血管内皮抑制因子IFN-α和IFN-γ对淋巴管内皮细胞生成的影响
发布时间：2018-08-07 14:21
【摘要】：目的 探讨IFN-α(α-干扰素)和IFN-γ(γ-干扰素)对淋巴管内皮细胞增殖和游走的影响及作用机制。旨在找到安全、有效、实用的淋巴管生成抑制剂。 材料和方法 本实验所用淋巴管内皮细胞来自猪的胸导管。1、淋巴管内皮细胞的鉴定:应用LYVE-1(淋巴管内皮透明质酸受体1)和VEGFR-3(血管内皮生长因子受体-3)特异性抗体进行鉴定。2、光镜和电镜观察淋巴管内皮细胞的形态和超微结构。3、抑制实验:本实验分为两大组:IFN-α组和IFN-γ组。每大组分别采用划线法和MTT法观察两种因子对淋巴管内皮细胞增殖和游走的影响。(1)划线法:IFN-α组和IFN-γ组分别包括各自的对照组和3种浓度的实验组。(2)MTT法:IFN-α组和IFN-γ组包括对照组和6种浓度的实验组。4、细胞凋亡实验:应用Caspase染色法和Hoechst染色法进行细胞凋亡实验。5、干扰素活体实验:在活体兔的两个后肢上切断淋巴管并分别注射IFN-α和生理盐水,以观察淋巴管的再生情况。 结果 1、淋巴管内皮细胞的鉴定:经LYVE-1鉴定和VEGFR-3鉴定,为典型淋巴管内皮细胞。2、光镜和电镜观察:光镜下淋巴管内皮细胞呈特征性“鹅卵石状”或“铺路石状”镶嵌排列;电镜下观察到淋巴管内皮细胞的超微结构,细胞间有类似桥粒样结构。3、划线法:与对照组相比,当IFN-α和IFN-γ的浓度达到1000ng/ml时,两者对淋巴管内皮细胞的增殖和游走都有明显的抑制作用(P0.01)。4、MTT法:当IFN-α和IFN-γ的浓度达到2000ng/ml时,两者对内皮细胞的增殖具有显著的抑制作用(P0.01)。5、凋亡实验:(1)Caspase染色法证实,经IFN-α和IFN-γ处理后的淋巴管内皮细胞,阳性表达为胞浆呈黄色或棕黄色染色。(2)Hoechst染色证实,经IFN-α和IFN-γ处理后的淋巴管内皮细胞,可观察到其核周围有凋亡小体。6、干扰素活体实验:手术切断兔后肢淋巴管后16天,生理盐水侧淋巴管已愈合,无染料渗漏,IFN-α侧仍有大量染料渗漏。 结论 1、IFN-α和IFN-γ对淋巴管内皮细胞的增殖和游走具有明显的抑制作用。2、IFN-α和IFN-γ具有促进淋巴管内皮细胞凋亡的作用。
[Abstract]:Objective to investigate the effect and mechanism of IFN- 伪 and IFN- 纬 on proliferation and migration of lymphatic endothelial cells. Aim to find safe, effective, and practical lymphangiogenesis inhibitors. Materials and methods Lymphatic endothelial cells from porcine thoracic ducts were identified by LYVE-1 (lymphatic endothelial hyaluronic acid receptor-1) and VEGFR-3 (vascular endothelial growth factor receptor -3) specific antibodies. The morphology and ultrastructure of lymphatic endothelial cells were observed by light microscope and electron microscope, and the inhibition experiment was carried out. The experiment was divided into two groups: group 1: IFN- 伪 and group IFN- 纬. The effects of two kinds of factors on the proliferation and migration of lymphatic endothelial cells were observed by underlined method and MTT method respectively. (1) the two groups included their respective control groups and three experimental groups with different concentrations. (2) the MTT method: IFN- 伪 group and IFN- 纬 group were divided into two groups: (1) the control group and the IFN- 纬 group, respectively. (2) the MTT method: IFN- 伪 group and IFN- 纬 group, respectively. The apoptosis test was carried out by Caspase staining and Hoechst staining. Interferon assay was performed in vivo. The lymphatic vessels were severed on the hind limbs of living rabbits and IFN- 伪 and normal saline were injected respectively. To observe the regeneration of lymphatic vessels. Results 1. The identification of lymphatic endothelial cells: by LYVE-1 and VEGFR-3 identification, they were typical lymphatic endothelial cells. Light and electron microscope observation showed that the lymphatic endothelial cells were cobblestone or paving stone mosaic. The ultrastructure of lymphatic endothelial cells was observed under electron microscope, and the desmosome like structure was observed between the cells. The results showed that compared with the control group, the concentration of IFN- 伪 and IFN- 纬 reached the level of 1000ng/ml, and the expression of IFN- 伪 and IFN- 纬 was similar to that of the control group. When the concentration of IFN- 伪 and IFN- 纬 reached 2000ng/ml, both of them could significantly inhibit the proliferation of endothelial cells (P0.01) .5. the apoptosis experiment: (1) Caspase staining showed that, when the concentration of IFN- 伪 and IFN- 纬 reached the level of 2000ng/ml, both of them could inhibit the proliferation of endothelial cells significantly (P0.01) .5. the apoptosis experiment: (1) Caspase staining confirmed that the proliferation of endothelial cells was inhibited by IFN- 伪 and IFN- 纬 (P0.01). The lymphatic endothelial cells treated with IFN- 伪 and IFN- 纬 were positive for yellow or brown cytoplasm staining. (2) Hoechst staining confirmed that the lymphatic endothelial cells were treated with IFN- 伪 and IFN- 纬. It was observed that there were apoptotic corpuscles. 6 around the nucleus. Interferon in vivo experiment: 16 days after the operation, the lymphatic vessels of the hind limbs of rabbits were healed, and a large amount of dye leakage was still found in the IFN- 伪 side of IFN- 伪 without dye leakage. Conclusion (1) IFN- 伪 and IFN- 纬 can significantly inhibit the proliferation and migration of lymphatic endothelial cells. (2) IFN- 伪 and IFN- 纬 can promote the apoptosis of lymphatic endothelial cells.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329
