Objective To isolate and purify Sertoli cells and spermatogonia from rat testis,and to study the proliferation and differentiation of the spermatogonia Co-cultured with sertoli cells. Methods After enzymatic digestions of rat testis,the suspension passed the BSA uncontinuous gradient medium in a gravity unit by velocity sedimentation.Then the spermatogonia were further purified by different times of attachment culture.Results The purities of Sertoli cells and spermatogonia were 92.73% and 78.36% respectively after velocity sedimentation separation.The semi-anchored spermatogonial cells were usually round or oval in outline,singly scattered or existed as aligned,clumped populations,while the outline of the attached Sertoli cells in plate was fibroblast-like cells with little protrusions.Conclusion The results suggest that the spermatogonial cells can survive and proliferate for some time in the culture medium containing EGF,bFGF and GDNF,while Sertoli cells can be more prolific and enhance the mitosis and proliferation of the spermatogonial cells.The confluent monolayer of Sertoli cells can be used as feeding layers for Co-culture with spermatogonial cells.

We reconstructed and compared 3D digital models of basal ganglia of human and rat. After selecing the sections con-taining basal ganglia from sterotoxic atlases of human and rat, 3D digital model of basal ganglia of human and rat was recon-structed by using general-purpose 3D modeling and animation software 3D Studio MAX and its 3D loft function. Several modifi-cation processes were done then to make the digital model more smooth. The 3D digital models of basal ganglia of human and ratwere successfully constructed and the comparative neuroanatomy study was conducted. The virtual reality technics was then in-troduced into this study, using internet browser, digitalized basal ganglia could be rotated, detached and resembled arbitrarily.Our study showed: (1) No matter on their morohology or their components, there were little differeces.(2) Due to ortho-statism, the rostro-caudal axis of human brain was rotated with certain angle, as the result, the relative positions among nucleiof human basal ganglia did differ from that of rat. (3) The projection fibers arrived the correspoding part of their target nucleiwith the shortest way, which might become the basis of the basal ganglia intrinsic topological projection.

Objective To determine the ultrastructural localization of MDR1 and GFAP in the surgically resected brain tissues from intractable epilepsy patients. Methods Expression of MDR1 and GFAP in brain tissues was examined by using PAG immunolabeling technique for electron microscopy. Results The MDR1 and GFAP labeled by gold particles were only detected at some reactive astrocytes. The positive gold particles were mainly located in the astrocytic cytoplasm and their membrane, but not in the nucleus.Conclusion The expression of MDR1 and GFAP in the brain of patients with clinically intractable epilepsy were mainly located at the cytoplasm and membrane of certain reactive astrocytics.;

Aim To study the effect of cyclosporin A and tetrand ri ne on P-glycoprotein (P-gp)of bovine brain capillary endothelial cell. M ethods The fluorescent dye, rhodamine-123 (Rh-123) was used to evaluate t he functional activity of the P-glycoprotein (P-gp) efflux transport system in primary cultured bovine brain capillary endothelial cell (BCEC) monolayer. Results Rhodamine-123 accumulation was increased significantly in monola yer treated with the P-gp modifying agent, cyclosporin A and tetrandrine. Conclusion The observation suggests that this Rh-123 method is sens itive, stable to evaluate the function of P-gp of blood-brain barrier (BBB). R h-123 accumulation is also increased by tetrandrine in dose-dependent manner.

Objective To investigate the role of asctrocytes in the process of chronic degeneration of dopaminergic neurons in intracephalic inflammation rat model induced by intracerebroventricularly injection of lipopolysaccharide.Methods Sixty healthy male SD rats were assigned into lipopolysaccharide group or saline control group randomly.All injections were made intracerebroventricularly on right side of the rats.Ethovison software was used to measure the movement distance of rats within 30 minutes.Specific antibody for glial fibrillary acid protein (GFAP) was used in immunohistochemistry stain to detect the changes of asctrocytes in the substantia nigra of rats.Results Movement distance of lipopolysaccharide-injected rats decreased by 21.2% compared with saline-injected rats at 16 weeks after injection (t=2.54,P<0.05)by 27.0% (t=3.55,P<0.01) at 24 weeks and by 31.4% (t=3.91,P<0.01) at 28 weeks after lipopolysaccharide injection.The asctrocytes were activated obviously in the substantia nigra of lipopolysaccharide-injected group at 2 weeks,while the numbers of GFAP-positively stained cells (228.60 + 22.35) increased significantly compared with saline-injected group ( 165.20 ± 25.97) (t = 4.14,P< 0.05).The activation of asctrocytes was not found in lipopolysaccharide-injected group at 4 weeks and 12 weeks.The asctrocytes were re-activated in the substantia nigra of lipopolysaccharide-injected group at 24 weeks,while the numbers of GFAP-positively stained cells (220.00±21.01 ) increased significantly compared with saline-injected group (169.00± 19.00) (t= 4.03,P<0.05).The activation of asctrocytes was not seen at any time point in saline-injected group.Conclusions Intracephalic inflammation induces chronic degeneration of substantia nigral dopaminergic neurons in rats.The asctrocytes exhibite "acute activation-quiescing-reactivation" state,indicating that they might be involved in the mechanism of dopaminergic neurons degeneration.

