Myelin-associated glycoprotein inhibits the differentiation and neurite growth of neural stem cells

AIM: To observe the characterization in neural cells derived from the hippocampus of embryonic rats and to examine the effect of myelin-associated glycoprotein (MAG) on the proliferation, differentiation and neurite growth of neural stem cells (NSCs). METHODS: The hippocampus cells of embryonic rats were isolated and cultured in vitro. The expressions of nestin and doublecortin, the marks of NSCs, were observed by immunocytochemical method. The rate of proliferating cells was examined by BrdU immunocytochemistry. The average neuronal neurite length and the percentage of differentiated neurons were detected by immunocytochemistry staining. RESULTS: The hippocampus cells of 16 days old embryonic rats had the characteristics of NSCs. The percentage of differentiated neurons (β-tubulin Ⅲ-positive cells) was 18.17%±2.79% and the average neuronal neurite length was (136.27±33.66)μm, seven days after the differentiation initiated in vitro in control group. After NSCs were treated with MAG-Fc (200 μg/L), the percentage of differentiated neurons and the average neurite length were decreased, respectively, to 10.05%±3.42% (P<0.01) and (84.87±24.94)μm (P<0.01). The results from proliferation test indicated that the rate of BrdU incorporation did not changed after the treatment of MAG-Fc with different dosages (P>0.05). CONCLUSION: MAG-Fc inhibits the differentiation and neurite growth of the NSCs, but has no effect on the proliferation.     

Influence of murine cytomegalovirus infection on the differentiation and the differentiation genes expression in neural stem cells

AIM: The influence of MCMV infection on differentiation and differentiation gene expression in neural stem cells(NSCs) in vitro were investigated for studying the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection.METHODS: NSCs were separated from fetal BALB/C mouse, and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunofluorescence. The NSCs infected by MCMV at dosage of MOI(multiplicity of infection) equaled to 5, 1 and 0.1,respectively, were cultured in differentiation medium. The morphological changes of infected cells were observed under inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expressions of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence(MOI=1). The expression of early antigen(EA) of MCMV was detected to observe the infection process. Real-time RT-PCR method was employed to measure the expression levels of the key genes Neurog2, Myc and Ccnd1 in Wnt signal pathway of NSCs at early stage of differentiation culture.RESULTS: NSCs isolated from embryonic mouse brains proliferated to form neurospheres, strongly expressed nestin and differentiated into NF-200 positive neurons or GFAP positive astrocytes. The infected NSCs did not adhere to the wall and appeared differentiation growths, but showed swollen gradually after differentiation culture. The nestin expression in the infected cells downregulated slowly and was higher than that in control groups(P<0.05). The GFAP and NSE expressions of the infected cells were lower than those in control groups(P<0.05). The early antigen(EA) of MCMV was always detected in the cells in infected groups. The ratios of nestin positive cells in infected groups were higher than those in control groups, but the ratios of GFAP and NSE positive cells of former were lower than that of the latter from 3rd to 9th d after differentiation culture(P<0.05). The levels of Neurog2 mRNA and Myc mRNA in infected groups were markedly lower than those in normal control groups on 1st d and from 1st to 4th d after differentiation culture, respectively(P<0.05). The levels of Ccnd1 mRNA of infected groups were obviously lower than those in normal control groups from 12th h to 1st d(P<0.05). These changes in infected groups became more obvious as MCMV MOI increased.CONCLUSION: MCMV significantly inhibits differentiation of NSCs to neurons and astrocytes, and leads to the decrease in differentiated cells. MCMV inhibits or interferes with the gene expression of Neurog2, Myc and Ccnd1 in Wnt signal pathway of NSCs. The effect that MCMV inhibits the expressions of differentiation and the differentiation genes in NSCs shows dose-dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering with the expression of differentiation gene in NSCs, which is possibly the one of primary causes of brain development disorders induced by congenital CMV infection.     

