本文選題：單核細胞趨化蛋白-1　切入點：趨化因子受體2　出處：《遵義醫(yī)學(xué)院》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:研究趨化因子受體2(CCR2)在單核細胞趨化蛋白-1(MCP-1)誘導(dǎo)人臍靜脈內(nèi)皮細胞株(CRL-1730)凋亡中的作用并初步探討蛋白激酶C(PKC)信號傳導(dǎo)通路對MCP-1誘導(dǎo)人臍靜脈內(nèi)皮細胞凋亡的影響。 方法:第一部分:培養(yǎng)并鑒定人臍靜脈內(nèi)皮細胞;用不同濃度單核細胞趨化蛋白-1(0.1ng/ml,1.0ng/ml,10ng/ml,100ng/ml)對其作用24h后,Western blot法檢測CCR2蛋白表達量的變化;設(shè)計CCR2正義寡核苷酸鏈、反義寡核苷酸鏈及與人類基因非同源性的寡核苷酸序列,運用反義寡核苷酸技術(shù)以脂質(zhì)體為載體將CCR2正義寡核苷酸鏈、反義寡核苷酸鏈及與人類基因非同源性的寡核苷酸序列轉(zhuǎn)染人臍靜脈內(nèi)皮細胞,Western Blot險測轉(zhuǎn)染后MCP-1誘導(dǎo)的CCR2蛋白的表達、流式細胞術(shù)檢測細胞凋亡率。第二部分:加入CCR2的阻斷劑RS102895后,用MCP-1作用細胞,激光掃描共聚焦顯微鏡原位檢測細胞凋亡;Annexin V-FITC/PI雙染流式細胞術(shù)觀察加入不同劑量RS102895后MCP-1誘導(dǎo)的細胞凋亡率。第三部分:用不同濃度的PKC激動劑PMA與PKC抑制劑chelerythrine分別作用人臍靜脈內(nèi)皮細胞后,再用10ng/ml的MCP-1誘導(dǎo),流式細胞術(shù)檢測細胞凋亡率。 結(jié)果:培養(yǎng)的人臍靜脈內(nèi)皮細胞株經(jīng)免疫熒光鑒定Ⅷ抗體陽性,證明為內(nèi)皮細胞;MCP-1(0.1ng/ml,1.0ng/ml,10ng/ml,100ng/ml)能誘導(dǎo)人臍靜脈內(nèi)皮細胞中受體CCR2蛋白的表達,其效應(yīng)隨濃度的增加而增加;熒光倒置顯微鏡與流式細胞術(shù)確定0.4μg/μl FITC標記的CCR2反義寡核苷酸作用CRL-1730細胞48h為最佳轉(zhuǎn)染效率;CCR2反義寡核苷酸轉(zhuǎn)染后可明顯抑制CCR2蛋白表達(P＜0.05),對MCP-1誘導(dǎo)的hUVECs凋亡有明顯抑制作用。激光掃描共聚焦顯微鏡觀察到加入CCR2阻斷劑組比只加入MCP-1作用組細胞凋亡數(shù)明顯減少;流式細胞技術(shù)檢測加入CCR2阻斷劑對MCP-1誘導(dǎo)的CRL-1730細胞凋亡有明顯的抑制作用,并隨RS102895劑量的增加細胞的凋亡率逐漸降低(P＜0.01,P＜0.01,P＜0.01).不同濃度的PMA(1x10~(-4)mmol/L、1x10~(-3)mmol/L)能增強MCP-1誘導(dǎo)的人臍靜脈內(nèi)皮細胞凋亡,凋亡效應(yīng)隨劑量增加而增強(P＜0.05,P＜0.01)。不同濃度的PKC抑制劑chelerythrine并非都能抑制MCP-1誘導(dǎo)的人臍靜脈內(nèi)皮細胞凋亡,在抑制劑濃度為1x10~(-6)mmol/L作用12h后凋亡效應(yīng)最弱(P＜0.05)。 結(jié)論:CCR2介導(dǎo)了MCP-1誘導(dǎo)的CRL-1730細胞凋亡;MCP-1通過與CCR2結(jié)合誘導(dǎo)CRL-1730細胞凋亡,此結(jié)合位點與MCP-1發(fā)揮趨化作用時與CCR2結(jié)合的位點相同。PKC信號通路參與了MCP-1誘導(dǎo)的CRL-1730細胞凋亡。
[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，

本文編號：1616156