Objective To investigate the influence of recombinant neuregulin-1 beta (rhNRG-1β) on neural stem cell proliferation through extracellular regulated protein kinases (ERK) signaling pathway in oxygen and glucose deprivation (OGD) environment.Methods Neural stem cells were obtained from embryonic brain of mice pregnant for 14-17 d,cultured and identified by immunochemical staining through detection of the indicator nestin using the SABC-FITC (POD)double standard kit.Neural stem cells were divided into three experiment groups (OGD group,control group and OGD + rhNRG-1β group).Control group:identified neural stem cells,2 × 107,were cultured for 3 h in the 24-hole culture plate with DMEM/F12 complete culture medium;OGD group:neural stem cells,2 × 107,were cultured in the 24-hole culture plate deprived glucose DMEM/F12 in a wet airtight container (37 ℃ constant temperature),cells were cultured with mixed gas of nitrogen (950 ml/L) and oxygen (50 ml/L) for 1 h,and then the culture medium was replaced with complete culture medium and cultured for 3 h;OGD + rhNRG-1β group:before OGD intervention,100 μg/L rhNRG-1β was given for 3 h.Neurospheres formation:the three groups of stem cells were dispersed into single cells,1 × 106/ml cells were inoculated to culture plates containing cover slips coated with poly lysine,and cultivated for 7 d,and neurospheres formation of the 3 groups of neural stem cells was observed under microscope,which was aimed to record neural stem cells proliferation changes.Colony formation:the three groups of stem cells,vaccinated in 60 mm in a petri dish,2 × 107 in number,were cultivated in complete culture medium for 24 h.The colony formation of the three groups of cells was observed under microscope,and neural stem cells proliferation changes were observed.Western blotting:the change of phosphorylation ERK (pERK) protein of the three groups of stem cells was determined,and the effect of pERK protein expression regulated by rhNRG-1β in mice neural stem cells proliferation through ERK signaling pathway was observed.Results Microscopically the primary cultured stem cells grew in single or in pairs,in a round shape;neural stem cell proliferated in clumps or colony;neural stem cells expressed the specificity of nestin protein markers with fluorescent yellow-green color.The differences in the aspects of the average diameter of neurospheres and neurospheres quantity in the neural stem cells between groups were statistically significant (F =693.66,1 002.09,all P ＜ 0.01),and among them the neuropheres formation of OGD group was significantly suppressed.Formation quantity and average diameter in OGD group [(88.78 ± 7.14) numbers,(62.12 ± 2.52) μm] were significantly lower than those in the control group [(246.34 ± 8.67) numbers,(128.45 ± 2.33) μm] and those in OGD + rhNRG-1β group [(237.87 ± 6.61) numbers,(118.37 ± 2.71) μm,all P ＜ 0.01].The difference of colony formation rate of neural stem cell was statistically significant (F =132.03,P ＜ 0.01),and among them colony formation of OGD group significantly suppressed.Formation rate in OGD group [(11.65 ± 0.94)％] was significantly lower than that in the control group [(33.23 ± 2.93)％] and that in OGD + rhNRG-1β group [(31.42 ± 2.61)％,all P ＜ 0.01].Western blotting showed that the difference of pERK protein expression of neural stem cells between groups was statistically significant (F =63.76,P ＜ 0.01).Relative expression of the pERK protein in OGD group (0.487 ± 0.072) was significantly lower than that in the control group (1.013 ± 0.112) and that in OGD + rh-NRG-1β group (1.752 ± 0.278,all P ＜ 0.01).Conclusion rhNRG-1β preserves neural stem cell proliferation with phosphorylation ERK protein expression up-regulated in oxygen and glucose deprivation environment.

Objective To investigate the variation and correlation between tumor necrosis factor-?(TNF-?)and transforming growth factor-?1(TGF-?1)in children with Henoch-Schonlein purpura nephritis(HSPN)in plasma and explore their effects on kidney lesion in children with Henoch-Schonlein purpura(HSP).Methods Plasma TNF-? and TGF-?1 were determined with enzyme-linked immunosorbent assay(ELISA)in 30 cases with HSP,38 cases with HSPN and 30 normal controls,urinary protein excretion with urinary analyze method in these children.Renal biopsies were performed and renal biopsy specimens were observed by light,immunofluorescence and electron microscopy in 32 out of 38 cases with HSPN.The SPSS 11.0 software was used to analyze the data.Results 1.Comparing with normal controls,the plasma level of TNF-? and TGF-?1 in children with HSP increased with significant difference in statistics(Pa

Objective The purpose of this study was to determine how to preserve the remaining pancreatic body and tail in the pancreatectomy. Methods In seven cases of pancreatectomy, three of them were the rupture of pancreatojejunal anastomosis, and four of them were the pancreatectomy for tumor in the pancreatic neck or body. During operations, a bridge internal drainages was used to drain the pancreatic juice into the adjacent jejunum. After the operations, the supportive treatment, continuous irrigation of peritoneal cavity and pancreatic enzyme inhibition were used. Results In all seven patients, the remaining pancreatic body and tail were successfully preserved. The endocrine functions of these patients recovered to nearly normal level and patients were discharged. Conclusions In preserving the remaining pancreatic body or tail, the bridge internal drainage has its advantage of convenience. It effectively preserves the exocrine of pancreas as well as its endocrine

