Research progress on function and molecular mechanism of transcription factor ELK1 of Ets / 中国生物制品学杂志

@#Ets transcription factor ELK 1,a member of the ternary complex factor(TCF) subfamily in the Ets family,is directly downstream signal of MAPK/ERK pathway and is activated by phosphorylation to execute the function of ERK signal.ELK1,which is highly expressed and phosphorylated in stem cells and tumor cells,plays a role in promoting proliferation,inhibiting apoptosis and differentiation in stem cells.In tumor cells,ELK1 has shown to promote proliferation,migration,and inhibit apoptosis.In neural cells,ELK1 is involved in long-term memory,learning,addiction and other functions.In this paper,the research progress on the main structure and basic biological characteristics of ELK1,the function and mechanism of ELK 1 in tumor cells,stem cells and nerve cells are reviewed in order to provide new ideas for the follow-up research.

Effects of Cyclic Strain on Neural Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells / 医用生物力学

Objective To study the mechanical effects of cyclic strain on neural differentiation of rat bone marrow mesenchymal stem cells (rBMSCs). Methods The rBMSCs were subjected to cyclic strain for 24 hours andthen cultured for 5 days. The expression of neural markers and the phosphorylation of relative signaling pathway proteins were evaluated. The stress distribution on cell surface was analyzed by finite element method. The differentially expressed genes induced by strain were identified by RNA sequencing analysis. Results The 0. 5 Hz strain with 5% magnitude could significantly induce higher expression of neural markers and elevated phosphorylation level of extracellular-signal-regulated kinase (ERK), protein kinase B (AKT) and mammalian target of rapamycin ( mTOR). KEGG pathway analysis showed that the focal adhesion and ECM-receptor interaction were significantly enriched under cyclic strain. Conclusions Cyclic strain could change the interaction of cells with the extracellular matrix ( ECM) and enhance the AKT/ mTOR and ERK pathway, finally promote rBMSC neural differentiation. Knowledge about the impact of mechanical stimulation on BMSC neural differentiation is expected to improve the efficiency of stem cell differentiation, shed light on device design for tissue engineering, and promote clinical application of mesenchymal stem cells in neural issue repair and regeneration.

Effects of Dianxianqing granules on tau protein in P301S mice by regulating mitophagy / 中国药房

OBJECTIVE To study the effects of Dianxianqing granules on the tau protein in P301S mice by regulating mitophagy. METHODS Totally 36 P301S mice were randomly divided into model group， Dianxianqing granule group （12.48 g/kg）， donepezil hydrochloride group （positive control， 1.3 mg/kg）， with 12 mice in each group； another 10 C57BL6 mice were selected as control group. Administration groups were given relevant drug solutions intragastrically， and control group and model group were given constant volume of water intragastrically. The gavage volume was 20 mL/kg， once a day， for consecutive 5 months. During the experiment， the general condition of mice was observed in each group. After the last medication， the learning and memory ability was determined by Y maze test and Morris water maze test； HE staining was used to observe the morphological changes in brain tissue， and Nissl staining was used to observe the structure of neural cells and the number of Nissl bodies in cerebral tissue. Immunohistochemistry was used to detect the expressions of phospho-tau serine 202/threonine 205 （abbreviated as AT8） in brain tissue. Western blot assay was used to determine the expressions of mitophagy-associated proteins [PTEN-induced putative kinase-1 （PINK1）， Parkin， microtubule-associated protein 1 light chain 3B （LC3B）， p62]， synaptic-associated proteins [postsynaptic density protein-95 （PSD-95）， synaptophysin （SYP）， and growth-associated protein-43 （GAP-43）] and the phosphorylation of tau protein [expressed by the phosphorylation levels of serine 199 （Ser199） and Ser202] in brain tissue. RESULTS The mice in E-mail：lnzyxyqy2003@163.com model group showed symptoms such as white hair， decreased body mass， and lower limb paralysis， with incomplete hippocampal structures in their brain tissue， as well as incomplete cell membrane edges and cell structures； the spontaneous alternating response rate， the times of crossing platform， the number of Nissl bodies， the protein expressions of PINK1， Parkin， LC3B， SYP， GAP-43， and PSD-95 were decreased significantly， compared with control group； swimming latency （fourth and fifth day）， the protein expressions of AT8 and p62，the phosphorylation levels of Ser199 and Ser202 were increased or lengthened significantly， compared with control group （P＜0.05 or P＜0.01）. Compared with model group， the above symptoms and indexes of mice were improved significantly in administration groups （P＜0.05 or P＜0.01）. CONCLUSIONS Dianxianqing granules can effectively improve cognitive impairment in P301S mice，the mechanism of which may be associated with inducing mitochondrial autophagy， reducing the hyperphosphorylation of tau protein， up-regulating the expression of synaptic-associated proteins in brain tissue，and repairing damaged neural cells.

