[Neuroprotective effect and mechanism of cPLA2 inhibitor increases autophagic flux on spinal cord injury].

OBJECTIVE: To investigate the mechanism of cytosolic phospholipase A2(cPLA2) inhibitor to improve neurological function after spinal cord injury (SCI). METHODS: Thirty-six 3 months old female SD rats, with body mass (280±20) g, were divided into three groups (n=12):sham group, SCI group, and SCI+ arachidonyl trifluoromethyl ketone(AACOCF3) group. Balloon compression SCI model was established in all three groups. In the sham model group, the spinal cord compression model was created after the balloon was placed without pressure treatment, and the remaining two groups were pressurized with the balloon for 48 h. After successful modeling, rats in the SCI+AACOCF3 group were injected intraperitoneally with AACOCF3, a specific inhibitor of cPLA2. The remaining two groups of rats were injected intraperitoneally with saline. The animals were sacrificed in batches on 7 and 14 days after modeling, respectively. And the damaged spinal cord tissues were sampled for pathomorphological observation, to detect the expression of cPLA2 and various autophagic fluxPrelated molecules and test the recovery of motor function. RESULTS: Spinal cord histomorphometry examination showed that the spinal cord tissue in the sham group was structurally intact, with normal numbers and morphology of neurons and glial cells. In the SCI group, spinal cord tissue fractures with large and prominent spinal cord cavities were seen. In the SCI+AACOCF3 group, the spinal cord tissue was more intact than in the SCI group, with more fused spinal cord cavities, more surviving neurons, and less glial cell hyperplasia. Western blot showed that the sham group had the lowest protein expression of LC3-Ⅱ, Beclin 1, p62, and cPLA2 compared with the SCI and SCI+AACOCF3 groups (P<0.05) and the highest protein expression of LC3-Ⅰ (P<0.05). P62 and cPLA2 expression in the SCI group were higher than in the SCI+AACOCF3 group (P<0.05). Behavioral observations showed that the time corresponding to BBB exercise scores was significantly lower in both the SCI and SCI+AACOCF3 groups than in the sham group (P<0.05). Scores at 3, 7, and 14 days after pressurization were higher in the SCI+AACOCF3 group than in the SCI group (P<0.05). CONCLUSION: cPLA2 inhibitors can reduce neuronal damage secondary to SCI, promote neurological recovery and improve motor function by improving lysosomal membrane permeability and regulating autophagic flux.

Is coronal imbalance in degenerative lumbar scoliosis patients associated with the number of degenerated discs? A retrospective imaging cross-sectional study.

BACKGROUND: Degenerative lumbar scoliosis (DLS) is a common degenerative disease of the spine, that predominates in the elderly, and causes spinal deformities along with severe pain and reduced quality of life. The relationship between DLS and degenerated discs is now a new direction of research. Our study aimed to the relationship between the imaging parameters of coronal imbalance and the number of degenerated discs in patients with degenerative lumbar scoliosis and analyzed the segmental distribution of the degenerated discs in patients with DLS. METHODS: We performed a retrospective analysis of the imaging of 40 patients who met the inclusion criteria who attended our outpatient clinic between April 2021 and July 2021, measuring the intervertebral space height of the AV (high side and low side), Cobb angle, and AVT (Apical vertebral translation) from coronal X-ray. Degenerated discs were evaluated by the Pfirrmann score based on T2-weighted magnetic resonance images. We record the number of degenerated discs (Graded as Grade III, Grade IV or Grade V by the Pfirrmann score) and the segments in which they are located. Finally, we explore the relationship between the imaging parameters of coronal imbalance and the number of degenerated discs in patients with DLS. RESULT: Among the 40 patients with DLS in our study, all patients had degenerated discs in the lumbar spine, 95% of patients had degenerated discs(Pfirrmann score Grade III, Grade IV or Grade V) in 2 or more segments, with the L4-L5 segment being the most involved segment with the most degenerated discs, followed by the L3-L4 segment and the L5-S1 segment. There was no statistically significant relationship between the number of degenerated discs and the coronal imbalance in patients with DLS. CONCLUSION: Our results showed an association between DLS and degenerated discs, but there was no statistically significant relationship between imbalance in the coronal plane of the lumbar spine and the number of degenerated discs in patients with DLS. The distribution of degenerated disc segments in patients with DLS showed a higher likelihood of disc degeneration in 2 or more segments, and a higher frequency of disc degeneration in the inferior disc and in the adjacent segments of the AV.

