| Background: Intestinal trefoil factor (ITF, also known as TFF3), a member of the trefoil factor family (TFF), is a small peptide secreted by intestinal goblet cells. ITF is composed of one structurally characteristic trefoil domain. A trefoil domain is defined as a sequence of 38 or 39 amino acid residues in which 6 cysteine residues are linked in the configuration 1-5, 2-4, 3-6, thus forming a characteristic three-leaved structure. ITF is essential for protecting the epithelial layer of the gastrointestinal tract from damage and repairing epithelium after injury. ITF is attracting the researchers'attention for its potential pharmacological value. Though ITF is a novel polypeptide with potential pharmacological value, its production is too limited for to be clinical useful. Gene delivery is the process in which plasmid DNA is introduced into target cells, transcribed and the genetic information ultimately translated into the corresponding protein. If hITF gene therapy was used, the major drawbacks limiting its clinical use would be overcomed. Objective: To select an optimal hITF gene delivery systems, and to explore the effect of ITF gene therapy on the treatment of gut derived infection.Methods:1. hITF gene (73 aa), encoding both a signal peptide and a mature secretory peptide, was generated by RT-PCR technology. After digestion with XhoI and EcoRI, the gene was inserted into the multiple cloning sites of the E. coli vector, pEGFP-N1, to construct a GFP N-terminal tagged recombinant plasmid. Following confirmation by restriction analysis plasmid DNA was sent to Shanghai Invitrogen Corp for DNA sequencing.2. Chitosan nanoparticles were prepared by a complex coacervation technique, and its size and morphology was assessed using TEM. Zeta potentials were determined using a Zetasizer Nano. Gel retarding analysis and DNA integrity analysis were assayed by gel electrophoresis. In addition, DNA loading efficiency and loading capacity were evaluated and in vitro release of DNA from the chitosan nanoparticles was analysed.3. Gene transfer capability was assessed in HEK293 cells, and transfection efficiency was evaluated by fluorescence microscopy and flow cytometry. The transcription and expression of hITF was assessed by RT-PCR and Western Blot.4. After digestion with Bgl?and Not?, the gene was inserted into the multiple cloning sites of pAdTrack-CMV, to construct recombinant vector.Then the recombinant vector was co-transfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying hITF was generated with homologous recombinantion in bacteria. The adenoviral plasmid was transfected in 293 cells and the recombinant adenovirus Ad-hITF was packed in 293 cells.5. Ad-hITF was transfected to human colon adenocarcinoma cell line (HT-29, Caco-2), and the expression of hITF mRNA and protein was detected by RT-PCR ,Western blot and immunofluorescence. Then, wounds were established in confluent monolayers of HT-29 cells infected with pAd-hITF, and wounded monolayers were cultured for 24 h after addition of DMEM. The width of the wound border was measured in a blind manner. 6. Kunming mice were inflicted with 30 % total body surface area full thickness burns and administrated with Ad-hITF orally. Injury index, villous height, crypt depth, LPS, intestinal bacterial translocation rate and so on were determined on 1,3 ,5and 7 PBDs.Results:1. As a result of RT-PCR, a 240bp DNA fragment was obtained and identified by gel electrophoresis. Then, the DNA fragment was inserted into plasmid pEGFP-N1 to construct recombinant vector. The recombinant vector was confirmed by restriction analysis and gene sequencing to be the same as the original sequences of hITF gene from GenBank(Accession No. NM003226), and the recombinant vector was named pEGFP-N1-hITF.2. The chitosan nanoparticles were successfully prepared by a complex coacervation technique with sizes 300-500 nm and positive zeta potentials. The nanoparticles could protect DNA from nuclease degradation, and release profiles of DNA were dependent on N/P ratios.The loading efficiency of chitosan nanoparticles increased depending on the N/P ratio, while the loading capacity of nanoparticles was lower with decreasing N/P ratios.3. The transfection efficiency was studied by fluorescence microscopy and flow cytometry. The results showed that the transfection efficiencies of chitosan nanoparticles were about 80%, similar to the transfection efficiency of Lipofectamine. RT-PCR and Western Blot assay demonstrated that hITF was expressed successfully and the proteins had much good antigenicity and specificity.4. The recombinant vector pAdTrack-CMV-hITF was constructed.After digestion with restriction enzyme Pme?, linearized recombinant vector was transformed into BJ5183 bacteria and the adenoviral plasmid Ad-hITF carrying hITF was generated with homologous recombination. The mature viruses would be packaged in 293 cells. The titer of virus was 2×109pfu/ml by TCID50 method.5. Transfection efficiency was about 100% as the MOI of the recombinant adenoviruses was 50.RT-PCR and Western Blot assay demonstrated that hITF was transcribed and expressed successfully. hITF was proved to distribute in cytoplasm by immunofluorescence assay.In addition, Ad-hITF enhanced migration activity of HT-29 cells obviously.6. Ad-hITF treatment significantly decreased the injury index, increased villous height and crypt depth, alleviated pathological damage, lowered LPS and intestinal bacterial translocation rate in comparison with the control group.Conclusion:1. Recombinant eukaryotic expression vector pEGFP-N1-hITF was constructed successfully.2. The chitosan nanoparticles were successfully prepared by a complex coacervation technique with sizes 300-500 nm and positive zeta potentials and could protect DNA from nuclease degradation.3. The chitosan nanoparticles could be transfected to HEK293 cells with high transfection efficiency, and hITF was expressed in the cultures.4. The recombinant adenovirus Ad-hITF carrying hITF was constructed successfully.5. Ad-hITF could infect human colon adenocarcinoma cell line(HT-29, Caco-2) with transfection efficiency close to 100%, and hITF was expressed in the cultures. Ad-hITF has the ability to enhance cell migration activity.6. Ad-hITF treatment significantly alleviated intestinal mucosal damage, promoted the healing of mucosa and had efficient therapeutic effect on gut derived infection. |
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