| ObjectiveTIPE2 is an essential protein for the maintenance of immune homeostasis.It was identified as a highly expressed protein in immune cells,immune tissues and inflamed nervous tissue.TIPE2 is thought to contain sixαhelices and a DED through which it could bind to proteins that contain DED such as Caspase 8.The high-resolution crystal structure of TIPE2 reveals a large,hydrophobic,central cavity that is poised for cofactor binding.Deletion of TIPE2 mice leads to multiorganic inflammation indicating that it is a novel negative regulator of immune response.TIPE2 is mainly expressed on immune cells,such as lymphocyte and monocyte.It is still unknown that whether nonimmune cells can also express TIPE2.The investigation of the relationship between TIPE2 and diseases is only about experimental autoimmune encephalomyelitis until now.Atherosclerosis is generally believed as a chronically inflammatory disease of artery wall.Mounts of inflammatory cells and factors involve all the stages of atherosclerosis from lipid streak to unstable plaque formation.As TIPE2 is a novel negative regulator of immune response,whether it involves in the development of atherosclerosis and what's it function in this process? As there is ample evidence that the dysfunction of endothelial cell contributes to the pathogenesis of this disease,however,the expression pattern of TIPE2 on endothelial cell is unknown.Many researches indicate that endothelial dysfunction triggers the beginning of atherosclerosis which includes increase of adhesion molecular,abnormal apoptosis,and decrease of NO production.Then the full understanding of biologic and immunologic characters in endothelial cell would be helpful for the research of mechanism in atherosclerosis.In order to study the relationship of TIPE2 and atherosclerosis,we detect the expression of TIPE2 in the atherosclerosis mouse model and analyse the relationship of TIPE2 and other inflammatory cytokines.Then we find TIPE2 is expressed not only on immunocells but also on human vascular endothelial cells.To further study the biologic features and function of human TIPE2,we have successfully produced rabit antihuman-TIPE2 polyclonal antibody in a company and then detect its expression on many human normal tissues and colon tissues of patients with ulcerative colitis.Methods1.The expression of TIPE2 and inflammatory factors in the mouse atherosclerosis model plaqueApoE-/- mouse are used to establish atherosclerosis model and wild type C57 is the control group.The formation of atherosclerosis plaque is observed under microscopes after HE and oil red O staining and then the expression of TIPE2 and inflammatory factors such as IL-6,IL-12p40,TGF-β,and TNF-αare detected by reverse-transcript polymerase chain reaction.After quantization,their linear correlation is analyzed.2.The expression of TIPE2 in HUVEC and its influencing factorsHuman umbilical vein endothelial cell is digested through 0.125%trypsin + 0.01%EDTA,and then cultured in M199 containing 20%PBS.After identification of the cultured cell as endothelial cell through immunochemistry ofⅧ-related antigen,it is treated with TNF-αand oxLDL.The expression of TIPE2, ICAM-1 and VCAM-1 is detected by RT-PCR.The apoptosis of endothelial cell is detected by Hoechst,Annexin V and PI.The apoptosis of endothelial cell is induced by deprivation of serum,and the TIPE2 expression is examined in this process.3.The preparation and application of rabit antihuman-TIPE2 polyclonal antibodyBased on the crystal structure of TIPE2 and its homology anolysis,we choose the specific peptides to produce antihuman-TIPE2 polyclonal antibody in a company. Then the TIPE2 expression in many normal human tissues is examined through immunochemistry.We also collect colorectal tissues of ulcerative colitis patients in Qilu Hospital of Shandong University and the expression of TIPE2 in patients with ulcerative colitis and normal human is evaluated by a standard immunohistochemical procedure.Results1.The expression and function of TIPE2 and inflammatory factors in the development of atherosclerosis1.The establishment of mouse atherosclerosis model8 week-old ApoE-/- mouse with high-fat feeding until 16 or 24 weeks old, atherosclerosis plaque is detected in the artery of ApoE-/- mouse,while the age-matched control group C57 is negative.2.The expression of TIPE2 in the development of mouse atherosclerosisThe expression of TIPE2 at the aorta of ApoE-/- mouse is significantly higher at no plaque stage(8 weeks),early stage(16 weeks) and middle-advanced stage(24 weeks) than that of the age-matched control group C57.3.The relationship of the expression of TIPE2 and inflammatory factorsWhen there is no plaque at the aorta,the expressions of IL-12p40,TGF-βand TNF-αare significantly higher(P＜0.05).During the early stage the expression of TGF-βand TNF-αis still significantly higher(P＜0.05).Through further analysis,we find the expression of TIPE2 is positively correlated with that of TNF-αbut is not irrelevant to the expression of IL-6,IL-12p40 and TGF-β.Our results indicate that TIPE2 seems to participate in the formation of atherosclerosis plaque,and TNF-αcan induce the expression of TIPE2.Further studies are necessary to determine the specific mechanisms.