【作者】 王慧；

【导师】 李卓娅；

【作者基本信息】 华中科技大学 ， 分子免疫学， 2007， 博士

【摘要】 肝脏缺血再灌注（Ischemia and reperfusion,I/R）损伤的核心是过度的炎症反应。该损伤分为两个时相:I相主要为激活的枯否细胞（Kupffer cell, KC）连同其分泌的细胞因子（如TNF-α、IL-1β等）引起的损伤;II相则是在KC,趋化性细胞因子、粘附分子等共同作用下,导致中性粒细胞（Polymorph nuclear leukocyte,PMN）趋化、黏附、聚集、活化所介导的损伤。后者是肝脏I/R损伤的关键,其结果是肝细胞的坏死和凋亡,最终引起肝功能衰竭、甚至全身多脏器功能障碍综合征。Toll样受体4 （Toll-like receptor 4,TLR4）是新近发现的启动机体急性炎症反应的受体,在天然免疫中起重要作用。通过该受体可诱导促炎细胞因子的产生,启动机体的炎症防御机制。已证明TLR4参与肝脏I/R损伤。TLR4分布于肝窦内皮细胞（Liver sinusoidal endothelia cells,LSEC）、KC及PMN,虽然肝细胞也表达TLR4,但一般认为该细胞的TLR4不参与肝脏I/R损伤。有人证实在同一脏器组织中,不同细胞表达的TLR4在急性炎症过程中的作用不同。用氯化钆阻断肝脏KC细胞功能后,虽然可减轻肝I/R损伤,但是与假手术对照比较,仍有明显损伤,提示除KC外,其它细胞如PMN及LSEC表达的TLR4也可能参与启动肝脏I/R的炎症过程。因此,本研究目的在于观察肝脏内不同细胞表面TLR4在肝I/R损伤中的作用,阐明其通过招募、活化PMN而参与肝脏I/R II期损伤的机制。一、骨髓移植小鼠模型的建立及鉴定将两种纯系雄鼠C3H/HeJ (TLR4^Mut/Mut)及C3H/HeN (TLR4^+/+)的骨髓分别移植于同系雌鼠或交叉移植于相应品系的雌鼠,骨髓移植10周后,通过比较接受及未接受骨髓移植小鼠的死亡率,用FISH和PCR检测受者的Y染色体及SRY基因,证实骨髓移植成功。故本研究获取了以下2种纯合体小鼠和2种嵌合体小鼠,为后续实验研究不同细胞表面TLR4在肝脏I/R损伤中的作用奠定基础。小鼠名称供体基因型受体基因型表型WT/WT TLR4^+/+ TLR4^+/+源细胞TLR4 ^+/+/非髓源细胞TLR4 ^+/+ Mut/Mut TLR4^Mut/Mut TLR4^Mut/Mut源细胞TLR4^Mut/Mut/非髓源细胞TLR4^Mut/Mut WT/Mut TLR4^+/+ TLR4^Mut/Mut源细胞TLR4 ^+/+/非髓源细胞TLR4^Mut/Mut Mut/WT TLR4Mut/Mut TLR4^+/+源细胞TLR4^Mut/Mut/非髓源细胞TLR4 ^+/+二、比较髓源及非髓源细胞TLR4在肝脏I/RⅡ期损伤中的作用用上述4种类型的小鼠建立肝脏部分缺血再灌注损伤动物模型。各组动物模型中门静脉血清无内毒素污染,外周血谷丙转氨酶（Alanine aminotransferase,ALT）水平明显上升,HE及电镜显示肝细胞受损,炎性细胞浸润,且均在肝脏恢复灌注6-12小时最为显著。四组比较,在恢复灌注6小时及12小时,Mut/Mut小鼠肝损伤最轻,而髓源细胞TLR4突变者组（Mut/WT）小鼠肝组织损伤较髓源细胞TLR4野生型组（WT/Mut）轻（P <0.05）,提示髓源及非髓源细胞TLR4均参与了肝脏I/RⅡ期损伤,但髓源细胞TLR4的作用更大。三、髓源及非髓源细胞TLR4参与肝脏I/RⅡ期损伤的机制研究为进一步探讨髓源及非髓源细胞TLR4参与肝脏I/RⅡ期损伤的机制,我们主要观察上述4种动物肝脏缺血再灌注Ⅱ期损伤时中性粒细胞的变化,证实: 1.髓源及非髓源细胞细胞TLR4在肝脏I/R时对PMN聚集的影响:用特异性酯酶D对中性粒细胞进行特异性染色和记数,并测定其产生的髓过氧化物酶。结果证实在恢复灌注后3小时,各组肝组织间隙内PMN水平明显上升,6至12小时仍维持在较高水平。四组比较,恢复灌注6小时,WT/WT组PMN水平最高,Mut/Mut最低,髓源细胞TLR4野生型组（WT/Mut）高于髓源细胞TLR4突变型（Mut/WT）（p<0.05）;恢复灌注12小时,WT/WT组与髓源细胞TLR4野生型组（WT/Mut）的PMN聚集均高于髓源细胞TLR4突变型（Mut/WT）组及^Mut/Mut组（p<0.05）。提示在肝脏I/RⅡ期损伤中,PMN的聚集程度与TLR4相关,且髓源细胞表面TLR4的作用较大。2.髓源及非髓源细胞TLR4在肝脏I/R时对参与PMN募集相关因素的影响:（1）促炎细胞因子:用ELISA检测证实在恢复灌注后3小时各组小鼠产生TNF-α和IL-1β达高峰。四组组间比较,WT/WT组的TNF-α,IL-1β水平最高,其次为髓源细胞TLR4野生型组（WT/Mut）,均高于髓源细胞TLR4突变型组（Mut/WT）,而^Mut/Mut组水平最低,组间差异有显著性。（2）趋化性细胞因子:用ELISA检测发现各组内MIP-2水平在再灌注3小时及6小时最高,四组组间差异与促炎细胞因子相似。（3）黏附分子:用用Western证实Mac-1/CD11b在I/R损伤晚期明显升高,其组间差异与上相同。ICAM-1的表达亦在I/R损伤晚期明显升高,与Mac-1/CD11b不同的是,虽然WT/WT组ICAM-1表达最强,^Mut/Mut组最弱,但髓源细胞TLR4突变型（Mut/WT）组表达强于髓源细胞TLR4野生型组（WT/Mut）。ICAM-1免疫组化结果与Western结果一致,且在再灌注12小时,WT/WT组及髓源细胞TLR4突变型组（Mut/WT）肝细胞ICAM-1染色呈强阳性表达,而髓源细胞TLR4野生型组（WT/Mut）及^Mut/Mut小鼠肝细胞ICAM-1染色仅呈弱阳性。上述结果提示TLR4影响肝脏缺血再灌注损伤中促炎细胞因子、趋化性细胞因子和粘附分子的表达,进而影响PMN在肝脏I/RⅡ期损伤中的效应。髓源细胞TLR4主要促进促炎细胞因子、趋化性细胞因子及Mac-1的表达,而非髓源细胞TLR4对ICAM-1表达的影响则更大。四、PMN/TLR4体外对肝脏I/R局部介质的应答（1）TLR4对肝脏I/R的肝匀浆液趋化PMN的影响:我们在体外用Transwell观察缺血1小时再灌注3小时肝组织匀浆液对TLR4^+/+ PMN及TLR4^Mut/MutPMN迁移及穿越内皮能力的影响,结果证实WT/WT匀浆液组趋化作用最强,髓源细胞TLR4野生型组（WT/Mut）强于髓源细胞TLR4突变型组（Mut/WT）,^Mut/Mut组作用最弱;此外,每组匀浆液对TLR4^+/+PMN的趋化作用均强于趋化TLR4^Mut/Mut PMN。提示髓源细胞TLR4不仅可通过影响趋化物质的表达量来影响PMN的迁移及内皮穿越功能,而且PMN表面功能性TLR4尚可增强PMN对趋化物质的敏感性。（2）TLR4对MIP-2介导PMN穿越内皮能力的影响:用MIP-2中和抗体可显著抑制肝脏I/R的肝匀浆液诱导TLR4^+/+PMN的内皮穿越能力,对TLR4^Mut/Mut PMN的影响力较弱,提示PMN的TLR4能增强其对MIP-2趋化能力的敏感性。（3）TLR4对CD11b介导PMN粘附及内皮穿越能力的影响:用黏附实验及Transwell实验证实CD11b中和抗体可明显抑制TLR4^+/+PMN的粘附能力及肝脏I/R的肝匀浆液诱导TLR4^+/+PMN的定向运动能力及,且此影响均强于TLR4^Mut/MutPMN,提示PMN的TLR4能增强其对CD11b的反应性。