Ratiometric Fluorescence Probes for In Situ Imaging of Membrane Tension in Live Cells.

Membrane tension is an important physical parameter of describing cellular homeostasis, and it is widely used in the study of cellular processes involving membrane deformation and reorganization, such as cell migration, cell spreading, and cell division. Despite the importance of membrane tension, direct measurement remains difficult. In this work, we developed a ratiometric fluorescent probe sensitive to membrane tension by adjusting the carbon chain structure based on polarity-sensitive fluorophores. The probe is sensitive to changes in membrane tension after cells were subjected to physical or chemical stimuli, such as osmotic shock, lipid peroxidation, and mechanical stress. When the polarity of the plasma membrane increases (the green/red ratio decreases) and the membrane tension increases, the relative magnitude of the membrane tension can be quantitatively calculated by fluorescence ratio imaging. Thus, the probe proved to be an efficient and sensitive membrane tension probe.

In Situ Quantitative Imaging of Plasma Membrane Stiffness in Live Cells Using a Genetically Encoded FRET Sensor.

Cell membrane stiffness is critical for cellular function, with cholesterol and sphingomyelin as pivot contributors. Current methods for measuring membrane stiffness are often invasive, ex situ, and slow in process, prompting the need for innovative techniques. Here, we present a fluorescence resonance energy transfer (FRET)-based protein sensor designed to address these challenges. The sensor consists of two fluorescent units targeting sphingomyelin and cholesterol, connected by a linker that responds to the proximity of these lipids. In rigid membranes, cholesterol and sphingomyelin are in close proximity, leading to an increased FRET signal. We utilized this sensor in combination with confocal microscopy to explore changes in plasma membrane stiffness under various conditions, including differences in osmotic pressure, the presence of reactive oxygen species (ROS) and variations in substrate stiffness. Furthermore, we explored the impact of SARS-CoV-2 on membrane stiffness and the distribution of ACE2 after attachment to the cell membrane. This tool offers substantial potential for future investigations in the field of mechanobiology.

Electrochemiluminescence-Based Single-Particle Tracking of the Biomolecules Moving along Intercellular Membrane Nanotubes between Live Cells.

Electrochemiluminescence (ECL) imaging, a rapidly evolving technology, has attracted significant attention in the field of cellular imaging. However, its primary limitation lies in its inability to analyze the motion behaviors of individual particles in live cellular environments. In this study, we leveraged the exceptional ECL properties of quantum dots (QDs) and the excellent electrochemical properties of carbon dots (CDs) to develop a high-brightness ECL nanoprobe (CDs-QDs) for real-time ECL imaging between living cells. This nanoprobe has excellent signal-to-noise ratio imaging capabilities for the single-particle tracking (SPT) of biomolecules. Our finding elucidated the enhanced ECL mechanism of CDs-QDs in the presence of reactive oxygen species through photoluminescence, electrochemistry, and ECL techniques. We further tracked the movement of single particles on membrane nanotubes between live cells and confirmed that the ECL-based SPT technique using CD-QD nanoparticles is an effective approach for monitoring the transport behaviors of biomolecules on membrane nanotubes between live cells. This opens a promising avenue for the advancement of ECL-based single-particle detection and the dynamic quantitative imaging of biomolecules.

Accurate Cancer Screening and Prediction of PD-L1-Guided Immunotherapy Efficacy Using Quantum Dot Nanosphere Self-Assembly and Machine Learning.

Accurate quantification of exosomal PD-L1 protein in tumors is closely linked to the response to immunotherapy, but robust methods to achieve high-precision quantitative detection of PD-L1 expression on the surface of circulating exosomes are still lacking. In this work, we developed a signal amplification approach based on aptamer recognition and DNA scaffold hybridization-triggered assembly of quantum dot nanospheres, which enables bicolor phenotyping of exosomes to accurately screen for cancers and predict PD-L1-guided immunotherapeutic effects through machine learning. Through DNA-mediated assembly, we utilized two aptamers for simultaneous ultrasensitive detection of exosomal antigens, which have synergistic roles in tumor diagnosis and treatment prediction, and thus, we achieved better sample classification and prediction through machine-learning algorithms. With a drop of blood, we can distinguish between different cancer patients and healthy individuals and predict the outcome of immunotherapy. This approach provides valuable insights into the development of personalized diagnostics and precision medicine.

