siRNA抑制CTGF表达对高糖诱导肾小管上皮细胞肥大和转分化的影响及机制
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摘要
研究背景
糖尿病肾病(diabetic nephropathy,DN)是糖尿病的常见并发症之一,也是终末期肾功能衰竭的主要原因之一。肾脏肥大是DN早期最重要的病理生理学特征,晚期则表现为纤维化。肾小管体积约占整个肾脏体积的90%,因而肾小管肥大在肾脏肥大中占有十分重要的地位。肾小管间质纤维化的进展程度则与肾功能的进行性下降密切相关。肾小管上皮细胞作为构成肾小管间质结构的主要细胞之一,在DN肾小管肥大及间质纤维化过程中扮演了重要的角色。肾小管上皮细胞的肥大及向间充质细胞转分化(epithelial mesenchymal transition,EMT)现象是引起肾小管肥大,间质纤维化的重要原因。
(一)肾小管上皮细胞肥大机制
目前关于DN时肾小管上皮细胞肥大的具体机制仍不清楚。已知细胞的增生及肥大往往与细胞周期的转换密切相关。正常情况下肾小管上皮细胞均处于静息期即G_0期。当细胞需要增殖时即进入G_1期,如果某些因素导致细胞停滞在G_1期无法进入S期,则会引起蛋白在细胞内聚积,细胞肥大。作为细胞周期素激酶抑制剂(CDK inhibitors,CKIs)的CIP/KIP家族具有抑制细胞周期转换的作用,因此其在DN发病早期肾脏固有细胞肥大中的作用倍受关注。CIP/KIP家族包括p21~(kip)、p27~(kip)、p57~(kip)三个成员。肾小管上皮细胞主要以表达p27~(kip),p21~(kip)为主,p57~(kip)则尚未见文献报道。
体外及体内研究提示在高糖及糖尿病状态时肾小管上皮细胞p27~(kip)蛋白表达增加,并与细胞肥大呈正比。然而DN时p27~(kip)表达上调的具体机制仍不清楚。Jau-Shyang Huang发现高糖引起体外培养的肾小管上皮细胞肥大,p21~(kip)和p27~(kip)表达升高的同时伴有p42/p44丝裂原活化蛋白激酶(mitogen-activated proteinkinase,MAPK)又称细胞外信号调节激酶1/2(extracellular signal-regulatedkinase 1/2,ERK1/2)的磷酸化激活,提示MAPK信号途径可能是高糖刺激肾小管上皮细胞表达p27~(kip)的细胞内机制。
MAPK信号途径如何被激活,其具体机制是什么还有待探索。近年来结缔组织生长因子(connective tissue growth factor,CTGF)由于在DN的发生和发展中起了重要作用而倍受关注。有实验证明CTGF可激活肾小球系膜细胞和肾间质细胞的ERK1/2通路引起细胞形态功能变化;新近研究又发现CTGF是血管紧张素Ⅱ诱导人近端肾小管上皮细胞肥大的重要介质。因此我们推测CTGF可能通过激活ERK1/2通道来调节p27~(kip)的表达进而诱导肾小管上皮细胞肥大,这一推论有待证实。
(二)肾小管上皮细胞EMT机制
肾小管上皮细胞EMT是肾间质纤维化的重要原因。正常状态下肾小管上皮细胞通过细胞间黏附机制紧密连接在一起,E-钙黏蛋白(E-cadherin)是组成细胞间紧密连接的重要成分,在保持细胞完整性和极性中起重要作用。肾小管上皮细胞表型转化发生的基本步骤之一,就是E-cadherin表达减少,上皮细胞丧失黏附特性,从基底膜上脱落下来,胞浆内细胞骨架重排,表达间充质细胞表型α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)。上皮细胞转分化后形成的间质成纤维细胞可大量合成细胞外基质(Extracellar matrix,ECM)成分,引起间质纤维化。CTGF是引起肾间质纤维化的重要细胞因子。体外及体内实验均证明高糖及糖尿病状态可刺激肾小管上皮细胞表达CTGF,后者是公认的能促进肾小管EMT的因素之一。但CTGF引起EMT的具体机制仍不明确。Janus激酶(Januskinase,JAK)/信号转导与转氯?钜蜃?signal transduction and activators oftranscription,STAT)是一条重要的介导细胞因子和生长因子信号传导的通路。近来有研究揭示高糖刺激可引起体外培养的肾小管上皮细胞内JAK2、STAT1和STAT3激活,伴有ECM成分合成增多。但其在肾小管上皮细胞EMT过程中的作用如何,文献尚未见报道。对上皮细胞癌的研究表明,JAK2/STAT3信号通路参与调节上皮细胞之间的黏附,可能与恶性病变中EMT有关。那么JAK2/STATs信号途径是否也参与了DN时肾小管上皮细胞的EMT呢?前已述CTGF是DN时肾小管EMT的重要因素,那么CTGF是否能通过激活JAK2/STATs信号途径引起EMT呢?这些问题都有待探索。
因为CTGF在DN发展过程中占有重要地位,且作用单一,在生理情况下表达很低,所以被认为是一个可选择的治疗DN的靶点。但对于CTGF在糖尿病肾病中的具体作用机制仍不完全明了,且缺乏有效、安全、实用的干预方式,故目前在这方面的治疗研究仍无大的进展。小分子干扰RNA(small interfering RNA,siRNA)是近年出现的一种新的抑制基因表达的技术方法。与传统的反义寡核苷酸、基因敲除相比,具有特异、高效且易操作等优点,已成为研究基因沉默在临床应用的前沿热点。
基于上述背景,本研究设想,通过检测高糖条件下培养的人肾小管上皮细胞CTGF、p27~(kip)的mRNA及蛋白表达水平,ERK1/2、JAK2、STAT3磷酸化水平,α-SMA、E-cadherin的蛋白水平,细胞总蛋白含量及周期变化,探讨高糖诱导肾小管上皮细胞肥大及EMT的作用途径及可能的发生机制;通过构建可高效抑制CTGFmRNA表达的siRNA载体,在体外转染肾小管上皮细胞,观测抑制CTGF表达后对细胞肥大及EMT相关指标的影响,进一步探寻CTGF对肾小管上皮细胞肥大及EMT的诱导机制,为针对CTGF的RNA干扰(RNA interference,RNAi)治疗提供一定的实验基础。
第一章CTGF基因特异性siRNA的设计、合成及表达载体的构建和鉴定
一、研究目的
构建并鉴定稳定表达针对CTGF基因的siRNA的质粒。
二、研究方法
1.根据人CTGF mRNA基因编码区序列,利用计算机辅助设计软件设计3条针对CTGF的siRNAs(siRNA-CTGF-1、siRNA-CTGF-2及siRNA-CTGF-3),和1条作为阴性对照的无关序列(siRNA-CTGF-neg)。并合成分别包含这4条siRNAs的短发夹状RNA(shRNA)的模板脱氧寡核苷酸序列。
2.将上述shRNA模板脱氧寡核苷酸序列分别插入pGenesil-1质粒,构建分别表达3种特异性siRNA的质粒(pshRNA-CTGF-1、pshRNA-CTGF-2、pshRNA-CTGF-3)和1种表达无关序列的质粒pshRNA-CTGF-neg。
3.重组质粒转化DH5αE.coli菌液,挑取单克隆菌落,摇菌,小量提取质粒后进行酶切及基因测序鉴定。对鉴定正确的细菌克隆进行大量质粒提取。
三、结果
提取pshRNA-CTGFs质粒进行SalI酶切鉴定。因在插入的目的基因片段里,我们设计了一个SalI的酶切位点,重组后的质粒被SalI酶切出一条约400bp的DNA小带,提示目的基因接入正确,质粒重组成功。
