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
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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。
Slit protein family，a kind of secreted glycoprotein, consisting of threeSlit (Slit1、Slit2、Slit3) members in vertebrates，were originally identified inthe nervous system. Slit in mammals contains four leucine-rich regions (it isthe necessary area which Slit bind its receptor Robo), nine epidermal growthfactor repeats, a laminin G-domain, and a C-terminal cysteine-rich domain.Slit in the process of hydrolysis can be cleaved into the N-and C-terminalfragments, and only full- length and the N-terminus of Slit can bind cellmembrane receptor Robos then working.
Slit protein is be expressed in the human variety of tissues and the celllines, and it is known than Slit-Robo signaling pathways play an importantrole of guiding axons growth and repelling nerve cell migration in thedeveloping of the nervous system. Nonetheless, reports on roles of Slit-Robosignaling pathways in other tissues and systems are controversial. In recentyears, studies have found that Slit-Robo signaling pathways have relationwith proliferation and migration of vascular endothelial cell. A report ofWang et al has shown that Slit2 is expressed in endothelial cells of human malignant melanoma and tumor microvascular density is reduced by blockingbind domain of Slit and Robo1, which suggesting that the Slit2-Robosignaling pathways may speed up the development of cancer by promotingcancer angiogenesis．In contrast, a report of study has shown that Slit2-Robosignaling pathways inhibit pathological corneal neovascularization, at thesame time, and the study of Seth et al has also confirmed that Silt2-Robo4signaling pathways inhibit migration of HUVECS.
Slit1 of Slit protein family is mainly expressed in nervous tissues; Slit2and Slit3 are also expressed vascular endothelial cell besides expressed innervous tissues. Research of Wu et al has proved that the Slit2 and Slit3 wereexpressed in rat aortic endothelial cell lines and Slit2 was also expressed inthe arterioles and venules of human kidney. Report of Zhang et al has foundSlit3 was expressed endothelial cells and smooth muscle cells of in vascularsystem. A experiment has proved that inflammation factors (TNF-α)upregulated Slit2 mRNA expression, and it can make endothelial cellsactivation hyperplasia then take park in formation and development ofcoronary atherosclerosis. It is no report so far at home and abroad that Slit2 iswhether expressed in mouse myocardial microvascular endothelial cells, sowe first examine expressed situation of Slit2 in mouse normal ventriclemuscle microvascular tissues, and examine expression of Slit2 mRNA in mouse cardiac microvascular endothelial cells through using primary cultureof the mouse cardiac microvascular endothelial cells, at the same time, anddetect expression of Slit2 mRNA and secretion of Slit2 protein using thedifferent concentration of TNF-αto stimulate mouse cardiac microvascularendothelial cells. Second, we explore potential effects of Slit2 differentconcentration in regulating proliferation and migration of mouse cardiacmicrovascular endothelial cell by proliferation and migration experiment ofvascular endothelial cells, so as to provide experimental evidence in studyingfunctions of Slit-Robo signaling pathways in microvascular endothelial cellsof myocardial ischemia, angiogenesis and coronary atherosclerosis caused byinflammation factor TNF-α.
Objective: 1. To examine expression of Slit2 protein in mouse normalventricle muscle microvascular tissues and expression of Slit2 mRNA inmouse normal cardiac microvascular endothelial cells. 2. To examineexpression of Slit2 mRNA and secretion of Slit2 protein, using the differentconcentration of TNF-αto stimulate mouse cardiac microvascular endothelialcells. 3. To explore potential effects of different concentration of Slit2 proteinin proliferation and migration of mouse cardiac microvascular endothelialcells. 4. To explore potential effects of different concentration Slit2 protein inmigration of mouse cardiac microvascular endothelial cells, under the action of VEGF.
