【作者】 顾蓓蓓；

【导师】 邹思湘；

【作者基本信息】 南京农业大学 ， 动物医学生物学， 2010， 博士

【摘要】 本实验选用健康SD大鼠,利用LPS诱发试验性大鼠乳腺炎模型,研究多功能营养素视黄醇对大鼠乳腺防御机能的影响和保护作用；并建立原代培养的大鼠乳腺上皮细胞炎症反应模型,进一步在体外研究了视黄醇对大鼠乳腺上皮细胞炎症反应的调节机制。1视黄醇对试验性乳腺炎大鼠的保护作用研究为研究视黄醇对LPS诱发的试验性乳腺炎大鼠的保护作用,40只清洁级SD孕鼠随机分为正常对照组(NC,n=8)、阳性对照组(PC,n=8)和试验组(T,n=24)。自怀孕第十天起,试验组分别灌胃视黄醇(溶解于大豆油)4000 I.U/kg·d(L, n=8)、8000 I.U/kg·d(M, n=8)、16000 I.U/kg·d (H, n=8),对照组灌胃等量的大豆油,连续灌胃十天。产后72 h分别灌注灭菌生理盐水和10μg/侧LPS到大鼠第四对乳腺(两侧)内,12 h处死大鼠,收集血清及乳腺组织样品。LPS灌注乳腺12 h后,乳腺组织中髓过氧化物酶(myeloperoxidase, MPO)、N-乙酰-β-D-氨基葡萄糖苷酶(N-acetyl-β-D-glucosaminidase, NAGase)活性,肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)、白细胞介素-8(interleukin-8, IL-8)均显著升高,血清中MPO、超氧化物歧化酶(superoxide dismutase, SOD)活性、总抗氧化能力(total anti-oxidizing capability, T-AOC)及乳腺组织中白细胞介素-2 (interleukin-2, IL-2)水平显著降低。与阳性对照组相比,灌胃视黄醇显著降低乳腺组织中MPO、NAGase的活性及TNF-α水平,显著提高血清中IL-2水平；低、中剂量视黄醇能降低中性粒细胞(neutrophil,PMN)在乳腺组织中的浸润,提高血清SOD活性及T-AOC,改善因LPS引起的外周血T淋巴细胞CD4+与CD8+比值的失衡。结果证实视黄醇对LPS诱发的试验性乳腺炎大鼠有一定的保护作用。2视黄醇对试验性乳腺炎大鼠炎性细胞因子释放的影响为探讨视黄醇对试验性乳腺炎大鼠炎性细胞因子释放的调节作用机制,72只清洁级SD怀孕大鼠被随机分成对照组(C,n=36)和试验组(T,n=36)。怀孕第十天起,每天灌胃视黄醇8000I.U/kg·d(溶解于大豆油),连续灌胃十天；对照组灌胃等体积的大豆油。产后72 h经乳头管灌注LPS 10μg/侧到第四对乳腺(两侧)内,分别于灌注前(定义为0 h)及灌注后2、4、8、16和24 h(n=6)颈静脉放血处死动物,采集样品。试验结果显示,视黄醇处理显著降低了LPS灌注后乳腺组织中Toll样受体-4(toll-like receptor-4, TLR-4)、核转录因子-κB (nuclear factor-κB, NF-κB) p65 mRNA表达,显著抑制NF-κB与DNA的结合活性,显著降低乳腺组织中(?)(?)NF-α与白细胞介素-1β(interleukin-1β, IL-1β)的释放。结果表明,灌胃视黄醇通过降低TLR-4 mRNA的表达,抑制NF-κB p65 mRNA表达及NF-κB与DNA的结合活性,抑制其下游相关炎症因子TNF-α及IL-1β的过度释放,减轻炎症因子对乳腺组织的损伤。3视黄醇对试验性乳腺炎大鼠中性粒细胞活性的影响为探讨视黄醇对试验性乳腺炎大鼠中性粒细胞活性的影响,72只清洁级SD怀孕大鼠被随机分为对照组(C,n=36)和试验组(T,n=36)。怀孕第十天起,每天灌胃视黄醇8000 I.U/kg·d(溶解于大豆油),连续灌胃十天；对照组灌胃等体积的大豆油。产后72 h经乳头管灌注LPS 10μg/侧到第四对乳腺(两侧)内。分别于灌注前(定义为O h)及灌注后2、4、8、16和24 h(n=6)颈静脉放血处死动物,采集样品。MPO活力及组织病理学观察显示,视黄醇加快了LPS诱发炎症的组织修复进程,降低PMN在组织中的积聚与持续激活,显著降低LPS灌注后IL-8的释放,降低乳腺组织与血清中NAGase活力,促进细胞间粘附分子-1 (intercellular adhesion molecule-1, ICAM-1)的优先表达,降低外周血PMN活性氧的释放。试验结果表明,灌胃视黄醇能调节PMN活性,加快PMN的动态迁徙,降低其活性氧的过度释放,对PMN引起的氧化应激具有一定的保护作用。4大鼠乳腺上皮细胞的优化培养及其炎症反应模型的建立乳腺上皮细胞是乳腺组织的功能细胞,在受到病原微生物刺激时,乳腺上皮细胞也可产生“自主”抵抗。本研究旨在优化大鼠乳腺上皮细胞培养体系,并建立其炎症反应模型,为进一步深入研究动物乳腺防御机制奠定基础。选择妊娠15-18天的健康SD大鼠,无菌采集乳腺组织,分离大鼠乳腺上皮细胞。结果表明通过胶原酶和透明质酸酶联合消化能成功分离大鼠乳腺上皮细胞,并且在添加了胰岛素、转铁蛋白、表皮生长因子、氢化可的松等营养因子的DMEM/F12培养液中生长情况良好。对角蛋白及β-酪蛋白的鉴定结果表明,通过此法分离培养的大鼠乳腺上皮细胞纯度在95%以上,并具备较好的生物学功能。LPS刺激细胞24 h,TNF-α、IL-1p及诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS) mRNA表达均极显著升高,并且LPS刺激浓度在1-10μg/mL范围内呈现剂量依赖效应,由于细胞毒性的作用20μg/mL LPS处理时上述细胞因子的表达有轻微下降,但仍显著高于对照组。10μg/mL LPS刺激显著提高TNF-αIL-1β、一氧化氮(nitric oxide, NO)(?)的释放。结果表明,本试验成功建立了大鼠乳腺上皮细胞原代培养体系及其炎症反应模型。5视黄醇对试验性诱发的大鼠乳腺上皮细胞炎症反应的调节及机制研究为进一步研究视黄醇对原代培养的大鼠乳腺上皮细胞炎症反应的调节及机制,分离并原代培养大鼠乳腺上皮细胞,待细胞铺满整个培养瓶的90%,分为对照组和试验组,试验组于基础培养液中添加1μmol/L视黄醇(溶解于DMSO),对照组于基础培养液中添加等量的DMSO,处理24 h后更换培养液,用10μg/mL的LPS处理细胞,分别于不同时间点收集细胞。LPS能显著上调大鼠乳腺上皮细胞TLR-4蛋白水平及NF-κB与DNA的结合活性,而视黄醇处理能显著降低LPS处理后TLR-4蛋白水平,极显著降低NF-κB与DNA的结合活性。LPS处理大鼠乳腺上皮细胞后引起各个时间点炎性因子TNF-α、IL-1β、中性粒细胞趋化因子-1 (cytokine-induced neutrophil chemoattractant-1, CINC-1)、iNOS、ICAM-1 mRNA表达极显著的升高,视黄醇能显著下调2.4,8,12h TNF-αmRNA表达,2,4,8hIL-1βmRNA表达及8,12 h iNOS、ICAM-1 mRNA表达,而对CINC-1 mRNA表达无显著影响。试验结果表明,视黄醇预处理通过下调TLR-4/NF-κB信号途径减轻LPS介导的大鼠乳腺上皮细胞的炎症反应。综上所述,视黄醇能增强大鼠乳腺防御机能,通过下调TLR-4/NF-κB信号途径,抑制中性粒细胞在组织中持续激活,减轻炎性相关因子造成的乳腺组织损伤。本研究证实,视黄醇作为营养免疫生理调节剂在动物乳腺炎防控中有潜在的应用前景。更多还原

