Microarray analysis of lncRNAs and mRNAs in spinal cord contusion rats with iPSC-derived A2B5+ oligodendrocyte precursor cells transplantation.

Spinal cord injury (SCI) is a severe complication of spinal trauma with high disability and mortality rates. Effective therapeutic methods to alleviate neurobehavioural deficits in patients with SCI are still lacking. In this study, we established a spinal cord contusion (SCC) model in adult Sprague Dawley rats. Induced pluripotent stem cell-derived A2B5+ oligodendrocyte precursor cells (iP-A2B5+OPCs) were obtained from mouse embryonic fibroblasts and injected into the lesion sites of SCC rats. Serological testing and magnetic resonance imaging were employed to determine the effect of iP-A2B5+OPCs cell therapy. The Basso-Beattie-Bresnahan score and inclined plane test were performed on days 1, 3, 7, and 14 after cell transplantation, respectively. Differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) were detected by microarray analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to analyse the biological functions of these lncRNAs and mRNAs. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to verify variations in the expression of crucial target genes. The results demonstrated that induced pluripotent stem cells exhibited embryonic stem cell-like morphology and could differentiate into diverse neural cells dominated by oligodendrocytes. The neurobehavioural performance of rats treated with iP-A2B5+OPCs transplantation was better than that of rats with SCC without cell transplantation. Notably, we found that 22 lncRNAs and 42 mRNAs were concurrently altered after cell transplantation, and the key lncRNA (NR_037671) and target gene (Cntnap5a) were identified in the iP-A2B5+OPCs group. Moreover, RT-qPCR revealed that iP-A2B5+OPCs transplantation reversed the downregulation of NR_037671 induced by SCC. Our findings indicated that iP-A2B5+OPCs transplantation effectively improves neurological function recovery after SCC, and the mechanism might be related to alterations in the expression of lncRNAs and mRNAs, such as NR_037671 and Cntnap5a.

Corrigendum: Geminicoccus flavidas sp. nov. and Geminicoccus harenae sp. nov., two IAA-producing novel rare bacterial species inhabiting desert biological soil crusts.

[This corrects the article DOI: 10.3389/fmicb.2022.1034816.].

Pangenome analysis of the genus Herbiconiux and proposal of four new species associated with Chinese medicinal plants.

Five Gram-stain-positive, aerobic, non-motile actinobacterial strains designated as CPCC 205763T, CPCC 203386T, CPCC 205716T, CPCC 203406T, and CPCC 203407 were obtained from different ecosystems associated with four kinds of Chinese traditional medicinal plants. The 16S rRNA gene sequences of these five strains showed closely related to members of the genus Herbiconiux of the family Microbacteriaceae, with the highest similarities of 97.4-99.7% to the four validly named species of Herbiconiux. In the phylogenetic trees based on 16S rRNA gene sequences and the core genome, these isolates clustered into the clade of the genus Herbiconiux within the lineage of the family Microbacteriaceae. The overall genome relatedness indexes (values of ANI and dDDH) and the phenotypic properties (morphological, physiological and chemotaxonomic characteristics) of these isolates, readily supported to affiliate them to the genus Herbiconiux, representing four novel species, with the isolates CPCC 203406T and CPCC 203407 being classified in the same species. For which the names Herbiconiux aconitum sp. nov. (type strain CPCC 205763T = I19A-01430T = CGMCC 1.60067T), Herbiconiux daphne sp. nov. (type strain CPCC 203386T = I10A-01569T = DSM 24546T = KCTC 19839T), Herbiconiux gentiana sp. nov. (type strain CPCC 205716T = I21A-01427T = CGMCC 1.60064T), and Herbiconiux oxytropis sp. nov. (type strain CPCC 203406T = I10A-02268T = DSM 24549T = KCTC 19840T) were proposed, respectively. In the genomes of these five strains, the putative encoding genes for amidase, endoglucanase, phosphatase, and superoxidative dismutase were retrieved, which were classified as biosynthetic genes/gene-clusters regarding plant growth-promotion (PGP) functions. The positive results from IAA-producing, cellulose-degrading and anti-oxidation experiments further approved their potential PGP bio-functions. Pangenome analysis of the genus Herbiconiux supported the polyphasic taxonomy results and confirmed their bio-function potential.

Corrigendum: Pangenome analysis of the genus Herbiconiux and proposal of four new species associated with Chinese medicinal plants.

[This corrects the article DOI: 10.3389/fmicb.2023.1119226.].

Geminicoccus flavidas sp. nov. and Geminicoccus harenae sp. nov., two IAA-producing novel rare bacterial species inhabiting desert biological soil crusts.