Methods: 1. To examine expression of Slit2 protein in mouse normalventricle muscle microvascular tissues by immunohistochemistry method. 2.To examine expression of Slit2 mRNA in mouse normal cardiacmicrovascular endothelial cells by RT-PCR technology. 3. Mouse cardiacmicrovascular endothelial cells are divided into two groups. Normal controlgroup (ordinary culture without TNF-αstimulation) and five experimentalgroups (0.01, 0.1, 1, 10, 100ng/ml TNF-αwas respectively added into cellsolution). To detect expression of Slit2 mRNA in mouse cardiacmicrovascular endothelial cells by RT-PCR technology and secretion of Slit2protein in cell superate by ELISA kit respectively. 4. Mouse cardiacmicrovascular endothelial cells are divided into two groups. Normal controlgroup (ordinary culture without Slit2 protein stimulation) and fiveexperimental groups (50, 75, 100, 125, 150ng/ml Slit2 was respectively addedinto cell solution). To detect potential effects of two groups cell solution inregulating proliferation of mouse cardiac microvascular endothelial cells byCCK-8 experiment. 5. Mouse cardiac microvascular endothelial cells aredivided into two groups. Normal control group (ordinary culture without Slit2protein stimulation) and six experimental groups A(25, 50, 75, 100, 125,150ng/ml Slit2 was respectively added into cell solution). To detect potential effects of two groups cell solution in regulating migration of mouse cardiacmicrovascular endothelial cells by transwell experiment. 6. Mouse cardiacmicrovascular endothelial cells are divided into three groups. Normal controlgroup (ordinary culture without Slit2 protein stimulation), positive controlgroup (ordinary culture with VEGF stimulation) and six experimental groupsB (25, 50, 75, 100, 125, 150ng/ml Slit2 was respectively added into cellsolution that has been added VEGF stimulation). To detect potential effects ofthree groups cell solution in migration mouse cardiac microvascularendothelial cells by transwell experiment.
Results: 1. Expression of Slit2 in mouse normal ventricle musclemicrovascular tissues. 2. Expression of Slit2 mRNA in mouse cardiacmicrovascular endothelial cells. 3. Five experimental groups compared withnormal control group, the difference of grey value increasing has statisticalsignificance (P<0.05), and meanwhile statistical analysis shows that thedifference of OD value increasing has statistical significance (P<0.05).Expression of Slit2 mRNA and secretion of Slit2 protein have maximum levelat 10ng/ml TNF-α. 4. As compared with normal control group，OD valuedifference of five experimental groups was not statistical significance(P>0.05). 5. As compared with normal control group，the numbers differenceof cell migration in six experimental groups was not statistical significance (P>0.05). 6. Positive control group and six experimental groups B comparedwith normal control group, the numbers difference of cell migration hasstatistical significance (P<0.05). Three experimental groups B at 100, 125,150ng/ml Slit2 protein compared with positive control group and the othergroups (at lower than 100ng/ml Slit2) result in numbers of cell migration havestatistically significant difference (P<0.05), but numbers of endothelial cellmigration in the three groups have no statistically significant difference (P>0.05).
Conclusion: 1.There are expression and release of Slit2 in normalcardiac microvascular endothelial cells. 2. With TNF-αstimulates mousecardiac microvascular endothelial cells, expression of Slit2 mRNA increases,at the same time, secretion of Slit2 protein also increases, Slit2 proteinsecretion has maximum level at 10ng/ml TNF-α. 3. Slit2 may not play animportant role in regulating proliferation of mouse cardiac microvascularendothelial cells; 100ng/ml Slit2 protein can inhibit migration of mousecardiac microvascular endothelial cells mediated by 10ng/ml VEGF.
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。
Slit protein family，a kind of secreted glycoprotein, consisting of threeSlit (Slit1、Slit2、Slit3) members in vertebrates，were originally identified inthe nervous system. Slit in mammals contains four leucine-rich regions (it isthe necessary area which Slit bind its receptor Robo), nine epidermal growthfactor repeats, a laminin G-domain, and a C-terminal cysteine-rich domain.Slit in the process of hydrolysis can be cleaved into the N-and C-terminalfragments, and only full- length and the N-terminus of Slit can bind cellmembrane receptor Robos then working.
Slit protein is be expressed in the human variety of tissues and the celllines, and it is known than Slit-Robo signaling pathways play an importantrole of guiding axons growth and repelling nerve cell migration in thedeveloping of the nervous system. Nonetheless, reports on roles of Slit-Robosignaling pathways in other tissues and systems are controversial. In recentyears, studies have found that Slit-Robo signaling pathways have relationwith proliferation and migration of vascular endothelial cell. A report ofWang et al has shown that Slit2 is expressed in endothelial cells of human malignant melanoma and tumor microvascular density is reduced by blockingbind domain of Slit and Robo1, which suggesting that the Slit2-Robosignaling pathways may speed up the development of cancer by promotingcancer angiogenesis．In contrast, a report of study has shown that Slit2-Robosignaling pathways inhibit pathological corneal neovascularization, at thesame time, and the study of Seth et al has also confirmed that Silt2-Robo4signaling pathways inhibit migration of HUVECS.
