AML1-ETO 真核表达载体的构建及对 U937细胞增殖和分化的影响

目的 构建pcDNA3.1-AML1-ETO真核表达载体,并观察AML1-ETO融合蛋白在U937细胞内的表达并检测其对细胞增殖与分化的影响.方法 以原核表达载体pCMV5-AML1-ETO为模板扩增AML1-ETO目的 片段,将该基因重组于pcDNA3.1/V5-His-TOPO真核表达载体上,用脂质体转染技术将其导入 U937细胞,经G418筛选获得稳定转染的克隆,PCR检测AML1-ETO基因的整合,RT-PCR及 Western blot检测AML1-ETO mRNA和蛋白的表达,用锥虫蓝拒染人工计数法观察细胞增殖活性,流式细胞术检测髓系分化抗原表达的变化,瑞特染色法观察细胞形态改变.结果 pcDNA3.1-AML1-ETO经酶切鉴定及DNA测序证实序列完全正确,筛选出AML1-ETO基因高表达的亚克隆,证实AML1-ETO基因稳定转染到U937细胞中并得到表达;转染细胞增殖受抑(P<0.05),髓系分化抗原CD11b表达阳性率[(4.17±0.31)%]低于转染空载体和未转染对照组[(11.40±0.17)%、(11.03±0.15)%](P<0.001),形态呈低分化表现.转染细胞经TPA处理后,CD11b表达无明显变化(P>0.05).结论 成功构建pcDNA3.1-AML1-ETO表达载体,并在真核细胞中得到了正确表达,AML1-ETO 基因能抑制U937细胞的增殖与分化,为进一步研究该基因致白血病的机制奠定了基础.

Abstract：

Objective To construct a pcDNA3. 1 -AML1-ETO expression vector and investigate its effects on proliferation and differentiation of U937 leukemic cells. Methods AML1 -ETO gene was amplified by PCR from pCMV5-AML1-ETO and inserted into eukaryotic expression plasmid pcDNA3. 1/V5-His-TOPO. The recombinant plasmid was transfected into U937 cells by Lipofectamin 2000. Individual clones selected with G418 were isolated. The integration and the expression levels of AML1-ETO in transfectants were determined by PCR, RT-PCR and Western blot analysis respectively. Trypan blue refusal staining method was used to detect the proliferation of U937 cells. Light microscope was applied to observe the morphologic changes of the cell. The expression of myeloid cell differentiation antigen was detected using flow cytometry. Results The recombinant pcDNA3. 1-AML1-ETO was confirmed by enzyme digestion and sequencing. The highly expressing AML1-ETO subclone was established. AML1-ETO was expressed in U937 cells transfected with pcDNA3.1 -AML1-ETO. The growth of the monoclonal cells was inhibited evidently (P < 0. 05). The expression of CD11b in transfected group [(4. 17±0. 31)%] was lower than that in empty plasmid transfected group and non-transfected group [(11.40 ± 0. 17)% and (11.03 ± 0. 15) %] respectively (P < 0.001). Transfected cells displayed morphology of less differentiation. The expression level of CD11b was unchanged in transfected cells treated with TPA (P > 0. 05). Conclusion The eukaryotic expression vector for AML1ETO gene was successfully constructed and expressed in U937. AML1-ETO inhibits the proliferation and differentiation of transfected cells. It provides the basis for further study of mechanisms of AML1 -ETO in leukemogenesis.     

上海市闸北区初中生自我意识状况调查

目的：了解上海市闸北区初中生自我意识状况。方法抽取上海市闸北区2018名初中生采用Piers‐Harris儿童自我意识量表进行测评分析。结果入组初中生Piers‐Harris儿童自我意识量表总分为（52．83±12．62）分,自我意识偏低检出率为46．6％;不同性别初中生总分及行为、智力与学校、焦虑、合群因子分比较差异有极显著性（P＜0．01）,不同年龄总分及行为、智力与学校、焦虑、幸福与满足因子分比较差异有极显著性（P＜0．01）;男生自我意识偏低检出率显著高于女生（P＜ 0．01）,不同年龄初中生自我意识偏低检出率比较差异有极显著性（P＜0．01）。结论初中生自我意识偏低,存在性别及年龄差异,学校应予以有针对性的引导与教育。     

