Melanoma cell-derived vascular endothelial growth factor induces endothelial tubulogenesis within fibrin gels by a metalloproteinase-mediated mechanism

Angiogenesis is a process required not only for embryonal development but is encountered in wound healing and in pathological situations such as tumour growth. In vitro, formation of capillary-like structures can be induced by seeding human microvascular endothelial cells (HDMECs) on top of a fibrin matrix in the presence of phorbol 12-myristate 13-acetate (PMA) as a stimulating agent. In this study, we show that supernatants collected from high-invasive melanoma cells (BLM) induce the formation of tubular structures similar to PMA treatment whereas supernatants from low-invasive cells (WM164) did not. Analysis of proteins secreted into the supernatant of both melanoma cell lines identified differential expression of several pro-angiogenic proteins in high- and low-invasive melanoma cells. Vascular endothelial growth factor (VEGF) was strongly expressed by high- but not by low-invasive melanoma cells. Neutralisation of VEGF as well as inhibition of matrix metalloproteases (MMPs) using the broad spectrum MMP inhibitor 1,10-phenanthroline, both strongly reduced the melanoma-induced tube formation. PMA treatment of HDMECs on a fibrin matrix stimulated MT1-MMP synthesis, indicating that this protease is involved in PMA-induced angiogenesis. In addition, stimulation of HDMECs by supernatants of BLM melanoma cells resulted in a strong induction of ADAM-15, which is known to act as a metalloproteinase. In conclusion, these results show that VEGF released by melanoma cells is an important mediator of neo-vascularisation and that this process depends on the presence of metalloproteinases.     

Hepatocyte-derived fibrinogen-related protein-1 is associated with the fibrin matrix of a plasma clot

In order to study the multiple functions of fibrinogen and fibrin, we are investigating which proteins bind to the fibrin matrix of a plasma clot by using a proteomic approach. Extracts from washed plasma clots were analysed by 2-D gel electrophoresis. A relatively abundant spot was identified as hepatocyte-derived fibrinogen-related protein-1 (HFREP-1) by MALDI-TOF analysis, molecular mass (34 kDa), iso-electric point (pI 5.5) as well as by Western blot analysis. HFREP-1 in plasma almost completely bound to the fibrin matrix during clot formation. Several purified fibrinogen preparations proved to be contaminated with HFREP-1. It is concluded that HFREP-1 (also named hepassocin), a protein with liver cell growth regulatory properties, occurs in plasma and strongly associates with fibrin and possibly fibrinogen.     

纤维蛋白胶一药物缓释系统的研究进展

纤维蛋白胶作为一种生物蛋白制剂,除了应用于伤口止血、封闭组织缺损、防止组织粘连之外,也可以作为药物缓释载体。本文就纤维蛋白胶-药物缓释系统的研究及临床应用作以综述。     

以可注射性纤维蛋白封闭剂为支架体内构建组织工程软骨

目的: 研究以纤维蛋白封闭剂（FS）为载体复合人胚关节软骨细胞体内构建可注射性组织工程软骨的可行性。方法:常规分离消化,体外单层培养胎儿关节软骨细胞,观察软骨细胞的生物学特性。分别将1×107、2×107、3×107第4代软骨细胞与FS混合接种于裸鼠皮下, 并于第10周取材判断体内形成软骨的能力。结果: 3~4代软骨细胞保持了很高的增殖和分泌基质的能力。软骨细胞与FS的复合物体内接种后各组均可形成软骨样组织块,其湿重、GAG含量随着接种细胞数量的增多而增高,各组之间差异具有显著性（p<0.05）。3×107细胞组GAG含量与正常人胚关节软骨没有差异（p>0.05）。组织切片显示软骨细胞位于类似正常软骨组织的陷窝中,阿尔新蓝染色及II型胶原表达阳性,细胞内富含高尔基体、粗面内质网及大量分泌泡。结论: FS和人胚关节软骨细胞可以作为理想的支架材料和种子细胞应用于可注射软骨组织的构建。     

Streptococcus uberis plasminogen activator (SUPA) activates human plasminogen through novel species-specific and fibrin-targeted mechanisms