Ⅱ.The expression of TIPE2 in human umbilical vein endothelial cell and its influenting factorsWe have found that the expression of TIPE2 at the aorta of ApoE-/- mouse is significantly higher and positively correlated with that of TNF-α.Then we wonder what the function of TIPE2 in human beings is? We culture the HUVEC and investigate its function in endothelial cell,the important part of vessel wall.1.The cultivation and identification of HUVECHuman umbilical vein endothelial cell is digested through 0.125%trypsin + 0.01%EDTA,and then cultured in M199 containing 20%FBS.The cutured cells are fusiform or orbicular-ovate,aligning as cobblestone,with plenty of cytoplasm and round nuclei.There are claybank particles in the plasma of the cultured cell through immunochemistry ofⅧ-related antigen,especially around the nuclei.The cultured cells are confirmed as endothelial cell.2.The expression of TIPE2 in HUVECThe expression of TIPE2 is detected by RT-PCR,with human smooth muscle cell as negative control and human colon as the positive control.The results show that TIPE2 expresses in endothelial cell,but compared to its expression in colon its expression level in endothelial cell is low.3.Factors influencing of TIPE2 expression in HUVECThe expression of TIPE2 increases after the treatment of different level of TNF-α(control group,12.5μg,25μg,50μg,75μg and 100μg groups).After 6h of treatment with different concentration oxLDL(control group,12.5μg,25μg,50μg,75μg and 100μg groups),the expression of TIPE2 significantly increases in 12.5μg and 25μg groups,compared with control group,but the expression of TIPE2 gradually decreases along with the increase of the concentration of oxLDL.The expression of ICAM-1 and VCAM-1 also increases after the treatment of TNF-αand oxLDL.In addition,we induce apoptosis of endothelial cells by the serum deprivation, and observe the effect of the apoptosis in the expression of TIPE2.After 3h of serum deprivation,intracellular granules increases and cell starts to die.Hoechst shows cell nuclear pyknosis and apoptosis bodies.The rates of apoptosis cell also increase through PI staining.The expression of TIPE2 decreases at the early stage of apoptosis and then gradually increases,but the expression of ICAM-1 and VCAM-1 decreases after the deprivation of serum.These results confirm that many factors can effect the expression of TIPE2 in endothelial cells.Ⅲ.The preparation of rabit polyclonal antihuman TIPE2 antibody1.The selection of the polypeptide segment of the rabit polyclonal antihuman TIPE2 antibodyBased on the crystal structure of TIPE2 and homology analysis of TIPE family,the peptides that we choose to produce antibody in a company are RSVAHLFID.The content of the antibody is 200μg/ml through their detection and it has a strong specificity.It could only bind to TIPE2 not to other members of TIPE family.2.The expression of TIPE2 in plenty of human normal tissuesThe expressions of TIPE2 in many normal tissues are detected through immunohistochemistry in tissue array.2.1.The expression of TIPE2 in human nervous systemWe firstly investigate that TIPE2 are highly expressed in the neuron of cerebrum and brain stem,but the expression in cerebellum is negative.Further studies are necessary to determine the significance of the expression.2.2.The expression of TIPE2 in human digestive systemThe expression of TIPE2 is detected in squamous epithelial cell and plasmocyte of esophagus,lymphocyte of liver,granular of gastric,and lymphocyte and glandular epithelium cells of appendix in different degrees. Meanwhile the expression in hepatocyte and pancreas is negative.2.3.The expression of TIPE2 in other tissuesBesides,we find that TIPE2 are also expressed in lymphoid vessel, lymphoid nodule,lymphocyte of tonsil and hairfolliclegland of skin,but not in thyroid gland and lung.In conclusion,human TIPE2 is detected in both immunocyte and non-immunocyte.3.The expression of TIPE2 in ulcerative colitisUlcerative colitis is believed to be an abnormal mucosal inflammatory,and data from various sources suggest that immune dysfunction can contribute to disease pathogenesis of progression.To explore the relationship of TIPE2 and ulcerative colitis,we examine the expression of TIPE2 in ulcerative colitis and normal tissue of colon.The expression of TIPE2 in colon of ulcerative colitis patients is much higher than that of normal tissues,especially in the mucosa approaching to enteric cavity and lymphocyte of proper layer.This indicates that TIPE2 may involve in the pathogenesis of ulcerative colitis.Conclusions1.The expression of TIPE2 at the aorta of ApoE-/- mouse is significantly higher than that of wild type,and positively correlated with that of TNF-α.2.We detect that endothelial cell expresses TIPE2,and the expression of TIPE2 increases after the treatment of TNF-α,oxLDL and apoptosis of endothelial cells.3.We have successfully prepared antihuman-TIPE2 polycolony antibody and find both immunocytes and non-immunocytes express TIPE2 which is different from the expression pattern of TIPE2 in mouse.4.Finally,we find TIPE2 is highly expressed in mucosa colon of ulcerative colitis patients.Innovation and significances1.TIPE2 is a newly identified gene and it's unknown its role in many human diseases.We have first detected the increase of TIPE2 on aorta of atherosclerosis prone mice,and it is positively correlated with TNF-α.Besides,we also find human vascular endothelial cell expresses TIPE2 and could be induced by TNF-α, oxLDL and apoptosis.2.Now the research about TIPE2 is mainly in mice,there are still many unanswered questions about characters of human TIPE2.We have first finds the differences of TIPE2 in human and in mice.Human TIPE2 not only expresses on immune cells,but also expresses on nonimmune cells.We also detect the increase of TIPE2 on colonic mucosa epithelial cell.3.We have first prepared antihuman-TIPE2 polyclonal antibody and it is helpful for future research of TIPE2. Limitations1.TIPE2 is a newly identified gene and there is no specific antibody,so the reseach is extremely limited.2.As the prepared antibody arrived recently,we have detected many normal human tissues but there are still many works to do.
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