白细胞介素1促进外周神经再生作用的形态学研究

目的 从形态学角度了解白细胞介素1局部应用对外周神经损伤后神经再生的作用。并通过对脊髓前角神经元的研究，初步探讨其有关机制。方法 用昆明小鼠单侧坐骨神经切割伤后一期端端缝合为损伤模型。按是否给予白细胞介素1分为实验组和对照组，分别在术后28d和42d切取坐骨神经和相应脊髓节段，测定神经纤维面数密度比，神经纤维断面面积，髓鞘厚度及脊髓前角神经元计数，脊髓前角神经元等效圆直径，等效圆面积，作定量比较。结果 术后28d，神经纤维面数密度比，神经纤维断面面积实验组均优于对照组，髓鞘厚度无显著性；术后42d，除近端神经纤维断面面积两组差异无显著性外，余各指标实验组均优于对照组。损伤侧脊髓前角神经元计数。等效圆直径，等效圆面积在28d和42d，实验组均优于对照组。结论 外周神经损伤局部应用白细胞介素1能促进神经再生，保护脊髓前角神经元。     

坐骨神经预损伤后miR-124-3p调节GAP-43表达并促进轴突生长的实验研究

 目的 通过研究坐骨神经预损伤后背根神经节中microRNA表达谱变化,探讨坐骨神经预损伤促进脊髓后索损伤修复机制。方法 利用微阵列芯片技术和生物信息学方法,研究与坐骨神经预损伤促进脊髓后索损伤修复有关的microRNA,并用RT-qPCR技术、Western blot技术、免疫荧光染色、反义miRNA寡核苷酸抑制剂等技术验证。结果 单纯脊髓后索损伤组miR-124-3p在大鼠脊髓后索损伤后7 d、14 d明显上调;坐骨神经预损伤组,脊髓后索损伤后7 d、14 d背根神经节中miR-124-3p明显下调。与正常背根神经节神经元相比,抑制miR-124-3p后GAP-43表达增加,神经元轴突延长。与对照组相比,各组各时间窗STAT3 mRNA表达差异无统计学意义,坐骨神经预损伤组STAT3蛋白在脊髓后索损伤后7 d和14 d表达上调而单纯脊髓后索损伤组脊髓后索损伤后7 d和14 d STAT3蛋白表达下调。与正常背根神经节神经元相比抑制miR-124-3p的神经元STAT3免疫荧光增强、轴突延长,p-STAT3、GAP-43蛋白表达明显上调。与正常背根神经节神经元相比AG490+AMO-124共同处理的神经元轴突长度无明显差异,STAT3表达明显上调,而p- STAT3和GAP-43表达无明显差异。结论 背根神经节神经元中miR-124-3p下调通过STAT3蛋白的上调促使GAP-43蛋白表达增加是坐骨神经预损伤促进脊髓后索损伤修复的机制之一。     

兔坐骨神经损伤修复后骶交感神经节内c-ret mRNA表达的变化

目的　探讨兔坐骨神经损伤修复后骶交感神经节内c-retmRNA表达的变化。方法　用组织原位杂交方法观察兔坐骨神经损伤修复 1 ,3,7,1 4 ,30 ,6 0d后c -retmRNA在骶交感神经节内的表达。结果　兔坐骨神经损伤修复 3d后 ,在损伤侧骶交感神经节内c -retmRNA表达明显上调 ,在损伤后 7d达高峰 ,损伤后 1 4d与 30d仍维持较高水平 ,损伤后 6 0d基本恢复到正常水平。结论　C -retmRNA在兔坐骨神经损伤修复后骶交感神经节内表达的变化 ,提示在坐骨神经损伤修复后给予外源性胶质源性神经营养因子 (GDNF)可能有利于交感神经纤维的再生。     

