| Objective:In this study,firstly to establish the primary culture of rat astrocytes and oligodendrocyte precursor cells.And then,to study the biological characteristics of Telomerase reverse transcriptase(TERT)-overexpressing astrocytes and the effects of their conditioned medium on apoptosis,proliferation,and differentiation of rat oligodendrocyte precursor cells.Furthermore,to detect protein changes in TERT-overexpressing astrocyteconditioned medium,and to screen differentially expressed proteins related to oligodendrocyte precursor cells differentiation and neuroprotection,then to explore the role of differentially expressed pleiotrophin(PTN)in regulating the differentiation and apoptosis of oligodendrocytes.Materials and Methods:The cerebral cortex of neonatal SD rats was digested using a combination of papain and DNase and then separated to obtain primary astrocytes.A constant temperature shaker was used to shake the sample continuously for18–20 h followed by washing with phosphate-buffered saline to remove microglia,oligodendrocytes,and other impurities.Cell growth status was observed using an optical microscope.Immunofluorescence staining was used to detect glial fibrillary acidic protein(GFAP)expression to identify the purity of the obtained astrocytes.Next,the cerebral cortex of neonatal SD rats was digested using a combination of papain and DNase and A2B5 positive cells were separated using A2B5 immunomagnetic beads.Platelet derived growth factor(PDGF),basic fibroblast growth factor(b FGF),triiodothyronine(T3),and ciliary neurotrophic factor(CNTF)were added to promote proliferation and differentiation of oligodendrocyte precursor cells.Cell morphology was observed at different stages under an inverted optical microscope,and immunofluorescence staining was used to detect the expression of specific molecular markers,O4 and myelin basic protein(MBP),at different stages to identify the purity of the extracted oligodendrocytes and cell differentiation.Astrocytes were transfected with lentivirus to overexpress TERT,and a negative control was set simultaneously.Puromycin was used for resistance screening to obtain stably transfected cells,and q PCR,immunofluorescence staining,and western blotting were used to detect TERT expression.For astrocytes stably transfected with TERT and their controls,real-time fluorescent quantitative PCR was used to detect telomere length,telomerase repeat amplification protocol and enzyme-linked immunosorbent assays were used to detect telomerase activity,and the Brd U assay was used to detect cell proliferation.Astrocytes,negative control virus-transfected astrocytes,and TERT-overexpressing astrocytes were cultured without serum for 18 h,and the conditioned media were collected and named ACM,NC,and TERT,respectively.Oligodendrocyte precursor cells were insulted with oxygenglucose deprivation(OGD),and conditioned media were used to treat the cells.Immunofluorescence staining was used to detect the apoptosis,proliferation,and differentiation of oligodendrocytes in each group.In the last part,the control group and the TERT-overexpressing astrocyte-conditioned media,named NC and TERT,respectively,were collected for proteomic detection which was completed by Shanghai Luming Biotechnology Co.,Ltd.,China.Tandem mass tag(TMT)technology and bioinformatics were used for qualitative and quantitative protein analysis and for analysis of the function of differentially expressed proteins,respectively.Screening of differentially expressed proteins related to oligodendrocyte precursor cells differentiation and neuroprotection indicated that PTN may be an effector molecule related to oligodendrocyte precursor cells differentiation and neuroprotection.q PCR was performed to verify the transcription level of PTN.Finally,the rat oligodendrocyte precursor cells were insulted with OGD,and to verify PTN function,PTN recombinant protein was added to detect oligodendrocyte precursor cell differentiation and apoptosis.Results:After primary extraction and shaking-purification,astrocytes showed good cell growth,irregular morphology,and multiple cell protrusions under an inverted optical microscope.Immunofluorescence staining showed that the proportion of positive GFAP cells reached 97%.In primary isolated and cultured rat oligodendrocyte precursor cells,observation under a light microscope showed that the cells