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Objective To investigate the characteristics of the bone marrow mesenchymal stem cell(MSC)in children with aplastic anemia(AA)in vitro,and the expressions of tumor necrosis factor -α-induced protein -8 -like 2(TIPE2)in the bone marrow,and the correlation between the level of TIPE2 mRNA with γ-interferon(IFN -γ)and IL -6 in AA patients.Methods Bone marrow samples were collected from 1 8 children with AA(AA group)and 8 children with bone injury (control group)who were hospitalized in Jinan Children′s Hospital from January 201 2 to June 201 5.MSC were isolated and cultured.The morphology of MSC was observed and immune phenotype was detected.The TIPE2 mRNA was detected by using real -time fluorescence quantitative PCR,and the levels of IFN -γand IL -6 were detected by using enzyme linked immunosorbent assay.Results Different sizes had been presented in the primi-tive MSC of AA patients,but the third passage MSC until 80% confluence had manifested the uniform convergence with long spindle and swirl distribution.In the sixth passage,cells showed degenerative change.The primitive and first pa-ssage MSC in patients with AA was longer than that in the controls.CD73 ,CD1 05 ,CD44 and CD90 were expressed in MSC,while CD34 ,CD45 ,CD271 expressed rarely.The level of TIPE2 mRNA in AA patients (5.29 ±1 .56)was obviously lower than that of the control group(8.68 ±2.00),and the difference was significant(t =-4.48,P 0.05).Conclusions The proliferation of MSC is significantly reduced in patients with AA.TIPE2,as an important role to stabilize the immune system,plays an important role in the occurrence of AA by its low expression and up -regula-ting the expression of inflammatory factors.
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BACKGROUND:General assessment of viability of umbilical cord mesenchymal stem cel s during transport is not considered at home and abroad. OBJECTIVE:To explore the effects of different transportation conditions such as albumin and transport time on survival rate of umbilical cord mesenchymal stem cel s. METHODS:Umbilical cord mesenchymal stem cel s cultured in vitro were divided into experimental and control groups. Each of group contained 3.15×109/L cel s in 3 mL normal saline. The experimental group contained 1%albumin in dark environment. The control group included two subgroups:dark preservation group with the absence of albumin and light preservation group with the presence of albumin. Cel counting, trypan blue staining and cel survival rate were compared at 0, 2, 4, 8, and 24 hours. RESULTS AND CONCLUSION:Compared with the control group, no cel loss was observed in experimental group (with the presence of albumin in the dark) and a 90%viability ratio was achieved at 8 hours. In the control group without albumin, loss rate reached 38.5%and survival rate reached 86%at 8 hours. Results revealed that 1%albumin predominantly improved cel survival rate in long-distance transport of umbilical cord mesenchymal stem cel s. When transport time was more than 8 hours, cel loss rate increased and cel survival rate downregulated. Our experimental data demonstrated that umbilical cord mesenchymal stem cel s preserved in normal saline consisting of 1%albumin placing in dark environment at 16 ℃ apply to clinical application criterion in 8 hours.
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ObjectiveTo evaluate bacteria contamination during collection,processing and storage of cord blood to gain insight into contamination mechanism and direct prevention.MethodsFresh cord blood was separated by hydroxyethyl starch (HES) to harvest nucleated cells.The bacteria contamination was tested by culturing 10 ml plasma-red cells with BacT/ALERT 3D-480 automatic blood culture system.Total 87 positive samples were further identified for bacteria species.Ninety six cord blood nucleated cells concentrate with bacteria positive stored in liquid nitrogen(LN2) for 6-7 years were thawed at 37 C and re-cultured for bacteria analysis.ResultsWe collected 19 062 umbilical cord blood.Among them,336 was bacteria positive ( contamination rate 1.8 ％ ).Eighty-seven positive samples were further investigated with facultative bacteria 58 (66.7 ％ ),aerobic 38(43.7％ ) and anaerobic 17( 19.5％ ),Gram- negative accounted for 68％ while positive 32％.The most frequent bacteria were Escherichia coli ( 25.3％ ),Streptacoccus intermediate ( 14.9％ ) and Chromobacteria violaceum(9.2％ ).Ninety-six nucleated cells concentrate with bacteria positive were cryopreserved at liquid nitrogen for researching.Of them,83 samples( 86％ ) showed positive of bacteria culture after deep-low temperature storage for 6-7 years.ConclusionsBacteria contamination rate of the cord blood collection,processing and storage in 2000 ～ 2007 was 1.8％.Stored in liquid nitrogen for 6-7 years,the viability of bacteria was 86％.The aseptic procedures of cord blood collection in delivery room should be intensified.The bacteria re-culture following thawing of cord blood cells is necessary before clinical transfusion.
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Objective To explore the dynamic expressions of autophagia and apoptosis associated protein Beclin-1, Cathepsin B and Bcl-2 in hippocampus and the intervention efficacy of cathepsin-B inhibitor (CBI) after recurrent neonatal seizure. Method Ninty 6-day-old SD rats were randomly (random number) divided into the recurrent neonatal seizure group (RS group, n = 30), CBI-treated seizure group (CBI group, n = 30) and the control group (n = 30). Rats in RS group were subjected to 55 attacks of seizure induced by using flurothyl during the consecutive 9 days beginning on the 6 th postnatal day (P6). In CBI group, CBI (2 μL, 0.5 μg/μL) was administered every day before seizures induced. Western blot was employed to determine the protein level at different intervals (1.5 h,3 h,6 h,24 h) after the last convulsion.Results There were higher expressions of Beclin-1 and Cathepsin B, and lower expressions of Bcl-2 expression in RS group(1. 5 h,3 h,6 h and 24 h) than those at the same time in control group (P ＜ 0.05). Beclin1 and Cathepsin B expressions were lower while Bcl-2 expressions were higher in CBI group at the intervals of 1.5 h,3 h,6 h and 24 h compared with those in RS group (P ＜ 0. 05). Conciusions Autophagic and apoptotic pathways were activated immediately after recurrent neonatal seizures as indicated by expression changes of Beclin-1, Cathepsin B and Bcl-2 in hippocampus, which suggests a synergistic effect of the two pathways in the pathophysiology of the long-term brain damage of neonateε resulted from the adverse effects of recurrent neonatal seizures.
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Objective To explore the intervention effect of cathepsin B inhibitor (CBI) and its signaling pathways after flurothyl inducing brain injury in rats with recurrent seizures. Methods 6-day-old (P6) SD rats were randomly divided into: recurrent neonatal seizure group ( RS group, n = 30), CBI-treated seizure group ( CBI group, n=30) and the control group( CONT group). Rats in RS group were subjected to 5 seizures with flurothyl during the first 14 days of life. In CBI group,CBI was injected each day before seizures were induced. Then examined development indexes such as the physical growth, neural reflex, neural behavior and cognitive emotional competence. Western blot was employed to determine Cathepsin B expression at different time points ( 1.5h,3h,6h,24h and P35) after the last convulsion. Results The weights of rats in RS group( (27.28 ± 1.6) kg) were lighter than CONT group( ( 33.45 ± 1.57 ) kg). They had significant difference (P＜ 0.01 ) at the age of p14. The time of cliff avoidance in RS group( (2. 10 ± 1.45 ) s) was longer than CBI group( ( 1.05 ± 0. 32) s). It showed a statistically significant (P ＜ 0.05 ) in p12. In the open-field behavior test: the activities of RS group (20.00 ± 13.08 )were markedly reduced than CONT group ( 57.83 ± 33.22 ) in the horizontal movements, the RS group ( 2.50 ±2.43 ) were significantly decreased than the CONT group and CB1 group( ( 22.17 ± 10.74), (9.33 ± 5.39 ) ) in the vertical motions (P ＜ 0. 05 ). Cathepsin-B expression in RS group( 1.5h, 3h,6h and 24h )was significantly higher than that at the same time in CONT group(P＜ 0. 05 ). Cathepsin-B expression of CBI group was significantly decreased compared with that in RS group (P＜ 0. 05 ) at the time point (1.5h ,3h,6h and 24h). There were no significant differences among three groups at P35(P＞0.05 ). Conclusions CBI can improve brain injury and regulate the expression of abnormal molecules.