Smad3沉默对IGFBPrP1诱导的肝星状细胞作用的研究
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摘要
研究背景：
各种病因可引起肝脏局部炎症,继而由此导致的以胶原为主的细胞外基质过度沉积,进而形成肝纤维化。肝纤维化是各种慢性肝病发展至肝硬化的必经阶段和中心环节,属于可逆性病变。近年来,有关肝纤维化发病机制的研究已取得了长足的进展,大量研究表明,肝星状细胞(hepatic stellate cells, HSC)是细胞外基质(extracellular matrix, ECM)的主要细胞来源,在肝纤维化形成中起关键性作用。
肝纤维化是一个有多种细胞因子和多条细胞信号通路共同参与的复杂病理过程。参与肝纤维化的信号转导通路有：Smad信号通路、JAK/STAT信号通路、NF-κB信号通路以及MAPK信号通路等,其中Smad信号通路是目前研究较为清楚的信号转导通路。有研究显示,动物肝纤维化往往伴随HSC数量进行性增多,Smad3 mRNA表达量显著增高,提示Smad信号通路在肝纤维化发生发展中起着至关重要的作用。
RNA干扰(RNA interference, RNAi)是生物界广泛存在的保守防御反应,是近年来发现的一种重要的转录后基因沉默方式,它通过一些小的双链RNA阻断特定基因的表达,诱导mRNA的降解,从而达到“基因沉默”的功效。
胰岛素样生长因子结合蛋白相关蛋白1(insulin-like growth factor binding protein related protein 1, IGFBPrP1)是一种相对分子质量为31 KD的可溶性分泌蛋白。导师刘立新等的研究发现IGFBPrP1可能是肝纤维化发生机制中新的致病因子。肝纤维化患者肝组织中IGFBPrP1的表达与TGFβ1及胶原的表达具有正相关关系。重组IGFBPrP1可诱导HSC活化并使活化的HSC合成ECM增多,而抗IGFBPrP1抗体能减少肝纤维化小鼠肝组织中ECM的沉积,且ECM与Smad3的表达变化具有正相关关系。为了明确IGFBPrP1对ECM的影响是否通过Smad3信号通路来实现,并探讨IGFBPrP1对HSC中TGFβ1表达的影响,设计了本课题。
第一部分siRNA Smad3对肝星状细胞Smad3基因表达的影响
目的：
探讨针对大鼠Smad3基因的siRNA对肝星状细胞Smad3基因表达的影响。
方法：
1.转染细胞：采用小分子RNA干扰(RNAi)技术,将非特异带荧光标记的siRNA(NC siRNA,与Smad3 mRNA无同源性的21ntsiRNA)转染体外培养的HSC-T6细胞株,并在荧光显微镜下检测转染效率。
2.筛选有效的SiRNA:实验分为4组：阴性对照组、2对siRNA组及空白对照组。应用实时定量PCR(RT-PCR)和Western blot法分析siRNA对HSC-T6 Smad3 mRNA及蛋白的阻抑效率。
结果：
1.将转染后24h的细胞在荧光显微镜下观察,可见绿色荧光,说明转染成功,转染效率约为70%。
2.转染48h后,RT-PCR及Western blot检测结果显示,siRNA1, siRNA2转染组对Smad3 mRNA抑制率分别为32%和41%,siRNA2转染组Smad3蛋白表达水平抑制率为61.5%,与阴性对照组相比,差异有显著意义(P<0.01),而siRNAl转染组Smad3蛋白表达水平抑制率为7.5%,与阴性对照组相比无显著差异。提示siRNA2对Smad3表达有较好的抑制效率,以后实验均用siRNA2进行。
结论：针对Smad3的siRNA可以有效地抑制HSC-T6细胞株中Smad3 mRNA及蛋白的表达。
第二部分IGFBPrP1对沉默了Smad3的肝星状细胞分泌细胞外基质的影响
目的：明确胰岛素样生长因子结合蛋白相关蛋白1 (IGFBPrPl)是否通过Smad3
响HSC分泌细胞外基质。
方法：
1.选择体外培养的肝星状细胞株(HSC-T6)作为研究对象,分别设立30μg/L IGFBPrPl处理组和空白对照组(加入等量磷酸盐缓冲溶液(PBS)),干预因素处理48h后收集细胞培养上清液,应用Western blot法检测经IGFBPrP1刺激HSC后,上清液中CollagenI和FN的表达。
2.选择体外培养的肝星状细胞株(HSC-T6)作为研究对象,分别设立阴性对照组(转染NC siRNA)、Smad3 RNAi组(转染Smad3 siRNA2)、Smad3 RNAi+IGFBPrPl组(Smad3 siRNA2转染24h后,加30μg/L IGFBPrP1作用48h)及IGFBPrP1组(培养液中加30μg／LIGFBPrPl作用48h)。分别提取各组细胞总蛋白并收集培养上清液,采用(?)Vestern blot法检测各组Smad3、Collagen I和FN的表达。
结果：
1. Western blot结果显示,IGFBPrP1作用于HSC后,Collagen I和FN的表达均较空白对照组显著增高(0.74±0.09 vs 0.53±0.07；0.63±0.07 vs 0.46±0.07,P<0.01),差异具有统计学意义。2. Smad3 RNAi组Smad3的表达较阴性对照组显著降低(0.14±0.02 vs 0.33±0.06,P<0.01)。另一组实验先对HSC-T6进行Smad3 RNA干扰,然后再加入IGFBPrP1进行刺激,Collagen I和FN的表达较IGFBPrP1组明显降低(0.42±0.05 vs 0.74±0.09;0.34±0.06 vs 0.63±0.07,P<0.01)。
结论：
IGFBPrP1可使ECM的重要组成成分Collagen I和FN的分泌增加,该作用的机制之一是通过Smad3信号通路来实现的。
第三部分IGFBPrP1对肝星状细胞TGFβ1表达影响的初探
目的：
观察胰岛素样生长因子结合蛋白相关蛋白1 (IGFBPrP1)对HSC中转化生长因子β1(transforming growth factor beta 1, TGFβ1)表达的影响,以判定IGFBPrP1是否可作为TGFβ1产生的原因之一。
方法：
选择体外培养的肝星状细胞株(HSC-T6)作为研究对象,分别设立IGFBPrP1 10μg/L 20μg/L、30μg/L处理组和空白对照组(加入等量磷酸盐缓冲溶液(PBS)),干预因素处理24h后收集细胞爬片及培养上清液,采用免疫细胞化学染色观察HSC中TGFβ1的表达变化；酶联免疫吸附实验(ELISA)检测细胞培养上清液中TGFβ1的含量。同时用Western blot法检测经30μg/L IGFBPrP1刺激HSC后,培养上清液中TGFβ1的表达。结果：