取各组抽提质粒送上海生工公司进行测序鉴定,测序结果表明分别插入siRNA-CTGF-1、siRNA-CTGF-2、siRNA-CTGF-3和siRNA-CTGF-neg的质粒插入片段与设计片段完全一致,表明重组成功。
四、结论
利用pGenesil-1质粒成功设计并构建了表达针对CTGF mRNA的特异性siRNA的质粒载体。
第二章siRNA表达载体转染人肾小管上皮细胞抑制CTGF表达的实验研究
一、研究目的
研究针对CTGF的siRNA表达载体转染人肾小管上皮细胞株HK-2后对CTGF mRNA及蛋白表达的抑制效果。
二、研究方法
1.HK-2细胞分高糖组及正常对照组分别在含葡萄糖4.5g/L及1g/L的DMEM/F12完全培养基中传代培养,并于第24h、48h、72h、96h和20d收集细胞。以RT-PCR检测CTGF的mRNA表达水平,免疫细胞化学、Western blot检测CTGF蛋白表达水平。
2.以阳离子化合物jetPEI~(TM)为介导,将重组质粒分别转染入高糖组培养20天后的细胞,用空质粒载体做空白对照。各组细胞继续培养于高糖培养基中,分别于24、48、72、96h收集细胞,以RT-PCR检测CTGF的mRNA表达水平,免疫细胞化学、Western blot检测CTGF蛋白表达水平。挑选抑制效果最好的siRNA表达载体进行后续实验。
三、结果
1.HK-2细胞在正常情况下表达CTGF极低,高糖刺激可显著上调细胞CTGF的mRNA及蛋白表达水平。
2.将表达siRNA-CTGF-1～3的质粒分别转染长期(20天)高糖培养的HK-2细胞,48h后测定CTGF mRNA表达,结果提示3条siRNAs均能显著抑制CTGFmRNA表达,但以siRNA-CTGF-1的抑制效率最高,转染细胞后CTGF mRNA水平同另外2条比较显著降低。
3.转染siRNA-CTGF-1的细胞24h时CTGF mRNA水平已出现降低,随着时间延长,抑制率随之增加,48h逐达到最大抑制效果,96h时仍显著低于空白对照组。
4.转染siRNA-CTGF-1的细胞CTGF蛋白表达水平随时间延长进行性下降。
四、结论
高糖刺激可显著上调HK-2细胞CTGF的mRNA及蛋白表达水平。我们设计的3条针对CTGF基因的siRNA均能特异和高效地抑制高糖诱导的这一变化。其中以干扰靶基因序列436-454位点siRNA-CTGF-1的抑制效率最高。
第三章高糖诱导HK-2细胞肥大和EMT的机制及siRNA沉默CTGF表达对其的影响
一、研究目的
研究高糖诱导HK-2细胞肥大和EMT的作用途径及可能机制,并观察细胞转染siRNA-CTGF-1后对这一过程的影响。
二、研究方法
1.HK-2细胞分6组培养:(1)正常对照组:细胞在含葡萄糖1g/L的DMEM/F12完全培养基中传代培养。(2)高糖组:细胞在含葡萄糖4.5g/L的DMEM/F12完全培养基中传代培养。(3)等渗对照组:细胞在含葡萄糖1g/L,甘露醇3.Sg/L的DMEM/F12完全培养基中传代培养;(4)高糖+空白对照组:细胞转染空质粒pGenesil-1后培养于高糖DMEM/F12完全培养基中。(5)高糖+阴性对照组:细胞转染pshRNA-CTGF-neg后培养于高糖DMEM/F12完全培养基中。(6)高糖+siRNA-CTGF-1组:细胞转染pshRNA-CTGF-1后培养于高糖DMEM/F12完全培养基中。
2.分别于各组培养24、48、96h时收集细胞以Real-time荧光定量PCR检测各组CTGF、p27~(kip)的mRNA表达水平。
3.分别于各组培养24、48、96h时收集细胞,以Western blot方法检测CTGF、p27~(kip)、α-SMA、E-cadherin的蛋白表达水平。
4.分别收集各组培养30min,1h,24h,48h时的细胞,以Western blot方法检测磷酸化ERK1/2、磷酸化JAK2、磷酸化STAT3的水平。
5.MTT法检测各组细胞培养24、48、96h时的细胞增殖活力。
6.考马斯亮蓝法测定各组细胞培养24、48、96h时的细胞内总蛋白含量。
7.流式细胞分析技术检测各组细胞培养24、48、96h时的细胞周期分布。
三、结果
1.高糖刺激可引起HK-2细胞的CTGF、p27~(kip)的mRNA及蛋白表达水平显著升高;ERK1/2磷酸化激活;越来越多的细胞被阻滞于G_1期;细胞增殖活力受抑制;细胞肥大主要指标胞内蛋白总含量进行性增高。
2.转染siRNA-CTGF-1能显著抑制高糖诱导HK-2细胞的CTGF、p27~(kip)的mRNA及蛋白高表达,使磷酸化ERK1/2水平降低,更多的细胞由G_1期进入S期,细胞增殖活力增强,细胞肥大主要指标胞内蛋白总含量降低。
3.高糖刺激可呈时间依赖性降低HK-2细胞的E-cadherin蛋白表达水平,与此同时出现α-SMA蛋白表达水平进行性升高,提示高糖可直接诱导肾小管上皮细胞发生EMT。
4.高糖刺激30min时即已显著升高磷酸化JAK2/STAT3水平,1h达峰值,且一直持续至48小时仍高于正常对照组。转染siRNA-CTGF-1能显著降低HK-2细胞的磷酸化JAK2/STAT3水平,同时伴有细胞EMT过程受抑。
四、结论
证实了CTGF是介导高糖诱导肾小管上皮细胞肥大的重要因子,而这一作用是通过激活ERK1/2信号转导,上调p27~(kip)转录水平,使增殖细胞周期停滞于G_1期,从而抑制细胞的增殖引起的。针对CTGF的siRNA能通过降低ERK1/2活性,抑制p27~(kip)表达,从而提高细胞增殖活力,明显减轻高糖诱导的肾小管上皮细胞肥大。
证实高糖可以通过上调CTGF表达,继而激活JAK2/STAT3途径,引起肾小管上皮细胞发生EMT。而通过特异性siRNA抑制CTGF表达后可减轻高糖引起的JAK2/STAT3磷酸化激活,进而抑制肾小管上皮细胞EMT。
Background
Diabetic nephropathy(DN)is a very common complication of diabetes,and it has become one of the main cause of end-stage renal failure now.The most important pathophysiologic characteristic of early-stage of DN is renal hypertrophy,then progress to renal fibrosis in end-stage of DN.About 90%of renal volume is occupied by tubulus,so tubular hypertrophy plays an important role in renal hypertrophy.In addition,tubulointerstitial fibrosis correlates closely to renal disfuction.Being the main structure of tubulointerstitium,renal tubular epithelial cell contributes greatly to tubular hypertrophy and interstitial fibrosis through hypertrophy growth and epithelial mesenchymal transition(EMT).
1.The mechanisms of renal tubular epithelial hypertrophy.