Objective To investigate whether there is any functional link between p27~(Kip1) function and all-trans retinoic acid (RA) in the control of neuronal differentiation of immortalized human neural progenitor cells (hSN12W-TERT cells). To investigate the mechanism by which p27~(Kip1) regulates the differentiation of immortalized human neural progenitor cells. Methods hSN12W-TERT cells were derived from the striatums of human embryos at 12 weeks gestation and cultured with serum-free medium in presence of EGF and bFGF. At the appropriated time, hSN12W-TERT cells were exposed to 1μmol/L RA for 3, 5, 7 days respectively. The experiment was repeated there times. Cell cycle analysis was performed by flow cytometry analysis (FACS). The expression of p27~(Kip1), p21~(cip1), cyclin-dependent kinase 2 (cdk2), p-cdk2 and S-phase kinase-associated protein 2 (skp2) in hSN12W-TERT cells before and after RA treatment cells were determined by using Western blotting analysis. Results FACS result showed that 77.25% of proliferating hSN12W-TERT cells were in the G1/G0-phase while 9.38% of cells in the S-phase. Following RA treatment, cell growth was arrested, and 85.68% of cells accumulated in G1/G0-phase while 8.57% of cells in the S-phase. Western blotting analysis demonstrated that the levels of p27~(Kip1) in the hSN12W-TERT cells increased following 3 days' treatment with RA compared with those of normal untreated cells, with a peak at 5 days (P<0.05). The similar results were acquired both in nuclear proteins and in cytoplasm proteins of hSN12W-TERT cells. The expression level of p21~(cip1) decreased in response to RA treatment. RA did not affect the expression of cdk2, but the expression of p-cdk2, which represented the activity of cdk2, was markedly decreased in response to RA treatment. Skp2, which was required for the ubiquitin-mediated degradation of p27~(Kip1), was detected in proliferating hSN12W-TERT cells. The expression of skp2 reduced dramatically in response to RA treatment in a time-dependent manner.Conclusion There is a functional link between RA and p27~(Kip1) function in the control of neuronal differentiation in hSN12W-TERT cells. P27~(Kip1) plays a key role during neuronal differentiation. Moreover, high levels of p27~(Kip1) are associated with its degradation inhibiting through reducing proteasome-dependent proteolysis.

he herpes simplex virus type1 (HSV 1) was used for study on tracing neuronal connections of the cerebellum in the rat. The anterior or posterior lobe of the cerebellum in total 23 rats was injected by HSV 1 (HK 2 strain, from Institute of Virology, Chinese Academy of Preventive Medicine) with the titer of 10 -7 in a volume of 10 15?l for each case. Following a postoperative survival period of 1 5 or 10 days, the animals were perfused and their brains and spinal cords were sectioned and stained immunohistochemically by polycolonal antibodies raised in rabbit against HSV 1. Under the LM, the HSV 1 labelled neurons were shown to be a Golgi like appearance, with clearly labelled cell body and dendrites. It showed that the distributions of the labelled neurons in the CNS depended on the postoperative periods and injection sites. 1. After posterior lobe injection, the labelling was limited in the cerebellum, especially in Purkinje cells and the deep nuclei in 1 day of survival time. Two days later, the labelling could be seen in the vestibular, pontive nuclei and inferior olive nucleus. Anterogradely transneuronal labelling in the ventrolateral thalamic nuclus became apparant 3 days after injetions; 2. After anterior lobe injection, the red nucleus, cuneate nucleus, cuniform nucleus and interstitial nucleus of cajal were labelled after 3 days, in addition to the labelling as shown in those cases with injections in the posterior lobe. The results proved that HSV 1 (HK 2 strain) could be transported both retrogradely and anterogradely in CNS, while the transneuronal transport would mainly occur anterogradely. The distances of HSV 1 transport in neuronal pathways would depend upon the postoperative survival times. This indicates that HSV 1 (HK 2 strain) is a powerful tool for demonstrating the chains of synaptically connected neurons in CNS.

