An Experimental Study On The Protection Of Intestinal Mucosa Against Hypoxic/Ischemic Injury By HSP70 Gene Transfection Following Severe Burn

Background: The Hypoxic/Ischemic injury post severe burn can cause multiple organs dysfunction or even failure. The injury of intestinal mucosal barrier, especially the mechanical one is the key factor leading to Systematic Inflammatory Response Syndrome(SIRS) and Multiple Organs Dysfunction Syndrome(MODS).It is therefore important to find an effctive way to attenuate the intestinal Hypoxia/Ischemia injury following severe burn. Heat Shock Proteins (HSPs) conserved molecules present in all prokaryotes and eukaryotes studied so far.Under normal physiological conditions,the expression of these proteins is very low. In stress situations, a very strong synthesis of these proteins has been observed. The main function of HSPa is to operate as intracellular chaperones for aberrantly foled or mutated proteins and to provide cytoprotection against the stress conditions.One of the major HSPs is HSP70,named after its molecular mass of approximately 70 kDa. The protection of HSP70 is positive related to its upregulation in several organs.There are usually two ways to improve HSP70 contents: induction of endogeneic HSP70 and transfection of exogeneic HSP70 gene. To improve the overexpression of HSP70 gene we employed adenovirus as the vector to transfect HSP70 gene into cells and rats. We intended to find the role of transfected gene in the protection of postburn intestinal hypoxia/ischemia injury after severe burn in ratsNevertheless, adenovirus has not been applied successfully in clinic due to its disadvantages, and now microcapsules are highlighted in gene therapy and transplantation. Therefore we constructed the microcapsules containing adenovirus which we had prepared in the first part of our research to avoid kickbacks of adenovirus.Objective: To research on the effectiveness of HSP70 gene transfection in intestinal mucosaagainst hypoxic/ischemic injury following severe burn, and to detective the effection of microcapsulation of these adenovirus, then try to explore a new way in gene therapy in clinic.Methods: In the first part we got the HSP70 gene and then cloned into the cosmid vector pAxCAwt , co-transfected with adenovirus DNA-TPC into 293 cells. The recombinant adenovirus (Ad-HSP70) was constructed successfully by the clones selection and the titer detected was high enough for the transfection .In the second part the recombinant adenovirus Ad-HSP70 was transfected into the intestinal epithelial cells in vitro and then suffered from 1 hour of hypoxia followed by 1 hour of reoxygenation, the effects of transfected HSP70 gene over expression during varied transfection times (24h, 48h, 72h) were detected. The cell viability was detected by MTT method, and the apoptosis and necrosis cells ratio were evaluated with Annexin-V-FLUOS staining kit in vitro.In the third part we prepared the microcapsules containing the adenovirus vectors Ad-HSP70 according to the references of the patents .The microcapsulation can theoretically protect the adenovirus avoid the injury of the acid and enzymes in stomach and then will be absorbed in the intestine.At last we transfect Ad-HSP70 into severe burned rats in order to find the protection in intestinal mucosa against the hypoxia/ischemia injury. We also studied on the protection against hypoxic/ischemic injury in intestinal mucosa in rats who took the microcapsules orally following severe burn . Results:1.The HSP70 gene was cloned successfully and after the repair of the mutation we got the Hsp70 cDNA whose sequence was same as which was reported in GenBank.2. The HSP70 gene was cloned into the recombinant adenovirus vector pAxCAwt , then the recombinant adenovirus Ad-HSP70 was constructed which can only be replicated in 293 cells containing the E1 gene. The titer of the prepared adenovirus was 3.57 ×1010 (pfu/ml) which was high enough to transfect in vitro and in vivo.3. The transfection of Ad-HSP70 into intestinal epithelial cells could successfully induce the overexpression of HSP70 gene .4. The HSP70 transfection in vitro...