BACKGROUND:Basic fibroblast growth factor(bFGF)belongs to active peptide,which is an effective mitogenic factor.OBJECTIVE:To investigate the effect of bFGF on proliferation and differentiation of monkey bone marrow mesenchymal stem cells(BMMSCs)into neuronal precursor cells.METHODS:Monkey BMMSCs were in vitro cultured by density gradient centrifugation,and then divided into 4 groups after passaged,namely,control,bFGF with low,medium and high concentration groups.In the bFGF groups,0,3,6,10 μ/L bFGFwere applied.The proliferation of BMMSCs in each group were observed.The 5th BMMSCs were cultured with serum free L-DMEM culture medium containing 20 mg/L cryptotanshinone to differentiated into neuraMike cells.The expression of positive-nestin protein was detected by immunohistochemical method.RESULTS AND CONCLUSION:Compared with the control group,proliferation rate of BMMSCs in the bFGF groups were accelerated(P ＜ 0.05),which showed a positive correlation to the concentration of bFGF.The positive-nestin protein could be found in the low and medium concentration groups at 0.5 hours after induction,and reached a peak at 1.5 hours,which increased obviously in the low concentration group than that of the high concentration group(P ＜ 0.05).bFGF can promote BMMSCs proliferation in vitro,enhance inducing ratio of prophase neuron-like cells at lower concentration but inhibit differentiation at high level.

Hypoxia-inducible factor-1 (HIF-1), as a nuclear transcription regulator of the hypoxic response, is up-regulated during hypoxia, and it regulates a series of downstream target gene expression, such as vascular endothelial growth factor, glucose transporter and erythropoietin through binding with hypoxia response element. It plays important roles in angiogenesis, anerobic metabolism, cell survival, proliferation, migration, and differentiation.This article reviews the structure, function and activity regulation of HIF-1 and its roles in acute ischemic brain injury.

AIM:To evaluate the influence of scutellarin on the expression of vascular endothelial growth factor ( VEGF) in high glucose-treated human retinal pigment epithelial cell line ARPE-19 and to observe the effects of scutellarin on the protein expression of VEGF, p-ERK and VEGFR2 in the retinas of type II diabetic rats.METHODS: Cultured ARPE-19 cells were divided into normal control group, scutellarin group, high glucose group and high glucose+scutellarin group.The protein levels of VEGF, p-ERK and VEGFR2 were measured by Western blot.The VEGF release in ARPE-19 cells was detected by ELISA.Normal rats were randomly divided into normal control group and scutellarin group.Diabetic rat model was established by feeding with high-fat diet and injecting with streptozocin, and randomly divided into diabetes group and diabetes treated with scutellarin group.After 16 weeks, the eyes were removed.The morphological changes of the retinas were observed under light microscope with HE staining, and histopathological score was recorded.The expres-sion of VEGF in the retinas was observed by the method of immunohistochemistry.RESULTS:Compared with normal con-trol group, the protein levels of VEGF, p-ERK and VEGFR2 in the ARPE-19 cells decreased in scutellarin group, but in-creased in high glucose group.The histopathological score of the retinas showed significant difference among diabetes group, diabetes treated with scutellarin group and normal control group, and no significant difference between normal con-trol group and scutellarin group was observed.The expression of VEGF increased in diabetic group and was significantly higher than that in scutellarin treatment group (P<0.05).CONCLUSION:Scutellarin inhibits the increased protein le-vels of VEGF, p-ERK and VEGFR2 in ARPE-19 cells, and decreases the expression of VEGF in the retinas of diabetic rats.The suppression of the diabetic retinopathy development by scutellarin may be partly involved in the ERK/MAPK pathway.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can proliferate and differentiate into marrow and non-marrow cell types, such as adipocytes, chondrocytes, myocytes, and so on. In recent years, many researchers have studied whether MSCs are capable of differentiation into neurons in vivo and ex vivo. The result that MSCs-derived neurons express NSE and NF, but don't express GFAP suggests MSCs can differentiate into neurons, some researchers have achieved success in promoting functional recovery in Pakinsons and transactional spinal cord injury rat models by use of MSCs-derived neurons. Therefore, MSCs-derived neurons will play an important role in the therapy for a variety of diseases of the nervous system. [

AIM: To explored the potential role of HIF-1α in reducing the neuronal apoptosis and promoting the neuronal proliferation after stroke in rats. METHODS: The bone marrow-derived mesenchymal stem cells (BMSCs) were lentivirally transduced to express the stable form of HIF-1α. Ischemic stroke was induced by permanent middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. Neurological function was evaluated by modified neurological severity score (mNSS). Cerebral infarct volume was measured by TTC staining. Immunohistochemistry and terminal deoxynucleotidyltransferase mediated dUTP nick end labeling (TUNEL) method were performed to detect neuronal proliferation and apoptosis. RESULTS: Significant improvement of neurological deficits was found in BMSCs-mHIF-1α rats as compared to the control animals at 14th d and 28th d after MCAO (P<0.05). Significant reduction of infarct volume was observed in rats in BMSCs-mHIF-1α group at 3rd day after MCAO (P<0.05). Histological evaluation showed that BMSCs-mHIF-1α treatment significantly promoted neuronal survival and proliferation in the ischemic boundary area. CONCLUSION: Constitutive expression of HIF-1α in BMSCs reduces the neuronal apoptosis and promotes the neuronal proliferation after stroke in rats.

