分离纯化大鼠肺泡Ⅱ型细胞中水通道蛋白-4的表达

目的 观察经分离纯化的大鼠肺泡Ⅱ型上皮细胞水通道蛋白-4(AQP4)的表达。方法 对大鼠肺泡Ⅱ型上皮细胞进行分离、纯化,经过特殊染色及电镜鉴定,用免疫细胞化学及免疫电镜方法检测水通道蛋白-4在该单细胞上的表达。结果 大鼠肺泡Ⅱ型细胞免疫细胞化学水通道蛋白-4呈强阳性,免疫电镜结果细胞膜上可见高电子密度阳性反应。结论 水通道蛋白-4存在于分离的大鼠肺泡II型细胞膜上,对呼吸系统水转运可能起到重要作用。     

大鼠油酸性急性肺损伤初期水通道蛋白4在肺水代谢的实验研究

目的 测定油酸造成急性肺损伤(ALI)初期损伤程度及肺泡Ⅱ型上皮细胞(ATⅡ)上水通道蛋白4(AQP-4)的表达量以评估病理状态下ATⅡ细胞水通道水转运能力的状况.方法 血气分析、观察肺组织病理学、用重力法测定血管外肺水(EVLW)含量检测早期肺损伤的程度;急性分离纯化大鼠油酸ALI模型的ATⅡ细胞,显微镜及电子显微镜观察形态病理变化:免疫组化了解肺组织AQP-4的表达,采用逆转录-聚合酶链反应(RT-PCR)检测肺泡Ⅱ型上皮细胞AQP-4的m-RNA表达的情况.结果 血气分析、光镜下肺损伤评分及EVLW油酸组严重程度显著高于对照组,显微镜下肺泡Ⅱ型上皮细胞的数量较对照组无明显减少,但电镜下细胞的超微结构改变,板层小体排空,线粒体损伤等改变;肺组织免疫组化观察到AQP-4明显表达增强,肺泡Ⅱ型上皮细胞AQP-4的m-RNA表达增多,AQP-4由胞浆向胞膜转运,胞膜的表达增多(P<0.05).结论 在肺损伤初期就有了肺损伤水肿的病理生理的改变,AQP-4在肺泡Ⅱ型上皮细胞上m-RNA及细胞膜的表达上调.很大程度调节肺泡腔及肺泡上皮细胞的水转运,在钠通道功能下降的情况下发挥了重要的代偿作用.     

急性分离的肺泡Ⅱ型上皮细胞钠通道亚基mRNA表达

目的 研究急性大鼠肺泡Ⅱ型细胞钠通道各亚基的表达.方法 20只健康SD大鼠,对其肺泡Ⅱ型上皮细胞进行分离、纯化,逆转录-聚合酶链反应(RT-PCR)技术半定量检测ENaC三个亚单位α、β、γmRNA水平.结果 急性分离纯化大鼠肺泡Ⅱ型上皮细胞ENaC三个亚单化均有表达,其中α-业基mRNA表达最强,显著高于β-和γ-业基,β-和γ-亚基mRNA表达无显著性差别.结论 大鼠肺泡Ⅱ型细胞钠通道在DNA水平上表达以α-亚基为主,α-亚基在肺水清除中起主要作用.     

ARDS大鼠肺泡Ⅱ型上皮细胞的分离纯化及电镜下特征

目的观察急性呼吸窘迫综合征(ARDS)大鼠肺泡Ⅱ型上皮细胞超微结构的改变。方法建立大鼠油酸性ARDS模型,对其肺泡Ⅱ型上皮细胞进行分离、纯化,电镜鉴定并观察其超微结构的变化。结果电镜下ARDS大鼠Ⅱ型上皮细胞胞浆内板层小体排空明显而致空泡化,细胞分离后IgG免疫黏附方法纯化可使纯度提高15%。结论先分离纯化细胞再进行电镜观察更易观察到Ⅱ型细胞的形态学改变,从板层小体的变化最为突出可观察到肺泡Ⅱ型上皮细胞在ARDS中的重要作用。     

急性呼吸窘迫综合征大鼠肺泡Ⅱ型上皮细胞的超微结构观察

目的 观察急性呼吸窘迫综合征(ARDS)大鼠肺泡Ⅱ型上皮细胞的超微结构。方法 建立大鼠油酸性ARDS模型，对其肺泡Ⅱ型上皮细胞进行分离、纯化，电镜鉴定并观察超微结构的变化。结果 电镜下ARDS大鼠Ⅱ型上皮细胞胞质内板层小体排空明显而致空泡化。结论 分离纯化细胞后更易观察到Ⅱ型细胞的形态学改变，板层小体的变化最为突出提示肺泡Ⅱ型上皮细胞在ARDS中可能起重要作用。     

