Studies Of Expression Of Slit2 In Mouse Cardiac Microvascular Endothelial Cells And The Effects On Proliferation And Migration Of Cardiac Microvascular Endothelial Cells

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.