Dopaminergic neuronal differentiation of neural stem cells in rats at different gestational ages / 中国组织工程研究

BACKGROUND: Differentiation inducing factors and gestational age influence the differentiation potential of embryonic neuralstem cells.OBJECTIVE: This study was designed to observe the differentiation potential of rat mesencephalic neural stem cells at differentgestational ages towards dopaminergic neurons.DESIGN: A randomized controlled observation.SETTING: Department of Neurosurgery, First Affiliated Hospital of Sun Yat-sen University, Guangzhou City, GuangdongProvince, China.MATERIALS: This study was performed at the Laboratory of the First Affiliated Hospital of Sun Yat-sen University between Marchand September 2007. Thirty adult gestational SD rats, weighing 350 400 g, were provided by the Laboratory Animal Center of SunYat-sen University (Permission No. 2007-0034). The protocol was performed in accordance with ethical guidelines stated in Guide forthe use and care of laboratory animals, approved by the Committee on the Care and Use of Laboratory Animals of the Institute ofLaboratory Animal Resources Commission on Life Sciences, National Research Council, China (1985). DMEM/F12 serum-free medium,B27 additives, epidermal growth factor, basic fibmblast growth factor, and fetal bovine serum (volume fraction:0, 1) were purchased fromGibco Company, British; Interleukin lα, interleukin 11, and glial cell-derived neurotrophic factors were purchased from R&D Company,USA; In addition, leukaemia inhibitory factor (Perpotech, British), tyrosine hydroxylase(Santa Cruz, USA), nidogen antibody,microtubule-associated protein 2 antibody, and glial fibrillary acidic protein antibody(Chemicon, USA) were also used.METHODS: Six rats were randomly selected at each time point (on days 10,12,14,16, and 18 after gestation). After anesthesia, therats were sacrificed. Under the aseptic condition, fetal rat was harvested. Rat mesencephalic ventral brain tissue was isolated forculture of neural stem cells. Different gestational ages of rat brain-derived neural stem cells were separately cultured in theserum-free medium containing epidermal growth factors and basic fibroblast growth factors. After passage and amplification, theneural stem cells were induced to differentiate towards dopaminergic neurons in the medium containing interleukin lu, interleukin11, leukaemia inhibitory factors, glial cell-derived leukaemia inhibitory factors. On day 6 after induction and differentiation, thedopaminergic neurons were observed and identified by immunocytochemistry. After labeled by tyrosine hydroxylase, thedifferentiated dopaminergic neuron proportion was detected by a flow cytometer.MAIN OUTCOME MEASURES: The growth state of differentiated rat neural stem cells at different gestational ages and theimmunocytochemistry results. The tyrosine hydroxylase staining-positive neural stem cell proportion after induction anddifferentiation.RESULTS: Rat mesencephalic neural stem cell spheres on days 10,12, 14, 16, and 18 after gestation adhesively grew in thedifferentiation-inducing medium. The neural stem cells in the spheres gradually grew in radial tendency. On day 6 afterdifferentiation, most of the neural stem cells exhibited 1-2 long processes or several short processes. After nidogenimmunocytochemical staining, most of neural stem cells exhibited cytoplasm-positive. After culture for 6 days in the differentiationinducing medium, rat mesencephalic neural stem cells at gestational 10,12, 14, 16, and 18 days were detected by a flow cytometer.Results demonstrated that the proportion of tyrosine hydroxylase-positive cells was (10.3±2.5)%, (21.6±3.4)%, (16.7±2.8)%,(14.2±3.2)%, and (8.9±1.8)%, respectively. There was a significant difference in the proportion of tyrosine hydroxylase-positivecells among the cells at different gestational days (P < 0.05). Rat neural stem cells at gestational 12 days could be induced todifferentiate into dopaminergic neurons at the highest proportion.CONCLUSION: Mesencephalic neural stem cells of rats at different gestational days have different capabilities to differentiatetowards dopaminergic neurons. The proportion of dopaminergic neurons is the highest when mesencephalic neural stem cells ofrats at gestational 12 days.

