Neuronal-like differentiation of single versus multiple treatments with human amnion-derived mesenchymal stem cells induced by basic fibroblast growth factor

BACKGROUND： Cultures from multiple portions of umbilical cord blood mesenchymal stem cells have been shown to undergo more rapid proliferation and attachment than single portions. OBJECTIVE： To observe growth of basic fibroblast growth factor （bFGF）-induced cultures of human amnion-derived mesenchymal stem cells （AMSCs） and differentiation into neuronal-like cells. DESIGN, TIME AND SETTING： Comparative observation. The study was performed at the Laboratory of Microbiology and Immunology, Basic Medical School of Zhengzhou University from January to May 2008. METHODS： Amnia from full-term, uterine-incision delivery were donated by 12 healthy women. AMSCs were obtained by cell separation and culture techniques, and were passaged and induced by bFGF. From the third passage, a total of 1 mLAMSCs, at a density of 1.0 × 10^4/mL, was separately harvested from six samples, which served as group A. A total of 1 mL AMSCs, at a density of 1.0 × 10^4/mL, was harvested separately from the remaining six samples, which served a group B. A total of 0.5 mL from the six samples of group A and 0.5 mL from the six samples of grot, B were combined to form group C. MAIN OUTCOME MEASURES： Differences in cell quantity among the three groups were compare by cell quantification and 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） analysis. Expression of a glial cell marker, neuron-specific enolase, and nestin was detected in the three groups by immunocytochemistry. RESULTS： Cell quantification and MTT analysis of live cells, as well as AMSC absorbance, were significantly greater in group C compared with groups A and B at 18 days of culture （P 〈 0.05）, anc no significant difference was observed between groups A and B. Glial fibrillary acidic protein, neuron-specific enolase, and nestin were expressed in all groups following bFGF induction. CONCLUSION： Mixed AMSC cultures promoted proliferation, and bFGF-induced AMSCs differentiated into neuronal-like cells.     

碱性成纤维细胞因子在局灶性脑缺血后内源性神经干细胞增殖过程中对β-catenin的影响

BACKGROUND： The Wnt/β-catenin signaling pathway plays an important role in neural development. ,β-catenin is an important component of the Wnt/β-catenin signaling pathway. The Wnt signaling pathway has been shown to regulate the interaction of neural stem cells with the extracellular matrix.
OBJECTIVE： To investigate the effects of basic fibroblast growth factor （bFGF） on β-catenin protein and mRNA expression, and on hippocampal neural stem cell proliferation in a rat model of cerebral ischemia/reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, neurobiology experiment was performed in Shenyang Medical College between August 2006 and August 2008. MATERIALS： A total of 72 healthy male Wistar rats, aged 3 months, were used in this study. bFGF was provided by Beijing SL Pharmaceutical Co.,Ltd., China. METHODS： Rats were randomly divided into 3 groups： sham-operated, ischemia/reperfusion, and bFGF-treated （n = 24 per group）. Focal cerebral ischemia/reperfusion was induced in rats from the ischemia/reperfusion group and the bFGF-treated group by 2 hour right middle cerebral artery occlusion and 2 hour restoration of blood flow using the suture method. The ischemia/reperfusion and bFGF-treated groups were intraperitoneally administered 500 IU/mL of bFGF, or the same volume of physiological saline, once a day at postoperative days 1 3, and once every 3 days thereafter. Simultaneously, the sham-operated group underwent experimental procedures identical to the ischemia/reperfusion and bFGF-treated groups, with the exception of ischemia/reperfusion induction and drug administration. At 2 hours, 2, 6, 13, and 20 days after ischemiaJreperfusion induction, 50 mg/kg bromodeoxyuridine （BrdU） was administered to each group, twice daily, to label proliferating neural stem cells. MAIN OUTCOME MEASURES： The effects of bFGF on BrdU labeling, and ,8 -catenin mRNA and protein expression, in neural stem cells were examined by immunohistochemistry, Western blot, RT-PCR, and in situ hybridization techniques. RESULTS： In the sham-operated group, only a few BrdU-immunoreactive neural stem cells were found. In the ischemia/reperfusion group, BrdU-immunoreactive cells began to increase from 3 days after ischemia/reperfusion induction, reached a peak level at 7 days, and gradually reduced from 21 days. At 3, 7, 14, and 21 days after ischemia/reperfusion induction, the numbers of BrdU-immunoreactive cells were significantly greater in the bFGF-treated group than in the ischemia/reperfusion group. The sham-operated group exhibited slight expression of β-catenin and β-catenin mRNA. In the ischemia/reperfusion group, the expression of β-catenin and β-catenin mRNA gradually increased with reperfusion time, peaked at 14 days after reperfusion, and gradually decreased thereafter; by 21 days, the expression was markedly lower. Following bFGF injection, the expression of hippocampal BrdU, β-catenin, and β-catenin mRNA had apparently increased in each group. CONCLUSION： bFGF promotes neural stem cell proliferation, and the expression of β-catenin and β-catenin mRNA in the ischemic brain tissue. These findings indicate that bFGF promotion of neural stem cell proliferation may be mediated by Wnt/β-catenin signaling pathway.     

