大鼠硅肺纤维化组织cDNA消减文库的构建和差异表达基因的克隆
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摘要
前言:肺纤维化是一组由多种病因引起的肺破坏性疾病,肺纤维化过程包括肺组织的炎症损伤、组织结构破坏以及随后伴有的肺间质细胞积聚的组织修复过程。在此过程中,肺泡巨噬细胞、肺泡上皮细胞、肺成纤维细胞等效应细胞通过分泌细胞因子、炎症介质等生物活性物质,发挥直接或间接的作用,从而使这些参与肺纤维化的多种细胞共同构成了一个复杂的细胞网络,彼此间相互影响,共同促进了病变发展。但目前对该疾病发生发展起关键作用的细胞分子机制尚不清楚,迄今为止仍未找到有效的防治措施。目前肺纤维化的动物研究模型大多数仅限于在病变早期,尚未形成典型的肺纤维化病变,因此也不能分析肺纤维化病变与其他疾病的关系。目前认为慢性阻塞性肺疾病、间质性肺病(石棉肺、硅肺、特发性间质性肺纤维化等)均为肺癌的危险因素,间质性肺纤维化引发肺癌的机制目前尚不清楚,大多数观点认为,间质性肺纤维化由于炎症损伤和修复的相互作用可导致局部上皮反复的细胞及基因损伤,通过连续的细胞形态学改变如化生,异常增生,非典型增生而最终导致肿瘤发生。故加深对肺纤维化发病机制的认识,并在此基础上发展新的治疗策略已成为更加迫切的需要。
目的:本研究拟通过建立大鼠慢性硅肺纤维化动物模型,利用SSH技术,筛选和鉴定硅肺纤维化大鼠与正常大鼠之间肺组织差异表达基因,旨在获得与肺纤维化相关的特异表达基因,为肺纤维化发生机制及与其他疾病的关系提供实验依据。
方法:本研究以SD大鼠为实验对象,采用气管插管灌注二氧化硅粉尘,经过360天,建立硅肺肺纤维化动物模型;应用抑制消减杂交技术(SSH),结合T/A克隆技术和蓝白筛选构建了硅肺肺纤维化正反向差异表达消减cDNA文库(SNA,SNB);经过菌液PCR技术初步验证,插入片段大小,采用半定量RT—PCR技术证实部分基因确实存在差异表达,随机挑取部分含有插入片段的克隆进行测序分析及生物信息学分析。
结果:
1、360天,X线显示大鼠肺纹理增粗,肺野有散在分布的高密度影出现。肉眼见肺组织表面及切面有散在分布的灰白色小结节,结节大小不等;显微镜下肺内有典型的纤维性硅结节形成,在部分硅结节周围肺泡上皮细胞和管上皮明显增生,部分细胞显不同程度不典型增生,肺间质纤维化明显。
2、分别以肺纤维化肺组织cDNA为实验方(Tester),以正常肺组织为对照方(Driver),以正常肺组织cDNA为实验方(Tester),以肺纤维化肺组织cDNA为对照方(Driver)构建了反向差异表达消减cDNA文库(SNB)。经过菌液PCR技术初步验证,插入片段大小主要分布于200-600bp之间。随机挑取正向和反向消减文库共52个含有插入片段的克隆进行测序分析,共获得47个有效差异表达cDNA片段。为证实这些基因确实存在差异表达,采用半定量RT—PCR技术对随机挑选的硅肺纤维化肺组织消减文库中的SNA—11、SNA—28、SNA—43、SNA—48,正常肺组织消减文库中的SNB—13、SNB—30、SNB—42进行RT—PCR分析,结果显示它们在硅肺纤维化肺组织和正常肺组织中的表达存在差异,根据图像扫描定量分析,在两组肺组织中基因表达量相差在2—5倍以上。
3、生物信息学分析表明双向消减cDNA文库测序得到的47个阳性克隆代表了35个基因,包含2个假想蛋白、4个推测的相似蛋白。其中大多数功能已知基因在细胞或细胞外基质结构/运动、细胞/机体防御、物质转运、翻译/表达调控、代谢等方面起重要作用。表明SSH技术是筛选差异表达基因的有效方法,对这些基因功能的步研究,有利于从另外的角度了解肺纤维化的机制。
结论:
1成功建立了典型大鼠慢性硅肺纤维化动物模型。
2建立了较高质量的硅肺纤维化正反差异消减cDNA文库。
3获得部分与肺纤维化相关的基因,对这些基因的进一步研究,有利于从不同的角度了解肺纤维化的机制。
4证实硅肺与肺癌有关,可能是肺癌发生的危险因素之一。
Introduction: Pulmonary fibrosis is a sort of destructive lung diseases induced by multiple agents, which has poor prognosis. The pathological processes of pulmonary fibrosis include inflammatory injury, tissue damage and repair. Many kinds of cells such as alveolar macrophages, epithelial cells and fibroblasts et al play important roles in the progress of pulmonary fibrosis. These cells educe direct or indirect effects by secreting many bioactive compounds such as cytokines and mediators of inflammation and constitute a complicate cellular network in which the cells interacting each other and promoting the development of pulmonary fibrosis. However, the critical molecular mechanism in the development of the pulmonary fibrosis is still unknown, it is vital to search for better treatment plan of pulmonary fibrosis, based on the understanding of the pathogenesis of these diseases. As the developing of strategies and methods of identification of novel genes, various methods to compare patterns of gene expression have been described, it is becoming possible to identify some novel genes related with pulmonary fibrosis. The aim of our studies is to screen and identify differentially expressed gene of pulmonary fibrosis.
Methods: The SD (Sprage-Dawley) rats were poured into suspension of crystalline silica (200mg/kg body weight) through trachea cannula to construct the animal model of silicosis. The suppression subtractive hybridization was performed; two different forward and reverse subtractions were performed to compare gene expression between lung tissue with silicosis and normal lung tissue. The SSH PCR products from the library were cloned into pMD20-T vector; the positive clones were randomly selected, sequenced and compared to the database in GenBank of the differentially expressed gene fragments from the libraries. Seven novel cDNA sequences were examined by reverse transcription- polymerase chain reaction (RT-PCR) in order to confirm the differentially expression of cDNA fragments in rat's lung tissue with silicosis and normal lung tissue.
Results:
1. After 360 days, radiograph of chest showed that rat's pulmonary markings was thickened, some scattered high-density shadows appeared in lung field. From the surface and cross section of lung, some scattered nodules could be seen whose sizes were inequable. Under microscope, typical fibrosing siliconic nodules were formed in lung, alveolar epithelial cells and bronchial epithelial cells proliferated around partial fibrosing siliconic nodules, and some cells of which showed atypical hyperplasia. Diffused pulmonary interstitial fibrosis also could be seen.
2. Two different forward and reverse subtractions were performed to compare gene expression between lung tissue with silicosis and normal lung tissue. For the lung tissue with silicosis vs. normal lung tissue comparison forward subtraction (named as SNA), lung tissue with silicosis was the tester and normal lung tissue the driver. For the reverse subtraction (named as SNB), normal lung tissue was the tester and lung tissue with silicosis the driver. With the methods of T/A cloning and blue-white screening, the cDNA fragments generated by SSH were cloned into a T/A cloning plasmid (pMD20-T), the subtracted cDNA libraries being constructed. The polymerase chain reaction (PCR) using liquid which containing competent bacteria JM109 was initially used to validate the insert cDNA fragments, many of which contained 200-600bp inserts. seven novel cDNA sequences(named as SNA-11, SNA-28, SNA-43, SNA-48, SNB-13, SNB-30, SNB-42) were examined by reverse transcription- polymerase chain reaction(RT-PCR), the results showed that the expressions of these seven cDNA sequences in rat's lung tissue with silicosis were all different from which in normal lung tissue(Fig. 7). According to the quantitative analysis of image scanning, the gene expression quantity was 2-5 times between two lung tissues.
3. Bioinformatics analysis showed that 47 positive clones represented 35 genes containing 2 putative proteins and 4 predicted similar proteins. Most genes whose function known had important roles in the structure and movement of cell or extracellular matrix, the defense of cell and body, material transportation, the regulation of expression and metabolism.
Conclusions:
1. The animal model of silicosis is successfully constructed.
2. Two different forward and reverse subtraction libraries of silicosis are successfully constructed.
3. Some genes may be related to pulmonary fibrosis are cloned, if we can study these genes further, it is useful to understand the mechanism of pulmonary fibrosis.
4. It is confirmed that silicosis is related to lung cancer and may be a dangerous cause of lung cancer.
目的:本研究拟通过建立大鼠慢性硅肺纤维化动物模型,利用SSH技术,筛选和鉴定硅肺纤维化大鼠与正常大鼠之间肺组织差异表达基因,旨在获得与肺纤维化相关的特异表达基因,为肺纤维化发生机制及与其他疾病的关系提供实验依据。
方法:本研究以SD大鼠为实验对象,采用气管插管灌注二氧化硅粉尘,经过360天,建立硅肺肺纤维化动物模型;应用抑制消减杂交技术(SSH),结合T/A克隆技术和蓝白筛选构建了硅肺肺纤维化正反向差异表达消减cDNA文库(SNA,SNB);经过菌液PCR技术初步验证,插入片段大小,采用半定量RT—PCR技术证实部分基因确实存在差异表达,随机挑取部分含有插入片段的克隆进行测序分析及生物信息学分析。
结果:
1、360天,X线显示大鼠肺纹理增粗,肺野有散在分布的高密度影出现。肉眼见肺组织表面及切面有散在分布的灰白色小结节,结节大小不等;显微镜下肺内有典型的纤维性硅结节形成,在部分硅结节周围肺泡上皮细胞和管上皮明显增生,部分细胞显不同程度不典型增生,肺间质纤维化明显。
2、分别以肺纤维化肺组织cDNA为实验方(Tester),以正常肺组织为对照方(Driver),以正常肺组织cDNA为实验方(Tester),以肺纤维化肺组织cDNA为对照方(Driver)构建了反向差异表达消减cDNA文库(SNB)。经过菌液PCR技术初步验证,插入片段大小主要分布于200-600bp之间。随机挑取正向和反向消减文库共52个含有插入片段的克隆进行测序分析,共获得47个有效差异表达cDNA片段。为证实这些基因确实存在差异表达,采用半定量RT—PCR技术对随机挑选的硅肺纤维化肺组织消减文库中的SNA—11、SNA—28、SNA—43、SNA—48,正常肺组织消减文库中的SNB—13、SNB—30、SNB—42进行RT—PCR分析,结果显示它们在硅肺纤维化肺组织和正常肺组织中的表达存在差异,根据图像扫描定量分析,在两组肺组织中基因表达量相差在2—5倍以上。
3、生物信息学分析表明双向消减cDNA文库测序得到的47个阳性克隆代表了35个基因,包含2个假想蛋白、4个推测的相似蛋白。其中大多数功能已知基因在细胞或细胞外基质结构/运动、细胞/机体防御、物质转运、翻译/表达调控、代谢等方面起重要作用。表明SSH技术是筛选差异表达基因的有效方法,对这些基因功能的步研究,有利于从另外的角度了解肺纤维化的机制。
结论:
1成功建立了典型大鼠慢性硅肺纤维化动物模型。
2建立了较高质量的硅肺纤维化正反差异消减cDNA文库。
3获得部分与肺纤维化相关的基因,对这些基因的进一步研究,有利于从不同的角度了解肺纤维化的机制。
4证实硅肺与肺癌有关,可能是肺癌发生的危险因素之一。
Introduction: Pulmonary fibrosis is a sort of destructive lung diseases induced by multiple agents, which has poor prognosis. The pathological processes of pulmonary fibrosis include inflammatory injury, tissue damage and repair. Many kinds of cells such as alveolar macrophages, epithelial cells and fibroblasts et al play important roles in the progress of pulmonary fibrosis. These cells educe direct or indirect effects by secreting many bioactive compounds such as cytokines and mediators of inflammation and constitute a complicate cellular network in which the cells interacting each other and promoting the development of pulmonary fibrosis. However, the critical molecular mechanism in the development of the pulmonary fibrosis is still unknown, it is vital to search for better treatment plan of pulmonary fibrosis, based on the understanding of the pathogenesis of these diseases. As the developing of strategies and methods of identification of novel genes, various methods to compare patterns of gene expression have been described, it is becoming possible to identify some novel genes related with pulmonary fibrosis. The aim of our studies is to screen and identify differentially expressed gene of pulmonary fibrosis.
Methods: The SD (Sprage-Dawley) rats were poured into suspension of crystalline silica (200mg/kg body weight) through trachea cannula to construct the animal model of silicosis. The suppression subtractive hybridization was performed; two different forward and reverse subtractions were performed to compare gene expression between lung tissue with silicosis and normal lung tissue. The SSH PCR products from the library were cloned into pMD20-T vector; the positive clones were randomly selected, sequenced and compared to the database in GenBank of the differentially expressed gene fragments from the libraries. Seven novel cDNA sequences were examined by reverse transcription- polymerase chain reaction (RT-PCR) in order to confirm the differentially expression of cDNA fragments in rat's lung tissue with silicosis and normal lung tissue.
Results:
1. After 360 days, radiograph of chest showed that rat's pulmonary markings was thickened, some scattered high-density shadows appeared in lung field. From the surface and cross section of lung, some scattered nodules could be seen whose sizes were inequable. Under microscope, typical fibrosing siliconic nodules were formed in lung, alveolar epithelial cells and bronchial epithelial cells proliferated around partial fibrosing siliconic nodules, and some cells of which showed atypical hyperplasia. Diffused pulmonary interstitial fibrosis also could be seen.
2. Two different forward and reverse subtractions were performed to compare gene expression between lung tissue with silicosis and normal lung tissue. For the lung tissue with silicosis vs. normal lung tissue comparison forward subtraction (named as SNA), lung tissue with silicosis was the tester and normal lung tissue the driver. For the reverse subtraction (named as SNB), normal lung tissue was the tester and lung tissue with silicosis the driver. With the methods of T/A cloning and blue-white screening, the cDNA fragments generated by SSH were cloned into a T/A cloning plasmid (pMD20-T), the subtracted cDNA libraries being constructed. The polymerase chain reaction (PCR) using liquid which containing competent bacteria JM109 was initially used to validate the insert cDNA fragments, many of which contained 200-600bp inserts. seven novel cDNA sequences(named as SNA-11, SNA-28, SNA-43, SNA-48, SNB-13, SNB-30, SNB-42) were examined by reverse transcription- polymerase chain reaction(RT-PCR), the results showed that the expressions of these seven cDNA sequences in rat's lung tissue with silicosis were all different from which in normal lung tissue(Fig. 7). According to the quantitative analysis of image scanning, the gene expression quantity was 2-5 times between two lung tissues.
3. Bioinformatics analysis showed that 47 positive clones represented 35 genes containing 2 putative proteins and 4 predicted similar proteins. Most genes whose function known had important roles in the structure and movement of cell or extracellular matrix, the defense of cell and body, material transportation, the regulation of expression and metabolism.
Conclusions:
1. The animal model of silicosis is successfully constructed.
2. Two different forward and reverse subtraction libraries of silicosis are successfully constructed.
3. Some genes may be related to pulmonary fibrosis are cloned, if we can study these genes further, it is useful to understand the mechanism of pulmonary fibrosis.
4. It is confirmed that silicosis is related to lung cancer and may be a dangerous cause of lung cancer.
引文
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