Shizhong Zhang

Human umbilical vein-derived dopaminergic-like cell transplantation with nerve growth factor ameliorates motor dysfunction in a rat model of Parkinson's disease.

Mesenchymal stem cells are capable of differentiating into dopaminergic-like cells, but currently no report has been available to describe the induction of human umbilical vein mesenchymal stem cells (HUVMSCs) into dopaminergic-like cells. In this study, we induced HUVMSCs in vitro into neurospheres constituted by neural stem-like cells, and further into cells bearing strong morphological, phenotypic and functional resemblances with dopaminergic-like cells. These HUVMSC-derived dopaminergic-like cells, after grafting into the brain of a rat model of Parkinson’s disease (PD), showed a partial therapeutic effect in terms of the behavioral improvement. Nerve growth factor was reported to improve the local microenvironment of the grafted cells, and we therefore further tested the effect of dopaminergic-like cell grafting combined with nerve growth factor (NGF) administration at the site of cell transplantation. The results showed that NGF administration significantly promoted the survival of the grafted cells in the host brain and enhanced the content of dopaminergic in the local brain tissue. Behavioral test demonstrated a significant improvement of the motor function of the PD rats after dopaminergic-like cell grafting with NGF administration as compared with that of rats receiving the cell grafting only. These results suggest that transplantation of the dopaminergic-like cells combined with NGF administration may represent a new strategy of stem cell therapy for PD.

Expression of cytokines in rat brain with focal cerebral ischemia after grafting with bone marrow stromal cells and endothelial progenitor cells

BACKGROUND AIMS This study aimed to observe nine factors expressed in rat ischemic brain after transplantation of bone marrow stromal cells (BMSC) and/or endothelial progenitor cells (EPC). These factors were vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF-l), transforming growth factor-β (TGF-β), platelet-derived growth factor-BB (PDGF-BB), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF). METHODS Adult Wistar rats were divided randomly into four groups: a vehicle group, BMSC group, EPC group and BMSC combined with EPC group. The rats were subjected to middle cerebral artery occlusion (MCAO) then implanted intravenously with 3 × 10(6) BMSC, EPC, BMSC/EPC or phosphate-buffered saline (PBS) 24 h after MCAO. Neurologic functional deficits were measured on days 1, 7, 14, 28 after transplantation. On day 7 after transplantation, quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and Western blot were employed to detect the expression of VEGF, SDF-1, bFGF, IGF-l, TGF-β, PDGF-BB, BDNF, GDNF and NGF. RESULTS The neurologic evaluation found that the neurologic severity scores were no different between the four groups on day 1, and the scores of rats in the BMSC/EPC group were significantly lower than those of rats in the other groups on days 7, 14 and 28 after transplantation. The expressions of bFGF, VEGF and BNDF were significantly higher in the BMSC/EPC group compared with the other groups. CONCLUSIONS The intravenous transplantation of BMSC combined with EPC could promote the functional rehabilitation of rats with focal cerebral ischemia, and the mechanism may be related to the enhanced expression of factors.

The Therapeutic Effects of Tyrosine Hydroxylase Gene Transfected Hematopoetic Stem Cells in a Rat Model of Parkinson’s Disease

AimsTo investigate the therapeutic effects of tyrosine hydroxylase (TH)-transfected neuronal stem cells derived from bone marrow stem cells (NdSCs-D-BMSCs) on Parkinson’s disease (PD) through different transplantation protocols, including microinjection into the cerebral ventricles (CV) and the striatum (ST).MethodsAfter identification by enzyme digestion, the constructed plasmid pEGFP-C2-TH was transfected into 8-day-cultured NdSCs-D-BMSCs by electroporation resulting in the coexpression of green fluorescent protein (GFP) and TH. The TH-transfected cells were injected into either the right ST or CV of PD rats. The changes in locomotor activity of PD rats and the migration of transplanted cells in cerebral tissue were monitored and cerebral DA levels were assayed by high performance liquid chromatography (HPLC).ResultsFive days after plasmid pEGFP-C2-TH transfection into NdSCs-D-BMSCs GFP was expressed in 62.1% of the cells and the rate of co-expression with TH was 83.5%. Ten weeks following transplantation, the symptoms of PD rats in both groups were significantly improved and DA levels were restored to 46.6% and 33% of control. The transferred cells showed excellent survival rates in PD rat brains and distant migration was observed.ConclusionBoth CV and ST transplantation of TH-transfected NDSCs-D-BMSCs has obvious therapeutic effects on PD rats. This study could provide evidence for future transplantation route selection, possibly leading to stem cell transplantation through lumbar puncture.

