[Effects of let-7a on proliferation, osteogenic differentiation and apoptosis of human dental pulp stem cells].

PURPOSE: To study the effect and possible mechanism of let-7a on proliferation, differentiation and apoptosis of human dental pulp stem cell (hDPSCs). METHODS: The cells were divided into four groups: overexpression control (let-7a control/let-7a agomir control), overexpression let-7a (let-7a mimics/let-7a agomir), knockdown let-7a control (let-7a inhibitor control) and knockdown let-7a (let-7a inhibitor). Cell counting kit-8 assay(CCK-8) was used to detect the proliferation of cells at 24 hours, 48 hours and 72 hours after transfection. Calcified nodules were detected by Alizarin red staining. The protein expression of alkaline phosphatase (ALP), osteopontin (OPN), 4E-binding protein 1 (4EBP1), p-4EBP1, mammalian target of rapamycin (mTOR) and p-mTOR were detected by Western blot. Annexin V-APC/7-AAD cell apoptosis detection kit was used to detect the level of apoptosis after transfection. Statistical analysis was performed using GraphPad Prism 5.0 software. RESULTS: Let-7a inhibited proliferation of hDPSCs and promoted odontoblast differentiation and apoptosis. Let-7a down-regulated the expression of 4EBP1, p-4EBP1, mTOR and p-mTOR. CONCLUSIONS: Let-7a may inhibit proliferation of hDPSCs and promote their differentiation and apoptosis by inhibiting mTOR-4EBP1 molecular pathway.

Transplantation of adipocyte-derived stem cells in a hydrogel scaffold for the repair of cortical contusion injury in rats.

Adipocyte-derived stem cells have emerged as a novel source of stem cell therapy for their autologous and readily accessible and pluripotent potential to differentiate into different lineages such as neural stem cells (NSCs) and endothelial progenitor cells (EPCs). Transplantation of NSCs and EPCs has been promising for the repair of brain injury. We explored using co-transplanted hydrogel scaffold to improve the survival of the transplanted cells and recovery of neurological function. Adult Wistar rats were transplanted with EPC-hydrogel, NSC-hydrogel, NSC-EPC-hydrogel, EPC only, or NSC only 7 days after cortical contusion injury. Behavioral tests were performed to evaluate neurological function before, and 1, 2, 3, and 4 weeks after transplantation. Size of injury, extent of vascularization, as well as the survival and differentiation of the transplanted EPCs and NSCs, were evaluated at week 5. All transplantation groups displayed significantly better neurological function compared with the control groups. Improved neurological function correlated with significantly smaller injury volumes than that of the saline group. Using immunostaining, we have shown that while transplanted NSCs differentiated into both neurons and astrocytes, the EPCs were incorporated into vessel epithelia. The extent of reactive gliosis (based on glial fibrillary acidic protein immunostaining) was significantly reduced in all treatment groups (NSC-EPC-hydrogel, NSC-hydrogel, and EPC-hydrogel) when compared with the saline group, with the highest reduction in the NSC-EPC-hydrogel transplantation group. Thus, co-transplantation of hydrogel scaffold provides a more conducive environment for the survival and differentiation of NSCs and EPCs at the site of brain injury, leading to improved vascularization and better recovery of neurological function.

Up-regulation of the transient A-type K+ current (IA) in the differentiation of neural stem cells of the early postnatal rat hippocampus.

BACKGROUND: Neural stem cells (NSCs) not only are essential to cell replacement therapy and transplantation in clinical settings, but also provide a unique model for the research into neurogenesis and epigenesis. However, little attention has been paid to the electrophysiological characterization of NSC development. This work aimed to identify whether the morphological neuronal differentiation process in NSCs included changes in the electrophysiological properties of transient A-type K(+) currents (I(A)). METHODS: NSCs were isolated from early postnatal rat hippocampus and were multiplied in basic serum-free medium containing basic fibroblast growth factor. Potassium currents were investigated and compared using whole-cell patch-clamp techniques and one-way analysis of variance (ANOVA), respectively. RESULTS: Compared with NSC-derived neurons, cloned NSCs (cNSCs) had a more positive resting membrane potential, a higher input resistance, and a lower membrane capacitance. Part of cNSCs and NSC-derived neurons possessed both delayed-rectifier K(+) currents (I(DR)) and I(A), steady-state activation of I(A) in cNSCs (half-maximal activation at (21.34 +/- 4.37) mV) occurred at a more positive voltage than in NSC-derived neurons at 1-6 days in vitro (half-maximal activation at (12.85 +/- 4.19) mV). CONCLUSIONS: Our research revealed a developmental up-regulation of the I(A) component during differentiation of postnatal NSCs. Together with the marked developmental up-regulation of I(DR) in vitro neuronal differentiation we have previously found, the voltage-gated potassium channels may participate in neuronal maturation process.

