Parthenogenetic activation of rabbit oocytes and effects of different additives on development of activated embryos / 中华神经医学杂志

Objective To investigate an appropriate activation method and culture system in vitro of rabbit parthenogenetic embryos to make an experimental foundation for therapeutic cloning.Methods Mature rabbit oocytes with polar body 1 (PB 1) were divided randomly into four groups and accepted,respectively,parthenogenetic activation with 1.2 kV/cm,1.6 kV/cm and 2.0 kV/cm electricity stimulation or 5 μg/mL ionomycin (ION) chemical treatment; the effects of these different treatments on activation efficiency were compared.The activated oocytes were cultured,respectively,in M199 supplemented with various concentrations of leukaemia inhibitory factor (LIF,0,10,20,40 and 80 ng/mL) or insulin+transferrin+sodium selenite (ITS,0×,1 × and 2×); and then,the rate of cleaved oocytes,percentage of morulas/blastocysts and total number of blastocysts were compared between each two groups.Results (1) Different treatments on the oocytes showed significantly different activation effects.The survival rate of oocytes activated in 5 μg/mL ION for 4 min were significantly higher than that of ones stimulated by 2.0 kV/cm electricity treatment (P=0.016).The percentages of cleaved oocytes,developing 8-16 cells and morulas/blastocysts in the ION treatment group were also significantly higher as compared with those in the 1.2 kV/cm electric treatment group (P=0.000,P=0.002 andP=0.026,respectively).(2) Significant difference in the percentage of morulas/blastocysts was noted between 20 ng/mL and 80 ng/mL of LIF supplementation (P=0.003); in addition,the total number ofblastocysts in the medium supplemented with 20 ng/mL LIF was significantly different to those with 40 ng/mL and 80 ng/mL,respectively (P=0.011,P=0.002); In experiment of ITS supplementation,significant difference in the percentage ofmorulas/blastocysts was shown between 1 × and 2× ITS supplementation (P=0.003); in addition,the total number of blastocysts in the medium supplemented with 0× or 1 × ITS (166 and 147 cells) was significantly different as compared with that with 2× ITS (78 cells,P=0.015,P=0.044).Conclusion The parthenogenesis activation of the New Zealand rabbit's oocytes treated with ION+6-DMAP shows a higher rate of cleaved oocytes and blastocysts than that with electricity stimulation; the subsequent development of parthenogenetic embryos in vitro could be significantly improved by supplementing with 20 ng/mL LIF and 1× ITS.

Scriptaid improves early development potential of mouse somatic cell nuclear transfer embryos / 中华神经医学杂志

Objective To examine the effect of Scriptaid,a histone deacetylase inhibitor and a kind of anti-cancer drug,on development of mouse somatic cell nuclear transfer (SCNT) embryos in vitro and explore a new strategy to improve the efficiency of SCNT. Methods SCNT was carried out by pizeo-activated micromanipulator in C57/BL6 mouse,from which the oocytes were chsoen as recipients and the cumulus cells as donors.The rcconstructcd embryos were randomly divided into 5 groups and activated in calcium-free activators with 0 mmol/L (negative control group),and 50,100,250 and 500 mmol/L Scriptaid for 6 h, respectively; and then, they were transferred into KSOM medium with corresponding concentrations of Scriptaid for 4 h.The cloned embryos were finally cultured in KSOM medium for 96 h.The development (form rate) of cloned embryos and the count of blastocysts cells were recorded. Results No significant differences on the activated rate of the reconstructed embryos and the 2-cell cleavage rate were noted between each 2 groups (P＞).05).However,the form rate (24.2％) and cell numbers (56.27±2.43) of blastocysts in 250 mmol/L Scriptaid activation group were significantlyhigher as compared with those in the negative control group, and 50, 100 and 500 mmol/L Scriptaid activation groups (form rates:5.3％,6.5％,9.4％ and 6.9％; cell numbers:44.67±1.53,50.25±1.26,52.33±2.50 and 50.75±1.50,respectively,P＜0.05). Conclusion The early development potential of mouse SCNT embryos in vitro can be dramatically improved by 250mmol/L Scriptaid.