The molecular mechanism of renal tubular epithelial hypertrophy in DN is not fully understood.Cellular proliferation and hypertrophy usually correlate intimately with the progression of cell cycle.In normal condition,tubular epithelia usually stay in GO phase.Only in proliferation,cells enter the G1 phase,then to S phase.If there are some factors make the cells arrest in G1 phase,protein will accumulate in cell and lead to hypertrophy.The CIP/KIP family attracts great interests in the research of cellular hypertrophy in early stage of DN for their inhibitive effect on cell cycle. There are three members in the CIP/KIP family include p21~(kip),p27~(kip),p57~(kip).Renal tubular epithelia only express p21~(kip)and p27~(kip),there are no reports for p57~(kip).
Experiments in vitro demonstrated that the expression of p27~(kip)increased significantly in tubular epithelial cells under high ambient glucose and positively correlated with the cellular hypertrophy.The similar results were observed in vivo. The specific mechanism of up-regulation of p27~(kip)in DN still remain unclear. Jau-Shyang Huang reported that high glucose could induce hypertrophy of tubular epithelia with up-regulated p21~(kip)and p27~(kip),simultaneously with the activation of p42/p44 mitogen-activated protein kinase(p42/p44 MAPK),also called extracellular signal-regulated kinase 1/2(ERK1/2).His report presumed that MAPK may be a mediator of up-regulation of p27~(kip)induced by high glucose in tubular epithelia.
The pathway through which the MAPK is activated needs to be further studied.For the past few years,connective tissue growth factor(CTGF)received extraordinary attention in DN research field.It is generally accepted that CTGF plays a very important role in the genesis and progress of DN.There were some experiments demonstrated that CTGF could induce morphological and functional changes in mesangial cells and interstitial cells through activating ERK1/2 pathway.Meanwhile it was reported that CTGF was a key mediator of tubular epithelia hypertrophy induced by angiotensinⅡ.Hence we presume that CTGF may mediate tubular epithelia hypertrophy through activating ERK1/2 to up-reg??e p27~(kip)expression. This presumption needs to be testified.
2.The mechanisms of EMT of renal tubular epithelial cells.
EMT of renal tubular epithelia is the main cause of renal interstitial fibrosis.In normal condition,tubular epithelia adhere together closely through tight junctions, E-cadherin is the main constituent of the tight junctions.One of the basic steps of EMT is E-cadherin decreasing.Tubular epithelia lose their adherent abilities and fall off from basement.The cytoskeletons are rearranged and a-smooth muscle actins (a-SMA)presented which was only expressed by mesenchymal cells.The mesenchymal fibroblasts transformed by tubular epithelia can synthesise abundant extracellular matrix(ECM)and lead to interstitial fibrosis.We had found that tubular epithelia have higher CTGF expression under high ambient glucose in vitro or in vivo, and CTGF was an acknowledged effective fibrotic inducer.CTGF can induce EMT and cause interstitial fibrosis.Nevertheless,how EMT is induced by CTGF still need to be explored.Janus kinase(JAK)/signal transduction and activators of transcription(STAT)are very important signal conductive pathways of cytokines and growth factors.Recently,it was reported that high glucose could activate JAK2、STAT1 and STAT3 in tubular epithelia in vitro,accompanied with synthesis of ECM. But there are no reports about the role of JAK2/STATs in EMT.In the study on epithelial cell carcinoma,it is reported that JAK2/STAT3 could regulate the adherence of epithelia,maybe contribute to the EMT in malignant process.Dose JAK2/STAT pathway contribute to the EMT of tubular epithelia in DN?We had known that CTGF is an important mediator of EMT in DN,does CTGF induce EMT through JAK2/STAT pathway?All these questions still need to be answered.
Although CTGF is a very important factor in DN,it's biological activity is comparatively simple and it's expression is usually very little on normal conditions. CTGF may be a promising therapeutic target for DN.However,the detailed role of CTGF in DN process is not fully understood.We yet need effective,safe and pragmatic methods to interfere the expression of CTGF.That is a long way to go.The new technique of small interfering RNA(siRNA)shed light on the research. Compared with the traditional techniques of antisense oligonucleotides and gene knock-out,siRNA has the superioriti??f high performance,specificity,easy manipulation and economy.It has become the focus of clinical application research on gene silence.
In this study,in order to investigate the mechanisms of tubular epithelial hypertrophy and EMT induced by high glucose,we detected the mRNA and protein levels of CTGF and p27~(kip),protein levels of phospho-ERK1/2,phospho-JAK2, phospho-STAT3,a-SMA and E-cadherin,the total cellular protein contents,and cell cycle distribution of human tubular epithelial cells cultured in high glucose medium. Further more,we inhibited the CTGF expression of tubular epithelia by siRNA,and observed the changes of hypertrophy and EMT induced by high glucose.Through these studies,we try to elucidate the pathways through which CTGF mediate the tubular epithelial hypertrophy and EMT under high glucose.Meanwhile,give some information for clinical application research on CTGF RNA interference.
ChapterⅠConstruction and identification of Specific CTGF siRNA expression vector
Objective
To construct and identify siRNA expression plasmid vectors targeting to CTGF mRNA.
Methods
1.According to CTGF mRNA sequence in Genebank of NCBI and using the computer aided design software,we designed 3 siRNA sequences targeting to CTGF mRNA(siRNA-CTGF-1,siRNA-CTGF-2,siRNA-CTGF-3)and 1 non-sense sequence(siRNA-CTGF-neg),then synthesized 4 short hairpin RNA(shRNA) oligonucleotide fragments include the 3 siRNAs and non-sense sequence respectively.
2.pGenesil-1 plasmids were used to construct the specificity RNA interfering vectors contain the siRNAs aim at CTGF gene respectively(named pshRNA-CTGF-1, pshRNA-CTGF-2,pshRNA-CTGF-3)and negtive control vector(named pshRNA-CTGF-neg)contains the non-sense sequence.
3.The recombinant plasmids were transformed into DH5αcompetence germ for amplification.Bacterial clones possibly containing recombinant pshRNA-CTGFs were selected and amplified for extracting plamid.The recombinant plasmids were certificated by SalⅠenzymic digestion and nucleotide sequence analysis.
Result
In the inserted oligonucleotide fragments,we designed SalⅠenzymic digestion sites,if the plasmids were recombined correctly,400bp band were detected by electrophoresis after digestion.The result showed the 4 kinds of plasmids were all recom??d correctly.
Nucleotide sequence analysis showed all the plasmids were constructed exactly as being designed.
Conclusion
We successfully designed and constructed specific siRNA expression plasmids targeting to CTGF mRNA.
hapterⅡExperimental studies on inhibition of CTGF expression by siRNAs in human renal tubular epithelial cells
Objective
To observe the inhibitive effects of siRNA expression vectors on CTGF mRNA and protein levels in human renal tubular epithelial cells line HK-2 cells.
Methods
1.The HK-2 cells were cultured in high glucose(4.5g/L)and normal glucose (1g/L)DMED/F12 medium respectively.Collected the cells on 24h,48h,72h,96h and 20d,detected the levels of CTGF mRNA by RT-PCR,the levels of CTGF protein by immunocytochemistry and Western blot.
2.HK-2 cells cultured in high glucose for 20d were transfected with combined plasimids by jetPEI~(TM).Use the empty plasmids as blank control.All the transfected cells were sequentially cultured in high glucose medium,collected cells on 24h,48h, 72h and 96h.Detected the levels of CTGF mRNA by RT-PCR,the levels of CTGF protein by immunocytochemistry and Western blot.Selected the most effective siRNA expression vector on inhibiting the expression of CTGF for the next experiments.
