Research of autophagy activity between rat bone marrow mesenchymal stem neural differentiation / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To study the autophagy activity between rat bone marrow stem cells (BMSCs) neural differentiation in order to explore the mechanism involve in this process.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Autophagy is increased after rat BMSC neural differentiation.</p>

Wnt/β-catenin pathway might underlie the MET in trans- differentiation from MSC to MSC-derived neuron / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To observe MET-associated alteration during the trans-differentiation from MSCs to neuron-like cells, and to explore the possible molecular mechanism.</p><p><b>METHODS</b>Bone marrow MSCs were isolated from rat femur and purified in continuous cell culture. After induced differentiation to neuron-like cells by the combination of butylated hydroxyanisole (BHA) and dimethyl sulfoxide (DMSO), cells were tested by comparative polymerase chain reaction (PCR) for the relative expression of MET biomarkers and transcription factors, and for cell cycle by flow cytometry. Meanwhile, target genes of Wnt/β-catenin pathway were also analyzed by comparative PCR to determine the possible involvement.</p><p><b>RESULTS</b>In MSC-induced neuron-like cells, MET-associated transcription factors such as Snail, Slug, ZEB1, ZEB2, and Twist were significantly attenuated in expression level. The Mesenchymal marker Vimentin expression level was increased. Membrane protein E-cad was slightly down-regulated, while N-cad level was marginally elevated. Percentage of proliferating cells (S phase in cell cycle) markedly shrank from 40.42% for MSCs to 6.76% for MSC-derived neuron. Additionally, Wnt/β-catenin target genes β-catenin and c-myc were decreasingly expressed.</p><p><b>CONCLUSION</b>Chemically induced trans-differentiation from MSC to neuron caused similar MET-featured alteration in gene expression and proliferation to known MET, which might be underlied by deactivation of Wnt/β-catenin pathway.</p>

Construction of inducible lentiviral vector containing human Notch1 and EGFP gene and its expression in PC12 cells / 中国应用生理学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Effect of proteasome inhibitor bortezomib on proliferation, apoptosis and XIAP expression in K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of proteasome inhibitor bortezomib on proliferation, apoptosis of K562 cells and the expression of XIAP.</p><p><b>METHODS</b>K562 cells were treated with bortezomib at different concentration. Cell proliferation was analyzed by WST-1 assay, cell apoptosis by flow cytometry and TUNEL, XIAP mRNA expression from 5 - 100 nmol/L by RT-PCR, and XIAP protein expression by SP immunohistochemistry.</p><p><b>RESULTS</b>K562 cells were treated with bortezomib at different concentrations for 24 h respectively, the cells growth was significantly inhibited with inhibition rates from (13. 6 ± 0. 2)% to (81. 4 ± 0. 1)%, respectively, being markedly higher than that of control (1. 2 ± 0. 1)% (P < 0.05). IC(50) was 24. 6 nmol/L of bortezomib treated for 24 h. When K562 cells were treated with 30 nmol/L of bortezomib for 12 - 48 h, the inhibition rates were (29. 1 ± 0. 9)% to (59. 8 ± 1. 2)%, respectively, the differences being statistically significant (P < 0.05) between 12 h group and 24 h group, while there was no statistical difference between 24 h, 36 h and 48 h groups. K562 cells treated with 30 nmol/L bortezomib for 24 h showed nuclear condensation, nuclear margination, nuclear fragmentation, cytoplasmic vacuoles and a large number of apoptotic body formation. The apoptotic cells rate was 83. 67% in bortezomib treated group, and 2. 33% in untreated group (P < 0.05). The expression of XIAP mRNA was decreased in a dose-dependent manner, and the expression of its protein was down-regulated.</p><p><b>CONCLUSION</b>Bortezomib can inhibit the proliferation of K562 cells, and induce apoptosis by down-regulating the expression of XIAP, providing the laboratory evidence for the targeted therapy in acute leukemia.</p>

Abeta(25-35) and ginsenoside Rb1 influence on the expression of GSK-3beta, CDK-5 and PP2A in differentiated neural stem cells of rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Influence of gemcitabine on expression of C-myc gene and its apoptosis-inducing effect on HL-60 cells / 中国实验血液学杂志

