凋亡蛋白抑制因子livin基因siRNA表达载体的构建与转染

目的:构建3个针对凋亡蛋白抑制因子livin基因mRNA的小干扰RNA(siRNA)表达载体,然后转染HCT-8/V细胞。为研究livin基因沉默对HCT-8/V细胞耐长春新碱特性的影响奠定基础。方法:合成3对靶基因livin的siRNA寡核苷酸序列通过退火生成转录模板,与线性pGCsi-H1/Neo/GFP质粒连接,转化DH5α钙化菌抽提重组质粒,测序鉴定。用脂质体2000将重组质粒转染HCT-8/V细胞并用G418筛选。结果:转化大肠杆菌涂布平板长出阳性菌落,测序结果与所设计、合成的livin的siRNA转录模板序列一致;在荧光显微镜下观察到HCT-8/V细胞表达绿色荧光蛋白(GFP)证实重组质粒已转染入细胞并用G418剔除了未转染质粒的细胞。结论:成功构建livin基因的siRNA表达载体,转染并筛选出转染了livin siRNA表达载体的HCT-8/V细胞。     

咪达普利与氯沙坦对糖尿病大鼠心肌间质重构的对比研究

目的:研究在不同水平阻断肾素-血管紧张素系统(RAS)对糖尿病大鼠心肌间质重塑过程的影响及保护机制。方法:50只健康雄性SD大鼠随机分为空白对照组(10只)和糖尿病组(40只),糖尿病组用链脲佐菌素(STZ)50 mg/kg腹腔注射建立实验性糖尿病大鼠模型,将建模成功的39只大鼠随机分为咪达普利组10只、氯沙坦组10只、联合用药组10只及模型对照组9只,给药9周后麻醉处死,免疫组化SP法检测肿瘤坏死因子-α(TNF-α)、核因子-κB(NF-κB)、基质金属蛋白酶-9(MMP-9)的蛋白表达,RT-PCR法测NF-κB、MMP-9和TNF-α的mR-NA表达。VG染色观察心肌胶原容积分数(CVF)变化。结果:与空白对照组相比,糖尿病组大鼠心脏指数、左心室指数、心肌CVF明显升高,心肌间质发生重构,TNF-α、MMP-9、NF-κB含量和TNF-αmRNA、MMP-9 mRNA和NF-κB mRNA的表达均增高。咪达普利、氯沙坦及联合用药干预后,上述指标表达均明显降低,但咪达普利和氯沙坦的改善程度无明显差异,联合用药明显优于单独用药。结论:咪达普利和氯沙坦可能通过影响TNF-α、MMP-9的表达来改善糖尿病心肌间质重构,NF-κB参与了这一过程。     

肺动脉平滑肌细胞上TMEM16A的表达及其阻滞剂对细胞增殖的作用

目的：研究TMEM16A在大鼠肺动脉平滑肌细胞(PASMCs)上的表达及其阻滞剂(T16Ainh-A01)对PASMCs增殖的作用。方法组织块贴壁法分离正常大鼠PASMCs,原代培养。光镜下观察细胞形态,细胞免疫化学和细胞免疫荧光进行鉴定。细胞免疫荧光检测TMEM16A在PASMCs上表达。原代培养的3~6代PASMCs分别在10μmol/L T16Ainh-A01、20μmol/L T16Ainh-A01和50μmol/L T16Ainh-A01下培养24 h,用CCK-8法检测细胞增殖情况。结果倒置显微镜下PASMCs以长梭形为主,细胞核卵圆形,部分细胞重叠区域呈典型的“峰-谷”状结构。免疫学检测显示胞浆内与细胞长轴平行的肌丝被染成棕黄色,标记肌丝的红色荧光也与细胞长轴平行,标记细胞膜上的TMEM16A的绿色荧光可见表达,而细胞核不染色。CCK-8实验检测,相对于对照组,10μmol/L T16Ainh-A01组细胞活力均数为(96.74±0.92)%,20μmol/L T16Ainh-A01组均数为(92.21±1.25)%,50μmol/L T16Ainh-A01组均数为(87.41±3.27)%,差异均具有统计学意义(P<0.01)。结论正常大鼠PASMCs表达TMEM16A;T16Ainh-A01可抑制PASMCs增殖,其抑制作用呈浓度依赖性;TMEM16A可能参与调控PASMCs增殖。     

