Study On The Effect Of New Immune Molecule TIM-4 On Biological Activity Of Murine Macrophage Cell Line RAW264.7

Aim:Tim is a new gene family,which relates with asthma and allergic diseases. Murine Tim-4,one member of Tim family,is only expressed in APC(antigen presenting cell).In the present study,we investigated the effect of Tim-4 on biological activity of murine macrophage cell line RAW264.7 with antibody blocking and Tim-4 overexpression cell model.The study not only provides a new idea for further studying biological activity of macrophage but a new target for therpy and preventing of diseases that macrophages involved in.Methods:1.The expression of Tim-4 in murine macrophage cell line RAW264.7 and effect of anti-Tim-4 blocking on biological activity of macrophage RAW264.7.Macrophages were cultivated in 24 wells-plate,24 hours later,LPS(100 ng/mL)was added.48 hours later,cells were collected.Tim-4 mRNA was measured by Reverse Transcription-Polymerase Chain Reaction(RT-PCR)and Tim-4 protien was detected by flow cytometer method(FCM).RAW264.7 cells were cultivated in 96 wells-plate and divided into 4 groups.24 hours later,two groups were separately added with goat IgG and anti-Tim-4(final concentration were 5μg/mL).1 hour later,lipopolysaccharide(LPS,final concentration was 100 ng/mL)was added.In another two groups,one group was added with the same concentration of LPS,and the other group was given PBS.After 48 hours,the medium was collected to detect the secretion of nitric oxide(NO)using Griess reaction and cells were harvested to extract the total RNA for RT-PCR to analyze the expression of iNOS mRNA.2.Establishment of RAW264.7-pcDNA3.0-Tim-4(RAW264.7-T4)cell line with stable and high expression of murine Tim-4Peritoneal macrophages were regularly separated from mice.RT-PCR was used to amplify the full gene fragment of murine Tim-4,with which to construct pcDNA3-Tim-4 eukaryotic expression vector.Restriction endonucluease digestion, PCR and sequencing were used to verify its correctness.To transfect RAW264.7 cells with pcDNA3 and pcDNA3-Tim-4 separately by liposome.After screening with high level of G418,a new cell line expressing stable and high Tim-4 was established.The mRNA and protein level were further measured by RT-PCR,FCM and immunochemistry staining.3.The effect of Tim-4 overexpression on biological activity of macrophage RAW264.7Two macrophage cell Iines(RAW264.7-P,RAW264.7-T4)with different Tim-4 expression level were cultivated in 96 wells-plate with or without LPS stimulation.48 hours later,medium and cells were collected seperately.The medium was used to detect the secretion of NO using Griess reaction and the cells were harvested to extract the total RNA for RT-PCR to analyze the expression of iNOS and TNF-αmRNA.The expression level of MHC-Ⅱ,CD80,CD86 and phagocytic activity of FITC-dextran were evaluated in RAW264.7-P and RAW264.7-T4 cell lines by FCM.Results:1.The expression of Tim-4 in murine macrophage cell line RAW264.7 and effect of anti-Tim-4 blocking on biological activity of macrophage RAW264.7Tim-4 mRNA level of macrophages with LPS stimulation was significantly higher than that of unstimulation group.Tim-4 protein expression in RAW264.7 was improved with LPS stimulation in a dose-dependent method.The secretion of NO and the expression of iNOS mRNA in anti-Tim-4 blocking group was significantly higher than control group with or whithout LPS stimulation.2.Establishment of RAW264.7-pcDNA3.0-Tim-4(RAW264.7-T4)cell line with stable and high expression of murine Tim-4Products of RT-PCR amplification were digested with BarnHⅠand HindⅢ,then Tim-4 fragment was cloned into pcDNA3 and pcDNA3-Tim-4 recombinant was constructed.The result was insistent with sequence of GenBank after digestion,PCR and sequencing.After transfection with liposome and screening with G418,new macrophage cell line RAW264.7-pcDNA3.0-Tim-4(RAW264.7-T4)and corresponding control RAW264.7-pcDNA3.0(RAW264.7-P)were established.mRNA level of Tim-4 in RAW264.7-T4 cells was significantly higher than that of control group,so did protein expressed in these cells,which was proved by FCM and immunochemistry staining.Tim-4 protein was mainly expressed on the surface of macrophage,and cytoplasmic expression was also found.3.The effect of Tim-4 overexpression on biological activity of macrophage RAW264.7Statistic analysis showed that the secretion of NO and the expression of iNOS mRNA in RAW264.7-T4 cells was lower than RAW264.7-P cells with or whithout LPS stimulation,and the expression of TNF-αmRNA was also significantly lower in RAW264.7-T4 cells without LPS stimulation.There was significant difference in the expression level of MHCⅡ,CD80,CD86 between RAW264.7-P and RAW264.7-T4;there was no difference in MHC-Ⅱexpression level and phagocytic activity between those groups(P＞0.05).Conclusion:LPS upregulates the expression of Tim-4 in murine macrophage cell line RAW264.7.Tim-4 down-regulates secretion of NO and expression of iNOS mRNA, TNF-αmRNA,CD80,CD86 molecules in RAW264.7,whlile Tim-4 has no influence on MHC-Ⅱexpression and phagocytic activity.These results indicate that Tim-4 could negatively regulate biological activity of murine macrophage cell line RAW264.7.