（4）TLR4对TNF-α介导PMN聚集的影响:用TNF-α抗体中和各组肝组织匀浆液的TNF,结果对TLR4^+/+PMN及TLR4^Mut/Mut PMN的趋化能力无影响;但TNF-α却能激活PMN,增强TLR4^+/+PMN及TLR4^Mut/Mut PMN穿越内皮的能力,但两者无显著性差异。提示TNF-α可促进PMN内皮穿越能力,但该作用与TLR4无关。用TNF-α激活假手术TLR4^+/+及TLR4^Mut/Mut鼠的PMN,RT-PCR检测证实TNF-α可促进PMN CD11bmRNA及MIP-2 mRNA转录,但该作用与PMN的TLR4无关。（5）用RT-PCR证实缺血再灌注刺激可增强TLR4^+/+及TLR4^Mut/Mut PMN转录CD11b及MIP-2 mRNA ,但TLR4^+/+PMN转录这两种因子mRNA水平均高于TLR4^Mut/Mut PMN,提示TLR4可上调缺血再灌注损伤诱导PMN对这两因子的表达。本研究通过体内实验证实TLR4,特别是髓源细胞TLR4在肝脏I/R时,通过促进促炎细胞因子、趋化性细胞因子及粘附分子的表达来导致PMN在肝脏的聚集和活化,进而介导I/RⅡ期损伤;体外实验进一步证实TLR4不仅可上调缺血再灌注诱导CD11b和MIP-2的表达量来影响PMN的活化、粘附及穿越内皮等功能,同时PMN表面有功能的TLR4可增强其对MIP-2的敏感性。更多还原

【Abstract】 An excessive inflammatory response was clearly recognized as a key mechanism of liver ischemia and reperfusion （I/R） injury. Liver I/R injury could be divided into two phase:Ⅰphase, Kupffer cells（KC） played a central role as the initial cytotoxic cell type as a souce of many inflammatory mediators, such as TNF-α,IL-1βet.Ⅱphase: polymorph nuclear leukocyte（PMN） were activated and recruited into the liver stimulated by KC ,procytokines, chemokines as well as adhesion molecules. The fate was the necrosis and apoptosis of hepatocytes, which further resulting in failure of liver function, even multiple organ dysfunction syndrome（MODS）.Toll like receptor 4（TLR4） was one of the newly found receptor to activate acute inflammatory response, which played important role in signal transduction of the innate immune response. It was this receptor who initiated the production of cytokines and initiated the defence mechanism of the body in early stage. It had been demonstrated that TLR4 was involved in liver I/R injury. Liver sinus endothelia cells（LSEC）,KC and PMN could all express TLR4.Although hepatocytes could also express TLR4, it was generally believed that TLR4 on hepatocytes was not implicated in liver I/R injury. Andoneui G et reported that TLR4 on different cells were responsible for different effects during acute inflammatory response. Liver I/R injury could be alleviated by depletion of KC using Gadolinium, however, the injury could not be prevented completely, which suggested that besides TLR4 on KC, TLR4 on some other cell types might also play important role in liver I/R injury. Our aim in this study was to determine whether the TLR4-dependent injury requires TLR4 expression on different cells in liver. Because PMN was the key players or I/R injury, we would elucidate the following specific role of TLR4 on different cells: role on the sequestion, activation, adhesion as well as transmigration of PMN.1. Establishing and identification of Chimeric mice as well as homozygous miceTwo genetic type of mice were used for establishing chimeric and homozygous mice model, one is C3H/HeJ with genotype of TLR4^Mut/Mut, the other is C3H/HeN with genotype of TLR4^+/+.Ten weeks after transplantation, The following methods were used to confirm successful engraftment: （1） Death rate of control group; （2） FISH （fluorescence in situ hybridization）: PY3.4 probes for Y chromosome which has been labeled with PY3 was used to hybridize to metaphase chromosomal slides of mice; （3） DNA was extracted from peripheral blood of those mice and PCR was performed to amplify SRY gene which is located on Y chromosome. After transplantation, we successfully established 2 kinds of chimeric mice