TLR3基因缺陷的肝纤维化小鼠血生化指标、肝组织纤维化标志物及炎性因子变化

目的分析Toll样受体3(TLR3)基因缺陷的肝纤维化小鼠血生化指标、肝组织纤维化标志物及炎性因子变化。方法将18只野生型雄性小鼠随机分为空白造模组和空白对照组,两组各9只;将18只TLR3基因缺陷型雄性小鼠随机分为TLR3造模组和TLR3对照组,两组各9只。空白造模组和TLR3造模组均注射四氯化碳,而空白对照组与TLR3对照组均注射玉米油。造模2个月后,采集血液标本,通过全自动生化分析仪对血清白蛋白(ALB)、总胆红素(TBIL)、天门冬氨酸氨基转移酶(AST)及丙氨酸氨基转移酶(ALT)水平进行测定。比较各组小鼠肝组织纤维化标志物(Ⅰ型胶原)表达水平和肝组织炎性因子如白介素6(IL-6)、肿瘤坏死因子-α(TNF-α)、单核细胞趋化因子-1(MCP-1)的表达水平。结果空白造模组ALB水平较空白对照组明显降低,TBIL、AST及ALT等血生化指标的水平较空白对照组均显著升高(P0.01);TLR3造模组ALB水平较空白对照组明显降低,TBIL、AST及ALT等血生化指标的水平较TLR3对照组均显著升高(P0.01),但与空白造模组比较,均无显著差异(P0.05)。空白造模组肝组织纤维化标志物(Ⅰ型胶原)表达水平较空白对照组显著升高(P0.01);TLR3造模组Ⅰ型胶原表达水平较空白造模组、TLR3对照组均显著升高(P0.01)。空白造模组IL-6、TNF-α及MCP-1等肝组织炎性因子表达水平较空白对照组显著升高(P0.05);TLR3造模组炎性因子水平较空白造模组、TLR3对照组均显著升高(P0.05)。结论 TLR3基因缺陷的肝纤维化小鼠经四氯化碳诱导后,Ⅰ型胶原等肝纤维化标志物的水平明显升高,并且IL-6、TNF-α及MCP-1等肝组织炎性因子表达亦明显上升,提示TLR3可能是一种保护性基因,在肝纤维化疾病发生及发展中起到阻滞肝组织炎性因子等重要作用。     

铁调素在肝纤维化中的表达特点及对肝星状细胞的作用

目的 观察铁调素在肝纤维化过程中的表达特点,评估铁调素对肝星状细胞(HSC)的作用及其机制。方法腹腔注射四氯化碳与橄榄油混合物诱发肝纤维化,于第0、4、8、12周后处死大鼠,动态观察铁调素在血清和肝组织中的表达特点。用不同浓度的铁调素-25多肽刺激大鼠星状细胞系(HSC-T6) 48 h,检测纤维化相关指标如α-平滑肌肌动蛋白(α-SMA)、金属蛋白酶组织抑制因子-1(TIMP-1)和I型胶原在基因和蛋白质水平的表达。数据比较采用单因素方差分析与Tukey检验。结果 成功建立四氯化碳诱导的大鼠肝纤维化模型。对铁调素的检测发现,随着纤维化进展,肝组织中铁调素的基因水平在四氯化碳注射后逐渐降低(P 0. 05);四氯化碳混合物注射后第4、8、12周铁调素的相对表达分别为0. 75±0. 12、0. 52±0. 20、0. 20±0. 10。血清中铁调素在四氯化碳混合物注射0、4、8、12周分别为(351. 45±51. 12) pg/ml、(254. 24±49. 41) pg/ml、(211. 45±42. 28) pg/ml、(189. 23±30. 24) pg/ml。体外实验表明,外源性补充铁调素(10 ng/ml、100 ng/ml)可以直接抑制HSC中纤维化相关基因(α-SMA,TIMP-1和I型胶原)的表达,且具有剂量依赖性。进一步我们发现铁调素可以通过抑制TGFβ1信号中SMAD4的表达阻断TGFβ1诱导的星状细胞活化和I型胶原的分泌。结论 铁调素水平与纤维化进展负相关,体外补充铁调素能够抑制HSC的活化并减少I型胶原的分泌。     

