| Inflammation and immune response is one of the most common pathological processes, and various human diseases are related to it. In addition to well-known infectious diseases, rheumatoid arthritis, rheumatoid disease, systemic lupus erythematosus, liver fibrosis, atherosclerosis, myocardial infarction, Alzheimer's disease, as well as a variety of malignant diseases are closely related to inflammation, which involved in the occurrence, development and prognosis in the whole disease process. Macrophage is an important kind of cell in immune system, which not only has a strong phagocytic function, but also be main antigen-presenting cell, and play a key role in a variety of inflammatory and immune responses. Activated macrophage can secrete a variety of bioactive substances, such as nitric oxide, interleukine-1, tumor necrosis factor-α, reactive oxygen species. Inducible cyclooxygenase (COX), COX-2 can be highly expressed in macrophage. Many studies have suggested that COX-2 is not specific to participate in inflammatory response, but play an important role in protection of gastric mucosa, maintenance of renal function, learning and memory, so other subtypes of COX might exist. A large number of inflammatory factors form the complex inflammation-immune network, which interact, co-regulate and control the process of inflammatory response and prognosis at different stages. To search specific factors for the regulation of inflammatory response will be far-reaching significance to a deep understanding of the pathophysiological mechanisms of inflammation, as well as the treatment of inflammatory diseases.Methods1. To design the degenerate primers base on the consensus sequence of different cyclooxytenases isoforms from various species.2. Acute peritonitis model was established in rats using intraperitoneal injection of zymosan A. Total RNA was extracted from peritoneal macrophages, and then purified PCR products were ligated into T vector and sequenced with automated technology. 3. Full-length sequence of the novel gene was obtained by 3'and 5'rapid amplification of cDNA ends technique.4. The mRNA expression level of CNT2 and the novel gene CNT2b in different tissues in rats and temporal expression pattern in macrophages from the zymosan A-induced acute peritonitis of rat were detected by Realtime RT-PCR technique.5. The effects of anti-inflammatory drug aspirin (12mg/kg), celecoxib (0.13mg/kg) and prednisone (0.4mg/kg) on CNT2b mRNA and CNT2 mRNA expression in rat peritoneal macrophages were measured by Realtime RT-PCR.6. siRNA interference sequence was designed according to CNT2b sequence and transfected into NR8383 rat macrophages using liposomes in vitro.7. After siRNA interference, mRNA expression of inflammation related factors in NR8383 rat macrophages, such as IL-1α, IL-1β, IL-6, TNF-α, CNT2, CNT2b, COX-2, IL-10 were measured by Realtime RT-PCR.Results1. Two conservative amino acid sequences were observed in different species of COX-1, COX-2 and COX-3 sequences.2. A novel mRNA sequence, 13330nt long, was found and cloned from rat peritoneal macrophages, which corresponding DNA is located at rat chromosome 3q35. BLAST analysis and sequence alignment indicated that the novel gene had high homology with Na+-dependent nucleoside transporters (Na+/nucleoside cotransporter, CNT) 2, which retained the intron 1,7-14,16,17 and a 3197nt sequences was appendixed to the last exon of CNT2. Since the novel gene and CNT2 were both complete transcriptions, which contained tailing signal and poly A tail, it was identified as the splicing isomers of CNT2 and named as CNT2b.3. CNT2 mRNA was expressed in different tissues and the level was higher in heart, liver, skeletal muscle and spleen; CNT2b mRNA expression level in normal organs was far less than that of CNT2, and the level was slightly higher in heart, skeletal muscle and liver.4. The significant phase characteristics of CNT2b mRNA expression was observed in peritoneal macrophages from peritonitis rat. After zymosan injection, CNT2b mRNA expression level was significantly increased and reached the peak point at 2h; subsequently, the expression level was gradually decreased, and returned to their levels at 0.5h until 24h. At 36th hour after inflammatory stimulation, CNT2b mRNA expression level was significantly increased again. The expression level of CNT2b mRNA was decreased by about 53% at 48th hour after inflammatory stimulation by comparison of that at 36th hour.5. After treated with aspirin, prednisone and celecoxib, respectively, CNT2b mRNA expression decreased significantly, in which the effect of prednisone was strongest and celecoxib weakest.6. Suppression of CNT2b expression resulted in significant increase of mRNA expression of IL-1β, IL-6, TNF-αand COX-2, as well as obvious reduction of IL-10 in NR8383 cells.Conclusion1. A novel gene CNT2b was found and cloned in rat peritoneal macrophages.2. CNT2b was high-expression gene induced by inflammation, which not significant increase in normal tissues but in macrophages stimulated with inflammation. During the course of acute peritonitis, CNT2b mRNA expression in peritoneal macrophages showed obvious temporal and bimodal characteristics. Anti-inflammatory drugs could decrease the expression level of CNT2b mRNA.3. Suppression of CNT2b expression resulted in significant increase of pro-inflammatory factor expression and reduction of IL-10 expression.4. CNT2b involved in regulation process of inflammatory response, and its function was related to suppressing inflammation response.
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