凝血酶与神经细胞骨架蛋白的研究

目的：观察凝血酶与神经细胞骨架蛋白的关系及使用凝血酶抑制剂（如水蛭素）及钙离子拮抗剂（尼莫地平）是否能减轻凝血酶对脑细胞的毒性损伤，是否存在治疗的有效时间。方法：分别以不同剂量的凝血酶注入SD大鼠尾状核，建立SD大鼠模，采用免疫组化法观察神经细胞骨架蛋白的改变。SD大鼠随机分为6组。1组：生理盐水组；2组：小剂量凝血酶；3组：大剂量凝血酶组；4组：小剂量凝血酶与小剂量水蛭素同时注入组；5组：大剂量凝血酶与大剂量水蛭素同时注入组；6组：尼莫地平组。每组每时相（4h,24h,48h,3d,7d）和5只SD大鼠。结果：大剂量凝血酶的早期（4h）即能引起细胞骨架蛋白的变化，24h-48h，可进一步导致可逆或不可逆损伤，3至7d则主要造成神经细胞不可逆损伤即坏死，而生理盐水，小剂量凝血酶无此作用。水蛭素可拮抗凝血酶的这种作用，尼莫地平组4h神经骨架蛋白无明显变化，24h-48h可导致神经细胞可逆损伤，3至7d则主要造成神经细胞不可逆损伤，结论：钙离子拮抗剂（尼莫地平）能减轻或延缓因酶对脑细胞的毒性作用，有保护神经元的作用。为进一步治疗嬴得时间，由以上结果可推测早期脑水肿是由于凝血酶对脑细胞的毒性作用所致，这种作用（可能通过凝血酶受体）可引起神经细胞突起的改变而造成细胞损伤。     

天然、重组水蛭素对随意皮瓣淤血模型血管内皮细胞生长因子的影响

背景：随意皮瓣移植后易发生皮瓣静脉淤血,甚至坏死,临床与动物实验证明水蛭素能够提高淤血皮瓣的成活率。目的：观察水蛭素对大鼠随意皮瓣淤血模型成活保护作用。方法：Wistar大鼠30只背部设计随意皮瓣10cm×3cm制成淤血模型,随机分为天然水蛭素组、重组水蛭素组和对照组,分别于皮下注射5U天然水蛭素、5U重组水蛭素和生理盐水。术后测定血管内皮细胞生长因子含量及血管内皮细胞生长因子mRNA表达量,计算皮瓣成活率。结果与结论：天然水蛭素组与重组水蛭素组皮瓣成活率比对照组高（P〈0.05）。天然水蛭素组与重组水蛭素组比对照组新生毛细血管血管内皮细胞生长因子表达多（P〈0.05）,且天然水蛭素组最多。提示天然、重组水蛭素均能够促进随意皮瓣血管内皮细胞生长因子表达,有利于新生血管的生成,能够提高随意皮瓣的成活率,且天然水蛭素效果优于重组水蛭素。     

脑微血管内皮细胞缺氧模型转化生长因子β1表达与脑溢安的干预

背景：临床及动物实验证实脑溢安能促进脑出血急性期血肿吸收及神经功能恢复,提高生活质量。目的：观察脑溢安血清对体外缺氧培养的大鼠脑微血管内皮细胞转化生长因子β1mRNA表达的影响。方法：用分离培养的大鼠脑微血管内皮细胞移入厌氧培养箱培养18h建立缺氧损伤模型,并随机分为正常组、模型组、正常血清组及含脑溢安血清组。正常组为同一批正常培养的脑微血管内皮细胞;模型组将正常培养的细胞放入厌氧培养箱内培养18h;正常血清对照组细胞在培养液中加5%正常血清后,放入厌氧培养箱中培养18h;脑溢安血清组细胞在培养液中加5%脑溢安血清后,放入厌氧培养箱内培养18h。结果与结论：缺氧培养使脑微血管内皮细胞存活数量减少,其表达的转化生长因子β1mRNA增强,而脑溢安血清能明显增加脑微血管内皮细胞存活数量,降低转化生长因子β1mRNA表达水平（P〈0.05）。说明脑溢安血清下调转化生长因子β1mRNA的表达可能是其抑制脑微血管内皮细胞的凋亡对缺氧损伤脑微血管内皮细胞的保护作用机制之一。     

