第一部分 大鼠肺Ⅱ型上皮细胞的分离培养 目的：探讨一种简单分离成年大鼠肺Ⅱ型上皮细胞的方法，建立大鼠肺Ⅱ型上皮细胞(alveoli type Ⅱ epithelial cells，AT-Ⅱ)原代培养模型，为体外研究肺部疾病提供实验手段。 方法：大鼠麻醉抗凝后，0.9%生理盐水灌洗气道和肺循环，分别采用0.1%、0.25%胰蛋白酶(简称“胰酶”)和0.02%EDTA、弹性蛋白酶消化液气道灌注消化150-250gSD大鼠肺组织，并用IgG包被的培养皿粘附纯化，碱性磷酸酶染色和电镜鉴定AT-Ⅱ。 结果：0.1%胰酶消化提取的AT-Ⅱ产量为2.2±0.5×107/鼠，纯度为94+1.2%，活力为97±0.4%； 0.25%胰酶消化提取的AT-Ⅱ产量为2.4±0.6×107/鼠，纯度为88±2.5%，活力为90±1.6%；弹性蛋白酶消化提取的AT-Ⅱ产量为2.5±0.5×107/鼠，纯度为95±1.5%，活力为98±0.5%。三种方法所提取的细胞产量没有明显差异，但0.25%胰酶所提的AT-Ⅱ活力和纯度明显低于0.01%胰酶和弹性蛋白酶。 结论：0.1%胰酶和0.02%EDTA混合消化加IgG免疫粘附纯化可以简单有效的分离AT-Ⅱ，有利于原代培养AT-Ⅱ技术的推广。弹性蛋白酶消化食一种理想的方法，但价格昂贵。 关键词：上皮细胞；原代培养；胰蛋白酶；弹性蛋白酶 第二部分 谷氨酰胺通过提高谷胱甘肽水平抑制内毒素诱导的大鼠肺泡Ⅱ型上皮细胞炎性因子释放 目的：肺泡Ⅱ型上皮细胞(AT-Ⅱ)细胞在受到内毒素(LPS)、氧化刺激时，可以分泌炎症介质，从而在急性呼吸窘迫综合症(ARDS)的发生发展中起到重要作用。还原型/氧化型谷胱甘肽(GSH/GSSG)是AT-Ⅱ内完美的抗氧化系统，可以通过两者的转换，影响细胞的炎症反应、应激反应、细胞的增殖和凋亡等。谷氨酰胺(Gln)是GSH的前体物质，已有研究证实，在炎症刺激时，Gln可以提高GSH的水平从而调节细胞的炎症反应。然而，对于Gln对LPS诱导的大鼠肺泡上皮细胞的GSH水平的影响及其对肿瘤坏死因子-α(TNF-α)、白介素(IL)-6分泌的调节以及其作用机制仍未见报道。 方法 原代培养大鼠肺泡上皮细胞，分为0、0.5、2.0、10.0mMGln及0、10.0mMGln共六个组，作用2、8、16、24.0小时后，前四组1μ g/mlLPS刺激24小时；另分为六组，前四组1μg/mlLPS刺激4小时后，不同浓度Gln作用20小时.Tietze还原酶法测定细胞内GSH的表达；确定LPS处理前8小时是Gln促进GSH合成的最佳时间点。分为 0、0.5、2.0、10.0mMGln 及 0、10.0mMGln 共六个组，作用 8小时后，前四组1μg/mlLPS刺激24小时，测定细胞内GSH浓度、活性氧(ROS)及TNF- α、IL-6水平；同时10mM Gln与GSH合成限速酶γ-谷氨酰半胱氨酸合成酶(γ-GCS)抑制剂丁硫氨酸亚砜胺(BSO)相互作用，分为 100μ MBSO、BSO(1、10、100μM)+10.0mM Gln及0、10.0mMGln六个组，作用8小时后，1μg/ml LPS刺激24小时。测定细胞内GSH浓度、ROS水平及TNF-α、IL-6水平。分为对照、10mMGln、100μMBSO， BSO+Gln及空白对照五个组，作用8小时后，前四组1μg/mlLPS刺激24小时。电泳迁移实验(EMSA)法测定细胞内NF-κB活性； 结果 补充谷氨酰胺可以提高LPS诱导大鼠肺泡Ⅱ型上皮细胞GSH的浓度，降低细胞内ROS水平，降低TNF-α和IL-6的水平，其作用随浓度的增加而增加，在10mM浓度最为显著，在LPS处理前8小时是Gln促进GSH合成的最佳时间点；谷氨酰胺可以抑制LPS诱导大鼠肺泡上皮细胞NF-κB活性；谷氨酰胺的作用可以被10μM浓度以上的BSO阻断。 结论 Gln对LPS刺激的AT-Ⅱ细胞的炎症调控和保护作用可能是通过促进GSH的合成而介导的. 关键词：内毒素；谷氨酰胺；谷胱甘肽；肿瘤坏死因子；白介素 第三部分 谷氨酰胺通过提高谷胱甘肽水平保护内毒素诱导的大鼠肺损伤 目的：谷氨酰胺(glutamine，Gln)是一种人体的条件必需氨基酸，也是人体内最丰富的游离氨基酸。谷氨酰胺作为一种免疫营养剂已为临床和动物实验所证实。在脓毒症时，Gin可以保护肠粘膜屏障、防止肺损伤和肺组织代谢异常的出现，降低脓毒症患者或大鼠的死亡率。 Gln同时也可以作为抗氧化剂谷胱甘肽(GSH)的前体物质之一，调节组织或细胞的氧化还原状态，影响组织或细胞的免疫反应。本实验用谷胱甘肽合成抑制剂丁硫氨酸亚砜胺(BSO)与谷氨酰胺相互作用，研究了谷氨酰胺对脓毒症大鼠肺损伤的保护作用，以及GSH在其中可能的介导作用。 方法：健康Sprague-Dawley成年大鼠(200-250g)随机分成5组，各为生理盐水对照组、Gln (2mmol/㎏)组、LPS(1 ㎎/ml/㎏)组、LPS+Gln组和LPS+Gln+BSO (0.75g/㎏)组。LPS通过气道内滴入，Gln通过尾静脉注射，BSO通过腹腔内注射给予。处理后2、18小时，取外周血流式细胞仪检测中性粒细胞CD11b，用生理盐水进行左肺灌洗，灌洗液(BALF)计数细胞总数，然后离心后上清测定总蛋白水平，细胞沉渣Wriht-Giemsa染色作中性粒细胞分类记数，取肺组织测IL-8、GSH和髓过氧化物酶(MPO)，取肺组织作病理学检查，并进行病理分级。 结果：Gln可以降低内毒素刺激的大鼠外周血中性粒细胞CD11b表达，在处理2小时后差异明显，但在处理18小时后各组间没有明显差异；Gln可以提高内毒素刺激的大鼠肺组织GSH水平，降低MPO水平，并减少IL-8的分泌；Gln可以保护内毒素刺激的大鼠气道屏障，减少炎性细胞的分泌；Gln可以减轻内毒素刺激的大鼠肺组织病理损害；GSH合成阻断剂可以阻断Gln的作用 结论：Gln对LPS刺激的大鼠肺组织的炎症调控和保护作用可能是通过促进GSH的合成而介导的. 关键词：内毒素；谷氨酰胺；谷胱甘肽；白介素；肺损伤；髓过氧化物酶

