低分子肝素雾化吸入对急性肺损伤治疗作用的实验研究

目的:急性肺损伤死亡率高且目前尚无有效可靠的治疗方法,本研究旨在探讨低分子肝素雾化吸入对急性肺损伤的治疗作用。方法:健康纯种白兔24只,随机分成3组(n=8):①正常对照组,②生理盐水雾化组,③低分子肝素雾化治疗组。各组分别测定动脉血气、肺干/湿重比、支气管肺泡灌洗液总蛋白含量和凝血功能。结果:与正常对照组相比,生理盐水雾化组和低分子肝素雾化治疗组动脉血氧合指数、肺干/湿重比值显著降低(P0.05),支气管肺泡灌洗液总蛋白含量显著升高(P0.05),而低分子肝素雾化治疗组较生理盐水雾化组动脉血氧合指数、肺干/湿重比值显著升高(P0.05),支气管肺泡灌洗液总蛋白含量显著降低(P0.05);凝血酶原时间以及活化部分凝血活酶时间生理盐水雾化组和低分子肝素雾化治疗组间没有显著差异(P0.05),但均较正常对照组延长(P0.05)。结论:低分子肝素雾化吸入治疗可以改善肺换气,提高氧合,降低肺泡渗出,并且对凝血功能没有明显的副作用,可以在一定程度上缓解急性肺损伤。本研究为急性肺损伤的治疗提供了新的思路和实验依据。     

低分子肝素的绿色荧光蛋白荧光标记及其活性检测

增强型绿色荧光蛋白（EGFP）是生物领域常用的标记物. 本实验首先利用高碘酸法活化低分子肝素（LMWH）,与EGFP连接得到LMWH EGFP.然后用Sephacryl S-200 HR对其进行初步分离,再用Sephadex G-10 HR进行脱盐纯化. 采用Sephacryl S 200 HR检测LMWH EGFP的纯度,为单一对称峰. 经检测,LMWH-EGFP具有良好的热稳定性和耐碱性. 通过荧光分光检测器检测LMWH-EGFP的λEx为488 nm,λEm为509 nm. 通过抗凝实验发现LMWH EGFP仍具有抗凝活性. 本实验建立了LWMH荧光标记的方法,为多糖的荧光标记提供了新的选择.     

Photochemical Preparation of a Novel Low Molecular Weight Heparin

Commercial low molecular weight heparins (LMWHs) are prepared by several methods including peroxidative cleavage, nitrous acid cleavage, chemical ?-elimination, and enzymatic β-elimination. The disadvantages of these methods are that strong reaction conditions or harsh chemicals are used and these can result in decomposition or modification of saccharide units within the polysaccharide backbone. These side-reactions reduce product quality and yield. Here we show the partial photolysis of unfractionated heparin can be performed in distillated water using titanium dioxide (TiO(2)). TiO(2) is a catalyst that can be easily removed by centrifugation or filtration after the photochemical reaction takes place, resulting in highly pure products. The anticoagulant activity of photodegraded LMWH (pLMWH) is comparable to the most common commercially available LMWHs (i.e., Enoxaparin and Dalteparin). (1)H NMR spectra obtained show that pLMWH maintains the same core structure as unfractionated heparin. This photochemical reaction was investigated using liquid chromatography/mass spectrometry (LC/MS) and unlike other processes commonly used to prepare LMWHs, photochemically preparation affords polysaccharide chains of reduced length having both odd and even of saccharide residues.     

Localization of Heat Shock Protein of Low Molecular Weight by Electromicroscopic Immunogold-Labelling

Inspire of the large amount of low molecular weight heat shock protein (LMW HSP) present in plant, its function has still not been clearly known. Understanding the distribution and movement of LMW HSP in cells could provide useful information about its biological functions. A 14 kD HSP was purified from the microsome isolated from the bean of a highly thermotolerant plant Phaseolus vulgaris. Antiserum against this protein was preparaed. The localization of the protein in the cell was analysed by means of electromicroscopic immunogold-labelling method. The images of electromicrograph showed that 14 kD HSP mainly existed in both cytoplasm and endoplasmic reticulum and that no labeled gold was found in tonoplasma, mitochondria or cell wall.     