天然、重组水蛭素对随意皮瓣淤血模型血管内皮细胞生长因子的影响

背景:随意皮瓣移植后易发生皮瓣静脉淤血,甚至坏死,临床与动物实验证明水蛭素能够提高淤血皮瓣的成活率。目的:观察水蛭素对大鼠随意皮瓣淤血模型成活保护作用。方法:Wistar大鼠30只背部设计随意皮瓣10cm×3cm制成淤血模型,随机分为天然水蛭素组、重组水蛭素组和对照组,分别于皮下注射5U天然水蛭素、5U重组水蛭素和生理盐水。术后测定血管内皮细胞生长因子含量及血管内皮细胞生长因子mRNA表达量,计算皮瓣成活率。结果与结论:天然水蛭素组与重组水蛭素组皮瓣成活率比对照组高(P<0.05)。天然水蛭素组与重组水蛭素组比对照组新生毛细血管血管内皮细胞生长因子表达多(P<0.05),且天然水蛭素组最多。提示天然、重组水蛭素均能够促进随意皮瓣血管内皮细胞生长因子表达,有利于新生血管的生成,能够提高随意皮瓣的成活率,且天然水蛭素效果优于重组水蛭素。     

凝血酶致脑水肿的实验观察

目的：探索凝血酶是否产生或促进水肿形成,以及由凝血酶引起的水肿能否被加入其特异性抑制剂水蛭素或钙拮抗剂尼莫地平所防止。方法：成熟SD大鼠分别接受立体定向尾状核注射,在指定时间点被断头处死,取出大脑作冠状切片,用于湿重法和显微镜(HE染色)观察脑水肿情况。结果：大剂量凝血酶注射后4小时即可产生明显脑水肿,与其余各组比水蛭素或尼莫地平治疗鼠脑水含量减少,脑肿胀减轻。结论：凝血酶直接涉及脑出血水肿形成的过程,水蛭素对脑出血后脑水肿治疗的有效性提供进一步依据证明凝血酶在实质性血块产生水肿病因中占重要地位,因而代表着治疗干预的潜在目标。钙离子拮抗剂尼莫地平能减缓凝血酶对脑细胞的毒性,起保护神经的作用。     

脑微血管内皮细胞缺氧模型转化生长因子β1表达与脑溢安的干预

背景:临床及动物实验证实脑溢安能促进脑出血急性期血肿吸收及神经功能恢复,提高生活质量。目的:观察脑溢安血清对体外缺氧培养的大鼠脑微血管内皮细胞转化生长因子β1mRNA表达的影响。方法:用分离培养的大鼠脑微血管内皮细胞移入厌氧培养箱培养18h建立缺氧损伤模型,并随机分为正常组、模型组、正常血清组及含脑溢安血清组。正常组为同一批正常培养的脑微血管内皮细胞;模型组将正常培养的细胞放入厌氧培养箱内培养18h;正常血清对照组细胞在培养液中加5%正常血清后,放入厌氧培养箱中培养18h;脑溢安血清组细胞在培养液中加5%脑溢安血清后,放入厌氧培养箱内培养18h。结果与结论:缺氧培养使脑微血管内皮细胞存活数量减少,其表达的转化生长因子β1mRNA增强,而脑溢安血清能明显增加脑微血管内皮细胞存活数量,降低转化生长因子β1mRNA表达水平(P<0.05)。说明脑溢安血清下调转化生长因子β1mRNA的表达可能是其抑制脑微血管内皮细胞的凋亡对缺氧损伤脑微血管内皮细胞的保护作用机制之一。     

