血循毒蛇咬伤激素用量研究

目的研究血循毒蛇咬伤患者激素使用最佳剂量。方法收集我院2007~2014年血循毒蛇咬伤住院患者339例,随机给予5~40mg不同剂量地塞米松治疗,比较其需要使用激素的时间、住院时间及其并发症发生率。结果 10、15mg剂量组需要使用激素时间分别为2.29、2.30天,相对于5、20、25、30、40mg剂量组(分别为5.30、3.22、3.50、3.34、3.25天)具有统计学意义(P0.05)。其中5mg剂量组的住院时间为7.58天,相对于10、15、20、25、30、40 mg剂量组(分别为4.01、3.94、3.90、3.88、3.91、4.30天)具有统计学意义(P0.05);10、15mg剂量组的并发症发生率分别为4.48%、4.00%,相对于5、20、25、30、40mg剂量组(分别为14.70%、10.42%、14.29%、17.50%、21.21%)具有统计学意义(P0.05)。结论地塞米松10、15mg剂量为血循毒蛇咬伤患者激素使用最佳用量。     

早期伤口切开对血循毒蛇咬伤患者预后的影响

目的探讨血循毒类毒蛇咬伤患者早期伤口切开对预后的影响。方法回顾性分析我院2011年3月~2014年3月诊治的血循毒类毒蛇咬伤患者223例的临床资料,总结并比较其临床特点及预后。结果本组患者轻度肿胀97例中,伤后12h内切开21例,未溃疡12例,Ⅰ°溃疡7例,Ⅱ°溃疡2例;晚期切开或未切开76例,未溃疡15例,Ⅰ°溃疡41例,Ⅱ°溃疡16例,Ⅲ°溃疡4例。中度肿胀患者85例中,伤后12h内切开32例,未溃疡8例,Ⅰ°溃疡21例,Ⅱ°溃疡2例,Ⅲ°溃疡1例;晚期切开或未切开53例,未溃疡6例,Ⅰ°溃疡16例,Ⅱ°溃疡24例,Ⅲ°溃疡7例。重度肿胀患者41例中,伤后12h内切开17例,Ⅰ°溃疡11例,Ⅱ°溃疡4例,Ⅲ°溃疡2例;晚期切开或未切开24例,Ⅰ°溃疡2例,Ⅱ°溃疡6例,Ⅲ°溃疡16例。在蛇伤后12h内切开的患者坏死溃疡的形成、肿胀程度的改善均明显优于晚期切开或未切开的患者,差异有显著统计学意义(P0.05)。结论随着患者凝血功能的改善,早期伤口切开对血循毒类毒蛇伤患者局部伤口状况有较好的改善作用。     

蜂蜇伤致MODS及骨筋膜室综合征1例合并文献复习

目的:提高对蜂蜇伤致多脏器功能衰竭(MODS)的认识并引起对该病的重视。方法:对1例蜂蜇伤致MODS及骨筋膜室综合症患者进行临床分析,并结合文献进行复习。结果:蜂蜇伤后同时或序贯出现2或2个以上系统器官的功能不全或衰竭的临床综合征,称为蜂蜇伤致MODS。病死率与衰竭器官数目有关。结论:治疗蜂蜇伤致MODS早期应采用血液净化措施,同时综合性保护肾、心、肝等多器官功能,促进多器官功能恢复是抢救治疗的关键。对于骨筋膜室综合症,应早期诊断、早期彻底筋膜室切开减压。     

222例血循毒和混合毒毒蛇咬伤患者的凝血功能分析

目的探讨血循毒和混合毒毒蛇咬伤患者凝血功能与致伤蛇种及就诊时间的相关性。方法对我院2010年7月～2012年7月收治的222例毒蛇咬伤患者的凝血功能(凝血酶原时间、活化部分凝血活酶时间、纤维蛋白原)及致伤蛇种、就诊时间进行回顾性分析。结果 (1)我院蛇伤(血循毒和混合毒)患者以竹叶青蛇、烙铁头蛇、五步蛇、眼镜蛇为常见,少数为乡城烙铁头蛇、白头蝰蛇、眼镜王蛇,其它蛇伤罕见。(2)五步蛇伤早期(6h内)容易出现凝血功能障碍。(3)除五步蛇、蝰蛇外,其它血循毒和混合毒毒蛇咬伤6h后就诊者凝血功能障碍机率高。结论我院蛇伤以竹叶青蛇、烙铁头蛇、五步蛇、眼镜蛇咬伤常见,五步蛇咬伤早期(6h内)易出现凝血功能障碍,其它血循毒、混合毒毒蛇咬伤6h后凝血功能障碍率高;早期凝血功能障碍对五步蛇咬伤有诊断参考价值。     