舒芬太尼对小鼠周围神经损伤后脊髓星形胶质细胞活化的影响

目的探讨舒芬太尼对小鼠单侧坐骨神经损伤后脊髓星形胶质细胞活化的影响。方法清洁级健康雄性BALB/c小鼠80只,6~8周龄,体重18~22g。采用随机数字量表法分为四组:舒芬太尼高剂量组(H组)、舒芬太尼中剂量组(M组)、舒芬太尼低剂量组(L组)和模型组(MO组),每组20只。制备单侧坐骨神经离断损伤模型后,H、M、L组分别腹腔注射舒芬太尼10μg/kg、5μg/kg、2.5μg/kg,MO组腹腔注射等容量生理盐水,连续注射3d,每天1次。于4、8、12周检测小鼠坐骨神经功能指数(SFI)。于2、4、8、12周每组随机处死的5只小鼠,取其损伤同侧L4~L6脊髓节段。光学显微镜下观察病理学结果。采用免疫组化法检测脊髓各时点GFAP的表达。结果 H、M组SFI明显高于L、MO组(P0.05),L、MO组SFI差异无统计学意义;H、M组脊髓前角神经元形态较好,L、MO组脊髓前角神经元形态较差。与MO组比较,各时点H、M、L组GFAP的表达上调(P0.05);与L组比较,各时点H、M组GFAP的表达上调(P0.05)。结论舒芬太尼促进小鼠周围神经损伤后脊髓星形胶质细胞活化,利于周围神经损伤后的修复。     

兔坐骨神经损伤修复后骶交感神经节内c-ret mRNA表达的变化

目的 探讨兔坐骨神经损伤修复后骶交感神经节内c-ret mRNA表达的变化。方法 用组织原位杂交方法观察兔坐骨神经损伤修复1，3，7，14，30，60d后c-ret mRNA在骶交感神经节内的表达。结果 兔坐骨神经损伤修复3d后，在损伤侧骶交感神经节内c-ret mRNA表达明显上调，在损伤后7d达高峰，损伤后14d与30d仍维持较高水平，损伤后60d基本恢复到正常水平。结论 C—ret mRNA在兔坐骨神经损伤修复后骶交感神经节内表达的变化，提示在坐骨神经损伤修复后给予外源性胶质源性神经营养因子(GDNF)可能有利于交感神经纤维的再生。     

中枢和外周神经损伤后再生早期神经元胞体基因表达的差异

目的:基因芯片技术在进行功能基因组的研究方面具有高通量、大规模、相关性分析等其他方法所不可比拟的优势.我们应用CLONTECH公司的AtlasTM 1.2大鼠cDNA microarray试剂盒及Image 3.0软件分析了大鼠脊髓和坐骨神经损伤后神经元分布区组织的基因表达谱,发现有多种结构和功能基因表达存在差异.我们将筛选出的部分基因用RT-PCR的方法在动物模型中进行了验证.方法:雄性Wistar大鼠,体重200±20克,随机分为坐骨神经夹伤和脊髓半横断损伤两组.伤后7d取相应的神经元胞体分布区神经组织,提取总RNA;根据脊髓损伤和坐骨神经夹伤后神经元胞体区神经组织cDNA microarray分析筛选的结果,选择的基因有即刻早期基因(Fos,Jun-B)、与信号转导有关的分子(14-3-3PGS,GPR12,FRAG1)、与细胞增殖有关的分子(PC-NA)、参与细胞凋亡调节的分子(Bcl-2,Bcl-X)、与神经细胞可塑性有关的分子(NCAM)以及胶质细胞来源的分子(MBP).用RT-PCR的相对定量的方法检测不同损伤模型中胞体区神经组织基因表达的水平,并进行分析对比.结果:(1)筛选出的基因在坐骨神经损伤后表达呈增加趋势的多与促进生长有关,包括GAP43,PCNA,NCAM,Bcl-2等;(2)脊髓损伤后其相应的皮层感觉运动区神经组织基因表达变化的种类与坐骨神经损伤后脊髓相应节段有所不同:即早基因Jun-B、与细胞内信号转导有关的分子14-3-3PGS、GPR12及FRAG1基因表达上调;(3)胶质细胞源性的分子BMP(myelin basic protein)在中枢和外周神经损伤后表达均呈上升趋势,提示轴突损伤后受损神经元胞体周围的胶质细胞也受到影响.结论:脊髓和坐骨神经损伤后相应胞体分布区神经组织基因表达确实存在差异,进一步动态观察这些基因在整体和离体损伤模型中表达的变化规律、进行多基因相关性的分析以及进行功能研究将有可能使我们更加明确这些基因变化在神经再生调控中的作用.     

