半乳糖凝集素3对内皮细胞生长、迁移及炎症反应的影响

Effects of galectin-3 on growth,migration and inflammation of endothelial cells

目的:探讨半乳糖凝集素3(GAL-3)对人脐静脉内皮细胞(HUVECs)生长、迁移及炎症反应的影响及其作用机制。方法:体外培养HUVECs,给予GAL-3重组蛋白2 mg/L或GAL-3短发夹RNA(shRNA)慢病毒载体处理,实验分为正常对照组、GAL-3重组蛋白处理组、阴性对照组和GAL-3-shRNA组。通过实时荧光定量PCR检测GAL-3、单核细胞趋化蛋白1(MCP-1)、IL-6、基质金属蛋白酶9(MMP-9)和cyclin D1的mRNA水平;利用Western blot检测GAL-3、MCP-1和IL-6的蛋白表达;利用ELISA检测MCP-1和IL-6在培养基中的水平;通过CCK-8实验和划痕实验检测HUVECs的活力和迁移能力;利用Western blot检测信号分子热休克蛋白90(HSP90)、ERK1/2、p-ERK1/2、JNK和p-JNK的蛋白水平。结果:首先,给予GAL-3重组蛋白处理后,GAL-3、MCP-1和IL-6的mRNA及蛋白水平,MMP-9和cyclin D1的mRNA水平,MCP-1和IL-6在培养基的分泌水平均明显高于正常对照组;而GAL-3-shRNA感染细胞后,上述分子的表达水平均低于正常对照组和阴性对照组。其次,GAL-3重组蛋白处理后,细胞活力和迁移能力明显高于正常对照组;而GAL-3-shRNA感染后,细胞活力和迁移能力均低于正常对照组和阴性对照组。此外,GAL-3重组蛋白处理组中,p-ERK1/2和HSP90的蛋白水平高于正常对照组;而GAL-3-shRNA组中,p-ERK1/2和HSP90的蛋白水平均低于正常对照组和阴性对照组。p-JNK的蛋白水平在各组间比较,差异无统计学显著性。结论:GAL-3促进血管内皮细胞的生长、迁移及炎症反应的发生,其机制可能与HSP90-ERK1/2信号通路有关。

AIM： To explore the effects of galectin-3 （ GAL-3） on the viability, migration and inflammation of human umbilical vein endothelial cells （HUVECs） and the mechanisms. METHODS ： The HUVECs were cultured in vitro and treated with GAL-3 recombinant protein at 2 mg/L or GAL-3 short hairpin RNA （ shRNA） . The HUVECs were divided into normal group, recombinant GAL-3 group, shControl group and GAL-3-shRNA group. The mRNA expression of GAL- 3, monocyte chemotactic protein （MCP）-l, IL-6 , matrix metalloproteinase （MMP）-9 and cyclin D1 was detected by real-time quantitative PCR, and the protein expression of GAL-3, IL-6 and MCP-1 was detected by Western blot. The secretion levels of MCP-1 and IL-6 in the culture medium were measured by ELISA. The viability and the ability of migration of the HUVECs were examined by CCK-8 assay and wound healing assay. The protein levels of heat shock protein 90 （HSP90 ） , ERK1/2, p-ERKl/2, JNK and p-JNK were determined by Western blot. RESULTS： The expression of GAL-3, MCP-1 and IL-6 at mRNA and protein levels, the mRNA expression of MMP-9 and cyclin D1, and the secretion levels of MCP-1 and IL-6 in the culture medium were significantly higher than those in normal group （P 〈 0. 05 ） after the HUVECs were treated with GAL-3 recombinant protein. However, these molecules mentioned above in GAL-3-shRNA group were signifi-cantly lower than those in normal group and negative control group （P 〈0. 05）. Compared with normal group, the viability and migration ability of the HUVECs in recombinant GAL-3 group were significantly increased, but the viability and migra-tion ability of the HUVECs in GAL-3-shRNA group were lower than those in normal group and shControl group （P 〈 0. 05 ） . In addition, the protein levels of p-ERKl/2 and HSP90 in recombinant GAL-3 group were higher than those in nor-mal group （P 〈0. 05） , but those in GAL-3-shRNA group were lower than those in normal group and shControl group （P 〈 0. 05 ）. The protein level of p-JNK was not oviously changed among the 4 groups. CONCLUSION ： GAL-3 is involved in regulating the cell growth, migration and the release of inflammatory cytokines in vascular endothelial cells, which may be mediated by HSP90-ERK1/2 signaling pathway.