【作者】 张哲；

【导师】 李红；

【作者基本信息】 浙江大学 ， 内分泌与代谢病学， 2008， 博士

【摘要】 第一部分Decorin重组腺病毒改善STZ诱导糖尿病大鼠的肾脏结构与功能目的越来越多的证据表明,转化生长因子β1(Transforming Growth Factorβ1,TGFβ1)在糖尿病肾病的发生发展中起重要作用。以TGFβ1为靶点的治疗模式可能缓解糖尿病肾病(Diabetic Nephropathy,DN)发生发展。核心蛋白聚糖(Decorin,DCN)是一种体内天然存在的TGFβ1拮抗剂,已经证实,DCN可以拮抗TGFβ1所致的纤维化。本研究通过基因转染技术在链脲佐菌素(Streptozocin,STZ)诱导的糖尿病大鼠肾脏内过度表达DCN,观察DCN过表达对糖尿病大鼠肾脏结构与功能的影响。方法1.载体构建:由本研究组成员前期构建大鼠DCN重组腺病毒载体(Adenovirus vector-DCN,AD-DCN)和LacZ重组腺病毒载体(Adenovirus vector-LacZ,AD-LacZ),经鉴定AD-DCN能够高效表达完整而有活性的DCN核心蛋白。2.动物模型的建立与分组:采用一次性腹腔注射STZ 60mg/kg的方法建立Sprague—Dawley(SD)大鼠糖尿病模型。造模成功后第8周将糖尿病大鼠随机分为3组,进行下述处理:1)DCN治疗组(n=12):分别在8周及12周腹腔内注射AD-DCN 0.6ml(1×10~9PFU/ml)。2)AD-LacZ转染组(n=12):分别在8周及12周腹腔内注射AD-LacZ 0.6ml(1×10~9PFU/ml),该组作为重组腺病毒载体转染对照组。3)糖尿病对照组(n=12):分别在8周及12周腹腔注射PBS0.6ml。另设正常对照组(n=12),分别在8周及12周腹腔注射PBS0.6ml。其中12周先处死大鼠,然后对剩余的大鼠作腹腔注射。3.标本留取:糖尿病模型确立后第10,12,16周(即AD-DCN初次转染后2周、4周、8周)每组各取4只大鼠,收集次尿,然后称体重,取股动脉血后处死。取出双侧肾脏,左肾皮质-80℃冷冻保存,右肾称重后福尔马林浸泡固定,制成蜡块。4.观测指标:尿白蛋白排泄率(The Rate of Urinary Albumin Excretion,UAER)以尿白蛋白比尿肌酐(ug/mg)表示,尿白蛋白采用酶免法测定,尿肌酐采用碱性苦味酸法测定。肾脏肥大指数(Kidney Hypertrophy Index,KHI)以肾重/体重(g/kg)表示。血浆血糖,血肌酐和谷丙转氨酶(Glutamate Pyruvate Transaminase,GPT)采用全自动生化仪测定。上述的石蜡切片进行常规高碘酸-希夫(PeriodicAcid-Schiff Staining,PAS)染色以及Masson染色,并进行TGFβ1和肾脏细胞外基质(Extracellular Matrix,ECM)的重要成分Ⅳ型胶原的免疫组化染色。所有染色在HPIAS-1000型高清晰度彩色病理图文报告分析系统中进行半定量分析。结果1.实验大鼠的一般情况:糖尿病大鼠较正常对照组血糖增高,体重增长缓慢(P＜0.01)。AD-LacZ转染组和DCN治疗组没有改善糖尿病大鼠的血糖,也没有缓解糖尿病大鼠体重增长缓慢的状况。整个实验过程中,各组大鼠的死亡未见统计学差异。所有糖尿病大鼠(含AD-LacZ转染组和DCN治疗组)均未出现GPT,血肌酐异常。2.实验大鼠UAER和KHI的改变:10周至16周,糖尿病组大鼠UAER始终较正常对照组增高(P＜0.05),AD-LacZ转染组的情况与糖尿病组相似(P＞0.05)。DCN治疗后糖尿病大鼠UAER在各观察时点都有下降,在16周时与糖尿病组间有统计学意义(P＜0.05),JIDCN治疗组与正常对照组没有统计学差异。糖尿病组,AD-LacZ转染组和DCN治疗组大鼠的KHI明显高于正常组(P＜0.01),DCN治疗组使糖尿病大鼠KHI略有下降,但并未见统计学差异。3.PAS染色和Masson染色PAS染色发现糖尿病组和AD-lacZ转染组大鼠肾小球系膜区,基底膜有PAS阳性物质沉积,并且随病程延长逐渐增多(P＞0.05)。10-16周,AD-DCN治疗可以明显减少PAS阳性物质沉积(P＜0.01)。Masson染色中糖尿病组和AD-lacZ转染组大鼠肾小球系膜区可见Ⅰ型和Ⅲ型胶原纤维异常沉积,AD-DCN治疗同样使Ⅰ型和Ⅲ型胶原纤维异常沉积减少。4.免疫组化研究:免疫组化研究结果提示,糖尿病组与AD-LacZ转染组大鼠肾皮质Ⅳ型胶原的表达较正常对照组增高(P＜0.01)。外源DCN抑制Ⅳ型胶原在糖尿病大鼠肾皮质中的表达(P＜0.05),10周时DCN的抑制效应最强(P＜0.05)。与正常对照组相比,糖尿病组与AD-LacZ转染组大鼠肾皮质TGF-β1表达增高。外源DCN能够抑制糖尿病大鼠肾皮质TGF-β1表达(P＜0.05),在第10周时,该抑制效应最强(P＜0.05)。结论1.STZ诱导的糖尿病大鼠肾脏ECM过度沉积,UAER增高。腺病毒载体本身不能改善上述变化。2.腺病毒载体介导的DCN基因转染能抑制糖尿病大鼠肾皮质TGF-β1的表达,缓解糖尿病大鼠UAER增加,减轻肾脏局部ECM堆积。第二部分Decorin重组腺病毒对糖尿病大鼠肾脏TGFβ1/Smads信号传导系统的影响目的经典的信号传导通路中TGFβ1通过Smad2,3蛋白磷酸化以及核转位将信号传递至靶基因。AD-DCN转染糖尿病大鼠后肾脏TGFβ1的表达有所减少,究竟外源性DCN转入对TGFβ1/Smads信号传导系统影响如何?本研究检测了糖尿病大鼠肾皮质DCN,TGFβ1以及磷酸化Smad2,3(Phosphorylation Smad2,3,pSmad2,3)与Smad2,3的比值,阐明上述问题。方法1.