| Background and objective:Macrophages play a significant role in innate immunity and specific immune responses.when inflammatory or tissue damage occurs,macrophages can engulf and remove extracellular substances,apoptotic cells and debris In condition of nonspecific immunity;In condition of specific immunization,macrophages can present antigens and secrete a variety of cytokines,growth factors and chemokines,which can stimulate the activation of T lymphocytes and regulate immune response.MicroRNAs are widely exist in eukaryotes,they can conbine target gene that 3'UTR of miRNA sequence by complementation and pairing.The function of this miRNA is to degrade target gene miRNAs or inhibitie the translation of target gene miRNAs,thereby negatively regulating the expression of post-transcriptional level genes.miR-15a/16-1 knockout mice were introduced from the American jackson laboratory for further study the relationship of MiR-16 and macrophage lineage.This study was to investigate how lacking of miR-15a/16-1 can affect on the phenotype and function of macrophages in hepatocellular carcinoma and enteritis mice and its molecular mechanism,further more the study can provide new ideas for the development of immunotherapy strategies about targeting macrophages.Methods1.Detection of macrophage subsets in miR-15a/16-1-/-mice in different states1.1 Study on the frequency and phenotype of macrophages in spleen of mice in physiological state We took the peripheral blood of miR-15a/16-1+/+?WT?and miR-15a/16-1-/-?KO?C57 female mice in physiological state and detected the levels of IL-10 and IL-12 by ELISA.Then two groups mice were sacrificed respectively.The spleen of mice was removed and made into single cells.The frequency and phenotype of F4/80+,F4/80+CD16/32+,F4/80+CD206+ in the spleen were detected by flow cytometry.1.2 Study on the frequency and phenotype of macrophages in mice in H22 hepatocellular carcinoma state H22 hepatocellular carcinoma cell was cultured in RIPA1640 containing 10%bovine serum.The culture conditions were 37? and 5%CO2.Equivalent H22 cell were injected subcutaneously into miR-15a/16-1+/+?WT?and miR-15a/16-1-/-?KO?C57 female mice in the left armpit.On the 7th day,the tumor growth was observed at the end of the 15th day.The tumor growth of the two groups was measured at the same time every day.Draw the tumor growth curve.On the 15th day of tumor formation,the mice were sacrificed and the tumor size and morphology of the two groups were observed visually.Tumortissue?fixed by 10%formalin?were screened by HE staining to observe the tumor morphology.The remaining part of the tumor tissue and spleen are made of single cell suspension,the frequency and phenotype of F4/80+,F4/80+CD 16/32+and F4/80+CD206+cells in the spleen and tumor of the two groups were detected by flow cytometry.1.3 Study on the frequency and phenotype of macrophages in mice in dextran sulfate?DSS?enteritis MiR-15a/16-1+/+?WT?and miR-15a/16-1-/-?KO?C57 female mice were given 2%DSS liquor.Mice were weighed daily and the mouse weight curve was drawn.After one week,mice have bloody phenomenon and were sacrificed.The spleen and part of the intestinal tissue were removed and the spleen was made into single cell suspension.The frequency and phenotype of F4/80+,F4/80+CD16/32+ and F4/80+CD206+ cells were detected by flow cytometry.The positive rate of CD68+,CD86+ and CD206+ in intestinal tract?fixed with 10%formalin?was detected by immunohistochemistry.2.Study on the effect of miR-15a/16-1 on the mechanism of macrophage differentiation in mice2.1 Study on level of M-CSF and the expression of M-CSF-R in mice in H22 hepatocacinoma state miR-15a/16-1+/+?WT?and miR-15a/16-1-/-?KO?C57 female mice were taken blood from Orbital of eyes on 15th days.After centrifugation,the levels of M-CSF in the supernatant of mice were detected by ELISA.In addition,the mice were sacrificed and the spleen was removed and made into a single cell suspension,the expression of F4/80+CD115+cells were detected by flow cytometry.2.2 Study on the expression of PD-L1 in mice in H22 hepatocellular carcinoma state and DSS enteritis state After 15 days tumor-bearing,miR-15a/16-1+/+?WT?and miR-15a/16-1-/-?KO?C57 female tumor bearing mice were sacrificed,tumor and spleen were made to a single cell suspension.The expression of PD-L1 + in the spleen and tumor was detected by flow cytometry.The two groups of mice were fed with 2%DSS solution respectively.After one week,there were hematochezia in cage,then the mice were sacrificed.The spleen and part of the intestinal tissue were removed,the spleen was made into a single cell suspension,and the expression of PD-L1+ in the spleen was detected by flow cytometry.2.3 Study on the expression levels of signaling molecules of STAT3,SOCS3,NF-?B in macro-phages of spleen of miR-15a/16-1-/-?KO?mice in physiology condition The miR-15a/16-1+/+?WT?and miR-15a/16-1-/-?KO?C57 female mice were sacrificed in physiological state condition,then spleen was take out from