NICD inhibits cell proliferation and promotes apoptosis and autophagy in PC12 cells.

Pheochromocytoma is a tumor of the adrenal medulla for which surgical resection is the only therapy. Though the Notch1 signaling pathway has been suggested as a target for pheochromocytoma treatment, the effect of Notch1 intracellular domain (NICD) on pheochromocytoma cell growth remains unknown. In the present study, the effect of NICD on pheochromocytoma cell growth was examined, by use of a tetracycline­inducible system for NICD overexpression in the PC12 pheochromocytoma cell line. Flow cytometry was used to determine the effect of NICD on cell cycle phase distribution and apoptosis in PC12 cells. Protein expression levels of microtubule associated protein 1 light chain 3 B (LC3B), Beclin 1, autophagy­related (ATG) 5 and ATG7 were examined using western blot analysis. Untreated PC12 cells lack NICD expression, while treatment with doxycycline resulted in a significant NICD overexpression. NICD overexpression promoted cell apoptosis and suppressed cell proliferation via regulating S­phase arrest. In addition, NICD overexpression stimulated the expression of autophagy­related proteins LC3B, Beclin 1, ATG5 and ATG7. In conclusion, NICD promoted cell apoptosis, suppressed cell proliferation, and stimulated autophagy­related protein expression in PC12 cells. The present data indicate that overexpression of NICD may be a promising potential therapy for pheochromocytoma.

The Effect of Advanced Motherhood on Newborn Offspring's Hippocampal Neural Stem Cell Proliferation.

Objective. To investigate the effect of advanced motherhood on rat hippocampal neural stem cell proliferation. Methods. Female parents were subdivided into control and old mother group by age, and neural stem cells were cultured from hippocampal tissues for 24 h newborn offspring. The diameter and numbers of neurospheres were examined by microscopy, and differences in proliferation were examined by EdU immunofluorescence, CCK-8 assay, and cell cycle analysis. Results. The number of neurospheres in the old mother group after culture was lower than the control group. Additionally, neurospheres' diameter was smaller than that of the control group (P < 0.05). The EdU positive rate of the old mother group was lower than that of the control group (P < 0.05). CCK-8 assay results showed that the absorbance values for the old mother group were lower than that of the control group at 48 h and 72 h (P < 0.05). The proportions of cells in the S and G2/M phases of the cell cycle for the older mother group were less than that found for the control group (P < 0.05). Conclusion. The proliferation rates of hippocampal NSCs seen in the older mother group were lower than that seen in the control group.

Role of autophagy on bone marrow mesenchymal stem­cell proliferation and differentiation into neurons.

The purpose of the present study was to investigate the role of autophagy on rat bone marrow mesenchymal stem cell (BMSC) proliferation, apoptosis and differentiation into neurons. After treatment with rapamycin, 3-methyladenine (3-MA) or chloroquine, the cell cycle, apoptosis, expression of neuron-specific enolase (NSE) and the mean fluorescence intensity (MFI) of Notch1 in BMSCs were examined by flow cytometry. The expression of microtubule-associated protein 2 (MAP2), Notch1 and Hes1 was investigated by western blot analysis. The results showed that after induction of autophagy using rapamycin, the proliferation of BMSCs was inhibited. Furthermore, the S-phase population was significantly decreased compared to that in the control group (P<0.05). In addition, the percentage of NSE-positive cells and the expression of MAP2 were significantly increased compared to those in the control group (P<0.05). The MFI of Notch1 was markedly upregulated compared to that in the control group (P<0.05). When autophagy was inhibited by 3-MA or chloroquine, the percentage of apoptotic cells and NSE-positive cells as well as the expression of MAP2 were markedly reduced compared to those in the control group (P<0.05). Furthermore, western blot analysis showed that Notch1 and Hes1 were decreased in the rapamycin-treated group, while they were not affected by 3-MA or chloroquine. The present study indicated that induction of autophagy in BMSCs decreased their S-phase population, promoted their differentiation into neurons and promoted the expression of NSE and MAP2. The mechanisms underlying this process may be linked to the regulation of autophagy-induced inhibition of the Notch1 signaling pathway.

