ER stress) in endothelial cells stimulated with cigarette smoke extract (CSE).Human umbilical vein endothelial cells (HUVECs) were cultured and divided into 3 groups:CSE-stimulated group,CSE-stimulated with 4-PBA group,and negative control group.HUVECs were cultured and stimulated with CSE at concentrations of 5％,10％ and 20％,respectively,mRNA of CXCL-8 and GRP78 was detected by real-time PCR.ELISA was performed to test the expression of CXCL-8 protein,and neutrophils migration was detected by Transwell board test.The NF-κB,ERK,p38MAPK and transforming growth factor beta (TGF-β) were detected by flow cytometry.The mRNA of CXCL-8 and GRP78 increased in CSE-stimulated HUVECs (P＜0.05).Furthermore,it was concentration-dependent.4-PBA significantly reduced the expression of CXCL-8 protein (P＜0.05) and neutrophil migration (P＜0.05).The TGF-β,rather than the NF-κB,ERK and P38MAPK pathway might be involved in ER stress stimulated by CSE.CSE induced neutrophils migration by increasing the expression of CXCL-8 in endothelial cells.ER stress might play a role in the effect of neutrophils migration stimulated with CSE,and TGF-β pathway may contribute to the ER stress in HUVECs.

Preparation and Identification of scFv and bsFv against Transferrin Receptor / 华中科技大学学报（医学）（英德文版）

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complemenary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease Sill, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB 1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively.Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.