| Monocytes and momocyte-derived macrophages plays an important role in the detection of invading pathogens, especially takeing in the celluar defense for pathgens. Both of them have different morphology, function and metabolism in the different environment and different stages of differentiation, which express TLR on their surface, recognising and binding these PAMP and initiating a signalling pathway that stimulates the host defences. Until now, there have been reported the partical gene sequence on the TLR1-10. Therefore it can be used in our experiment.This experiment do the exam about the Toll-like receptor family and cytokines expression pattern on monocyte and monocyte-derived macrophage in calves, and the responses by the LPS stimulation.Experiment1:The methods of isolation and culture monocytes and monocyte-derived macrophage from peripheral jugular vein blood.After isolating Mo and Mod-Mφ, we use Flow cytometric and magenetic cell sorting to determine the result we have collected. The purity of Mo and Mod-Mφis 97.9% and 73.9%, respectively. In this experiment, we cultured Mod-Mφin the Repcell other than Taflon bag method which is more simple and easy, shorting the steps in the experiment.Experiment2: The TLRs mRNA expression pattern on Mo and Mod-Mφ.Using RT-PCR and NIH image system we get the result of TLRs mRNA expression pattern on Mo and Mod-Mφwith or without the stimulation of LPS, This results show that difference responses between Mo and Mod-Mφ. The PBMC were stimulated by LPS for 24 h and the mRNA expression pattern of TLR and cytokines in monocytes-derived macrophages (Mod-Mφ) was analyzed. Results showed that LPS stimulation of Mod-Mφcould increase the mRNA levels of the genes of TNF-α, IL-6 and IL-8. In addition, the mRNA levels of the genes of TNF-αand IL-6 in the group of LPS stimulation were most significantly (P<0.01) higher than those in control group, which expression were 2.02±0.48 and 1.66±0.39, respectively. Meanwhile, IL-8was relatively higher than the control one, which was 2.14±0.52. The mRNA levels of TLR1, TLR3, TLR5, TLR8and TLR10 were significantly (P<0.05) decreased after LPS stimulation. There were no difference in the mRNA expressions of TLR2, 4, 6 and 7 between the groups of the control and LPS stimulation. At the same time, the TLRs and cytokines expression on Mo were stable after LPS stimulation. TLR1,TLR2, TLR3,TLR4,TLR7,TLR8 and TLR10 experssion were higher than1.0 fold, IL-8 and IL-6 experssion were higher thanTNF-α, which were1.38±0.41,1.47±0.17 and 1.21±0.51, respectively. Besides, there was no expression of TLR9 in Mo and Mod-Mφ. It suggested that Mod-Mφcould respond to LPS and they might take an important role in the innate immunity. The important function of the cells might contribute to better disease treatment.Experiment3: The TLRs protein level expression pattern onMo and Mod-MφIn order to find the mainly function of Mo and Mod-Mφ, we use the fluorescent immunostaining, flow cytometric and cofouse telescrope to analyse the protein level expression of TLR2 and TLR4 with or without LPS stimulation. The results show that TLR2 and TLR4 protein level experssion pattern on Mo and Mod-Mφafter LPS stimulation were34.1%,47.1% and 22.4%,10.1%, respectively. In addition, the results make us understand the main functions of Mo and Mod-Mφmore deeply.
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