Objective The in situ hybridization method was used with signal amplification system for detecting G-protein expression in taste buds. Methods The gene of gustducin and ?- rod transducin in rat are cloned by RT- PCR. Their sense and antisense probes were labeled with digoxigenin. About 150 taste buds in rat vallate and foliate papillae were analyzed. Results The results showed that there were 1.2 transducin-positive cells and 6.2 gustducin-positive cells counted in each taste bud profile on average. The number of gustduin-positive cells was approximately five times more than transducin-positive cells in taste buds. There were 4. 1% gustducin-positive cells and 0.8% transducin-positive cells in all taste cells. It could not be seen any cross reation of transducin probe to gustducin target. The high expression of transducin in retina was also observed but no gustducin expression could be detected. Conclusion The result of present study proved that both transducin and gustducin were taste G-protein and gustducin was high expressed. Both of them might be involved in taste signal transduction.

The origin of neuropeptide Y(NPY)-like immunoreactive(IR)fibers in the lateral and basolateral amygdaloid nuclei was examined by using indirect immunofluorescence and retrograde tracing method in the rat.1njection of a retrograde tracer.fluorogold(FG),into the lateral and basolateral amygdaloid nuclei,labeled many neurons in the ipsilateral anterior amygdaloid area,and simultaneous treatment with antiserum against NPY stained some of these neurons.Destruction of the anterior amygdaloid area caused an ipsilateral decrease of NPY-IR fibers in the lateral and basolateral amygdaloid nuclei.These findings indicate that NPY-IR neurons in the anterior amygdaloid area project ipsilaterally to the lateral and basolateral amygdaloid nuclei.In addition,the present study also shows that NPY-IR neurons located in the lateral and basolateral amygdaloid nuclei are intrinsic to the amygdaloid complex,since after destruction of the anterior amygdaloid area,some of NPY-IR fibers still can be found in lateral and baso lateral nuclei,and transection of the two major amygdalofugal system,stria terminalis and ventral amygdalofugal pathway,failed to cause the accumulation of NPY-like immunoreactivity in the axons proximal to the section.

Aim To investigate whether Nurr1 gene,a member of the nuclear receptor superfamily of transcription factors,contributed to 6-OHDA-induced DAergic neurons insults by comparing the effect of 6OHDA on neuroblastoma cells SK-N-SH and SK-N-SH cells overexpressing Nurr1gene(SK-N-SH/Nurr1).Methods The effect of Nurr1 gene on cell proliferation of SK-N-SH cells was investigated,then SK-N-SH and SK-N-SH/Nurr1 cells were exposed to 6-OHDA(5～100 ?mol?L~(-1))for 1～24 h,cells' morphology was observed under phase-contrast microscope.The cells viability was analyzed via MTT assay.Apoptosis was detected using Hoechst33342 staining.Results The Nurr1-overexpressing SK-N-SH cells showed significantly slow growth rate compared with that of SK-N-SH cells.Treatment with 50 ?mol?L~(-1) 6-OHDA changed SK-N-SH-Nurr1 cells' morphology within 1 h,accompanied by retraction of processes,shrinkage of cell body,whereas the morphology of SK-N-SH cells changed after treated with 50?mol?L~(-1) 6-OHDA for 2 h.The result of MTT assay indicated that exposure to 100 ?mol?L~(-1) 6-OHDA for 24 h induced a significant decrease in SK-N-SH/Nurr1 cells survival compared with SK-N-SH cells at various time points with an IC_(50) value of 75 ?mol?L~(-1) and 50 ?mol?L~(-1) respectively in SK-N-SH and SK-N-SH/Nurr1 cells.The evidence that treatment with 75 ?mol?L~(-1) 6-OHDA for 12 h induced typical apoptosis in SK-N-SH/Nurr1 cells was found in morphological features provided by Hoechst33342 staining,including chromatin condensation and nuclear fragmentation,and the percentages of apoptosis in SK-N-SH/Nurr1 cells was about 22%～24%.In contrast,no morphological characteristics of apoptosis in SK-N-SH cells were observed.Conclusions The present study suggests that Nurr1 gene renders SK-N-SH cells more vulnerable to neurotoxic insults. The mechanism of such action is probanly that normal Nurr1 gene function can stimulate apoptotic pathway induced by 6-OHDA,and play a major role in the high sensitivity of DArgic neurons to 6-OHDA insults as a vulnerable factor.