Association of NOD1 and NOD2 genes polymorphisms with Helicobacter pylori related gastric cancer in a Chinese population

AIM:To investigate the association between the tag single nucleotide polymorphisms(TagSNPs) of NOD1 and NOD2 and the risk of developing gastric cancer.METHODS:We conducted a hospital-based case-control study including 296 incident gastric cancer patients and 160 gastritis controls.Eight TagSNPs in the NOD1 and NOD2 genes were selected from the Hapmap database using the haploview software and genotyped by the Sequenom MassArray system.The serum levels of anti-Helicobacter pylori(H.pylori) IgG were measured by enzyme-linked immunosorbent assay to indicate H.pylori infection.The odds ratios(OR) and 95% confidence intervals(CI) were calculated by unconditional logistic regression,including sex and age as confounding factors.RESULTS:The NOD1 rs2907749 GG genotype showed a decreased risk for gastric cancer(OR 0.50,95% CI:0.26-0.95,P = 0.04) while the rs7789045 TT genotype showed an increased risk(OR 2.14,95% CI:1.20-3.82,P = 0.01).An elevated susceptibility to gastric cancer was observed in the subjects with H.pylori infection and the NaOD1 rs7789045 TT genotype(OR 2.05,95% CI:1.07-3.94,P = 0.03) or the NOD2 rs7205423 GC genotype(OR 2.52,95% CI:1.05-6.04,P = 0.04).Haplotype analysis suggested that the distribution of AGT(rs2907749,rs2075820 and rs7789045) in NOD1 between the cases and control groups was significantly different(P corrected:0.04),and the diplotype AGT/AGT was associated with an elevated gastric cancer risk(OR 1.98,95% CI:1.04-3.79,P = 0.04).The association of the NOD1 rs7789045 TT genotype and the diplotype AGT/AGT was significant with H.pylori-related diffuse-type gastric cancer(OR 3.00,95% CI:1.38-6.53,P = 0.01;OR 4.02,95% CI:1.61-10.05,P < 0.01,respectively).CONCLUSION:Genetic polymorphisms in NOD1 and NOD2 may interact with H.pylori infection and may play important roles in promoting the development of gastric cancer in the Chinese population.

Clinical significance of NOD2/CARD15 and Toll-like receptor 4 gene single nucleotide polymorphisms in inflammatory bowel disease

AIM: To evaluate the role of genetic factors in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC), we investigated the single nucleotide poly- morphisms (SNPs) of NOD2/CARD15 (R702W, G908R and L1007fi nsC), and Toll-like receptor 4 (TLR4) genes (D299G and T399I) in a selected inflammatory bowel disease (IBD) population coming from Southern Italy. METHODS: Allele and genotype frequencies of NOD2/ CARD15 (R702W, G908R and L1007finsC) and TLR4 (D299G and T399I) SNPs were examined in 133 CD pa-tients, in 45 UC patients, and in 103 healthy controls. A genotype-phenotype correlation was performed. RESULTS: NOD2/CARD15 R702W mutation was sig-nificantly more frequent in CD (9.8%) than in controls (2.4%, P = 0.001) and in UC (2.3%, P = 0.03). No sig-nificant difference was found between UC patients and control group (P > 0.05). In CD and UC patients, no signifi cant association with G908R variant was found. L1007f insC SNP showed an association with CD (9.8%) compared with controls (2.9%, P = 0.002) and UC patients (2.3%, P = 0.01). Moreover, in CD patients, G908R and L1007finsC mutations were significantly associated with different phenotypes compared to CD wild-type patients. No association of IBD with the TLR4 SNPs was found in either cohort (allele frequencies: D299G-controls 3.9%, CD 3.7%, UC 3.4%, P > 0.05; T399I-controls 2.9%, CD 3.0%, UC 3.4%, P > 0.05). CONCLUSION: These findings confirm that, in our IBD patients selected from Southern Italy, the NOD2/ CARD15, but not TLR4 SNPs, are associated with in-creased risk of CD.

NOD2/CARD15 gene polymorphism in patients with inflammatory bowel disease: Is Hungary different?