肾皮质DCN,TGFβ1 mRNA的检测:采用逆转录聚合酶链反应(Reverse Transcription Polymerase ChainReaction,RT-PCR)方法检测四组大鼠肾脏DCN,TGFβ1 mRNA的表达。用Trizol试剂提取肾皮质mRNA。逆转录后合成的第一条cDNA链为模板进行目的基因PCR扩增,以βactin为内参。扩增产物经电泳后,在紫外光下进行凝胶成像。测定各产物与βactin的密度值(Image Density View,IDV)。目的基因的mRNA水平=目的基因mRNA的IDV/βactin基因mRNA的IDV。2.肾皮质DCN,TGFβ1,pSmad2,3与Smad2,3蛋白的测定:采用免疫印迹法(Western blot)检测肾组织DCN,TGFβ1,pSmad2,3与Smad2,3的表达。首先从100mg肾组织中提取组织蛋白,然后进行SDS-PAGE电泳,转膜。转膜成功后先封闭非特异性免疫反应,然后浸于封闭液稀释的一抗中,4℃反应过夜。将PVDF膜,浸泡在用TBST稀释二抗反应液中,室温反应2小时。经TBST浸洗,进行显色反应。最后通过凝胶成象分析,测定目的蛋白和actin蛋白的IDV。目的蛋白的表达量=目的蛋白IDV/actin IDV。结果1.AD-DCN促进糖尿病大鼠肾皮质DCN转录,进一步增强DCN蛋白的表达。与正常对照组相比较,糖尿病组,AD-LacZ转染组大鼠肾皮质DCN mRNA和蛋白水平的表达均增高(分别P＜0.01或P＜0.05),后两组之间未见明显统计学差异。AD-DCN治疗后肾皮质DCN表达较糖尿病组更高,10周最为明显,其中DCNmRNA增幅达20%(P＜0.01);而DCN蛋白的表达增加110%(P＜0.01)。12,16周,DCNmRNA的表达已经与糖尿病组无差别,而DCN蛋白水平虽然随着时间的推移逐渐下降,但始终高于糖尿病组(P＜0.05)。2.AD-DCN治疗可以抑制大鼠肾皮质TGF-β1的表达糖尿病组及AD-LacZ转染组大鼠肾皮质TGF-β1 mRNA和蛋白表达增高(P＜0.01)。AD-DCN治疗抑制糖尿病大鼠肾皮质TGF-β1 mRNA和蛋白表达(10周时,P＜0.05:12,16周,P＞0.05)。3.AD—DCN明显抑制Smad2,3磷酸化糖尿病时大鼠肾皮质pSmad2,3与Smad2,3比值增高,提示TGFβ1/Smad2,3信号系统激活。AD-DCN阻止了Smad2,3的激活,这一作用一直延续至16周(P＜0.05),这与DCN蛋白水平的变化一致。结论1.糖尿病大鼠肾脏局部的DCN表达增高,TGFβ1/Smad2,3信号系统活跃。2.AD-DCN转染可以进一步糖尿病大鼠促进肾脏局部DCN转录,提高DCN蛋白水平。同时抑制TGFβ1的高表达,减少Smad2,3分子的磷酸化。第三部分Decorin重组腺病毒对糖尿病大鼠肾脏细胞凋亡与Bax/Bcl-2表达的影响目的DN早期肾脏体积增大主要因为肾脏细胞周期紊乱导致的异常增生、肥大:DN晚期肾小球硬化主要由于肾小球细胞数减少和细胞外基质的增多。从细胞增生变为细胞减少的过程伴有细胞凋亡的增加。细胞凋亡即程序性细胞死亡,线粒体凋亡是其中重要的途径之一。Bax与Bcl-2作为一对促凋亡因子和细胞生存基因,两者的比例调控细胞凋亡过程,是线粒体凋亡过程的重要调控基因。本研究检测AD-DCN转染糖尿病大鼠肾脏凋亡细胞数目,同时采用免疫组化的方法测定Bax与Bcl-2的表达,研究外源性DCN对糖尿病大鼠肾脏组织细胞凋亡的影响。方法采用免疫组化的方法测定大鼠肾组织中Bax与Bcl-2的表达,在HPIAS-1000型高清晰度彩色病理图文报告分析系统中进行半定量分析。采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminaldeoxynucleotidyl transferase-mediated dUTP nick end labeling,TUNEL)检测肾脏凋亡细胞,并在高倍镜下(×400)统计阳性细胞核的数目,凋亡指数以每个视野下阳性细胞核数/总细胞核数表示。分别采用Spearman法和Pearson法将凋亡指数,Bax/Bcl-2以及UAER作两两相关分析。结果1.AD-DCN减少糖尿病大鼠肾组织的细胞凋亡正常对照组大鼠肾组织内凋亡细胞很少,主要分布于远端肾小管细胞,糖尿病组与AD-lacZ转染组肾组织内凋亡细胞增多,可见于肾远曲小管,近曲小管,肾间质以及肾小球,并且随糖尿病病程延长而增加(P＜0.05)。而AD-DCN转染使糖尿病大鼠肾组织中凋亡的细胞数显著减少(P＜0.01),但未能恢复到正常对照组水平(P＜0.01)。2.AD-DCN可以调节Bax和Bcl-2在肾脏的表达糖尿病组和AD-lacZ组大鼠肾脏Bax的表达较正常对照组增强,Bcl-2的表达亦增强,且Bax/Bcl-2比值增高,诱导肾组织中的细胞凋亡。随着病程延长,Bax进一步增高,而Bcl-2的增高趋势有所缓解,故Bax/Bcl-2的比值进一步增高(P＜0.01)。糖尿病大鼠接受AD-DCN治疗后,Bax的表达减弱(P＜0.05),而Bcl-2的表达有所增强,因此Bax/Bcl-2比值下降甚至低于正常对照组,阻断细胞的凋亡。3.Bax/Bcl-2比值与肾脏细胞凋亡指数以及UAER的相关性分析。10周、12周、16周肾组织凋亡指数与Bax/Bcl-2比值显著正相关(P＜0.01)。UAER增高与肾凋亡指数正相关(P＜0.05),与Bax/Bcl-2比值仅在10周时正相关(P＜0.05)。结论1.糖尿病大鼠肾组织中凋亡细胞增多,AD-DCN降低糖尿病大鼠肾组织Bax/Bcl-2比值的同时减少了细胞的凋亡。Bax/Bcl-2的比值与肾组织细胞凋亡指数正相关。2.AD-DCN减少糖尿病大鼠UAER的效应部分与DCN减少肾组织细胞的凋亡有关。总结STZ诱导的糖尿病大鼠肾脏ECM过度沉积,UAER增高,这可能是因为肾脏局部TGFβ1/Smad2,3信号系统活跃;肾组织中Bax/Bcl-2比值增高诱导细胞凋亡。腺病毒载体本身不能改善上述变化。而腺病毒载体介导的DCN基因转染能缓解糖尿病大鼠UAER增加,减轻肾脏局部ECM堆积。AD-DCN的这种肾脏保护作用可能源于:AD-DCN能促进糖尿病大鼠肾脏局部DCN转录,提高肾脏局部DCN蛋白水平,从而抑制肾皮质TGFβ1的高表达,减少Smad2,3分子的磷酸化,抑制TGFβ1/Smad2,3信号系统在肾组织中的传导:同时AD-DCN可以降低糖尿病大鼠肾组织Bax/Bcl-2比值,减少了细胞的凋亡。更多还原

