Study of angiogenesis induced by metastatic and non-metastatic liver cancer by corneal micropocket model in nude mice

AIM To study the angiogenesis induced by liver cancer with different metastatic potentials using corneal micropocket model in nude mice.METHODS Corneal micropockets were created in nude mice. Tumor tissues and liver tissues were implanted into the corneal micropockets. Angiogenesis was observed using a digital camera under slit-lamp biomicroscope, and compared among different grafts and incision alone. Vascular responses were recorded in regard to the range, number and length of new blood vessels toward the grafts or incisions.RESULTS Vascular responses induced by tumor tissues were greater than those by incision alone and liver tissue grafts. LCI-D20 induced more intensive angiogenesis than LCI-D35.CONCLUSION Highly metastatic liver cancer LCI D20 was more angiogenic than low metastatic cancer LCI D35 and liver tissue. Micropocket was a useful model to study dynamic process of angiogenesis in vivo.

Corneal Neovascularization Suppressed by TIMP2 Released from Human Amniotic Membranes

Purpose: To investigate the effects of culture medium of human amniotic membrane (AM) on corneal neovascularization (CNV) induced by basic fibroblast growth factor (bFGF) in mice.Methods: Culture medium of amniotic membrane was prepared by cultivating AM (with epithelium side up) in EGM basic medium for 3 days, and was collected separately to three groups, e.g. Control (EGM only), AM with epithelium (AM) and AM without epithelium (De-AM). Corneal neovascularization was induced in mice by using micropocket assay with Hydron polymer pellets containing 100 ng bFGF. Migration and proliferation of human umbilical cord vein endothelial cells (HUVEC) were performed in Boyden chambers and by using the CyQUANT fluorescence binding assay respectively.The levels of tissue inhibitors of metalloproteinase 1 and 2 (TIMP1, TIMP2) in culture medium were determined by ELISA assay.Results: CNV induced by bFGF was significantly suppressed by culture medium of amniotic membrane. When the medium was applied as an eyedrop 4 times a day for 7 days,the area of CNV was (2.48±0.76) mm2,(0.64±0.52) mm2 and (1.96±0.65) mm2 incontrol, AM and De-AM group respectively. The migration and proliferation of HUVEC were strongly inhibited by culture medium of AM with epithelium, while the De-AM had no effect on the migration of HUVEC cells. The high level of TIMP2 was found in AM group, but not in De-AM group, while there was no difference in the amount of TIMP1 in medium among three groups.Conclusion: Culture medium of amniotic membrane significantly suppresses the corneal nevovascularization induced by bFGF. The mechanism of which at least in part is that high level of TIMP2 protein secreted or released into the culture medium of AM and inhibition of migration and growth of vascular endothelial cells.

1-磷酸鞘氨醇对角膜血管新生的影响

目的评价1-磷酸鞘氨醇(S1P)对大鼠角膜新生血管(CNV)的影响。方法 20只SD大鼠随机分成对照组和实验组,制做角膜微囊袋并分别植入空白缓释微粒体和含S1P的微粒体,术后1d,7d,14d观察各组大鼠角膜新生血管的生长情况。结果对照组未见明显新生血管生长而实验组新生血管生长明显,差异有统计学意义(P<0.05),实验组CNV的面积、累积钟点数和血管长度随观察时间推移而增多(P<0.05)。结论 1-磷酸鞘氨醇可促进角膜的血管增生,作用机理可能与激活角膜缘附近血管内皮细胞S1PR通路有关。

大鼠角膜微囊袋植入微粒体诱导新生血管模型的研究

目的:探讨大鼠角膜微囊袋植入微粒体诱导新生血管模型的制作技巧和特点。方法:9只SD大鼠手术显微镜下做角膜基质微囊袋并植入含有VEGF的缓释微粒体。术后1 d、3 d、5 d、7 d采用裂隙灯显微镜观察角膜新生血管的生长情况。结果:术后1 d大鼠角膜缘血管网充血扩张,无新生血管生长;3 d可见毛刷状新生血管自角膜缘伸入角膜,面积为(2.23±0.59)mm^2;5 d新生血管垂直于角膜缘向微粒体方向束状延伸,面积为(6.81±1.35)mm^2;7 d新生血管继续向前生长,部分新生血管顶端互相吻合成袢状,面积为(8.92±1.79)mm^2。所有术眼均未见前房积血、角膜溃疡、微粒体脱出等并发症。结论:大鼠角膜微囊袋植入微粒体诱导的新生血管生长稳定、无并发症、适用于定量研究。

Slit3/Robo4在大鼠角膜新生血管中的表达

目的：通过研究神经轴突导向因子Slit3及Robo4受体在大鼠正常角膜与新生血管化角膜中的差异性表达,探讨其在角膜新生血管（ corneal neovascularization,CNV）形成中的作用。 方法：采用角膜微囊袋法建立以碱性成纤维细胞生长因子（ bFGF）诱导的大鼠CNV模型,采用眼前段照相,免疫组化染色、彩色图像分析系统分别计算1,4,7,10,14d共5个时间点的CNV面积和Slit3, Robo4的平均光密度值。结果：Slit3及Robo4在新生血管化角膜中表达增加,在第7 d达到最高,且二者的表达水平与 CNV 的面积除第1 d外,在其他各时间点均呈正相关（r=0.84~0.91,P〈0.05）。结论：Slit3/Robo4与CNV形成明显相关,其可能成为抑制CNV的重要靶点。

利用裸鼠角膜微囊观察人肝癌诱导肿瘤血管形成

目的观察不同转移潜能的肝癌组织诱导肿瘤血管的能力。方法用裸鼠角膜制作微囊,将来自不同转移潜能肝癌组织LCID20(高转移)和LCID35(低转移)植入微囊。在裂隙灯下观察肿瘤血管形成情况。结果 LCID20诱导的肿瘤血管在范围和条数上均高于LCID35。结论高转移肝癌诱导肿瘤血管能力高于低转移肝癌,角膜微囊模型是研究肝癌肿瘤血管形成的影响因素和干预治疗的较理想模型。