溃疡性结肠炎患者TLR9表达变化的研究

Expression of Toll-like Receptor 9 in Patients with Ulcerative Colitis

目的从Toll样受体9(TLR9)的角度探讨溃疡性结肠炎(ulcerative colitis,UC)的发病机制和微生态制剂治疗UC的作用机制。方法采用随机、对照设计,将30例轻、中度活动期的UC患者分为:美常安联合柳氮磺胺吡啶(SASP)组(A组)10例、丽珠肠乐联合SASP组(B组)10例、SASP组(C组)10例,疗程均为4周;正常对照组10例(肠镜及结肠病检均示正常)。于治疗前后取病变肠组织,用免疫组化法检测TLR9的表达水平,以逆转录聚合酶链反应(RT-PCR)检测TLR9mRNA的表达,比较UC患者与正常对照组TLR9蛋白和mRNA在结肠组织中的表达水平,及UC患者治疗前后TLR9蛋白和mRNA在结肠组织中的表达变化,并对3组UC患者治疗前后的Sutherland疾病活动性指数(DAI)、肠镜分级积分、病理组织学分级积分、大便菌群变化进行比较。结果 1免疫组化显示TLR9在UC结肠上皮和炎症细胞中的表达均高于正常对照组(P<0.01)。RT-PCR检测UC患者结肠组织可见TLR9mRNA表达,而正常对照组未检出TLR9mRNA的表达。且TLR9与Sutherland DAI、肠镜分级积分、病理组织学分级积分及大便菌群积分呈正相关性。2免疫组化及RT-PCR检测显示A、B、C 3组治疗前病变肠组织中TLR9及TLR9mRNA均为阳性表达,治疗后均为弱阳性或阴性表达,各组治疗前后3组组内比较差异有统计学意义(P<0.05);3组组间比较,A、B组间无差异,C组与A、B组比较,差异均有统计学意义(P<0.01)。33组治疗前后Sutherland DAI、肠镜分级积分、病理组织学分级积分变化均具有统计学意义(P<0.05或P<0.01);3组(组间)比较:Sutherland DAI、肠镜分级积分、病理组织学分级变化差值A、B组间差异无统计学意义,与C组比较差异均有统计学意义(P<0.01)。大便菌群变化A、B组治疗前后具有统计学意义,C组治疗前后大便菌群变化无统计学意义。结论 TLR9失调可能参与溃疡性结肠炎发病机制;微生态制剂能有效降低UC患者的TLR9表达水平,TLR9下调可能是益生菌治疗UC的作用机制之一。

Objective To examine the expression of Toll-like receptor 9（TLR9） in patients with ulcerative colitis（UC））in order to explore the pathogenesis of UC and the action mechanism of microecologics in the treatment of UC.Methods Thirty patients with mild or moderate UC（active phase）were randomly divided into groups A（n=10）,B（n=10）and C（n=10）.The patients in group A were treated with Medilac-S and SASP,those in group B with bifidobacterium capsule and SASP,and those in group C,serving as the controls,were only treated with SASP.Subjects in the normal control group had normal colonoscopic results.The treatment lasted four weeks.Colonic biopsies were taken before and 4 weeks after treatment in order to immunohistochemically detect the expression of TLR9 protein.The mRNA levels of TLR9 were assessed by reverse transcriptase polymerase chain reaction（RT-PCR）.The Sutherland disease active index（DAI）,score of colonoscopic and histological grades,score of fecal microbial population were compared among the groups.Results ①Immunohistochemistry showed that the expression of TLR9 was much higher in the colon epithelia and inflammatory cells in UC groups than in normal control group（P〈0.01） 0 TLR9 was expressed at mRNA level in UC patients but not in the normal healthy controls.The expression of TLR9 was positively correlated with the DAI,score of colonoscopic and histological grades and score of fecal microbial population.②Immunohistochemistry and RT-PCR showed that TLR9 was intensely expressed in groups A,B and C before treatment and weakly or negatively expressed in those groups after treatment.There was significant difference in the expression of TLR9 in those groups between before and after treatment（P〈 0.05）.Comparison among the three groups showed no significant difference in the TLR9 expression between groups A and B（P〉0.05）and significant difference existed between group A or B and group C（P〈 0.01）.③There was significant difference in the Sutherland DAI,score of colonoscopic and histological grades in groups A,B and C between before and after treatment（P〈0.05 or P〈0.01）.No significant difference was found in these indexes between groups A and B but between group A or B and group C（P〈0.01）.Additionally,the changes of the fecal microbial population score were significant in groups A and B（P〈0.01）but not in group C after treatment.Conelusion The disregulation of TLR9 may be involved in the pathogenesis of UC.Microecologics can effectively reduce the expression level of TLR9 in UC patients,which may be the action mechanism of microecologics in the treatment of UC.