油酸肺损伤时肺泡Ⅱ型上皮细胞钠水通道的研究

目的测定急性呼吸窘迫综合征（ARDS）状态下肺泡Ⅱ型上皮细胞（ATⅡ）上AOPs的表达量以及全细胞钠电流的大小．以评估病理状态下ATⅡ细胞钠水主动转运能力的状况。方法急性分离纯化油酸ARDS模型大鼠的ATⅡ细胞．用免疫细胞化学、免疫电镜以及Western blotting方法测定AQ1l的表达;用膜片钳技术的全细胞模式测定全细胞钠电流并观察应用特布他林对它的影响。结果ARDS模型组ATⅡ细胞免疫细胞化学AQP1呈阳性,免疫电镜结果细胞膜上可见高电子密度阳性反应。Western blotting分析显示AQP1蛋白条带较对照组明显减弱。全细胞钠电流较对照组明显降低．但对特布他林仍旧敏感。结论ARDS状态下,ATⅡ细胞的钠水主动转运能力明显受损,但并未完全丧失,钠通道激动剂特布他林可以促进钠的转运。ATⅡ细胞钠水通道在肺水的清除中都起着重要的作用。     

大鼠溺死时肺组织水通道蛋白1的变化

目的 观察溺死大鼠肺组织中水通道蛋白1(aquaporin AQF1)的变化。方法 取对照组、实验组大鼠肺组织,用免疫组织化学法和免疫电镜胶体金法,观察及定位肺组织中水通道蛋白1。结果 免疫组化显示溺死时肺间质的毛细血管内皮、支气管上皮及肺泡上皮AQP1的阳性表达,积分光密度值差别有统计学意义。免疫电镜显示肺泡(型上皮细胞AQP1的胶体金数的差别有统计学意义。结论 AQP1在大鼠溺死时肺组织中表达有阳性意义且定位在Ⅰ型上皮细胞膜上。     

急性呼吸窘迫综合征大鼠肺泡Ⅱ型上皮细胞的超微结构观察

目的观察急性呼吸窘迫综合征(ARDS)大鼠肺泡Ⅱ型上皮细胞的超微结构。方法建立大鼠油酸性ARDS模型,对其肺泡Ⅱ型上皮细胞进行分离、纯化,电镜鉴定并观察超微结构的变化。结果电镜下ARDS大鼠Ⅱ型上皮细胞胞质内板层小体排空明显而致空泡化。结论分离纯化细胞后更易观察到Ⅱ型细胞的形态学改变,板层小体的变化最为突出提示肺泡Ⅱ型上皮细胞在ARDS中可能起重要作用。     

新生小鼠肺泡Ⅱ型上皮细胞的分离方法

目的提取新生小鼠的原代肺泡Ⅱ型上皮细胞(AECⅡ),并进行培养和鉴定。方法应用酶消化和免疫磁珠分选方法分离并纯化C57B/L6新生小鼠AECⅡ。根据AECⅡ特异的细胞形态,利用透射电镜进行鉴定。正置荧光显微镜观察AECⅡ特异性肺泡表面活性蛋白-C前体(proSP-C)及表面活性蛋白-B (SP-B)的表达。免疫印记技术检测新生小鼠AECⅡ中特异性肺泡表面活性蛋白-A (SP-A)及proSP-C的表达。结果每只新生小鼠可获得(2±0.5)×105AECⅡ,纯度>90%。电镜下可见丰富的AECⅡ及其特异结构板层小体。正置免疫荧光显微镜观测AECⅡ中proSP-C和SP-B蛋白的表达,免疫印迹结果显示细胞内SP-A和proSP-C蛋白表达呈阳性。结论利用酶消化和免疫磁珠分选的方法可成功分离出高产量、高纯度的新生小鼠AECⅡ。     

