miR-20a-5p调控结核分枝杆菌诱导巨噬细胞凋亡相关基因表达的研究

目的 明确小非编码RNA(microRNA,miR-20a-5p)对结核分枝杆菌(MTB)诱导人巨噬细胞凋亡相关基因表达的调控作用。方法 构建可表达或抑制miR-20a-5p、转染miR-20a-5p抑制剂(miR-20a-5p-inhibitor,简称“miR-20a-5p-inh”)、作为慢病毒载体的阴性对照(LV1-NC)的慢病毒载体,由oligo-dT引物通过固相亚磷酰胺法合成茎环寡核苷酸,并克隆到含绿色荧光蛋白(GFP)的慢病毒载体pGLV3-GFP内,将构建的miR-20a-5p、miR-20a-5p-inh、LV1-NC转染THP-1人巨噬细胞3h,培养72h后用流式细胞仪分选出GFP+THP-1细胞,并分别采用减毒MTB菌株(H37Ra)感染8h和过氧化氢(H₂O₂)处理30min 两种方法诱导。通过实时定量PCR技术测定细胞中线粒体相关抗凋亡基因Bcl-2,以及促凋亡基因Bax、Bim和Bad的转录水平。同时,用蛋白免疫印迹法检测细胞裂解物中相关蛋白的表达。结果 慢病毒转染细胞后,在无刺激的THP-1细胞中miR-20a-5p的相对荧光强度值为12.21±1.29、miR-20a-5p-inh为9.68±1.38、LV1-NC为10.64±0.96,三者的表达差异均无统计学意义(q=1.815,P=0.385;q=2.072,P=0.602);但在H37Ra和H₂O₂的刺激后,miR-20a-5p过表达时其相对荧光强度值分别为7.20±0.53、8.55±0.82,明显高于LV1-NC (4.46±0.07、5.49±0.44)(q=50.250,P=0.007;q=1.041,P<0.01),miR-20a-5p受抑制时(1.88±0.08、1.44±0.21)明显低于LV1-NC(q=3.457,P=0.031;q=4.384,P=0.001)。在感染miR-20a-5p慢病毒的THP-1细胞中,H37Ra诱导Bcl-2的表达增加(相对荧光强度值由10.67±0.89增至14.98±0.88)(q=1.064,P=0.008),而感染miR-20a-5p-inh慢病毒时Bcl-2表达减少(由10.67±0.89降至6.49±0.47)(q=3.518,P=0.003);在miR-20a-5p过表达的THP-1细胞中Bim的转录水平减少(由1.22±0.05降至0.98±0.04)(q=1.240,P=0.011),当miR-20a-5p受抑制时Bim的转录水平增加(由1.22±0.05增至1.51±0.08)(q=2.460,P=0.021)。蛋白印迹法检测凋亡相关基因Bcl-2和Bim的蛋白表达水平,结果与基因转录水平相符。 结论 miR-20a-5p的表达水平影响MTB诱导的巨噬细胞凋亡相关基因Bcl-2和Bim的表达,并且与促凋亡基因Bim的水平呈负相关,提示miR-20a-5p可能通过调控Bim的表达而影响细胞凋亡。     

干扰趋化因子Fractalkine的表达对胰腺癌细胞株生物学功能的影响

目的 探讨阻断趋化因子Fractalkine(FKN)对胰腺癌细胞株SW-1990和PNAC-1生物学功能的影响.方法 腺病毒作为载体包装FKN-小干扰RNA(siRNA),转染人胰腺癌细胞株SW-1990和PNAC-1.应用克隆形成实验、MTT法和细胞侵袭实验检测FKN-siRNA转染与否、胰腺癌细胞增殖和侵袭力的变化.Western印迹法和RT-PCR检测转染FKN-siRNA的胰腺癌细胞FKN、TNF-α和IL-6蛋白和mRNA的表达.统计学处理采用单因素方差分析.结果 FKN-siRNA转染人胰腺癌细胞株SW-1990和PNAC-1后,与对照组的克隆数[分别为(1.97％±0.25％)和(1.77％±0.25％)]和阴性FKN-siRNA组的克隆数[分别为(2.10％±0.30％)和(1.97％±0.25％)]相比,细胞克隆增大,克隆数[分别为(5.27％±0.35％)和(4.60％±0.30％)]增多,差异均有统计学意义(F=113.51、103.86,P值均＜0.05).MTT实验48 h和72 h后,转入FKN-siRNA的转染人胰腺癌细胞株SW-1990和PNAC-1吸光度(48 h时分别为1.28±0.07和1.19±0.14;72 h时分别为1.49±0.11和1.52±0.16)高于对照组([48 h时分别为0.80±0.03和0.74±0.11;72 h时分别为0.89±0.03和0.93±0.04)和阴性FKN-siRNA组(48 h时分别为0.85±0.02和0.76±0.05;72 h时分别为0.89±0.02和1.07±0.09),差异均有统计学意义(F=83.80、71.99、17.19、23.51,P值均＜0.05).细胞侵袭实验中,转入FKN-siRNA的细胞侵袭力强于对照组和阴性FKN-siRNA组,差异有统计学意义(F=37.37、9.08,P值均＜0.05).FKN-siRNA转染后,SW-1990和PNAC-1细胞中,FKN蛋白(F=118.93、88.62)和mRNA(F=47.91、72.59)表达水平下降,同时TNF-α和IL-6的蛋白(FTNF-α=112.90、77.88,FIL-6=165.27、286.49)和mRNA(FTNFα=47.93、45.19,FIL-6=36.41、23.67)表达水平增高,差异均有统计学意义(P均＜0.05).结论 应用siRNA技术静默趋化因子FKN的功能后,胰腺癌细胞株的增殖活性和侵袭力增强,表明FKN可能抑制了胰腺癌细胞的生物学活性.     

