炎性相关细胞因子和心肌梗死微循环再灌注状态的关系

目的　观察急性心肌梗死 (AMI)患者梗死相关血管 (IRA)开通前后炎性细胞因子的动态变化及其与心肌组织水平灌注状态的关系。方法　 (1)测定 8例健康人和 2 2例AMI患者急诊冠状动脉介入治疗术 (PCI)前即刻 ,术后 12、2 4h ,血浆白细胞介素 (IL) 1β、肿瘤坏死因子 (TNF)α、IL 10的变化。 (2 )按照再灌注后 2h心电图ST段回落是否 >70 % ,将 2 2例AMI患者分为 :A组 (ST回落≥ 70 % )12例和B组 (ST回落 <70 % ) 10例 ,比较两组患者IL 1β、TNFα、IL 10的变化幅度。 结果　 (1)治疗前A、B两组AMI患者血浆TNFα、IL 10略高于健康对照组 ,但差异无统计学意义 (P >0 0 5 ) ;而IL 1β显著高于健康对照组 (P <0 0 5 ) ;再灌注后 12、2 4hA、B两组血浆IL 1β和TNFα均较术前显著增高 (P <0 0 1,P <0 0 5 ) ,B组血浆IL 10较术前显著增高 (P <0 0 5 ) ,A组则无此变化 (P >0 0 5 )。 (2 )A、B两组间比较 ,治疗前TNFα、IL 1β、IL 10差异均无显著性 (P >0 0 5 ) ;成功PCI、IRA血流达TIMI 3级者 ,B组患者血浆IL 1β、TNFα、IL 10 ,在再灌注 12h显著高于A组 (P <0 0 1,P <0 0 5 ,P <0 0 5 ) ,再灌注 2 4h ,IL 1β、IL 10仍然高于A组 (P <0 0 5 )。 (3)A、B两组患者抗炎因子IL 10的升高幅度均显著低于致炎     

顽固性室性早搏的导管标测与射频消融治疗

采用射频导管消融术对症状明显、药物无效的１０例顽固性室性早搏（简称室早）进行治疗。将消融电极送至右室流出道区域，以Ｓ１Ｓ１或ＲＳ２早搏刺激标测到与体表１２导联心电图记录的自发室早ＱＲＳ波群图形完全相同，并且激动标测时自发室早的局部电图较体表心电图ＱＲＳ波群提前３０ｍｓ以上的部位为消融靶点。以室早在放电后１０ｓ内消失，维持稳定窦性心律３０～６０ｍｉｎ为即刻成功标准。９例患者经１０～２０Ｗ、消融６０～１８０ｓ，早搏和短阵室速完全消失；１例失败。平均随访１１个月，未服任何抗心律失常药物症状消失，复查心电图和动态心电图，９例中８例无早搏、１例为偶发室早，均无并发症。提示射频导管消融术是治疗某些右室流出道早搏的可行方法。     

Mouse model of post-infarct ventricular rupture: time course, strain- and gender-dependency, tensile strength, and histopathology

OBJECTIVE: Recent studies on mice with surgically induced acute myocardial infarction (AMI) have documented the frequent occurrence of ventricular rupture, an event not previously reported in other laboratory species. We have examined the natural history, histopathology and myocardial mechanical strength in mice with AMI. METHODS: AMI was induced by coronary artery occlusion and animals were monitored for fatal events. Gross and histological examinations were undertaken. RESULTS: Rupture occurred in the left ventricular free wall at 2-6 days after AMI. Incidence of rupture in male mice varied among three strains studied (3% for FVB/N, 27% for C57B/6J, and 59% for 129sv, P<0.05) and was lower in female than male mice (23% vs. 59%, P<0.05). Histologically, ruptured hearts had rapid-occurring and severe infarct expansion, multifocal intramural hemorrhage and leucoyte infiltration at the border zone and infarcted zone. In vitro, infarcted left ventricles demonstrated a 50-60% reduction in muscle tensile strength. This reduction preceded the onset of rupture and was related to the time-window of rupture and to infarct size. CONCLUSION: LV wall rupture in the mouse occurs within a narrow time-window after AMI and is strain- and gender-dependent. Infarct expansion, regional hemorrhage with formation of hematoma and leuocyte accumulation are important pathological changes leading to reduced myocardial tensile strength.     

