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Objective:To investigate the changes of cognitive function in non-fatal drowning rats after blast-induced traumatic brain injury (bTBI).Methods:Eighty SD rats were divided into normal group, bTBI group, drowning group and bTBI plus drowning group according to the random number table, with 20 rats per group. Rats in normal group were not injured. In bTBI group, bTBI was established in a BST-I biological shock tube with a pressure of 4.0 MPa in the driving section. In drowning group, rats were subjected to non-fatal drowning by falling into the water with temperature of 18 ℃ and depth of 30 cm from the height of 1 m and were taken out quickly after swimming to exhaustion. After being injured in a biological shock tube, rats in bTBI plus drowning group were immediately forced to drowning using the same method. On day 3 post-injury, the neurocognitive function was evaluated by elevated plus maze and Morris water maze tests. Morphological changes of neurons in CA1 and CA3 regions of hippocampus were observed by Nissl staining, and the number of surviving neurons were counted. The concentrations of hippocampal neurotransmitters glutamate, γ-aminobutyric acid (GABA), glycine and endoplasmic reticulum stress (ERS) related glucose-regulated protein 78 (GRP78) and caspase-12 were examined by ELISA analysis. Levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated protein (Bax) and caspase-3 were detected by Western blotting. The ratio of Bcl-2 to Bax was calculated as well.Results:In elevated plus maze test, the percentage of open arm entry and number of head-dipping behaviour were decreased in bTBI plus drowning group compared with normal and bTBI groups at 3 days after injury ( P<0.05 or 0.01), with no statistical difference from those in drowning group ( P>0.05). The number of head-dipping behaviour in drowning group was lower than that in bTBI group ( P<0.05). In Morris water maze test, bTBI plus drowning group showed increased target latency on the third and fourth days of spatial acquisition training and decreased number of crossing the target area and percentage of swimming time in the target quadrant during probe trials as compared with normal group ( P<0.05 or 0.01), but there was no statistical difference among bTBI, drowning and normal groups (all P>0.05). Nissl staining showed that the neurons in the CA1 and CA3 regions of hippocampus in normal group were arranged neatly with clear Nissl bodies at 3 days after injury, while the other groups showed different degrees of injury. In contrast with normal group, the neurons in the CA1 and CA3 regions of hippocampus in all other groups were decreased with the lowest number in bTBI plus drowning groups ( P<0.05 or 0.01). In ELISA analysis, the level of hippocampal glutamate in bTBI plus drowning group was higher than that in all other groups at 3 days after injury and the level in bTBI injury and drowning groups was higher than that in normal group ( P<0.05 or 0.01); the level of hippocampal glycine in bTBI plus drowning group was lower than that in normal group ( P<0.05), but there was no statistical difference among bTBI, drowning or normal groups (all P>0.05); the concentration of hippocampal GABA had no statistical difference among all groups (all P>0.05). In addition, the concentration of GRP78 in bTBI injury, drowning and bTBI injury plus drowning groups were increased compared with normal group ( P<0.05 or 0.01), but did not statistically differ from each other (all P>0.05). The concentration of caspase-12 in drowning and bTBI plus drowning groups were increased compared with normal group ( P<0.05 or 0.01), but was not statistically different from each other ( P>0.05), and its concentration in bTBI plus drowning group was increased compared with bTBI group ( P<0.05). In Western blotting, the level of Bcl-2 in bTBI plus drowning group was decreased compared with all other groups at 3 days after injury, and the level in bTBI and drowning groups were decreased compared with normal group, but a much lower level was observed in drowning group than that in bTBI group ( P<0.05 or 0.01); the level of Bax in bTBI plus drowning group was increased compared with all other groups at 3 days after injury, and the level in drowning group was increased compared with normal group ( P<0.05 or 0.01), with no statistical difference between bTBI and drowning groups ( P>0.05). The ratio of Bcl-2 to Bax in bTBI plus drowning group was decreased compared with all other groups, while the ratio in bTBI and drowning groups were decreased compared with normal group, showing a much lower level in drowning group than that in bTBI group ( P<0.05 or 0.01). Also, the level of caspase-3 in drowning and bTBI plus drowning groups were increased compared with normal and bTBI groups ( P<0.05 or 0.01), but there was no statistical difference between drowning and bTBI plus drowning groups ( P>0.05). Conclusions:Non-fatal drowning can aggravate hippocampal neuron damage in bTBI rats and cause memory, emotion and other cognitive dysfunction. The mechanism may involve the imbalance of hippocampal neurotransmitters glutamate and glycine, which activates the downstream pro-apoptotic pathway through ERS in the early stage of injury to induce hippocampal neuron apoptosis.