Proliferation characteristics of adipose-derived stem cells from neonatal suckling rats and adult ones / 中华神经医学杂志

Objective To investigate the proliferative differences of adipose-derived stem cells (ADSCs) from neonatal suckling SD rats (5-d-old) and adult ones under the same culture condition.Methods ADSCs were isolated from the subcutaneous adipose tissues of neonatal suckling SD rats and adult ones,and then,type Ⅰ collagenase digestion was employed to obtain the ADSCs; the morphology of these cells was detected.The expressions of such cell surface markers as CD45,CD29 and CD90 were observed. The number of ADSCs on the 4th d of culture under the same condition and with the same planted density was compared between the neonate and adult rats. In vitro culture of the second generation of ADSCs was performed in the 96-well plates, and CCK-8 and alamar blue kit were employed to compare and quantitate the proliferative differences; optical density was observed by microplate reader. Results The ADSCs from neonatal SD rats and adult ones expressed the stem cell biomarkers: the expression of CD45 was negative, and that of CD29 was 98.04％ and 93.17％,respectively,and that of CD90 was 94.92％ and 93.3％,respectively,for neonate SD rat and adult ones.The cell counting results indicated that the number of ADSCs from neonatal rats ([8.87±0.13]×105 cells) was larger than that of adult ones ([4.51±0.36]×105 cells) after being cultured under the same condition and at the same planted density. The optical density value of ADSCs in neonatal rats was significantly higher than that in adult ones on the 6th and 7th d of culturing detected by CCK-8 kit and on the 2nd-7th d of culturing by alamar blue assay. Conclusion The proliferative ability of ADSCs from neonatal rats is greater than that of adult ones.

Blockage of transforming growth factor-β signaling pathway inhibits vasculogenic mimicry in gliomas / 中华神经医学杂志

Objective To study the effect of transforming growth factor-β (TGF-β) signaling pathway blockage on vasculogenic mimicry (VM) in gliomas and explore its possible mechanism.Methods Three-dimentional culture was performed on the glioma cell lines U251 and SHG44; the effects of U251 culture supematant and TGF-β on VM formation of SHG44 cells were observed; the capability of VM formation of U251 and SHG44 cells after being treated with 0 μg/mL (PBS group),15 μg/mL TGF-β neutralizing antibody (Ab15 group) and 30 μg/mL TGF-β neutralizing antibody (Ab30 group) was evaluated.ELISA was used to detect the concentrations of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in the supematant of U251 cells from the blank group,PBS group,Ab 15 group and Ab30 group and the concentrations of VEGF and PDGF in the supernatant of SHG44 cells from the blank group,TGF-β treatment group,PBS group,Ab15 group and Ab30 group.Results VM was formed in the U251 cells while not in the SHG44 cells during the three-dimentional culture; SHG44 cells could only gather into colonies of different sizes.U251 culture supernatant could induce SHG44 cells to form VM,enjoying the most obvious effect at 24-48 h of culture; TGF-β could not induce SHG44 cells to form VM.The number of U251 cells annulation in PBS group,Ab15 group and Ab30 group decreased in sequence with significant difference (P＜0.05).The number of U251 cells armulation in SHG44 cells cultured in U251 culture supematant from the PBS group,Ab15 group and Ab30 group decreased in sequence after being added TGF-β antibody with significant difference (P＜0.05).As compared with that in the blank group and PBS group,significant decrease of VEGF and PDGF concentrations in the U251 cells from Ab15 group and Ab30 group was noted (P＜0.05); as compared with that in the blank group and TGF-β treatment group,significant increase of VEGF and PDGF concentrations in the SHG44 cells from PBS group,Ab15 group and Ab30 group was noted.Conclusion Blockage of TGF-β signaling pathways inhibits VM in glioma,and it maybe probably due to the decrease of VEGF and PDGF expressions..

A novel method for successive acquisition of high purity and yield of microglia / 中华神经医学杂志

Objective To establish an easy culture method of successively getting high purity and yield of microglia. Methods Cortices of neonatal Wistar rats (1-3 days old) were employed in this experiment. The first-generation microglial cells were isolated from the mixed glial culture by mechanical means (gently shaking and blowing with pipette). After the mixed glial cells being passaged at a density third generations ofmicroglial cells were harvested. CD1 lb/c, CD45, CD80, CD86 and GFAP were employed as the identification markers in detecting the phenotypes and purity of different generation of microglial cells by scanning electron microscope and flow cytometry. Immunofluorescence staining and CCk8 vitality measurement were used to judge the expression of CD11b/c and detect the proliferation of microglia cells. Microglial phagocytotic function was evaluated by phagocytosis of fluorescent microspheres. Results High yield and purity of microglial cells were stably obtained in this experiment. CD11b/c, CD45, CD80 and CD86 positive expressions were noted in the first and third generations of microglial cells by flow cytometry; CD1 1b/c positive expression was noted in the first,second and third generations of microglial cells by immunofluorescence staining. No obvious differences in the 3 different generations of microglia cells were found on proliferation ability by CCk8 vitality measurement, and on morphology and phenotypes by scanning electron microscope; no obvious differences in the first and third generations of microglia cells were found on phagocytic ability (P＞0.05).Conclusion High yield and purity of microglial cells can successively obtain through the above method;no significant differences are noted among different generations of microglia cells on purity, morphology,phenotypes, proliferation activity and phagocytic ability.

