介导可溶性MHCⅠ产生的去整合素金属蛋白酶初步筛选研究

目的:以HEK293细胞为研究模型,筛选介导可溶性MHCⅠ产生的去整合素金属蛋白酶。方法:将ADAM8、ADAM10、ADAM15和ADAM17cDNA分别克隆入pcDNA3.1/V5载体,并用TransIT-LT1转染试剂将构建好的载体转染入HEK293细胞,用潮霉素B筛选ADAMs蛋白稳定表达克隆;用SDS-PAGE、免疫印迹结合化学发光法检测ADAMs在HEK293细胞中的表达;用细胞表面生物素标记、免疫沉淀、SDS-PAGE、免疫印迹结合化学发光检测ADAMs过表达对可溶性MHCⅠ产生的影响。结果:成功获得稳定表达ADAMs的HEK293细胞;过表达ADAM10和ADAM17可增强HEK293细胞产生可溶性MHCⅠ。结论:ADAM10和AD-AM17具介导可溶性MHCⅠ产生的潜能。     

三氧化二砷减轻支气管哮喘小鼠气道重塑及基质金属蛋白酶-9表达

气道重塑是支气管哮喘的典型特征之一,主要因气道壁结构的改变,形成不可逆的气流阻塞和持续性气道高反应,导致肺功能下降,故预防和逆转哮喘的气道重塑具有重要意义.     

1,25-二羟维生素D3对哮喘小鼠气道重塑及基质金属蛋白酶-9表达的影响

目的:观察1,25-二羟维生素D3(VD)对哮喘小鼠气道重塑及其肺组织中基质金属蛋白酶-9(MMP-9)表达的影响,探讨VD在哮喘治疗中的作用.方法:BALB/c小鼠随机分为对照组、哮喘组及VD组.卵蛋白致敏和激发建立慢性哮喘小鼠模型;HE染色观察各组气道结构改变情况;采用计算机图像分析系统评价各组气道重塑情况;采用RT-PCR法检测各组的MMP-9mRNA表达水平.结果:①HE染色提示哮喘组与对照组相比出现炎性细胞浸润增多、上皮细胞脱落及平滑肌细胞层增厚等气道重塑改变,而VD组可部分逆转上述病理改变;②VD组的支气管内壁厚度、平滑肌层厚度和平滑肌细胞核数显著低于哮喘组,但仍高于对照组(P<0.05);③VD组肺组织MMP-9mRNA表达水平均明显低于哮喘组,但仍高于对照组(P<0.05).结论:VD干预可明显减轻慢性哮喘气道重塑的病理改变;并可通过部分抑制肺内MMP-9的表达来延缓气道重塑进程.     

麻杏石甘汤对哮喘模型小鼠气道重塑及肺组织MMP-9和TIMP-1表达的影响

目的:观察麻杏石甘汤对哮喘模型小鼠气道重塑及肺组织基质金属蛋白酶9(MMP-9)和金属蛋白酶组织抑制物1(TIMP-1)表达的影响,探讨其治疗哮喘的可能机制。方法:将72只健康雌性BALB/c小鼠随机分为:空白对照组,模型组,麻杏石甘汤低剂量组、中剂量组和高剂量组,阳性对照组。采用卵清蛋白致敏激发建立小鼠哮喘模型。空白对照组和模型组于激发前30 min以生理盐水灌胃;麻杏石甘汤低剂量组、中剂量组和高剂量组分别于激发前30 min以麻杏石甘汤按5.0 g/kg、10.0 g/kg和20.0 g/kg剂量灌胃;阳性对照组于激发前30 min以地塞米松按0.005 g/kg剂量灌胃。连续给药7 d后,观察气道反应性、支气管肺泡冲洗液(BALF)中嗜酸性粒细胞(EOS)计数、杯状细胞百分比和胶原沉积的变化;ELISA法检测MMP-9和TIMP-1水平;Western blot法检测MMP-9和TIMP-1的蛋白表达;RT-qPCR法分析检测MMP-9和TIMP-1的mRNA表达。结果:与空白对照组比较,模型组的气道反应性、杯状细胞百分比、胶原沉积、BALF中EOS计数及肺组织MMP-9和TIMP-1的mRNA和蛋白水平均显著升高(P 0.01);与模型组比较,麻杏石甘汤低剂量组、中剂量组和高剂量组及阳性对照组上述指标则明显降低(P 0.05或P 0.01)。结论:麻杏石甘汤可能通过降低MMP-9和TIMP-1的表达改善哮喘模型小鼠气道重塑状态。     