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@#Objective To explore the expression and the changes of microtubule, aquaporin-4 (AQP4) and potassium ion channel 4.1 (Kir4.1) after spinal cord injury in rats.Methods Ninety female adult Sprague-Dawley rats were randomly divided into sham operation group (n=30) and injury group (n=60). The injury group was divided into six hours, one day, three days, five days and seven days subgroups, with twelve rats in each subgroup. Spinal cord injury at T10 was established with modified Allen's method (20 g×25 mm) in the injury group. The water content of spinal cord was measured at each time point after injury. Then, the pathology was observed with HE staining, the expression of α-Tubulin, AQP4 and Kir4.1 was detected and analyzed with immunohistochemical staining and Western blotting.Results The water content of the spinal cord was higher in the injured group than in the sham operation group (P<0.05), and was highest on the fifth day. HE staining showed that the gray matter hemorrhage at six hours after injury; one day after injury, the gray matter bled seriously, and neuron swelling was aggravated; three days after injury, the area of gray matter necrosis increased, and the edema phenomenon was obvious; five days and seven days after injury, the gray matter necrosis and the edema phenomenon were more serious. Western blotting and immunohistochemistry showed that the expression of AQP4 gradually increased after injury, and raised at peak on the fifth day; the expression of α-Tubulin and Kir4.1 was similar, and the expression gradually decreased after injury, especially on the fifth day.Conclusion The expression of α-Tubulin and Kir4.1 is similar after spinal cord injury, and is contrary to the expression of AQP4. α-Tubulin, AQP4 and Kir4.1 may be related after injury and may participate in the formation of spinal cord edema.
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@#Objective To observe the effects of total saponins of Astragalus (TSA) on neural stem cells (NSCs) differentiating to neuron in vitro and the best concentration, as well as the role of Wnt/β-catenin signaling pathway in it. Methods NSCs from the cerebral cortex of a neonatal rat were cultured and identified. The possible effective concentration of TSA was screened with Cell Counting Kit-8 (CCK-8). The third generation of NSCs was induced as normal group (without TSA) and various concentration of TSA groups for seven days. The expression of microtubule-associated proteins (MAP-2) and glial fibrillary acidic protein (GFAP) was detected with indirect immunofluorescence and Western blotting to detect the ratio of neuron and astrocyte. Then, the third generation of NSCs was induced as normal group (without TSA), the best concentration of TSA group, TSA group and inhibitor ICG-001 group, and ICG-001 group, for seven days, and the expression of Wnt3/3a, β-catenin and neurogenin 1 (Ngn1) was detected with Western blotting. Results TSA promoted the NSCs proliferation in the concentration of 1×10-4 mol/L, 1×10-5 mol/L and 1×10-6 mol/L (P < 0.001), and increased the proportion of neurons, especially in the concentration of 1×10-5 mol/L (P < 0.05). TSA increased the expression of Wnt3/3a, β-catenin and Ngn1. Conclusion TSA could promote NSCs differentiating into neurons in vitro, which may associate with the activation of the Wnt/β-catenin signaling pathway and the expression of differentiation-promoting target protein Ngn1. TSA can do it best in the concentration about 1×10-5 mol/L..
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Objective To explore how and where ski expresses under lipopolysaccharide (LPS) in rats' astrocytes. Methods Astrocytes were obtained from cerebral cortex of a newborn (within 3 days) Sprague-Dawley rat and cultured in vitro. Astrocytes were cultured with LPS in concentration of 0μg/ml, 0.001μg/ml, 0.01μg/ml, 0.1μg/ml, 1μg/ml, 10μg/ml and 100μg/ml for six hours;and cultured with LPS in concentration of 0.1μg/ml for 0 day, 2 days, 4 days, 6 days and 8 days. The level of ski was determined with Western blotting, and the lo-cation of ski was detected with indirect immunofluorescent staining. Results The expression of ski was induced by LPS, especially in the concentration of 0.1μg/ml. The expression of ski induced with 0.1μg/ml LPS peaked at 4 days of inducement and then decreased. Ski was mainly observed in nuclear in the normal astrocytes and the astrocytes induced with 0.1μg/ml LPS for 6 days. However, it was observed in cytoplasm 2 and 4 days of inducement. Conclusion LPS could induce the expression of ski in rats' astrocytes, which may participate in in-flammation.
