| Background and Objective:The naked mole rat is a kind of animals which live in the ground and is eusocial animal at the age of life,and are distributed in Kenya,Ethiopia and other African regions.In 1967,naked mole rats were introduced lab by foreign scholars.Only for half a century,foreign scholars have launched a series of studies,and research reports have been published in the journal Nature,Science,and so on.In existing reports,the domestic and foreign scholars have studied naked mole rats more focused on the respect such as its long life,resistant to low oxygen,lack of p substance,little spontaneous tumor,but the study of the immune system is little.Immune system is an important index of biomedical research,which is not only for animal physiological state judgment and feeding management,also is the important basis of clinical medical treatment and scientific research.Current study reveals the relationship between the immune system and cancer.Along with the aging of the body,the immune system is gradually deteriorating,the process is called immune senescence,and immune senescence is related to systemic inflammation,chronic inflammatory diseases and many cancers.Therefore,this research is the first time to preliminary study on the naked mole rats immune system,in order to interpret naked mole rats immunology mechanism,provide theoretical basis for the mechanism of geriatric diseases,and new train of thought and ideas for diseases treatment,such as inflammatory diseases,diseases of old age and tumor.Methods:This study selected the body's largest immune organ spleen and important immune cell macrophages to carry out a preliminary study of the naked mole rats immune system.First of all,separate naked mole rats and mice spleen lymphocytes,and use flow cytometry to compare the number and proportion of immune cells in the naked mole rats and mice spleens.Second,establish separation and culture method of naked mole rats bone marrow macrophage,and use the phagocytosis red cell method and flow cytometry to compare macrophage phagocytosis between naked mole rats and mice bone marrow.Again,infect bone marrow macrophage with polyI:C simulating virus,and use flow cytometry to compare apoptosis between naked mole rats and mice bone marrow macrophage before and after the infection.Extract RNA and use Real-time PCR to make a comparison of TLR3 and IFN-beta expression quantity changes between naked mole rats and mice bone marrow macrophages before and after the infection.Further we extract protein and compare TLR3 and NF-?B expression changes of naked mole rats and mice bone marrow macrophage before and after the infection with western-blot technique.All are for study the mechanism of naked mole rats macrophages in resistance to virus invasion.Finally,invade the bone marrow macrophage by using LPS simulation gram-negative bacteria,and compare apoptosis of naked mole rats and mice bone marrow macrophage before and after the attack by using flow cytometry.Extract RNA of cells,and use Real-time PCR technology to compare TLR3 and IFN-? expression quantity changes before and after the attack in naked mole rats and mice bone marrow macrophages.To further compare TLR3 and NF-?B expression changes in naked mole rats and mice bone marrow macrophage before and after the attack using the western-blot technology.All are for study the mechanism of macrophages resist bacteria invasion in naked mole rats.Results: Comparison of percentage of immune cell in spleen lymphocyteNaked mole rats and mice spleen lymphocytes were extracted and the proportions of various types of immune cells were detected by the way of using flow cytometry.The results are as follows: in mouse,the proportion of macrophages was 15.54±2.12%,the proportion of B cell was 43.21±9.34%,the proportion of T cells was 37.54±4.50%,the proportion of NK cells was 6.06±0.23%;and in naked mole rats spleen,the proportion of macrophages was 34.96 ± 5.85%,the proportion of B cell was 2.34 ± 0.67%,the proportion of T cells was 32.14±4.66%,the proportion of NK cells was 3.09±0.16%.Compared with mice,naked mole rats spleen lymphocyte has the higher percentage of macrophages,but the lower proportion of B cells,and there is no difference in the proportion of T cells and NK cells.Establishment of cultivation method of bone marrow-derived macrophage and comparison of phagocytosisWith reference to the classic method of mice bone marrow-derived macrophage,establish the method of cultivation and separation naked mole rats bone marrow stem cells,and then use M-CSF(macrophage colony stimulating factor)induced hematopoietic stem cells into the macrophages.And use flow cytometry to identify the purity of macrophages by detecting proportion of CD11 b which is the cell surface protein.The proportion of CD11 b is as high as 90%,so the cells separated and cultured are macrophages.Phagocytosis red cells method and flow cytometry were used respectively to detect phagocytosis rate of naked mole rats and mice bone marrow macrophages.The naked mole rats bone marrow macrophage phagocytosis rate was 99.81 ± 0.13%,and the mice macrophage phagocytosis rate was 88.79±0.90%.The results showed that the naked mole rats macrophage phagocytosis was higher than mice macrophage.Comparison before and after polyI:C infected macrophagesThe same concentration of polyI:C simulation virus infected naked mole rats and mice bone marrow macrophages,after a certain period of time to detect cell apoptosis.And then extracted RNA and protein respectively and tested the expression of TLR3,IFN-?,and NF-?B.The results showed that the apoptosis of naked mole rats and mice bone marrow macrophage after polyI:C infection were increased,and the expression of TLR3,IFN-?,and NF-?B were increased.Compared with mice,the increase of naked mole rats bone marrow macrophage apoptosis was not significant,TLR3 expression in cells increase was not obvious,but there was the marked increase in the expression of IFN-?.Comparison of before and after LPS invasive macrophagesBacteria invaded naked mole rats and mice bone marrow macrophage with the same concentration of LPS,after a certain period of time to detect cell apoptosis.Then extracted RNA and protein,respectively detected the expression of TLR4,TNF-?,and NF-?B.The results showed that after LPS attack naked mole rats bone marrow macrophage apoptosis has failed to rise,but mice bone marrow macrophage apoptosis rate increased.In the cell,the expression of TLR4,TNF-? and NF-?B were increased.Compared with mice,naked mole rats bone marrow-derived macrophages higher amount of TLR4 protein expression was not significant,while the NF-?B protein expression in cell markedly rose,and the expression of TNF-? significantly increased.Conclusion:Using flow cytometry confirmed that the ratio of macrophages in naked mole rats spleen is higher,and then compared the macrophage function between naked mole rats and mice.Flow cytometry detection results showed that the naked mole rats macrophages were of stronger phagocytosis than the macrophages in mice.Using Real-time PCR and western-blot studied the possible mechanism of immune response in macrophages.When virus or bacteria invasion,naked mole rats activated TLRs of macrophages,activating the NF-?B signaling pathways,leading cells to secretion of various cytokines to resist the invasion of exogenous substances.Above all the research,we could infer that naked mole rats macrophages faster produce more cytokines,rapidly induce immune response to protect the body,it may be the reasons for long life and less spontaneous tumor in naked mole rats. |
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