JNK1与TIPE2抑制剂改善感染后膈肌无力的分子机制

Sepsis is one of the crucial factors for diaphragmatic weakness, but little has been elucidated for this aspect, not to say the molecular mechanism. Prelimanary experiments indicated that PKR and its upstream stimulator MEK3/6 were significantly increased after exposing the mice to LPS treatment. This changing module is consistent with that of caspase pathway activation. We will establish the infection models for both murine diaphragm and skeletal muscle cells, apply Western Blot, enzyme acivity detection, flow cytometry and force generation measurement to explore the relashionship between the decrease of diaphragmatic force generation and the signal molecules such as caspases, TIPE2, PCNP and especially JNK1. Furthermore chemical and genetic inhibitors for JNK1 will be tested in curing diaphragmatic dysfunction. Because JNK1 is so crictical to cellular growth, the silencing of JNK1 with siRNA will cuase the growth stop at maturation of C2C12 cells, delaying the study to follow. To overcome this difficulty, we designed a new strategy of gene silencing at cellular level and knockout in vivo, which placed the 4 stop codon sequences in between 2 Loxps in front of dominant negative(DN) construct of JNK1. The expression of Cre protein by tamoxifen will delete the loxps, then remove the inhibition of DN by stop codons and facilitate the expression of DN-JNK1. The time and site dependent control of DN-JNK1 expression surmounted the problem of JNK1-siRNA and opened a new path to investigate the key genes in muscle cells and in vivo. It was anticipated that the mechanism of diaphragmatic weakness and the stratagies to improve diaphragmatic force generation would be identified after the relationship of JNK1, PCNP and TIPE2 with the malfunction of diaphragmatic cells was determined in sepsis.

感染是膈肌无力的主因之一，但认识匮乏，分子机制鲜有研究。预实验表明内毒素处理小鼠后膈肌活性PKR和上游MAPK激活剂MEK3/6显著增加，而这种变化方式与Caspase转导通路的激活一致。拟建立小鼠膈肌和肌细胞感染模型，采用Western印迹，酶活性测定，流式细胞检测和膈肌肌力测量等方法，探讨感染致膈肌肌力下降与Caspase、TIPE2、JNK等分子的关系；运用JNK化学和基因抑制剂进一步确认其在肌肉功能障碍中的作用。JNK与细胞生长有关，直接用siRNA沉默后肌细胞无法分化成熟，影响进一步研究。拟培育新一代JNK转基因小鼠，将终止密码、Cｒｅ-Lｏｘｐ与负显性基因有机结合，定时定点控制肌细胞DN基因表达, 克服RNAi之不足，开创肌细胞模型中重要基因功能研究的新路径。预期找到感染造成的膈肌细胞功能障碍与JNK及TIPE2通路的关系，从分子水平探讨感染后膈肌肌力下降的机理和治策。

感染是膈肌无力的主要原因之一，但长期以来对该问题的认识较为匮乏，分子机制也鲜有研究。本课题组的实验表明：内毒素处理小鼠后膈肌活性PKR和上游MAPK激活剂MEK3/6显著增加，而这种变化方式与Caspase转导通路的激活一致。我们假设：ＪＮＫ１通路能够通过影响Caspase造成膈肌收缩蛋白的降解，进而引起膈肌无力，出现呼吸功能障碍。为验证该假设，我们建立了小鼠膈肌和肌细胞感染模型，采用Western印迹，酶活性测定，流式细胞检测和膈肌肌力测量等方法，探讨了感染致膈肌肌力下降与Caspase、TIPE2、JNK等分子的关系；并运用JNK化学和基因抑制剂进一步确认其在肌肉功能障碍中的作用。研究结果表明，JNK与细胞生长有关，直接用siRNA沉默后肌细胞无法分化成熟，影响进一步研究。为此我们又进一步培育成功了新一代JNK转基因小鼠，将终止密码、Cｒｅ-Lｏｘｐ与负显性基因有机结合，定时定点控制肌细胞DN基因表达, 克服RNAi之不足，开创肌细胞模型中重要基因功能研究的新路径。多种方法证实，感染造成的膈肌细胞功能障碍与JNK及TIPE2通路具有密切的关系，本研究为临床改善膈肌无力，特别是感染引起的膈肌功能下降找到了潜在的分子靶点，并从分子水平上阐明了感染后膈肌肌力下降的机理和治疗对策，下一步我们将把该成果运用于更大规模的动物实验，　并在大动物如狗、猪、猴等体内进行验证，为最终把ＪＮＫ１抑制剂和ＴＩＰＥ２作为药品在临床使用奠定基础。