in the proliferation stage had a typical bipolar or tripolar morphology with a strong refractive index.Cells in the differentiation stage had multiple filamentous protrusions,which were connected to each other and were radially surrounded by elliptical or spindleshaped cell bodies.Immunofluorescence staining showed that 92% cells in the proliferation stage were positive for O4 staining and were co-stained with DAPI.Cells in the differentiation stage expressed MBP,which surrounds the cell body and presents radially distributed filamentous protrusions.After three days of infection,observation under an inverted fluorescence microscope showed that the expression of the green fluorescent label protein reached 80%.After adding puromycin to obtain stably transfected cells,the green fluorescent protein expression reached more than 90%.Real-time fluorescent quantitative PCR,immunofluorescence staining,and western blotting showed that the expression levels of TERT m RNA and protein in the astrocytes stably transfected with overexpression of TERT were higher than those in the negative control group(P < 0.05).Telomere length of TERToverexpressing astrocytes was prolonged(P < 0.05).And telomerase was activated,however,it was significantly weaker than that of tumor cells(P <0.05).The cell proliferation rate did not increase significantly,and it had no effect on the activation of astrocytes(P > 0.05).After 2 h of OGD insult,the cell survival rate of oligodendrocyte precursor cells decreased significantly(approximately 48.3%),number of apoptotic cells increased,proliferation was inhibited,and the differentiation ability decreased(P < 0.05).ACM-and NCtreated groups showed no significant changes in the survival rate,apoptosis,proliferation,and differentiation of OGD-treated oligodendrocyte precursor cells(P > 0.05).The TERT-treated group showed increased survival of OGDtreated oligodendrocyte precursor cells(P < 0.05),reduced cell apoptosis(P< 0.05),no significant changes in proliferation ability(P > 0.05),and enhanced differentiation ability(P < 0.05).TMT-labeled quantitative proteomic analysis was performed on the NCand TERT-treated groups.Based on the criteria of fold change(FC)= 1.5 time and P < 0.05,236 differentially expressed proteins were identified in the two groups,of which 131 proteins were down-regulated and 105 proteins were upregulated.The gene ontology(GO)analysis of the biological processes of the differential proteins between the NC-and TERT-treated groups revealed a total of 1431 GO entries,involving cell response to hypoxia,inflammatory response,brain development,cell differentiation,apoptotic processes,aging,and biological processes such as oxidative stress.Using the Kyoto Encyclopedia of Genes and Genomes database to analyze the differential proteins between the NC-and TERT-treated groups,211 pathways were enriched,including necroptosis,oxidative phosphorylation,HIF-1,IL-17,AMPK,MAPK,and m TOR signaling pathways;cytokine/cytokine receptor interaction;Huntington’s,Parkinson’s,and Alzheimer’s disease;and amyotrophic lateral sclerosis.The difference in protein PTN between the NCand TERT-treated groups was FC = 2.725016(P < 0.05).In the GO enrichment analysis,PTN was involved in dendritic regeneration,brain development,cell proliferation,oligodendrocyte differentiation,neurotrophic factors,cell response to hypoxia,positive regulation of neuron projection development,and many other biological processes.q PCR analysis showed that compared with the NC-treated group,in the TERT-treated group,the expression of PTN m RNA in astrocytes was upregulated by 86.3451-fold(P<0.001).After using 100 ng/m L recombinant PTN protein,the expression of MBP in the OGD PTN group increased(P < 0.05),and cell apoptosis reduced(P < 0.05)in comparison with those in the OGD negative control group.Conclusion:Cultured primary rat astrocytes and oligodendrocyte precursor cells have high purity,and the A2B5 positive cells have the ability to differentiate into mature oligodendrocytes.Successful establishment of TERT-overexpressing astrocytes and their conditioned medium can protect oligodendrocytes from hypoxia and ischemia,and PTN may be the main effector molecule of the TERT-overexpressing astrocyte-conditioned medium to modulate the function of oligodendrocytes after hypoxia-ischemia insult. |
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