五指山小型猪近交系主动脉内皮细胞体外培养模型的建立和TLR2介导炎症相关因子的表达

Establishment of Aortic Endothelial Cells Model in Vitro from Wuzhishan of Inbred Miniature Pig and Expression of In2ammatory Cytokines Mediated by TLR2

【目的】探讨五指山小型猪近交系主动脉内皮细胞体外分离培养和鉴定的方法,研究LPS对内皮细胞TLR2表达的影响及TLR2介导内皮细胞炎症因子的表达情况。【方法】以3—4月龄的五指山小型猪近交系为试验材料,取其胸腹主动脉,采用0.1%I型胶原酶消化分离主动脉内皮细胞。利用细胞吸收DiI-Ac-LDL试验和流式细胞仪检测CD31的特异表达两种方法进行内皮细胞的鉴定。1μg.mL-1LPS刺激内皮细胞0、6、8和12 h,实时荧光定量PCR检测TLR2和TLR4的表达量;1μg.mL-1LPS刺激内皮细胞12 h后,10μg.mL-1LTA孵育0、6和12 h,实时荧光定量PCR检测炎症因子IL-6、IL-8和黏附分子ICAM-1的表达量。【结果】分离得到的主动脉内皮细胞呈铺路石样整齐排列,且生长状态良好;细胞膜上表达CD31分子和Ac-LDL的受体,证实分离得到的细胞是血管内皮细胞。LPS刺激后细胞TLR2 mRNA水平的表达量明显升高(P<0.05),而TLR4 mRNA水平表达量无明显变化。添加LTA孵育后,细胞炎症因子IL-6、IL-8和黏附分子ICAM-1的mRNA水平表达量较对照明显升高(P<0.05)。【结论】成功建立了五指山小型猪近交系主动脉内皮细胞体外培养模型,并检测到TLR2在LPS刺激内皮细胞后表达量升高,能够介导细胞炎症相关因子的表达。

【Objective】 This study aimed to explore a protocol of isolation,culture and identification of Wuzhishan Inbred Miniature Pig aortic endothelial cells（EC）,and to study the regulation of Toll-like receptor 2（TLR2） expression by LPS in EC with the expression of in2 ammatory cytokines mediated by TLR2.【Method】Thoracoabdominal aortic were isolated from 3-4 month-old Wuzhishan Inbred Miniature Pig,disassociated with 0.1% collagenase I.Aortic EC were identified with the methods of uptake of fluorescence-labeled acetylated low density lipoproteins（DiI-Ac-LDL） and detecting of CD31 expression in the cells by flow cytometry.After stimulation of aortic derived EC with 1 μg·mL-1 lipopolysaccharide（LPS） for 0,6,8 and 12 h,the level of TLR2 and TLR4 mRNA was detected by real-time PCR.Furthermore,we detected the influence of lipoteichoic acid（LTA） on IL-6,IL-8 and ICAM-1 expression in aortic EC pretreated for 12 h with 1 μg·mL-1 LPS.【Result】 Obtained Wuzhishan Inbred Miniature Pig aortic EC displayed paving stone-like pattern with high vitality.The cultured cells expressed CD31 and Ac-LDL Receptor in membrane,demonstrating that they were indeed EC.Stimulation of aortic derived EC with LPS induced a strong increase in TLR2 mRNA but not in TLR4 mRNA.LTA potentiated the increase of the in2 ammatory cytokines IL-6,IL-8 and ICAM-1 expression in EC pretreated with LPS,but not in EC without LTA.【Conclusion】The present study successfully isolated and identified Wuzhishan Inbred Miniature Pig aortic EC in vitro culture system.LPS stimuli induced a high TLR2 expression in EC,which mediated the expression of inflammatory cyokines.