【作者】 丁露露；

【导师】 孔维信；

【作者基本信息】 江苏大学 ， 内科学， 2008， 硕士

【摘要】 足细胞(Podocyte)即肾小球脏层上皮细胞(Glomerular Visceral Epithelial Cell),是一种具有高度特异性且终级分化的细胞,不具有增殖特性。足细胞损伤、脱落或凋亡是肾小球硬化起始和进展的主要原因,而介导足细胞凋亡的启动因子及发病机制尚不清楚。环氧合酶-2(Cyclooxygenase-2,COX-2)是前列腺素合酶的同工酶,其表达部位在肾小球皮质的致密斑、髓质间质细胞及亨氏襻升支粗段。有研究报道在人类肾小球足细胞可检测到COX-2表达,且在肾大部切除、Thy-1肾炎、糖尿病肾病大鼠模型中足细胞COX-2的表达显著增强。已有动物实验表明特异性COX-2抑制剂对许多慢性肾脏疾病有减轻蛋白尿,延缓肾损害进展的作用,但作用机制尚未完全阐明。本研究旨在通过特异性COX-2抑制剂塞来昔布(Celecoxib)对嘌呤霉素氨基核苷(Puromycin Aminonucleoside,PA)诱导的足细胞凋亡的影响,进一步探讨特异性COX-2抑制剂对足细胞保护作用的分子机制,为临床对慢性进行性肾损害的防治探索出新的治疗靶点。方法1.将体外培养的条件永生性小鼠足细胞复苏并传代培养,并用PA干预足细胞诱导其凋亡,采用Western blot法分析COX-2在凋亡足细胞中的表达。为进一步研究特异性COX-2抑制剂塞来昔布对PA诱导的足细胞凋亡的影响建立实验基础。2.以条件永生性小鼠足细胞株为研究对象,实验分为8组:正常对照组(CON)、嘌呤霉素氨基核苷组(PA)、地塞米松组(DEX)、地塞米松+嘌呤霉素氨基核苷组(DEX+PA)、氯沙坦组(LOS)、氯沙坦+嘌呤霉素氨基核苷组(LOS+PA)、塞来昔布组(CEL)和塞来昔布+嘌呤霉素氨基核苷组(CEL+PA),在0h,8h,24h和48h对体外培养的足细胞分别用MTT比色法和Hoechst 33258荧光染色法检测足细胞活性和凋亡水平;用Caspase-3活性检测试剂盒检测凋亡过程中caspase-3蛋白酶的活性水平;用Western blot技术检测凋亡过程中p53及COX-2水平。结果1.在33℃增殖状态下,足细胞呈鹅卵石状,在37℃分化状态下的足细胞呈分叉状。在37℃分化14d后,用低血清培养液培养后,给予PA干预后,随时间延长,PA诱导的足细胞凋亡逐渐增多,在24h及48h COX-2表达明显增加。2.与正常对照组比较,LOS、CELE、DEX组足细胞凋亡率无明显变化(P＞0.05),PA组凋亡率在24h及48h明显增高(P＜0.05),而LOS+PA、CELE+PA、DEX+PA组凋亡率在24h及48h比正常对照组升高,但较PA组明显下降(P＜0.05)。3.与正常对照组比较,LOS、CELE、DEX组足细胞增殖活性无明显变化(P＞0.05),PA组增殖活性在24h及48h明显下降(P＜0.05),而LOS+PA、CELE+PA、DEX+PA组增殖活性在24h及48h比正常对照组下降,但较PA组明显升高(P＜0.05)。4.各组caspase-3活性无明显差异(P＞0.05)。5.在24h及48h时,PA组p53表达较正常对照组明显增多,用LOS、CELE、DEX干预后明显降低(P＜0.05)。6.正常对照组表达COX-2,用PA诱导凋亡后,24h及48h时COX-2表达明显增加,用LOS、CELE、DEX干预后COX-2表达水平有所减少(P＜0.05)。结论1.COX-2在凋亡足细胞中的表达呈时间依赖性。2.塞来昔布可以抑制PA诱导的足细胞凋亡,p53参与了PA诱导的足细胞凋亡过程,塞来昔布抑制PA诱导的足细胞凋亡的可能与足细胞所表达的COX-2水平有一定相关性。更多还原