低分子肝素与普通肝素在介入手术中安全性与疗效的比较

肝素已经普遍用于冠状动脉介入术中,但是由于在应用过程中抗凝效果个体差异大、出血风险相对较高、并发症等问题的日益显现,已经引起了医生们的注意。相对而言,低分子肝素具有抗凝作用强、出血风险低、并发症发生率相对较低、无需实验室监测等优点,进而越来越广泛的在冠状动脉介入术中担当重要角色。普通肝素与低分子肝素在冠状动脉介入术中的有效性与安全性的比较是值得我们去探讨的,知道普通肝素与低分子肝素的优缺点后,可使我们在冠状动脉介入术中更好的选择抗凝药物,使手术的成功率大大提高,降低术后并发症的发生,减轻患者的痛苦。本文结合普通肝素与低分子肝素的药理作用及相关临床研究的结果,分析比较了普通肝素与低分子肝素在冠状动脉介入术中的有效性与安全性。     

Neurofibromatosis Type 2 Tumor Suppressor Protein,NF2, Induces Proteasome-Mediated Degradation of JC Virus T-Antigen in Human Glioblastoma

Neurofibromatosis type 2 protein (NF2) has been shown to act as tumor suppressor primarily through its functions as a cytoskeletal scaffold. However, NF2 can also be found in the nucleus, where its role is less clear. Previously, our group has identified JC virus (JCV) tumor antigen (T-antigen) as a nuclear binding partner for NF2 in tumors derived from JCV T-antigen transgenic mice. The association of NF2 with T-antigen in neuronal origin tumors suggests a potential role for NF2 in regulating the expression of the JCV T-antigen. Here, we report that NF2 suppresses T-antigen protein expression in U-87 MG human glioblastoma cells, which subsequently reduces T-antigen-mediated regulation of the JCV promoter. When T-antigen mRNA was quantified, it was determined that increasing expression of NF2 correlated with an accumulation of T-antigen mRNA; however, a decrease in T-antigen at the protein level was observed. NF2 was found to promote degradation of ubiquitin bound T-antigen protein via a proteasome dependent pathway concomitant with the accumulation of the JCV early mRNA encoding T-antigen. The interaction between T-antigen and NF2 maps to the FERM domain of NF2, which has been shown previously to be responsible for its tumor suppressor activity. Co-immunoprecipitation assays revealed a ternary complex among NF2, T-antigen, and the tumor suppressor protein, p53 within a glioblastoma cell line. Further, these proteins were detected in various degrees in patient tumor tissue, suggesting that these associations may occur in vivo. Collectively, these results demonstrate that NF2 negatively regulates JCV T-antigen expression by proteasome-mediated degradation, and suggest a novel role for NF2 as a suppressor of JCV T-antigen-induced cell cycle regulation.     

Comparative Studies on Chitosan and Polylactic-co-glycolic Acid Incorporated Nanoparticles of Low Molecular Weight Heparin