黄芪诱导大鼠骨髓间充质干细胞向神经类细胞分化

BACKGROUND： Chemical induction has been shown to be effective at promoting the differentiation of bone marrow-derived mesenchymal stem cells （MSCs）. However, these inductors have cytotoxicity side effects that may damage cells over time. Traditional Chinese medicines avoid this disadvantage while still producing effective induction.
OBJECTIVE： To investigate the influence of RadixAstragafi （Huangql） on the differentiation of MSCs.
DESIGN, TIME AND SETTING： In vitro study of traditional Chinese medicine in neural stem cell differentiation. The experiment was performed at the Central Laboratory of Hebei North University between April and June 2007.
MATERIALS： Radix Astragafi solution （lot No. 060105; license No. Z53021585） was purchased from Dali Pharmaceutical Co., Ltd., China; rabbit anti-rat nestin, rabbit anti-rat neuron-specific enolase （NSE）, mouse anti-rat microtubule-associated protein 2, and rabbit anti-rat glial fibrillary acidic protein were purchased from Wuhan Boster, China.
METHODS： Whole bone marrow was isolated from the femur and tibia of 6-week-old male Wistar rats and subcultured. The fourth passage of MSCs were harvested and induced by different concentrations （50, 100, 200, 400 g/L） of Radix Astragali.
MAIN OUTCOME MEASURES： Hematoxylin-eosin staining was used to observe MSC morphology after 24 hours of induction. Immunocytochemistry was employed to observe the expression of NSE （specific neuronal marker）, nestin （marker of neural stem cell）, glial fibrillary acidic protein and microtubule-associated protein 2 （markers of astrocytes）.
RESULTS： Following Radix Astragali treatment, changes occurred in cell morphology including： cell body pyknosis; thin and long processes formed in some cells, with growth corresponding to drug concentration and induction time; and the formation of network-like connections between some cells. With increasing drug concentration and induction time, nestin expression was upregulated, and the number of positive cells increased; cells produced NSE, glial fibrillary acidic protein and microtubule-associated protein 2; nestin was expressed earlier than glial fibrillary acidic protein and microtubule-associated protein 2 expression. In addition, the number of NSE-positive cells was increased significantly more than glial fibrillary acidic protein-positive cells.
CONCLUSION： Radix Astragafi promoted process formation in stem cells. It may induce the differentiation of MSCs into neural stem cells, and subsequently into neuronal- and glial-like cells. Radix Astragafi exhibits stronger inductive effect on neuronal differentiation than glial differentiation of MSCs.     

嗅鞘细胞对神经干细胞增殖与分化的影响

BACKGROUND： Olfactory ensheathing cells can promote oriented differentiation and proliferation of neural stem cells by cell-secreted neural factors. OBJECTIVE： To observe the effect of olfactory ensheathing cells on the differentiation and proliferation of neural stem cells. DESIGN, TIME AND SETTING： Cytology was performed at the Department of Neurology, Tongji Medical College, Huazhong University of Science and Technology, China, from September 2007 to October 2008. MATERIALS： Mouse anti-nestin polyclonal antibody （Chemicon, USA）, mouse anti-glial fibrillary acidic protein （GFAP） IgG1, mouse anti-2＇, 3＇-cyclic nucleotide 3＇-phosphodiesterase （CNPase） IgG1, mouse anti-Tubulin Class-Ill IgG1 （Neo Markers, USA）, Avidin-labeled Cy3 （KPL, USA）, and goat anti-mouse IgGl： fluorescein isothiocyanate （FITC） （Serotec, UK） were used in this study. METHODS：Tissues were isolated from the embryonic olfactory bulb and subependymal region of Wistar rats. Serum-free DMEM/F12 culture media was used for co-culture experiments. Neural stem cells were incubated in serum-free or 5% fetal bovine serum-containing DMEM/F12 as controls. MAIN OUTCOME MEASURES： After 7 days of co-culture, neural stem cells and olfactory ensheathing cells underwent immunofluorescent staining for nestin, tubulin, glial fibrillary acidic protein, and CNPase. RESULTS： Olfactory ensheathing cells promoted proliferation and differentiation of neural stem cells into neuron-like cells, astrocytes and oligodendrocytes. The proportion of neuron-like cells was 78.2%, but the proportion of neurons in 5% fetal bovine serum DMEM/F12 was 48.3%. In the serum-free DMEM/F12, neural stem cells contracted, unevenly adhered to the glassware wall, or underwent apoptosis at 7 days. CONCLUSION： Olfactory ensheathing cells promote differentiation of neural stem cells mainly into neuron-like cells, and accelerate proliferation of neural stem cells. The outcome is better compared with serum-free medium or medium containing 5% fetal bovine serum.     

