BACKGROUND: Pleiotropia and indeterminateness of the differentiation of neural stem cells (NSCs) cause great difficulties for clinical application.Therefore,it is the key to solving the problem to investigate the proliferation and differentiation condition of NSCs.OBJECTIVE:To observe the effects of epidermal growth factor(EGF)and basic fibroblast growth factor (bFGF) on the proliferation and differentiation of neural stem cells of human embryonic brain.DESIGN: A randomised and controlled experiment taking cultured human embryonic stem cells as subjects.SETTING: Department of Pediatrics of Zhujiang Hospital, the First Military Medical University of Chinese PLA.MATERIALS:This experiment was conducted at the Central Laboratory of the Department of Pediatrics of Chinese PLA from January to May 2004.Two 16-week embryos from induced labor by water bags voluntarily were chosen at random from the Department of Obstetrics in a tertiary hospital in Guangzhou(Informed consent was obtained from the parents of the fetuses).Then,cells in the subventricular zone were cultured in serum-free medium and serum medium, respectively.METHODS:Primary cells of subventricular zone of human embryonic brain were cultured with serum-free nedium containing EGF,bFGF and EGF + bFGF.The concentration of two growth factors was both 20 μg/L;experiment of differentiation was performed on the cell neurospheres cultured from the primary generation with serum culture medium;differentia tive cells were detected with immonohistochemical technique.MAIN OUTCOME MEASURES:The number of neurospheres and the change of neurons and gliocytes from neurospheres in each group.RESULTS:① There was no significant difference in the number of primary neurospheres between bFGF group and EGF+bFGF group [(150.3±14.9) /well vs (173.6±26.4)/we11, P ＞ 0.05] , but the number in the two groups was both more than that in EGF group [(99.5±14.9)/we11, P ＜ 0.01].② The neurons differentiated from neurospheres in bFGF group and EGF+bFGF group outnumbered those in EGF group, but gliocytes in EGF group outnumbered those in EGF + bFGF group.CONCLUSION:① bFGF can promote the proliferation of neural stem cells of subventricular zone of human embryonic brain,and the formed neurospheres can differentiate more neurons.② Combination of EGF and bFGF is not superior to single bFGF in effect,suggesting that there is no synergistic effect.

BACKGROUND: Neural stem cells(NSCs) have been used to treat brain injury or some degenerating diseases of nervous system. Since in vitro culture conditions for NSCs differ from normal physiological conditions, whether the properties of the cultured cells are consistent with those of cells under physiological conditions? Therefore, inducing endogenous NSCs to proliferate and differentiate may be more promising for practise of NSCs.OBJECTIVE: This study was designed to investigate the developmental properties of NSCs in frontal cortex of embryonic human brain at various ages.DESIGN: It was a randomized experimental study.SETTING: This study was conducted at Department of Pediatrics, Zhujiang Hospital, Southern Medical University.PARTICIPANTS: Totally 90 16-to-36-week-old fetuses underwent inducing abortion by water bag were selected at the Obstetrics & Gynecology Department of Zhujiang Hospital Affiliated to Southern Medical University from October 2003 to March 2004. Brain tissue was taken from the frontal cortex of the aborted fetuses. All the mothers had normal physical examination findings. The informed consents on inducing abortion by water bag had been obtained from relatives and the mothers. The study was conducted with a prior permission from the competent department of the First Military Medical University. According to their ages, the fetuses were divided into 6 groups,16-week group, 20-week group, 24-week group, 28-week group and 36-week group, each group containing 15 cases.METHODS: After inducing abortion by water bag, under axenic conditions, the aborted fetus was dissected, with the scalp excisd, the skull opened and the membrane covering brain pull apart. Then the frontal cerebral cortex was taken out, fixed and sliced. Employing immunohistochemical staining and light microscope, distribution, morphological features, phenotypes, growth patterns and quantity of NSCs in the frontal cortex were observed. Morphological features of the cells and expressions of markers in the cells were examined under light microscope. Negative control was set according to the substitution method. Under a × 400 field of microscope, some nestin-positive cells with speckled brown cytoplasmic staining were defined as NSCs. Two slides of each sample were detected and 10 fields of each slide were observed. Based on these observations, in each group, the total number of cells and the number of positive-stained cells in 300 fields were counted. The rates of nestin-positive cells were calculated.MAIN OUTCOME MEASURES: Morphological features, quantitative assessment and developmental features of the nestin-positive NSCs in frontal cortex of embryonic human brain at various ages were main outcome measurements in this study.RESULTS: NSCs were found in frontal cortex of embryonic human brain. They mainly were distributed in the pyramidal layer and the internal granular layer. They were small round-or oval-shaped, most were small round-shaped. These cells had a relatively large vacuolar nucleus with 1 - 3 nucleoli, loose chromatin and marked cytoplasmic staining. Some of the round-shaped cells were mitral cells with short neurites. The oval-shaped cells had 2 neurites. A distinct territorial distribution of NSCs could be observed. Some colonies, consisting of a few NSCs and looked like the neurospheres in in vitro culture, could be seen. Occasionally, symmetrical division of NSCs could be observed. In all the groups, 16-week, 20-week, 24-week,28-week, 32-week and 36-week group, the rates of nestin-positive NSCs in frontal cortex of embryonic human brain decreased with the increase of age (15.59%, 13.48%, 11.62%, 10.52% ,9.87% ,6.68% ,X2 = 1 265. 152, P＜ 0.01).CONCLUSION: The distribution, morphological features, phenotypes, growth pattern and quantity of the NSCs in frontal cortex of embryonic human brain at various ages are different and auto-developmental features exist. The number of these cells decreases with the increase of age.

