人骨关节炎滑膜组织基因表达谱分析
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摘要
骨关节炎是一组由多种原因导致的以关节软骨退行性变为主要病理特征的临床综合征,是一种常见病,多发生于中年以后,是一种慢性、进展性关节病变。主要发生在关节软骨。其表现主要是关节软骨受到破坏,软骨表面失去均质性、中断、斑片凹陷、断裂和溃疡。近来一些研究表明骨关节炎的发生与遗传因素有关。滑膜对于维持关节的正常功能,并且在骨关节炎疾病的发生发展过程中起着重要的作用。有研究表明:滑膜细胞在受到病理因素刺激下,可通过凋亡形式而死亡。
本课题分别选择人骨关节炎与正常骨关节的滑膜组织,构建5'端非偏性cDNA文库,对每个文库大规模EST测序。本文在获得的大规模EST数据基础上进行数据分析、挖掘,以期发现与骨关节炎发病相关的重要功能基因,并获取基因表达的组织差异和特异信息。这些基因的功能和表达信息获得,对骨性关节炎发病机理以及有效预防有重要价值。
1.cDNA文库构建
cDNA文库构建是基因克隆的重要方法之一,从cDNA文库中能够筛选到所需的目的基因,并直接用于该目的基因的表达,它是发现新基因和研究基因功能的基础工具。文库的滴度、重组率及插入片段的大小是鉴定cDNA文库质量的重要指标。本文库原始文库的滴度为1.45×106pfu/mL,总克隆数为4.6×105,重组率为96.2%,扩增后文库总滴度为6.4×109pfu/mL,插入片段多分布在0.5～2.6kb之间,文库质量鉴定结果表明,构建骨关节炎滑膜组织cDNA文库具有较好的库容量、较高的重组率以及较大的插入片段。平均长度为1.5kb,达到了平均插入片段长度大于1kb的要求。
2.大规模EST测定及初步分析
正常滑膜组织3989个合格的EST序列,骨关节炎滑膜组织4278个合格的EST序列。用Phrap软件进行拼接,正常滑膜组织获得1273个非冗余序列(UniGene),其中包括611个重盈群(contig), 662个独立EST (Singleton )。骨关节炎滑膜组织获得1287个非冗余序列(UniGene),其中包括607个重盈群(contig), 680个独立EST (Singleton )。正常滑膜组织EST序列有59.99%是未知功能序列,骨关节炎滑膜组织EST序列有59.80%是未知功能序列。
3.基于EST数据的基因表达谱聚类方法
本文采用基于大规模EST数据的基因表达谱聚类法,探讨在测序数据大量获得时从中提取有价值信息的数据分析方法。通过确定基因与重盈群相关系数之间或者样本与文库相关系数之间的距离,通过显著性分析比较组织间差异表达基因,获得骨关节炎与正常骨关节的滑膜组织基因表达显著不同的基因。
4.正常骨关节滑膜组织与骨关节炎滑膜组织基因表达差异
本试验研究了显著性表达差异4类基因,筛选出了25个差异表达基因,从基因表达水平研究了骨关节炎的发病机理。免疫相关的基因有11条,其中上调占57%,下调占43%。在这些基因中, NM-005402表现为下调,可减轻OA滑膜的增生。Toll样受体2基因(NM-003264)是防御感染的先天免疫基因表现为下调。髓鞘磷脂磷酸二酯酶1,酸性溶酶体(酸性髓磷脂酶)基因(NM-000543)表现为上调,可减轻关节炎症状,表现为下调的自体炎性综合征1基因(AF-410477)在TNF-α和IL-1β信号途径上会聚,可控制炎症的反应。与能量相关基因8条,其中上调占25%,下调占75%,本次实验中与能量相关基因大部分表现为下调。
总之,本次实验提示骨关节炎的差异基因表达谱主要集中于代谢类尤其是免疫类、细胞代谢和信号转导基因,这些基因的表达异常可能是它发病的分子生物学基础;某些与代谢、免疫、信号转导相关的多生物学过程基因与骨关节炎发病的相关性值得进一步研究。
Osteoarthritis is a clinical syndrome which is caused by various factors and pathologically characterized as articular cartilage retrograde affection. It is a chronic, progressive, common disease with high frequency within populations after middle ages. This disease occurred mostly in the articular cartilage. Its symptoms mainly include destruction of articular cartilage, loss of isotropy, breaks, sunken patch, disruption and ulcer. Recently, it is reported that the development of osteoarthritis was related to genetic factors. Synovium is important in maintaining regular articular function and plays an important role in the developing process of osteoarthritis. It was demonstrated that synovium cell would die by programmed cell death under the pathological stimulations.