颈动脉支架置入术会取代颈动脉内膜切除术吗?

多年来,颈动脉内膜切除术(CEA)一直被视为重度颈动脉狭窄患者治疗的"金标准",但随着颈动脉支架置入术(CAS)的发展,逐渐使CEA的"金标准"地位受到挑战,甚至有人提出用CAS代替CEA.近年来陆续发表了有关两种治疗方式的对比研究,但对相关研究结果的解读,以及各项研究间的结果却并不一致.文章就这些方面的争议进行了分析.     

支气管镜在儿童呼吸系统疾病应用的安全性研究进展

随着支气管镜技术的不断发展和完善,支气管镜在儿科领域的应用日益广泛.同时,由于儿童自身的特殊性,支气管镜在儿科应用的安全性问题一直都备受关注.与儿童支气管镜诊疗安全性相关的研究及观察也逐渐成为热点.为了降低不良反应和并发症的发生率,术前应仔细评估并进行积极的心理护理.术中应选择适合的麻醉方式、持续给氧、熟练快捷地完成操作并应密切监护患者的生命体征.尤其是对于小年龄组患者更要引起高度重视.为了避免患者发生不必要的医源性感染,还应完善严格执行消毒隔离制度.同时也要保护好医护人员的安全.     

Retransplantation for severe accelerated coronary artery disease in heart transplant recipients

Development of accelerated coronary artery disease (CAD) in the cardiac allograft is one of the major causes of late graft failure in heart transplant recipients. At the Stanford University Medical Center 356 heart transplant procedures were performed in 329 patients by the end of January 1985. Eighty-nine of these patients developed evidence of transplant CAD. Twenty retransplant procedures, including 2 third transplants, were performed in 19 of the 89 patients because of transplant CAD. The graft survival rates after the second transplant were 55%, 25% and 10% after 1, 2 and 5 years, respectively. Nine of these retransplant patients currently survive, the longest for 5.5 years. To examine potential risk factors for development of severe transplant CAD, these 20 retransplant procedures were compared with 113 transplant recipients who had no evidence of transplant CAD on annual coronary arteriograms. An excess of rejection episodes (3 +/- 2 vs 2 +/- 1 episodes/patient, p = 0.02), elevated total cholesterol (266 +/- 78 vs 225 +/- 47 mg/dl, p = 0.002) and higher low-density lipoprotein levels (176 +/- 88 vs 137 +/- 46 mg/dl, p = 0.009) were noted in the transplant CAD retransplant group. Five of 11 retransplant recipients who survived greater than 1 year again developed transplant CAD. Characteristic morphologic features and rapid progression of CAD in the second graft were similar to those in the primary graft.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetra-fluorinated aromatic azide for highly efficient bioconjugation in living cells

We developed a fast strain-promoted azide–alkyne cycloaddition reaction (SPAAC) by tetra-fluorinated aromatic azide with a kinetic constant of 3.60 M⁻¹ s⁻¹, which is among the fastest SPAAC ligations reported so far. We successfully employed the reaction for covalent labelling of proteins with high efficiency and for bioimaging of mitochondria in living cells. The reaction could be a generally useful toolbox for chemical biology and biomaterials.

A fast strain-promoted azide–alkyne cycloaddition based on tetra-fluorinated aromatic azide was developed and applied to label proteins and living cells with high efficiency.

