人α-防御素-1(α-HNP-1)基因真核表达载体的构建及在大鼠骨髓间充质干细胞中的转染表达

目的构建人α-防御素-1(α-HNP-1)基因的真核表达载体,并且转染大鼠骨髓间充质干细胞。方法提取人外周血粒细胞中的总RNA,反转录为cDNA作为模板,采用PCR的方法扩增得到人α-防御素-1(α-HNP-1)的基因片断。将扩增产物连接入pGEM-T载体,转化大肠杆菌DH5α感受态细胞,蓝白筛选,对PCR及酶切鉴定含有目的片断的克隆进行测序。经测序证实无误后,将获得的pGEM-T-HNP-1重组质粒上的α-HNP-1基因亚克隆到真核表达载体pcDNA3.1(-)上,构建HNP-1的真核表达载体pcDNA3.1-HNP-1。将真核表达载体pcDNA3.1-HNP-1用脂质体法转染原代大鼠骨髓间充质干细胞,用免疫组化法检测α-HNP-1的表达。结果获得预期大小为303bp的RT-PCR产物;经PCR、酶切鉴定和DNA测序分析证实重组质粒载体pcDNA3.1-HNP-1构建正确;免疫组化法显示转染细胞呈阳性反应。结论成功构建HNP-1基因的真核表达载体,并且在大鼠骨髓间充质干细胞中能成功表达。     

人α-防御素-1(α-HNP-1)基因乳腺定位表达载体的构建及表达

目的:将人α-防御素-1(α-HNP-1)基因与山羊β-乳球蛋白(Beta-Lac-toglobulin,BLG)基因5’调控区序列融合,构建α-HNP-1基因的乳腺定位表达载体并对其进行检测。方法:用PCR方法获得α-HNP-1基因片断并克隆于pMD18-T载体,经DNA测序证实α-HNP-1基因序列无误后,再将其亚克隆到真核表达载体pcDNA3.1(+),构建pcDNA3.1-HNP-1重组质粒。用PCR方法扩增出BLG基因5’区调控序列并克隆到pcD-NA3.1-HNP-1中,构建乳腺定位表达载体pcDNA3.1-BLG-HNP-1,该载体经脂质体包裹后,经乳腺导管注入妊娠中后期大鼠乳腺中进行表达。结果:经酶切鉴定和DNA测序分析证实重组质粒载体pcDNA3.1-BLG-HNP-1构建正确,经ELISA检测,大鼠乳汁中有α-HNP-1表达,其48h表达量为27.67ng/ml,72h表达量为49.00ng/ml。结论:成功构建了α-HNP-1基因乳腺定位表达载体并最终在乳腺获得一定量表达,说明乳腺导管注入法是一种较理想的评价乳腺特异表达载体的可行性方法。     

Notch4在溃疡性结肠炎中的表达与分析

目的研究Notch4在溃疡性结肠炎(ulcerative colitis,UC)中的表达、分布及意义。方法收集50例UC患者病变部结肠组织及32例结肠癌患者术后大体标本两端的正常黏膜组织。用免疫组化方法检测Notch4在50例UC及32例对照结肠黏膜组织中的表达情况。结果Notch4在UC患者结肠组织及对照的正常结肠组织中均有表达,两者之间的差异无统计学意义(P>0.05)。Notch4的表达与UC患者的性别、年龄及UC病变程度无统计学意义(P>0.05)。结论Notch4在UC患者结肠黏膜组织与正常结肠黏膜组织中的表达相似,差异无统计学意义。     

Paneth细胞分泌的α-防御素的免疫印迹鉴定

帕内特 (Paneth)细胞 (肠内的嗜酸性细胞 )可合成、分泌具有广谱高效抗微生物活性的阳离子抗菌肽 ,α 防御素(α defensin)为其中的一类 ,它是构成机体的天然免疫系统第一道防线的组成成分之一[1] 。小肠黏膜中的α 防御素分子量小、含量微 ,对其提纯和鉴定的难度较大。Selsted     

IL-1α及LPS 刺激Caco-2细胞防御素表达的差异性

目的 比较细胞因子及LPS组对Caco-2细胞防御素表达的差别。方法用LPS和rhIL-1α刺激Caco-2细胞，采用半定量RT-PCR技术观察防御素HD-5、HD-6、hBD-1和hBD-2 mRNA的表达差异。结果经IL-1α刺激后，hBD-1的表达量有所增加，同时hBD-2、HD-5和HD-6也能表达；但LPS刺激后，hBD-1的表达量增加不明显，HD-5、HD-6和hBD-2仍未表达。结论Caco-2细胞对LPS的刺激产生“哑”反应，推测肠上皮对LPS存在一定的适应性，这对维持一定的正常菌群有益。     

大鼠不同发育阶段β-防御素-2基因在肺组织的表达研究

目的：探讨大鼠β-防御素-2基因在肺组织表达的发育调控。方法：根据Genbank大鼠β-防御素-2的cDNA序列,设计一对特异引物。采用RT-PCR技术在大鼠不同发育阶段的肺组织总RNA中,扩增其特异cDNA片段,并进行DNA序列测定。借助Genbank中BLASI、程序,将从该cDNA序列推导的氨基酸序列与人的hBD-2和大鼠的rBD-1做相似性分析。     

