蛋白酶活化受体-2激活剂在诱导人脐静脉内皮细胞释放炎症因子中的作用
发布时间：2018-11-10 14:52
【摘要】： 位于血液与组织之间的血管内皮细胞(以下简称内皮细胞)不仅是血管内外物质交换的屏障,而且被认为是多种生理和病理生理过程的积极参与者。在炎症过程中,内皮细胞的主要作用表现为介导炎症细胞(如嗜酸性粒细胞、单核细胞、淋巴细胞)从血液循环迁移至炎症组织,释放多种炎症介质(如IL-1、IL-8、INF-α、MCP-1)、调节血管的通透性和参与血管新生等。 PAR-2是蛋白酶活化受体(proteinase-activated receptors, PARs)家族的成员之一。在炎症反应中,PAR-2参与调控血管内皮细胞的多种功能。如:PAR-2能够调节内皮细胞的屏障功能、增加炎症细胞对血管内皮的黏附、诱导视网膜血管的新生、刺激组织因子的表达。此外,TNF-α和内毒素能够放大PAR-2激活肽SLIGKV诱导的IL-6产生。尽管PAR-2涉及血管内皮细胞的多种生理和病理生理作用,但是对胰蛋白酶(trypsin, EC 3.4.21.4)诱导内皮细胞的炎症因子释放所知不多。内皮细胞表达PAR-2和胰蛋白酶,表明内皮细胞表面的PAR-2能够直接与胰蛋白酶相互作用。为此,我们推测胰蛋白酶可能诱导内皮细胞释放炎症因子,以调节其自身或周围细胞的功能。 本研究用0.1% I型胶原酶分离人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)、培养体系为含内皮细胞生长添加物endothelial cell growth supplement (12.5μg/ml)和肝素(100μg/ml)的20% FBS-M199培养液,用CD105和CD31(platelet-endothelial cell adhesion molecule-1,PECAM-1)鉴定分离、培养HUVECs的纯度,采用RT-PCR和流式细胞术检测HUVECs的PAR-2 mRNA和蛋白质水平表达。用胰蛋白酶和其他PAR-2激活剂诱导HUVECs表达炎症因子。抗体芯片和ELISA检测诱导的HUVECs培养上清中的炎症细胞因子水平。 结果表明,本研究分离、培养的HUVECs纯度约95%。HUVECs在mRNA和蛋白质水平表达PAR-2。抗体芯片检测显示1μg/ml胰蛋白酶诱导释放32种炎症因子,而100μM SLIGKV-NH_2诱导释放16种炎症因子。在SLIGKV-NH_2诱导释放的16种炎症因子中,15种炎症因子也被胰蛋白酶诱导产生。在芯片结果中,胰蛋白酶和SLIGKV-NH_2均显著诱导IL-1α、IL-8、IL-10和IL-12释放,因此这些细胞因子被进一步研究。诱导细胞16 h后,胰蛋白酶能够显著刺激HUVECs释放IL-1α、IL-10、IL-12和IL-8 (P均 0.05)。这些细胞因子的升高水平分别约为HUVECs基础释放水平的4.8、4.3、4.1和1.8倍。10μM和100μM SLIGKV-NH_2及10μM tc-LIGRLO-NH_2也能够诱导IL-8水平升高(P均 0.05)。胰蛋白酶抑制剂(大豆蛋白酶抑制剂,α1-抗胰蛋白酶)或PAR-2抑制肽FSLLRY-NH_2均能够显著抑制胰蛋白酶诱导的IL-1α, IL-10、IL-12和IL-8释放(P均 0.05)。相关性分析表明,SLIGKV-NH_2和胰蛋白酶诱导培养HUVECs 8 h和16 h后,IL-8水平之间的相关性显著(P 0.05)。 总之,胰蛋白酶能够诱导内皮细胞释放多种炎症细胞因子,胰蛋白酶的作用很可能通过激活PAR-2途径。这表明胰蛋白酶和内皮细胞PAR-2相关机制参与人类炎症性疾病的发生、发展。
[Abstract]:Vascular endothelial cells (VECs) located between blood and tissue are not only the barrier of material exchange in and out of blood vessels, but also considered to be active participants in many physiological and pathophysiological processes. In the process of inflammation, endothelial cells play a major role in mediating the migration of inflammatory cells (such as eosinophils, monocytes, lymphocytes) from blood circulation to inflammatory tissues, releasing a variety of inflammatory mediators (such as IL-1,IL-8,). INF- 伪, MCP-1, regulate vascular permeability and participate in angiogenesis. PAR-2 is a member of the protease activated receptor (proteinase-activated receptors, PARs) family. In inflammatory response, PAR-2 is involved in the regulation of many functions of vascular endothelial cells. For example, PAR-2 can regulate the barrier function of endothelial cells, increase the adhesion of inflammatory cells to vascular endothelium, induce retinal angiogenesis and stimulate the expression of tissue factor. In addition, TNF- 伪 and endotoxin can amplify IL-6 production induced by PAR-2 activated peptide SLIGKV. Although PAR-2 involves many