Slit1 of Slit protein family is mainly expressed in nervous tissues; Slit2and Slit3 are also expressed vascular endothelial cell besides expressed innervous tissues. Research of Wu et al has proved that the Slit2 and Slit3 wereexpressed in rat aortic endothelial cell lines and Slit2 was also expressed inthe arterioles and venules of human kidney. Report of Zhang et al has foundSlit3 was expressed endothelial cells and smooth muscle cells of in vascularsystem. A experiment has proved that inflammation factors (TNF-α)upregulated Slit2 mRNA expression, and it can make endothelial cellsactivation hyperplasia then take park in formation and development ofcoronary atherosclerosis. It is no report so far at home and abroad that Slit2 iswhether expressed in mouse myocardial microvascular endothelial cells, sowe first examine expressed situation of Slit2 in mouse normal ventriclemuscle microvascular tissues, and examine expression of Slit2 mRNA in mouse cardiac microvascular endothelial cells through using primary cultureof the mouse cardiac microvascular endothelial cells, at the same time, anddetect expression of Slit2 mRNA and secretion of Slit2 protein using thedifferent concentration of TNF-αto stimulate mouse cardiac microvascularendothelial cells. Second, we explore potential effects of Slit2 differentconcentration in regulating proliferation and migration of mouse cardiacmicrovascular endothelial cell by proliferation and migration experiment ofvascular endothelial cells, so as to provide experimental evidence in studyingfunctions of Slit-Robo signaling pathways in microvascular endothelial cellsof myocardial ischemia, angiogenesis and coronary atherosclerosis caused byinflammation factor TNF-α.
Objective: 1. To examine expression of Slit2 protein in mouse normalventricle muscle microvascular tissues and expression of Slit2 mRNA inmouse normal cardiac microvascular endothelial cells. 2. To examineexpression of Slit2 mRNA and secretion of Slit2 protein, using the differentconcentration of TNF-αto stimulate mouse cardiac microvascular endothelialcells. 3. To explore potential effects of different concentration of Slit2 proteinin proliferation and migration of mouse cardiac microvascular endothelialcells. 4. To explore potential effects of different concentration Slit2 protein inmigration of mouse cardiac microvascular endothelial cells, under the action of VEGF.
Methods: 1. To examine expression of Slit2 protein in mouse normalventricle muscle microvascular tissues by immunohistochemistry method. 2.To examine expression of Slit2 mRNA in mouse normal cardiacmicrovascular endothelial cells by RT-PCR technology. 3. Mouse cardiacmicrovascular endothelial cells are divided into two groups. Normal controlgroup (ordinary culture without TNF-αstimulation) and five experimentalgroups (0.01, 0.1, 1, 10, 100ng/ml TNF-αwas respectively added into cellsolution). To detect expression of Slit2 mRNA in mouse cardiacmicrovascular endothelial cells by RT-PCR technology and secretion of Slit2protein in cell superate by ELISA kit respectively. 4. Mouse cardiacmicrovascular endothelial cells are divided into two groups. Normal controlgroup (ordinary culture without Slit2 protein stimulation) and fiveexperimental groups (50, 75, 100, 125, 150ng/ml Slit2 was respectively addedinto cell solution). To detect potential effects of two groups cell solution inregulating proliferation of mouse cardiac microvascular endothelial cells byCCK-8 experiment. 5. Mouse cardiac microvascular endothelial cells aredivided into two groups. Normal control group (ordinary culture without Slit2protein stimulation) and six experimental groups A(25, 50, 75, 100, 125,150ng/ml Slit2 was respectively added into cell solution). To detect potential effects of two groups cell solution in regulating migration of mouse cardiacmicrovascular endothelial cells by transwell experiment. 6. Mouse cardiacmicrovascular endothelial cells are divided into three groups. Normal controlgroup (ordinary culture without Slit2 protein stimulation), positive controlgroup (ordinary culture with VEGF stimulation) and six experimental groupsB (25, 50, 75, 100, 125, 150ng/ml Slit2 was respectively added into cellsolution that has been added VEGF stimulation). To detect potential effects ofthree groups cell solution in migration mouse cardiac microvascularendothelial cells by transwell experiment.