蜂蛰伤致横纹肌溶解症强化治疗策略的初步研究

目的:研究以肌酸激酶升高值结合SOFA评分为时机,进行包括激素冲击、适时血液净化和局部持续湿敷的强化方案对蜂蛰伤造成的横纹肌溶解症预后的影响。方法:收集整理2009年1月至2015年12月我院急诊科收治的蜂蛰伤患者的临床资料,按肌酸激酶升高值分为对照组和治疗组,对照组进行常规治疗,治疗组进行早期激素冲击、以SOFA评分为时机进行血液净化和局部持续治疗。比较入院后两组患者横纹肌损伤程度和基本治疗情况,比较两组患者治疗1周后脏器功能情况,比较两组患者严重并发症和预后。结果:治疗组横纹肌溶解程度较对照组重(P0.05),其补液量、补碱量、激素用量及循环负荷均多于对照组(P0.05),SOFA评分高于对照组(P0.05);治疗1周后治疗组脏器功能情况较对照组明显改善(P0.05);治疗组患者急性肾损伤、高钾血症、过敏性休克和多器官功能障碍发生率及病死率低于对照组(P0.05)。结论:以肌酸激酶升高程度结合SOFA评分作为依据,进行早期激素冲击、适时血液净化和局部持续治疗,这种强化治疗方案有利于脏器功能改善和预后。     

Bioluminescent Assay of Total and Brain-Specific Creatine Kinase Activity in Cerebrospinal Fluid

Abstract: A bioluminescent assay based on the firefly luciferase reaction has been used for determination of creatine kinase activity in CSF. Activities as low as 0.1 U/L can be measured. The coefficient of variation at an activity level of 0.3–0.4 U/L was between 5 and 6%. The assay conditions optimized for serum specimens can be used for CSF. The adenylate kinase activity is almost completely inhibited, which simplifies the procedure. The creatine kinase (CK) isoenzyme distribution was obtained using the bioluminescent assay in combination with immunoinhibition or ion exchange chromatography. All specimens contained both MM and BB activity, but no MB was found. The study indicates that the bioluminescent assay is useful in the determination of CK isoenzymes in CSF. The clinical importance of the observed CK levels will be reported in a separate communication.     

黄河裸裂尻鱼肌酸激酶M-CK cDNA的克隆及组织表达分析

肌酸激酶(CK)能够催化磷酸基在二磷酸腺苷(ADP)和磷酸肌酸之间的可逆性转移,在细胞能量代谢过程中发挥重要作用。本研究利用RT-PCR和RACE方法克隆了黄河裸裂尻鱼(Schizopygopsis pylzovi)肌酸激酶基因全长c DNA序列,并进行了生物信息学分析和系统发育关系研究。黄河裸裂尻鱼肌酸激酶c DNA全长1 599 bp,开放阅读框(ORF)1 143 bp,编码380个氨基酸,具有典型的N-端结构域和C-端催化结构域。氨基酸序列同源性分析表明,黄河裸裂尻鱼肌酸激酶与斑马鱼(Danio rerio)和鲤(Cyprinus carpio)M3-CK有较高的同源性,其序列一致度达94%以上。系统发育分析显示,黄河裸裂尻鱼肌酸激酶与斑马鱼和鲤的M3-CK聚在一支,形成一个单系群,初步判定克隆获得的黄河裸裂尻鱼肌酸激酶基因可能与鲤和斑马鱼M3-CK是直系同源基因。利用Real-time PCR方法检测和分析了黄河裸裂尻鱼主要组织肌酸激酶m RNA表达水平,肌肉、肠、眼、心中肌酸激酶转录本表达较高,在肝胰脏和脑中表达微弱。肌酸激酶在肌肉、肠、和心中高表达与其能量代谢功能相适应,而在眼组织中的高表达是否与肌酸激酶的其他功能相关,有待于进一步研究。     

人类线粒体肌酸激酶uMtCK的底物结合位点分析

研究人类线粒体肌酸激酶u Mt CK的结合位点,将其与底物肌酸和ATP结合有关的关键氨基酸进行突变,并对突变体进行酶动力学和圆二色谱数据分析,探讨这些关键氨基酸在底物识别和催化过程中的作用。结果显示,与野生酶相比,突变体Q313A和R336A的K_m~(Cr)分别提高了2.6和2.9倍,k_(cat)下降了19%和55%;同样地,与ATP结合相关的突变体R125A和R287A分别使得K_m~(ATP)升高了3.2和4.2,k_(cat)下降了72%和38%。以上结果表明突变体R125A、R287A、Q313A和R336A影响对底物的结合,同时也降低了酶促反应的速度。利用圆二色谱比较野生酶与不同突变体的二级结构并无明显变化,但进一步的结构模拟表明底物结合位点氨基酸在与底物之间的氢键对底物的识别和酶催化过程中发挥着重要作用。     