地塞米松下调哮喘大鼠肺组织bFGF的表达

支气管哮喘(简称哮喘)是由多种细胞和细胞组分参与的慢性炎症性疾病,反复炎症刺激导致气道高反应和气道重塑,目前认为气道重塑是难治性哮喘及哮喘气道不可逆损伤的主要原因。研究表明,碱性成纤维细胞生长因子(basic fibrob last growth factor,bFGF)参与了哮喘气道重塑过程,但目前国内外对哮喘时bFGF表达变化的研究多集中在慢性哮喘气道重塑方面,而对于哮喘急性期bFGF的表达变化却鲜见报道。本实验通过建立哮喘大鼠模型,研究bFGF蛋白和mRNA在哮喘急性期的表达并观察地塞米松治疗对其影响,探讨早期地塞米松干预抑制气道重塑的可能机制…     

解整合素-金属蛋白酶33基因T1位点多态性与毛细支气管炎的相关性研究

目的 探讨在广州地区汉族儿童中解整合素-金属蛋白酶33(ADAM33)基因中的T1位点不同基因型及等位基因的分布频率与毛细支气管炎的相关性.方法 采用聚合酶链反应结合限制性片段长度多态性(PCR-RFLP)方法,检测60例毛细支气管炎儿童(试验组)及60例对照儿童(对照组)T1位点单核甘酸多态性.结果 在广州地区儿童中可检测到Tl位点的3种基因型(TT CT和CC),试验组3种基因型的分布频率分别为78.3％、16.7％和5％,对照组分别为88.3％、10％和1.7％,两组分布频率差异无统计学意义(-2.36,P＞0.05);试验组的T等位基因和C等位基因频率分别为86.7％和13.3％,对照组分别为93.3％和6.7％,两组分布差异也无统计学意义(x2=2.27,P＞0.05).结论 在广州地区汉族儿童中,ADAM33基因Tl位点基因多态性与毛细支气管炎发病无关.     

红花多糖对荷瘤小鼠肿瘤组织基质金属蛋白酶-9、组织金属蛋白酶抑制剂-1mRNA表达影响的研究

目的 探讨红花多糖(safflower polysaccharide,SPS)对荷瘤小鼠肿瘤组织中基质金属蛋白酶(matrix metalloproteinases,MMP)-9、组织金属蛋白酶抑制剂1(tissue inhibitors of metalloproteinase,TIMP-1)mRNA表达的影响.方法 BABL/c小鼠腋下接种S180肉瘤,腹腔注射SPS连续10 d,给药结束24 h后取肿瘤组织Real time-PCR法测MMP-9、TIMP-1 mRNA表达水平.结果 SPS低剂量组、中剂量组和高剂量组肿瘤组织中MMP-9的表达量分别是模型对照组的0.452、0.204、0.026倍,TIMP-1的表达量分别是模型对照组的3.4、5.2、10.0倍.结论 SPS能够抑制肿瘤组织中MMP-9基因的表达,促进TIMP-1基因的表达,具有抑制肿瘤及其转移的作用.     