Results: 1. Expression of Slit2 in mouse normal ventricle musclemicrovascular tissues. 2. Expression of Slit2 mRNA in mouse cardiacmicrovascular endothelial cells. 3. Five experimental groups compared withnormal control group, the difference of grey value increasing has statisticalsignificance (P<0.05), and meanwhile statistical analysis shows that thedifference of OD value increasing has statistical significance (P<0.05).Expression of Slit2 mRNA and secretion of Slit2 protein have maximum levelat 10ng/ml TNF-α. 4. As compared with normal control group，OD valuedifference of five experimental groups was not statistical significance(P>0.05). 5. As compared with normal control group，the numbers differenceof cell migration in six experimental groups was not statistical significance (P>0.05). 6. Positive control group and six experimental groups B comparedwith normal control group, the numbers difference of cell migration hasstatistical significance (P<0.05). Three experimental groups B at 100, 125,150ng/ml Slit2 protein compared with positive control group and the othergroups (at lower than 100ng/ml Slit2) result in numbers of cell migration havestatistically significant difference (P<0.05), but numbers of endothelial cellmigration in the three groups have no statistically significant difference (P>0.05).
Conclusion: 1.There are expression and release of Slit2 in normalcardiac microvascular endothelial cells. 2. With TNF-αstimulates mousecardiac microvascular endothelial cells, expression of Slit2 mRNA increases,at the same time, secretion of Slit2 protein also increases, Slit2 proteinsecretion has maximum level at 10ng/ml TNF-α. 3. Slit2 may not play animportant role in regulating proliferation of mouse cardiac microvascularendothelial cells; 100ng/ml Slit2 protein can inhibit migration of mousecardiac microvascular endothelial cells mediated by 10ng/ml VEGF.
引文
[1] Fujiwara M, Ghazizadeh M, Kawanami Q, et al. Potential role of the Slit/Robo signalpathway in angiogenesis[J]. Vasc Med,2006,11(2):115-121.
[2] Li-Jing W, Yuan Zhao, Bing Han, et al. Targeting Slit–Roundabout signaling inhibitstumor angiogenesis in chemical-induced squamous cell carcinogenesis[J]. CancerSci,2008,99(3):510-517.
[3] Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switchduring tumorigenesis[J]. Cell,1996,86(3):353-64.
[4] Carmeliet P. Angiogenesis in life, disease and medicine[J]. Nature,2005,438(7070):932-6.
[5] Carmeliet P, Tessier-Lavigne M. Common mechanisms of nerve and blood vesselwiring[J]. Nature,2005,436(7048):193-200.
[6] Holash J, Thurston G, Rudge JS, et al. Inhibitors of growth factor receptors, signalingpathways and angiogenesis as therapeutic molecular agents[J]. Cancer MetastasisRev,2006,25(2):243-52.
[7] Folkman J. Angiogenesis[J]. Annu Rev Med,2006,57(-):1–18.
[8] Ferrara N. Role of vascular endothelial growth factor physiologic and pathologicangiogenesis: therapeutic implications[J]. Semin Oncol,2002,29(16):10-14.
[9] Bicknell R, Harris AL. Novel angiogenic signalling pathways and vascular targets[J].Annu Rev Pharmacol Toxicol,2004,44(-):219-238.
[10]Suchting S, Bicknell R, Eichmann A. Neuronal clues to vascular guidance[J]. ExpCell Res,2006,312(5):668-675.
[11]Itoh A, Miyabayashi T, Sakano S. Cloning and expression of three mammalianHomologues of Drosophila slit suggest possible roles of Slit in the formation andMaintenance of the nervous system[J]. Mol Brain Res,1998,62(2):175-86.
[12]Brose K, Bland KS, Wang KH, et al. Slit proteins bind Robo receptors and have anevolutionarily conserved role in repulsive axon guidance1[J].Cell,1999,96(6):795-806.
[13]Li HS,Chen JH,Wu W, et al. Vertebrate slit, a secreted ligand for the transmembraneprotein roundabout, is a repellent for olfactory bulb axons[J]. Cell,1999,96(6):807-18.
[14]Kim T, Nguyen Ba-Charvet, Katja Brose, et al. Diversity and Specificity of Actionsof Slit2 Proteolytic Fragments in Axon Guidance[J]. Neurosci,2001,21(12):4281-4289.
[15]Rothberg JM, Jacobs JR, Goodman CS, et al. Slit: an extracellular protein necessaryfor development of midline glia and commissural axon pathways contains both EGFand LRR domains[J]. Genes Dev,1990,4(12A):2169-87.
[16]Katoh Y, Katoh M. Comparative genomics on SLIT1, SLIT2, and SLIT3 orthologs[J].Oncol Rep,2005,14(5):1351-5.
[17]Chen JH,Wen L,Dupuis S, et al. The N-terminal leucine2rich regions in Slit aresufficient to repel olfactory bulb axons and subventricular zone neurons[J]. JNeurosci,2001,21(5):1548-1556.
