ObjectiveTo explore the effects of hypoxia on the migration ability of human oligodendrocyte precursor cells （hOPCs） and its regulatory mechanisms. MethodsBased on the variations in oxygen concentration within the culture system， three experimental groups were set up： the 21%O₂ group （normoxic control group）， the 5%O₂ group， and the 2%O₂ group. The migration ability of hOPCs under normoxia （21%O₂）， 5%O₂， and 2%O₂ conditions was detected through the Transwell migration assay. RT-qPCR， transcriptome sequencing， and flow cytometry were used to detect the expression changes of genes and proteins such as hypoxia-inducible factor 1 alpha （HIF-1α） and chemokine （C-X-C Motif） receptor 4 （CXCR4）. Bioinformatics analysis was combined to analyze the KEGG pathways related to migration， so as to explore the effects of different oxygen concentrations on the migration ability of hOPCs and their possible mechanisms. ResultsHypoxia treatments at concentrations of 5%O₂ and 2%O₂ could both promote the in vitro migration of hOPCs， and the promoting effect of migration was more significant at the 2%O₂ concentration （P<0.001）. After hypoxia treatment， the mRNA expression levels of HIF-1α， CXCR4， etc. in hOPCs significantly increased （P<0.001）. Compared with the 5%O₂ concentration， the expression of CXCR4 in cells was higher at the 2%O₂ concentration （P<0.000 1）. Flow cytometry analysis detection showed that the expression of CXCR4 increased significantly after hypoxia treatment （P<0.01）， and with the decrease of oxygen concentration， its expression level further increased （P<0.000 1）. Ordinary transcriptome sequencing analysis indicated that hypoxia treatment could activate the PI3K-Akt signaling pathway and the Axon guidance pathway. ConclusionHypoxia treatment can enhance the in vitro migration ability of hOPCs， and this effect is negatively correlated with the oxygen concentration. Its mechanism may be related to the up-regulation of the expression of genes such as HIF-1α and CXCR4， and the activation of the migration related signaling pathway including PI3K-Akt signaling pathway and axon guidance pathway.

This report describes two cases of severe immune-mediated thrombocytopenia after allogeneic hematopoietic stem cell transplantation (HSCT) who were treated with umbilical cord mesenchymal stem cells (UC-MSCs). Case 1 was a child with severe aplastic anemia who underwent haploidentical bone marrow and peripheral blood HSCT, with a chimerism rate of 99.8% on day +25 and severe immune-mediated thrombocytopenia on day +60. After intravenous immunoglobulin (IVIG) pulse therapy, platelet count increased temporarily but then decreased, while cyclosporine, methylprednisolone, and rituximab had a poor therapeutic effect. Case 2 was a child with Gaucher's disease who underwent unrelated umbilical cord blood HSCT, with a chimerism rate of 96.35% on day +41 and severe immune-mediated thrombocytopenia on day +153. After three sessions of IVIG pulse therapy, the platelet count increased initially but subsequently decreased. Therapies with dexamethasone, prednisone, cyclosporine, and recombinant human thrombopoietin also yielded a poor response. Both children received three sessions of UC-MSCs infusion, and platelet counts increased and were subsequently maintained within the normal range. Case 1 has been followed up for 10 years and remains in disease-free survival. UC-MSCs infusion may be effective for severe immune-mediated thrombocytopenia that is unresponsive to first- and second-line therapies after HSCT and could potentially improve the quality of life and disease-free survival rate.
Child ; Humans ; Hematopoietic Stem Cell Transplantation/adverse effects* ; Mesenchymal Stem Cell Transplantation ; Purpura, Thrombocytopenic, Idiopathic/etiology* ; Thrombocytopenia/therapy* ; Transplantation, Homologous ; Umbilical Cord/cytology*