大鼠β-防御素rBD-2的分子克隆及其在皮肤组织的诱导表达

本应用RT-PCR技术,根据Genbank公布的Noreway大鼠β-防御素-2cDNA序列设计引物,从Wistar大鼠皮肤组织总RNA中扩增出-cDNA片段,经核苷酸序列测定证实为全长β-防御素-2cDNA,,本实验还显示大鼠β-防御素rBD-2基因在大肠杆菌感染性损伤皮肤组织中的表达明显增强,与人hBD-2的表达相似。     

溃疡性结肠炎患者血清25(OH)维生素D水平与疾病活动性的相关性

目的检测溃疡性结肠炎患者血清25(OH)维生素D水平,并分析25(OH)维生素D水平与疾病活动性的关系,探讨其临床意义。方法选择40例溃疡性结肠炎患者和30例健康对照者为研究对象,采用ELISA法检测溃疡性结肠炎患者和健康体检者血清中25(OH)维生素D水平,应用Mayo评分法对UC病人的疾病活动度进行分级,将维生素D水平与UC患者临床资料以及疾病活动度进行相关性分析。结果溃疡性结肠炎患者血清25(OH)维生素D水平(15.8±12.3)ng/ml明显低于健康对照组(42.5±26.7)ng/ml,p0.05,血清25(OH)维生素D水平与患者年龄、性别、病程、是否吸烟、抗生素的应用、5-氨基水杨酸的应用及非甾体类抗炎药的应用无相关性,但与皮质类固醇激素的应用及溃疡性结肠炎的活动性相关。结论溃疡性结肠炎患者低储备维生素D水平与疾病活动性及皮质类固醇激素的应用相关。     

A rare variant in CYP27A1 and its association with atopic dermatitis with high serum total IgE

This study investigated rare variants associated with atopic dermatitis. We performed exome analyses on 37 patients who were diagnosed with atopic dermatitis by board‐certified dermatologists and had total serum IgE levels greater than 1000 IU/ml. The exome analysis identified seven variants with <1% allele frequency in Asian (ASN) population of 1000 Genomes Project phase 1 data and >5% allele frequency in the atopic dermatitis exome samples. We then conducted a replication study using 469 atopic dermatitis patients with total serum IgE ≥1000 IU/ml and 935 Japanese controls to assess the presence of these 7 candidate variants. The replication study confirmed that CYP27A1 rs199691576 (A/G) was associated with atopic dermatitis with high serum IgE levels (P = 0.012, odds ratio = 2.1). CYP27A1 is involved in the metabolism of vitamin D3, which plays important roles in modulating immune function. Previous studies have reported polymorphisms in vitamin D pathway genes that are associated with allergy‐related phenotypes. Our data confirm the importance of genes regulating the vitamin D pathway in the development of atopic dermatitis.     

Confirmation of association between multiple sclerosis and CYP27B1

Multiple sclerosis, MS (OMIM No. 126200), is a complex inflammatory disease that is characterized by lesions in the central nervous system. Both genes and other environmental factors influence disease susceptibility. One of the environmental factors that has been implicated in MS and autoimmune disease, such as type 1 diabetes, is vitamin D deficiency, in which patients have lower levels of 25-hydroxyvitamin D3 (25-OHD₃) in blood than do controls. Previtamin D₃ is produced in the skin, and turned into 25-OHD₃ in the liver. In the kidney, skin and immune cells, 25-OHD₃ is turned into bioactive 1,25(OH)₂D₃ by the enzyme coded by CYP27B1 (cytochrome P450 family 27 subfamily B peptide 1) on chromosome 12q13.1–3. 1,25(OH)₂D₃ binds to the vitamin D receptor, expressed in T cells and antigen-presenting cells. 1,25(OH)₂D₃ has a suppressive role in the adaptive immune system, decreasing T-cell and dendritic cell maturation, proliferation and differentiation, shifting the balance between T-helper 1 (Th1) and Th2 cells in favor of Th2 cells and increasing the suppressive function of regulatory T cells. Rs703842 in the 12q13–14 region was associated with MS in a recent study by the Australian and New Zealand Multiple Sclerosis Genetics Consortium (ANZgene). We show associations with three SNPs in this region in our Swedish materials (2158 cases, 1759 controls) rs4646536, rs10877012 and rs10877015 (P=0.01, 0.01 and 3.5 × 10⁻³, respectively). We imputed rs703842 SNP and performed a joint analysis with the ANZgene results, reaching a significant association for rs703842 (P=5.1 × 10⁻¹¹; odds ratio 0.83; 95% confidence interval 0.79–0.88). Owing to its close association with 25-OHD₃, our results lend further support to the role of vitamin D in MS pathology.     