本文编号：2170303
[Abstract]:Objective to investigate the effect and mechanism of IFN- 伪 and IFN- 纬 on proliferation and migration of lymphatic endothelial cells. Aim to find safe, effective, and practical lymphangiogenesis inhibitors. Materials and methods Lymphatic endothelial cells from porcine thoracic ducts were identified by LYVE-1 (lymphatic endothelial hyaluronic acid receptor-1) and VEGFR-3 (vascular endothelial growth factor receptor -3) specific antibodies. The morphology and ultrastructure of lymphatic endothelial cells were observed by light microscope and electron microscope, and the inhibition experiment was carried out. The experiment was divided into two groups: group 1: IFN- 伪 and group IFN- 纬. The effects of two kinds of factors on the proliferation and migration of lymphatic endothelial cells were observed by underlined method and MTT method respectively. (1) the two groups included their respective control groups and three experimental groups with different concentrations. (2) the MTT method: IFN- 伪 group and IFN- 纬 group were divided into two groups: (1) the control group and the IFN- 纬 group, respectively. (2) the MTT method: IFN- 伪 group and IFN- 纬 group, respectively. The apoptosis test was carried out by Caspase staining and Hoechst staining. Interferon assay was performed in vivo. The lymphatic vessels were severed on the hind limbs of living rabbits and IFN- 伪 and normal saline were injected respectively. To observe the regeneration of lymphatic vessels. Results 1. The identification of lymphatic endothelial cells: by LYVE-1 and VEGFR-3 identification, they were typical lymphatic endothelial cells. Light and electron microscope observation showed that the lymphatic endothelial cells were cobblestone or paving stone mosaic. The ultrastructure of lymphatic endothelial cells was observed under electron microscope, and the desmosome like structure was observed between the cells. The results showed that compared with the control group, the concentration of IFN- 伪 and IFN- 纬 reached the level of 1000ng/ml, and the expression of IFN- 伪 and IFN- 纬 was similar to that of the control group. When the concentration of IFN- 伪 and IFN- 纬 reached 2000ng/ml, both of them could significantly inhibit the proliferation of endothelial cells (P0.01) .5. the apoptosis experiment: (1) Caspase staining showed that, when the concentration of IFN- 伪 and IFN- 纬 reached the level of 2000ng/ml, both of them could inhibit the proliferation of endothelial cells significantly (P0.01) .5. the apoptosis experiment: (1) Caspase staining confirmed that the proliferation of endothelial cells was inhibited by IFN- 伪 and IFN- 纬 (P0.01). The lymphatic endothelial cells treated with IFN- 伪 and IFN- 纬 were positive for yellow or brown cytoplasm staining. (2) Hoechst staining confirmed that the lymphatic endothelial cells were treated with IFN- 伪 and IFN- 纬. It was observed that there were apoptotic corpuscles. 6 around the nucleus. Interferon in vivo experiment: 16 days after the operation, the lymphatic vessels of the hind limbs of rabbits were healed, and a large amount of dye leakage was still found in the IFN- 伪 side of IFN- 伪 without dye leakage. Conclusion (1) IFN- 伪 and IFN- 纬 can significantly inhibit the proliferation and migration of lymphatic endothelial cells. (2) IFN- 伪 and IFN- 纬 can promote the apoptosis of lymphatic endothelial cells.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329
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