血管内皮生长因子与促血管生成素1对大鼠血管内皮细胞的作用

背景：血管内皮生长因子、促血管生成素1是血管形成过程中始动并且使之持续的重要因子,研究其对血管内皮细胞的作用具有重要的意义。目的：观察血管内皮生长因子与促血管生成素1对培养血管内皮细胞迁移与增殖能力的影响,并探讨其在血管生成方面的作用机制。方法：在大鼠脐静脉内皮细胞内单独或联合加入血管内皮生长因子、促血管生成素1后,划痕实验和MTT检测对细胞迁移与增殖的影响,观察内皮细胞形态、活性、迁移能力。结果与结论：划痕实验显示单独血管内皮生长因子作用时,与空白对照组细胞迁移无明显差异,单独促血管生成素1作用时,不仅不能增加细胞的迁移作用,反较空白对照组有所减弱,当血管内皮生长因子与促血管生成素1联合作用时,细胞迁移较空白对照组明显增强;MTT实验结果表明：单纯加入血管内皮生长因子或促血管生成素1,均不能起到有效促进内皮细胞增殖的作用;联合应用血管内皮生长因子及促血管生成素1可有效促进增殖。结果可见当血管内皮生长因子与促血管生成素1联合应用时,才能有效促内皮细胞迁移与增殖,发挥促血管生成作用。     

脑微血管内皮细胞对体外缺氧神经干细胞增殖及凋亡的影响

目的:观察脑微血管内皮细胞对体外培养缺氧神经干细胞增殖、凋亡的影响。方法:实验于1996-01/1996-12在佳木斯大学神经科学所完成。实验材料:同一基因背景新生的Wistar大鼠由哈尔滨医科大学实验动物学部提供(黑动字第99102001)。实验方法:①取新生24hWistar大鼠大脑培养神经干细胞,采用免疫组织化学方法鉴定神经干细胞。②取出生1～7dWistar大鼠大脑培养脑微血管内皮细胞,采用免疫细胞化学方法鉴定。③将培养传代纯化的脑微血管内皮细胞,用0.25%胰酶消化成单细胞液后接种于涂有多聚赖氨酸包被的盖玻片上,接种传3代后用胰酶消化的神经干细胞,以1∶10接种比例加入神经细胞完全培养液共培养。④用无菌石蜡油覆盖于低糖细胞培养液表面,制备低氧低糖溶液。对照组:正常神经干细胞置于37℃、体积分数为0.05的CO2饱和湿度培养。缺氧共培养组:取共培养3d细胞,吸出培养液,用无菌Hank’s缓冲液清洗2次,加入低氧低糖溶液共同作用2,4,8,16h后换液,用PBS缓冲液冲洗3次后,换神经细胞完全培养液,置于细胞培养箱中复氧培养。缺氧单纯神经干细胞组:取传3代神经干细胞,用无菌Hank’s缓冲液清洗2次后,方法同上。实验评估:免疫组织荧光鉴定缺氧条件下神经干细胞的增殖与凋亡。结果:①免疫组织化学方法检测Nestin及Ⅷ因子相关抗原分别鉴定为神经干细胞及脑微血管内皮细胞。②对照组细胞胞体饱满,折光性强,周围有光晕。缺氧单纯神经干细胞组细胞胞体肿胀,折光性差,有大量细胞碎屑,仅有少量活细胞,并出现悬浮死细胞。缺氧共培养组细胞形态有所改善,细胞折光性仍较好,大部分细胞突起仍未见回缩,仅少数细胞肿胀,坏死。③随着缺氧时间的延长,细胞死亡明显增多,存活数明显下降。缺氧共培养组细胞存活数明显高于缺氧单纯神经干细胞组与对照组(321.76±22.20,180.00±17.89,255.33±10.33,P<0.01)。④缺氧条件下脑微血管内皮细胞对神经干细胞增殖及凋亡的影响:缺氧共培养组细胞存活数明显高于缺氧单纯神经干细胞组,细胞凋亡细胞数低于缺氧单纯神经干细胞组(P<0.05)。结论:脑微血管内皮细胞可以促进缺氧神经干细胞的增殖、减少其凋亡。     

