不同浓度血管抑素抑制大鼠角膜血管的新生

背景：血管抑素在角膜中是否抑制组织中的新生血管生长尚不清楚。 目的：观察血管抑素溶液对大鼠角膜新生血管生长的作用。 方法：24只大鼠制备左眼碱烧伤角膜新生血管模型,随机抽签法分为4组,对照组给予生理盐水,25,50,75 mg/L AS组分别给与25,50,75 mg/L AS血管抑素溶液滴眼,4次/d至实验结束。 结果与结论：造模后第3天各组角膜均可见少量血管新生,但对照组角膜新生血管生长较其他3组明显。3~7 d,各组角膜新生血管生长速度加快,对照组角膜新生血管粗大。烧伤后14 d,对照组角膜新生血管仍然粗大未见明显消退,各浓度组较前消退明显。碱烧伤后不同时间点各浓度组角膜新生血管面积较对照组少(P < 0.05),75mg/L AS组碱烧伤14 d各时间新生血管面积较其他3组减少(P < 0.05)。Real-Time PCR结果显示：各浓度组间CD31 mRNA表达差异均有显著性意义(P < 0.01)。Western blot结果显示血管抑素作用大鼠后,各浓度组角膜组织的CD31表达均较对照组降低,且随着血管抑素溶液浓度的增加而减少。提示血管抑素能够阻止角膜新生血管的生长。     

重组IFN-α蛋白联合endostatin基因对碱烧伤诱导角膜新生血管的抑制作用

目的探讨重组IFN-α蛋白联合endostatin基因对碱烧伤诱导角膜新生血管的抑制作用。方法兔眼球结膜下注射包有绿色荧光蛋白表达载体的脂质体，3d后用激光共聚焦显微镜观察角膜绿色荧光蛋白表达。利用碱烧伤法制备兔眼角膜新生血管模型，球结膜下联合注射重组IFN-α蛋白及包有endostatin真核表达载体的脂质体，用裂隙灯显微镜观察对新生血管的抑制作用。结果实验组角膜有很强的绿色荧光，而在对照组则无荧光。联合应用重组IFN-α蛋白和endostatin基因治疗，第7、10、13天角膜新生血管长度、面积明显小于重组IFN-α蛋白和endostatin基因的单独治疗组（P〈0．05）。结论重组IFN-α蛋白联合endostatin基因可有效抑制碱烧伤诱导角膜新生血管的生长。     

色素上皮衍生因子对角膜碱烧伤后新生血管形成的抑制

背景：角膜新生血管导致角膜透明性降低,造成严重的视觉障碍。色素上皮衍生因子是一种内源性血管生成抑制剂,其对于角膜新生血管是否具有抑制作用尚不清楚。 目的：探索局部运用色素上皮衍生因子对大鼠角膜碱烧伤后角膜新生血管的抑制作用。 方法：将20只大鼠随机分为生理盐水组与色素上皮衍生因子组,每组10只。用NaOH溶液将大鼠右眼角膜烧伤诱导产生新生血管。碱烧伤后2组每日分别给予生理盐水和色素上皮衍生因子点眼,并采用裂隙灯显微镜观察和测量各组角膜新生血管生长情况。碱烧伤后12 d处死大鼠,将角膜组织固定切片,行苏木精-伊红染色观察,并进行免疫组织化学染色检测各组大鼠角膜血管内皮生长因子和CD31的表达。 结果与结论：大鼠角膜碱烧伤后3,7,12 d,色素上皮衍生因子组大鼠角膜新生血管面积均小于生理盐水组(P < 0.05)。角膜碱烧伤后12 d,苏木精-伊红染色显示生理盐水组大鼠角膜产生大量新生血管,角膜组织结构紊乱;色素上皮衍生因子组新生血管较少,角膜组织结构趋于整齐。角膜碱烧伤后12 d,免疫组织化学染色示生理盐水组大鼠角膜上皮和基质层可见血管内皮生长因子大量表达,角膜基质层可见血管内皮生长因子和CD31大量表达;色素上皮衍生因子组新生血管稀少,CD31表达较弱。证实局部应用色素上皮衍生因子可有效抑制大鼠角膜化学伤后的血管新生。     

