CAJ | 학술논문

目的观察胃生长素对晚期糖基化终产物(AGE)致人脐静脉内皮细胞(HUVEC)损伤的影响.方法体外分离及培养HUVEC细胞株,制备糖基化牛血清白蛋白(AGE-BSA).处理细胞时AGE-BSA终浓度为200 mg/L,胃生长素预作用时间为24h.将细胞分为无血清组、不同浓度胃生长素组(0.01,0.1,1和10 μmol/L)、AGE-BSA组和不同浓度胃生长素(0.01,0.1,1和10 μmol/L)+ AGE-BSA组,AGE-BSA处理48 h后采用噻唑蓝法检测细胞存活率;将细胞分无血清组、AGE-BSA组和不同浓度胃生长素(0.01,0.1,1和10 μmol/L)+ AGE-BSA组,AGE-BSA处理48 h后Annexin V/PI双染法检测细胞凋亡情况;将细胞分为无血清组、AGE-BSA组和胃生长素(1μmol/L)+AGE-BSA组,AGE-BSA处理24 h后,电镜观察细胞超微结构变化;将细胞分为无血清组、胃生长素组(1 μmol/L),AGE-BSA组和胃生长素(1μmol/L)+AGE-BSA组,检测细胞内活性氧簇(ROS)水平.结果不同浓度胃生长素组中,胃生长素剂量依赖性促进细胞增殖,0.1,1和10 μmol/L胃生长素组与无血清组细胞存活率比较差异均有统计学意义[(109±18)％、(113±15)％、(115±14)％比(100±11)％,P＜0.05];胃生长素预处理抑制AGE-BSA所致HUVEC存活率下降,呈剂量依赖性,1和10 μmol/L胃生长素+AGE-BSA组与AGE-BSA组比较差异有统计学意义[(87±18)％、(97±19)％比(45±10)％,P＜0.05].胃生长素剂量依赖性抑制AGE-BSA所致细胞凋亡,0.1,1和10 μmol/L胃生长素+AGE-BSA组细胞凋亡数量与AGE-BSA组比较差异有统计学意义[(14.7±9.5)％、(5.2±1.9)％、(4.5±2.1)％比(19.4±5.3)％,P＜0.05].电镜下见AGE-BSA组凋亡细胞数量增多,细胞核呈致密浓染,或呈碎块状,线粒体呈絮状变;胃生长素+ AGE-BSA组细胞凋亡明显减轻.无血清组、胃生长素组、AGE-BSA组和胃生长素+AGE-BSA组ROS水平分别为(1.00±0.10)、(0.75±0.07)、(1.50±0.17)、(1.10±0.14),其中胃生长素组低于无血清组(P＜0.05),AGE-BSA组明显高于无血清组(P＜0.01),胃生长素+ AGE-BSA组明显低于AGE-BSA组(P＜0.01).结论胃生长素能抑制AGE诱导脐静脉内皮细胞凋亡及ROS的增加. 目的觀察胃生長素對晚期糖基化終產物(AGE)緻人臍靜脈內皮細胞(HUVEC)損傷的影響.方法體外分離及培養HUVEC細胞株,製備糖基化牛血清白蛋白(AGE-BSA).處理細胞時AGE-BSA終濃度為200 mg/L,胃生長素預作用時間為24h.將細胞分為無血清組、不同濃度胃生長素組(0.01,0.1,1和10 μmol/L)、AGE-BSA組和不同濃度胃生長素(0.01,0.1,1和10 μmol/L)+ AGE-BSA組,AGE-BSA處理48 h後採用噻唑藍法檢測細胞存活率;將細胞分無血清組、AGE-BSA組和不同濃度胃生長素(0.01,0.1,1和10 μmol/L)+ AGE-BSA組,AGE-BSA處理48 h後Annexin V/PI雙染法檢測細胞凋亡情況;將細胞分為無血清組、AGE-BSA組和胃生長素(1μmol/L)+AGE-BSA組,AGE-BSA處理24 h後,電鏡觀察細胞超微結構變化;將細胞分為無血清組、胃生長素組(1 μmol/L),AGE-BSA組和胃生長素(1μmol/L)+AGE-BSA組,檢測細胞內活性氧簇(ROS)水平.