 PartⅠ Isolation of type Ⅱ epithelial cells from rat lungs with different enzymes Objective : Compare the yields, viability and purity of alveoli type Ⅱ (AT-Ⅱ) epithelial cells from rat lungs digested by different concentration of trypsin and elastase. To establish an effective primary culture model of AT-Ⅱ for studying the pulmonary disease in vitro. Methods : After anesthesia and anti-coagulation by ketamine 100㎎/㎏ and heparin 800u, the trachea and pulmonary circulation vessels were washed by 0.9% NS. Then digesting enzymes of 0.1%, 0.25% trypsin added with 0.02% ethylenediamine tetraacetic acid (EDTA) and elastase were infused into airway of Sprague-Dawley (SD) rat lung respectively. The lung was put into water-bath box for 20 minutes at 37 degrees. After the lung was cut into pieces, filtered with strainer meshes and centrifuged, the cell suspension was incubated into rat IgG-coated dish for purification. Alkaline phosphatase (AKP) staning and electron microscope were used to identify AT-Ⅱ. Results: The yield of AT-Ⅱ of 0.1% typsin was 2.2±0.5×10⁷/rat, purity was 94±1.2% and viability was 97 ±0.4%; The yield of AT-Ⅱ of 0.25% typsin was 2.4±0.6×10⁷/rat, purity was 88±2.5% and viability was 90± 1.6%; The yield of AT-Ⅱ of elatase was 2.5±0.5×10⁷/rat, purity was 95±1.5% and vialility was 95±1.5%. The three methods have no significant difference for yields, while the viability and purity of AT-Ⅱ digested by 0.25% trypsin were lower than 0.1% trypsin and elastase. Conclusion: 0.1% trypsin added with 0.02% EDTA and purified by IgG immune adherence which can isolate AT-Ⅱ effectively and help to spread the method of primary culture of AT-Ⅱ. The elatase digestion also is a possible way, but its too expensive and hard to obtain. Key words: epithelial cells, primary culture, trypsin, elastase Part Ⅱ Glutamine prevents inflammatory factors release of LPS-induced alveolar type Ⅱ epithelial cells through enhancement of glutathione synthesis Objectives: To investigated the role of glutathione (GSH) synthesis in the regulation on nuclear factor (NF)- κB activity and tumor necrosis factor-alpha (TNF-α) release by glutamine (Gln) in lipopolysaccharide (LPS)-stimulated from rat lungs. Methods: Primary cultured AT-Ⅱ cells divided into 0、0.5、2.0、10.0mM and 0、10.0mM Gln six groups. 2, 8, 16, 24 hours later, the first four groups were treated with 1 μg/mlLPS for another 24 hours. In another set, the first four groups were treated with1μg/mlLPS for 4 hours then the cells were added with various concentration of Gln for 20 hours. That aimed to make sure that 8 hours before LPS stimulation was the best time point for Gln to enhance the GSH synthesis. Then, after treated with various doses of Gln at the 8 hours time point before LPS stimulation, the GSH and ROS levels in cells and TNF-α and IL-6 levels in supernatant were measured. Various doses of L-buthionine-(S, R)-sulfoximine (BSO), an inhibitor of GSH synthesis, were added with 10mM Gln. Then the cells were stimulated with 1 μg/ml LPS for 24 hours. The cells were obtained for GSH and ROS measurement. TNF-α level in the supernatant was