Objective To search for a good method for generation of hematopoietic stem cells (HSC) by embryonic stem cells (ESC),and to investigate the potential of HSC derived from ESC to reconstruct hematopoiesis in vivo.Methods Using a three-step method to induce a mice ESC line, E14.1-into HSC.And identifying HSC by flow cytometry analysis cell markers with CD34 +/Sca-1+, then HSC (1×109L-1)from ESC differentiation were injected into severe combined immunodefieency (SCID) mice for observing teratoma formation.To validate function of HSC by colonngenic cells assay and to reconstitute the hematopoiesis in lethally irradiated mice.Results Combining to use more hematopoietic stimulating factor to availably promote the El4.1 cell into embryoid body (EBs) with abundant hematopoietic progenitors.EBs were induced after 14 day to fast differentiate for HSC with peak percentages of CD34+/Sca-1+ cells reached to (13.72±2.07)%.To harvested ceils from EBs by day 14 for second -step HSC differentiation, percent of CD34+/Sca-1+ cells rise to(24.62±2.50) % after day 16 induction.Cloning forming units (CFU) analysis showed that more Erythro -myeloid cloning generation was observed at this period.Cells obtained in the second step are subsequently plated on monolayer of mice bone marrow stromal cells, in the presence of TPO, FLt3 ligand and superoatant of mice fetal liver stromal cells, cultured for additional 15 days, followed fast expansion of CD34+/Sca-1+ to maximally (58.64±4.20 ) % with more CFU-E, CFU-GM and CFU-GEMM population.Wright-Giemsa stain showed that its had the character of hematopoietic progenitors.Cells from the third-step were injected into SCID mice, but no teratomas were recovered in 2 mices after 6 weeks.Positive selection of CD34+/Sca-1+ cells by magnetic sorting from the third - step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 days after transplantation, with 71.4% successful engraftment rate.And 3 recipients showed that the cell population of the peripheral blood leukocytes,red cells, hemoglobin approached normal index at 40 days after transplant, hut followed relative'slow renew in platelet count.Survival rate of transplant group is 43.0%, compared to 100% mortality in control mice.Karyotyping assays confirmed female mice with XY.Conclusions The results showed that the three-step differentiation and the culture conditions described here good support differentiation of ESC into HSC.HSC derived from ESC were safe without teratomas formation in body, and its can reconstruct hematopoiesis.

Adult ; Bronchoalveolar Lavage ; methods ; Humans ; Male ; Pulmonary Surfactants ; therapeutic use ; Silicosis ; therapy

Objective To determine the effect of knocking down zebrafish faf1 gene by CRISPR/Cas9 editing technique.Methods gRNA was designed and prepared for the faf1 gene of zebrafish,and gRNA was mixed with Cas9 mRNA by microinjection into zebrafish single cell embryos.The mutant F0 generation zebrafish was screened out by enzyme digestion and gene sequencing.The mutant F0 was genetically outcrossed with the wild-type zebrafish to get the F1 heterozygous zebrafish,and the genotype of zebrafish was detected by microscopic observation.Results The faf1 gRNA and Cas9 mRNA were successfully prepared.The gRNA (gRNA6) located in the exon 6 of faf1 could shift the faf1 gene into frameshift mutations.The mutation type MU1 was screened out and the somatic cytochrome deposition delay was observed in this heterozygous zebrafish.At 4 d post fertilization (dpf),there were sarcomeric dysplasia and head shrinkage,increased hyoid angle and other craniofacial cartilage deformities.And the zebrafish died at 8 ～9 dpf.Conclusion CRISPR/Cas9 knocking out thefaf1 gene produces a new phenotype for zebrafish,with delayed pigment deposition and nodule-like change in tail muscle section.

A safe and effective anesthesia technique is necessary in ensuring a successful surgical operation in rabbit experiments.A variety of anesthesiamethod have been reported, yet, no one matured and widely accepted anesthesiamethod is available so far.This article aims to provide an information basis for further research on general anesthesia in rabbits by reviewing the literature on single and combined anesthesia techniques in rabbits reported in the last decade.