兔角膜碱烧伤模型房水中肿瘤坏死因子α及血管内皮生长因子表达与姜黄素的抑制

背景：稳定的角膜新生血管动物模型是研究角膜新生血管调控机制,姜黄素对碱烧伤角膜新生血管具有抑制作用和保护作用。 目的：探讨姜黄素对碱烧伤角膜新生血管模型中肿瘤坏死因子α及血管内皮生长因子表达的影响,为防治角膜新生血管提供理论依据。 方法：纳入33只新西兰大耳白兔,随机取3只设为正常组,其余30只建立兔角膜碱烧伤诱发角膜新生血管模型,右眼设为对照组给予生理盐水,左眼设为干预组给予姜黄素,裂隙灯观察角膜新生血管生长及角膜混浊情况,酶联免疫吸附实验检测肿瘤坏死因子α和血管内皮生长因子在房水中的表达。 结果：正常组没有角膜新生血管生成。与对照组比较,干预组角膜新生血管受到抑制且角膜混浊较轻(P < 0.05)。房水肿瘤坏死因子α和血管内皮生长因子在3组中均有表达,对照组和干预组明显高于正常组,但干预组低于对照组(P < 0.05)。说明姜黄素可以有效降低角膜碱烧伤后房水肿瘤坏死因子α及血管内皮生长因子的表达进而抑制兔角膜碱烧伤后角膜新生血管的生长。     

miR-184在碱烧伤大鼠角膜新生血管中的表达及靶基因预测分析

目的 初步探miR-184在碱烧伤诱导大鼠角膜新生血管形成过程中的作用和基因调控机制.方法 碱烧伤诱导大鼠角膜新生血管形成.基质胶上建立人脐静脉血管内皮细胞体外三维培养体系.应用qRT-PCR检测miR-184的体内外表达.生物信息学方法预测并分析miR-184靶基因.结果 初步建立起血管新生的体内外研究模型,miR-184在碱烧伤大鼠角膜新生血管角膜中的表达(0.145±0.013)较正常角膜(1.015±0.189)显著下降(P＜0.01),而在人脐静脉血管内皮细胞中相对表达量极少(0.007±0.004) (P ＜0.05).预测的靶基因与VEGF血管生成信号通路有关.结论 新生血管进程中伴随着miR-184表达量降低,提示其与血管新生密切相关,可能通过VEGF信号通路参与碱烧伤角膜新生血管形成.     

激光和缝线血管吻合术对血管吻合口耐压强度影响的对照研究

目的：观察激光血管吻合和缝线血管吻合术吻合血管后耐压强度的变化，探讨不同吻合方法对血管功能恢复的影响及其原因。材料和方法：家兔30只，右侧颈动脉采用缝合法，左侧颈动脉用激光法加以血管支撑物吻合血管。术后1d、3d、7d、14d、28d，分别观察血管通畅率和耐压强度变化。结果：激光吻合方法通畅率高于缝线吻合方法。激光吻合与缝线吻合的血管耐压强度无差异，趋势是激光法在术后前几天比缝线法低，从7d后耐压强度高于缝合组。结论：利用激光吻合血管有利于保持血管的长期畅通，但初始耐压强度低。应当研究激光吻合加强剂及合适的激光工作方式等提高激光组织吻合的初始耐压强度等力学参数。     

角膜裂伤的缝合体会

角膜裂伤是比较常见的眼科疾病,其轻者影响视力,重者毁坏眼球,故常需及时地缝合处理。然而不妥当的缝合又会影响角膜的正常愈合,故对此一定要保持慎重态度。下面就将根据本人的临床经历对角膜裂伤的缝合谈一些粗浅的体会。 一、角膜伤口的愈合:角膜伤口的愈合主要是创伤及炎性反应的刺激,成纤维细胞合成并分泌胶原单体及胶原单体聚合成胶原纤维的过程。其愈合的临床指征为:伤口收缩;缝线松动;伤口中出现白色半透明的瘢痕;实质层新生血管伸入。认识上述这些,对加强精细地缝合角膜裂伤并掌握拆线时机是十分重要的。一般的角膜裂伤缝合在11～14天拆线。     

不同激光诱导小鼠脉络膜新生血管模型探讨

目的以C57BL／6J小鼠为研究对象，探讨激光诱导脉络膜新生血管（CNV）模型的方法并评价其效果。方法分别以氩激光（514nm，100μm，0．1s，100mW）、氪激光（670nm，100μm，0．1s，100mW）诱导CNV。分别于激光照射后1、2、4周以荧光造影、光镜结果判断诱导CNV效果。结果激光照射1周后开始出现CNV。激光照射后1、2和4周，氩激光荧光渗漏及CNV形成率分别为33．33％、83.33％、58.33％和41．67％、8.33％、75．00％;氪激光荧光渗漏及CNV形成率分别为83.33％、91．67％、66．67％和83．33％、91．67％、83．33％。结论以激光诱导小鼠CNV模型是有效、可行的；激光诱导小鼠CNV模型,氪激光应为首选方式。     

血管内皮生长因子D在小鼠碱烧伤后角膜的表达及其作用

目的观察血管内皮生长因子D(VEGF-D)在小鼠角膜碱烧伤后不同时间角膜组织内的表达,探讨VEGF-D在小鼠角膜碱烧伤后新生淋巴管形成过程中的作用。方法制作小鼠角膜碱烧伤模型,分别于碱烧伤后1d、3d、5d、7d、12d和18d取材。应用免疫组化SP法观察VEGF-D在正常角膜和碱烧伤后不同时间角膜内的表达,应用淋巴管内皮透明质酸受体1(LYVE-1)标记淋巴管,观察小鼠碱烧伤角膜内新生淋巴管的形成情况。结果碱烧伤后1d、3d、5d,角膜内VEGF-D表达水平明显高于正常角膜(P<0.01),于碱烧伤后3d,表达达到高峰。碱烧伤后7d,VEGF-D的表达下降至正常水平。在碱烧伤角膜内,可见阳性表达LYVE-1的新生淋巴管。结论 VEGF-D过表达可能参与小鼠角膜碱烧伤后新生淋巴管形成过程。     

血管抑素抑制兔眼表碱烧伤角膜缘移植术后的角膜血管新生

目的:检测眼表碱烧伤角膜缘移植术后血管抑素抑制角膜新生血管的作用。方法:16只新西兰大白兔双眼制作碱烧伤模型1d后,双眼行角膜缘移植术,术后左眼局部应用血管抑素治疗2周,右眼作对照;观测4周,根据新生血管侵入角膜缘内的范围、角膜混浊与水肿程度进行分级并作统计学处理;同时测量术后7、14、21及28d的眼压。结果:术后4周时,应用血管抑素的左眼的新生血管的评分为1.19±0.10,而对照组为1.63±0.72,统计学处理显示左眼的新生血管较右眼的明显减少(P<0.05),角膜混浊与水肿程度亦明显下降。各时间点各术眼的眼压均在正常范围,无统计学差异。结论:局部应用血管抑素能有效抑制眼表碱烧伤角膜缘移植术后的新生血管增生。     

尼古丁通过调节巨噬细胞极化加重实验性脉络膜新生血管的形成

目的:探究巨噬细胞在尼古丁加重脉络膜新生血管(CNV)形成中的作用。方法:饮用尼古丁水溶液(100 mg/L)4周后,使用激光诱导小鼠CNV模型,7 d后用异硫氰酸荧光素标记的葡聚糖心脏灌注,测量CNV面积。分别于激光后1、3、7和14 d采用免疫荧光法检测巨噬细胞的时间和空间分布。采用RT-qPCR检测巨噬细胞相关分子标志物的mRNA表达。同时采用ELISA法检测激光后第1和3天视网膜色素上皮-脉络膜-巩膜组织中血管内皮生长因子(VEGF)、细胞间黏附分子1(ICAM-1)、肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的表达。结果:尼古丁显著增加CNV的渗漏程度及CNV的面积,尼古丁组的重度渗漏率和CNV面积分别为56.25%和(17 569.96±1 444.00)μm²,显著高于对照组的31.25%和(10 158.63±711.00)μm²(P<0.05)。尼古丁促进了M2型巨噬细胞的浸润,增加了M2/M1型巨噬细胞的比率(P<0.05)。尼古丁也促进了M2型巨噬细胞相关分子标志物的mRNA表达,并且促进了VEG...     

脂肪干细胞与外源性血管内皮生长因子及纤维蛋白胶复合物体内构建血管化组织工程脂肪

背景：血运重建机制是组织工程化脂肪组织成功构建的决定性因素。 目的：观察脂肪干细胞与外源性血管内皮生长因子和纤维蛋白胶复合物在体内构建血管化组织工程脂肪的可行性。 方法：从健康成年人吸脂术后的脂肪组织中分离脂肪干细胞并行原代及传代培养,将第3代经BrdU标记的脂肪干细胞向脂肪细胞定向诱导2周后,制成5×10¹⁰ L^-1细胞悬液。由0.5 mL细胞悬液、100 μL的血管内皮生长因子工作液或DMEM培养基与0.5 mL纤维蛋白胶组成实验组和对照组移植物,分别植入裸鼠背部皮下。 结果与结论：术后8周取材时：①实验组可见血管增生并长入材料,呈轻度纤维包裹;对照组有少量血管长入材料中,也有轻度纤维包裹现象。实验组新生组织湿质量大于对照组(P < 0.01)。②苏木精-伊红染色均可见移植物中有新生脂肪组织形成和不同程度的微血管长入;实验组微血管数多于对照组(P < 0.01)。③新生组织免疫荧光染色示,两组新生脂肪细胞的胞核及部分微血管内皮细胞的胞核呈现绿色荧光。结果说明脂肪干细胞与外源性血管内皮生长因子和纤维蛋白胶复合物在体内可构建血管化组织工程脂肪,脂肪干细胞与外源性血管内皮生长因子共同参与新生脂肪组织的血管化过程。     

Inhibition of blood vessel formation by a chondrocyte-derived extracellular matrix

In this study, the chondrocyte-derived extracellular matrix (CECM) was evaluated for its activity to inhibit vessel invasion in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) and rabbit chondrocytes were plated on a bio-membrane made of CECM or human amniotic membrane (HAM). The adhesion, proliferation, and tube formation activity of HUVECs and chondrocytes were examined. The CECM and HAM powders were then mixed individually in Matrigel and injected subcutaneously into nude mice to examine vessel invasion in vivo after 1 week. Finally, a rabbit model of corneal neovascularization (NV) was induced by 3-point sutures in the upper cornea, and CECM and HAM membranes were implanted onto the corneal surface at day 5 after suture injury. The rabbits were sacrificed at 7 days after transplantation and the histopathological analysis was performed. The adhesion and proliferation of HUVECs were more efficient on the HAM than on the CECM membrane. However, chondrocytes on each membrane showed an opposite result being more efficient on the CECM membrane. The vessel invasion in vivo also occurred more deeply and intensively in Matrigel containing HAM than in the one containing CECM. In the rabbit NV model, CECM efficiently inhibited the neovessels formation and histological remodeling in the injured cornea. In summary, our findings suggest that CECM, an integral cartilage ECM composite, shows an inhibitory effect on vessel invasion both in vitro and in vivo, and could be a useful tool in a variety of biological and therapeutic applications including the prevention of neovascularization after cornea injury.     

Membrane Type-1 Matrix Metalloproteinase Potentiates Basic Fibroblast Growth Factor-Induced Corneal Neovascularization

Corneal neovascularization is one of the leading causes of blindness. The aim of this study was to evaluate the pro-angiogenic role of corneal fibroblast-derived membrane type-1 matrix metalloproteinase (MT1-MMP) on basic fibroblast growth factor (bFGF)-induced corneal neovascularization in vivo and in vitro. Immunohistochemical studies demonstrated that MT1-MMP was expressed in keratocytes and immortalized corneal fibroblast cell lines. Vascular endothelial growth factor protein levels were increased after bFGF-stimulation of wild-type fibroblast cells compared with MT1-MMP knockout fibroblast cells. Corneal vascularization was significantly increased after a combination of bFGF pellet implantation and naked MT1-MMP DNA injection in wild-type mouse corneas compared with either bFGF pellet implantation or naked MT1-MMP DNA-injected corneas. Western blotting analysis of the phosphorylation levels of the key signaling molecules (p38, JNK, and ERK) demonstrated that phosphorylation levels of both p38 and JNK were diminished after bFGF stimulation of MT1-MMP knockout cells compared with wild-type and MT1-MMP knockin cells. These results suggest that MT1-MMP potentiates bFGF-induced corneal neovascularization, likely by modulating the bFGF signal transduction pathway.The cornea is typically avascular in its normal state. However, corneal neovascularization (NV) occurs in conjunction with several corneal diseases such as infection, injury, and autoimmune reactions and is one of the leading causes of blindness. Recent studies have identified several tyrosine kinases and their corresponding ligands that mediate NV, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF).^1,2,3bFGF was first identified as a pro-angiogenic factor and is studied extensively in corneal NV models because it is thought to be a major factor in the induction of corneal NV.^4,5,6 bFGF is secreted by corneal epithelial cells, stromal fibroblasts, and endothelial cells, and is localized to the corneal extracellular matrix.⁷ Low levels of bFGF are produced in unwounded corneas; however, enhanced bFGF production was detected in corneal epithelial cells after injury.⁸ VEGF was also shown to promote NV in corneal wounding models,⁹ and cross talk is thought to occur between bFGF and VEGF during corneal NV. For example, bFGF was shown to induce corneal NV by activating the VEGF/VEGFR system^10,11 and the systemic administration of anti-VEGF-A neutralizing antibodies dramatically reduces this effect.¹²Membrane type-1 matrix metalloproteinase (MT1-MMP) is the first transmembrane-containing matrix metalloproteinase to be identified.¹³ Based on previous reports using corneal wound-healing models, MT1-MMP mRNA is mainly localized to the corneal stroma.¹⁴ During NV, quiescent endothelial cells are activated and migration is facilitated by degrading the extracellular matrix through the action of specific proteases, including MT1-MMP.^15,16,17 The importance of the enzymatic function of MT1-MMP in corneal NV was shown using the corneal pocket assay in MT1-MMP-deficient mice.¹⁸ Interestingly, the expression of MT1-MMP is up-regulated by bFGF stimulation in prostate carcinoma cell lines,¹⁹ and it was also reported that MT1-MMP promotes VEGF secretion.^{20,21,22,23,24,25}In this study, we developed anti-MT1-MMP antibody to localize and characterize MT1-MMP protein in the mouse cornea. To assess the relationship between MT1-MMP and bFGF during corneal NV, we performed experiments that combined the corneal pocket assay using a bFGF pellet with the injection of naked MT1-MMP DNA. We observed an enhanced phosphorylation of MAP kinases in wild-type and MT1-MMP knockin (KI) cell lines over that of MT1-MMP knockout (KO) cell lines, suggesting a role of MT1-MMP in modulating bFGF-mediated signal transduction pathways.     

可吸收胶原蛋白线与丝线编织非吸收线在口腔种植中的应用

背景：胶原蛋白线由动物的胶原蛋白制备而成,由于其具备可降解、无排异、易于制备、使用方便等优点,现在已开始大量使用于临床。 目的：比较胶原蛋白线与丝线编织线对口腔种植手术切口愈合的影响。 方法：100例种植手术患者随机等分为可吸收胶原蛋白线组和丝线编织非吸收性缝线组,分别使用2-0带圆针可吸收胶原蛋白缝合线与4-0带圆针丝线编织非吸收性缝线对伤口进行间断无张力缝合。植入后3,5,7 d观察缝线及伤口愈合情况,植入后第7天拆线,植入后14 d复诊。 结果与结论：可吸收胶原蛋白线组患者切口甲级愈合率明显多于丝线编织非吸收性缝线组(P < 0.05)。可吸收胶原蛋白线组患者口腔切口中的2-0带圆针可吸收胶原蛋白缝合线在治疗7 d时大多数被吸收,而丝线编织非吸收线组患者口腔切口中4-0带圆针丝线编织非吸收性缝线未见吸收。且使用2-0可吸收胶原蛋白线的患者口内缝线未见污物附着,线体清洁。而使用4-0丝线编织非吸收线缝合的患者口内可见线体周围有软垢附着。提示胶原蛋白线比丝线编织线更适合口腔种植手术切口的无张力缝合,能够获得更好的愈合效果,且时间能够与伤口愈合时间匹配,并能维持更好的口腔卫生。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Composite surgical sutures with bioactive glass coating

A processing method was developed to coat polyglactin 910 (Vicryl) sutures with bioactive glass powder (45S5 Bioglass). High reproducibility and homogeneity of the coating in terms of microstructure and thickness along the suture length were achieved. Bioglass-coated sutures exhibited a high level of chemical reactivity in simulated body fluid (SBF), indicating their bioactive behavior. This was evident by the prompt formation of hydroxyapatite (HA) crystals on the surface after only 7 days of immersion in SBF. These crystals grew to form a thick HA layer (15 microm thickness) after 3 weeks in SBF. The tensile strength of the sutures was tested before and after immersion in SBF in order to assess the effect of the bioactive glass coating on suture degradation. The tensile strength of composite sutures was lower than that of as-received Vicryl sutures, 385 and 467 MPa, respectively. However, after 28 days of immersion in SBF the residual tensile strengths of coated and uncoated sutures were similar (83 and 88 MPa, respectively), indicating no negative effect of the HA layer formation on the suture strength. The effect of bioactive glass coating on the polymer degradation is discussed. The developed bioactive sutures represent interesting materials for applications in wound healing, fabrication of fibrous three-dimensional scaffolds for tissue engineering, and reinforcement elements for calcium-phosphate temporary implants.