VS-1基因转染对血管内皮细胞缺氧/复氧损伤的保护作用

目的构建Ad-VS-1基因转染HUVEC的体外缺氧/复氧模型,探讨VS-1转基因治疗对血管内皮细胞缺氧/复氧损伤的保护作用及其机制。方法①建立HUVEC缺氧/复氧（H/R）损伤模型,分为4组：空白组,H/R组,空载腺病毒转染＋H/R组,Ad-VS-1转染＋H/R组;②测定内皮细胞活力（MTT）,超氧化物歧化酶活力（SOD）、丙二醛（MDA）、eNOS、NO表达、炎症因子ICAM-1、VCAM-1、TNF-α的表达情况;③在Ad-VS-1转染＋H/R组基础上增设Hb、KT5823、SB203580和W ortm an in 4个信号抑制组,流式细胞仪测定细胞凋亡率进行统计学分析。结果①成功构建Ad-VS-1转染HUVEC表达后H/R模型;②与空白组比较,H/R组和空腺病毒感染＋H/R组的MTT、SOD含量、eNOS、NO表达显著降低,而MDA、ICAM-1、VCAM-1、TNF-α含量显著增加（P〈0.05）;与H/R组和空载腺病毒感染＋H/R组比较,VS-1转染＋H/R组MTT、SOD、eNOS、NO表达含量明显回升,而MDA、ICAM-1,TNF-α生成量明显回落（P〈0.05）;③与H/R组和空载腺病毒感染＋H/R组比较,VS-1转染组细胞凋亡率明显降低,而4组信号抑制剂组的细胞凋亡率较VS-1转染组均明显回升（P〈0.05）。结论Ad-VS-1成功转染HUVEC,并通过PI3K/Akt-eNOS-NO-cGMP-PKG和P38MPAK等信号途径,抑制氧化应激和炎症反应,产生显著的抗血管内皮细胞缺氧/复氧损伤作用,为今后VS-1抗心肌缺血/再灌注损伤研究奠定基础。     

姜黄素诱导卵巢癌细胞株A2780细胞周期阻滞和凋亡

目的 探讨姜黄素对卵巢癌细胞株A2780的生长抑制作用及其机制。方法 10一50μmol/L姜黄素作用6—24h后，MTT比色法检测细胞生长活性，流式细胞仪测定细胞周期时相变化，末端TdT酶标记技术和DNA Ladder检测细胞凋亡；透射电镜观察细胞超微结构变化。结果 姜黄素能显著抑制卵巢癌细胞株A2780的体外生长，呈时间与剂量依赖性；细胞周期阻滞于G0／Gl期；部分细胞出现凋亡形态学改变，凝胶电泳可见“梯形”条带，凋亡率为6．41％一28．48％(P＜o．01)。结论 姜黄素通过抑制细胞增殖、诱导细胞凋亡等机制，能显著地抑制卵巢癌细胞的体外生长。     

全反式维甲酸联合细胞因子诱导无血清培养的骨髓基质干细胞向神经细胞分化

目的研究全反式维甲酸与表皮生长因子、碱性成纤维细胞生长因子及神经生长因子等细胞因子联合诱导体外无血清培养的骨髓基质干细胞（BMSCs）向神经细胞分化的作用。方法用含2%Utroser G的UltraCULTURE无血清培养体系体外扩增大鼠BMSCs，免疫细胞化学染色鉴定其表面标志，用以全反式维甲酸与表皮生长因子、碱性成纤维细胞生长因子及神经生长因子为主要成分的复合诱导液诱导BMSCs向神经细胞方向分化，镜下观察细胞形态学变化，用抗神经元特异性烯醇化酶（NSE）抗体和抗胶质纤维酸性蛋白（GFAP）抗体进行免疫细胞化学染色鉴定诱导后的神经细胞。结果经全反式维甲酸、表皮生长因子、碱性成纤维细胞生长因子及神经生长因子联合诱导后的BMSCs表现为神经细胞形态特征，诱导后的细胞可存活14 d以上，免疫细胞化学染色显示NSE和GFAP均有阳性表达，前者阳性率为65.6%，后者阳性率为12.5%。结论全反式维甲酸与表皮生长因子、碱性成纤维细胞生长因子及神经生长因子联合应用可在体外高效、稳定地诱导无血清培养的BMSCs分化为神经细胞。     

焦虑情绪对老年慢性便秘患者自主神经功能的影响

目的 通过心理因素评价及心率变异性(HRV)的检测,探讨老年便秘患者焦虑症状与自主神经功能的关系及可能机制.方法 收集住院及门诊查体便秘患者资料共计52例,称为便秘组,对照组40例,均为健康老年男性,所有入选者行24h动态心电图检查,观察心率变异性指标SDNN、SDANN、RMSSD、TP、LF及HF并填写焦虑自评量表(SAS).结果 便秘组SAS标准分明显高于正常对照组,便秘组SDNN、RMSSD、TP、LF及HF值低于对照组,且便秘患者焦虑自评量表SAS标准分与SDNN、RMSSD、TP、LF和HF均呈显著负相关.结论 焦虑等心理因素在便秘的发病中起一定作用,焦虑症状的产生可能与便秘患者自主神经系统功能异常有关,尤以迷走神经为甚.     

