A Study On Changing Laws Of Neural Stem Cells In Human Fetal Brain And Hypoxic Ischemic Encephalopathy

Section OneA study on neural stem cells about developing laws and experimentalcloning in human fetal brainObjectives To investigate the features of neural stem cells(NSCs) from the developing fetal brain of human,the features including distribution, shape, growth mode and amount. To culture NSCs of fetal brain tissue from different gestational age and different location in vitro respectively and determine potency of proliferation and differentiation.Eventually,to find developing laws of NSCs, so as to provide experimental and theoretic evidences for further studying mechanism about controlling proliferation and differentiation in vivo and in vitro and treating regressional diseases of nervous system.Methods 90 cases of fetuses at gestational ages 16~36w and by induction of labor with water bag were collected and divided into six groups according to gestational age 16w,20w, 24w,28w,32w and 36w,each including 15 cases. Experimental materials including hippocampus, striatum, subventricular zone (SVZ),the frontal lobe,the temporal lobe,the parietal lobe and, the occipital lobe were cutted from brains of cases. Distribution,shape,growth mode and the number of NSCs in six groupsincluding seven locations were examined with hybridization in situ and immunohistochemical method under light microscope.NSCs in six groups including seven locations were isolated, cultured,passed and differentiated respectively with serum free medium containing basic fibroblast growth factor(bFGF) and epidermal growth factor(EGF) and single cell clone tecnique,then the cells including cultured NSCs and differentiated cells were identified with immunofluorescence staining.Results NSCs that included large or small round and elliptic,fusiform, cajal,triangle and multiangle cells were existed in fetal brain tissues in six groups including seven locations.NSCs had several growth modes including unsymmetral or symmetral cleavage,clony with two or eight cells,cluster and crowd,as if the clusters and crowds of NSCs settled down on the migration way,or they were cell generative centres.Enations of some NSCs extended to others,there,they forming synapse.There were some different items including distribution,shape,and growth mode at different location in different gestational age.The number of NSCs in the same region were decreasing with the increase of gestational age.NSCs of the same gestational age in hippocampus,striatum,SVZ,the frontal lobe,the temporal lobe,the parietal lobe and the occipital lobe decreased by returns,there was a significant differences among them.Astrocytes of NSCs expressed not only nestin but also GFAP.NSCs expressed nestin and CD34 existed in hippocampus and SVZ at gestational age 28w,they also existed in hippocampus,striatum and SVZ at gestational age 32w and 3 6w.There were NSCs expressed nestin and CD34 at gestational age 16w, 20w, 24w, 28w and 32w.The less the gestational age,the more the NSCs.The NSCs from brain tissues in six groups including seven locations were success fully isolated and cultured with serum free midium,they formed typical neuraospheres in suspension.These NSCs could be cloned and passed continuously,expressing nestin antigen.Serum midium induced these NSCs to differentiate and express specific antigens of neuron,astrocyte and oligodendrocyte.In cultured NSCs respectively,Therewere no significant differences on shape,proliferation and differentiation.Conclusions Our results suggest following conclusions:First,NSCs exist in locations of human fetal brain at gestational age 16-36w,the locations including hippocampus,striatum,SVZ,the frontal lobe,the temporal lobe,the parietal lobe and the occipital lobe.The second, Freezed and primary cells from fetal brain by induction of labor with water bag have be cultured with serum free midium,but there is an inverse relationship between the number of spheres with the time course of postmortem.The third,EGF and bFGF are the key growth factor for p NSCs' proliferation. The fourth,Nestin and CD 133 are the characteristic markerof NSCs,CD34 may be the c...