Two Gram-staining negative strains (CPCC 101082T and CPCC 101083T) were isolated from biological sandy soil crusts samples collected from Badain Jaran desert, China. Both isolates were heterotrophic phototroph, could produce indole-3-acetic acid. The 16S rRNA gene sequences of these two strains were closely related to the members of the family Geminicoccaceae, showing high similarities with Geminicoccus roseus DSM 18922T (96.9%) and Arboricoccus pini B29T1T (90.1%), respectively. In phylogenetic tree based on 16S rRNA gene sequences, strain CPCC 101082T and CPCC 101083T formed a robust distinct clade with Geminicoccus roseus DSM 18922T within the family Geminicoccaceae, which indicated that these two isolates could be classified into the genus Geminicoccus. The growth of strain CPCC 101082T occurred at 15-42°C and pH 4.0-10.0 (optima at 28-37°C and pH 6.0-8.0). The growth of strain CPCC 101083T occurred at 4-45°C and pH 4.0-10.0 (optima at 25-30°C and pH 6.0-8.0). The major cellular fatty acids of CPCC 101082T and CPCC 101083T contained C18:1 ω7c/C18:1 ω6c, cyclo-C19:0 ω8c, and C16:0. Q-10 was detected as the sole respiratory quinone. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, an unidentified phospholipid and an unidentified aminolipid were tested in the polar lipids profile. The genomes of the two isolates were characterized as about 5.9 Mbp in size with the G + C content of nearly 68%. The IAA-producing encoding genes were predicated in both genomes. The values of average nucleotide identity were 80.6, 81.2 and 92.4% based on a pairwise comparison of the genomes of strains CPCC 101082T and CPCC 101083T and Geminicoccus roseus DSM 18922T, respectively. On the basis of the genotypic, chemotaxonomic and phenotypic characteristics, the strains CPCC 101082T (=NBRC 113513T = KCTC 62853T) and CPCC 101083T (=NBRC 113514T = KCTC 62854T) are proposed to represent two novel species of the genus Geminicoccus with the names Geminicoccus flavidas sp. nov. and Geminicoccus harenae sp. nov.

Characterization of three Stenotrophomonas strains isolated from different ecosystems and proposal of Stenotrophomonas mori sp. nov. and Stenotrophomonas lacuserhaii sp. nov.

Stenotrophomonas spp. have primarily been reported as non-pathogenic, plant-probiotic bacteria, despite the presence of some opportunistic human pathogens in the genus. Here, three Gram-stain negative, rod-shaped, non-spore-forming bacteria, designated as strains CPCC 101365T, CPCC 101269T, and CPCC 101426 were isolated from surface-sterilized medicinal plant roots of a mulberry plant in Chuxiong of the Yunnan Province, freshwater from Erhai Lake in the Yunnan Province, and sandy soils in the Badain Jaran desert in Inner Mongolia Autonomous Region, China, respectively. The 16S rRNA gene sequences analysis of these isolates in comparison with sequences from the GenBank database indicated that they belong to the genus Stenotrophomonas, with nucleotide similarities of 96.52-99.92% to identified Stenotrophomonas members. Phylogenetic analysis based on 16S rRNA gene and genome sequences confirmed that the isolates are members of the genus Stenotrophomonas. Values for genomic average nucleotide identity (ANI; <95%) and digital DNA-DNA hybridization (dDDH; < 70%) indicated that strains CPCC 101365T and CPCC 101269T were well-differentiated from validly described Stenotrophomonas species, while strain CPCC 101426 shared high ANI (97.7%) and dDDH (78.3%) identity with its closest phylogenetic neighbor, Stenotrophomonas koreensis JCM 13256T. The three genomes were approximately 3.1-4.0 Mbp in size and their G + C content ranged in 66.2-70.2%, with values slightly differing between CPCC 101365T (3.4 Mbp; 70.2%), CPCC 101269T (4.0 Mbp; 66.4%), and CPCC 101426 (3.1 Mbp; 66.2%). Genes encoding enzymes involved in the biosynthesis of indole-3-acetic acid (IAA) and siderophores were identified in the genomes of the three isolates, suggesting that these strains might serve roles as plant-growth promoting microorganisms. The polar lipid fractions of the three isolates primarily comprised diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE). The predominant cellular fatty acid was iso-C15: 0, with moderate amounts of antesio-C15: 0, iso-C11: 0, iso C17: 1 É·9c/C16: 0 10-methyl, iso-C14: 0, and C16: 1 É·7c/C16: 1 É·6c. These results indicated that polyphasic characteristics of strains CPCC 101365T and CPCC 101269T differed from other identified Stenotrophomonas species and that strain CPCC 101426 was affiliated with the species Stenotrophomonas koreensis. Accordingly, two novel species of the genus Stenotrophomonas were consequently proposed, corresponding to Stenotrophomonas mori sp. nov. (type strain CPCC 101365T = DY006T = KCTC 82900T) and Stenotrophomonas lacuserhaii sp. nov. (type strain CPCC 101269T = K32T = KCTC 82901T). Highlights: Members of the genus Stenotrophomonas, and particularly Stenotrophomonas maltophilia, are opportunistic human pathogens, but not enough research has evaluated the identification of environmental Stenotrophomonas spp. However, most Stenotrophomonas spp. serves as plant-probiotic bacteria.In this study, we obtained and characterized three Stenotrophomonas strains from different ecosystems. Based on phenotypic differences, chemotaxonomic properties, ANI and dDDH identity values, and phylogenetic analyses, two novel Stenotrophomonas species are proposed for the strains identified here. The encoding genes related to plant-growth promotion in the genomes of the newly recovered Stenotrophomonas spp. were retrieved. Follow-on experiments confirmed that these strains produced the important plant hormone IAA. Thus, these Stenotrophomonas spp. could considerably contribute to shaping and maintaining ecological stability in plant-associated environments, particularly while acting as plant-probiotic microorganisms.