p27kip1和Skp2在大鼠坐骨神经切断后脊髓中的表达

目的 观察坐骨神经切断后大鼠脊髓中p27^kip1和Skp2的表达变化。方法 将成年SD大鼠随机分为正常对照组和实验组。对实验组实行坐骨神经切断术。用Western blot和免疫组织化学技术检测脊髓中p27^kip1和Skp2的表达变化。结果 Western blot结果表明坐骨神经切断后脊髓中p27^kip1表达持续下降，Skp2表达上调。免疫组织化学结合免疫荧光双标技术显示，在正常组和实验组，p27^kip1和Skp2在脊髓神经元和胶质细胞均有表达。在腹角神经元，损伤后p27^kip1 免疫染色减弱，Skp2增强。坐骨神经损伤前后脊髓腹角p27^kip1和Skp2阳性细胞数目改变呈负相关（r=-0．6892，P〈0．01）。结论 坐骨神经损伤可影响脊髓中p27^kip1和Skp2的表达水平及亚细胞定位，两者改变呈负相关。     

大鼠坐骨神经切断后L3～L6脊髓前角运动神经元凋亡及C-Jun表达的研究

目的　观察大鼠坐骨神经切断后L3 ～L6脊髓前角运动神经元的凋亡及C Jun表达。方法　在大鼠坐骨神经切断术后 12h、2 4h、3 6h、48h、3d、7d、9d、13d ,分别应用TUNEL及免疫组化技术检测神经元的凋亡和C Jun的表达。对照组动物接受同样的手术 ,但不切断神经 (假手术 )。结果　坐骨神经切断后 ,L3 ～L6脊髓前角运动神经元部分发生了凋亡 ,C Jun的表达呈双期性 ,在短暂、迅速的陡升期后接以平缓、持久的平台期。结论　研究结果提示外周神经切断后 ,部分神经元的死亡通过凋亡来介导 ,C Jun的早期表达与神经元的凋亡有关 ,而晚期则可能与神经再生相连。     

他克莫司对周围神经损伤后脊髓组织中肝细胞生长因子表达的影响

[目的]探讨他克莫司对大鼠周围神经损伤后脊髓组织中肝细胞生长因子 (HGF) 表达的影响.[方法]成年雄性大鼠 50 只,随机分为正常组、损伤组和治疗组,后两组行双侧坐骨神经切断.术后治疗组颈后皮下注射他克莫司 (1 mg/kg),损伤组注射生理盐水.术后不同时间点取 L4～6脊髓,应用 Western blotting 技术及免疫荧光标记对脊髓组织中HGF 的表达变化进行定量和定位检测.[结果]HGF 在脊髓灰质前角、中间带和后角的各类神经元中均有表达;损伤组与正常组大鼠脊髓组织中 HGF 的表达之间无显著性差异 (P>0.05),治疗组大鼠脊髓组织中 HGF 的表达在术后 7、14 d 时明显高于损伤组 (P< 0.05).[结论] (1) 周围神经损伤本身并不能使脊髓组织中 HGF 的表达上调;(2) 他克莫司能诱导周围神经损伤后内源性 HGF 的高表达,从而保护相应神经元,促进轴突生长.     

NUMBL在大鼠脊髓损伤过程中的表达变化

[目的]观察NUMBL在大鼠脊髓损伤过程中的表达情况,探讨其在脊髓损伤和修复过程中的生物学功能.[方法]制作脊髓损伤模型,利用western blot、免疫组织化学、免疫荧光等技术观察NUMBL在损伤脊髓中的表达变化、组织分布与细胞定位,在此基础上探讨NUMBL在脊髓损伤后的病理生理意义.[结果]western blot结果显示NUMBL经历了在损伤后8 h和1 d的显著增加后开始下降,直到损伤后2周仍没有明显上调的变化趋势.免疫组化结果显示在脊髓损伤后1 d,NUMBL的表达在临近损伤中心的脊髓中显著增加,而在脊髓损伤后1周,NUMBL的表达较1d时明显降低.免疫荧光结果显示NUMBL与神经元和少突胶质细胞存在共定位,而与星形胶质细胞无明显共定位.[结论]脊髓损伤后脊髓中NUMBL的表达上调参与脊髓损伤的病理过程.     

