恶性疟P190抗原保守区Ⅰ、Ⅴ基因的亚克隆及在大肠杆菌中高效表达

作者采用PCR方法克隆了我国海南省FCC1/HN株P190抗原两个保守区基因,分别定名为P190CRI和P190CRV。基因片段经纯化后连接到pUC18载体中进行DNA序列分析,结果显示:除了P190CRV中有5个碱基变换外,其余序列均与MAD20型序列一致。经序列分析的两个基因片段分别与pGEX-2T载体连接,经双酶切鉴定后转化感受态JM109(DE_3)大肠杆菌进行高效融合表达,并且用Sepharose 4B-谷胱甘肽层析柱进行亲和纯化,结果为:两个插入基因片段均得到高效融合表达,经一步亲和纯化后就取得高纯度的重组蛋白。     

Abrogation of IL-3 Requirements and Stimulation of Hematopoietic Cell Proliferationin Vitroandin Vivoby Carboxylic Acids

ABSTRACT: Short-chain fatty acids, such as butyrate and propionate, induce fetal globin gene expression and are under clinical investigation in the β-hemoglobinopathies. Limitations of the short-chain fatty acids as therapeutics include their rapid metabolism and a tendency to induce cell growth arrest if administered for prolonged periods. In studies described here, the cellular effects of other inducers of fetal globin, phenoxyacetic acid and derivatives of short-chain fatty acids and cinnamic acids, were investigated in the human erythroid cell line K562, the IL-3 dependent multi-lineage cell line (32D), and in mice and primates. Several test compounds supported 32D cell proliferation despite a 50-fold depletion of IL-3, which resulted in growth arrest and apoptotic death in control cells. The degree of proliferation induced by certain test compounds was similar to the degree of proliferation induced by Erythropoietin and G-CSF in the cells. Eight of ten compounds induced γ globin mRNA in K562 cells. A 2.5 to 6-fold increase in reticulocytosis was observedin vivoin mice treated with two prototype compounds. Pharmacokinetic studies of three prototype compounds demonstrated millimolar plasma concentrations after single oral doses for many hours in primates. These findings identify orally bioavailable compounds which induce γ globin gene expression and hematopoietic cell proliferation through an activity which partially abrogates requirements for IL-3. Such compounds provide potential for oral therapeutics which stimulate proliferation of hematopoietic cells of multiple lineages, as well as inducing fetal globin.     

Protective genes expressed in endothelial cells of second hamster heart transplants to rats carrying an accommodated first graft

Abstract: Accommodation refers to survival of a xenograft despite the presence of anti-donor organ antibodies and complement. We have recently shown that accommodation of a hamster heart transplanted to a rat receiving short-term cobra venom factor (CVF) and continuing cyclosporine A (CyA) therapy is associated with i) the expression in the endothelial cells (EC) and smooth muscle cells of the graft of a number of "protective" genes, ii) a prominent intragraft Th2 cytokine profile, and iii) the relatively heavy deposition of IgG2c antibodies on the EC of the graft. In contrast, rejecting grafts do not express the protective genes, have a Th1 cytokine profile, and apparently have lesser amounts of IgG2c. These findings are consistent with host factors (Th2 cytokines and IgG2c) contributing to xenograft accommodation. To test whether these host factors may predispose to the development of accommodation, we placed a second hamster heart into each of 12 rats carrying a surviving first heart; recipients were, at the time, receiving only CyA. Whereas first grafts transplanted to rats receiving only CyA survive for 3 to 4 days, 11 out of 12 second transplants survived more than 20 days, and the other survived for 7 days. Nine of the twelve were not rejected: of these, four were removed between day 35 and 132 for study, and the remainder are still beating at 35 to 52 days. The surviving second hearts we studied had accommodated in that the picture on immunopathology was the same as for surviving first hearts. We suggest that the Th2 cytokines and perhaps the IgG2c response are factors in allowing prolonged survival of the second grafts and, further, that these factors contribute to the expression in the EC and smooth muscle cells of the surviving second hearts of the protective genes.     

