| Part I Isolation and cultivation of rat splenic macrophageObjective: To isolate and culture the primary rat splenic macrophage .Method: Sprague-Dawley rats were given intraperitoneal chloral hydrate anaesthesia and spleen was resected aseptically. Spleen cells suspension were prepared and the erythrocytes in suspension were decomposed. RPIM-1640 medium with re-suspended sediments were inoculated into disposable plastic flasks added 50ml·l-1 CO2 kept in 100% humidity at 37℃and cultured for 1,2,3,6,12 and 24 hour respectively. Non-adherent cells were washed and splenic macrophage adherent cells were observed under phase contrast microscope to determine their optimum adherent time. Immunohistochemical methods were used to identify cells; trypan blue staining to identify the cell viability and Wright's staining used to identify cell purity.Results: 1.Large numbers of rat spleen cells were obtained from suspension after spleen tissue grinding which were enough to fulfil the requirements of further isolation and culture. 2. Observation of adherent cells found small amount of adherent cells in 1-3h, most of cells adhere in 12h and few adherent cells in 24h. 3. Adherent culture splenic macrophage were observed under phase contrast microscope; most were round or oval and a few were spindle or polygonal. It was observed that cells were large with intact cell membrane, clearly visible nuclei and cytoplasm with rich contents. Immunohistochemistry results showed positive expression of CD68 of adherent splenic macrophage suggesting that derived spleen cells are macrophages. Trypan blue staining showed cell viability> 95% and Wright stain identified cell purity> 90%.Conclusion: 1. Rat splenic macrophage were obtained by adherent culture method and their viability and purity meet the requirement of experiment. 2. Adherent for 12h is appropriate adherent time for rat splenic macrophage . Part II Effect of mild heat stress on immune function of rat splenic macrophageObjective:To investigate the effect of mild heat stress on immune function of rat splenic macrophage.Method:Rat primary splenic macrophages constantly kept at 37℃were assigned as control group. In the experimental group, rat splenic macrophage were placed in 41℃incubator for mild heat stress and temperature restored to 37℃after an hour. After being heat stressed cells were divided into five sub-groups of 0min, 30min, 60min, 120min and 180min ,with a total of six groups. The ability to engulf neutral red dye(OD540nm value) was used to detect macrophage phagocytosis, MTT assay was used to measured the macrophage cytocidal effect towards L1210 cells which determined the macrophage cytotoxicity ,macrophage Chemotaxis was observed in Costar Transwell chemotaxis chamber inserts, concentration changes of macrophage's secretions of TNF-a and IL-12 were measured by using Enzyme-linked Immunosorbnent Assay method .Results In experimental group, functions of macrophages were changed after mild heat stress (41℃for 1h) and changes were directly related to the recovery time after being mild heat stressed. In 0min group after mild heat stress, OD540nm value of macrophages increased from normal value 0.21±0.01 to 0.34±0.01 (p <0.05) and then continue to rise and reached to a maximum value of 0.81±0.04 (p <0.01) at 60min. Then OD540nm value decreased gradually and after 180min of mild heat stress was still on 0.47±0.03 levels (compared with the control group, p <0.05). Cytotoxicity of 0min group increased from normal value of 35.30±4.37% to 45.00±4.74% (p <0.05) after mild heat stress and reached to maximum of 82.07±5.17% (p <0.01) in 60min group and then decreased gradually. In 180min group Cytotoxicity returned to normal value of 37.27±5.11% (compared with the control group, p> 0.05).Macrophages secretion of cytokines TNF-αand IL-12 increased with the time after mild heat stress.Conclusion:After mild heat stress phagocytosis, cytotoxicity and Chemotaxis of rat splenic macrophage has increased and gradually restored to normal with the extended recovery time after mild heat stress. During experimental period; macrophages secretion of cytokines TNF-αand IL-12 increased with the time after mild heat stress, which suggested that mild heat stress has promoting role on immune function of rat splenic macrophage .Part III Expressions and role of Bip protein in rat splenic macrophage during mild heat stress.Objective:To investigate Bip protein expressions and its role in rat splenic macrophage functions changes after mild heat stress.Method:Rat splenic macrophage constantly kept at 37℃were assigned as control group. In the experimental group , rat splenic macrophage were placed in 41℃incubator for mild heat stress and temperature restored to 37℃after an hour. After being mild heat stressed cells were divided into five sub Groups of 0min, 30min, 60min, 120min and 180min. BipmRNA and Bip protein expression were detected in each group. At the same time phagocytosis, cytotoxicity and chemotaxis was measured in each group.Result : 1. There were a significant increase in expression of splenic macrophage BipmRNA after mild heat stress ,which reached to maximum in 30min group after stress and still remain higher in 60min and 120min group (p <0.01),BipmRNA level restored to normal in 180min group. Bip protein expression reached to peak levels in 60min group after stress and still remained higher in 120min and 180min groups (p <0.05). 