[18]Morlot C, Thielens N.M, Ravell R. Structural insights into the Slit-Robo complex[J].PNAS,2007,104(38):14923-14928.
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[25]Dickinson RE, Fegan KS, Hillier SG & Duncan WC. Steroid regulated expression ofthe SLIT/ROBO pathway in human ovarian surface epithelial cells and ovariancancer[J]. Proceedings of the Society for Gynecologic Investigation, 2009,56thAnnual Meeting, Glasgow, UK. Abstract 73.
[26]Kim HK, Zhang H, Li H, et al. Slit2 inhibits growth and metastasis of fibrosarcomaand squamous cellcarcinoma[J]. Neoplasia, 2008,10(12):1411-1420.
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[28]Qian, L, J Liu , R. Bodmer. Slit and Robo control cardiac cell polarity andmorphogenesis[J]. Curr Biol,2005,15(24):2271-2278 .
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[30]Abdollahi A, Schwager C, Kleeff J, et al. Transcriptional network governing theangiogenic switch in human pancreatic cancer[J]. Proc Natl Acad Sci U SA,2007,104(31):12890-12895.
[31]Kaur S, Samant GV, Pramanik K, et al. Silencing of directional migration inroundabout4 knockdown endothelial cells[J]. BMC Cell Biology,2008, 9:(61)1-12.
[32]Suchting S, Heal P, Tahtis K,et al. Soluble Robo4 receptor inhibits in vivoangiogenesis and endothelial cell migration [J]. Faseb J 2005,19(1):121-23.
[33]Hinck L. The versatile roles of“axon guidance”cues in tissue morphogenesis[J]．DevCen. 2004,7(6):783-793.
[34]Ypsilanti A＆Zagar Y Chedotal A．Moving away from the midline：newdevelopments for Slit and Robo[J]．Development, 2010,137(12):1939-1952.
[35]Grieshammer U，Le M，Plump AS，Wang F，et a1．Slit2 mediated Robo2signalingrestricts kidney induction to a single site[J]. Dev cell,2004, 6(5):709-717.
[36]Liu J，Zhang L，Wang D，Shen H，et a1．Congenital diaphragmatichernia,kidneyagenesis and cardiac defects associated with Slit3-deficiency in mice[J].Mech Dev. 2003;120(9):1059-1070．
[37]Augustin H. Methods in endothelial cell biology[M]. Springer Lab Maunal,2004,15-40.
[38]Mart in GM. Tissue and orginic culture of blood vesels [M ]. In Rolbbat G Hed. NewYork: Acadenic Press,1987:13-21.
[39]Yoshida S，Ono M, Shono T，et at. Involvement of interleukin-8, vascular endothelialgrowth factor, and basic fibroblast growth factor in tumor necrosis factoralpha-dependent angiogenesis[J]. Mol Cell Biol 1997,17(7):4015-23.
[40]Bussolino F, Camussi G, Baglioni C. Synthesis and release of platelet-activatingfactor by human vascular endothelial cells treated with tumor necrosis factor orinterleukin l alpha[J]. J Bioi Chem,1988,263(24):11856-1.
[41]Seth P, Lin Y, Hanai J, et al. Magic roundabout, a tumor endothelial marker:expression and signaling[J]. Biochem Biophys Res Commun,2005,332(2): 533-541.
[42]Park KW,Morrison CM,Sorensen LK,et al. Robo4 is a vascular-specific receptor thatinhibits endothelial cell migration[J]. Dev Biol,2003,261(1):251-267.
[43]Brantley-Sieders D, Dunaway C, Rao M, et al. Angiocrine Factors Modulate TumorProliferation and Motility through EphA2 Repression of Slit2 Tumor SuppressorFunction in Endothelium[J]. Cancer Res,2011,71(3):976-987.
[44]Abdollahi A, Schwager C, Kleeff J, et al. Transcriptional network governing theangiogenic switch in human pancreatic cancer[J]. Proc Natl Acad Sci U SA,2007,104(31):12890-12895.
[45]Latil A, Chene L, Cochant-Priollet B, et al. Quantification of expression of netrins,slits and their receptors in human prostate tumors[J]. Int J Cancer, 2003,103(97):306.
[46]Zhang B, Dietrich UM, Geng JG,et al. Repulsive axon guidance molecule Slit3 is anovel angiogenic factor[J]. Blood, 2009,10(19):182-1211.