[Research of autophagy activity between rat bone marrow mesenchymal stem neural differentiation].

OBJECTIVE: To study the autophagy activity between rat bone marrow stem cells (BMSCs) neural differentiation in order to explore the mechanism involve in this process. METHODS: BMSCs were passed by 3 generation, then was induced with the revulsant 2% (DMSO) + 200 µmol/L (BHA), NSE expression was detected by immunocytochemical stain, the mRNA expression of autophagy associated genes L3B, Beclinl, Atg5, Atg7, Atg10 were detected by RT-PCR, the autophagy protein LC3B was examined by Western blot and flow cytometry analysis. RESULTS: BMSCs were passed by 3 generation, the purity of BMSCs could reach more than 90%, the morphology of cells were like fibroblasts, after the revulsant 2% DMSO + 200 µmol/L BRA induced, cells were extended long neurites, like nerve cells, positive rate of NSE staining was (83±5) %, RT-PCR results showed that the expression of autophagy associated genes LC3B, Beclinl, Atg5, Atg7 Atg0 were rised after BMSCs neural differentiation, Western blot analysis showed that the LC3B-II protein expression was increased after neural differentiation and the MFI of L3B was highten by flow cytometry. CONCLUSION: Autophagy is increased after rat BMSC neural differentiation.

Effects of delayed motherhood on hippocampal gene expression in offspring rats.

While many studies have examined the pregnancy and health-related outcomes of delayed motherhood for women, less is known concerning the potential consequences for their children. This study aims to investigate the effect of delayed motherhood on the hippocampus at the whole genome level. Sprague-Dawley rat females, either at the age of 3 or 12 months, were individually housed with a randomly selected 3-month-old male. The rat whole genome expression chips were used to detect gene expression differences in the hippocampus of newborn rats. The gene expression profile was studied through gene ontology and signal pathway analyses. qRT-PCR was used to determine the mRNA expression of solute carrier family 2 (SLC2A1) and S-phase kinase-associated protein 2 (SKP2). Western blot was used to detect the protein expression of SKP2. Compared to the control group, 1291 differentially expressed genes were detected, including 635 up-regulated genes and 656 down-regulated genes. These differential expressed genes were involved in 110 significant biological process and nine significant signaling pathways, in which the pathway in cancer is the most changed pathway. For SKP2 (up-regulated) and SLC2A1 (up-regulated) genes which were relevant to the pathway in cancer, qRT-PCR results were consistent with gene chip assay results. The upregulation of SKP2 was also demonstrated at protein level. In conclusion, delayed motherhood led to unique patterns of hippocampal gene expression in offspring and the newly identified genes afford a quantitative view of the changes which enable deeper insights into the molecular basis underlying the role of delayed motherhood.

[Measurement of Foxp3 and NFAT1 in children with aplastic anemia].

OBJECTIVE: To study the expression of Foxp3 and NFAT1 protein in peripheral blood (PB) in children with aplastic anemia (AA) and their roles in the pathogenesis of AA. METHODS: The expression levels of Foxp3 and NFAT1 protein of mononuclear cells in PB were measured by Western blot in 68 children with AA before and after treatment and in 60 normal children (control group). The correlation between Foxp3 and NFAT1 protein expression and the correlation of the Foxp3 and NFAT1 protein expression with blood Hb, WBC and platelet levels were analyzed. RESULTS: The expression levels of Foxp3 and NFAT1 protein in PB in the acute phase in the AA group were significantly lower than in the control group (P<0.05). After treatment (recovery phase) the expression levels of Foxp3 and NFAT1 protein increased obviously compared with those in the acute phase (P<0.05). The Foxp3 protein level was positively correlated with the NFAT1 protein level (r=0.812, P<0.05). Both the Foxp3 and NFAT1 protein levels were positively correlated with blood Hb, WBC and platelet levels in children with AA in the recovery phase (r=0.537, 0.579, 0.655 respectively; P<0.05). CONCLUSIONS: The Foxp3 and NFAT1 protein levels in PB are reduced in children with AA, suggesting that they are involved in the pathogenesis of AA. The measurement of Foxp3 and NFAT1 protein levels may be useful in the severity evaluation of AA.