【Abstract】 In this study, effects of retinoid on mammary defense and the protective mechanism of retinoid on mammary gland were first evaluated on experimental mastitis induced by LPS in rats. Optimized primary culture of rat mammary epithelial cells and its inflammatory model was also established to discuss the protective mechanism of retinoid.1 Protective effect of retinoid against LPS-induced mastitis in ratsA LPS induced mastitis model in rats was used to study the protective effect of retinoid. Commencing at gestation day 10, retinoid (dissolved in soy oil) or an equal volume of soy oil was administered to rats daily by gavage until parturition. LPS or pyrogen-free physiological saline was inoculated into the mammary gland 72 h after parturition and the rats were euthanized at 12 h post-infection. Myeloperoxidase (MPO), N-acetyl-β-D-glucosaminidase (NAGase), tumor necrosis factor-alpha (TNF-α), interleukin-8 (IL-8) in mammary tissues and CD4+/CD8+ in peripheral blood were increased and serum MPO, superoxide dismutase (SOD), total anti-oxidizing capability (T-AOC) and interleukin-2 (IL-2) in mammary tissues were decreased 12 h after LPS infusion. Retinoid decreased MPO, NAGase, TNF-αin mammary tissue and increased IL-2 in serum. Four thousand and 8000 I.U/kg·d of retinoid significantly decreased the infiltration of PMN in mammary alveoli, ameliorated the imbalance of CD4+/CD8+ in peripheral blood and increased SOD and T-AOC in serum. These results suggest that retinoid protects against LPS-induced mastitis in a rat model.2 Effect of retinoid on inflammatory cytokines secretion in rats acute experimental mastitisIn this study, the putative protective effect of retinoid on inflammatory cytokines secretion was evaluated in a LPS-induced mastitis model in rats. Commencing at 10 d of gestation, retinoid (dissolved in soy oil) or an equal volume of soy oil were administered daily by gavage to pregnant rats until parturition. LPS or pyrogen-free physiological saline were infused into the mammary gland 72 h after parturition. At pre-infusion (defined as 0 h) and at 2,4,8,16 and 24 h post-infusion, six rats from each group were euthanized. The results showed that retinoid administration decreased toll-like receptor-4 (TLR-4) and nuclear factor-κB (NF-κB) p65 mRNA in mammary tissues, significantly decreased NF-κB and DNA binding activity, decreased the secretion of TNF-αand interleukin-1β(IL-1β) in mammary gland. Overall, the results suggest that retinoid protects the mammary gland through decreasion the activity of TLR-4 and NF-κB and inhibition the inflammtory factors secreting.3 Effect of retinoid on the activity of neutrophils in rats acute experimental mastitisActivated PMN are able to produce large quantities of bactericidal molecules such as reactive oxygen species (ROS) that are associated with tissue damage in models of inflammatory mastitis. In this study, the putative protective effect of retinoid was evaluated in a LPS induced mastitis model in rats. Commencing at 10 d of gestation, retinoid (dissolved in soy oil) or an equal volume of soy oil were administered daily by gavage to pregnant rats until parturition. LPS or pyrogen-free physiological saline were infused into the mammary gland 72 h after parturition. At pre-infusion (defined as 0 h) and at 2,4,8,16 and 24 h post-infusion, six rats from each group were euthanized. Retinoid administration decreased PMN accumulation in mammary alveoli, significantly decreased the level of TNF-a in mammary tissues and IL-8 in serum