消化道肿瘤环氧合酶-2表达与凋亡抑制蛋白及化疗药敏性的关系

目的 探讨消化道肿瘤环氧合酶(COX)-2表达与凋亡抑制蛋白及体外化疗药敏性的关系.方法 对84例胃癌、大肠癌标本进行噻唑蓝(MTT)比色法体外化疗药物敏感性实验,并进行COX-2、p53、Survivin、bel-2免疫组织化学染色.结果 肿瘤组织COX-2、p53、Survivin、bel-2表达率分别为70.3%、64.3%、89.3%、60.7%;COX-2与Survivin、bcl-2表达呈正相关(r=0.5072、0.3783,均P<0.01).在肿瘤COX-2强表达组,紫杉醇(PTX)、表阿霉素(eADM)、羟基喜树碱(OPT)对肿瘤细胞抑制率明显低于弱表达组(均P<0.05);p53强表达与PTX、顺铂(DDP)对肿瘤细胞的抑制率明显降低有关(均P<0.05);Survivin强表达时,长春新碱(VCR)、DDP对肿瘤细胞抑制率明显降低(均P<0.05);bcl-2强表达时,5-氟尿嘧啶(5-Fu)、VCR、eADM、奥沙利铂(OXA)对肿瘤细胞抑制率明显低于弱表达组(均P<0.05).结论 消化道肿瘤COX-2通过抑制肿瘤细胞凋亡参与了肿瘤的多药耐药.     

Endocrine Active Compounds: From Biology to Dose Response Assessment

Histone deacetylase (HDAC) inhibitors target key steps of tumor development: They inhibit proliferation, induce differentiation and/or apoptosis, and exhibit potent antimetastatic and antiangiogenic properties in transformed cells in vitro and in vivo. Preliminary studies in animal models have revealed a relatively high tumor selectivity of HDAC inhibitors, strenghtening their promising potential in cancer chemotherapy. Until now, preclinical in vitro research has almost exclusively been performed in cancer cell lines and oncogene-transformed cells. However, as cell proliferation and apoptosis are essential for normal tissue and organ homeostasis, it is important to investigate how HDAC inhibitors influence the regulation of and interplay between proliferation, differentiation, and apoptosis in primary cells as well. This review highlights the discrepancies in molecular events triggered by trichostatin A, the reference compound of hydroxamic acid-containing HDAC inhibitors, in hepatoma cells and primary hepatocytes (which are key targets for drug-induced toxicity). The implications of these differential outcomes in both cell types are discussed with respect to both toxicology and drug development. In view of the future use of HDAC inhibitors as cytostatic drugs, it is highly recommended to include both tumor cells and their healthy counterparts in preclinical developmental studies. Screening the toxicological properties of compounds early in their development process, using a battery of different cell types, will enable researchers to discard those compounds bearing undesirable adverse activity before entering into expensive clinical trials. This will not only reduce the risk for harmful exposure of patients but also save time and money.     

III型分泌系统及其抑制剂的研究进展

细菌分泌系统的发现是近年来细菌致病机制研究的重点。致病菌为在宿主体内生存、繁殖、扩散，会分泌一些蛋白性质的毒性因子。目前认为革兰阴性致病菌在长期进化过程中形成入侵宿主细胞的特异性分泌系统共有5种类型(I~V型)，其中最显著的是细菌Ⅲ型分泌系统(type three secretion system, T3SS)，与细菌致病性密切相关。T3SS抑制剂的策略是细菌分泌毒力蛋白，阻止其对宿主细胞的侵染。本文对细菌T3SS的组成、T3SS抑制剂筛选系统、T3SS抑制剂以及作用机制等进行综述，为深入研究以T3SS为靶点的药物设计提供参考。     

Turnover of 125I-labelled tissue kallikrein following intraduodenal or intravenous administration

