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.

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.

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 .

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.

Objective To study the effects of the monoclonal anti idiotypic antibody NP30 active immunization on egg granuloma formation and hepatic fibrosis in Schistosoma japonicum infection. Methods ICR mice were actively immunized with NP30 100 ?g ?3 ip. every 10 days while the mice in control group were injected with SP2/0 ascites ip. simultaneously. After cercariae challenging,the mice were killed at the 4th, 8th,12th, 16th, 20th and 24th week, respectively.Mouse livers were removed and stained histochemically with VG and subjected to immunohistochemical assay of collagen type Ⅰ,Ⅲ and fibronectin(FN).The volume of egg granulomas and the content of collagen type Ⅰ,Ⅲ and FN were determined quantitatively by NYD 1000 Image Analysis System. Results The volume of egg granulomas in NP30 immunized group was much smaller than that of control group from the 12th week after cercariae challenge. The cellular components of egg granulomas in NP30 immunized group were significantly different from those of the control group,exhibiting two types of atypical egg granulomas were found.VG stain revealed that the average optical density of collagen in hepatic granulomas of experimental group was lower than that of control group.Immunohistochemical assay revealed that the contents of collagen type Ⅰ,Ⅲ and fibronectin in egg granulomas of experimental group were lower than those of control group. Conclusion NP30 vaccination may induce both cellular and humoral protective immunity to modulate egg granulomas and suppress liver fibrosis of schistosomiasis japonica.

Objective] To amplify and sequence the light chain of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum. [Methods] By comparing the conserved regions at each end of the nucleotide sequences of murine germ line genes enco ding FR1 and FR4 regions of immunoglobulin light chain variable regions, we designed a set of primers for amplification of V L gene. The hybridoma cells secreting anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum were cultured and their genome DNAs were extracted and used as templates for PCR. The PCR product was then cloned into pUC19 vector. The recombinants were sequenced by Sanger′s method. The V L gene was compared with GenBank and published mouse V L genes. [Results] The full length of V L gene was 318 bp. The V L gene was a member of mouse Ig ? light chain subgroup IV and generated from rearrangement of germ line V and J? 4 genes. The V L gene sequence has been registered by GenBank(accession No. AF206720). [Conclusion] The obtained V L gene was a potentially functional gene of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum .

OBJECTIVETo investigate the protective immunity induced by the anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum in mice.

METHODSAn orthogonal table L(16) (4 x 2(12)) was selected as the experimental design. Eight-week-old Kunming outbred mice (male and female) were randomly divided into 16 experimental groups and 2 control groups. Control groups were injected with SP2/0 ascites intraperitoneally. Mice from each group were infected with 100 +/- 2 cercariae of Schistosoma japonicum in the abdominal skin and were sacrificed on the thirtieth day postchallenge. Adult worms were recovered and counted by perfusion of the left ventricle-portal vein. The SP2/0 ascites injected mice were used as controls and the percentage of protection was calculated.

RESULTSActive immunization of mice with NP30 could produce protection levels ranging from 22.36% to 50.46% depending on the different immunity protocols. The best immunization protocol was established from the results.

CONCLUSIONSActive immunization with NP30 can induce a degree of protection to infection with Schistosoma japonicum cercariae and NP30 is a potential vaccine candidate against Schistosoma japonicum.

Analysis of Variance ; Animals ; Animals, Outbred Strains ; Antibodies, Anti-Idiotypic ; immunology ; therapeutic use ; Antibodies, Monoclonal ; immunology ; therapeutic use ; Female ; Male ; Mice ; Schistosoma mansoni ; immunology ; Schistosomiasis mansoni ; immunology ; parasitology ; prevention & control ; Treatment Outcome ; Vaccination

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.
Embryonic Stem Cells ; metabolism ; Genetic Vectors ; genetics ; Glial Cell Line-Derived Neurotrophic Factor ; biosynthesis ; genetics ; Humans ; Lentivirus ; genetics ; Nerve Tissue ; cytology ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

