In this paper, further study was made on the teratogenicity and the morphological teratogenesis mechanism of neural tube defects (NTD) caused by cyclophosphamide(CP). Pregnant SD rats were given single intraperitoneal injection of CP 15 mg/kg on day 13 of gestation. The fetuses were removed on day 20 of gestation, weighed and examined for external malformations. Some embryos were removed respectively at 4, 8, 12, 24, 48 hr after administration of CP, and examined with light microscope and electron microscope. The results showed that CP has obvious embryotoxie and teratogenic effects. Of the survivings, 97.46％ showed external malformations including encephalocele, exencephaly, opened eyes, micrognathia, limb and digital defects etc. We considered that the possible way by which CP caused the malformation on developing embryos may involve the following aspects: (1) CP caused DNA-synthesising cells to degenerate and become necrosis. (2) The cellular organelle (mitochondria and endoplasmic reticulum, etc.) became irregular in shape and fragmented. (3) The mesenchyme surrounding the neural tube were also damaged by CP and therefore influenced the skull ossification.

Objective To study the time relationships between apoptosis of neurons and endotheliocytes with the expression of Bcl 2 and Bax after reperfusion of focal cerebral ischemia in rats. Methods Coronal sections of brain were analyzed using an in situ terminal deoxynucleotidyl transferase mediated biotinylated deoxyuridine triphosphate nick end labeling(TUNEL)and immunohistochemical staining methods to observe apoptosis of neurons and endotheliocytes,and expression of Bcl 2 and Bax after reperfusion(2,6,12,24hours and 2,3,7,14,21 days)of focal cerebral ischemia. Results 1.In the ischemic penumbra,apoptotic cells were increased at 2h after reperfusion,peaked at 12 24?h,then decreased successfully for 7 14?days.The time of apoptotic endothelial cells was 12?h later than that of apoptotic neurons.2.The expression of Bcl 2 protein began at 2?h after reperfusion,peaked at 12 24?h,and decreased for 7 14?days.3.Bax protein expressed from 6?h after reperfusion;peaked at 24 48?h,and lasted for 14days.4.The time phase of Bcl 2 expression was similar to the Bax is but later than it. Conclusion\ Apoptosis was a pattern of cell death after cerebral ischemia/reperfusion.The time of apoptotic endothelial cells was later than that of apoptotic neurons.Bcl 2 and Bax play a regulatory role in the apoptotic process.\;[

Objective To explore the inductive effect of striatal tissue on mouse embryonic stem cells and further analyse the cell source and inductive pattern of this inductive effect. Methods We employed striatal extracts、conditioned medium of striatal astrocytes and conditioned medium of striatal neurons to induce embryonic stem cell to differentiate directly. The differentiated cells were evaluated by morphological observation and TH immunocytochemical staining method. We also performed a quantitative analysis of the results. Results Striatal extracts and conditioned medium of striatal astrocytes had obvious inductive effect on embryonic stem cell.Percentages of three groups were 15%,15.2% and 3% respectively. Conclusions The astrocytes in striatum might have an inductive effect on dopaminergic neuronal differentiation of embryonic stem cells.The determination of inductive factor will be our next research aim.;

Objective To explore the optimal condition of direct differentiation into dopaminergic neurons of embryonic stem cells in serum-free and feeder layer cell free medium. Methods We used the method of phase induction to culture embryonic stem cells. At first, embryonic stem cells were cultured in the serum-free medium with bFGF and LIF so as to realize the direct differentiation from embryonic stem cells to neural precursors. Differentiated cells were determined by nestin immunocytochemical staining. On this basis, we transferred embryonic stem cells to the B27 serum-free medium with IL-1 so as to realize the direct differentiation from neural precursors to dopaminergic neurons. Differentiated cells were determined by TH immunocytochemical staining. Results Approximately 85 percent of cell masses were nestin immuno-positive. The differentiation ratio of dopaminergic neurons was 13%, which increased significantly in comparison with natural differentiation ratio of dopaminergic neurons.Conclusion Without serum and feeder layer cell, we can induce embryonic stem cells to differentiate into dopaminergic neurons effectively by adding different growth factors at different phases, which makes the inductive processes more easily.

30 sexually mature, virgin female SD rats, weighed 200-270 g were mated and used for the study of the teratogenic effect of N, N-methylene-bis (2-amino-1,3,4-thiadiazole) (Bis-A-TDA) on fetal neural tube formation and to explore the possible morphological mechanism of neural tube defects (NTD). In the morning of day 10 of gestation, the experimental group was administered with 10mg/kg body weight Bis-A-TDA mixed in peanut oil, and the control group with the same amount of peanut oil only. The results Showed that the incidence of NTD was 52.9% and the majority of NTD were excencephaly and encephalocele in the experimental group. In the early stage of NTD formation, some neuroepithelial cells showed vacuolated degeneration and necrosis, and the mitochondria became swollen and with indistinct or even disappeared crista. The intercellular spaces widened, and some cells escaped into the lumen of neural tube. The mitotic index of neuroepitbelial cells were sharply decreased. In the closure region of the telencephalon, similar changes of the neuroepithelium were present also, and decreased migration of mesodermal cells was noted. We consider the failure of cranial neural folds to approximate and closure was caused mainly by the damage of neuroepithelial cells, inhibition of cell proliferation, alteration of intercellular junctions and the changes of topographical arrangement of the neuroepithelium. The damage and delayed migration of mesodermal cells might also be involved in this event.

