Objective To establish human neural stem cells(HNSCs) model for further basic research and clinical application. Methods Cells from human fetal cerebral cortex were collected and cultured with serum free midium and then identified for nestin immunocytochemical staning;The cells were induced to differentiate by 5% fetal bovin serum and identified by neurofilament-200(NF-200) and glial fibrillary acidic protein(GFAP) immunocytochemical staning. Results The harvested cells appeared as clusters in suspension and both NF-200 and GFAP positive cells were observed after induction.After 12 generations of culture,these cells retained the main characteristics of NSCs.Conclusions The HNSCs were harvested from human fetal cerebral cortex and this HNSCs model can be used for futher basic research and clinical applications.;

Objective To study the effect of fluoride on bone metabolism in rats, and to understand the mechanism of pathogenesis of skeletal fluorosis. Methods A total of 80 Wistar rats were randomly divided into 4 groups that included control group (distilled water), low-dose group(NaF, 50 mg/L), medium-dose group (NaF,100 mg/L) and high-dose group(NaF, 150 mg/L), respectively. After being bred for 12 weeks, the rats were put to death (etherization). Incidence of dental fluorosis was estimated, and serum was collected. Radioimmunoassay was employed to detect the levels of osteocalcin (BGP), parathyroid hormone (PTH) and calcitonin (CT), respectively.Colorimetry method was employed to determine the levels of alkaline phosphatase (ALP) and acid phosphatase (ACP). Results Incidence of dental fluorosis between the four groups was significantly different statistically(x2 =82.81 ,P ＜ 0.01 ). The incidence was significantly different(x2 = 22.67, 40.00, 40.00, all P＜ 0.01 ) between low-dose ( 80%, 16/20), medium-dose ( 100%, 20/20), high-dose groups ( 100%, 20/20) and control group (0,0/20),respectively. Serum levels of BGP, PTH, CT were significantly different between the groups(F = 38.614, 20.778,3.023, P ＜ 0.01 or ＜ 0.05). There was no significant difference between the four.groups of ALP and ACP in serum (F = 0.609,2.895, all P ＞ 0.05 ). Serum BGP in low-dose, medium-dose and high-dose groups[ ( 19.60 ± 12.79),(33.41 ± 10.81 ), (39.46 ± 9.51 )mg/L, respectively] was significantly higher than that of the control group[ (7.35 ± 3.22)mg/L, all P ＜ 0.01 ]. Serum PTH in low-dose, medium-dose and high-dose groups[ (72.27 ± 25.38), (67.80 ± 12.01), (106.52 ± 36.37)pmol/L] was significantly higher than that of the control group[(47.08 ± 9.22)pmol/L,all P ＜ 0.01 ]. Serum PTH of the high-dose group was significantly higher than that of the low-dose and the mediumdose groups(all P ＜ 0.01 ). Serum CT in medium-dose and high-dose groups[ ( 13.39 ± 2.07), ( 15.05 ± 4.77)pmol/L ] was significantly lower than that of the control group[ (26.06 ± 28.31 ) pmol/L, all P ＜ 0.05 ] and also significantly lower than that of the low-dose group [ (24.49 ± 14. 10) pmol/L, all P ＜ 0.05 ]. Conclusions Fluoride affects bone metabolism in rats, BGP, PTH and CT play a key role in the pathogenesis of skeletal fluorosis.

Amyloidosis ; pathology ; Female ; Gadolinium ; Heart Diseases ; pathology ; Humans ; Magnetic Resonance Spectroscopy ; methods ; Male ; Middle Aged ; Myocardium ; pathology