Tissue kallikrein is released in the body both physiologically and in many inflammatory disorders. Little is, however, known about the turnover of released tissue kallikrein in humans. Approximately 1 mg of tissue kallikrein (mol wt 43 000 Da) was purified from 85 L human urine by: (1) ultracentrifugation, (2) filtration through an aprotinin-coupled Sepharose 4B column, followed by (3) gel filtration over a Sephadex G-75 column. The elimination, after intraduodenal or intravenous administration of purified tissue kallikrein radiolabelled with ¹²⁵I, was followed by collecting serial samples of plasma, urine and faeces from three volunteers. Within 72 h, about 96% of the intraduodenally administered radioactivity had been excreted in urine, and approximately 5.4% in faeces, mainly as ¹²⁵I. No intact ¹²⁵I-tissue kallikrein was found in plasma, urine or faeces after the intraduodenal instillation of the protein. The plasma half-life of ¹²⁵I-tissue kallikrein up to 3 h after intravenous injection was 9 min and, thereafter, 20 h. The ¹²⁵I-tissue kallikrein was quickly bound to a plasma protein with a mol wt of about 67 kDa, but some of the radioiodinated tissue kallikrein was still unbound 15 min after injection, judged by gel filtration on Sephadex G-200 columns. Most of the radioactivity was excreted in the urine as ¹²⁵I, but about 4- 6% was recovered as free ¹²⁵I-tissue kallikrein. Conclusion: The use of tissue kallikrein as an oral drug appears, therefore, to be useless. Tissue kallikrein released into plasma seems to be quickly bound to a protein with a mol wt of 67 kDa, probably kallistatin or Protein C inhibitor, but some tissue kallikrein seems to be unbound and may have some physiological or pathophysiological action. The unbound tissue kallikrein is, at least partly, cleared from the circulation by the kidneys, and tissue kallikrein in the urine may partly be derived from plasma.     

非经典型蛋白激酶C的研究进展

非经典型蛋白激酶C是G蛋白偶联受体系统中的效应物，其在细胞内信号传递过程中起重要作用，可介导细胞增殖和分化、影响细胞周期和细胞凋亡、参与代谢调节、促进肿瘤形成和转移等。因此，靶向非经典型蛋白激酶C的药物在疾病治疗中有广泛的应用前景。     

The rare coexistence of high titer inhibitor development and gastrointestinal stromal tumor in a patient with severe hemophilia: A case report

Hemophilia is a hereditary disease with impaired blood coagulation due to a genetic deficiency of blood coagulation factors. The development of inhibitors further complicates the course of the disease and management. The case is here reported of a haemophilia patient who presented with coexisting development of high titer inhibitor with Gastrointestinal Stromal Tumor (GIST) diagnosis and was admitted with upper gastrointestinal system bleeding. The patient had no prior history of inhibitor presence. During all procedures including surgery, excellent hemostasis was achieved with rFVIIa treatment and no hemorrhagic complication was observed. To the best of our knowledge, this constitutes the first reported case of GIST associated with inhibitor development in a hemophilia A patient.     

Tariquidar inhibits P-glycoprotein drug efflux but activates ATPase activity by blocking transition to an open conformation

P-glycoprotein (P-gp, ABCB1) is a drug pump that confers multidrug resistance. Inhibition of P-gp would improve chemotherapy. Tariquidar is a potent P-gp inhibitor but its mechanism is unknown. Here, we tested our prediction that tariquidar inhibits P-gp cycling between the open and closed states during the catalytic cycle. Transition of P-gp to an open state can be monitored in intact cells using reporter cysteines introduced into extracellular loops 1 (A80C) and 4 (R741C). Residues A80C/R741C come close enough (<7 Å) to spontaneously cross-link in the open conformation (<7 Å) but are widely separated (>30 Å) in the closed conformation. Cross-linking of A80C/R741C can be readily detected because it causes the mutant protein to migrate slower on SDS-PAGE gels. We tested whether drug substrates or inhibitors could inhibit cross-linking of the mutant. It was found that only tariquidar blocked A80C/R741C cross-linking. Tariquidar was also a more potent pharmacological chaperone than other P-gp substrates/modulators such as cyclosporine A. Only tariquidar promoted maturation of misprocessed mutant F804D to yield mature P-gp. Tariquidar interacted with the transmembrane domains because it could rescue a misprocessed truncation mutant lacking the nucleotide-binding domains. These results show that tariquidar is a potent pharmacological chaperone and inhibits P-gp drug efflux by blocking transition to the open state during the catalytic cycle.