CAJ | 학술논문

目的研究慢性肾衰竭大鼠血清对主动脉血管平滑肌细胞(VSMC)泛素-蛋白酶体系统、激活转录因子4(ATF4)及组蛋白乙酰转移酶p300表达的影响,初步探讨慢性肾衰竭血清对主动脉VSMC作用的可能机制.方法建立慢性肾衰竭大鼠模型,采集其血清;采用原代培养的大鼠主动脉VSMC,分正常组(正常大鼠血清刺激)和慢性肾衰竭血清组(慢性肾衰竭血清刺激).用不同大鼠血清刺激72 h后,分别采用实时定量PCR、Western印迹及特异底物反应法检测慢性肾衰竭血清对VSMC泛素、泛素激活酶(ubiquitin activating enzyme,简称E1)、β转导重复相容蛋白1(β-transducin repeat containing protein 1,β-TrCP1)、p300及ATF4mRNA表达,E1、β-TrCP1、p300及ATF4蛋白表达及蛋白酶体活性的影响.结果 (1)慢性肾衰竭血清使主动脉VSMC泛素、E1、β-TrCP1、p300及ATF4mRNA表达增加,与正常组相比,差异均有统计学意义(P＜ 0.01),随时间延长;慢性肾衰竭血清组泛素、E1、β-TrCP1、p300及ATF4 mRNA表达逐渐升高,差异有统计学意义(P＜0.01).(2)慢性肾衰竭血清使主动脉VSMC E1、β-TrCP1、p300及ATF4蛋白表达增加,与正常组相比,差异均有统计学意义(P＜0.01);随时间延长,慢性肾衰竭血清组E1、β-TrCP1及p300蛋白表达逐渐升高,差异有统计学意义(P＜0.01).ATF4表达有下降趋势,但差异无统计学意义(P＞0.05).(3)慢性肾衰竭血清刺激后蛋白酶体活性增强,与对照组相比,差异均有统计学意义(P＜ 0.01);随时间延长,慢性肾衰竭血清组蛋白酶体活性逐渐升高(P＜0.01).结论慢性肾衰竭血清可致大鼠主动脉VSMC泛素-蛋白酶体途径活化,但其对ATF4的降解受阻,可能和p300表达增高有关.这为进一步采取措施,防治慢性肾衰竭血管病变提供了一定的理论和实验依据.
목적 연구만성신쇠갈대서혈청대주동맥혈관평활기세포(VSMC)범소-단백매체계통、격활전록인자4(ATF4)급조단백을선전이매p300표체적영향,초보탐토만성신쇠갈혈청대주동맥VSMC작용적가능궤제.방법 건립만성신쇠갈대서모형,채집기혈청;채용원대배양적대서주동맥VSMC,분정상조(정상대서혈청자격)화만성신쇠갈혈청조(만성신쇠갈혈청자격).용불동대서혈청자격72 h후,분별채용실시정량PCR、Western인적급특이저물반응법검측만성신쇠갈혈청대VSMC범소、범소격활매(ubiquitin activating enzyme,간칭E1)、β전도중복상용단백1(β-transducin repeat containing protein 1,β-TrCP1)、p300급ATF4mRNA표체,E1、β-TrCP1、p300급ATF4단백표체급단백매체활성적영향.결과 (1)만성신쇠갈혈청사주동맥VSMC범소、E1、β-TrCP1、p300급ATF4mRNA표체증가,여정상조상비,차이균유통계학의의(P＜ 0.01),수시간연장;만성신쇠갈혈청조범소、E1、β-TrCP1、p300급ATF4 mRNA표체축점승고,차이유통계학의의(P＜0.01).(2)만성신쇠갈혈청사주동맥VSMC E1、β-TrCP1、p300급ATF4단백표체증가,여정상조상비,차이균유통계학의의(P＜0.01);수시간연장,만성신쇠갈혈청조E1、β-TrCP1급p300단백표체축점승고,차이유통계학의의(P＜0.01).ATF4표체유하강추세,단차이무통계학의의(P＞0.05).(3)만성신쇠갈혈청자격후단백매체활성증강,여대조조상비,차이균유통계학의의(P＜ 0.01);수시간연장,만성신쇠갈혈청조단백매체활성축점승고(P＜0.01).결론 만성신쇠갈혈청가치대서주동맥VSMC범소-단백매체도경활화,단기대ATF4적강해수조,가능화p300표체증고유관.저위진일보채취조시,방치만성신쇠갈혈관병변제공료일정적이론화실험의거.
Objective To investigate the effects of the rat serum with chronic renal failure (CRF) on ubiquitin-proteasome pathway,histone acetyltransferase p300 and activation of activating transcription factor 4(ATF4) of rat arterial vascular smooth muscle cells(VSMCs) cultured in vitro,and explore the possible mechanism.Methods To establish the rat model of CRF by 5/6 nephrectomy,VSMCs were incubated in the media with the 10％ of CRF serum or control serum in vitro.The mRNA expressions of ubiquitin(Ub),ubiquitin activating enzyme(E1),ubiquitin ligases enzymes (β-transducin repeat containing protein 1,β-TrCP1),p300 and ATF4 in the rat VSMCs were examined by using realtime PCR.Expressions of E1,β-TrCP1,p300 and ATF4 proteins in response to the CRF serum in VSMCs were determined by Western blotting analysis.The enzyme activities of 20S proteasomes in the total protein were examined by using three special fluorogenic peptide substrates.Results The CRF serum significantly promoted the mRNA expressions of Ub,E1,β-TrCP1,p300 and ATF4 in VSMCs in a time dependent manner.Compared with that in control serum group,the mRNA levels of Ub,E1,β-TrCP1,p300 and ATF4 in CRF serum group increased significantly (P ＜ 0.01).The CRF serum also increased the protein expressions of E1,β-TrCP1 and p300 in a time dependent manner.The expression of ATF4 was decreased,but the difference was not significant (P ＞ 0.05).Compared with that in control serum group,the protein expressions of E1,β-TrCP1,p300 and ATF4 in CRF serum group increased significantly (P ＜ 0.01).The activities of 20S proteasomes in the CRF serum group were significantly increased in a time dependent manner.Compared with that in control serum group,the activities of 20S proteasomes in the CRF serum group increased significantly (P ＜ 0.01).Conclusions The serum of CRF rat can effectively active the ubiquitin-proteasome pathway,but ATF4 ubiquitinylated degradation is blocked.The latter may be associated with increased expression of p300.