Mutant amyloid precursor protein and presenilin-1 genes effect on ischemia vulnerability via calcium homeostasis disturbance

BACKGROUND： Previous studies have demonstrated that mutant amyloid precursor protein （APP） or presenilin-1 （PS1） genes increase susceptibility to ischemic brain damage induced by middle cerebral artery occlusion. Possible mechanisms include over-production of beta-amyloid peptide （Aβ）. OBJECTIVE： Because Aβ is over-produced in the APP/PS1 double-transgenic mouse, the present study focused on mechanisms of increased ischemic damage due to mutant APP and PS1 genes by measuring oxidative stress, mitochondrial function, and calcium homeostasis. DESIGN, TIME AND SETTING： The non-randomized, controlled, in vivo and in vitro experiments were performed at the Medical Research Center, Second Clinical College, Jinan University between May and October 2008. MATERIALS： Male APP transgenic mice carrying the mutant 695swe gene and female PS1 transgenic mice carrying the mutant Leu235Pro gene were donated from the University of Hong Kong. SHSY5Y human neureblastoma cells were purchased from ATCC （Manassas, VA, USA）, and Aβ1-42 was obtained from Sigma-Aldrich （St. Louis, MO, USA）. METHODS： APP transgenic mice were mated with PS1 transgenic mice to produce APP/PS1 double-transgenic mice and wildtype littermates mice. The photothrombotic stroke model was induced in six APP/PS1 double-transgenic and 6 wildtype littermates mice. SHSY5Y human neuroblastoma cells were cultured in vitro, and were divided into 4 groups： Aβ group, cells were exposed to 5 pmol/L Aβ for 24 hours; oxygen-glucose deprivation （OGD） group, cells were exposed to OGD for 1 hour after treatment with sterile, ultra-pure water for 24 hours; OGD＋Aβ group, cells were exposed to OGD and Aβfor 1 hour after treatment with 5 pmol/L Aβ for 24 hours; sham control group： cells were exposed to sterile, ultra-pure water for 25 hours. OGD was achieved by exposing the cells to glucose-free DMEM and placing the cells in an anaerobic chamber flushed with 5% CO2 and 95% N2 （v/v） at 37 ℃ for 1 hour. MAIN OUTCOME MEASURES： TTC staining was used to measure infarct volume 7 days after photothrombotic stroke. Cell viability was evaluated using the MTT kit. Opening of the mitochondrial permeability transition pore, intracellular concentration of superoxide anion, and calcium after OGD were detected with fluorescence intensity of calcein-AM, hydroethidine, and fluo-3/AM. RESULTS： At 7 days after stroke, total infarct volume and cortical infarct volume were significantly greater in the APP/PS1 transgenic mice compared with the wildtype littermates mice （P 〈 0.01）. Aβ, OGD, and Aβ ＋ OGD significantly decreased cell viability and increased fluorescence intensity of hydroethidine and fluo-3/AM （P 〈 0.01）. Compared with the Aβ or OGD group, Aβ ＋ OGD significantly decreased cell viability （P 〈 0.01） and significantly increased fluorescence intensity of calcein-AM, hydroethidine, and fluo-3/AM （P 〈 0.01 or P 〈 0.05）. CONCLUSION： The APP/PS1 double-transgenic mice were more vulnerable to ischemia. The possible mechanisms included enhanced opening of the mitochondrial permeability transition pore, overproduction of superoxide anion due to pore opening, and disturbed calcium homeostasis induced by excess superoxide anion.     

Identification and analysis of a minority fraction in a 387 cell lineDo side-population cells represent bona fide stem cell-like cancer cells？