This study was performed to test the feasibility of chitosan and polylactic-co-glycolic acid (PLGA) incorporated nanoparticles as sustained-release carriers for the delivery of negatively charged low molecular weight heparin (LMWH). Fourier transform infrared (FTIR) spectrometry was used to evaluate the interactions between chitosan and LMWH. The shifts, intensity, and broadening of the characteristic peaks for the functional groups in the FTIR spectra indicated that strong interactions occur between the positively charged chitosans and the negatively charged LMWHs. Three types of LMWH nanoparticles (NP-1, NP-2, and NP-3) were prepared using chitosan with or without PLGA: NP-1 nanoparticles were formed by polyelectrolyte complexation after single mixing, NP-2 nanoparticles were prepared by polyelectrolyte complexation after single emulsion–diffusion–evaporation, and NP-3 nanoparticles were optimized by double emulsion–diffusion–evaporation. NP-3 nanoparticles of LMWH prepared by the emulsion–diffusion–evaporation method showed significant differences in particle morphology, size, zeta potential, and drug release profile compared to NP-1 nanoparticles formed by polyelectrolyte complexation. Another ionic complex of LMWH with chitosan-incorporated PLGA nanoparticles (NP-2) showed lower drug entrapment efficiency than that of NP-1 and NP-3. The drug release rate of NP-3 was slower than the release rates of NP-1 and NP-2, although particle morphology of NP-3 was similar to that of NP-2. Cell viability was not adversely affected when cells were treated with all three types of nanoparticles. The data presented in this study demonstrate that nanoparticles formulated with chitosan–PLGA could be a safe sustained-release carrier for the delivery of LMWH.Key words: chitosan, low molecular weight heparin, nanoparticles, PLGA     

低分子肝素钙对小血管损伤术后血液流变学影响的临床研究

目的：观察应用低分子肝素钙如何提高断指再植成活率的作用机制,为临床更好的应用低分子肝素钙提供理论基础。方法：通过临床随机对照试验设计,以30例断指再植患者为研究对象,30例健康成人为参照对象。观察30例断指再植患者术前术后不同时段血液流变学指标的变化。结果：断指患者受伤至术后72h血液流变学指标均明显升高,与对照组比较有显著差异（P〈0.01）。低分子肝素钙可以显著降低断指再植患者的血液粘度,改善血液不利于断指成活状态,提高断指成活率,降低其坏死率、致残率。结论：低分子肝素钙,毒副作用小,为防治断指再植术后并发症的一种良药,为临床断指成功再植提供了一种较为理想的治疗方法。     

A Low Molecular Weight Heparin Inhibits Experimental Metastasis in Mice Independently of the Endothelial Glycocalyx

Background

Some low molecular weight heparins (LMWHs) prolong survival of cancer patients and inhibit experimental metastasis. The underlying mechanisms are still not clear but it has been suggested that LMWHs (at least in part) limit metastasis by preventing cancer cell-induced destruction of the endothelial glycocalyx.

Methodology/Principal Findings

To prove or refute this hypothesis, we determined the net effects of the endothelial glycocalyx in cancer cell extravasation and we assessed the anti-metastatic effect of a clinically used LMWH in the presence and absence of an intact endothelial glycocalyx. We show that both exogenous enzymatic degradation as well as endogenous genetic modification of the endothelial glycocalyx decreased pulmonary tumor formation in a murine experimental metastasis model. Moreover, LMWH administration significantly reduced the number of pulmonary tumor foci and thus experimental metastasis both in the presence or absence of an intact endothelial glycocalyx.

Conclusions

In summary, this paper shows that the net effect of the endothelial glycocalyx enhances experimental metastasis and that a LMWH does not limit experimental metastasis by a process involving the endothelial glycocalyx.     

东菱克栓酶加低分子肝素治疗急性脑梗死的疗效及安全性研究

目的 观察东菱克栓酶加低分子肝素治疗急性脑梗死的疗效及安全性。方法 在东菱克栓酶常规疗程结构后。２８例人造病人加用低分子肝素皮下注射。结果 总有效率达８２．１４％,神经功能缺损评分显著进步（Ｐ〈０．０５）。血液流变学多项指标进一步改善;而且加用低分子肝素后ＰＣＴ、ＰＴ、ＫＰＴＴ的影响不大（Ｐ〉０．０５）。结论 严格人造标准和必要的凝血功能监测。加用低分子肝素能在一定程度上提高疗诳改善预后,临床出血     

