克罗恩病与IL-32的表达及基因多态性的相关性分析
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摘要
目的:
观察促炎性细胞因子IL-32在克罗恩病(Crohn’s disease)患者肠黏膜中的表达情况,研究CARD15、IRGM、ATG16L1、NKX2-3基因的单核苷酸多态性(single nucleotide polymorphisms, SNP)位点与中国人克罗恩病易感性的关系,为深入研究克罗恩病的发病机制奠定基础,亦为该病的防治提供实验依据。
方法:
⑴收集临床确诊的克罗恩病患者(47例)及其健康对照(16名)的组织标本,经病理检验,通过免疫组化以及western blot方法观察IL-32在克罗恩病患者肠黏膜内的表达情况,检验IL-32在克罗恩病患者肠黏膜中的表达与意义。⑵提取克罗恩病患者(32例)和健康对照个体(30名)的外周血,抽提白细胞基因组DNA,检测CARD15_rs2066842、IRGM_rs13361189、ATG16L1_rs2241880以及NKX2-3_ rs10883365的SNP多态性与克罗恩病的关系,根据靶序列片段设计相应的引物,PCR扩增目的片段,经纯化后测序,测序结果分组做统计分析。
结果:
⑴与对照相比,经免疫组化法和western blot法检测的IL-32在克罗恩病患者肠黏膜中表达上调,二者的表达量有显著性差异(P﹤0.05)。⑵克罗恩病病例组和健康对照组基因频率和基因型频率的组间比较显示: CARD15_rs2066842、IRGM_rs13361189、ATG16L1_rs2241880以及NKX2-3_rs10883365的SNP分布频率的差异均无统计学意义(P﹥0.05)。提示这四个基因多态性位点可能与中国人克罗恩病的易感性无关。
结论:
⑴IL-32无论在克罗恩病患者还是在健康对照个体的肠黏膜内均有表达,但在克罗恩病患者肠黏膜内IL-32表达上调,研究显示IL-32表达上调能加重克罗恩病的炎症反应。⑵国外研究报道的与高加索人克罗恩病相关的CARD15_rs2066842,IRGM_ rs13361189、ATG16L1_rs2241880以及与日本人克罗恩病易感性的NKX2-3_rs10883365的SNP位点可能与我国克罗恩病患者的易感性无关,这些SNP的变异至多是增加了中国人罹患克罗恩病的风险。
Objective:
To explore the expression of interleukin-32(IL-32), a novel pro-inflammatory cytokine in the intestinal mucosa of patients with Crohn’s disease (CD). Nowadays, it has been reported that some sequence variants and other multiple replicating loci contribute to Crohn’s disease susceptibility, such as CARD15(NOD2), IRGM, ATG16L1, NKX2-3, etc. We study the correlations of these single nucleotide polymorphisms(SNPs ) associated with Chinese CD, in order that study the pathogenesis of CD in-depth, and provide experimental basis for prevention and treatment of this disease.
Methods:
The biopsy specimens of clinical diagnosis of CD patients (47 cases) and healthy controls (16 cases) were collected, after pathological examinations, the expressions of IL-32 were detected by immunohistochemistry (IHC) and western blot, the results were used for analysis. To evaluate the relations between gene polymorphisms and CD, peripheral blood from CD patients (32 cases) and healthy controls (30 cases)were extracted, genomic DNA was isolated from leukocyte in the peripheral blood, to estimate the relationships of SNPs about CARD15_rs2066842, IRGM_ rs13361189, ATG16L1_rs2241880 and NKX2-3_rs10883365 mutations associated with CD, matched primers were designed according to the target sequences, the fragments of target sequences were amplified by PCR, after purified, the PCR products were sequencing, and the sequencing results were compared, and make statistical analysis.
Results:
Compared to controls, the expression of IL-32 in intestinal mucosa with CD patients were up-regulation, and the protein levels of these two groups were significant difference(P﹤0.05). Gene frequencies and genotype frequencies between CD and healthy control groups comparisons showed that: the frequency distributions of the SNPs about CARD15_rs2066842, IRGM_rs13361189, ATG16L1_rs2241880 and NKX2-3 _rs10883365 were no significant difference. It is suggesting that these SNPs of genes variants with Chinese CD patients susceptibility has no correlations.