融合蛋白AML1-ETO对C/EBPα基因表达的影响

背景：已有研究显示AML1-ETO可以通过包括抑制C/EBPα的功能在内的机制引起分化的阻滞。目的：观察AML1-ETO对髓系分化相关基因C/EBPα表达的影响,探讨其在白血病发生中的作用。方法：应用流式细胞术检测急性单核细胞白血病细胞株U937、U937-MOCK和U937-A/E1细胞表面抗原CD11b、CD14的表达;荧光实时定量PCR检测C/EBPαmRNA的表达;免疫印迹法检测C/EBPα蛋白的表达;染色质免疫沉淀技术研究转染细胞中AML1-ETO与C/EBPα基因启动子之间直接的相互作用情况。结果与结论：AML1-ETO转染细胞的细胞表面分化抗原CD11b和CD14的表达明显下降,且转染了AML1-ETO的U937细胞系中,C/EBPα的mRNA与蛋白水平均下调,转染细胞沉淀富集的DNA中含有C/EBPα基因的启动子序列。结果提示C/EBPα是AML1-ETO的直接靶基因,AML1-ETO能下调C/EBPα的表达。     

PGC-1α基因多态性与冠心病的相关性分析

目的研究过氧化物酶体增殖物激活受体7共激活因子1α（PGC-1α）基因的单核苷酸多态性（single-nucleotidepolymorphism,SNP）住点rs2970847与中国汉族人群冠心痛（coronaryheartdisease,CHD）的相关性。方法本研究共选取中国汉族冠心病患者231人（男性151,女性80）,正常对照组478人（男性297,女性181）。采用限制性酶切片段长度多态性（Restriction Fragment Length Polymorphism,RFLP）的方法对rs2970847进行基因分型。并测定所有入选对象的体重指数、血糖、总胆固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白等指标。结果冠心病患者中,rs2970847位点的CC基因型为144人（62．3％）,CT基因型为75人（32．5％）,TT基因型为12人（5．2％）;而正常对照中,CC基因型为235A-（49．2％）,CT基因型为201人（42．1％）,TT基因型为42人（8．8％）。rs2970847位点与冠心痛显著相关（P=0．0009）,纯合子CC基因型可以增加冠心病的患病风险（ORCC：2．14,95％CI=1．09-4．21）。结论PGC—1α基因多态位点rs2970847与冠心痛具有显著相关性,其CC基因型是冠心病的危险因素。     

Chemical composition and antiproliferative activity of essential oil from the leaves of a medicinal herb, Schefflera heptaphylla

Schefflera heptaphylla (L.) Frodin is a medicinal herb widely used as a main ingredient of the popular health tea formulation against infections in Southern China. Twenty-seven volatile compounds were identified by GC-MS analysis from the essential oil obtained from the leaves of S. heptaphylla, and 17 of them belonged to monoterpenes or sesquiterpenes. The main volatile constituent in S. heptaphylla was found to be a monoterpene, beta-pinene, comprising about 22% of the total volatile components. The essential oil showed significant antiproliferative activity against three cancer cell lines, MCF-7, A375 and HepG2 cells, with IC50 values of 7.3 microg/mL, 7.5 microg/mL and 6.9 microg/mL, respectively. The result of the cytotoxicity assay indicates that (-)-beta-pinene and (+)-beta-pinene (commercially available from Sigma) also possessed antiproliferative activity against the cancer cells MCF-7, A375 and HepG2 with IC50 values ranging from 147.1 to 264.7 microm.     