with bone marrow derived cells alternately expressing wild TLR4 and mutant TLR4, 2 kinds of homozygous mice with wild TLR4 and mutant TLR4 alternately were also established as well. Genotypes of mice could be expressed as follows:（1） WT/WT group: mice with genotype of“bone marrow derived cells TLR4^+/+ / endothelium TLR4^+/+”,（2） WT/Mut group: mice with genotype of“bone marrow derived cells TLR4^+/+ / endothelium TLR4^Mut/Mut”,（3） Mut/WT group: mice with the genotype of“bone marrow derived cells TLR4^Mut/Mut / endothelium TLR4^+/+”,（4） ^Mut/Mut mice with the genotype of“bone marrow derived cells TLR4^Mut/Mut / endothelium TLR4^Mut/Mut”.2. Establishing the model of liver partial I/R injury and analysis of the injury of four groups of miceEach chimeric and homozygous mouse was underwent partial liver ischemia/reperfusion procedure,endotoxin in each mice remained the normal level, suggesting no endotoxemia was developed in each mice during I/R injury. Homozygous mice with wild TLR4 presented the most serious injury, homozygous mice with mutant TLR4 presented the mildest injury, while the injury of chimeric mice with wild TLR4 on bone marrow derived cells was more serious than that of chimeric mice with mutant TLR4 on bone marrow derived cells. These results suggested that liver I/R injury was associated with functional TLR4. TLR4 mutation could protect liver from I/R damage. Furthermore,TLR4 on bone marrow derived cells played more important role in liver I/R injury compared with TLR4 on other cells.3. Mechanism about TLR4 on bone marrow derived or non-bone marrow derived cells in liver I/R injuryTo further study the mechanism of TLR4 on bone marrow derived cells or non-bone marrow derived cells in liver I/R injury, we focused on the variation of function of PMN in liver I/R phaseⅡ, which indicated that:1. The effects of bone marrow derived or non-bone marrow derived TLR4 on PMN accumulation in liver I/R: On the basis of mice model established in part 2, PMN number was counted in I/R liver tissue by chloroacetate esterase staining methods, MPO of each group was also determined to quantify the accumulation of PMN in liver tissue. Chloroacetate asterase staining showed that at 3 h after reperfusion,the numbers of PMN in liver tissues had elevated dramatically, the numbers of PMN reached the highest level at 6 h after reperfusion and remained high till 12 h. Of those 4 groups at 6 h, there achieved the largest PMN accumulation in homozygous mice with wild TLR4, the least PMN accumulation in homozygous mice with mutant TLR4. Strong PMN accumulation could be observed in chimeric mice with wild TLR4 on bone marrow derived cells compared with another kind of chimeric mice. However, at 12 h, differences between homozygous mice with wild TLR4 and chimeric mice with wild TLR4 on bone marrow derived cells weren’t significant; both of them achieved more PMN accumulation than that of chimeric mice with mutant TLR4 on bone marrow derived cells and homozygous mice with mutant TLR4. The differences of the later two groups weren’t significant as well. The results of MPO were identical to those of chloroacetate asterase staining. Results suggested that functional TLR4 appeared to be important for the PMN accumulation in liver in the