| BackgroundOsteopetrosis is a class of disease of bone resorption disorder featured by abnormally increased bone density and abnormal numbers or functions of osteoclast.Based on genetic pattern of genotype,it can be classified into three types:autosomal recessive osteopetrosis,autosomal dominant,and X-linked osteopetrosis.In the case of autosomal recessive genetic osteopetrosis,some of them occurred in infancy,which showed a bone resorption disorder and even with a squeezed bone marrow cavity,lethal reduced whole blood and high fatality rate as severe symptoms.It has been reported that about 50%of autosomal recessive genetic osteopetrosis has mutations of tcirgl and there is no good treatment only by bone marrow transplantation can patient’s condition be alleviated.In recent years,it has become a research focus to study gene regulation,explore protein function,explore pathogenesis and construct disease model by using zebrafish as a model organism,which has obvious advantages in external fertilization and development,transparent embryo,high genomic homology especially the disease-causing genes compared with human,numerous and mature operating techniques on embryo and genetics.Zebrafish belongs to vertebrate whose hematopoietic development is conservative.Osteoclasts,derived from monocytes from hematopoietic system,have a common precursor to macrophages and gradually have the function of bone resorption through a series of proliferative fusion.The skeletal development of vertebrates is in a dynamic equilibrium in which bone resorption(osteoclast)and bone formation(osteoblast)play a role simultaneously.It can cause serious and even fatal diseases if the balance is broken.Using zebrafish also provides the basis for us to study the function of osteoclasts.The vertebrate bone remodeling is in a homeostatic balance with bone resorption(osteoclasts)and bone formation(osteoblasts).Breaking one’s balance can cause serious or even fatal diseases.We built a mutant of tcirg1 in zebrafish to simulate the pathological phenotype as well as pathogenesis of human osteoaclerosis and expected to achieve the ultimate goal of medicine screening for the treatment of osteoaclerosis by zebrafish model.Objective:The aim of this research is to construct a mutant of tcirg1 in zebrafish,simulating the pathological phenotype and pathogenesis of human osteosclerosis.The model was used to screen medicines for alleviating or treating osteosclerosis,and further explain its therapeutic principle.Method:The mutants of tcirg1b were obtained by ENU(ethylnitrosourea)mutagenesis,and the expression of specific gene markers was clarified by in situ hybridization.Then bone density was scanned and the phenotype of bone was studied by chemical staining in adult fish,which was compared with the clinical diagnostic criteria of the human osteosclerosis simultaneously.Results:Compared with sibling,we found that the head of the microglia of the head was absence in the embryo of homozygous tcirg1b-/-zebrafish mutants by Neutral Red staining in 4dpf(day post fertilization).And morphology of these microglial cells were found to be abnormal when the head of tcirg1b-/-mutants were observed under high magnification microscope.At the same time,skeleton observation of the fish showed a phenotype of osteosclerosis including obviously increased bone density,unchanged diameter of vertebral canal,decreased neural tube area and blood vertebral canal area.It was found that in tcirglb-/-mutant,TRAP+ osteoclasts increased by acid phosphatase TRAP(tartrate-resistant acid phosphatase)staining,while the number of osteoclast was not significantly changed.Conclusions:In tcirg1b-/-mutant,the main embryonic phenotype was abnormal morphology of microglia,which suggested that the function of microglia might be impaired.And the results of bone mineral density scanning and chemical staining in adulthood showed tcirg1b-/-mutants have skeletal phenotypes similar to human osteosclerosis caused by tcirg1 mutation. |
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