β-arrestin1通过活化p38信号转导通路激活肝星状细胞促进肝纤维化

目的 探讨β-arrestin1(ARRB1)在肝纤维化中的作用，阐明ARRB1和p38信号转导通路诱导肝星状细胞活化从而促进肝纤维化的机制。方法 选取C57BJ/6L背景的雄性小鼠12只，随机分为对照组和模型组，每组6只小鼠，采用浓度为20%的四氯化碳诱导肝纤维化小鼠模型，并于临床收集肝硬化患者的肝组织及健康对照志愿者的肝组织样本。采用HE和天狼星红染色来评估肝纤维化情况。通过免疫组织化学法、蛋白免疫印迹法及实时荧光定量PCR法检测ARRB1、p38、磷酸化p38(pp38)及纤维化相关指标α平滑肌肌动蛋白（α-SMA）和Ⅰ型胶原蛋白的表达水平。在人肝星状细胞LX2中使用转化生长因子-β(TGF-β)细胞因子诱导其活化，并分析ARRB1的表达水平；进而利用小干扰RNA和质粒沉默或者过表达ARRB1，同时配合p38特异性抑制剂（SB203580），揭示两者的调控关系。结果 在肝纤维化组织中ARRB1表达升高，p38信号活化增强（P均<0.05）。在肝星状细胞LX2中，TGF-β可以激活肝星状细胞并表达ARRB1和增强p38信号。ARRB1表达的升高或抑制可以相应地影响p38信号从...     

GFAP启动子介导EGFP靶向肝星状细胞治疗肝纤维化的价值探索

目的探索体内特异性靶向肝星状细胞(HSC)治疗肝纤维化的技术方法。方法建立四氯化碳诱导的小鼠肝纤维化模型,构建胶质纤维酸性蛋白(GFAP)启动子调控增强绿色荧光蛋白(EGFP)表达载体,采用尾静脉高压注射方法将EGFP表达裸质粒转染到纤维化小鼠的肝脏。荧光显微镜观察EGFP在肝脏的表达及免疫荧光共定位,观测EGFP与HSC标志蛋白(DESMIN)和α-平滑肌肌动蛋白(α-SMA)的共定位。结果 EGFP与DESMIN和α-SMA共定位说明EGFP主要在HSC中表达,GFAP启动子能够调控外源基因在体内靶向HSC。结论 GFAP启动子介导体内靶向HSC,治疗肝纤维化策略具有临床应用的潜力。     

DMN肝纤维化模型肝组织I、Ⅲ型胶原和TIMP-1 mRNA表达及复方861的干预作用

目的　研究二甲基亚硝胺 (DMN)肝纤维化模型I型胶原 (CoL -1)、Ⅲ型胶原 (CoL -Ⅲ )和基质金属蛋白酶组织抑制因子 (TIMP -1)mRNA表达以及复方 86 1的干预作用。材料和方法　采用 1%DMN 1ml /kg腹腔注射模型组大鼠 ,复制DMN诱导的大鼠肝纤维化模型 ,同时复方 86 1灌胃 3g/kg ,共 8周 ,以RT -PCR法观察肝纤维化模型肝组织CoL -Ⅰ、CoL -Ⅲ及TIMP -1mRNA表达水平。结果　DMN大鼠肝纤维化模型组CoL -I、CoL -Ⅲ及TIMP-1mRNA分别为 0 82 7± 0 0 94、 0 95 3± 0 0 33及 0 92 4± 0 110 ,明显高于正常对照组 (P <0 0 1) ;复方 86 1治疗组CoL -I、CoL -Ⅲ及TIMP -1mRNA为 0 4 97± 0 0 5 7、 0 85 1± 0 0 6 5和 0 6 48± 0 10 7,明显低于模型对照组 (P <0 0 1或P <0 0 5 )。结论　DMN肝纤维化模型肝组织CoL -Ⅰ、CoL -Ⅲ及TIMP -1mRNA表达水平增高 ,复方 86 1能够抑制肝组织CoL -Ⅰ、CoL -Ⅲ及TIMP -1mRNA的表达 ,从而发挥其抗肝纤维化的作用。     