AIM: To analyse the impact of NOD2/CARD15 mutations on the clinical course of Crohn's disease patients from an eastern European country (Hungary).METHODS: We investigated the prevalence of the three common NOD2/CARD15 mutations (Arg702Trp, Gly908Arg,1007finsC) in 148 patients with Crohn's disease, 128patients with ulcerative colitis and 208 controls recruited from the University of Szeged, Hungary. In patients with Crohn's disease, the prevalence of NOD2/CARD15 mutations was correlated to the demographical and clinical parameters.RESULTS: In total, 32.4% of Crohn's disease patients carried at least one mutant allele within NOD2/CARD15compared to 13.2% of patients with ulcerative colitis (P = 0.0002) and to 11.5% of controls (P＜0.0001). In Crohn's disease patients, the allele frequencies for Arg702Trp,Gly908Arg and 1007finsC were 7.1%, 3.0% and 10.8%respectively. Interestingly, only the 1007finsC mutation was associated with a distinct clinical phenotype. The patients positive for the 1007finsC mutation suffered more frequently from stenotic disease behaviour (P = 0.008). Furthermore,51.9% of patients positive for the 1007finsC mutation underwent a surgical resection within the ileum compared to only 17.4% of patients without the 1007finsC mutation (P = 0.001). With respect to the other two mutations (Arg702Trp and Gly908Arg), no associations were found with all investigated clinical parameters.CONCLUSION: NOD2/CARD15 mutations are frequently found in Crohn's disease patients from Hungary. The 1007finsC mutation is associated with stenotic disease behaviour and frequent ileal resections.

Polymorphisms in interleukin-10 gene according to mutations of NOD2/CARD15 gene and relation to phenotype in Spanish patients with Crohn's disease

瞄准：检验 interleukin-10 (IL-10 ) 的贡献基因多型性到 Crohnos 疾病(CD ) 显型，和在 IL-10 基因多型性和 CARD15/NOD2 基因变化之间的可能的基因排泄制止。方法：有 Crohn 从一个单个中心招募的疾病的 205 个西班牙的无关的病人的一个队被学习。所有病人是严厉地 phenotyped 并且为至少 3 年跟随起来(吝啬的时间， 12.5 年) 。临床的显型在 genotyping 以前被建立。结果：遗传型的关联 -- 维也纳分类组证明回肠结肠地点显著地在 NOD2/CARD15 变化积极的病人与 -1082G 等位基因被联系(RR=1.52， 95%CI， 1.21 ～ 1.91， P=0.008 ) 。多，变量分析证明在 NOD2/CARD15 变化的 IL-10 G14 微卫星等位基因积极病人与二个风险因素被联系，阑尾切除术的历史(RR=2.15， 95%CI=1.1-4.30， P=0.001 ) 并且在诊断的吸烟习惯(RR=1.29， 95%CI=1.04-4.3， P=0.04 ) 。结论：在从马德里的西班牙的人口，在带至少一个 NOD2/CARD15 变化的 CD 病人， -1082G 等位基因与回肠结肠疾病被联系， IL-10G14 微卫星等位基因在诊断与阑尾切除术和吸烟习惯的以前的历史被联系。这些数据为 CD 的临床的异质的一个基因基础提供进一步分子的证据。

NOD2/CARD15基因突变与中国人克罗恩病相关性的研究

背景:近年多项研究证明NOD2/CARD15基因序列的单核苷酸多态性(SNP)与西方白种人克罗恩病(CD)明显相关,其中3个SNP(R702W、G908R和3020insC)与CD的相关性尤为显著。目的:探讨NOD2/CARD15基因SNP与中国人CD发病的相关性及其与CD临床特点的关系。方法:选取临床资料完整的CD患者48例、溃疡性结肠炎(UC)患者和健康对照者各50例,提取人血白细胞基因组DNA,经聚合酶链反应(PCR)扩增NOD2基因全部12对外显子,纯化后直接测序,根据结果分析其突变与CD病变特点的关系。结果:CD组、UC组和健康对照组均未检出3个西方人常见的NOD2/CARD15基因多态性位点。CD组的P268S突变率显著高于UC组和健康对照组(P<0.05)。5例P268S突变的CD患者病变均位于回肠(P<0.01),4例发病年龄≤20岁(P<0.01),且均并发肠腔狭窄(P<0.01)。结论:中国人CD患者中存在NOD2/CARD15基因P268S突变,且与患者的发病年龄、病变部位和并发症相关,有必要对其功能作进一步探讨。

