Human Osteoarthritis Synovum Gene Expression Profiling Analysis

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 librarycDNA 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 analysisThe 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 dataWith 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 osteoarthritisWe 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.