Slit2蛋白在小鼠心肌微血管内皮细胞的表达及对心肌微血管内皮细胞增殖迁移影响的实验研究

发布时间：2018-01-01 22:25

本文关键词：Slit2蛋白在小鼠心肌微血管内皮细胞的表达及对心肌微血管内皮细胞增殖迁移影响的实验研究　出处：《川北医学院》2012年硕士论文　论文类型：学位论文

【摘要】：Slit蛋白家族最早是在中枢神经系统中被发现的，Slit蛋白是一类分泌性糖蛋白，在脊椎动物中Slit蛋白家族包括三个成员（Slit1、Slit2、Slit3）。哺乳动物中的Slit蛋白结构是由一个N-端信号肽，四个富含亮氨酸的重复序列(LRRs)，九个EGF样重复序列和一个C-端的半胱氨酸结四部分组成的，而LRRs是Slit蛋白与其受体Robo结合必需的区域。Slit蛋白在水解过程中产生N-端和C-端两个片段，只有全长的和N-末端的Slit蛋白能与细胞膜受体Robo结合发挥作用。 Slit蛋白在人类多种组织及细胞系中均有表达，已知Slit-Robo信号通路在神经系统发育中具有导向神经轴突生长和排斥神经细胞迁移的重要作用。而关于Slit-Robo信号通路在其它组织和系统中的作用报道则是不一致的。近年来的研究发现Slit-Robo信号通路与血管内皮细胞增殖、迁移有关。Wang等报道显示Slit2表达在人类恶性黑色素瘤的血管内皮细胞上，可以通过阻断Slit2与受体Robol的结合降低肿瘤微血管密度，说明Slit2-Robo信号通路有促进肿瘤微血管生成的作用。与之相反的是，研究报道显示Slit2-Robo信号通路对病理性角膜新生血管的生成起抑制作用，同时Seth等也证实Silt2-Robo4信号通路抑制HUVEC的迁移。 Slit蛋白家族中Slit1主要表达在神经组织，Slit2、Slit3除了表达在神经组织中外，还表达在血管内皮细胞。Wu等报道Slit2和Slit3表达在大鼠主动脉的内皮细胞系中,同时也证实了Slit2表达在人类肾脏的小动脉和小静脉上。Zhang等报道Slit3表达在血管系统的内皮细胞和平滑肌细胞上。有实验证实炎症因子TNF-α可以上调Slit2 mRNA的表达，而且TNF-α能活化内皮细胞并参与动脉粥样硬化的发生发展。目前国内外尚无报道在小鼠心肌微血管内皮细胞上是否有Slit2表达，因此我们首先对Slit2在正常小鼠心室肌微血管组织中表达情况进行检测，并通过小鼠心肌微血管内皮细胞的原代培养，检测Slit2 mRNA是否在小鼠心肌微血管内皮细胞的表达，同时用不同浓度的TNF-α刺激小鼠心肌微血管内皮细胞后，检测Slit2 mRNA的表达及Slit2蛋白的分泌情况。其次通过血管内皮细胞的增殖和迁移实验，探讨不同浓度的Slit2蛋白对小鼠心肌微血管内皮细胞增殖及迁移的影响，为今后研究Slit-Robo信号通路在缺血心肌微血管内皮细胞迁移、血管新生以及炎症因子TNF-α致动脉粥样硬化中的作用提供实验依据。研究目的1.检测Slit2在正常小鼠心室肌微血管组织中的表达以及Slit2 mRNA在正常小鼠心肌微血管内皮细胞的表达。2.检测不同浓度的TNF-α刺激小鼠心肌微血管内皮细胞后Slit2 mRNA的表达及Slit2蛋白的分泌情况。3.探讨不同浓度的Slit2蛋白对小鼠心肌微血管内皮细胞增殖以及迁移的影响。4.探讨在VEGF作用下不同浓度的Slit2蛋白对小鼠心肌微血管内皮细胞迁移的影响。研究方法1.免疫组织化学法检测正常小鼠心室肌微血管组织中Slit2的表达。2. RT-PCR技术检测Slit2 mRNA在正常小鼠心肌微血管内皮细胞中的表达。3.小鼠心肌微血管内皮细胞分为正常对照组和实验组2组。正常对照组：无添加TNF-α的细胞悬液；实验组：分别含有TNF-α（0.01、0.1、1、10、100ng/ml）5个不同浓度的细胞悬液组。RT-PCR技术检测2组细胞Slit2 mRNA的表达，，ELISA技术检测2组细胞上清液中Slit2蛋白的分泌。4.小鼠心肌微血管内皮细胞分为正常对照组和实验组2组。正常对照组：无添加Slit2的细胞悬液；实验组：分别含有Slit2蛋白(50、75、100、125、150ng/ml) 5个不同浓度的细胞悬液组。CCK-8实验检测2组细胞悬液对小鼠心肌微血管内皮细胞增殖的影响。5.小鼠心肌微血管内皮细胞分为正常对照组和实验A组2组。正常对照组：无添加Slit2的细胞悬液；实验A组：分别含有Slit2蛋白(25、50、75、100、125、150ng/ml) 6个不同浓度的细胞悬液组。Transwell实验检测2组细胞悬液对小鼠心肌微血管内皮细胞迁移的影响。6.小鼠心肌微血管内皮细胞分为正常对照组、阳性对照组和实验B组3组。正常对照组：无添加Slit2的细胞悬液；阳性对照组：含有10ng/ml VEGF的细胞悬液。实验B组：分别含有Slit2蛋白(25、50、75、100、125、150ng/ml)+10ng/ml VEGF的6个不同浓度的细胞悬液组。Transwell实验检测3组细胞悬液对小鼠心肌微血管内皮细胞迁移的影响。研究结果1.正常小鼠心室肌微血管组织中有Slit2的表达。2. Slit2mRNA表达于小鼠心肌微血管内皮细胞。3. 5个实验组与正常对照组相比，灰度值升高，差异有统计学意义(P＜0.05)，同时OD值升高，差异有统计学意义(P＜0.05)，10ng/ml TNF-α导致的Slit2 mRNA和Slit2蛋白水平达顶峰。4. 5个实验组与正常对照组的OD值相比，差异无统计学意义（P＞0.05）。5. 6个实验A组与正常对照组的细胞迁移数目相比，差异无统计学意义（P＞0.05）。6.阳性对照组和6个实验组与正常对照组对比，细胞迁移数目差异有统计学意义（P＜0.05）。Slit2蛋白浓度(100、125、150ng/ml)的3个实验B组与阳性对照组和低于此3组浓度的Slit2蛋白3个实验B组相比，细胞迁移数目差异有统计学意义（P＜0.05），但此三组之间的细胞迁移数目差异无统计学意义（P＞0.05）。研究结论1.正常小鼠心肌微血管内皮细胞存在并分泌Slit2蛋白。2. TNF-α刺激小鼠心肌微血管内皮细胞后Slit2 mRNA的表达上调Slit2蛋白的分泌增加，浓度为10ng/ml TNF-α刺激内皮细胞后Slit2蛋白的分泌水平达峰值。3. Slit2蛋白对小鼠心肌微血管内皮细胞的增殖无影响，100ng/ml Slit2蛋白即可抑制10ng/ml VEGF诱导的微血管内皮细胞的迁移。
