Endogenously, VEGF initiates angiogenesis, then later Angiopoietin (Ang)-1 matures vessels. We hypothesized that multigene therapy of VEGF before Ang1 to ischemic hindlimb tissue would result in persistent angiogenesis. At 2, 4 and 8 wks after inducing ischemia, blood flow was assessed by contrast-enhanced ultrasound. Animals were treated with VEGF at 2 wks, VEGF/Ang1 at 2 wks, or VEGF at 2 wks and Ang1 at 4 wks. In untreated controls, blood flow remained reduced. After VEGF delivery, resting flow and vessel density increased; however, flow reserve remained reduced, and vasculature was capillary-rich and eventually regressed. After VEGF/Ang1 co-delivery, flow increased marginally, flow reserve improved and vascular architecture remained normal. After separated VEGF and Ang1 delivery, flow, vessel density and flow reserve increased and were sustained, while vascular architecture remained normal. In conclusion, temporally separated VEGF and Ang1 delivery promotes sustained angiogenesis and improved vessel functionality.

(<特集>悪性脳腫瘍の病態と治療) (<SPECIAL ISSUE>Pathology and Treatment of Malignant Brain Tumors)

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references.

Osteoporotic fractures are still the major health concerns in many developed societies especially when the incidence of that tremendously increased with the aging population. However, the outcomes of osteoporotic fracture treatment have not been entirely satisfactory due to the poor quality of bone substance. Inspiringly, bone morphogenetic protein 2 (BMP2) with the ability to accelerate bone formation showed advantages over the conventional treatment. The only problem needed to overcome is its short half-life which resulted in the requirement of readministration and extremely high cost. As a solution to that, gene therapy provides a promising way to sustainably release this protein at the regeneration site. Since viral vectors have been hampered by genetic toxicity and immunogenicity, nanoscaled non-viral vectors offer an attractive means for gene delivery. Chitosan as non-viral vector has been widely investigated for its excellent biocompatibility. Most efforts have been given to improve its low transfection efficiency. In this study, chitosan was first modified with octaarginine, one of cell membrane penetrating peptides, and showed enhanced transfection activity, but which was not significant as expected. Following that, low molecular weight polyethyleminine (PEI) was introduced to modify chitosan through bioreducible disulfide linkage, denoted as Chitosan-ss-PEI. PEI is an efficient non-viral vector but hampered by molecular-weight dependent toxicity. The developed Chitosan-ss-PEI showed good biocompatibility in MTT assay in three different cell lines, during which cells were maintained 80% of viability when the concentration of this vector was up to 100 μg/mL. The optimal transfection efficiency of Chitosan-ss-PEI was higher than that of PEI 25k and comparable to Lipofectamine in delivering luciferase reporter gene. GFP expression mediated by Chitosan-ss-PEI also showed similar results. Chitosan-ss-PEI was then applied to deliver BMP2 gene to skeletal system cells and exhibited the osteogenic ability. For C2C12 myoblast cells, this system inhibited their myoblast differentiation and induced the osteogenic differentiation. It also showed stronger effect in promoting the differentiation of immature osteblast-like MG63 cells and in inducing C3H10T1/2 mesenchymal stem cells osteogenic differentiation in term of ALP activity and mineralization ability compared with other commercial available non-viral vectors. Primary MSCs such as bone marrow stromal cells (BMSC) and human umbilical cord blood mesenchymal stem cells (hUCB-MSC), are usually more difficult to transfect, but they showed stronger osteogenic differentiation ability induced by this system comparing with the cell lines. BMP2 usually requires extremely high concentration to realize its function. Through the investigation of BMP2 signaling regulation in this study, it was found that parathyroid hormone (PTH) could increase the access of BMP2 ligands to their receptors by negatively influencing BMPs antagonist network, resulted in enhanced BMP2 activity in bone remodeling and in promoting the commitment of MSC to osteoblast lineage both in vitro and in vivo. This course involved the endocytosis of PTHR with a complex of LRP6, which organized antagonist network on the cell surface to shield the BMPs receptors. Novel approaches are expected to be developed based on this mechanism with the purpose of intensifying the therapeutic effect of BMPs. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Non-viral vectors have been used to deliver therapeutic genes to treat different diseases. There are a variety of non-viral vectors such as liposomes, cationic polymers and peptides. Among all, pH responsive peptides showed excellent DNA transfection efficiency in many types of cell. These peptides are capable of changing their structural conformation as pH decreases, adopting a disordered structure which can destabilize endosomal membrane and therefore enhancing the release of DNA from endosomes into cytosol. Traditional pH responsive histidine-rich peptides showed good DNA transfection efficiency and low toxicity to the cells when compared with other non-viral vectors. However, their low pKa value restricted these peptides to be protonated only at late endosomal stage, in which DNA is extremely susceptible to endosomal degradation. This hindered the DNA to be released to the cytosol efficiently and therefore reduced DNA transfection efficiency. In response to this, it is of great interest to probe into the insertion of either 2,3-diaminopropionic acid (Dap) or methylated-2,3-diaminopropionic acid Dap(Me) to the peptide as alternative pH sensitive components. The pKa values for both Dap and Dap(Me) peptides are higher than that of histidine. It is anticipated that the higher pKa value, the protonation of peptide could be happened at an earlier stage of endosomal maturation. Such protonation of peptide destabilizes the endosome membrane rapidly, causing the release of DNA to the cytosol effectively and hence improving DNA transfection efficiency. In this experiment, LADap(Me)4-L1 peptide was the optimal candidate within the series. It showed good DNA transfection efficiency and cell viability in A549 cells among all Dap and Dap(Me) peptides. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