解整合素-金属蛋白酶12在子痫前期患者胎盘组织中的表达及意义

目的：探讨子痫前期患者胎盘组织中解整合素-金属蛋白酶12(a disintegrin and metalloproteinase 12,ADAM12)蛋白和mRNA表达水平变化及其意义。方法 采用免疫组织化学方法检测31例子痫前期患者（子痫前期组包括子痫前期重度16例、子痫前期轻度15例）和20例正常足月孕妇（对照组）胎盘组织中ADAM12的蛋白表达水平。采用杂交组化方法检测各组孕妇胎盘组织中ADAM12-S和ADAM12-L的mRNA表达水平。结果：子痫前期组孕妇胎盘组织中ADAM12蛋白表达水平为0.56±0.14,对照组的为0.39±0.14,两者相比,差异有统计学意义（P<0.05）;子痫前期重度组与轻度组比较,差异没有统计学意义（P>0.05）。子痫前期组孕妇胎盘组织中ADAM12-S mRNA水平为0.52±0.09,对照组为0.40±0.15,两者相比,差异有统计学意义（P<0.01）;子痫前期重度组与轻度组比较,差异有统计学意义（P<0.05）;子痫前期组孕妇胎盘组织中ADAM12-L mRNA水平为0.51±0.09,对照组为0.42±0.18,两者相比,差异有统计学意义（P<0.05）;子痫前期重度组与轻度组比较,差异没有统计学意义（P>0.05）。结论：ADAM12蛋白和mRNA水平在胎盘组织中的表达增高可能在子痫前期的发病过程中起重要作用。     

组织蛋白酶D在小鼠肺腺癌发生发展中的表达特点及其意义

目的探索组织蛋白酶D(CTSD)在肺腺癌发生发展中的表达特点及其意义。方法使用氨基甲酸乙酯腹腔注射建立BALB/c小鼠肺腺癌模型;蛋白质组放射性核素相对标记和绝对定量(i TRAQ)分析筛选差异表达蛋白;免疫组织化学和Western blotting检测蛋白在肺腺癌不同病理时期的表达变化特点。结果 CTSD蛋白在肺腺癌早期表达较对照组明显增强;免疫组织化学和Western blotting检验分析发现,随着肺腺癌病程进展,CTD表达呈上升趋势,在肺腺癌晚期,实验组为对照组13.7倍;出现肝转移实验组小鼠肺组织CTSD呈现不同异构体。结论CTSD是肺组织癌变过程中的重要蛋白,其异常表达和修饰可能在肺腺癌发生、发展和转移中起重要作用,可能成为肺腺癌早期诊断的分子指标。     

CpG ODN和地塞米松对哮喘气道的重塑及MMP-9、TIMP-1表达的影响

目的： 探讨非甲基化胞嘧啶-鸟嘌呤的寡核苷酸(CpG ODN)和糖皮质激素对哮喘气道重塑和肺内MMP-9及TIMP-1表达的影响。方法： 以卵蛋白致敏激发的慢性哮喘小鼠模型为对象，测定正常对照组、哮喘组、地塞米松组及CpG ODN组小鼠支气管上皮黏膜层面积(WAmuc)、平滑肌面积(WAmus)、气管内壁面积(WAi),并用支气管基底膜周径(Pbm)标准化。天狼猩红染色测定气道Ⅰ、Ⅲ型胶原的面积。免疫组化方法测定气道基质金属蛋白酶-9（MMP-9）及其抑制剂TIMP-1，行免疫组化图像分析。结果： (1) CpG ODN组和地塞米松组的WAmuc/Pbm、WAmus/Pbm、WAi/Pbm显著大于对照组而显著小于哮喘组（P<0.05），而CpG ODN组和地塞米松组间则无显著差异（P>0.05）;(2) CpG ODN组和地塞米松组Ⅰ、Ⅲ型胶原面积较大于对照组而明显低于哮喘组(P<0.05)；(3) CpG ODN组和地塞米松组MMP-9、 TIMP-1表达明显高于对照组而显著小于哮喘组(P<0.05)，而CpG ODN组和地塞米松组间则无显著差异（P>0.05）。结论： 慢性哮喘小鼠气道壁明显增厚，而早期行CpG ODN和地塞米松干预则可通过抑制肺内MMP-9、TIMP-1的表达而减轻气道重塑。     

