核因子-κB亚单位p65短发夹状干扰RNA对肝星状细胞凋亡的影响
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摘要
慢性肝脏疾病是一类世界性的严重危害人类健康的主要疾病,其中肝纤维化是这个过程的中间及关键环节。肝星状细胞(hepatic stellate cell, HSC)是启动整个事件的开端,在肝纤维化过程中扮演着重要的角色,诱导活化HSC的凋亡可使肝纤维化发生逆转。越来越多的研究认为HSC活化和增殖与其凋亡受到抑制有关,而核因子-κB(nuclear factor kappa B, NF-κB)与细胞凋亡关系密切。NF-κB是一种能与免疫球蛋白κ轻链基因的增强子κB序列特异结合的蛋白因子,由Rel蛋白家族的成员以同源或异源二聚体形式组成,目前发现的哺乳动物的Rel蛋白包括p65 (RelA, NF-κB3)、p50 (NF-κB1)、Rel (c-Rel)、RelB和p52 (NF-κB2),其中p65/p50是发现最早的,分布最广泛,主要发挥生理作用的NF-κB。NF-κB能与多种细胞基因启动子或增强子序列特定位点发生特异性结合而促进转录和表达,与炎症反应、免疫应答以及细胞的增生、转化和凋亡等重要的病理生理过程密切相关。近年RNA干扰(RNA interference, RNAi)技术的出现为抑制NF-κB的基因表达开辟了新的途径。RNA干扰是指将与内源性mRNA互补的双链RNA导入细胞后,引起该mRNA特异性降解,导致mRNA编码的基因不能表达,发生基因沉默,提示我们可以通过RNA干扰技术沉默NF-κB的基因表达,从而使HSC的凋亡增加,减缓肝纤维化的进程。
目的:本实验通过设计短发夹状干扰RNA(short hairpin RNA, shRNA)对NF-κBp65进行基因干扰,研究其对脂多糖(lipopolysaccharide, LPS)激活的肝星状细胞凋亡的影响。
方法:体外培养大鼠肝星状细胞,进行以下分组:①空白对照组;②LPS组:经LPS处理;③阴性组:经阴性干扰RNA与LPS处理;④阳性组:经NF-κBp65 shRNA与LPS处理;⑤脂质体2000组:经脂质体2000与LPS处理。应用western blot检测其NF-κBp65、p50及IκBα的蛋白表达,反转录聚合酶链反应(reverse transcription polymerase chain reaction, RT-PCR)检测NF-κBp65与Ⅰ型前胶原mRNA的表达,流式细胞术、末段脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末段标记法( terminal deoxynucleotidy1 transferase-mediated dUTP nick end labeling, TUNEL)检测细胞凋亡的变化,酶谱法测定明胶酶活性。
结果:
1 siRNA转染HSC的效率:
将荧光素标记的对照siRNA转染HSC,通过荧光显微镜观察转染效率,结果显示33nmol/LsiRNA组转染72小时效率最高,可达90%以上。
2 LPS激活HSC后细胞核中NF-κBp65蛋白的表达:
western blot结果显示同一浓度的LPS激活HSC后,HSC细胞核中NF-κBp65蛋白表达增加,于1小时细胞核中NF-κBp65蛋白表达最高,并且浓度为1000ng/mlLPS激活HSC1小时后表达NF-κBp65蛋白增加(232.15%±5.28%)比100ng/mlLPS激活HSC后表达NF-κBp65蛋白增加(168.18%±3.39%)明显。
3 NF-κBp65 shRNA基因干扰LPS激活的HSC后细胞核中NF-κBp65蛋白及mRNA的表达减低:
western blot结果显示阳性组HSC细胞核中NF-κBp65蛋白的表达减低,24小时比LPS组减低72.41%±1.22%,48小时减低77.45%±1.23%,72小时减低86.34%±1.01%,于72小时减低最高,而阴性组及脂质体2000组与LPS组比较无明显减低。RT-PCR结果显示,阳性组NF-κBp65 mRNA表达明显减低,而阴性组及脂质体2000组与LPS组比较无明显减低。此结果说明实验构建了针对大鼠HSC的NF-κBp65 mRNA靶序列1543–1561的阳性shRNA,将其转染HSC有效地干扰了NF-κBp65的mRNA及蛋白发达,并于72小时干扰效率达到最高。
4 NF-κBp65 shRNA基因干扰LPS激活的HSC72小时后NF-κBp50和IκBα蛋白表达减低:
western blot结果显示经阳性组HSC细胞核中NF-κBp50蛋白的表达减低,比LPS组减低75.45%±0.93%,而阴性组及脂质体2000组与LPS组比较无明显减低。阳性组细胞浆中IκBα蛋白减低,比LPS组减低70.61%±2.19%,而阴性组及脂质体2000组与LPS组比较无明显减低,说明NF-κBp65 shRNA转染HSC后,干扰NF-κBp65的mRNA及蛋白发达的同时,NF-κBp50及IκBα蛋白表达随之降低。
5 NF-κBp65 shRNA基因干扰LPS激活的HSC后HSC凋亡增加:
流式细胞法结果显示阳性组细胞凋亡较LPS组增加,凋亡率为15.40%±0.72%,而阴性组及脂质体2000组与LPS组比较无明显增加。TUNEL法结果显示经阳性组HSC凋亡较LPS组增加,凋亡率为33.33%±1.06%,而阴性组及脂质体2000组与LPS组比较无明显增加。
6 NF-κBp65 shRNA基因干扰LPS激活的HSC后Ⅰ型前胶原mRNA表达减低:
RT-PCR结果显示,经阳性组Ⅰ型前胶原mRNA表达明显减低,较LPS组减低56.84%±1.89%,而阴性组及脂质体2000组与LPS组比较无明显减低,表明经NF-κBp65 shRNA基因干扰后Ⅰ型前胶原mRNA表达减低。
7 NF-κBp65 shRNA基因干扰LPS激活的HSC后明胶酶活性增高:
酶谱法测定结果显示,阳性组明胶酶活性明显升高,阴性组及脂质体2000组与LPS组比较无明显变化。
结论:
1本实验构建了针对HSC的NF-κBp65 mRNA靶序列1543–1561的阳性shRNA,将其转染HSC,有效地干扰了NF-κBp65的基因发达,与此同时,NF-κB p50及IκBα蛋白表达减低。
2 LPS可激活HSC中NF-κBp65的表达,使其蛋白表达增多。
3抑制NF-κBp65的基因发达可促进HSC的凋亡,同时Ⅰ型前胶原合成减少,明胶酶活性增强,延缓肝纤维化的发展。
The chronic hepatic disease is one of the important cosmopolitan diseases that are severely harmful to the health of human. Hepatic fibrosis is the middle and key element of this process. Hepatic stellate cell(HSC) is the beginning of whole thing and plays an important role in the process of the hepatic fibrosis. Inducing the apoptosis of the activated hepatic stellate cell can reverse the process of hepatic fibrosis. The increasing research has indicated that the activation and proliferation of HSC were related with the inhibition of apoptosis. Nuclear factor-κB (NF-κB) has a close relation with apoptosis. NF-κB, a protein that binds specifically to an enhancerκB sequence ofκimmunoglobin light chain is generated via homodimeric and heterodimeric interactions of Rel subunits. Mammalian Rel proteins found by now include p65 (RelA,NF-κB3), p50 (NF-κB 1) ,Rel (c-Rel),RelB和p52 (NF-κB2), and p65/p50 are found earliest, distribute most widely, and are the NF-κB that play chief physiological functions. NF-κB can induce transcription and expression of multiple cellular cytokines by sequence specifically binding to their promoter or enhancer region, thus promotes inflammation, immunological response, and has been closely correlated with certain important pathological and physical process including cell proliferation, transformation and apoptosis. Recently the appearance of RNA interference opens a new way to inhibit the gene expression.RNA interference is a process that after double-stranded RNA, complementary to the endogenous mRNA, transfected into cells, the mRNA degrades specifically, so that the gene the mRNA codes can’t express, and gene silences, showing us that we can silence the gene expression of NF-κB through the application of RNA interference, which can increase the apoptosis of the HSC and slow down the process of the hepatic fibrosis.
Objective: Through RNA interference by designing short hairpin RNA on the NF-κBp65, the test investigates the effects on apoptosis of lipopolysaccharide (LPS) - stimulated HSC.
Methods: Rat HSC was cultured in vitro and divided into 5 groups①blank control group;②LPS group: dealing with LPS;③negative group: dealing with negative siRNA and LPS;④positive group: dealing with NF-κBp65 shRNA and LPS;⑤lipidosome 2000 group: dealing with lipidosome 2000 and LPS. The expression levels of NF-κBp65, p50 and IκBαprotein were detected by western blot, the expression level of NF-κBp65 and procollagenⅠmRNA was detected by reverse transcription polymerase chain reaction (RT-PCR), and the change of apoptosis of HSC was detected by flow cytometry method and the method of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL).
Results:
1 SiRNA to monitor efficiency in HSC: A fluorescein-labeled non-special siRNA was used to monitor efficiency in HSC, demonstrating nealy 90% transfection efficiency after transfected with 33nmol/L siRNA at 72h.
2 The expression of NF-κBp65 protein increased in the cell nucleus after LPS stimulated HSC: the result of the western blot displayed that NF-κBp65 protein expression in the cell nucleus increased after LPS stimulated HSC. The expression was highest at one hour after the stimulation, and the increase of the expression with 1000ng/ml stimulating(232.15%±5.28%) was higher than that with 100ng/ml stimulating(168.18%±3.39%). The result showed us that LPS could stimulate the expression of NF-κBp65 in HSC, the expression arrived at the peak at one hour after LPS stimulated HSC and then fell off to the normal, and the expression of NF-κBp65 increased while the concentration of LPS increased.
3 NF-κBp65 protein and mRNA expression decreased in the cell nucleus after NF-κBp65 shRNA transfected into the HSC: the result of the western blot displayed that NF-κBp65 protein expression of the positive group in the cell nucleus decreased. The expression at 24 hours after the interference decreased by 72.41%±1.22% than the LPS group, the expression at 48 hours after the interference decreased by 77.45%±1.23% than the LPS group, and the expression at 72 hours after the interference decreased by 86.34%±1.01% than the LPS group, and the decrease was highest at 72 hours after the interference. The expression of the negative group and lipidosome 2000 group had not marked change.The result of the RT-PCR displayed that NF-κBp65 mRNA expression of the positive group in the cell decreased obviously. The expression of the negative group and lipidosome 2000 group had not marked change. The results showed us that the experiment constructed the short hairpin RNA which aimed at the 1543-1561 of the NF-κBp65 mRNA in rat HSC, the short hairpin RNA interfere effectively the expression of NF-κBp65 mRNA and protein after the short hairpin RNA was transfected into the HSC, and the efficiency of the interference arrived at the peak at 72 hours after the interference.
4 The expression of NF-κBp50 and IκBαprotein after NF-κBp65 shRNA transfected into the HSC: the result of the western blot displayed that NF-κB p50 protein expression of the positive group in the cell nucleus decreased, and the expression at 72 hours after the interference decreased by 75.45%±0.93% than the LPS group. And the expression of the negative group and lipidosome 2000 group had not marked change. IκBαprotein expression of the positive group in the cell cytoplasm decreased, and the expression at 72 hours after the interference decreased 70.61%±2.19% than the LPS group. And the expression of the negative group and lipidosome 2000 group had not marked change. The result showed us that the expression of NF-κBp50 and IκBαprotein decreased while NF-κBp65 shRNA interfered the expression of mRNA and protein of NF-κBp65 after it transfected into HSC.
5 The apotosis of HSC increased after interfering with the gene expression of NF-κBp65: the result of the flow cytometry method displayed that the apoptosis of HSC of the positive group increased, and the apoptosis at 72 hours after the interference was higher than the LPS group, the apoptosis ratio increased by 15.40%±0.72%. And the apoptosis of the negative group and lipidosome 2000 group had not marked change. The result of the TUNEL assay displayed that the apoptosis of HSC of the positive group increased after NF-κBp65 shRNA interfered the LPS-stimulated HSC, and the apoptosis at 72 hours after the interference was higher than the LPS group, the apoptosis ratio increased by 33.33%±1.06%. And the apoptosis of the negative group and lipidosome 2000 group had not marked change.
6 ProcollagenⅠmRNA expression in the cell decreased after interfering with the gene expression of NF-κBp65: the result of the RT-PCR displayed that procollagenⅠmRNA expression of the positive group in the cell decreased by 56.84%±1.89%. The expression of negative group and lipidosome 2000 group had not marked change. The result showed us that mRNA expression of procollagenⅠdecreased after interfering with the gene expression of NF-κBp65.
7 The avtivity of gelatinase increased after interfering with the gene expression of NF-κBp65: the result of the MMPs zymography showed that the avtivity of gelatinase of the positive group increased. The avtivity of negative group and lipidosome 2000 group had not marked change.
Conclusion:
1 The experiment constructed the short hairpin RNA which aimed at the 1543-1561 of the NF-κBp65 mRNA in rat HSC. And the short hairpin RNA interferes effectively the expression of NF-κBp65 mRNA and protein after the short hairpin RNA was transfected into the HSC. At the same time, the expression of NF-κB p50 and IκBαprotein decreased.
2 The expression of NF-κBp65 protein increased after LPS stimulated HSC.
3 The apoptosis of HSC increased after NF-κBp65 shRNA interfere effectively the expression of NF-κBp65 mRNA and protein, and the expression of ProcollagenⅠmRNA decreased and the activity of gelatinase increased, which can delay the process of hepatic fibrosis.
目的:本实验通过设计短发夹状干扰RNA(short hairpin RNA, shRNA)对NF-κBp65进行基因干扰,研究其对脂多糖(lipopolysaccharide, LPS)激活的肝星状细胞凋亡的影响。
方法:体外培养大鼠肝星状细胞,进行以下分组:①空白对照组;②LPS组:经LPS处理;③阴性组:经阴性干扰RNA与LPS处理;④阳性组:经NF-κBp65 shRNA与LPS处理;⑤脂质体2000组:经脂质体2000与LPS处理。应用western blot检测其NF-κBp65、p50及IκBα的蛋白表达,反转录聚合酶链反应(reverse transcription polymerase chain reaction, RT-PCR)检测NF-κBp65与Ⅰ型前胶原mRNA的表达,流式细胞术、末段脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末段标记法( terminal deoxynucleotidy1 transferase-mediated dUTP nick end labeling, TUNEL)检测细胞凋亡的变化,酶谱法测定明胶酶活性。
结果:
1 siRNA转染HSC的效率:
将荧光素标记的对照siRNA转染HSC,通过荧光显微镜观察转染效率,结果显示33nmol/LsiRNA组转染72小时效率最高,可达90%以上。
2 LPS激活HSC后细胞核中NF-κBp65蛋白的表达:
western blot结果显示同一浓度的LPS激活HSC后,HSC细胞核中NF-κBp65蛋白表达增加,于1小时细胞核中NF-κBp65蛋白表达最高,并且浓度为1000ng/mlLPS激活HSC1小时后表达NF-κBp65蛋白增加(232.15%±5.28%)比100ng/mlLPS激活HSC后表达NF-κBp65蛋白增加(168.18%±3.39%)明显。
3 NF-κBp65 shRNA基因干扰LPS激活的HSC后细胞核中NF-κBp65蛋白及mRNA的表达减低:
western blot结果显示阳性组HSC细胞核中NF-κBp65蛋白的表达减低,24小时比LPS组减低72.41%±1.22%,48小时减低77.45%±1.23%,72小时减低86.34%±1.01%,于72小时减低最高,而阴性组及脂质体2000组与LPS组比较无明显减低。RT-PCR结果显示,阳性组NF-κBp65 mRNA表达明显减低,而阴性组及脂质体2000组与LPS组比较无明显减低。此结果说明实验构建了针对大鼠HSC的NF-κBp65 mRNA靶序列1543–1561的阳性shRNA,将其转染HSC有效地干扰了NF-κBp65的mRNA及蛋白发达,并于72小时干扰效率达到最高。
4 NF-κBp65 shRNA基因干扰LPS激活的HSC72小时后NF-κBp50和IκBα蛋白表达减低:
western blot结果显示经阳性组HSC细胞核中NF-κBp50蛋白的表达减低,比LPS组减低75.45%±0.93%,而阴性组及脂质体2000组与LPS组比较无明显减低。阳性组细胞浆中IκBα蛋白减低,比LPS组减低70.61%±2.19%,而阴性组及脂质体2000组与LPS组比较无明显减低,说明NF-κBp65 shRNA转染HSC后,干扰NF-κBp65的mRNA及蛋白发达的同时,NF-κBp50及IκBα蛋白表达随之降低。
5 NF-κBp65 shRNA基因干扰LPS激活的HSC后HSC凋亡增加:
流式细胞法结果显示阳性组细胞凋亡较LPS组增加,凋亡率为15.40%±0.72%,而阴性组及脂质体2000组与LPS组比较无明显增加。TUNEL法结果显示经阳性组HSC凋亡较LPS组增加,凋亡率为33.33%±1.06%,而阴性组及脂质体2000组与LPS组比较无明显增加。
6 NF-κBp65 shRNA基因干扰LPS激活的HSC后Ⅰ型前胶原mRNA表达减低:
RT-PCR结果显示,经阳性组Ⅰ型前胶原mRNA表达明显减低,较LPS组减低56.84%±1.89%,而阴性组及脂质体2000组与LPS组比较无明显减低,表明经NF-κBp65 shRNA基因干扰后Ⅰ型前胶原mRNA表达减低。
7 NF-κBp65 shRNA基因干扰LPS激活的HSC后明胶酶活性增高:
酶谱法测定结果显示,阳性组明胶酶活性明显升高,阴性组及脂质体2000组与LPS组比较无明显变化。
结论:
1本实验构建了针对HSC的NF-κBp65 mRNA靶序列1543–1561的阳性shRNA,将其转染HSC,有效地干扰了NF-κBp65的基因发达,与此同时,NF-κB p50及IκBα蛋白表达减低。
2 LPS可激活HSC中NF-κBp65的表达,使其蛋白表达增多。
3抑制NF-κBp65的基因发达可促进HSC的凋亡,同时Ⅰ型前胶原合成减少,明胶酶活性增强,延缓肝纤维化的发展。
The chronic hepatic disease is one of the important cosmopolitan diseases that are severely harmful to the health of human. Hepatic fibrosis is the middle and key element of this process. Hepatic stellate cell(HSC) is the beginning of whole thing and plays an important role in the process of the hepatic fibrosis. Inducing the apoptosis of the activated hepatic stellate cell can reverse the process of hepatic fibrosis. The increasing research has indicated that the activation and proliferation of HSC were related with the inhibition of apoptosis. Nuclear factor-κB (NF-κB) has a close relation with apoptosis. NF-κB, a protein that binds specifically to an enhancerκB sequence ofκimmunoglobin light chain is generated via homodimeric and heterodimeric interactions of Rel subunits. Mammalian Rel proteins found by now include p65 (RelA,NF-κB3), p50 (NF-κB 1) ,Rel (c-Rel),RelB和p52 (NF-κB2), and p65/p50 are found earliest, distribute most widely, and are the NF-κB that play chief physiological functions. NF-κB can induce transcription and expression of multiple cellular cytokines by sequence specifically binding to their promoter or enhancer region, thus promotes inflammation, immunological response, and has been closely correlated with certain important pathological and physical process including cell proliferation, transformation and apoptosis. Recently the appearance of RNA interference opens a new way to inhibit the gene expression.RNA interference is a process that after double-stranded RNA, complementary to the endogenous mRNA, transfected into cells, the mRNA degrades specifically, so that the gene the mRNA codes can’t express, and gene silences, showing us that we can silence the gene expression of NF-κB through the application of RNA interference, which can increase the apoptosis of the HSC and slow down the process of the hepatic fibrosis.
Objective: Through RNA interference by designing short hairpin RNA on the NF-κBp65, the test investigates the effects on apoptosis of lipopolysaccharide (LPS) - stimulated HSC.
Methods: Rat HSC was cultured in vitro and divided into 5 groups①blank control group;②LPS group: dealing with LPS;③negative group: dealing with negative siRNA and LPS;④positive group: dealing with NF-κBp65 shRNA and LPS;⑤lipidosome 2000 group: dealing with lipidosome 2000 and LPS. The expression levels of NF-κBp65, p50 and IκBαprotein were detected by western blot, the expression level of NF-κBp65 and procollagenⅠmRNA was detected by reverse transcription polymerase chain reaction (RT-PCR), and the change of apoptosis of HSC was detected by flow cytometry method and the method of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL).
Results:
1 SiRNA to monitor efficiency in HSC: A fluorescein-labeled non-special siRNA was used to monitor efficiency in HSC, demonstrating nealy 90% transfection efficiency after transfected with 33nmol/L siRNA at 72h.
2 The expression of NF-κBp65 protein increased in the cell nucleus after LPS stimulated HSC: the result of the western blot displayed that NF-κBp65 protein expression in the cell nucleus increased after LPS stimulated HSC. The expression was highest at one hour after the stimulation, and the increase of the expression with 1000ng/ml stimulating(232.15%±5.28%) was higher than that with 100ng/ml stimulating(168.18%±3.39%). The result showed us that LPS could stimulate the expression of NF-κBp65 in HSC, the expression arrived at the peak at one hour after LPS stimulated HSC and then fell off to the normal, and the expression of NF-κBp65 increased while the concentration of LPS increased.
3 NF-κBp65 protein and mRNA expression decreased in the cell nucleus after NF-κBp65 shRNA transfected into the HSC: the result of the western blot displayed that NF-κBp65 protein expression of the positive group in the cell nucleus decreased. The expression at 24 hours after the interference decreased by 72.41%±1.22% than the LPS group, the expression at 48 hours after the interference decreased by 77.45%±1.23% than the LPS group, and the expression at 72 hours after the interference decreased by 86.34%±1.01% than the LPS group, and the decrease was highest at 72 hours after the interference. The expression of the negative group and lipidosome 2000 group had not marked change.The result of the RT-PCR displayed that NF-κBp65 mRNA expression of the positive group in the cell decreased obviously. The expression of the negative group and lipidosome 2000 group had not marked change. The results showed us that the experiment constructed the short hairpin RNA which aimed at the 1543-1561 of the NF-κBp65 mRNA in rat HSC, the short hairpin RNA interfere effectively the expression of NF-κBp65 mRNA and protein after the short hairpin RNA was transfected into the HSC, and the efficiency of the interference arrived at the peak at 72 hours after the interference.
4 The expression of NF-κBp50 and IκBαprotein after NF-κBp65 shRNA transfected into the HSC: the result of the western blot displayed that NF-κB p50 protein expression of the positive group in the cell nucleus decreased, and the expression at 72 hours after the interference decreased by 75.45%±0.93% than the LPS group. And the expression of the negative group and lipidosome 2000 group had not marked change. IκBαprotein expression of the positive group in the cell cytoplasm decreased, and the expression at 72 hours after the interference decreased 70.61%±2.19% than the LPS group. And the expression of the negative group and lipidosome 2000 group had not marked change. The result showed us that the expression of NF-κBp50 and IκBαprotein decreased while NF-κBp65 shRNA interfered the expression of mRNA and protein of NF-κBp65 after it transfected into HSC.
5 The apotosis of HSC increased after interfering with the gene expression of NF-κBp65: the result of the flow cytometry method displayed that the apoptosis of HSC of the positive group increased, and the apoptosis at 72 hours after the interference was higher than the LPS group, the apoptosis ratio increased by 15.40%±0.72%. And the apoptosis of the negative group and lipidosome 2000 group had not marked change. The result of the TUNEL assay displayed that the apoptosis of HSC of the positive group increased after NF-κBp65 shRNA interfered the LPS-stimulated HSC, and the apoptosis at 72 hours after the interference was higher than the LPS group, the apoptosis ratio increased by 33.33%±1.06%. And the apoptosis of the negative group and lipidosome 2000 group had not marked change.
6 ProcollagenⅠmRNA expression in the cell decreased after interfering with the gene expression of NF-κBp65: the result of the RT-PCR displayed that procollagenⅠmRNA expression of the positive group in the cell decreased by 56.84%±1.89%. The expression of negative group and lipidosome 2000 group had not marked change. The result showed us that mRNA expression of procollagenⅠdecreased after interfering with the gene expression of NF-κBp65.
7 The avtivity of gelatinase increased after interfering with the gene expression of NF-κBp65: the result of the MMPs zymography showed that the avtivity of gelatinase of the positive group increased. The avtivity of negative group and lipidosome 2000 group had not marked change.
Conclusion:
1 The experiment constructed the short hairpin RNA which aimed at the 1543-1561 of the NF-κBp65 mRNA in rat HSC. And the short hairpin RNA interferes effectively the expression of NF-κBp65 mRNA and protein after the short hairpin RNA was transfected into the HSC. At the same time, the expression of NF-κB p50 and IκBαprotein decreased.
2 The expression of NF-κBp65 protein increased after LPS stimulated HSC.
3 The apoptosis of HSC increased after NF-κBp65 shRNA interfere effectively the expression of NF-κBp65 mRNA and protein, and the expression of ProcollagenⅠmRNA decreased and the activity of gelatinase increased, which can delay the process of hepatic fibrosis.
引文
1 Reeves HL, Friedman SL. Activation of hepatic stellate cells-akey issue in liver fibrosis. Front Biosci, 2002, 7: 808~826
2 Zhau XY, Murphy FR, Gehdu N, et al. Engagement of αvβ3 integrin regulates proliferation and apoptosis of hepatic stellate cells. J Biol Chem, 2004, 279 (23): 23996~24006
3 Oakley F, Meso M, Iredale JP, et al. Inhibition of IκB kinases stimulates hepatic stellate cell apoptosis and accelerates recovery from liver fibrosis. Gastroenterology, 2005, 128: 108~120
4 Mello CC, Conte DJ. Revealing the world of RNA interference. Nature, 2004, 431: 338~342
5 Verma NK, Dey CS. RNA-mediated gene silencing: mechanisms and its therapeutic applications. J Clin PharmTher, 2004, 29: 395~404
6 Li Y, Wasser S, Lim SG, et al. Genome-wide expression profiling of RNA interference of hepatitis B virus gene expression and replication. Cell Mol Life Sci, 2004, 61: 2113~2124
7 Daniel D, M. Vitorial BB, Ram IM. Effect of iNOS and NF-κB gene silencing on β-cell survival and function. Journal of Drug Targeting, June 2007, 15(5): 358~369
8 Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell, 1995, 81 (4): 611~620
9 Fire A, Xu S, Montgomery MK,et al. Potent and specific genetic interference by double-stranded RNA in C. elegans. Nature, 1998, 391(6669): 806~811
10 Baulcombe D. Viruses and gene silencing in plants. Archives Virology Suppl, 1999, 15: 189~201
11 Tavernarakis N, Wang S L, Dorovkov M , et al. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nature Genetics, 2000, 24: 180~183
12 Ui Jin Lee, So-Rim Choung, K. V. Bhanu Prakash, et al. Dual knockdown of p65 and p50 subunits of NF-kappaB by siRNA inhibits the induction of inflammatory cytokines and significantly enhance apoptosis in human primary synoviocytes treated with tumor necrosis factor-alpha. Mol Biol Rep, 2007, 5: 203~211
13 Ghosh S, M. Karin. Missing pieces in the NF-kappaB puzzle. Cell, 2002, 109: S81~S96
14 Saile B, DeRocco P, Dudas J, et al. IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells (HSC) but DNA synthesis and proliferation in rat liver myofibroblasts( rMF) . LabInvest, 2004, 84: 1037~1049
15 Thirunavukkarasu C, Watkins S, Harvey R. Superoxide-induced apoptosis of activated rat hepatic stellate cell s. J Hepatol, 2004, 41: 567~575
16 Montiel-Duarte C, Ansorena E, Lopez-Zabalza MJ, et al. Role of reactive oxygen species, glutathione and NF-kappaB in apoptosis induced by 3, 4-methylenedioxy Methampheta-mine (”Ecstasy”) on hepatic stellate cells. Biochem Pharmacol, 2004, 67 (6): 1025~1033
17 Lang A, Schoonhovan R, Tuvia S, et al. Nuclear factor kappaB in proliferation, activation and apoptosis in rat hepatic stellate cells. J Hepatol 2000, 33: 49~58
18 Wang CY, Mayo MW, Baldwin AS. TNF and cancer therapy induce apoptesis: potentiation by inhibition of NF Kappa B. Science, 1996, 274: 784~787
19 Saile B, Matthes N, El Armouche H, et al. The bcl, NF-kappaB and p53/ p21WAF1 systems are involved in spontaneous apoptosis and in the anti-apoptotic effect of TGF-beta or TNF-alpha on activated hepatic stellate cells. Eur J Cell Biol. 2001 Aug, 80 (8) : 554~561
20 Wang CY, Mayo MW, Komeluk RG, et al. NF-κappa Banti-apoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science, 1999, 281 (5383):1680 ~1693
21 Yasui H, Adachi M, Hamada H, et al. Adenovirus-mediated gene transfer of caspase-8 sensitizes human adeno-carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand. Int J Oncol, 2005, 26 (2): 537~ 544
1 Henkel T, Zabel U, van Zee K, et al. Intramolecular masking of the nuclear location signal and dimerization domain in the p recursor for the p50 NF-kappa B subunit. Cell, 1992, 68(6) : 1121~1133
2 Ghosh S, M. Karin. Missing pieces in the NF-kappaB puzzle. Cell, 2002, 109 :S81~S96
3 Cohen L, Henzel WJ, Baeuerle PA. IKAP is a scaffold protein of the IkappaB kinase complex. Nature, 1998, 395 (6699): 225~226
4 Jun NT, Kazuya K, Atsushi H, et al. The kinase TAK1 can active the NIK-IκB as well as the MAP kinase cascade in the IL-1 sigalling pathway. Nature, 1999, 398 (6724): 252~256
5 Arsura M, Fitz G, Fausto N. Nuclear factor Kappa B/Rel bloxks transforming growth factor beta 1 induced apoptosis of murine hepatocyte cell lines. Cell GROWTH Differ, 1997, 8 (10): 1049~1059
6 Verma UN, Yamamoto Y, Prajapati S, et al. Nuclear role of Ikappa B Kinase-gamma/ NF-kappa B essential modulator (IKKgamma/NEMO) in NF-kappa B-dependent gene expression. JBiol Chem, 2004, 279 (5): 3509~3515
7 PrendesM, Zheng Y, Beg AA. Regulation of developing B cell survival by RelA-containing NF-kappa B complexes. J Immunol, 2003, 171(8): 3963~3969
8 Vasudevan KM, Gurumurthy S, RangnekarVM. Suppressionof PTEN expression by NF-kappa B prevents apoptosis. Mol Cell Biol, 2004, 24 (3): 1007~1021
9 Nagaki M, Naiki T, Brenner DA, et al. Tumor necrosis factor alpha prevents tumor necrosis factor receptor-mediated mouse hepatocyte apoptosis, but not fas-mediated apoptosis: role of nuclear factor-κappa B . Hepatology, 2000, 32 (6): 1272 ~ 1279
10 Biffl WL, Moore EE, Moore FA, et al. Interleukin-6 delays neutrophil apoptosis. Arch Surg, 1996, 131 (1): 24 ~29
11 Hong F, Kim WH, Tian Z, et al. Elevated interleukin-6 during ethanol consumption acts as a potential endogenous protective cytokine against ethanol-induced apoptosis in the liver: involvement of induction of Bcl-2 and Bcl-x L proteins. Oncogene, 2002, 21(1): 32 ~ 43
12 Wang CY, Guttridge DC, Mayo MW, et al. NF-κappa B induces expression of the Bcl-2 homologue Al/ Bfl-1 to preferentially suppress chemotherapy-induced apoptosis. Mol Cell Biol, 1999, 19 (9): 5923 ~ 5929
13 Lee HH, Dadgostar H, Cheng Q, et al. NF-κappa B-mediated up-regulation of Bcl-x and Bfl/ Al is required for CD40 survival signaling in B lymphocytes. Proc Natl Acad Sci USA, 1999, 96(19): 9136 ~9141
14 Lee EG, Boone DL, Chai S, et al. Failure to regulate TNF-induced NF-κappa B and cell death responses in A202deficient mice. Science, 2000, 289 (5488): 2350 ~ 2354
15 Wang CY, Mayo MW, Komeluk RG, et al. NF-κappa B anti-apoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science, 1999, 281 (5383): 1680 ~1693
16 Yasui H, Adachi M, Hamada H, et al. Adenovirus-mediated gene transfer of caspase-8 sensitizes human adeno-carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand. Int J Oncol, 2005, 26 (2): 537 ~ 544
17 Kuhnel F, Zender L, Paul Y, et al. NF-κB mediates apoptosis through transcriptional activation of Fas (CD95) in adenoviral hepatitis. J Bio Chem, 2000, 275 (9): 6421 ~ 6427
18 Hsu YL, Kuo PL, Chiang LC, et al. Involvement of p53, nuclear factorκappa B and Fas/ Fas ligand in induction of apoptosis and cell cycle arrest by saikosaponin d in human hepatoma cell lines. Cancer Lett, 2004, 213 (2): 213 ~ 221
19 Gil J, Alcami J, Esteban M, et al. Induction of apoptosis by double- stranded-RNA dependent protein kinase (PKR) involves the a subunit of eukaryotic translation initiation factor 2 and NF-κB. Mol Cell Biol, 1999, 19(7): 4653 ~ 4663
20 Saile B, DeRocco P, Dudas J, et al. IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells ( HSC) but DNA synthesis and proliferation in rat liver myofibroblasts( rMF). LabInvest, 2004, 84: 1037~1049
21 Thirunavukkarasu C, Watkins S, Harvey R. Superoxide-induced apoptosis of activated rat hepatic stellate cells. J Hepatol, 2004, 41: 567~575
22 Oakley F, Meso M, Iredale JP, et al. Inhibition of IκB kinases stimulates hepatic stellate cell apoptosis and accelerates recovery from liver fibrosis. Gastroenterology, 2005, 128: 108~120
23 Papa S, Zazzeroni F, Bubici C, et al. Gadd45 beta mediates the NF-kappa B suppression of JNK signaling by targeting MKK7/JN KK2. Nat Cell Biol, 2004, 6: 146~153
2 Zhau XY, Murphy FR, Gehdu N, et al. Engagement of αvβ3 integrin regulates proliferation and apoptosis of hepatic stellate cells. J Biol Chem, 2004, 279 (23): 23996~24006
3 Oakley F, Meso M, Iredale JP, et al. Inhibition of IκB kinases stimulates hepatic stellate cell apoptosis and accelerates recovery from liver fibrosis. Gastroenterology, 2005, 128: 108~120
4 Mello CC, Conte DJ. Revealing the world of RNA interference. Nature, 2004, 431: 338~342
5 Verma NK, Dey CS. RNA-mediated gene silencing: mechanisms and its therapeutic applications. J Clin PharmTher, 2004, 29: 395~404
6 Li Y, Wasser S, Lim SG, et al. Genome-wide expression profiling of RNA interference of hepatitis B virus gene expression and replication. Cell Mol Life Sci, 2004, 61: 2113~2124
7 Daniel D, M. Vitorial BB, Ram IM. Effect of iNOS and NF-κB gene silencing on β-cell survival and function. Journal of Drug Targeting, June 2007, 15(5): 358~369
8 Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell, 1995, 81 (4): 611~620
9 Fire A, Xu S, Montgomery MK,et al. Potent and specific genetic interference by double-stranded RNA in C. elegans. Nature, 1998, 391(6669): 806~811
10 Baulcombe D. Viruses and gene silencing in plants. Archives Virology Suppl, 1999, 15: 189~201
11 Tavernarakis N, Wang S L, Dorovkov M , et al. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nature Genetics, 2000, 24: 180~183
12 Ui Jin Lee, So-Rim Choung, K. V. Bhanu Prakash, et al. Dual knockdown of p65 and p50 subunits of NF-kappaB by siRNA inhibits the induction of inflammatory cytokines and significantly enhance apoptosis in human primary synoviocytes treated with tumor necrosis factor-alpha. Mol Biol Rep, 2007, 5: 203~211
13 Ghosh S, M. Karin. Missing pieces in the NF-kappaB puzzle. Cell, 2002, 109: S81~S96
14 Saile B, DeRocco P, Dudas J, et al. IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells (HSC) but DNA synthesis and proliferation in rat liver myofibroblasts( rMF) . LabInvest, 2004, 84: 1037~1049
15 Thirunavukkarasu C, Watkins S, Harvey R. Superoxide-induced apoptosis of activated rat hepatic stellate cell s. J Hepatol, 2004, 41: 567~575
16 Montiel-Duarte C, Ansorena E, Lopez-Zabalza MJ, et al. Role of reactive oxygen species, glutathione and NF-kappaB in apoptosis induced by 3, 4-methylenedioxy Methampheta-mine (”Ecstasy”) on hepatic stellate cells. Biochem Pharmacol, 2004, 67 (6): 1025~1033
17 Lang A, Schoonhovan R, Tuvia S, et al. Nuclear factor kappaB in proliferation, activation and apoptosis in rat hepatic stellate cells. J Hepatol 2000, 33: 49~58
18 Wang CY, Mayo MW, Baldwin AS. TNF and cancer therapy induce apoptesis: potentiation by inhibition of NF Kappa B. Science, 1996, 274: 784~787
19 Saile B, Matthes N, El Armouche H, et al. The bcl, NF-kappaB and p53/ p21WAF1 systems are involved in spontaneous apoptosis and in the anti-apoptotic effect of TGF-beta or TNF-alpha on activated hepatic stellate cells. Eur J Cell Biol. 2001 Aug, 80 (8) : 554~561
20 Wang CY, Mayo MW, Komeluk RG, et al. NF-κappa Banti-apoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science, 1999, 281 (5383):1680 ~1693
21 Yasui H, Adachi M, Hamada H, et al. Adenovirus-mediated gene transfer of caspase-8 sensitizes human adeno-carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand. Int J Oncol, 2005, 26 (2): 537~ 544
1 Henkel T, Zabel U, van Zee K, et al. Intramolecular masking of the nuclear location signal and dimerization domain in the p recursor for the p50 NF-kappa B subunit. Cell, 1992, 68(6) : 1121~1133
2 Ghosh S, M. Karin. Missing pieces in the NF-kappaB puzzle. Cell, 2002, 109 :S81~S96
3 Cohen L, Henzel WJ, Baeuerle PA. IKAP is a scaffold protein of the IkappaB kinase complex. Nature, 1998, 395 (6699): 225~226
4 Jun NT, Kazuya K, Atsushi H, et al. The kinase TAK1 can active the NIK-IκB as well as the MAP kinase cascade in the IL-1 sigalling pathway. Nature, 1999, 398 (6724): 252~256
5 Arsura M, Fitz G, Fausto N. Nuclear factor Kappa B/Rel bloxks transforming growth factor beta 1 induced apoptosis of murine hepatocyte cell lines. Cell GROWTH Differ, 1997, 8 (10): 1049~1059
6 Verma UN, Yamamoto Y, Prajapati S, et al. Nuclear role of Ikappa B Kinase-gamma/ NF-kappa B essential modulator (IKKgamma/NEMO) in NF-kappa B-dependent gene expression. JBiol Chem, 2004, 279 (5): 3509~3515
7 PrendesM, Zheng Y, Beg AA. Regulation of developing B cell survival by RelA-containing NF-kappa B complexes. J Immunol, 2003, 171(8): 3963~3969
8 Vasudevan KM, Gurumurthy S, RangnekarVM. Suppressionof PTEN expression by NF-kappa B prevents apoptosis. Mol Cell Biol, 2004, 24 (3): 1007~1021
9 Nagaki M, Naiki T, Brenner DA, et al. Tumor necrosis factor alpha prevents tumor necrosis factor receptor-mediated mouse hepatocyte apoptosis, but not fas-mediated apoptosis: role of nuclear factor-κappa B . Hepatology, 2000, 32 (6): 1272 ~ 1279
10 Biffl WL, Moore EE, Moore FA, et al. Interleukin-6 delays neutrophil apoptosis. Arch Surg, 1996, 131 (1): 24 ~29
11 Hong F, Kim WH, Tian Z, et al. Elevated interleukin-6 during ethanol consumption acts as a potential endogenous protective cytokine against ethanol-induced apoptosis in the liver: involvement of induction of Bcl-2 and Bcl-x L proteins. Oncogene, 2002, 21(1): 32 ~ 43
12 Wang CY, Guttridge DC, Mayo MW, et al. NF-κappa B induces expression of the Bcl-2 homologue Al/ Bfl-1 to preferentially suppress chemotherapy-induced apoptosis. Mol Cell Biol, 1999, 19 (9): 5923 ~ 5929
13 Lee HH, Dadgostar H, Cheng Q, et al. NF-κappa B-mediated up-regulation of Bcl-x and Bfl/ Al is required for CD40 survival signaling in B lymphocytes. Proc Natl Acad Sci USA, 1999, 96(19): 9136 ~9141
14 Lee EG, Boone DL, Chai S, et al. Failure to regulate TNF-induced NF-κappa B and cell death responses in A202deficient mice. Science, 2000, 289 (5488): 2350 ~ 2354
15 Wang CY, Mayo MW, Komeluk RG, et al. NF-κappa B anti-apoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science, 1999, 281 (5383): 1680 ~1693
16 Yasui H, Adachi M, Hamada H, et al. Adenovirus-mediated gene transfer of caspase-8 sensitizes human adeno-carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand. Int J Oncol, 2005, 26 (2): 537 ~ 544
17 Kuhnel F, Zender L, Paul Y, et al. NF-κB mediates apoptosis through transcriptional activation of Fas (CD95) in adenoviral hepatitis. J Bio Chem, 2000, 275 (9): 6421 ~ 6427
18 Hsu YL, Kuo PL, Chiang LC, et al. Involvement of p53, nuclear factorκappa B and Fas/ Fas ligand in induction of apoptosis and cell cycle arrest by saikosaponin d in human hepatoma cell lines. Cancer Lett, 2004, 213 (2): 213 ~ 221
19 Gil J, Alcami J, Esteban M, et al. Induction of apoptosis by double- stranded-RNA dependent protein kinase (PKR) involves the a subunit of eukaryotic translation initiation factor 2 and NF-κB. Mol Cell Biol, 1999, 19(7): 4653 ~ 4663
20 Saile B, DeRocco P, Dudas J, et al. IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells ( HSC) but DNA synthesis and proliferation in rat liver myofibroblasts( rMF). LabInvest, 2004, 84: 1037~1049
21 Thirunavukkarasu C, Watkins S, Harvey R. Superoxide-induced apoptosis of activated rat hepatic stellate cells. J Hepatol, 2004, 41: 567~575
22 Oakley F, Meso M, Iredale JP, et al. Inhibition of IκB kinases stimulates hepatic stellate cell apoptosis and accelerates recovery from liver fibrosis. Gastroenterology, 2005, 128: 108~120
23 Papa S, Zazzeroni F, Bubici C, et al. Gadd45 beta mediates the NF-kappa B suppression of JNK signaling by targeting MKK7/JN KK2. Nat Cell Biol, 2004, 6: 146~153