腺病毒载体介导的RNAi抑制肺间质纤维化大鼠TGF-β1基因表达的研究
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摘要
背景
纤维化可导致器官结构破坏和功能减退、乃至衰竭。肺间质纤维化(pulmonary fibrosis,PF)是由多种因素引起的一组肺间质弥漫性疾病,是异质性疾病,也是多种肺疾病的共同结局,其发病机制复杂,研究证实肺间质纤维化的发生、发展与转化生长因子-β1(TGF-β1)的高表达密切相关。
转化生长因子-β1(TGF-β1)是公认的肺间质纤维化形成和发展中的“开关性”细胞因子,作为一种多功能及强效的致纤维化因子,参与调控细胞的分化、增殖及细胞外基质(extracellular membrane)分泌,增加ECM的聚积并抑制其降解,在肺纤维化形成过程中主要作用于胶原的转录和翻译过程,诱导前胶原mRNA的产生,促进胶原蛋白的形成和沉积,是纤维化发生过程中最直接的一种细胞因子。抑制TGF-β1基因表达显然有助于延缓肺纤维化进程,而应用常规抗纤维化药物抑制其表达的效果并不理想。
RNA干扰(RNA interference, RNAi)是新近出现的一种分子生物学技术,它通过导入由对应的正义RNA和反义RNA组成的双链RNA(double strand RNA, dsRNA),诱导受体细胞中与其有同源序列的特异性靶mRNA降解,阻止蛋白质的翻译过程,其作用类似于基因敲除,但基因表达并未永远消除,因而又被称为“基因沉默”。
目的
本研究针对大鼠TGF-β1的mRNA序列设计、合成并构建具有特异性阻断大鼠转化生长因子β1(TGF-β1)基因的TGF-β1 siRNA Shuttle载体,然后重组入腺病毒载体,包装成转化生长因子受体β1 (TGF-β1) siRNA的腺病毒,将腺病毒载体局部转染肺间质纤维化大鼠,观察并分析其对TGF-β1mRNA表达的抑制作用,为肺纤维化的防治提供实验依据。
材料和方法
60只SD雄性大鼠,生理盐水对照组、重组复制缺陷型TGF-β1腺病毒治疗组、空载体对照组,第0d经气管滴入0.3ml的博来霉素(BLM3mg/kg)建立肺间质纤维化模型,空白对照组大鼠滴注等体积生理盐水,治疗组于滴入博来霉素24h后经鼻滴入重组复制缺陷型TGF-β1腺病毒(AdCMV TGF-β1)10×109空斑形成单位(pfu)(100μl／鼠),生理盐水组于造模后24h经鼻滴入同体积的生理盐水,同时设立无外源活性基因的复制缺陷型腺病毒(AdCMVNull)空载体对照组,空白对照组于24h给予气管内滴入同体积生理盐水。各组动物于7、14、28d随机处死5只,取肺组织肺行苏木精伊红(HE)染色；免疫组化法检测肺组织TGF-β1的蛋白表达水平；荧光定量PCR检测肺组织中TGF-βmRNA的表达水平；ELISA法检测血清中TGF-β的蛋白的表达水平,评价其对全身的影响。
结果
HE染色示治疗组各时期肺泡炎和肺纤维化程度均较生理盐水对照组及空载体对照组轻；免疫组化结果显示治疗组与生理盐水及空载体对照组相比肺组织TGF-β1蛋白表达水平降低,差异有统计学意义(P<0.05)；实时荧光定量PCR结果显示治疗组TGF-β1mRNA表达水平与生理盐水及空载体对照组比较降低,差异有统计学意义(P<0.05)；而血清中TGF-β1各组各时期蛋白表达量无统计学差异(P>0.05)。
结论
腺病毒介导的RNAi可以抑制肺纤维化鼠肺组织TGF-β1的表达,减轻肺纤维化大鼠肺泡炎和肺纤维化,对血清中的TGF-β1表达影响较小,有望成为防治肺间质纤维化的一种手段。
Background
Fibrosis could lead to disorganization and hypofunction, even failure of organ. Pulmonary interstitial fibrosis (PF) is a series of diffused pulmonary interstitial disease caused by many factors, and is a heterogeneous disease as well as the common end of many pulmonary diseases.The pathogenesis was complex,and research shew the occurrence and development of pulmonary interstitial fibrosis were closely correlated with high expression of TGF-β1.
Transforming growth factor-β1 known as the key cell factor in the formation and progression of pulmonary interstitial fibrosis,could regulate differentiation hyperplasia and excretion of extracellular membrane (ECM) as a multifunctional and super effective cell factor, and increase accumulation and inhibit degradation of ECM. TGF-β1 plays an important role in transcription and translation on the processes of collagens,and induce the production of pre-collagen mRNA,advance formation and deposition of collagen protein. it is the most direct cell factor in pulmonary fibrosis. It's obvious that inhibiting the expression of TGF-β1 is helpful to delay the process of pulmonary fibrosis, but the effect is still unsatisfactory by conventional anti-fibrosis drugs to inhibit the expression of TGF-β1。
RNA interference is a new molecular biological technology, and it can import the corresponding sense RNA and antisense RNA which are called double strand RNA (dsRNA)to induce specific target mRNA with homological sequences to degrade in the recipient cell, and then to prevent the translation process of protein.The effect is similar to gene knock-out, but the disappearance of genetic expression is temporary,also called "gene silencing"
Objective
To construct the specific small interfereing RNA(siRNA) Duplication-deficiency adenovirus vector that can block the rat TGF-β1 gene according to the mRNA of rat TGF-(31,To investigate the inhibitory effect of TGF-β1 transgenic expression on bleomycin-induced pulmonary fibrosis in mice.
Materials and methods
60 male Sprague-Dawley (SD) rats were treated with bleomycin by 5mg per kg weight via trachea on day 0 in the normal saline group, therapeutic group (AdCMV TGF-β1) and sham recombinant adenoviruses(AdCMVNull),Adenoviral vector with murine TGF-β1SiRNA (AdCMVTGF-β1)10×109plague forming unit (pfu) was administrated via nostril instillation 24h after bleomycin treatment in the therapeutic group, Mice treat Fluorescence Quantitative PCR and the TGF-β1 level in the blood serum was quantitatively determined by EnzymeLinked immunosorbent Assay.ed with a same volume of normal saline (NS) and a same dosage of sham recombinant adenoviruses (AdCMV Null) served as controls.The animals were killed on day 7,14,28,The right lung was stained with either hematoxylin-eosin,and the expression of TGF-β1 in the lung was detected by immunohistochemical technique;the TGF-β1mRNA expression in lung was detected by Real-time.
Result
The alveolitis and fibrosis of the treatment group were less than that in normal saline group and sham recombinant adenoviruses group in HE sections.The immunohistochemistry expression of TGF-β1 in treatment group was significantly lower than the normal saline group and sham recombinant adenoviruses group (P<0.05);the TGF-β1 mRNA expression was significantly lower than other normal saline group and sham recombinant adenoviruses group (P<0.05),the TGF-β1 expression in blood serum was no statistical significance in all the groups(P>0.05).
Conclusion
The RNA interference mediated by Adenovirus vector can inhibit the expression of TGF-beta1 in lungs of mice with pulmonary fibrosis;reduce the alveolitis and fibrosis of pulmonary in rats with pulmonary fibrosis.but the expression of TGF-beta1 in serum were not impacted.
纤维化可导致器官结构破坏和功能减退、乃至衰竭。肺间质纤维化(pulmonary fibrosis,PF)是由多种因素引起的一组肺间质弥漫性疾病,是异质性疾病,也是多种肺疾病的共同结局,其发病机制复杂,研究证实肺间质纤维化的发生、发展与转化生长因子-β1(TGF-β1)的高表达密切相关。
转化生长因子-β1(TGF-β1)是公认的肺间质纤维化形成和发展中的“开关性”细胞因子,作为一种多功能及强效的致纤维化因子,参与调控细胞的分化、增殖及细胞外基质(extracellular membrane)分泌,增加ECM的聚积并抑制其降解,在肺纤维化形成过程中主要作用于胶原的转录和翻译过程,诱导前胶原mRNA的产生,促进胶原蛋白的形成和沉积,是纤维化发生过程中最直接的一种细胞因子。抑制TGF-β1基因表达显然有助于延缓肺纤维化进程,而应用常规抗纤维化药物抑制其表达的效果并不理想。
RNA干扰(RNA interference, RNAi)是新近出现的一种分子生物学技术,它通过导入由对应的正义RNA和反义RNA组成的双链RNA(double strand RNA, dsRNA),诱导受体细胞中与其有同源序列的特异性靶mRNA降解,阻止蛋白质的翻译过程,其作用类似于基因敲除,但基因表达并未永远消除,因而又被称为“基因沉默”。
目的
本研究针对大鼠TGF-β1的mRNA序列设计、合成并构建具有特异性阻断大鼠转化生长因子β1(TGF-β1)基因的TGF-β1 siRNA Shuttle载体,然后重组入腺病毒载体,包装成转化生长因子受体β1 (TGF-β1) siRNA的腺病毒,将腺病毒载体局部转染肺间质纤维化大鼠,观察并分析其对TGF-β1mRNA表达的抑制作用,为肺纤维化的防治提供实验依据。
材料和方法
60只SD雄性大鼠,生理盐水对照组、重组复制缺陷型TGF-β1腺病毒治疗组、空载体对照组,第0d经气管滴入0.3ml的博来霉素(BLM3mg/kg)建立肺间质纤维化模型,空白对照组大鼠滴注等体积生理盐水,治疗组于滴入博来霉素24h后经鼻滴入重组复制缺陷型TGF-β1腺病毒(AdCMV TGF-β1)10×109空斑形成单位(pfu)(100μl／鼠),生理盐水组于造模后24h经鼻滴入同体积的生理盐水,同时设立无外源活性基因的复制缺陷型腺病毒(AdCMVNull)空载体对照组,空白对照组于24h给予气管内滴入同体积生理盐水。各组动物于7、14、28d随机处死5只,取肺组织肺行苏木精伊红(HE)染色；免疫组化法检测肺组织TGF-β1的蛋白表达水平；荧光定量PCR检测肺组织中TGF-βmRNA的表达水平；ELISA法检测血清中TGF-β的蛋白的表达水平,评价其对全身的影响。
结果
HE染色示治疗组各时期肺泡炎和肺纤维化程度均较生理盐水对照组及空载体对照组轻；免疫组化结果显示治疗组与生理盐水及空载体对照组相比肺组织TGF-β1蛋白表达水平降低,差异有统计学意义(P<0.05)；实时荧光定量PCR结果显示治疗组TGF-β1mRNA表达水平与生理盐水及空载体对照组比较降低,差异有统计学意义(P<0.05)；而血清中TGF-β1各组各时期蛋白表达量无统计学差异(P>0.05)。
结论
腺病毒介导的RNAi可以抑制肺纤维化鼠肺组织TGF-β1的表达,减轻肺纤维化大鼠肺泡炎和肺纤维化,对血清中的TGF-β1表达影响较小,有望成为防治肺间质纤维化的一种手段。
Background
Fibrosis could lead to disorganization and hypofunction, even failure of organ. Pulmonary interstitial fibrosis (PF) is a series of diffused pulmonary interstitial disease caused by many factors, and is a heterogeneous disease as well as the common end of many pulmonary diseases.The pathogenesis was complex,and research shew the occurrence and development of pulmonary interstitial fibrosis were closely correlated with high expression of TGF-β1.
Transforming growth factor-β1 known as the key cell factor in the formation and progression of pulmonary interstitial fibrosis,could regulate differentiation hyperplasia and excretion of extracellular membrane (ECM) as a multifunctional and super effective cell factor, and increase accumulation and inhibit degradation of ECM. TGF-β1 plays an important role in transcription and translation on the processes of collagens,and induce the production of pre-collagen mRNA,advance formation and deposition of collagen protein. it is the most direct cell factor in pulmonary fibrosis. It's obvious that inhibiting the expression of TGF-β1 is helpful to delay the process of pulmonary fibrosis, but the effect is still unsatisfactory by conventional anti-fibrosis drugs to inhibit the expression of TGF-β1。
RNA interference is a new molecular biological technology, and it can import the corresponding sense RNA and antisense RNA which are called double strand RNA (dsRNA)to induce specific target mRNA with homological sequences to degrade in the recipient cell, and then to prevent the translation process of protein.The effect is similar to gene knock-out, but the disappearance of genetic expression is temporary,also called "gene silencing"
Objective
To construct the specific small interfereing RNA(siRNA) Duplication-deficiency adenovirus vector that can block the rat TGF-β1 gene according to the mRNA of rat TGF-(31,To investigate the inhibitory effect of TGF-β1 transgenic expression on bleomycin-induced pulmonary fibrosis in mice.
Materials and methods
60 male Sprague-Dawley (SD) rats were treated with bleomycin by 5mg per kg weight via trachea on day 0 in the normal saline group, therapeutic group (AdCMV TGF-β1) and sham recombinant adenoviruses(AdCMVNull),Adenoviral vector with murine TGF-β1SiRNA (AdCMVTGF-β1)10×109plague forming unit (pfu) was administrated via nostril instillation 24h after bleomycin treatment in the therapeutic group, Mice treat Fluorescence Quantitative PCR and the TGF-β1 level in the blood serum was quantitatively determined by EnzymeLinked immunosorbent Assay.ed with a same volume of normal saline (NS) and a same dosage of sham recombinant adenoviruses (AdCMV Null) served as controls.The animals were killed on day 7,14,28,The right lung was stained with either hematoxylin-eosin,and the expression of TGF-β1 in the lung was detected by immunohistochemical technique;the TGF-β1mRNA expression in lung was detected by Real-time.
Result
The alveolitis and fibrosis of the treatment group were less than that in normal saline group and sham recombinant adenoviruses group in HE sections.The immunohistochemistry expression of TGF-β1 in treatment group was significantly lower than the normal saline group and sham recombinant adenoviruses group (P<0.05);the TGF-β1 mRNA expression was significantly lower than other normal saline group and sham recombinant adenoviruses group (P<0.05),the TGF-β1 expression in blood serum was no statistical significance in all the groups(P>0.05).
Conclusion
The RNA interference mediated by Adenovirus vector can inhibit the expression of TGF-beta1 in lungs of mice with pulmonary fibrosis;reduce the alveolitis and fibrosis of pulmonary in rats with pulmonary fibrosis.but the expression of TGF-beta1 in serum were not impacted.
引文
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