CAJ | 학술논문

目的评价氢气对脂多糖(LPS)诱导人脐静脉内皮细胞损伤的影响.方法人脐静脉血管内皮细胞培养于含10％胎牛血清的DMEM培养基,以2×106个/ml的密度接种于6孔培养板,2ml/孔.采用随机数字表法,将细胞分为4组(n=35):对照组(C组)、氢气组(H2组)、LPS组和L胳+H2组.C组和LPS组用正常培养基培养,H2组和LPS+ H2组用含饱和氢气培养基培养.LPS组和LPS+H2组分别加入LPS1 μg/ml孵育,C组和H2组加入等容量生理盐水.分别于孵育6、12和24h时,取5孔细胞,采用瑞姬氏染色观察细胞损伤情况;分别于孵育6、12和24h时,取5孔细胞,采用Western blot法测定血管内皮细胞钙粘连蛋白和β-连环蛋白的表达.于孵育24h时,取5孔细胞,采用免疫荧光法测定血管内皮细胞钙粘连蛋白和p连环蛋白的表达.结果 LPS组内皮细胞出现病理学损伤;与LPS组比较,LPS+ H2组内皮细胞病理学损伤减轻.与C组比较,LPS组和LPS+ H2组血管内皮细胞钙粘连蛋白表达下调(P＜0.05),β-连环蛋白表达差异无统计学意义(P＞0.05);与LPS组比较,LPS+ H2组血管内皮细胞钙粘连蛋白表达上调(P＜0.05),p连环蛋白表达差异无统计学意义(P＞0.05).结论氢气可有效减轻LPS诱导人脐静脉内皮细胞损伤,与抑制血管内皮细胞钙粘连蛋白表达下调有关.
목적 평개경기대지다당(LPS)유도인제정맥내피세포손상적영향.방법 인제정맥혈관내피세포배양우함10％태우혈청적DMEM배양기,이2×106개/ml적밀도접충우6공배양판,2ml/공.채용수궤수자표법,장세포분위4조(n=35):대조조(C조)、경기조(H2조)、LPS조화L각+H2조.C조화LPS조용정상배양기배양,H2조화LPS+ H2조용함포화경기배양기배양.LPS조화LPS+H2조분별가입LPS1 μg/ml부육,C조화H2조가입등용량생리염수.분별우부육6、12화24h시,취5공세포,채용서희씨염색관찰세포손상정황;분별우부육6、12화24h시,취5공세포,채용Western blot법측정혈관내피세포개점련단백화β-련배단백적표체.우부육24h시,취5공세포,채용면역형광법측정혈관내피세포개점련단백화p련배단백적표체.결과 LPS조내피세포출현병이학손상;여LPS조비교,LPS+ H2조내피세포병이학손상감경.여C조비교,LPS조화LPS+ H2조혈관내피세포개점련단백표체하조(P＜0.05),β-련배단백표체차이무통계학의의(P＞0.05);여LPS조비교,LPS+ H2조혈관내피세포개점련단백표체상조(P＜0.05),p련배단백표체차이무통계학의의(P＞0.05).결론 경기가유효감경LPS유도인제정맥내피세포손상,여억제혈관내피세포개점련단백표체하조유관.
Objective To evaluate the effects of hydrogen gas (H2) on lipopolysaccharide (LPS)-induced damage to human umbilical vein endothelial cells (HUVECs) in vitro.Methods HUVECs were cultured in DMEM culture medium containing 10％ fetal bovine serum.The cells were seeded in 6-well plates (2 ml/hole) at a density of 2 × 106 cells/ml and randomly divided into 4 groups (n =35 each) using a random number table:control group (group C),group H2,LPS group and LPS + H2 group.The cells were cultured in the plain culture medium in C and LPS groups or in hydrogen-saturated culture medium in H2 and LPS + H2 groups.In addition,LPS 1 μg/ml was added simultaneously in LPS and LPS + H2 groups,and the equal volume of normal saline was added instead in C and H2 groups.The cells were incubated for 24 h.At 6,12 and 24 h of incubation,the cells of 5 wells were chosen and stained with Wright-Giemsa to observe the destruction of HUVEC barrier.At 6,12 and 24 h of incubation,the cells of 5 wells were chosen to detect the expression of VE-cadherin and β-catenin using Western blot.At 24 h of incubation,the cells of 5 wells were chosen to detect the expression of VE-cadherin and βcatenin using immunofluorescence.Results Pathological changes of endothelial cells were observed in LPS group.The pathological changes of the cells were lighter in LPS + H2 group than in LPS group.Compared with group C,VE-cadherin expression was significantly down-regulated (P ＜ 0.05),while no significant change was found in β-catenin expression in LPS and LPS + H2 groups (P ＞ 0.05).Compared with group LPS,VE-cadherin expression was significantly up-regulated (P ＜ 0.05),while no significant change was found in ~catenin expression in group LPS + H2 (P ＞ 0.05).Conclusion H2 can effectively reduce LPS-induced damage to HUVECs through inhibiting down-regulation of VE-cadherin expression.