Up-regulation of the transient A-type K+ current (IA) in the differentiation of neural stem cells of the early postnatal rat hippocampus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Neural stem cells (NSCs) not only are essential to cell replacement therapy and transplantation in clinical settings, but also provide a unique model for the research into neurogenesis and epigenesis. However, little attention has been paid to the electrophysiological characterization of NSC development. This work aimed to identify whether the morphological neuronal differentiation process in NSCs included changes in the electrophysiological properties of transient A-type K(+) currents (I(A)).</p><p><b>METHODS</b>NSCs were isolated from early postnatal rat hippocampus and were multiplied in basic serum-free medium containing basic fibroblast growth factor. Potassium currents were investigated and compared using whole-cell patch-clamp techniques and one-way analysis of variance (ANOVA), respectively.</p><p><b>RESULTS</b>Compared with NSC-derived neurons, cloned NSCs (cNSCs) had a more positive resting membrane potential, a higher input resistance, and a lower membrane capacitance. Part of cNSCs and NSC-derived neurons possessed both delayed-rectifier K(+) currents (I(DR)) and I(A), steady-state activation of I(A) in cNSCs (half-maximal activation at (21.34 +/- 4.37) mV) occurred at a more positive voltage than in NSC-derived neurons at 1-6 days in vitro (half-maximal activation at (12.85 +/- 4.19) mV).</p><p><b>CONCLUSIONS</b>Our research revealed a developmental up-regulation of the I(A) component during differentiation of postnatal NSCs. Together with the marked developmental up-regulation of I(DR) in vitro neuronal differentiation we have previously found, the voltage-gated potassium channels may participate in neuronal maturation process.</p>

Immunogenicity of recombinant DNA vaccine that coded for the neurite growth inhibitors / 中华神经医学杂志

Objective To detect the immunogenicity of the recombinant DNA vaccine that encoded for neurite growth inhibitors: Nogo-A, oligodendrocyte myelin glycoprotein (OMgp), tenascin-R (TN-R) and myelin-associated glycoprotein (MAG) after the nerve injury under the help of pAdEasy, a kind of adenovirus plasmid being the vector of the DNA. Methods Sixteen 5-w-old Lewis rats were randomized into DNA vaccination group (vaccine group) and pAdEasy group. Rats in the vaccine group were immunized once weekly for a consecutive 8 w by bilateral injection of the recombinant plasmid into the musculus tibialis. The immunized animals in the 2 groups were exsanguinated each time before the vaccination for sera collection, and the qualitation and quantitation of the antibodies in the serum were detected by Dot-blot analysis and ELISA. Results The vaccine group could produce fusion-protein antibodies against Nogo-A, MAG, OMgp and TN-R at the 6th w of vaccine injection, while pAdEasy group could not. The valency of antiserum was shown by ELISA as 1:1 000 000 at the 6th w of vaccine injection and kept this level stably. Conclusion The DNA vaccine exclusively induces the generation of the fusion-protein antibodies against Nogo-A, MAG, OMgp and TN-R in vivo, which controls the favorable immunogenicity.

Establishment of synapses between rat cortical neurons and Neuron-like cells derived from bone marrow stromal cells in vitro / 中华神经医学杂志

Objective To investigate the establishment of synapses between the cortical neurons and the neuron-like cells difierentiated from the marrow stromal cells(BMSCs)in a simulated transplantation system in vitro.Methods The BMSCs from green fluorescent protein(GFP)transgenic mice(GFP-GM-BMSCs) were isolated, cultured and purified in vitro.The third passage of GFP-GM-BMSCs were co-cultured with primary cultured cortical neurons and gliai cells in a simulated transplantation system in serum-free medium conmining 2%B27 supplemented with 20 ng/mL basic fibroblast growth factor(bFGF)and 20 ng/mL epidermal growth factor(EGF).On day 10 of the co-culture,FM1-43,a fluorescent dye specific to active synaptic vesicles,was used to observe synapses formation between the cells under fluorescence microscope. Results The GFP.GM-BMSCsco-cultured with the neural cells in the Serum-free medium containing bFGF and EGF differentiated into neuron-like cells 7 days after the co-culture.On day 10 ofthe co-culture,FM1-43 dye-positive synaptic vesicles were foundin the cell culture,locating mostly in the cell body,processes and terminal sffuctures ofthe neuron-like cells. Conclusions The neuron-like cells derived from GFP-GM-BMSCs can form synapses with the coRical neurons in the simulated cell transplantation system in vitro.

