肝海绵状血管瘤捆扎术

目的 观察,总结和分析捆扎疗法治疗肝海绵状血管瘤的临床疗效。方法 选择总结了１４２例肝海绵状血管瘤病例,术中对瘤体地完整缝扎,术后９７例随访１－１９年,分组评定疗效。结果 显著疗效病例占６５．９％,有效病例占１２．５％,无效或暂无变化病例占２１．６％,结论 捆扎术治疗肝海绵状血管瘤,确为一种简便易行,损伤小,安全和病人恢复快,疗效好的方法。     

Carbohydrate-dependent binding of human myeloid leukemia cell lines to neoglycoenzymes,matrix-immobilized neoglycoproteins,and bone marrow stromal cell layers

Summary The presence of sugar receptors on human myeloid leukemia cells was comparatively assessed by a highly sensitive binding assay, employing a panel of 14 types of neoglycoenzymes (chemically glycosylatedEscherichia coli -galactosidase). The selected carbohydrate ligands mainly encompass common components of natural glycoconjugates as mono- or disaccharides. The monocytoid cells of the THP-1 line, the very young myeloblasts and the myeloblasts of the lines KG-1a and KG-1, the promyelocytes of the HL-60 line, and the early myeloblasts/erythroblasts of the K-562 line displayed a nonuniform pattern of specific binding with quantitative differences at a fixed, nonsaturating concentration of the probes. Scatchard analysis in four cases corroborated the indication of cell-type-related differences between the various cell lines. To test whether the detectable cellular sugar-binding sites can mediate adhesion to glycoligands, a rather simple model matrix of nitrocellulose-immobilized neoglycoproteins was first used. In comparison to the carbohydrate-free carrier protein significant cell adhesion was observed primarily with neoglycoproteins that exposed galactose,N-acetylgalactosamine,N-acetylglucosamine, mannose, and fucose moieties among the 11 tested types of carbohydrate residue. Subsequently, human bone marrow stromal cell layers were tested as a model matrix with increased levels of physiological relevance and complexity. Mixtures of carbohydrate and neoglycoprotein were employed as inhibitors of an interaction via lectins between the stromal and the tumor cells. The carbohydrate-dependent alterations of this parameter revealed cell-type-associated properties. Tumor cell binding was significantly decreased for not more than two lines with the effective sugars, namelyN-acetylgalactosamine, mannose, fucose, and sialic acid.     

内皮细胞中剪切型XBP1的过表达对动脉粥样硬化形成的影响

目的探讨血管壁内皮细胞中剪切型XBP1的过表达对动脉粥样硬化形成的影响。方法从产后的健康新生儿脐带获取脐静脉内皮细胞,脐静脉内皮细胞传代至第3代后,以不同浓度(10mg/L,20mg/L)的氧化胆固醇(7-酮基胆固醇,7-KC)温育脐静脉内皮细胞,应用Western Blot的方法,检测脐静脉内皮细胞中剪切型XBP1的蛋白表达水平,凋亡蛋白Caspase3的活性,应用流式细胞学的方法检测脐静脉内皮细胞的凋亡率和继发性细胞坏死阳性率。结果 随着7-KC温育浓度的增加,脐静脉内皮细胞中剪切型XBP1的蛋白表达水平增加,Caspase3的活性增加,脐静脉内皮细胞的凋亡率和继发性细胞坏死阳性率增加。结论血管壁内皮细胞S-XBP1的表达可能会激活凋亡途径,使内皮细胞发生凋亡,启动AS的形成。     

High‐throughput genotyping of mannose‐binding lectin variants using high‐resolution DNA‐melting analysis

High Resolution Melting Analysis (HRMA) is a rapid and sensitive method for single nucleotide polymorphism (SNP) analysis. In the present study we present a novel HRMA assay to detect three SNPs in close proximity of each other in the first exon of the gene encoding mannose‐binding lectin (MBL), a key molecule of innate immunity. These SNPs have been selected for their known biological and clinical relevance. The three SNPs in MBL2 were simultaneously determined in sixty‐nine human DNA samples using HRMA and a single non‐fluorescent melting probe, without any post‐PCR processing of samples. Combining analyses from amplicon melting and probe melting, we have been able to discriminate ten exon 1 MBL2 genotypes with HRMA, making it a suitable tool for MBL genotyping. A second HRMA assay is presented to detect a relevant polymorphism (Y/X SNP) in the MBL2 promoter region. In conclusion, HRMA is a closed tube assay that is easy to setup and lends itself perfectly for high throughput genotyping of MBL2 variants. The present study thereby facilitates further clinical studies into the role of MBL in inflammatory and infectious disease. © 2010 Wiley‐Liss, Inc.     