C-fos expression in the rat nucleus basalis upon excitotoxic lesion with quisqualic acid: a study in adult and aged animals

Summary. A unilateral quisqualic acid lesion was placed in the nucleus basalis magnocellularis of 3- and 24-month-old rats, and the animals were sacrificed at different times post-surgery. The morphology and the number of the cholinergic neurons of the nucleus basalis were analyzed by means of immunohistochemistry for cholineacetyltransferase, in order to evaluate the size and severity of the lesion. Immunohistochemistry for the immediate early gene c-fos was also performed in order to clarify its role in the process of neurodegeneration following the excitotoxin injection. The DNA laddering and TUNEL techniques were used to define the type of cell death involved. At short times (4 hr) the lesion induced alterations in the morphology of cholinergic neurons of the nucleus basalis. Subsequently, a significant decrease in the number of neurons was found in comparison to the contralateral unlesioned side. In the older animals the loss of cholineacetyltransferase immunoreactivity had an earlier onset (4 hr) than in the young (24 hr). C-fos expression was induced by the lesion and not by saline injection in the nucleus basalis and in neighbouring areas of the brain as early as 4 hr after surgery. The c-fos protein was no longer present by 24 hr. Furthermore, the c-fos gene product was consistently absent from the nuclei of cholinergic cells. The aged animals exhibited a slower and smaller increase in c-fos as measured by counting the labelled nuclei in the injected area. Analysis of DNA fragmentation did not provide any evidence for apoptosis as the type of cell death involved in the cholinergic degeneration. These results indicate that the c-fos protein might have a protective role in the response to excitotoxic lesions. Furthermore, we have shown that the aged brain displays a reduced ability to produce a c-fos-mediated plastic response to the lesion. Received December 17, 1997; accepted February 17, 1998     

HLA-DR, DQ genotypes of celiac disease patients and healthy subjects from the West of Ireland

Celiac disease (CD) has one of the strongest class II HLA associations of any human illness. We used DNA-RFLP typing to study the class II HLA genotypes of celiac disease patients from the West of Ireland, the geographic area with the highest rate of celiac disease in the world. We confirmed the high frequency of HLA-DR3 in this population, and we were also able to demonstrate the additional risk of developing celiac disease imparted by HLA-DR7. This was done by clearly distinguishing DR7, DQ2 haplotypes from DR7, DQ9 haplotypes, and by "subtraction analysis" of haplotype frequencies. As reported in other populations, most of the patients without DR3 were heterozygous for DR7 and DR11 or 12 (DR5), or had DR4. We used PCR-RFLP and direct sequencing of amplified DNA to examine HLA-DR4 subtypes. The frequency of HLA-DR4 was markedly decreased in patients compared with controls (p=0.000001) and there was a significant alteration of DR4 subtypes of the patients compared with controls (p=0.0227). Moreover, all of the CD patients (5 of 5) with DR4 had a haplotype associated with the DQB1*0302 allele compared with only 11 of 23 control subjects with DR4. Our results in this population with exceptionally high risk of CD strongly support the DQ heterodimer hypothesis and suggest that the recently described sequence difference between the DQB1*02 alleles of DR3 and DR7 may contribute to a synergistic increased risk when these haplotypes are inherited together. In addition, our findings suggest a role for HLA-DQ in DR4-associated CD.     