【Abstract】 PARTⅠRecombinant Adenovirus-Mediated Decorin Gene Transfection Has The Effect of Anti-proteinuria and Anti-Fibrosis on STZ-induced Diabetic RatsBackgroundAccumulating evidence suggests that TGFβ1 is a key mediator in diabetic nephropathy.One of the approaches down-regulating TGFβ1 expression is to use proteoglycanⅡ（decorin）,a natural inhibitor of TGFβ1.In the present study,a recombinant adenovirus expressing decorin（AD-DCN）was utilized to investigate the effects of decorin over-expression on kidney of streptozocin（STZ）induced diabetic rats.Material and MethodsAD-DCN was constructed according to our previous research and confirmed to express intact and active rat decorin.Recombinant adenovirus vector of LacZ gene （AD-lacZ）was constructed in the same way,used as transfection control.Diabetic Sprague-Dawley rats was induced by once intraperitoneal injection of STZ（60mg/kg）.8 weeks after the diabetic model had succeeded,diabetic rats were divided into three groups randomly:diabetic group treated with PBS 0.6ml by intraperitoneal injection at8th and 12^thweek;AD-DCN group and AD-lacz group treated with AD-DCN or AD-lacZ（0.6ml,1×10⁹pFU/ml）respectively,by intraperitoneal injection at 8^thand 12^thweek.Moreover,12 SD rats were treated with PBS 0.6ml by intraperitoneal injection at 8^thand 12^thweek as normal control.At the 10^th,12^thand 16^thweek,which was 2,4,8 weeks after the first injection of AD-DCN respectively,four rats in every group were scarified.Body weight and blood glucose concentration was monitored.Urinary albumin excretion（UAER）was expressed as the ratio of urinary albumin to urinary creatinine（ug/mg）.Blood samples were analyzed for glucose,GPT,creatinine（enzymatic assay）on Hitachi -7170 automated chemistry analyzer.Left kidney were weighed and kept at -80℃for Reverse Transcription Polymerase Chain Reaction（RT-PCR）,and Westen-Blot. Kidney hypertrophy index（KHI）was estimated by comparing the weight of right kidney to body weight.The right kidney was fixed in neutral buffered formalin, embedded in paraffin,sectioned,stained for periodic acid schiff（PAS）,Masson and performed immumohistochemistry for typeⅣcollagen and TGFβ1.Results1.Diabetic rats,including diabetic group,AD-DCN group and AD-laeZ group,had high levels of serum glucose