[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.

[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.

[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.

[Effects of fenofibrate on the proliferation and apoptosis and nitric oxide synthase expression of cultured human umbilical vein endothelial cells induced by lysophosphatidylcholine].

OBJECTIVE: To investigate the effects of fenofibrate on the proliferation and apoptosis and endothelial nitric oxide synthase (eNOS) mRNA expression of cultured human umbilical vein endothelial cells (HUVECs) induced by lysophosphatidylcholine (LPC). METHODS: HUVECs were cultured in vitro. The study was designated to 5 groups according to fenofibrate concentration: control group, LPC group, LPC + low-concentration fenofibrate (10 micromol/L), LPC + middle-concentration fenofibrate (50 micromol/L), and LPC + high-concentration fenofibrate (100 micromol/L). The study was designated to 6 groups according to the intervention time: control group, LPC group, LPC + fenofibrate (50 micromol/L) 6 h, LPC + fenofibrate 12 h, LPC + fenofibrate 24 h, and LPC + fenofibrate 48 h. The proliferation and apoptosis of HUVECs were evaluated by MTT assay, flow cytometry and fluorescence microscopy, respectively. eNOS mRNA were assayed by real time-PCR. RESULTS: Compared with the control group, LPC could inhibit the proliferation and induce apoptosis, and downregulate eNOS mRNA expression and decrease NO production of HUVECs. Fenofibrate could increase the proliferation and decrease the apoptosis, and up-regulate eNOS mRNA expression and enhance NO production in HUVECs. CONCLUSION: Fenofibrate could improve the proliferation and inhibit the apoptosis, and up-regulate eNOS mRNA expression of HUVECs induced by LPC, which may be responsible for fenofibrate to prevent and treat atherosclerosis.

[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.

[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.

[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.

[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.

[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.

[Expressions of Tau protein during the differentiation process of mesenchymal stem cells into neural cells].

AIM: To observe expressions and changes of Tau protein, pSer202 and Tau protein's contents during the differentiation process of bone-marrow mesenchymal stem cells (MSCs) into neural cells, and discuss Tau's effects on it. METHODS: EGF and bFGF were combined for the induction of 4th, 8th, and 12th-MSCs into neural cells. Expressions of Tau protein and pSer202 were tested by immunocytochemistry. ELISA assay was applied for testing Tau protein's contents during differentiation process. RESULTS: Positive rates of Tau protein in uninduced MSCs of 4th, 8th, and 12th-MSCs were under < 6%; After 14-day induction, the cellular morphologic characteristics in different passages were very similar to neurons, positive rates of Tau protein had no significant differences between passages (P > 0.05), but had differences with their uninduced groups (P < 0.05). There hadn't had expression of pSer202 in uninduced and induced groups of passages. ELISA assay indicated that there was an upward tendency in Tau protein's contents during the 14-day induction process, those in the 14th day had no significant differences between passages too (P > 0.05). CONCLUSION: The increase in Tau protein's expressions and its non-phosphorylated state may make for MSCs differentiating into normal neural cells and formation of neuronal processes.

Effects of EGF and bFGF on expression of microtubule-associated protein tau and MAP-2 mRNA in human umbilical cord mononuclear cells.

The aim of this study was to explore the regulatory effects of cytokines, such as EGF and bFGF, on expression of the neural-specific molecules tau and MAP2 mRNA in mononuclear cells (MNCs) derived from human umbilical cord blood (UCB). Phenotypic changes were monitored by inverse phase-contrast microscopy. Tau and MAP2 mRNA were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Tau and MAP2-positive cells were determined by immunocytochemistry. The expression of tau mRNA was negative in uncultured cells, but MAP2 mRNA was positive; in cultured cells, tau protein mRNA expression was positive, MAP2 mRNA expression was upregulated by EGF+bFGF, EGF and bFGF compared to the control group (no cytokines). EGF+bFGF had a greater effect on MAP2 mRNA expression than EGF or bFGF alone. The same upregulatory tendency was noted for tau mRNA expression. It is concluded that MNCs derived from human UCB cells may express some neural specific molecules that can be upregulated by cytokines, especially EGF and bFGF together.