Result
1.The levels of CTGF mRNA and protein were very low in HK-2 cells under normal ambient glucose.High glucose could significantly up-regulate the levels of CTGF mRNA and protein.
2.We detected the CTGF mRNA levels on 48h after transfection with siRNA-CTGF-1,siRNA-CTGF-2,siRNA-CTGF-3 expressional plasmids.All the 3 kinds of siRNA could significantly inhibit the expression of CTGF mRNA,but siRNA-CTGF-1 had the highest effici??.
3.The CTGF mRNA decreased significantly after transfected with siRNA-CTGF-1 for 24h,and down to the lowest level after 48h,the inhibitive effect still remained satisfactory till 96h.
4.The expressions of CTGF protein in siRNA-CTGF-1 transfected cells decreased progressively with the time past.
Conclusion
High glucose can significantly up-regulate the levels of CTGF mRNA and protein in HK-2 cells.The 3 siRNAs targeting to CTGF mRNA can inhibit CTGF mRNA and protein expression in HK-2 efficiently.siRNA-CTGF-1 has the highest inhibitive efficiency.The interferential site is 436-454 in CTGF mRNA gene sequence.
ChapterⅢThe mechanisms of tubular epithelial hypertrophy and EMT induced by high glucose and the effects of inhibiting CTGF by siRNA
Objective
To elucidate the mechanisms of tubular epithelial hypertrophy and EMT induced by high glucose,and to observe the changes after transfected with siRNA-CTGF-1.
Methods
1.HK-2 cells were cultured in 6 groups:
(1)Normal control group:cells were cultured in DMEM/F12 medium containing 1g/L glucose.
(2)High glucose group:cells were cultured in DMEM/F12 medium containing 4.5g/L glucose.
(3)Iso-osmia control group:cells were cultured in DMEM/F12 medium containing 1g/L glucose and 3.5g/L mannitol.
(4)High glucose+blank control group:cells were transfected with blank plasmids (pGenesil)and cultured in high glucose medium.
(5)High glucose+negative control group:cells were transfected with pshRNA-CTGF-neg and cultured in high glucose medium.
(6)High glucose+siRNA-CTGF-1 group:cells were transfected with pshRNA-CTGF-1 and cultured in high glucose medium.
2.Collection of cells of every group after being cultured for 24h,48h and 96h. Detection of the mRNA levels of CTGF and p27~(kip)by real-time PCR.
3.Collection of cells of every group after being cultured for 24h,48h and 96h. Detection of the protein levels of CTGF,p27~(kip),a-SMA and E-cadherin by Western blot analysis.
4.Collection of cells of every group after being cultured for 30min,1h,24h and 48h.Detection of the protein levels of phospho-ERKl/2,phospho-JAK2 and phospho-STAT3 by Western blot analysis.
5.Observation of the cellular proliferative activities of every group after being cultured for 24h,48h and 96h by MTT.
6.Detection of the total cellular protein contents of every group after being cultured for 24h,48h and 96h by Bradford method.
7.Analysis of the cell cycle process of every group after cultured for 24h,48h and 96h by flow cytometry.
Result
1.High glucose could significantly up-regulate the mRNA and protein levels of CTGF and p27~(kip),activate ERK1/2 signal conductive pathway in HK-2 cells.The cellular proliferative activities degraded progressively after being exposed to high glucose,more and more cells were arrested in G1 phase with increasing total cellular protein content reflecting cellular hypertrophy.
2.Transfection with siRNA-CTGF-1 could significantly inhibit the up-regulation of mRNA and protein levels of CTGF and p27~(kip)induced by high glucose, accompanied with decreasing level of phospho-ERK1/2.The cellular proliferative activities improved,more cells could entered S phase successfully,total cellular protein contents decreased reflecting cellular hypertrophy were lightened.
3.High glucose could significantly down-regulate the levels of E-cadherin in HK-2 cells by time dependent way,and up-regulate the levels of a-SMA inversely.These results show that high glucose can induce EMT in HK-2 cells directly.
4.Phospho-JAK2 and phospho-STAT3 increased significantly in HK-2 cells after being cultured in high glucose for 30 min,rose to peak level in 60min and constantly maintained higher than normal control group after 48h.Transfection with siRNA-CTGF-1 could significantly inhibit the increasement of phospho-JAK2 and phospho-STAT3 induced by high glucose,accompanied with inhibiting EMT process of tubular epithelia.
Conclusion
CTGF is an important mediator of tubular epithelial hypertrophy induced by high glucose.The mechanism is that CTGF up-regulates the p27~(kip)expression through activating ERK1/2 pathway.Up-regulated p27~(kip)lead proliferative cell to be arrested in G1 phase and cause cellular hypertrophy.Transfected with siRNA targeting to CTGF mRNA can repress ERK1/2 activating and the up-regulation of p27~(kip)induced by high glucose,thus improve the cellular proliferative activities and cellular hypertrophy.
High glucose induce EMT in tubular epithelia through up-regulating CTGF mRNA and protein,and then activating JAK2/STAT3 pathway.Transfected with siRNA targeting to CTGF mRNA can repress the activation of JAK2/STAT3 induced by high glucose,then inhibit EMT of tubular epithelial cells.
