柴朴汤对哮喘小鼠支气管上皮细胞炎症因子及HMGB1/TLR4/NF-κB的影响

目的探讨柴朴汤对哮喘小鼠支气管上皮细胞高迁移率族蛋白B1(HMGB1)、Toll样受体4(TLR4)、核因子-κB(NF-κB)及下游炎症介质单核细胞趋化蛋白-1(MCP-1)、白细胞介素-6(IL-6)的影响。方法将小鼠支气管上皮细胞分为对照组(Con组,空白血清培养)、Model组(哮喘小鼠血清培养)和Chaipu组(柴朴汤药血清培养)。采用qRT-PCR和Western blotting检测小鼠支气管上皮细胞中HMGB1、TLR4、NF-κB、MCP-1、IL-6的mRNA及蛋白表达情况,ELISA法测定细胞培养液中MCP-1和IL-6的水平。结果与Con组相比,Model组小鼠支气管上皮细胞HMGB1、TLR4、NF-κB、MCP-1、IL-6表达均明显升高(P<0.05);Model组细胞培养液中MCP-1、IL-6也显著升高(P<0.05)。与Model组相比,Chaipu组小鼠支气管上皮细胞HMGB1、TLR4、MCP-1、IL-6 mRNA和蛋白表达降低,NF-κB m RNA表达亦降低(P<0.05);Chaipu组细胞培养液中MCP-1、IL-6也显著降低(P<0.05)。结论柴朴汤可下调哮喘血清诱导的炎症因子的表达,可能与其抑制HMGB1/TLR4/NF-κB炎症通路有关。

无赖氨酸激酶4在小鼠气管上皮细胞液体转运中的作用

该研究探讨了无赖氨酸激酶4(with no lysine kinase 4, WNK4)对于小鼠气管上皮细胞液体转运中的调节作用。在原代培养的小鼠气管上皮细胞中,应用siRNA特异性沉默WNK4基因,半定量PCR和Western blot实验验证沉默效率;随后应用尤斯灌流装置和Western blot实验记录该激酶的低表达对小鼠气管上皮细胞的短路电流及钠离子通道α-亚基蛋白表达水平的影响。半定量PCR和Western blot结果显示,该研究选用的siRNA序列可以沉默WNK4的表达。尤斯灌流和Western blot结果显示,沉默该激酶后,小鼠气管上皮细胞的阿米洛利敏感性电流和钠离子通道α-亚基蛋白表达明显增加。该研究表明,降低WNK4基因表达能增加小鼠气管上皮细胞的上皮钠离子通道α-亚基蛋白表达,促进钠离子转运,此过程可能参与相关水肿性肺疾患的修复。

TEMPORAL EXPRESSION OF NOTCH RECEPTORS DURING LUNG DEVELOPMENT IN RAT

Objective To investigate the temporal expression of Notch receptors in developing lungs of rats and to explore the regulating role of Notch in lung development. Methods We studied the expression of Notch1,2,3 isforms in embryonic days 18,20,21 and postnatal days 1,4,7,14, 21 rat lungs. Six rats of each group were used to assess lung histologic changes by HE staining and expression of Notch in lungs by immunohistochemistry. Total RNA was extracted by Trizol reagent from the frozen lung tissues. mRNA levels of Notch were measured by reverse transcription polymerase chain reaction (RT-PCR). Results It is showed that Notch_ 1-3 mainly localized in the airway surface epithelium、alveolar epithelium during the psdueoglandular stage, and reached the peaks at canalicular period. The expression patterns of Notch_ 1-3 were changed with the fetal age. Conclusion These results support multiple roles for Notch1,2,and 3 receptor activation during lung development, probably not only modulating the process of branching morphogenesis but also involved in determining the cell differentiation fate in fetal alveolar epithelium.

流感病毒对囊性纤维化跨膜转导调节因子影响的研究

目的 探讨原代培养的小鼠气管单层细胞培养方法及流感病毒对原代培养的小鼠气管单层细胞囊性纤维化跨膜转导调节因子的影响，明确流感病毒破坏呼吸系统水盐转运平衡的影响机制。方法 应用倒置显微镜记录细胞形态，应用电阻计测量跨上皮电阻以评估细胞间的紧密连接状态，尤斯灌流装置记录原代培养的小鼠气管单层细胞短路电流。结果 200倍焦距的光镜下培养的原代小鼠气管上皮细胞形态呈紧密连接的铺路石状，跨上皮电阻在培养6 d后大于1 000 Ω；在细胞膜完整的条件下，流感病毒能降低囊性纤维化跨膜转导调节因子短路电流至（52.77±10.30）%，在基底膜通透的细胞单层中，流感病毒能降低囊性纤维化跨膜转导调节因子短路电流至（41.50±1.09）%，而囊性纤维跨膜转导调节因子在调节呼吸道水盐转运平衡中有重要作用。结论 气液交界面模式培养小鼠气管上皮细胞非常高效，并且接近小鼠正常的生理状态；流感病毒可能通过抑制囊性纤维化跨膜转导调节因子的功能，阻滞阴离子分泌，进而诱发慢性阻塞性肺病以及哮喘等肺部相关性疾病。