at the different time points. ROS release was significantly increased after LPS infusion and was reduced by retinoid at 16 h PI. Retinoid reduced NAGase activity in both serum and mammary tissue at 8 h PI. Intercellular adhesion molecule 1 (ICAM-1) mRNA expression reached its peak value earlier in retinoid treated rats than in the control group. Overall, the results suggest that activated PMN play an important role in the pathogenesis of acute mastitis and retinoid administration can adjust PMN activity, may be an effective tool for protecting mammary tissue against PMN-induced oxidative stress during LPS-induced acute mastitis.4 Optimized culture and inflammatory model establishment of rat mammary epithelial cellsIn this study, rat mammary epithelial cells were cultured and the inflammatory model was established. Primary culture of rat mammary epithelial cells (rMEC) were separated through digestion by collagenase and hyaluronidase, cells grown well in DMEM/F12 when supplemented with insulin, transferrin, epidermal growth factor (EGF), hydrocortisone and other nutritional factors. Cells were identified by cytokeratin andβ-casein, the results shown that the cell purity was up to 95%, and had perfect biological function. Stimulation of rMEC with LPS for 24 h elicited a marked increase in mRNA expression for TNF-α, IL-1βand inducible nitric oxide synthase (iNOS), the expression showed a dose-dependent manner from 1 to 10μg of LPS per ml,20μg LPS stimulation showed a slightly decrease in those genes expression due to the cell toxicity effect. Secretion of TNF-α, IL-1βand nitric oxide (NO) was also increased when induced by LPS, and with a maximal level when stimulated with 10μg of LPS per ml. In this study, we successfully established the inflammatory models of primary culture rat mammary epithelial cells which would be important in further investigation.5 Modulation and mechanism of retinoid in LPS-induced inflammation in primary rat mammary epithelial cellsIn order to study the protective mechanism of retinoid on mammary gland, LPS induced inflammatory model in rat mammary epithelial cells was used in this study. Primary culture of rat mammary epithelial cells were separated through digestion by collagenase and hyaluronidase, cells were pretreated with 1μmol/L retinoid before stimulation with 10μg/mL LPS. The result showed that TLR-4 protein expression and NF-κB DNA binding activity was significantly decreased in primary rMEC pretreated with 1μmol/L retinoid at 1 h post LPS stimulation. LPS stimulation significantly increased the TNF-α, IL-1β, cytokine-induced neutrophil chemoattractant (CINC)-1, iNOS and ICAM-1 gene expression, and retinoid treatment could down-regulate TNF-αgene expression at 2,4, 8,12 h, IL-1βat 2,4,8 h and 8,12 h iNOS, ICAM-1 gene expression. These findings demonstrate that direct action by retinoid leads to attenuate LPS-induced inflammatory responses by suppressing TLR-4/NF-κB signaling system, thereby providing a novel explanation for some of underlying effect proposed for retinoid in the protection of mammary tissue during LPS-induced acute mastitis.In conclusion, retinoid may protect the mammary gland by down-regulating TLR-4/NF-κB signaling pathway and inhibiting the over activation of PMN. The results suggest that retinoid can be used as a potential candidate to prevent mastitis.更多还原