BACKGROUND： Overwhelming evidence suggests that tumor bulks are comprised of differentiated tumor cells and cancer stem cells （CSCs）. The stem cell-like side-population （SP） cells account for a minor fraction of the total tumor cells, yet are apparently the cells capable of tumor initiation, growth, maintenance, and recurrence. OBJECTIVE： To identify potential stem cell-like cancer cells in a U87 human brain glioma cell line on the basis of dye efflux, clone formation, and multi-drug resistance capacity. DESIGN, TIME AND SETTING： The cellular and molecular biology experiment was performed at the Laboratory of Shanghai Institute of Hematology and Laboratory of Shanghai Institute of Endocrinology in Ruijin Hospital; in vivo contrast observational animal trial was performed at Experimental Animal Center, School of Medicine, Shanghai Jiao Tong University from June 2007 to May 2008. MATERIALS： The U87 cell line was provided by the Shanghai Institute of Cancer Research, Chinese Academy of Science; DMEM/F12 （1 ： 1） and fetal bovine serum were purchased from Gibco Invitrogen, USA; human recombinant basic fibroblast growth factors were purchased from BD Bioscience, USA; Hoechst 33342, Verapamil, and methyl thiazolyl tetrazolium were purchased from Sigma, USA; phycoerythrin-labeled anti-human-CD133 was purchased from Milteny Biotec, Germany; SYBR PrimeScriptTM RT-PCR kit was purchased from TaKaRa Biotechnology, Dalian, China. METHODS： Monolayer cultured cells were harvested by 0.25% Trypsin-EDTA and suspended at a 1 ×10^6/mL dilution in PBS containing 2% FBS, and were stained with Hoechst 33342 dye, either alone or in combination with Verapamil. Following fluorescence-activated cell sorting, SP and non-SP subsets were cultivated with serum-containing （DMEM plus 10% fetal bovine serum） or serum-free culture medium [DMEM/F12 （1： 1） ＋ 1× B27 supplement ＋ 10 ng/mL basic fibroblast growth factors ＋ 1× L-glutamine] to determine growth characteristics in vitro. Finally, single free U87 cells and subsets （SP or non-SP cells） were subcutaneously injected into the backs of 5-week-old nude mice for in vivo tumorigenicity. MAIN OUTCOME MEASURES： Cell morphology and clonogenicity were observed under inverted microscope; SP phenotype and fluorescent antibody labeling were analyzed by MoFIoTM flow cytometry; ABC transporter mRNA expression was evaluated by semi-quantitative real-time RT-PCR; efflux capacity for anti-neoplastic drugs from the U87 cell line and subsets was measured with the MTT assay, then detected by enzyme-linked immunosorbent assay at a wavelength of 490 nm; in vivo tumorigenicity in immunodeficient nude mice was evaluated by diameter size. RESULTS： During in vitro passages, human U87 cells maintained a stable SP fraction profile and exhibited the ability to form neurosphere-like clones. SP cell proliferation decreased compared with non-treated U87 cells. CD133 expression was reduced in the SP and non-SP cells. Freshly sorted SP fractions expressed higher levels of ABC drug transporter genes, and exhibited increased potential for cytotoxic drug resistance. The in vivo malignancy of U87 cells was largely dependent on non-SP cells in nude mice, and tumors that formed from the non-SP fraction developed faster and larger compared with tumors from the SP fraction. CONCLUSION： The SP cell component was a key factor that influenced mRNA expression and cytotoxic drug resistance. In particular, cancer stem cells or tumor-initiating cells were not exclusively enriched in the SP subset of the U87 cell line, and non-SP cells were even more tumorigenic.     

Inhibitory effect of synthetic small interfering RNAs on glial fibrillary acidic expression in astrocytes*☆

BACKGROUND： Glial fibritlary acidic protein （GFAP） expression highly correlates with spinal glial scar formation, and is regarded as an important target for scar therapy. Efficient inhibition of expression could benefit recovery from spinal cord injury. OBJECTIVE： To investigate the inhibitory effects of synthetic small interfering RNAs （siRNAs） on astrocytic GFAP expression in rats. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment at the cellular and molecular level was performed at the First Hospital of Dalian Medical University between June 2005 and February 2006. MATERIALS： A total of 100 seven-day-old, Sprague Dawley rats were selected. GAPDH siRNA was purchased from Ambion, USA, And TransMessengerTM Transfection Reagent from DAKO, Carpinteria, CA. METHODS： Rat astrocytes were isolated and cultured. Three pairs of 21-nucleotide （nt） siRNAs specific to rats GFAP mRNA, 401,404 and 854, were synthesized and transfected in primary astrocytes at 1, 2, 3, and 4 g/L using TransMessengerTM Transfection Reagent. Non-transfected astrocytes served as the blank group. Cells transfected with siRNA were regarded as the negative control group, with GAPDH siRNA as the positive control group, and 401 siRNA, 404 siRNA, and 854 siRNA as experimental groups. MAIN OUTCOME MEASURES： GFAP mRNA and protein expression were assessed by RT-PCR and Western blot, respectively, at 24, 48, and 72 hours of culture. RESULTS： GFAP mRNA expression in the positive control group was significantly less than the negative control group （P 〈 0.01）. GFAP mRNA expression in astrocytes from three pairs of siRNA was significantly less than the blank group after 48 hours （P 〈 0.01 ）, while no differences were detected between the negative control and blank groups （P 〉 0.05）. GFAP protein expression was remarkably less in siRNA-transfected astrocytes compared to the blank control （P 〈 0.01 ）. CONCLUSION： Transfected siRNAs could significantly inhibit GFAP gene expression in astrocyt     

