A Preliminary Study On Macrophage Polarization In The Peak Period Of Rat Models Of Tuberculosis Wound

Objective:To investigate the infiltrations of different phenotypes of macrophages in the peak period of tuberculosis wound and to discuss the relationship between IFN-?/IL-10cytokines and macrophage polarization by successfully constructing rat models of tuberculosis wounds.Methods:1.Thirty-five SD rats(female,8 weeks old)were injected with purified protein derivative(PPD),then 30 rats were selected,which were no inflammatory reaction or weak reaction in PPD experiment.The 30 rats were divided into experimental group(n=15)and negative control group(n=15)according to the random number table method.Thirty rats had been immunized and allergized with Freund's complete adjuvant 6 weeks previously.BCG was intradermal injection to each experiment rat with the doses of 0.2ml,5×10~7CFU/ml.PBS was intradermal injection to each rat in negative control group.2.The changes of wound were observed broadly.At 2,6 and 11 days after injection,paraffin-embedded skin section was did in each group(each time took five rats from each group),and hematoxylin-eosin staining was used to observe wound healing and infiltration of inflammatory cells in each group.The pathogenic microorganism was used to detect and identify strains.3.Six days after injection,immunohistochemical staining was did,CD68-labeled macrophages,subtypes of M1 macrophages iNOS markers and M2 macrophages CD206markers were used to analyze the expression of M1 and M2 macrophages by immunohistochemistry in the experimental group and the negative control group.4.Six days after injection,the expression levels of IFN-?,IL-10 were determined by real-time PCR in the experimental group and the negative control group.5.All data were analyzed by t-test.Results:1.Apparent red,liquefaction,necrosis and ulceration were produced in the skin of rats injected with BCG.2.The appearance of tuberculosis wound from ulceration to healing was observed by the use of hematoxylin-eosin staining.The result of acid-fast staining was positive.3.The macrophage differentiation markers were counted by immunohistochemical staining in the peak period of tuberculosis wound:CD68,iNOS and CD206 in the experiment group were(84.8±3.4)/HP,(60.8±2.8)/HP and(13.2±2.2)/HP respectively,which were significantly higher than those in the control group(1.4±0.5)/HP,(0.4±0.50)/HP and(0.6±0.50)/HP,the difference were statistically significant(t=93.2,95.5,28.2,with P values below 0.05).iNOS[(60.8±2.8)/HP]in the experiment group were significantly higher than CD206[(13.2±2.2)/HP]in the peak period of tuberculosis wound,the difference were statistically significant(t=27.6,P<0.05).4.The mRNA levels of IFN-?and IL-10 derived from peak period of tuberculosis wound by RT-PCR showed that:IFN-?mRNA expression was significantly up-regulated in the experiment group when compared to the negative control group(t=23.9,P<0.05),while the expression of IL-10 mRNA was significantly down-regulated in the experiment group(t=16.2,P<0.05).Conclusion:1.After sensitization of rats injected with BCG(5×10~7CFU/ml,0.2ml)can effectively induce liquefaction and necrosis that can constructing rat models of tuberculosis wounds successfully.2.When IFN-?pro-inflammatory cytokine dominated,the local microenvironment of wound tissue at the peak of liquefaction necrosis in tuberculosis wounds can induce the changes of macrophages to M1 type,which is beneficial to the effect of macrophages in killing BCG.