| Objective: Using mouse embryos from Kunming as experimental materials,this experiment is aimed to research into the source,migration,proliferation and morphological changes of stem cells in gonads,skin and liver at different developmental stages of mouse embryos with the methods of Hematoxylin-eosin staining,Masson staining,PAS staining,immunohistochemical staining and etc.In this experiment,the mechanism of skin and liver development is the histological basis for the deeper study of the molecular level regulation mechanism of embryonic development.Methods:1.Bouin fixative,which has the advantages of fast infiltration,uniform fixation,small tissue shrinkage,and good preservation of cells and tissue microstructures;2.Dehydration of 30% alcohol,starts with which is a low concentration to prevent excessive contraction of embryonic tissue;3.Xylene,which can control the transparent speed and prevent the embrittlement of embryonic tissue.It is more suitable for the preparation of embryonic tissue sections than toluene;4.After about 17 days,the preparation of tissue sections is divided from the midline of the embryo to the sagittal plane,which is more conducive to preserving the fine structure of the embryonic organ.Results:1.Developmental morphology and immunohistochemical study of germ cell differentiation of mouse embryonic stem cells1)Gonadal development begins at the inner side of the embryonic urine gonads of 10-day-old mice,gonad crests appear at 11-day-old,and undifferentiated gonads form takes shape at 12-day-old.13-day-old embryo specimen sections can be distinguished between male and female;Testicular structure appears in male gonads after 14 days.The 16-day-old female gonads begin to develop into ovaries,and the 20-day-old Panwan spermatogenic tubules differentiate into two layers of cells.This moment the underlying level branches,and many primitive follicles appear in the ovarian cortex.2)At different stages of embryonic development,the expression of Oct-4 in gonadal stem cells changes dynamically.Moreover,the expression of Oct-4 positive cells gradually increases and decreases,while the semin-iferous tubules developing.There are two Oct-4 positive stem cells in old mouse embryo testes from 13 to 16 days,one of which is strong positive precursor cells and the other is weak positive spermatogonia.After 17 days,the expression of Oct-4 in the gonads gradually decreased.2.Morphology and immunohistochemical study of the development of skin cells differentiated from mouse embryonic stem cellsAs the HE staining reveals,the embryonic skin of 10-day-old mice consisted of a layer of flat cells with no obvious dividing lines between the cells.11-12 day old skin is composed of bilayer cells.At 13 days of age,the epidermis is divided into three layers,during which the epidermis is rough,collagen is synthesized,and loose connective tissue appears in the dermis.At 14 days of age,the hair follicle primordia form,the dermis thickens,and the epidermis is in close contact with the dermis.The epidermis,dermis,and subcutaneous tissues of 15-16 day old mouse embryos can be distinguished from the skin,and the skin tissue structure gradually matures.The 17-day-old mice have structural characteristics of adult skin tissue in their embryonic skin.After 18 days,the skin tissue structure remained unchanged.Immunohistochemical results shows that Nesitn is strongly expressed in the skin of mouse embryos at different developmental stages.It is expressed first in epidermal precursor cells,then in basal cells,and finally migrates and remains in hair follicle precursor cells and some basal stem cells.In addition,Nesitn is also strongly expressed in myoblasts around 13 days old.3.Morphology and immunohistochemical research of mouse embryonic stem cell differentiated hepatocytesMorphological observations demonstrates that a hepatic primitive with a blind sac structure is formed at the end of the foregut of the 9-day-old mouse embryo.At 10 days of age,liver cells are arranged irregularly,connected to each other into a network,and some hematopoietic cells exists in the blood sinus.12-13 days is the peak period of liver development.The liver cells increase in terms of number and bulk,and there are central veins and megakaryocytes coming into being.At 14 days of age,embryonic liver lobules appear,which are immature,however.At 15 to 16 days of age,the hepatic lobule further matures and the primary portal area appears.At the age of 17 to 18 days,the second hepatic hilum appeared in the liver,and glycogen synthesis reached its peak.Oct-4 immunohistochemical research showed that Oct-4 positive cells begin to appear in 11-day-old mouse embryos,but they are scattered and irregularly distributed.Oct-4 is expressed as oval cells at 12 days of age.With the development of the portal area,oct-4 positive oval cells gradually migrate to the portal area.About 17 days,it is basically limited to the interlobular bile duct,around the blood vessels and around the central vein.Conclusion:1.specimen preparation and observation of rat whole embryo tissue sectionThrough different staining methods,the improved method of tissue section specimen preparation is beneficial to display the changes of cell and tissue morphology and some chemical components perfectly.Above all,it keeps each organ in the embryo relatively fixed.2.Morphological and immunohistochemical research of gonad cells differentiated from mouse embryonic stem cells1)Germ cells exist at the early stage of embryogenesis,and the migration,proliferation and differentiation of germ cells are closely related to the development of spermatogenic tubules.2)dynamic changes of Oct-4 expression in gonad stem cells.Oct-4 can be used as a specific surface marker protein.It is strongly positive in pro-embryo cells and weakly positive in spermatogonia cells.With the maturation and differentiation of renal tubules,the expression of Oct-4 gradually decreases and disappears3.Morphological and immunohistochemical studies on the development of skin cells differentiated from mouse embryonic stem cells1)the most active period of skin development is the 13-14 days of embryonic development of mice,and the growth of epidermis,dermis and hair follicle precursor cells has reached an unprecedented active state.2)the expression of Nesitn is closely related to the development of epidermis and dermis and the migration of skin stem cells.4.Morphological and immunohistochemical researches of hepatocytes differentiated from mouse embryonic stem cellsDuring embryonic development of mice,liver stem cells are mainly concentrated around interlobular bile ducts,blood vessels and central veins,which may be the storage area of liver stem cells.Oct-4 may play an important role in maintaining the undifferentiated and multidirectional differentiation potential of liver stem cells during embryonic liver development. |
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