phaseⅡliver I/R injury, especially for functional TLR4 on bone marrow derived cells.2. The effects of bone marrow derived or non-bone marrow derived TLR4 on the factors in PMN accumulation in liver I/R injury:（1） Level of pro-cytokines: At the indicated time points after restoration of blood supply, which were 0 h, 3 h, 6 h, 12 h respectively, the levels of TNF-α, IL-1β, as well as the level of MIP-2 in ophthalmic artery were tested by ELISA. Three hours after reperfusion, the levels of TNF-α, IL-1βreached their peak in each group. Among the four groups, homozygous mice with wild TLR4 presented the highest levels of the two pro-cytokines, homozygous mice with mutant TLR4 presented the lowest level, while between the two chimeric groups, chimeric mice with wild TLR4 on bone marrow derived cells mice expressed higher levels of TNF-α, IL-1βcompared with another kind of chimeric mice and the differences were significant.（2） Level of chemokines: The level of chemokine MIP-2 reached its peak at both 3 h and 6 h after blood restoration in each group. At 3 h and 6 h, homozygous mice with wild TLR4 expressed the highest level of MIP-2, homozygous mice with mutant TLR4 mice expressed the lowest level, chimeric mice with wild TLR4 on bone marrow derived cells expressed higher level of MIP-2 than that of another kind of chimeric mice.（3） Expression of adhesion molecules: Western-blot analysis for ICAM-1 and Mac-1 were also performed. Immunohistochemical staining method was performed to compare the expression and distribution of ICAM-1 on mice with different genotypes.As the progression of reperfusion, the expression of ICAM-1 and Mac-1 was increased. The variation of Mac-1 expression in four groups paralleled with those pro- cytokines, PMN accumulation. It’s not the same with the variation of ICAM-1 expression. Homozygous mice with wild TLR4 expressed the highest level of ICAM-1 and homozygous mice with mutant TLR4 mice expressed the lowest level, while in two chimeric groups, chimeric mice with mutant TLR4 on bone marrow derived cells expressed higher level of ICAM-1 than that of another kind of chimeric mice. Results of immunohistochemical staining for ICAM-1 were consistent to results of western-blot. At 12 h after reperfusion hepatocytes were strongly stained in homozygous mice with wild TLR4 mice and chimeric mice with mutant TLR4 on bone marrow derived cells, while hepatocytes of chimeric mice with wild TLR4 on bone marrow derived cells and homozygous mice with mutant TLR4 showed only weakly positive stained.These results suggested that functional TLR4 appeared to be important for expression of cytokines, chemotactic factors and adhesion factors, which further influence PMN accumulation in ischemic liver in the phaseⅡof liver I/R injury. TLR4 on bone marrow derived cells were mainly involved in expression of cytokines, chemotactic factors and Mac-1, while expression of ICAM-1 was mainly affected by TLR4 on non-bone marrow derived cells.4. In vitro experiments elucidating the mechanism about PMN accumulation in liver I/R influenced by bone marrow derived TLR4（1） The effects of TLR4 on chemotactic activity of homogenate in I/R liver: we performed migration and transmigration assays to observe the effects of homogenate of I/R 