低分子肝素钠预防过敏性紫癜肾炎机制研究

目的:探讨低分子肝素钠预防患儿过敏性紫癜肾炎的临床机制.方法:将60例患儿随机分为治疗组(30例)和对照组(30例).治疗组在常规治疗基础上给予低分子肝素钠.观察两组临床症状消失时间及尿常规异常情况.比较两组疗效、肾损害的情况及比较治疗前、治疗1个月、3个月后复查尿微量白蛋白(Alb)、32-微球蛋白(32-mG)含量的变化以及血清IL-8的水平.结果:治疗组临床症状消失时间,尿常规异常明显低于对照组.两组疗效比较,差异无统计学意义(P＞0.05).治疗组肾损害发生率低于对照组.治疗1个月后、3个月后治疗组尿Alb及β2-rG和血清IL-8水平均明显低于对照组(P＜0.05).结论:低分子肝素钠能预防或减少HSPN的发生,具有显著的肾脏保护作用,可能与其降低血清IL-8水平有关.     

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.^1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.^4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.⁶ Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.^7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.^10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.     

Identification of the Low Molecular Weight Copper Protein from Copper-intoxicated Mung Bean Plants

Mung bean plants (Wilczek) accumulate increasingly greater amounts of buffer-extractable copper in both their shoots and roots when grown in liquid medium containing greater than 2 micrograms per milliliter copper (31.4 micromolar) as cupric sulfate. This increase in soluble copper is accompanied by an increase in the relative amount of low molecular weight (7,000 to 20,000) macromolecular-bound copper and a decrease in the relative amount of high molecular weight (greater than 20,000) copper. The major low molecular weight copper protein has been isolated from copper-intoxicated mung bean plants by a combination of ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. It was identified as mung bean plastocyanin on the basis of its molecular weight, optical behavior, and amino acid composition. No evidence was found for a low molecular weight copper-binding protein corresponding to mammalian thionein or chelatin.     

High Temperature Stress Resistance of Escherichia coli Induced by a Tobacco Class I Low Molecular Weight Heat-Shock Protein

TLHS1 is a class I low molecular weight heat-shock protein (LMW HSP) of tobacco (Nicotiana tabacum). For a functional study of TLHS1, a recombinant DNA coding for TLHS1 with a hexahistidine tag at the aminoterminus was constructed and expressed in Escherichia coli. An expressed fusion protein, H₆TLHS1, was purified using a Ni²⁺ affinity column and a Sephacryl S400 HR column. A polyclonal antibody against H₆TLHS1 was produced to follow the fate of H₆TLHS1 in E. coli. The fusion protein in E. coli maintained its solubility at a temperature of up to 90°C and most of the proteins in the E. coli cell lysate with H₆TLHS1 were prevented from thermally induced aggregation at up to 90°C. We compared the viability of E. coli cells expressing H₆TLHS1 to the E. coli cells without H₆TLHS1 at a temperature of 50°C. After 8 h of high temperature treatment, E. coli cells with H₆TLHS1 survived about three thousand times more than the bacterial cells without H₆TLHS1. These results showed that a plant class I LMW HSP, TLHS1, can protect proteins of E. coli from heat denaturation, which could lead to a higher survival rate of the bacterial cells at high temperature.     

Microbial Detection with Low Molecular Weight RNA

The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%. Received: 13 March 2001 / Accepted: 3 May 2001     

Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress

Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death.     

Chemical and Immunological Characterization of a Low Molecular Weight Outer Membrane Protein of Salmonella typhi

A new immunogenic outer membrane protein, Omp-28 (MW 28,000 and pI 4.6), was isolated from smooth Salmonella typhi cells by the use of an extracting medium containing 6 m urea, 1% deoxycholate and 5 mM EDTA. The purification of Omp-28 was performed by gel filtration and fast ion exchange chromatography. This protein showed to be the prevalent component isolated by the latter methodology. Omp-28 is formed by three identical subunits (MW 9,000), not linked by disulfide bonds. The partial N-terminal amino acid sequence of Omp-28 presented great homology with part of the sequence of an Escherichia coli protein found in a precursor whose sequence was predicted by c-DNA. ELISA and Western blotting identified Omp-28 as the major antigenic protein present in the outer membrane protein fraction, isolated by gel filtration. Antibodies against Omp-28 were detected by ELISA in 43% of 28 sera from typhoid fever convalescent patients. The antisera from mice immunized with Omp-28 and the highest positive typhoid fever convalescent serum gave a positive bactericidal test, killing 50% of Salmonella typhi cells in serum dilutions of 1/80 and 1/320, respectively. These results indicate the immunogenic importance of Omp-28 isolated from Salmonella typhi outer membrane and strongly suggest it should be used in further studies of animal protection against the disease caused by this pathogenic bacteria.     