Effect of VEGF on proliferation and differentiation of neural stem cells from the hippocampal gyrus of rat in vitro

AIM: To observe the effects of vascular endothelial growth factor (VEGF) on the proliferation and differentiation of neural stem cells (NSCs) of rats in vitro.METHODS: NSCs isolated from the hippocampal gyrus of SD rats were primary cultured and subcultured,and then divided into two groups: (1) the cells in VEGF group were treated with 150 μg/L VEGF in the culture system,and VEGF was removed at the 7 th day;(2) control group (without VEGF treatment).The cellular morphology of two groups was observed by contrast phase microscope.Nestin and NF-200 expressing cells were detected via immunofluorescence method.The percentages of the immunostaining positive cells in each group at the 7 th day and at the 11 th day were determined.RESULTS: At the 7 th day,the percentage of nestin positive cells in VEGF group was 52.19%±7.95%,vs 29.26%±4.12% in control group (P<0.01).The percentage of NF positive cells in VEGF group was 22.33%±4.13%,vs 38.62%±5.31% in control group (P<0.01).At the 3 th day after VEGF was removed,the percentage of NF positive cells in VEGF group was 43.10%±3.70%,vs 30.56%±4.16% in control group (P<0.01).CONCLUSION: VEGF stimulates the proliferation of neural stem cells and inhibits their differentiation.     

Effects of Aβ1-42-induced microglial cells on survival of neural stem cells in vitro

AIM: To explore the effects of β-amyloid protein 1-42 (Aβ_1-42)-induced microglia on the survival of cultured neural stem cells (NSCs) in vitro . METHODS: Using the Transwell chambers to build a coculture system of NSCs and microglia, we detected the proliferation, differentiation and apoptosis of the NSCs with the microglia before and after induction by Aβ_1-42. RESULTS: Compared with non-intervention group, the proliferation rate of NSCs in Aβ_1-42 intervention coculture group decreased, as well as the positive expression rates of microtubule-associated protein 2 (MAP-2) and choline acetyltransferase. CONCLUSION: The inflammation mediated by Aβ_1-42 inhib their the proliferation of NSCs and induces their apoptosis. Inflammation also significantly reduces the ratio of NSCs differentiating to neurons, especially to cholinergic neurons.     

Survival and neuronal differentiation of transplanted stem cells derived from embryonic hippocampus in adult rats with stroke lesions

AIM: To study whether exogenous neural stem cells (NSCs) derived from embryonic hippocampus differentiates into the neurons after transplanted into the infarct periphery of the brain in a stroke model and to further investigate the behavioral improvement in the rats.METHODS: The NSCs were prepared after isolated from the embryonic hippocampus of green fluorescent protein (GFP)-transgenic rats and cultured. The NSCs were identified using nestin and doublecortin(DCX) as markers. The cortical infarction in rats was induced using photochemical method, named photothrombotic cortical injury (PCI). Twenty adult rats were randomly divided into NSC transplantation group (NSC group) and control group. The cultured NSCs were transplanted into the infarct periphery of the rats in NSC group, and nothing in control group at first day was applied after PCI. The locomotor behavior of animals was checked using the rotarod test at 1st, 7th, 14th and 21st d after PCI. The survival and differentiation of transplanted NSCs were evaluated by immunocytochemical method at 12th week after PCI.RESULTS: The cells derived from the embryonic hippocampus significantly expressed the markers of NSCs. The grafted cells survived at least 12 weeks in the infarct periphery of adult rats and differentiated into mature glial cells and neurons. The density of NeuN+/GFP+ and volume of grafts at 12th week were less than those at 3rd week after transplantation (P<0.05). The time standing on the rod was longer in NSC group than that in control group at 7th, 14th and 21st d after PCI (P<0.01).CONCLUSION: The NSCs derived from embryonic hippocampus survive in the infarct periphery of adult rats up to 12 weeks and differentiate into mature neurons, which might be associated with the improvement of locomotor behavior of stroke animals. The neuronal replacement of neurons differentiated from NSCs may be the underlying mechanism.     