重组人纽兰格林对心力衰竭大鼠线粒体功能的影响

目的观察重组人纽兰格林(neuregulin,NRG)对心力衰竭(HF)大鼠线粒体功能的影响,并探讨其机制。方法雄性Wister大鼠40只,结扎大鼠左冠状动脉建立缺血性HF模型。4周后,建模成功的24只大鼠随机分为NRG组[NRG 10μg/(kg·d)],AG825组[AG825 50μg/(kg·d)]及HF组,各组8只,另6只大鼠作为假手术组(Sham组)。各组干预10 d,于实验结束时提取左心室梗死区外心肌线粒体,测定线粒体膜电位(MMP)、呼吸控制率(RCR)及组织ATP酶活性,同时测定活性氧水平及谷胱甘肽过氧化物酶(GSHPX)mRNA水平。结果与Sham组比较,HF组大鼠线粒体功能受损,MMP下降、RCR及ATP酶活性降低,活性氧生成增加,GSHPX mRNA表达减少。与HF组比较,NRG组大鼠MMP增高,线粒体RCR改善,ATP酶活性增多,活性氧生成减少,GSHPXmRNA表达增加,而AG825组RCR、MMP和ATP酶活性均明显降低(P<0.05,P<0.01)。结论 NRG明显改善衰竭大鼠线粒体功能,NRG对衰竭心肌线粒体功能的保护作用,可能与其抗氧化应激作用有关。     

肝纤维化相关细胞因子双重RNA干扰质粒的构建及转染肝星状细胞研究

目的构建以大鼠CTGF和TIMP-1基因为靶点的双重RNA干扰表达载体质粒,以检测其转染肝星状细胞的效率.方法筛选出对CTGF和TIMP-1基因最有效的RNA干扰靶位,各设计1对含有短发夹结构的RNA 干扰靶点序列,分别克隆到质粒载体psiRNA-DUO-GFPzeo,构建含目的靶基因片段的重组质粒载体psiRNA-GFP-CT-GF、psiRNA-GFP-TIMP-1和psiRNA-GFP-Com（含有CTGF和TIMP-1）,进行酶切和测序鉴定.将构建成功的重组质粒psiRNA-GFP-CTGF、psiRNA-GFP-TIMP-1和psiRNA-GFP-Com转染大鼠肝星状细胞,在荧光显微镜下观察质粒转染情况,用流式细胞仪检测转染效率.结果酶切与测序结果提示重组质粒 psiRNA-GFP-CTGF、psiR-NA-GFP-TIMP-1和psiRNA-GFP-Com成功构建;成功将重组质粒psiRNA-GFP-CTGF、psiRNA-GFP-TIMP-1和psiRNA-GFP-Com转染肝星状细胞,在24小时,空质粒组、CTGF组、TIMP-1组和CTGF ＋TIMP-1组转染效率分别为15±2%、13±1%、15±1%和14±2%,均低于质粒psiRNA-GFP-Com转染组（Com组,20±2%,P〈0.05）;在48小时,空质粒组、CTGF组、TIMP-1组和CTGF＋TIMP-1组转染效率分别为10±2%、9±1%、8±2%和10±1%,均低于Com组的15±2%（P〈0.05）.结论成功构建靶向大鼠CTGF和TIMP-1最有效的RNA干扰靶位的双重RNA干扰表达质粒,能转染至肝星状细胞,并且psiRNA-GFP-Com转染效率最高.     

磷酸二酯酶5的短发夹RNA对肌细胞纤维化的作用抑制研究

目的 通过短发夹RNA(shRNA)延长对磷酸二酯酶5(PDE5)的抑制作用,探索shRNA-PDE5对细胞存活率的影响以及对心肌梗死后心功能及心室重构的作用及其机制.方法 (1)细胞实验:分离SD大鼠骨髓间充质干细胞(MSCs),将Ad-shRNA-PDE5和Ad-Null分别感染体外培养的MSCs,嘌呤霉素筛选稳定...     