miR-29a activates Hes1 by targeting Nfia in esophageal carcinoma cell line TE-1.

MicroRNA (miR)-29a has been associated with carcinogenesis in humans; however, its functional significance in esophageal squamous cell carcinoma (ESCC) is yet to be determined. In the present study, the expression of miR-29a was markedly downregulated in ESCC tissue and the ESCC TE-1 cell line, compared with normal esophageal tissue and cells. Furthermore, the present study identified that the forced expression of miR-29a in TE-1 cells significantly reduced cell proliferation and migration. miR-29a overexpression did not affect the expression of Notch1, however, it did increase the gene expression levels of hairy and enhancer of split 1 (Hes1), which is the key effector of the Notch signaling pathway. Direct targeting by miR-29a resulted in the downregulation of nuclear factor 1 A (Nfia), which represses the transcriptional activity of the Hes1 promoter. Furthermore, knockdown of Nfia increased Hes1 expression and inhibited cell growth in TE-1 cells. These results indicate that a low level of miR-29a expression is involved in ESCC tumorigenesis, and exogenous expression of miR-29a may repress cancer cell growth by downregulating Nfia and activating the Notch signaling pathway.

miR-29a modulates neuronal differentiation through targeting REST in mesenchymal stem cells.

OBJECTIVE: To investigate the modulation of microRNAs (miRNAs) upon the neuronal differentiation of mesenchymal stem cells (MSCs) through targeting RE-1 Silencing Factor (REST), a mature neuronal gene suppressor in neuronal and un-neuronal cells. METHODS: Rat bone marrow derived-MSCs were induced into neuron-like cells (MSC-NCs) by DMSO and BHA in vitro. The expression of neuron specific enolase (NSE), microtubule-associated protein tau (Tau), REST and its target genes, including synaptosomal-associated protein 25 (SNAP25) and L1 cell adhesion molecular (L1CAM), were detected in MSCs and MSC-NCs. miRNA array analysis was conducted to screen for the upregulated miRNAs after neuronal differentiation. TargetScan was used to predict the relationship between these miRNAs and REST gene, and dual luciferase reporter assay was applied to validate it. Gain and loss of function experiments were used to study the role of miR-29a upon neuronal differentiation of MSCs. The knockdown of REST was conducted to show that miR-29a affected this process through targeting REST. RESULTS: MSCs were induced into neuron-like cells which presented neuronal cell shape and expressed NSE and Tau. The expression of REST declined and the expression of SNAP25 and L1CAM increased upon the neuronal differentiation of MSCs. Among 14 upregulated miRNAs, miR-29a was validated to target REST gene. During the neuronal differentiation of MSCs, miR-29a inhibition blocked the downregulation of REST, as well as the upregulation of SNAP25, L1CAM, NSE and Tau. REST knockdown rescued the effect of miR-29a inhibition on the expression of NSE and Tau. Meanwhile, miR-29a knockin significantly decreased the expression of REST and increased the expression of SNAP25 and L1CMA in MSCs, but did not significantly affect the expression of NSE and Tau. CONCLUSION: miR-29a regulates neurogenic markers through targeting REST in mesenchymal stem cells, which provides advances in neuronal differentiation research and stem cell therapy for neurodegenerative diseases.

[Construction of inducible lentiviral vector containing human Notch1 and EGFP gene and its expression in PC12 cells].