AIM:We investigated the survival,migration and differentiation abilities of human oligodendro-cyte precursor cells(hOPC)in the brains of mice with vascular dementia(VaD),the effects of hOPC on cerebral white matter,and the underlying mechanisms.METHODS:Mouse VaD model was constructed using the bilateral common ca-rotid artery stenosis method,and the mice were randomly divided into sham,VaD and hOPC groups.Eight weeks after model establishment,the mice in VaD and hOPC groups received equal volume of vehicle(PBS)and hOPC solution,re-spectively,through the corpus callosum.Survival,migration and differentiation of hOPC in the brain were observed by im-munofluorescence staining at 4 and 12 weeks after transplantation.Twelve weeks after transplantation,the effects of hOPC on mouse brain white matter were detected by immunofluorescence staining of myelin basic protein(MBP),myelin-associ-ated glycoprotein(MAG),neurofilament protein 200(NF200)and non-phosphorylated neurofilament H(using monoclo-nal antibody SMI32),and by water maze experiments.Paracrine signaling by hOPC was explored using immunofluores-cence staining for vascular endothelial growth factor(VEGF).RESULTS:The hOPC survived in the brains of VaD mice for 12 weeks,migrated to damaged white matter areas,and partially differentiated into mature oligodendrocytes(approxi-mately 64%).Twelve weeks after transplantation,hOPC significantly increased the fluorescence intensity of MBP,MAG,and NF200(P<0.05 or P<0.01)and decreased the fluorescence intensity of SMI32(P<0.01).The VEGF expression in hOPC-treated mice was significantly higher than that in sham and VaD groups(P<0.01).The difference in water maze test performance between hOPC and sham groups was not statistically significant(P>0.05).The mice in hOPC group had a shorter latency than those in VaD group(P<0.05 or P<0.01),and performed more platform crossings than those in VaD group(P<0.05).CONCLUSION:The hOPC can survive,migrate and differentiate in the brains of VaD mice,attenuate cerebral white matter lesions,and improve cognitive function.These improvements may be attributed to cell replacement and paracrine effects.

BACKGROUND:Oligodendrocyte precursor cells are seed cells for the treatment of white matter damage diseases.Establishing an efficient and stable in vitro differentiation method is an important prerequisite for clinical translational research.OBJECTIVE:To investigate the effect of fibronectin on biological characteristics such as proliferation,migration,and differentiation of oligodendrocyte precursor cells derived from human neural stem cells.METHODS:Human neural stem cells cultured in suspension were digested into single cells using Accutase.The expression of specific markers Nestin,Sox2,Vimentin,CD133,and Musashi was detected by flow cytometry.The single cells of human neural stem cells were resuspended in oligodendrocyte precursor cell medium and seeded in six-well plates coated with different concentrations of fibronectin(0,1,2.5,5,and 10 μg/mL).Accutase digestion was performed after 7 days of culture.Cells were counted by trypan staining.Fibronectin-coated group with the strongest amplification ability and the oligodendrocyte precursor cells without fibronectin-coated group were selected for further tests.The migration ability of the two groups of cells was detected by Transwell.Flow cytometry was used to detect the expression of Olig2,Sox10,and PDGFR-α.Oligodendrocyte precursor cells were induced to differentiate into oligodendrocytes for 3 weeks,and the expression of Galc in differentiated cells was detected by immunofluorescence staining.RESULTS AND CONCLUSION:(1)H uman neural stem cells grew in suspension spheres.Flow cytometry showed that human neural stem cells highly expressed Nestin,Sox2,Vimentin,CD133,and Musashi.(2)The cell bodies of oligodendrocyte precursor cells induced by human neural stem cells were round or oval,with strong refractive nature and bipolar or tertiary protrusions.Compared with the 0 μg/mL fibronectin coating group,there was a significant difference in the amplification ability of oligodendrocyte precursor cells in the 2.5,5,and 10 μg/mL fibronectin coating groups(P＜0.05).The amplification ability of oligodendrocyte precursor cells was the strongest when the fibronectin concentration was 10 μg/mL.(3)Flow cytometry results showed that the oligodendrocyte precursor cell markers 0Iig2,Sox10,and PDGFR-α were highly expressed in the 0 and 10 μg/mL fibronectin coating groups,and there was no significant difference between the two groups(P＞0.05).(4)Transwell chamber assay results showed that compared with the 0 μg/mL fibronectin-coated group,the migration ability of oligodendrocyte precursor cells in the 10 μg/mL fibronectin-coated group was increased(P＜0.01).(5)After 3 weeks of differentiation into oligodendrocytes,oligodendrocyte precursor cells showed complex morphology with multiple branches,grids or membrane sheets.Immunofluorescence staining results showed that there was no statistical difference in the Galc positive rate of oligodendrocytes between the two groups(P＞0.05).These findings indicate that when the concentration of fibronectin coated well plate is 10 μg/mL,the proliferation and migration of oligodendrocyte precursor cells are the strongest,but it does not affect the expression of oligodendrocyte precursor cells-specific markers Olig2,Sox10,and PDGFR-α and their differentiation into oligodendrocytes.

