Function Study Of Mil/S1Pr2 And Vmhc Genes And Zebrafish Models For Drug Heart Toxicity

Cardiovascular disease is the most common disease for human health and caused huge economic losses. Developing new drugs of cardiovascular disease has been the focus of pharmaceutical research. However, the support of early drug toxicity screening and new prediction models still fall behind relatively. Withdrawing of a drug from the market due to its cardiovascular toxicity, is not only for life lost, but also has led to huge waste of human resouce, substance and time. Thus, it is very important and necessary to establish an in vivo evaluation system for drug cardiac safety, which can be used in the test early, sensitively, accurately and in high throughput.Zebrafish is a small vertebrate animal model, It not only has very strong reproductive ability, in vitro fertilization and development, also it has a high similarity to human in growth and development genome sequencing showed that both the gene and protein structure and function are highly conserved.In addition, experimental techniques have been developed,which contain cell markers of zebrafish,gene activity transfer and mutation and microinjection techniques.Therefore, zebrafish is a properate model animal to research the mechanism of human diseases, prevention and treatment, and drug development.The heart of zebrafish, which can be observed clearly and easily, only have two chambers-an atrium and a ventricle, but is similar to human’s heart in the development route and genetic regulation mechanism. In embyo and larve periods, the heart lies in the surface, thus making it quite easy to be observed. Therefore, we chose zebrafish to establish an animal model of drug cardiac toxicity testing. The main results are as follows:For the first time, we clone the promoter regulatory sequences of Sphingosine 1-phosphate receptor 2(mil/slpr2), and though gene upstream segments deletion, gene knock-down and knock-up experiments, we found that mil Gene impose very important bioological functions on zebrafish cardiovascular system, nervous system, lateral line system and hearing, etc. the disfunction of mil Gene will result in malformation or abnormality. More importantly, we found that the disorder of auditory associated gene and the damage of hair cell are affected by mil/slpr2.zebrafish ventricular myosin heavy chain (vmhc) gene regulatory sequences has been cloned and recombinated to green fluorescent protein vector, whose CMV promoter has been deleted. Transgenic zebrafish model that EGFP was regional-specific expression in the heart has been prepared, and positive transgenic F2 generation has been received. Preliminary results show that the research has received strains of transgenic zebrafish with heart marked.The zebrafish evaluation model for cardiac drug toxicity was established. the representative drugs, such as terfenadine, verapamil, moxifloxacin, vitamin A (Va), were given to zebrafish embryo and larve respectively. Basing on the correlation among the phenotype, cardiac rhythm and gene expression, we attain the primary criteria to evaluate the drugs toxity on zebrafish heart.In summary, this paper focuses on the gene function of the mil/slpr2,establishes the, transgenic zebrafish model that heart specific marked by EGFP, and attains zebrafish drug evaluation model of cardiac toxicity..