Not available / text

published_or_final_version / Medicine / Master / Master of Philosophy

The Best MPhil Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize / published_or_final_version / Pathology / Master / Master of Philosophy

No description available.

As a consequence of safety issues encountered by the use of viral vectors in gene therapy, there has been a steady increase in the development and application of non-viral vectors, especially liposomes. Cationic liposome mediated delivery is one of the most promising nonviral delivery methods. These liposomes are prepared from synthetic lipids, are positively charged and interact favourably with DNA through electrostatic interactions. Cationic liposomes have also shown immense potential in the targeting of specific cell types such as HepG2 (hepatocellular carcinoma) cells, a model in vitro gene delivery system for the study of hepatocyte function. However, these liposomes also have a number of limitations in vivo. In an attempt to overcome these restrictions, a hydrophilic polymer, polyethylene glycol (PEG) is incorporated into the cationic liposome. This covalent attachment of (PEG) to the liposomal surface is thought to sterically stabilise liposomes, promote biological stability, inhibit aggregation, decrease toxicity and immunogenicity, prevent interaction with serum proteins and complement and thus prolonging the circulation time of liposomes in vivo. The versatility and simplicity of cationic liposomes have made them vitally significant non-viral gene delivery vehicles for human gene therapy. In this investigation novel untargeted and targeted glycosylated liposomes with and without PEG were synthesised to evaluate their gene transfer activities in vitro to potentially develop a suitable gene delivery system for future in vivo applications. A constant molar quantity of the cationic cholesterol derivative, 3 [N-(N’, N’-dimethylaminopropane)-carbamoyl] cholesterol (CHOL-T) was mixed with dioleoylphosphatidylethanolamine (DOPE) and a galactose/glucose derivative to produce targeted cationic liposomes. PEG liposomes were prepared in the same way with the addition of distearoylphosphoethanolamine polyethylene glycol 2000 (DPSE-PEG2000), 2% on a molar basis. Supported by transmission electron microscopy characterisation, we present evidence that the pegylation of liposomes affects the DNA binding capability and transfection efficiencies of the cationic liposomes in addition to protecting the plasmid DNA in lipoplexes from serum nuclease degradation. Optimal DNA : liposome binding ratios were obtained from gel retardation studies and confirmed by ethidium bromide intercalation assays. These complexes were then tested on the human hepatoma cell line, HepG2, to determine toxicity and assess transfection efficiencies. From results obtained in this study, it appears that both cationic and pegylated cationic liposomes are well tolerated by cells in vitro. The results further suggest that targeting by use of glycolipids incorporated into the structure of the liposome increases transfection, while pegylation of cationic liposomes marginally decreases the transfection efficiency of the lipoplexes to HepG2 cells in vitro. / Thesis (M.Sc.)-University of KwaZulu-Natal, 2009.