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Objective To explore the localization and expression of dopaminergic neurons in olfactory bulb of cynomolgus monkeys damaged by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).Methods Three adult cynomolgus monkeys were injected with MPTP to induce the damage of dopamine neurons ( MPTP group ) and three adult cynomolgus monkeys were as a control group .Immunohistochemical staining was performed to examine the localization and expression of dopaminergic neurons in the olfactory bulb in normal and MPTP group monkeys .The numbers of DA-positive and DARPP32-positive cells were counted and the average absorbance was measured in normal and MPTP group .Results DA and DARPP32 positive neurons were concentrated in the glomerular layer ( GL) of olfactory bulb.DA positive nerve fibers were distributed in the GL while DARPP 32 positive nerve fibers appeared in all layers , and most nerve fibers were in GL and external plexiform layers (EPL).After MPTP injury, compared with the normal control group , DA and DARPP32 positive neurons and nerve fibers decreased in MPTP group and DA neurons and nerve fibers decreased significantly . Conclusions DA neurons and nerve fibers are in the GL of cynomolgus monkey olfactory bulb .DA neurons and fibers are significantly reduced in the olfactory bulb of cynomolgus monkeys damaged by MPTP , which may be associated with the dysosmia in Parkinson ’ s disease .
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BACKGROUND: Neural stem cells are always derived from animals, and unsuitable for human transplantation treatment. OBJECTIVE: To explore the in vitro culture methods of human embryonic striatum-derived neural stem cells, and to observe the biological characteristics. METHODS: The human embryonic striatum were separated from the embryo at a gestational age of 8-16 weeks that received induction of labor with water bag, and then the embryonic striatum was in vitro cultured in the serum-free Dulbecco’s modified Eagle’s medium. The cells were passaged after neurospheres formation, and then the cells were induced to differentiation with the Dulbecco’s modified Eagle’s medium/F12 containing 10% fetal bovine serum. RESULTS AND CONCLUSION: The in vitro cultured human embryonic striatum-derived neural stem cells grew rapidly and could express nestin. Colony formation assay showed the cel clone formation rate was 6.0%-7.0%. 5-Bromodeoxyuridine incorporation assay showed the cel proliferation rate was 37.9%. Immunofluorescence staining showed that the cells after induction and differentiation could express Tuj-1, glial fibril ary acidic protein and nestin, but not express myelin basic protein. The results indicate that human embryonic striatum-derived neural stem cells cultured in the serum-free medium can maintain their biological characteristics and have self-renewal capacity, and the cells can differentiate into the neurons and astrocytes induced by the fetal bovine serum.
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Objective To explore the effects of intraventricular administration of insulin on the expressions of Bcl-2,Bax mRNA and neuronal hippocampus apoptosis in rats after cardiopulmonary resuscitation (CPR).Methods This experiment was implemented in the animal Laboratory center of Xuanwu Hospital of Capital Medical University.Thirty male SD rats were randomly (random number)divided into three groups:control group (n=6),CPR group (n=12),insulin treated group ( n =12).CPR was performed at 6 minutes after ventricular fibrillation induced by transesophageal overdrive pacing.Resuscitation procedures lasted until restoration of spontaneous circulation (ROSC).ROSC was defined as the recovery of the supraventricular heart rates and the increase of mean arterial pressure (MAP) ＞ 60mmHg for more than 10 minutes.Ten minutes after ROSC in rats,12.5 μL ( 1 U) regular insulin was injected into the left ventricle in the insulin group,and 12.5 μL isotonic saline was injected the control and CPR groups at least 10 minutes.Real-time PCR was used to observe the expressions of Bcl-2,Bax mRNA in hippocampus CAI after reperfusion 24 h and 72 h.TUNEL staining was used to observe the neuronal apoptosis in all groups after reperfusion 7 days.Blood glucose was monitored in rats before and after CPR.Results ① The Bcl-2mRNA in insulin groups were significantly higher than those in the CPR group after 24 h and 72 h (P ＜0.01 ).The expression of Bcl-2 mRNA in 24 h insulin group were significantly higher than those in 72 h insulin group ( P ＜ 0.01 ) ; There were no significantly different in the Bax mRNA between insulin groups and the CPR and the control group after 24 h and 72 h ( P ＞ 0.05 ) ; ②After CPR 7 d,the apoptotic neurons of hippocampal CA1 area in the CPR group ( 124.75 ± 17.35 ) were significantly higher than those in the control group (5.12 ± 3.26) ( P ＜ 0.01 ) and the insulin group (92.79 ± 7.35 )(P ＜0.01 ); the apoptotic neurons in the insulin group were higher than those in the control group (P ＜0.0l ),and the differences were statistically significant.③There were no significant difference in venous blood glucose in the CPR and insulin groups (P ＞ 0.05).Conclusions Insulin may regulate Bcl-2mRNA expression in hippocampus,inhibit neuronal apoptosis and protect neurons after CPR in rats.
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We transfected human neural stem cells using lentiviral vectors encoding glial cell line derived neurotrophic factor (GDNF) to study its expression level in vitro and to get a stable cell line expressing GDNF. First, GDNF gene was sub-cloned into the lentiviral transfer vectors. Then, the recombinant lentiviral supernatants were packaged by 293T cells through three plasmids transient co-transfection method using standard lipofectamine reagent. The viral titers were tested by the transfection efficiency of 293T cells. At the same time, human neural stem cells (hNSC) were transfected under different multiplicity of infection. GDNF gene expression level and protein secretion level of hNSC were tested by real-time PCR and ELISA methods after transfection. Lentiviral vectors encoding GDNF were constructed. Using lentiviral vectors encoding GDNF we successfully transfected human neural stem cells, and got a stable neural stem cell lines over-expressing GDNF. Furthermore, the results indicated that GDNF expression was influenced by the multiplicity of infection. Human neural stem cells could over-express GDNF through lentivial vectors tranfection. Its gene expression level and protein expression level correlate with the multiplicity of infection.
Subject(s)
Humans , Embryonic Stem Cells , Metabolism , Genetic Vectors , Genetics , Glial Cell Line-Derived Neurotrophic Factor , Genetics , Lentivirus , Genetics , Nerve Tissue , Cell Biology , Recombinant Proteins , Genetics , TransfectionABSTRACT
Objective To establish methods for examining expression of clock genes including BMAL1,CLOCK,CRY1and CRY2,and to figure out expression profile of these genes in single cell derived from embryoid body(EB).Methods Total RNA isolated from EB was subjected to reverse transcription and amplification with clock genes specific primers to determine thermo-cycle condition,which was used consequently to examine the expression profiles of these clock genes in single cells isolated with patch clamp from embryoid bodies.Results Parameters in amplification and detection were determined.No unspecific band was amplified.At least 2 molecules could be detected with established systems.In the differentiating EB cells,co-expression of these clock genes was rare.Conclusion Theses systems are sensitive enough to detect expression of clock genes in single cells.Transcription and translation loop among clock genes are not intact in differentiating EB cells,and the clock genes may play a role inthe early development and differentiation.