【作者】 黄长文；

【导师】 傅华群；

【作者基本信息】 南昌大学 ， 外科学， 2009， 博士

【摘要】 第一部分大鼠脾脏巨噬细胞的分离与培养目的:分离培养并鉴定大鼠脾脏巨噬细胞。方法:SD大鼠,腹腔注射水合氯醛麻醉,无菌取脾脏,制备成脾脏组织细胞混悬液,裂解脾脏组织细胞混悬液中的红细胞;用RPIM-1640培养基重悬沉淀,接种于一次性塑料培养瓶中,在37℃,50ml·l-1CO2,100%湿度中分别培养1、2、3、6、12、24h,洗涤未贴壁细胞,相差显微镜下分别观察脾脏巨噬细胞的贴壁情况,确定最佳细胞贴壁时间。应用免疫组织化学等方法进行细胞的鉴定;台盼蓝染色鉴定细胞活力,瑞氏染色鉴定细胞纯度。结果: 1.大鼠脾脏组织经过研磨等处理后,得到大量的脾细胞混悬液,可以满足进一步的分离培养的需要;2.观察贴壁细胞, 1-3h细胞少量贴壁,12h左右的贴壁细胞增多, 24h的贴壁细胞减少。12h是贴壁细胞合适时间; 3.相差显微镜下观察到贴壁培养的脾脏巨噬细胞多为圆形或椭圆形,少数呈梭形或多边形。镜下观察细胞体积较大,细胞膜完整,细胞核清晰可见,胞浆内容物丰富;免疫组织化学结果显示贴壁的脾脏巨噬细胞CD68表达阳性,提示所得的细胞是脾脏巨噬细胞,台盼蓝染色显示细胞活力>95%,瑞氏染色鉴定细胞纯度>90%。结论:1分离培养出活力和纯度符合实验要求的大鼠脾脏巨噬细胞;2.贴壁12h是大鼠原代脾脏巨噬细胞的合适贴壁培养时间。第二部分轻度热应激对大鼠脾脏巨噬细胞免疫功能的影响目的:探讨轻度热应激对大鼠脾脏巨噬细胞免疫功能的影响。方法:以始终处于37℃的大鼠原代脾脏巨噬细胞作为对照组,实验组将大鼠原代脾脏巨噬细胞置于41℃恒温箱中,使细胞轻度热应激1h,然后恢复到37℃,分成应激后0min,30min,60min,120min,180min五个亚组组,总共六个组;以吞噬中性红能力(OD540nm值)检测巨噬细胞的吞噬功能、噻唑蓝法测定巨噬细胞对L1210细胞的杀伤作用来反应巨噬细胞的杀伤活性,Transwell小室趋化装置观察巨噬细胞趋化活性变化;采用双抗夹心ABC-ELISA法检测大鼠脾脏巨噬细胞分泌的TNF-a,IL-12浓度的变化。结果:轻度热应激(41℃、1h)后,实验组大鼠脾脏巨噬细胞的功能均发生了变化,而且与应激后恢复时间有关。热应激后0min,巨噬细胞的OD540nm值由正常的0.21±0.01上升到0.34±0.01(p<0.05),然后继续上升,至60min达最高值0.81±0.04(p<0.01),随后逐渐下降,应激后180min仍然有0.47±0.03(与对照组比较,p<0.05);杀伤活性由正常的35.30±4.37﹪上升到应激后0min的45.00±4.74﹪(p<0.05),在60min分钟达到最大值82.07±5.17﹪(p<0.01),随后逐渐下降,180min后杀伤活性基本恢复正常,为37.27±5.11﹪(与对照组比较,p>0.05);趋化作用也呈现大致相同的变化趋势,大鼠脾脏巨噬细胞正常趋化活性为23.40±5.32/HP;热应激1h后0min为34.60±5.22/HP(p<0.05),在60min分钟达到最大值60.80±4.02/HP(p<0.01)后逐渐下降,180min仍为31.60±4.21/HP(与对照组比较,p<0.05);在实验时间内,大鼠脾脏巨噬细胞分泌的TNF-α和IL-12均随着应激后时间的延长而增多,180min达到最高(与对照组比较,P < 0.01)。结论:轻度热应激后,大鼠脾脏巨噬细胞的吞噬功能、杀伤活性、趋化活性都有上调;在实验时间内,大鼠脾脏巨噬细胞分泌的细胞因子TNF-α和IL-12在轻度热应激后随着时间的延长而增加。提示轻度热应激对大鼠脾脏巨噬细胞的免疫功能有促进作用。第三部分Bip蛋白在轻度热应激大鼠脾脏巨噬细胞中的表达与意义目的:探讨轻度热应激大鼠脾脏巨噬细胞Bip蛋白的表达及其对细胞功能改变的影响。