阳离子聚合物载体介导脑源性神经营养因子基因转染大鼠骨髓间充质干细胞

BACKGROUND： Gene therapy is an effective expression of genes within target cells after transferring exogenous target genes. Both vector selection and transfection method are important factors for gene transfection. An ideal gene vector is required for a high transfusion of target gene and an exact introduction of target gene into specific target cells so as to express gene products.
OBJECTIVE： To study the expression of mRNA and protein after transfecting rat bone marrow mesenchymal stem cells （BMSCs） with brain-derived neurotrophic factor （BDNF） genes based on cationic polymer vector.
DESIGN, TIME AND SETTING： A randomized, controlled in vitro study using gene engineering, performed at the Neurobiology Laboratory, Xuzhou Medical College between October 2007 and April 2008.
MATERIALS： PcDNA3.1 BDNF was obtained from Youbiai Biotechnological Company, Beijing and cationic polymer vector used was the SofastTM gene transfection reagent that was made by Taiyangma Biotechnological Co., Ltd., Xiamen.
METHODS： BMSCs extracted from six Sprague Dawley （SD） rats aged 1 month were isolated and cultured in vitro. Third passage BMSCs were inoculated on a 6-well culture plate at the density of 1×106 cells/L. At about 80% confluence, BMSCs were transfected with PcDNA3.1-BDNF （2 μg） combined with SofastTM gene transfection reagent （6 μg） （BDNF group） or with PcDNA3.1 （2 μg） combined with SofastTM gene transfection reagent （6 μg） （blank vector group）. Cells that were not transfected with any reagents but still cultured under primary culture conditions were used as a non-transfection group.
MAIN OUTCOME MEASURES： Enzyme linked immunosorbent assay was used to measure time efficiency of BMSC-secreted BDNF protein. Twenty-four hours after gene transfection, RT-PCR was used to detect expression of BDNF mRNA in the BMSCs. Immunohistochemistry was used to determine expression of BDNF protein in the BMSCs.
RESULTS： BDNF protein expression was detected at day 1 after gene transfection, rapidly increased after 5–9 days and gradually increased after 11–15 days in the BDNF group; moreover, BDNF protein expression was higher than that in the non-transfection group and the blank vector group at different time points （P 〈 0.01）. Additionally, BDNF mRNA expression in the BDNF group was higher than that in the blank vector group and the non-transfection group （P 〈 0.01）.
CONCLUSION： A cationic polymer vector can effectively mediate the BDNF gene to transfect BMSCs; genetically modified BMSCs can express BDNF protein effectively for a long term.     

Induced neuronal differentiation of neural stem cells by a combination of cytokines One-step versus two-step methods

BACKGROUND： A combination of basic fibroblast growth factor （bFGF）, platelet-derived growth factor （PDGF）, human heregulin-beta-1, beta-mercaptoethanol retinoic acid and forskolin has been reported to induce the differentiation of rat bone marrow stromal cells into myelinating Schwann-like cells. OBJECTIVE： To investigate the inducing effects of a combination of bFGF, PDGF, human heregulin-beta-1, beta-mercaptoethanol retinoic acid and forskolin on neural stem cell differentiation by one- and two-step methods. DESIGN, TIME AND SETTING： A cytobiology experiment was performed at the Department of Histology and Embryology, Medical School of Nantong University, and Jiangsu Province Key Laboratory of Neuroregeneration, China, between August 2005 and January 2007. MATERIALS： A total of 30 healthy Sprague Dawley rat embryos at gestational days 14-16 were selected, bFGF, PDGF, human heregulin-beta-t, beta-mercaptoethanol, retinoic acid, and forskolin were purchased from Sigma, USA. METHODS： Passage 3 rat neural stem cells were cultured by a one-step method in serum-free medium plus 10 ng/m/bFGF, 5 ng/mL PDGF, 200 ng/mL heregulin-beta-1,35 ng/mL all-trans retinoic acid, and 5 pmol/L forskolin or by a two-step method in serum-free medium plus 35 ng/m/ all-trans retinoic acid for 72 hours, followed by serum-free medium plus 10 ng/mL bFGF, 5 ng/mL PDGF, 200 ng/mL heregulin-beta-t and 5 μmol/L forskolin. The control condition consisted of 10% fetal bovine serum alone or 20 ng/mL bFGF alone. MAIN OUTCOME MEASURES： Differentiated cells were identified by immunocytochemical staining for microtubule associate protein-2 （MAP2） and St 00 protein. Geometric parameters and sodium ion currents of the differentiated cells were measured by image analysis and whole-cell patch-clamp techniques, respectively. RESULTS： Compared with the two-step culture method, neuronal-like cells exhibited longer processes and a similar appearance to mature neurons using the one-step method. The percentage of MAP2 positive cells induced by the one-step method was significantly greater than the serum-alone group （P 〈 0.05）. Furthermore, the MAP2 positive cells induced by the one-step method had greater surface areas, cell body perimeters, and longer process than cells induced by serum-alone and bFGF-alone （P 〈 0.05）. There were no significant differences in these parameters between the one-step and two-step methods （P 〉 0.05）. In addition, 80% of the induced neuronal-like cells from the one-step method and 20% from the two-step method displayed inwardly-evoked currents. CONCLUSION： The combination of bFGF, PDGF, human heregulin-beta-t, beta-mercaptoethanol retinoic acid and forskolin successfully induced neuronal differentiation from neural stem cells, with the one-step induction being more effective than the two-step method.     