AIM:We investigated the survival,migration and differentiation abilities of human oligodendro-cyte precursor cells(hOPC)in the brains of mice with vascular dementia(VaD),the effects of hOPC on cerebral white matter,and the underlying mechanisms.METHODS:Mouse VaD model was constructed using the bilateral common ca-rotid artery stenosis method,and the mice were randomly divided into sham,VaD and hOPC groups.Eight weeks after model establishment,the mice in VaD and hOPC groups received equal volume of vehicle(PBS)and hOPC solution,re-spectively,through the corpus callosum.Survival,migration and differentiation of hOPC in the brain were observed by im-munofluorescence staining at 4 and 12 weeks after transplantation.Twelve weeks after transplantation,the effects of hOPC on mouse brain white matter were detected by immunofluorescence staining of myelin basic protein(MBP),myelin-associ-ated glycoprotein(MAG),neurofilament protein 200(NF200)and non-phosphorylated neurofilament H(using monoclo-nal antibody SMI32),and by water maze experiments.Paracrine signaling by hOPC was explored using immunofluores-cence staining for vascular endothelial growth factor(VEGF).RESULTS:The hOPC survived in the brains of VaD mice for 12 weeks,migrated to damaged white matter areas,and partially differentiated into mature oligodendrocytes(approxi-mately 64%).Twelve weeks after transplantation,hOPC significantly increased the fluorescence intensity of MBP,MAG,and NF200(P<0.05 or P<0.01)and decreased the fluorescence intensity of SMI32(P<0.01).The VEGF expression in hOPC-treated mice was significantly higher than that in sham and VaD groups(P<0.01).The difference in water maze test performance between hOPC and sham groups was not statistically significant(P>0.05).The mice in hOPC group had a shorter latency than those in VaD group(P<0.05 or P<0.01),and performed more platform crossings than those in VaD group(P<0.05).CONCLUSION:The hOPC can survive,migrate and differentiate in the brains of VaD mice,attenuate cerebral white matter lesions,and improve cognitive function.These improvements may be attributed to cell replacement and paracrine effects.

BACKGROUND:Oligodendrocyte precursor cells are seed cells for the treatment of white matter damage diseases.Establishing an efficient and stable in vitro differentiation method is an important prerequisite for clinical translational research.OBJECTIVE:To investigate the effect of fibronectin on biological characteristics such as proliferation,migration,and differentiation of oligodendrocyte precursor cells derived from human neural stem cells.METHODS:Human neural stem cells cultured in suspension were digested into single cells using Accutase.The expression of specific markers Nestin,Sox2,Vimentin,CD133,and Musashi was detected by flow cytometry.The single cells of human neural stem cells were resuspended in oligodendrocyte precursor cell medium and seeded in six-well plates coated with different concentrations of fibronectin(0,1,2.5,5,and 10 μg/mL).Accutase digestion was performed after 7 days of culture.Cells were counted by trypan staining.Fibronectin-coated group with the strongest amplification ability and the oligodendrocyte precursor cells without fibronectin-coated group were selected for further tests.The migration ability of the two groups of cells was detected by Transwell.Flow cytometry was used to detect the expression of Olig2,Sox10,and PDGFR-α.Oligodendrocyte precursor cells were induced to differentiate into oligodendrocytes for 3 weeks,and the expression of Galc in differentiated cells was detected by immunofluorescence staining.RESULTS AND CONCLUSION:(1)H uman neural stem cells grew in suspension spheres.Flow cytometry showed that human neural stem cells highly expressed Nestin,Sox2,Vimentin,CD133,and Musashi.(2)The cell bodies of oligodendrocyte precursor cells induced by human neural stem cells were round or oval,with strong refractive nature and bipolar or tertiary protrusions.Compared with the 0 μg/mL fibronectin coating group,there was a significant difference in the amplification ability of oligodendrocyte precursor cells in the 2.5,5,and 10 μg/mL fibronectin coating groups(P＜0.05).The amplification ability of oligodendrocyte precursor cells was the strongest when the fibronectin concentration was 10 μg/mL.(3)Flow cytometry results showed that the oligodendrocyte precursor cell markers 0Iig2,Sox10,and PDGFR-α were highly expressed in the 0 and 10 μg/mL fibronectin coating groups,and there was no significant difference between the two groups(P＞0.05).(4)Transwell chamber assay results showed that compared with the 0 μg/mL fibronectin-coated group,the migration ability of oligodendrocyte precursor cells in the 10 μg/mL fibronectin-coated group was increased(P＜0.01).(5)After 3 weeks of differentiation into oligodendrocytes,oligodendrocyte precursor cells showed complex morphology with multiple branches,grids or membrane sheets.Immunofluorescence staining results showed that there was no statistical difference in the Galc positive rate of oligodendrocytes between the two groups(P＞0.05).These findings indicate that when the concentration of fibronectin coated well plate is 10 μg/mL,the proliferation and migration of oligodendrocyte precursor cells are the strongest,but it does not affect the expression of oligodendrocyte precursor cells-specific markers Olig2,Sox10,and PDGFR-α and their differentiation into oligodendrocytes.