Autoimmune diseases are characterized by the presence of various autoantibodies and may cause injuries to multiple organs,with kidney as the most common and important organ involved.Autoantibodies are of great importance in the diagnosis,treatment and prognosis of autoimmune renal diseases.Lupus nephritis,anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis and antiglomerular basement membrane (GBM) disease are the most common autoimmune renal diseases.Anti-C1q antibody,ANCA and anti-GBM antibody play important roles in those diseases,respectively.Appropriate and steady detecting methods are crucial to clinicians,and the results should also be interpreted with great cautions.

Objective To determine the pathogen profile and antibiotic resistance in aerobic isolates from blood cultures of neonates. Methods All blood culture reports (n=28120) from newborns admitted to the Department of Neonatology during 2002-2012 were analyzed, and the sensitivity patterns were recorded. Results A total of 1665 bacteria were isolated from 1606 blood culture-positive samples and the positive rate of blood cultures was 5.7%(1606/28120). Gram-positive bacteria were iso-lated in 1336 cases, with Staphylococcus epidermidis (902 cases) and Staphylococcus haemolyticus (206 cases) being the com-mon bacteria. Klebsiella pneumoniae (108 cases), followed by Escherichia coli (73 cases), were the major Gram-negative bacte-ria (235 cases). The determination of the antibiotic resistance of aerobic isolates was performed in 2012. Most Gram-positive iso-lates were sensitive to vancomycin and moxifloxacin, and more than 90%were resistant to penicillin while most of Gram-nega-tive isolates were sensitive to amikacin and imipenem. Conclusions Staphylococcus epidermidis, Staphylococcus haemolyticus, Klebsiella pneumoniae and Escherichia coli remain to be the principal organisms responsible for neonatal sepsis.

Objective To study the development of neural stem cells from human fetal brains at different developmental stages. Methods A total of 100 cases of embryos at 16-32 gestational weeks by induction of labor with water bag were collected for the determination of the distribution, forms, existing modes, and the number of neural stem cells in the hippocampus by SABC immunohistochemical method and light microscopy. Results Neural stem cells were found in the hippocampus at different fetal ages and located in the polymorphic layer, pyramidal, granular and molecular layers of hippocampus, mainly in polymorphic layer, pyramidal layer, and granular layer. Neural stem cells in hippocampus were round, ellipse, triangle, and stellate, particularly round and ellipse. No obvious enation was found. Neural stem cells had plenty of cytoplasm. The nuclei were round and ellipse with rare chromatin and nucleoli from 2 to 6. Most of neural stem cells were distributed among other neurons, and symmetric cleavage was found in some of them, but some neural stem cells were distributed in cluster and nest. The number of neural stem cells in hippocampus were different between groups and gradually decreased with the increasing gestational age. Conclusion Neural stem cells exist widely in the hippocampus at different gestational ages. There are differences in distribution, forms, existing modes, and number of neural stem cells in hippocampus at different gestational ages. Hippocampus may be the new originating region of neural stem cells.

Objective To discuss the application value of the combination detection of serum cystatin C(CysC),serum amyloid A (SAA)and urinary microalbumin(malb)for the early diagnosis of diabetic nephropathy(DN).Methods 112 cases of diabetic melli-tus(DM)were selected and divided into the non-DN group(malb<25 mg/L)and the early DN group(malb 30-299 mg/L)according to the urinary microalbumin level.50 cases of healthy subjects were selected as the control group.The levels of serum CysC and malb were measured by immuno-scatter turbidmetry and SAA was measured by enzyme-linked immunosorbent assays(ELISA).At the same time,the biochemical indexes of GLU,Cr,HbA1C were detected too.Results The levels of CysC,SAA and malb in the DM two groups were significantly higher than those in the healthy control group with statistical differences(P <0.05 ),moreover which were increased with the renal damage aggravation;the levels in the early DN group were significantly higher than those in the non-DN group with statistical differences(P <0.05).The levels of GLU and HbA1C in the DM two groups had statistically signifi-cant differences compared with the healthy control group(P <0.05).Conclusion The levels of serum CysC and SAA in the DM pa-tients are increased with the urinary malb level increase.The combined detection of CysC,SAA and malb could increase the diagnos-tic rate of the renal damage in early DN.