Efficacy of Tyrosine Hydroxylase gene modified neural stem cells derived from bone marrow on Parkinson's disease – a rat model study

The aim of this study is to determine the efficacy of injecting adult bone marrow derived stem cells (BMSCs) transfected with a pEGFP-C2 plasmid containing the gene for Tyrosine Hydroxylase (TH) into the lateral ventricle for treating rats with Parkinsons Disease (PD) induced by injections into the Substantia Nigra pars compacta (SNc) with 6-hydroxydopamine (6-OHDA), a potent and selective neurotoxin for catecholamine expressing neurons. BMSCs were obtained from the femur of rats; transfected with plasmid constructed with TH and green fluorescent protein (GFP) (with about 85% co-transfection efficiency rate) and then cultured with neuronal differentiation media. Eighty rats were injected into the SNc with 6-OHDA and tested behaviorally to verify the model was induced. Then, 12 PD rats were injected into the anterior horn of the lateral ventricle with x10(5) cells, while 12 more rats were given saline as control. We found that 10 days after transplantation there was a significant (P<0.01) reduction in Apomorphine induced rotations in rats receiving transplanted cells. Also, combined SNc and Striatal dopamine contents (microg/g wet tissue weight) in transplanted rats were greater than controls (0.19+/-0.06 vs 0.63+/-0.14 P<0.01). Immunohistological examination found GFP expression, indicating the presence of transplanted cells within the brain, some of which had migrated through the nerve fibers along the ventricular wall. We feel this study shows the efficacy of genetically engineered BMSCs in the treatment of a rat model of PD. However, future experiments are needed to determine the mechanisms.

MiR-124-5p inhibits the growth of high-grade gliomas through posttranscriptional regulation of LAMB1

BACKGROUND Glioblastoma multiforme (GBM) is the most aggressive form of human brain tumor. It was previously shown that high levels of laminin-8 expression were a predictor of tumor recurrence and patient survival. It is thus important to elucidate the mechanism by which laminin-8 expression is regulated and determine how this contributes to glioma progression. This study investigated the mechanism of regulation of LAMB1, which encodes the β1 chain of laminin-8, in glioma cells lines and in a mouse model of GBM. METHODS The expression levels of LAMB1 and miR-124-5p were examined in glioma cell lines (U87 and U251) and GBM tissue samples by quantitative PCR and Western blotting. The potential regulation of LAMB1 by miR-124-5p was investigated by assessing the effects of restored miR-124-5p expression on cell proliferation, colony formation, and tumor growth and angiogenesis. The effects of inhibiting LAMB1 on tumor growth and angiogenesis were also assessed. RESULTS The upregulation of LAMB1 expression was highly correlated with the downregulation of miR-124-5p. LAMB1 protein expression was suppressed by miR-124-5p. The restoration of miR-124-5p expression suppressed glioma growth by inhibiting angiogenesis, effects that were also observed upon LAMB1 knockdown. CONCLUSIONS The findings indicate that miR-124-5p functions as a tumor suppressor and could serve as a molecular marker for glioma diagnosis and as a potential therapeutic target in GBM treatment.

Functional Analysis of Neuron-like Cells Differentiated from Neural Stem Cells Derived from Bone Marrow Stroma Cells in vitro

The transversal differentiation of bone marrow stroma cell (BMSCs) into neural stem cells (NSCs) has attracted much attention in recent years because of their therapeutic potential. However, the problem in therapeutic application of NSCs was how to confirm whether neuron-like cells differentiated from bone marrow stroma cell-derived neural stem cells (BMSCs-D-NSCs) possess corresponding functions of neurochemistry and electrophysiology. In the present study, we tried to affirm the function of neuron-like cells differentiated from BMSCs-D-NSCs in vitro. The BMSCs were harvested by gradient centrifugation in Ficoll-Paque and cultured in “NSCs medium”. Immunocytochemistry was used to detect positive expression of neuron-specific nuclear protein (NeuN) in neuron-like cells derived from the BMSCs-D-NSCs. High-pressure liquid chromatography (HPLC) was used to identify neuron-like cells by detecting excitable amino acids [aspartic acid (Asp), glutamic acid (Glu)], inhibited amino acids [glycine (Gly), gamma (γ) -aminobutyric acid (GABA), alanine (Ala)] or monoamines [noradrenaline (NE), 5-hydroxytryptamine (5-HT), dopamine (DA)]. Electrophysiological properties of the neuron-like cells were also examined using patch clamp analysis to verify their neuron-like functions. It was found that the neuron-like cells differentiated from the BMSCs-D-NSCs could express positive NeuN, synthesize and excrete amino acids, and show some typical electrophysiological properties including the typical Na+ and K+ ion channel membrane current under the voltage patch clamp condition, the typical static electrical membrane potential under the current patch clamp condition, and the differential membrane capacitance and resistance values in series between undifferentiated BMSCs-D-NSCs and differentiated neuron-like cells under the whole-cell patch clamp condition. The neuron-like cells differentiated from BMSCs-D-NSCs exhibit both neuron-like biochemical function and some corresponding electrophysiological properties.