Strain-promoted azide–alkyne cycloaddition (SPAAC) as an efficient bioconjugation tool has recently found a wide range of applications in chemical biology, materials science and medical science.^1–3 In 2001, Sharpless et al., first introduced the concept of click chemistry that emphasized on the rapid synthesis of useful new compounds and combinatorial libraries through heteroatom links (C–X–C).⁴ To overcome the limitations for classical copper-catalyzed azide–alkyne cycloaddition (CuAAC),⁵ Bertozzi improved the method using strained cyclooctynes to react with azide in 2004, which effectively avoids the transition metal catalysts (such as Cu) and makes it more general for bioconjugations.^6,7 It gave birth to the development of SPAAC. But one of the limitations is the relative low reaction rate compared with other biorthogonal reactions and a high rate could be beneficial for labelling low abundance or transient structures with short-lived signal molecules, such as radioisotopes for imaging.⁸ During the past decade, efforts have been made to further improve the method. Structure modified cyclooctynes, including aliphatic cyclooctynes⁹ and (di)benzoannulated cyclooctynes¹⁰ have been investigated to accelerate the reaction, and recently the modified azides was also reported to remarkably increase SPAAC rate.¹¹We have been interested in the development of fast chemoselective reactions for bioconjugations.¹² We reported o,o′-difluorinated aromatic azide can accelerate both the reaction rate significantly on SPAAC and H₂S-mediated reduction of the azide.^12c Therefore, we envision that multi-fluorinated aromatic azide may have interesting properties for fast and convenient bifunctional conjugation. 4-Azido-2,3,5,6-tetrafluorobenzoic acid (1) was selected for investigation. Here, we report the SPAAC kinetic properties of 1 and its applications for protein labelling in vitro and for bioimaging of subcellular organelles in living cells (Fig. 1).Open in a separate windowFig. 1Rational design of SPAAC reaction based on multi-fluorinated aromatic azide. (a) SPAAC reaction based on o,o′-difluorinated aromatic azide reported in our previous work; (b) SPAAC reaction based on tetra-fluorinated aromatic azide for faster and bifunctional ligation.To test our idea, the reaction between 1 and [1R,8R,9S]-bicyclo[6.1.0]non-4-yn-9-yl methanol (2) was studied for SPAAC reaction, monitored with time-dependent ¹H NMR. To our delight, the reaction could finish within 5 min. As shown in Fig. 2, the ¹H-NMR signal of 2 (40 mM) completely changed to the product (3) after adding 1 for 5 min, and there''s no further change at 15 min and 2 hours in NMR spectra. The resulted product was also confirmed by high resolution mass spectrum. This result implies that the 4-azido-2,3,5,6-tetrafluorobenzoic acid derivatives can be used for this fast SPAAC reaction. But NMR is limited by its relative low sensitivity for accurate testing the kinetic rate at micromolar concentration.Open in a separate windowFig. 2 ¹H NMR analysis of the reaction between 1 (120 mM) and 2 (40 mM). The reaction was carried out in CD₃OD.In order to quantify the kinetic rate, we designed a procedure based on fluorescence resonance energy transfer (FRET) method to monitor this fast SPAAC reaction. We synthesized the azide compound 4 conjugated to an azo-quencher and the cyclooctyne compound 5 conjugated to a BODIPY-dye (Fig. 3). All the compounds were isolated and characterized by NMR and HRMS. After the reaction between 4 and 5, 6 could formed in which BODIPY fluorescent signal was quenched due to the FRET effect. Such fluorescence change could be employed to monitor the reaction in a real-time.Open in a separate windowFig. 