Bacterial plasminogen (Pg) activators generate plasmin to degrade fibrin blood clots and other proteins that modulate the pathogenesis of infection, yet despite strong homology between mammalian Pgs, the activity of bacterial Pg activators is thought to be restricted to the Pg of their host mammalian species. Thus, we found that Streptococcus uberis Pg activator (SUPA), isolated from a Streptococcus species that infects cows but not humans, robustly activated bovine but not human Pg in purified systems and in plasma. Consistent with this, SUPA formed a higher avidity complex (118-fold) with bovine Pg than with human Pg and non-proteolytically activated bovine but not human Pg. Surprisingly, however, the presence of human fibrin overrides the species-restricted action of SUPA. First, human fibrin enhanced the binding avidity of SUPA for human Pg by 4-8-fold in the presence and absence of chloride ion (a negative regulator). Second, although SUPA did not protect plasmin from inactivation by α(2)-antiplasmin, fibrin did protect human plasmin, which formed a 31-fold higher avidity complex with SUPA than Pg. Third, fibrin significantly enhanced Pg activation by reducing the K(m) (4-fold) and improving the catalytic efficiency of the SUPA complex (6-fold). Taken together, these data suggest that indirect molecular interactions may override the species-restricted activity of bacterial Pg activators; this may affect the pathogenesis of infections or may be exploited to facilitate the design of new blood clot-dissolving drugs.     

Non-pesticide alternatives for reducing feeding damage caused by the large pine weevil (Hylobius abietis L.)

The large pine weevil (Hylobius abietis L.) is an important pest of young forest stands in Europe. Larvae develop under the bark of freshly cut pine and spruce stumps, but maturing weevils feed on the bark of coniferous seedlings. Such seedlings frequently die because of bark consumption near the root collar. We tested the effect of three treatments (the insecticide alpha cypermethrin, a wax coating and a glue coating) on the feeding damage caused by H. abietis on Douglas fir (Pseudotsuga menziesii) and Norway spruce (Picea abies) seedlings under semi-natural conditions. In two experiments (one in 2016 and another in 2017) seedlings in cages were subjected to pine weevil feeding for 16 weeks under shaded outdoor conditions. The experiment in 2016 compared insecticide and wax treatments and an untreated control on Douglas fir and Norway spruce, and the experiment in 2017 compared insecticide, wax and glue treatments and an untreated control on Norway spruce. In both experiments, all treatments significantly reduced H. abietis feeding damage at week 8 at the end of both experiments (week 16); the effect of treatments was significant only on spruce seedlings. The damages on Douglas fir seedlings was less on treated seedlings than on untreated control seedlings but differences were not significant. Coating stems with glue and especially with wax was generally effective at reducing weevil damage and in most cases provided control that was not significantly different from that provided by insecticide treatment. Our results suggest that a wax coating has the potential to replace the protection of seedlings provided by insecticides.     

创伤性颅脑损伤患者血清Trx1、FGL2水平与GCS评分及预后的关系研究

摘要 目的：研究创伤性颅脑损伤（TBI）患者血清硫氧还蛋白1（Trx1）、纤维蛋白原样蛋白2（FGL2）水平与格拉斯哥昏迷指数（GCS）评分及预后的关系。方法：将从2017年2月～2019年2月广州中医药大学第一附属医院收治的120例TBI患者纳入研究。将其按照入院即刻GCS评分分成轻度组（13～15分）46例,中度组（9～12分）38例,重度组（3～8分）36例。另取同期于广州中医药大学第一附属医院进行体检的健康志愿者40例作为对照组。比较各组人员血清Trx1以及FGL2水平,并作相关性分析。此外,将重度组患者按照预后结果的不同分成死亡组以及存活组。比较两组血清Trx1以及FGL2水平,以受试者工作特征（ROC）曲线分析血清Trx1以及FGL2对重度TBI患者预后的预测价值。结果：轻度组、中度组及重度组患者的血清Trx1水平均低于对照组,且重度组低于中度组,中度组又低于轻度组（P＜0.05）;轻度组、中度组及重度组患者的血清FGL2水平均高于对照组,且重度组高于中度组,中度组又高于轻度组（P＜0.05）。经Pearson相关性分析可得：TBI患者血清Trx1水平与GCS评分呈正相关关系（r/P=0.554/0.001）,而血清FGL2水平与GCS评分呈负相关关系（P＜0.05）。死亡组患者血清Trx1水平低于存活组（P＜0.05）,而FGL2水平高于存活组（P＜0.05）。经ROC曲线分析发现：血清Trx1联合FGL2预测重度TBI患者预后的曲线下面积、敏感度以及特异度均高于上述两项指标单独检测。结论：Trx1以及FGL2在TBI患者血清中存在明显的异常表达,且与GCS评分相关,可能作为临床TBI诊断、病情判断以及预后评估的辅助生物学标志物。     