缩胆囊素-A受体基因多态性与焦虑症的相关性

目的探讨焦虑症(AD)与缩胆囊素-A(CCK-A)受体基因多态性的相关性.方法运用PCR技术检测88例AD患者和87例健康对照(NC)组缩胆囊素-A受体基因多态性,并加以对照分析.结果 CCK-A受体基因的3种基因型(A1/A1、A1/A2和A2/A2),在AD组的分布分别为5%、41%和54%,在NC组分别为1%、44%和55%,两组间无显著性差异(P＞0.05);A1和A2等位基因频率,在广泛性焦虑(GAD)组分别为34%、66%,惊恐障碍(PD)组分别为18%和82%,两组间均有显著性差异(P＜0.05);A1/A1、A1/A2、A2/A2基因型分布频率,GAD组分别为5%、59%、36%,PD组分别为6%、24%和70%,两组间均有显著性差异(P＜0.05).结论 CCK-A受体基因多态性与AD之间无显著关联;AD两种亚型间CCK-A受体基因多态性有显著性差异.     

关节腔注射氨甲环酸对全膝关节置换术后的影响

目的：探讨关节腔注射氨甲环酸（ TXA）对全膝关节置换术（ TKA）术后出血量、术后功能恢复及术后并发症的影响。方法因骨性关节炎（ OA）行全膝关节置换术的患者46例,随机分为氨甲环酸组（ A组,23例）,对照组（ B组,23例）,A组于手术缝皮结束后关节腔注射氨甲环酸（2．0 g氨甲环酸溶入20 mL生理盐水）,B组注射20 mL生理盐水,均夹闭引流管2 h。比较两组术后可见失血量、输血例数、输血量、术后血红蛋白（Hb）及红细胞比容（HCT）,术后6周随访膝关节HSS评分及下肢深静脉血栓（ DVT）或肺栓塞发生情况。结果两组患者术后可见失血量、输血例数、输血量、术后Hb及HCT比较差异均有统计学意义（ P＜0．05）。比较两组术中、术后3小时纤维蛋白原（Fg）、凝血酶原时间（PT）和活化部分凝血活酶时间（APTT）差异无统计学意义（P＞0．05）。两组患者膝关节HSS评分差异无统计学意义（P＞0．05）。46例患者术后2周行下肢血管多普勒超声检查未发现DVT形成,术后6周随访未发现下肢DVT或肺栓塞发生。结论关节腔注射氨甲环酸在全膝关节置换术后能明显降低患者术后失血量及输血率,并且没有增加下肢DVT形成的风险。     

胆囊癌组织中蛋白赖氨酸乙酰化的变化及临床意义

目的:探讨蛋白赖氨酸乙酰化在胆囊癌组织中的变化情况。方法:采用Western 免疫印迹法,分别检测30例胆囊癌组织和20例慢性胆囊炎组织中蛋白赖氨酸乙酰化的变化。结果:与慢性胆囊炎组织相比,胆囊癌组织中的蛋白赖氨酸乙酰化显著增加（P < 0.01）,乙酰化的显著增加主要发生在相对分子质量为10 000~36 000的蛋白。结论:利用赖氨酸乙酰化单克隆抗体特异性能够检测到胆囊组织蛋白的赖氨酸乙酰化,且相对分子质量为10 000~36 000蛋白的赖氨酸乙酰化增加显著。     

Cryo-EM structure of DNA-bound Smc5/6 reveals DNA clamping enabled by multi-subunit conformational changes

Structural maintenance of chromosomes (SMC) complexes are essential for chromatin organization and functions throughout the cell cycle. The cohesin and condensin SMCs fold and tether DNA, while Smc5/6 directly promotes DNA replication and repair. The functions of SMCs rely on their abilities to engage DNA, but how Smc5/6 binds and translocates on DNA remains largely unknown. Here, we present a 3.8 Å cryogenic electron microscopy (cryo-EM) structure of DNA-bound Saccharomyces cerevisiae Smc5/6 complex containing five of its core subunits, including Smc5, Smc6, and the Nse1-3-4 subcomplex. Intricate interactions among these subunits support the formation of a clamp that encircles the DNA double helix. The positively charged inner surface of the clamp contacts DNA in a nonsequence-specific manner involving numerous DNA binding residues from four subunits. The DNA duplex is held up by Smc5 and 6 head regions and positioned between their coiled-coil arm regions, reflecting an engaged-head and open-arm configuration. The Nse3 subunit secures the DNA from above, while the hook-shaped Nse4 kleisin forms a scaffold connecting DNA and all other subunits. The Smc5/6 DNA clamp shares similarities with DNA-clamps formed by other SMCs but also exhibits differences that reflect its unique functions. Mapping cross-linking mass spectrometry data derived from DNA-free Smc5/6 to the DNA-bound Smc5/6 structure identifies multi-subunit conformational changes that enable DNA capture. Finally, mutational data from cells reveal distinct DNA binding contributions from each subunit to Smc5/6 chromatin association and cell fitness. In summary, our integrative study illuminates how a unique SMC complex engages DNA in supporting genome regulation.

