人Ism1表达载体的构建及其在293T细胞的表达

目的:构建人Ism1基因真核表达载体,并验证其在真核细胞中的表达,为相关研究奠定基础。方法:采用高保真DNA聚合酶和GC缓冲液从A549细胞扩增获取人Ism1编码序列全长,经酶切、连接、转化后,挑取阳性克隆进行菌液PCR、双酶切,及测序鉴定;用构建成功的Ism1表达载体转染293T细胞,用实时定量RT-PCR检测Ism1表达水平。结果:构建的Ism1载体经菌液PCR,双酶切鉴定后,测序结果完全正确,载体转入293T细胞后,Ism1表达量显著上升。结论:成功构建能在真核细胞中表达的人Ism1基因载体,将为相关研究提供便利。

Ism1 deficiency in mice exacerbates bleomycin-induced pulmonary fibrosis with enhanced cellular senescence and delayed fibrosis resolution

Idiopathic pulmonary fibrosis(IPF)is a progressive fibrosing interstitial lung disease with high morbidity and mortality but unclear etiology and incomplete pathophysiological understandings,making the discovery of effective therapeutics arduous.Currently,two drugs,nintedanib and pirfenidone,are available for IPF treatment which can slow down the fibrotic scarring in the lung but are unable to provide disease resolution.Hence,further revelation of the molecular mechanisms of IPF is critical for the development of novel therapeutics.Isthmin-1(ISM1)is a secreted anti-inflammatory protein highly expressed in the mouse and human lung.Ism1^(-/-)mice presented spontaneous and progressive lung emphysema,as well as heightened acute lung injury(ALI)upon lipopolysaccharide(LPS)treatment with an accompanied increase of post-LPS-ALI pulmonary fibrosis.ISM1 is important for lung homeostasis with airway-delivered recombinant ISM1(rISM1)suppressing cigarette smoke-induced emphysema,LPS-ALI,and house-dust mites(HDM)-induced asthma-like symptoms in mice.However,the role of ISM1 in pulmonary fibrosis is yet to be clearly understood.In this work,we show that Ism1^(-/-)mice presented heightened bleomycin-induced pulmonary fibrosis(BIPF),with enhanced immune cell infiltration,myofibroblast accumulation,and collagen deposition.ISM1 deficiency also led to increased cellular senescence in mouse lungs,isolated primary alveolar type II epithelial cells,and primary lung fibroblasts upon bleomycin treatment.Ism1^(-/-)mice also showed delayed resolution of pulmonary fibrosis with reduced lipofibroblasts and downregulation of lipid synthesis-related genes.These results are in congruence with the RNA-seq data which demonstrated gene expression alterations in Ism1^(-/-)lung that are linked to predisposition to pulmonary fibrosis and dysregulation of lipid metabolism pathways.Gene expression analyses showed that Ism1 is similarly dysregulated in the lungs of BIPF and human IPF patients.These findings reveal an anti-fibrotic role of ISM1 in mouse lungs and provide the foundation to further investigate possible therapeutic applications of ISM1 for pulmonary fibrosis in the future.

isthmin 1对斑马鱼胚胎汇聚延伸运动的影响

目的在原肠期,斑马鱼胚胎细胞进行着外包(epiboly)、内卷(involution)以及汇聚延伸(convergence and extension)运动,对于胚胎的形态发生起关键作用。本文旨在研究isthmin 1(ism1)在斑马鱼胚胎发育中的表达图谱,并进一步探讨其在原肠期胚胎汇聚延伸运动中的功能。方法通过原位杂交技术,检测ism1在斑马鱼早期胚胎发育中的表达图谱;利用过表达和特异敲降等实验技术,活体拍照及原位杂交检测ism1对胚胎汇聚延伸运动的影响。结果整胚原位杂交实验结果显示ism1是合子表达的基因,在30%外包时期开始表达于胚胎背部,原肠期表达在整个胚胎边缘区,尤其在胚盾部位表达更为明显。活体拍摄及原位杂交结果表明,与野生型斑马鱼胚胎相比,过表达或敲低ism1均会使胚胎的汇聚延伸运动受到影响。结论 ism1是调控斑马鱼胚胎原肠期汇聚延伸运动的重要基因。