冠状动脉粥样硬化性心脏病患者心外膜脂肪组织的生物信息学研究

背景 心血管疾病（CVD）是常见病和多发病,患病率和死亡率呈快速上升趋势。动脉粥样硬化（AS是缺血性CVD的病理基础,研究表明心外膜脂肪组织（EAT）通过分泌外泌体（EXO）和生物活性物质促进AS进展,但其作用机制仍需进一步研究。目的 通过生物信息学方法挖掘冠状动脉粥样硬化性心脏病（CAD）患者EAT中的关键基因,探讨免疫细胞浸润情况,联合CAD患者EXO间差异表达基因（DEGs）推测EAT来源EXO间DEGs并进行验证,从细胞及分子水平上探讨EAT在CAD疾病过程中的作用机制。方法 从基因表达综合数据库（GEO）中下载关于EAT的数据集GSE64554、GSE120774,根据临床信息将EAT的测序数据分为CAD组和健康对照组,使用R语言及相关软件包进行生物信息学分析。首先使用R语言筛选CAD组与健康对照组EAT间DEGs,并进行GO富集分析和KEGG通路富集分析,构建蛋白质-蛋白质相互作用（PPI）网络,评估所选基因的生物学功能及可能参与其调控的转录因子。构建GSE64554数据集中EAT的加权基因共表达网络（WGCNA）,获取同CAD表型相关的基因模块,将所获EAT间DEGs与模...     

急性脑梗死患者血清CTRP-3、D-二聚体、sTREM2水平及相关临床特征与溶栓后出血性转化的关系

目的 探讨急性脑梗死患者血清补体C1q/肿瘤坏死因子相关蛋白3（CTRP-3）、D-二聚体、可溶性髓样细胞触发受体2（sTREM2）水平及相关临床特征与溶栓后出血性转化（HT）的关系。方法 回顾性分析2018年9月—2022年9月在青海省人民医院接受溶栓治疗的120例急性脑梗死患者的临床资料,根据患者溶栓后是否发生HT分为HT组（30例）、非HT组（90例）。比较两组患者的临床资料及血清CTRP-3、D-二聚体、sTREM2水平。采用多因素逐步Logistic回归分析急性脑梗死患者溶栓后发生HT的危险因素;绘制受试者工作特征（ROC）曲线,分析急性脑梗死患者溶栓后HT预测模型预测HT发生的价值。结果 HT组心房颤动（以下简称房颤）、大面积脑梗死、入院NIHSS评分≥ 15分占比高于非HT组（P <0.05）,血清CTRP-3水平低于非HT组（P <0.05）,D-二聚体、sTREM2水平高于非HT组（P <0.05）。血清CTRP-3、D-二聚体、sTREM2水平预测急性脑梗死患者溶栓后发生HT的敏感性分别为66.7%（95% CI：0.598,0.756）、70.0%（95% CI：0.607,0.812）、80.0%（95% CI：0.714,0.889）,特异性分别为73.3%（95% CI：0.636,0.821）、86.7%（95% CI：0.778,0.923）、86.7%（95% CI：0.747,0.942）。多因素Logistic逐步回归分析结果显示,房颤［O^R=1.237（95% CI：1.103,1.387）］、大面积脑梗死［O^R=2.338（95% CI：1.292,4.231）］、入院NIHSS评分≥ 15分［O^R=2.087（95% CI：1.231,3.538）］、CTRP-3 ≤ 269.265 μg/L ［O^R=3.006（95% CI：1.508,5.992）］、D-二聚体≥ 2.625 mg/L ［O^R=2.649（95% CI：1.374,5.107）］、sTREM2 ≥ 314.675 ng/L ［O^R=2.328（95% CI：1.411,3.841）］是急性脑梗死患者溶栓后发生HT的危险因素（P <0.05）。根据多因素Logistic逐步回归分析结果建立急性脑梗死患者溶栓后HT预测模型,Logit（P） = -33.887 + 0.213×房颤+ 0.849×大面积脑梗死+0.736×入院NIHSS评分+ 1.101×CTRP-3 + 0.974×D-二聚体+ 0.845×sTREM2;ROC曲线分析结果表明,预测模型预测HT发生的敏感性为93.3%（95% CI：0.841,0.991）,特异性为87.8%（95% CI：0.808,0.976）。结论 血清CTRP-3、D-二聚体、sTREM2水平与急性脑梗死患者溶栓后HT有关,预测价值较高,且急性脑梗死患者溶栓后HT预测模型预测HT优于各项指标单独预测。