The Study In The Mechanisms Of Myocardial Injury Of Endotoxemia-induced Rats And The Establishment Of Human IPSCs Line From The Patient With Endotoxemia-induced Lung Injury

Background and ObjectivesEndotoxemia is induced by endotoxin or lipopolysaccharide (LPS), a major component in the outer monolayer of the outer membrane of most Gram-negative bacteria, which is important pathogens responsible for endotoxemic shock, multiple organ failure and myocardial dysfunction during endotoxemia. The heart is one of the most common organs adversely affected by endotoxemia. The myocardial injury,an important clinical manifestations during endotoxemia is the major cause of mortality in intensive care units. However, the detailed mechanisms remains incompletely defined.MicroRNAs (miRNAs, miRs) are small(～21-25 nucleotides in length), endogenous, non-coding RNA molecules in eukaryotic organisms. They have the ability to bind mature mRNA molecules and affect their translation, thus usually act as endogenous repressors of target genes by either inhibiting translation or by promoting degradation of the mRNA. It has recently been shown that miRNAs be considered as key regulators of the cardiovascular functions,novel therapeutic targets and/or diagnostic markers for cardiovascular diseases.Recently, numerous studies suggest miRNAs play important roles in cardiac disease, including cardiac hypertrophy, myocardial infarction and cardiac arrhythmia and so on.Therefore, the mechanism of studying miRNAs in cardiac disease has important significance and application value for unveiling the molecular mechanisms of cardiac disease, guiding the development of new therapeutic agents in the treatment of cardiac disease from miRNAss target, and providing and opening the way to a new line of new potential biomarkers to diagnose and prognose for cardiac disease.The cardiac damage in rats with endotoxemia is researched by useing molecular biotechnology and bioinformatics methods, to unveil the potential mechanisms of endotoxemia-caused cardiac damage, and to provide new strategies of treatment for the prevention and treatmen of cardiac damage in clinical and new potential molecular targets of drugs. We analysis the changes of micrornas differentially gene expression profiles in the myocardial tissue of rats with endotoxemia, and demonstrate and prognoste miRNAs triggering target genes for the new molecular target of therapeutic potential of in heart damage.The research of mechanisms in disease are most commonly used for animal modeling.However, there aremajor differences in the gene expression and physiology between animals and humans. Although useful insights have been gained, experimental findings from animal models have not always extrapolated to human disease presentation.The establishments of novel and powerful method and human models in vitro provide important research tools for understanding its pathophysiological mechanisms and drug screening. It is also important to the biomedical research of lung disease.Induced pluripotent stem cells (IPSCs) are a type of pluripotent stem cell like embryonic stem cells(ESCs) derived from adult somatic cells,with their ability of indefinite self-renewal and capability to differentiate into cell types derivatives of all three germlayers,and without the ethical challenges associated with hESCs and the risk of immune rejection.IPSCs can offer a powerful research tool in developmental biology for drug screening, disease modelling and drug toxicology, and potentially cell replacement therapy.The specific IPSCs for a variety of disease have been successfully established at presents, but we rarely found some reports that IPSCs be established successfully from somatic cells in patient with endotoxemia-induced lung injury. We successfully establish IPSCs from patient with endotoxemia-induced lung injury by using Oct4, Sox2, Klf4, Nanog gene to reprogramme IPSCs from patients dermal fibroblasts in vitro,aiming to prove a pluripotent patient-specific cell line and a better cells resource of autologous transplant for appling to drug screening and the development of cellular transplantation therapies, and for clarifing the mechanism of endotoxemia-induced lung injury.Methods1. Sprague-Dawley rats were randomly divided into 2 groups: lipopolysaccharide (LPS) group (n=10) and control group (n=10). LPS group were intraperitoneally injected with 10mg/kg LPS(Escherichia coli,0111:B4, Sigma), while the control group were