結果不同濃度胃生長素組中,胃生長素劑量依賴性促進細胞增殖,0.1,1和10 μmol/L胃生長素組與無血清組細胞存活率比較差異均有統計學意義[(109±18)％、(113±15)％、(115±14)％比(100±11)％,P＜0.05];胃生長素預處理抑製AGE-BSA所緻HUVEC存活率下降,呈劑量依賴性,1和10 μmol/L胃生長素+AGE-BSA組與AGE-BSA組比較差異有統計學意義[(87±18)％、(97±19)％比(45±10)％,P＜0.05].胃生長素劑量依賴性抑製AGE-BSA所緻細胞凋亡,0.1,1和10 μmol/L胃生長素+AGE-BSA組細胞凋亡數量與AGE-BSA組比較差異有統計學意義[(14.7±9.5)％、(5.2±1.9)％、(4.5±2.1)％比(19.4±5.3)％,P＜0.05].電鏡下見AGE-BSA組凋亡細胞數量增多,細胞覈呈緻密濃染,或呈碎塊狀,線粒體呈絮狀變;胃生長素+ AGE-BSA組細胞凋亡明顯減輕.無血清組、胃生長素組、AGE-BSA組和胃生長素+AGE-BSA組ROS水平分彆為(1.00±0.10)、(0.75±0.07)、(1.50±0.17)、(1.10±0.14),其中胃生長素組低于無血清組(P＜0.05),AGE-BSA組明顯高于無血清組(P＜0.01),胃生長素+ AGE-BSA組明顯低于AGE-BSA組(P＜0.01).結論胃生長素能抑製AGE誘導臍靜脈內皮細胞凋亡及ROS的增加.
목적 관찰위생장소대만기당기화종산물(AGE)치인제정맥내피세포(HUVEC)손상적영향.방법 체외분리급배양HUVEC세포주,제비당기화우혈청백단백(AGE-BSA).처리세포시AGE-BSA종농도위200 mg/L,위생장소예작용시간위24h.장세포분위무혈청조、불동농도위생장소조(0.01,0.1,1화10 μmol/L)、AGE-BSA조화불동농도위생장소(0.01,0.1,1화10 μmol/L)+ AGE-BSA조,AGE-BSA처리48 h후채용새서람법검측세포존활솔;장세포분무혈청조、AGE-BSA조화불동농도위생장소(0.01,0.1,1화10 μmol/L)+ AGE-BSA조,AGE-BSA처리48 h후Annexin V/PI쌍염법검측세포조망정황;장세포분위무혈청조、AGE-BSA조화위생장소(1μmol/L)+AGE-BSA조,AGE-BSA처리24 h후,전경관찰세포초미결구변화;장세포분위무혈청조、위생장소조(1 μmol/L),AGE-BSA조화위생장소(1μmol/L)+AGE-BSA조,검측세포내활성양족(ROS)수평.결과 불동농도위생장소조중,위생장소제량의뢰성촉진세포증식,0.1,1화10 μmol/L위생장소조여무혈청조세포존활솔비교차이균유통계학의의[(109±18)％、(113±15)％、(115±14)％비(100±11)％,P＜0.05];위생장소예처리억제AGE-BSA소치HUVEC존활솔하강,정제량의뢰성,1화10 μmol/L위생장소+AGE-BSA조여AGE-BSA조비교차이유통계학의의[(87±18)％、(97±19)％비(45±10)％,P＜0.05].위생장소제량의뢰성억제AGE-BSA소치세포조망,0.1,1화10 μmol/L위생장소+AGE-BSA조세포조망수량여AGE-BSA조비교차이유통계학의의[(14.7±9.5)％、(5.2±1.9)％、(4.5±2.1)％비(19.4±5.3)％,P＜0.05].전경하견AGE-BSA조조망세포수량증다,세포핵정치밀농염,혹정쇄괴상,선립체정서상변;위생장소+ AGE-BSA조세포조망명현감경.무혈청조、위생장소조、AGE-BSA조화위생장소+AGE-BSA조ROS수평분별위(1.00±0.10)、(0.75±0.07)、(1.50±0.17)、(1.10±0.14),기중위생장소조저우무혈청조(P＜0.05),AGE-BSA조명현고우무혈청조(P＜0.01),위생장소+ AGE-BSA조명현저우AGE-BSA조(P＜0.01).결론 위생장소능억제AGE유도제정맥내피세포조망급ROS적증가.