determined by enzyme-linked immunosorbent assay (ELISA). For NF-κB activity measurement, the cells were divided into LPS、 10mMGln+LPS、100μMBSO+LPS、BSO+Gln+LPS and control five groups. The activity was assessed by electrophoretic mobility shift assay (EMSA). Results: 8 hours before LPS exposure was the best time point for Gln's enhancing GSH synthesis. LPS could decrease the GSH level, increase ROS level, NF- κB activation and TNF-α, IL-6 release in AT-Ⅱ cells significantly. Supplementation of Gln could increase the GSH and ROS levels and attenuate the release of TNF-α and IL-6 in LPS-stimulated AT-Ⅱ cells in a dose-dependant manner. And NF- κB activation also could be prevented by Gln. BSO could block the effect of Gln. Conclusions: As a precursor of GSH, glutamine could prevent the NF-κ B activation and attenuate the release of TNF-α and IL-6 in LPS-stimulated AT-Ⅱ cells and the effect may be mediated via GSH synthesis. Key words: glutamine; glutathione; NF-κB; tumor necrosis factor; lipopolysaccharide Part Ⅲ Glutamine attenuats acute lung injury through enhancement of glutathione synthesis Objectives: It has been reported that glutamine (Gln) can attenuate the acute lung injury (ALI) following sepsis. Gln also is thought as a precursor of glutathione (GSH) synthesis. By using a GSH synthesis blocker, L-buthionine-(S,R)-sulfoximine (BSO), We investigated the role of GSH synthesis in the protective effect of Gln on ALI. Methods: In this study, we used an ALI model by lipopolysaccharide (LPS) (1 ㎎/ml/㎏) intratracheally injection. Gln (0.75g/㎏, intraveinous) and BSO (2mmol/㎏, intraperitoneal) were administrated simultaneously. At 2 and 18 hours time point after the disposal the rats were sacrificed by right ventricular puncture and bronchoalveolar lavage (BAL) was done. Low right lung was excised for histological examination. Total protein concentration, total cell and neutrophils count in the BAL fluid (BALF) were detected. CD11b expression in blood was determined via flow cytometry. We also analyzed the MPO activity, GSH and interleukin (IL)-8 levels in lung tissues. Results: Gln supplement reduced the total protein concentration, total cells and neutrophils count in BALF after LPS challenge. Gln could enhanced GSH synthesis and attenuated the IL-8 release and MPO activity in lung tissues. Gln also could decrease the CD11b expression and prevented lung histological changes. BSO could abolish the Gln' s effect and then abrogate its protection on acute lung injury. Conclusions: These results indicate that Gln could prevent neutrophils' recruitment and infiltration, protect alveolar barrier and attenuate inflammatory injury during sepsis. And this effect may relate to the enhancement of GSH synthesis. Key words: glutamine; acute lung injuiy; glutathione; lipopolysaccharide; bronchoalveolar lavage