Association of ADAM33 gene polymorphisms with asthma in Indian children

Asthma is the most common chronic disorder in childhood, and asthma exacerbation is an important cause of childhood morbidity and hospitalization. In the present study, the relationship between single-nucleotide polymorphisms (SNPs) of the ADAM33 gene and asthma in Indian children has been examined using a case-control study. Five SNPs of the ADAM33 gene, F+1(rs511898) G/A, S2 (rs528557) G/C, ST+4 (rs44707) A/C, ST+5 (rs597980) C/T and V4 (rs2787094) C/G, were analyzed in 211 asthma cases and 137 controls aged 1-15 years using the PCR-restriction fragment length polymorphism method. Data were statistically analyzed using the χ(2)-test and logistic regression model. Haplotype estimation and linkage disequilibrium were conducted using the expectation-maximization algorithm. The genotypes and allele frequencies of SNPs S2 and ST+5 of the ADAM33 gene were significantly associated with asthma risk (P = 0.020 - < 0.001), whereas F+1, ST+4, V4 homozygous mutant genotypes and mutant alleles were significantly associated with increased asthma risk (P = 0.031 - < 0.001). A positive association was also found with haplotypes AGCCT, GGACT and AGCCC (P = < 0.001, odds ratio (OR) = 6.10-6.50), whereas ACAGT, AGCGC, AGCGT, GCAGC and GCCGT showed protective association with asthma (P = 0.019-0.000, OR = 0.50-0.20). Taken together, out results suggest that ADAM33 gene polymorphisms may modify individual susceptibility to develop childhood asthma in the Indian population.     

ADAM33: a newly identified gene in the pathogenesis of asthma

There is much to find out about this fascinating and complex molecule in relation to the development and progression of asthma. Added to it are three further new asthma/allergy genes identified by positional cloning: PDH Finger Protein II (PHF11) on chromosome 13q14, which encodes NY-REN-34 a protein first described in patients with renal cell carcinoma [67]; Dipeptidyl diptidase 10 (DDP10) on chromosome 2q14 [68]; and G protein-coupled receptor for asthma susceptibility (GPRA) on chromosome 7p [69]. For each of these genes, as is the case for ADAM33, determining their normal function(s) and how these become disordered in asthma is the future challenge.     

Polymorphisms of the ADAM33 gene are associated with accelerated lung function decline in asthma

BACKGROUND: Asthma is a genetically complex disease characterized by respiratory symptoms, intermittent airway obstruction and airway hyper-responsiveness due to airway inflammation and remodelling. The ADAM33 gene is associated with asthma and airway hyper-responsiveness and is postulated as a gene for airway remodelling. OBJECTIVE: To investigate whether polymorphisms of the ADAM33 gene are associated with accelerated lung function decline in patients with asthma. METHODS: In a cohort of 200 asthma patients followed over 20 years, eight single nucleotide polymorphisms of the ADAM33 gene were analysed to estimate their effect on annual FEV(1) decline. RESULTS: The rare allele of the S_2 polymorphism was significantly associated with excess decline in FEV(1) (P<0.05). CONCLUSION: These findings suggest that a variant in ADAM33 is not only important in the development of asthma but also in disease progression, possibly related to enhanced airway remodelling.     

Association of a disintegrin and metalloprotease 33 (ADAM33) gene with asthma in ethnically diverse populations

BACKGROUND: Asthma is a complex genetic disease characterized by reversible intermittent airway obstruction and respiratory symptoms primarily caused by acute and chronic bronchial inflammation. Recently, a gene potentially involved in airway remodeling, a disintegrin and metalloprotease 33 (ADAM33), was implicated in asthma susceptibility. OBJECTIVE: We sought to determine whether polymorphisms in ADAM33 are associated with asthma or closely related phenotypes in 4 different asthma populations. METHODS: Eight single nucleotide polymorphisms (SNPs) were evaluated in the 3' portion of ADAM33 in 4 unique asthma populations (African American, US white, US Hispanic, and Dutch white). These SNPs were previously reported to be associated with asthma in white populations from the United States and United Kingdom. RESULTS: Significant associations were observed with at least one SNP and asthma in each population (P =.0009-.04). Related phenotypes that included total serum IgE levels and skin test responsiveness were also associated (P =.003-.05). However, no single SNP was associated across all populations. Additionally, haplotype analysis revealed that no single haplotype accounted for asthma susceptibility risk, although potential risk haplotypes existed within some of the populations. CONCLUSION: Replication of the original ADAM33 findings in these 4 additional asthma populations suggests that this gene (and perhaps others that interact with it) is important in the development and pathogenesis of asthma.     

