CCR2在MCP-1诱导人内皮细胞凋亡中的作用

发布时间：2018-03-15 13:06

  本文选题：单核细胞趋化蛋白-1　切入点：趋化因子受体2　出处：《遵义医学院》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:研究趋化因子受体2(CCR2)在单核细胞趋化蛋白-1(MCP-1)诱导人脐静脉内皮细胞株(CRL-1730)凋亡中的作用并初步探讨蛋白激酶C(PKC)信号传导通路对MCP-1诱导人脐静脉内皮细胞凋亡的影响。 方法:第一部分:培养并鉴定人脐静脉内皮细胞;用不同浓度单核细胞趋化蛋白-1(0.1ng/ml,1.0ng/ml,10ng/ml,100ng/ml)对其作用24h后,Western blot法检测CCR2蛋白表达量的变化;设计CCR2正义寡核苷酸链、反义寡核苷酸链及与人类基因非同源性的寡核苷酸序列,运用反义寡核苷酸技术以脂质体为载体将CCR2正义寡核苷酸链、反义寡核苷酸链及与人类基因非同源性的寡核苷酸序列转染人脐静脉内皮细胞,Western Blot险测转染后MCP-1诱导的CCR2蛋白的表达、流式细胞术检测细胞凋亡率。第二部分:加入CCR2的阻断剂RS102895后,用MCP-1作用细胞,激光扫描共聚焦显微镜原位检测细胞凋亡;Annexin V-FITC/PI双染流式细胞术观察加入不同剂量RS102895后MCP-1诱导的细胞凋亡率。第三部分:用不同浓度的PKC激动剂PMA与PKC抑制剂chelerythrine分别作用人脐静脉内皮细胞后,再用10ng/ml的MCP-1诱导,流式细胞术检测细胞凋亡率。 结果:培养的人脐静脉内皮细胞株经免疫荧光鉴定Ⅷ抗体阳性,证明为内皮细胞;MCP-1(0.1ng/ml,1.0ng/ml,10ng/ml,100ng/ml)能诱导人脐静脉内皮细胞中受体CCR2蛋白的表达,其效应随浓度的增加而增加;荧光倒置显微镜与流式细胞术确定0.4μg/μl FITC标记的CCR2反义寡核苷酸作用CRL-1730细胞48h为最佳转染效率;CCR2反义寡核苷酸转染后可明显抑制CCR2蛋白表达(P＜0.05),对MCP-1诱导的hUVECs凋亡有明显抑制作用。激光扫描共聚焦显微镜观察到加入CCR2阻断剂组比只加入MCP-1作用组细胞凋亡数明显减少;流式细胞技术检测加入CCR2阻断剂对MCP-1诱导的CRL-1730细胞凋亡有明显的抑制作用,并随RS102895剂量的增加细胞的凋亡率逐渐降低(P＜0.01,P＜0.01,P＜0.01).不同浓度的PMA(1x10~(-4)mmol/L、1x10~(-3)mmol/L)能增强MCP-1诱导的人脐静脉内皮细胞凋亡,凋亡效应随剂量增加而增强(P＜0.05,P＜0.01)。不同浓度的PKC抑制剂chelerythrine并非都能抑制MCP-1诱导的人脐静脉内皮细胞凋亡,在抑制剂浓度为1x10~(-6)mmol/L作用12h后凋亡效应最弱(P＜0.05)。 结论:CCR2介导了MCP-1诱导的CRL-1730细胞凋亡;MCP-1通过与CCR2结合诱导CRL-1730细胞凋亡,此结合位点与MCP-1发挥趋化作用时与CCR2结合的位点相同。PKC信号通路参与了MCP-1诱导的CRL-1730细胞凋亡。
[Abstract]:Aim: to investigate the role of CCR2 in the apoptosis of human umbilical vein endothelial cell line CRL-1730 induced by monocyte chemoattractant protein-1 (MCP-1), and to explore the effect of protein kinase CK-PKC signal transduction pathway on the apoptosis of human umbilical vein endothelial cells induced by MCP-1. Methods: the first part: culture and identification of human umbilical vein endothelial cells (HUVEC). The expression of CCR2 protein was detected by Western blot assay 24 hours after the treatment with different concentrations of monocyte chemoattractant protein -0.1 ng / ml ~ (-1) ng / ml ~ (-1) ng / ml ~ (-1) ng / ml ~ (10) ng / ml ~ (-1) / ml ~ (10) ng / ml). Antisense oligonucleotide chains and oligonucleotide sequences not homologous to human genes were used to carry the sense oligonucleotide chain of CCR2 with liposome as carrier by using antisense oligonucleotide technique. Transfection of antisense oligonucleotide chains and oligonucleotide sequences not homologous to human genes into human umbilical vein endothelial cells (HUVEC) was performed to detect the expression of CCR2 protein induced by MCP-1. Flow cytometry was used to detect the apoptosis rate. Part two: after adding RS102895, an antagonist of CCR2, the cells were treated with MCP-1. In situ detection of apoptosis by laser scanning confocal microscopy Annexin V-FITC / Pi double staining flow cytometry was used to observe the apoptotic rate induced by MCP-1 after adding different doses of RS102895. Part 3: using different concentrations of PKC agonist PMA and PKC inhibitor chelerythrine scores. After not acting on human umbilical vein endothelial cells, Then 10 ng / ml MCP-1 was used to induce apoptosis and flow cytometry was used to detect the apoptosis rate. Results: the cultured human umbilical vein endothelial cells (HUVECs) were confirmed to be positive for 鈪，

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