【作者】 王艳艳；

【导师】 谌贻璞；

【作者基本信息】 中国协和医科大学 ， 内科学， 2009， 博士

【摘要】 研究背景含马兜铃酸（AA）成分的中草药可引起马兜铃酸肾病,其发病机理尚未完全明确。目前的研究表明肾小管上皮细胞是AA的主要作用靶点之一,AA可导致肾小管上皮细胞坏死、凋亡、活化或转分化。但AA是如何对细胞产生作用?是通过与受体结合的方式发挥作用,还是直接进入细胞发挥作用?如果进入细胞发挥作用的话,又是通过何种途径呢?我们利用体外细胞生物学实验对上述问题进行了研究。研究目的1)以人肾小管上皮细胞（HKC）为研究对象,观察AA是否能进入HKC细胞;2)观察AA通过何种途径进入HKC细胞;3)观察抑制AA进入细胞对其刺激CTGF表达作用的影响。研究方法1)采用液相色谱-串联质谱（LC-MS/MS）技术检测HKC细胞裂解液中AA。2)利用乳酸脱氢酶（LDH）释放实验,①检测有机阴离子转运蛋白（OATs）抑制剂丙磺舒和有机阳离子转运蛋白（OCTs）抑制剂四乙基铵对AA细胞毒性作用的影响;②构建pCMV5-HA-hOAT1/3表达质粒并进行转染,检测过量表达hOAT1/3对AA细胞毒性作用的影响。3)实时荧光定量聚合酶链反应和免疫印迹法检测OATs抑制剂丙磺舒对HKC细胞CTGF mRNA和蛋白表达的影响。研究结果1) AA-Na刺激组细胞破碎液中可检测到AA,对照组检测不到;依据峰面积粗略估算AA-Na刺激组细胞破碎液中AA浓度可达到1×10^-3h/L。2) 60mg/L AA-Na可致HKC细胞LDH释放率显著升高,OATs抑制剂丙磺舒可抑制上述LDH释放（抑制率达27.5%,P＜0.05）,而OCTs抑制剂四乙基铵无此抑制作用。3) pCMV5-HA-hOAT1/3表达质粒转染HKC细胞后,AA对HKC的细胞毒性作用增强,且细胞毒性作用随转染质粒量的增大而增强。4) 40mg/L AA-Na能显著上调HKC细胞CTGF mRNA及蛋白表达,OATs抑制剂丙磺舒可抑制上述CTGF高表达（mRNA及蛋白表达抑制率分别为16%和21%,P＜0.05）。结论1) AA可以进入HKC细胞。2) AA可能通过hOAT1/3进入HKC细胞,AA细胞毒性作用与hOAT1/3表达水平相关。3) OATs抑制剂丙磺舒可抑制AA对HKC的细胞毒性作用,并可抑制AA诱导的HKC细胞CTGF高表达。更多还原

【Abstract】 BackgroundAristolochic acids(AA) belong to a family of structurally related nitrophenanthrene carboxylic acids.The consumption of herbs containing AA has been associated with severe nephropathy which is called aristolochic acid nephropathy(AAN).AAN is characterized by a paucicelluar progressive and extensive interstitial fibrosis without prominent glomerular lesions and inflammatory cell infiltration.Clinical and in vitro findings have suggested that the proximal tubule was the major site of AA-induced renal injury.We have reported AA-induced cytotoxic and fibrogenic effects on proximal tubule epithelial cells.However the potential pathogenic mechanism of AAN has not been clarified.Could AA enter the proximal tubular epithelial cells? From the structural point of view,AA are classified into a diverse group of organic compounds of low molecular weight.For the entry of these compounds,special membrane proteins such as transporters are required, because they cannot penetrate the lipid bilayer of the cell membrane.In the kidney, organic anion transporters,which are expressed in the apical and basolateral membranes of tubular epithelial cells,are responsible for tubular secretion of organic anions including drugs,toxins and endogenous compounds.The mRNA levels of hOAT1 and hOAT3 were much higher than those of other organic ion transporters in the human kidney cortex.These findings suggest that hOAT1 and hOAT3 play important roles in the tubular uptake of various drugs from the circulation.Under such circumstance,we decided to investigate whether AA could enter the proximal tubular epithelial cells and if organic anion transporters are involved in this process.Objectives1) To investigate whether AA could enter the proximal tubular epithelial cells (HKC).2) To investigate which transporters are involved in the process of AA entering HKC.3) To investigate whether decreased intracellular AA could down-regulate its cytotoxic and fibrogenic effects on HKC.Methods1) Liquid chromatography-tandem mass spectrometry was applied to measure intracellular AA.2) The release of lactate dehedrogenase(LDH) induced by AA in the presence of organic anion transporter inhibitor(probenecid) or organic cation transporter inhibitor(tetraethylammonium) was evaluated.3) After HKC were transiently transfected with pCMV5-HA-hOAT1/3,the cytotoxic effect of AA on HKC was assessed by the release of LDH.4) The effects of probenecid on AA induced mRNA and protein expression of connective tissue growth factor(CTGF) were also determined by real time polymerase chain reaction and Western blots,respectively.Results1) Intracellular AA of HKC,which was not detected in the control group,were detected and measured in the suspension of the broken HKC cells.2) The LDH releasing from HKC stimulated by 60mg/L AA-Na was significantly inhibited by 1 mM probenecid(P＜0.05),but not by 1mM tetraethylammonium.3) The cytotoxic effect of AA on the HKC cells was increased when HKC were transiently transfected with pCMV5-HA-hOAT1/3.4) The up-regulated mRNA and protein expression of CTGF in HKC stimulated by 40mg/L AA-Na was significantly inhibited by 1mM probenecid(P＜0.05) with a inhibition rate of 16%and 21%,respectively.Conclusions1) The results suggest that AA could be transported into HKC through organic anion transporters. 2) AA can be transported into HKC and then exerts its biological response and cytotoxic effect,which can be inhibited by probenecid.3) The cytotoxic effect of AA on HKC was increased with overexpressed hOAT1/3 in a dose-dependend manner.更多还原