重组水蛭素在大鼠体内的药动学研究

目的研究重组水蛭素iv给药在大鼠体内的药动学。方法采用125-标记的放射性同位素法(RA法)、三氯醋酸沉淀结合放射性检测法(TCA-RA法)研究重组水蛭素iv给药在大鼠体内的药动学。结果大鼠iv0.5、1.0和2.0mg/kg重组水蛭素,以RA法检测,该药的消除半衰期(t1/2ke)分别为162.8、147.5和172.8min,AUC分别为264.9、429.9和1112.9(μg·min)/mL;以TCA-RA法检测,该药的t1/2ke分别为100、101和107min,AUC分别为160.7、327.7和551.3(μg·min)/mL。两种方法所得结果均证明AUC与剂量呈正相关,相关系数均大于0.99。大鼠iv重组水蛭素1.0mg/kg后用RA法和TCA-RA法测定不同组织的药物质量浓度,在各时间点以肾脏含药量显著高于其他组织,肺、胃、脾、心、肝、性腺等组织次之,脑、脂肪最少。大鼠iv重组水蛭素1.0mg/kg后,用RA法测定不同时间段内尿、粪的放射性总量,在96h内70.16%放射性从尿中回收,1.35%从粪中回收到,24h内胆汁排泄量为3.46%。结论RA法和TCA-RA法测得重组水蛭素在大鼠体内的药动学结果不同,但整体趋势一致;重组水蛭素主要由尿排泄。     

甘油对毕赤酵母高密度发酵表达重组水蛭素Ⅱ的影响

目的：考察甘油对毕赤酵母生长和重组水蛭素Ⅱ表达的影响。方法：在摇瓶和IOL发酵罐中用含有不同浓度甘油的培养基培养毕赤酵母，测定毕赤酵母的生长和重组水蛭素Ⅱ的表达。结果：发酵培养基中初始甘油浓度为5g／L时，毕赤酵母生长速度明显加快；补料过程中甘油浓度大于70g／L时，毕赤酵母生长受到抑制。与不加甘油相比较，诱导表达期维持培养基中甘油浓度在5g/L以下，重组水蛭素表达可提前4h，诱导30h其活性达13000 ATU／ml，比仅用甲醇诱导时效价提高了7．7％。结论：降低初始培养基中甘油浓度有利于毕赤酵母生长，补料过程中培养基甘油浓度应维持在押制毕赤酵母生长的浓度之下，诱导阶段培养基中少量的甘油有利于水蛭素的表达.     

重组水蛭素治疗兔弥散性血管内凝血

目的:观察重组水蛭素对治疗脂多糖致兔弥散性血管内凝血模型的抗凝疗效。方法:建立兔脂多糖弥散性血管内凝血模型,18只新西兰大白兔随机分为3组,静脉注射脂多糖的同时分别予生理盐水、普通肝素和重组水蛭素,于注射脂多糖前,注射后2,4,6h检测血小板计数、活化部分凝血活酶时间、抗凝血酶Ⅲ、纤维蛋白原、血清纤维蛋白降解产物(FDP);并观察心、肺脏器的病理变化。结果:模型组兔活化部分凝血活酶时间明显延长,血小板计数和纤维蛋白原显著降低,抗凝血酶Ⅲ活性明显下降,与肝素组相比重组水蛭素组血小板和纤维蛋白原消耗减少,抗凝血酶Ⅲ活性下降程度减轻。所有实验动物血清FDP变化无统计学差异。病理检查模型组心、肝、肺、肾、脾脏可见微血栓形成,重组水蛭素组均未见微血栓形成。结论:重组水蛭素具有明显的抗凝作用,可有效治疗弥散性血管内凝血。     