【Abstract】 The visceral glomerular epithelial cell,also called the podocyte,is a highly specilized and terminally differentiated cell which plays a major role in establishing the glomerular filtration barrier.Podocytes therefore have limited ability to be replaced if lost. Podocytes injury as a result of apoptosis and/or detachment of cells from the golmerular basement membrane and the inability of podocytes to replicate is a major cause of progressive glomerular damage,but underlying fators of the predisposition of podocytes to injury and the mechanisms that mediate the injury remain unclear.Cyclooxygenase-2 is the key enzyme in the synthesis of prostaglandins(PGs)from arachidonic acid.In normal adult mammalian kidney COX-2 expression is localized to the macula densa and associated cortical thick ascending limb of Henle(cTAL)in the cortex and to a subset of medullary interstitial cells near the papillary tip.COX-2 is detectable in the podocytes of human kidney.Increased podocyte COX-2 expression in rats has been reported in models of subtotal renal ablation,Thy,1 nephritis,and diabetic nephropathy,and selective COX-2 inhibitors have been shown to decrease proteinuria and retard progressive glomerular injury in models of renal ablation,diabetes,and salt- sensitive hypertension. The underlying mechanisms of which the specific cyclooxygenase-2(COX-2)inhibitors have been shown to decrease proteinuria and retard progressive glomerular injury in some animal models have not been addressed formally.Accordingly,in this study we investigated the direct effect of celecoxib,a specific COX-2 inhibitors,on podocyte apoptosis induced by puromycin aminonucleoside(PA)and the mechanisms that underlie this effect in order to further search for clinical therapeutic target on chronic progressive glomerular injury.Methods1.Experiments were performed using early-passage growth-restricted,conditionally immortalized mouse podocytes.The expression of COX-2 in apoptotic podocytes was measured by use of Western blot analysis.The effects of specific COX-2 inhibitors celecoxib on podocyte apoptosis induced by PA will be examined on the basis of podocyte culture.2.The conditionally immortalized mouse podocytes were studied and divided into eight groups.Control group(CON);puromycin aminonucleoside group(PA);losartan group (LOS);dexamethasone group(DEX);celecoxib group(CEL);losartan + puromycin aminucleoside group(LOS + PA);dexamethasone + puromycin aminucleoside group (DEX + PA);celecoxib + puromycin aminudeoside group(CEL + PA).Apoptosis was measured by staining with Hoechst 33258 at 0,8,24 and 48 h.To determine the activity of caspase-3,we used the BD ApoAlert Caspase Colormettic Assay Kit, according to the manufacturer’s instructions at 0,8,24 and 48 h.Western blot analysis was performed to measured the pretein levels of p53 and theprotein levels of COX-2 at 24 h.Results1.At the permissive temperature of 33℃,the conditionally immortalized mouse podocyte grew in a cobblestone morphology.They are grown arborized under nonpermissive conditions at 37℃for 14 days.The COX-2 expression in apoptotic podocytes induced by puromycin aminonucleoside increased over time.The highest level was found at 24 hour.Afterwards it decreased gradually.2.The percentage of apoptotic cells significantly increased in the podocytes that were exposed to PA for 24 h compared with control group.No significant change was found in LOS,CEL and DEX groups.In contrast,losartan,dexamethasone and celecoxib significantly reduced PA-induced apoptosis(P＜0.05).3.The proliferetion activity significantly increased in the podocytes that were exposed to PA for 24 h compared with control group.No significant change was found in LOS, CEL and DEX groups.Losartan,dexamethasone and celecoxib significantly increased the proliferetion activity of apoptotic podocytes induced by PA(P＜0.05).4.There was no difference in the activity of caspase-3 among each group(P＞0.05).5.Western blot analysis showed that PA increased the protein levels for p53 at 24 h in arborized podocytes.The protein levels were reduced by LOS,DEX and CEL (P＜0.05).6.Western blot analysis showed that COX-2 expression was seen in control group.It was elevated profoundly after 24 hours induced by PA and reduced by use of LOS, DEX and CEL(P＜0.05).ConclusionsThe COX-2 expression in apoptotic podocytes was time-dependent.Celecoxib prevents podocyte apoptosis induced by PA,and it is p53 dependent.The preventing effect of celecoxib on podocyte apoptosis may be potential interactions with COX-2 expression.更多还原