细胞共培养技术在医药研究中的应用

细胞共培养由于能更好地模拟体内环境,便于更好的观察细胞与细胞、细胞与培养环境之间的相互作用以及探讨药物的作用机制和可能的作用靶点,填补了单层细胞培养与整体动物实验研究的鸿沟,近年来倍受医药领域的关注,成为药物研发、生物制药领域的研究热点.细胞共培养方法包括直接共培养和间接共培养,主要用于疾病病理基础、新型治疗手段以及药物活性筛选的研究.现有的细胞共培养技术主要用于单一药物的药效学研究,用于联合药物相互作用的研究甚少.中药复方之间协同配伍,减毒增效.细胞共培养技术符合中药多成分、多靶点的作用特点,这对于未来探讨中药联合用药对机体的作用及机制的研究具有一定参考价值,为中药及复方研究提供了一种新的手段.该文就细胞共培养技术的方法与运用进行了概述,并对该技术运用于中药及复方的研究进行了展望.     

经颅多普勒超声在儿童颅内压增高性疾病中的应用

经颅多普勒超声(transcranial doppler ultrasonography,TCD)是一种非侵入性评价颅内动脉的检测手段,自上世纪80年代问世以来,已被广泛性应用于临床工作的各个领域.由于其操作简便、重复性好,可以对患者进行床旁连续、动态观察,尤其适用于监测危重症患者.颅内压增高是儿童致病致死的再要原因之一,它可使脑血流灌注下降,造成脑功能障碍等严重后果,因此颅内压监测有重要临床意义.TCD作为一种无创性监测工具,可根据其血流速度、相关参数及血流频谱的变化对颅内压增高患者脑血流动力学进行动态监测与评估,从而达到监测颅内压改变的目的.本文着重阐述TCD在几种常见的儿童颅内高压性疾病中的应用进展.     

NSE和S-100在先天性胆管扩张症发病机制中的协同作用

目的 探讨神经细胞特异性烯醇化酶(NSE)和S-100蛋白在先天性胆管扩张症(CBD)发病中的作用及临床意义。方法 2007～2009年连续CBD患儿36例,均行囊肿和胆囊切除、肝门空肠吻合术,切取囊肿远、近端囊壁和胆囊壁为实验组。选取非正常怀孕或因其母亲特殊原因需终止妊娠胎儿15例的肝外胆管和胆囊为对照组。每组标本分别固定、包埋,免疫组织化学染色检测对比其NSE和S-100表达,比较其染色结果和分布特点,并对囊肿直径和胆道不同部位NSE和S-100的表达进行相关分析。结果 NSE和S-100在囊肿远端表达(1.86±1.29,1.81±1.04)均弱于囊肿近端表达(3.61±1.92,3.58±1.95)(P＜0.05);囊肿远端与胆囊(3.42±1.99,3.72±2.08)间差异有统计学意义(P＜0.05),囊肿近端与胆囊间差异无统计学意义(P＞0.05)。胎儿胆管(4.86±2.97,5.14±2.73)和胎儿胆囊(3.71±2.14,4.00±1.63)间NSE和S-100差异无统计学意义(P＞0.05)。组间比较,囊肿远端与胎儿胆管、胆囊间NSE和S-100差异均有统计学意义(P＜0.05);囊肿近端与胎儿胆管、胆囊间差异无统计学意义(P＜0.05);两组胆囊间差异无统计学意义(P＞0.05)。囊肿直径和胆道不同部位NSE和S-100的表达均呈负相关(P＜0.05)。结论 胆道远端神经节细胞和神经纤维的发育和分布受限与CBD发病关系密切,与其囊肿大小呈负相关;NSE与S-100表达在CBD发病中协同互补,可为CBD术中囊肿切除范围的正确判断提供理论依据。     