3(a) Chemical structures of 4 and 5 and their SPAAC reaction to produce 6. (b) Time-dependent fluorescence spectra of 5 (2 μM) upon treated with 4 (30 μM) in PBS (50 mM, pH 7.4, containing 70% CH₃CN) at room temperature (excitation, 473 nm). The reaction time is shown inset. (c) The linear relationship between the concentration of 4 and k_obs. The slope of the best linear fitting gives the reaction rate k₂ (M⁻¹ s⁻¹).The reaction between 4 and 5 was set up in PBS buffer (2 μM 5, 50 mM, pH 7.4) and the maximum emission at 511 nm was monitored with the excitation at 473 nm. The pseudo-first-order rate k_obs was determined by fitting the data with a single exponential function. The linear fitting between k_obs and the concentrations of 4 gave the reaction rate (k₂) as 3.60 M⁻¹ s⁻¹. It''s two-fold faster than o,o′-difluorinated aromatic azide that we reported earlier, 1500-fold faster than the original SPAAC reaction and among the fastest SPAAC reactions reported so far.With this highly efficient reaction, covalent protein labelling was first tested as its application. N-Hydroxysuccinimide ester of 4-azido-2,3,5,6-tetrafluorobenzoic acid was synthesized as bifunctional labelling compound (7) for amide and SPAAC reactions. We chose bovine serum albumin (BSA) and lysozyme as model proteins considering their different sizes and functions.⁸ As shown in Fig. 4a, we tested to label protein first with 7 and then conjugated a dye to the protein using fluorescent cyclooctyne (5). BSA or lysozyme was treated with 0.5 mM 7 in PBS buffer (50 mM, pH 8.5, containing 10% DMSO) for 2 h to incorporate tetra-fluorinated aromatic azide into the protein. After removing of small molecules, the azide labelled protein was incubated with 1 mM 5 for another 2 h to achieve SPAAC protein fluorescent dye labelling. As a control, the azide labelled protein was also treated with Na₂S for 10 minutes to reduce azide into amine before incubating with 5, so to prove the reaction specificity. The labelled proteins were analysed with SDS-PAGE either stained by Coomassie blue or excited under UV lamp to visualize the desired protein. The results are shown in Fig. 4b. The strong fluorescent labelled BSA (lane 1) and lysozyme (lane 4) could be observed after the reaction with tandem addition of 7 and 5, while the controls of 5 only (lane 2 and 5) and Na₂S treated labelling (lane 3 and 6) did not show any fluorescent signal. The results indicated that tetra-fluorinated aromatic azide-cyclooctyne is a highly efficient SPAAC reaction for protein labelling.Open in a separate windowFig. 4Fluorescence labeling of BSA and lysozyme via the SPAAC reaction. (a) The fluorescence labelling strategy. (b) 15% SDS-PAGE of BSA (lane 1–3) and lysozyme (lane 4–6) was imaged under UV lamp (right) and then stained by Coomassie blue (left).To future explore the biocompatibility of this SPAAC reaction in living cells, we tested it for bioimaging of the subcellular organelle mitochondria. Triphenylphosphines are known to enrich into mitochondria mainly due to its positive charged character.¹³ We designed and synthesized compound 8, with the azide conjugated to a triphenylphosphine. Upon incubating 8 with the cells, we expected it would target mitochondria, so we can use SPAAC reaction to label the cells with fluorescent compound 5 (Fig. 5a). Human embryonic kidney cells 293 (HEK 293) were chosen for the experiment. The cells were first treated with 8 (10 μM) for 20 min to accumulate tetra-fluorinated aromatic azide into mitochondria, and then incubated with 5 μM 5 for another 30 min. After the non-specific fluorescent signal was washed away, the cells were imaged under the excitation of 488 nm. The experimental conditions and results were shown in Fig. 5b. The cells could only be fluorescence-labelled when treated with both 8 and 5, and the imaging signal all localized on mitochondria. The result proved both the feasibility and biocompatibility for the application of this improved SPAAC conjugation at cellular level. Its superior kinetic character (k₂ of 3.60 M⁻¹ s⁻¹) may even enable in vivo applications for pretargeted imaging,¹⁴ which is currently under investigation.Open in a separate windowFig. 5Fluorescence labelling of mitochondria in living cells via the SPAAC reaction. (a) The fluorescence labelling strategy. (b) Fluorescence images of HEK293 cells. HEK293 cells were treated for 30 min only with 10 μM of 5 (left); 20 min only with 10 μM of 8 (middle); 20 min with 10 μM of 8 followed by 5 μM of 5 for 30 min (right).