低分子肝素与普通肝素对经皮冠脉介入手术的疗效和安全性的比较

目的:回顾性比较冠心病患者接受经皮冠脉介入(PCI)治疗时分别使用低分子量肝素(LMWH)和普通肝素(UFH)的疗效和安全性的差异。方法:选择本院2011-01至2012-12接受PCI术患者共200例,根据PCI使用肝素不同分为LMWH组(n=117),UFH组(n=83)。按常规方法,完成PCI术后,根据患者术中靶血管TIMI血流评价各组疗效,根据术中、后72h内出血/血肿、死亡、其他(心包填塞、胸痛、心源性休克、心脏破裂、室间隔穿孔、室速室颤、心脏骤停,阿斯发作、亚急性支架内血栓形成等)不良事件的发生率,比较各组安全性。结果:(1)各组患者临床基线资料差异无统计学意义(P0.05)。(2)各组患者PCI术中支架植入后靶血管血流改变存在统计学差异(P0.05),LMWH在疗效上优于UFH。(3)各组间死亡发生上无统计学差异(P0.05),在出血/血肿及其他(心包填塞、胸痛、心源性休克、心脏破裂、室间隔穿孔、室速室颤、心脏骤停,阿斯发作、亚急性支架内血栓形成等)的发生上存在统计学差异(P0.05),LMWH的不良反应少,安全性更高。结论:LMWH在PCI术中疗效更为显著,且较UFH不良反应少、安全性高,更适用于PCI术的抗凝治疗。     

应用PCR技术定向引入DNA小片段的策略

描述一种应用PCR技术定向引入DNA小片段和特异酶切位点的方法。为了获得m2/loxp66EGFPloxp71基因片段。根据EGFP基因序列,设计一对特异引物,上、下游引物分别引入m2/loxp66、loxp71序列和Xhol、Mlu1酶切位点。以pEGFPN1质粒为模板,采用PCR扩增以合成DNA双链,插入到克隆载体pMD18T。对重组子测序结果表明,实现了DNA小片段和酶切位点的定向引入。     

血凝酶对髋关节置换术低分子肝素抗凝患者出血量及凝血功能的影响

目的:观察注射用血凝酶对髋关节置换术低分子肝素抗凝患者术中出血量及凝血功能的影响。方法:选取收治的髋关节置换术的患者共161例,采用双盲、随机的方法分为实验组83例和对照组78例,所有患者均为我院收治的行髋关节置换术的病例。对照组采用术前给予低分子肝素处理,观察组患者术前给予注射用血凝酶及低分子肝素处理。术后对两组患者术中出血量、术后24 h引流量、术后24 h红细胞(RBC)、血红蛋白(Hb)、纤维蛋白原(Fib)、术后24 h部分凝血活酶时间(APTT)、凝血酶原时间(PT)等情况进行分析。结果:实验组患者术中出血量、术后24 h引流量值均低于对照组,且差异有显著性(P0.05)。两组患者术后24 h红细胞RBC、血红蛋白Hb均下降,但是组间比较差异无统计学意义(P0.05)。术后纤维蛋白原Fib值均上升,组间比较差异无统计学意义(P0.05)。实验患者术后24 h部分凝血活酶时间APTT、凝血酶原时间PT均较术前有所降低,但是于术前及组间比较差异无统计学意义(P0.05)。结论:全髋置换术中使用注射用血凝酶不受低分子肝素的抑制,且安全有效,值得临床进一步研究和应用。