intraperitoneally injected with equivalent amount of sterile saline solution. All the rats were killed 24h after LPS administration and removed the myocardial tissue under aseptic operation.The expression of TLR4, TNF-α and IL-1β in myocardium were determined by real-time PCR. The cardiac ultrastructure was observed under light microscope and electron microscope.The differentially expressed miRNAs in cardiac tissue were detected by miRNAs array. Selected the common differentially expressed miRNAs and verified their actual expression by real-time PCR. The expression level candidated miRNAs verified by real-time PCR. miRNAs target genes were predicted by bioinformatics. The combination level of miRNAs with target genes were verified by dual luciferase activity method.2. In the present study, we obtained IPSCs by forced expression of a cocktail of transcription factors (Oct4, Sox2, Klf4 and Nanog) into dermal fibroblasts from patient with endotoxemia-induced lung damage resulting in reprogramming to a pluripotent state. The IPSCs generated from this study were analyzed in many aspects, including colony morphology, alkaline phosphatase (AP) activity, surface antigens, and differentiation ability.Results1. The expression level of TLR4,TNF-α and IL-1β in the myocardial tissue were high, the damage changes of cardiac morphology and ultrastructure and cardiac mitochondrial damage were observed.2. Significant differences expression of miRNA in endotoxemia-induced myocardial tissue were found:miR-194-3p, miR-21-3p, miR-344a-3p, miR-465-3p, miR-501-5p, miR-3596c, miR-185-3p, miR-186-3p, miR-877 and miR-3592(ten of up-expresion miRNAs); miR-208b-3p, miR-547-3p, miR-141-3p, miR-28-5p, miR-335, miR-199a-5p, miR-322-5p, miR-3585-5p, miR-532-5p and miR-339-3p(ten of down-expresion miRNAs).3. Seven of up-expresion miRNAs and five of down-expresion miRNAs were found in twenty of miRNAs screening, which show that twelve of miRNAs are involved in the damage changes of cardiac morphology and ultrastructure and cardiac mitochondrial damage.4. In twenty of miRNAs screening, miR-194 of up-expresion miRNAs and miR-141 of down-expresion miRNAs in endotoxemia-caused myocardial tissue.5. The expresion of Fmrland Sumo2 inhibited by miR-194 in transcriptional level was demonstrate.6. The expresion of Myh10 and Cyp26b1 inhibited by miR-141 in transcriptional level was demonstrate.7. The ES like cell clones appear in primary culture of human skin fibroblasts,and gain reprogramming into IPSCs taked on AP staining positive.8. The pluripotent gene marker NANOG,SSEA3 and Tra-1-81 of human embryonic stem cells expressing IPSCs were dentified by cellular immunofluorescence staining method.9. The elevated expression levels of endogenous pluripotent gene Oct4,Sox2,Nanog and Cripto expressing IPSCs were detected by Real-time PCR method.10. Teratoma tissure differentiating into the three germ layers were detected by experiment in vivo.Conclusions1. The expression level of TLR4,TNF-α and IL-1β in the myocardial tissue were high, the damage changes of cardiac morphology and ultrastructure and cardiac mitochondrial damage were observed. The cardiac damage model in rats with endotoxemia variety have been successfully established.2. To our knowledge, in significant differences expression of miRNA from endotoxemia-induced myocardial tissue, miR-194 of up-expresion miRNAs and miR-141 of down-expresion miRNAs were found for the first time. miR-194 and miR-141 may play an important role in cardiac damage caused by endotoxemia and might be looked as new potential molecular targets to cure it.3. Skin fibroblast cells of patient with endotoxemia-induced lung injury can be reprogrammed to induced pluripotent stem cells by lentivirus vector carrying vector, Sox2, Oct4, Nanog and Klf4 four reverse transcription factor. Reprogrammed IPSCs in gene expression, the surface antigen, clone and alkaline phosphatase activity with human ES cells in such aspects as highly similar.4. Subcutaneous injection of IPSCs into immunodeficient (SCID) mice resulted in tumors containing a variety of tissues from all three germ layer.5. This is the first report showing that endotoxemia-induced lung injury patient-specific IPSCs line is successfully established, which can serve as a valuable model to prove a better cells resource of autologous transplant for applying to drug screening and the development of cellular transplantation therapies, and for clarifying the mechanism of endotoxemia-caused lung injury.