方法:原代培养大鼠脾脏巨噬细胞,将细胞置于41℃恒温箱中,使细胞轻度热应激,1h后恢复到37℃,分别检测应激后0min,30min,60min,120min,180min巨噬细胞BipmRNA、Bip蛋白的表达;同时段分别检测巨噬细胞吞噬功能、杀伤活性和趋化作用。结果:(1)轻度热应激后,大鼠脾脏巨噬细胞BipmRNA的表达明显增加,30min后到达高峰,60min、120min时仍高于对照组(p<0.01),180min后恢复至正常水平。Bip蛋白的表达在轻度热应激60min后到达高峰,120min、180min时仍高于对照组(p<0.05)。(2)轻度热应激后,大鼠脾脏巨噬细胞的吞噬功能、杀伤活性和趋化作用均明显增强,且与BipmRNA、Bip蛋白表达的改变基本同步。结论:轻度热应激大鼠脾脏巨噬细胞BipmRNA、Bip蛋白的表达与巨噬细胞功能发生同步改变,提示Bip蛋白表达上调与轻度热应激大鼠脾脏巨噬细胞功能增强可能有关。第四部分BiP反义寡核苷酸对轻度热应激大鼠脾脏巨噬细胞免疫功能的影响目的:探讨Bip反义寡核苷酸对体外轻度热应激大鼠脾脏巨噬细胞功能的影响,进一步证实Bip在轻度热应激大鼠脾脏巨噬细胞功能改变中的作用。方法:设计特异性Bip寡核苷酸,反义序列为5’- CTTTGTCCTAGCCGCTCG - 3’;错义序列为5’- CTTTGTCCTAGCCGCTCG - 3’。通过脂质体包裹后将其转染至小鼠脾脏巨噬细胞,观察Bip反义寡核苷酸转染后及未转染组、Bip错义寡核苷酸转染后大鼠脾脏巨噬细胞BipmRNA的改变;然后分别观察正常对照组、轻度热应激组和Bip反义寡核苷酸转染并轻度热应激后脾脏巨噬细胞功能的改变。结果:Bip反义寡核苷酸转染大鼠脾脏巨噬细胞后,与未转染组和错义组比较,轻度热应激后BipmRNA的表达明显减少;大鼠脾脏巨噬细胞吞噬功能、趋化活性和杀伤活性明显回落(P<0.01)。结论:Bip反义寡核苷酸可显著抑制大鼠脾脏巨噬细胞BipmRNA的表达,并可降低轻度热应激大鼠脾脏巨噬细胞吞噬功能、趋化活性和杀伤活性。Bip对轻度热应激大鼠脾脏巨噬细胞的免疫功能有促进作用。第五部分P38MAPK信号途径在轻度热应激大鼠脾脏巨噬细胞免疫功能改变中的作用目的:研究MAPK信号途径在Bip蛋白介导的体外轻度热应激大鼠脾脏巨噬细胞免疫功能改变中的作用。方法:P38MAPK抑制剂预处理大鼠脾脏巨噬细胞,将细胞置于41℃恒温箱中,使细胞轻度热应激,1h后恢复到37℃(抑制组),以未应激(对照组)和单纯41℃热应激1h后60min大鼠脾脏巨噬细胞(应激组)为对照,分别检测三组大鼠脾脏巨噬细胞吞噬、杀伤、趋化功能。同时检测P38MAPK蛋白和Bip蛋白的表达。结果:轻度热应激后60min,应激组大鼠脾脏巨噬细胞吞噬、趋化和杀伤活性增强,与对照组、抑制组比较差异有显著性(P<0.01)。P38MAPK抑制剂预处理大鼠脾脏巨噬细胞,与应激组比较,热应激后巨噬细胞吞噬、趋化和杀伤活性明显降低(P<0.01),与对照组比较无显著差异(P>0.05)。应激组P38MAPK蛋白表达明显上调,与对照组和抑制组比较差异有显著性(P<0.01);P38MAPK抑制剂预处理后,抑制组P38MAPK蛋白表达受到抑制,与应激组比较(P<0.01)。Bip蛋白的表达也因P38MAPK抑制剂预处理而发生改变,由应激组的1.2702±0.5345(Bip/β-actin)下降到抑制组的1.0281±1.0614(P<0.05)。结论:轻度热应激可增强大鼠巨噬细胞吞噬、趋化和杀伤作用的同时,P38MAPK、Bip蛋白表达上调。P38MAPK抑制剂可显著抑制大鼠巨噬细胞吞噬、趋化和杀伤功能以及P38MAPK、Bip蛋白的表达。P38 MAPK信号途径参与Bip蛋白介导的轻度热应激后大鼠脾脏巨噬细胞免疫功能的调节。更多还原

【Abstract】 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 .更多还原