[1] Fujiwara M, Ghazizadeh M, Kawanami Q,. Potential role of the Slit/Robo signalpathway in angiogenesis. Vascular Medicine, 2006,11: 115–121.
[2] Carmeliet P, Tessier-Lavigne M. Common mechanisms of nerve and blood vesselwiring. Nature, 2005,436: 193–200.
[3] Ferrara N. Role of vascular endothelial growth factor physiologic and pathologicangiogenesis: therapeutic implications. Semin Oncol, 2002, 29: 10–14.
[4] Suchting S, Bicknell R, Eichmann A. Neuronal clues to vascular guidance. Exp CellRes, 2006,312:668–675.
[5] Whitford KL, Marillat V, Stein E, et al. Regulation of cortical dendrite developmentby Slit–Robo interactions. Neuron, 2002; 33: 47–61.
[6] Wang B, Xiao Y, Ding BB, et al. Induction of tumor angiogenesis by Slit-Robosignaling and inhibition of cancer growth by blocking Robo activity. CancerCell,2003,4:19-29.
[7] Santiago-Martinez , E, N.H. Soplop , S.G. Kramer. Lateral positioning at the dorsalmidline: Slit and Roundabout receptors guide Drosophila heart cell migration. ProcNatl Acad Sci U S A,2006, 103:12441–12446.
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[11]Itoh A, Miyabayashi T, Sakano S. Cloning and expression of three mammalianHomologues of Drosophila slit suggest possible roles of Slit in the formation andMaintenance of the nervous system. Mol Brain Res,1998,62:175-186.
[12]Li HS,Chen JH, Wu W, et al. Vertebrate slit, a secreted ligand for thetransmembrane protein roundabout, is a repellent for olfactory bulb axons.Cell,1999,96:807-18.
[13]Morlot C, Thielens NM, Ravell R . Structural insights into the Slit-Robo complex.PNAS, 2007,104:14923–14928.
[14]Nguyen Ba-Charvet KT, Brose K, Ma L, et al. Diversity and specificity of actions ofSlit2 proteolytic fragments in axon guidance. J Neurosci,2001,21:4281-4289.
[15]Song HJ,Ming GL,Poo MM. cAMP-induced switching in turning direction of nervegrowth cones. Nature,1997,388:275-279.
[16]Battye R, Stevens A, Perry RL, et al. Repellent signaling by Slit requires theleucine-rich repeats. J Neurosci,2001,21:4290-4298.
[17]Hohenester E. Structural insight into Slit–Robo signaling[J]. Biochem Soc Trans,2008,36:251–256.
[18]Kidd T,Brose K,Mitchell KJ, et al. Roundabout controls axon crossing of the CNSmidline and defines a nove subfamily of evolutionarily conserved guidance receptors.Cell,1998,92:205-15.
[19]Huminiecki L, Gorn M, Suchting S,et al. Magic roundabout is a new member of theroundabout receptor family that is endothelial specific and expressed at sites of activeangiogenesis. Genomics, 2002, 79:547–542.
[20]Park KW, Morrison CM, Sorensen LK, et al. Robo4 is a vascular-specific receptorthat inhibits endothelial cell migration. Dev Biol,2003, 261:251-267.
[21]Kidd T,Bland KS,Goodman CS. Slit is the midline repellent for the robo receptor inDrosophila. Cell,1999,96:785-794.
[22]Latil A,Chene L,Cochant-Priollet B, et al. Quantification of expression of netrins,slits and their receptors in human prostate tumors. Int J Cancer,2003,103:306
[23]Wu JY,Feng L,Park HT,et al. The neuronal repellent Slit inhibits leukocytechemotaxis induced by chemotactic factors. Nature,2001,410:948-952.
[24]Abdollahi A, Schwager C, Kleeff J, et al. Transcriptional network governing theangiogenic switch in human pancreatic cancer[J]. Proc Natl Acad Sci U SA ,2007,104:12890–12895.
[25]MacMullin A, J.R. Jacobs. Slit coordinates cardiac morphogenesis inDrosophila.Deve Biol ,2006,293:154–164.
[26]Zhang B, Dietrich UM, Geng JG ,et al. Repulsive axon guidance molecule Slit3 is anovel angiogenic factor. Boold, 2009,114(19):4300-9.
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[30]Hohenester E. Structural insight into Slit–Robo signaling. Biochemical SocietyTransactions,2008,36:251–256.
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[2] Li-Jing W, Yuan Zhao, Bing Han, et al. Targeting Slit–Roundabout signaling inhibitstumor angiogenesis in chemical-induced squamous cell carcinogenesis[J]. CancerSci,2008,99(3):510-517.
[3] Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switchduring tumorigenesis[J]. Cell,1996,86(3):353-64.
[4] Carmeliet P. Angiogenesis in life, disease and medicine[J]. Nature,2005,438(7070):932-6.
[5] Carmeliet P, Tessier-Lavigne M. Common mechanisms of nerve and blood vesselwiring[J]. Nature,2005,436(7048):193-200.
[6] Holash J, Thurston G, Rudge JS, et al. Inhibitors of growth factor receptors, signalingpathways and angiogenesis as therapeutic molecular agents[J]. Cancer MetastasisRev,2006,25(2):243-52.
[7] Folkman J. Angiogenesis[J]. Annu Rev Med,2006,57(-):1–18.
[8] Ferrara N. Role of vascular endothelial growth factor physiologic and pathologicangiogenesis: therapeutic implications[J]. Semin Oncol,2002,29(16):10-14.
[9] Bicknell R, Harris AL. Novel angiogenic signalling pathways and vascular targets[J].Annu Rev Pharmacol Toxicol,2004,44(-):219-238.
[10]Suchting S, Bicknell R, Eichmann A. Neuronal clues to vascular guidance[J]. ExpCell Res,2006,312(5):668-675.
[11]Itoh A, Miyabayashi T, Sakano S. Cloning and expression of three mammalianHomologues of Drosophila slit suggest possible roles of Slit in the formation andMaintenance of the nervous system[J]. Mol Brain Res,1998,62(2):175-86.
[12]Brose K, Bland KS, Wang KH, et al. Slit proteins bind Robo receptors and have anevolutionarily conserved role in repulsive axon guidance1[J].Cell,1999,96(6):795-806.
[13]Li HS,Chen JH,Wu W, et al. Vertebrate slit, a secreted ligand for the transmembraneprotein roundabout, is a repellent for olfactory bulb axons[J]. Cell,1999,96(6):807-18.
[14]Kim T, Nguyen Ba-Charvet, Katja Brose, et al. Diversity and Specificity of Actionsof Slit2 Proteolytic Fragments in Axon Guidance[J]. Neurosci,2001,21(12):4281-4289.
[15]Rothberg JM, Jacobs JR, Goodman CS, et al. Slit: an extracellular protein necessaryfor development of midline glia and commissural axon pathways contains both EGFand LRR domains[J]. Genes Dev,1990,4(12A):2169-87.
[16]Katoh Y, Katoh M. Comparative genomics on SLIT1, SLIT2, and SLIT3 orthologs[J].Oncol Rep,2005,14(5):1351-5.
[17]Chen JH,Wen L,Dupuis S, et al. The N-terminal leucine2rich regions in Slit aresufficient to repel olfactory bulb axons and subventricular zone neurons[J]. JNeurosci,2001,21(5):1548-1556.
[18]Morlot C, Thielens N.M, Ravell R. Structural insights into the Slit-Robo complex[J].PNAS,2007,104(38):14923-14928.
[19]Nguyen Ba-Charvet KT,Brose K,Ma L, et al. Diversity and specificity of actions ofSlit2 proteolytic fragments in axon guidance[J]. J Neurosci,2001, 21(12):4281-4289.
[20]Wu W, Wong K, Chen JH, et al. Directional guidance of neuronal migration in theolfactory system by the protein Slit[J]. Nature,1999,400(6742):331-6.
[21]Whitford KL, Marillat V, Stein E et al. Regulation of cortical dendrite developmentby Slit–Robo interactions[J]. Neuron,2002,33(1):47-61.
[22]Wu JY,Feng L,Park HT, et al. The neuronal repellent Slit inhibits leukocytechemotaxis induced by chemotactic factors[J]. Nature,2001,410(6831):948-952.
[23]Wang B,Xiao Y,Ding BB, et al. Induction of tumor angiogenesis by Slit-Robosignaling and inhibition of cancer growth by blocking Robo activity[J]. CancerCell,2003,4(1):19-29.
[24]Dickinson RE, and Duncan WC. The SLIT–ROBO pathway: a regulator of cellfunction with implications for the reproductive system[J]. Reproduction,2010,139(4):697-704.
[25]Dickinson RE, Fegan KS, Hillier SG & Duncan WC. Steroid regulated expression ofthe SLIT/ROBO pathway in human ovarian surface epithelial cells and ovariancancer[J]. Proceedings of the Society for Gynecologic Investigation, 2009,56thAnnual Meeting, Glasgow, UK. Abstract 73.