OBJECTIVE: To construct inducible lentiviral vector containing human Notch1 intracellular domain (NICD) gene and enhanced green fluorescent protein (EGFP), and to study its expression in PC12 cells. METHODS: NICD cDNA was amplified by RT-PCR from human placenta tissue. EGFP gene was amplified by PCR from pEGFP-C1. Both NICD and EGFP were cloned into pcDNA 3.1 (+) plasmid to form pcDNA3.1-Notch1-EGFP. Then the Notch1-EGFP fragment was separated and cloned into pLVX-Tight-puro to form pLVX-Notch1-EGFP. The lentivirus were packaged and harvested, which were used to infect PC12 cells. After antibody selection for 2 weeks, the PC12 cells were induced by doxycycline (Dox). The expression of Notch1-EGFP was detected by fluorescence microscope and flow cytometry. RESULTS: The recombinant inducible lentiviral vectors (pLVX-Notch1-EGFP) were success fully constructed. The EGFP positive cell percentage was over 90% in transfected PC12 cells after 500 ng/ml Dox induction for 36 h. The expression of Notch1 was posited correlated to the Dox concentration. The expression of Notch1 increased with the duration of Dox induction, which got the peak at 36 h after Dox induction. CONCLUSION: The recombinant inducible lentiviral vectors containing Notch1 and EGFP gene are successfully constructed, which provides an effective and simple method to regulate the expression of Notch1 in PC12 cells.

CXCR7 silencing attenuates cell adaptive response to stromal cell derived factor 1α after hypoxia.

Previous studies have shown that chemotactic factor stromal-cell derived factor 1α (SDF1α) promotes cell recovery from hypoxic injury via its main receptor C-X-C chemokine receptor type (CXCR) 4. However, the role of its new receptor CXCR7 on cell repair against hypoxia and cell response to SDF1α remains largely unknown. In this study, neurons induced from hippocampal progenitor cells were pre-conditioned in hypoxia for 4 h and subsequently monitored to investigate the function of SDF1α on cell repair after hypoxia. Neurons were assessed for their cell morphology, actin filament polymerization and migration capability. SDF1α protein levels increased significantly 1 h after hypoxia compared to control (P<0.01), and it reached a peak at 24 h after hypoxia. Moreover, addition of SDF1α promoted neurite outgrowth and actin filament polymerization both in normoxic and hypoxic cells compared to untreated cells. Cell migration showed a time-dependent increase with SDF1α stimulation in both groups, and hypoxic cells illustrated a significant augment at 0.5 h, 1 h and 12 h after SDF1α application compared to normoxic cells (P<0.01). CXCR7 expression also increased with time dependence after hypoxia and demonstrated a two-fold upregulation compared to control at 24 h after hypoxia. With CXCR7 silencing, axon elongation and actin filament polymerization induced by SDF1α were inhibited sharply both in normoxic and hypoxic cells. CXCR7 silencing also leads to reduced hypoxic cell migration at 0.5 h, 1 h, 12 h, 24 h and 36 h after SDF1α application (P<0.01), but it failed to reduce normoxic cell migration induced by SDF1α at 0.5 h, 1 h and 12 h (P>0.05). 24 h SDF1α stimulation led to higher ERK1/2 phosphorylation compared to control, and ERK1/2 phosphorylation increased more in hypoxic cells than that in normoxic cells. This study suggested that CXCR7 plays an important role on cell repair processing induced by SDF1α, and CXCR7 silencing attenuates cell adaptive response to acute SDF1α stimulation (≤12 h) after hypoxia.

Effect of neuronal induction on NSE, Tau, and Oct4 promoter methylation in bone marrow mesenchymal stem cells.

Cell differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. The differentiation potential differences in DNA methylation patterns might function in pluripotency restriction, while tissue-specific differences might work in lineage restriction. To investigate the effects of neuronal induction on promoter methylation pattern in rat bone marrow mesenchymal stem cells (MSCs), we used bisulfite sequencing to analyze the methylation status of the promoter regions in neuron-specific enolase (NSE), microtubule-associated protein Tau, and Oct4 genes in MSCs pre- and post-chemical induction. Neurocytes from the newborn rat brains were used as control. Data showed that NSE and Tau were abundantly expressed in the brain cells and MSC-derived neurocyte-like cells as well but not in the MSCs. However, both NSE promoter (-214~+57 bp) and Tau promoter (-239~+131 bp) were hypomethylated (<4 % CpG methylation). Oct4 was expressed in MSCs, and the Oct4 promoter (-293~-85 bp) was hypermethylated (>79 % CpG methylation). Interestingly, it was found that the methylation of the locus -113 bp upstream of Oct4 transcription start site was specifically enhanced in the process of MSCs' neuronal differentiation. Further experiments in hepatocytes derived from MSCs and hepar tissue proved that the -113 bp locus methylation increased also in non-neurogenic lineages. Tfsitescan prediction showed that AP-2-alpha/gamma and Sp1 might regulate Oct4 transcription upon MSC differentiation by binding the -113 bp locus. So, we conclude that promoter methylation modifies pluripotency-specific gene, rather than regulates the expression of neural-specific genes when MSCs differentiate into neurocyte-like cells.

VEGF-A-induced immature DCs not mature DCs differentiation into endothelial-like cells through ERK1/2-dependent pathway.

Dendritic cells (DCs) are professional antigen-presenting cells that play an important role in anti-tumour immunity. Endothelial-like differentiation of DCs is an interesting phenomenon. The specific role of vascular endothelial growth factor-A (VEGF-A) on the differentiation of immature DCs (iDCs) and mature DCs (mDCs) is worth further research. Here, we show that VEGF-A can induce iDCs to differentiate into endothelial-like cells (ELCs). But it has no obvious influence on mDCs. In the process of endothelial-like differentiation of iDCs, a sustained activation of extracellular signal-regulated kinase (ERK1/2) and cAMP response element binding protein (CREB) was detected. VEGF-A induced the activation of ERK1/2, and led to the nuclear translocation of phosphorylation ERK1/2. Incubation of iDCs with the ERK1/2 upstream kinase MEK1/2 inhibitor PD98059, blocked the phosphorylation of ERK1/2 and CREB as well as the endothelial-like differentiation of iDCs. These data suggest that VEGF-A induces endothelial-like differentiation of iDCs not mDCs through ERK1/2 signalling pathway.

VEGF-A not Ang2 mediates endothelial-like differentiation of immature DCs by ERK1/2 signaling in the microenvironment of human colon adenocarcinoma.

Endothelial-like differentiation of dendritic cells (DCs) is an interesting and significant phenomenon, which is worth further investigation. Here, we show that the tumor microenvironment derived from the supernatant of the SW620 human colon adenocarcinoma cell line and colon adenocarcinoma tissue homogenate can promote immature DCs (iDCs) to differentiate from the DC pathway toward endothelial cells, while the peri-carcinoma homogenate supernatant does not have this role. Inhibition of angiopoietin-2 (Ang2) in the supernatant by its antibody has no obvious influence on the endothelial-like differentiation. In contrast, inhibition of vascular endothelial growth factor-A (VEGF-A) blocked the differentiation. During the course of differentiation, a sustained activation of ERK1/2 was detected. PD98059 blocked the phosphorylation of ERK1/2 as well as the endothelial-like differentiation of iDCs. Inhibition of VEGF-A also blocked the phosphorylation of ERK1/2. These data suggest that VEGF-A not Ang2 mediates endothelial-like differentiation of iDCs by ERK1/2 signaling in the microenvironment of human colon adenocarcinoma.

[Abeta(25-35) and ginsenoside Rb1 influence on the expression of GSK-3beta, CDK-5 and PP2A in differentiated neural stem cells of rats].

OBJECTIVE: To explore the expression of GSK-3beta, CDK-5 and PP2A and the regulation of them by Abeta(25-35) and ginsenoside Rb1 after neural stem cells (NSCs) are transformed into neurons. METHODS: To culture NSCs from the dentate gyrus of newborn rats(24 h) hippocampus in vitro. NSCs of the third passage were induced towards neurons; the expressions of GSK-3beta(pTyr279,216), PP2A and the regulation of them by Abeta(25-35) and ginsenoside Rb1 were tested by the immunofluorescence cytochemical staining after NSCs had been induced for one week; The expressions of GSK-3beta, CDK-5, PP2A and the regulation of them by Abeta(25-35) and ginsenoside Rb1 were detected with RT-PCR. RESULTS: Immunofluorescence cytochemisty showed that neural cells from NSCs which had been differentiated after one week could express GSK-3j (pTyr279,216)and PP2A. Abeta(25-35) could enhance the expression of GSK-3beta(pTyr279,216), meanwhile it also restrained the expression of PP2A. Moreover ginsenoside Rb1 could reverse the affect of Abeta(25-35). RT-PCR found that neural stem cells which had been differentiated after one week could express GSK-3beta, CDK-5, PP2A . The expression of GSK-3beta and CDK-5 rose up and the expression of PP2A weakened when they were treated by Abeta(25-35). However, the effect of Abeta(25-35) was restrained when they were pretreated by ginsenoside Rb1. CONCLUSION: These observations indicated that NSCs which were cultured and induced in vitro can express GSK-3beta, CDK-5 and PP2A; moreover Abeta(25-35) and ginsenoside Rb1 can regulate the expressions of GSK-3beta, CDK-5 and PP2A. It hints that cells which differentiated from neural stem cells in vitro have protein phosphorylation regulation system of normal cells.

MAPK/ERK1/2 signaling mediates endothelial-like differentiation of immature DCs in the microenvironment of esophageal squamous cell carcinoma.

Endothelial-like differentiation of dendritic cells (DCs) is a new phenomenon, and the mechanism is still elusive. Here, we show that the tumor microenvironment derived from the human esophageal squamous cell carcinoma (ESCC) cell line EC9706 can induce immature DCs (iDCs) differentiate toward endothelial cells, and become endothelial-like cells, but it has no obvious influence on mature DCs. During the course of endothelial-like differentiation of iDCs, a sustained activation of mitogen-activated protein kinase/extracelluar signal-regulated kinase1/2 (MAPK/ERK1/2) and cAMP response element-binding protein (CREB) was detected. Incubation of iDCs with MEK phosphorylation inhibitor PD98059 blocked the MAPK/ERK1/2 and CREB phosphorylation as well as the endothelial-like differentiation of iDCs. Inhibition of vascular endothelial growth factor-A (VEGF-A) in the microenvironment with its antibody blocked the endothelial-like differentiation and the phosphorylation of MAPK/ERK1/2 and CREB. These data suggest that MAPK/ERK1/2 signaling pathway activated by VEGF-A could mediate endothelial-like differentiation of iDCs in the ESCC microenvironment.

[The change of potassium current of neural stem cells cultured in vitro from newborn rat hippocampus].

AIM: To observe the change of potassium current on cultured neurons differentiated from hippocampus neural stem cells of the newborn rat. METHODS: Neural stem cells from newborn rat hippocampus were cultured in vitro and passaged continuously. Differentiation of the cell was induced by serum and removing mitogens. After differentiation cells were plated on plastic dishes and cultured for 1 d, 7 d, 14 d and 21 d. Whole-cell voltage patch clamp recording was used respectively to detect voltage-dependent K+ current. RESULTS: After 1 d culture, no current was detected, and on the 7th d, 14th d, 21st d after differentiation, the amplitude of K+ currents was (18.077 +/- 2.789)pA/pF, (13.099 +/- 2.742)pA/pF, (34.045 +/- 8.067)pA/pF at +50 mV. The recorded K+ current included two components that could be blocked by TEA and 4-AP separately, assumed the slowly inactivating delayed rectifier K+ current (IK) and the fast inactivating transient outward K+ current (IA). CONCLUSION: The function of potassium channels on the hippocampus neural stem cells of the newborn rat approaches mature gradually when the time of differentiation becomes longer in vitro.

[Expression changes of Notch-related genes during the differentiation of human mesenchymal stem cells into neurons].

The Notch signaling pathway has been implicated in the regulation of cell-fate decisions such as differentiation of embryo stem cells and neural stem cells into neurons. We cultured human mesenchymal stem cells (hMSCs) in vitro and induced hMSCs to differentiate into neural cells by beta-mercaptoethanol (beta-ME), DMSO and 3-tert-butyl-4-hydroxyanisole (BHA). Immunocytochemistry was utilized to detect neuron-specific enolase (NSE) and Nissl body, and flow cytometry was used to determine cell growth phases. The expressions of signal molecules involved in the Notch pathway such as Notch1, Jagged 1 (JAG1), presenilin 1 (PS1) and hairy and enhancer of split 1(HES1) were observed by RT-PCR and immunofluorescent techniques. The results were as follows: (1) Before induction, the percentage of hMSCs at G(0)/G(1) was 58.5%, and the percentage at S+G(2)/M was 41.5%. After induction, the percentage of hMSCs at G(0)/G(1) increased to 73.1%, 76.2% and 78.1%, respectively on days 2, 4 and 6, and the percentage at S+G(2)/M decreased to 26.8%, 24.8% and 21.9%, respectively; The percentage of NSE-positive cells reached (77+/-0.35) %; Nisslos staining was positive in cytoplasm. (2) Notch1 and JAG1 were both expressed in hMSCs before and after induction, but the mRNA expressions of both Notch1 and JAG1, detected by RT-PCR, decreased obviously after induction(P<0.05). Notch1 mRNA/beta-actin was 1.157, 0.815, 0.756 and 0.570, and JAG1 mRNA/beta-actin was 0.437, 0.350, 0.314 and 0.362, respectively, on days 0, 2, 4 and 6 after induction. The Notch pathway activation participant PS1 mRNA and Notch pathway target gene HES1 mRNA also decreased apparently after induction (P<0.05), and their mRNA/beta-actin was 0.990, 0.449, 0.441, 0.454 and 0.370, 0.256, 0.266, 0.240 on days 0, 2, 4 and 6, respectively. These observations indicate that the expressions of Notch signal molecules were suppressed when hMSCs were induced to differentiate into neural cells. Based on these findings, we propose that low level of Notch signaling activation may contribute to neural cell differentiation.

[Human bone marrow mesenchymal stem cells differentiated into dopaminergenic neurons in vitro].

Midbrain dopamine (DA) neurons play an essential role in modulating motor control. Defects in central DA neurons affect a wide range of neurological disorders including Parkinson's disease (PD). The greatest motivation in the field has been the potential use of DA neurons for cell transplantation therapy in Parkinsonian patients. Recent studies indicated that BMSCs could differentiate into DA neurons in vitro as neural stem cells (NSC) and embryonic stem cells (ESC) could. However, there are no direct evidences about functional DA neurons derived from BMSCs. According to the protocols which had been applicated in inducing neuronal stem cells and embryonic stem cells differentiate into DA neurons in vitro, the present study provides a protocol by using 50 micromol/L brain derived neurotrophy factor (BDNF), 10 micromol/L forskolin (FSK) and 10 micromol/L dopamine (DA) to induce BMSCs differentiate into DA neurons. After 2 weeks of differentiation, the cells expressed the character of neurons in ultrastructure. RT-PCR discovered mRNA of NSE (neuron specific enolase), Nurr1, Ptx3, Lmx1b and Tyrosine hydroxylase (TH) were positive. Immunocytochemistry staining indicated the ratio of TH-positive neural cells was significantly increased after induced 2 weeks (24.80 +/- 3.36) % compared to that of induction of 3 days (3.77 +/- 1.77) %. And the DA release was also different between differentiated and undifferentiated cells detected by high performance liquid chromatography (HPLC). That is to say BDNF and FSK and DA can induce BMSCs differentiate into DA neurons in vitro, and the transdifferentiated cells express mature neurons characters. BMSCs might be a suitable and available source for the in vitro derivation of DA neurons and cell transplantation therapy in some central neural system diseases such as PD.