【作者】 陶光利；

【导师】 张璟；

【作者基本信息】 第三军医大学 ， 内科学， 2011， 硕士

【摘要】 背景和目的:肾间质纤维化是各种慢性肾脏疾病的最终共同途径。近来发现,肾小管上皮细胞间质转分化(epithelial to mesenchymal transition,EMT)是肾间质纤维化发生发展的重要机制。肾小管上皮细胞转分化是一个有序的发生过程,E-cadherin介导的细胞-细胞的粘附功能丧失被认为是EMT的关键步骤。EMT最早期改变就是E-cadherin的表达抑制,随着E-cadherin的丢失,细胞间的紧密连接遭到破坏,使上皮细胞失去粘附特性。Snail、Slug基因属于锌指转录因子超家族成员,可以通过与靶基因的E-box结构域结合从而抑制其转录。在多个细胞系统研究中发现,Snail通过与E-cadherin启动元件E-box结合,对E-cadherin的转录进行直接抑制,下调其表达,从而使上皮细胞间失去粘附,促进EMT的发生,参与胚胎发育、肿瘤转移及组织纤维化过程。EMT信号系统中多个重要信号途径如转化生长因子-β1-Smad、Wnt/β-catenin/淋巴增强因子、Ras/MAPK/细胞外调节蛋白激酶通路等均可调节调节Snail与Slug基因在细胞内的表达表达。但Snail及Slug在人肾小管EMT过程中作用机制的研究尚未见报道。本研究拟采用siRNA技术,探讨slug与snail基因在EMT中的作用机制,为防治肾纤维化提供新的思路。方法:1.针对Snail、Slug基因mRNA的RNA干扰质粒的构建根据人Snail、Slug基因的mRNA,采用RNAi设计软件,进行全基因组扫描和序列同源性分析,设计针对Genbank中Snail、Slug基因的特异siRNA,同时设计序列相同的阴性对照组。采用siRNA表达载体的方法体外制备人类Snail基因与Slug基因的siRNA真核表达载体。用阳性脂质体LipofectamineTM2000进行HKCs的siRNA转染,倒置显微镜下观察细胞形态。2.实验分组:将HKCs分组如下:①对照组(C组):用无血清培养基(free serum medium,FSM)培养48h;②TGF-β1培养处理HKCs组(T组):用含TGF-β1(20ng/ml)的FSM培养HKCs 48h;③TGF-β1培养处理HKCs-siRNA组(T+siR组):用含TGF-β1(20ng/ml)的FSM培养HKCs-snail-siRNA 48h为T+siRa亚组,用含TGF-β1(20ng/ml)的FSM培养HKCs-slug-siRNA 48h为T+siRb亚组;④TGF-β1培养处理HKCs-siC组(T+siC组):用含TGF-β1(20ng/ml)的FSM培养HKCs-snail-siC 48h为T+siCa亚组,用含TGF-β1(20ng/ml)的FSM培养HKCs-slug-siC 48h为T+siCb亚组。3.RT-PCR方法各组细胞α-SMA、E-cadherin、Snail及Slug基因表达水平。4.Western blot方法检测各组细胞α-SMA、E-cadherin、Snail及Slug在蛋白水平的表达。5.免疫荧光检测各组HKCs的E-cadherin及α-SMA表达。结果:1.分别成功构建Snail、Slug基因的siRNA重组质粒,用阳性脂质体Lipofect amineTM2000转染细胞,转染效率达60%。2.构建TGF-β1诱导的EMT体外细胞培养模型,倒置显微镜下观察细胞形态:C组呈铺路石样紧密排列生长;T组与siC组细胞均出现显著形态改变,类似于成纤维细胞呈长梭形,细胞间间距增大,多数细胞呈离散型生长;T+siRa组及T+siRb组细胞形态改变不明显,仅有少量细胞呈现长梭形;提示沉默Snail及Slug基因可抑制EMT的发生。3.RT-PCR、Western blot方法检测各组细胞α-SMA、E-cadherin、Snail及Slug的表达:C组E-cadherin在基因和蛋白水平均有较强表达,α-SMA无表达,Snail与Slug表达均较弱;T组与siC组E-cadherin表达减弱,Snail、Slug表达明显增强,α-SMA表达明显增强;T+siRa组及T+siRb组E-cadherin表达增强,α-SMA表达较弱;提示抑制Snail及Slug基因可重建E-cadherin表达。4.免疫荧光观察细胞E-cadherin、α-SMA的表达:C组HKCs细胞膜和胞浆E-cadherin表达均为阳性,而α-SMA表达为阴性;T组与siC组均可见E-cadherin表达显著下降,而α-SMA表达为明显增加;T+siRa组与T+siRb组E-cadherin表达增强,α-SMA表达较弱。结论:1.RNA干扰技术可沉默Snail、Slug基因的表达,从而阻断其生物学作用;2.Snail、Slug基因与EMT密切相关,抑制Snail、Slug基因的表达可显著上调E-cadherin的表达,从而抑制EMT发生;3.Snail、Slug基因可作为新靶点,在EMT防治中起重要作用。更多还原

【Abstract】 Background and objective:Renal interstitial fibrosis is the final common pathway of all chronic kidney disease. Recently, emerging evidences indicated that the epithelial-mesenchymal transition （EMT） of renal tubular epithelial cells were key mechanism in the development of renal interstitial fibrosis. The EMT of renal tubular epithelial cells is considered to be an orderly process, and the loss of E-cadherin-mediated intercellular adhesion function is considered to be a key step in EMT. The earliest change of EMT is the inhibition of E-cadherin expression, as the loss of E-cadherin, intercellular tight junctions would be destroyed which could ultimately lead to the lose of adhesion properties of epithelial cells. Snail and Slug gene are members of zinc-finger transcription factors family, and combined with E-box to inhibit transcription of target genes. Studies in multiple cell systems found that Snail could combine with E-cadherin starting element E-box to directly inhibit the transcription of E-cadherin, and down-regulate its expression, then lead to the loss of adhesion among epithelial cells, ultimately promote the occurrence of EMT, and participate in the process of embryonic development, tumor metastasis as well as tissue fibrosis.Many important signaling pathway in the signaling systems of EMT affected the expressions of Snail and Slug genes in cells. For example, it has been demonstred that transforming growth factor-β1 （TGF-β1） - Smad, Wnt/β- catenin/LEF and Ras/MAPK/ERK pathways could regulate the transcription level of Snail and Slug genes although the mechanisms remain unclear. As two members in the same family, although there is a high degree of homology between Snail and Slug, their functions were different in different organs. Up to date, there was no report about the mechanisms of Snail and Slug in human renal tubular EMT. In this study siRNA technology was applied to examine the role and related mechanisms of Snail and Slug genes in EMT.Methods:1. Based on the sequence of human Snail and Slug mRNA, we designed Snail-siRNA and Slug-siRNA by using the software of RNAi Designer for genome wide scanning and sequence homology analysis. A negative control with identical sequence but not targeting at Snail and Slug was also included. The method of expression vector was used to construct the eukaryotic expression vector of Snail-siRNA and Slug-siRNA in vitro. LipofectamineTM2000 was used to transfected siRNA into HKCs. MorpHological characteristics were observed under the inverted microscope.2. Human renel tubular epithelial cells（HKCs） were divided into four groups:①Control group（C）:The HKCs were treated with FSM for 48 hours;②TGF-β1 treated HKCs group（T）:The HKCs were co-incubated with TGF-β1（20ng/ml） and FSM for 48 hours;③TGF-β1 treated HKCs-siRNA group（T+siR）: The HKCs-snail-siRNA were co-incubated with TGF-β1（20ng/ml） and FSM for 48 hours（T+siRa）; The HKCs-slug-siRNA were co-incubated with TGF-β1（20ng/ml） and FSM for 48 hours （T+siRb）;④TGF-β1 treated HKCs-siC group（T+siC）: The HKCs-snail-siC were co-incubated with TGF-β1（20ng/ml） and FSM for 48 hours（T+siCa）; The HKCs-slug-siC were co-incubated with TGF-β1 （20ng/ml） and FSM for 48 hours （T+siCb）3. The mRNA expression of E-cadherin,α-SMA, Snail and Slug was determined by reverse transcription polymerase chain reaction （RT-PCR）.4. The protein expression of E-cadherin,α-SMA, Snail and Slug was determined by Western blot.5. The expression of E-cadherin andα-SMA were observed by confocal laser scanning microscope after immunofluorescence.Results:1. The siRNA eukaryotic expression vector of Snail and Slug gene were constructed successfully which confered by Lipofectamine^TM2000 transfect HKCs with a high efficiency of transfection （60%）.2. The cell model of EMT was induced by TGF-β1 in vitro, MorpHological characteristics were observed under the inverted microscope: C group: the shape of cells showed a classic cobblestone morpHology and grew in a compact mode; T group and T+siC group cells becoming elongated in shape, and losing their cobblestone monolayer pattern; the shape of cells changes indistinctively in T+siRa and T+siRb group, cells showed a classic cobblestone morpHology, similar to Control group. The results showed that inhibition of the expression of Snail and Slug gene could prevent the EMT progress.3. The levels of E-cadherin,α-SMA, Snail and Slug were determined by RT-PCR and Western blot. The results showed that the expression of Snail, Slug andα-SMA was significantly increased and the expression of E-cadherin was significantly decreased in T group and T+siC group vs C group; while the expression of Snail, Slug andα-SMA were significantly decreased and E-cadherin expression was significantly increased in T+siRa and T+siRb group. The results also conviced that expression of Snail and Slug were negative to the expression of E-cadherin.4. The expression of E-cadherin andα-SMA were further detected by immunofluorescence:α-SMA was significantly increased and the expression of E-cadherin was significantly decreased in the T group vs C group and T+siC group; whileα-SMA expression was significantly decreased and E-cadherin expression was significantly increased in T+siRa and T+siRb group.Conclusion:1. The expression of Snail and Slug gene can be silenced by RNA intereference, also their biological function were bloked.2. Snail and Slug gene were highly associated with EMT; the silencence of Snail and Slug gene could prevent the EMT progress and its downstream effects.3. Snail and Slug gene could be a new target for the prevention and treatment of renal fibrosis.更多还原