Chaetomium thermophilum CT2 can produce extracellular cellulase with industrial value. We designed two degenerate primers to amplify catalytic domain sequence of cellobiohydrolase II ( CBH II). Full length of cDNA was obtained by rapid amplification of cDNA ends technologies. DNA sequencing revealed that cbh2 has an open reading frame of 1428bp, which encodes a putative polypeptide of 476 amino acids. The deduced amino acid sequence shows that the predicted molecular mass is 53 kD and the cbh2 consists of a fungal-type carbohydrate binding domain (CBD) separated from a catalytic domain by a linker region rich in proline/serine/threonine. PCR product consisting of the entire CBH II coding region without its signal sequences was cloned into the yeast secretive plasmid pPIC9K, which was then transformed into Pichia pastoris GS115. Highly efficient production of the cellobiohydrolase II was achieved in P. pastoris under the control of the AOX1 promoter, and the expressing level was 1.2 mg/mL by small-scale culturing. The recombinant cellobiohydrolase II was purified by using ammonium sulfate fraction, DEAE-Sepharose Fast flow chromatography. A molecular mass of the purified enzyme is 67 kD determined by SDS-PAGE and this is similar to the native cellobiohydrolase II purified from C. thermophilum CT2. The recombinant enzyme exhibited optimum catalytic activity at pH 4.0 and 50 degrees C respectively. It was thermostable at 50 degrees C and retained 50% of its original activity after 30 min at 70 d degrees C . The high level of fully active recombinant cellobiohydrolase II got from P. pastoris makes this expression system attractive for fermentor and industrial applications.
Amino Acid Sequence ; Base Sequence ; Cellulose 1,4-beta-Cellobiosidase ; biosynthesis ; genetics ; Chaetomium ; enzymology ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Fungal Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Open Reading Frames ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVEIn order to get the method for improving the salt resistance of Hedysarum polybotrys seeds and seedlings under different salt-alkaline stress, the seed germination and physiological characteristics of H. polybotrys seedlings were studied.

METHODSeveral physiological indexes of H. polybotrys seeds under different salt-alkaline stress, such as the germination vigor, germination rate, relative germination rate, relative salt damage rate were measured. And others indexes of the seedlings like chlorophyll contents, soluble protein contents, the permeability of plasmalemma, the activities of POD and SOD were also measured.

RESULTDifferent salt-alkaline stress decreased the germination rate, vigor of germinate, germination index, while relative salt damage rate increased. With the increased salt-alkaline concentration, the adverse effects became more obvious. The strength of the salts: Na2CO3 > Na2SO4 > NaCl. With the increase of the salt-alkaline concentration, the chlorophyll contents and the soluble protein contents decreased, but the permeability of plasmalemma increased. The change trend of SOD and POD activity was similar, it is increased firstly, and then decreased as the stress intensity extended, the most significant increase of Na2SO4 and Na2CO3 in the concentration of salt-alkaline was 25 mmol x L(-1), but NaCl was 50 mmol x L(-1).

CONCLUSIONThe seeds and seedlings inhibition of the salts was Na2CO3 > Na2SO4 > NaCl.

Alkalies ; metabolism ; Fabaceae ; growth & development ; metabolism ; physiology ; Seedlings ; growth & development ; metabolism ; physiology ; Seeds ; growth & development ; metabolism ; physiology ; Sodium Chloride ; metabolism ; Stress, Physiological

Background Epidemiological surveys showed that the types of bacteria are different in the conjunvical sac from different nationalities,which possibly is associated with living environment.To characterize the types of conjunctival bacteria involved is important for the prevention and treatment of infectious eye diseases.Objective The present survey was to obtain data about bacterial species in the conjunctival sac in the Tibetan minority population aged over 40 years old and compared with the matched Han nationality population.Methods The standardized training and protocol were performed before this survey.A total of 290 eyes of 145 individuals from the Tibetan minority and 346 eyes of 173 subjects from the Han nationality were enrolled in this study in Ganzi Autonomous Prefecture,who had received questionnaire surveys and ophthalmological examinations.The secretion of the inferior palpebral conjunctival sac was embrocated and inoculated and grown on blood plates for 48-72 hours.The bacteria were isolated and identified.This study was approved by the Medical Ethic Committee of the Sichuan People Hospital.Oral informed consent was obtained from the subjects.Results No significant differences were seen in the constituent ratio of the gender as well as the age between the Tibetan minority and Han nationality in this study (x2 =0.987,P=0.3202;t=1.142,P=0.254).There was a significant difference in the proportions of farmers and herdsmen between the two groups(x2 =8.557,P =0.000).The positive rate of bacterial cultivation in Tibetan individuals was 50.74％,showing a statistically significant decrease in comparison with the Han people(60.4％)(x2=6.042,P=0.014).There was no statistical difference in the multiple bacterial species between the two groups (11.0％ in Tibetan,11.6％ in Han people)(x2 =0.0271,P =0.869).The rate of staphylococcus epidemics was 26.6％ in the Tibetan minority and that of Han population was 33.2％,without a significant difference between them (x2 =3.350,P=0.060).No significant difference was seen in the ratio of corynbacterium infection between the two population(15.9％ vs.17.3％)(x2 =0.248,P =0.618).Conclusions The ratio of bacterial cultivation in Tibetans is statistically lower than that of the Han people.The types and distribution of bacteria are similar in the Tibetan and Han nationality.