Postoperative changes in plasma tissue-type plasminogen activator and type I plasminogen activator inhibitor

To clarify the changes which occur postoperatively in intravascular fibrinolysis, plasma levels of tissue-type plasminogen activator (t-PA) antigen, the total plasminogen activator inhibitor type-1 (PAI-1) antigen, and the t-PA-PAI-1 complexes were assayed in this study. Blood samples were taken the morning before surgery, then at 0, 12, 24, 36, 60, 108, and 156h postoperatively in ten patients who underwent radical surgery for thoracic esophageal cancer. The plasma levels of the t-PA and total PAI-1 antigens, and the t-PA-PAI-1 complexes were then measured by enzyme immunoassay. The plasma t-PA and total PAI-1 levels increased significantly in the immediate postoperative period, the percent increase of the latter being much greater than that of the former. Moreover, the calculated free t-PA antigen level was decreased throughout the postoperative period, suggesting postoperative hypofibrinolysis. The platelet count and neutrophil elastase level were significantly correlated with the free t-PA antigen level atr = 0.630,P < 0.001, andr = -0.447,P < 0.01, respectively. The results of this study indicated that postoperative hypofibrinolysis caused by the increased synthesis of PAI-1 may enhance postoperative hypercoagulability, and this may lead to the development of organ damage. Thus, the concentration of the PAI-1 antigen may be a potentially important index for the prediction of postoperative illness.     

Tissue plasminogen activator and penetrating keratoplasty

We report a patient who developed severe intraocular fibrin formation following penetrating keratoplasty and vitrectomy surgery. The fibrin response worsened despite aggressive treatment with topical steroids. On the second postoperative day, 25 micrograms of intracameral tissue plasminogen activator was administered, resulting in rapid resolution of the fibrin response. The graft remained clear. We believe tissue plasminogen activator may be useful in selected cases of severe, recalcitrant postkeratoplasy fibrin formation.     

Peroxynitrite inactivates tissue plasminogen activator

Tissue plasminogen activator (tPA) has a prominent role in physiological fibrinolysis in vivo. Thrombosis has been associated with clinical scenarios (e.g., atherosclerotic disease) known to involve local decreases in tPA activity with concomitant formation of reactive nitrogen species such as peroxynitrite (OONO(-)), a molecule formed from nitric oxide and superoxide. We hypothesized that exposure of tPA to OONO(-) would result in a decrease in tPA activity. OONO(-) was generated with 3-morpholinosydnonimine (SIN-1), a molecule that produces both nitric oxide and superoxide. Recombinant tPA was incubated at 37 degrees C for 60 min with 0 microM SIN-1; 100 microM SIN-1; 100 microM SIN-1 and 4000 U/mL recombinant human superoxide dismutase; or 4000 U/mL recombinant human superoxide dismutase (n = 8 separate reactions per condition). Changes in tPA activity were assessed by addition of tPA samples to tissue factor-exposed human plasma and measuring clot fibrinolysis with a thrombelastograph. Exposure to SIN-1 resulted in a decrease in tPA-mediated fibrinolysis (<1% activity of tPA not exposed to SIN-1) that was significantly (P < 0.001) different from the other three conditions. There were no significant differences between the other conditions. We conclude that tPA is inhibited by OONO(-), and that OONO(-) may have a role in clinical thrombotic scenarios. IMPLICATIONS: Tissue plasminogen activator (tPA) has a prominent role in fibrinolysis in vivo. Thrombosis has been associated with clinical scenarios involving decreases in tPA activity with concomitant formation of the oxidant peroxynitrite. We determined that peroxynitrite decreased tPA activity via thrombelastography. Peroxynitrite-mediated tPA inactivation may have a role in thrombotic states.     

纤溶酶原激活及抑制因子在人蜕膜腺体与间质细胞中的生成及调节

为了探讨人早孕蜕膜腺体与间质细胞离体培养下释放活性纤蛋白溶酶原激活及抑制因子（ＰＡ，ＰＡＩ）的平衡关系与激素调节。应用琼脂糖纤蛋白铺盖及反向铺盖技术，测得腺体及间质细胞均仅释放尿激酶型ＰＡ（ＵＰＡ），而不释放组织型ＰＡ（ｔＰＡ），并同时生成Ⅰ型ＰＡ抑制因子（ＰＡＩ－１）。雌二醇（Ｅ２）及ＲＵ４８６刺激ｕＰＡ、抑制ＰＡＩ－１活性，孕酮（Ｐ）的作用则相反，提示Ｐ在人早孕蜕膜中的作用之一是使ＰＡ－ＰＡ１的动态平衡趋于纤溶活性受抑的安静状态，而ＲＵ４８６激活蜕膜纤溶过程，从而增强细胞外基质蛋白水解，可能是其抗早孕作用机理之一。     

