| Background: Axolotls have a remarkable ability to regenerate damaged tissue,which has fascinated scientists for centuries and is often used as a model organism to reveal the secrets of regeneration.Previous studies have found that salamander regeneration is closely related to inflammation.Macrophages,as key immune cells affecting tissue regeneration,regulate tissue regeneration by promoting wound detoxification and sequentially producing chemokines,proinflammatory and anti-inflammatory cytokines.Macrophages have a certain phenotypic plasticity and can be polarized into two different functional states in response to activated local microenvironment signals.Among them,M1 macrophages express CD86,secrete pro-inflammatory cytokines and perform pro-inflammatory functions.M2 macrophages express CD163,secrete anti-inflammatory cytokines and perform tissue repair functions.However,the mechanism of different phenotypes of macrophages in axolotl limb regeneration is still unclear,and further research is needed.Methods: In this study,a limb regeneration model of axolotl was established.Morphological changes in the tissue during regeneration were analyzed by HE and Masson staining.Then,primers were designed according to the axolotl gene bank,and the open reading frame sequences of CD86 and CD163 were amplified by PCR.Then,analysis was carried out by sequencing and phylogenetic tree construction.The q RT-PCR was used to detect the expression of CD86 and CD163 genes.Prokaryotic expression vectors were constructed by molecular cloning technology,and recombinant CD86 and CD163 were induced by IPTG.After their purification,monoclonal antibodies were prepared by immunizing mice as antigens.The specificity of antibodies was identified by indirect ELISA,cellular immunofluorescence and Western blot.After purifying antibody by saturated ammonium sulfate,we first verified its function for dissolving macrophages through the classical complement activiation pathway.Meanwhile,we injected antibody in the end of amputated axolotl limbs to observe the regeneration by HE,Masson staining and q PCR.The experiment was divided into 4 groups,respectively.They were anti-CD86 antibody injection group,anti-CD163 antibody injection group,anti-CD86+163 antibody injection group,PBS control group.The antibody injection does is 125 μg/100 g,6individuals in each group.Results: The process of limb regeneration of axolotl was observed: wound epithelium formed at 1 dpa,blastema appeared at 7 dpa,and limb regeneration was complete at 42 dpa.At the same time,the transcription levels of CD86 and CD163 genes in axolotl were the highest at 7 dpa.The prokaryotic expression vectors p ET28a-sumo/CD86 and p ET28asumo/CD163 were successfully constructed,the recombinant proteins CD86 and CD163 were induced.And monoclonal antibodies against CD86 and CD163 were successfully prepared,which had strong specific binding with recombinant proteins,endogenous proteins and peritoneal macrophages.Anti-CD86 and CD163 monoclonal antibodies were proved to have strong lysis function on macrophages in vitro.In vivo injection experiments showed that the axolotls injected with anti-CD163 or anti-CD86+CD163 antibody failed to regenerate,while the axolotls injected with anti-CD86 antibody had normal limb regeneration and promoted regeneration to a certain extent.The expression level was analyzed at 30 dpa and 40 dpa,and it was found that compared with the control group,the expression level of CD86 in the anti-CD86 antibody injection group decreased at 30 dpa with statistical difference.The expression level of CD163 in the anti-CD163 antibody injection group decreased at 30 dpa with significant difference,and also decreased at 42 dpa with statistical difference.Conclusion: This study confirms that M1 and M2 macrophages play a key role in axolotl limb regeneration.Studying the regulation of M1 and M2 macrophages under inflammatory conditions and during limb regeneration will help to improve healing and propose regenerative therapy strategies. |
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