中国汉族人群NOD2、IRGM、ATG16L1和STAT4基因多态性与克罗恩病的相关性研究

背景:克罗恩病(CD)的病因和发病机制尚未完全阐明,近年国外研究发现NOD2、IRGM、ATG16L1、STAT4基因突变与CD相关。目的:分析NOD2、IRGM、ATG16L1、STAT4基因多态性与中国汉族人群CD发病的相关性。方法:连续纳入2007年1月～2010年1月苏州市立医院中国汉族CD患者66例,66名健康体检者作为正常对照,以PCR联合基因测序检测4种基因相应单核苷酸多态性(SNP)位点的基因型,分析各基因型和等位基因频率。结果:CD组和正常对照组NOD2基因rs2066842位点、IRGM基因rs13361189位点、ATG16L1基因rs2241880位点和STAT4基因rs7574865位点基因型和等位基因频率分布均符合Hardy-Weinberg遗传平衡定律,两组间4种基因相应SNP位点的基因型和等位基因频率差异均无统计学意义。结论:NOD2、IRGM、ATG16L1和STAT4基因多态性与中国汉族人群CD发病不相关。

P268S突变型NOD2/CARD15真核表达载体的构建及其体外表达

背景:NOD2/CARD15基因序列单核苷酸多态性(SNP)与欧美人群的克罗恩病(CD)明显相关,其中R702W、G908R和3020insC3个SNP位点与CD的相关性尤为显著。而日本、韩国以及我国香港和浙江地区的研究均未发现上述3个SNP的改变,但最近研究发现了可能与中国人CD相关的P268S突变。目的:构建P268S突变型NOD2/CARD15真核表达载体和体外转染体系,为研究突变型NOD2/CARD15的功能提供实验基础。方法:应用定点诱变技术构建P268S突变型NOD2/CARD15真核表达载体,以阳离子脂质体介导体外转染技术瞬时转染人胚肾细胞HEK293T,以蛋白质印迹法和逆转录聚合酶链反应(RT-PCR)检测HEK293T细胞NOD2/CARD15的表达。结果:经克隆、酶切、测序证实获得P268S突变型NOD2/CARD15基因,突变载体转入HEK293T细胞后,NOD2/CARD15有效表达。结论:成功构建了P268S突变型NOD2/CARD15真核表达载体,阳离子脂质体是人胚肾细胞有效的体外转染体系。

Association between polymorphisms in the Toll-like receptor 4,CD14,and CARD15/NOD2and inflammatory bowel disease in the Greek population

AIM: Crohn's disease(CD)and ulcerative colitis(UC)are multifactorial diseases with a significant genetic background.Apart from CARD15/NOD2 gene, evidence is accumulating that molecules related to the innate immune response such as CD14 or Toll-like receptor 4 (TLR4), are involved in their pathogenesis. In further exploring the genetic background of these diseases, we investigated the variations in the CARD15/NOD2 gene (Arg702Trp,Gly908Arg and Leu1007fsinsC), and polymorphisms in the TLR4 gene (Asp299Gly and Thr399Ile) as well as in the promoter of the CD14 gene (T/C at position -159) in Greek patients with CD and UC.METHODS: DNA was obtained from 120 patients with CD,85 with UC and 100 healthy individuals. Genotyping was performed by allele specific PCR or by PCR-RFLP analysis.RESULTS: The 299Gly allele frequency of the TLR4 gene and the T allele and TT genotype frequendes of the CD14 promoter were significantly higher in CD patients only compared to healthy individuals (P = 0.026＜0.05; P = 0.0048＜0.01 and P= 0.047＜0.05 respectively). Concerning the NOD2/CARD15mutations the overall presence in CD patients was significantly higher than that in UC patients or in controls.Additionally, 51.67% of the CD patients were carriers of a TLR4 and/or CD14 polymorphic allele and at least one variant of the NOD2/CARD15, compared to 27% of the UC patients. It should be pointed out that both frequencies significantly increased as compared with the 10% frequency of multiple carriers found in healthy controls. A possible interaction of the NOD2/CARD15 with TLR4 and especially CD14, increased the risk of developing inflammatory bowel disease (IBD).CONCLUSION: Our results indicate that co-existence of a mutation in either the TLR4 or CD14 gene, and in NOD2/CARD15is associated with an increased susceptibility to developing CD compared to UC, and to developing either CD or UC compared to healthy individuals.

