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ENOS Gene Transfer Into Lower Esophageal Sphincter For Treatment Of Experimental Achalasia

Posted on:2004-01-23Degree:DoctorType:Dissertation
Country:ChinaCandidate:S B NingFull Text:PDF
GTID:1104360095961437Subject:Internal Medicine
Abstract/Summary:PDF Full Text Request
[Background and aim] Achalasia is an idiopathic esophageal motor disorder characterized by absent peristalsis in the smooth muscle part of the esophageal body, incomplete relaxation of the lower esophageal sphincter (LES) in response to swallowing and often by an elevated resting pressure of LES. The primary symptom is dysphagia, which usually occurs with both solids and liquids. It has been demonstrated that the neurons containing nitric oxide synthase (NOS) are specifically absent but the cholinergic nerves are intact in the sphincter region, and the smooth muscle of LES is normal in a state of contraction and relaxation. Thus, the deficiency of inhibitory neurotransmitters nitric oxide (NO) is the chief cause of the spasm of LES. The treatment for achalasia includes drug treatment, pneumatic balloon dilatation, intrasphincteric injection of botulinum toxin ( BT ) , and surgical treatment (mostly laparoscopic) with Heller myotomy. It is clear that all of these therapies are palliative, and have their limitations respectively. So it is necessary to establish an optimal method for achalasia treatment. Based on the hypothesis that the absence of nitric oxide synthase (NOS), which can convert L-Arg to NO, in the LES is the primary cause of achalasia, we attempted to transfer gene of NOS into LES with adenoviral vector in order to develop a optimal therapy for patients with achalasia.[Method] 1. Generated recombinant adenovirus carrying endothelial nitric-oxide synthase (eNOS) gene 4.1kb fragment of eNOS cDNA was liberated from plasmid eNOS-pcDNA3 and subcloned into shuttle plasmid pAdTrack-CMV, forming the transfer plasmid pAdTrack-CMV-eNOS. The plasmid pAdTrack-CMV-eNOS and adenoviral backbone plasmid pAdEasy-1 were cotransformed into E coli. BJ5 183 by electroporation, then an efficient homologous recombination event that took place in the bacterial resulted to the production of recombinant adenovirus plasmid pAd-eNOS. pAd-eNOS was transferred into HEK293 cells to produce adenovirus particles Ad-eNOS. Several rounds of amplification of the adenovirus were achieved by infection of HEK293 with a low passage virus stock in order to get high titer of the virus. Determined the titer of the adenovirus. Constructed the control adenovirus Ad-GFPwith the pAdTrack-CMV and pAdEasy-1 through the same way. The PCR technique was used to detect target gene and replication-competent adenovirus contamination. 2. Transfected cultured LES smooth muscle cells (SMCs) of feline with Ad-eNOS LES smooth muscle was isolated and then digested by collagenase to get single cells. The freshly isolated cells were cultivated in DMEM medium with high glocose (12mmol/L) and 15% calf serum. The cultured cells were validated by immunohistochemical staining and electron microscope observation. Infected cultured SMCs with Ad-eNOS and Ad-GFP respectively, detected the expression of e-NOS in the SMCs by western blot assay, RT-PCR technique and immunohistochemical staining and measured the activity of eNOS and NO output. 3. Generated animal model of feline achalasia Domestic cats 28 randomly divided into two groups, trial group(BAC group, n=20) and control group(NS group, n=8). Benzyldimethyltetradecylammonium Chloride (BAG, 4mmol/L) of 2ml were injected into feline LES circumferentially through endoscope as well as saline of 2ml into feline LES of the control group. 8 weeks after injection, measured the LES pressure and evaluated LES relaxation function. Investigated the retention of barium during esophageal barium meal examination and the changes of ingestion and body weight of pre or post-intervention. Studied the in vitro and hi vivo responses of LES to L-Arginine, N - nitro-L-arginine (L-NAME) and Sodium nitroprusside(SNP) respectively. Muscle strips of LES were used to the electric field stimulation (EPS) studies. Immunohistochemically inspected the influence of BAC on the distribution of NOS positive neurons in the LES tissue. 4. Effect of gene transfer of eNOS on achalasia models 18 alchalsia models were randomly divided into treatme...
Keywords/Search Tags:achalasia of cardia, feline, animal model, eNOS gene adenoviral vector, gene therapy
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