The Effect Of The Reconstructed Multigenic Expression Vector Of HD5 And GLP2 On Gut Original Infection After Radiation-burn Combined Injury In Mice

Background: Gut is the largest "germ reservoir" and "endotoxin pool" of human body. When severe injury occurs, germs and endotoxin disperse rapidly from gut and cause gut original infection. The intestinal impairments are as follows: 1. The widespread necrosis of villus epithelium can cause mechanical barrier of gut damaged. 2. The quantity and dysfunction of immune cells of epithelium may decrease. 3. Dysbacteriosis can damage the microecosystem. The harmful germs may impose another heavy blow on human body and cause multi-organ dysfunction syndrome (MODS). Control of gut original infection was critical to severely injured patients' prognosis. There hasn't had any effective strategy so far, because all the studies just emphasized on one aspect. If we can find a way of gene therapy, which not only restores the structure and function of intestinal epithelium but also enhances mucosa immune effect, the gut original infection can be cured ultimately.Objective: By constructing a multigenic expression vector expressing GLP-2 (a peptide accelerating the proliferation of epithelial cells) and HD-5 (a natural component of intestinal immune system), and transfecting it to intestinal epithelium by chitosan-DNA nanoparticles, we investigated the influence of intestinal expressions of GLP-2 and HD-5 on gut original infection after severe trauma in order to cure radically gut original infection and the other kind of gut impairment, and avoid the occurrence and development of MODS.Methods: Clone technology was used to mutate GLP-2 site-directedly to prevent the impact of DPP-IVase. LoxP sequences were inserted into the either side of GLP-2 and HD-5 to control the gene expression as one's demand. This recombinant fragment was then inserted into multigenic expression vector, and the recombined plasmid was transfected to intestinal epithelium in vivo mediated by oral delivery of chitosan-DNA nanoparticles. The expression of mRNA of GLP-2 and HD-5 in the intestinal epithelium was determined by RT-PCR and the structural changes were detected by scanning and transmission electron microscopy. Translocated germs in lymph node, liver and blood as well as the concentration of plasma endotoxin were detected to judge the effect. The study of ketamine was alsoperformed in the animal model on research its effect on intestinal structure and function. Results:1. The gene of proglucagon cDNA was obtained from the brain of epileptic by RT-PCR.2. The gene of interest HD-5 and GLP-2 were obtained by PCR technique and confirmed by dideoxy-mediated-chain termination. Signal peptides and mature peptides of GLP-2 and HD-5 had been pieced up respectively. The sequence of LoxP was inserted into the upstream of HD-5 sequence and downstream of GLP-2 sequence. The result of sequencing showed the gene, which coded the second amino acid in the N-terminal of GLP-2, had already been mutated from GCT (coding alanine) to GGT (coding glycosthene). The success of site-directed mutation provided basis for the further study.3. The orientation of the fragment inserted into the recombined expression plasmid 'pVITRO3-HD5-GLP2' was confirmed by restriction enzyme digestion. Dideoxy-mediated- chain termination showed the sequence was absolutely correct.4. The recombined plasmid was transfected to caco-2 by liposome and the expression of HD-5 and GLP-2 mRNA could be detected by RT-PCR. MTT showed that rGLP-2 could accelerate the proliferation of caco-2 cell significantly.5. Chitosan could form equirotal nanoparticles and envelop DNA very well to resist DNAase I. After pEGFP was transfected to caco-2 by chitosan-DNA nanoparticles, green fluorescence protein could be observed under fluorescence microscope. But the expression was lower than that of control group, which was transfected by liposome.6. After taking chitosan-DNA nanoparticles orally, HD-5 and GLP-2 mRNA can be detected in the mice's intestinal epithelium by RT-PCR. The results of Microscopy as well as scanning and transmission electron microscopy showed that the stru...