NOD2/CARD15基因突变与中国人克罗恩病相关性研究

背景NOD2/CARD15基因是人类克罗恩病(Crohn′sdisease,CD)第一个易感基因,既往研究发现P268S可能与中国人CD发病及临床特征相关。目的本研究旨在证实P268S与中国人CD发病及其临床特征的相关性。方法血样来自临床确诊的50例CD患者,60例溃疡性结肠炎(ulcerativecolitis,UC)患者及100例健康体检者(healthycontrols,HC)。提取人血白细胞基因组DNA,PCR扩增目的片段,PCR-RFLP发现突变位点,DNA测序证实突变位点。结果共有8例CD患者发现有P268S改变,而在UC患者和HC中分别发现2例和3例P268S改变,CD组明显高于UC和HC组(χ2=10.829,P=0.004),而UC组和HC组无明显差异。8例有P268S改变的CD患者临床特征包括病变多位于回肠,发病年龄轻(6例<20岁),常并发肠腔狭窄而需手术治疗,中-重度患者比例高。结论P268S可能是NOD2/CARD15基因中与中国人CD相关的SNP。P268S与CD患者发病年龄、病变部位及并发症及病情严重程度可能相关。

NOD2基因对舌鳞癌Tca8113细胞增殖和凋亡的影响

目的:观察NOD2基因对舌鳞癌Tca8113细胞增殖、凋亡的影响。方法:构建NOD2表达载体(NOD2-pEZ-M29)和NOD2-shRNA表达载体,分别转染舌鳞癌Tca8113细胞48 h后,RT-PCR和Western blot法检测NOD2和HBD-2分子及蛋白表达,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡比例。结果:转染NOD2表达载体的Tca8113细胞NOD2和HBD-2表达较对照组显著增高,细胞增殖速度较对照组慢,凋亡较对照组高;转染NOD2-shRNA载体的Tca8113细胞NOD2和HBD-2表达较对照组显著降低,细胞增殖速度较对照组快,凋亡较对照组低。结论:NOD2可促进舌鳞癌细胞凋亡,抑制其增殖。在舌鳞癌细胞中,NOD2与HBD-2的表达呈正相关。

TLR2、TLR4和NOD2/CARD15基因多态性与溃疡性结肠炎相关性的meta分析

背景:近年我国溃疡性结肠炎(UC)的患病率明显增高,Toll样受体2(TLR2)、TLR4和NOD2/CARD15基因多态性与UC的发生、发展可能密切相关。目的:探讨TLR2、TLR4和NOD2/CARD15基因多态性对UC发生的影响。方法:计算机检索PubMed、中国生物医学文献、中国知网、万方数据库、重庆维普等数据库中所有TLR2、TLR4和NOD2/CARD15基因多态性与UC相关性的研究。按照纳入与排除标准筛选文献、评价质量并提取数据,采用RevMan 5.3软件进行meta分析。结果:共纳入15项研究。Meta分析结果显示,TLR2 Arg753Gln基因多态性与UC发生风险无关(P>0.05)。除隐性模型外,TLR4 Asp299Gly基因多态性可显著增加UC的发生风险(P<0.05),TLR4 Thr399Ile基因超显性模型可导致UC风险增加(P<0.05),但显性模型和隐性模型与UC无关(P>0.05)。NOD2/CARD15(Arg702Trp、Gly908Arg、Leu1007fsinsC)基因多态性均与UC无关(P>0.05)。结论:NOD2/CARD15(Arg702Trp、Gly908Arg、Leu1007fsinsC)、TLR2(Arg753Gln)与UC发生风险无关,TLR4(Asp299Gly、Thr399Ile)可增加UC的发生风险。