The aim of this study was to investigate the effects of gemcitabine(GEM) on apoptosis and c-myc gene expression of HL-60 cells, and feasibility of using GEM in therapy of leukemia. The HL-60 cells were cultured in vitro. The expressions of the c-myc mRNA and C-MYC protein were detected by RT-PCR and Western-blot respectively. The cell apoptosis was analyzed by TUNEL staining. The results showed that after the HL-60 cells were treated with 1.0 microg/ml GEM for 12, 24, 36 and 48 hours, the expression of c-myc mRNA was inhibited to various degree. This inhibitory effect displayed time-dependent manner and the most optimal effective time was 24 hours. Compared GEM group with Ara-C group and blank control group, there were statistical differences (p<0.05). After the HL-60 cells were treated with 1.0 microg/ml GEM for 24, 48, 72 hours, C-MYC protein significantly decreased, and the expression of C-MYC protein reached to lowest level at 48 hours after treating with GEM, and with inhibition rate of 94.16%. Compared GEM group with Ara-C group and blank control group, the differences were significant (p<0.01). There was significant difference between cells treated with GEM for 24, 48 and 72 hours (p<0.01). After the HL-60 cells were treated with 1.0 microg/ml GEM for 24 hours, the apoptotic cells increased obviously. The positive rate was 83.67% in GEM-treated group. Compared GEM group with Ara-C group (positive rate 10.67%) and untreated group (positive rate 3.00%), the differences had statistical significance (p<0.01). It is concluded that GEM can induce the apoptosis and down-regulate c-myc gene expression significantly in HL-60 cells and it may be used as a new therapeutic drug for leukemia.

Effect of gemcitabine on granulocyte colony-stimulating factor receptor and bcr/abl mRNA in patients with chronic myeloid leukemia / 中国实验血液学杂志

This study was purposed to investigate the effect of gemcitabine (GEM) on granulocyte colony-stimulating factor receptor (G-CSFR) and bcr/abl mRNA in patients with chronic myeloid leukemia (CML). 23 cases of CML in chronic phase, 8 cases of CML in blastic phase and 10 cases of non-hematologic diseases with normal bone marrow were enrolled in this study. The bone marrow from all these cases was collected and divided into 2 group: GEM group (bone marrow cells of CML patients and normal bone marrow cells were cultured with 10 µg/ml GEM for 48 hours) and control group (above-mentioned bone marrow cells were cultured without GEM for 48 hours). The expression of G-CSFR was detected by flow cytometry, the expression of bcr/abl mRNA was assayed by RT-PCR. The results showed that the G-CSFR expression rates of bone marrow in CML chronic phase and blastic phase as well as normal bone marrow in GEM group were (50.72±8.57)%, (36.32±4.25)% and (59.42±7.62)% respectively, while the G-CSFR expression rates of above-mentioned bone marrow in control group were (45.42±6.52)%, (30.58±5.68)% and (58.56±5.54)% respectively. The comparison of G-CSFR expression rates between bone marrow of CML and normal bone marrow, between bone marrow of chronic phase and blastic phase and between bone marrow of GEM group and control group all demonstrated significant difference (p<0.05). The RT-PCR assay showed that the expressions of bcr/abl mRNA in CML chronic and blastic phases of GEM group were (0.59±0.15)% and (0.60±0.13)% respectively, while above-mentioned indicators of control group were (0.60±0.10)% and (0.63±0.11)%; there was no significant difference on expression of bcr/abl mRNA between GEM and control groups (p>0.05). The negative correlation of G-CSFR expression rate with bcr/abl mRNA expression level was observed in GEM and control groups as well as in CML chronic phase and blastic phase of GEM group (r=-0.747, p<0.01; r=-0.803, p<0.01 respectively). It is concluded that the GEM can in vitro enhance the expression rate of bone marrow G-CSFR in CML patients at chronic or blastic phases, but no significant effect on expression of bcr/abl mRNA. The negative correlation of G-CSFR expression rate with bcr/abl mRNA expression level exists in CML patients at chronic or blastic phases.

The change of potassium current of neural stem cells cultured in vitro from newborn rat hippocampus / 中国应用生理学杂志

<p><b>AIM</b>To observe the change of potassium current on cultured neurons differentiated from hippocampus neural stem cells of the newborn rat.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Expression changes of Notch-related genes during the differentiation of human mesenchymal stem cells into neurons / 生理学报

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.

Expressions of Tau protein during the differentiation process of mesenchymal stem cells into neural cells / 中国应用生理学杂志

<p><b>AIM</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>