The Investigation on Mechanical Performances of High-Strength Steel Reinforced Concrete Composite Short Columns under Axial Load

At present, the existing standards (AISC360-16, EN1994-1-1:2004, and JGJ138-2016) lack relevant provisions for steel-reinforced concrete (SRC) composite columns with high-strength steel. To investigate the axial compressive mechanical performance of short high-strength steel-reinforced concrete (HSSRC) columns, the axial load test was conducted on 12 short composite columns with high-strength steel and ordinary steel. The influences of steel strength, steel ratio, and the section form of steel on the failure modes, bearing capacity, and ductility of the specimens were studied. Afterward, the experimental data were compared with the existing calculation results. The results show: compared with the specimens with Q235 steel, the bearing capacity of the specimens with Q460 steel increases by 7.8–15.3%, the bearing capacity of the specimens with Q690 steel increases by 13.2–24.1%, but the ductility coefficient increases by 15.2–202.4%; with the increase of steel ratio, the bearing capacity and ductility of specimens are significantly improved. A change of the steel cross-section could influence the ductility of SRC columns more than their bearing capacity. Moreover, the calculation results show that present standards could not predict the bearing capacity of HSSRC columns. Therefore, a modified method for determining the effective strength of steel equipped in HSSRC columns was proposed. The results of the ABAQUS simulation also showed that the addition of steel fibers could significantly improve the bearing capacity of Q690 HSSRC columns. The research results provide a reference for engineering practices.     

Superior Proton Exchange Membrane Fuel Cell (PEMFC) Performance Using Short-Side-Chain Perfluorosulfonic Acid (PFSA) Membrane and Ionomer

Optimization of the ionomer materials in catalyst layers (CLs) which sometimes is overlooked has been equally crucial as selection of the membranes in membrane electrode assembly (MEA) for achieving a superior performance in proton exchange membrane fuel cells (PEMFCs). Four combinations of the MEAs composed of short-side-chain (SSC) and long-side-chain (LSC) perfluorosulfonic acid (PFSA) polymers as membrane and ionomer materials have been prepared and tested under various temperatures and humidity conditions, aiming to investigate the effects of different side chain polymer in membranes and CLs on fuel cell performance. It is discovered that SSC PFSA polymer used as membrane and ionomer in CL yields better fuel cell performance than LSC PFSA polymer, especially at high temperature and low RH conditions. The MEA with the SSC PFSA employed both as a membrane and as an ionomer in cathode CL demonstrates the best cell performance amongst the investigated MEAs. Furthermore, various electrochemical diagnoses have been applied to fundamentally understand the contributions of the different resistances to the overall cell performance. It is illustrated that the charge transfer resistance (R_ct) made the greatest contribution to the overall cell resistance and then membrane resistance (R_m), implying that the use of the advanced ionomer in CL could lead to more noticeable improvement in cell performance than only the substitution as the membrane.     

RAB5A基因对大肠癌细胞侵袭能力的影响

目的研究RAB5A基因转染后对大肠癌细胞侵袭能力的影响.方法用脂质体将RAB5A反义寡核苷酸转染人大肠癌细胞LS174T,48h后transwell小室法观察其对细胞转移能力的影响.结果 RAB5A反义寡核苷酸转染后48h,transwell小室结果可见转染后大肠癌细胞LS174T的侵袭和运动能力明显低于对照组(P《0.01).结论 RAB5A在人大肠癌细胞的侵袭及转移特性的形成中具有重要的作用,RAB5A的反义分子能够有效地阻断该基因表达的翻译过程,在细胞内发挥预期作用.     

星形胶质细胞形态学观察在高通量药物筛选水通道蛋白4抑制剂中的应用

目的:探讨星形胶质细胞形态学观察在高通量药物筛选水通道蛋白4(AQP4)抑制剂中的应用,为脑水肿和其他水转运障碍疾病的治疗提供理论依据.方法:应用药物库和原代培养的人星形胶质细胞,以AQP4 siRNA转染细胞后的形态学改变和阻抑蛋白表达作参照,观察药物对星形胶质细胞形态学改变的影响,Western blot方法检测能明显改变细胞形态的靶药物对AQP4蛋白表达的影响,并测定靶药物作用剂量下细胞ATP活性.结果:洛伐他汀、胍那苄和米安色林在12～24 h内能明显改变人星形胶质细胞形态,减低AQP4蛋白表达,与AQP4的siRNA转染细胞4 d后的形态改变和蛋白阻滞效果类似,靶药物作用剂量下对细胞ATP活性无影响.结论:星形胶质细胞的形态学观察可作为大规模药物筛选的初步指标,本研究筛选出的靶药物可能是潜在的AQP4抑制剂.