Establishment Of The Phenotypes Of Human Keratin 1 Transgenic Mice And Investigation The Effect Of Human Keratin 1 Expression On The Mice With Experimental Colitis

Background:Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disorder of the colonic mucosa, which starts in the rectum and generally extends proximally in a continuous manner through part of, or the entire, colon. The clinical course is marked by alternating periods of exacerbation and remission. The precise etiology of UC remains unknown. Our group screened differential genes and proteins expressed in UC patients using gene microarray, two-dimensional gel electrophoresis and mass spectrum methods and found keratin 1(KRT1) expression was down-regulated in both gene and protein level. The outcome of immunohistochemistry showed that KRT1 expression in UC mucosa was correlated with disease severity. But the precise pathogenesis of KRT1 in UC remains unknown. Based on publicly available data, aiming to validate the underlying impact of KRT1 in UC.Objective:To establish heterozygous transgenic mice with human keratin 1. Observating the effect of KRT1 on dextran sodium sulfate (DSS)-induced colitis to define the relationship between KRT1 and UC.Method:We used microinjection to establish transgenic mice with human keratinl, Offspring were investigated by RT-PCR to define the expression of human KRT1. C57BL/6 mice were used for this study. The experimental colitis model were established by administration of 4%DSS for 7 days. Mice were randomly grouped as follows:model group: ①TG DSS:transgenic mice(4% DSS, n=15),②WT DSS: wild-type mice(4% DSS, n=15); control group: ③TG control:transgenic mice (distilled water, n=15),④WT control:wild-type mice (distilled water, n=15). Animals were sacrificed at 8 day. Severity of colitis was evaluated by body weight changes, disease activity index (DAI), colon length/weight, spleen length/weight, colonic pathological changes and pathology score. TNF-α and IL-1β concentrations in colon homogenates were detected using ELISA method. Statistics analysis was performed using SPSS 21.0.Results:(1)Establishment the phenotypes of human keratin 1 transgenic mice. Identifying heterozygous transgenic mice with human keratin 1 for follow-up study.(2)Establishment of DSS-induced colitis in mice.(3) The results showed that body weight had significantly greater loss in the model group compared with those of the control group both in transgenic mice and wild-type mice (P<0.05). And transgenic mice had significantly greater loss than wild-type mice in the model group (P<0.05)(4) Both in transgenic mice and wild-type mice, mice in the model group had a higher DAI than those of control group(p<0.05). And DAI in transgenic mice were higher than wild-type mice in the model group, but there was no statistical significance (P> 0.05).(5) Both in transgenic mice and wild-type mice, there was significant difference in colon length between the model group and the control group (P<0.05), And colon weight in the model group were also significantly reduced than those of the control group (P<0.05). However, induced by DSS, transgenic mice had significantly greater loss in colon weight compared with those of wild-type mice.(6) Comparing spleen weight in the experimental groups (TD DSS,WT DSS.TG control,WT control), more significant increase in spleen weight were observed in model group compared with those of the control group (P<0.05)(7)Pathology scores observed by eye and microscope in the model group were significantly higher than those of the control group(p<0.05). In the model group, a large number of inflammatory cell infiltrated into the submucosa. Congestion and edema of the colon wall were much severity in TG DSS (P<0.05). Besides, colon in the model group displayed severer loss of the mucosa and crypt destruction in histological lesions. Wild-type mice in control group showed that the mucosa was normal without histological lesion. But transgenic mice in the control group were found that a few lympholeukocyte infiltrated into the mucosal layer.(8) Both in transgenic mice and wild-type mice, TNF-a and IL-1β concentrations of colon homogenates in the model group were higher than those in the control group (P <0.05). Transgenic mice had significantly higher expression of TNF-a and IL-1β than wild-type mice in the model group (P<0.05)Conclusion:Our findings indicated that human KRT1 transgenic mice increased sensitivity to DSS induced colitis, And suggested that a functional protein of keratinl was needed for efficient protection in the colonic epithelia. Abnormal gene coding sequence and protein structure of KRT1 can cause and aggravate the intestinal inflammation.