physiological and pathophysiological effects of vascular endothelial cells, little is known about the release of inflammatory factors induced by trypsin (trypsin, EC 3.4.21.4. The expression of PAR-2 and trypsin in endothelial cells showed that PAR-2 on endothelial cells could interact with trypsin directly. Therefore, we speculate that trypsin may induce endothelial cells to release inflammatory factors to regulate their own or peripheral cell function. In this study, the (human umbilical vein endothelial cells, HUVECs), culture system of human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVECs),) was isolated by 0.1% collagenase from human umbilical vein. It was used as 20% FBS-M199 medium containing endothelial cell growth supplement (12.5 渭 g/ml and heparin (100 渭 g/ml). The purity of HUVECs was identified by CD105 and CD31 (platelet-endothelial cell adhesion molecule-1,PECAM-1). The expression of PAR-2 mRNA and protein in HUVECs was detected by RT-PCR and flow cytometry. The expression of inflammatory factors in HUVECs was induced by trypsin and other PAR-2 activators. The levels of inflammatory cytokines in the supernatant of HUVECs culture induced by antibody chip and ELISA were detected. The results showed that the purified HUVECs expressed PAR-2. at the level of mRNA and protein. Antibody chip analysis showed that 1 渭 g/ml trypsin induced the release of 32 inflammatory factors and 100 渭 M SLIGKV-NH_2 induced the release of 16 inflammatory factors. Of the 16 inflammatory factors induced by SLIGKV-NH_2, 15 were also induced by trypsin. In the microarray results, both trypsin and SLIGKV-NH_2 significantly induced the release of IL-1 伪, IL-8,IL-10 and IL-12, so these cytokines were further studied. After induction for 16 h, trypsin significantly stimulated the release of IL-1 伪, IL-10,IL-12 and IL-8 by HUVECs (all P 0.05). The elevated levels of these cytokines were 4.34.1 and 1.8 times of the basal release level of HUVECs, respectively. 10 渭 M, 100 渭 M SLIGKV-NH_2 and 10 渭 M tc-LIGRLO-NH_2 could also induce the increase of IL-8 level (P 0.05). Trypsin inhibitor (daidzein inhibitor, 伪 1-antitrypsin) or PAR-2 inhibitory peptide FSLLRY-NH_2 could significantly inhibit the release of IL-1 伪, IL-10,IL-12 and IL-8 induced by trypsin. The correlation analysis showed that there was a significant correlation between IL-8 level in SLIGKV-NH_2 and trypsin induced HUVECs cultured for 8 h and 16 h (P 0.05). In conclusion, trypsin can induce endothelial cells to release a variety of inflammatory cytokines. Trypsin probably activates the PAR-2 pathway. This suggests that the mechanisms associated with trypsin and endothelial cell PAR-2 are involved in the occurrence and development of human inflammatory diseases.