增殖性糖尿病视网膜病变多次手术临床分析

目的：探讨增殖性糖尿病视网膜病变玻璃体切割术后多次手术的原因及处理。方法：回顾性分析3a来因增殖性糖尿病视网膜病变行玻璃体切割术后需要再次手术的患者。结果：患者189眼术后需要再次进行手术的为24例26眼（占总观察眼数13.8%）。分别是：玻璃体再积血3眼,前房积血1眼,视网膜脱离11眼（占总观察眼数5.8%,占再手术眼42.3%,其中3眼合并新生血管性青光眼）,白内障6眼（占总观察眼数3.2%）,单纯硅油取出5眼。189眼中有6眼发生了新生血管性青光眼（3.2%）,6眼均为玻璃体晶状体联合手术或玻璃体切割术后又摘除白内障病例。2次手术19眼,3次或以上手术7眼（其中5眼合并视网膜脱离）。结论：发生视网膜脱离是糖尿病视网膜病变玻璃体切割术后需要多次手术的主要原因;也占据需要3次或以上手术的主要部分。新生血管性青光眼的发生值得重视,出现视网膜脱离时要提高警惕,需要摘除晶状体时必须慎重。     

妊娠早期阻断协同刺激分子诱导自然流产模型孕鼠脾脏细胞母-胎免疫耐受的研究

目的　探讨阻断协同刺激分子———CD80 和CD86对自然流产模型孕鼠妊娠结局及孕鼠脾脏免疫细胞对父系抗原免疫耐受状态的影响。方法　将雌性小鼠 (CBA/J)分别与BALB/c及DBA/2两种雄性小鼠合笼交配 ,分别建立正常妊娠模型CBA/J×BALB/c( 2 0只 ,对照组 )和自然流产模型CBA/J×DBA/2 ( 2 0只 ,研究组 )。CBA/J小鼠于妊娠第 4天 (着床期 )腹腔分别注射大鼠同型IgG 0 2mg( 10只 ) ,或大鼠抗小鼠CD80 和CD86单克隆抗体 ( 10只 )。妊娠第 9天 ,采用单向混合淋巴细胞反应 ,分析孕鼠脾脏免疫细胞对父系抗原的增殖能力 ,并测定细胞培养上清液中白细胞介素 2(IL 2 )水平 ,以研究脾脏细胞母 胎免疫耐受状态 ;妊娠第 14天观察两组的胚胎吸收率。结果　 ( 1)研究组中 ,腹腔注射大鼠IgG的孕鼠胚胎吸收率为 2 4 3% ,而注射大鼠抗小鼠CD80 和CD86单克隆抗体的孕鼠胚胎吸收率为 9 8% ,两者比较 ,差异有显著性 (P <0 0 5 )。 ( 2 )应用大鼠抗小鼠CD80 和CD86单克隆抗体 ,使妊娠 9d的孕鼠脾脏免疫细胞对父系抗原的增殖能力及IL 2水平显著下降(P <0 0 5 )。结论　孕早期阻断协同刺激分子 ,可诱导产生孕鼠脾脏免疫细胞对父系抗原的免疫耐受 ,从而使自然流产模型孕鼠的妊娠结局达到正常妊娠水平。     

卵巢肿瘤内部血流阻力指数与Ki67及Snail表达的关系

目的 研究卵巢肿瘤内部血流阻力指数(RI)与肿瘤组织内的Ki67及Snml表达间的关系.方法 选择2005年10月至2006年10月复旦大学附属妇产科医院妇科收治的住院手术卵巢肿瘤患者共96例,术前应用彩色多普勒超声血流显像(CDFI)检测肿瘤内部RI,术后应用免疫组织化学方法检测其中36例卵巢良性肿瘤和47例卵巢上皮性癌中Ki67及Snail的表达.结果 良性卵巢肿瘤内部RI显著高于卵巢上皮性癌(0.61±0.19对0.38±0.09.P<0.01).Ki67及Snail在卵巢上皮性癌中的表达要显著高于良性肿瘤(分别为70.2%时13.9%,55.3%对8.3%,P<0.01),且在内部血流RI<0.4的卵巢肿瘤中的表达要高于RI≥0.4者[分别为58.0%对27.3%(P<0.01),44.0%对21.2%(P<0.05)].结论 卵巢肿瘤内部血流RI与肿瘤组织内的Ki67及Snail表达有关;RI<0.4的肿瘤为恶性的可能性要大于RI≥0.4者.     

