| [Background and Objective]Achalasia is an idiopathic esophageal motor disorder characterized by aperistalsis in the smooth muscle esophagus and failure of the LES to relax completely with swallowing. The smooth muscle relaxes mediated both by nitric oxide and vasoactive intestinal polypeptide, and contracts by acetylcholine which is hydrolysised by acetylcholinesterase (AChE), which maintain Basal LES pressures from 10 to 20 mmHg in vivo, the reduction of AChE in LES results in the reduction of acetylcholine hydrolysised, which causes increase of LES pressures, otherwise, LES pressures decrease.Recombinant adenoviruses are currently used for gene transfer in vitro and gene therapy. Several features of adenovirus biology have been made as vectors of choice for these applications. For example, adenoviruses can transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, high titers of viruses and high levels of transgene expression generally can be obtained. AdEasy?system has more advantages over the other methods in constructing recombinant adenoviruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria based on AdEasy?system rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral porduction is conveniently followed with the aid of green fluorescent protein (GFP), encoded by a gene incorporated into the viral backbone, which can be allowed directly to observe the efficiency of transfection and infection. Homogeneous viruses can be obtained from this procedure without plaque purification. All of these can significantly decrease the time required to generate viruses.The aim of this study was to develop an achalasia model in the cats using the surfactant benzyldimethyltetradecylammonium chloride (BAC), and further evaluatethe mechanism of achalasia and the effect of AChE on achalasia. Then a kinds of replication-deficient recombinant adenoviruses by AdEasy?system was constructed rapidly, AdAChET inserted with AChEx cDNA and they would be used for gene transfer in smooth muscle cell and the genes delivery in experimental achalasia, which could provide a new pathway for the combination of gene therapy in achalasia. [Methods]1. effect of AChE on development of experimental achalasiaTwenty four cats were randomized into two groups and injected with BAC in models group or saline in controlours in the distal esophagus by endoscopy respectively, 8 weeks later, manometry, barium esophagrams, HE, AChE immunohistochemistry and activity were performed. The morphology of the neurons with the myenteric plexus was studied by electron microscope.2. Construction of recombinant adenoviruses carrying AChET and investigation of AChET effect on smooth muscle cellThe AChEx cDNA were obtained from the plasmids by digestion or amplification, The shuttle plasmids- pAdTrack-CMV- AChEx were established by ligation. Then the linearized shuttle plasmids were transformed into colibacillus BJ5183 with backbone vector AdEasy-1 to obtain the recombinant adenoviral plasmids-pAdAChEx by homologous recombination. After packed in 293 cells, the recombinant adenoviruses-AdAChEx were generated. And AdGFP (without exogenous gene, only GFP expressed) were generated by the same way.After primary culture of smooth muscle cell derived from cats, AChEx gene was delivered into the smooth muscle cell. The expression of AChEx in smooth muscle cell was detected by RT-PCR technique, western blot and AChE activty on the third day of post-infected by the adenoviruses.3. Treatment of achalasia model by Acetylcholinesterase gene deliveryThirty six cats were randomized into 4 groups, each group composed 9 cats. Group 1 was served as a control injected with saline in the distal esophagus by endoscopy, and the cats in next three groups were achalasia models injected with BAC in the distal esophagus by endoscopy respectively, After 8...
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