Early lesions of the colonic mucosa possibly developing into ulcers of ulcerative colitis

Biopsy specimens from the colonic mucosae of patients with ulcerative colitis (UC) were examined using both transmission and scanning electron microscopes (TEM and SEM) in order to elucidate the fine pathological changes in the mucosae preceding obvious ulcers. The interglandular surface epithelium exhibited distension of the intercellular spaces, where both blood cells and plasma had infiltrated. Partial destruction of epithelial cells was also seen. Thick fibrous layers of the basement membranes of the interglandular epithelium were rough and porous, with fenestrations. Partial loss of the fibrous layers was also seen, which suggested mucosal ulcers nearby. In the lamina propria, congested blood vessels with ruptured walls containing blood cells were frequently seen. The perivascular tissues were edematous with large amounts of exudate, containing many blood cells. The subsequent, longtermed intramucosal retention of infiltrated blood may thus lead to mucosal lesions, including cell death and destruction, and, as a result, prolong the severe lesions in the mucosae with inflammation. This study was presented in part at the 20th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Fukuoka, September 1, 1988.     

HLA基因多态性与溃疡性结肠炎遗传易感关系的研究

目的:研究HLA-DRB1等位基因多态性与我国汉族溃疡性结肠炎遗传易感的相关性,并分析其与疾病临床分型的相关性。方法:应用基因芯片技术分析了本地区汉族溃疡性结肠炎60例患者和健康对照者DRB1的基因分型,采用Fisher’s精确概率法比较了两组各位点等位基因频率分布的差异。结果:溃疡性结肠炎患者DR2和DRB1*15基因表达频率分别为45%和41.7%,较对照组(23.3%和21.7%)明显升高,OR分别为2.688和2.582(均P<0.05)。慢性持续型UC的DRB1*15等位基因频率较相应其他型升高更为显著(P<0.05)。结论:DR2或DRB1*15等位基因可能是我国汉族人群UC的易感基因。HLA-DR基因多态性与UC的临床分型密切相关。     

Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not involved in the risk of recurrent pregnancy loss

The etiology of recurrent pregnancy loss (RPL) remains unclear, but it may be related to a possible genetic predisposition together with involvement of environmental factors. We examined the relation between RPL and polymorphisms in four genes, human aryl hydrocarbon (Ah) receptor, cytochrome P450 (CYP) 1A1, CYP1A2 and CYP1B1, which are involved in the metabolism of a wide range of environmental toxins and carcinogens. All cases and controls were women resident in Sapporo, Japan and the surrounding area. The Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotypes were assessed in 113 Japanese women with recurrent pregnancy loss (RPL) and 203 ethnically matched women experiencing at least one live birth and no spontaneous abortion (control). No significant differences in Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotype frequencies were found between the women with RPL and the controls [Ah receptor: Arg/Arg (reference); Arg/Lys and Lys/Lys, odds ratio (OR)=0.67; 95% confidence interval (CI)=0.40-1.11, CYP1A1: m1m1 (reference); m1m2 and m2m2, OR = 0.86; 95% CI = 0.53-1.40, CYP1A2: C/C and C/A (reference); A/A, OR = 1.16; 95% CI = 0.71-1.88, CYP1B1: Leu/Leu (reference); Leu/Val and Val/Val, OR = 1.18; 95% CI = 0.68-2.02]. The present study suggests that the Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not major genetic regulators in RPL.     

溃疡性结肠炎患者PBMCs体外sIL-2R诱生水平的研究

目的探讨sIL-2R在溃疡性结肠炎（UC）中的表达水平。方法采用特异性ELISAsIL-2R检测试剂盒，对25例溃结患者PBMCs体外PHA诱导产生的sIL-2R水平进行测定。结果溃结患者PHA诱导48h的细胞培养上清液中sIL-2R的含量为319.63±164.70U/ml，明显低于正常对照组的451.15±246.93U/ml(P<0.05);而未经PHA诱导的sIL-2R含量，两组无明显差异（UC组263.78±115.05U/ml，对照组236.47±139.10U/ml，P＞0.05）。结论溃结患者体外PHA诱导的sIL-2R水平下降，提示其淋巴细胞的免疫活化功能障碍。     

The relationship between the expression of the glucocorticoid receptor in biopsied colonic mucosa and the glucocorticoid responsiveness of ulcerative colitis patients

The objective of this study was to clarify the relationship between the frequency of infiltrating cells expressing the glucocorticoid receptors (GR) α and β in biopsied colonic mucosa and the glucocorticoid (GC) responsiveness of ulcerative colitis (UC) patients. Active UC patients (n = 38) were divided into GC-sensitive and GC-resistant groups. GRβ⁺ cells were significantly higher in the GC-resistant group than in the GC-sensitive and control groups. GRα mRNA was expressed in all UC patients, while GRβ mRNA was expressed in only 1 patient in the GC-sensitive group (n = 6) and 7 patients in the GC-resistant group (n = 8). Double-positive cells for GRβ and CD4 or CD19 were frequently observed. The Foxp3⁺ cell count was significantly higher in the GC-sensitive group than in the GC-resistant group, but double Foxp3⁺GRβ⁺ cells were not observed. These results indicated that the sensitivity of GC therapy could probably be predicted by immunostaining biopsy specimens for GRβ and Foxp3.