大鼠TIMP1 RNAi表达载体的构建及其对肝纤维化大鼠的修复

肝纤维化形成过程中涉及多种细胞及细胞因子之间的作用，肝脏星状细胞（HSC）的激活是肝纤维化发生、发展的中心环节，而TGF-β1和TIMPs是HSC的早期“启动”的后期的“持久化”过程中关键因子，Kim KH等应用TGF-β1 siRNA修复小鼠肝纤维化模型，使工型胶原和α-平滑肌肌动蛋白表达下降。     

CHFR介导的蛋白PAR化参与肝纤维化DNA损伤应答的分子机制研究

目的探讨泛素连接酶蛋白(CHFR)介导的蛋白PAR化参与肝纤维化DNA损伤应答的分子机制。方法制备和野生型C57BL/6小鼠同背景的CHFR基因敲除(CHFR-/-)小鼠构建四氯化碳肝纤维化(CCl4)模型,饲养4周后处死,通过免疫组化方法以及Western blotting方法验证小鼠肝纤维模型中DNA的损伤标记物γH2AX表达变化以及对小鼠肝星状细胞(HSC)进行激光照射诱导DNA双链断裂(DSBs)损伤,在荧光显微镜下观察CHFR和γH2AX的定位,采用sh RNA降低小鼠HSC中CHFR蛋白表达,进行放射线照射后,进行单细胞凝胶电泳(Comet)实验。结果①Western blotting和免疫组化结果均显示,正常肝组织中未见γH2AX,肝纤维化组织表达明显;②荧光显微镜下显示,HSC细胞核出现γH2AX与CHFR荧光表达,CHFR显著定位于DNA损伤部位,且CHFR与γH2AX表达趋势一致,同时表达于细胞核相同部位;③与野生型C57BL/6小鼠对比,CHFR-/-小鼠γH2AX阳性细胞数明显增多,放射线照射后,CHFR-/-小鼠"彗星尾巴"增加,DNA修复减弱。结论 CHFR可能通过参与DNA损伤修复过程介导HSC活化继而参与肝纤维过程。     

Toll样受体4在四氯化碳致大鼠慢性肝损伤中的作用机制研究

目的:动态观察肝Kupffer细胞(KCs)Toll样受体4(TLR4)mRNA及蛋白在四氯化碳(CCl4)致大鼠慢性肝损伤中的表达及作用.方法:以CCl4诱导慢性肝损伤大鼠模型,于0、4、6、8、10周采集标本,分离肝组织KCs并以逆转录聚合酶链反应检测TLR4mRNA的表达;用基质显色法测定大鼠血清内毒素水平:放射免疫法测定血清肿瘤坏死因子-α(TNF-α)和白细胞介素1-β(IL1-β)水平;TLR4蛋白和核因子kB(NF-kB)蛋白采用免疫组化SP法检测.结果:0周组肝组织TLR4蛋白、KCs,TLR4 mRNA几乎无表达,NF-kB蛋白和炎症积分水平也处于较低水平;4、6、8、10周组肝组织TLR4蛋白、NF-kB蛋白、炎症积分和KCs TLR4 mRNA以及血清内毒素、TNF-α、IL-1β水平明显升高,同0周组比较差异有显著性(P<0.001).NF-kB蛋白、TLR4蛋白、KCs TLR4 mRNA和TNF-α在第10周时较第8周有轻度下降:肝KCs TLR4 mRNA的表达与血浆内毒素水平呈正相关(r=0.845,P<0.001).结论:CCl4诱导的慢性肝损伤中,内毒素可上调KCs TLR4的表达,而TLR4的高表达进一步造成肝脏的损伤.     