Isolation and culture of adipose-derived stromal cells and a two-step protocol to induce their differentiation into neural cells / 中华神经医学杂志

Objective To explore a new method for in vitro culture and differentiation induction of rat adipose-derived stromal cells(ADSCs)into neural cells.Methods The retroperitoneal adipose tissue of adult SD rats was carefully dissected and digested using type Ⅰ collagenase.After centrifugation.the stromal cell pellet was resuspended in DMEM/F12 containing 10%fetal bovine serum (FBS).The ADSCs in passage 5 were harvested and cultured in serum-free Neurobasal (NB)medium supplemented with 20 ng/mL basic fibroblast growth factor(bFGF),20 ng/mL of epidermal growth factor (EGF)and N2(1:100)to induce neurosphere formation.The neurospheres in passage 2 were incubated in NB medium supplemented with 0.5 μmol/L all-trails-retinoic acid(RA),1%FBS,5%horse serum,and 50 ng/mL of brain-derived neurotrophic factor (BDNF)to induce the their differentiation into neuron-and glial-like cells.Immunofluorescent staining was performed to identify the differentiated cells. Results Early after inoculation,the spherical ADSCs were suspended in the medium and began to adhere to the wall of the culture flask 24 h after the inoculation.With the increase of the cell density in the cell culture,a fibroblast-like cell monolayer occurred.Neurosphere formation was observed 5-7 days after culturing the ADSCsin serum-free NB medium,and the neurospheres rapidly proliferated and reached a diameter of 100μm at 14 days of culture.The neurospheres further terminally differentiated into neuron-and glial-like cells with spherical morphology and clearly visible cell nuclei.The differentiated cells extended long and thin cell processes or reticular cell processes,and numerous cells established networks through the cell processes.Immunofluorescent staining showed that the neurospheres were positive for nestin (with a positivity rate of 75.62%±1.34%),and 57.62%±4.92%of the terminally differentiated cells expressed β-tubulin Ⅲ,a marker of immature neurons,and 11.25%±3.87%were positive for MAP2ab,a marker of mature neurons.Some of the differentiated cells were positive for glial fibrillary acidic protein (GFAP)expression(18.34%±3.87%)and galactoeerebroside(GalC)(14.35%±3.98%). Conclusions ADSCs from adult rat adipose tissue Can produce a large quantity of stable multipotent daughter cells.The two-step method for inducing the differentiation of ADSCs Can yield high rates of nestin-and β-tubulin Ⅲ-positive cells and some MAP2ab-positive cells.

Direct induction versus neurosphere induction of neural cells from rat adipose stromal cells: a comparative study / 中华神经医学杂志

Objective To enhance the differentiation efficiency of adipose-derived stromal cells (ADSCs) into neural cells. Methods ADSCs were isolated from the adipose tissue of SD rats and induced to differentiate into neural cells using two different protocols, namely direct induction (group A) and neurosphere induction (group B) protocols. For direct induction, the ADSCs were cultured in Neurobasal medium containing fetal bovine serum (FBS) and neurotrophic factors (NTs). Neurosphere induction protocol was carried out by culturing the ADSCs in serum-free Neurobasal medium to induce the formation of neurospheres, which were further induced in serum- and NT-containing Nenrobasal medium into neural cells. Morphological observation and immunohistochemistry for nestin, β-tubulin Ⅲ, MAP2ab and GFAP were used to identify the differentiated cells. Results Primary cultured ADSCs appeared polymorphous, from which the mixed cells were removed by adherent culture. The fifth passage of ADSCs showed homogenous morphology in regular alignment, and the phenotype could be maintained after further passaging. Immunohistochemistry showed that the two induction protocols both resulted in high expression of nestin and β-tubulin Ⅲ in the induced ADSCs, and nestin expression in group A reached the peak level of(73.8±6.5)% at 6 h of the induction, followed by rapid reduction to (50.3±3.8)% at 24 h and (10.5±30)% at 72 h, becoming almost undetectable afterwards;β-tubulin Ⅲ expression occurred at 24 h following the induction [(33.5±6.6)%] and reached the peak level of (84.3±33)% at 1 week. In group B, high nestin expression persisted throughout the neurosphere stage, while low levels of β-tubulin Ⅲ expression was detected during the neurosphere stage [(14.1±3.3)% at 7 days after neurosphere formation], but its expression increased obviously with time after neurosphere differentiation [(46.4±6.1)% at 3 days after the differentiation. In group B, 24.5% of the cells were found positive for MAP2ab after the differentiation of the neurospheres into neural cells. Conclusion ADSCs can be induced into neural cells under appropriate conditions. Both of the induction protocols can obtain high rate of nestin- and β-tubulin Ⅲ -positive cells, and the neurosphere induction protocol produces more stable nestin expression in the differentiated cells, which contain a proportion of MAP2ab-positive cells.