The protective effect of l-carnosine on neurotoxicity of deguelin-induced SH-SY5Y cells / 中国药理学通报

Aim: To study the protective effect of L-carnosine on deguelin-induced neurotoxicity. Methods: SH-SY5Y cells were treated with 1, 10, 20, 40, 60, 80, 100 mmol · L" L-carnosine for 24 h; cell viability was detected by CCK-8 assay. SH-SY5Y cells were respectively treated with 30 mmol · L-1 L-carnosine, 20 μmol · L-1 deguelin, 20 μmol · L-1 deguelin with 30 mmol · L-1 L-carnosine for 24 h; AO/EB method was employed for observing the morphology and apoptotic morphology of treated cells. SH-SY5Y cells were respectively treated with 8 μmol · L-1 deguelin, 8 μmol · L-1 deguelin with 3 mmol · L-1 L-carnosine, 8 μmol · L-1 deguelin with 30 mmol · L-1 L-carnosine, 30 mmol · L-1 L-carnosine, 20 μmol · L-1 deguelin, 20 μmol · L-1 deguelin with 3 mmol · L-1 L-carnosine, 20 μmol · L-1 deguelin with 30 mmol · L-1 L-carnosine, 50 μmol · L-1 deguelin, 50 μmol · L-1 deguelin with 3 mmol · L-1 L-carnosine, 50 μmol · L-1 deguelin with 30 mmol · L-1 L-carnosine for 24 h. Cell proliferation was detected by CCK-8 assay; apoptotic rate of treated cells was determined by flow cytometry; the reactive oxygen species (ROS) of treated cells was detected by DCFH-DA staining flow cytometry. Results: After 30 mmol · L-1 L-carnosine was co-treated with 20 μmol · L-1 and 50 μmol · L-1 of deguelin, the cell inhibitory rate decreased by 9. 07% and 6. 1%, the number of early apoptotic cells decreased, and the early apoptotic rate decreased by 9. 35%. The total apoptotic rate decreased by 10. 7%, and the ROS level was lower than that of the deguelin alone treatment group. The difference was statistically significant (P < 0. 05). Conclusions: L-carnosine can effectively reduce the neurotoxicity of deguelin-induced SH-SY5Y cells, which may be related to the reduction of oxidative stress levels, thereby inhibiting apoptosis and protecting cells.

Effects of Bcl-2 associated with athanogene-1 gene silencing on heat shock protein 70 expression and human neuroblastoma cell apoptosis induced by hypoxia / 中国中西医结合急救杂志

Objective To investigate the protective effects of Bcl-2 associated with athanogene-1 (BAG-1) gene on human neuroblastoma cells (SH-SY5Y) injury induced by hypoxia/reoxygenation,and its influence on heat shock protein 70 (HSP70) expression.Methods The SH-SYSY cells at logarithmic growth phase were collected.Lenti virus mediated RNA interference (RNAi) technology was used to suppress the BAG-1 expression.The cells screened out can be divided into four groups:the cell control group with no lentivirus infection,lentivirus control group (containing only fluorescein protein lentivirus infection),BAG-1 siRNA group (BAG-1 siRNA silencing group),including BAG-1 siRNA-α group and BAG-1 siRNA-β group with lentivirus containing fluorescein protein (GFP) but at different BAG-1 siRNA target sites of silencing.Western Blot was used to detect the protein expression of BAG-1 and HSP70 in target cells after infectious recombination lentivirus for 72 hours;the Cell Counting Kit-8 (CCK-8) was used to detect the activity of four different group cells after hypoxia;the flow cytometry was used to detect the cell apoptosis;the HSP70 mRNA transcription level were detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-qPCR) respectively in each group.Results After lentiviral infection for 72 hours,the Western Blot results showed that in the two BAG-1 siRNA silencing groups,the interference effect of BAG-1 siRNA-β group was superior to that of BAG-1 siRNA-α group.The cell viability of each group showed an increase followed by a decrease with the prolongation of hypoxia time,and reaching the peak at 8 hours.After hypoxia for 8 hours being given,the cell viability in BAG-1 siRNA-β group was significantly lower than that of the cell control group,lentivirus control group and BAG-1 siRNA-α group (A value:0.59 ±0.09 vs.0.94±0.12,0.90± 0.11,0.91± 0.14,P ＜ 0.01);the cell apoptosis rate was obviously higher in BAG-1 siRNA-β than that in the above three groups [(34.63 ± 3.46)％ vs.(14.83 ± 3.75)％,(19.93 ± 6.49)％,(16.40± 1.18)％,all P ＜ 0.01].There were no statistically significant differences in the HSP70 protein level and mRNA transcription level between BAG-1 siRNA-3 grroup,and the cell control group,lentivirus control group and BAG-1 siRNA-α group respectively (all P ＞ 0.05).Conclusion BAG-1 gene can play a role in protection of hypoxia nerve cells,reduce the apoptosis,and its protective effect can be independent of HSP70 gene.

Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

Variation in Growth Pattern and Morphological Appearance of Primary Monolayer Cultures of Chondrocytes and Neural Cells Isolated from the Chick Embryo at Different Stages.

Aim: This study was an attempt at investigating the variation in growth and morphology of primary avian chondrocyte and neural cell cultures established under standard laboratory conditions. Methods: For establishing chondrocyte cultures, the tibia were dissected from chick embryos that were 12 (E12), 14 (E14) and 15 (E15) days old. For the neural cell culture initiation, the two lobes of the forebrain were dissected from chick embryos that were 4 (E4), 8 (E8) and 12 (E12) days old. The final characterization of the cells was performed by H&E and CFV staining. Results: In the chondrocyte cultures, two forms of morphology were observed which is reversible in process.The primary variation evaluated in chondrocytes cultures was the effect of the media on the state of the cells. Based on whether the cells were cultured in DMEM or MEM, there was a difference in the transformation of the attached cells from the differentiated to de-differentiated forms. The primary variation examined in neuron cultures was the embryonic age of the tissue and the effect that it had on the proliferation and neurite formation of the cells. E8 embryo neural cells cultures result in well-developed cells with neurite formation along with axonal and dendritic outgrowths with highly complex interconnections between them. Conclusion: Ultimately, this study demonstrated the composition of culture media had an effect on the morphological appearance of the chondrocytes, as well as, confirmed that the culture of primary neurons is best performed using cells from an E8 embryo.

Effect of lavage with artificial cerebrospinal fluid on neural cell apoptosis and the ERK pathway after rat traumatic brain injury / 中南大学学报(医学版)

Objective: To explore the effect of lavage with artiifcial cerebrospinal lfuid on neural cell apoptosis and the extracellular regulated kinase (ERK) pathway atfer traumatic brain injury. Methods: A total of 192 SD rats were randomly divided into a sham group, a traumatic brain injury model group, a local artiifcial cerebrospinal lfuid group, and a local saline group. Each group was divided into 4 sub-groups by the sacriifced time at 6 h, 12 h, 1 d and 3 d atfer the operation. hTe phosphorylation of extracellular regulated kinase 2 (P-ERK2), TNF-α and cellular apoptosis were examined. Results: hTe levels of P-ERK2 protein and TNF-α protein, as well as the number of apoptotic cellsat each time point in the local artiifcial cerebrospinal lfuid group were lower than those in the model group or in the saline group (P<0.05). Conclusion: Lavage with artificial cerebrospinal fluid can reduce apoptosis of neural cells after brain injury through the ERK pathway.

Human SNF2L Gene Is Regulated Constitutively and Inducibly in Neural Cells via a cAMP-Response Element