【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363
本文编号：2322766
[Abstract]:Vascular endothelial cells (VECs) located between blood and tissue are not only the barrier of material exchange in and out of blood vessels, but also considered to be active participants in many physiological and pathophysiological processes. In the process of inflammation, endothelial cells play a major role in mediating the migration of inflammatory cells (such as eosinophils, monocytes, lymphocytes) from blood circulation to inflammatory tissues, releasing a variety of inflammatory mediators (such as IL-1,IL-8,). INF- 伪, MCP-1, regulate vascular permeability and participate in angiogenesis. PAR-2 is a member of the protease activated receptor (proteinase-activated receptors, PARs) family. In inflammatory response, PAR-2 is involved in the regulation of many functions of vascular endothelial cells. For example, PAR-2 can regulate the barrier function of endothelial cells, increase the adhesion of inflammatory cells to vascular endothelium, induce retinal angiogenesis and stimulate the expression of tissue factor. In addition, TNF- 伪 and endotoxin can amplify IL-6 production induced by PAR-2 activated peptide SLIGKV. Although PAR-2 involves many physiological and pathophysiological effects of vascular endothelial cells, little is known about the release of inflammatory factors induced by trypsin (trypsin, EC 3.4.21.4. The expression of PAR-2 and trypsin in endothelial cells showed that PAR-2 on endothelial cells could interact with trypsin directly. Therefore, we speculate that trypsin may induce endothelial cells to release inflammatory factors to regulate their own or peripheral cell function. In this study, the (human umbilical vein endothelial cells, HUVECs), culture system of human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVECs),) was isolated by 0.1% collagenase from human umbilical vein. It was used as 20% FBS-M199 medium containing endothelial cell growth supplement (12.5 渭 g/ml and heparin (100 渭 g/ml). The purity of HUVECs was identified by CD105 and CD31 (platelet-endothelial cell adhesion molecule-1,PECAM-1). The expression of PAR-2 mRNA and protein in HUVECs was detected by RT-PCR and flow cytometry. The expression of inflammatory factors in HUVECs was induced by trypsin and other PAR-2 activators. The levels of inflammatory cytokines in the supernatant of HUVECs culture induced by antibody chip and ELISA were detected. The results showed that the purified HUVECs expressed PAR-2. at the level of mRNA and protein. Antibody chip analysis showed that 1 渭 g/ml trypsin induced the release of 32 inflammatory factors and 100 渭 M SLIGKV-NH_2 induced the release of 16 inflammatory factors. Of the 16 inflammatory factors induced by SLIGKV-NH_2, 15 were also induced by trypsin. In the microarray results, both trypsin and SLIGKV-NH_2 significantly induced the release of IL-1 伪, IL-8,IL-10 and IL-12, so these cytokines were further studied. After induction for 16 h, trypsin significantly stimulated the release of IL-1 伪, IL-10,IL-12 and IL-8 by HUVECs (all P 0.05). The elevated levels of these cytokines were 4.34.1 and 1.8 times of the basal release level of HUVECs, respectively. 10 渭 M, 100 渭 M SLIGKV-NH_2 and 10 渭 M tc-LIGRLO-NH_2 could also induce the increase of IL-8 level (P 0.05). Trypsin inhibitor (daidzein inhibitor, 伪 1-antitrypsin) or PAR-2 inhibitory peptide FSLLRY-NH_2 could significantly inhibit the release of IL-1 伪, IL-10,IL-12 and IL-8 induced by trypsin. The correlation analysis showed that there was a significant correlation between IL-8 level in SLIGKV-NH_2 and trypsin induced HUVECs cultured for 8 h and 16 h (P 0.05). In conclusion, trypsin can induce endothelial cells to release a variety of inflammatory cytokines. Trypsin probably activates the PAR-2 pathway. This suggests that the mechanisms associated with trypsin and endothelial cell PAR-2 are involved in the occurrence and development of human inflammatory diseases.
【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363
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3 张均克,邓耀祖;血管新生研究进展[J];江汉大学学报(自然科学版);2005年02期
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