人NOD2基因启动子的绿色荧光蛋白表达载体的构建

人NOD2基因启动子的绿色荧光蛋白表达载体的构建。以人基因组DNA为模板,PCR扩增含有人NOD2基因启动子不同长度的4段序列,利用特定的限制性内切酶位点,以切除启动子的pEGFP-N3作为框架结构,将这4个序列片段进行酶切并插入表达载体pEGFP-N3中,用Vsp I和Pst I双酶切和PCR鉴定重组质粒,再将重组质粒进行DNA序列分析。构建的重组质粒经脂质体LipofectamineTM2000介导转染HTK293T细胞,转染24 h后加入TNF-α持续刺激细胞24 h或不加TNF-α刺激,在倒置荧光显微镜下观察其能否在NOD2基因启动子的调控下表达报告基因绿色荧光蛋白(green fluores-cent proteins,GFP)。结果:pEGFP-N3-NOD2(617 bp)、pEGFP-N3-NOD2(747 bp)、pEGFP-N3-NOD2(1 136 bp)、pEG-FP-N3-NOD2(1 387 bp)分别经酶切鉴定和序列测定证实目的基因已插入重组质粒;细胞转染结果表明,构建的4段重组质粒pEGFP-N3-NOD2(617 bp)、pEGFP-N3-NOD2(747 bp)、pEGFP-N3-NOD2(1 136 bp)、pEGFP-N3-NOD2(1 387 bp)转染的HTK293T均能表达绿色荧光,其中构建pEGFP-N3-NOD2(1 387 bp)重组质粒经TNF-α持续刺后激绿色荧光表达最强。4段不同长度的人NOD2基因启动子绿色荧光蛋白表达载体成功构建,这为进一步研究NOD2基因表达及调控机制奠定了良好的基础。

Nod2基因突变与小肠CD相关性的研究

目的探讨Nod2基因与中国人小肠CD发病的相关性及其可能作用机制。方法通过双气囊小肠镜(DBE)进行病变部位组织取材,经病理学检查确诊的组织标本小肠CD 24份,结肠CD 44份,UC 40份,健康对照50份。提取组织标本基因组DNA,经聚合酶链反应(PCR)扩增Nod2基因全部12对外显子,纯化后直接测序,根据结果分析其突变与CD病变的关系。检测各组突变的Nod2mRNA及其编码的NF-κB表达。结果小肠CD组、结肠CD组、UC组和健康对照组均未检出西方人常见的3个Nod2基因多态性位点。小肠CD组的P268S突变率高于结肠CD组。UC组和健康对照组未显示该突变。小肠CD组Nod2突变标本其mRNA和NF-κB蛋白表达增加大于结肠CD突变标本,但差异无统计学意义(P>0.05),而小肠和结肠CD组无突变的CD患者与UC和健康者表达也无明显差异(P>0.05),但Nod2突变的小肠和结肠CD标本mRNA表达大于任何无突变的标本,差异具有统计学意义(P<0.05)。结论中国人小肠和结肠CD患者中存在Nod2基因P268S突变,它可能通过调控NF-κB蛋白表达,导致细胞功能的改变,促进CD的发生。

Nod2与克罗恩病相关性研究的进展

Nod2(Card15)及其相关的Nod1(Card4)都属于近年来研究较多的Nod分子家族。Nod蛋白最初被描述为细胞内Caspase和核因子-κB(NF-κB)信号转导通路的活化因子。随着分子生物学和遗传学研究的深入,NOD2(CARD15)基因与克罗恩病(CD)易患体质的关系逐渐明确。与此同时,生物化学研究证实Nod1及Nod2是细菌肽聚糖成分的细胞内识别分子。作者就Nod蛋白介导的细胞内细菌识别与CD的相关性研究作一综述。