Objective To investigate the main cell types expressed brain-derived neurotrophic factor (BDNF) in the posterior cortical infarction area in cerebral infarction rats after early vein allograft of bone marrow mesenchymal stem cells (BM-MSCs) and the effect of BM-MSCs transplantation on their ce11 numbers and percentages.Methods (1) Fifteen SD rats were randomly divided into sham-operated group 1,ischemia control group 1,and BM-MSCs transplantation group 1 (n=5);distal middle cerebral artery occlusion (dMCA) models were used in the later two groups;1 × 106 CM-DiI labeled BM-MSCs were intravascularly transplanted into the tail vein of rats from the transplantation group 1 at one h after ischemia;all rats were sacrificed 48 h after ischemia;BM-MSCs with co-existence of CM-Dil and BDNF in the ischemia cortex areas were detected by immunofluorescence staining.(2)Fifteen SD rats were randomly divided into sham-operated group 2,ischemia control group 2,and BM-MSCs transplantation group 2 (n=5);dMCAO models were used in the later two groups;1 ×106 non-labeled BM-MSCs were intravascularly transplanted into the tail vein of rats from the transplantation group 2 at one h after ischemia;48 h after ischemia onset,all rats were sacrificed;the number of BDNF+ and CD68+ microglia cells,BDNF+ and Iba-1+ microglia cells,and BDNF+ and neuron-specific nucleoprotein (NeuN)+ neurons were measured by immunofluorescence staining.Results (1) CM-Dil red fluorescence labeled allogeneic BM-MSCs were only found in BM-MSCs transplantation group 1;the labeled cells scattered in the infarct and peri-infarct cortices;9.70％±3.47％ CM-Dil labeled BM-MSCs expressed BDNF,accounting for 13.32％±4.48％ of all BDNF+ cells in the infarct brain cortex.(2) In the brain tissues of cortex infarct area of BM-MSCs transplantation group 2,38.40％±9.04％ BDNF+ cells were Iba-1+ microglia cells,11.65％±2.76％ BDNF+ cells were CD68+ microglia cells,and 28.96％±6.99％ BDNF+ cells were NeuN+ neurons;the Iba-1+ cell numbers and Iba-1+/BDNF+ double positive cell percentages in the BM-MSCs transplantation group 2 ([92.06±36.52]/mm2 and 79.21％±12.27％) were significantly increased as compared with those in the ischemia control group 2 ([31.13±10.23] mm2 and 60.15％±28.20％,P＜0.05).Conclusion Allogeneic BM-MSCs is capable of migrating into the infarct cortex when intravenous transplantation of BM-MSCs is performed at the early stage after ischemia;the main sources of BDNF in these areas are microglias cells and neurons;these BM-MSCs increase both number and percentage of Iba-1+/BDNF+ double positive cells,which may be one of the underlying mechanisms of therapeutic effects.

BACKGROUND: Syringomyelia is a progressive chronic disease that leads to nerve pain, sensory dissociation, and dyskinesia. Symptoms often do not improve after surgery. Stem cells have been widely explored for the treatment of nervous system diseases due to their immunoregulatory and neural replacement abilities. METHODS: In this study, we used a rat model of syringomyelia characterized by focal dilatation of the central canal to explore an effective transplantation scheme and evaluate the effect of mesenchymal stem cells and induced neural stem cells for the treatment of syringomyelia. RESULTS: The results showed that cell transplantation could not only promote syrinx shrinkage but also stimulate the proliferation of ependymal cells, and the effect of this result was related to the transplantation location. These reactions appeared only when the cells were transplanted into the cavity. Additionally, we discovered that cell transplantation transformed activated microglia into the M2 phenotype. IGF1-expressing M2 microglia may play a significant role in the repair of nerve pain. CONCLUSION Cell transplantation can promote cavity shrinkage and regulate the local inflammatory environment.Moreover, the proliferation of ependymal cells may indicate the activation of endogenous stem cells, which is important for the regeneration and repair of spinal cord injury.