ABSTRACT
BACKGROUND: Oligodendrocyte precursor cell transplantation is one of the keys to the treatment of white matter damage in premature infants. At present, there is a lack of research on the comparison of oligodendrocyte precursor cells induced by neural stem cells derived from human fetal brain cultured in vitro in different vessels worldwide. OBJECTIVE: To observe the morphology of human oligodendrocyte progenitor cells and pre-oligodendrocytes in different cell culture vessels (6-well plates, 24-well plates and T25 flasks). METHODS: The 6-well plates, 24-well plates and T25 flasks were used as culture vessels to culture human oligodendrocyte progenitor cells and pre-oligodendrocytes. The characteristics of human oligodendrocyte progenitor cells were identified by immunofluorescence staining and flow cytometry. The morphology of cells was observed by an ordinary light microscope. Cell counts were performed according to cell morphology and statistical analysis was performed. RESULTS AND CONCLUSION: (1) The oligodendrocyte progenitor cell body was round and the bipolar protrusions were bead-like; the pre-oligodendrocyte protrusions were more than two poles, and did not bifurcate. (2) The ratios of oligodendrocyte progenitor cell morphology in the oligodontia lines were significantly higher in the 6-well plates than those in the 24-well plates and T25 flasks (P < 0.05), followed by T25 flasks and 24-well plates. Morphological ratios of pre-oligodendrocytes were significantly higher in the 24-well plates compared to the 6-well plates and T25 flasks (P < 0.01), followed by T25 flasks and 6-well plates. (3) The cells cultured in the 6-well plate had fewer dregs, and the morphology, vigor and growth were better than those of the other culture vessels. (4) According to morphological view, 6-well plates are more suitable for oligodendrocyte progenitor cell growth, and 24-well plates are more suitable for pre-oligodendrocytes growth.
ABSTRACT
BACKGROUND: Oligodendrocyte precursor cells are seed cells for the treatment of white matter injury. The establishment of an efficient and stable in vitro culture method is an important prerequisite for clinical transformation. OBJECTIVE: To investigate the morphological changes of oligodendrocyte precursor cells during passage. METHODS: Four batches of human oligodendrocyte progenitor cells were subcultured from the second generation (P2) to the seventh generation (P7) in vitro. Five pictures of 200-fold field were taken under an optical microscope before each passage. According to the cell morphology, oligodendrocyte precursor cells were divided into three types: bipolar cells (oligodendrocyte precursor cells), multipolar cells (late oligodendrocyte precursor cells) and supra-polar bifurcated cells (immature oligodendrocyte cells). The proportion of oligodendrocyte progenitor cells to the total number of cells was calculated, so as to compare the difference of cell morphology among different generations. RESULTS AND CONCLUSION: In the process of passage from P2 to P7, oligodendrocyte progenitor cells included three types: bipolar cells, multipolar cells and supra-polar bifurcated cells. Among them, bipolar cells and multipolar cells were the main part, and a small number of supra-polar bifurcated cells could be seen in the rest. There were no significant differences in the proportion of bipolar cells, multipolar cells and supra-polar bifurcated cells among P2-P7 (P > 0.05). The cell morphology classification and counting method can be used to preliminarily evaluate that oligodendrocyte progenitor cells have no change in morphology during culture.
ABSTRACT
Objective To investigate the mechanism of white matter damage (WMD) and the neuroprotective effect of Xenon on neonates with WMD.Methods Three-day-old SD rat pups (n =96) were randomly divided into the blank control group (n =24),the WMD control group (n =24),the Xenon intervention group A (n =24) and the Xenon intervention group B (n =24) by random number method according to their birth time.WMD rat models were successfully established by giving intraperitoneal injection of lipopolysaccharide(LPS) 0.05 mg/kg combined with carotid artery ligation and hypoxia for 1 hour in the WMD control group and the Xenon intervention groups.In the control group,only 9 g/L saline (0.05 mg/kg) was injected intraperitoneally,while carotid artery ligation and hypoxia were not administered.Rats in Xenon intervention group A and group B were given inhalation of 500 mL/L Xenon for 3 hours at 0 and 2 hours respectively after establishment of the models.Six rats in each group were randomly selected and decapitated at 0,24,48 and 72 hours after the intervention.The brain white matter on the right was analyzed by using HE staining and myelin basic protein(MBP) immunofluorescence staining,and real-time quantitative polymerase chain reaction was used to detect the expressions level of CLIC4 mRNA.Results (1) Brain tissue pathology:compared with the blank control group,the brain white matter on the right of the WMD control group and the Xenon intervention group A and group B had loose and disordered structure,nuclear pyknosis and cytoplasm loosening.However,the lesions in both Xenon intervention group A and group B were significantly less than those in the WMD control group,and there was no significant difference between the Xenon intervention group A and group B.(2) MBP measurement:the number of MBP-positive cells in the brain white matter on the right of WMD control group was significantly lower than that in the blank control group,while compared with WMD control group,they were significantly higher in Xenon intervention group A and group B.(3) CLIC4 mRNA expression level:compared with blank control group,the expressions levels of CLIC4 mRNA at most time point were higher both in the WMD control group and the Xenon intervention group A and group B (all P ＜ 0.05),except the time point 24 h in the Xenon intervention group A.The expressions of CLIC4 mRNA in group A and group B were significantly decreased compared with those in the WMD control group (all P ＜ 0.05).However,there were no significant differences between Xenon intervention group A and group B (P ＞ 0.05).Conclusions The expressions of CLIC4 mRNA in brain tissues on neonatal rats with WMD significantly increased,indicating that the mitochondrial pathway could be one of the pathological processes of WMD.Early Xenon intervention may reduce neonatal WMD by reducing the expression of CLIC4 mRNA,which plays a neuroprotective role.