Effects of induction time on dopaminergic neuronal differentiation from rat neural stem cells cultured in vitro / 中国组织工程研究

BACKGROUND: Recently, several scientists have found that differentiation of neural stem cells (NSCs) towards dopaminergic neurons may be increased in vitro by combination of some special cytokines. They have also found that dopaminergic neurons differentiated from NSCs can be used for the treatment of Parkinsn's disease. To improve the therapeutic effects of/n vitro transplantation, we should further study the biologic characteristics of NSCs at the induction and differentiation.OBJECTIVE: To explore the differentiation of NSCs which were incubated in differentiation solution for different time towards dopaminergic neurons in vitro.DESIGN: Single sample observation.SETTING: Department of Neurosurgery, First Affiliated Hospital of Sun Yat-sen University.MATERIALS: This study was performed at the Department of Ncurosurgery, First Affiliated Hospital of Sun Yat-sen University from May to October 2007. Six healthy Sprague Dawley (SD) rats, gestational age 14 days, of clean grade, weighing 350-400 g,were provided by the Laboratory Animal Center, Sun Yat-sen University[permission No. SCXK (yue)2007-0034]. The protocol was performed in accordance with ethical guidelines for the use and care of animals.METHODS: NSCs derived from rat embryonic mesencephalon were cultured in serum-free culture medium containing epidermal growth factors and basic fibroblast growth factors. After passage, the NSCs were induced to differentiate towards dopaminergic neurons in the differentiation medium supplemented with interleukin 1o, interleukinl 1, human leukaemia inhibitory factors, and glial cell line-derived neurotrophic factors. The percentage of tyrosine hydroxylase positive neurons in differentiated ceils was detected with flow cytometer when NSCs were cultured in differentiation solution for 2, 4, 6, 8 and 10 days, respectively.MAIN OUTCOME MEASURES: Cellular morphological alteration of rat NSCs after differentiation. The percentage of tyrosine hydroxylase positive neurons in differentiated cells derived from NSCs.RESULTS: In differentiation medium, NSC spheres attached the bottom of plates and began to collapse. Cells inside the spheres grew out gradually and became irregular in shape. Six days later, most of the cells had I or 2 long processes and a few short processes. The percentage of tyrosine hydroxylase positive neurons in differentiated cells was respectively (3.2_+_0.9)%, (6.8 +1.6)%, (16.7-+2.6)%, (14.8_+1.8)% and (12.2_+2.5)% after culture for 2, 4, 6, 8 and 10 days, with significant differences (F =26.449, P ＜ 0.05).CONCLUSION: Induction time influences the differentiation of NSCs towards dopaminergic neurons in vitro. The percentage of dopaminergic neurons is the highest in differentiated cells derived from NSCs which are cultured in differentiation solution for 6 days.

Antimicrobial Resistance of Pathogens Isolated from Diabetic Foot Patients / 中华医院感染学杂志

OBJECTIVE To study the antimicrobial sensitivity of pathogens isolated from diabetic foot patients. METHODS Totally 102 diabetic foot patients were enrolled from Jun 2000 to Dec 2007 in our hospital.Specimens such as pus and wound exudate were collected for culture.Pathogenic spectrum and antimicrobial sensitivities were investigated. RESULTS From 70 cases 109 strains of pathogenic bacteria were isolated,of which 61 were Gram-positive bacteria,43 Gram-negative bacteria and 5 fungi.Thirty-seven patients were with single microbial infection and 33 patients with polymicrobial infection and 28 of 33 patients were with Wagner′s grade 3 and upwards.According to susceptibility test,multi-drug resistance was found.Gram-negative bacteria were sensitive to imipenem and ?-lactamases inhibitor,and Gram-positive bacteria were sensitive to vancomycin,chloramphenicol,and cephalosporin. CONCLUSIONS The pathogenic bacteria in diabetic foot infection distribute extensively and some of them are multi-drug resistant.The key to the treatment of diabetic foot infection is early combination application of sensitive antimicrobial agents.

Primarily reconstruction for the scalp,skull and dura defects of type D craniopagus / 中华显微外科杂志

Objective To discuss the methods of primarily repairing scalp,skull and dura defects of separated type D craniopagus Methods The huge defects in craniopagus was primarily repaired by the application of scalp expander, transferring scalp flap, repairing dura with pedicle periosteums, repairing skull defects with Titanium plates Results The survival baby has a good sharp of skull and well healing of scalp postoperatively Conclusions Defects of separated type D craniopagus can be primarily repaired

Alteration in peripheral blood CD_5~+B cells is associated with disease activity in Graves′ disease / 中华内分泌代谢杂志

Peripheral blood CD_5~+B cell was detected by flow cytometry in patients with Graves′ disease (GD) before and after treatment. As compared with normal controls, peripheral blood CD_5~+B cells in a group of 43 patients with GD showed a significant increase in number [(17.0+5.1)% vs (39.5+12.4)%, P