Objective The aim of the study was to observe the survival and differentiation of embryonic neuroepithelial cells in lateral ventricle of rats after transplantation. Methods Embryonic rats(E12d) were obtained from the pregnant Sprague\|Dawley rats;neuroepithelial cells were dissociated from embryonic neural tube and treated with 0\^25% trypsin,then transplanted into the lateral ventricle of father rat.The hosts were anesthetized and transcardially perfused with PBS followed by 4% paraformaldehyde fixative by 10?d,14?d,21?d and 28?d transplantation respectively.Coronal sections of the brain were cut with in cryostat microtome.Using the techniques of histology staining,Nissl staining and immunocytochemistry, survival of neuroepithelial cells transplant and neuro\|specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression of some stem cells were examined. Results Some grafts were found survival which attached to the ventricle by day 10.Some grafts could migrate into the third ventricle and grew into a mass of cells.Some stem cells were able to differentiate into pyramidal cells.By immunocytochemistry,a mass of NSE\|positive cells and GFAP\|positive cells could be detected in the core of graft region.Around intracerebral grafts,some astrocytes showed immunoreactive for glial fibrillary acidic protein.Conclusion\ These findings demonstrate that embryonic stem cells dissociated from neural tube can survive and differentiate into neurons and astrocytes.

Objective To carry out construction of chimeras from embryonic stem cells of outbred KM mice. Methods We isolated embryonic stem cells from inner cell mass of KM mice blastocysts. Then we transferred embryonic stem cells into blastocoele of C57BL/6J inbred mice by microinjection in order to construct chimeric mice. Results The cells isolated from inner cell mass have typical characteristics,i e positive alkaline phophatase staining,normal karyotype,forming embryoid body. Now,we have constructed one chimeric mice successfully. Conclusion Embryonic stem cells isolated from inner cell mass can be used for the chimeras production successfully,which forms the substantial base of transgenic animal model by the way of using embryonic stem cells.

The animal experiment studies have demonstrated that hyperthermia is a strong teratogen to many kinds of animal with high incidence of neural tube defects(NTD).Epidermiological investigation showed that hyperthermia was closely related to the acencephaly and excenphaly in human being,but little was known about the distinct mechanism of NTD induced by hyperthermia.In order to study the developmental mechanism of NTD induced by hyperthermia,we observed the effects of hyperthermia on the neuroepithelial cells in primary culture.The neural tubes of the hamster embryos on 10 d after fertilization were obtained.the neuroepithelia were dissociated and then seeded at the density of 1×106 cells per well.The cells were divided into experimental and control groups randomly.The experimental groups were exposed to 42 C for 20 min,whereas groups exposed to 37 Cserved as control.After treatment of hyperthermia,the cells were incubated continuously at 37 C in a 95%air/5%CO2 humidified incubator,the culture was terminated after different intervals.Phase-contrast microscopy.scanning electron microscopy.transmission electron microscopy,MTT assay,agarose gel electrophoresis analysis,TUNEL detection,immunocytochemistryand image analysis were made.The results of the experimental groups indicated that the floating cells and apoptotic cells increased in number,the neurites of the cells were shortened even disappeared,the survival cells decreased in number,the ultrastructure of the cells demonstrated distinct abnormal changes,the function of mitochondria was impaired,the expressions of both bcl-2 and bax were abnormal.The above results suggest that hyperthermia may induce apoptosis of neuroepithelial cells,duringwhich bcl-2 and bax genes may play important regulatory roles.

Objective Constructing golden hamster neural tube cDNA library in order to detect new genes associated with development of neural tube and neural tube defects. Methods Total RNAs were isolated from neural tube using TRIZOL Reagent; Doublestranded cDNAs were synthesized from total RNAs using LD-PCR method; The dsc DNAs were digested by proteinase K and Sfi Ⅰ, less than 500?bp fragments were separated by CHROMA SPIN-400, and then the cDNAs were ligated to ? TriplEX-2 vector.After packaging,the neural tube cDNA library was constructed. Results The titer of neural tube cDNA library was 1.5?10 6?pfu/ml.The recombination rat was 99%.The average insert was larger than 1?kb.Conclusion Our successfully constructed cDNA library is a full-length library with high efficiency,and can be used to detect the genes related to development of neural tube and neural tube defects.

Objective To explore the survival,migration and differentiation of embryonic stem cells after transplantation into striatum and provide advantageous data for feasibility and safety of therapeutic transplantation. Methods We transplanted embryonic stem cells(undifferentiated ESCs and ESCs that had already developed into the stage of neural progenitor cells respectively) into striatum of the rat.Then differentiated cells were determined by morphological observation,Nissl's staining,TH and BrdU immunocytochemistry. Results We found that after transplanted 4 weeks,partially differentiated ESCs could survive and migrate into the surrounding host tissue.Some of them differentiated into TH-positive cells which had Nissl's bodies in cytoplasm.Whereas undifferentiated ESCs couldn't differentiate into TH-positive cells effectively and have the tendency to form tumor.Conclusion When conducting transplantation experiments of ESCs,it's better for ESCs to be induced into the stage of neural progenitor cells first and then transplanted.;