糖尿病肾病(diabetic nephropathy,DN)是糖尿病的常见并发症之一,也是终末期肾功能衰竭的主要原因之一。肾脏肥大是DN早期最重要的病理生理学特征,晚期则表现为纤维化。肾小管体积约占整个肾脏体积的90%,因而肾小管肥大在肾脏肥大中占有十分重要的地位。肾小管间质纤维化的进展程度则与肾功能的进行性下降密切相关。肾小管上皮细胞作为构成肾小管间质结构的主要细胞之一,在DN肾小管肥大及间质纤维化过程中扮演了重要的角色。肾小管上皮细胞的肥大及向间充质细胞转分化(epithelial mesenchymal transition,EMT)现象是引起肾小管肥大,间质纤维化的重要原因。
(一)肾小管上皮细胞肥大机制
目前关于DN时肾小管上皮细胞肥大的具体机制仍不清楚。已知细胞的增生及肥大往往与细胞周期的转换密切相关。正常情况下肾小管上皮细胞均处于静息期即G_0期。当细胞需要增殖时即进入G_1期,如果某些因素导致细胞停滞在G_1期无法进入S期,则会引起蛋白在细胞内聚积,细胞肥大。作为细胞周期素激酶抑制剂(CDK inhibitors,CKIs)的CIP/KIP家族具有抑制细胞周期转换的作用,因此其在DN发病早期肾脏固有细胞肥大中的作用倍受关注。CIP/KIP家族包括p21~(kip)、p27~(kip)、p57~(kip)三个成员。肾小管上皮细胞主要以表达p27~(kip),p21~(kip)为主,p57~(kip)则尚未见文献报道。
体外及体内研究提示在高糖及糖尿病状态时肾小管上皮细胞p27~(kip)蛋白表达增加,并与细胞肥大呈正比。然而DN时p27~(kip)表达上调的具体机制仍不清楚。Jau-Shyang Huang发现高糖引起体外培养的肾小管上皮细胞肥大,p21~(kip)和p27~(kip)表达升高的同时伴有p42/p44丝裂原活化蛋白激酶(mitogen-activated proteinkinase,MAPK)又称细胞外信号调节激酶1/2(extracellular signal-regulatedkinase 1/2,ERK1/2)的磷酸化激活,提示MAPK信号途径可能是高糖刺激肾小管上皮细胞表达p27~(kip)的细胞内机制。
MAPK信号途径如何被激活,其具体机制是什么还有待探索。近年来结缔组织生长因子(connective tissue growth factor,CTGF)由于在DN的发生和发展中起了重要作用而倍受关注。有实验证明CTGF可激活肾小球系膜细胞和肾间质细胞的ERK1/2通路引起细胞形态功能变化;新近研究又发现CTGF是血管紧张素Ⅱ诱导人近端肾小管上皮细胞肥大的重要介质。因此我们推测CTGF可能通过激活ERK1/2通道来调节p27~(kip)的表达进而诱导肾小管上皮细胞肥大,这一推论有待证实。
(二)肾小管上皮细胞EMT机制
肾小管上皮细胞EMT是肾间质纤维化的重要原因。正常状态下肾小管上皮细胞通过细胞间黏附机制紧密连接在一起,E-钙黏蛋白(E-cadherin)是组成细胞间紧密连接的重要成分,在保持细胞完整性和极性中起重要作用。肾小管上皮细胞表型转化发生的基本步骤之一,就是E-cadherin表达减少,上皮细胞丧失黏附特性,从基底膜上脱落下来,胞浆内细胞骨架重排,表达间充质细胞表型α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)。上皮细胞转分化后形成的间质成纤维细胞可大量合成细胞外基质(Extracellar matrix,ECM)成分,引起间质纤维化。CTGF是引起肾间质纤维化的重要细胞因子。体外及体内实验均证明高糖及糖尿病状态可刺激肾小管上皮细胞表达CTGF,后者是公认的能促进肾小管EMT的因素之一。但CTGF引起EMT的具体机制仍不明确。Janus激酶(Januskinase,JAK)/信号转导与转氯?钜蜃?signal transduction and activators oftranscription,STAT)是一条重要的介导细胞因子和生长因子信号传导的通路。近来有研究揭示高糖刺激可引起体外培养的肾小管上皮细胞内JAK2、STAT1和STAT3激活,伴有ECM成分合成增多。但其在肾小管上皮细胞EMT过程中的作用如何,文献尚未见报道。对上皮细胞癌的研究表明,JAK2/STAT3信号通路参与调节上皮细胞之间的黏附,可能与恶性病变中EMT有关。那么JAK2/STATs信号途径是否也参与了DN时肾小管上皮细胞的EMT呢?前已述CTGF是DN时肾小管EMT的重要因素,那么CTGF是否能通过激活JAK2/STATs信号途径引起EMT呢?这些问题都有待探索。
因为CTGF在DN发展过程中占有重要地位,且作用单一,在生理情况下表达很低,所以被认为是一个可选择的治疗DN的靶点。但对于CTGF在糖尿病肾病中的具体作用机制仍不完全明了,且缺乏有效、安全、实用的干预方式,故目前在这方面的治疗研究仍无大的进展。小分子干扰RNA(small interfering RNA,siRNA)是近年出现的一种新的抑制基因表达的技术方法。与传统的反义寡核苷酸、基因敲除相比,具有特异、高效且易操作等优点,已成为研究基因沉默在临床应用的前沿热点。
基于上述背景,本研究设想,通过检测高糖条件下培养的人肾小管上皮细胞CTGF、p27~(kip)的mRNA及蛋白表达水平,ERK1/2、JAK2、STAT3磷酸化水平,α-SMA、E-cadherin的蛋白水平,细胞总蛋白含量及周期变化,探讨高糖诱导肾小管上皮细胞肥大及EMT的作用途径及可能的发生机制;通过构建可高效抑制CTGFmRNA表达的siRNA载体,在体外转染肾小管上皮细胞,观测抑制CTGF表达后对细胞肥大及EMT相关指标的影响,进一步探寻CTGF对肾小管上皮细胞肥大及EMT的诱导机制,为针对CTGF的RNA干扰(RNA interference,RNAi)治疗提供一定的实验基础。
第一章CTGF基因特异性siRNA的设计、合成及表达载体的构建和鉴定
一、研究目的
构建并鉴定稳定表达针对CTGF基因的siRNA的质粒。
二、研究方法
1.根据人CTGF mRNA基因编码区序列,利用计算机辅助设计软件设计3条针对CTGF的siRNAs(siRNA-CTGF-1、siRNA-CTGF-2及siRNA-CTGF-3),和1条作为阴性对照的无关序列(siRNA-CTGF-neg)。并合成分别包含这4条siRNAs的短发夹状RNA(shRNA)的模板脱氧寡核苷酸序列。
2.将上述shRNA模板脱氧寡核苷酸序列分别插入pGenesil-1质粒,构建分别表达3种特异性siRNA的质粒(pshRNA-CTGF-1、pshRNA-CTGF-2、pshRNA-CTGF-3)和1种表达无关序列的质粒pshRNA-CTGF-neg。
3.重组质粒转化DH5αE.coli菌液,挑取单克隆菌落,摇菌,小量提取质粒后进行酶切及基因测序鉴定。对鉴定正确的细菌克隆进行大量质粒提取。
三、结果
提取pshRNA-CTGFs质粒进行SalI酶切鉴定。因在插入的目的基因片段里,我们设计了一个SalI的酶切位点,重组后的质粒被SalI酶切出一条约400bp的DNA小带,提示目的基因接入正确,质粒重组成功。
取各组抽提质粒送上海生工公司进行测序鉴定,测序结果表明分别插入siRNA-CTGF-1、siRNA-CTGF-2、siRNA-CTGF-3和siRNA-CTGF-neg的质粒插入片段与设计片段完全一致,表明重组成功。
四、结论
利用pGenesil-1质粒成功设计并构建了表达针对CTGF mRNA的特异性siRNA的质粒载体。
第二章siRNA表达载体转染人肾小管上皮细胞抑制CTGF表达的实验研究
一、研究目的
研究针对CTGF的siRNA表达载体转染人肾小管上皮细胞株HK-2后对CTGF mRNA及蛋白表达的抑制效果。
二、研究方法
1.HK-2细胞分高糖组及正常对照组分别在含葡萄糖4.5g/L及1g/L的DMEM/F12完全培养基中传代培养,并于第24h、48h、72h、96h和20d收集细胞。以RT-PCR检测CTGF的mRNA表达水平,免疫细胞化学、Western blot检测CTGF蛋白表达水平。
2.以阳离子化合物jetPEI~(TM)为介导,将重组质粒分别转染入高糖组培养20天后的细胞,用空质粒载体做空白对照。各组细胞继续培养于高糖培养基中,分别于24、48、72、96h收集细胞,以RT-PCR检测CTGF的mRNA表达水平,免疫细胞化学、Western blot检测CTGF蛋白表达水平。挑选抑制效果最好的siRNA表达载体进行后续实验。
三、结果
1.HK-2细胞在正常情况下表达CTGF极低,高糖刺激可显著上调细胞CTGF的mRNA及蛋白表达水平。
2.将表达siRNA-CTGF-1～3的质粒分别转染长期(20天)高糖培养的HK-2细胞,48h后测定CTGF mRNA表达,结果提示3条siRNAs均能显著抑制CTGFmRNA表达,但以siRNA-CTGF-1的抑制效率最高,转染细胞后CTGF mRNA水平同另外2条比较显著降低。
3.转染siRNA-CTGF-1的细胞24h时CTGF mRNA水平已出现降低,随着时间延长,抑制率随之增加,48h逐达到最大抑制效果,96h时仍显著低于空白对照组。
4.转染siRNA-CTGF-1的细胞CTGF蛋白表达水平随时间延长进行性下降。
四、结论
高糖刺激可显著上调HK-2细胞CTGF的mRNA及蛋白表达水平。我们设计的3条针对CTGF基因的siRNA均能特异和高效地抑制高糖诱导的这一变化。其中以干扰靶基因序列436-454位点siRNA-CTGF-1的抑制效率最高。
第三章高糖诱导HK-2细胞肥大和EMT的机制及siRNA沉默CTGF表达对其的影响
一、研究目的
研究高糖诱导HK-2细胞肥大和EMT的作用途径及可能机制,并观察细胞转染siRNA-CTGF-1后对这一过程的影响。
二、研究方法
