蛋白酶活化受體-2激活劑在誘導(dǎo)人臍靜脈內(nèi)皮細(xì)胞釋放炎癥因子中的作用
發(fā)布時(shí)間：2018-11-10 14:52
【摘要】： 位于血液與組織之間的血管內(nèi)皮細(xì)胞(以下簡(jiǎn)稱內(nèi)皮細(xì)胞)不僅是血管內(nèi)外物質(zhì)交換的屏障,而且被認(rèn)為是多種生理和病理生理過(guò)程的積極參與者。在炎癥過(guò)程中,內(nèi)皮細(xì)胞的主要作用表現(xiàn)為介導(dǎo)炎癥細(xì)胞(如嗜酸性粒細(xì)胞、單核細(xì)胞、淋巴細(xì)胞)從血液循環(huán)遷移至炎癥組織,釋放多種炎癥介質(zhì)(如IL-1、IL-8、INF-α、MCP-1)、調(diào)節(jié)血管的通透性和參與血管新生等。 PAR-2是蛋白酶活化受體(proteinase-activated receptors, PARs)家族的成員之一。在炎癥反應(yīng)中,PAR-2參與調(diào)控血管內(nèi)皮細(xì)胞的多種功能。如:PAR-2能夠調(diào)節(jié)內(nèi)皮細(xì)胞的屏障功能、增加炎癥細(xì)胞對(duì)血管內(nèi)皮的黏附、誘導(dǎo)視網(wǎng)膜血管的新生、刺激組織因子的表達(dá)。此外,TNF-α和內(nèi)毒素能夠放大PAR-2激活肽SLIGKV誘導(dǎo)的IL-6產(chǎn)生。盡管PAR-2涉及血管內(nèi)皮細(xì)胞的多種生理和病理生理作用,但是對(duì)胰蛋白酶(trypsin, EC 3.4.21.4)誘導(dǎo)內(nèi)皮細(xì)胞的炎癥因子釋放所知不多。內(nèi)皮細(xì)胞表達(dá)PAR-2和胰蛋白酶,表明內(nèi)皮細(xì)胞表面的PAR-2能夠直接與胰蛋白酶相互作用。為此,我們推測(cè)胰蛋白酶可能誘導(dǎo)內(nèi)皮細(xì)胞釋放炎癥因子,以調(diào)節(jié)其自身或周圍細(xì)胞的功能。 本研究用0.1% I型膠原酶分離人臍靜脈內(nèi)皮細(xì)胞(human umbilical vein endothelial cells, HUVECs)、培養(yǎng)體系為含內(nèi)皮細(xì)胞生長(zhǎng)添加物endothelial cell growth supplement (12.5μg/ml)和肝素(100μg/ml)的20% FBS-M199培養(yǎng)液,用CD105和CD31(platelet-endothelial cell adhesion molecule-1,PECAM-1)鑒定分離、培養(yǎng)HUVECs的純度,采用RT-PCR和流式細(xì)胞術(shù)檢測(cè)HUVECs的PAR-2 mRNA和蛋白質(zhì)水平表達(dá)。用胰蛋白酶和其他PAR-2激活劑誘導(dǎo)HUVECs表達(dá)炎癥因子�？贵w芯片和ELISA檢測(cè)誘導(dǎo)的HUVECs培養(yǎng)上清中的炎癥細(xì)胞因子水平。 結(jié)果表明,本研究分離、培養(yǎng)的HUVECs純度約95%。HUVECs在mRNA和蛋白質(zhì)水平表達(dá)PAR-2�？贵w芯片檢測(cè)顯示1μg/ml胰蛋白酶誘導(dǎo)釋放32種炎癥因子,而100μM SLIGKV-NH_2誘導(dǎo)釋放16種炎癥因子。在SLIGKV-NH_2誘導(dǎo)釋放的16種炎癥因子中,15種炎癥因子也被胰蛋白酶誘導(dǎo)產(chǎn)生。在芯片結(jié)果中,胰蛋白酶和SLIGKV-NH_2均顯著誘導(dǎo)IL-1α、IL-8、IL-10和IL-12釋放,因此這些細(xì)胞因子被進(jìn)一步研究。誘導(dǎo)細(xì)胞16 h后,胰蛋白酶能夠顯著刺激HUVECs釋放IL-1α、IL-10、IL-12和IL-8 (P均 0.05)。這些細(xì)胞因子的升高水平分別約為HUVECs基礎(chǔ)釋放水平的4.8、4.3、4.1和1.8倍。10μM和100μM SLIGKV-NH_2及10μM tc-LIGRLO-NH_2也能夠誘導(dǎo)IL-8水平升高(P均 0.05)。胰蛋白酶抑制劑(大豆蛋白酶抑制劑,α1-抗胰蛋白酶)或PAR-2抑制肽FSLLRY-NH_2均能夠顯著抑制胰蛋白酶誘導(dǎo)的IL-1α, IL-10、IL-12和IL-8釋放(P均 0.05)。相關(guān)性分析表明,SLIGKV-NH_2和胰蛋白酶誘導(dǎo)培養(yǎng)HUVECs 8 h和16 h后,IL-8水平之間的相關(guān)性顯著(P 0.05)。 總之,胰蛋白酶能夠誘導(dǎo)內(nèi)皮細(xì)胞釋放多種炎癥細(xì)胞因子,胰蛋白酶的作用很可能通過(guò)激活PAR-2途徑。這表明胰蛋白酶和內(nèi)皮細(xì)胞PAR-2相關(guān)機(jī)制參與人類炎癥性疾病的發(fā)生、發(fā)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- 偽, 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- 偽 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 偽, 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 偽, 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, 偽 1-antitrypsin) or PAR-2 inhibitory peptide FSLLRY-NH_2 could significantly inhibit the release of IL-1 偽, 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.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363
本文編號(hào)：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- 偽, 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- 偽 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 偽, 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 偽, 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, 偽 1-antitrypsin) or PAR-2 inhibitory peptide FSLLRY-NH_2 could significantly inhibit the release of IL-1 偽, 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.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 王海燕,何韶衡,付意玲,方澤曼;人肺上皮細(xì)胞表達(dá)蛋白酶激活受體[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2005年17期
2 牛青霞,何韶衡;蛋白酶活化受體-2的研究進(jìn)展[J];生理科學(xué)進(jìn)展;2003年04期
3 張均克,鄧耀祖;血管新生研究進(jìn)展[J];江漢大學(xué)學(xué)報(bào)(自然科學(xué)版);2005年02期
，本文編號(hào)：2322766
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2322766.html
最近更新
教材專著
