变态反应性疾病儿童血浆肥大细胞羧肽酶和类糜蛋白酶含量的检测

目的：测定变态反应性疾病患儿血浆中肥大细胞羧肽酶和类糜蛋白酶的含量,评价其在变态反应性疾病诊断中的意义。方法：采用ELISA法检测59例变态反应性疾病儿童和53例健康儿童血浆中肥大细胞羧肽酶和类糜蛋白酶水平。结果：变态反应性疾病患儿血浆羧肽酶和类糜蛋白酶含量分别为1.089±0.752 ng/mL、0.905(0.375~2.318)ng/mL,显著高于健康儿童［0.593±0.380 ng/mL、0.454(0.097~1.077) ng/mL］,差异有统计学意义（P<0.05)。变态反应性疾病患儿血浆羧肽酶水平与类糜蛋白酶水平呈显著正相关(r=0.684,P<0.01)。结论：变态反应性疾病儿童血浆中肥大细胞羧肽酶和类糜蛋白酶水平增高,提示二者可作为变态反应性疾病的诊断的有意义的指标。     

An improved procedure for the development of human mast cells from dispersed fetal liver cells in serum-free culture medium

The in vitro development of human mast cells from fetal liver cells with recombinant human stem cell factor in serum-containing RPMI was compared to that in AIM-V media with and without serum. Compared to serum-containing media, AIM-V medium caused mast cells to develop earlier and in greater numbers. By 2 weeks, about 60% of cells in serum-free AIM-V medium were phenotypic mast cells, 2 times the percentages in serum-containing media. By 6 weeks the percentages of mast cells were ≥80% under all conditions, but the number of mast cells was 3–4-fold greater in serum-free AIM-V medium than in serum-supplemented media. Mast cells obtained in serum-free AIM-V medium exhibited rounded nuclei, like tissue-derived mast cells; mast cells obtained in serum-supplemented media had segmented nuclei. By 10–12 weeks of culture about 40% of the AIM-V-derived cells showed strong chymase immunocytochemical staining, a pattern observed for only 14% of the cells in serum-containing media. AIM-V medium is a suitable medium for the development of human mast cells in vitro, and permits an earlier, more selective and greater expansion of mast cells than serum-containing media.     

Human gastroepiploic artery has greater chymase activity than the internal thoracic artery

Objective: Recent reports have demonstrated that long-term patency of the gastroepiploic artery (GEA) in coronary artery bypass grafting (CABG) is less satisfactory compared with the internal thoracic artery (ITA). However, the reason has not been fully elucidated. Angiotensin II is known to play an important role in the development of intimal hyperplasia, we hypothesized that the GEA is different from the ITA with respect to angiotensin II-forming ability. Accordingly, we measured activities of angiotensin II-forming enzymes, angiotensin-converting enzyme (ACE) and chymase, in human GEA and ITA. Methods: Remnant of the GEAs and ITAs were obtained from 24 patients who underwent CABG in which both conduits were used simultaneously. Activities of ACE and chymase were measured by using the extract form the GEA or ITA. Sections of the GEA or ITA were immunohistochemically stained with anti-human chymase antibody. Results: The ACE activity of the GEA (0.28 ± 0.16 mU/mg protein) was greater than that of the ITA (0.18 ± 0.11, p < 0.001). The chymase activity of the GEA (11.11 ± 7.15 mU/mg protein) was also greater than that in the ITA (7.13 ± 4.89, p < 0.001). The density of chymase-positive cells in the GEA (3.8 ± 4.2 cells/mm²) was greater than that in the ITA (1.1 ± 1.2, p < 0.01). Conclusion: Activities of both ACE and chymase were significantly greater in the GEA compared with the ITA. The GEA may be different from the ITA with respect to potential ability of angiotensin II-formation.     

抑制肾素-血管紧张肽系统药物研究

肾素-血管紧张肽系统(RAS)是药物治疗心血管系统疾病的关键作用靶点。目前的主要药物是血管紧张肽转化酶抑制剂(ACEI)和血管紧张肽Ⅰ受体拮抗剂(ARBs),因此被认为是作用于RAS治疗心血管疾病的经典药物,但是还有许多能够通过其他途径而产生对RAS的抑制作用。本文综述了目前发现的一些其他途径以及和经典途径的联系以及阻断它们产生的可能效应,包括ACEI抑制剂、ARBs、肾素抑制剂、糜蛋白酶抑制剂、血管紧张肽转化酶2、中性肽链内切酶抑制剂、醛固酮抑制剂以及针对RAS的基因治疗和免疫治疗。     

