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Objective To explore the value of amplitude-integrated electroencephalography(aEEG) in diagnosis and prognosis in term newborns with brain injury.Methods One hundred and thirty consecutive patients with brain injury admitted in NICU were prospectively enrolled in the study from Nov 2013to Apr 2015.The monitoring of aEEG was done at 1d,4d,7d,respectively.Clinical data were collected and com-pared with the result of aEEG.Results All the cases of newborns with abnormal aEEG background:discon-tinuous voltage(109cases),continuous low voltage(12cases),flat(4cases).The epileptic activity were re-corded in 33cases,and 15cases showed burst-suppression;sleep wake cycle:mature(32cases),immature (54cases),no sleep wake cycle(39cases).The judgment of abnormal level:70cases had mildly abnormal aEEG,severe abnormalities 60cases,and no significant difference in different types of diseases(x2=6.176, P=0.19).Recent prognosis:the death of mild and severe abnormalities were 1case,12cases,respectively, there were significant differences(x2=12.76,P﹤0.001).Developmental quotient(DQ)of these newborns were followed up for more than 6months,and there were significant differences in mild,severe abnormal aEEG in them with DQ≥85and DQ﹤85(x2=33.195,P﹤0.001).The sensitivity of aEEG in severe abnor-mal aEEG was 68.75%,the specificity was 78.68%,and the positive and negative predictive values of aEEG were 77.19%and 70.58%,respectively.The results of aEEG classification and sleep wake cycle were corre-lated with the prognosis of the patients(r=0.505,0.507,respectively,P﹤0.001).Conclusion aEEG can be used to monitor brain function,and it is helpful to evaluation of early diagnosis and prognosis.
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BACKGROUND:Currently,there is not a standard method for in vitro culture and large scale amplification of umbilical cord blood mesenchymal stem cells(UCB-MSCs).OBJECTIVE:To investigate the isolation,purification and culture of UCB-MSCs in vitro,and to detect its surface marker variation.METHODS:The monocytes were harvested from UCB using 1.077 g/cm3 lymphocytes separating solution and density gradient centrifugation,followed by incubation in an incubator containing 5%CO2 at 37℃.The cell morphological changes were observed at different time points and the expression of surface marker was detected using flow cytometry.RESULTS AND CONCLUSION:The monocytes isolated from the UCB grew initially into numerous hematopoietic cell clones,most of which were granulocyte/macrophage colony-forming units and burst forming unit-erithroid,increasing by(37.1±2.3)and (10.4±1.7),respectively.Switzerland staining showed most of them were granulocyte clones(80,1±85.2)%,next was erythroid clones(14.2±1.8)%.At 7 days after culture,some shuttle fibroblast-like cells and fiat osteogenic-like cell spread the whole plastic well.At 14 days after culture,flow cytometry showed CD38+ cells accounted for 1.64%,and CD34+/CD38+ cells accounted for 1,71%,and CD34+/CD38- were 0.55%.PI+ and Annexin-V+ cells accounted for 0.05% and 0.18% respectively.At 21 days after culture,CD38+,CD34+/CD38+ and CD34+/CD38- cells were 74.32%,1.61%,and 0.24%.The results reveled that UCB-MSCs can be isolated and cultured in vitro.