肺泡Ⅱ型上皮细胞染色体脆性位点基因WWOX与特发性肺间质纤维化的关系

目的研究肺间质纤维化模型大鼠肺泡Ⅱ型上皮细胞染色体脆性位点基因WWOX表达的改变。方法 SD雄性大鼠随机分为假手术组、模型组及川芎嗪预防用药组,每组6只,分别于给药7、14、28 d后提取肺泡Ⅱ型上皮细胞体外培养,免疫组织化学法检测WWOX蛋白表达。结果与假手术组比较,注射博莱霉素7 d后模型组大鼠肺泡Ⅱ型上皮细胞染色体脆性位点基因WWOX蛋白表达无显著变化。用药14、28 d后,WWOX蛋白表达则明显下降。而川芎嗪预防用药组WWOX蛋白表达比模型组有显著上调。结论肺间质纤维化过程中可能存在染色体脆性位点基因WWOX的改变,川芎嗪可以在一定程度上抑制这种改变而发挥防治作用。     

Aquaporin and Na+ transport of alveolar type II cells in rats with oleic acid-induced acute respiratory distress syndrome]

OBJECTIVE: To evaluate the capacity of active water and sodium transport of alveolar type II (AT II cells in rats with acute respiratory distress syndrome (ARDS). METHODS: AT II cells were isolated and purified from rats with ARDS, and the distribution of aquaporin-1 (AQP-1) on the cells was observed by immunocytochemistry, immunoelectron microscopy and Western blotting. The sodium currents were detected by patch-clamp technique in whole-cell recording mode. RESULTS: Immunocytochemistry showed staining for AQP-1 in rat AT II cells, and immunoelectron microscope demonstrated the presence of AQP-1, which was decreased as shown by Western blotting in comparison with the control group. The whole-cell currents were also decreased, but remained sensitive to terbutalin treatment. CONCLUSIONS: The capacity of active water and sodium transport is damaged, but not totally lost in AT II cells of ARDS rats. Terbutalin can increase the currents even if in pathological conditions. AQP-1 and sodium transport of AT II cells play an important role in pulmonary liquid clearance.     

Decreased expression of AQP1 and AQP5 in acute injured lungs in rats

Objective To determine if aquaporin1 (AQP1) and aquaporin5 (AQP5) are expressed in the alveolar capillary membrane in rats. Moreover, to investigate the alteration of AQP1 and AQP5 in acute injured lungs. Methods The distribution of AQP1 and AQP5 in alveolar capillary membrane were investigated by immunohistochemistry and immunoelectron microscopy with affinity- purified antibodies to human AQP1 and AQP5. To study the possibility that alveolar capillary membrane AQP1 and AQP5 undergo altered regulation, we established a rat model using alveolar instillation of lipopolysaccharide (LPS). Results Immunolabelling showed AQP1 was stained primarily in the microvascular endothelia of normal lungs, while AQP5 was expressed in type Ⅰ pneumocytes. Immunohistochemical analysis showed a significant decrease in the expression of AQP1 and AQP5 in injured lungs at 4h-48h after LPS instillation. AQP1 protein was resumed partly at 24h after LPS instillation and steroid administration, whereas AQP5 was unchanged. Conclusion The decreased expressions of AQP1 and AQP5 in injured lungs suggest that both of them may play a role in abnormal fluid transportation.     

Ultrastructure of type II alveolar epithelial cells in rats with acute respiratory distress syndrome.

OBJECTIVE: To observe the ultrastructure of type II alveolar epithelium cells of rats with experimental acute respiratory distress syndrome (ARDS). METHODS: Rat models of ARDS were established via oleic acid injection, and the type II alveolar epithelium was isolated and purified to observe the changes in the ultrastructure of the cells under transmission electron microscope. RESULTS: Observation under transmission electron microscope revealed evidently decreased lamellar bodies in type II alveolar epithelial cells leading to vacuolation in rats with ARDS. CONCLUSION: After proper isolation and purification, the morphological changes in type II alveolar epithelial cells can be more easily observed, and the changes in the lamellar bodies suggest that type II cells play an important role in ARDS.     

Primary Culture of Alveolar Epithelial Type Ⅱ Cells and Its Bionomic Study

To establish a better method of primary culture for alveolar epithelial type Ⅱ cells (AEC Ⅱ) and to study its bionomics, alveolar epithelial type Ⅱ cells were isolated by digestion with tryp- sin and collagenase, which were then purified by plated into culture flask coated with rat immu- noglobulin G. The purified AEC Ⅱ were identified by alkaline phosphatase staining, electron mi- croscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expres- sion and transfection characteristics were compared with those of A549 cell line. The results showed that AEC Ⅱ could be isolated by digestion with trysin and collagenase and purified by adhesive pu- rification by using IgG, with a yield of about 2–3×107, and a purity of about 75%–84 %. Cells could be quickly identified with AKP staining. AECⅡ were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AECⅡ, and AKP staining is simple in cell identification. AECⅡ can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.     

