Impact Of Exogenous Slit3 And Its Receptor Robo1 On Proliferation And Migration Of Human Brain Vascular Smooth Muscle Cells And Its Regulation Mechanism Exploration

At present, cardiovascular disease is the major disease that threatens the people’s health around the world, and atherosclerosis is one of the most important cardiovascular diseases. Atherosclerosis pathogenesis is very complexly, there have been a variety of theories from different perspectives to elaborate it. At present, research suggests that vascular smooth muscle cells(VSMCs) play a very important role in the formation of atherosclerosis. That the abnormal proliferation and migration of VSMCs and secretion of extracellular matrix promote lipid streaks into the mature fiber plaque is an essential feature of atherosclerosis occurrence and development. Studies confirm that the proliferation and migration of VSMCs are a variety of common regulation of inflammatory cytokines and growth factors. Recent studies have found that neural axon guidance molecules Slit3 is expressed by both endothelial cells(ECs) and VSMCs. As a novel vascular active factor,Slit3 may regulate the proliferation and migration of ECs by via interaction with Robo4.While whether Slit3 could regulate VSMCs with similar way, it is currently a lack of experimental data to confirm. In this study, the human brain vascular smooth muscle cells(HBVSMCs) were selected as experimental subjects, detecting the expression of Slit3 and its receptor Robo,observing the impact of exogenous recombinant human Slit3 protein on VSMCs proliferation and migration, exploring the possible receptor Robo that Slit3 combine with and the molecular mechanism to regulate the proliferationand migration of HBVSMCs, laying the theoretical foundation for further research to explore the relationship between Slit3 and pathogenesis of atherosclerosis. The experiment was divided into three parts as follows:Part One The expression of Slit3/Robo genes and proteins in HBVSMCsObjectives: Detect the expression of Slit3 and Robo genes and proteins in HBVSMCs.Methods: Culture HBVSMCs with good growth conditions in vitro,extract total RNA and total protein and then detect the expression of slit3 and robo by RT-q PCR and WB assay.Results: We detected that the axon guidance molecules Slit3, Robo1,Robo4 genes and proteins express in HBVSMCs by RT-q PCR and WB assay.Conclusion: Slit3, Robo1, Robo4 genes and proteins express in HBVSMCs.Part Two Impact of exogenous Slit3 on the proliferation and migration ofHBVSMCsObjectives: Detect the impact of exogenous recombinant human Slit3 protein on HBVSMCs proliferation and migration.Methods: Set the negative control group containing BSA86ng/m L,experimental group containing Slit3 0ng/m L, 30ng/m L, 60ng/m L, 90ng/m L,120ng/m L and Positive control group containing PDGF 10ng/m L. Observe effects of HBVSMCs proliferation and migration upon exogenous Slit3 stimulation by CCK-8 and transwell chambers.Results: Exogenous Slit3 could promote the proliferation and migration activity of HBVSMCs, the best activity was obtained when Slit3 90ng/m L, it has the statistically significant differences compared with negative controlBSA(*P<0.05), it has no statistical difference compared with positive control PDGF(△P>0.05)Conclusion: Exogenous Slit3 could promote HBVSMCs proliferation and migration.Part Three Exploring the receptor Robo1 that Slit3 combine with and themolecular mechanism to regulate the proliferation and migration ofHBVSMCsObjectives: 1.Explore the possible transmission mechanism of Slit3/Robo regulating HBVSMCs proliferation and migration. 2. Explore the possible receptor Robo1 or(and) Robo4 that Slit3 combines with.Methods: 1.HBVSMCs, stimulated by Slit3 with concentration 90ng/m L were collected when the stimulation time was 0min,5min,15 min,30min,60 min for extracting cell protein, then detect the expression of active Rac1 and Rho A by Western Blot. 2.The construction and transfection of Robo1 si RNA and Robo4 si RNA:(1)Select HBVSMC as target cells,Robo1 and Robo4 as target genes, CY3 used to mark target gene sequence. Search Robo1 m RNA and Robo4 m RNA whole genome sequences from gene bank, all searched sequences were homology analyzed by BLAST software.(2) Take the Selected Robo1 m RNA and Robo4 m RNA whole genome sequences as template, according to the design principle of si RNA, design three gene sequences that could express specific Robo1 and Robo4 sh RNA, andsynthesize three double-stranded si RNA by chemical synthesis methods. set up negative control group, NC-si RNA group, and si RNA Robo1/ 4 transfected group.(3) si RNA was transfected into HBVSMCs by Liposome-mediated method, observed under fluorescent microscope. Estimate the transfection efficiency and screen optimal transfection concentration.(4)The expression of Robo1 and Robo4 was detected by RT-q PCR and Western Blot to screen optimal transfection fragmen. 3.Set up following groups :(1) blank control group(2)NC-si RNA group(3)Robo1si RNA group(4)Robo4si RNA group(5)Robo1si RNA+Robo4si RNA group. Add serum-free DMEM medium containing Slit3 with concentration 90ng/m L, detect the activity of cell proliferation and migration by CCK-8 and Transwell method as the second part after performed for 48 h. 4. Set up experiment group and add Slit3 as the before, after Slit3 performing for an hour, detect the expression of Rac1 and Rho A by Western Blot method.Results: After Slit3 stimulating HBVSMCs, the expression of a-Rac1 and a-Rho A increased with time duration of Slit3 acting, and reached a maximum level when performed 60 min.2.A little weak red fluorescence could be observed in the HBVSMCs cytoplasm under fluorescent microscope after transfection for 48 h, this described successful transfection. It was observed when the concentration of CY3-si RNA was 50 n M the fluorescence was brighter, the most transfected cells, and he maximum transfection efficiency was about 80%. 3. si RNA Robo1 and si RNA Robo4 could respectively inhibit Robo1 and Robo4 gene expression, it has the statistically significant differences compared with negative control group(P<0.05). 4. Detection of transfected HBVSMCs proliferation and migration: the activity of proliferation and migration was suppressed in Robo1 si RNA transfectedgroup, Robo1 si RNA and Robo4 si RNA both transfected group, it has the statistically significant differences compared with blank control group(P<0.05),while Robo4 si RNA transfected group had no influence on HBVSMCs. 5.The expression of a-Rac1 was obviously suppressed in Robo1 si RNA transfected group, Robo1 si RNA and Robo4 si RNA both transfected group, it has the statistically significant differences compared with blank control group(P<0.05),while Robo4 si RNA transfected group had no influence on a-Rac1. The expression of a-Rho A was suppressed in Robo4 si RNA transfected group, Robo1 si RNA and Robo4 si RNA both transfected group, it has the statistically significant differences compared with blank control group(P<0.05), while Robo1 si RNA transfected group had no influence on a-Rho A.Conclusion: 1.Exogenous Slit3 could stimulate the expression of active Rac1 and Rho A of GTPase family. 2. The optimal concentration of CY3-si RNA was 50 n M. 3. Robo1 si RNA and Rob4 si RNA transfection fragments synthesize could down-regulate the expression of Robo1 and Robo4 genes. 4. Neural axon guidance molecules Slit3 promote the proliferation and migration of HBVSMCs via interaction with Robo1. 5. Slit3 interacts with Robo1 to activate Rac1 of GTPase family to modulate HBVSMCs proliferation and migration activity. 6. Slit3 may interact with Robo4 to activate Rho A of GTPase family to regulate other biological activities of HBVSMCs.