中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2014年
5期
370-376
,共7页
彭隽%贾汝汉%党建中%兰天飙%任星峰
彭雋%賈汝漢%黨建中%蘭天飆%任星峰
팽준%가여한%당건중%란천표%임성봉
抗原,CD36%肾小球系膜细胞%细胞凋亡%氧化性应激
抗原,CD36%腎小毬繫膜細胞%細胞凋亡%氧化性應激
항원,CD36%신소구계막세포%세포조망%양화성응격
Antigens,CD36%Mesangial cells%Apoptosis%Oxidative stress
目的 观察高糖对大鼠肾小球系膜细胞CD36抗原表达的影响,探讨其与系膜细胞氧化应激和凋亡的关系.方法 大鼠肾小球系膜细胞分为对照组(5.6 mmol/L葡萄糖培养基),甘露醇组(24.2 mmol/L甘露醇+5.6 mmol/L葡萄糖培养基),高糖组(30 mmol/L葡萄糖MEM培养基)、anti-CD36受体封闭组(anti-CD36+30 mmol/L葡萄糖MEM培养基).采用激光共聚焦显微镜检测细胞内活性氧(Reactive Oxygen Species,ROS)水平.收集细胞培养上清液,测定丙二醛(MDA)、8-羟基脱氧尿苷(8-OHDG)及谷胱甘肽过氧化酶(GSH-PX)含量.流式AnnexinV-FITC/PI双染法检测细胞凋亡.用RT-PCR和Western印迹法检测肾小球系膜细胞CD36、促凋亡基因Bax、抑凋亡基因Bel-2的mRNA及蛋白的表达.结果 大鼠肾小球系膜细胞表达CD36抗原,高糖培养24 h时CD36蛋白表达水平最高.对照组与甘露醇组的ROS水平、细胞上清液中MDA、8-OHDG、GSH-PX水平、早晚期凋亡率、CD36抗原、Bax及Bel-2的mRNA及蛋白表达差异无统计学意义(均P＞ 0.05).与对照组相比,高糖组ROS水平、细胞上清液中MDA、8-OHDG水平、早晚期凋亡率、CD36和Bax的mRNA及蛋白表达水平增高,而细胞上清液中GSH-PX水平、Bcl-2的mRNA及蛋白表达降低,差异均有统计学意义(均P＜0.05).与高糖组相比,anti-CD36受体封闭组CD36的mRNA及蛋白表达差异无统计学意义(P＞0.05),ROS水平、细胞上清液中MDA、8-OHDG水平、早晚期凋亡率、Bax mRNA及蛋白表达减少,而细胞上清液GSH-PX水平、Bcl-2 mRNA及蛋白表达增高,差异有统计学意义(均P＜0.05).相关分析证实细胞内ROS水平与细胞凋亡率及CD36、Bax蛋白表达均呈正相关,与Bcl-2蛋白表达呈负相关.结论 肾小球系膜细胞表达CD36抗原,且高糖可以诱导CD36抗原的表达,其表达水平与细胞内氧化应激水平成正相关,CD36抗原参与高糖诱导的肾小球系膜细胞凋亡,机制可能是通过高糖环境中增加细胞内氧化应激水平来介导的.
目的 觀察高糖對大鼠腎小毬繫膜細胞CD36抗原錶達的影響,探討其與繫膜細胞氧化應激和凋亡的關繫.方法 大鼠腎小毬繫膜細胞分為對照組(5.6 mmol/L葡萄糖培養基),甘露醇組(24.2 mmol/L甘露醇+5.6 mmol/L葡萄糖培養基),高糖組(30 mmol/L葡萄糖MEM培養基)、anti-CD36受體封閉組(anti-CD36+30 mmol/L葡萄糖MEM培養基).採用激光共聚焦顯微鏡檢測細胞內活性氧(Reactive Oxygen Species,ROS)水平.收集細胞培養上清液,測定丙二醛(MDA)、8-羥基脫氧尿苷(8-OHDG)及穀胱甘肽過氧化酶(GSH-PX)含量.流式AnnexinV-FITC/PI雙染法檢測細胞凋亡.用RT-PCR和Western印跡法檢測腎小毬繫膜細胞CD36、促凋亡基因Bax、抑凋亡基因Bel-2的mRNA及蛋白的錶達.結果 大鼠腎小毬繫膜細胞錶達CD36抗原,高糖培養24 h時CD36蛋白錶達水平最高.對照組與甘露醇組的ROS水平、細胞上清液中MDA、8-OHDG、GSH-PX水平、早晚期凋亡率、CD36抗原、Bax及Bel-2的mRNA及蛋白錶達差異無統計學意義(均P＞ 0.05).與對照組相比,高糖組ROS水平、細胞上清液中MDA、8-OHDG水平、早晚期凋亡率、CD36和Bax的mRNA及蛋白錶達水平增高,而細胞上清液中GSH-PX水平、Bcl-2的mRNA及蛋白錶達降低,差異均有統計學意義(均P＜0.05).與高糖組相比,anti-CD36受體封閉組CD36的mRNA及蛋白錶達差異無統計學意義(P＞0.05),ROS水平、細胞上清液中MDA、8-OHDG水平、早晚期凋亡率、Bax mRNA及蛋白錶達減少,而細胞上清液GSH-PX水平、Bcl-2 mRNA及蛋白錶達增高,差異有統計學意義(均P＜0.05).相關分析證實細胞內ROS水平與細胞凋亡率及CD36、Bax蛋白錶達均呈正相關,與Bcl-2蛋白錶達呈負相關.結論 腎小毬繫膜細胞錶達CD36抗原,且高糖可以誘導CD36抗原的錶達,其錶達水平與細胞內氧化應激水平成正相關,CD36抗原參與高糖誘導的腎小毬繫膜細胞凋亡,機製可能是通過高糖環境中增加細胞內氧化應激水平來介導的.