and slow gain in body weight compared to normal control.There was no difference of body weight and serum glucose among three groups.The level of GPT and serum cretinine as well as the mortality in four groups had no statistical difference.2.UAER increased in diabetic rats.Adenovirus itself had no effect on albuminuria, while exogenous decorin attenuated albuminuria.There were no difference of KHI among diabetic group,AD-DCN group and AD-lacZ group.These groups had higher KHI than normal control. 3.PAS staining and Masson staining suggested ECM over-expression in kidney of diabetic group and AD-laeZ group.AD-DCN inhibited abnormal deposition of ECM.4.TGFβ1 and typeⅣcollagen expression was stronger in kidney of diabetic rats than that of normal control,which was reduced by AD-DCN rather than AD-lacZ.ConclusionsSTZ-induced diabetic rats had ECM over-deposition and albuminuria increment. Adenovirus itself had no effect on these change.Decorin gene transfection mediated by a recombinant adenovirus had anti-fibrotic and anti-proteinuric effect on STZ-induced diabetic rats,without blood glucose improvement.The renal protective effect of deeorin might be due to the down-regulation of TGFβ1 expression. BackgroundIn previous study,we found that AD-DCN could prevent abnormal deposition of ECM in kidney and albuminuria of diabetic rats through TGFβ1 down-regulation.But the mechanism of renal protection of DCN is still unknown.TGFβ1 is known as the common mediator in final stage of kidney fibrosis.P-Smad2 and 3,the active form of Smad2 and 3,are important factors in TGFβ1/Smads signal system.So we detected the levels of DCN,TGFβ1,Smad2,3 and p-Smad2,3 by RT-PCR and Western-Blot,in order to investigate the effect of DCN on TGFβ1/Smads signal system in diabetic kidney.Material and MethodsAll renal tissue was acquired as part 1.Total RNA was extracted from renal tissue using Trizol Reagent.One microgram of each RNA sample were reverse transcribed by RevertAid^TMH Minus M-MuLV Reverse Transcriptase according to the manufacturer’s instruction.The PCR products（5ul）were separated by 1.5%（w/v） agarose gel electrophoresis and then visualized by ethidium bromide staining.The final results were measured in image density view（IDV）with Bio-Rad digital image system.The target gene was estimated by the ratio of target gene IDV toβ-actin IDV.The protein levels of DCN,TGFβ1,Smad2,3 and p-Smad2,3 were detected by Western-Blot,using total protein extracted from 100mg renal tissue.Results1.The expression of