1.HK-2细胞分6组培养:(1)正常对照组:细胞在含葡萄糖1g/L的DMEM/F12完全培养基中传代培养。(2)高糖组:细胞在含葡萄糖4.5g/L的DMEM/F12完全培养基中传代培养。(3)等渗对照组:细胞在含葡萄糖1g/L,甘露醇3.Sg/L的DMEM/F12完全培养基中传代培养;(4)高糖+空白对照组:细胞转染空质粒pGenesil-1后培养于高糖DMEM/F12完全培养基中。(5)高糖+阴性对照组:细胞转染pshRNA-CTGF-neg后培养于高糖DMEM/F12完全培养基中。(6)高糖+siRNA-CTGF-1组:细胞转染pshRNA-CTGF-1后培养于高糖DMEM/F12完全培养基中。
2.分别于各组培养24、48、96h时收集细胞以Real-time荧光定量PCR检测各组CTGF、p27~(kip)的mRNA表达水平。
3.分别于各组培养24、48、96h时收集细胞,以Western blot方法检测CTGF、p27~(kip)、α-SMA、E-cadherin的蛋白表达水平。
4.分别收集各组培养30min,1h,24h,48h时的细胞,以Western blot方法检测磷酸化ERK1/2、磷酸化JAK2、磷酸化STAT3的水平。
5.MTT法检测各组细胞培养24、48、96h时的细胞增殖活力。
6.考马斯亮蓝法测定各组细胞培养24、48、96h时的细胞内总蛋白含量。
7.流式细胞分析技术检测各组细胞培养24、48、96h时的细胞周期分布。
三、结果
1.高糖刺激可引起HK-2细胞的CTGF、p27~(kip)的mRNA及蛋白表达水平显著升高;ERK1/2磷酸化激活;越来越多的细胞被阻滞于G_1期;细胞增殖活力受抑制;细胞肥大主要指标胞内蛋白总含量进行性增高。
2.转染siRNA-CTGF-1能显著抑制高糖诱导HK-2细胞的CTGF、p27~(kip)的mRNA及蛋白高表达,使磷酸化ERK1/2水平降低,更多的细胞由G_1期进入S期,细胞增殖活力增强,细胞肥大主要指标胞内蛋白总含量降低。
3.高糖刺激可呈时间依赖性降低HK-2细胞的E-cadherin蛋白表达水平,与此同时出现α-SMA蛋白表达水平进行性升高,提示高糖可直接诱导肾小管上皮细胞发生EMT。
4.高糖刺激30min时即已显著升高磷酸化JAK2/STAT3水平,1h达峰值,且一直持续至48小时仍高于正常对照组。转染siRNA-CTGF-1能显著降低HK-2细胞的磷酸化JAK2/STAT3水平,同时伴有细胞EMT过程受抑。
四、结论
证实了CTGF是介导高糖诱导肾小管上皮细胞肥大的重要因子,而这一作用是通过激活ERK1/2信号转导,上调p27~(kip)转录水平,使增殖细胞周期停滞于G_1期,从而抑制细胞的增殖引起的。针对CTGF的siRNA能通过降低ERK1/2活性,抑制p27~(kip)表达,从而提高细胞增殖活力,明显减轻高糖诱导的肾小管上皮细胞肥大。
证实高糖可以通过上调CTGF表达,继而激活JAK2/STAT3途径,引起肾小管上皮细胞发生EMT。而通过特异性siRNA抑制CTGF表达后可减轻高糖引起的JAK2/STAT3磷酸化激活,进而抑制肾小管上皮细胞EMT。
Background
Diabetic nephropathy(DN)is a very common complication of diabetes,and it has become one of the main cause of end-stage renal failure now.The most important pathophysiologic characteristic of early-stage of DN is renal hypertrophy,then progress to renal fibrosis in end-stage of DN.About 90%of renal volume is occupied by tubulus,so tubular hypertrophy plays an important role in renal hypertrophy.In addition,tubulointerstitial fibrosis correlates closely to renal disfuction.Being the main structure of tubulointerstitium,renal tubular epithelial cell contributes greatly to tubular hypertrophy and interstitial fibrosis through hypertrophy growth and epithelial mesenchymal transition(EMT).
1.The mechanisms of renal tubular epithelial hypertrophy.
The molecular mechanism of renal tubular epithelial hypertrophy in DN is not fully understood.Cellular proliferation and hypertrophy usually correlate intimately with the progression of cell cycle.In normal condition,tubular epithelia usually stay in GO phase.Only in proliferation,cells enter the G1 phase,then to S phase.If there are some factors make the cells arrest in G1 phase,protein will accumulate in cell and lead to hypertrophy.The CIP/KIP family attracts great interests in the research of cellular hypertrophy in early stage of DN for their inhibitive effect on cell cycle. There are three members in the CIP/KIP family include p21~(kip),p27~(kip),p57~(kip).Renal tubular epithelia only express p21~(kip)and p27~(kip),there are no reports for p57~(kip).
Experiments in vitro demonstrated that the expression of p27~(kip)increased significantly in tubular epithelial cells under high ambient glucose and positively correlated with the cellular hypertrophy.The similar results were observed in vivo. The specific mechanism of up-regulation of p27~(kip)in DN still remain unclear. Jau-Shyang Huang reported that high glucose could induce hypertrophy of tubular epithelia with up-regulated p21~(kip)and p27~(kip),simultaneously with the activation of p42/p44 mitogen-activated protein kinase(p42/p44 MAPK),also called extracellular signal-regulated kinase 1/2(ERK1/2).His report presumed that MAPK may be a mediator of up-regulation of p27~(kip)induced by high glucose in tubular epithelia.
The pathway through which the MAPK is activated needs to be further studied.For the past few years,connective tissue growth factor(CTGF)received extraordinary attention in DN research field.It is generally accepted that CTGF plays a very important role in the genesis and progress of DN.There were some experiments demonstrated that CTGF could induce morphological and functional changes in mesangial cells and interstitial cells through activating ERK1/2 pathway.Meanwhile it was reported that CTGF was a key mediator of tubular epithelia hypertrophy induced by angiotensinⅡ.Hence we presume that CTGF may mediate tubular epithelia hypertrophy through activating ERK1/2 to up-reg??e p27~(kip)expression. This presumption needs to be testified.
2.The mechanisms of EMT of renal tubular epithelial cells.
EMT of renal tubular epithelia is the main cause of renal interstitial fibrosis.In normal condition,tubular epithelia adhere together closely through tight junctions, E-cadherin is the main constituent of the tight junctions.One of the basic steps of EMT is E-cadherin decreasing.Tubular epithelia lose their adherent abilities and fall off from basement.The cytoskeletons are rearranged and a-smooth muscle actins (a-SMA)presented which was only expressed by mesenchymal cells.The mesenchymal fibroblasts transformed by tubular epithelia can synthesise abundant extracellular matrix(ECM)and lead to interstitial fibrosis.We had found that tubular epithelia have higher CTGF expression under high ambient glucose in vitro or in vivo, and CTGF was an acknowledged effective fibrotic inducer.CTGF can induce EMT and cause interstitial fibrosis.Nevertheless,how EMT is induced by CTGF still need to be explored.Janus kinase(JAK)/signal transduction and activators of transcription(STAT)are very important signal conductive pathways of cytokines and growth factors.Recently,it was reported that high glucose could activate JAK2、STAT1 and STAT3 in tubular epithelia in vitro,accompanied with synthesis of ECM. But there are no reports about the role of JAK2/STATs in EMT.In the study on epithelial cell carcinoma,it is reported that JAK2/STAT3 could regulate the adherence of epithelia,maybe contribute to the EMT in malignant process.Dose JAK2/STAT pathway contribute to the EMT of tubular epithelia in DN?We had known that CTGF is an important mediator of EMT in DN,does CTGF induce EMT through JAK2/STAT pathway?All these questions still need to be answered.
Although CTGF is a very important factor in DN,it's biological activity is comparatively simple and it's expression is usually very little on normal conditions. CTGF may be a promising therapeutic target for DN.However,the detailed role of CTGF in DN process is not fully understood.We yet need effective,safe and pragmatic methods to interfere the expression of CTGF.That is a long way to go.The new technique of small interfering RNA(siRNA)shed light on the research. Compared with the traditional techniques of antisense oligonucleotides and gene knock-out,siRNA has the superioriti??f high performance,specificity,easy manipulation and economy.It has become the focus of clinical application research on gene silence.
In this study,in order to investigate the mechanisms of tubular epithelial hypertrophy and EMT induced by high glucose,we detected the mRNA and protein levels of CTGF and p27~(kip),protein levels of phospho-ERK1/2,phospho-JAK2, phospho-STAT3,a-SMA and E-cadherin,the total cellular protein contents,and cell cycle distribution of human tubular epithelial cells cultured in high glucose medium. Further more,we inhibited the CTGF expression of tubular epithelia by siRNA,and observed the changes of hypertrophy and EMT induced by high glucose.Through these studies,we try to elucidate the pathways through which CTGF mediate the tubular epithelial hypertrophy and EMT under high glucose.Meanwhile,give some information for clinical application research on CTGF RNA interference.
ChapterⅠConstruction and identification of Specific CTGF siRNA expression vector
Objective
To construct and identify siRNA expression plasmid vectors targeting to CTGF mRNA.
Methods
1.According to CTGF mRNA sequence in Genebank of NCBI and using the computer aided design software,we designed 3 siRNA sequences targeting to CTGF mRNA(siRNA-CTGF-1,siRNA-CTGF-2,siRNA-CTGF-3)and 1 non-sense sequence(siRNA-CTGF-neg),then synthesized 4 short hairpin RNA(shRNA) oligonucleotide fragments include the 3 siRNAs and non-sense sequence respectively.
2.pGenesil-1 plasmids were used to construct the specificity RNA interfering vectors contain the siRNAs aim at CTGF gene respectively(named pshRNA-CTGF-1, pshRNA-CTGF-2,pshRNA-CTGF-3)and negtive control vector(named pshRNA-CTGF-neg)contains the non-sense sequence.
3.The recombinant plasmids were transformed into DH5αcompetence germ for amplification.Bacterial clones possibly containing recombinant pshRNA-CTGFs were selected and amplified for extracting plamid.The recombinant plasmids were certificated by SalⅠenzymic digestion and nucleotide sequence analysis.
Result
In the inserted oligonucleotide fragments,we designed SalⅠenzymic digestion sites,if the plasmids were recombined correctly,400bp band were detected by electrophoresis after digestion.The result showed the 4 kinds of plasmids were all recom??d correctly.
Nucleotide sequence analysis showed all the plasmids were constructed exactly as being designed.
Conclusion
We successfully designed and constructed specific siRNA expression plasmids targeting to CTGF mRNA.
hapterⅡExperimental studies on inhibition of CTGF expression by siRNAs in human renal tubular epithelial cells
Objective
To observe the inhibitive effects of siRNA expression vectors on CTGF mRNA and protein levels in human renal tubular epithelial cells line HK-2 cells.
Methods
1.The HK-2 cells were cultured in high glucose(4.5g/L)and normal glucose (1g/L)DMED/F12 medium respectively.Collected the cells on 24h,48h,72h,96h and 20d,detected the levels of CTGF mRNA by RT-PCR,the levels of CTGF protein by immunocytochemistry and Western blot.
2.HK-2 cells cultured in high glucose for 20d were transfected with combined plasimids by jetPEI~(TM).Use the empty plasmids as blank control.All the transfected cells were sequentially cultured in high glucose medium,collected cells on 24h,48h, 72h and 96h.Detected the levels of CTGF mRNA by RT-PCR,the levels of CTGF protein by immunocytochemistry and Western blot.Selected the most effective siRNA expression vector on inhibiting the expression of CTGF for the next experiments.
Result
1.The levels of CTGF mRNA and protein were very low in HK-2 cells under normal ambient glucose.High glucose could significantly up-regulate the levels of CTGF mRNA and protein.
2.We detected the CTGF mRNA levels on 48h after transfection with siRNA-CTGF-1,siRNA-CTGF-2,siRNA-CTGF-3 expressional plasmids.All the 3 kinds of siRNA could significantly inhibit the expression of CTGF mRNA,but siRNA-CTGF-1 had the highest effici??.
3.The CTGF mRNA decreased significantly after transfected with siRNA-CTGF-1 for 24h,and down to the lowest level after 48h,the inhibitive effect still remained satisfactory till 96h.
4.The expressions of CTGF protein in siRNA-CTGF-1 transfected cells decreased progressively with the time past.
Conclusion
High glucose can significantly up-regulate the levels of CTGF mRNA and protein in HK-2 cells.The 3 siRNAs targeting to CTGF mRNA can inhibit CTGF mRNA and protein expression in HK-2 efficiently.siRNA-CTGF-1 has the highest inhibitive efficiency.The interferential site is 436-454 in CTGF mRNA gene sequence.
ChapterⅢThe mechanisms of tubular epithelial hypertrophy and EMT induced by high glucose and the effects of inhibiting CTGF by siRNA
Objective
To elucidate the mechanisms of tubular epithelial hypertrophy and EMT induced by high glucose,and to observe the changes after transfected with siRNA-CTGF-1.
Methods
1.HK-2 cells were cultured in 6 groups:
(1)Normal control group:cells were cultured in DMEM/F12 medium containing 1g/L glucose.
(2)High glucose group:cells were cultured in DMEM/F12 medium containing 4.5g/L glucose.
(3)Iso-osmia control group:cells were cultured in DMEM/F12 medium containing 1g/L glucose and 3.5g/L mannitol.
(4)High glucose+blank control group:cells were transfected with blank plasmids (pGenesil)and cultured in high glucose medium.
(5)High glucose+negative control group:cells were transfected with pshRNA-CTGF-neg and cultured in high glucose medium.
(6)High glucose+siRNA-CTGF-1 group:cells were transfected with pshRNA-CTGF-1 and cultured in high glucose medium.
