| Atherosclerosis is a chronic inflammatory disease of arteriers.Vascular endothelial cells morphology and function have changed by local and circulating inflammatory factors,such as TNF alpha,this pathological process promote atherosclerotic plaque formation. A lot of studies have shown that TNF alpha plays a vital role in the process of atherosclerosis,and another study reported that axon guidance molecules slit2 plays as an anti-inflammatory factor in inflammatory disease. In this study Human umbilical vein endothelial cells(HUVECs) have stimulated by TNF alpha to simulate the endonthelial cells inflammatory reaction model,then slit2 were used as an anti-inflammatory factor in the model, the gene and protein expression of IL-8 were detected by real-time PCR and Elisa; the gene and protein expression of VCAM-1、ICAM-1 were detected by real-time PCR and Western Blot. The purpose of this study is to investigate the role of Slit2 in the inflammatory response of endothelial cells mediated by TNF alpha.PRAT 1:the expression of slit2,robo1 and robo4 in humam umbilical vein endothelial cells.Objective:To detect the gene and protein expression of Slit2, Robo1 and robo4 on HUVECs. Methods: the gene and protein expression of slit2、robo1 and robo4 were detected by Semi-quantitative RT PCR(RT PCR) and Western Blot. Results:This study detected the gene and protein expression of slit2、 robo1 and robo4 on human umbilical vein endothelial cells in vitro. Conclusion: Umbilical vein endothelial cells express slit2、robo1 and robo4 gene and protein.PRAT 2: Effects of slit2 on TNF alpha-induced gene and protein expression of IL-8 、ICAM-1、VCAM-1 in humam umbilical vein endothelial cellsObjective:To observe the role of Slit2 in inflammation. Methods:1. Human umbilical vein endothelial cells were divided into normal control group,Positive control group and experimental group, normal control group(NC group): medium containing no TNF alpha; Positive control group(TNF alpha group): TNF alpha concentration in medium was 1μg/L、10μg/L、100μg/L; experimental group(TNF alpha +slit2 group):medium containing 1 0μg/L TNF alpha and different concentration gradient of Slit2(25μg/L、50μg/L、100μg/L、200ug/L), respectively. After 24 hours, the gene and protein expression of IL-8 were detected by real-time PCR and Eliasa. 2. Human umbilical vein endothelial cells divided into three groups :normal group(NC group) : medium containing no TNF alpha and slit2; Positive control group(TNF alpha group): medium containing 10μg/L TNF alpha; experimental group(TNF alpha +slit2 group): medium containing 10μg/L TNF alpha and 100μg/L slit2. After 24 hours, the gene and protein expression of VCAM-1、ICAM-1 were detected by real-time PCR and Western Blot. Results:1. Compared with NC group, 10μg/L TNF alpha increased the gene and protein expression of IL-8( P(27)01.0); 2. 50μg/L、100μg/L、200μg/L slit2 decreased the TNF alpha-induced gene and protein expression of IL-8( P(27)05.0); 3. 100μg/L slit2 decreased the TNF alpha-induced gene and protein expression of VCAM-1 and ICAM-1( P(27)01.0).Conclusion: 1.Slit2 can inhibit the TNF alpha-induced secretion of IL-8 in the inflammation of Human umbilical vein endothelial cells; 2. TNF alpha-induced secretion of inflammatory cytokine ICAM-1、VCAM-1 in Human umbilical vein endothelial cells depressed by Slit2.PRAT 3: the possible mechanism of Slit2-Robo4 signal pathway inhibiting the expression of inflammatory cytokine induced by TNF alpha in human umbilical vein endothelial cellsObjective:1.To study the receptor of Slit2 in the TNF alpha mediated inflammatory response of human umbilical vein endothelial cells.2. To explore the possible mechanism of Slit2 inhibiting the inflammatory response mediated by TNF alpha in human umbilical vein endothelial cells.Methods:1. Using si RNA to silence the expression of Robo1 or robo4 in endothelial cells, this study designed three different si RNA sequences,named robo1-si RNA-1, robo1-si RNA-2, robo1-si RNA-3, robo4-si RNA-1, Robo4-si RNA-2 robo4-si RNA-3.Human umbilical vein endothelial cells were divided into the following three groups: Normal control group(NC group), negative control group(NT-si RNA group):cell transfection negative control si RNA;experimental: the cells were transfected with robo1-si RNA or robo4-si RNA, respectively.After 48 hours, the gene and protein expression of Robo1,and robo4 were detected by real-time PCR and Blot Western. 2. The transfected cells were divided into the following three groups:(1) transfected with NT-si RNA groups:(1)negative control group: culture medium does not contain TNF alpha and Slit2;(2)TNF alpha Group: culture medium containing 10μg/L TNF alpha;(3)TNF alpha+ Slit2 group: culture medium containing 10μg/L TNF alpha and 100μg/L Slit2;(2) transfected robo1-si RNA group: culture medium containing 10μg/L TNF alpha and 100μg/L Slit2;(3) transfected robo4-si RNA group: culture medium containing 10μg/L TNF alpha and 100μg/L slit2.After 24 hours, the gene and protein expression of ICAM-1 and VCAM-1 were detected by real-time PCR and Blot Western. 3.The possible mechanism of Slit2 inhibiting TNF alpha mediated endothelial inflammatory response: The cells were divided into three groups:normal group(NC group) : medium containing no TNF alpha and slit2; Positive control group(TNF alpha group): medium containing 10μg/L TNF alpha; experimental group(TNF alpha +slit2 group): medium containing 10μg/L TNF alpha and 100μg/L slit2. The protein of IKBa、p-IKBa、NF-k B、p-NF-k were detected by Blot Western after 1 hours, 2 hours and 4 hours,respectively.Results: 1.The expression of Robo1 and robo4 protein can be silenced by robo1-si RNA-1 or robo4-si RNA-2, respectively in vitro. 2.after transfection of Robo1-si RNA, the expression of ICAM-1 and VCAM-1 gene and protein expression induced by TNF alpha can inhibited by Slit2 in comparison with TNF alpha group( P(27)05.0);3.Compared with TNF alpha group, the gene and protein expression of ICAM-1 and VCAM-1 in endothelial cells was not significantly different after transfection of Robo4-si RNA( P(29)05.0); 4. This study detected the protein expression of IKBa, p-IKBa, NF-k B, p-NF-k B after added slit2. comparised with TNF alpha group,There was no significant difference at 1h and 4h( P(29)05.0);But P-IKBa and p-NF-k B expression was significantly reduced at 2h, the difference was statistically significant( P(27)05.0).Conclusion: 1.Slit2-robo4 pathway can inhibit the release of ICAM-1 and VCAM-1induced by TNF alpha in Human umbilical vein endothelial cells.2. Slit2 signaling pathway can reduce the phosphorylation of IKBa and NF-Kb, In TNF alpha –induced inflammation. |
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