[Objective]To explore the protective effect of glucocorticoid-induced transcription factor 1(GLCCI1)on cognitive dysfunction in diabetic mice and its mechanism.[Methods]Twenty-four C57BL/6J mice were randomly divided into 4 groups,namely Control,DM,DM+AAV-Glcci1,and DM+AAV-NC.The Control group was intraperitoneally injected with saline,while the other groups were all injected with streptozotocin(STZ).Two weeks after successful modeling,the DM+AAV-Glcci1 group was brain stereotactic injected with Glcci1 overexpressing adeno-associated virus,and the DM+AAV-NC group was stereoscopically injected with the control virus.After 12 weeks,the Morris water maze test was used to evaluate the learning and memory abilities of mice in each group.Subsequently,the localized expression of GLCCI1 in the hippocampus were determined by immunofluorescence and immunohistochemistry experiments.The myelin morphology in the hippocampus was observed by LFB staining,the neuronal morphology was observed by Nissl staining,and the myelin-related proteins MBP and CNPase were stained by immunohistochemistry.Molecular docking was used to predict the interaction between GLCCI1 and HSPA5.The expression of endoplasmic reticulum stress-related proteins was detected by Western blot.[Results]The results of the behavioral experiment showed that compared with the mice in the Control group,DM mice exhibited obvious cognitive dysfunction behaviors(P＜0.000 1),and the learning and memory abilities of mice improved after overexpression of Glcci1(P=0.000 7).The results of immunofluorescence and immunohistochemistry showed that GLCCI1 was expressed in hippocampal neuron cells.Compared with Control mice,the expression level of GLCCI1 in DM mice was significantly downregulated(P＜0.000 1).The molecular docking results revealed that GLCCI1 interacts with HSPA5.The Western blot results indicated that,compared with the Control group,the expression levels of endoplasmic reticulum stress-related proteins HSPA5(P＜0.000 1),ATF4(P＜0.000 1),ATF6(P=0.001 1),and p-ELF2α/elF2α(P=0.000 1)in the DM group were significantly increased;Compared with the DM group,the expression of the corresponding protein HSPA5(P＜0.000 1),ATF4(P＜0.000 1),ATF6(P=0.000 2),and p-ELF2α/elF2α(P=0.000 1)was significantly down-regulated after overexpression of Glcci1.LFB staining showed that compared with the Control group,the myelin integrity of DM mice decreased significantly(P=0.010 3),the expressions of myelin-related proteins MBP and CNPase decreased significantly(P=0.000 4,P=0.000 2),and Nissl staining observed disordered neuronal arrangement.Compared with the mice in the DM group,the myelin integrity in the hippocampal region significantly increased after overexpression of Glcci1(P=0.000 3),the expressions of myelin-related proteins MBP and CNPase significantly increased(P=0.001 4,P=0.000 1),and the ordered arrangement of neurons was observed by Nissl staining.[Conclusion]The down-regulation of GLCCI1 expression in hippocampal neurons promotes demyelination of hippocampal neurons and thereby induces diabetic cognitive dysfunction.The specific mechanism may be related to endoplasmic reticulum stress.

Objective To establish the UPLC fingerprint chromatogram combined with chemometric analysis for the quality evaluation of classical formula Linggui Zhugan Decoction.Methods SHIMADZU Shim-Pack GIST C18 column(100 mm×2.1 mm,2.0 μm)was used with acetonitrile-0.1%phosphoric acid aqueous solution as mobile phase,gradient elution;flow rate was 0.2 mL/min;the detection wavelength was 266 nm for the first 30 minutes and 235 nm for the last 36 minutes;the column temperature was 30℃.The UPLC fingerprint of Linggui Zhugan Decoction was established by Similarity Evaluation System for Chromatographic Fingerprint of TCM(2012.130723 version),and the common peak was determined and the similarity evaluation was carried out.Based on the peak area determination results of the common peak of the fingerprint,the quality of different batches of Linggui Zhugan Decoction was evaluated by chemometrics such as clustering analysis and principal component analysis.Results A total of 24 common peaks were confirmed and 14 components were identified by using reference substances.The similarity of 10 batches of Linggui Zhugan Decoction samples was greater than 0.950,which could be divided into two categories by chemometrics,and the principal component 1-4 were the main factors affecting its quality evaluation.OPLS-DA identified 6 differential markers.Conclusion The fingerprint research method established in the study is simple,reliable and reproducible.Through the method of fingerprint combined with chemometrics analysis,the differences between Linggui Zhugan Decoction from different origins of medicinal materials are identified,which provides a reference for the internal quality evaluation of Linggui Zhugan Decoction.