大鼠肺泡Ⅱ型上皮细胞的体外培养及内毒素攻击模型的建立

目的探讨大鼠肺泡Ⅱ型上皮细胞（ATⅡ）体外培养的方法及建立脂多糖（LPS）攻击ATⅡ的模型模拟内毒素性肺损伤。方法分离、纯化、鉴定得到大鼠ATⅡ,分别用01、1、10μg／ml的LPS刺激ATⅡ,并利用细胞增殖／毒性检测试剂在刺激2、4、6h后分别检测ATⅡ的抑制率,从而探索内毒素性肺损伤细胞模型中最适的LPS浓度和刺激时间。结果用碱性磷酸酶染色及电镜观察进行细胞纯度判断,未用差异贴壁去除成纤维细胞和IgG免疫黏附去除巨噬细胞纯化前,纯度达（70．4±4．8）％,纯化后可提高至（90．2±3．4）％;而用LPS干预ATⅡ,ATⅡ出现明显的细胞抑制。其中刺激浓度为1μg／ml时与其他3个实验组相比,细胞毒性反应最大（P〈0．01）,且在各个浓度的不同刺激时间里,4h时,细胞毒性反应最大（P〈0．01）。结论细胞分离后采用差异贴壁法和免疫黏附法进行纯化可使细胞纯度提高18％左右l细胞培养时接种密度达到1X107／ml以上有利于细胞的贴壁和生长,而用1μg／ml LPS刺激体外培养的大鼠ATⅡ4h能建立较为合理的内毒素性肺损伤细胞模型。     

Polymorphism of apolipoprotein A5 is a risk factor for cerebral infarction in type 2 diabetes

Summary To establish a better method of primary culture for alveolar epithelial type II cells (AEC II) and to study its bionomics, alveolar epithelial type II cells were isolated by digestion with trypsin and collagenase, which were then purified by plated into culture flask coated with rat immunoglobulin G. The purified AEC II were identified by alkaline phosphatase staining, electron microscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expression and transfection characteristics were compared with those of A549 cell line. The results showed that AEC II could be isolated by digestion with trysin and collagenase and purified by adhesive purification by using IgG, with a yield of about 2–3 × 10⁷, and a purity of about 75%–84%. Cells could be quickly identified with AKP staining. AEC II were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC II, and AKP staining is simple in cell identification. AEC II can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression. This project was supported by grants from the National Natural Sciences Foundation of China (No. 30500224 and No. 30400193).     

急性肺损伤大鼠呼吸膜AQP1和AQP5的表达

目的确定水通道蛋白AQP1及AQP5是否在大鼠肺泡毛细血管膜(呼吸膜)表达,进而研究急性肺损伤(ALI)大鼠AQP1和AQP5的表达调节及激素干预的作用。方法采用亲和的抗人AQP1和AQP5抗体,应用免疫组化及免疫电镜的方法研究AQP1及AQP5在呼吸膜的分布。选用肺泡内灌注脂多糖(LPS)制作大鼠ALI动物模型,研究ALI时呼吸膜AQP1及AQP5的变化。结果免疫染色显示AQP1主要表达于正常肺组织的微血管内皮,而AQP5主要表达于肺泡I型上皮细胞。免疫组化分析进一步表明LPS灌注后4h~48h AQP1及AQP5在呼吸膜的表达均下降;AQP1蛋白于LPS灌注后24h及激素干预后有部分恢复(P0.05),而AQP5无这种恢复现象。结论 ALI时AQP1及AQP5在呼吸膜的表达减少,提示ALI时AQP1和AQP5的下降表达可能与其液体转运的异常有关。     

Temporal Expression of Notch in Preterm Rat Lungs Exposed to Hyperoxia

Notch signaling is essential in tissue and organdevelopment. Signals transmitted through theNotch receptor, in combination with other factorsinfluence cell differentiation, proliferation and ap optotic events at all stages of development and re habilitation or remolding of tissues[1]. Previous re searches revealed that Notch receptor/ligand per sistently exists over the whole period of fetal lungdevelopment[2], but it is still unknown how theywork during the chronic lun…