Conclusion:
In spite of the pro-inflammatory cytokine IL-32 was expressed in intestinal mocusa of CD patients and controls, the expression levels of this protein in CD was increased, our studies show that IL-32 up-expression could aggravate chronic inflammatory response in CD. The abroad researchers have reported the SNPs about CARD15_rs2066842, IRGM_rs13361189, ATG16L1_rs2241880 and NKX2-3_ rs10883365 associated with the Caucasians or Japanese CD, but we found that the SNPs of these gene sites variants associated with susceptibility of Chinese CD might not be related, and the SNPs variants at most increased the risk of suffering from CD.
观察促炎性细胞因子IL-32在克罗恩病(Crohn’s disease)患者肠黏膜中的表达情况,研究CARD15、IRGM、ATG16L1、NKX2-3基因的单核苷酸多态性(single nucleotide polymorphisms, SNP)位点与中国人克罗恩病易感性的关系,为深入研究克罗恩病的发病机制奠定基础,亦为该病的防治提供实验依据。
方法:
⑴收集临床确诊的克罗恩病患者(47例)及其健康对照(16名)的组织标本,经病理检验,通过免疫组化以及western blot方法观察IL-32在克罗恩病患者肠黏膜内的表达情况,检验IL-32在克罗恩病患者肠黏膜中的表达与意义。⑵提取克罗恩病患者(32例)和健康对照个体(30名)的外周血,抽提白细胞基因组DNA,检测CARD15_rs2066842、IRGM_rs13361189、ATG16L1_rs2241880以及NKX2-3_ rs10883365的SNP多态性与克罗恩病的关系,根据靶序列片段设计相应的引物,PCR扩增目的片段,经纯化后测序,测序结果分组做统计分析。
结果:
⑴与对照相比,经免疫组化法和western blot法检测的IL-32在克罗恩病患者肠黏膜中表达上调,二者的表达量有显著性差异(P﹤0.05)。⑵克罗恩病病例组和健康对照组基因频率和基因型频率的组间比较显示: CARD15_rs2066842、IRGM_rs13361189、ATG16L1_rs2241880以及NKX2-3_rs10883365的SNP分布频率的差异均无统计学意义(P﹥0.05)。提示这四个基因多态性位点可能与中国人克罗恩病的易感性无关。
结论:
⑴IL-32无论在克罗恩病患者还是在健康对照个体的肠黏膜内均有表达,但在克罗恩病患者肠黏膜内IL-32表达上调,研究显示IL-32表达上调能加重克罗恩病的炎症反应。⑵国外研究报道的与高加索人克罗恩病相关的CARD15_rs2066842,IRGM_ rs13361189、ATG16L1_rs2241880以及与日本人克罗恩病易感性的NKX2-3_rs10883365的SNP位点可能与我国克罗恩病患者的易感性无关,这些SNP的变异至多是增加了中国人罹患克罗恩病的风险。
Objective:
To explore the expression of interleukin-32(IL-32), a novel pro-inflammatory cytokine in the intestinal mucosa of patients with Crohn’s disease (CD). Nowadays, it has been reported that some sequence variants and other multiple replicating loci contribute to Crohn’s disease susceptibility, such as CARD15(NOD2), IRGM, ATG16L1, NKX2-3, etc. We study the correlations of these single nucleotide polymorphisms(SNPs ) associated with Chinese CD, in order that study the pathogenesis of CD in-depth, and provide experimental basis for prevention and treatment of this disease.
Methods:
The biopsy specimens of clinical diagnosis of CD patients (47 cases) and healthy controls (16 cases) were collected, after pathological examinations, the expressions of IL-32 were detected by immunohistochemistry (IHC) and western blot, the results were used for analysis. To evaluate the relations between gene polymorphisms and CD, peripheral blood from CD patients (32 cases) and healthy controls (30 cases)were extracted, genomic DNA was isolated from leukocyte in the peripheral blood, to estimate the relationships of SNPs about CARD15_rs2066842, IRGM_ rs13361189, ATG16L1_rs2241880 and NKX2-3_rs10883365 mutations associated with CD, matched primers were designed according to the target sequences, the fragments of target sequences were amplified by PCR, after purified, the PCR products were sequencing, and the sequencing results were compared, and make statistical analysis.
Results:
Compared to controls, the expression of IL-32 in intestinal mucosa with CD patients were up-regulation, and the protein levels of these two groups were significant difference(P﹤0.05). Gene frequencies and genotype frequencies between CD and healthy control groups comparisons showed that: the frequency distributions of the SNPs about CARD15_rs2066842, IRGM_rs13361189, ATG16L1_rs2241880 and NKX2-3 _rs10883365 were no significant difference. It is suggesting that these SNPs of genes variants with Chinese CD patients susceptibility has no correlations.