Objective To investigate the expressions of TK1 (thymidine kinase 1) in PHC (primary hepatic carcinoma). Methods TK1 and AFP in serum of 33 cases of PHC (primary hepatic carcinoma), 38 cases of hepatic cirrhosis,36 cases of hepatitis and 35 cases of healthy people were detected by means of Western blot—enhanced chemiluminecence and electrochemiluminescence. Results The difference of TK1 level in PHC group indicated significance when compared with that in hepatic cirrhosis group , hepatitis group and control group (U value was 436.4, 352.1, 163.6, respectively, all P < 0.01). TK1 level in patients with PHC was related with differentiation (χ2 = 7.476,P < 0.05) and TNM stage (χ2 = 7.227,P < 0.05),but not with sex, age, tumor diameter, number of tumors, vascular invasion, lymph node metastasis (P > 0.05). Kaplan-Meier curve analysis revealed that PHC patients with TK1≤ 2.0 pmol/L had a significantly shortened overall survival when compared with those with TK1 > 2.0 pmol/L (χ2 = 3.954,P < 0.05). Multivariable Cox regression analysis indicated that the level of TK1 and TNM stage were the independent risk factors for patients with PHC (all P <0.05). Conclusions The detection of TK1 has a certain clinical value in the diagnosis, monitoring and evaluation of the prognosis of the PHC.

Objective To study the role of deferoxamine(DFO)on regulating hypoxia-inducible factor 1α(HIF-1α)expression after hypoxia-ischemia brain damage(HIBD)in neonatal rats,to explore the therapeutic strategy for HIBD. Methods Postnatal day 10 SD rats were divided into four groups: hypoxia-ischemia(HI)group,DFO-treated group,normal saline(NS)-treated group,and sham operation group. HIBD model was established by the ligation of right common carotid artery following the inhalation of nitrogen-oxygen mixtures containing 92% nitrogen and 8% oxygen. DFO-treated group and NS-treated group were treated by intraperitoneal injection of DFO or NS respectively. The brains were collected at 4 h,8 h,and 24 h after hypoxia. HIF-1α protein expression was detected by Western blot analysis,and HIF-1α mRNA expression was detected by using RT-PCR at each time point. Results The synthetic level of HIF-1α protein increased significantly at 4 h,peaked at 8 h,and decreased at 24 h after HI in HI group,as well as NS-treated group. However,in DFO-treated group HIF-1α protein was peaked at 4 h,maintained higher level at 8 h and 24 h after HI. The level of HIF-1α protein was much higher in HI and DFO-treated groups than those in sham controls(P ＜ 0.05). The synthetic level of HIF-1α protein were higher in DFO-treated groups than those in HI groups at each time point(P ＜ 0.05). HIF-1α mRNA expression was higher in DFO-treated groups than those in HI groups at each time point. Conclusions DFO upregulate HIF-1α protein and mRNA expression in neonatal rats with HIBD. The peak of HIF-1α protein expression are also more advanced and lasted longer after DFO-treatment.

Objective:To study the curative efficacy of budesonide combined with huaiqihuang granules in the treatment of children with asthma and its effects on the serum leukotriene D4(LTD4),nerve growth factor (NGF),tissue inhibitor of metalloproteinase-1 (TIMP-1) levels.Methods:90 cases of children with asthma who were treated from March 2014 to March 2015 in our hospital were selected and divided into the observation group (n=45) and the control group (n=45) according to the random number table.Both groups of children were given bronchial antispasmodic agent,oxygen,antibiotics,glucocorticoid and other conventional treatment after admission,the control group was given budesonide atomization inhalation treatment on the basis of conventional treatment,0.5 mg budesonide were add in 3ml saline for continuous inhalation,twice a day,while the observation group was treated with Huaiqihuang Granules on the basis of the control group,twice a day.After 12 weeks of treatment,the curative effect,disappearance time of wheezing,cough relief,pulmonary rales,wheezing and other symptoms,forced vital capacity (FVC),serum LTD4,NGF,TIMP-1 levels,CD3+,CD4+,CD8+ of both groups were compared.Results:After treatment,the total effective rate of observation group was significantly higher than that of the control group [93.33％(42/45) vs 77.77％(35/45)](P＜0.05);disappearance time of breath,cough,moist rales and wheezes were significantly shorter than those of the control group[(4.32± 1.03)d vs (6.08± 1.24)d,(5.60± 1.12)d vs (7.21± 1.30)d,(3.19± 0.98)d vs (4.98± 1.02)d,(3.25± 1.03)d vs (5.89± 1.35)d](P＜0.05);the FEV1,FEV1/FVC were significantly higher than those of the control group [(92.63± 10.01)L/s vs (78.36± 9.19)L/s,(95.37± 11.72)％ vs (80.19± 10.23)％](P＜0.05);the serum LTD4,NGF,TIMP-1 levels were significantly lower than those of the control group [(7.24± 0.86)ng/mL vs (12.68± 1.01vng/mL,(68.18± 9.01)pg/mL vs (80.78± 10.24)pg/mL,(34.16 ± 5.06)ng/mL vs (49.76 ± 5.47)ng/mL] (P ＜0.05);the CD3+,CD4+ were significantly higher than those of the control group [(66.15± 7.20)％ vs (62.03± 6.85)％,(45.13± 7.90)％ vs (37.42± 7.06)％],CD8+ was significantly lower than that of the control group[(34.16± 5.06)％ vs (49.76± 5.47)％] (P＜0.05).Conclusion:Budesonide combined with Huaiqihuang granules was effective for children with asthma,which could enhance the immune function,improve the lung function,reduce the serum levels of LTD4,NGF,TIMP-1.