To assess the reliability, validity and responsiveness of the Chinese version of the atopic dermatitis control tool (ADCT). After this study obtained authorization for the Chinese version of the ADCT scale. 114 patients with atopic dermatitis were enrolled from the Department of Dermatology, Peking University First Hospital using convenience sampling from October 2022. Patients were surveyed using the General Information Questionnaire, Chinese version of ADCT, patient-oriented eczema measure (POEM),peak pruritus numerical rating scale (PP-NRS),dermatology life quality index (DLQI) and the global patient self-assessment for disease severity. Mann-Whitney rank sum test and Spearman correlation analysis were used for item analysis; content validity was assessed using content validity index (CVI); exploratory factor analysis was used to assess structural validity; Cronbach' alpha coefficient was used to assess internal consistency; Spearman correlation analysis was used to assess the correlation of ADCT with other scales to assess external responsiveness. The results showed that all items were retained by item analysis. I-CVI was 0.9-1, and S-CVI/Average was 0.983; the scale extracted one common factor by factor analysis, the cumulative variance explanation rate was 77.927%; the Cronbach' alpha coefficient of the scale was 0.937; the correlation coefficients of the Chinese version of ADCT with POEM, PP-NRS, and DLQI were 0.805, 0.861, and 0.709 respectively. In conclusion, the Chinese version of the ADCT has adequate reliability, validity and responsiveness, and is suitable for measuring disease control in Chinese patients with atopic dermatitis.
Humans ; Dermatitis, Atopic ; Quality of Life ; Reproducibility of Results ; Surveys and Questionnaires

This study aimed to investigate the effect and molecular mechanism of sinomenine on proliferation, apoptosis, metastasis, and combination with inhibitors in human hepatocellular carcinoma HepG2 cells and SK-HEP-1 cells. The effect of sinomenine on the growth ability of HepG2 and SK-HEP-1 cells were investigated by CCK-8 assay, colony formation assay, and BeyoClick~(TM) EdU-488 staining. The effect of sinomenine on DNA damage was detected by immunofluorescence assay, and the effect of sinomenine on apoptosis of human hepatocellular carcinoma cells was clarified by Hoechst 33258 staining and CellEvent~(TM) Cystein-3/7Green ReadyProbes~(TM) reagent assay. Cell invasion assay and 3D tumor cell spheroid invasion assay were performed to investigate the effect of sinomenine on the invasion ability of human hepatocellular carcinoma cells in vitro. The effect of sinomenine on the regulation of protein expression related to the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription 3(STAT3) signaling pathway in HepG2 and SK-HEP-1 cells was examined by Western blot. Molecular docking was used to evaluate the strength of affinity of sinomenine to the target cysteinyl aspartate specific proteinase-3(caspase-3) and STAT3, and combined with CCK-8 assay to detect the changes in cell viability after combination with STAT3 inhibitor JSI-124 in combination with CCK-8 assay. The results showed that sinomenine could significantly reduce the cell viability of human hepatocellular carcinoma cells in a concentration-and time-dependent manner, significantly inhibit the clonogenic ability of human hepatocellular carcinoma cells, and weaken the invasive ability of human hepatocellular carcinoma cells in vitro. In addition, sinomenine could up-regulate the cleaved level of poly ADP-ribose polymerase(PARP), a marker of apoptosis, and down-regulate the protein levels of p-Akt, p-mTOR, and p-STAT3 in human hepatocellular carcinoma cells. Molecular docking results showed that sinomenine had good affinity with the targets caspase-3 and STAT3, and the sensitivity of sinomenine to hepatocellular carcinoma cells was diminished after STAT3 was inhibited. Therefore, sinomenine can inhibit the proliferation and invasion of human hepatocellular carcinoma cells and induce apoptosis, and the mechanism may be attributed to the activation of caspase-3 signaling and inhibition of the Akt/mTOR/STAT3 pathway. This study can provide a new reference for the in-depth research and clinical application of sinomenine and is of great significance to further promote the scientific development and utilization of sinomenine.
Humans ; Carcinoma, Hepatocellular/genetics* ; Proto-Oncogene Proteins c-akt/metabolism* ; Caspase 3/metabolism* ; Liver Neoplasms/genetics* ; Molecular Docking Simulation ; Sincalide/pharmacology* ; Cell Line, Tumor ; Cell Proliferation ; Hep G2 Cells ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis