【作者】 黄庆伦；

【导师】 谢勇；

【作者基本信息】 南昌大学 ， 内科学， 2008， 硕士

【摘要】 研究目的:探讨TLR/NF-κB信号通路在IBD致病中的作用及其抑制因子SIGIRR对LPS/TLR致炎信号通路负性调控的作用机制。为IBD的治疗提供新的靶点和方向。研究方法:⑴TLR4/NF-κB信号通路在溃疡性结肠炎患者肠黏膜中的变化及其相互之间关系的研究:采用SP免疫组化二步法检测了68例内镜活检的肠黏膜内TLR4和NF-κBP65的表达。68例包括:UC 53例(根据Tmelove-Richards分级分为:Ⅰ级9例,Ⅱ级15例,Ⅲ级19例)及正常对照者15例。⑵SIGIRR对巨噬细胞LPS/TLR信号通路的影响:①以未转染SIGIRR基因的巨噬细胞株Raw264.7为SIGIRR低表达株,采用脂质体转染技术将pUNO-mSIGIRR转入Raw264.7细胞,构建SIGIRR高表达细胞株;②分以下6组:对照组1:Raw264.7;对照组2: Raw264.7 + pUNO(空质粒);对照组3: Raw264.7 cell + pUNO-mSIGIRR;实验组1:Raw264.7 cell +LPS;实验组2: Raw264.7 + pUNO (空质粒) + LPS;实验组3 : Raw264.7 cell + pUNO-mSIGIRR + LPS;实验组在质粒转染后,加入10ng/ml LPS培养12h;③用RT-PCR方法检测各组细胞SIGIRR、TLR2、TLR4、MyD88、IRAK-1和TRAF6 mRNA的表达。研究结果:⑴TLR4/NF-κB信号通路在溃疡性结肠炎患者肠黏膜中的变化及其相互之间关系的研究:①TLR4和NF-κB P65在UC患者肠黏膜中的表达分别为50/53(94.3%)和46/53(86.8%)显著高于正常对照者的6/1(540%)和5/15(33.3%)(P<0.01),并且随着病变严重程度加重而增高;②UC患者肠黏膜内TLR4和NF-κB p65的表达呈显著正相关(r =0.923,P<0.01)。⑵SIGIRR对巨噬细胞LPS/TLR信号通路的影响:①转染了SIGIRR的Raw264.7细胞内SIGIRR mRNA的表达为0.470±0.045显著高于未转染和转染空质粒的Raw264.7细胞(0.210±0.021,0.212±0.027,P<0.001)。②无论是转染了还是未转染SIGIRR的Raw264.7细胞,经LPS作用后其SIGIRR mRNA的表达均显著低于对照组(0.114±0.052 vs 0.210±0.021, 0.109±0.047 vs 0.212±0.027, 0.302±0.036 vs 0.470±0.045)(P<0.001)。③LPS对转染和未转染SIGIRR的Raw264.7细胞TLR2 mRNA的表达均无影响(0.222±0.037 vs 0.218±0.036, 0.220±0.039 vs 0.215±0.035, 0.217±0.032 vs 0.205±0.029)(P>0.05);同时SIGIRR对Raw264.7细胞内TLR2 mRNA的表达也无影响(P>0.05);④无论是转染了还是未转染SIGIRR的Raw264.7细胞,经LPS作用后其TLR4 mRNA的表达均显著高于对照组(0.587±0.103 vs 0.309±0.046 , 0.564±0.092 vs 0.291±0.050, 0.558±0.085 vs 0.210±0.062)(P<0.001);SIGIRR对Raw264.7细胞内TLR4 mRNA的表达无影响(P>0.05);⑤未转染SIGIRR的Raw264.7细胞,经LPS作用后其MyD88 mRNA的表达均显著高于对照组(0.509±0.134 vs 0.890±0.144, 0.480±0.134 vs 0.974±0.166)(P<0.001),而转染SIGIRR的Raw264.7细胞其MyD88 mRNA的表达在两组之间无显著差异(0.478±0.151 vs 0.341±0.130)(P>0.05)。在LPS作用组转染了SIGIRR的Raw264.7细胞其MyD88 mRNA的表达(0.341±0.130)显著低于未转染(0.890±0.144)和转染空质粒(0.974±0.166)的Raw264.7细胞(P<0.001),但在对照组SIGIRR对MyD88 mRNA的表达无影响(0.509±0.134, 0.480±0.134, 0.478±0.151)(P>0.05);⑥未转染SIGIRR的Raw264.7细胞,经LPS作用后其IRAK1 mRNA的表达均显著高于对照组(0.964±0.208 vs 0.522±0.096, 1.042±0.256 vs 0.491±0.121)(P<0.001),而转染SIGIRR的Raw264.7细胞IRAK1 mRNA的表达在两组之间无显著差异(0.440±0.114 vs 0.530±0.134)(P>0.05);在LPS作用组转染了SIGIRR的Raw264.7细胞其IRAK1 mRNA的表达(0.440±0.114)显著低于未转染(0.964±0.208)和转染空质粒(1.042±0.256)的Raw264.7细胞(P<0.001),但在对照组SIGIRR对IRAK1 mRNA的表达无影响(0.522±0.096, 0.491±0.121, 0.530±0.134)(P>0.05);⑦无论是转染了还是未转染SIGIRR的Raw264.7细胞,经LPS作用后其TRAF-6 mRNA的表达均显著高于对照组(0.458±0.039 vs 0.263±0.051, 0.444±0.049 vs 0.268±0.043, 0.320±0.061 vs 0.247±0.034) (P<0.001或P<0.01);在LPS作用组转染了SIGIRR的Raw264.7细胞其TRAF-6 mRNA的表达(0.320±0.061)显著低于未转染(0.458±0.039)和转染空质粒(0.444±0.049)的Raw264.7细胞(P<0.001),但在对照组SIGIRR对TRAF-6 mRNA的表达无影响(0.263±0.051, 0.268±0.043, 0.247±0.034)(P>0.05)。结论1、UC患者肠黏膜TLR4和NF-κB p65的表达均显著上调,这可能增加肠上皮细胞、固有层单个核细胞对肠腔内的细菌和内毒素的敏感性,产生过激的免疫炎症反应,导致IBD的发生。2、UC患者肠黏膜TLR4和NF-κB p65的表达随着患者肠黏膜病理分级加剧而增加,提示TLR4/ NF-κB通路参与了UC的发生发展。3、LPS可显著抑制巨噬细胞Raw264.7内SIGIRR的表达,同时上调TLR4和其信号通路的下游分子(MyD88、IRAK-1和TRAF6)的表达。4、给予外源性SIGIRR基因可阻断LPS对TLR信号通路的激活,下调MyD88、IRAK-1和TRAF6的表达,这可能是其对TLR信号通路的负性调控机制之一。5、给予外源性SIGIRR基因对无LPS作用的巨噬细胞Raw264.7的TLR信号通路无影响,但是可阻断LPS对TLR信号通路的激活,从而抑制LPS诱导的免疫炎症反应,因此,SIGIRR有望作为IBD乃至其它炎症性疾病的治疗靶点。更多还原

【Abstract】 Objective:To discuss the role of TLR/NF-κB signal pathway in IBD and the mechanism of negative regulatory of its inhibitor SIGIRR on LPS/TLR inflammatory signal pathway. To provide new way of treatment of IBD.Methods:⑴The changes of TLR/NF-κB signal pathway in the intestinal mucosa of patients with ulcerative colitis and their relations: The protein expressions of TLR4 and NF-κB P65 in 68 biopsy intestinal mucosa samples were determined by immunohistochemistry, 68 biopsy samples including: UC 53 (according to Tmelove-Richards Classification: grades I: 9, grades II: 15, grades III: 19) and the control group 15.⑵The effect of SIGIRR on LPS/TLR signal pathway of macrophage cells:①Macrophage cells Raw264.7 non-transfected with SIGIRR was used as SIGIRR low expressing cell strain. Whereas macrophage cells Raw264.7 were transfected with pUNO-mSIGIRR using LipofectamineTM2000 to construct SIGIRR high expressing cell strain.②Groups: Control group 1:Raw264.7;Control group 2: Raw264.7 + pUNO ( blank plasmid); Control group 3: Raw264.7 cell + pUNO-mSIGIRR; LPS Group 1:Raw264.7 cell +LPS;LPS Group 2: Raw264.7 + pUNO(blank plasmid) + LPS;LPS Group 3: Raw264.7 cell + pUNO-mSIGIRR + LPS;LPS groups were incubated with 10 ng/ml LPS medium for 12 hours after transfection;③The mRNA expressions of SIGIRR, TLR2, TLR4, MyD88, IRAK-1 and TRAF6 were determined by RT-PCR.Results:⑴The changes of TLR/NF-κB signal pathway in the intestinal mucosa of patients with ulcerative colitis and their relations:①The expressions of TLR4 and NF-κB P65 in the intestinal mucosa of patients with ulcerative colitis were 50/53(94.3%) and 46/53(86.8%), respectively, which were higher than those in control groups 6/15(40%)和5/15(33.3%), respectively, (P<0.01). And the expressions were higher when the pathological changes were more serious;②The expressions of TLR4 and NF-κB P65 in the intestinal mucosa of patients with ulcerative colitis were significantly positive correlated (r =0.923,P<0.01).⑵The effect of SIGIRR on LPS/TLR signal pathway in macrophage cells:①The mRNA expression of SIGIRR in Raw264.7 cells transfected with SIGIRR was 0.470±0.045, which was significantly higher than those in cells non-transfected or transfected with pUNO (0.210±0.021 and 0.212±0.027,P<0.001).②In Raw264.7 cells transfected or non-transfected with SIGIRR, the mRNA expressions of SIGIRR in cells stimulated with LPS were significantly lower than those in control groups (0.114±0.052 vs 0.210±0.021, 0.109±0.047 vs 0.212±0.027, 0.302±0.036 vs 0.470±0.045)(P<0.001).③The mRNA expressions of TLR2 in Raw264.7 cells transfected or non-transfected were not affected with LPS (0.222±0.037 vs 0.218±0.036, 0.220±0.039 vs 0.215±0.035, 0.217±0.032 vs 0.205±0.029);and the mRNA expressions of TLR2 in Raw264.7 cells were not affected with SIGIRR (P>0.05).④In Raw264.7 cells transfected or non-transfected with SIGIRR, the mRNA expressions of TLR4 in cells stimulated with LPS were significantly higher than those in control groups (0.587±0.103 vs 0.309±0.046 , 0.564±0.092 vs 0.291±0.050, 0.558±0.085 vs 0.210±0.062)(P<0.001); the mRNA expressions of TLR4 of Raw264.7 cells were not affected with SIGIRR (P>0.05);⑤In Raw264.7 cells non-transfected with SIGIRR, the mRNA expression of MyD88 of cells stimulated with LPS were significantly higher than those in control groups (0.509±0.134 vs 0.890±0.144, 0.480±0.134 vs 0.974±0.166)(P<0.001), Whereas in Raw264.7 cells transfected with SIGIRR, the mRNA expressions of MyD88 were not significantly different in those two groups (0.478±0.151 vs 0.341±0.130)(P>0.05). In LPS groups, the mRNA expressions of MyD88 in Raw264.7 cells transfected with SIGIRR (0.341±0.130) were significantly lower than those in cells non-transfected (0.890±0.144) or transfected with pUNO (0.974±0.166) (P<0.001) Whereas in control groups, the mRNA expressions of MyD88 were not affected with SIGIRR (0.509±0.134, 0.480±0.134, 0.478±0.151) (P>0.05).⑥In Raw264.7 cells non-transfected with SIGIRR, the mRNA expressions of IRAK1 of cells stimulated with LPS were significantly higher than those in control groups (0.964±0.208 vs 0.522±0.096, 1.042±0.256 vs 0.491±0.121) (P<0.001),Whereas in Raw264.7 cells transfected with SIGIRR, the mRNA expressions of IRAK1 were not significantly different in those two groups (0.440±0.114 vs 0.530±0.134) (P>0.05); In LPS groups, the mRNA expressions of IRAK1 in Raw264.7 cells transfected with SIGIRR (0.440±0.114) were significantly lower than those in cells non-transfected (0.964±0.208) or transfected with pUNO (1.042±0.256) (P<0.001), Whereas in control groups, the mRNA expressions of IRAK1 were not affected with SIGIRR (0.522±0.096, 0.491±0.121, 0.530±0.134) (P>0.05).⑦In Raw264.7 cells transfected or non-transfected with SIGIRR, the mRNA expressions of TRAF-6 in cells stimulated with LPS were significantly higher than those in control groups (0.458±0.039 vs 0.263±0.051, 0.444±0.049 vs 0.268±0.043, 0.320±0.061 vs 0.247±0.034) (P<0.001 or P<0.01); In LPS groups, the mRNA expressions of TRAF-6 in Raw264.7 cells transfected with SIGIRR (0.320±0.061) were significantly lower than those in cells non-transfected (0.458±0.039) or transfected with pUNO (0.444±0.049) (P<0.001), Whereas in control groups, the mRNA expressions of TRAF-6 were not affected with SIGIRR (0.263±0.051, 0.268±0.043, 0.247±0.034) (P>0.05)Conclusion⑴The expressions of TLR4 and NF-κB p65 in the intestinal mucosa of patients with UC were significantly upregulated, which may increase the sensibilities of intestinal epithelial cells and lamina propria mononuclear cells to bacteria and endotoxin in intestine, and generate excessive immune inflammatory response, thus cause IBD.⑵The expressions of TLR4 and NF-κB P65 in the intestinal mucosa of patients with ulcerative colitis were higher when the pathological changes were more serious, which indicate that TLR4/ NF-κB signal pathway participate in the occurrence and development of UC.⑶LPS can significant inhibit the expression of SIGIRR in macrophage cells Raw 264.7, and upregulate the expressions of TLR4 and the downstream molecules (MyD88、IRAK-1 and TRAF6) of the signal pathway.⑷SIGIRR provided extrinsically can block the activation LPS/TLR signal pathway, and downregulate the expression of MyD88、IRAK-1 and TRAF6, which may be one of the mechanisms of negative regulatory to the TLR signal pathway. ⑸SIGIRR provided extrinsically has no effect on the TLR signal pathway of macrophage cells Raw264.7 not-stimulated with LPS, but can block the activation of LPS to the TLR signal pathway, thus inhibit the immune inflammatory response induced by LPS. Therefore, SIGIRR may be one of therapeutic targets of IBD and other inflammatory diseases.更多还原