Comprehensive mutation screening for 10 genes in Chinese patients suffering very early onset inflammatory bowel disease

AIM: To perform sequencing analysis in patients with very early-onset inflammatory bowel disease(VEO-IBD) to determine the genetic basis for VEO-IBD in Chinese pediatric patients.METHODS: A total of 13 Chinese pediatric patients with VEO-IBD were diagnosed from May 2012 and August 2014. The relevant clinical characteristics of these patients were analyzed. Then DNA in the peripheral blood from patients was extracted. Next generation sequencing(NGS) based on an IlluminaMiseq platform was used to analyze the exons in the coding regions of 10 candidate genes: IL-10, IL-10 RA, IL-10 RB, NOD2, FUT2, IL23 R, GPR35, GPR65, TNFSF15, and ADAM30. The Sanger sequencing was used to verify the variations detected in NGS.RESULTS: Out of the 13 pediatric patients, ten were diagnosed with Crohn's disease, and three diagnosed with ulcerative colitis. Mutations in IL-10 RA and IL-10 RB were detected in five patients. There were four patients who had single nucleotide polymorphisms associated with IBD. Two patients had IL-10 RA andFUT2 polymorphisms, and two patients had IL-10 RB and FUT2 polymorphisms. Gene variations were not found in the rest four patients. Children with mutations had lower percentile body weight(1.0% vs 27.5%, P = 0.002) and hemoglobin(87.4 g/L vs 108.5 g/L, P = 0.040) when compared with children without mutations. Although the age of onset was earlier, height was shorter, and the response to treatment was poorer in the mutation group, there was no significant difference in these factors between groups.CONCLUSION: IL-10 RA and IL-10 RB mutations are common in Chinese children with VEO-IBD. Patients with mutations have an earlier disease onset, lower body weight and hemoglobin, and poorer prognosis.

Risk factors and gene polymorphisms of inflammatory bowel disease in population of Zhejiang,China

AIM:To identify the risk factors and three single nucleotide polymorphisms(SNPs) of NOD2/CARD15 gene in inflammatory bowel disease(IBD) of the population in Zhejiang,China.METHODS:A case-control study was conducted using recall questionnaire to collect data on demographic,socioeconomic,lifestyle characteristics and dietary behaviors from 136 determined IBD patients and 136 paired healthy controls.COX regression method was used to screen the statistically significant risk factors for IBD.The polymorphisms of NOD2/CARD15 gene Arg702Trp,Gly908Arg and Leu1007fsinsC were genotyped and further compared between 60 patients with IBD and 60 healthy controls by polymerase chain reaction and restriction fragment length polymorphism.RESULTS:IBD occurred primarily in young and middle-aged people.The mean age for IBD patients was 42.6 years.The ratio of males to females was 1.23:1.COX regression indicated a higher statistical significance in milk,fried food and stress compared with the other postulated risk factors for IBD.None of the patients with IBD and healthy controls had heterozygous or homozygous SNPs variants.CONCLUSION:Milk,fried food and stress are associated with increased risk of IBD.The common variants in NOD2/CARD15 gene are not associated with IBD in China's Zhejiang population.

NOD2^(-/-)小鼠H37Ra感染后对肺组织Toll样受体表达的影响

目的探讨NOD2(nucleotide-binding oligomerization domain 2)基因缺陷(NOD2^(-/-))小鼠感染结核分枝杆菌H37Ra后Toll样受体(toll like receptor,TLR)2、TLR4的变化。方法通过PCR技术鉴定NOD2^(-/-)小鼠和野生型C57BL/6(WT)小鼠,气管滴注分枝杆菌减毒株H37Ra建立肺部感染模型,滴注等量生理盐水(NS)为阴性对照。感染1、8周后,无菌取肺脏。抗酸染色观察模型构建是否成功、HE染色观察小鼠肺部病变程度;利用荧光定量PCR、Western blot技术检测小鼠肺组织中TLR2、TLR4的表达。结果H37Ra感染小鼠1周,NOD2^(-/-)组与WT组小鼠肺组织均出现少量结核分枝杆菌菌体,感染8周,出现结核分枝杆菌聚集成团现象;1周感染组的TLR2和TLR4的基因与蛋白表达与生理盐水组相比,差异均无统计学意义(P均>0.05),8周NOD2^(-/-)与WT感染组TLR2和TLR4的基因与蛋白表达均高于对照组(P均<0.05),NOD2^(-/-)小鼠TLR2和TLR4的基因与蛋白水平高于WT小鼠(P均<0.05)。结论H37Ra感染NOD2^(-/-)小鼠后TLR2、TLR4表达升高。