Engineered Lipidic Nanomaterials Inspired by Sphingomyelin Metabolism for Cancer Therapy.

Sphingomyelin (SM) and its metabolites are crucial regulators of tumor cell growth, differentiation, senescence, and programmed cell death. With the rise in lipid-based nanomaterials, engineered lipidic nanomaterials inspired by SM metabolism, corresponding lipid targeting, and signaling activation have made fascinating advances in cancer therapeutic processes. In this review, we first described the specific pathways of SM metabolism and the roles of their associated bioactive molecules in mediating cell survival or death. We next summarized the advantages and specific applications of SM metabolism-based lipidic nanomaterials in specific cancer therapies. Finally, we discussed the challenges and perspectives of this emerging and promising SM metabolism-based nanomaterials research area.

Artificial Cells for Cellular Behavior Mimicry Induced by Sphingomyelin Degradation.

Sphingomyelinase (SMase), a hydrolase of sphingomyelin (SM) enriched in the outer leaflet of the plasma membrane of mammalian cells, is closely associated with the onset and development of many diseases, but the specific mechanisms of SMase on the cell structure, function, and behavior are not yet fully understood due to the complexity of the cell structure. Artificial cells are minimal biological systems constructed from various molecular components designed to mimic cellular processes, behaviors, and structures, which are excellent models for studying biochemical reactions and dynamic changes in cell membranes. In this work, we presented an artificial cell model that mimics the lipid composition and content of the outer leaflet of mammalian plasma membranes for studying the effect of SMase on cell behavior. The results confirmed that the artificial cells can respond to SM degradation by producing ceramides that enrich and alter the membrane charge and permeability, thus inducing the budding and fission of the artificial cells. Thus, the artificial cells developed here provide a powerful tool to study the mechanism of action of cell membrane lipids on cell biological behavior, paving the way for further molecular mechanism studies.

Neuroprotective effect and mechanism of cPLA2 inhibitor increases autophagic flux on spinal cord injury / 中国骨伤

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.

ADP-Ribosylation Factor Like GTPase 4C (ARL4C) augments stem-like traits of glioblastoma cells by upregulating ALDH1A3.

Glioma cells with stem cell-like properties are crucial for tumor initiation, progression and therapeutic resistance. Therefore, identifying specific factors in regulating stem-like traits is critical for the design of novel glioma therapeutics. Herein, we reported that ADP-Ribosylation Factor Like GTPase 4C (ARL4C) was highly expressed in glioma stem-like cells (GSLCs). GSLCs, determined by the efficiency of sphere formation in vitro and tumor growth in vivo, was increased by overexpression of ARL4C. ARL4C induced the tumorigenesis through ALDH1A3. Analyses of 325 patient specimens showed that ARL4C was highly expressed in glioblastoma (GBM) as compared with lower grade gliomas. In addition, higher level ARL4C expression in glioma was correlated with poorer progression-free survival and overall survival of patients. Therefore, ARL4C may act as a novel prognostic marker and a therapeutic target for GBM.

Measurement of Foxp3 and NFAT1 in children with aplastic anemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>

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>

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>

[Study on the correlation of the levels of serum IFN-γ and RANTES and rabbit vulnerable atherosclerotic plaque].

AIM: To investigate the correlation between the levels of serum IFN-γ and RANTES (regulated upon on activation on normal T cell expressed and secreted) and rabbit atherosclerotic plaque stability. METHODS: 30 male New Zealand rabbits were randomly divided into three groups: control group, stable atherosclerotic plaque group(AS group) and vulnerable atherosclerotic plaque group(VAP group), with 10 rabbits in a group. The control group was given normal diet while the AS group and the VAP group were given high-fat diet for 12 weeks. According to literature method pharmacological triggers inducing the vulnerable atherosclerotic plaque formation in the VAP group. Fasting blood was extracted from ear medium artery about 2 mL at week-0 and the 12th week, serum total cholesterol, triglyceride and high density lipoprotein cholesterol were detected by enzymatic method, and low density lipoprotein cholesterol was calculated by Friedwald formula. All animals were executed after they were fed by injected excessive Phenobarbital sodium at ear vein for 12 weeks. Before executed, ear medium artery blood was taken to detect the levels of serum IFN-γ and RANTES by ELISA.The corrected plaque area and vulnerability index were calculated by Masson staining and its correlation coefficient with inflammatory factors were measured. RESULTS: The level of serum IFN-γ at the AS group and the VAP group, compared with the control group, was significantly increased(all P<0.01), at the same time, the level of serum IFN-γ in the VAP group was significantly higher than in the AS group(P<0.01). About the level of serum RANTES there was no obvious difference when the AS group was compared with the control group(P>0.05). The level of serum RANTES in the VAP group was higher than in the AS group and the control group(all P<0.01). Regarding corrected plaque area and vulnerability Index, the VAP group was significantly higher than the AS group (P<0.01). In the AS group and the VAP group, the serum concentrations of IFN-γand RANTES exhibited a positive correlation with corrected plaque area and vulnerability Index (P<0.05). CONCLUSION: IFN-γ and RANTES may be inflammatory marker about atherosclerotic vulnerable plaque, and their serum concentration can be used to evaluate the atherosclerotic plaque instability.