2. After mild heat stress phagocytosis, cytotoxicity and Chemotaxis of splenic macrophage were significantly increased with synchronous increasing levels of BipmRNA and Bip protein.Conclusion:Changes in BipmRNA and Bip protein expression and functions of mild heat stressed splenic macrophage happened at the same pace. It suggested that after mild heat stress increasing Bip expression and enhanced splenic macrophage function are closely related. Part IV. Effect of BiP antisense oligonucleotide on immune functions of mild heat stressed rat splenic macrophageObjective:To investigate Bip antisense oligonucleotides role on immune functions of mild heat stressed rat splenic macrophage and to determine the Bip role on immune function changes of mild heat stressed rat splenic macrophage in vitro.Methods:Bip-specific oligonucleotide were designed with antisense sequence and mismatch sense sequence of 5'-CTTTGTCCTAGCCGCTCG-3' and 5'- CTTTGTCCTAGCCGCTCG-3' respectively. Liposome-mediated transfection method was used for transfection of them to ret splenic macrophage. Bip mRNA levels were observed in rat splenic macrophage of Bip antisense oligonucleotide transfected group(Bip AS--ODN) ,Bip mismatch sense oligonucleotide transfected group(Bip MS-ODN) and non transfected group(control group). Then functional changes of rat splenic macrophage were observed in control group, non transfested mild heat stressed group and Bip antisense oligonucleotide transfected mild heat stressed group.Results : As compared to control group and Bip mismatch sense oligonucleotide transfected group, significant reduction in expressions of BipmRNA and phagocytosis, cytotoxicity and Chemotaxis of rat splenic macrophage were observed in Bip antisense oligonucleotide transfected mild heat stressed group (P <0.01).Conclusion:Bip antisense oligonucleotide can significantly inhibit splenic macrophage BipmRNA expressions and decrease phagocytosis, cytotoxicity and chemotaxis of mild heat stressed rat splenic macrophage ;Bip play promoting role on immune function of mild heat stressed rat splenic macrophage. Part V. The role of P38MAPK in immune functional changes of heat stressed rat splenic macrophageObjective: Research on MAPK signaling role in Bip protein-mediated immune functional changes of mild heat stressed rat splenic macrophage in vitro.Methods: Rat splenic macrophage were pretreated with P38MAPK inhibitor and then placed in 41℃incubator for mild heat stress,after 1h temerature restored to 37℃in inhibition group. Non stressed rat splenic macrophage were assigned as control group , macrophages which simply heat stressed at 41℃for 1h as 60min group(stress group) used as control ,too. Three groups were detected for macrophage phagocytosis, cytotoxicity and chemotaxis. At the same time P38MAPK protein and Bip protein expressions were detection.Results: After 60min of mild heat stress; macrophages phagocytosis, cytotoxicity and chemotaxis in stress group greately increased which was significantly different from the control group and inhibition group (P <0.01). P38MAPK inhibitor pretreated rat splenic macrophage , when compared with the stress group, phagocytosis, cytotoxicity and chemotaxis significantly lowered after heat stress (P <0.01). P38MAPK inhibitor pretreated group when compared with the control group , there was no significant difference (P> 0.05). In stress group P38MAPK protein expressions were significantly increased and were significantly different from inhibition control group (P <0.01).As compared with the stress group, P38MAPK protein expressions were inhibited after P38MAPK inhibitor pretreatment in inhibition group (P <0.01) . P38MAPK inhibitor pretreatment also caused changes in Bip protein expressions i.e. in the stress group from 1.2702±0.5345 (Bip /β-actin)dropped to 1.0281±1.0614 in inhibition group (P <0.05).Conclusion: Mild heat stress can enhance phagocytosis, cytotoxicity and chemotaxis of rat splenic macrophage and at the same time promote their P38MAPK and Bip protein expressions. P38 inhibitors can significantly inhibit mild heat stressed rat splenic macrophage phagocytosis, cytotoxicity and chemotactic functions as well as these inhibit P38MAPK and Bip protein expressions. P38MAPK may be involved in Bip protein-mediated immune modulation of mild heat stressed rat splenic macrophage .
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