Differentiation of neurons from mouse induced pluripotent stem cells in vitro

AIM: To study the induction method of mouse induced pluripotent stem cells (iPSCs) that differentiate into neurons in vitro. METHODS: Mouse iPSCs were cultured in non-adherent culture dishes for 2 d to form embryoid bodies (EBs). The EBs were cultured for consecutive 2 d in the presence of retinoic acid (RA), and then were plated in the serum-free medium for adherent culture. Seven days later, Pasteur pipette was used to detach the differentiated cells around adherent EBs into “fragment” cell colonies with the help of dissecting microscopes, and these “fragments” were transferred to culture dishes with neural stem cell medium. Another 7 days later, the cells were plated onto the culture dishes using differentiation medium containing fetal bovine serum (FBS) and RA. The morphological changes of the cells were observed under inverted microscope. The iPSCs markers Oct4, Sox2 and SSEA1, the neural stem cell (NSC) marker nestin, the neuronal marker microtubule-associated protein 2 (MAP-2), the astrocyte marker glial fibrillary acidic protein (GFAP) and oligodendrocyte marker myelin basic protein (MBP) were detected by immunofluorescence method. The mRNA expression of GFAP, nestin, β3-tubulin, MAP-2 and MBP was detected by RT-RCR. MAP-2 gene sequence was identified. The proportions of NSCs differentiated from iPSCs and neurons from NSCs were detected by flow cytometry. RESULTS: Mouse iPSCs strongly expressed Oct4, Sox2 and SSEA1, and formed spherical EBs by suspended culture. The EBs were induced by RA and serum-free medium in adherent culture for 2 d, and rosette structure was observed under the microscope. “Fragments” separated by Pasteur pipette from the rosette structure formed neurosphere-like colonies. After the colonies were cultured in adherent condition for 5 d to 7 d in the presence of RA and FBS, the typical neurite was observed under the microscope. The neurospheres expressed nestin and their differentiated derivatives expressed MAP-2, GFAP and MBP, respectively. RT-PCR analysis and gene sequencing showed that the neurons were induced successfully. The results of flow cytometry demonstrated that 63.93%±1.47% of iPSCs differentiated into NSCs and 21.4%±1.70% of NSCs differentiated into neurons. CONCLUSION: Mouse iPSCs proliferate stably and differentiate into neurons in vitro, which provide a reliable source for the treatment of spinal cord injury.     

Hepatocyte growth factor enhances the neural differentiation from human embryonic stem cells

AIM: To investigate the possibility that hepatocyte growth factor (HGF) directly induces differentiation of human embryonic stem cells (hESCs) into neural progenitors (NPs). METHODS: hESCs colonies were induced to form the embryoid body (EB). Four-day-old EBs were randomly divided into 4 groups: control group (EBs were cultured in neural induction medium); G5 supplement group (EBs were cultured in neural induction medium supplied with G5 supplement); HGF group (EBs were cultured in neural induction medium supplied with 10 mg/L HGF), and HGF+G5 group (EBs were cultured in neural induction medium supplied with 10 mg/L HGF and G5 supplement). After induced in suspension system for 7 days, EBs with various treatments were cultured in poly-D-lysine/laminin-coated plates for 7-10 days for selection of NPs. NPs were gathered by 0.3 g/L dispase treatment and characterized by immunofluorescence staining. The percentages of the nestin+ cells in NPs in various groups were detected by fluorescent activated cell sorter (FACS). The multipotency of NPs was determined by immunofluorescence staining after the NPs were cultured without G5 and HGF for 7 days. The expression of region markers of neural progenitors treated with sonic hedgehog (Shh) protein (one of the neural inductive signals), was detected by RT-PCR. RESULTS: HGF+G5 supplement induced hESCs differentiation into neural progenitors. Immunofluorescence staining indicated that NPs differentiated from hESCs expressed NP markers including nestin, Pax6 and musashi-1. FACS data showed that the proportion of nestin positive cells in HGF+G5 supplement group (87.3%±3.9%) was the highest in all treatment groups. The time of HGF and G5 supplement treatment was important to differentiate into NPs, the maximal effect was observed at 7th day. After treated with Shh, the expression of ventral forebrain/hindbrain marker genes (Nkk2.1, and Nkk2.2) and hindbrain progenitor marker gene Gbx2 in NPs were upregulated, while the forebrain progenitor marker genes Otx2 and Bf1 were downregulated. CONCLUSION: The neural induction system containing HGF and G5 supplement effectively induces the differentiation of hESCs into NPs, which might be a potent model for investigating the mechanism of neural development and differentiation.     