多孔自凝固磷酸钙/纤维蛋白1∶1比例血管化的分析研究

目的:观察复合纤维蛋白的多孔自凝固磷酸钙的理学性能及体内血管化情况,探讨其各组份对材料生物学特性的影响,为其临床应用提供实验数据.方法:1)采用凝固时间测定和电镜观察材料表面和断面等方法对复合支架材料构造和理学性能进行分析;2)将24只新西兰白兔随机分为3组,每组8只,分别在每只实验兔腰背筋膜下植入1:1、CPC两种比例材料各一枚.术后2、4、8周进行取材,对材料及其周围组织进行组织学观察、微血管情况定量分析.结果:1)CPC/FG复合支架材料以1∶1(g/ml)混合后,与单纯的CPC相比,初凝时间延长,而终凝时间没有明显的统计学差异;电镜观察发现多孔自凝固磷酸钙复合了纤维蛋白胶之后,纤维蛋白胶分布均匀贯穿多孔自凝固磷酸钙晶体之间,并将其紧密相连;2)术后2周材料外周可见幼稚的微血管.1∶1组高于CPC组(P＜0.05).4周微血管密度达到高峰,相对于2周时有明显差异.1∶1组高于CPC组(P＜0.05).8周时微血管密度相对4周没有显著差异.各组微血管密度与4周时没有明显变化(P＞0.05).结论:复合支架材料具有合适的凝固时间和较好的结构,纤维蛋白胶及其降解产物影响复合材料在体内的微血管密度,使复合材料血管化能力提高,其可作为细胞载体和骨缺损修复支架材料应用于临床和实验中.     

生物蛋白胶封堵合并宫颈环扎术治疗胎膜早破的疗效观察

目的:观察生物蛋白胶封堵合并宫颈环扎术治疗胎膜早破,对孕妇、胎儿及新生儿的影响,明确该技术治疗胎膜早破的疗效。方法:我院产科共收治妊娠<30周胎膜早破患者48例,采用生物蛋白胶封堵合并宫颈环扎术治疗胎膜早破,观察治疗后孕妇羊水指数,胎儿双顶径,新生儿出生时体重,及孕妇的副反应。结果:在本次封堵治疗过程中均一次封堵成功,没有不良反应发生,所有孕妇术后阴道流水情况均明显改善,其中有2例孕妇术后1周阴道流水停止。所有孕妇术后羊水漏出量均明显减少,B超检查显示羊水指数均在(6±2)cm,胎儿每周双顶径增长均达到(0.15±0.02)cm,延长孕周最短(5±2)w,10例早产,38例继续妊娠至37 w。新生儿出生时体重为(2000±260)g,无1例发生NRDS。同期没有进行宫颈内封堵仅行药物保胎治疗的10例孕妇,胎龄延长最长达到10 d,最短者24 h内出现早产分娩,早产儿平均体重1400 g,均发生NRDS,有2例放弃治疗,4例在NICU行治疗,最终因各种并发症死亡。48例行封堵治疗孕妇未发生过敏反应,无阴道出血及产后出血和感染等。结论:应用生物蛋白胶结合宫颈环扎术治疗胎膜早破,手术操作简单,可延长了孕周,提高了新生儿成活率,无不良反应,具有广泛的应用前景。