Conclusion:
In spite of the pro-inflammatory cytokine IL-32 was expressed in intestinal mocusa of CD patients and controls, the expression levels of this protein in CD was increased, our studies show that IL-32 up-expression could aggravate chronic inflammatory response in CD. The abroad researchers have reported the SNPs about CARD15_rs2066842, IRGM_rs13361189, ATG16L1_rs2241880 and NKX2-3_ rs10883365 associated with the Caucasians or Japanese CD, but we found that the SNPs of these gene sites variants associated with susceptibility of Chinese CD might not be related, and the SNPs variants at most increased the risk of suffering from CD.
引文
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4 Dinarello CA, Kim SH. IL-32, a novel cytokine with a possible role in disease[J]. Ann Rheum Dis,2006,65: 61-64.
5 Novick D, Rubinstein M, Azam T et al. Proteinase 3 is an IL-32 binding protein[J]. Proc Natl Acad Sci USA ,2006;103 (9) :3316-3321.
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8 Shoda H, Fujio K, Yamaguchi Y et al. Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases[J]. Arthritis Res Ther,2006,8(6):1-13.
9 Müller A, Voswinkel J, Gottschlich S et al. Human Proteinase 3 (PR3) and Its Binding Molecules Implications for Inflammatory and PR3-Related Autoimmune Responses[J]. Ann. N.Y. Acad Sci,2007,1109: 84-92.
10 Netea M G, Azam T, Ferwerda G et al. IL-32 synergizes with nucleotide oligomerization domain(NOD)1 and NOD2 ligands for IL-1βand IL-6 production through a caspase 1-dependent mechanism[J]. Proc Natl Acad Sci,2005,102(45): 16309-16314.
11 Kundu M, Joyoti Basu. IL-32: an emerging player in the immune response network against tuberculosis[J]. PLoS Med,2006,8(3):1210-1211.
12 Goda C, Kenaji T, Kenaji S et al. Involvement of IL-32 in activation-induced cell death in T cells[J]. International Immunology,2006,18(2):233-240.
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15 Mun SH, Kim JW, Nah SS et al. Tumor necrosis factorαinduced interleukin-32 is positively regulated via the Syk/protein kinase Cδ/JNK pathway in rheumatoid synovial fibroblasts[J]. Arthritis &Rheumatism,2009,60(3):678-685.
16 Netea MG, Lewis EC, Azam T et al. Interleukin-32 induces the differentiation of monocytes into macrophage-like cells. Proc Natl Acad Sci,2008;105(9):3515- 3520.
17 Netea MG, Azam T, Lewis EC et al. Mycobacterium tuberculosis induces interleukin-32 production through a Caspase-1/IL-18/Interferon-γ-dependent mechanism[J]. PLoS Med,2006,3(8):1310-1319.
18 Barksby HE, Nile CJ, Jaedicke KM et al. Differential expression of immuno- regulatory genes in monocytes in response to P. gingivalis and E. coli LPS[J],2009 156(3):479-487.
19 Rasool S.T, Tang H, Wu JM et al. increased level of IL-32 during human immunodeficiency virus infection suppresses HIV replication[J]. Immunolology Letters,2008,117:161-167.
20 Li W, Liu Y, Mukhtar MM et al. Activation of interleukin-32 pro-inflammatory pathway in response to influenza A virus infection[J]. PLoS One,2008,3(4):1-9.
2 Sandler RS, Eisen GM. Epidemiology of bowel inflammatory disease. In: Kirsner JB eds. bowel inflammatory disease. 5th ed. Philadelphia: WB Saunders Company, 2000,89-112.
3 Loftus EV, Sandborn WJ, Epidemiology of bowel inflammatory disease. Gastroe- nterol Clin Nam, 2002,31(1):1-20.
4 Mowat AH. Anatomical basis of tolerance and immunity to intestinal antigens. Nat Rev Immunol, 2003,3:331-341.
5 Van Kruiningen HJ, Ganley LM, Freda BJ. The role of Peyer’s patches in the age- related incidence of Crohn’s disease[J]. J Clin Gastroenterol,1997,25:470-475.
6 Barreau F, Meinzer U, Chareyre F, et al. CARD15/NOD2 is required for Peyer’s patches homeostasis in mice[J]. Plosone, 2007, 2:e523.
7 Ahmad T, Satsangi J, McGovern D, et al. The genetics of bowel inflammatory disease[J]. Aliment Pharmacol Ther,2001,15:731-748.
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