TLR4受体拮抗剂对Aβ1-42诱导的小胶质细胞炎性因子分泌的影响

目的探讨体外培养条件下Toll样受体4(TLR4)拮抗剂对β淀粉样蛋白1-42片段(Aβ1-42)诱导的小胶质细胞炎性因子分泌的影响。方法用不同浓度的Aβ1-42(终浓度为0、1、5、10、20、40、80μmol/L)及TAK-242(终浓度1μmol/L)处理小鼠小胶质细胞(BV2),24 h后取其上清干预小鼠海马神经元(HT22)。48 h后,应用CCK8方法检测HT22细胞的存活,ELISA方法检测BV2细胞上清液中TNF-α、IL-1β水平。结果与空白对照组相比,Aβ1-42直接作用于体外培养的小胶质细胞后,以剂量依赖的方式诱导TNF-α、IL-1分泌的增加(P0.05);与空白对照组相比,Aβ1-42诱导组培养液干预的HT22细胞存活率显著下降(P0.05)。给予TLR4拮抗剂TAK-242干预后,Aβ1-42诱导TNF-α、IL-1分泌的作用被抑制,细胞分泌TNF-α、IL-1β水平较Aβ1-42诱导组显著降低(P0.05),HT22细胞存活率也显著提高(P0.05)。结论阻断TLR4可抑制Aβ1-42诱导的小胶质细胞炎性因子的分泌,并减轻活化的小胶质细胞对海马神经元的损害。     

黄芪联合干扰素α对CCl_4诱导的大鼠肝纤维化的保护作用

目的观察黄芪联合干扰素α对四氯化碳(CCl4)诱导的大鼠纤维化病变的干预效果及可能的作用机制。方法采用诱导CCl4肝纤维化,造模6周,SD大鼠随机分为5组:正常对照组、模型组、黄芪组、干扰素α组和黄芪+干扰素α联合治疗组。采用放免法检测大鼠血清透明质酸(HA)、层黏连蛋白(LN)、Ⅲ型前胶原(PC-III)、Ⅳ型胶原(IV-C);同时使用Jamall法测定肝组织羟脯氨酸(HYP)的水平;采用ELISA检测大鼠肝组织中基质金属蛋白酶组织抑制剂-1(MMP-1)和基质金属蛋白酶抑制因子-1(TIMP-1)含量的变化。结果黄芪和干扰素均能显著降低肝纤维化大鼠血清中HA、LN、PC-III和IV-C的含量(P0.01);而且与单一药物治疗组相比,联合药物组治疗后大鼠HA和IV-C的表达水平降低更明显(P0.05)。黄芪和干扰素联合用药可以导致肝纤维化大鼠TIMP-1表达水平的降低和MMP-1表达水平的增高(P0.05)。结论黄芪联合干扰素α对CCl4诱导的大鼠肝纤维化具有保护作用,这种保护作用的产生可能与调节肝组织中TIMP-1、MMP-1表达水平有关。     

Guns and Knives in New Mexico: Patterns of Penetrating Trauma, 1978–1993

Objective: To identify patterns of nonfatal and fatal penetrating trauma among children and adults in New Mexico using ED and medical examiner data.
Methods: The authors retrospectively sampled in 5-year intervals all victims of penetrating trauma who presented to either the state Level-1 trauma center or the state medical examiner from a 16-year period (1978–1993). Rates of nonfatal and fatal firearm and stabbing injury were compared for children and adults.
Results: Rates of nonfatal injury were similar (firearm, 34.3 per 100,000 person-years; stabbing, 35.1). However, rates of fatal injury were significantly different (firearm, 21.9; stabbing, 2.7; relative risk: 8.2; 95% confidence interval: 5.4, 12.5). From 1978 to 1993, nonfatal injury rates increased for children (p = 0.0043) and adults (p < 0.0001), while fatal penetrating injury remained constant. The increase in nonfatal injury in children resulted from increased firearm injury rates. In adults, both stabbing and firearm nonfatal injury rates increased.
Conclusions: Nonfatal injury data suggest that nonfatal violence has increased; fatal injury data suggest that violent death rates have remained constant. Injury patterns vary by age, mechanism of trauma, and data source. These results suggest that ED and medical examiner data differ and that both are needed to guide injury prevention programs.     