人正常胚胎和葡萄胎绒毛中纤蛋白溶酶原激活与抑制因子及其尿激酶受体的表达

本研究应用原位杂交技术检测人正常胚胎及葡萄胎绒毛中纤蛋白溶酶原激活与抑制因子（ｔＰＡ,ｕＰＡ,ＰＡＩ－１）及其尿激酶受体（ｕＰＡ－Ｒ）的表达。结果表明,人正常胚胎绒毛的合体滋养层和细胞滋养层细胞中均含有ｔＰＡ、ｕＰＡ、ＰＡＩ－１及ｕＰＡ－ＲｍＲＮＡ,葡萄胎绒毛中ｔＰＡ、ＰＡＩ－１及ｕＰＡ－ＲｍＲＮＡ表达明显增强。提示：（１）ＰＡ－ＰＡＩ的协同作用在维持妊娠期间正常的纤蛋白溶解可能是重要的;（２）纤溶激活与抑制在葡萄胎的发展过程中可能起作用     

Expression of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and plasminogen activator inhibitors in patients with renal cell carcinoma: correlation with tumor associated macrophage and prognosis

PURPOSE: Urokinase-type plasminogen activator (uPA) has an important role in tumor progression through the degradation of extracellular matrix. In addition, uPA receptor (uPAR) and plasminogen activator inhibitors (PAIs), composed of PAI-1 and 2, are also known to affect such activities. Tumor associated macrophage (TAM) is an important regulator of tumor progression that is associated with the uPA system in various cancers. However, to our knowledge the clinical significance of PAI-2 and the relationship between the uPA system and TAM in human renal cell carcinoma (RCC) tissues have not been investigated. We investigated and clarified these issues. MATERIALS AND METHODS: The subjects of the current study were 106 consecutive surgically resected specimens from patients with RCC. The expression of uPA, uPAR, PAI-1 and PAI-2 was determined by immunohistochemistry. We also examined the relationships among these molecules, survival and TAM. RESULTS: The mean immunoreactive scores (range 0 to 6) of uPA, uPAR, PAI-1 and PAI-2 were 3.09, 2.22, 1.99 and 0.56, respectively. These scores correlated with the grade and presence of metastasis. The expression of uPA, uPAR and PAI-1 but not PAI-2 correlated negatively with cause specific survival. Of uPA family members multivariate analysis showed that PAI-1 independently influenced cause specific survival. TAM counts correlated with PAI-1 only (p <0.001). CONCLUSIONS: Our results suggest that PAI-1 is an important regulator of tumor progression and survival, and PAI-1 may modulate them via TAM. On the other hand, PAI-2 has a minimum role in survival. Our results may help discussions of treatment strategy in patients with RCC.     

Differential effects of acute and chronic wound fluids on urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and tissue-type plasminogen activator in cultured human keratinocytes and fibroblasts

The effect of wound fluids collected from acute well-healing wounds and chronic nonhealing venous leg ulcers on the plasminogen activation system of keratinocyte and fibroblast cell cultures was studied in a simplified wound-healing model. Acute wound fluid was collected from donor sites of split skin grafts at different time points representing the progressive healing of the wound. Urokinase-type plasminogen activator, tissue-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen activator inhibitor 1 expression were studied. The methods used were immunocapture assay and immunocytochemistry. The results indicated that the later the acute wound fluid was collected, the greater the urokinase-type plasminogen activator and the lower the plasminogen inhibitor-1 level in treated cells. In contrast, the level of urokinase-type plasminogen activator receptor remained stable irrespective of wound fluid treatment. Immunostaining for urokinase-type plasminogen activator of acute wound fluid-treated cells showed a disseminated punctate pattern over the cell surface, but with chronic wound fluid, urokinase-type plasminogen activator was localized to focal contacts. Our findings support the view that in the acute wound environment the plasminogen activator system is proteolytically active and that in chronic leg ulcers urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor may also be organized for cell adhesion and migration.