Inhibitory effect of all-trans retinoic acid on proliferation of microvascular endothelial cells

AIM: To evaluate the effects of all-trans retinoic acid (atRA) on the proliferation in cultured mouse cerebral microvascular endothelial cells (bEnd.3). METHODS: Cultured cells were divided into five groups randomly, one as control group, the other four groups were 10-9, 10-8, 10-7 and 10-6 mol/L group. Effects of atRA on proliferation in bEnd.3 cells were detected by flow cytometry and immunocytochemitry of PCNA and MTT at 24 h, 48 h and 72 h. The effects of atRA (10-6 mol/L group) on the expressions of angiogenic genes in bEnd.3 cells were studied using microarray. RESULTS: The results of MTT and flow cytometry showed that all-trans retinoic acid at concentration of 10-6 mol/L significantly inhibited the proliferation of bEnd.3 cells. Immunocytochemical staining showed the expression of PCNA was markedly decreased in bEnd.3 cells at 24 h after treatment with atRA. Microarray results demonstrated that there were 11 down-regulated angiogenic genes and 2 up-regulated angiogenic genes in 10-6mol/L atRA group. CONCLUSION: All-trans retinoic acid at concentration of 10-6mol/L may significantly inhibit the proliferation of bEnd.3 cells treated for 24 h in vitro via down-regulation of angiogenic genes and PCNA expression.     

NT-3 genetically modified Schwann cells promote neural stem cells to differentiate into neuron-like cells

AIM: To explore the effects of neurotrophin-3 (NT-3)-genetically modified Schwann cells (NT-3-SCs) on differentiation of neural stem cells (NSCs) into the neuron-like cells. METHODS: The NSCs were co-cultured with NT-3-SCs. Report gene LacZ genetically modified Schwann cells (LacZ-SCs) and normal SCs respectively in vitro. 7 d later, the differentiation of NSCs was studied by immunohistochemistry, and the percentage of neuron-like cells was calculated. RESULTS: NSCs differentiated to the GFAP-positive cells (glial-like cells) and NF-positive cells (neuron-like cells) in vitro. Compared to the normal SCs, NT-3-SCs more efficiently promoted NSCs to differentiate into the neuron-like cells. The effect of LacZ-SCs was as the same to the normal SCs. CONCLUSION: NT-3-SCs promote NSCs to differentiate into the neuron-like cells.     

Differentiation of neural stem cells after transplanted into vitreous and the effects on the regeneration of retina ganglion cells

