Gene Therapy Of Hepatocyte Growth Factor Mediated By Attenuated Salmonella Typhimurium For TNBS-induced Ulcerative Colitis In Rats

Objectives:To explore the therapeutic effects of human hepatocyte growth factor (HGF)gene mediated by attenuated salmonella typhimurium on ulcerative colitis(UC) in rats,which was induced by TNBS.Methods:1.Respectively with 1×10⁹cfu the attenuated Salmonella typhimurium (Ty),carrying green fluorescent protein attenuated Salmonella typhimurium (Typl-GFP),with hepatocyte growth factor gene attenuated Salmonella typhimurium (Typl-HGF),10%NaHCO3 0.3ml section 0,14,28 days gavage immune mice,at the end of the 7d after immunization,the animals were collected and blood anticoagulant used anticoagulant flow detection of CD4 +,CD8 +,IgM,IgGl,IgA,IgM in serum by ELISA,IgGl,IgA immune globulin,MTT assay of B and T lymphocyte proliferation2.40 ulcerative colitis model rats were induced by TNBS enema,and randomly divided into 4 groups:simple attenuated salmonella typhimurium(Ty) control group,attenuated salmonella typhimurium carrying hepatocyte growth factor gene(Typl-HGF)treatment group,attenuated salmonella typhimurium carrying green fluorescent protein gene(Typl-GFP)treatment group and model control group,10 rats each.Another 10 rats which were intrarectally administered with normal saline as normal control group.On the 3rd after TNBS administration,rats were treated by intragastric administration with 1×10⁹cfu of Ty,Typl- HGF,Typl-GFP,respectively, 0.5ml each animal,once another day.Same volume 10%NaHCO₃ was administrated to the model and normal control rats.All rats' general state of health and fecal character were observed.After treatments for 3 or 6 times,the colons were removed and scored by appearance,its specimens were subjected to H.E staining and then the mucosa damage was scored.The expression of target gene in colon was observed by fluorescence microscope.Detected by flow cytometry peripheral blood CD4 +,CD8 +, IgM,IgGl,IgA.Results:1.Mice at the end of the 7 d after immunization,the experimental group Typl-HGF CD+4,CD8,CD+4/CD +8 ratio(p＜0.01,p＜0.05,p＜0.01).IgM,IgA no statistical significance,IgGl significantly increased p＜0.01.2.3,6 gavage model group and the normal group of CD4+T cells compared to no significant changes,but CD8+T cells decreased significantly(P＜0.01), three times the treatment group than CD4 + T model group P＜0.05,3,6 gavage model group,CD4+/CD8+T ratio was higher than normal(P＜0.05);6th gavage treatment group CD4 +,CD8 + model group were significantly lower than the control group (P＜0.01,P＜0.01,P＜0.05,P＜0.05),CD4+/CD8+T ratio compared to the model,the control group(P＜0.05).3,6 gavage model group and the normal group IgM,IgGl, lgA increased significantly in the control group and 3,6 model than the same period IgM was increased significantly(P＜0.01),while the treatment group and six of the model group than IgM,IgGl decreased significantly(P＜0.05).3.The weight of rats treated for 3 times with Typl-HGF had a little increase (5.31±1.13g),but no significant difference between Typl-HGF group and model control group(P＞0.05),at this time,the common symptoms of colitis were slightly ameliorated.The weight of rats treated for 6 times with Typl-HGF was significantly higher(9.75±2.07g)than that of in model control group(P＜0.05),the common symptoms of colitis were obviousaly ameliorated,but no evidently ameliorated in Ty group and model control group.Grossly,mucosal hyperemia and thickening of colonic wall were evidently ameliorated,and the loss of mucosa was superficial and small,the colon score of rats treated with Typl-HGF for 3 or 6 times were 2.20±0.90 and 1.70±1.10,respectively,were significantly lower than that of treated with Ty and NaHCO₃(P＜0.01),and there was a significant difference between rats treated with Typl-HGF for 3 and 6 times(P＜0.05).Histopathologically,in colon sections of Typl-HGF treated for 3 times group,the extent of inflamed mucosa was mild,the area of ulcer was shorter,and the mucosa hyperblastosis was observed,the score was 3.87±0.76,but no significant difference with Ty treatment and model control group(P＞0.05).The sections of Typl-HGF treated for 6 times was significantly better than that of 3 times,new granulation tissue was observed in ulcer margin,even in some samples ulcer and mucosa damage was completely repaired,the section score was 1.60±0.30,but the score of sections in model control group was 3.50±0.70,there was a significant difference between them(P＜0.05).But no significant difference between sections in Ty treatment group and model control group(P＞0.05). GFP expression was observed in colon tissue after intragastric administration with Typl-GFP.Conclusion:1.Oral Typl-HGF can reduce the mice cells and humoral immunity, immune to normal mice inhibited.2.Typl-HGF could dramatically ameliorate mucosa imflammation and symptoms of TNBS-induced colitis in rats,and promote the repair of mucosa damage and ulcer,At the same time in the pathogenesis of experimental colitis in immune function in a state of hyperthyroidism,Typl-HGF can inhibit the proliferation of T and B lymphocytes produce antibodies,it maybe an alterative strategy of gene therapy for ulcerative colitis in clinical practice.