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AIM:To explore the time-dependent change of Ski protein expression in normal and activated astrocytes in rats.METHODS:The astrocytes were obtained from rat cerebral cortex and cultured in vitro.The astrocytes were treated with LPS and scratch injury for activation.Western blot analysis was used to determine glial fibrillary acidic protein (GFAP) and Ski protein levels in activated astrocytes at a series of time points.The indirect immunofluorescence staining method was performed to detect the location of Ski protein in the astrocytes.RESULTS:The protein of GFAP was naturally expressed in the astrocytes, beginning to increase after treated with LPS and scratch injury.Little protein expression of Ski in the normal astrocytes was observed.The Ski protein expression began to increase after treated with 1 mg/L LPS, peaked at 4 d (P<0.05) and then deceased, but was stills higher than that in the normal cells.The protein expression level of Ski after scratch injury was highly consistent with above mentioned.Ski was mainly observed in the nucleus of the normal cells and the cells treated with LPS for 6 d, while it was observed in the cytoplasm 2 and 4 d after treated with LPS.CONCLUSION:The protein of Ski is expressed in the astrocytes, and the expression level is increased in activated astrocytes,mainly located in the nucelus.Ski may plays an essential roles in the processes of activation and proliferation of astrocytes.
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Objective To explore the expression and change of ski-interacting protein (SKIP) in rats after spinal cord injury. Methods A total of 60 adult female Sprague-Dawley rats were randomly divided into sham group (n=30) and spinal cord injury (SCI) group (n=30), each group was further divided into five time points including one day, three days, five days, seven days, and 14 days with six rats in each time points. The model was established at T10 with modified Allen's technique, and the sham group only bit the lamina of rats. The hindlimbs behavior was assessed with Basso-Beattie-Bresnahan (BBB) score at each time point. The pathological changes of spinal cord neurons were detected with Nissl staining. The expression of SKIP were observed with immunofluorescence staining. Results The BBB scores were signif-icantly lower in each time point in SCI group than in the sham group (t>48.267, P<0.001). Compared with the sham group, Nissl bodies in the cytoplasm of spinal cord neurons began to disintegrate, coalesce and irregularly distribute, the neurons began to degenerate and die on the fifth day, and the damage deteriorated on the 14th day. Immunofluorescence staining showed that SKIP expression was mainly expressed in the gray matter of the spinal cord and little expressed in the white matter. The expression of SKIP gradually increased after SCI, and reached a peak on the fifth day (t=-17.035, P<0.001) and decreased significantly on the 14th day (t=3.853, P<0.05). Conclusion SKIP may be a new signaling molecule, which play an important role in neuronal apoptosis after SCI.
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Objective To investigate the effect of ski gene in migration process of astrocytes in rats. Methods Astrocytes were obtained from rats' cerebral cortex and cultured in vitro. siRNA targeting ski gene and negative control sequences were prepared. The ski-siRNA group, siRNA negative control group and untreated group were set in this experiment. The specific siRNA targeting ski gene was transfected into astrocytes with Lipofectamine?RNAiMAX Reagent. Then the ski protein levels were determined with Western blotting. After transfec-tion, the changes in migration of astrocytes were measured with wound scratch assay and Transwell migration assay. Results Western blot-ting showed that the expression of ski protein was significantly lower in the ski-siRNA group than in the siRNA negative control group and untreated group (F=132.957, P47.197, P69.187, P<0.001). Conclusion Ski knocked down by siRNA could inhibit the migration ability of astrocytes. It is a reminding that ski may take part in the migration process of astrocytes, and moreover, ski may play an important role in the formation of glial scar.