Velcade影响K562细胞基因表达谱的分子机制

目的 应用生物信息学方法探索Velcade影响K562细胞基因表达谱的分子机制.方法 提取并扩增Velcade处理组和溶剂对照组的K562细胞RNA,与22KAgilent Human 1A基因芯片杂交,用Agilent Feature Extraction软件采集扫描数据,再以GeneSifter,GATHER等基因芯片数据分析工具,对差异表达基因进行基因本体分类、KEGG通路分析、蛋白互作网络分析和文献挖掘.结果 获得228个差异表达基因,其中上调84个,下调144个.下调糜酶1基因幅度最大.对数比值达10.80倍,干扰素α-21基因也下调2.31倍.本体分类显示,衰老过程、白细胞活动等过程显著增强:KEGG通路分析显示,JAK-STAT信号通路,自然杀伤细胞介导的细胞毒作用和抗原处理与提呈等通路受显著影响;蛋白互作网络揭示泛素依赖的蛋白质降解通路、抗原提呈和免疫反应、JAK-STAT信号通路等处于网络中重要位置;文献挖掘显示差异表达基因与白血病、细胞凋亡、细胞周期、蛋白酶体、抑制剂、衰老和IκB等关键词高度关联.结论 Velcade可能通过抑制NF-κB和JAK-STAT等促进细胞存活的信号通路,增强细胞毒作用,诱导肿瘤细胞凋亡;Velcade还可能参与抗原加工与提呈、免疫反应、炎症反应等过程;糜酶1基因可能是Velcade发挥抗肿瘤作用的关键靶标.     

Application of novel tissue microarrays to investigate expression of tryptase,chymase and KIT protein in placental mast cells

Introduction Tissue microarrays comprise numerous small representative tissue samples from hundreds of different cases assembled on a single histologic slide, and therefore allow high throughput analysis of multiple specimens at the same time. Mast cells are paracrine cells found ubiquitously in connective tissue. Expression of the serine proteases tryptase and chymase, as well as KIT protein, the receptor for stem cell factor (SCF), has been demonstrated in mast cells. Because little is known about the role of mast cells in the placenta, we investigated the number and expression of chymase, tryptase, and KIT protein in placental mast cells using newly developed tissue microarrays.Materials and methods Tissue microarrays were prepared from archival paraffin tissue blocks of 90 placentae, including 15 normal ones as a control group. Gestational age of the placentae ranged from 7 to 42 weeks. Sections of formalin-fixed paraffin-embedded material were prepared on chemically activated cover-slides. The slides were cut in 4-mm² squares containing representative areas, and transferred to a tissue microarray. Hematoxylin and eosin (H&E), chloroacetate esterase (CAE), toluidine blue, periodic acid–-Schiff (PAS), and immunohistochemical staining were performed. The number of mast cells and expression of chymase, tryptase, and KIT protein were evaluated in each case.Results Mast cell numbers in placentae with inflammation/abortion exceeded that of normal placentae. Although statistically not significant, we furthermore observed an increase in chymase-positive mast cells in the group of placentae associated with fetal malformations/chromosomal aberrations compared with normal placentae.Discussion Novel tissue microarray technique has been introduced into placental research, and allows multiple placental tissue samples to be effectively analyzed simultaneously. This study indicated an increased number of chymase-positive mast cells in placentae with fetal malformation/chromosomal aberration. Activation of angiotensin II by chymase may play a role in fetal malformation. Moreover, it has been speculated that mast cells may only express chymase (MC_C). Our findings denote the presence of placental MC_C. However, further studies are needed to elucidate more precisely the role of mast cell chymase in the placenta.     

Application of a Chymase Inhibitor,NK3201, for Prevention of Vascular Proliferation

NK3201 is an orally active chymase inhibitor. Its inhibitory activity leads to formation of acyl‐intermediate between active serine residue of the enzyme and di‐ketone structure of NK3201. NK3201 inhibits human, dog and hamster chymases with IC₅₀ of 2.5, 1.2, and 28 nM, respectively. On the other hand, NK3201 does not inhibit other types of serine proteases, tryptase, thrombin, elastase, plasmin, and plasminogen activator. In dogs, at 8 h after oral administration of NK3201, 1 mg/kg, the drug levels in plasma, heart, and aorta reached 470, 195, and 78 nM, respectively. In a dog model NK3201, 5 mg/kg/day, increased chymase activity in grafted veins, and suppressed vascular proliferation. After balloon injury in dog vessels, chymase activity was increased locally, in the injured artery, and NK3201, 1 mg/kg/day was effective in preventing vascular proliferation. On the other hand, NK3201, unlike angiotensin converting enzyme inhibitors or angiotensin II receptor blockers, did not affect blood pressure. These findings indicate that local angiotensin II production by chymase is involved only in vascular proliferation, as seen in the injured vessels. Therefore, NK3201 may be useful for preventing vascular proliferation without affecting blood pressure.     