Highly Sensitive Immunoassay for Rat Brain-Type Creatine Kinase: Determination in Isolated Purkinje Cells

Ultrasensitive enzyme immunoassay method for the measurement of rat brain-type creatine kinase BB (CK-BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK-BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK-B), showing about 10% cross-reactivity with CK-MB, but it did not cross-react with CK-MM and neuron-specific gamma gamma enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK-BB, being sufficiently sensitive for the measurement of CK-B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK-B in a single Purkinje cell was 1.64 pg. The CK-B concentration in rat cerebellum (about 22 micrograms/mg protein) was about twofold higher than that (about 13 micrograms/mg protein) in the cerebrum. High levels (greater than 5 micrograms/mg protein) of CK-B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK-B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.     

2-氯汞-4-硝基苯酚对人肌肌酸激酶巯基的修饰

本文用含汞试剂MNP修饰人肌肌酸激酶,结果表明,人肌肌酸激酶有6个可反应巯基。MNP首先修饰了一对与活力无关的非必需巯基,增大MNP摩尔比,则进一步修饰另外四个与活性有关的巯基。修饰酶的差吸收光谱、荧光光谱表明这三对巯基的微环境各不相同。其中第二对巯基很可能是位于活性部位的必需巯基,而第三对巯基则是由于第二对巯基,也就是必需巯基,被修饰后,微区构象发生改变而暴露出来的。比较MNP修饰人肌肌酸激酶、鸡胸脯肌肌酸激酶、兔肌肌酸激酶的结果,探讨了MNP对肌酸激酶的修饰以及人肌肌酸激酶可反应巯基的化学微环境。     

肌酸激酶同工酶电泳对窒息新生儿心肌脑组织损伤的意义

目的:探讨血清肌酸激酶同工酶电泳对于新生儿窒息后发生心肌、脑组织损伤中的应用价值。方法:回顾性分析2009年1月至12月我院儿科ICU送检的CK同工酶电泳检测结果,并按不同疾病诊断进行分组统计。结果:在HIE、新生儿窒息患儿血清中CK-BB分别占总CK的13.9±16.8%、2.6±3.1%,CK-MB分别占总CK的2.5±2.0%、8.2±7.5%;CK-MM分别占总CK的91.4±14.1%、94.0±5.8%。新生儿窒息组可见CK-MB增加,显著高于对照及HIE组(P<0.01);HIE组患儿血清中可见CK-BB增加,与对照组、新生儿窒息组比较均有显著性差异(P<0.01)。结论:CK同工酶电泳对诊断新生儿窒息后引起的心脑损伤有重要价值。     

Interaction of Creatine Kinase from Monkey Brain with Substrate: Analysis of Kinetics and Fluorescence Polarization

Abstract: Titrimetric determination of the dissociation constants for the binding of substrates to creatine kinase from monkey brain reveals 13-fold and 4-fold synergism in the forward and reverse directions, respectively. This synergism is expressed as a decrease in the K_D for a given substrate in the ternary complex compared with the binary complex and may be a reflection of substrate-induced conformational change. Creatine kinase labeled with two molecules of 5′-iodoacetamidofluorescein displays a blue shift and a decrease in fluorescence intensity upon binding of MgADP, indicative of movement of the dye into a more hydrophobic environment and quenching of the extrinsic fluorescense. Rotational relaxation times determined from analysis of fluorescence polarization of dansylated brain creatine kinase decrease from 212 ± 7 ns to 189 ± 6 ns upon MgADP binding. Dansylated creatine kinase in 0.5% sodium dodecyl sulfate has a rotational relaxation time of 135 ± 6 ns. The rotational relaxation time of dansylated muscle-type isoenzyme is unaffected by MgADP and has the same value as the brain isoenzyme-MgADP complex. Polarization values at 25°C for muscle and brain enzyme labeled with 3 - (4 - maleimidylphenyl) - 7 - diethylamino - 4 - methylcoumarin compared with limiting polarization and polarization of the free dye suggest that the dye rotation is severely restricted in the muscle form, but possesses freedom of rotation in the brain form. These results support the conclusion that compared with the muscle isoenzyme, the brain isoenzyme is more open at the active site and more flexible overall. Binding of MgADP by brain creatine kinase produces a protein more compact across one or both of its rotational axes, thus resembling the conformation of the muscle isoenzyme. It is probable that creatine kinase in the brain, unlike that from muscle, is subject to kinetic regulation accompanied by conformational modification. This suggests that the neurobiochemical role of the brain isoenzyme is distinct from the metabolic function of the muscle isoenzyme.     