Passive immunization with LINGO-1 polyclonal antiserum afforded neuroprotection and promoted functional recovery in a rat model of spinal cord injury.

LINGO-1 (leucine-rich repeat and Ig domain-containing, Nogo receptor-interacting protein) is an important component of the NgR receptor complex involved in RhoA activation and axon regeneration. The authors report on passive immunization with LINGO-1 polyclonal antiserum, a therapeutic approach to overcome NgR-mediated growth inhibition after spinal cord injury (SCI). The intrathecally administered high-titer rabbit-derived antiserum can be detected around the injury site within a wide time window; it blocks LINGO-1 in vivo with high molecular specificity. In this animal model, passive immunization with LINGO-1 antiserum significantly decreased RhoA activation and increased neuronal survival. Adult rats immunized in this manner show recovery of certain hindlimb motor functions after dorsal hemisection of the spinal cord. Thus, passive immunotherapy with LINGO-1 polyclonal antiserum may represent a promising repair strategy following acute SCI.

[Prokaryotic expression, purification of human LINGO-1(aa76-319) and preparation of its polyclonal antibody].

OBJECTIVE: To express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb). METHODS: The 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting. RESULTS: The prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity. CONCLUSION: The fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.

Human mesenchymal stem cells-like cells as cellular vehicles for delivery of immunotoxin in vitro.

Human mesenchymal stem cells-like cells (hMSCs-like cells) were used as a tumor treatment platform for the systemic delivery of immunotoxin genes. VEGF165-PE38 recombinant immunotoxin served as the model system. hMSCs-like cells were isolated, expanded, and electroporated with the pIRES2-VEGF165PE38-EGFP plasmid. RT-PCR and ELISA were used to confirm the expression of VEGF165-PE38 in the transfected hMSCs-like cells. These cells released 1390 +/- 137 pg VEGF165-PE38/10(4)cells over 48 h into the culture medium and the supernatant was capable of selectively killing human umbilical vein endothelial cells (HUVECs) and increasing apoptosis in these cells. In contrast, RPMI8226 was not inhibited by identical supernatants. Thus, these results lay the foundation for further studies on the potential role of hMSCs-like cells as a targeted therapeutic delivery vehicle for immunotoxins.

Genetic analysis of interleukin-1A C(-889)T polymorphism with Alzheimer disease.

Neuroinflammation has been implicated in the etiology of Alzheimer's disease (AD). Many studies have suggested that C(-889) T promoter polymorphism in one of the proinflammatory cytokine interleukin-1 (IL-1) encoding gene IL-1A may be associated with AD pathogenesis. To determine whether the polymorphism contributes to the risk for late-onset AD (LOAD) in Chinese, we carried out our investigation in 344 sporadic LOAD patients and 224 healthy controls. No statistical significant association was obtained between IL-1A C(-889) T polymorphism and LOAD and no statistical difference was found between cases and controls after stratification for apolipoprotein E allele 4 (APOE epsilon4) status. The results reveal that it is not likely that the IL-1A C(-889) T polymorphism is involved in AD pathogenesis in the Chinese population. Further studies of the associations between other IL-1A genetic polymorphisms and AD should be performed in a larger population and biologic functional analysis of IL-1A gene is required to verify the underlying roles of IL-IA in LOAD.

Adenoviral-mediated interleukin-18 expression in mesenchymal stem cells effectively suppresses the growth of glioma in rats.