脑源性神经营养因子与心脏微血管内皮细胞的管状形成

背景：研究发现脑源性神经营养因子可促进内皮细胞的存活,诱导血管新生,但其促进血管新生的细胞与分子机制尚不清楚。目的：观察脑源性神经营养因子在3D体外管状形成模型中对心脏微血管内皮细胞管状结构形成的影响。方法：分离提取大鼠心脏微血管内皮细胞并培养使之形成细胞小球,将心脏微血管内皮细胞小球接种于Ⅰ型胶原-甲基纤维素的3D环境中培养,然后分别加入50,70,100μg/L的脑源性神经营养因子继续培养,于培养24,48h观察并测量心脏微血管内皮细胞的分枝长度和分枝条数。结果与结论：经分离的大鼠心脏微血管内皮细胞小球在Ⅰ型胶原-甲基纤维素3D环境中培养24h,均可见管状分枝,且脑源性神经营养因子可促进管状分枝生长,以100μg/L的脑源性神经营养因子的促生长效果最明显,分枝也最多。培养至48h,心脏微血管内皮细胞小球的分枝更长。说明脑源性神经营养因子在3D体外管状形成模型中可促进大鼠心脏微血管内皮细胞发芽及管状结构的形成,且具有剂量依赖性。     

黄芩苷作用于脑微血管内皮细胞和星形胶质细胞对神经干细胞分化的影响

背景:实验室前期研究表明黄芩苷可以提高体外培养的神经干细胞分化为神经元的比例.但考虑到体内实验中血脑屏障的存在,黄芩苷能否通过血脑屏障主要细胞成分而发挥诱导神经干细胞定向分化为神经元的能力还不清楚.目的:分别将大鼠脑微血管内皮细胞、星形胶质细胞与神经干细胞共培养,观察在模拟体内复杂微环境条件下,黄芩苷能否定向诱导神经干细胞向神经元分化并促进分化神经元的成熟.设计、时间及地点:细胞水平的体外对照观察,于2007-09/2008-03在天津中医药大学中医药研究院中药药理学重点实验室和南开大学医学院完成.材料:取孕14 d SD大鼠,用以分离培养神经干细胞.方法:利用Transwell装置,分别将脑微血管内皮细胞、星形胶质细胞和神经干细胞共培养.用含有10 μmol/L黄芩苷的培养基作用 7 d,并设置空白对照组.以β-tubulinⅢ标记未成熟神经元,MAP-2标记成熟神经元,胶质纤维酸性蛋白标记星形胶质细胞.主要观察指标:应用细胞免疫荧光化学染色检测神经干细胞分化后β-tubulinⅢ、MAP-2和胶质纤维酸性蛋白阳性细胞比例,以实时荧光定量反转录-聚合酶链反应技术检测黄芩苷对脑微血管内皮细胞和星形胶质细胞血管内皮细胞生长因子、神经生长因子和血小板衍生生长因子mRNA表达的影响.结果:与脑微血管内皮细胞共培养条件下,与空白对照组比较,黄芩苷可显著增加β-tubulinⅢ阳性细胞比例(P<0.05).与星形胶质细胞共培养条件下,与空白对照组比较,黄芩苷对β-tubulinⅢ、MAP-2和胶质纤维酸性蛋白阳性细胞比例均无明显影响(P>0.05).与脑微血管内皮细胞、星形胶质细胞共培养条件下,与空白对照组比较,黄芩苷可显著增加MAP-2阳性细胞比例(P<0.01).黄芩苷作用于脑微血管内皮细胞48 h,可以显著上调血小板衍生生长因子基因表达(P<0.01);作用72 h可显著上调星形胶质细胞血管内皮细胞生长因子、神经生长因子和血小板衍生生长因子基因表达(P<0.01).结论:黄芩苷作用于脑微血管内皮细胞可诱导神经干细胞向神经元分化,黄芩苷同时作用于脑微血管内皮细胞和星形胶质细胞可诱导神经干细胞向神经元定向分化并促进其成熟,可能与黄芩苷调控脑微血管内皮细胞和星形胶质细胞生长因子分泌,改善微环境有关.     

