1．Expression Of Human Single-chain Antibody Against Hepatocellular Cancer, And The Function Study Of The Scfv Protein 2. Potential Involvement Of The Cyclooxygenase-2 Pathway In Hepatocellular Carcinoma-associated Angiogenesis

The use of monoclonal antibodies (mAbs) by hybridma fusion as therapeutic agents is gaining importance in the prevention, diagnosis and treatment of various diseases. But it is severely limited in their clinical applications. Phage display technology ,which is an important tool for the high affinity Antibody, can make human antibody without immunization. This kind of antibody can avoid the side-effect caused by human antimouse antibody (HAMA). A single-chain antibody (ScFv) which is specific for hepatocellular cancer was selected and obtained from the human ScFv phage display library in our laboratory previously. AimTo construct the protein expression system in E.coli, express the single-chain antibody (ScFv) which is specific for hepatocellular cancer, and characterize the affinity and specific of ScFv to the HCC.Methods:Expression as inclusion bodies in E.coli and correctly folding of human ScFv against human Hepatocarcinoma;and characterize it's affinity and specific binding to the HCC.1. The VL and VH genes were amplified by PCR with the template of pDAN5/ScFv-D25 which was selected and obtained from the human ScFv phage display library. The complementary DNAs encoding the variable regions of the light chain (VL) and heavy chain (VH) were connected by a (Gly4Ser)3 linker, using a splicing by overlap extension polymerase chain reaction. The resultant ScFv gene was cloned into pGEM-T Vector and sequenced,After digestion by restriction endonuclease, the ScFv genes were ligated to prokaryotic expression vector (pET28a+) digested with the same restriction endonucleases,Then, recombinant vectors were transformed to E.coli BL21(DE3)and expressed induced by IPTG. The expressed protein was checked by SDS-PAGE electrophoresis analysis, After extraction from the E. coli cells, the inclusion bodies were purified and finally renatured by gradient dialysis. The refolded product was identified and analyzed by SDS-PAGE electrophoresis analysis and ELISA.2. secretory express the human ScFv against human Hepatocarcinoma in E.coli, and characterize it's affinity and specific binding to the human tissue of Hepatocarcinoma. the pGEM/D25 vector were digested with the restriction enzymes, the segment ScFv genes were then subcloned into the expression vector (pET32a+) containing the His tag. Bacterial clones were cultured and induced with IPTG. The ScFv antibody fragments were harvested from the periplasm as soluble. The expressed protein was checked by SDS-PAGE electrophoresis analysis, Tissue microarray was used in 63 cases of human hepatocellular cancer,5 cases of normal hepatic tissue,and 3 cases pancreatic cancer tissue.The ability to bind specifically with hepatocellular cancer were detected using immunohistochemical method. Results1.The prokaryotic expression vector pET28a/ScFv-D25 was successfully constructed, The target protein was expressed by IPTG induction in E.coli BL21(DE3), the expression product exists mainly in the form of inclusion body, and the relative molecular weight is proximately 32 KD, and the quantity was about 26% of the total bacterial proteins. the result of ELISA, indicated that the renatured ScFv-D25 could bind with the SMMC-7721 cell lines with high specificity and acidity,the affinity constant of the ScFv was 3.6×107L/mol.2 the results of restrictive enzyme digestion show that the expression vector pET32/ScFv-D25 has been constructed successfully, After recombinant vector was transformed into E.coli BL21 trexB, and induced by PPTG, the soluble expression products mainly existed in periplasmic space of E.coli. SDS-PAGE electrophoresis showed that the molecular weight of interested protein was approximately 42KD, In hepatocellular cancer,the positive binding with the ScFv rates were 68.3%(43/63) vs. 0%(0/5)in normal hepatic tissue and 0%(0/3)in pancreatic cancer(p<0.05).ConclusionsThe single-chain antibody (ScFv) which is specific for hepatocellular cancer was expressed successfully in the in E.coli, and the single-chain antibody (ScFv) can bind with the SMMC-7721 cell lines and hepatocellular cancer tissue with high specificity and acidity。