Dissociable correlates of two classes of retrieval processing in prefrontal cortex

Although substantial evidence suggests that the prefrontal cortex (PFC) implements processes that are critical for accurate episodic memory judgments, the specific roles of different PFC subregions remain unclear. Here, we used event-related functional magnetic resonance imaging to distinguish between prefrontal activity related to operations that (1) influence processing of retrieval cues based on current task demands, or (2) are involved in monitoring the outputs of retrieval. Fourteen participants studied auditory words spoken by a male or female speaker and completed memory tests in which the stimuli were unstudied foil words and studied words spoken by either the same speaker at study, or the alternate speaker. On "general" test trials, participants were to determine whether each word was studied, regardless of the voice of the speaker, whereas on "specific" test trials, participants were to additionally distinguish between studied words that were spoken in the same voice or a different voice at study. Thus, on specific test trials, participants were explicitly required to attend to voice information in order to evaluate each test item. Anterior (right BA 10), dorsolateral prefrontal (right BA 46), and inferior frontal (bilateral BA 47/12) regions were more active during specific than during general trials. Activation in anterior and dorsolateral PFC was enhanced during specific test trials even in response to unstudied items, suggesting that activation in these regions was related to the differential processing of retrieval cues in the two tasks. In contrast, differences between specific and general test trials in inferior frontal regions (bilateral BA 47/12) were seen only for studied items, suggesting a role for these regions in post-retrieval monitoring processes. Results from this study are consistent with the idea that different PFC subregions implement distinct, but complementary processes that collectively support accurate episodic memory judgments.     

Interprofessional approaches to creating safe,high quality health care

Three supplementary perspectives are presented arguing that interprofessional collaboration is both necessary and desirable. Nonetheless, there are often too many serious intra-professional barriers and obstacles to interprofessional collaboration to make it successful. Some of these barriers, it is argued and illustrated, are found in the multiple ways in which professional identity is tacitly acquired and embodied in the practitioners' habitual, everyday practice. The paper then explores ways in which reflection, especially Second order reflection, can help to elucidate and overcome these obstacles, as well as increasing professional adaptability and competence.     

Systematic Reviews Published in the January 2011 Issue of The Cochrane Library

ABSTRACT

The Cochrane Library of Systematic Reviews is published quarterly as a DVD and monthly online. The January 2011 issue (first quarterly DVD for 2011) contains 4515 complete reviews, 1985 protocols for reviews in production, and 13,521 one-page summaries of systematic reviews published in the general medical literature. In addition, there are citations of 641,000 randomized controlled trials, and 14,018 cited papers in the Cochrane methodology register. The health technology assessment database contains over 9300 citations. One hundred and seven new reviews have been published in the last 3 months, of which five have potential relevance for practitioners in pain and palliative medicine.     

Modified micro-superior percutaneously-assisted total hip: early experiences & case reports

Because of the extensile nature and familiarity of the standard posterior-lateral approach to the hip, a family of "micro-posterior" approaches has been developed. This family includes the Percutaneously-Assisted Total Hip (PATH) approach, the Supercapsular (SuperCap) approach and a newer hybrid approach, the Supercapsular Percutaneously-Assisted Total Hip (SuperPATH) approach. Such approaches should ideally provide a continuum for the surgeon: from a "micro" (external rotator sparing) posterior approach, to a "mini" (external rotator sacrificing) posterior approach, to a standard posterior approach. This could keep a surgeon within his comfort zone during the learning curve of the procedure, while leaving options for complicated reconstructions for the more practiced micro-posterior surgeons. This paper details one author's experiences utilizing this combined approach, as well as permutations of this entire micro-posterior family of approaches as applied to more complex hip reconstructions.     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

Structure and function of "metalloantibiotics"

Although most antibiotics do not need metal ions for their biological activities, there are a number of antibiotics that require metal ions to function properly, such as bleomycin (BLM), streptonigrin (SN), and bacitracin. The coordinated metal ions in these antibiotics play an important role in maintaining proper structure and/or function of these antibiotics. Removal of the metal ions from these antibiotics can cause changes in structure and/or function of these antibiotics. Similar to the case of "metalloproteins," these antibiotics are dubbed "metalloantibiotics" which are the title subjects of this review. Metalloantibiotics can interact with several different kinds of biomolecules, including DNA, RNA, proteins, receptors, and lipids, rendering their unique and specific bioactivities. In addition to the microbial-originated metalloantibiotics, many metalloantibiotic derivatives and metal complexes of synthetic ligands also show antibacterial, antiviral, and anti-neoplastic activities which are also briefly discussed to provide a broad sense of the term "metalloantibiotics."