DCN mRNA and protein were elevated in diabetic group and AD-lacZ group.AD-DCN further increased DCN mRNA by 20 percent and DCN protein by 100 percent compared to diabetic group at the 10^thweek.The increment of DCN protein lasted until the 16^thweek.2.AD-DCN attenuated TGFβ1 mRNA and protein over-expression in diabetic renal, but it was only found at the 10^thweek.3.The ratio of p-Smad2,3 to Smad2,3 was elevated in diabetic group and AD-lacZ group.AD-DCN decreased this ratio at all time points in accordance with the change of DCN protein.ConclusionsAD-DCN could elevate the levels of DCN protein,attenuate expression of TGFβ1 and persistently prevent activation of Smad2,3 in diabetic kidney. BackgroundIn the progress of diabetic nephropahy,number of cells increases at early stage and decreases at end stage,which is accompanied by cell apoptosis.Bax and Bcl-2 are a pair of contrl genes in apoptosis.The former causes apoptosis and the latter inhibits apoptosis.The ratio of Bax to Bcl-2 is important to apoptosis.In this study,we want to investigate the effect of AD-DCN on apoptosis and expression of Bax/Bcl-2 in the kidney of diabetic rats.Materials and methodsWe measured bax and bcl-2 by immunohistochemistry.Apoptosis was evaluated by means of terminal deoxynucleotidyl transferase mediated d-UTP nick end labelling （TUNEL）.Index of apoptosis was measured by the ratio of positive cell to total cells under a high-power microscope perspective（×400）.Pearson assay or Spearman assay in Spssl 3.0 was used for correlation analysis.Results1.More cells underwent apoptosis in diabetic kidney compared to normal control, appearing in renal distal convoluted tubules,the proximal tubules and renal glomerular.The index of apoptosis gradually increased from 10^thweek to 16^thweek. AD-DCN reduced the index of apoptosis.2.The expression of Bax and Bcl-2 increased in diabetic kidney,but the level of Bax was higher than that of Bcl-2.As a result,the ratio of Bax to Bcl-2 elevated.AD-DCN decreased the level of Bax while elevated that of Bcl-2,thus reduced the ratio of Bax to Bcl-2.3.There was positive correlation between the ratio of Bax to Bcl-2 and index of apoptosis.The latter was also correlated with albuminuria. Conclusions1.The index of apoptosis and the ratio of Bax to Bcl-2 increased in kidneys of diabetic rats.2.AD-DCN reduced the ratio of Bax to Bcl-2 and inhibited cell apoptosis in kidneys of diabetic rats.3.The index of apoptosis correlated with the ratio of Bax to Bcl-2 and albuminuria更多还原