放血疗法适宜病症初探

探讨放血疗法的适宜病症,以指导临床更好地应用该疗法.采用回顾性期刊文献系列研究,检索中国知网中国期刊全文数据库、维普中文科技期刊全文数据库、万方数据库、中国生物医学文献数据库等4个数据库的放血疗法所有临床研究文献,统计放血疗法治疗疾病的文献频次、病例总数,运用临床流行病学和循证医学的原则和方法对文献进行分类分级,按照世界卫生组织关于疾病和有关健康问题的国际统计分类(ICD10)对疾病进行分类.检出有效文献1149篇,共计病例98 526例,共涉及18大类系统,261个病种.结果表明放血疗法的适宜病症广,值得推广,特别是对带状疱疹、痤疮、麦粒肿、颈椎病、口腔溃疡有明显的治疗优势.     

Clinical features of intracerebral hemorrhage in patients with colorectal cancer and its underlying pathogenesis

BACKGROUNDThe diagnosis of both cancer and intracerebral hemorrhage (ICH) in the same patient is not uncommon, but the clinical features and pathogenesis of patients with colorectal cancer (CRC) and ICH are still not well known.AIMTo investigate the clinical features and underlying pathogenesis of ICH in patients with CRC. METHODSA retrospective review of CRC patients complicated with ICH from three centers between January 2014 and December 2020 was performed. Clinical data such as laboratory examinations, imaging features, prognosis, and underlying pathogenesis were analyzed.RESULTSOf 16673 identified CRC patients, 20 (0.12%) suffered from ICH. There were 13 males and 7 females, with an average age (mean ± SD) of 68.45 ± 10.66 years. Fourteen patients (70%) had distant metastases and most patients (85%) showed an elevation of one or more cancer biomarkers. The hemorrhagic lesions in 13 patients (65%) were in the intracerebral lobe. Four patients were completely dependent and 4 died within 30 days after hemorrhage. Intratumoral hemorrhage (50%) and coagulopathy (50%) accounted for the majority of hemorrhages.CONCLUSIONPatients with ICH and CRC often have clinical features with lobar hemorrhage, distant metastases and poor prognosis. Intratumoral hemorrhage and coagulopathy are the main causes of ICH in patients with CRC.     

TGF-β1/Smad3信号转导通路与创伤后瘢痕形成

目的对近年TGF-β1/Smad3信号转导通路与创伤后瘢痕形成的相关研究作一综述。方法广泛查阅国内外近年有关TGF-β1/Smad3信号转导通路及与创伤后瘢痕形成的文献,并进行综述。结果 TGF-β1是纤维化疾病的重要影响因子,通过TGF-β1/Smad3信号通路转导产生其生物学效应。该途径受多种因子调控并在细胞及分子水平与其他信号通路串话。该途径参与创伤后早期炎性反应、创面愈合及后期病理性瘢痕的形成。在分子水平干预转导途径的各个环节,可以影响纤维化及细胞外基质沉着的进程。结论 TGF-β1/Smad3信号转导途径是影响创伤后瘢痕形成及细胞外基质沉着的重要途径。对该途径进行深入研究,可为临床促进创面的愈合及病理性瘢痕的防治奠定理论基础。     

Facile synthesis of photoactivatable adenosine analogs

Adenosine and its derivatives are important building blocks of the biological system. They serve as the universal energy currency, amplify intracellular signals for various signal transduction pathways, and can also be used as the co-substrates for enzymatic transformations. The synthesis and regulation of adenosine and its analogs rely on the adenosine binding proteins (ABPs). Dysregulated ABP activity contributes to numerous diseases such as cancer, metabolic disorders, and neurodegenerative diseases. Presently, there is intense interest in targeting ABPs for therapeutic purposes. A large fraction of the human ABP family remains poorly characterized. The need for innovative chemical probes to investigate ABP function in the native biological matrix is apparent. In this study, an adenosine analog, probe 1, with a photoaffinity group and biotin tag was synthesized using concise synthetic strategies. This probe was able to label and capture individual recombinant ABPs with good target selectivity. Probe 1 was also evaluated for its ability to label spiked ABP in complex cell lysates. This chemical probe, together with the labeling and enrichment assay, is of great value to interrogate the biological functions of ABPs and to elucidate their diversity under different physiological conditions.

Photoactivatable adenosine analog-enabled capture and enrichment of adenosine binding protein (ABP).