[Nifedipine regulated expression of bcl-2 in human gingival epithelial cells in vitro].

PURPOSE: To investigate the effect of nifedipine(NIF) on transcription of bcl-2 in human gingival epithelial cells(HGECs) in vitro and to study the pathogenesis of epithelial thickening in drug-induced gingival overgrowth(DGO). METHODS: The gingival tissues obtained from periodontal surgeries were digested with enzyme and HGECs were cultured in vitro; HGECs were identified by immunohistochemistry; bcl-2 mRNA levels were quantitated by Real-time PCR 24 hours and 48 hours after cells stimulated by NIF with different concentration (0microg/ml, 1microg/ml,2microg/ml,3microg/ml), in which 0microg/ml NIF as blank control. The data was analyzed by one-way ANOVA using SPSS 11.0 software package. RESULTS: HGECs cultured in vitro showed keratin positive signal and vimentin negative signal; the level of bcl-2 mRNA increased with NIF 3microg/ml after 24 hours treatment, which appeared significant increase compared with blank control (P<0.05); after 48 hours treatment the level of bcl-2 mRNA in the groups of 2microg/ml and 3microg/ml showed significant increase compared with blank control (P<0.05). CONCLUSION: NIF regulates the level of bcl-2 mRNA.

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>

[Amelogenin expression in cultured human dental pulp and periodontium related cells].

PURPOSE: To investigate gene expression of amelogenin (Am) in human gingival epithelial cells(HGEC) and also other oral ectomesenchyme cells (human gingival fibroblasts, human periodontal ligament fibroblasts and human pulp cells). METHODS: The amelogenin mRNA expression patterns were determined by the reverse transcription polymerase chain reaction (RT-PCR),and the protein expression was studied with Western blotting. RESULTS: There was no amelogenin expression detected in any of the cells. CONCLUSIONS: These findings suggest that amelogenin expression could not be detected in cultured human periodontium-related cells. Supported by National Natural Science Foundation of China (Grant No.30672315) and Research Fund of Science and Technology Commission of Shanghai Municipality(Grant No.08DZ2271100).

[Development of a multiplex PCR-suspension array for simultaneous detection of five bioterrorism bacteria].

OBJECTIVE: To develop a rapid, high-throughput screening method of gene suspension array technique to simultaneously detect five bioterrorism bacteria: Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp. and Burkholderia pseudomallei. METHODS: Highly validated specific primers were used to amplify diagnostic regions unique to each pathogen. Biotin labelled PCR products were hybridized to corresponding probes coupling on the unique sets of fluorescent beads. The hybridized beads were processed through the Bio-plex, which identified the presence of PCR products. RESULTS: Multiplex PCR-suspension array can detect five bioterrorism bacteria correctly with high specificity and high sensitivity, the results suggest the utility of suspension array system for high-throughput screening of bioterrorism samples. CONCLUSION: A multiplex PCR-suspension array for rapid detection of five bioterrorism bacteria was established.

[Development of a universal primers PCR-coupled liquid bead array to detect biothreat bacteria].

OBJECTIVE: To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria. METHODS: 16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. RESULTS: After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. CONCLUSION: The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.

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>

Human bone marrow mesenchymal stem cells differentiated into dopaminergenic neurons in vitro / 生物工程学报

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.

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.