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The Effect of the Reconstructed Multigenic Expression Vector of HD5 and GLP2 on Gut Original Infection After Radiation-burn Combined Injury in Mice

Author: TanHu
Tutor: YangTianDe
School: Third Military Medical University
Course: Anesthesiology
Keywords: Enterogenic infection Human defensins 5 Human pancreatic glucagon -like peptide- 2 Recombinant Expression of multiple genes Intestinal epithelial Endotoxin Bacterial translocation Ketamine Chitosan Nanoparticles Cre recombinase LoxP sites
CLC: R644
Type: Master's thesis
Year: 2004
Downloads: 75
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Abstract

Background: The intestinal tract is the body's largest reservoir bacteria \Intestinal injury after trauma to the following three aspects: 1, the epithelial cells of the intestinal villi extensive necrosis, intestinal mechanical barrier damage; 2, reduce the number of intestinal mucosal immune cell function suppression; 3, intestinal flora, microecological severely damaged; causing a large number of intestinal bacteria invasion of the body caused by the two-hit, to procure secondary to multiple organ dysfunction syndrome (MODS) form. Therefore, intestinal infection is under control will directly affect the prognosis of patients with severe trauma. In this regard, there is no effective treatment measures. Previous studies were concentrated to proceed from a single, rarely consider the combination therapy, it has made a certain effect, but it is not ideal, can do when infection control enterogenic, \recovery, while enhancing the immune function of the intestinal mucosa and prevention of intestinal damage, you can fundamentally. Objective: This issue through constructing double gene expression plasmid, after the Chitosan oral transfected to intestinal epithelial injury partial expression of it to promote value-added effect of glucagon-like peptide-2 (glucagon-like peptid-2, GLP-2) to observe the impact of post-traumatic enterogenic infection and anti-infective effect of human defensin 5 (human defensin 5; HD5), aims to establish a prevention and treatment of intestinal-borne infections, and other similar causes intestinal damage ways to avoid the occurrence and development of MODS. Methods: This topic is selected promote intestinal epithelial cell proliferation specific regulatory factors GLP-2 and intestinal innate immune component HD-5; using cloning technology first GLP-2, site-directed mutagenesis to prevent DPP-Ⅳ enzymatic deactivation and enhance its activity; design the sequence of the Cre recombinase LoxP sites, and GLP-2, and both ends of the HD-5 for timely Gene Expression Close; then \5 gene-LoxP \Animal intestinal tissue mRNA expression of the transfected gene was detected by RT-PCR method, by optical microscopy, scanning electron microscopy and transmission electron microscopy system observed before and after the treatment of intestinal epithelial morphology changes, and mesenteric lymph nodes, liver and blood detection bacterial translocation and blood endotoxin concentration. On this basis, the Third Military Medical University, a master's degree thesis further studied and the effects of ketamine anesthesia in combination with the treatment method. Results: 1 by RT-PCR method, total RNA from the brain tissue of patients with epilepsy glucagon original gene sequence, sequencing results showed that the correct sequence. Obtained by PCR recombination technology, HD-5 target gene fragment sequencing results show has successfully splicing together the sequence of the signal peptide and mature skin and join the Lox P sequence. GLP-2 is also obtained the same results, and encoding the second amino acid position of the N-terminal of the mature peptide sequence by alanine (GCT) was mutated to glycine (GGT), prompted the successful site-directed mutagenesis. 3, recombinant expression plasmid pvrrR03 HDS a GLPZ digestion analysis prompted to insert the correct direction of fragment sequencing results showed that the correct sequence, gene therapy experiments can be used in the next step. 4, intestinal epithelial cells the Cac small 2 recombinant plasmid liposome transfection, RT-PCR showed positive clones HD-5 and GLP 2 mRNA expression, suggesting that the recombinant plasmid successful successfully transcription; M Bamboo found to contain 2 culture supernatant can significantly promote rGLP of Cac. A 2 cell proliferation. 5, the size of the chitosan nanoparticles formed uniformly, the DNA encapsulation efficiency, can effectively resist the degradation of the DNA enzyme I; mediated pEGFP gene transfer, by fluorescence microscopy can be observed to the green fluorescent protein produced, but the expression amount is lower than liposome transfected with the control group. 6, the oral recombinant plasmid chitosan nanoparticles, RT-PCR method to detect experimental mouse intestinal epithelial tissue the HD and GLP of 2 nlRNA expression; optical microscopy, scanning electron microscopy and electron microscopy were prompt, recombinant plasmid therapy intestinal epithelial morphology repair group compared with the control group significantly advance performance for the villus height increases, the density increases, arranged in neat rows, each organelle damage is significantly reduced; the Shift bacteria and endotoxin detection prompted a shift to the lymph nodes, liver, blood, bacteria significantly reduced plasma endotoxin concentrations were significantly lower. Plus ketamine no significant impact on the treatment effect. Conclusion: 1. By PCR recombination technology can successfully encoded signal peptide sequence and mature skin stitching together apply to secretory carrier. 2 double cistron Construction of multi-gene expression plasmid can be successful strategy to carry multiple genes and can also efficiently expressed, suitable for the expression of multiple genes. Chitosan nanoparticles can be used for the oral route of intestinal gene transfection. Chitosan nanoparticles can express rHD-5 and rGLP-2 gene expression plasmid transfer to the intestinal epithelial effective to alleviate post-traumatic intestinal damage, reduce the incidence of intestinal-borne infections. 5. Ketamine had no significant effect on intestinal structure and function, together with the gene therapy method can be safely used for the treatment of trauma patients.

The Effect of the Reconstructed Multigenic Expression Vector of HD5 and GLP2 on Gut Original Infection After Radiation-burn Combined Injury in Mice

Abstract

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