[Abstract]:The Slit protein family was first found in the central nervous system, Slit protein is a kind of secretory glycoprotein in vertebrate Slit protein family consists of three members (Slit1, Slit2, Slit3). The Slit protein structure in mammals is composed of a N- terminal signal peptide, four leucine rich repeats. (LRRs), cysteine nine EGF like repeats and a C- terminal node is composed of four parts, and the LRRs is the Slit protein and its receptor Robo binding region required.Slit protein N- end and C- produced in the hydrolysis process of end of two fragments, only Slit and N- at the end of the full length protein binds to cells Robo membrane receptors play a role.
Slit protein in many human tissues and cell lines were expressed, known Slit-Robo signaling pathway has an important role in guiding neurite outgrowth and rejection of neuronal migration in the developing nervous system. And the report about the role of Slit-Robo signaling pathway in other tissues and in the system is not consistent. Recent studies have found that the proliferation of Slit-Robo signal pathway and vascular endothelial cell migration, the.Wang reported that the expression of Slit2 in human malignant melanoma on vascular endothelial cells, reduced tumor microvessel density by blocking Slit2 and Robol receptors, indicating that Slit2-Robo signaling pathway plays an important role in tumor angiogenesis. On the contrary, the research report shows that inhibition the role of Slit2-Robo signal pathway on pathological corneal neovascularization, and at the same time, Seth also confirmed that inhibition of Silt2-Robo4 signaling Migration of HUVEC.
Slit2 in nerve tissue, the main expression of Slit1 Slit protein family, in addition to the expression of Slit3 and in neural tissue, also expressed in vascular endothelial cells.Wu reported that Slit2 and Slit3 expression in rat aortic endothelial cell lines, also confirmed in human renal arterioles and venules of.Zhang reported the expression of Slit2 Slit3 in vascular endothelial cells and smooth muscle cells. It has been confirmed that TNF- can up regulate the expression of inflammatory factor alpha Slit2 mRNA, and TNF- alpha can activate endothelial cells and involved in the occurrence and development of atherosclerosis. At home and abroad has not been reported whether in mouse cardiac microvascular endothelial cells on the expression of Slit2, so we first on the expression of Slit2 in normal mouse ventricular muscle microvascular tissue was detected, and through the primary mouse cardiac microvascular endothelial cell culture, Slit2 mRNA is not detected The expression of mouse cardiac microvascular endothelial cells, and with different concentrations of TNF- stimulated mouse cardiac microvascular endothelial cells after the detection of the expression of mRNA and Slit2 secreted Slit2 protein. Then through the proliferation and migration of vascular endothelial cells, the effects of different concentrations of Slit2 protein on microvascular endothelial cell proliferation and migration mouse cardiac microvascular endothelial cell migration, for the future research of Slit-Robo signaling pathway in myocardial ischemia, to provide experimental basis for angiogenesis and inflammatory factor TNF- alpha induced atherosclerosis in rats.
