CAJ | 학술논문

目的观察氧化低密度脂蛋白(oxidized LDL,Ox-LDL)刺激对体外培养的小鼠肾小球足细胞清道夫受体A (scavenger receptor A,SR-A)表达的影响,并初步探讨其可能的分子机制.方法体外培养小鼠条件永生性足细胞,以不同浓度Ox-LDL刺激足细胞24 h及以20 mg/L Ox-LDL干预不同时间,酶法检测细胞内胆固醇含量,采用实时荧光定量PCR、Western印迹法检测正常对照组、Ox-LDL刺激组SR-A、nephrin、PTEN的mRNA和蛋白表达水平;DiI标记的氧化低密度脂蛋白(DiI-Ox-LDL)与足细胞共孵育,免疫荧光观察足细胞对脂质的摄取情况.进一步采用PTEN抑制剂bpv(hOpic) [dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate(Ⅴ)]、PTEN siRNA以及PTEN腺病毒(adPTEN)双向调控PTEN的表达,观察SR-A、nephrin的表达、足细胞对脂质的摄取情况及细胞内胆固醇含量的变化.结果小鼠足细胞表达SR-A受体并介导脂质摄取;与对照组相比,Ox-LDL刺激上调足细胞SR-A表达的同时伴有PTEN、nephrin表达下降(均P＜0.05),且与Ox-LDL的浓度及作用时间相关;增加PTEN表达能抑制Ox-LDL诱导的SR-A表达水平上调和脂质摄取(均P＜0.05),降低细胞内胆固醇含量(P＜0.05),上调裂孑隔膜蛋白nephrin的表达(均P＜0.05);与之相反,抑制PTEN表达能进一步上调Ox-LDL诱导的SR-A表达,促进脂质摄取,增加细胞内胆固醇含量(均P＜0.05),并进一步降低nephrin的表达(均P＜0.05).结论 Ox-LDL可能通过PTEN调控SR-A表达进而调控脂质摄取,导致足细胞损伤.
목적 관찰양화저밀도지단백(oxidized LDL,Ox-LDL)자격대체외배양적소서신소구족세포청도부수체A (scavenger receptor A,SR-A)표체적영향,병초보탐토기가능적분자궤제.방법 체외배양소서조건영생성족세포,이불동농도Ox-LDL자격족세포24 h급이20 mg/L Ox-LDL간예불동시간,매법검측세포내담고순함량,채용실시형광정량PCR、Western인적법검측정상대조조、Ox-LDL자격조SR-A、nephrin、PTEN적mRNA화단백표체수평;DiI표기적양화저밀도지단백(DiI-Ox-LDL)여족세포공부육,면역형광관찰족세포대지질적섭취정황.진일보채용PTEN억제제bpv(hOpic) [dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate(Ⅴ)]、PTEN siRNA이급PTEN선병독(adPTEN)쌍향조공PTEN적표체,관찰SR-A、nephrin적표체、족세포대지질적섭취정황급세포내담고순함량적변화.결과 소서족세포표체SR-A수체병개도지질섭취;여대조조상비,Ox-LDL자격상조족세포SR-A표체적동시반유PTEN、nephrin표체하강(균P＜0.05),차여Ox-LDL적농도급작용시간상관;증가PTEN표체능억제Ox-LDL유도적SR-A표체수평상조화지질섭취(균P＜0.05),강저세포내담고순함량(P＜0.05),상조렬혈격막단백nephrin적표체(균P＜0.05);여지상반,억제PTEN표체능진일보상조Ox-LDL유도적SR-A표체,촉진지질섭취,증가세포내담고순함량(균P＜0.05),병진일보강저nephrin적표체(균P＜0.05).결론 Ox-LDL가능통과PTEN조공SR-A표체진이조공지질섭취,도치족세포손상.
Objective To evaluate the effect of oxidized LDL (Ox-LDL) on scavenger receptor A (SR-A) expression in mouse podocytes and to explore the possible mechanism.Methods The conditionally immortalized mouse podocyte cell line was cultured in vitro and exposed to Ox-LDL of different concentrations for 24 h, or 20 mg/L Ox-LDL for different hours.Cell cholesterol accumulation was examined.Real-time quantity PCR and Western blotting were used to analyze the expression level of PTEN, SR-A and nephrin.Podocytes were incubated with DiI labeled Ox-LDL for 4 h and immunofluorescence was used to analyze lipid uptaking.To further confirm the relationship between PTEN and SR-A, PTEN inhibitor bpv (hOpic) [dipotassium bisperoxo (5-hydroxypyridine-2-carboxyl)oxovanadate (Ⅴ)], PTEN siRNA and PTEN adenovirus (adPTEN) were used to dual-directional regulate PTEN expression, so as to observe the change of SR-A, nephrin, cell cholesterol accumulation and lipid uptake.Results SR-A was expressed on mouse podocyte and mediated podocyte lipid uptake.Compared with control group, Ox-LDL increased cell cholesterol accumulation, and up-regulated SR-A expression along with inhibited expression of PTEN and nephrin (P ＜ 0.05), which were correlate with dose and exposure time of Ox-LDL.Expression of PTEN significantly inhibits the expression level of SR -A and lipid uptake induced by Ox-LDL (P ＜ 0.05), thereby decreasing cell cholesterol accumulation,but up-regulating nephrin level (P ＜ 0.05).However, down-regulation of PTEN could cause opposite effect.Conclusion Ox-LDL up-regulates SR-A through decreasing the expression of PTEN, and contributes to podocyte injury.