PURPOSE: SNF2L belongs to Imitation Switch family and plays an essential role in neural tissues and gonads. In our previous studies, we have demonstrated that the basal transcription of human SNF2L gene is regulated by two cis-elements, cAMP response element (CRE)- and Sp1-binding sites. Recent studies suggested that cyclic adenosine monophosphate (cAMP) stimulation significantly up-regulated SNF2L expression in ovarian granulose cells. These data suggested that protein kinase-mediated signal pathways might also regulate SNF2L expression in neural cells. We therefore investigated the effects of agents that activate protein kinases A on SNF2L gene expression in neural cells. MATERIALS AND METHODS: To increase intracellular cAMP levels, all neural cells were treated with forskolin and dbcAMP, two cAMP response activators. We exmined the effects of cAMP on the promoter activity of human SNF2L gene by luciferase reporter gene assays, and further examined the effects of cAMP on endogenous SNF2L mRNA levels by qPCR. RESULTS: Transient expression of a luciferase fusion gene under the control of the SNF2L promoter was significantly increased by treatment of rat primary neurons with forskolin or dbcAMP, but not PC12, C6 and SH-SY5Y cells. Consistently, treatment with forskolin or dbcAMP could enhance endogenous SNF2L mRNA levels also only in rat primary neurons. CONCLUSION: These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation and regulation by cAMP in neural cells.

Effects of nerve growth factor on the neural cells and Egr-1 gene expression in the brain of childhood intractable epileptics / 临床儿科杂志

Objective To investigate the effect of nerve growth factor (NGF) on the neural cells and expression of early growth response gene-1 (Egr-1 gene) in the hippocampus discharge zone of childhood intractable epileptics. Methods Acutely dissociated cell suspensions of childhood hippocampus discharge zone with non specific pathological changes (n = 16) were exposed to NGF group and control group respectively. There were three subgroups exposed to NGF at the levels of 12.5, 50, 100 ng/ml in NGF group. After 4 hr culturing, the astrocytes and neurons were lablled by Bb immunostain with the specific markers such as GFAP and MAP2. The total number of neural cells was counted under inversion fluorescence microscope and the Egr-1mRNA expression was detected by semiquantitative RT-PCR analysis. Results There were significant differences of the numbers of neural cells survived in hippoeampus region between the NGF group and the non-added NGF group (P < 0.01). Among the different levels of NGF 12.5, 50, 100 ng/ml, the number of the total cultural neurons and GFAP(+) astrocytes, MAP_2(+) neurons were increased with ascending levels of NGF (P < 0.05). At the same time, the expression of Egr-1mRNA (A_(Egr-1)/A_(β-actin)) in the NGF groups was also increased (P < 0.01). There was positive correlation between the number of the survivable neural cells and Egr-1mRNA quantity (r = 0.780, P < 0.01) among the three NGF groups. Conclusions NGF can protect the neural cells of epileptic discharge zone by promoting the expression of Egr-1 gene.

Prowess in the study on induction of bone marrow stromal cells into mature functional neural cells / 国际生物医学工程杂志

Bone marrow stromal cells(BMSCs) is a kind of adult stem cells which exists widely in human mesenchymal tissues and have active abilities of self-replication and multi-differentiation. When intervened individually or jointly by certain inducing agents and by the kind of genes, it can differentiate into specific lineage of cells in vitro and the capacity of multilineage differentiation and proliferation does not change. At the same time, with the unique immunological characteristics,it is an ideal seed cell for tissue engineering. In recent years, a lot of studies have proved that BMSCs can not only be induced and differentiate into nerve cells under certain conditions but have a certain structure and function of the nerve cells, which provide a new way in the treatment of nerve system diseases.This paper gives a review of studies on induced differentiation of BMSCs into nerve cells and the functions.

Correlation between the changes of neural cell apoptosis and caspase-3 gene expression after the brain traumatic injury in rats / 中华急诊医学杂志