重组水蛭素经蟾蜍肺泡膜的转运研究

 目的考察重组水蛭素跨膜转运的基本特点和影响因素,对转运的机制进行初步的探讨,为重组水蛭素肺部给药的体内评价打下理论基础。方法将蟾蜍肺泡膜作为肺液上皮体外模型,装在水平扩散池上,向给药池中加入荧光标记的重组水蛭素(FTTC-rHV₂),在不同的温度、不同的给药池初始浓度、不同的转运方向的条件下测定表观渗透系数,同时考察给药池溶液中高渗透压、酸性pH、加入渗透促进剂或者代谢抑制剂时的转运情况。同时监测膜电阻的变化。结果FTTC-rHV₂的表观渗透系数具有浓度非依赖性、温度非依赖性和方向非依赖性。酸性pH环境、高渗透压和吸收促进剂使表现渗透系数明显增加,而代谢抑制剂对袁观渗透系数没有明显的影响。3h内膜电阻没有明显的变化,说明膜的完整性得以保持。结论重组水蛭素经蟾蜍肺泡膜的转运是一个非能量依赖、非载体依赖的被动扩散过程。     

Assessment of Multiplate platelet aggregometry using citrate,heparin or hirudin in Rhesus macaques

Electrical impedance aggregometry (EIA) has gained popularity in clinical and research applications. Nonhuman primates are used to study disease and drug-related mechanisms that affect hemostasis, therefore establishing normal EIA parameters are necessary. The anticoagulants sodium heparin, hirudin and sodium citrate and three agonists, ADP, ASPI, and collagen were evaluated. Whole blood from 12 adult male rhesus macaques was collected to evaluate anticoagulants, sodium heparin, hirudin and sodium citrate using three agonists (ADP, ASPI and collagen), on the Multiplate® 5.0 Analyzer. Platelet function was reported for three parameters: Area under the curve (AUC), aggregation, and aggregation velocity. There was a significant difference in mean AUC between citrate and heparin samples, and citrate and hirudin samples regardless of the agonist used. There was no difference in AUC between heparin and hirudin. ADP-activated samples showed an increase in impedance with hirudin samples compared to citrate. Furthermore, heparin and hirudin out-perform citrate as the anticoagulant for EIA in the macaque. Finally, this study demonstrates the utility of the Multiplate® system in this model and provides important insight into anticoagulant choice when using EIA.     

Effects of Hirudin on High Glucose-Induced Oxidative Stress and Inflammatory Pathway in Rat Dorsal Root Ganglion Neurons

Objective: To investigate protective effects of hirudin on oxidative stress and apoptosis of spinal dorsal root ganglion cells in high-glucose rats at the cellular and molecular level. Methods: Dorsal root ganglion neurons(DRGn) were harvested from embryonic day in 15 SD rats, purified and identificated after primary culture. They were divided into the normal control group, high-glucose(HG) group, positive control(alpha-lipoic acid, ALA) group, low-dose hirudin group(H1), medium-dose hirudin group(H2) and high-dose hirudin group(H3). The control group was cultured by neuron specific culture medium, while the HG group was cultured by neuron specific culture medium and 20 mmol/L glucose(HG medium). The hirudin groups were cultured by HG medium+0.25 IU/mL hirudin(H1), HG medium+0.5 IU/mL hirudin(H2) and HG medium+1 IU/mL hirudin(H3). The ALA group was cultured by HG medium+100 μmol/L ALA. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylt etrazolium bromide(MTT) assay was used to explore the optimum concentration and intervention time. Flow cytometry assay was used to detect the level of reactive oxygen series(ROS). Western blot and quantificational realtime polymerase chain reaction(qRT-PCR) were used to detect the expression of protein and mRNA of nuclear factor erythroid 2-related factor 2(Nrf-2), hemeoxygence-1(HO-1), nuclear factor-κB(NF-κB) and Caspase-3. TUNEL assay was used to test the apoptosis rate of different groups. Results: After 24 h of culture, the cell activity of hirudin and ALA groups were higher than that of HG group, and there was a statistical difference between the H1 group and HG group(P0.05). In hirudin groups, the apoptosis rate of cells, the expression of activated Caspase-3 protein and Caspase-3 mRNA were lower than those of HG group(P0.01), higher than those of ALA group(P0.01 or P0.05). The ROS level of hirudin groups was higher than that of ALA group(P0.01), lower than that of HG group(P0.01 or P0.05). The expression of NF-κB(P65) protein in H3 group were lower than those of HG group(P0.05). The expression of Nrf-2 protein in hirudin groups was higher than that of HG group(P0.01), lower than that of ALA group(P0.01 or P0.05). The expression of HO-1 protein in hirudin groups was lower than that of ALA group(P0.01 or P0.05), higher than that of HG group(P0.01 or P0.05). Conclusions: The activity of DRGn cells can be promoted by hirudin under HG conditions. The effects of hirudin on the inhibition of HG on DRGn cells damage mainly include scavenging ROS, up-regulating Nrf-2/HO-1 pathway, inhibiting activation of NF-κB pathway, down-regulating the expression of and Caspase-3 and reducing DRGn cell apoptosis.     

水蛭素对增生性瘢痕基质金属蛋白酶-2、9表达作用的影响

目的 研究水蛭素对增生性瘢痕组织基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)的影响,探讨水蛭素对增生性瘢痕作用机理.方法 取烧伤后6个月内的皮肤增生性瘢痕,体外培养成纤维细胞,ELISA法测定水蛭素作用后MMP-2、MMP-9表达含量.结果 水蛭素作用后增生性瘢痕组织内MMP-2、MMP-9含量增加,分别以0.5 U/mL和1 U/mL组作用最明显,与对照组比较差异均有统计学意义(P＜0.01).结论 水蛭素能促进MMP-2、MMP-9表达,有抑制增生性瘢痕的作用.     

疏血通注射液对2型糖尿病患者炎性因子的影响

目的观察疏血通注射液对2型糖尿病患者炎性细胞因子影响。方法 175例2型糖尿病患者随机分为对照组和治疗组,分别给予双胍类和双胍类基础上加疏血通注射液。入选后第2天和第16天分别测定肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、C反应蛋白、纤溶酶原激活抑制物-1(PAI-1)和脂联素。结果治疗16d后,治疗组TNF-α、IL-6、C反应蛋白、PAI-1和脂联素与对照组显著差异(P<0.05)。结论疏血通注射液对2型糖尿病患者炎性细胞因子调节有一定效果。     

水蛭素通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化

摘要：目的 观察水蛭素对人骨髓间充质干细胞（bone marrow mesenchymal stem cell,BMSC）成骨分化的影响。方法 BMSCs细胞分为正常培养的对照组、成骨诱导的诱导组以及加入不同浓度（1、10、20 ATU/mL）处理的水蛭素组。MTT检测细胞增值并筛选水蛭素最适作用浓度。流式细胞仪检测细胞凋亡。RT-PCR和Western blot分别检测成骨基因Runx2、Osterix、COL1A1的mRNA和蛋白表达。BCIP/NBT染色法检测细胞中的碱性磷酸酶水平。茜素红染色检测矿化结节。检测VEGF、Notch1、Jagged1和CBF1的mRNA和蛋白表达。结果 骨髓间充质干细胞经成骨诱导细胞增殖显著增加,中高浓度的水蛭素可以不同程度促进成骨诱导的BMSCs细胞增殖（P＜0.05）,并筛选出20 ATU/mL作为水蛭素的使用浓度。水蛭素抑制成骨诱导的BMSCs细胞凋亡,上调Runx2、Osterix、COL1A1的mRNA和蛋白表达,增加碱性磷酸酶水平,促进细胞中矿化结节的生成,并提升BMSCs细胞中VEGF、Notch1、Jagged1和CBF1的表达（P＜0.05）。结论 水蛭素可能通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化。     

仿生诱导高效提取水蛭素及分离纯化的研究

目的:找出一种高效快速提取水蛭素的方法,避免传统方式的低效以及杀死水蛭造成的浪费。方法:采用仿生诱导的方法对活体水蛭进行诱导,使其分泌唾液。利用超滤技术对水蛭唾液进行快速膜分离,再经离子交换层析和凝胶过滤层析进行中间纯化,最后利用HPLC进行分离纯化得到高纯度的水蛭素。结果:经过分离纯化可以得到纯度为92%的水蛭素。结论:仿生诱导的方法简便可行,超滤作为粗级分离有利于水蛭素后期分离,且超滤能进行大量的分离,有利于产业化。