BACKGROUND:Hyperbranched cationic amylopectin is a kind of nonviral gene vectors with low toxicity and good transfection efficiency. However, searching for more efficient methods to delivery it into the body and making the genes expressed are being explored.
OBJECTIVE:To study the expression of DMAPA-Amp wrapped green fluorescent protein (GFP) transferred by modified carotid injection into cerebral ischemic area.
METHODS:Male Sprague-Dawley rats subjected to middle cerebral artery infarction were randomly divided into two groups after 24 hours:experimental group (injected with GFP entrapped DMAPA-Amp via the internal carotid artery) and control group (injected with GFP entrapped DMAPA-Amp via the tail vein). These rats were put to death and their brain tissue was removed after 7 days. The expression of GFP was detected by quantitative PCR and western blot assay, and immunofluorescence staining was performed to detect the expression of GFP located near cerebrovascular endothelial cel s by frozen section.
RESULTS AND CONCLUSION:Compared with the control group, the expression of GFP was much higher in the experimental group detected by quantitative PCR and western blot (P< 0.05). Additional y, the expression of GFP located near cerebrovascular endothelial cel s by frozen section was also higher than that in the control group. Modified carotid injection could significantly promote the expression of hyperbranched cationic amylopectin derivates and GFP in the brain tissue of Sprague-Dawley rats undergoing middle cerebral artery infarction compared with tail vein injection, which indicates DMAPA-Amp and modified carotid injection may cast new lights on the therapy for angiogenesis of ischemic stroke.

AIM:To explore the survivorship and the mechanism of the intravenous administration of bone marrow stromal stem cells(BMSCs) for treating permanent focal cerebral ischemia in rats.METHODS:After purified,proliferated,and marked with BrdU,the BMSCs were injected intravenously into rats 1 d after focal cerebral ischemia.Modified neurological severity score(mNSS) was evaluated before and following 1,7,14 and 28 d after middle cerebral artery occlusion(MCAO).Rats were executed at 1,7,14 and 28 d after MCAO.Brain sections were stained with hematoxylin and eosin(HE) for determining the infarct volume.Slides were stained by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling(TUNEL) and immunostaining for cleaved caspase-3 method for apoptosis detection and mechanism exploration in situ.RESULTS:mNSS in BMSCs-transplanted group at 14th day and 28th day of MCAO was significantly lower than that in control group(P

AIM: To construct a recombinant adenovirus expression vector containing CTLA4Ig gene.METHODS: The CTLA4Ig gene derived from the plasmid PCDNA3.0/CTLA4Ig by using polymerase chain reaction (PCR) was inserted into the backward position of cytomegalovirus (CMV) immediate early promoter of the shuttle plasmid (pAdTrack-CMV). After being identified by endonuclease, PCR and sequencing, the recombinant shuttle plasmid pAdTrack-CTLA4Ig was co-transformed into E.coli. BJ5183 cells with the adeoviral backbone plasmid pAdEasyl-1 to obtain the homologous recombination. The adenovirus was generated in 293 cells. A series methods such as PCR and fluorescence microscope was employed to identify the generated recombinant adenovirus. RESULTS: Recombinant CTLA4Ig adenoviruses were constructed and the titer of virus was generally up to 1.65?10 12 phaque forming units per liter (PFU/L). CONCLUSION: Success in constructing recombinant pAdTrack-CTLA4Ig will be the base of the further research on its expression in the mammalian cells, and be potenially used in the prevention of transplant rejection and autoimmunity diseases.

AIM: To investigate the feasibility and infection efficiency of MSCs as the target cells of gene delivery mediated by adenoviral vector carrying CTLA4Ig gene, and to study the mechanism of transgenic MSC to inhibit immune response ex vivo. METHODS: The recombinant adenovirus containing CTLA4Ig gene was constructed, by which rat MSCs with various multiplicity of infection (MOI) were conducted. The infection efficiency was analyzed with FACS and fluorescence microscope. The expression of CTLA4Ig protein in transgenic MSCs was detected by FACS and western blot. Co-culturing the transgenic MSCs with mixed lymphocytes, the inhibitory effect of transgenic MSCs on lymphocyte proliferation was also observed. RESULTS: The adenoviral vector delivered CTLA4Ig gene with high efficiency to MSCs. The expression of CTLA4Ig protein was detected in transgenic MSCs. The gene modified MSCs inhibited the proliferation of mixed lymphocytes and maximal inhibition rate was observed on day 4 of MLR. The inhibition induced by CTLA4Ig was donor-specific. CONCLUSION: MSCs is a promising target cell for gene delivery. The expressed CTLA4Ig specifically inhibits the lymphocyte proliferation ex vivo.

AIM:To explored the potential role of HIF-1? in reducing the neuronal apoptosis and promoting the neuronal proliferation after stroke in rats. METHODS:The bone marrow-derived mesenchymal stem cells (BMSCs) were lentivirally transduced to express the stable form of HIF-1?. Ischemic stroke was induced by permanent middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. Neurological function was evaluated by modified neurological severity score (mNSS). Cerebral infarct volume was measured by TTC staining. Immunohistochemistry and terminal deoxynucleotidyltransferase mediated dUTP nick end labeling (TUNEL) method were performed to detect neuronal proliferation and apoptosis. RESULTS:Significant improvement of neurological deficits was found in BMSCs-mHIF-1? rats as compared to the control animals at 14th d and 28th d after MCAO (P