Objective To observe the correlation between the changes of neural cell apoptosis arid caspase-3 gene expression in brain tissues following acute severe traumatic injury to brain(TIB).Method A total of 120 adult Spraque-Dawley rats were divided into a control group(n=8)，TIB group(n=56)and TIB with administration of caspase-3 inhibitor group(n=56).TIB models of rats were made with Feeney's method.The z-DEVDfmk(5 μg)，caspase-3 inhibitor，was administered by intracerebral infusion，and the rats were sacrificed 1，6，24，48 hours and 3，7，14 days postinjury(n=8 for each interval).The specimens of the injured cerebral cortex，suhcerticai white matter，hippocampus，dentate gyrus and contrahteral corresponding brain tissues were taken for detecting apoptesis of neural cells by the terminal deoxynucleotidyl transferase mediated DUTP nick end labeling (TUNEL)methods and flow cytomeay.Caspase-3 mRNA and protein expression were detected by using RT-PCR，immunohistochemistry and western blot analysis.The caspase-3 activity was detected by using caspase-3 fluorescent assay kit.Student t-test and Spearman correlation analysis were used to analyze the data with SPSS version 10.1 software package.Results Apoptesis indexes(AI)and the apoptesis percentage(AP)of neural cells in the injured brain regions increased quickly after injury，and reached its peak 24 to 48 hours later，then decreased slowly,but it remained at higher level above that of normal till 14 days later(P＜0.01).The levels of caspase-3 mRNA,eastme-3 protein and caspase-3 activity were increased significantly post injury，and reached its peak at 24 to 48 hours，then it gradually decreased.Compared with control group，the levels ofoptical density of caspase-3 proteins in the injured hippocampus and subcortical white matter at 24 and 48 hours post injury increased 1484% and 1690%，caspase-3 mRNA expressiom increased 1043%and 1180%，and the degreas of caspase-3 activity increased 148% and 183%，respectively.The expression of caspase-3 proenzyme and its P17 subarrit increased.After trealment with caspase-3 inhibitor z-DEVD-fmk，the levels of caspase-3 mRNA，protein expression and caspase-3 activity were significantly decreased.and AI and AP were significantly decreased as well.The correlation between caspase-3 mRNA and level of neural apoptesis was positive(r=0.821，P＜0.01)，and it was likewise between caspase-3 protein and level of neural apoptosis(r=0.638.P＜0.01).Interestingly enough，a positive correlation was found between caspase-3 mRNA and easpase-3 proteins(r=0.945，P＜0.01).Conclusions The activation of caspase-3 leads to apoptosis of neural cells after acute TIB.The expression of caspase-3 are consistent with apoptosis of neural cells following TIB.The regulation of caspase-3 induced by TIB occurs at a ceriain critical link before transduction.Caspase-3 inhibitor can efficiently inhibit apoptosis of neural cells following TIB.

Progress in the study on neuronal differentiation of bone marrow-derived mesenchymal stem cells in vitro / 国际生物医学工程杂志

Bone marrow-derived mesenchymal stem cells(BMSCs)exist in and can be isolated from the bone marrow of adult human being and animals and can be induced to differentiate into different cells.They are easy to be isolated,cultivated,and amplified.They are also less immunogenic in the body and can avoid ethical dispute when applied in transplantation.Studies have shown that BMSCs can be induced to differentiate into neural cells under certain conditions in vitro.This article reviewed the recent development in the research on the induction protocols for neural differentiation of BMSCs and the possible mechanisms.

Differentiation of Mouse Embryonic Stem Cells into Neural Cells on Silk Fibroin Scaffolds / 中国生物工程杂志

Induced the mouse embryonic stem(ES)cells into neural cells on silk fibroin via the improved 4-/4+ RA method to explore the effect of the silk fibroin to the ES-derived neurons' growth,adherence and differentiation.Suspended the ES cells into EBs and then transferred them to three different substrates-coated 35 mm dishes including gelatin,Bombyx mori silk fibroin(SF) and Tussah silk fibroin(TSF) to identify the adherence and proportion of ES cells-derived neurons under these three substrates.The results showed that the EBs adhered to the gelatin and TSF are faster than to the SF.The average adhesive rate on gelatin and TSF are 90.3% and 84.4% respectively,and only 38.5% on SF,all the proportion of ?-Ⅲ-Tubulin positive cells is approximately 40%.It may provide important experimental information for tissue engineering,in which ES cells-derived neuron cells and silk fibroin materials are scaffolds,and also offer a source for cell therapy research of neurodegenerative disease.

Experimental study on cultivation of adult human retinal neural cells / 眼科

Objective To establish a culture system in vitro of adult human retinal neural cells for providing a model for the research of retinal neural cells. Design Experimental study. Participants Cultured adult human retinal neural cells. Methods The isolated cells from adult human postmortem retina (20?40 years old) were cultured, then cells of different stages were identified with immunocytochemical staining and judged with phase contrast microscopy and electron microscopy. Main Outcome Measures Cellular morphology and structure. Results (1) The results of cell culture: the adult retinal neural cells could survive in vitro under some conditions and were identified as NSE positive mostly. (2) The results of electron microscopy: most cultured cells were photoreceptors, bipolar cells, horizontal cells and some were glial cells with scanning electron microscopy. Conclusions Under feasible conditions, the adult human retinal neural cells could be cultured and maintained effectively in vitro.