Nod2基因在肺炎克雷伯菌肝脓肿中的作用研究

目的建立肺炎克雷伯菌(K.pneumoniae)感染Nod2基因肝组织条件敲除小鼠模型,初步探究Nod2基因在K.pneumoniae感染引起肝脓肿过程中的作用及机制。方法将引进的Nod2^(flox/+)小鼠自交获得Nod2^(flox/flox)小鼠,同时将Alb-Cre^(+)小鼠与Nod2^(flox/+)进行杂交获得Nod2^(flox/+);Alb-Cre^(+)的子代小鼠;将上述两种基因型的子代小鼠进行杂交,获得Nod2基因肝脏条件性敲除小鼠(Nod2^(flox/flox);Alb-Cre^(+))和同窝阴性对照组小鼠(Nod2^(flox/flox))。提取小鼠脚趾基因组DNA,经PCR扩增,通过琼脂糖凝胶电泳鉴定子代小鼠基因型并提取小鼠肝脏,利用RT-qPCR和Western blot验证Nod2基因在肝脏中的敲除效果。实验组和对照组小鼠分别感染K.pneumoniae,观察不同时间点小鼠生存率及肝组织病理变化;利用RT-qPCR检测实验组和对照组小鼠感染K.pneumoniae 48 h后肝组织内肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和趋化因子CXC配体1(CXCL1)的mRNA的表达水平。结果Nod2^(flox/flox);Alb-Cre^(+)小鼠肝脏NOD2 mRNA表达量降低(P<0.05),Western blot结果显示NOD2蛋白表达量降低;与对照组小鼠比较,实验组小鼠在感染K.pneumoniae后生存率降低(中位生存时间60.5 h,P=0.0469),肝组织出现了更为严重的病理损伤;实验组小鼠肝组织中TNF-α、IL-1β和CXCL1的mRNA表达水平降低,差异有统计学意义(P<0.05)。结论NOD2在K.pneumoniae感染诱导肝脓肿的过程中起保护作用。

克罗恩病的发病机制和治疗进展

克罗恩病(Crohn's disease,CD)是一种以小肠为主,累及全层节段性全胃肠道的非特异性炎症性肠道疾病(inflam-matory bowel disease,IBD)。该病须与溃疡性结肠炎、急性感染性结肠炎等鉴别诊断。该病在亚洲的发病率远低于西方国家,近10年来在我国的发病率有明显上升趋势。CD的病因与发病机制尚不明确,临床表现也复杂多样,且容易误诊,复发率高。在过去的10多年里,遗传和肠道免疫领域取得的研究成果为我们在研究IBD病因以及治疗上提供了新的思路。本文就CD的发病机制和治疗进展作一综述。

早发型结节病临床及基因突变分析

目的:探讨伴随血压升高的1例早发型结节病患儿临床表现及CARDl5基因突变情况。方法:提取患儿、其父母及102例健康人对照组外周血DNA,PCR扩增CARDl5基因所有编码区外显子,测序结果与正常序列对比,寻找致病的突变位点。结果:患儿临床及皮损组织病理表现均符合结节病,并随疾病发展出现血压增高。基因检测发现其CARDl5/NOD2基因4号外显子发生c.1000C>T杂合突变(p.R334W),其父母及102例健康对照者的相应外显子测序均未发现该突变。结论:该例早发型结节病患儿存在CARDl5/NOD2基因p.R334W热点突变,因此基因诊断可能是辅助诊断早发型结节病的有效手段。