There are many physical factors affecting the development of cartilage tissue,the mechanical con-dition is the main important one that particularly act.The mechanical conditions used in engineering cartilage tissue,such as compressive and shear force,fluid flow,hydrostatic pressure and tissue deformation or with some of them combined,were reviewed.From the standpoint of bionics,the mechanical environments ap-plied on tissue engineering should work in three aspects:providing adequately mechanical stimuli to the cells seeded in 3-D scaffold;ensuring the efficient mass-transport of the nutrients and waste products in the cells:promoting the development of functionally extracellular matrix in 3-D scaffold.The mechanical environments currently used only represented the part of mechanical conditions of in vive articular cartilage will be reviewed.In our view that rolling depression load may achieve the fit mechanical environment for cultivation of functional cartilage constructs in vitro.

There are many physical factors affecting the development of cartilage tissue,the mechanical con-dition is the main important one that particularly act.The mechanical conditions used in engineering cartilage tissue,such as compressive and shear force,fluid flow,hydrostatic pressure and tissue deformation or with some of them combined,were reviewed.From the standpoint of bionics,the mechanical environments ap-plied on tissue engineering should work in three aspects:providing adequately mechanical stimuli to the cells seeded in 3-D scaffold;ensuring the efficient mass-transport of the nutrients and waste products in the cells:promoting the development of functionally extracellular matrix in 3-D scaffold.The mechanical environments currently used only represented the part of mechanical conditions of in vive articular cartilage will be reviewed.In our view that rolling depression load may achieve the fit mechanical environment for cultivation of functional cartilage constructs in vitro.

OBJECTIVETo investigate the inhibitory effect of the small interfering RNA targeting mdm2 gene on the growth of osteosarcoma cells.

METHODSPGCsilencerTM-mdm2 siRNA was constructed and transfected into the osteosarcoma cell line U2OS cells. The inhibitory effects on mdm2 were determined by RT-PCR and Western blot analysis. The cell growth activity was determined by MTT assay, and the cell apoptosis was examined by flow cytometry. The therapeutic effects of simdm2 was assessed on the nude mouse model of transplanted tumor.

RESULTSThe simdm2 plasmid was successfully constructed. After simdm2 being transfected into the U2OS cells, the expressions of mdm2 gene and protein were significantly inhibited. The ability of cell growth activity decreased greatly and cell apoptosis occurred apparently. There was no significant difference between the negative control group and non-transfected group. The growth of xenograft tumor in simdm2 transfected nude mice was inhibited and the expressions of mdm2 gene and protein were down-regulated remarkably.

CONCLUSIONsiRNA targeting mdm2 gene inhibits the mdm2 expression in osteosarcoma U2OS cells and the growth of osteosarcoma in nude mice.

Animals ; Apoptosis ; Bone Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Female ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Osteosarcoma ; metabolism ; pathology ; Plasmids ; Proto-Oncogene Proteins c-mdm2 ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; Tumor Burden

AIMTo investigate the paclitaxel-loaded nanoparticles of poly(ethylene glycol)-b-poly(D,L-lactic acid) amphiphilic diblock copolymer (PMT).

METHODSPMT was prepared by solid dispersion technique. The average size and size distribution were determined by dynamic light scattering (DLS). The morphology was characterized by transmission electron microscopy (TEM) and 1HNMR. The influences of the copolymer molecular weight and the paclitaxel-fed amount on PMT were studied. Therapeutic effect of PMT was studied on Kunming mice liver cancer H22.

RESULTSPMT showed nanometer size and spherical morphology with core and shell. The sizes of PMT increased with increasing the molecular weight of the hydrophobic segment in PEDLLA or increasing the drug-loaded amount. The tumour inhibiting effect of PMT was similar with that of Taxol.

CONCLUSIONIt will provide an experiment basis for the development of new kind of intravenous administration of paclitaxel.

Animals ; Antineoplastic Agents, Phytogenic ; administration & dosage ; pharmacology ; Delayed-Action Preparations ; Drug Carriers ; Drug Delivery Systems ; Lactic Acid ; Liver Neoplasms, Experimental ; pathology ; Mice ; Microspheres ; Nanotechnology ; Paclitaxel ; administration & dosage ; pharmacology ; Particle Size ; Polyethylene Glycols