Propofol increases in vitro proliferation of cultured rat hippocampal precursor cells inhibited by corticosterone

BACKGROUND： Previous studies have shown that propofol enhances proliferation of cultured hippocampal precursor cells in vitro and increases proliferation of cultured hippocampal precursor cells inhibited by corticosterone. Because gamma-aminobutyric acid A （GABA-A） receptor is the functional target for propofol, the proliferative effects of propofol are thought to take place through GABA-A receptor. OBJECTIVE： To determine whether propofol enhances proliferation of rat hippocampal precursor cells inhibited by corticosterone by upregulating expression of GABA-A receptor. DESIGN, TIME AND SETTING： A comparative, observational, in vitro experiment was performed at the Beijing Institute of Pharmacology and Toxicology from April 2005 to April 2006. MATERIALS： Propofol was purchased from AstraZeneca, italy; corticosterone was purchased from Sigma, USA; bicuculline was purchased from Alexis, Switzerland. METHODS： Hippocampal precursor cells were isolated from newborn Wistar rats and cultured in vitro. The second passage of precursor cells was grouped according to the various drugs added to the culture medium： 0.5 μmol/L propofol; 2.5 pmol/L propofol; 100 μmol/L corticosterone; 10 μmol/L bicuculline; 100 μmol/L corticosterone and 0.5 μmol/L propofol; 100 μmol/L corticosterone and 2.5 μmol/L propofol; 100 μmol/L corticosterone, 10 μmol/L bicuculline, and 0.5 μmol/L propofol; 100 μmol/L corticosterone, 10 μmol/L bicuculline, and 2.5 μmol/L propofol; 100 μmol/L corticosterone and 10 pmol/L bicuculline. The cells were cultured for 24 hours with medium containing the respective concentration of drug. The control group consisted of precursor cells absent of drug treatment. MAIN OUTCOME MEASURES： The MTT and ^3H-TdR incorporation assays were used to detect proliferative effects of propofol and bicuculline on cultured rat hippocampal precursor cells inhibited by corticosterone. Immunocytochemistry was used to detect GABA-A receptor expression. Enzyme-linked irnmunosorbent assay was used to quantify GABA-A receptor expression. RESULTS： Propofol, at a concentration of 0.5 and 2.5 μmol/L, increased proliferation of cultured rat hippocampal precursor cells inhibited by corticosterone, while bicuculline antagonized the effects of propofol （P 〈 0.05 or P 〈 0.01 ）. Corticosterone （100μmol/L） decreased expression of GABA-A receptor in the hippocampal precursor cells （P〈 0.05）, and GABA-A receptor expression was upregulated when propofol （2.5μmol/L） was added to the culture medium （P〈 0.05）. CONCLUSION： Low concentrations of propofol increased expression of GABA-A receptor. These results suggest that GABA-A receptor is involved in increased proliferation of cortisone-inhibited rat hippocampal precursor cells in vitro.     

TAT-PDX1融合蛋白诱导人胎肝间充质干细胞向胰岛β细胞分化*☆◆

BACKGROUND： In vitro cultures of neural stem cells have shown that estrogen can regulate beta-amyloid precursor protein （β-APP） metabolism and reduce amyloid-beta production.
OBJECTIVE： To investigate the effects of long-term oral administration of compound nylestriol or low-dose 17beta-estradiol on β-APP and mRNA expression in the hippocampus of ovariectomized （OVX） rats.
DESIGN, TIME AND SETTING： This randomized and controlled experiment was performed at the Animal Laboratory and Laboratory of Endocrine and Metabolic Disease, Xiangya Second Hospital of Central South University between April 2003 and May 2004. MATERIALS： According to body mass, 50 six-month-old female Sprague-Dawley rats were randomly divided into five groups （n = 10 per group）： normal control, sham operation, OVX model, 17beta-estradiol （Sigma, USA）, and compound nylestriol tablet （Laboratory of Endocrine and Metabolic Disease, Xiangya Second Hospital of Central South University） groups.
METHODS： Rats in OVX plus 17beta-estradiol and OVX plus compound nylestriol tablet groups underwent ovariectomy. On the second day after surgery, rats were intragastrically given 17beta-estradiol （100 μg/kg）, once per day or compound nylestriol tablet （0.5 mg/kg） and levonorgestrel （0.15 mg/kg） every 2 days. MAIN
OUTCOME MEASURES： β-APP expression in the hippocampus of OVX rats was determined using immunohistochemistry （SABC method） and β-APP mRNA expression was analyzed by in situ hybridization. The results were quantitatively analyzed using cell counting and average optical density.
RESULTS： The number and optical density of β-APP-positive neurons in every subregion of the hippocampus of OVX rats was dramatically increased compared with normal and sham operation groups following 35 weeks of administration （P 〈 0.05）. Levels of β-APP were decreased following oral administration of compound nylestriol or 17beta-estradiol. In situ hybridization showed that long-term estrogen deficiency and oral administration of compound nylestriol or 17beta-estradiol did not alter the number of β-APP mRNA-positive neurons.
CONCLUSION： The results show that long-term estrogen deficiency results in an increase of expression of β-APP though no changes in the expression of β-APP mRNA are detected. Replacement of estrogen with low-dose 17 beta-estradiol or compound nylestriol tablet inhibits the expression of β-APP in the hippocampus to the same extent.     

Environmental factors in the development and progression of late-onset Alzheimer＇s disease

Late-onset Alzheimer＇s disease （LOAD） is an age-related neurodegenerative disorder characterized by gradual loss of synapses and neurons, but its pathogenesis remains to be clarified. Neurons live in an environment constituted by neurons themselves and glial cells. In this review, we propose that the neuronal degeneration in the AD brain is partially caused by diverse environmental factors. We first discuss various environmental stresses and the corresponding responses at different levels. Then we propose some mechanisms underlying the specific pathological changes, in particular, hypothalamic-pituitary adrenal axis dysfunction at the systemic level; cerebrovascular dysfunction, metal toxicity, glial activation, and Aβ toxicity at the intercellular level; and kinase-phosphatase imbalance and epigenetic modification at the intracellular level. Finally, we discuss the possibility of developing new strategies for the prevention and treatment of LOAD from the perspective of environmental stress. We conclude that environmental factors play a significant role in the development of LOAD through multiple pathological mechanisms.     

阿尔茨海默病病因新进展——线粒体老化

随着人口老龄化进程加快,阿尔茨海默病已成为一个研究热点。结合现有神经病理学发现,围绕阿尔茨海默病病因先后提出淀粉样蛋白级联假说,氧化应激假说、炎症假说及细胞凋亡假说等。这些假说均从一定角度,揭示了部分阿尔茨海默病病因,其中以淀粉样蛋白级联假说最为经典,一度成为阿尔茨海默病病因学的核心。     

Total saponins of Panax ginseng effects on proliferation and differentiation of human embryonic neural stem cells and in a Parkinson’s disease mouse model

BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.     

Targeting 13-secretase with RNAi in neural stem cells for Alzheimer＇s disease therapy

There are several major pathological changes in Alzheimer＇s disease, including apoptosis of cho- linergic neurons, overactivity or overexpression of 13-site amyloid precursor protein cleaving enzyme 1 （BACE1） and inflammation. In this study, we synthesized a 19-nt oligonucleotide targeting BACE1, the key enzyme in amyloid beta protein （AI3） production, and introduced it into the pSilenCircle vector to construct a short hairpin （shRNA） expression plasmid against the BACE1 gene. We transfected this vector into C17.2 neural stem cells and primary neural stem cells, resulting in downregulation of the BACE1 gene, which in turn induced a considerable reduction in reducing AI3 protein production. We anticipate that this technique combining cell transplantation and gene ther- apy will open up novel therapeutic avenues for Alzheimer＇s disease, particularly because it can be used to simultaneously target several pathogenetic changes in the disease.     