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Objective To investigate the effects of sperm DNA fragmentation (SDF)and sperm morphology on the fertilization and embryo development in ICSI.Methods SDF and sperm morphology were detected in the meanwhile of taking eggs in 1 45 ICSI treatment cycle.On the basis of SDF index (DFI)divided into group A (DFI≤30%)and group B (DFI >30%).According to the normal sperm morphology divided into group C (NMSR≥4%), group D (1 %≤NMSR 30% and NMSR 0.05).(2)The normal fertilization rate was statistically significant in group C,group D,group E (χ2 =34.5,65.8,11 .8,all P 0.05).(3 )The normal fertilization rate,embryo utilization rate,good quality embryo rate,implantation rate and clinical pregnancy rate in group F were significantly higher than those in group G,and normal fertilization rate,embryos utilization rate,good quality embryo rate had statistically significant differences (χ2 =37.5,1 1 .0,4.3,all P <0.05). Conclusion Sperm abnormal morphology has negative effect on fertilization,and the high DNA fragments have negative effects on fertilization and embryo development.
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Objective To investigate the treatment outcome of modified intracytoplasmic sperm injection (ICSI)using zona pellucida(ZP)-bound sperm.Methods 82 patients with less,weak,abnormal sperm disease who were conformed to ICSI,were divided into traditional ICSI group and the group of modified ICSI using ZP-bound sperm according to ICSI case number.The results of normal fertilization rate,cleavage rate,high-quality embryo rate, planting rate,clinical pregnancy rate and early abortion rate were compared.Results The women's age,the sterility year,mature egg rate,normal fertilization rate,cleavage rate in the two groups had no statistically significant differ-ences (all P>0.05).The planting rate and clinical pregnancy rate of the observation group (46.6%,63.3%)were higher than those of the control group(38.5%,53.6%),but there were no statistically significant differences(all P>0.05).The using embryo rate and high-quality embryo rate of the observation group (73.9%,51.0%)were signifi-cantly higher than those of control group(65.8%,38.6%),the differences were statistically significant(χ2 =5.84,χ2 =11.6,all P<0.05).Conclusion Modified ICSI using ZP-bound sperm can effectively improve the embryos quality in ICSI.
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AIM: To observe the expression change of growth and differentiation factor 10 ( GDF10 ) in the spinal cord of the rats with neuropathic pain .METHODS:Male SD rats (n=60) were used.The neuropathic pain was induced by ligation of left L 5 spinal nerves of the animals .The paw withdrawal threshold was detected 1 d before surgery , and 0 d, 1 d, 3 d, 10 d and 21 d after surgery.The changes of GDF10 in the dorsal horn of L5 spinal cord were detected by immunofluorescence staining and Western blot .RESULTS:The paw withdrawal threshold of the rats with spinal nerve ligation was decreased from 1 d after surgery until 3 d with obvious difference compared with the na ve rats ( P<0.05 ) , continuously decreased until 10 d, and then stabilized at 21 d.The GDF10 was located in the cytoplasm of the neurons in the dorsal horn of L5 spinal cord detected by immunofluorescence staining .The expression of GDF10 in L5 dorsal horn de-tected by immunofluorescence staining was reduced after surgery , significantly decreased from 10 d ( P<0.05) until more than 21 d after surgery in spinal nerve ligation group compared with na ve group.GDF10 in L5 spinal cord detected at 10 d after surgery by Western blot was significantly down-regulated in spinal nerve ligation group compared with na ve group (P<0.05).CONCLUSION:Spinal nerve ligation induces the decrease in GDF 10 expression in spinal dorsal horn .The down-regulation of GDF10 may contribute to the regulation of hyperpathia caused by mechanical stimulation after the injury of spinal nerve .
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Objective To investigate the correlation between recurrent spontaneous abortion and karyotype correlation,and provide reference for clinical diagnosis and treatment of spontaneous abortion.Methods 51 couples peripheral blood lymphocyte were collected and cultured from March 2010 to March 2013 in the region.Karyotype analysis were performed to observe the distribution of the rate of chromosomal abnormalities situation.Meanwhile the correlation between the number of abortions and chromosomes were compared.Results Among the 102 subjects,32 cases(31.37%)of them were found chromosomal abnormalities in RSA.The ratio of polymorphic mutation accounted for 56.25 % of the chromosome abnormality.accounting for 17.65 % the total.Which was significantly higher than other chromosomal abnormalities(P<0.05).Among the RSA and chromosomal karyotype cases,females accounted for 68.75 %,which was significantly higher than males (31.25 %)(P<0.05).46XY(Y≥18)has the highest incidence in chromosome polymorphism,accounting for 33.33 %,while abortions accounting for 30.77 %.RSA number and incidence of chromosomal abnormalities to miscarriages three times the highest rate of abnormal,accounting for 43.48%.Conclusion Chromosomal abnormalities is an important factor in recurrent spontaneous abortion.Recurrent abortion is not only related to the structure of chromosomes,but also has a certain relationship with the variation of chromosome polymorphism.
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@#Amyotrophic lateral sclerosis (ALS), including the familial and the sporadic, accounts for the most proportion of motor neuron disease. The pathogenesis of ALS covers gene mutation, oxidative stress, excitotoxicity, mitochondrial dysfunction, immune and inflammatory, and so on. With interplay and interrelation, these mechanisms, finally, caused multisystem lesion especially motor neural system.
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Dysfunction after spinal cord injury mainly focused on the loss of motor function and sensory function and its complications instead of cognitive impairment. In this paper, the relevant reports of cognitive impairment in patients with spinal cord injury were collected. The influencing factors mainly contained emotion, traumatic brain injury, alcohol intake, drug abuse and educational level, etc. The possible mechanisms included traumatic brain injury, structure change of brain, brain damage and functional change, and inappropriate treatment, etc.
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Objective To explore the writing quality and rational use of drugs for outpatient prescription of western medicine in Yanqing District Hospital of Beijing.Methods From January 2011 to December 2015, 12 000 prescriptions of western medicine were selected randomly.The prescriptions were analyzed by Excel and the Pareto diagram analysis was used to find out the main reasons of irrational prescription.Results From 2011 to 2015, the basic indicators of prescription of western medicine were reasonable:the average number of drugs on each pre-scription was 2.31,the utilization rate of injections was 15.91%,the utilization rate of essential drugs was 38.00%, the utilization rate of antibacterial was 14.56%.The unreasonable rate of prescription was 6.32%,and the main irra-tional reason were dosage inappropriate,indications inappropriate,inappropriate selection of drugs and combination therapy was not suitable.Conclusion By prescription review,we have listed the problems,and provide the basis for rational drug use and improvement measures.
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Ski, as an evolutionary conserved protein, is widely involoved in the proliferation and differentiation of many kinds of cells in different species. Ski also plays an irreplaceable role in many physiological and pathological processes of nervous system, including em-bryonic nervous system development, central and peripheral nervous system diseases, and so on, which may be assiciated with the signal pathways of transforming growth factor-beta and another family member bone morphogenetic protein.
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@#Objective To observe the effect of astragalus polysaccharide on motor function and pathology after spinal cord injury in rats. Methods Seventy adult Sprague-Dawley rats were selected as normal group (n=10), injury group (n=30) and treatment group (n=30), and the injury group and the treatment group were divided into 7 days, 14 days, 28 days subgroups. The injury group and the treatment group were modeled with Allen's mode at T10 (10 g×25 mm). The treatment group was injected with astragalus polysaccharide 10 mg/kg per day after injury. They were rated with Basso-Beattie-Bresnahan (BBB) scores 7 days, 14 days and 28 days after injury, while the morphology was observed with HE staining, Nylon body was observed with Nissl staining, and myelin sheath was observed with eriochrome cyanine staining. Results The BBB score was significantly higher in the treatment group than in the injury group at each time point (P<0.05). There was a large number of necrotic tissue in the injured cords and cystic cavity began to form 7 and 14 days after injury. Cystic cavity formed basically and surrounded with dense scar 28 days after injury. The necrosis and cystic cavity alleviated in the treatment group at each time point. Demyelination and myelin sheaths loose were found 7 days after injury, and aggravated with the time. There was a cystic cavity in trauma center 28 days after injury. The demyelination and myelin sheaths loose relieved at each time point in the treatment group. Nissl bodies began to coalesce 7 days after injury, and aggravated 14 days after injury, coalesced completely 28 days after injury. Nissl body coalesced alleviatively in the treatment group at each time point. Conclusion Astragalus polysaccharide may reduce the damage and promote the recovery of motor function after spinal cord injury in rats.
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Objective To explore the expression and the changes of ski with time in the injured spinal cord in rats. Methods Sixty adult female Sprague-Dawley rats were randomly divided into sham group (n=30) and injury group (n=30), each group were further divided into 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks subgroups, with 6 rats in each subgroup. Spinal cord injury at T10 was established with modi-fied Allen's technique (10 g × 25 mm) in the injury group. The hindlimbs behavior of rats was rated with Basso-Beattie-Bresnahan (BBB) scores 1 day, 3 days, 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks after spinal cord injury. Three rats in each subgroup were stained with HE staining to observe the pathological changes of the spinal cord and the formation of cavity. The other 3 rats were analyzed with im-munofluorescence staining of ski and semi quantitative analysis. Results The BBB scores of each time point were less in the injury group than in the sham group (P<0.05). Necrosis was the major pathological change in the injury groups 1 and 2 weeks after injury;cystic cavity completely formed 4 weeks after injury, with dense scar tissue around it;there was no significant change in the cavity and scar 8 and 12 weeks after injury, however, the adjacent spinal cord was obviously thinner. Ski expressed little in the normal spinal cord, and expressed more and more after injury, peaked at 8 weeks and decreased then. Ski was mainly observed in white matter in the sham group and 12 weeks injury subgroup, which was in gray matter 2, 4 and 8 weeks after injury. Ski was highly expressed around the cavity in injury center and formed high expression band. Conclusion Ski expresses after spinal cord injury in rats, that may be associated with the activation and prolif-eration of astrocytes and the formation of glial scar.
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In recent decades, with the development of electrophysiological and neuroimaging techniques, the researchers found that spi-nal cord injury (SCI) not only caused pathological changes in the spinal cord, but also in the brain. This paper reviewed the influence of SCI on the structure and function of the brain in terms of brain neuron degeneration caused by SCI, the changes of neurotrophic factor in brain, and the side-effects of medicine.
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Objective To study the differentiation and proliferation ability of the spinal neural stem cells (NSCs) at different gestational ages in fetal rats. Methods Sprague-Dawley fetal rats were divided into group A (12 days of pregnancy), group B (14 days of pregnancy) and group C (16 days of pregnancy). NSCs were separated with enzyme-assisted microdissection. The diameter and numbers of NSCs balls were measured at different time. The cell growth curve was drawn with CCK8 colorimeter. NSCs were identified with BrdU/Nestin immuno-histochemical staining. They were induced with 10%fetal bovine serum for 10 days, and the expression ofβ-tubulinⅢand glial fibrillary acidic protein was detected with immunocytochemistry. Results There were cells expressed BrdU, Nestin,β-tubulinⅢand GFAP in all the group. The most cells (22.74±0.79%) expressedβ-tubulinⅢin the group B, but no significant difference between group B and group C. The cell vitality on the 5th day of third-generation neural stem cells was the most in group B. Conclusion For enzyme-assisted microdissection, it may obtain more neurons to isolate the neural stem cells from 14 days of pregnancy pregnant rats.
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@#Objective To study the differentiation and proliferation ability of the spinal neural stem cells (NSCs) at different gestational ages in fetal rats. Methods Sprague-Dawley fetal rats were divided into group A (12 days of pregnancy), group B (14 days of pregnancy) and group C (16 days of pregnancy). NSCs were separated with enzyme-assisted microdissection. The diameter and numbers of NSCs balls were measured at different time. The cell growth curve was drawn with CCK8 colorimeter. NSCs were identified with BrdU/Nestin immunohistochemical staining. They were induced with 10% fetal bovine serum for 10 days, and the expression of β-tubulinⅢ and glial fibrillary acidic protein was detected with immunocytochemistry. Results There were cells expressed BrdU, Nestin, β-tubulinⅢ and GFAP in all the group. The most cells (22.74±0.79%) expressed β-tubulinⅢ in the group B, but no significant difference between group B and group C. The cell vitality on the 5th day of third-generation neural stem cells was the most in group B. Conclusion For enzyme-assisted microdissection, it may obtain more neurons to isolate the neural stem cells from 14 days of pregnancy pregnant rats.