This project took human synovium tissue with and without osteoarthritis, respectively, as starting materials, constructed 5’non–bias cDNA library, and performed large scale EST sequencing for both libraries. Based on the large scale EST data, we performed comprehensive data analysis to find important functional genes related to osteoarthritis and to obtain information on tissue differentiation and specificity of gene expression. This information on gene function and expression will be of important value to pathogenesis of osteoarthritis and effective prophylaxis.
1.Construction of cDNA library
cDNA library construction is one the important methods in molecular cloning. By screening target gene(s) directly from the library, and by using them directly for gene expression, it is a elementary tool to find new gene(s) and to study their function(s). Titre, recombination rate and the size of the inserting fragment are important index for the quality of the cDNA library. The original titre of cDNA library is 1.45×106pfu/mL, the total clone number is 4.6×105, and the recombination rate is 96.2%; the titre after the library amplification is 6.4×109pfu/ml,and the size of the inserting fragments ranges between 0.5kb and 2.6kb. The identification data of the library demonstrated that osteoarthritis synovum tissue cDNA library has better library capacity, higher recombination rate and bigger inserting fragments. The average size of the inserting fragments is 1.5kb, which is bigger than the required size of 1kb inserting fragment.
2.Large scale EST sequencing and primary analysis
The synovum cell without osteoarthritis has 3989 qualified EST sequences, and the synovum cell with osteoarthritis has 4278 qualified EST sequences. Splicing with the Phrap software, the synovum cell without osteoarthritis has 1273 non-redundancy sequence (UniGene) which include 611 contig and 662 independent EST (Singleton ). The synovum cell with osteoarthritis has 1287 non-redundancy sequence (UniGene) which include 607 contig and 680 independent EST (Singleton ). Among the EST sequences of the synovum cell without osteoarthritis, 59.99% is functionally unknown; but among the EST sequences of the synovum cell with osteoarthritis, 59.80% is functionally unknown.
3.Gene expression map clustering method based on EST data
With the gene expression map clustering method based on large scale EST data, we explored the data analysis method to screen valuable information while obtaining the large volume of sequencing date. By defining the distance between genes or samples with the correlation coefficient of (contig)or sample (library), we identified the gene(s) with marked expression differentiation between synovum cells without and with osteoarthritis.
4 . Gene expression differentiation between synovum cells without and with osteoarthritis
We compared 4 categories of gene with remarkable differential expression, screened 25 genes with differential expression, and studied the pathogenesis of osteoarthritis at the gene expression level. There are 11 immune related genes, among which 57% with increased expression and 43% with decreased expression. Within this immune related genes, the expression level of NM-005402 decreased, which relieved the hyperplasia of OA synovum. Toll-like acceptor 2 gene (NM-003264) is the natural immune gene with the function of infection prevention, and its expression level decreased. The expression level of sphingomyelin phosphodiesterase 1, acid lysosomal (acid sphingomyelinase()NM-000543)increased, which could relieve the symptom of arthritis; decreased expressed AF-410477 assembled in TNF-αand IL-1βsignal pathway, which could control the infection reaction. Most of the 8 energy-related genes showed decreased expression, 75% of them decreased, but 25% increased.
In summary, gene expression differentiation of osteoarthritis mainly concentrated in the metabolitic category, especially immune related genes, cell metabolism genes and signal transduction genes, and the abnormal expression of these genes is the possible molecular biology basis of osteoarthritis; The relativity between some metabolism, immune and signal transduction related multiple biological process genes and osteoarthritis need further investigation.
本课题分别选择人骨关节炎与正常骨关节的滑膜组织,构建5'端非偏性cDNA文库,对每个文库大规模EST测序。本文在获得的大规模EST数据基础上进行数据分析、挖掘,以期发现与骨关节炎发病相关的重要功能基因,并获取基因表达的组织差异和特异信息。这些基因的功能和表达信息获得,对骨性关节炎发病机理以及有效预防有重要价值。
1.cDNA文库构建
cDNA文库构建是基因克隆的重要方法之一,从cDNA文库中能够筛选到所需的目的基因,并直接用于该目的基因的表达,它是发现新基因和研究基因功能的基础工具。文库的滴度、重组率及插入片段的大小是鉴定cDNA文库质量的重要指标。本文库原始文库的滴度为1.45×106pfu/mL,总克隆数为4.6×105,重组率为96.2%,扩增后文库总滴度为6.4×109pfu/mL,插入片段多分布在0.5～2.6kb之间,文库质量鉴定结果表明,构建骨关节炎滑膜组织cDNA文库具有较好的库容量、较高的重组率以及较大的插入片段。平均长度为1.5kb,达到了平均插入片段长度大于1kb的要求。
2.大规模EST测定及初步分析
正常滑膜组织3989个合格的EST序列,骨关节炎滑膜组织4278个合格的EST序列。用Phrap软件进行拼接,正常滑膜组织获得1273个非冗余序列(UniGene),其中包括611个重盈群(contig), 662个独立EST (Singleton )。骨关节炎滑膜组织获得1287个非冗余序列(UniGene),其中包括607个重盈群(contig), 680个独立EST (Singleton )。正常滑膜组织EST序列有59.99%是未知功能序列,骨关节炎滑膜组织EST序列有59.80%是未知功能序列。
3.基于EST数据的基因表达谱聚类方法
本文采用基于大规模EST数据的基因表达谱聚类法,探讨在测序数据大量获得时从中提取有价值信息的数据分析方法。通过确定基因与重盈群相关系数之间或者样本与文库相关系数之间的距离,通过显著性分析比较组织间差异表达基因,获得骨关节炎与正常骨关节的滑膜组织基因表达显著不同的基因。
4.正常骨关节滑膜组织与骨关节炎滑膜组织基因表达差异
本试验研究了显著性表达差异4类基因,筛选出了25个差异表达基因,从基因表达水平研究了骨关节炎的发病机理。免疫相关的基因有11条,其中上调占57%,下调占43%。在这些基因中, NM-005402表现为下调,可减轻OA滑膜的增生。Toll样受体2基因(NM-003264)是防御感染的先天免疫基因表现为下调。髓鞘磷脂磷酸二酯酶1,酸性溶酶体(酸性髓磷脂酶)基因(NM-000543)表现为上调,可减轻关节炎症状,表现为下调的自体炎性综合征1基因(AF-410477)在TNF-α和IL-1β信号途径上会聚,可控制炎症的反应。与能量相关基因8条,其中上调占25%,下调占75%,本次实验中与能量相关基因大部分表现为下调。
总之,本次实验提示骨关节炎的差异基因表达谱主要集中于代谢类尤其是免疫类、细胞代谢和信号转导基因,这些基因的表达异常可能是它发病的分子生物学基础;某些与代谢、免疫、信号转导相关的多生物学过程基因与骨关节炎发病的相关性值得进一步研究。
Osteoarthritis is a clinical syndrome which is caused by various factors and pathologically characterized as articular cartilage retrograde affection. It is a chronic, progressive, common disease with high frequency within populations after middle ages. This disease occurred mostly in the articular cartilage. Its symptoms mainly include destruction of articular cartilage, loss of isotropy, breaks, sunken patch, disruption and ulcer. Recently, it is reported that the development of osteoarthritis was related to genetic factors. Synovium is important in maintaining regular articular function and plays an important role in the developing process of osteoarthritis. It was demonstrated that synovium cell would die by programmed cell death under the pathological stimulations.