Leflunomide therapy for adult patients with steroid-dependent minimal change disease or primary focal segmental glomerulosclerosis

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狼毒大戟中松香烷型二萜内酯的抗肿瘤活性研究

摘 要 目的： 对狼毒大戟中松香烷型二萜内酯类化学成分进行分离和结构鉴定,将得到的单体化合物与6种不同的人肿瘤细胞株相互作用探讨其抗肿瘤活性。方法: 通过硅胶、ODS等柱色谱与1D、2D-NMR波谱学测试技术结合,从狼毒大戟中分离并确定4个松香烷型二萜内酯类化合物：jolkinolide A (1)、jolkinolide B (2)、17 hydroxyjolkinolide A (3)、17-hydroxyjolkinolide B (4)。采用噻唑蓝比色法（MTT法）分别测定其与6种人肿瘤细胞株：肝癌HepG-2、乳腺癌MCF-7、胃癌SGC 7901、胃癌BGC-823、胃癌MGC-803、宫颈癌Hela相互作用结果,并计算抑制率与半抑制率(IC₅₀)。结果: 化合物2对肝癌HepG-2、乳腺癌MCF-7、宫颈癌Hela细胞的增殖有显著得抑制作用IC₅₀均＜25 μmol·L^-1,化合物4对乳腺癌MCF 7细胞的增殖有显著抑制作用IC₅₀＜30 μmol·L^-1。结论: 狼毒大戟中松香烷型二萜内酯类化合物具有显著的抗肿瘤活性。     

Analysis of circRNA‐mRNA expression profiles and functional enrichment in diabetes mellitus based on high throughput sequencing

To study the pathogenesis of diabetes mellitus (DM) and identify new biomarkers, high‐throughput RNA sequencing provides a technical means to explore the regulatory network of MD gene expression. To better elucidate the genetic basis of DM, we analysed the circRNA and mRNA expression profiles in serum samples from diabetic patients. The circRNAs and mRNAs with abnormal expression in the DM group and non‐diabetic group (NDM) were classified by RNA sequencing and differential expression analysis. The circRNA‐miRNA‐mRNA regulatory network reveals the mechanism by which competitive endogenous RNAs (ceRNAs) regulate gene expression. The biological functions and interactions of circRNA and mRNA were analysed by gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Differential expression analysis showed that 441 circRNAs (366 up‐regulated, 75 down‐regulated) and 683 mRNAs (354 up‐regulated, 329 down‐regulated) were significantly differentially expressed in the DM group compared with the NDM group. Screening of the differential genes at the nodes of the interaction network showed that a single circRNA could interact with multiple miRNAs and then jointly regulate more mRNAs. In addition, the expressions of circRNA CNOT6 and AXIN1 as well as mRNA STAT3, MYD88, and B2M were associated with the progression of diabetes. Enrichment pathway analysis indicated that differentially expressed circRNA and mRNA may participate in Nod‐like receptor signalling pathway, insulin signalling pathway, sphinolipid metabolism pathway, and ribosome pathway, and play a role in the pathogenesis of diabetes. This study provides a theoretical basis for elucidating the molecular mechanism of DM occurrence and development at circRNA and mRNA levels.     

Gut-derived lipopolysaccharide promotes alcoholic hepatosteatosis and subsequent hepatocellular carcinoma by stimulating neutrophil extracellular traps through toll-like receptor 4

Background/AimsBinge drinking leads to many disorders, including alcoholic hepatosteatosis, which is characterized by intrahepatic neutrophil infiltration and increases the risk of hepatocellular carcinoma (HCC). Molecular mechanisms may involve the migration of bacterial metabolites from the gut to the liver and the activation of neutrophil extracellular traps (NETs).MethodsSerum samples from both binge drinking and alcohol-avoiding patients were analyzed. Mouse models of chronic plus binge alcohol-induced hepatosteatosis and HCC models were used.ResultsA marker of NETs formation, lipopolysaccharide (LPS), was significantly higher in alcoholic hepatosteatosis and HCC patients and mice than in controls. Intrahepatic inflammation markers and HCC-related cytokines were decreased in mice with reduced NET formation due to neutrophil elastase (NE) deletion, and liver-related symptoms of alcohol were also alleviated in NE knockout mice. Removal of intestinal bacteria with antibiotics led to decreases in markers of NETs formation and inflammatory cytokines upon chronic alcohol consumption, and development of alcoholic hepatosteatosis and HCC was also attenuated. These functions were restored upon supplementation with the bacterial product LPS. When mice lacking toll-like receptor 4 (TLR4) received chronic alcohol feeding, intrahepatic markers of NETs formation decreased, and hepatosteatosis and HCC were alleviated.ConclusionsFormation of NETs following LPS stimulation of TLR4 upon chronic alcohol use leads to increased alcoholic steatosis and subsequent HCC.