AIM: To investigate the differentiation of neural stem cells (NSCs) after transplanted into vitreous and the effects on the regeneration of retina ganglion cells (RGCs) after optic nerve microcrushed.METHODS: After optic nerve microcrushed in adult rat,2×10⁴/2 μL NSCs or 2 μL 0.1 mol/L PBS was injected into vitreous.Animals were divided into control group (MC group,MC+PBS group) and experiment group (MC+NSCs).Animals in each group were allowed to survive for 3,4,5 weeks,respectively.The regenerating RGCs were labeled retrogradely with granular blue,and the numbers of regenerating RGCs in each retina were observed under fluorescent microscope.In addition after 5 animals in MC+NSCs group survived for 4 weeks,rat eyeballs were removed and prepared as freezing microtome sections for observing the migration of NSCs and NF,GFAP,CNP immumodetection.RESULTS: Compared the mean numbers of regenerating RGCs between experiment group and control group at 3,4,5 weeks,the difference was significant (P<0.01).NSCs expressed NF,GFAP and CNP at 4 weeks and were not found to incorporate into retina.CONCLUSION: It suggests that NSCs enhance the RGCs regeneration after ON microcrushed and differentiate into neurons,astrocytes and oligodendrocytes.     

Enhanced proliferation and neuronal differentiation of neural stem cells by bone marrow stromal cells

AIM:To evaluate whether bone marrow stromal cells (BMSCs) could provide a supportive microenvironment for proliferation and differentiation of neural stem cells (NSCs). METHODS:The proliferation and differentiation of NSCs were compared, in media without growth factors or in BMSCs-conditioned media. RESULTS:The proportion of neurons to total NSCs was significantly increased when NSCs were cultured in BMSCs-conditioned media (41.1%±3.2% vs 23.3%±16.5%, P<0.05), while the proportion of astrocytes was significantly decreased (33.8%±4.9% vs 65.0%±10.4%, P<0.01), as compared with the NSCs cultured in media without growth factors. The proportion of proliferated cells was also significantly increased (74.7%±4.7% vs 51.4%±12.3%, P<0.01). CONCLUSION:These finding indicates that BMSCs support the proliferation and neuronal differentiation of NSCs and suggests that their addition may be useful to improve outcomes of NSCs transplantation.     

Effect of heat treatment on striatum neurons

AIM: To determine the effect of heat treatment on rat primary cultured neurons, and give fundamental research for candidate molecule to protect the neurons from heat injury. METHODS: Neurons from rat striatum were primary cultivated in D-MEM with 15% horse serum, and when got mature, cells were identified by immuno-cytochemical staining with neurofilament protein (NF), tyrosine hydroxylase (TH) and neuron specific enolase (NSE) antibodies. Cells in heat treatment groups were put in an 43 ℃ CO₂ incubator for 1 h, and the control groups at 37 ℃ as normal. Striatum neurons were stained with trypan blue and dual fluorscence dye (PI/H33258) immediately followed heat treatment, and necrosis rate of neurons was estimated. At the same time, activated caspase-3 immuno-cytochemical and TdT TUNEL^ methods were applied to determine apoptosis rate, and cell volume was also identified with micro-photography. RESULTS: During day 7 to day 9, the cultured striatum neurons got mature, and many neuronal fibers starched out and formed neuron network, NF, TH, and NSE staining positive. Treatment at 43 ℃ for 1 h, cell number decreased greatly, while NF⁺ percentage kept unchanged, and the heat treatment survived neurons were processing cell necrosis and apoptosis, but necrosis percentage was much greater than that of apoptosis. While cell volume kept unchanged after heat treatment. CONCLUSION: Heat treatment greatly affects the growth and survival of the cultured striatum neurons, and the injury effect is most due to cell necrosis process.     

The correlation between differentiation and apoptosis in the process of differentiation from adult rat mesenchymal stem cells into neuron-like cells

AIM:To supply the theoretic evidences of elongating the lifetime of neuron-like cells differentiated from adult rat mesenchymal stem cells, we investigated the relationship between the differentiation and apoptosis in the process of induction. METHODS: The mesenchymal stem cells(MSCs) were isolated primarily from rat bone marrow, and purified by passage culture. The 5th passage of MSCs was induced by β-mercaptoethanol and all-trans-retinoic acid (ATRA). After 1 h, 3 h and 5 h of induction, the cells were stained immunocytochemically with anti-MAP-2 and anti-GFAP antibodies, respectively. In addition to counting the ratio of neuron-like cells in MSCs, DAPI staining was employed to identify whether the differentiated cells have an apoptotic morphological changes. The ratio of apoptotic cells at 1 h, 3 h and 5 h after induction were detected by flow cytometry (FCM). CONCLUSIONS:1. β-mercaptoethanol and ATRA had the different ability that induced MSCs to differentiate to neuron-like cells. 2. Apoptosis was also initiated in the process of differentiation, and there is positive correlation between the ratio of differentiation and apoptosis.     