The thrombin antagonist hirudin fails to inhibit endotoxin-induced leukocyte/endothelial cell interaction and microvascular perfusion failure

Besides its central role in coagulatory pathways, thrombin is known to be a key mediator of macrophage and granulocyte activation in vitro. During recent years the concept of thrombin inhibition by the specific thrombin inhibitor, hirudin, has been established to treat septic disorders. Since basic mechanisms of sepsis include leukocyte/endothelial cell interaction and deterioration of capillary perfusion, we hypothesized that hirudin modulates leukocyte activation and microvascular injury. Severe endotoxemia was induced in Syrian hamsters by intravenous administration of endotoxin (lipopolysaccharide [LPS], E. coli, 2mg/kg) at 0 h. Hirudin (0.25 mg/kg/h) was substituted intravenously during the 4 h after the induction of endotoxemia (n = 7, hirudin). In control animals (n = 6, control) LPS was given without hirudin substitution. In skinfold chamber preparations leukocyte/endothelial cell interaction and functional capillary density (FCD, measure of capillary perfusion) were analyzed during a 24-h period after LPS injection using intravital fluorescence microscopy. Hirudin effectively normalized thromboplastin time and antithrombin activity when compared to controls (P < 0.05, ANOVA). However, hirudin did not attenuate LPS-induced arteriolar and venular leukocyte adherence, and even tended to increase leukocyte adherence after 24 h (P > 0.05, MANOVA). In parallel, addition of hirudin led to a significant deterioration of FCD over time when compared to controls (hirudin: baseline = 171 +/- 19 cm(-1) versus 16 +/- 9 at 24 h; control: baseline = 150 +/- 20 cm(-1) versus 62 +/- 18 at 24 h; P < 0.05). The fall in FCD in hirudin animals was associated with a significant increase of wet-to-dry weight ratios in lung, kidney, muscle, and small intestine (P < 0.05 versus control, ANOVA). Thus our study does not indicate a protective effect of hirudin on microcirculation during endotoxemia, despite an improvement of coagulatory parameters. This result may at least in part explain the lack of efficacy of hirudin on lethality during endotoxemia and sepsis.     

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins.

We constructed a hirudin cDNA cassette, HV-1.1, that encodes mature hirudin variant-1 fused to the signal peptide of human tissue-type plasminogen activator (t-PA). The cassette was subcloned into retroviral vectors and used to transduce human vascular endothelial cells in vitro. Hirudin antigen and activity were measured by ELISA and thrombin inhibition assays, respectively. Transduced cells secreted up to 35 +/- 2 ng/10(6) cells/24 h of biologically active hirudin; expression was stable for at least 7 weeks. Recombinant hirudin, expressed from the HV-1.1 cassette, had a specific activity of 7.1 +/- 0.2 antithrombin units per microgram (ATU/microgram), compared with specific activities of approximately 12 ATU/microgram for both native leech hirudin and recombinant hirudin produced in yeast. Protein sequencing and mass spectroscopic analysis revealed the presence of an extra N-terminal serine residue, indicating aberrant cleavage of the t-PA signal peptide and likely accounting for the diminished activity. We therefore constructed a second cDNA cassette, HV-1.2, in which hirudin secretion was directed by the signal peptide of human growth hormone. Hirudin expressed from the HV-1.2 cassette had a specific activity of 13.5 +/- 0.2 ATU/microgram. Protein sequencing and mass spectroscopic analysis demonstrated proper cleavage of the growth hormone signal peptide. Thus, we achieved high level retrovirus-mediated secretion of biologically active hirudin from endothelial cells in vitro. Use of these vectors may permit sustained local antagonism of thrombin activity in vivo.     

水蛭素融合蛋白研究进展

水蛭素是一种高效特异的凝血酶抑制剂，临床上用于血栓病的预防和治疗，但它半衰期短、出血副作用强和功能单一。近年来，通过把水蛭素与某些功能蛋白融合表达来解决这些问题，得到的融合蛋白或在体内延长水蛭素的半衰期，或降低其出血副作用，或带来新的功能，如：溶栓、抑制血小板凝集、特异寻靶等。本文对近年来国内外水蛭素融合蛋白研究状况进行综述。     

Hirudin ameliorates intestinal radiation toxicity in the rat: support for thrombin inhibition as strategy to minimize side-effects after radiation therapy and as countermeasure against radiation exposure.

BACKGROUND: The small bowel is a dose-limiting normal tissue in radiation therapy of malignancies in the abdomen and pelvis, as well as an important determinant of survival after non-therapeutic radiation exposure. Irradiation of normal tissues, including intestine, causes loss of vascular thromboresistance and upregulation of thrombin receptors. Radiation-induced endothelial dysfunction is thought to be involved in both early and delayed radiation responses. Hence, thrombin may be a potential target for ameliorating normal tissue radiation toxicity. OBJECTIVE: To assess direct thrombin inhibition as a protective strategy against small bowel radiation toxicity. METHODS: Rat small intestine was exposed to localized orthovoltage X-radiation. Recombinant hirudin, a direct thrombin inhibitor, or vehicle was infused from 2 days before irradiation to 14 days after irradiation. Structural, cellular, and molecular aspects of intestinal radiation injury were assessed at 2 weeks (early toxicity) and 26 weeks (chronic toxicity) after irradiation. RESULTS: Compared with unirradiated intestine, irradiated intestine showed increased expression of tissue factor, increased immunoreactivity for enzymatically active thrombin, and increased extravascular fibrin(ogen) deposition. Hirudin treatment significantly attenuated radiation-induced mucosal damage (P = 0.04), reactive intestinal wall thickening (P = 0.02), transforming growth factor-beta immunoreactivity levels (P = 0.0002), and collagen III deposition (P = 0.003). The differences between hirudin-treated and control rats were more pronounced at 2 weeks than at 26 weeks after irradiation. Hirudin treatment did not affect postradiation granulocyte infiltration. CONCLUSIONS: Short-term thrombin inhibition attenuates important aspects of intestinal radiation toxicity. Thrombin is a promising target for minimizing normal tissue injury after radiation therapy of cancer, as well as for protecting normal tissues from the adverse effects of non-therapeutic radiation exposure.     

Preparation, characterization and in vivo observation of phospholipid-based gas-filled microbubbles containing hirudin

The objective of this work was to prepare echogenic phospholipid-based gas-filled microbubbles (PGM) and investigate their physical characteristics, echogenicity and loading ability of hirudin under various NaCl concentrations. PGM were prepared by a sonication-lyophilization method. Hirudin was used as a model drug to evaluate the drug encapsulation efficiency of the PGM. PGM loaded with hirudin were prepared by dissolving lyophilized powder with hirudin solution. The morphology, particle size and microbubble concentration of PGM were measured. The hirudin encapsulation efficiency as a function of NaCl concentration was determined. The mean particle size and microbubble concentration of PGM were unchanged by the presence of hirudin for at least 60 min after preparation. Hirudin encapsulation quantity was proportional to the hirudin concentration until saturation occurred at high concentration, and the encapsulation efficiency had an inverse relationship. Hirudin encapsulation efficiency was affected by NaCl concentration. When NaCl concentration was increased from 10 mg mL(-1) to 20 mg mL(-1) in PGM solution, hirudin encapsulation efficiency decreased from 35.8 to 26.7%, and microbubble concentration decreased from 2.7 x 10(8) to 1.7 x 10(8) microbubbles per mL. The PGM were shown easily to be visible in in vivo rabbit liver. There was no difference in echogenicity between the loaded and unloaded bubbles. PGM prepared by the sonication-lyophilization method exhibited satisfactory physical characteristics and loading ability and are suitable for use in imaging and ultrasound-triggered delivery.     