BACKGROUND:In vitro isolation and purity technique of stem cells mostly depends on the identification of cell surface marker,such as monoclonal antibody adherent spreading method,flow cell sorting method and immunomagnetic beads sorting method,but the operation was complicated and the price was high.OBJECTIVE:To observe the biological characteristics of human amniotic fluid-derived embryonic mesenchymal stem cells,which were isolated and cultured using the two-step method.DESIGN,TIME AND SETTING:The opening study was conducted at the Stem Cell Research Room of Xinjiang Medical University from March 2008 to March 2009.MATERIALS:Totally 10 amniotic fluid specimens were obtained from pregnant women who underwent prenatal diagnosis following 16-22 weeks of gestation or voluntarily induced abortion.With ultrasonic guidance,amniocentesis was performed to collect 20-40 mL amniotic fluid.METHODS:Human amniotic fluid-derived embryonic mesenchymal stem cells were isolated and cultured using the two-step method.Amniotic fluid was first centrifuged and incubated till spindle-shape cells were seen,with the presence of flbroblast-tike cell colonies.Supematant was moved to a new 25 cm~2 culture flask for further culture till spindle-shape fibroblast-like mesenchymal stem cell colonies.When 70% confluence,cells were digested,and incubated in α-MEM,supplemented with basic fibroblast growth factor,served as the first passage.MAIN OUTCOME MEASURES:Morphological changes in human amniotic fluid-derived embryonic mesenchymal stem cells of primary culture and subculture were measured.Karyotype,cycle,growth curve and colony formation ability of human amniotic fluid-derived embryonic mesenchymal stem cells were measured.Surface antigen and cytokine were examined using flow cytometry,immunofluorescence and RT-PCR.RESULTS:Human amniotic fluid-derived embryonic mesenchymal stem cells were successfully isolated and subcultured.During metaphase,primarily cultured amniotic fluid cells presented scattered spindle cells and flbroblast-like mesenchymal stem cell colonies every 7 days.Passaged cells completely adhered in 12 hours.Following 1 or 2 days of latent period,cells proliferated rapidly.About 90% confluence was observed following 6 or 7 days of culture.Cell arranged regularly,showing whirlpool-shape,radiated shape.Cells were spindle-shape,with unclear boundary.Chromosome karyotype of human amniotic fluid-derived embryonic mesenchymal stem cells was normal diploid.Growth curve showed "S" shape,but the two-step method reached a peak at (6.1±0.5) days,which was significantly rapid compared with the one-step method (7.2±0.6) days (P=0.035).Flow cytometry analyses showed that P3 cells at S phase took up (14±2.3)% using the two-step method,which was more than the one-step method (9.0±1.4)% (P=0.031).Low-density human amniotic fluid-derived embryonic mesenchymal stem cells were incubated for 7 days prior to cells formed scattered cell colonies.However,colony forming efficiency using the two-step method (15.0±2.3)% were significantly more than the one-step method (10.0±1.8)% (P=0.021).Flow cytometry results showed that human amniotic fluid-derived embryonic mesenchymal stem cells expressed CD44,CD29 and CD105,but were negatively for CD45,CD34,HLA-DR.Immunofluorescence suggested that Oct-4-positive cells were observed in amniotic fluid.However,the proportion of Oct-4-positive cells using two-step method (1.2±0.3)% was significantly greater than the one-step method (0.9±0.2)% (P=0.041).RT-PCR suggested that human amniotic fluid-derived embryonic mesenchymal stem cells obtained using the two methods expressed Oct-4.CONCLUSION:Human multipotent mesenchymal stem cells are present in human amniotic fluid.The two-step culture protocol could be a kind of high performance and simple protocol which may not interfere with the normal prenatal diagnosis procedure.