最大等速离心运动诱发肌肉损伤程度与肌酸激酶水平的关系

为探讨人体进行最大等速离心运动(ECC)诱发血液肌酸激酶(CK)水平变化、血清肌酸激酶水平与肌肉损伤(EIMD)的关系,本研究筛选出150名"缺乏运动"的健康大学生为受试者,进行血样采集,进行前测包括血清肌酸激酶(CK)、最大等长肌力(MVC)、肘关节活动角度(ROM)、上臂围(CIR)、肌肉感受(VAS)。受试者进行5组×12次最大等速离心运动,运动后恢复期,将全部受试者血清肌酸激酶值进行排序:血清肌酸激酶值最高和最低20%样本,高肌酸激酶水平组(HCK组)和低肌酸激酶水平组(low LCK组),利用SPSS18.0统计学软件,以方差分析和多元回归分析进行统计分析。本研究发现全部受试者、高肌酸激酶水平组、低肌酸激酶水平组在最大等速离心运动后各评估指标均显著高于比前测结果,p<0.05。全部受试者、高肌酸激酶水平组受试者在最大等速离心运动后各指标变化皆明显大于低肌酸激酶水平组受试者,p<0.05。受试者血清肌酸激酶峰值与最大等长肌力、肘关节活动角度、上臂围、肌肉感受最大变化值有相关,p<0.05。本研究认为肌肉损伤程度与肌酸激酶水平具有显著相关,尤其高血清肌酸激酶水平者肌酸激酶水平较大程度反映肌肉损伤程度趋势。本研究表明,肘关节活动角度、上臂围具有预测肌酸激酶峰值的效果。     

Interaction of Brain-Type Creatine Kinase with its Transition State Analog: Kinetics of Inhibition and Conformational Changes

Abstract

The effects of components of the transition state analog (creatine, MgADP, planar anion) on the kinetics and conformation of creatine kinase isozyme BB from monkey brain was studied. From analysis of the reaction time course using the pH stat assay, it was shown that during accumulation of the reaction products (ADP and creatine phosphate), among several anions added, nitrate proved the most effective in inhibiting catalytic activity. Maximum inhibition (77%) was achieved with 50 mM nitrate. The K_m for ATP was 0.48 mM and in the presence of 2.5 mM nitrate, 2.2 mM; for ATP in the presence of the dead-end complex, creatine and ADP, the apparent Km was 2.0 mM and theK wasO.16mM; in the presence of the transition state analog, MgADP + NO₃” + creatine, the K was estimated to be 0.04 mM.

Ultraviolet difference spectra of creatine kinase revealed significant differences only in the presence of the complete mixture of the components of the transition state analog. Comparison of gel nitration elution profiles for creatine kinase in the absence and presence of the complete mixture of components of the transition state analog did not reveal any differences in elution volume. Addition of components of the transition state analog to creatine kinase resulted in only a marginal change in intrinsic fluorescence. The presence of the components of the transition state analog increased the rate of reactivity of the enzyme with trinitrobenzenesulfonic acid from k = 6.06 ±0.05M^?1min to 6.96 ± 0.11 M^?1min^?1.

This study provides evidence that, like the muscle isozyme of creatine kinase, the brain form is effectively inhibited by the transition state analog. However, the inhibition is accompanied by small changes in the overall conformation of the protein. This adds to the evidence that the functional differences of the isozymic forms of creatine kinase cannot be attributed to differences in kinetic properties.     

鳜肌酸激酶M-CK cDNA的克隆与组织表达分析

利用RT–PCR和cDNA末端快速扩增法（RACE）克隆了鳜（Siniperca chuatsi）肌酸激酶（creatine kinase,CK）cDNA序列,并分析了该基因的结构特征和系统关系。鳜CK cDNA序列全长1586 bp,包括5′端非翻译区92 bp,3′端非翻译区348 bp和开放阅读框（ORF）1 146 bp,共编码381个氨基酸。鳜CK具有脊椎动物CK共有的保守结构域和肌型肌酸激酶（M-CK）同工酶的特异识别位点;氨基酸序列与M-CK型的相似度最高,而与脑型肌酸激酶（B-CK）和线粒体型肌酸激酶（Mi-CKs）的相似度较低;在CK系统关系树中鳜CK与M-CK群聚类。这些均表明,鳜CK属脊椎动物M-CK型。RT-PCR分析表明,鳜M-CK在成体不同组织中的表达量不同,其中,在皮肤、卵巢、肾脏、胃、肌肉和心脏中表达较强;而在眼和脑、肝胰脏中表达较弱。