Activation of Nrf2 by 18α-GA affects proliferation of adult neural stem cells

AIM: To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) activation by 18α-glycyrrhetinic acid (18α-GA) on the proliferation and self-renewal of adult neural stem cells (aNSCs), and to explore an effective way of maintaining the viability of aNSCs. METHODS: NSCs were dissociated from subventricular zone of the mice at postnatal days 0, 60, and 300. The expression levels of Nrf2 in the NSCs at various ages were compared. After treatment with 18α-GA, the expression of Nrf2 was examined by real-time PCR and Western blot. shRNA lentiviral vector (LV) carrying green fluorescent protein (GFP) gene was constructed to knock down Nrf2 expression. The knockdown efficiency in the aNSCs was detected by real-time PCR and Western blot. Subsequently, the aNSCs were divided into DMSO group, 18α-GA group, LV-GFP group and LV-Nrf2-shRNA group. BrdU incorporation assay, Tuj1 staining, CCK-8 assay, Hoechst 33342/PI staining and detection of reactive oxygen species (ROS) were performed to analyze the proliferation, differentiation, viability, apoptosis and oxidative stress levels of the NSCs. RESULTS: The mRNA expression level of Nrf2 in adult and aged NSCs was significantly lower than that in newborn NSCs (P<0.01), while the ROS level of aNSCs was significantly higher (P<0.05). After treatment with 18α-GA, the expression level of Nrf2 in the aNSCs was significantly up-regulated as compared with DMSO group (P<0.01). Increased number of BrdU⁺ and Tuj1⁺ cells was observed in 18α-GA group, indicating that 18α-GA-treated cells had higher viability (P<0.05). Meanwhile, there were fewer apoptotic cells and lower ROS level in 18α-GA group than those in DMSO group (P<0.05). After knockdown of Nrf2 in aNSCs and then treated with 18α-GA, there were less BrdU⁺ and Tuj1⁺ cells, as well as the aNSCs with lower viability in LV-Nrf2-shRNA group (P<0.05). Moreover, the ROS level was increased in LV-Nrf2-shRNA group as compared with LV-GFP group (P<0.05). CONCLUSION: Activation of Nrf2 by 18α-GA elevates the antioxidant capacity of aNSCs, thus ameliorating the cell proliferation and differentiation potentials.     

Effect of Xiaoyaosan on proliferation and differentiation of hippocampus nerve precursor cells under high corticosterone condition

AIM:To investigate the effect of Xiaoyaosan (XYS) drug-containing serum on proliferation and differentiation of hippocampus nerve precursor cells (HNPC) under high corticosterone (CORT) concentration condition. METHODS:Serum-free culture in vitro was adopted to culture HNPC. CORT at concentration of 120 μmol/L was used to establish the high CORT concentration condition. XYS is composed of Bupleurum Chinese DC, Chinese angelica root, Paeonia lactiflora Pal1, India bread, Largehead atractylodes rhizome, peppermint herb, fresh ginger and Licorice root. XYS drug-containing serum of different dose were prepared.10% drug-containing serum and 10% serum were adopted. RU38486, an antagonist of CORT, was used as positive control. MTT method was used to detect proliferation rate of HNPC, the methods of immunofluorescence ambi-tagged by BrdU with TUNEL, β-tubulin-Ⅲ and glial fibrillary acidic protein (GFAP) with TUNEL respectively were adopted to detect the proliferation and differentiation of HNPC. RESULTS:CORT at concentration of 120 μmol/L degraded the proliferation rate of HNPC significantly (P<0.01), which was reversed by RU38486 (P<0.05). 10% XYS drug-containing serum of each dose enhanced the proliferation rate (P<0.05 or P<0.01). After 120 μmol/L CORT processed, the staining fluorescence intensity ratio of BrdU/TUNEL of HNPC degraded significantly (P<0.01), while increased after treatment with RU38486 and 10% XYS drug-containing serum of each dose (P<0.01). Under high CORT concentration condition, the apoptotic rates of glial cells and neurons differentiated from HNPC increased significantly (P<0.01). RU38486 and 10% XYS drug-containing serum of each dose inhibited the apoptotic rates of glial cells and neurons (P<0.05 or P<0.01). CONCLUSION:Under high CORT concentration condition, XYS promotes the proliferation of HNPC, and inhibits the apoptotic rates of glial cells and neurons differentiated from HNPC, which act as the effect of anti-damage of CORT to hippocampus in stress state.     

Mesenteric lymph drainage alleviates spleen injury in hemorrhagic shock rats

AIM：To observe the effects of post-shock mesenteric lymph (PSML) drainage on histopathology, apoptosis, cell cycle and proliferation of the spleen in rats with hemorrhagic shock. METHODS：Eighteen Wistar rats were randomly divided into sham, shock and shock+drainage groups (n=6 in each group). The hemorrhagic shock model was established in the shock and shock+drainage groups. Fluid resuscitation for 30 min was performed 1.5 h after hypotension, and PSML was drained in the rats in shock+drainage group from 1 h after hypotension to 3 h after resuscitation finished. The fixed spleen tissue was harvested from each rat for histological observation with HE staining. The apoptosis of splenocytes was observed by Hoechst 33258 staining. The expression of Bcl-2 and Bax proteins was detected by immunohistochemical staining. The cell cycle and the expression of p53 protein were measured by flow cytometry, and the proliferation index (PI) was calculated. RESULTS：Compared with sham group, splenic tissue injury appeared in the shocked rats. The apoptotic cells and the expression of Bax and p53 in shock group were increased, while Bcl-2 expression was decreased. The percentage of G2/M cells in shock group was decreased. Compared with shock group, the splenic tissue damage in shock+drainage group was significantly attenuated. Moreover, the number of apoptotic cells, the percentage of G0/G1 cells, and the expression of Bax and p53 were obviously decreased, and the G2/M cells, Bcl-2 protein expression and PI were significantly increased in shock+drainage group. CONCLUSION: PSML drainage alleviates splenic injury in hemorrhagic shock rats, which may be related to reducing the apoptosis of splenocytes.     

In vitro differentiation of exocrine pancreatic cells from mouse embryonic stem cells

AIM: To explore the effects of sodium butyrate, activin A and dexamthasone on inducing mouse embryonic stem (ES) cells to differentiate into exocrine pancreatic cells in vitro. METHODS: E14 mouse ES cells were cultured in suspension to form embryonic bodies (EBs). The EBs were cultured with differentiating medium containing different concentrations of sodium butyrate, and the spontaneously differentiated ES cells were used as control. Exocrine pancreatic genes such as amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected by RT-PCR at different time points to determine the optimal concentration and exposure time of sodium butyrate. Furthermore, activin A or dexamthasone was also used to explore the effects on exocrine differentiation. After that, the combination of sodium butyrate, activin A and dexamthasone was used to promote the differentiation of exocrine pancreatic cells from ES cells. During the differentiation course, the gene expressions of amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected by RT-PCR. Morphological changes were investigated by phase contrast microscopy. Amylase expression was examined by immunofluorescence staining. RESULTS: Exocrine pancreatic gene expressions such as amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected in spontaneously differentiated EBs. A relatively lower concentration of sodium butyrate with a shorter exposure time significantly promoted those above gene expressions as compared to that of spontaneously differentiated EBs. Activin A and dexamethasone induced upregulation of exocrine gene expression. The combination of activin A, sodium butyrate and dexamethasone significantly enhanced the mRNA levels of amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase. Under the treatment of activin A, sodium butyrate and dexamethasone, differentiated cells were polygonal in shape with large, round, and center-situated nuclei. According to the observation of immunofluorescence staining, amylase was positive expressed at the final stage. CONCLUSION: These data indicate that exocrine pancreatic differentiation of ES cells is induced by sodium butyrate, activin A and dexamethasone. The combination of pancreatic inducing factors improves the differentiating efficiency.     

Effects of human bone morphogenetic protein 2 and 9 on proliferation,apoptosis and migration of human gastric carcinoma MNK-45 cells

AIM: To investigate the effects of human bone morphogenetic protein 2 (BMP2) and BMP9 on the proliferation, apoptosis and migration of human gastric carcinoma cell line MNK-45. METHODS: Immunocytochemical staining, MTT assay, wound-healing test, Transwells migration test, Hoechst 33258 staining and flow cytometry (FCM) were used to determine the infection of AdBMP2 and AdBMP9 on the proliferation, apoptosis and migration of MNK-45 cells. The expression of GSK-3β (including p-GSK-3β and total GSK-3β) and β-catenin in MNK-45 cells was also detected by Western blotting. RESULTS: The proliferation of MNK-45 cells was inhibited from the third day on and in a time-dependent manner after infected with AdBMP2 and AdBMP9. The results of Hoechst 33258 staining and FCM proved that apoptosis rates in BMP2 group and BMP9 group were higher than that in GFP group. Both wound-healing test and Transwell experiment indicated that up-regulating the expression of BMP2 and BMP9 inhibited the migration of MNK-45 cells. The phosphorylation levels of GSK-3β in BMP2 group and BMP9 group were higher than that in GFP group. However, no significant change of β-catenin among groups was observed. CONCLUSION: Up-regulation of BMP2 and BMP9 expression inhibits the proliferation of MNK-45 cells.     

Effect of SCF and G-CSF pretreatment on the proliferation and the differentiation of bone mesenchymal stem cells

AIM: To investigate the effect of pretreatment of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) on the proliferation and the differentiation of mesenchymal stem cells (MSCs) into cardiomyogenic cells. METHODS: The MSCs, isolated primarily from bone marrow, and purified by passage culture, were obtained from the adult rats of four groups: the rats were pretreated by 5 daily injections of SCF; the rats were pretreated with G-CSF; the rats were pretreated with SCF and G-CSF; the rats were treated without any intervention. The 4th passage of MSCs was labeled by DAPI and cellular cycle analysis was conducted by flow cytometry before co-culture. The neonatal rat cardiomyocytes cultured for 3 days were co-cultured with DAPI-MSCs. The percentage of the differentiation of MSCs into cardiomyogenic cells during the five co-culture days was analyzed. The morphologic changes of MSCs and the proteins expression of cardiac myosin heavy chain (MHC) and troponin T (TnT) were recorded respectively with digital microscope camera system and immunofluorescence technique. The percentage of the differentiation of MSCs into cardiomyogenic cells was also calculated. RESULTS: The percentage of MSCs in G₀/G₁ phase in SCF/G-CSF group was significantly lower than that in SCF group, G-CSF group and the control group. The percentage of MHC protein-positive MSCs in SCF/G-CSF group was markedly higher than that in SCF group, G-CSF group and the control group, and that in SCF group and G-CSF group was significantly higher than control group. The percentage of TnT protein-positive MSCs in SCF/G-CSF group, SCF group and G-CSF group was significantly higher than that in control group.CONCLUSION: SCF and G-CSF show the ability to stimulate the proliferation of MSCs and induce MSCs to differentiate into cardiomyocytes. The combination of using SCF and G-CSF is more effective than using only SCF or G-CSF.