The study laid a solid foundation for its further application. Hepatocellular carcinoma (HCC) is one of the most common causes of malignancy-related death in Africa and Asia. Although several treatment options such as surgical resection, transcatheter arterial embolization are available, the prognosis of HCC remains poor. Poor results of conventional treatments underscore the importance of developing alternative approaches that target molecular events of liver tumorigenesis.AimAngiogenesis plays a crucial role in tumor development and growth. The present study was carried out to investigate the potential involvement of the cyclooxygenase-2 (COX-2) pathway in the regulation of angiogenesis in hepatocellular carcinoma (HCC).Matheds1. COX-2 siRNA template DNA sequences were designed and synthesized.The annealed siRNA template was inserted into pSIREN-Shuttle vector, Similarly, negative control siRNA expression vector was generated. The recombinant plasmid (pSIREN/COX-2 siRNA and pSIREN/negative siRNA) was transformed into DH5αstrain and identified by restrictive enzyme digestion. HuH-7 cell lines were transfected with plasmid pSIREN/COX-2 siRNA and pSIREN/negative siRNA. COX-2 expression in stable transfected cells was measured by RT-PCR and Western blot。The PGE2 level in conditioned media was determined using a specific immunoassay system.2. To observe the 22 angiogenesis associated genes exprssion changs in HUH-7 cells after treatment with SC-58635 by microarray techinque. According to the method of microarray, mRNA was drawn and transcriped into cDNA probe and hybridized with gene chip of GEArray Q Series Human Cancer Pathway Finder Gene Array, the results were analyzed.3.We inhibited COX-2 expression in HCC cell line HuH-7 by selective COX-2 inhibitor (SC-58635) or COX-2 siRNA. Conditioned medias (CMs) from HuH-7 cells were used to study the effect of proliferation, migration, and differentiation of human umbilical vein endothelial cells (HUVECs) in vitro and neovascularization in vivo.Results1. It was confirmed by restrictive enzyme digestion that the recombinant plasmid of COX-2 siRNA and negetive siRNA was cloned. After transfected to HuH-7 cell lines, the mRNA and protein of COX-2, but not COX-1, were effectively inhibited by COX-2 siRNA. Furthermore, COX-2 inhibition by COX-2 siRNA resulted in an approximately 72% reduction in PGE2 production.2. The gene expression profiles of HuH-7 cells after treatment with SC-58635 were assessed by cDNA microarray. Among the 22 angiogenesis associated genes in the GEArray Q Series Human Cancer Pathway Finder Gene Array, proangiogenic factors such as Vascular endothelial growth factor (VEGF), Hepatocyte growth factor (HGF), Fibroblast growth factor 2 (FGF2), angiopoietin-1 (ANGPT1), and angiopoietin-2 (ANGPT2) showed more than 2-fold down-regulation. VEGF had the highest fold changes. There was 4.5-fold down regulation of VEGF by treatment with SC-58635.3. CMs from SC-58635 and COX-2 siRNA treated HuH-7 dramatically suppressed the proliferation, migration, and differentiation of human umbilical vein endothelial cells (HUVECs) in vitro and neovascularization in vivo. These inhibitory effects could be partially reversed by the addition of exogenous PGE2 to CMs.Conclusionwe demonstrated that the inhibition of COX-2 by COX-2 inhibitors or COX-2 siRNA suppressed HCC-associated angiogenesis in vitro and in vivo. PGE2 produced by COX-2 represented a vital angiogenic factor which acts directly on endothelial cells to promote HuH-7-stimulated angiogenic process. Moreover, the COX-2/PGE2/VEGF pathway possibly also contributes to tumor angiogenesis in HCC. Our resulis strongly support the involvement of the COX-2 pathway in HCC-associated angiogenic process. These findings suggest that COX-2 inhibitors could be effective candidates for the treatment or chemoprevention of HCC.