Oligodendrocyte precursor cell transplantation promotes functional recovery following contusive spinal cord injury in rats and is associated with altered microRNA expression.

It has been reported that oligodendrocyte precursor cells (OPCs) may be used to treat contusive spinal cord injury (SCC), and may alter microRNA (miRNA/miR) expression following SCC in rats. However, the association between miRNA expression and the treatment of rats with SCC with OPC transplantation remain unclear. The present study transplanted OPCs into the spinal cord of rats with SCC and subsequently used the Basso, Beattie and Bresnahan (BBB) score to assess the functional recovery and pain scores. An miRNA assay was performed to detect differentially expressed miRNAs in the spinal cord of SCC rats transplanted with OPCs, compared with SCC rats transplanted with medium. Quantitative polymerase chain reaction was used to verify significantly altered miRNA expression levels. The results demonstrated that OPC transplantation was able to improve motor recovery and relieve mechanical allodynia in rats with SCC. In addition, through a miRNA assay, 45 differentially expressed miRNAs (40 upregulated miRNAs and 5 downregulated miRNAs) were detected in the spinal cord of rats in the OPC group compared with in the Medium group. Differentially expressed miRNAs were identified according to the following criteria: Fold change >2 and P<0.05. Furthermore, quantitative polymerase chain reaction was used to verify the most highly upregulated (miR­375­3p and miR­1­3p) and downregulated (miR­363­3p, miR­449a­5p and miR­3074) spinal cord miRNAs that were identified in the miRNA assay. In addition, a bioinformatics analysis of these miRNAs indicated that miR­375 and miR­1 may act primarily to inhibit cell proliferation and apoptosis via transcriptional and translational regulation, whereas miR­363, miR­449a and miR­3074 may act primarily to inhibit cell proliferation and neuronal differentiation through transcriptional regulation. These results suggested that OPC transplantation may promote functional recovery of rats with SCC, which may be associated with the expression of various miRNAs in the spinal cord, including miR­375­3p, miR­1­3p, miR­363­3p, miR­449a­5p and miR­3074.

MicroRNA expression profiling of intestinal mucosa tissue predicts multiple crucial regulatory molecules and signaling pathways for gut barrier dysfunction of AIDS patients.

Human immunodeficiency virus­1 (HIV­1) infection severely damages the gut­associated lymphoid tissue (GALT), the immune system and the gut barrier, which leads to accelerating the disease progression for patients with acquired immune deficiency syndrome (AIDS). Dysregulation of microRNAs (miRNAs) may contribute to this process. However, few studies have investigated the importance of miRNAs in AIDS pathogenesis and progression. The whole miRNA profile of patients with HIV infection from southwest P.R. China and the mode of interaction between HIV­1 and miRNAs remains to be elucidated. Colon mucosal samples were collected from HIV+ patients and HIV­ healthy individuals, miRNAs were isolated and subjected to array hybridization in the present study. A total of 476 human and virus­derived microRNAs were significantly altered in the HIV+ group when compared with the control group (P<0.05), which may be involved in the progression to AIDS. Target genes of the significantly altered miRNAs were predicted using the TargetScan, miRbase and miRanda databases and the 10 shared target genes of upregulated miRNAs and the 391 target genes of downregulated miRNAs were selected. As only 10 target genes were predicted for upregulated miRNAs, subsequent GO and KEGG pathway analyses were focused on the 391 target genes of the downregulated miRNAs. The findings of the present study identified a series of crucial pathways, including cell­extracellular matrix interaction and chemokine regulation, which indicated close affinity with CD4+ T cell activation. These pathways, involving genes such as integrin α5, led to a gut barrier dysfunction of patients with HIV. Important miRNAs include hsa­miRNA­32­5p, hsa­miRNA­195­5p, hsa­miRNA­20b­5p, hsa­miRNA­590­5p. The expression levels of the miRNAs and their target genes were confirmed using RT­qPCR. Taking into previous observations, the findings of the present study identified the importance of miRNAs for regulating gut barrier dysfunction via multiple regulatory molecules and signaling pathways, which elucidated the underlying molecular mechanism of gut barrier dysfunction in patients with HIV.