Prokaryotic expression, purification of human LINGO-1(aa76-319) and preparation of its polyclonal antibody / 南方医科大学学报

<p><b>OBJECTIVE</b>To express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb).</p><p><b>METHODS</b>The 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting.</p><p><b>RESULTS</b>The prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity.</p><p><b>CONCLUSION</b>The fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.</p>

Feasibility of local LINGO-1 polyclonal antibody treatment for spinal cord injury in adult rats / 中华神经医学杂志

Objective To analyze the feasibility of local LINGO-1 polyclonal antibody administration for treatment of spinal cord injury in adult rats. Methods Twenty-four adult female SD rats were randomized into sham-operated group, rabbit IgG group and LINGO-1 antibody group. In the latter two groups, partial transaction of the T9 segment of the spinal cord was performed to completely sever the dorsal eorticospinal tract, followed immediately by administration of rabbit IgG and anti-LINGO polyclonal antibody via a mini-osmotic pump, respectively. At 3 and 28 days after the operation, the T8～10 segments of the spinal cord were harvested to prepare cryosections, and immunofluorescence staining was used to analyze the penetration of LINGO-1 polyclonal antibody into the spinal cord tissue and its specific binding to LINGO-1 molecules. Results In LINGO-1 antibody group, the presence of rabbit antibodies was detected at the injured sites of the spinal cord at 3 and 28 days after the operation. The mean immunofluorescence density was significantly lower in L1NGO-1 antibody group than in rabbit IgG group at 3 days after the operation (P<0.05). In rabbit IgG group, the mean immunofluorescence density for LINGO-1 in the crysections pre-treated with LINGO-1 polyclonal antibody was significantly lower than that in sections pre-treated with rabbit IgG(P<0.05). Conclusion Locally administered LINGO-1 polyclonal antibody can penetrate into the injured sites in the spinal cord in a wide time window and recognizes LINGO-1 molecule specifically, suggesting the feasibility of passive immunotherapy for spinal cord injury.

Identification of brain tumor stem cells and research on their characters of proliferation and drug-resistance / 中华神经医学杂志

Objective To isolate and culture brain tumor stem cells (BTSCs) from glioma tissues and explore the biological characteristics of BTSCs. Methods Different grade glioma tissues were obtained from 20 clinical cases. After tumors were dissociated, the sample was triturated into the single cells and then filtered. The primary glioma cells were collected and cultured in the DMEM/F12 medium containing epidermal growth factor (EGF), leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF), in order to promote the proliferation of BTSCs. CD133 + cells were separated by immunomagnetic bead method and identified by testing the expressions of CD133, NSE and GFAP using immunocytochemistry. CCK8 was employed to assay the proliferating situation of CD133+ cells in the different grade gliomas, and to compare the drug resistance between the CD133+ and CD133- cells in the medium containing VM-26. Results CD133+ cells were successfully separated from glioma tissues.CD133+ cells proliferated by self-renewal, then differentiated into NSE+ cells and GFAP+ ones respectively. CD133+ cells in the high grade gliomas showed the faster generation than the ones in the low grade gliomas. CD133+ cells survived more easily than the CD133- cells in the medium containing VM-26. Conclusions BTSCs exist in the glioma tissues, and possess the more tolerant to the VM-26.CD133+ cells in the high grade glioma can proliferate much more easily.