CT抗原KM-HN-1 HLA-A * 0201限制性表位预测及其与HLA-A * 0201结合强度分析

目的通过对CT抗原（cancer-testis antigen）KM-HN-1进行HLA-A＊0201限制性表位预测,并对候选表位肽与HLA-A＊0201分子结合亲和力及复合物稳定性进行分析,为探索基于KM-HN-1的免疫治疗奠定基础。方法利用基于蛋白酶体剪切位点特异性的算法PAProc及基于肽MHC-I结合的算法BIMAS和SYFPEITHI对KM-HN-1进行HLA-A＊0201限制性表位预测．合成KM-HN-1相关候选表位肽KM-HN-I321-329（KLLPFRETV）,KM-HN-I303-211,（FLPTAPPNV）,KM-HN-I629-637。（TLLQIIETV）,KM-HN-I87-95（ILNKSIIEV）,KM-HN-I538-596。（QMMEALDQL）及阳性对照肽HBVcAg18-27（FLPSDFFPSV）;对这些合成肽与HIA-A＊0201分子结合亲和力及其复合物稳定性根据文献报道的方法进行分析。结果KM-HN-I321-329（KLLPERETV）结合亲和力最低,KM-HN—I203-211（FLPTAPPNV）结合亲和力最高,其余3条肽结合亲和力介于2者之间;稳定性实验（DC50）结果显示：KM-HN-I538—546（QMMEALDQL）DC50小于2h,KM—HN-I321-329（KLLPERETV）的DC50介于2～4h之间,KM-HN-I87-95。（ILNKSIIEV）的DC50介于6～8h之间,KM-HN-I233-211（HLPTAPPNV）及KM-HN-I629—633（TLLQIIETV）的DC50均大于8h。结论基于蛋白酶体剪切位点特异性的算法及基于肽MHC-I结合的算法对KM-HN-1进行HLA-A＊0201限制性表位预测,结合候选表位肽与HLA-A＊0201分子结合的亲和力与复合物稳定性实验分析,为该抗原HLA-A＊0201限制性表位的鉴定奠定了基础。     

脑膜炎大肠埃希菌侵袭素IbeA结合多肽的初步研究

目的脑膜炎大肠埃希菌（E．coli）侵袭素IbeA通过与脑微血管内皮细胞表面受体结合,从而介导细菌穿透血脑屏障引起新生儿细菌性脑膜炎。为通过肽库筛选获得与IbeA蛋白结合的多肽,建立噬菌体随机肽库筛选IbeA结合多肽模体的方法。方法首先制备靶分子,用PCR扩增出全长ibeA基因序列,克隆至表达载体pET28a中,转化大肠埃希菌BL21（DE3）．经IPTG诱导表达出带有His-tag的目的蛋白,经变性复性后通过Ni^2＋-NTA亲和层析柱得到纯度达90％的重组蛋白。然后以固相化IbeA重组蛋白为靶亲和筛选噬菌体线性12肽库。结果经过3轮淘选,得到能和IbeA结合的24个重组噬菌体克隆;挑选15个亲和力高的克隆进行DNA测序,对比分析所得的短肽序列。固相Fmoc法合成短肽,并进行结合实验、阻断实验和竞争抑制实验。绪论以重组表达的IbeA融合蛋白为靶筛选得到的重组噬菌体十二肽克隆,能够和IbeA蛋白结合,为IbeA蛋白结合多肽。结果提示所筛选的阳性噬菌体克隆及其合成肽可能模拟IbeA受体表位的结构。     

Role of different receptors and actin filaments on Salmonella Typhimurium invasion in chicken macrophages

Bacterial attachment to host cell is the first event for pathogen entry. The attachment is mediated through membrane expressed adhesins present on the organism and receptors on the cell surface of host. The objective of this study was to investigate the significance of Fc receptors (FcRs), actin filament polymerization, mannose receptors (MRs), carbohydrate moieties like N-linked glycans and sialic acid on chicken macrophages for invasion of S. Typhimurium. Opsonisation of S. Typhimurium resulted in three folds more invasion in chicken monocyte derived macrophages. Cytochalasin D, an inhibitor of actin filament polymerization prevented uptake of S. Typhimurium. Pre-incubation of macrophages with cytochalasin D, showed severe decrease (28 folds) in S. Typhimurium invasion. Next we attempted to analyse the role of carbohydrate receptors of macrophages in S. Typhimurium invasion. Treatment of macrophages with methyl α-d-mannopyranoside, PNGase F and neuraminidase, showed 2.5, 5 and 2.5 folds decrease in invasion respectively. Our data suggest that deglycosylation of N-linked glycans including sialic acid by PNGase F is more effective in inhibition of S. Typhimurium invasion than neuraminidase which removes only sialic acid. These findings suggested FcRs, actin filament polymerization, MRs, N-linked glycans and sialic acid may act as gateway for entry of S. Typhimurium.     

Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP)

The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (E_Man) was examined. While the C4BP concentration for inhibiting 50% (IC₅₀) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05 nM), 3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81 nM) and E_Man (42.66 nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000–431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven α-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79 ± 0.60) when the C4b density on E_Man was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a 7- to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions.