人脐静脉血管内皮细胞培养、冻存、复苏与鉴定

目的:探讨人脐静脉血管内皮细胞(HUVEC)体外分离、培养、冻存、复苏与鉴定的可靠方法。方法:采用改良的胶原酶灌注消化法对HUVEC体外分离、培养,按慢冻速融原则对第3代细胞进行液氮冻存、复苏。采用免疫组化Ⅷ因子相关抗原染色和活体细胞荧光D iI-Ac-LDL鉴定。结果:分离的原代HUVEC总数平均为1×106个细胞,最多达3×106个细胞;两种鉴定结果HUVEC均为95%以上阳性;冻存后分3批复苏细胞活性均超过90%。结论:成功建立了人脐静脉血管内皮细胞体外培养、冻存、复苏与鉴定方法,获得了足够数目和纯度的血管内皮细胞,保持了细胞的同源同代性。

Protective effects of icariin on human umbilical vein endothelial cell injured by angiotensin Ⅱ

To investigate the effects of icariin （ICA） on angiotensin Ⅱ（Ang Ⅱ）-induced injury in human umbilical vein endothelial cells line （ECV-304）. The ECV-304 cells were cultured in vitro. After 24 h incubating with icariin, the model of AngⅡ-induced injury in ECV-304 was established. The cell viability （MTT method）, Lactate dehydrogenase （LDH） release and Nitric oxide （NO） production in the medium, the capacity of scavenging superoxide anion radicals （O2^-） and hydroxyl radicals （.OH） were measured. The activities of superoxide dismutase （SOD）, total nitric oxide synthase （T-NOS）, inducible nitric oxide synthase （iNOS） and constitutive nitric oxide synthase （cNOS） in the cells were determined. Compared with the Ang Ⅱ-treated group, ICA can significantly raise the viability of EC, increase the activities of SOD, T-NOS and cNOS, increase the production of NO, enhance the capacity of scavenging superoxide anion radicals （ O2^- ） and hydroxyl radicals（.OH）, and lower LDH leakage and iNOS activity. The results suggest that ICA can protect endothelial cells （ECV-304） from Ang II-induced injury.

The Inhibitory Effects of Arresten Protein on Tumor Formation

Objective To examine the inhibitory effects of recombinant purified arresten on tumor formation. Methods Purified arresten protein was incubated with human umbilical vein endothelial cells (HUVECs) and HeLa cells in vitro. The effect on proliferation of HUVECs and HeLa cells was examined using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide assay, and apoptosis of these cells monitored by flow cytometry. The effect on migration of HUVECs and HeLa cells was examined by Boyden chamber. Twenty colon carcinoma-bearing C67BL/6 mice were used to investigate the antitumor effects of arresten protein. The mice were randomly divided into arresten treatment group (n=10) and control group (n=10). The microvessel densities of the tumors were measured by immunohistochemical staining with anti-CD31 monoclonal antibody. Results Arresten inhibited the proliferation and migration of HUVECs in a dose-dependent manner while promoting apoptosis. However, arresten had no significant effects on the proliferation and apoptosis of HeLa cells. The migration of HeLa cells was modestly inhibited by arresten. The arresten treatment group of mice showed no weight loss or unusual behavior during the course of treatment, and the tumor growth was significantly decreased; in contrast, the control group of mice exhibited rapidly growing tumors and cachexia. A dramatically decreased microvessel density in tumor tissues was found in arresten-treated mice compared with that in the control mice. Conclusion Arresten can inhibit tumor growth through inhibition of tumor angiogenesis.

Extract of Meretrix meretrix Linnaeus induces angiogenesis in vitro and activates endothelial nitric oxide synthase

Meretrix meretrix Linnaeus has long been used as traditional Chinese medicine in oriental medicine.The angiogentic activity of the extract of M.meretrix was investigated in this study,using human umbilical vein endothelial cells(HUVECs).Extract of M.meretrix Linnaeus(AFG-25) was prepared with acetone and ethanol precipitation,and further separated by Sephadex G-25 column.The results show that AFG-25 promoted proliferation,migration,and capillary-like tube formation in HUVECs,and in the presence of eNOS inhibitor NMA,the tube formation induced by AFG-25 is inhibited significantly.Moreover,AFG25 could also promote the activation of endothelial nitric oxide synthase(eNOS) and the resultant elevation of nitric oxide(NO) production.The results suggested that M.meretrix contains active ingredients with angiogentic activity and eNOS/NO signal pathway is in part involved in the proangiogenesis effect induced by AFG-25.

Inhibition of angiogenesis by DADAG in vivo and in vitro

1,2,5,6-Dianhydro-3,4-diacetyl-galactitol （DADAG）, an alkylating sugar alcohol derivative, has been shown effective against tumor growth. In this research, we explored the effect of DADAG on angiogenesis in chick chorioallantoic membrane （CAM） model and on the proliferation and migration of human umbilical vein endothelial cells （HUVECs）. We also studied the possible mechanism of the anti-angiogenesis effect of DADAG. The results showed that DADAG （100, 500 and 1000μnol/L） inhibited angiogenesis in CAM model dose-dependently. Sulforhodamine B （SRB） assay indicated that DADAG （45, 90, 135, 180 and 225 μmol/L） suppressed HUVECs proliferation in a dose-dependent and time-dependent manner. High Content Screening （HCS, Cellomics） assay, in which the influence of cell proliferation on migration could be excluded, indicated that DADAG （45, 135 and 225 ~xmol/L） directly inhibited the motility ofHUVECs. Immunofiuorescence assay suggested that DADAG inhibited angiogenesis possibly by decreasing vascular endothelial growth factor （VEGF） expression in HUVECs. Our findings reveal that DADAG show anti-angiogenic activity in vivo and in vitro, which is related to the downregulation of VEGF expression in endothelial cells.

A novel synthetic small molecule YF-452 inhibits tumor growth through antiangiogenesis by suppressing VEGF receptor 2 signaling

Tumor angiogenesis is characterized by abnormal vessel morphology, endowing tumor with highly hypoxia and unresponsive toward treatment. To date, mounting angiogenic factors have been discovered as therapeutic targets in antiangiogenic drug development. Among them, vascular endothelial growth factor receptor 2 （VEGFR2） inhibitors exerts potent antiangiogenic activity in tumor therapy. Therefore, it may provide a valid strategy for cancer treatment through targeting the tumor angiogenesis via VEGFR2 pathway. In this study, we established a high-profile compounds library and certificated a novel compound named N-（N-pyrrolidylacetyl）-9-（4-bromobenzyl）-l,3,4,9-tetrahydro-^-carboline （YF-452）, which remarkably inhibited the migration, invasion and tube-like structure formation of human umbilical vein endothelial cells （HUVECs） with little toxicity invitro. Rat thoracic aorta ring assay indicated that YF-452 significantly blocked the formation ofmicrovascular exvivo. In addition, YF-452 inhibited angiogenesis in chick chorioallantoic membrane （CAM） and mouse corneal micropocket assays. Moreover, YF-452 remarkably suppressed tumor growth in xenografts mice model. Furthermore, investigation of molecular mechanism revealed that YF-452 inhibited VEGF-induced phosphorylation of VEGFR2 kinase and the downstream protein kinases including extracellular signal regulated kinase （ERK）, focal adhesion kinase （FAK） and Src. These results indicate that YF-452 inhibits angiogenesis and may be a potential antiangiogenic drug candidate for cancer therapy.