In vitro culture and identification of neural stem cells derived from the olfactory bulb of adult mice / 中华神经医学杂志

Objecfive To establish a method for in vitro culture and identification of neural stem cells(NSCs)derived from the olfactory bulb(OB)of adult mice and test the possibility of the OB as a new source of seed cells of adult NSCs. Methads NSCs were isolated from the OB of adult mice and cultured in serum-free medium.Clonal culture and BrdU incorporation assay were performed to assess the self-renewal and proliferative activities of the NSCs.Fluorescence immunocytochemistry was carried out to examine the expression of the NSC markers nestin and SOX2,neuronal marker Tujl,astrocyte marker GFAP and oligodendroeyte marker 04. Results NSCs possessing self-renewal and proliferative capacities were obtained from the OB of adult mice,and the cells grew in the form of floating neurospheres in the medium.The neurospheres consisted of cells were positive for NSC markers nestin and SOX2,which Were able to differentiate into Tuj1-positive neurons,GFAP-positive astrocytes and 04-positive oligodendrocytes. Conclusion NSCs are present in the OB of adult mice,and the NSCs isolated from the OB can proliferate and differentiate in vitro with obvious stem cell properties.suggesting the feasibility of using OB as anew source of adult NSCs.

Culture and identification of neural stem cells derived from the subventricular zone of adult mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a method for culturing and identifying neural stem cells (NSCs) derived from the subventricular zone (SVZ) in adult mice.</p><p><b>METHODS</b>NSCs were isolated from the SVZ of adult mouse brain and cultured in serum-free medium. Cell cloning and BrdU incorporation were performed to identify the self-renewal and proliferative capacity of the NSCs. Fluorescence immunocytochemistry was used to examine the expressions of the NSC markers nestin and SOX2, neuronal marker Tuj1, astrocyte marker GFAP and oligodendrocyte marker NG2. The expressions of nestin and SOX2 were further examined by Western blotting and RT-PCR.</p><p><b>RESULTS</b>NSCs with self-renewal and proliferative capacity were obtained from the SVZ of adult mice and grown as floating neurospheres. The NSCs expressed nestin and SOX2 and could differentiated into Tuj1-positive neurons, GFAP-positive astrocytes and NG2-positive oligodendrocytes.</p><p><b>CONCLUSION</b>This method allows simple and stable culture of NSCs from the SVZ of adult mice.</p>

Photodynamic effect mediated with 5-aminolevulinic acid on U251 human glioma cells / 中华神经医学杂志

Objective To investigate the photodynamic effect mediated with 5-aminolevulinic acid (5-ALA) on U251 human glioma cells. Methods Fluorescence microscope and confocal laser scanning microscope were used to detect the localization of Pp Ⅸ in U251 human glioma cells. The cells with/without 5-ALA were irradiated at the wavelength of 635 nm. MTT assay was used to measure the cell survival after laser irradiation. Results 5-ALA cocultured with U251 cells successfully produced endogenous Pp Ⅸthat was observed distributively in the cytoplasm, but not in nuclear region. The overall survival rates of the U251 glioma cells photodamaged by ALA-PDT decreased as the incubation time went by or the 5-ALA concentration increased, while peaked at the incubation time of 6 h and the 5-ALA concentration of 2.0mmol/L. Without one of 5-ALA and light irradiation, the survival rate of the cells had no significant difference compared with that of cells of the control group. Conclusion The 5-ALA-induced PDT appears to be a promising therapy for human glioma. The optimal incubation time may be 6 h and the optimal 5-ALA concentration be 2.0 mmol/L.

Construction of eukaryotic expression vector with brain-derived neurotrophic factor receptor trkB gene / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To construct an eukaryotic expression vector carrying rat brain-derived neurotrophic factor receptor trkB gene.</p><p><b>METHODS</b>Using the total RNA isolated from rat brain as template, the trkB gene was amplified by reverse-transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers which contained the restrictive sites of EcoR I and BamH I. The amplified fragment of trkB gene was digested with EcoR I and BamH I, and then subcloned into cloning vector pMD18-T and expression vector pEGFP-C2 respectively. The recombinant plasmids were identified by restriction endonuclease enzyme analysis and PCR.</p><p><b>RESULTS</b>The amplified DNA fragment was about 1461 bp in length. Enzyme digestion and PCR analysis showed that the gene of trkB had been successfully cloned into vector pMD18-T and pEGFP-C2.</p><p><b>CONCLUSIONS</b>The trkB gene of rat has been amplified and cloned into the eukaryotic expression vector pEGFP-C2.</p>