Glioma is the most common primary intracranial malignant tumor. Despite advances in surgical techniques and adjuvant radio- and chemotherapies, the prognosis for patients with glioma remains poor. We have explored the effects of using genetically modified mesenchymal stem cells (MSCs) to treat malignant glioma in rats. Mesenchymal stem cells isolated from Sprague-Dawley rats can directly suppress the growth of C6 cells in vitro. MSCs transplanted intratumorally can also significantly inhibit the growth of glioma and prolong survival in C6 glioma-bearing models. MSCs producing Interleukin-18 infected by adenoviral vector inhibited glioma growth and prolonged the survival of glioma-bearing rats. Transplantation of IL-18 secreting MSCs was associated with enhanced T cell infiltration and long-term anti-tumor immunity. Thus, IL-18 may be an effective adoptive immunotherapy for malignant glioma. When used in conjunction with MSCs as targeting vehicles in vivo, IL-18 may offer a promising new treatment option for malignant glioma.

[Culture and identification of neural stem cells derived from the subventricular zone of adult mice].

OBJECTIVE: To establish a method for culturing and identifying neural stem cells (NSCs) derived from the subventricular zone (SVZ) in adult mice. METHODS: NSCs were isolated from the SVZ of adult mouse brain and cultured in serum-free medium. Cell cloning and BrdU incorporation were performed to identify the self-renewal and proliferative capacity of the NSCs. Fluorescence immunocytochemistry was used to examine the expressions of the NSC markers nestin and SOX2, neuronal marker Tuj1, astrocyte marker GFAP and oligodendrocyte marker NG2. The expressions of nestin and SOX2 were further examined by Western blotting and RT-PCR. RESULTS: NSCs with self-renewal and proliferative capacity were obtained from the SVZ of adult mice and grown as floating neurospheres. The NSCs expressed nestin and SOX2 and could differentiated into Tuj1-positive neurons, GFAP-positive astrocytes and NG2-positive oligodendrocytes. CONCLUSION: This method allows simple and stable culture of NSCs from the SVZ of adult mice.

Construction of eukaryotic expression vector with brain-derived neurotrophic factor receptor trkB gene.

OBJECTIVE: To construct an eukaryotic expression vector carrying rat brain-derived neurotrophic factor receptor trkB gene. METHODS: Using the total RNA isolated from rat brain as template, the trkB gene was amplified by reverse-transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers which contained the restrictive sites of EcoR I and BamH I. The amplified fragment of trkB gene was digested with EcoR I and BamH I, and then subcloned into cloning vector pMD18-T and expression vector pEGFP-C2 respectively. The recombinant plasmids were identified by restriction endonuclease enzyme analysis and PCR. RESULTS: The amplified DNA fragment was about 1461 bp in length. Enzyme digestion and PCR analysis showed that the gene of trkB had been successfully cloned into vector pMD18-T and pEGFP-C2. CONCLUSIONS: The trkB gene of rat has been amplified and cloned into the eukaryotic expression vector pEGFP-C2.

[Induced differentiation of rabbit bone marrow stromal cells into neuron-like cells].

OBJECTIVE: To study whether the neuron-like cells derived from bone marrow stromal cells (BMSCs) may excrete amino acids with neurobiological activities and possess the biochemical characteristics of neurons. METHOD: Under sterile condition, BMSCs from New Zealand rabbits were purified by gradient density centrifugation, and were induced to differentiate into neural stem cells and neuronal-like cells in the culture medium for neural stem cells containing retinoic acid (RA, 0.5 microg/ml) and glial-derived neurotrophic factor (GDNF, 20 ng/ml). The differentiated cells were then examined with immunocytochemical method and high-performance liquid chromatograpy (HPLC). RESULTS: The round and enlarged BMSCs on day 10 of cell culture were positive for nestin, and on day 20, the cells with RA+GDNF stimulation differentiated into neuron-like cells with long protrusions and presented neuron-specific enolase (NSE) antigen. HPLC identified high levels of amino acids like Asp, Glu, Gly and Ala in the differentiated cells (P<0.01). CONCLUSIONS: Rabbit BMSCs may proliferate in vitro as from nestin-positive cells and differentiate into NSE-positive cells containing high levels of excitatory and inhibitory amino acid neurotransmitters. RA and GDNF are important promoters for in vitro differentiation of the BMSCs toward neural stem cells.

In vitro culture and induced differentiation of adult rat neural stem cells from the corpus striatum.

OBJECTIVE: To investigate the in vitro multipotential differentiation of neural stem cells from adult rat corpus striatum. METHODS: The neural stem cells isolated from adult rat corpus striatum were cultured in serum-free medium to obtain cell suspension before monoclonal subculturing and differential induction. Immunocytochemical staining and reverse transcriptional PCR (RT-PCR) were performed to identify the properties of the differentiated cells. RESULTS: Numerous cell clusters were formed in the phase of monoclonal culture, and different types of cells were observed 3 d after induction with fetal bovine serum. The differentiated cells contained cells positive for nestin, neuron-specific enolase (NSE) positive cells, and glial fibrillary acidic protein (GFAP) positive cells. RT-PCR identified expressions of the transcripts for neural cell-associated genes including brain factor-1, gamma-aminobutyric acid alpha-receptor gamma-subunit, tyrosine hydroxylase and tryptophan hydroxylase. CONCLUSION: The cells separated from adult rat corpus striatum possess the ability of self-proliferation and multipotential differentiation, and are identified as the stem cells of the central nervous system.

[Responses of neurons and astrocytes in rat hippocampus to kainic acid-induced seizures].

OBJECTIVE: To investigate the time course of the responses of neurons and astrocytes in rat hippocampus (HI) to kainic acid (KA)-induced seizures in various regions. METHODS: By means immunohistochemical staining for anti-Fos protein and anti-glial fibrillary acidic protein (GFAP), the regional distribution of reactive neurons and astrocytes in the HI was observed at different time points after a unilateral stereotaxic microinjection of KA into the lateral ventricle of rats to cause limbic and generalized convulsive seizures. RESULTS: The injection of KA triggered limbic motor seizures including immobilization, staring, facial and jaw clonus ect. followed by recurrent generalized convulsive seizures. After KA-induced seizures, the GFAP-positive astrocytes and Fos-positive neurons were markedly increased in the HI. The increase of GFAP immunoreactivity was observed 30 min after the seizure onset, reaching the maximum at 1 h; the increase of Fos immunoreactivity was detected at 1 h after the onset, peaking at 2 h. CONCLUSION: The neurons and astrocytes in rat HI are highly active during seizures and the reactive astrocytes might play an important role in epileptogenesis.

Effect of kainate on the synaptic transmission in hippocampal CA1 region.

OBJECTIVE: To investigate the effect of activated kainate receptor on both the excitatory and inhibitory synaptic transmission in the neurons in the hippocampal CA1 region. METHOD: Blind whole-cell voltage-clamp recordings were performed on the CA1 pyramidal cells in adult rat hippocampal slices to examine and analyze the effect of bath-applied kainate (10 micromol/L) on CA1 afferent fiber-evoked excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs), respectively. RESULTS: Activation of kainate receptor significantly depressed both IPSCs (P <0.01) and EPSCs (P <0.01) in neurons in the hippocampus CA1 region. CONCLUSION: Activation of kainate receptors directly inhibit excitatory and inhibitory input in those neurons, which contributes to the development of epilepsy in the hippocampus by affecting the dynamic balance of the hippocampal neurons.

Ultrastructrural observation of the membrane surface of kainic acid-treated hippocampal neurons by atomic force microscopy.

OBJECTIVE: To observe the three-dimensional morphological changes on the membrane surface of primary cultured rat hippocampal neurons in response to kainic acid (KA) exposure. METHODS: After isolation and primary culture, Wistar rat hippocampal neurons were treated with KA at the concentrations of 0, 25, and 250 micromol/L for different durations (10 and 100 min) to observe the subsequent changes in the membrane surface structure of the neurons by nano-scale scanning with an atomic force microscope (AFM). RESULTS: Normal neurons displayed smooth membrane surface with even and regular undulation, while the neurons treated with KA, in contrast, presented degenerative changes characterized by cell swelling and coarse membrane surface with processes and holes. As the treatment was prolonged and KA concentration increased, the changes became more evident. CONCLUSION: As a result of the toxic effect of KA, the membrane surface ultrastructure of rat hippocampal neurons undergo obvious changes, which can be clearly observed and quantitatively analyzed by means of AFM.