목적 관찰고당대대서신소구계막세포CD36항원표체적영향,탐토기여계막세포양화응격화조망적관계.방법 대서신소구계막세포분위대조조(5.6 mmol/L포도당배양기),감로순조(24.2 mmol/L감로순+5.6 mmol/L포도당배양기),고당조(30 mmol/L포도당MEM배양기)、anti-CD36수체봉폐조(anti-CD36+30 mmol/L포도당MEM배양기).채용격광공취초현미경검측세포내활성양(Reactive Oxygen Species,ROS)수평.수집세포배양상청액,측정병이철(MDA)、8-간기탈양뇨감(8-OHDG)급곡광감태과양화매(GSH-PX)함량.류식AnnexinV-FITC/PI쌍염법검측세포조망.용RT-PCR화Western인적법검측신소구계막세포CD36、촉조망기인Bax、억조망기인Bel-2적mRNA급단백적표체.결과 대서신소구계막세포표체CD36항원,고당배양24 h시CD36단백표체수평최고.대조조여감로순조적ROS수평、세포상청액중MDA、8-OHDG、GSH-PX수평、조만기조망솔、CD36항원、Bax급Bel-2적mRNA급단백표체차이무통계학의의(균P＞ 0.05).여대조조상비,고당조ROS수평、세포상청액중MDA、8-OHDG수평、조만기조망솔、CD36화Bax적mRNA급단백표체수평증고,이세포상청액중GSH-PX수평、Bcl-2적mRNA급단백표체강저,차이균유통계학의의(균P＜0.05).여고당조상비,anti-CD36수체봉폐조CD36적mRNA급단백표체차이무통계학의의(P＞0.05),ROS수평、세포상청액중MDA、8-OHDG수평、조만기조망솔、Bax mRNA급단백표체감소,이세포상청액GSH-PX수평、Bcl-2 mRNA급단백표체증고,차이유통계학의의(균P＜0.05).상관분석증실세포내ROS수평여세포조망솔급CD36、Bax단백표체균정정상관,여Bcl-2단백표체정부상관.결론 신소구계막세포표체CD36항원,차고당가이유도CD36항원적표체,기표체수평여세포내양화응격수평성정상관,CD36항원삼여고당유도적신소구계막세포조망,궤제가능시통과고당배경중증가세포내양화응격수평래개도적.
Objective To investigate the effects and underlying mechanism of the scavenger receptor CD36 in high glucose-induced rat glomerular mesangial cells apoptosis.Methods The mesangial cells of rats were divided into 4 groups:control group (5.6 mmol/L glucose),mannitol group (24.2 mmol/L mannitol+5.6 mmo]/L glucose),high glucose group (30 mmol/L glucose),CD36 monoantibody group (30 mmol/L glucose+CD36 mono-antibody).The intracellular ROS level was detected by confocal microscopy with fluorescent probe CM-H2DCFDA.MDA,GSH-PX,8-OHDGA in cell supernatant were detected.Apoptosis was determined by flow cytometry followed by Annexin V-FITC/PI double stains.The expression of CD36,Bax and Bcl-2 were detected by RT-PCR and Western blotting.Results The expression of CD36 was detected in glomerular mesangial cells.The highest level was found in high glucose group in 24 hours.There was no significant difference found between control group and mannitol group with respect to intracellular ROS generation,MDA,8-OHDG,GSH-PX level,apoptosis rate,expression of CD36,Bax and Bcl-2 (all P ＞ 0.05).There was no significant difference in the expression of CD36 between CD36 mono-antibody group and high glucose group (P ＞ 0.05).Compared to control group,the intracellular ROS generation,MDA and 8-OHDG levels,apoptosis rate,the expression of CD36 and Bax were significantly increased,the GSH-PX level and the expression of Bcl-2 were significantly lower in high glucose group (all P ＜ 0.05).Compared to the high glucose group,the intracellular ROS generation,MDA and 8-OHDG levels,apoptosis rate,the expression of Bax were suppressed but the GSH-PX level and the expression of Bcl-2 increased in CD36 mono-antibody group (all P ＜ 0.05).The intracellular ROS level was positively correlated with apoptosis rate,protein expression of CD36 and Bax gene,was negatively correlated with Bcl-2 protein expression.Conclusions CD36 was involved in the high glucose induced apoptosis of mesangial cells which was potentially mediated by an increased level of oxidative stress.
