sHLA-G1重链分子、β_2m的体外表达及纯化研究

目的获得sHLA-G1重链分子及轻链β2微球蛋白(β2m)基因体外表达并纯化的相关蛋白质。方法RT-PCR扩增可溶性HLA-G1重链分子及人β2m轻链的cDNA序列,构建表达载体pET28a(+)/sHLA-G1及pET28a(+)/β2m,导入大肠杆菌BL21(DE3),IPTG诱导sHLA-G1及β2m蛋白表达,并以Ni-NTA亲和层析和CM-FF弱阳离子柱分别纯化,透析后浓缩保存。SDS-PAGE,Western-Blot鉴定目的蛋白的表达和纯化。结果成功克隆了sHLA-G1及2βm基因并构建了pET28a(+)-sHLA-G1、pET28a(+)-β2m原核高效表达载体;表达产物以可溶性形式存在,表达量>30%,纯化后产物纯度达到95%。结论成功表达并纯化出的sHLA-G1重链分子及轻链β2m有助于阐明可溶性HLA-G1的功能。     

有机磷中毒家兔呼吸肌麻痹的病理机制和分子基础探讨

目的　探讨有机磷中毒家兔呼吸肌麻痹 (RMP)形成的分子基础和病理机制。方法　 2 4只青紫蓝家兔分 4组 :敌百虫 (10LD ,1112 0mg kg)组 ,久效磷 (10LD ,2 2 2 4mg kg)组 ,甲基对硫磷(10LD ,74 10mg kg)组及正常对照组 ,每组 6只。家兔中毒经腹部皮下给药 ,中毒后 6h、 2 4h取血及肋间外肌 ,苏木精 伊红染色观察肋间外肌病理改变。用酶抑制法测肋间外肌组织匀浆中游离有机磷毒质(FOP)含量。用放射免疫法测血浆中肿瘤坏死因子α (TNF 浕) ,白细胞介素 1 β (IL 1β)及血栓素B2(TXB2 )含量。结果　中毒后 2 4h ,肋间外肌组织出现严重的病理损伤 ,可见肌纤维变性、坏死、横纹消失 ,肌原纤维崩解断裂。同时 ,肋间外肌组织匀浆中可检出不同浓度的FOP ,血浆中TNF 浕及ＴＸＢ2 含量显著高于正常 (TNF 浕 :T =2 5 ,P <0 0 0 5 ;TXB2 :T =2 1,P <0 0 0 5 )。结论　重度有机磷中毒可致动物呼吸肌出现严重的病理损伤 ,FOP和TXA2及TNF 浕在呼吸肌损伤中起重要作用 ,这些改变对RMP的形成具有重要意义     

Direct in vivo gene transfer to healing rat patellar ligament by intra-arterial delivery of haemagglutinating virus of Japan liposomes

BACKGROUND: Manipulation of ligament healing has been a major focus of orthopaedic research. In recent years, gene transfer to healing ligament appears to be a feasible method for manipulating the healing process. In this study, we investigated the feasibility of gene transfer to healing rat patellar ligament by intra-arterial delivery. METHODS: An attempt was made to transfer a reporter gene (Escherichia coli, beta-galactosidase gene) to healing rat patellar ligament using the haemagglutinating virus of Japan (HVJ) liposome-mediated gene transfer method. Three days after cutting the patellar tendons of 25 14-week-old male Wistar rats, HVJ-liposome complexes containing beta-galactosidase (beta-gal) cDNA were injected into the femoral artery of 15 Wistar rats as the experimental group. HVJ liposomes without DNA were injected into the femoral artery of 10 Wistar rats as the control group. Three rats from the experimental group and two control rats were killed 3, 7, 14, 28 and 56 days after the injection. RESULTS: After X-gal staining, the rate of transfection in the experimental group (mean +/- SEM) was found to be 12.1% +/- 0.590%, 8.7% +/- 0.217%, 10.2% +/- 0.227%, 3.2% +/- 0.247% and 0.7% +/- 0.060% at post-injection days 3, 7, 14, 28 and 56 respectively. In control sections the number of blue-stained cells were very few at any point. CONCLUSION: We succeeded in introducing a reporter gene into healing rat patellar ligament by infra-arterial delivery of HVJ-liposome complexes. This method appears to have the potential to be applicable for soft-tissue healing studies and also healing studies of other tissues and organs.     