Lack of association between ADAM33 gene and asthma in a Chinese population

Asthma is a complex polygenic disease with gene-environment interactions being important. It has been previously suggested that ADAM33, which is a member of a gene family that encodes membrane-anchored proteins with a disintegrin and a metalloprotease domain, is primarily expressed in lung fibroblasts and bronchial smooth muscle cells and has been associated with airway remodelling and bronchial hyperresponsiveness. A significant association has previously been demonstrated between single nucleotide polymorphisms (SNPs) and haplotypes of the ADAM33 and asthma in ethnically diverse populations. To assess whether SNPs or haplotypes of ADAM33 are related to asthma in a Chinese Han population, we genotyped three SNPs of ADAM33 (7575G/A in intron 6, 11188A/T in intron 19, and 12433T/C in exon 20) in a case-control study involving 296 patients with asthma and 270 healthy controls. No significant association was detected between these three SNPs and asthma susceptibility in the Chinese population.     

The role of ADAM33 in the pathogenesis of asthma

While asthma is a disorder of the conducting airways characterised by Th2-directed inflammation, a second set of mechanisms is being increasingly recognised as fundamental to disease chronicity and severity, for which the term remodelling has been used. The cellular and mediator responses underpinning airway remodelling involve aberrant communication between the airway epithelium and underlying mesenchyme, involving the generation of growth factors that lead to proliferation of fibroblasts and smooth muscle and the deposition of matrix proteins to cause airway wall thickening linked to bronchial hyperresponsiveness and fixed airflow obstruction. The identification of ADAM33 on chromosome 20p13 from positional cloning as a novel candidate gene involved in the pathogenesis of these structural and functional changes has opened the way to further insight into these processes that contribute to corticosteroid refractoriness. The preferential expression of ADAM33 in mesenchymal cells and its multiple molecular actions provide ample opportunity for incriminating this molecule in chronic asthma. Its association with progressive asthma and in predicting reduced lung function in young children suggest that ADAM33 has an important role in the natural history and possibly the origins of asthma, a disease unique to humans.     

Mechanistic role of a disease-associated genetic variant within the ADAM33 asthma susceptibility gene

Background  

ADAM33 has been identified as an asthma-associated gene in an out-bred population. Genetic studies suggested that the functional role of this metalloprotease was in airway remodeling. However, the mechanistic roles of the disease-associated SNPs have yet to be elucidated especially in the context of the pathophysiology of asthma. One disease-associated SNP, BC+1, which resides in intron BC toward the 5' end of ADAM33, is highly associated with the disease.     

ADAM33 polymorphisms and phenotype associations in childhood asthma

BACKGROUND: A disintegrin and metalloproteinase (ADAM) 33 has been implicated as an asthma susceptibility gene by using a positional cloning approach. However, genetic linkage of asthma phenotypes to chromosome 20p13 (the location of ADAM33) has not been observed in most asthma genome scans, and it is unclear whether these associations with ADAM33 are broadly generalizable. OBJECTIVE: To examine whether ADAM33 is associated with asthma in a North American population of childhood asthmatic patients. METHODS: We performed a family-based association study by using 652 nuclear families ascertained through asthmatic subjects enrolled in a large randomized clinical trial. Seventeen ADAM33 single nucleotide polymorphisms (SNPs; including 9 associated with asthma in the initial report) were genotyped by mass spectrometry. Single-SNP and haplotype association analysis was performed. RESULTS: Among white and African American subjects, no single-SNP association with asthma was observed. However, a common 16-SNP haplotype (frequency, 14.6% in white subjects) was associated with asthma (P=.006). Two SNPs in strong linkage disequilibrium (T1 and T+1) were marginally associated with asthma in the Hispanic cohort (P=.04). These data provide marginal support for an asthma locus in the ADAM33 genomic region. However, the magnitudes of the observed associations are modest at best and are inconsistent with the original report. CONCLUSIONS: We conclude that either ADAM33 has only modest effects on asthma susceptibility, and the initial reports of association were a result of analysis in a selected population, or the initial findings were a result of chance. It is also possible that the true asthma susceptibility locus in this genomic region is near, but not at, ADAM33.