Immunoglobulin E and mast cell proteases are potential risk factors of impaired fasting glucose and impaired glucose tolerance in humans

Aim. Mast cells are important in experimental diabetes. Plasma levels of immunoglobulin E (IgE), tryptases, and chymases are inflammatory markers of human diabetes. Whether they also correlate with the risk of pre-diabetes, however, remains unknown.

Methods and results. A total of 260 subjects 55–75 years of age were grouped as normal glucose tolerance (NGT), isolated impaired fasting glucose (I-IFG), isolated impaired glucose tolerance (I-IGT), and mixed IFG/IGT. There were significant differences in plasma levels of high-sensitivity C-reactive protein (hsCRP) (P < 0.001) and IgE (P = 0.003) among all subgroups of pre-diabetes, and chymase in I-IGT (P = 0.043) and mixed IFG/IGT (P = 0.037) subgroups compared with NGT group. High-sensitivity CRP was a risk factor in all subgroups of pre-diabetes; IgE was a risk factor of mixed IFG/IGT; and chymase was a risk factor of I-IGT and mixed IFG/IGT. Interactions between hsCRP and high waist circumference (WC), waist-to-hip ratio (WHR), or HOMA-β index, and interactions between IgE and high WC or tryptase levels all increased further the risk of developing I-IFG, I-IGT, or mixed IFG/IGT.

Conclusion. Plasma hsCRP, IgE, and chymase levels associate with pre-diabetes status. While hsCRP, IgE, and chymase are individual risk factors of pre-diabetes, interactions with metabolic parameters increased further the risk of pre-diabetes.     

Increased mast cells in hepatocellular carcinoma and intrahepatic cholangiocarcinoma

BACKGROUND/AIMS: Human mast cells are categorized into those positive only for tryptase (MC(T)) and those positive for both tryptase and chymase (MC(TC)). METHODS: We investigated mast cells in "normal" livers (n=13), hepatocellular carcinoma (HCC) (n= 49) and intrahepatic cholangiocarcinoma (ICC) (n= 44) by double immunostaining and quantitative morphometry. RESULTS: In "normal" livers, mast cells were located in portal tracts, and to a lesser extent in the sinusoids. In HCC, mast cells were noted in tumoral sinusoids and fibrous septa. In ICC, many mast cells were present in tumoral stroma. Morphometry showed that densities of mast cells in HCC and ICC were significantly higher than those in "normal" livers. The density of mast cells in ICC (57.6+/-62.4/mm2) was significantly higher than that in HCC (9.32+/-12.9/mm2). The density of sinusoidal mast cells was significantly higher in HCC (1.79+/-2.35/mm2) than in "normal" livers (0.13+/-0.07/mm2). The density of stromal mast cells was significantly higher in ICC (57.6+/-62.4/mm2) than that of portal tracts in "normal" livers (28.4+/-7.0/mm2). MC(T) and MC(TC) were approximately 20% and 80%, respectively, being consistent in any anatomical compartments. CONCLUSIONS: Mast cells increase during carcinogenesis in HCC and ICC, and they may play a role in fibrosis or tumor immunology in HCC and ICC.     

慢性阻塞性肺疾病患者痰中肥大细胞和嗜酸粒细胞因子变化及其相关性研究

目的探讨慢性阻塞性肺疾病(COPD)患者类糜蛋白酶(chymase)活性,类胰蛋白酶(tryptase)、白细胞介素8(IL8)、嗜酸粒细胞趋化因子(eotaxin)水平和中性粒细胞(NEU)、嗜酸粒细胞(EOS)计数的相关性及临床意义。方法老年COPD患者73例(重度21例、中度21例、轻度31例),采用双抗体夹心酶联免疫吸附测定,检测诱导痰IL8、eotaxin水平。在UniCAP100全自动体外变应原检测仪上进行tryptase检测,Chymase活性测定使用琥珀酰丙氨酸丙氨酸脯氨酸苯丙氨酸酰苯氨(SAAPP)作为底物,采用酶标仪在410nm连续监测吸光度的变化。结果(1)老年急性加重期COPD患者(重、中、轻)痰tryptase的中位数分别为2840、2150、595ng/L,治疗后各组痰tryptase水平显著下降(分别为1510、920、33ng/L,P均<001)。加重期重度、中度与轻度患者比较差异均有显著性(P均=0)。急性加重期痰IL8、痰eotaxin中位数分别为12998、4549、787ng/L;227、151、74ng/L。重度、中度组痰IL8、eotaxin水平均高于轻度组(P均<001)。治疗后痰IL8、eotaxin中位数分别为10375、3266、679ng/L;79、63、68ng/L。重度、中度患者痰IL8、eotaxin水平均比治疗前显著降低(P均<001)。(2)重、中度COPD患者急性加重期痰chymase活性高于轻度组,黄豆胰蛋白酶抑制剂(SBTI)、α1抗胰蛋白酶(α1A