This project took human synovium tissue with and without osteoarthritis, respectively, as starting materials, constructed 5’non–bias cDNA library, and performed large scale EST sequencing for both libraries. Based on the large scale EST data, we performed comprehensive data analysis to find important functional genes related to osteoarthritis and to obtain information on tissue differentiation and specificity of gene expression. This information on gene function and expression will be of important value to pathogenesis of osteoarthritis and effective prophylaxis.
1.Construction of cDNA library
cDNA library construction is one the important methods in molecular cloning. By screening target gene(s) directly from the library, and by using them directly for gene expression, it is a elementary tool to find new gene(s) and to study their function(s). Titre, recombination rate and the size of the inserting fragment are important index for the quality of the cDNA library. The original titre of cDNA library is 1.45×106pfu/mL, the total clone number is 4.6×105, and the recombination rate is 96.2%; the titre after the library amplification is 6.4×109pfu/ml,and the size of the inserting fragments ranges between 0.5kb and 2.6kb. The identification data of the library demonstrated that osteoarthritis synovum tissue cDNA library has better library capacity, higher recombination rate and bigger inserting fragments. The average size of the inserting fragments is 1.5kb, which is bigger than the required size of 1kb inserting fragment.
2.Large scale EST sequencing and primary analysis
The synovum cell without osteoarthritis has 3989 qualified EST sequences, and the synovum cell with osteoarthritis has 4278 qualified EST sequences. Splicing with the Phrap software, the synovum cell without osteoarthritis has 1273 non-redundancy sequence (UniGene) which include 611 contig and 662 independent EST (Singleton ). The synovum cell with osteoarthritis has 1287 non-redundancy sequence (UniGene) which include 607 contig and 680 independent EST (Singleton ). Among the EST sequences of the synovum cell without osteoarthritis, 59.99% is functionally unknown; but among the EST sequences of the synovum cell with osteoarthritis, 59.80% is functionally unknown.
3.Gene expression map clustering method based on EST data
With the gene expression map clustering method based on large scale EST data, we explored the data analysis method to screen valuable information while obtaining the large volume of sequencing date. By defining the distance between genes or samples with the correlation coefficient of (contig)or sample (library), we identified the gene(s) with marked expression differentiation between synovum cells without and with osteoarthritis.
4 . Gene expression differentiation between synovum cells without and with osteoarthritis
We compared 4 categories of gene with remarkable differential expression, screened 25 genes with differential expression, and studied the pathogenesis of osteoarthritis at the gene expression level. There are 11 immune related genes, among which 57% with increased expression and 43% with decreased expression. Within this immune related genes, the expression level of NM-005402 decreased, which relieved the hyperplasia of OA synovum. Toll-like acceptor 2 gene (NM-003264) is the natural immune gene with the function of infection prevention, and its expression level decreased. The expression level of sphingomyelin phosphodiesterase 1, acid lysosomal (acid sphingomyelinase()NM-000543)increased, which could relieve the symptom of arthritis; decreased expressed AF-410477 assembled in TNF-αand IL-1βsignal pathway, which could control the infection reaction. Most of the 8 energy-related genes showed decreased expression, 75% of them decreased, but 25% increased.
In summary, gene expression differentiation of osteoarthritis mainly concentrated in the metabolitic category, especially immune related genes, cell metabolism genes and signal transduction genes, and the abnormal expression of these genes is the possible molecular biology basis of osteoarthritis; The relativity between some metabolism, immune and signal transduction related multiple biological process genes and osteoarthritis need further investigation.
引文
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