Objective To explore the effect of ghrelin on the injury of human umbilical vein endothelial cells (HUVECs) induced by advanced glycation end products (AGE).Methods The HUVECs were isolated and cultured in vitro,exposed to AGE-bovine serum albumin (BSA) with a terminal concentration of 200 mg/L for 24 or 48h with or without pretreatment with ghrelin for 24 h.The cells were divided into serum free group,different concentrations of ghrelin group (0.01,0.1,1 and 10 μmol/L),different concentrations of ghrelin (0.01,0.1,1 and 10 μmol/L) + AGE-BSA group,and AGE-BSA group;48 h after treatment with AGE-BSA,the cell viabilities were measured by thiazolyl blue assays.The cells were divided into serum free group,different concentrations of ghrelin (0.01,0.1,1 and 10 μmol/L) + AGE-BSA group,and AGE-BSA group.48 h after treatment with AGE-BSA,the cell apoptosis was measured by Annexin V-FITC/PI binding assays.The cells were divided into serum free group,AGE-BSA group and ghrelin (1 μmol/L) + AGE-BSA group.24 h after treatment with AGE-BSA,the cell ultrastructure was observed by electron microscopy.The cells were divided into serum free group,ghrelin(1 μmol/L) group,AGE-BSA group and ghrelin(1 μmol/L) + AGE-BSA group;the level of reactive oxygen species (ROS) in cells was measured.Results In different concentrations of ghrelin group,ghrelin promoted cell proliferation in a dose-dependent manner,with remarkable differences of viability between 0.1,1,10 μmol/L ghrelin group and serum free group [(109 ± 18) ％,(113 ± 15) ％,(115 ± 14) ％ vs (100 ± 11) ％,all P ＜ 0.05].The reduction of cell viability induced by AGE-BSA was inhibited by ghrelin pretreatment in a dose-dependent manner,with significant differences of viability between 1,10 μmol/L ghrelin + AGE-BSA group and AGE-BSA group [(87 ± 18) ％,(97 ± 19) ％,vs (45 ± 10) ％,P ＜ 0.05].The quantity of apoptosis cells was significantly inhibited by 0.1,1,10 μmol/L ghrelin pretreatment in a dose-dependent manner [(14.7 ±9.5)％,(5.2±1.9)％,(4.5 ±2.1)％ vs (19.4±5.3)％,P＜0.05].Under electron microscopy,the number of apoptosis cells was increased with cell nucleus condensed or fragmented and with mitochondria flocculent in AGE-BSA group,which could be improved significantly in ghrelin + AGE-BSA group.The ROS level was lower in ghrelin group (0.75 ± 0.07) and was higher in AGE-BSA group (1.50 ± 0.17) compared with that in serum free group (1.00 ±0.10);the ROS level in ghrelin + AGE-BSA group (1.10 ±0.14) was significantly lower than that in AGE-BSA group (all P ＜0.01).Conclusion Ghrelin can inhibit the apoptosis and ROS induced by AGE in HUVECs.