[26]Kim HK, Zhang H, Li H, et al. Slit2 inhibits growth and metastasis of fibrosarcomaand squamous cellcarcinoma[J]. Neoplasia, 2008,10(12):1411-1420.
[27]Han X, Zhang MC,et al. Potential anti-angiogenic role of Slit2 in cornealneovascularization[J]. 2010, 90(6):724-9.
[28]Qian, L, J Liu , R. Bodmer. Slit and Robo control cardiac cell polarity andmorphogenesis[J]. Curr Biol,2005,15(24):2271-2278 .
[29]Santiago-Martinez, E., N.H. Soplop, S.G. Kramer. Lateral positioning at the dorsalmidline: Slit and Roundabout receptors guide Drosophila heart cell migration[J]. ProcNatl Acad Sci U S A,2006,103(33):12441-12446.
[30]Abdollahi A, Schwager C, Kleeff J, et al. Transcriptional network governing theangiogenic switch in human pancreatic cancer[J]. Proc Natl Acad Sci U SA,2007,104(31):12890-12895.
[31]Kaur S, Samant GV, Pramanik K, et al. Silencing of directional migration inroundabout4 knockdown endothelial cells[J]. BMC Cell Biology,2008, 9:(61)1-12.
[32]Suchting S, Heal P, Tahtis K,et al. Soluble Robo4 receptor inhibits in vivoangiogenesis and endothelial cell migration [J]. Faseb J 2005,19(1):121-23.
[33]Hinck L. The versatile roles of“axon guidance”cues in tissue morphogenesis[J]．DevCen. 2004,7(6):783-793.
[34]Ypsilanti A＆Zagar Y Chedotal A．Moving away from the midline：newdevelopments for Slit and Robo[J]．Development, 2010,137(12):1939-1952.
[35]Grieshammer U，Le M，Plump AS，Wang F，et a1．Slit2 mediated Robo2signalingrestricts kidney induction to a single site[J]. Dev cell,2004, 6(5):709-717.
[36]Liu J，Zhang L，Wang D，Shen H，et a1．Congenital diaphragmatichernia,kidneyagenesis and cardiac defects associated with Slit3-deficiency in mice[J].Mech Dev. 2003;120(9):1059-1070．
[37]Augustin H. Methods in endothelial cell biology[M]. Springer Lab Maunal,2004,15-40.
[38]Mart in GM. Tissue and orginic culture of blood vesels [M ]. In Rolbbat G Hed. NewYork: Acadenic Press,1987:13-21.
[39]Yoshida S，Ono M, Shono T，et at. Involvement of interleukin-8, vascular endothelialgrowth factor, and basic fibroblast growth factor in tumor necrosis factoralpha-dependent angiogenesis[J]. Mol Cell Biol 1997,17(7):4015-23.
[40]Bussolino F, Camussi G, Baglioni C. Synthesis and release of platelet-activatingfactor by human vascular endothelial cells treated with tumor necrosis factor orinterleukin l alpha[J]. J Bioi Chem,1988,263(24):11856-1.
[41]Seth P, Lin Y, Hanai J, et al. Magic roundabout, a tumor endothelial marker:expression and signaling[J]. Biochem Biophys Res Commun,2005,332(2): 533-541.
[42]Park KW,Morrison CM,Sorensen LK,et al. Robo4 is a vascular-specific receptor thatinhibits endothelial cell migration[J]. Dev Biol,2003,261(1):251-267.
[43]Brantley-Sieders D, Dunaway C, Rao M, et al. Angiocrine Factors Modulate TumorProliferation and Motility through EphA2 Repression of Slit2 Tumor SuppressorFunction in Endothelium[J]. Cancer Res,2011,71(3):976-987.
[44]Abdollahi A, Schwager C, Kleeff J, et al. Transcriptional network governing theangiogenic switch in human pancreatic cancer[J]. Proc Natl Acad Sci U SA,2007,104(31):12890-12895.
[45]Latil A, Chene L, Cochant-Priollet B, et al. Quantification of expression of netrins,slits and their receptors in human prostate tumors[J]. Int J Cancer, 2003,103(97):306.
[46]Zhang B, Dietrich UM, Geng JG,et al. Repulsive axon guidance molecule Slit3 is anovel angiogenic factor[J]. Blood, 2009,10(19):182-1211.
[1] Fujiwara M, Ghazizadeh M, Kawanami Q,. Potential role of the Slit/Robo signalpathway in angiogenesis. Vascular Medicine, 2006,11: 115–121.
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