Objective: 1. expression of Slit2 was detected in normal mouse ventricular muscle microvascular tissues and Slit2 mRNA in normal mouse cardiac microvascular endothelial cells to detect the expression of.2. of different concentrations of TNF- stimulated mouse cardiac microvascular endothelial cells after.3. Slit2 hormones and the expression of mRNA and Slit2 protein of different concentrations of Slit2 on protein in the VEGF under the action of different concentration of Slit2 protein in mouse cardiac microvascular endothelial cell migration on mouse myocardial micro vascular endothelial cell proliferation and migration of.4..
Myocardial microvascular endothelial cells in.3. mice were divided into normal control group and experimental group 2 the expression of mRNA.2. RT-PCR Slit2 Slit2 detection technology research on detection method of normal mouse ventricular muscle 1. immunohistochemistry microvessel tissues in normal mouse cardiac microvascular endothelial cells. The normal control group: without adding TNF- alpha cells suspension; experimental group: respectively containing TNF- alpha (0.01,0.1,1,10100ng/ml) expression in 5 different concentrations of cell suspension liquid detection group.RT-PCR technology 2 group of cell Slit2 mRNA, Slit2 ELISA protein was detected in 2 groups of cells in the supernatant of the secretion of myocardial microvascular endothelial cells of.4. mice were divided into normal control group and experimental group 2 normal. Control group: no added Slit2 cell suspension; experimental group: Slit2 protein (50,75100125150ng/ml) containing 5 different concentrations of cell suspension group.CCK-8 test 2 groups of cells Effect of suspended on mouse cardiac microvascular endothelial cell proliferation of.5. mouse cardiac microvascular endothelial cells were divided into normal control group and experimental group A 2 group. Normal control group: no added Slit2 cell suspension; experimental group A: containing Slit2 protein (25,50,75100125150ng/ml) myocardial effects of.6. mice 6 different concentrations cell suspension group.Transwell test group 2 cell suspension of mouse cardiac microvascular endothelial cell migration of microvascular endothelial cells were divided into normal control group, positive control group and experimental group B 3 group. Normal control group: no added Slit2 cell suspension; positive control group: the cell suspension containing 10ng/ml VEGF experimental group B: Slit2 (25,50,75100125150ng/ml) protein respectively containing 6 different concentrations of +10ng/ml VEGF cell suspension group.Transwell test group 3 cell suspension of mouse cardiac microvascular endothelial cells The impact of migration.
The results of 1. normal mouse ventricular muscle microvascular tissues and the expression of Slit2.2. Slit2mRNA expressed in mouse cardiac microvascular endothelial cells.3. 5 experimental groups compared with normal control group, the gray value increased, the difference was statistically significant (P < 0.05), while the OD value increased, the difference was statistically significant (P < 0.05). Compared with the OD value of 10ng/ml TNF- alpha induced Slit2 mRNA and protein levels of Slit2 peaked 5.4. experimental group and normal control group, the difference was not statistically significant (P > 0.05) compared to the number of cell migration.5. 6 A experimental group and normal control group, the difference was not statistically significant (P > 0.05).6. positive control group and the 6 experimental groups compared with the normal control group, the number of migrated cells was statistically significant difference (P < 0.05).Slit2 protein concentration (100125150ng/ml) of the 3 B experimental group and positive control group and lower than the 3 group concentration of Slit2 protein of 3 B experimental group There was a significant difference in the number of cell migration (P < 0.05), but there was no significant difference in the number of cell migration between the three groups (P > 0.05).
The conclusion of the study 1. normal mice myocardial microvascular endothelial cells and the secretion of Slit2 protein.2. TNF- stimulated mouse cardiac microvascular endothelial cells secreting Slit2 mRNA up-regulated the expression of Slit2 protein increased, without affecting the concentration of 10ng/ml TNF- stimulated endothelial cell secretion after Slit2 protein reached the peak of.3. Slit2 protein of mouse cardiac microvascular endothelial cell proliferation, migration of 100ng/ml Slit2 protein can inhibit vascular endothelial cells 10ng/ml induced by VEGF.

【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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