the mice,third,macrophages were separed using the magnetic bead separation method.The expression of signal level in STAT3,SOCS3 and NF-?B in macrophages was detected by western blot.Result:1.Changes in the frequency,phenotype and function of macrophages in miR-15a/16-1-/-?KO?mice1.1 The frequency and phenotype of macrophages have no significant chage in miR-15a/16-1-/-?KO?mice in the physiological status The results of flow cytometry showed that,compared with miR-15a/16-1+/+?WT?mice,the expression of F4/80+,F4/80+CD 16/32+,F4/80+CD206+ cells had no significant changes in the miR-15a/16-1-/-?KO?mice,this result shows that M1 and M2 macrophages have no obvious changes in two groups mice.1.2 The frequency of macrophages,M1 macrophages and M2 macrophages were decreased slightly in miR-15a/16-1-/-?KO?mice in H22 hepatocellular carcinoma state Tumor growth curve showed that the miR-15a/16-1-/-?KO?mice had a slow tumor growth and smaller volume compared with miR-15a/16-1+/+?WT?mice.The results of flow cytometry showed that compared with miR-15a/16-1+/+?WT?mice,the frequency of F4/80+cells,F4/80+CD16/32+cells and F4/80+CD206+cells were decreased slightly in the spleen and tumor of miR-15a/16-1-/-?KO?mice.The deletion of miR-15a/16-1-/-gene in mice resulted in a slight reduce in the frequency of macrophages,Ml macrophages and M2 macrophages,and tumor suppression in mice status.1.3 The frequency of macrophages and M1 macrophages was increased,and the frequency of M2 macrophages did not change significantly in miR-15a/16-1-/-?KO?mice in DSS enteritis stateThe results of flow cytometry showed that the weight of miR-15a/16-1-/-?KO?mice was decreased compared with miR-15a/16-1+/+?WT?mice.The expression of F4/80+,F4/80+CD16/32+ in miR-15a/16-1-/-?KO?mice was significantly higher than that of miR-15a/16-1+/+?WT?mice in DSS enteritis,but the frequency of F4/80+CD206+cells did not change significantly.The results of immunohistochemistry showed that the expression of CD68+,CD86+cells in miR-15a/16-1-/-?KO?mice was significantly increased,and the expression of CD206+cells did not change significantly.The results showed that the deletion of miR-15a/16-1 gene in mice increased the frequency of macrophages,M1 macrophages and did not change the frequency of M2 macrophages;the mice are in a proinflammatory state.2.Study on the mechanism of macrophages differentiation in miR-15a/16-1-/-?KO?mice2.1 The level of M-CSF and the expression of M-CSF-R were not significantly changed in H22 hepatocellular carcinoma state The results of flow cytometry showed that the expression of F4/80+ M-CSF-R+ and the concentration of M-CSF in the peripheral blood in the two groups mice had no obvious change.The results showed that miR-15a/16-1 gene had no direct effect on M-CSF and M-CSF-R.2.2 The expression of PD-L1 in macrophages of miR-15a/16-1-/-?KO?mice was not significantly changed in H22 hepatocellular carcinoma state and DSS enteritis state Flow cytometry showed that compared with miR-15a/16-1+/+?WT?mice in H22 hepatocellular carcinoma state,F4/80 +PD-L1+ cells had no significant change in spleen and tumor of miR-15a/16-1-/-?KO?mice;compared to miR-15a/16-1+/+?/WT?mice in DSS enteritis,F4/80+PD-L1+ cells in spleen of miR-15a/16-1-/-?KO?mice had no significant change by Flow cytometry.The results showed that miR-15a/16-1 may not directly regulate PD-L1 in mice.2.3 The expression of STAT3 was increased,the expression of SOCS3 was decreased and the expression of NF-?B was not changed in macrophages of spleen in miR-15a/16-1-/-?KO?mice in physiological state The results of western blot showed that STAT3 signal protein in spleen of miR-15a/16-1-/-?KO?mice was increased,the expression of SOCS3 signal molecular protein was decreased,the expression of NF-?B signal molecular protein was not changed in physiological condition.The results suggest that miR-15a/16-1 may negatively regulate the expression of STAT3 signaling molecules.Conclusion1.In physiological state,the frequency and function of macrophages,M1 and M2 macrophages have no significant change in miR-15a/16-1-/-?KO?mice.2.In H22 hepatocellular carcinoma state,the frequency of macrophages,M1 macrophages and M2 macrophages were decreased slightly,the ratio of M1/M2 was increased and the effect of tumor growth was inhibited in miR-15a/16-1-/-?KO?mice.3.In DSS enteritis,the frequency of macrophages and M1 macrophage were increased and the frequency of M2 macrophage have no significant change in miR-15a/16-1-/-?KO?mice;the ratio of M1/M2 was increased,there is proinflammatory effect.4.In H22 hepatocellular carcinoma state,the levels of M-CSF,the expression of M-CSF-R of macrophages and the expression of PD-L1 of macrophages were not significantly changed in miR-15a/16-1-/-?KO?mice;in DSS enteritis state,the expression of PD-L1 of macrophages was not significantly changed.5.In physiological status,the function of miR-15a/16-1 may be related to STAT3 signaling molecules. |
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