Structural maintenance of chromosomes (SMC) complexes are essential genome regulators in both prokaryotes and eukaryotes. In eukaryotes, the cohesin and condensin SMC complexes organize DNA, while the Smc5/6 complex (referred to as Smc5/6) directly regulates DNA replication and repair (1). At the structural level, SMC complexes share similarities while possessing unique attributes (1). Each complex contains a pair of SMC subunits and a set of non-SMC subunits. The SMC subunits define the tripartite filamentous architecture of the complex: their approximal 50-nm long coiled coil arm region connects their dimerized hinge and adenosine triphosphatase (ATPase) head regions (1). A non-SMC kleisin subunit uses its N- and C-terminal domains to link the head of one SMC to the head-proximal arm region (neck) of another SMC, forming a trimeric SMC-kleisin structure. In cohesin and condensin, two large U-shaped HEAT (Huntington, elongation factor 3, PR65/A, TOR) repeat HAWK (HEAT proteins associated with kleisins) subunits attach to the middle region of the kleisin. By contrast, the Smc5/6 kleisin (Nse4) binds to smaller WH (winged helix)-containing KITE (kleisin interacting tandem WH elements) subunits (Nse1 and Nse3) (2).SMC-mediated functions depend on interactions with DNA. Recent cryogenic electron microscopy (cryo-EM) structures of DNA-bound cohesin and condensin revealed that their HAWK subunits and the SMC head-neck regions form a clamp to enclose a single DNA double helix (3–7). DNA clamping can be critical for cohesin and condensin to extrude DNA loops for chromatin folding (5, 7–9). DNA loop extrusion additionally requires arm bending at a region called the elbow, which is found in both cohesin and condensin (5, 7–9). By contrast, a lack of arm bending in Smc5/6 was suggested by negative stain EM and cross-linking mass spectrometry (CLMS) data (10–14). Since Smc5/6 does not contain HAWK proteins nor shows arm-bending, it has remained unclear how Smc5/6 engages DNA to accomplish its multiple functions.Here we address the molecular mechanisms by which this unique SMC complex binds DNA using an integrative approach, coupling a cryo-EM-based structural characterization with CLMS analyses and functional investigation. Our atomic structure of a DNA-bound Saccharomyces cerevisiae Smc5/6 complex reveals that the head-neck Smc5-6 regions and the Nse1-3-4 subcomplex together form a clamp entrapping the DNA helix. The structure further reveals protein subunit folds and association, as well as how the subunits collaborate to entrap DNA. Comparison of CLMS analyses of DNA-free Smc5/6 with the structure of the DNA-bound Smc5/6 unveils large scale, multi-subunit conformational changes that enable Smc5/6 to encircle DNA. Finally, our mutational data suggest distinct contributions from each of the DNA binding regions to Smc5/6 chromatin association and cellular fitness. Comparison of our findings with those of other SMCs reveals that diverse SMC complexes use a similar DNA clamping strategy despite structural differences, and that Smc5/6 possesses unique features distinct from cohesin, condensin, and prokaryotic SMCs. Our work lays the foundation for an in-depth understanding of how Smc5/6 fulfills unique roles in genome protection.     

大型公立医院社会责任评价指标体系构建

目的:制定社会责任评价指标体系,为大型公立医院社会责任改革实践提供管理工具。方法:应用文献法、德尔菲法,邀请专家对大型公立医院社会责任指标进行筛选和评估,通过层次分析法确定各级指标的权重。结果:开展3轮专家咨询,15位专家完成咨询。指标体系包括一级指标2项、二级指标15项及三级指标74项(约束性指标10项,推荐性指标64项)。专家权威系数0.92,两轮咨询积极系数均为100%,协调系数分别为0.25、0.29,差异均有统计学意义(P<0.01)。结论:专家积极性、权威程度、专家意见集中程度和协调性均较高,大型公立医院社会责任指标体系具有可信度,可作为评估大型公立医院履行社会责任状况的工具。     

食品中汞元素测定快速前处理方法研究

目的建立快速提取食品中汞元素的前处理方法。方法采用超声辅助HNO3-H2O2和控温加热辅助HNO3-HClO42种方式快速处理样品。结果超声辅助HNO3-H2O2和控温加热辅助HNO3-HClO4提取体系的测定结果满意,精密度2.2%～4.3%,加标回收率81.5%～90.7%。结论超声辅助HNO3-H2O2和控温加热辅助HNO3-HClO4提取样品60min可以作为测定食品中汞元素的快速前处理方法。