3h liver on the migration and transmigration of TLR4^+/+ PMN and TLR4^Mut/MutPMN. The results indicated that the homogenate of ischemic/reperfused liver tissues in homozygous mice with wild TLR4 appeared to be most potent in inducing PMN migration and transmigration, while that in homozygous mice with mutant TLR4 mice induced the least PMN migration and transmigration. Between the two mosaic mice, the homogenate of ischemic/reperfused liver tissues in chimeric mice with wild bone marrow derived TLR4 induced more PMN migration and transmigration than those induced by chimeric mice with mutant bone marrow derived TLR4. In addition, effects of each homogenate on TLR4^+/+PMN were stronger than those on TLR4^Mut/Mut PMN. Results suggested that functional bone marrow derived TLR4 adjust migration and transmigration of PMN not only by increasing the expression of chemotaxis substances, but also functional TLR4 on PMN can enhance sensitization of PMN to chemotaxis substances.（2） The effects of TLR4 on transmigration of PMN induced by MIP-2: when the homogenate was incubated with MIP-2 neutralizing antibody, numbers of PMN that had transmigrated would decrease obviously, especially the numbers of TLR4^+/+ PMN, which indicated that functional TLR4 on PMN could enhance sensitization of PMN chemotaxied by MIP-2.（3） The effects of TLR4 on adhesion and transmigration of PMN mediated by CD11b: When TLR4^+/+PMN and TLR4^Mut/MutPMN stimulated by liver I/R were incubated with CD11b neutralizing antibody, the adhesion and transmigration of PMN decreased, especially of TLR4^+/+PMN, which indicated that functional TLR4 on PMN could enhance reactivity of PMN to CD11b.（4） The effects of TLR4 on PMN accumulation mediated by TNF-α: There would be no change in the transmigration of PMN in the presence or absence of TNF-αneutralizing antibody, which suggests that TNF-αhad no direct chemotaxis activity. However, the transmigration of TLR4^+/+ PMN and TLR4^Mut/Mut PMN would increase to the same extent when PMN was stimulated with TNF-αin advance, which suggested that TLR4 appeared to have no influence on the increase of PMN transmigration caused by TNF-α. RT-PCR identified that stimulation with TNF-αfor 3 h could increase expression of CD11b and MIP-2 of TLR4^+/+PMN and TLR4^Mut/Mut PMN to almost the same extent.（5） RT-PCR identified: that process of I/R could increase more potential in the expression of CD11b and MIP-2 of TLR4^+/+PMN compared with TLR4^Mut/MutPMN, which indicated that TLR4 could up-regulate the expression of above two factors induced by liver I/R injury.In all, in this study, in vivo experiments indicated that functional TLR4 could adjust the infiltration of PMN in stageⅡliver I/R injury by regulating the expression of procytokines, chemokines and adhesion molecules. Furthermore, TLR4 on bone marrow derived cells were mainly involved in expression of cytokines, chemotactic factors and Mac-1, while expression of ICAM-1 was mainly affected by TLR4 on non-bone marrow derived cells. In vitro experiments indicated that functional TLR4 could adjust transmigration, activation, and adherence of PMN by increasing the expression of CD11b and MIP-2, furthermore, functional TLR4 on PMN can enhance the sensitization of PMN to MIP-2.更多还原