The effect of Shihusan on intracellular free calcium of human retinal neural cells / 眼科

Objective To explore the effects of Shihusan on the intracellular free calcium of human retina cells and the protection from the damage of glutamate. To investigate its function and mechanism in retinal degeneration diseases, and to offer the basis on which to treat the similar disease clinically. Design Experimental study. Participants Retinal cells from normal human eye. Methods Cultivation of human retinal neural cells was perfonned. The changes of fluorescent density of intracellular free calcium of cultured cells labeled with Fluo3/AM before and after adding Shihusan and verapamil were detected with laser scanning confocal microscopy. In addition, the effect of Shihusan on calcium overload provoked by glutamic acid was observed. Main Outcome Measures The level of intracellular free calcium of retinal cells. Results The retinal cells adhered, spread and grew well in vitro. The fluorescence intensity increased 88% in glutamic acid group, decreased 14.8% and 57.3% in Shihusan group and verapamil group respectively. Shihusan couldn't decrease the intracellular Ca2+ that was increased by glutamic acid, but could inhibit the increasing tendency induced by glutamic acid. The difference was significant compared to control group (P=0.000). Conclusions Shihusan might decrease the level of intracellular free calcium in cultured human retinal cells, and resist the damage of glutamate. Its mechanism was related possibly to resisting calcium overload and inhibiting apoptosis.

Expression of specific proteins of neural cell in amniotic epithelial cells in rats / 中国康复理论与实践

@#ObjectiveTo detect specific antigens of neural cells in amniotic epithelial cells(AECs) in rats. MethodsAECs were dissociated and purified from the amnion of pregnancy 12—14 d rats. The expression of specific markers of neural stem cells (Nestin, Musashi) and differentiated cells (MAP-2,NSE,GFAP) and ChAT, NT-3 in the AECs were detected by immunocytochemistry. ResultsThe cultured AECs displayed positive immunoreactivity to MAP-2, NSE, GFAP, Nestin and Musashi. In addition, the cells also demonstrated immunoreactivity to ChAT and NT-3. ConclusionAECs are similar with neural cells and it may be useful as a sustained source to improve outcome of neural stem cells transplantation.

Effects of environment in vivo and in vitro on the differentiation of bone mesenchymal stem cells into neural cells / 重庆医科大学学报

Objective:To investigate the effects of environment in vitro and in vivo on the differentiation of bone mesenchymal stem cells (MSCs) into neural cells.Methods:①MSCs were labeled with 4,6-diamidino-2-phenylindole (DAPI) and injected into the right brain of the rats(n=6),which had suffered from middle cerebral artery occlusion(MACO) a day before.Two weeks later,immunofluorescent staining was used to detect the expression of nestin,microtubule-associated protein 2 (MAP-2) and glial fibrillary acidic protein (GFAP).②In vitro,MSCs were cultured with modified neuronal medium(MNM)for 18 hours after being pretreated with 0.5umol/L all-trans retinoid acid (ATRA) for 24 hours.Immunocytochemistry was used to detect the expression of nestin,MAP-2 and GFAP.Results:①After being injected into rats for 2 weeks,MSCs survived in the ischemic areas and a few cells expressed nestin,MAP-2 and GFAP respectively.②In vitro,after ATRA and MNM treatment,the positive percentage of nestin and MAP-2 were (92.3?3.4)% and (89.6?3.3)% respectively,but no expression of GFAP was detected.Conclusion:The different ratio of MSCs differentiating into neural cells suggests that there is difference in the process and mechanism between environment in vitro and in vivo.

Effect of different dose of GM1 on inducing human adipose tissue-derived stromal cells into neural cells / 临床神经病学杂志

Objective To investigate the effect of different dose of GM1 on inducing human adipose tissue-derived stromal cells (ADSCs) into neural cells. Methods Human ADSCs were isolated from human liposuction tissues and cultured in NIM with different dose of GM1, which was used to induce ADSCs into neural cells. The experiment was separated into six groups: control group (?-MEM), Group A (NIM), group B (NIM+50 ?mol/L GM1), group C (NIM+100 ?mol/L GM1), group D (NIM+200 ?mol/L GM1) and group E (NIM+400 ?mol/L GM1). Cells' growth and morphologic changes were observed under invert phase-contrast microscopy. The expressions of Nestin, NSE, MAP2 and GFAP were identified by immunocytochemistry methods.Results (1)ADSCs could differentiate into neural cells in NIM with different dose of GM1. The number of neural cells differentiated from ADSCs increased in a certain period, especially in group E at each time point (all P