Disrupted structural and functional brain connectomes in mild cognitive impairment and Alzheimer＇s disease

Alzheimer＇s disease （AD） is the most common type of dementia, comprising an estimated 60-80% of all dementia cases. It is clinically characterized by impairments of memory and other cognitive functions. Previous studies have demonstrated that these impairments are associated with abnormal structural and functional connections among brain regions, leading to a disconnection concept of AD. With the advent of a combination of non-invasive neuroimaging （structural magnetic resonance imaging （MRI）, diffusion MRI, and functional MRI） and neurophysiological techniques （electroencephalography and magnetoencephaJography） with graph theoretical analysis, recent studies have shown that patients with AD and mild cognitive impairment （MCI）, the prodromal stage of AD, exhibit disrupted topological organization in large-scale brain networks （i.e., connectomics） and that this disruption is significantly correlated with the decline of cognitive functions. In this review, we summarize the recent progress of brain connectomics in AD and MCI, focusing on the changes in the topological organization of large-scale structural and functional brain networks using graph theoretical approaches. Based on the two different perspectives of information segregation and integration, the literature reviewed here suggests that AD and MCI are associated with disrupted segregation and integration in brain networks. Thus, these connectomics studies open up a new window for understanding the pathophysiological mechanisms of AD and demonstrate the potential to uncover imaging biomarkers for clinical diagnosis and treatment evaluation for this disease.     

Edema and neuronal apoptosis in the hippocampus and cortex of elderly rats following transient cerebral ischemia/reperfusion injury

BACKGROUND： Previous studies of cerebral ischemia have used young animals, with an ischemic time greater than 5 minutes （safe time limit）. Despite an increased understanding of neuronal apoptosis, it remains uncertain whether brief cerebral ischemic events of 5 minutes or less damage brain tissue in elderly rodents. OBJECTIVE： To investigate the effects of transient cerebral ischemia （5 minutes）/reperfusion injury on brain cortical and hippocampal edema, aquaporin-4 （AQP-4） expression, and neuronal apoptosis in aged rats, and to compare ischemic sensitivity between cortex and hippocampus. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Institute of Cerebrovascular Disease, Qingdao University Medical School from April 2008 to March 2009. MATERIALS： Rabbit anti-AQP-4 polyclonal antibody, TUNEL kit, and SABC immunohistochemistry kit were purchased from Wuhan Boster Bioengineering, China. METHODS： A total of 160 healthy, male, aged 19-21 months, Wistar rats were randomly assigned to 4 groups： sham-surgery, and ischemia 1-, 3-, and 5-minute groups, with 40 rats in each group. The global cerebral ischemia model was established using the Pusinelli four-vessel occlusion, and the three cerebral ischemia groups were subdivided into reperfusion 12-hour, 1-, 2-, 3-, and 7-day subgroups, with 8 rats in each subgroup. The sham-surgery group was subjected to exposure of the first cervical bilateral alar foramina and bilateral common carotid arteries. MAIN OUTCOME MEASURES： The dry-wet weight assay was used to measure brain water content and histopathology of the cortex and hippocampus was observed following hematoxylin-eosin staining. In addition, cortical and hippocampal AQP-4 expression was detected by streptavidin-biotin complex immunohistochemistry, and neuronal apoptosis was detected by the TUNEL method. RESULTS： There was no significant difference in brain water content or AQP-4 expression in the cortex and hippocampus between ischemia 1- and 3-minute groups and the sham-surgery group or brain water content or AQP-4 expression in the cortex between ischemia 5-minute group and sham-surgery group （P 〉 0.05）. However, brain water content and AQP-4 expression in the hippocampus after 5 minutes of cerebral ischemia were significantly increased compared with the sham-surgery group （P 〈 0.05 or P 〈 0.01）. Several TUNEL-positive cells were observed in the cortex and hippocampus of the sham-surgery group and ischemia 1-minute group, as well as in the cortex of the ischemia 3-minute group. In addition, the number of apoptotic neurons in the hippocampus of ischemia 3-minute group and in the cortex and hippocampus of ischemia 5-minute group was significantly increased （P 〈 0.05 or P 〈 0.01 ）. Neuronal apoptosis was increased after 12 hours of ischemia/reperfusion, and it reached a peak by 2 days （P 〈 0.01）. CONCLUSION： Transient cerebral ischemia （5 minutes） resulted in increased hippocampal edema, AQP-4 expression, and neuronal apoptosis. Moreover, cerebral ischemia had a greater effect on neuronal apoptosis than brain edema or AQP-4 expression, and the hippocampus was more sensitive than the cortex.     

阿尔茨海默病治疗的研究进展

阿尔茨海默病（AD）是一种隐匿性起病,进行性恶化的神经退行性疾病,临床最初表现为认知功能障碍,并有可能在5～10年内完全衰退。患者往往伴随严重的记忆力丧失、精神行为异常、人格改变、言语功能障碍,无法独立生活,最终近乎于植物状态。Ferri等采用DISMOD软件在全球60岁以上人群中估计,全球的痴呆患者人数到2040年将达到8llO万左右。