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BACKGROUND: Stem cells are relatively primitive cells possessing the capabilities of self-renewal, high proliferation and multi-potential differentiation in vivo under certain conditions. Pancreatic stem cells and umbilical cord blood mesenchymal stem cells (MSCs) may serve therapeutic purpose clinically, but they are still difficult to culture in vitro at present.OBJECTIVE: To explore the method for isolation, purification and culture of pancreatic stem cells and umbilical cord blood MSCs in vitro and observe their morphological changes during culture in vitro.DESIGN: Completely randomized experiment with repeated measurement.SETTING: Stem Cell Research Center, Teaching and Research Division of Physiology, Medical School of Zhengzhou University.MATERIALS: This experiment was conducted in the Stem Cell Research Center, Teaching and Research Division of Physiology, Medical College of Zhengzhou University, between April 2004 and January 2005. Ten to fifteen newborn SD rats (1-3 days) were selected for culture in vitro of pancreatic stem cells, and fresh umbilical cord blood was collected from healthy woman (24-35 years old, with informed consent) at full-term delivery for culture in vitro of umbilical blood SMCs.METHODS: The abdomen of the newborn SD rat was opened under aseptic condition to obtain the pancreas, which was cut into small tissue blocks and digested with type-V collagenase for islet isolation. The isolated islets were purified in continuous roller-bottle culture. Umbilical cord blood was freshly collected for isolating the monocytes by means of density gradient centrifugation in lymphocyte separation medium (with density of 1.077 g/cm3). The islet cells and umbilical cord blood monocytes were cultured in the incubator at 37 ℃ with 5% CO2. The morphological changes of the cells were observed at designed time points and flow cytometry was used to determine the expression of cell surface molecules.MAIN OUTCOME MEASURES: The isolation and culture of pancreatic stem cells and umbilical cord blood MSCs, and their morphological changes during culture in vitro.RESULTS: During culture in vitro, the fusiform islet progenitor cells showed adherent polar growth and continuous proliferation, which covered the whole bottom of the flask after 12-14 days and could be subcultured for passages. However round cells appeared after removal of the growth factor and serum in the culture medium. The monocytes isolated from the umbilical cord blood grew initially into numerous hematopoietic cell clones, most of which proved to be granulocyte clones by Switzerland staining. Seven days later, flat flask wall-adhering epithelial cells and long fusiform fibroblasts were observed mixed with a number of osteoclasts. As the cell culture was prolonged, the cell number increased steadily.CONCLUSION: Pancreatic stem cells and umbilical cord blood SMCs can be cultured in vitro for further experiments.
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AIM: To explore the regulatory effects of cytokines such as EGF, bFGF on expression of neural-specific molecules in mononuclear cells (MNCs) cultured in H-DMEM medium. METHODS: The umbilical cord blood samples were collected from health puerperal natural delivery. The mononuclear cells were isolated by centrifugation over Lymphoprep and planted in T-75 flasks containing H-DMEM medium with or without addition of EGF, bFGF or EGF plus bFGF at a final concentration of 20 ?g/L, respectively. Phenotypic changes were monitored by inverse phase-contrast microscopy. Tau and MAP2 mRNA were determined by reverse-transcript polymerase chain reaction (RT-PCR). Tau and MAP2-positive cell were determined by immunocytochemistry. RESULTS: The expression of tau protein mRNA was negative in uncultured cells, but MAP2 mRNA was positive. In cultured cells, tau protein mRNA expressed positively, MAP2 mRNA expression was upregulated by EGF+bFGF, EGF or bFGF compared with control group (no cytokines). The upregulatory capability of EGF+bFGF to MAP2 mRNA expression was stronger than that of EGF or bFGF alone. The same upregulatory tendency was noted in tau mRNA expression. In the group of control, bFGF, EGF, EGF+bFGF, the rate of MAP2-positive cells was 14.4%, 19.6%, 25.6%, 33.5%, respectively. Tau protein-positive cells were 13.5%, 15.3%, 21.4%, 29.8%, respectively. Under inverse microscopy, the freshly isolated MNCs were small and round, after culturing, the cells became larger with some big, long cytodenrites in the EGF+bFGF group, with 1 or 2 threadlike cytodenrites in the EGF group, or with some short multi-dendron like-astrocyte in the bFGF group and control group, but the number of astrocyte-like cells in the control group was less than that in bFGF group. CONCLUSION: MNCs derived from human umbilical cord blood cells express some neural specific molecules and are upregulated by cytokines, especially EGF and bFGF, which have the synergetic action. [