Lentiviral-Mediated Netrin-1 Overexpression Improves Motor and Sensory Functions in SCT Rats Associated with SYP and GAP-43 Expressions.

Spinal cord injury (SCI), as a major cause of disability, usually causes serious loss of motor and sensory functions. As a bifunctional axonal guidance cue, netrin-1 can attract axons via the deleted in colorectal cancer (DCC) receptors and repelling others via Unc5 receptors, but its exact role in the recovery of motor and sensory function has not well been studied, and the mechanisms remains elusive. The aim of this experiment is to determine whether lentiviral (LV)-mediated overexpression of netrin-1 or RNA interference (RNAi) can regulate the functional recovery in rats subjected to spinal cord transection (SCT). Firstly, two lentiviral vectors including Lv-exNtn-1 (netrin-1 open reading frame (ORF)) and Lv-shNtn-1 (netrin-1 sh) were constructed and injected into spinal cords rostral and caudal to the transected lesion site. Overexpressing netrin-1 enhanced significantly locomotor function, and reduced thermal and mechanical stimuli in vivo, compared with the control, while silencing netrin-1 did not significantly change the situation. Western blot and immunostaining analysis confirmed that netrin-1 ORF treatment not only effectively increased the expression level of netrin-1, also up-regulated the level of synaptophysin (SYP) in spinal cord rostral to the lesion, but also enhanced growth-associated protein-43 (GAP-43) expression in spinal cord caudal to the lesion site. Comparatively, knockdown of netrin-1 did not give rise to positive findings in our experimental condition. These findings therefore pointed that Lv-mediated netrin-1 overexpression could promote motor and sensory functional recoveries following SCT, and the underlying mechanisms were associated with SYP and GAP-43 expressions. The present study therefore provided a novel strategy for the treatment of SCI and explained the possible mechanisms for the functional improvement.

[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.

Wnt/ß-catenin pathway might underlie the MET in trans- differentiation from MSC to MSC-derived neuron.

OBJECTIVE: To observe MET-associated alteration during the trans-differentiation from MSCs to neuron-like cells, and to explore the possible molecular mechanism. METHODS: 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/ß-catenin pathway were also analyzed by comparative PCR to determine the possible involvement. RESULTS: 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/ß-catenin target genes ß-catenin and c-myc were decreasingly expressed. CONCLUSION: 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/ß-catenin pathway.

Lacking chloroplasts in guard cells of crumpled leaf attenuates stomatal opening: both guard cell chloroplasts and mesophyll contribute to guard cell ATP levels.

Controversies regarding the function of guard cell chloroplasts and the contribution of mesophyll in stomatal movements have persisted for several decades. Here, by comparing the stomatal opening of guard cells with (crl-ch) or without chloroplasts (crl-no ch) in one epidermis of crl (crumpled leaf) mutant in Arabidopsis, we showed that stomatal apertures of crl-no ch were approximately 65-70% those of crl-ch and approximately 50-60% those of wild type. The weakened stomatal opening in crl-no ch could be partially restored by imposing lower extracellular pH. Correspondingly, the external pH changes and K(+) accumulations following fusicoccin (FC) treatment were greatly reduced in the guard cells of crl-no ch compared with crl-ch and wild type. Determination of the relative ATP levels in individual cells showed that crl-no ch guard cells contained considerably lower levels of ATP than did crl-ch and wild type after 2 h of white light illumination. In addition, guard cell ATP levels were lower in the epidermis than in leaves, which is consistent with the observed weaker stomatal opening response to white light in the epidermis than in leaves. These results provide evidence that both guard cell chloroplasts and mesophyll contribute to the ATP source for H(+) extrusion by guard cells.

[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.

[Effect of proteasome inhibitor bortezomib on proliferation, apoptosis and XIAP expression in K562 cells].

OBJECTIVE: To investigate the effect of proteasome inhibitor bortezomib on proliferation, apoptosis of K562 cells and the expression of XIAP. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Effect of gemcitabine on granulocyte colony-stimulating factor receptor and bcr/abl mRNA in patients with chronic myeloid leukemia].

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.

[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.

[Influence of gemcitabine on expression of C-myc gene and its apoptosis-inducing effect on HL-60 cells].

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.

[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.