Background Treatment of corneal ulceration by transplanting drug-loaded amniotic membrane has been used widespreadly abroad,however,seldom study is found in China up to now.Objective This study was to explore the sustained release property and the efficacy of the drug-loaded amniotic membrane.Methods The bacteriostatic area of amniotic membrane fragments immersed with different concentrations (5,20,30 g/L) of levofloxacin for different time points (5,15,30,60 minutes) was evaluated by in vitro test.Bacterial corneal ulceration models were established in 20 rabbits by injected 0.7 MCF staphylococcus aureus suspension (0.1 ml) into the central corneal stroma to form the cloudy area of 4.0-6.0 mm.Then the rabbits were randomized into two groups.Regular amniotic membrane transplantation was performed laterally and 0.5％ levofloxacin drops was topically administered after operation in the amnion+levofloxacin drops group,and drug-loading amnion transplantation was used in the drug-loading amnion group.Aqueous humor of 0.1 ml was collected in 30 minutes,1 hour,2,3.5,5.5 hours after levofloxacin was administered and 1 day,3,7,10,14,21 days after operation for the detect of levofloxacin level with high-performance liquid chromatography.The corneal symptom was scored based on McNeill's criteria in 1 week,2 weeks and 4 weeks and the ulceration area was assessed under the slit lamp in the first week.The pathological examination was carried out in the fourth week after surgery.Results The mean bacteriostatic area was bigger with the increase of levofloxacin concentration,and bacteriostatic area in amnion immersed for 15 minutes was bigger than that of 5 minutes (P＜0.01).The levofloxacin concentration of aqueous humor after transplantation was decreased by extending the time,and that in 30 minutes and 5.5 hours after operation was (0.873±0.264) mg/L and (0.106±0.027) mg/L,respectively,in the amnion+levofloxacin drops group,and that in day 1,3,7 after surgery in the drug-loading amnion group was higher than at 30 minutes in the amnion+levofloxacin drops group,showing all significant differences (all P =0.00).In the first,second and fourth week after operation,the corneal symptom score was 1.7±0.6,1.3±0.5,0.2±0.4 in the drug-loading amnion group and 2.2±0.8,2.0±0.6,1.5±0.8 in the amnion+ levofloxacin drops group,with the significant differences among the different groups and time points (Fgroup =9.49,P =0.01 ;Ftime =22.96,P=0.01).The corneal ulceration area was (1.6±0.6) mm2 in the drug-loading amnion group and (3.2±0.8) mm2 in the amnion+levofloxacin drops group 1 week after operation,showing a significant difference between them (t =3.98,P =0.00).Histopathological revealed that the various layers of cornea tissue appeared irregular arrangement in the amnion + levofloxacin drops group 4 weeks after operation with 1-2 layers of new squamous epithelium.Disorder hypothallus structure,more inflammatory cells and residual vascular cavity were visible.However,new squamous epithelium of 4-5 layers was seen in the drug-loading amnion group,and inflammatory cells and residual vascular cavity were less than the amnion+levofloxacin drops group 4 weeks after operation.Conclusions Levofloxacin-loaded amniotic membrane can sustained release levofloxacin and maintain an effective drug concentration in aqueous humor,which improves the treating efficacy for staphylococcus aureus corneal ulceration.

Objective To isolate and identify exosomes from human serum , explore the feasibility of detecting exosomal miRNA in human serum.Methods Retrospective study.Serum samples from 10 healthy individuals in January 2013 were randomly selected.Besides, from January 2013 to December 2014, serum samples from prostate cancer(PCa) patients (n=20), benign prostatic hyperplasia(BPH) patients ( n=20 ) and healthy controls ( n=20 ) were selected.Exosomes were isolated from these serum samples using ExoQuick , and then identified by using transmission electron microscopy , NanoSight nano particle analyzer and Western Blot for morphology and molecular phenotype.The quality of exosomal RNA was analyzed using Agilent 2100 Bioanalyser.Then quantificational real-time polymerase chain reaction ( qRT-PCR) was carried out to detect miRNAs in different components of human serum ,and nonparametric tests were used for difference analysis.Results Exosomes isolated from human serum showed round or oval vesicles, mainly in diameter 40-100 nm, and with maximum peak distribution of 58 nm.Moreover, they expressed HSP70 and four transmembrane protein CD 63.Agilent 2100 Bioanalyzer results showed that the major RNA component of exosome was about 25nt small RNA.qRT-PCR confirmed that 4 normal miRNAs were expressed in human serum exosome , and the expression of miRNAs in exosome pellets were higher than the whole serum (miR-21, U =16,P =0.007 2; miR-16, U =3,P<0.000 1; miR-20a, U =2,P <0.000 1;let-7a, U=13,P=0.003 2) and exosome-depleted supernatant ( miR-21, U=15,P=0.006 5;miR-16, U=2,P<0.000 1;miR-20a, U=1,P<0.000 1;let-7a, U=10,P=0.002 8).miR-141, the molecular marker of prostate cancer ,were analyzed by qRT-PCR in whole serum samples and serum exosome pellets isolated from the same serum in a cohort of 20 PCa patients , 20 BPH patients and 20 healthy control people.The results showed that , in three groups , exosomal miR-141 expression were all significantly higher than serum circulating miR-141 (Control group, U=66,P=0.000 3; BPH group, U=83,P=0.001 6;PCa group, U=54,P<0.000 1).In addition, the expreession of exosomal miR-141 in PCa patients was significantly higher than BPH patients or healthy controls (3.85 fold, U=74,P=0.000 7 and 4.06 fold, U=70,P=0.000 5).Conclusion Exosome can be efficiently isolated from human serum.Compared with the whole serum , isolation of serum exosome may helpful to improve the detection of circulating miRNA.