免疫细胞化学染色结果显示：各组细胞胞浆内均可见棕黄色或棕褐色颗粒沉积,TGFβ1呈阳性表达。经图像分析显示,IGFBPrP1 10μg/L组(10.90±0.35)、20μg/L组(12.09±0.54)、30μg/L组(11.89±0.32)TGFβ1表达均较空白对照组(7.71±0.63)显著增强,且在一定范围内呈剂量依赖关系。ELISA检测结果显示：TGFβ1在空白对照组已有一定量的分泌,但20μg／L组(169.59±24.87)、30μg／L组(188.34±32.24)TGFβ1的含量仍较空白对照组(136.16±19.52)增高,差异具有统计学意义(P<0.05)。Western blot结果显示：IGFBPrP1组(0.46±0.05)TGFβ1的表达较空白对照组(0.30±0.02)增高,差异具有统计学意义(P<0.05)。
结论：
IGFBPrP1可使体外培养的HSC合成和分泌TGFβ1增加,且在一定剂量范围内随着IGFBPrP1剂量的增加TGFβ1的表达逐渐增强。
Background:
Hepatic fibrosis is the common result of liver injury from diverse origins. The sequence of response to injury is local inflammation followed by excessive deposition of extracellular matrix. Hepatic fibrosis is reversible and common pathological consequence of various chronic liver diseases which could eventually lead to hepatic cirrhosis. Up to date, hepatic stellate cells as the main matrix-producing cells and play a pivotal role in the process of liver fibrosis.
Hepatic fibrosis is a complex pathological process in which participate a variety of cytokines and multiple signaling pathways. The main signal transduction pathway of hepatic fibrosis have: Smad, JAK/STAT, NF-κB and MAPK signal transduction pathway. The Smad signaling pathway is the relatively clear signal transduction pathway. It has showed that hepatic fibrosis is often accompanied by progressive increasing HSC and Smad3 mRNA expression, suggesting the Smad signaling pathway plays the key role in the development of hepatic fibrosis.
RNAi is a widespread conservative defense response and an important post-transcriptional gene silencing approach. The double-stranded RNA induced the mRNA degradation by blocking specific gene expression to achieve "gene silencing" effectiveness.
Insulin-like growth factor binding protein related protein 1 is a secretory protein with a molecular mass of approximately 31 kDa. Professor Lixin Liu discovered that IGFBPrPl is probably a new pathogenic factor in the formation of liver fibrosis. In human livers with fibrosis and cirrhosis, the expression of IGFBPrPl has positive correlation with TGFβ1 and Collagen I. Recombinant-IGFBPrPl can activate HSC in vitro and can increase the synthesis of extracelluar matrix. Anti-IGFBPrPl antibody can reduce the deposition of extracellular matrix in liver tissue of mice with liver fibrosis and the expression of extracelluar matrix has positive correlation with Smad3. In order to identify whether IGFBPrP1 effect on the secretion of extracellular matrix in hepatic stellate cells through Smad3 signaling pathway and to explore the effect of IGFBPrP1 on the expression of TGFβ1 in activated hepatic stellate cells, we designed this study.
Part I The effect of siRNA Smad3 on Smad3 gene expression of hepatic stellate cells
Objective:
To investigate the effect of small interfering RNA targeting Smad3 gene on the expression of Smad3 in rat hepatic stellate cells.
Methods:
1. Nonspecific siRNA with fluorescence were transfected into cultured HSC-T6 cells by the RNA interfering technology. We observed the transfection efficiency using fluorescence microscopy.
2. Screening the effective siRNA. Groups in experiment:negative contol group, two pairs of Smad3 siRNA groups and blank group. The inhibition rate of siRNA on Smad3 expression of HSC-T6 was determined by Real time PCR and Western blot.
Results:
1. Under the fluorescence microscope, the green fluorescence with the cells could be seen after transfection, suggesting the transfection was successful. The transfection efficiency was 70%.
2. After 48 hours of transfecting siRNA 1 and siRNA2 into HSC-T6 cells, compared with negative control, the result of RT-PCR showed that the Smad3 mRNA inhibition rate was 32%and 41%, respectively. Western blot analysis revealed that the expression of Smad3 were down-regulated by 7.5%and 61.5%, respectively(P<0.01). siRNA2 inhibited Smad3 gene expression stronger than another siRNA.
Conclusions: siRNA against Smad3 could effectively inhibit the expression of Smad3 and the protein level of Smad3 on HSC-T6 cell line.
Part II The effect of IGFBPrPl with siRNA-mediated Smad3 silence on the secretion of extracellular matrix in activated hepatic stellate cells
Objective:
To identify whether IGFBPrP1 effect on the secretion of extracellular matrix in hepatic stellate cells through Smad3 signaling pathway.
Methods:
1. HSC-T6 cells were cultured in vitro and established respectively the groups treated with IGFBPrP1 30μg/L and the blank control group (incubated with equal phosphate buffer saline(PBS)), the supernate was collected after 48 hours. The expression of Collagen I and FN in HSC was confirmed by Western blot.
2. HSC-T6 cells were divided into four groups:Negative control group (transfection negative control siRNA), siRNA-Smad3 transfection group (transfection Smad3 siRNA2), siRNA-Smad3+IGFBPrP1 group (transfection Smad3 siRNA224 hours, adding IGFBPrP1 stimulating 48 hours), IGFBPrP1 group (adding IGFBPrP1 stimulating 48 hours). The expression of Smad3, Collagen I and FN were determined by Western blot.
Results:
1. The results of Western blot analysis:The expression of Collagen I and FN were significantly upregulated than that in the blank control group(0.74±0.09 vs 0.53±0.07; 0.63±0.07 vs 0.46±0.07) (P<0.01)
2. After transfection of siRNA2 Smad3, the protein expression of Smad3 significantly decreased compared with the negative control group(0.14±0.02 vs 0.33±0.06) (P<0.01). The protein expression of fibronectin and Collagen I in IGFBPrP1 stimulating HSC treated with siRNA2 Smad3 were significantly decreased compared to that in IGFBPrP1 stimulating HSC without siRNA2 Smad3(0.42±0.05 vs 0.74±0.09; 0.34±0.06 vs 0.63±0.07) (P<0.01).
Conclusions: The secretion of both Collagen I and FN which are the principal component of ECM were
increasing by IGFBPrP1, the one of the mechanisms of which is through Smad3 signaling
pathway.
Part III The effect of IGFBPrPl on the expression of TGFβ1 in activated stellate cells
Objective:
To observe the effect of IGFBPrP1 on the expression of TGFβ1 in hepatic stellate cells, and investigate whether IGFBPrP1 can be the one of the causes of TGFβ1.
Methods:
HSC-T6 was cultured in vitro and established respectively the groups treated with IGFBPrPl 10μg/L,20μg/L,30μg/L and the blank control group (incubated with equal phosphate buffer saline(PBS)), cell-coated dishes were attained after 24 hours, then the expression of TGFβ1 were detected by immunocytochemical staining and analyzed by Image-Pro image analysis system. Simultaneously, the supernatant were collected and the content of TGFp1 was measured by enzyme-linked immunosorbent assay (ELISA). The synthesis of TGFβ1 in HSC was confirmed by Western blot again.
Results:
The results of immunocyochemical staining:Each group had positive staining in cytoplasm showing some buffy or brown particles. The results of image analysis showed that the positive staining of TGFβ1 in IGFBPrPl treatment with 10μg/L group (10.90±0.35),20μg/L group(12.09±0.54)、30μg/L group(11.89±0.32)were significantly higher than that in the blank control group (7.71±0.63) (P<0.05). The results of ELISA:The content of TGFβ1 in IGFBPrP1 treatment with 20μg/L group (169.59±24.87)、30μg/L group (188.34±32.24) were significantly higher than that in the blank control group (136.16±19.52) (P<0.05). The results of Western blot analysis:The expression of TGFβ1 in IGFBPrPl group(0.46±0.05) were significantly upregulated than that in the blank control group(0.30±0.02) (P<0.05)
Conclusions:
The synthesis and secretion of TGFβ1 were increasing by IGFBPrP1 and the level of TGFβ1 gradually enhanced in a certain dose range of IGFBPrPl.
各种病因可引起肝脏局部炎症,继而由此导致的以胶原为主的细胞外基质过度沉积,进而形成肝纤维化。肝纤维化是各种慢性肝病发展至肝硬化的必经阶段和中心环节,属于可逆性病变。近年来,有关肝纤维化发病机制的研究已取得了长足的进展,大量研究表明,肝星状细胞(hepatic stellate cells, HSC)是细胞外基质(extracellular matrix, ECM)的主要细胞来源,在肝纤维化形成中起关键性作用。
肝纤维化是一个有多种细胞因子和多条细胞信号通路共同参与的复杂病理过程。参与肝纤维化的信号转导通路有：Smad信号通路、JAK/STAT信号通路、NF-κB信号通路以及MAPK信号通路等,其中Smad信号通路是目前研究较为清楚的信号转导通路。有研究显示,动物肝纤维化往往伴随HSC数量进行性增多,Smad3 mRNA表达量显著增高,提示Smad信号通路在肝纤维化发生发展中起着至关重要的作用。
RNA干扰(RNA interference, RNAi)是生物界广泛存在的保守防御反应,是近年来发现的一种重要的转录后基因沉默方式,它通过一些小的双链RNA阻断特定基因的表达,诱导mRNA的降解,从而达到“基因沉默”的功效。
胰岛素样生长因子结合蛋白相关蛋白1(insulin-like growth factor binding protein related protein 1, IGFBPrP1)是一种相对分子质量为31 KD的可溶性分泌蛋白。导师刘立新等的研究发现IGFBPrP1可能是肝纤维化发生机制中新的致病因子。肝纤维化患者肝组织中IGFBPrP1的表达与TGFβ1及胶原的表达具有正相关关系。重组IGFBPrP1可诱导HSC活化并使活化的HSC合成ECM增多,而抗IGFBPrP1抗体能减少肝纤维化小鼠肝组织中ECM的沉积,且ECM与Smad3的表达变化具有正相关关系。为了明确IGFBPrP1对ECM的影响是否通过Smad3信号通路来实现,并探讨IGFBPrP1对HSC中TGFβ1表达的影响,设计了本课题。
第一部分siRNA Smad3对肝星状细胞Smad3基因表达的影响
目的：
探讨针对大鼠Smad3基因的siRNA对肝星状细胞Smad3基因表达的影响。
方法：
1.转染细胞：采用小分子RNA干扰(RNAi)技术,将非特异带荧光标记的siRNA(NC siRNA,与Smad3 mRNA无同源性的21ntsiRNA)转染体外培养的HSC-T6细胞株,并在荧光显微镜下检测转染效率。
2.筛选有效的SiRNA:实验分为4组：阴性对照组、2对siRNA组及空白对照组。应用实时定量PCR(RT-PCR)和Western blot法分析siRNA对HSC-T6 Smad3 mRNA及蛋白的阻抑效率。
结果：
1.将转染后24h的细胞在荧光显微镜下观察,可见绿色荧光,说明转染成功,转染效率约为70%。
2.转染48h后,RT-PCR及Western blot检测结果显示,siRNA1, siRNA2转染组对Smad3 mRNA抑制率分别为32%和41%,siRNA2转染组Smad3蛋白表达水平抑制率为61.5%,与阴性对照组相比,差异有显著意义(P<0.01),而siRNAl转染组Smad3蛋白表达水平抑制率为7.5%,与阴性对照组相比无显著差异。提示siRNA2对Smad3表达有较好的抑制效率,以后实验均用siRNA2进行。
结论：针对Smad3的siRNA可以有效地抑制HSC-T6细胞株中Smad3 mRNA及蛋白的表达。
第二部分IGFBPrP1对沉默了Smad3的肝星状细胞分泌细胞外基质的影响
目的：明确胰岛素样生长因子结合蛋白相关蛋白1 (IGFBPrPl)是否通过Smad3
响HSC分泌细胞外基质。
方法：
1.选择体外培养的肝星状细胞株(HSC-T6)作为研究对象,分别设立30μg/L IGFBPrPl处理组和空白对照组(加入等量磷酸盐缓冲溶液(PBS)),干预因素处理48h后收集细胞培养上清液,应用Western blot法检测经IGFBPrP1刺激HSC后,上清液中CollagenI和FN的表达。
2.选择体外培养的肝星状细胞株(HSC-T6)作为研究对象,分别设立阴性对照组(转染NC siRNA)、Smad3 RNAi组(转染Smad3 siRNA2)、Smad3 RNAi+IGFBPrPl组(Smad3 siRNA2转染24h后,加30μg/L IGFBPrP1作用48h)及IGFBPrP1组(培养液中加30μg／LIGFBPrPl作用48h)。分别提取各组细胞总蛋白并收集培养上清液,采用(?)Vestern blot法检测各组Smad3、Collagen I和FN的表达。
结果：
1. Western blot结果显示,IGFBPrP1作用于HSC后,Collagen I和FN的表达均较空白对照组显著增高(0.74±0.09 vs 0.53±0.07；0.63±0.07 vs 0.46±0.07,P<0.01),差异具有统计学意义。2. Smad3 RNAi组Smad3的表达较阴性对照组显著降低(0.14±0.02 vs 0.33±0.06,P<0.01)。另一组实验先对HSC-T6进行Smad3 RNA干扰,然后再加入IGFBPrP1进行刺激,Collagen I和FN的表达较IGFBPrP1组明显降低(0.42±0.05 vs 0.74±0.09;0.34±0.06 vs 0.63±0.07,P<0.01)。
结论：
IGFBPrP1可使ECM的重要组成成分Collagen I和FN的分泌增加,该作用的机制之一是通过Smad3信号通路来实现的。
第三部分IGFBPrP1对肝星状细胞TGFβ1表达影响的初探
目的：
观察胰岛素样生长因子结合蛋白相关蛋白1 (IGFBPrP1)对HSC中转化生长因子β1(transforming growth factor beta 1, TGFβ1)表达的影响,以判定IGFBPrP1是否可作为TGFβ1产生的原因之一。
方法：
选择体外培养的肝星状细胞株(HSC-T6)作为研究对象,分别设立IGFBPrP1 10μg/L 20μg/L、30μg/L处理组和空白对照组(加入等量磷酸盐缓冲溶液(PBS)),干预因素处理24h后收集细胞爬片及培养上清液,采用免疫细胞化学染色观察HSC中TGFβ1的表达变化；酶联免疫吸附实验(ELISA)检测细胞培养上清液中TGFβ1的含量。同时用Western blot法检测经30μg/L IGFBPrP1刺激HSC后,培养上清液中TGFβ1的表达。结果：
免疫细胞化学染色结果显示：各组细胞胞浆内均可见棕黄色或棕褐色颗粒沉积,TGFβ1呈阳性表达。经图像分析显示,IGFBPrP1 10μg/L组(10.90±0.35)、20μg/L组(12.09±0.54)、30μg/L组(11.89±0.32)TGFβ1表达均较空白对照组(7.71±0.63)显著增强,且在一定范围内呈剂量依赖关系。ELISA检测结果显示：TGFβ1在空白对照组已有一定量的分泌,但20μg／L组(169.59±24.87)、30μg／L组(188.34±32.24)TGFβ1的含量仍较空白对照组(136.16±19.52)增高,差异具有统计学意义(P<0.05)。Western blot结果显示：IGFBPrP1组(0.46±0.05)TGFβ1的表达较空白对照组(0.30±0.02)增高,差异具有统计学意义(P<0.05)。
结论：
IGFBPrP1可使体外培养的HSC合成和分泌TGFβ1增加,且在一定剂量范围内随着IGFBPrP1剂量的增加TGFβ1的表达逐渐增强。
Background:
Hepatic fibrosis is the common result of liver injury from diverse origins. The sequence of response to injury is local inflammation followed by excessive deposition of extracellular matrix. Hepatic fibrosis is reversible and common pathological consequence of various chronic liver diseases which could eventually lead to hepatic cirrhosis. Up to date, hepatic stellate cells as the main matrix-producing cells and play a pivotal role in the process of liver fibrosis.
Hepatic fibrosis is a complex pathological process in which participate a variety of cytokines and multiple signaling pathways. The main signal transduction pathway of hepatic fibrosis have: Smad, JAK/STAT, NF-κB and MAPK signal transduction pathway. The Smad signaling pathway is the relatively clear signal transduction pathway. It has showed that hepatic fibrosis is often accompanied by progressive increasing HSC and Smad3 mRNA expression, suggesting the Smad signaling pathway plays the key role in the development of hepatic fibrosis.
RNAi is a widespread conservative defense response and an important post-transcriptional gene silencing approach. The double-stranded RNA induced the mRNA degradation by blocking specific gene expression to achieve "gene silencing" effectiveness.
Insulin-like growth factor binding protein related protein 1 is a secretory protein with a molecular mass of approximately 31 kDa. Professor Lixin Liu discovered that IGFBPrPl is probably a new pathogenic factor in the formation of liver fibrosis. In human livers with fibrosis and cirrhosis, the expression of IGFBPrPl has positive correlation with TGFβ1 and Collagen I. Recombinant-IGFBPrPl can activate HSC in vitro and can increase the synthesis of extracelluar matrix. Anti-IGFBPrPl antibody can reduce the deposition of extracellular matrix in liver tissue of mice with liver fibrosis and the expression of extracelluar matrix has positive correlation with Smad3. In order to identify whether IGFBPrP1 effect on the secretion of extracellular matrix in hepatic stellate cells through Smad3 signaling pathway and to explore the effect of IGFBPrP1 on the expression of TGFβ1 in activated hepatic stellate cells, we designed this study.
Part I The effect of siRNA Smad3 on Smad3 gene expression of hepatic stellate cells
Objective:
To investigate the effect of small interfering RNA targeting Smad3 gene on the expression of Smad3 in rat hepatic stellate cells.
Methods:
1. Nonspecific siRNA with fluorescence were transfected into cultured HSC-T6 cells by the RNA interfering technology. We observed the transfection efficiency using fluorescence microscopy.
2. Screening the effective siRNA. Groups in experiment:negative contol group, two pairs of Smad3 siRNA groups and blank group. The inhibition rate of siRNA on Smad3 expression of HSC-T6 was determined by Real time PCR and Western blot.
Results:
1. Under the fluorescence microscope, the green fluorescence with the cells could be seen after transfection, suggesting the transfection was successful. The transfection efficiency was 70%.
2. After 48 hours of transfecting siRNA 1 and siRNA2 into HSC-T6 cells, compared with negative control, the result of RT-PCR showed that the Smad3 mRNA inhibition rate was 32%and 41%, respectively. Western blot analysis revealed that the expression of Smad3 were down-regulated by 7.5%and 61.5%, respectively(P<0.01). siRNA2 inhibited Smad3 gene expression stronger than another siRNA.
Conclusions: siRNA against Smad3 could effectively inhibit the expression of Smad3 and the protein level of Smad3 on HSC-T6 cell line.
Part II The effect of IGFBPrPl with siRNA-mediated Smad3 silence on the secretion of extracellular matrix in activated hepatic stellate cells
Objective:
To identify whether IGFBPrP1 effect on the secretion of extracellular matrix in hepatic stellate cells through Smad3 signaling pathway.
Methods:
1. HSC-T6 cells were cultured in vitro and established respectively the groups treated with IGFBPrP1 30μg/L and the blank control group (incubated with equal phosphate buffer saline(PBS)), the supernate was collected after 48 hours. The expression of Collagen I and FN in HSC was confirmed by Western blot.
2. HSC-T6 cells were divided into four groups:Negative control group (transfection negative control siRNA), siRNA-Smad3 transfection group (transfection Smad3 siRNA2), siRNA-Smad3+IGFBPrP1 group (transfection Smad3 siRNA224 hours, adding IGFBPrP1 stimulating 48 hours), IGFBPrP1 group (adding IGFBPrP1 stimulating 48 hours). The expression of Smad3, Collagen I and FN were determined by Western blot.
Results:
1. The results of Western blot analysis:The expression of Collagen I and FN were significantly upregulated than that in the blank control group(0.74±0.09 vs 0.53±0.07; 0.63±0.07 vs 0.46±0.07) (P<0.01)
2. After transfection of siRNA2 Smad3, the protein expression of Smad3 significantly decreased compared with the negative control group(0.14±0.02 vs 0.33±0.06) (P<0.01). The protein expression of fibronectin and Collagen I in IGFBPrP1 stimulating HSC treated with siRNA2 Smad3 were significantly decreased compared to that in IGFBPrP1 stimulating HSC without siRNA2 Smad3(0.42±0.05 vs 0.74±0.09; 0.34±0.06 vs 0.63±0.07) (P<0.01).
Conclusions: The secretion of both Collagen I and FN which are the principal component of ECM were
increasing by IGFBPrP1, the one of the mechanisms of which is through Smad3 signaling
pathway.
Part III The effect of IGFBPrPl on the expression of TGFβ1 in activated stellate cells
Objective:
To observe the effect of IGFBPrP1 on the expression of TGFβ1 in hepatic stellate cells, and investigate whether IGFBPrP1 can be the one of the causes of TGFβ1.
Methods:
HSC-T6 was cultured in vitro and established respectively the groups treated with IGFBPrPl 10μg/L,20μg/L,30μg/L and the blank control group (incubated with equal phosphate buffer saline(PBS)), cell-coated dishes were attained after 24 hours, then the expression of TGFβ1 were detected by immunocytochemical staining and analyzed by Image-Pro image analysis system. Simultaneously, the supernatant were collected and the content of TGFp1 was measured by enzyme-linked immunosorbent assay (ELISA). The synthesis of TGFβ1 in HSC was confirmed by Western blot again.
Results:
The results of immunocyochemical staining:Each group had positive staining in cytoplasm showing some buffy or brown particles. The results of image analysis showed that the positive staining of TGFβ1 in IGFBPrPl treatment with 10μg/L group (10.90±0.35),20μg/L group(12.09±0.54)、30μg/L group(11.89±0.32)were significantly higher than that in the blank control group (7.71±0.63) (P<0.05). The results of ELISA:The content of TGFβ1 in IGFBPrP1 treatment with 20μg/L group (169.59±24.87)、30μg/L group (188.34±32.24) were significantly higher than that in the blank control group (136.16±19.52) (P<0.05). The results of Western blot analysis:The expression of TGFβ1 in IGFBPrPl group(0.46±0.05) were significantly upregulated than that in the blank control group(0.30±0.02) (P<0.05)
Conclusions:
The synthesis and secretion of TGFβ1 were increasing by IGFBPrP1 and the level of TGFβ1 gradually enhanced in a certain dose range of IGFBPrPl.
引文
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[1]Elbashir SM, Harborth J. Duplexes of 212 nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature,2001,411 (6836):494-498.
[2]严蓉,陈峰.Smad蛋白—肝纤维化时TGFβ1信号转导的关键物质.国际流行病学传染病学杂志,2007,34(3)：191-194.
[3]Bharathy S, Xie W, Yingling JM, Reiss M. Cancer-associated transforming growth factor β type Ⅱ
receptor gene mutant causes activation of bone morphogenic protein-Smads and invasive phenotype. Cancer Res.2008,68(6):1656-1666.
[4]Daly AC, Randall RA, Hill CS. Transforming growth factor binduced Smadl/5 phosphorylation in epithelial cells is mediated by novel receptor complexes and is essential for anchorageindependent growth. Mol Cell Biol,2008,28(22):6889-6902.
[5]Goumans MJ, Valdimarsdottir G, Itoh S, et al. Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFbeta/ALK5 signaling. Mol Cell,2003,12(4):817-828.
[6]Hill CS. Nucleocytoplasmic shuttling of Smad proteins. Cell Research,2009,19(1):36-46.
[7]Roberts AB, Piek E, Bottinger EP, et al. Is Smad3 a major player in signal transduction pathways leading to fibrogenesis? Chest,2001,120(1 Supp):43S-47S.
[8]Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol,2007, Suppl 1:S79-S84.
[9]Gauldie J, Bonniaud P, Sime P, et al. TGF-beta, Smad3 and the process of progressive fibrosis. Biochem Soc Trans,2007,35(Pt 4):661-664.
[10]Ashcroft GS, Yang X, Glick AB,et al. Mice lacking Smad3 show accelerated wound healing and an impaired local infl ammatory response. Nat Cell Biol,1999,1(5):260-266.
[11]Ashcroft GS, Roberts AB. Loss of Smad3 modulates wound healing. Cytokine Growth Factor Rev 2000, 11(1-2):125-131.
[12]Lakos G, Takagawa S, Chen SJ,et al. Targeted disruption of TGFbeta/Smad3 signaling modulates skin fibrosis in a mouse model of scleroderma. Am J Pathol,2004,165:203-217.
[13]Zhao J, Shi W, Wang YL, et al. Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol,2002,282(3):L585-593.
[14]Schnabl B, Kweon YO, Frederick JP, et al. The role of Smad3 in mediating mouse hepatic stellate cell activation. Hepatology,2001,34(1):89-100.
[15]Wang W, Koka V, Lan HY. Transforming growth factor-beta and Smad signalling in kidney diseases. Nephrology (Carlton),2005,10(1):48-56.
[16]Flanders KC. Smad3 as a mediator of the fibrotic response. Int J Exp Pathol 2004,85(2):47-64.
[17]Zimin Wang, Zhongyu Gao, Yi Shi, et al. Inhibition of Smad3 expression decreases collagen synthesis in keloid disease fibroblasts. J Plast Reconstr Aesthet Surg,2007,60(11):1193-1199.
[18]Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature,2001,409(6818):363～366.
[19]Lopez-Fraga M, Martinez T, Jimenez A. RNA interference technologies and trerapeutics:from basic research to products. BioDrugs,2009,23(5):305-332.
[20]Zheng R, Xiong Q, Zuo B, et al. Using RNA interference to identify the different roles of SMAD2 and SMAD3 in NIH/3T3 fibroblast cells.Cell Biochem Funct,2008,26(5):548-556.
[21]邓长柏,杨作成.阻断Smad3表达对转化生长因子β1诱导肌成纤维细胞增殖的影响.中国动脉硬化杂志,2008；16(4)：281-283.
[22]Liu XJ, Ruan CM, Gong XF, et al. Antagonism of transforming growth factor-β signaling inhibits fibrosis-related genes. Biotechnol Lett,2005,27(20):1609-1615.
[2]Liu H, Chen JL, Xiang GA. Signal transduction pathway in liver fibrosis regulated by human hepatocyte growth factor. Nan Fang Yi Ke Da Xue Xue Bao.2010,30(3):431-433.
[3]Liang TJ, Yuan JH, Tan YR, et al. Effect of ursodeoxycholic acid on TGF betal/Smad signaling pathway in rat hepatic stellate cells.Chin Med J(Engl),2009,122(10):1209-1213.
[4]Roberts AB, Russo A, Felici A. Flanders KC. Smad3:a key player in pathogenetic mechanisms dependent on TGF-p. AnnN Y Acad Sci,2003,995:1-10.
[5]田小霞,秦桂秀,负克明,刘立新(通讯作者),等.肝纤维化患者肝组织中胰岛素样生长因子结合蛋白的表达及意义[J].中华肝脏病杂志,2006,11(14)：859-861.
[6]Liu LX, Huang S, Zhang QQ, et al. Insulin-like growth factor binding protein-7 induces activation and transdifferentiation of hepatic stellate cells in vitro. World J Gastroenterol,2009,15(26):3246-3253.
[7]许君君,刘立新,张骞骞,等.抗胰岛素样生长因子结合蛋白相关蛋白1对小鼠肝纤维化的保护作用.中华肝脏病杂志,2009,17(6)：464-465.
[8]Bataller R, Brenner DA. Liver fibrosis. J Clin Invest,2005,115(2):209-218.
[9]Brenner DA.Molecular pathogenesis of liver fibrosis.Trans Am Clin Climatol Assoc,2009,120:361-368.
[10]Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroententerol Hepatol,2007, Suppll:S79-84.
[11]Liang TJ,Yuan JH, Tan YR, et al. Effect of ursodeoxycholic acid on TGF betal/Smad signaling pathway
in rat hepatic stellate cells.Chin Med J(Engl),2009,122(10):1209-1213.
[12]Gauldie J, Bonniaud P, Sime P, et al. TGF-beta, Smad3 and the process of progressive fibrosis. Biochem Soc Trans.2007,35(Pt 4):661-664.
[13]Sunde JS, Donninger H, Wu K, et al. Expression profiling identifies altered expression of genes that contribute to the inhibition of transforming growth factor-beta signaling in ovarian cancer. Cancer Res, 2006,66(17):8404-8412.
[14]Verrecchia F, Mauviel A. Transforming growth factor-beta signaling through the Smad pathway:role in extracellular matrix gene expression and regulation. J Invest Dermatol,2002,118(2):211-215.
[15]Chen Y, Blom IE, Sa S, et al. CTGF expression in mesangial cells:involvement of Smads, MAP kinase, and PKC. Kidney Int,2002,62(4):1149-1159.
[16]Ten Dijke P, Hill C S. New insights into TGF-beta-smad signaling. Trends Biochem Sci,2004,29(5): 265-273
[17]Flanders KC. Smad3 as a mediator of the fibrotic response. Int J Exp Pathol,2004,85(2):47-64.
[18]Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature.2001,411(6836):494-498.
[19]Moazed D. Small RNAs in transcriptional gene silencing and genome defence. Nature,2009,457(7228): 413-420.
[20]Holen T, Amarzguioui M, Wiiger MT, et al. Positonal effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Res.2002,30(8):1757-1766.
[21]Harborth J, Elbashir SM, Bechert K, et al. Identification of essential genes in cultured mammalian cells using small interfering RNAs. J Cell Sci.2001,114(Pt 24):4557-4565.
[22]Naqvi AR, Islam MN, Choudhury NR, et al. The fascinating world of RNA interference. Int J Biol Sci. 2009,5(2):97-117.
[23]Suzuki M, Zheng X, Zhang X, et al. Regulation of allergy with RNA interference. Crit Rev Immunol, 2009,29(6):443-468.
[24]Hong H, Zhang Y, Cai W. In vivo imaging of RNA interference. J Nucl Med,2010,51(2):169-172.
[25]Luo KQ, Chang DC. The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region. BiochemBiophys Res Commun.2004,318(1):303-10.
[26]Arbuthnot P, Ely A, Weinberg MS. Hepatic delivery of RNA interference activators for therapeutic application. Curr Gene Ther.2009,9(2):91-103.
[27]Jiao J, Friedman SL, Aloman C. Hepatic fibrosis. Curr Opin Gastroenterol.2009,25(3):223-229.
[28]Wells RG. Cellular sources of extracellular matrix in hepatic fibrosis. Clin Liver Dis.2008,12(4): 759-768.
[29]Plewka K, Szuster-Ciesielska A, Kandefer-Szerszen M. Role of stellate cells in alcoholic liver fibrosis. Postepy Hig Med Dosw(online),2009,63:303-317.
[30]Pinzani M, Marra F, Carloni V. Signal transduction in hepatic stellate cells. Liver,1998,18(1):2-13.
[31]Gressner AM, Weiskirehen R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and
TGF-beta as major players and therapeutic targets[J]. Cell Mol Med,2006,10(1):76-99.
[32]Ross S, Hill CS. How the Smads regulate transcription.Int J Biochem Cell Biol,2008,40(3):383-408.
[33]Dennler S, Goumans MJ, ten Dijke P. Transforming growth factorp signal transduction. J Leuk Biol, 2002,71 (5):731-740.
[34]Uemura M, Swenson ES, Gaca MD, et al. Smad2 and Smad3 play different roles in rat hepatic stellate cell function and a-smooth muscle actin organization. Mol Biol Cell,2005,16(9):4214-24.
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