Potential stem cell—Conditioned medium and their derived exosomes versus omeprazole in treatment of experimental model of gastric ulcer

Gastric ulcer is a common frequent clinical problem affecting all age and gender. This work aims to compare between the therapeutic effects of stem cell derived exosomes, stem cells conditioned medium and omeprazole on the healing of gastric ulcer model.Fifty rats were, assigned into 5 groups; control, gastric ulcer, omeprazole-treated, conditioned medium- treated, and exosomes-treated groups. Gastric ulcer was induced by aspirin dissolved in 1% carboxymethyl cellulose at a daily dose of 200 mg/kg for 5 consecutive days. Stomach specimens were obtained for histological, biochemical, and immunohistochemical assessments.The gastric ulcer group revealed widening of the fundic glands lumen containing, exfoliated dead cells. There was a remarkable distortion of the normal histological structure of the gastric mucosa with surface lining epithelial cell sloughing, vascular congestion and inflammatory cell infiltration. Both exosomes and conditioned medium treatments ameliorated almost all of the histopathological changes. Interestingly, the healing effect of exosomes was greater because it restored the histological architecture of gastric mucosa to nearly normal.In conclusion, this work may pave the future for using stem cell derived exosomes as a more convenient and effective adjuvant therapy in gastric ulcer.     

老年冠心病合并糖尿病患者血栓前状态分子标志物变化及意义

目的 观察老年冠心病合并非胰岛素依赖型糖尿病(NIDDM)患者血栓前状态分子标志物的变化及意义。方法 对观察组60例患者(稳定型心绞痛18例、不稳定型心绞痛19例、不稳定型心绞痛合并NIDDM23例)和对照组18例正常人体内血栓前状态分子标志物、血管内皮功能以及脂质过氧化物水平进行检测，并回归分析其之间的相关性。结果 老年冠心病合并NIDDM患者存在明显的血栓前状态分子标志物变化，并且这种变化与内皮功能失调以及脂质过氧化损伤密切相关。结论 老年冠心痛合并NIDDM患者存在明显的血栓前状态分子标志物变化，可能与血管内皮功能障碍及脂质过氧化损伤有关。     

MODY3基因在早发家族性2型糖尿病发病中的作用

目的评估MODY3（HNF-1a）基因在中国家族性早发2型糖尿病（T2DM）发病中的作用。方法收集100个早发T2DM家系,PCR扩增先证者MODY3基因的外显子和外显子/内含子拼接区,产物直接测序。对发现的SNPs进行病例对照研究。结果发现5个非编码区DNA变异IVS1-16G〉A、IVS2-23C〉T、IVS5＋9C〉G、IVS7＋7A〉G、IVS9-24C〉T,4个编码区的同义突变Leu17Leu、Gly288Gly、Thr515Thr、Leu459Leu,3个编码区的错义突变Pro379Ala、Ile27Leu、Ser487Asp,其中Pro379Ala突变在一个家系中与糖尿病共分离;Ile27Leu和Ser487Asp与糖尿病不相关。结论MODY3在中国早发家族性T2DM中患病率不超过1%,在中国家族性T2DM发病中不起主要作用。     

Phylogenetic and antigenic analyses of coxsackievirus A6 isolates in Yamagata,Japan between 2001 and 2017

Although coxsackievirus A6 (CV-A6) is generally recognized as a causative agent of herpangina in children, CV-A6 infections globally emerged as a new and major cause of epidemic hand-foot-and-mouth-diseases (HFMDs) around 2008. To clarify the longitudinal epidemiology of CV-A6, we carried out sequence and phylogenetic analyses for the VP1 and partially for the VP4-3D regions as well as antigenic analysis using 115 CV-A6 isolates and 105 human sera in Yamagata, Japan between 2001 and 2017. Phylogenetic analysis revealed that CV-A6 isolates were clearly divided into two clusters; strains in circulation between 2001 and 2008 and those between 2010 and 2017. Neutralizing antibody titers of two rabbit antisera, which were immunized with Yamagata isolates in 2001 and 2015, respectively, against 28 Yamagata representative strains as well as the prototype Gdula strain were 1:2560–1:5120 and 1:160–1:640, respectively. The neutralizing antibody titers among residents in Yamagata against the above two strains were similar. Our analyses revealed that there were cross-antigenicities among all analyzed CV-A6 strains, although the newly emerged strains were introduced into Yamagata around 2010 and replaced the previous ones. With regard to control measures, these findings suggest that we can prevent CV-A6 infections through the development of a vaccine that effectively induces neutralizing antibodies against CV-A6, irrespective of genetic cluster.