Forsythiaside A Exhibits Anti-inflammatory Effects in LPS-Stimulated BV2 Microglia Cells Through Activation of Nrf2/HO-1 Signaling Pathway

Abstract Inflammation and oxidative stress have been reported to play critical roles in the pathogenesis of neurodegenerative disease. Forsythiaside A, a phenylethanoside product isolated from air-dried fruits of Forsythia suspensa, has been reported to have anti-inflammatory and antioxidant effects. In this study, the anti-inflammatory effects of forsythiaside A on LPS-stimulated BV2 microglia cells and primary microglia cells were investigated. The production of inflammatory mediators TNF-α, IL-1β, NO and PGE2 were detected in this study. NF-κB, nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) expression were detected by western blot analysis. Our results showed that forsythiaside A significantly inhibited LPS-induced inflammatory mediators TNF-α, IL-1β, NO and PGE2 production. LPS-induced NF-κB activation was suppressed by forsythiaside A. Furthermore, forsythiaside A was found to up-regulate the expression of Nrf2 and HO-1. In conclusion, this study demonstrates that forsythiaside A inhibits LPS-induced inflammatory responses in BV2 microglia cells and primary microglia cells through inhibition of NF-κB activation and activation of Nrf2/HO-1 signaling pathway.

Cellular Immunologic Response to Primary Cryoablation of C6 Gliomas in Rats

The immunological consequences of cryoablation for gliomas are largely unknown. cryoablation is an attractive therapeutic option for tumors due to its minimally invasive nature. cryoablation is also potentially immunogenic. With an aim to explore changes in cellullar immunity following argon-helium cryosurgery, we established Wistar rat models bearing subcutaneous C6 glioma and divided the rats into the normal control (30 rats), sham-operated (33 rats), surgical resection (30 rats), and cryosurgery (33 rats) groups with corresponding treatments. The tumor cell morphology was observed, and changes in the T lymphocyte subset and NK lymphocyte subset and the ratio of Th1/Th2 were assessed with flow cytometry following the cryosurgery. The results showed that subcutaneous tumor implantation was successful in all cases and this was confirmed histologically. Compared with surgical resection that caused significant reduction in CD3+, CD4+, CD14+, CD16+56 cell percentages, cryosurgery resulted in significantly increased percentages of CD3+, CD4+, CD14+, CD16+56 cells (P < 0.05) with a increase of the Th1/Th2 ratio 7 days after the operation. These results demonstrate that in addition to tumor cell destruction, cryosurgery also results in enhanced cellular immunity, suggesting the great potential of argon-helium cryosurgery in clinical management of gliomas.

Gene therapy in hemiparkinsonian rhesus monkeys: long-term survival and behavioral recovery by transplantation of autologous human tyrosine hydroxylase-expressing neural stem cells

BACKGROUND AIMS Neural stem cells (NSC) derived from bone marrow stromal cells (BMSC) (BMSC-D-NSC) are remarkably versatile in response to environmental signals, which render them useful in the search for neurodegenerative disease treatments. METHODS We isolated NSC from rhesus monkey bone marrow (BM), transfected them with the human tyrosine hydroxylase (hTH) gene, and transplanted them into 1-methyl-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned hemiparkinsonian rhesus monkeys to determine changes in neural transmitter production and alterations in behavior. RESULTS hTH-expressing cells produced monoamine agents in vitro, such as noradrenalin and dopamine. After cell transplantation in the caudate nucleus and substantia nigra of the experimental monkeys, their disease symptoms and dysfunctional glucose metabolism and dopamine transport were ameliorated. CONCLUSIONS hTH-expressing BMSC-D-NSC survived in transplantation sites and assumed normal dopaminergic neuronal properties, playing an instrumental role in functional restoration.

Decreased percentages of regulatory T cells are necessary to activate Th1-Th17-Th22 responses during acute rejection of the peripheral nerve xenotransplantation in mice.

Background T cells have major functions in the initiation and perpetuation of nerve graft rejection. Our study aimed to investigate the function of regulatory T cells (Treg)-Th1-Th17-Th22 cells in the rejection of peripheral nerve xenotransplantation. Methods Adult male C57 BL/6 mice were used as the recipient for nerve xenotransplantation, and Sprague-Dawley rats were used as the donor. These nerve xenotransplanted mice were used as the experimental groups, and those that received autograft transplant were chosen as the control group. All of the animals were pretreated with interferon (IFN)-&ggr;, interleukin (IL)-17, and IL-22 before the experiment was conducted. The percentages of spleen Treg-Th1-Th17-Th22 cells were evaluated by flow cytometry 1, 3, 7, 14, and 28 days after transplantation. Serum levels of IFN-&ggr;, IL-17, and IL-22 were assessed by enzyme-linked immunosorbent assay. Statistical analysis was performed by Wilcoxon rank sum and Spearman correlation test. Results During acute rejection, the percentages of Th1-Th17-Th22 cells in the spleen and serum IFN-&ggr;, IL-17, and IL-22 levels in the experimental group increased compared with those in the control group. By contrast, CD4+CD25+Foxp3+ T cell level decreased. The rejection of xenograft was significantly prevented after the mice were treated with IL-17–neutralizing, IL-22–neutralizing, and IFN-&ggr;–neutralizing antibodies. Moreover, the percentage of CD4+CD25+Foxp3+ Treg was negatively correlated with the percentages of Th1-Th17-Th22 cells and levels of IL-17, IL-22, and IFN-&ggr;. Conclusion These results suggested that the Treg-Th1-Th17-Th22 cells involved in xenotransplant rejection and imbalance between Tregs and Th1-Th17-Th22 cells contribute to the acute rejection of peripheral nerve xenotransplant.