人体上皮细胞的原代培养

总结了人体上皮细胞的原代培养方法，详述了组织块培养法，单细胞分离培养法、成纤维细胞清除法及其关键技术，如以冷胰蛋白酶消化法制备各种上皮细胞悬液，用机械刮除加酶消法清除成纤维细胞。本资料有助于基础医学研究及在临床应用中获得理想的研究材料。     

T lymphocyte subset profile and serum alpha-1-antitrypsin in pathogenesis of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder characterized by the presence of non-fully reversible airflow limitation. The study was undertaken to investigate the involvement of alpha-1-antitrypsin (alpha(1)AT) and T lymphocyte subsets in the pathogenesis of COPD. Blood samples of 50 subjects, including 25 healthy volunteers and 25 patients with COPD, were analysed. Serum trypsin inhibitory capacity (STIC) was determined by enzymatic assay. CD4(+) and CD8(+) T lymphocytes were enumerated in heparinized blood using a fluorescence activated cell sorter counter. The STIC in COPD patients was found to be decreased significantly than in controls (P < 0.01). In COPD patients with lower expression levels of alpha(1)AT, a highly significant decrease in the number of CD4(+) T lymphocytes (P < 0.0009) and CD4/CD8 ratio was observed compared with control subjects (P < 0.008). The mean +/- standard error of CD8(+) lymphocytes was found to be little different (only marginally decreased) in COPD patients compared to healthy controls; however, an alteration in the individual count of CD8(+) lymphocytes cells was observed in COPD patients. Using linear regression analysis, a negative correlation was observed between STIC and CD4(+) lymphocytes and CD8(+) lymphocytes (r = -0.40, P < 0.04; r = -0.42, P < 0.03, respectively) in COPD patients. An alteration in alpha(1)AT and T lymphocyte subsets in COPD patients suggested that interplay of these factors may be responsible for the progression of COPD.     

Epitope selection and their placement for increased virus neutralization in a novel vaccination strategy for porcine epidemic diarrhea virus utilizing the Hepatitis B virus core antigen

Porcine epidemic diarrhea virus (PEDV) is a member of the Alphacoronaviridae genus within the Coronaviridae family. It is the causative agent of porcine epidemic diarrhea, a disease that can have mortality rates as high as 100% in suckling piglets. PEDV causes severe economic loss, and has been in existence for decades. A panzootic starting in 2010 renewed interest in the development of a universal vaccine toward PEDV. This report details several design changes made to a Hepatitis B virus core antigen (HBcAg)-based recombinant vaccine strategy, and their effect in vivo. Initially, several multi-antigen vaccine candidates were able to elicit antibodies specific to three out of four B-cell epitopes inserted into the chimeric proteins. However, a lack of virus neutralization led to a redesign of the vaccines. The focus of the newly redesigned vaccines was to elicit a strong immune response to the YSNIGVCK amino acid motif from PEDV. Genetically modified new vaccine candidates were able to elicit a strong antibody (Ab) response to the YSNIGVCK epitope, which correlated with an increased ability to neutralize the CO strain of PEDV. Additionally, the location of the inserted PEDV epitopes within the vector protein was shown to affect the immune recognition toward the native HBcAg during vaccination.     

Protective Effects of Urinary Trypsin Inhibitor on Systemic Inflammatory Response Induced by Lipopolysaccharide

Urinary trypsin inhibitor (UTI), a serine protease inhibitor, has been widely used in Japan as a drug for patients with acute inflammatory disorders such as disseminated intravascular coagulation (DIC), shock, and pancreatitis. Recent in vitro studies have demonstrated that serine protease inhibitors may have anti-inflammatory properties beyond their inhibition of neutrophil elastase at the site of inflammation. However, the therapeutic effects of UTI in vivo remain unclear. In this review, we introduce the roles of UTI in the experimental systemic inflammatory response induced by both intraperitoneal and intratracheal administration of lipopolysaccharide using UTI deficient and wild-type mice. Our experiments suggest that UTI can protect against systemic inflammatory response and subsequent organ injury induced by bacterial endotoxin, at least partly, through the inhibition of proinflammatory cytokine and chemokine expression. UTI may therefore present an attractive “rescue” therapeutic option for systemic inflammatory response syndromes such as DIC, acute lung injury, and multiple organ dysfunction.     

Measurements of exocrine proteins in the pig pancreas using microdialysis

The authors adapted microdialysis for the study of intrapancreatic exocrine proteins. Immunoreactive cationic and anionic trypsinogen were continuously measured after the insertion of one probe inside the pig pancreas and another placed under the peritoneum on the anterior surface of the gland. The concentration initially declined in the dialysate from the intrapancreatic probe, followed by an increasing tendency until 120 minutes after the insertion, after which a decline to a relative steady state after four to six hours was observed. A dialysate with a lower concentration of enzymes was obtained from the probe on the pancreatic surface. In order to study the turnover of an exogenously administered substance, a microdialysis probe was inserted into the pancreas and human recombinant pancreatic secretory trypsin inhibitor was injected into the pancreatic duct. The inhibitor was cleared from the pancreas with an intrapancreatic halflife of approximately 45 minutes. Membrane function, measured in vitro before and after use in vivo, was not significantly affected after seven hours in the pig pancreas. This work was supported by grants from the Swedish Medical Research Council (projects 03910 and 08715), the Swedish Cancer Society (project 1300), the Medical Faculty of the University of Lund, the Foundation of Malm? General Hospital against Cancer, the Foundation of Albert Pahlsson and the Foundation of Torsten and Ragnar S?derberg.     

不同试剂脱除猪胸主动脉壁细胞的对比研究

目的:用不同方法去除猪胸主动脉壁细胞,并比较去细胞效果,为组织工程瓣膜或带瓣管道提供良好的实验材料。方法:采用十二烷基硫酸钠、胆脂酸钠、曲拉通（TritonX100）制备猪胸主动脉脱细胞基质。将实验分为十二烷基硫酸钠组、胆脂酸钠、曲拉通组和空白对照组。标本进行大体、光镜、电镜观察,力学性能的检测并做比较。结果:胆脂酸钠无法完全脱除主动脉壁内细胞,十二烷基硫酸钠和曲拉通都能完全脱除猪胸主动脉细胞,曲拉通较好地保持了胶原纤维和弹性纤维的原有排列和分布,并有较好的力学性能。后两者脱细胞时间无显著差别。结论:曲拉通可以成功用于制备大血管脱细胞基质,为组织工程瓣膜或带瓣管道的研究提供材料。