Objective To construct and screen effective shRNA expression vectors targeting human AQP1 gene ,and evaluate the interference efficiency of the AQP1 shRNA recombinant plasmids ,thus provide basis for further exploration on the effect and mechanism of AQP1 gene on human breast cancer cells .Methods Four pairs of shRNA sequences targeting human AQP1 gene were designed and synthesized ,and then inserted into the GV115 vector .AQP1 shRNA and control shRNA plasmids were trans‐fected into human breast cancer MCF‐7 cells .The expression of AQP1 mRNA and protein were detected by real time PCR(RT‐PCR) and Western blot to evaluate the interfering efficiency .Results RT‐PCR demonstrated that AQP1 was expressed in human breast cancer MCF‐7 cells .Sequencing showed that the shRNA vectors targeting AQP1 were successfully constructed .48 h after the AQP1 shRNA transfection ,AQP1 mRNA and protein expression levels in MCF‐7 cells were reduced to a significant degree ,and the AQP1 shRNA 4 plasmid vector could inhibit the AQP1 most efficiently .Conclusion The AQP1 shRNA recombinant plasmids vectors were successfully constructed and can significantly inhibit the expression of AQP1 in MCF‐7 human breast cancer cells .

Humans ; Manipulation, Spinal ; methods ; Spondylosis ; surgery ; therapy

Adenocarcinoma ; drug therapy ; immunology ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; drug therapy ; immunology ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Killer Cells, Natural ; immunology ; Lung Neoplasms ; drug therapy ; immunology ; Male ; Middle Aged ; Phytotherapy ; Quality of Life ; T-Lymphocyte Subsets ; immunology

OBJECTIVETo compare the therapeutic effects between close reduction and plaster slab fixation combined with plaster external traction and operation for the treatment of pediatric closed Gartland type III supracondylar humerus fractures without neurovascular injury complications.

METHODSFrom June 2009 to June 2012, 151 children with closed Gartland III supracondylar humerus fractures were retrospectively studied and divided into two groups, including 87 boys and 64 girls, ranging in age from 1 to 12 years old with an average of 5.3 years old. Among them, 76 children (conservative group) were treated with close reduction and plaster slab fixation combined with plaster external traction; 75 children (operation group) underwent surgical operation. The time of elbow joint function exercise, the healing time of fracture, the function recovery of elbow joint and carrying angle was recorded and analyzed. The therapeutic effects were evaluated by the Flynn criteria system.

RESULTSAll patients were followed up from 6 to 36 months (18.3 months on average). The average time of fracture healing and elbow joint functional exercise of the conservative group was shorter than those of operation group (P < 0.001). Motion range of the elbows and carrying angle of two groups were no statistical difference (P > 0.05). According to Flynn criteria system, in conservative group, the result was excellent in 31 cases, good in 35, fair in 7, and poor in 3; in operation group, 27 in excellent, 30 in good, 17 in fair and 1 in poor; there was no significant difference between two groups in therapeutic effects (P > 0.05).

CONCLUSIONClose reduction and plaster slab fixation combined with plaster external traction in treatment of pediatric closed Gartland type III supracondylar humerus fractures without neurovascular injury complications,which has similar effect to surgical treatment, and the time of fracture healing and elbow joint function exercise are significantly shorter.

Case-Control Studies ; Casts, Surgical ; Child ; Child, Preschool ; External Fixators ; Female ; Fracture Healing ; Humans ; Humeral Fractures ; surgery ; Infant ; Male ; Traction ; methods

Objective To investigate the role of Catbindin-D28k in the kidney on calcium metabolism.Methods VDR/CaBP-D28k double knockout(KO)mice was made.Body weight,diet intake and serum,urinary parameters and length,density of the long bones,histological staining of the tibia of WT,CaBP-D28(-/-),VDR(-/-)and VDR(-/-)/CaBP-D28k(-/-)mice were determined on regular and high Ca-Lac diet.Results On a regular diet,the double KO mice were growth-retarded more and smaller than VDR KO mice.Compared with VDR KO mice,the double KO mice had higher urinary calcium excretion and rachitic skeletal phenotype,which were manifested with higher serum parathyroid hormone levels,lower bone mineral density,and more distorted growth plate with mole osteoid formation in the trabecular region.On high calcium and high lactose diet,blood-ionized calcium levels were normal in both VDR KO and the double KO mice.However,in contrast to VDR KO mice,the skeletal abnormalities were not completely corrected in the double KO mice.Conclusion These results directly demonstrate that CaBP-D28k plays a critical role in maintaining calcium homeostasis and skeletal mineralization and suggest that its caleemic role can be mostly compensated by CaBP-D9k.