2.Collection of cells of every group after being cultured for 24h,48h and 96h. Detection of the mRNA levels of CTGF and p27~(kip)by real-time PCR.
3.Collection of cells of every group after being cultured for 24h,48h and 96h. Detection of the protein levels of CTGF,p27~(kip),a-SMA and E-cadherin by Western blot analysis.
4.Collection of cells of every group after being cultured for 30min,1h,24h and 48h.Detection of the protein levels of phospho-ERKl/2,phospho-JAK2 and phospho-STAT3 by Western blot analysis.
5.Observation of the cellular proliferative activities of every group after being cultured for 24h,48h and 96h by MTT.
6.Detection of the total cellular protein contents of every group after being cultured for 24h,48h and 96h by Bradford method.
7.Analysis of the cell cycle process of every group after cultured for 24h,48h and 96h by flow cytometry.
Result
1.High glucose could significantly up-regulate the mRNA and protein levels of CTGF and p27~(kip),activate ERK1/2 signal conductive pathway in HK-2 cells.The cellular proliferative activities degraded progressively after being exposed to high glucose,more and more cells were arrested in G1 phase with increasing total cellular protein content reflecting cellular hypertrophy.
2.Transfection with siRNA-CTGF-1 could significantly inhibit the up-regulation of mRNA and protein levels of CTGF and p27~(kip)induced by high glucose, accompanied with decreasing level of phospho-ERK1/2.The cellular proliferative activities improved,more cells could entered S phase successfully,total cellular protein contents decreased reflecting cellular hypertrophy were lightened.
3.High glucose could significantly down-regulate the levels of E-cadherin in HK-2 cells by time dependent way,and up-regulate the levels of a-SMA inversely.These results show that high glucose can induce EMT in HK-2 cells directly.
4.Phospho-JAK2 and phospho-STAT3 increased significantly in HK-2 cells after being cultured in high glucose for 30 min,rose to peak level in 60min and constantly maintained higher than normal control group after 48h.Transfection with siRNA-CTGF-1 could significantly inhibit the increasement of phospho-JAK2 and phospho-STAT3 induced by high glucose,accompanied with inhibiting EMT process of tubular epithelia.
Conclusion
CTGF is an important mediator of tubular epithelial hypertrophy induced by high glucose.The mechanism is that CTGF up-regulates the p27~(kip)expression through activating ERK1/2 pathway.Up-regulated p27~(kip)lead proliferative cell to be arrested in G1 phase and cause cellular hypertrophy.Transfected with siRNA targeting to CTGF mRNA can repress ERK1/2 activating and the up-regulation of p27~(kip)induced by high glucose,thus improve the cellular proliferative activities and cellular hypertrophy.
High glucose induce EMT in tubular epithelia through up-regulating CTGF mRNA and protein,and then activating JAK2/STAT3 pathway.Transfected with siRNA targeting to CTGF mRNA can repress the activation of JAK2/STAT3 induced by high glucose,then inhibit EMT of tubular epithelial cells.
引文
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[4]Liu BC,Li MX,Zhang JD,et al.Inhibition of integrin-linked kinase via a siRNA expression plasmid attenuates connective tissue growth factor-induced human proximal tubular epithelial cells to mesenchymal transition.Am J Nephrol JT,2008,28(1):143-151.
[5]Burns WC,Twigg SM,Forbes JM,et al.Connective tissue growth factor plays an important role in advanced glycation end product-induced tubular epithelial-to- mesenchymal transition:implications for diabetic renal disease.J Am Soc Nephrol JT,2006,17(9):2484-2494.
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[1]Burns WC,Kantharidis P,Thomas MC.The role of tubular epithelial-mesen-chy -mal transition in progressive kidney disease.Cells Tissues Organs,2007,185(1-3):222-231
[2]Huang JS, Chuang LY, Guh JY, et al.Antioxidants attenuate high glucose-induced hypertrophic growth in renal tubular epithelial cells.Am J Physiol Renal Physiol.2007,293(4):1072-1082.
[3]Liu BC,Chen L,Sun J,et al.Connective tissue growth factor-mediated angiotensin Ⅱ-induced hypertrophy of proximal tubular cells.Nephron Exp Nephrol JT,2006,103(1):16-26.
[4]Liu BC,Li MX,Zhang JD,et al.Inhibition of integrin-linked kinase via a siRNA expression plasmid attenuates connective tissue growth factor-induced human proximal tubular epithelial cells to mesenchymal transition.Am J Nephrol JT,2008,28(1):143-151.
[5]Burns WC,Twigg SM,Forbes JM,et al.Connective tissue growth factor plays an important role in advanced glycation end product-induced tubular epithelial-to- mesenchymal transition:implications for diabetic renal disease.J Am Soc Nephrol JT,2006,17(9):2484-2494.
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[10]Liu BC,Huang HQ,Luo DD,et al.Connective tissue growth factor is associated with the early renal hypertrophy in uninephrectomized diabetic rats.Chinese Medical Journal,2006,119(12):1010-1016
[11]Awazu M,Omori S,Ishikura K,Hida M,Fujita H:The lack of cyclin kinase inhibitor p27(Kipl) ameliorates progression of diabetic nephropathy.J Am Soc Nephrol 2003,14:699-708.
[12]Haneda M,Araki SI,Togawa M,et al.Activation of mitogen-activated protein kinase cascade in diabetic glomeruli and mesangial cells cultured under high glucose conditions.Kidney Int Suppl,1997,60:S66-69.
[13]Zhang Y,Dong C.Regulatory mechanisms of mitogen-activated kinase signaling.Cell Mol Life Sci,2007,64(21) :2771-2789.
[14]Nishimoto S,Nishida E.MAPK signalling:ERK5 versus ERK 1/2.EMBO Rep,2006,7 (8) :782-786.
[15]Fujita H,Omori S,Ishikura K,et al.ERK and p38 mediate high-glucose-induced hypertrophy and TGF- expression in renal tubular cells.Am J Physiol Renal Physiol,2004,286(1):120-126.
[16]Wahab NA ,Mason RM.Connective tissue growth factor and renal diseases :some answers ,more questions.Curr Opin Nephrol Hypertens,2004,13 :53-58.
[17]Moussad EE,Brigstock DR.Connective tissue growth factor:what's in a name? Mol Genet Metab,2000,71:276-292.
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[19]Wang S,DenichiloM,Brubaker C et al.Connective tissue growth factor in tubulointerstitial injury of diabetic nephropathy.Kidney Int,2001;60 (1) :96~101.
[20]Wahab NA,Yevdokimova N,Weston BS,et al.Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy.Biochem J,2001,359:77-87.
[21]Ito Y,Bende RJ ,Oemar BS et al.Exp ression of connective tissue growth factor in human renal fibrosis.Kidney Int,1998;53 (4):853～ 861..
[22]Wahab NA,Weston BS,Roberts T,et al.Connective tissue growth factor and regulation of the mesangial cell cycle:role in cellular hypertrophy.J Am Soc Nephrol,2002,13:2437-2445.
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[28]Okada H,Kikuta T,Kobayashi T,et al.Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal fibrogenesis.J Am Soc Nephrol,2005,16(1):133-143.
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[32]Liu BC,Li MX,Zhang JD,et al.Inhibition of integrin-linked kinase via a siRNA expression plasmid attenuates connective tissue growth factor-induced human proximal tubular epithelial cells to mesenchymal transition.Am J Nephrol JT,2008,28(1):143-151
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