| Part one: The effects of silencing LMP2 gene on the expression of blood-brain barrier protein in rat cerebral microvascular endothelial cells under oxygen-glucose deprivation / reoxygenation ObjectivesTo investigate the effects of silencing LMP2(low molecular weight protein 2 LMP2,low molecular weight protein 2(also known as β1i)gene on the expression of Claudin-1,Occludin,ZO-1 and cell migration in rat brain microvascular endothelial cells(RBMVECs)under oxygen-glucose deprivation / reoxygenation.MethodsRBMVECs were resuscitation cultured and passaged.A stable and reliable oxygen-glucose deprivation/reoxygenation(OGD/R)model was established.CCK8 was used to detect cell viability.LMP2-si RNA and negative control si RNA were transfected with RBMVECs.Immunofluorescence staining(IF)and western blot(WB)was used to detect the expression of target protein.Cell scratch test was used to evaluate the migration ability of RBMVECs.Results(1)RBMVECs grew well after resuscitation,and the purity of cultured RBMECs identified by factor Ⅷ immunofluorescence staining was more than 90%.(2)A stable and reliable cell ischemia model of oxygen-glucose deprivation 1h/ reoxygenation for 24 hours(OGD/R)in vitro was established.(3)IF showed that the expression of LMP2 protein in OGD/R group was higher than that in normal conditioned culture group(Normal group),but the expression of ZO-1,Occludin and Claudin-1 protein was lower than that in Normal group.WB showed that the expression of LMP2 protein in OGD/R group was up-regulated,but the expression of ZO-1,Occludin and Claudin-1 protein was lower than that in Normal group,and there was significant difference compared with Normal group(P<0.001).(4)RBMVECs cultured under normal conditions(Normal group)showed obvious proliferation,migration and repairation at 48 hours after scratch injury,but the migration ability decreased significantly under OGD/R condition.(5)WB showed that the protein expression of ZO-1,Occludin and Claudin-1 in RBMVECs in OGD/R group was significantly lower than that in normal group,and the difference was statistically significant(P < 0.001).However,the expression of LMP2 gene silenced by LMP2-si RNA could reverse the down-regulation trend of these proteins.Compared with the negative control group(Control-si RNA group),the protein expression of ZO-1,Occludin and Claudin-1 in LMP2-si RNA group was significantly higher than that in LMP2-si RNA group(P < 0.001).(6)WB showed that compared with normal group,Bax and cleaved Caspase3 protein levels were up-regulated and Bcl-2 protein was significantly decreased in RBMVECs following OGD/R(P < 0.001).Compared with control-si RNA group,silencing LMP2 gene could reverse the down-regulation of Bcl-2 and reduce the expression of Bax and cleaved Caspase3 protein(P < 0.001).(7)Cell scratch test showed that compared with control-si RNA group and blank group,the proliferation and migration ability of RBMVECs transfected with LMP2-si RNA was significantly increased after OGD/R.ConclusionsSilencing the expression of LMP2 gene can up-regulate the expression of blood-brain barrier protein in RBMVECs under the condition of OGD/R,inhibit apoptosis and promote cell migration.Part two: The effect of silencing β-catenin gene on the expression of blood-brain barrier protein in rat brain microvascular endothelial cells under oxygen-glucose deprivation / reoxygenation.ObjectivesTo investigate the effects of silent β-catenin gene expression on the expression of Claudin-1,Occludin,ZO-1 and cell migration in rat brain microvascular endothelial cells(RBMVECs)under oxygen-glucose deprivation / reoxygenation.MethodsRBMVECs was resuscitated and passaged to establish a stable and reliable oxygen-glucose deprivation/reoxygenation(OGD/R)model.CCK8 was used to detect cell viability.Βeta-catenin-si RNA and negative control si RNA were transfected with RBMVECs.Immunofluorescence staining and western blot was detected the expression of target proteins.Cell scratch test was used to evaluate the migration ability of RBMVECs.Results(1)IF indicated that the expression of β-catenin protein in RBMVECs in OGD/R group was lower than that in normal conditioned group.WB showed that β-catenin protein level in OGD/R group was significantly lower than that in normal group(P < 0.001).(2)WB showed that compared with the normal group,the expression of ZO-1,Occludin and Claudin-1 protein in RBMVECs decreased significantly under OGD/R condition.The expression of β-catenin gene silenced by β-catenin-si RNA increased the down-regulation trend of these proteins.The ZO-1,Occludin and Claudin-1 protein levels in β-catenin-si RNA group was significantly lower than that in Control-si RNA group(P < 0.001).(3)WB showed that compared with the normal group,the expression of Wnt-3a and β-catenin protein in RBMVECs decreased significantly under OGD/R condition,but the down-regulation trend of these proteins could be reversed by silencing the expression of LMP2 gene in LMP2-si RNA group.The Wnt-3a and β-catenin protein levels inLMP2-si RNA group were significantly higher than that in control-si RNA group(P < 0.001).(4)WB showed that compared with the normal group,the levels of ZO-1,Occludin and Claudin-1 in RBMVECs decreased significantly following OGD/R(P<0.001),and the down-regulation of these proteins could be reversed by silencing the expression of LMP2 gene by transfection of LMP2-si RNA,but this effect was counteracted by co-transfection of β-catenin-si RNA(P < 0.001).(5)Cell scratch test indicated that the proliferation and migration ability of RBMVECs transfected with β-catenin-si RNA was significantly weakened under the condition of OGD/R compared with control-si RNA group,ConclusionsWnt/β-catenin pathway regulates the expression of blood-brain barrier protein in brain microvascular endothelial cells.Silencing LMP2 gene expression may up-regulate the expression of blood-brain barrier protein and promote cell migrationin brain microvascular endothelial cells maybe through activating Wnt/β-catenin pathway. |
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