| Cytokines are proteins that are produced by multiple immune cells acing on othercells. They have a significant role in starting and regulating the immune process.IL-10was first identified as an immunosuppressive cytokine in mouse that is secretedmainly by Th2. Besides, other immune cells such as monocyte, macrophage, dendriticcells, B cells, regulatory T cells, Th1, Th17as well as non-immune cells such asmastocyte, epithelial cells, keratinocyte and tumor cells can also produce a certainamount of IL-10. It can restrict and even terminate the immune response, regulatingdifferentiation and multiplication of regulatory T cells, B cells, and natural killer cells.But recent studies found that IL-10can not only act as a immunosuppressive factor,but can also promote immune process when inflammation is limited, eliminatinginfectious or non-infectious particles. IL-10has been cloned in various kinds ofvertebrates such as human, mouse, chicken, etc. Lots of studies have been shown onmammalian species, while studies on fishes are still inadequate.The study used part DNA sequence obtained from EST sequencing and labeledwith DIG used as a probe to filter common carp IL-10from peripheral bloodleukocyte cDNA library that was stimulated by mitogen. Then we got the full-lengthcDNA sequence of carp IL-10. GenBank registration number is JX524550. Thesequence is1204bp in length containing a ORF (open reading frame) and coding179amino acids. After analyzing with other putative IL-10proteins, we found that carpIL-10showed the highest identity with gold fish, which were88.3%and other fishspecies between45.5%~87.4%. Identified with bird species are from32.7%to33.2%and that of mammals are from25.6%to29.8%. We designed two pairs of primes inthe non-coding regions from the IL-10cDNA obtained and used spleen genomic DNAas plate to perform PCR. The two sequences are joint and IL-10genomic sequencewas obtained. It is2176bp in length including5exons and four introns which is inaccordance with other IL-10s and the splice sites of introns are all qualified with GT-AG regulation. In the third part, we stimulated carp peripheral blood leukocyteusing different types of mitogens and abstract RNA at different times after stimulation.Then, IL-10expressions in these groups were analyzed. The result showed that IL-10expressions in stimulated groups were markedly higher and LPS and PHAco-stimulated groups was stronger and longer than Con A groups, indicating thatIL-10started to perform its immune feature shortly after stimulation. In the last part ofthe experiment, we injected the carp with inactivated Aeromonas Hydrophila andextract RNA in5different organs and studied its expression change in different timepoints by using semi-PCR. The result demonstrated that expressions were mediate inhead kidney and spleen and even stronger after stimulation, followed by gill. Theweakest expressions were found in heart and liver. Then the IL-10expressions wereanalyzed in head kidney and spleen. We found the raising expression of IL-10wasmore obvious in head kidney after being stimulated with long duration, demonstratingthat head kidney has an significant role in the early immune in common carp. |
PDF Full Text Request