AIM To simultaneously identify several genes whose expression increase in fibroblast like synovial cells (FLS) of rheumatoid arthritis (RA) patients and to be related to the proliferation or invasion of FLS to cartila...AIM To simultaneously identify several genes whose expression increase in fibroblast like synovial cells (FLS) of rheumatoid arthritis (RA) patients and to be related to the proliferation or invasion of FLS to cartilage of RA patients. METHODS Total RNAs were prepared separately from FLS of RA patients and osteoarthritis (OA, as control) patients. The RNA samples were analyzed by using differentially display reverse transcription polymerase chain reaction (DDRT PCR). Complementary DNA (cDNA) fragments corresponding to differentially expressed messenger RNAs (mRNAs) were eluted, cloned, and sequenced. The obtained sequences were compared with those genes which entered into EMBL/Genebank database. RESULTS Seventy six differentially displayed fragments were obtained, but only 55% (42/76) were successfully cloned into plasmid vectors. Reverse dot blotting hybridization showed that of the 42 clones, only 22 clones were truely positive. The sequence analysis of the 19 clones showed that, their lengths were from 145 bp to 501 bp, most of which were about 300 bp in length. 13 sequences corresponded to known mRNAs, including cathepsin B. The other six sequences corresponded to transcripts of yet unidentified genes. Most of the novel sequences were too short and were not in the coding regions of mRNAs. CONCLUSION DDRT PCR method has many limitations on screening differentially expressed genes; cathepsin B might be related to the invasion of FLS in RA patients.展开更多
文摘AIM To simultaneously identify several genes whose expression increase in fibroblast like synovial cells (FLS) of rheumatoid arthritis (RA) patients and to be related to the proliferation or invasion of FLS to cartilage of RA patients. METHODS Total RNAs were prepared separately from FLS of RA patients and osteoarthritis (OA, as control) patients. The RNA samples were analyzed by using differentially display reverse transcription polymerase chain reaction (DDRT PCR). Complementary DNA (cDNA) fragments corresponding to differentially expressed messenger RNAs (mRNAs) were eluted, cloned, and sequenced. The obtained sequences were compared with those genes which entered into EMBL/Genebank database. RESULTS Seventy six differentially displayed fragments were obtained, but only 55% (42/76) were successfully cloned into plasmid vectors. Reverse dot blotting hybridization showed that of the 42 clones, only 22 clones were truely positive. The sequence analysis of the 19 clones showed that, their lengths were from 145 bp to 501 bp, most of which were about 300 bp in length. 13 sequences corresponded to known mRNAs, including cathepsin B. The other six sequences corresponded to transcripts of yet unidentified genes. Most of the novel sequences were too short and were not in the coding regions of mRNAs. CONCLUSION DDRT PCR method has many limitations on screening differentially expressed genes; cathepsin B might be related to the invasion of FLS in RA patients.