Evidence for thrombin enhancement of fibrin polymerization that is independent of its catalytic activity

Inhibition of thrombin proteolysis of fibrinogen with D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK) results in irreversible inactivation of the thrombin catalytic site, but the PPACK-inhibited thrombin, through its exosite, retains its ability to bind to fibrinogen or fibrin. Hirudin inactivates thrombin at the catalytic site and also inhibits thrombin exosite binding to fibrin or fibrinogen. PPACK or hirudin was added to a clotting mixture of fibrinogen and active thrombin (enzyme-to-substrate ratio = 1:400 at ionic strength of 0.14; 1:800 at ionic strength of 0.09) before the onset of gelation. Subsequent fibrin assembly was evaluated by turbidity measurements at 350 nm and by determining the fibrin and fibrinogen content of the clots that ultimately formed. Polymerization rates and the fibrin-fibrinogen content of the clots that formed were greater in the PPACK-inhibited system than in the hirudin-inhibited system, and the effect was amplified at the lower ionic strength. PPACK-thrombin also promoted the polymerization of native or prepared mixtures of fibrin and fibrinogen. The results suggest that in addition to its well-recognized role in the proteolytic conversion of fibrinogen to fibrin, thrombin functions through exosite binding to fibrin as a cofactor in fibrin polymerization by accelerating fibrin clot assembly.     

Thrombin induction of plasminogen activator-inhibitor in cultured human endothelial cells.

We have examined the effect of thrombin on the activity of plasminogen activator (PA) and plasminogen activator-inhibitor (PA-I) in medium conditioned by primary cultures of human umbilical vein endothelial cells. PA activity was measured by fibrinolytic and esterolytic assays, and total tissue-type PA (tPA) antigen by radioimmunoassay. Net PA-I activity was assayed by titration of human urokinase esterolytic activity. Incubation of confluent endothelial cell cultures with thrombin for 24 h caused a sixfold increase in PA-I activity. The effect of thrombin was half-maximal at approximately 0.4 U/ml (less than 4 nM), and required concomitant RNA and protein synthesis. The stimulation of PA-I activity required active alpha-thrombin and was not obtained with gamma-thrombin nor with thrombin catalytically inactivated with hirudin. Because of the excess of PA-I, PA activity was not measurable in either control or thrombin-treated cells. Thrombin did, however, increase medium concentration of tPA antigen by approximately fourfold. The thrombin-induced PA-I inhibited both tPA and urokinase, did not lose activity upon acidification, and was stable to sodium dodecyl sulfate and thiol reduction. We conclude that physiologic concentrations of thrombin increase both PA-I activity and tPA antigen in medium conditioned by human umbilical vein endothelial cells. Because there was always a several-fold increase in the net activity of free PA-I, these observations suggest that the net effect of thrombin is to decrease fibrinolytic activity in human endothelial cells. Thus, thrombin, in addition to its role in coagulation, may protect clots from premature lysis by increasing the amount of a specific fibrinolytic inhibitor.     

脑微血管内皮细胞黏附分子表达与缺血性脑损伤研究进展

脑微血管内皮表达的细胞黏附分子在生理上参与脑组织正常结构和功能的维持，保证内皮细胞层的连续性、通透血脑屏障等功能．在病理过程中具有介导炎性细胞向正常或炎症组织移动或定位等功能．其中由脑微血管内皮细胞表达的免疫球蛋白超家族和选择素家族两类黏附分子与缺血性脑损伤的发生、发展有密切关系。作者对脑微血管内皮细胞表达的黏附分子与缺血性脑损伤的研究进展进行了综述。