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 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 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 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.;

Objective To investigate the milieu-dependent differentiation of primordial germ cells(PEGs) in the acute damaged liver microenviroment. Methods After PGCs were cultured and proliferated,these cells were labelled with 5-bromo-2-deoxyuridine(BrdU),then transplanted into the acute damaged liver by CCl_4 through tail vein.Two and four weeks later,the liver was extracted and 10?m-cryostat continuous sections were obtained.The existing and differentiation of the transplanted cells were identified by immunohistochemistry,immunofluorescence double staining and histochemistry for BrdU and hepatic-specific ALB,and the glycogen. Results Transplanted PGCs were found to be incorporated into the acute damaged liver and differentiated into hepatocytes,compensating for acute liver failure.Conclusion PGCs can be induced to differentiate into hepatocytes in the acute damaged liver microenvironment,and can be used for cellular hepatoplasty to treat severe liver disease.

Objective\ To further clarify the biological effects of TGF?1 on blastocyst implantation. Methods We used female adult Kunming mice to mate with male for observing the distribution of TGF?1 and its receptor at different stages of implantation with immunohistochemical method. Results\ Compared with nonpregnancy,TGF?1 and its receptor immunostaining markedly increased in epithelial and glandual cells on day 4 of pregnancy.The moderate extracellular staining of TGF?1 was present around the fibroblasts.On day 5\|6 of pregnancy,strong immunostaining in intensity of TGF?1 and its receptor was noted in primary decidual zone.With blastocyst invasion,TGF?1 and its receptor immunostaining diffused in decidual cells. Conclusion\ TGF?1 might play an important role via its receptor during blastocyst implantation.\;

Objective\ Clone and sequence choriocarcinoma related gene T26. Method\ Choriocarcinoma related gene T26 was ligated into PGEM\|T vector using T\|A clone method.PGEM\|T\|T26 was digested with EcoRⅠ and sequenced,comparing the T26 sequence with GenBank. Results\ The sequence of choriocarcinoma related gene band T26 showed more than 99% identity with the 3' end of human scar,rps4 and ccg2 genes. Conclusion\ The gene T26 besides scar,rps4 and ccg2 genes were related with the oncogenesis of choriocarcinoma. [