Objective To evaluate the role of TCD examination in diagnosis of the early cerebrovascular diseases in aged diabetic patients.Methods 179 cases of aged type Ⅱ diabetic patients were divided into three groups(PDR,BDR and NDB)according to their retinopathy.TCD and retina examination were performed in all patients and the data be analysed. Results (1)The systolic peak flow velocity(Vp) of MCA、ACA、ICA and BA were significant increased in diabetic pa- tients than in normal control group(P

Objective To investigate the method of directed differentiation dendritic cells from embryonic stem cells(ESC) and to amplify high purity DCS in vitro for immunity therapy.Methods E14 ESC line were generated ESC-derived dendritic cells(ES-DC) in complete medium further supplemented with granulocyte-macrophage colony-stimulating factor(GM-CSF) and interleukin-3(IL-3).ES-DCs was used flow cytometry to determine CD11c,CD80,CD86,MHC-Ⅱ cell surface phenotype. Lipopolysaccharide (LPS) were added to induce the ES-DCs matured. The matured ES-DCs was harvested 24 hours later to be identified with morphology, transmission electron microscopy, analyzed by flow cytometry and compared with the immatured ES-DCs phenotype. The antigen presenting was evaluated by mixed lymphocyte responses.Results The ES-DC had obviously dendritic processes under scanning electron microscope . The immature DCs express low level of CD11c(4.33±0.23)%,CD80 (7.62±0.19) %, CD86 (4.77±1.22) % and MHC-Ⅱ (9.68±0.15) %, but the mature DCs express higher lerve of CD11c(47.36±2.68)%,CD80 (74.4±1.47) %, CD86 (29.77±2.00) % and MHC-Ⅱ (87.56±2.75) %. MLR showed that ES-DCs could effectively stimulate lymphocyte to proliferate.Conclusion These results provide evidence that DCs can be generated from E14 ESC with GM-CSF and IL-3, express high level of CD11c,CD80, CD86, MHC-Ⅱ and can effectively stimulate lymphocyte to proliferate. ES cells may become new origin for DCs which provided the immunotherapy.

Background Researches demonstrated that corneal dystrophy is associated with the mutation of transforming growth factor beta induced gene(TGFBI)located at chromosome 5q31 domine.Recent study showed that the gene mutation location is in R124H of TGFBI gene. Objective This study was to identify the mutation characteristics of TGFBI gene in a Chinese family with Avellino corneal dystrophy. Methods This Chinese family with Avellino corneal dystrophy were determined and surveyed in Peking University Third Hospital.Periphery blood from 8 patients with Avellino corneal dystrophy and 2 unaffected subjects were collected from a Chinese family with corneal dystrophy for the extraction of DNA.Exons 4,11,12 of the TGFBI gene were amplified by polymerase chain reaction(PCR),and the amplified products were sequenced directly and compared the gene sequence with that of TGFBI in GenBank.Written informed consent was obtained from each Subject prior to any medieal process. Results This family included 27 members of consecutive 4 generation.The hereditary pattern W88 in accordance with the autosomal dominant inheritance.Directly sequencing of 8 affected members revealed a G tO A transition at codon 124 (CGC to CAC),producing R124H mutation of TGFBI gene.Two synonymous single nucleotide polymorphism(SNP)of TGFBI gene occurred in the family.including a C to T transition at eodon 472(CTC to CTT)in 8 members,and a T to C transition at codon 540(TTT>TTC)in 9 members,which wag unrelated with disease. Conclusion R124H mutation of the TGFBI gene is found in this Chinese family with Avellino corneal dystrophy.

0.05)],which less than in control group [(4.614?1.683) IU/cm,(0.119?0.068) IU/cm,(564.2?53.8) ?m Pa

AIM To investigate the therapeutic effect of ethyl acetate extract from Pongamia pinnata roots (PREA) on ethanol-induced gastric lesions. METHODS The experimental gastric mucosal injuries were prepared by ig ethanol to rats, and the protective effect of PREA was evaluated by calculating lesion index, observing pathological changes, and measuring the contents of nitric oxide (NO) and malondialdehyde (MDA), and the activity of superoxide dismutase (SOD) from gastric mucosal tissue. In addition, gastric secretary and gastric wall adherent mucus were studied with the pylorus-ligation rat model. RESULTSCompared with the model control group, PREA (50, 150 and 450 mg·kg-1, ig) dose-dependently prevented the gastric mucosal damages induced by ethanol, its inhibition rates were 28.7%, 57.7% and 78.7 %, respectively. The pathomorphology lesions of mucosal tissue were obviously ameliorated. PREA obviously antagonized the ethanol-induced elevation of MDA content, and reduction of NO level and SOD activity of gastric mucosa. PREA significantly reduced gastric juice volume, free acidity, total acidity and total acid output, but didn′t affect the pepsin activity. Moreover, PREA obviously increased adherent mucus quantity of stomach wall, as well as free mucus quantity dissolved in gastric juice of pylorus-ligation rat. CONCLUSIONPREA has protective effect on ethanol-induced gastric mucosal injuries, which suggests that PREA may be used for protection or treatment of human ethanolinduced gastric lesions.

The study aims to establish a method for simultaneous determination of repaglinide and pravastatin sodium in rat plasma by LC-MS/MS and to study its pharmacokinetic interactions. Eighteen male SD rats were divided into repaglinide group, pravastatin sodium group and co-administration group. Blood samples were collected at different times after oral administration. Repaglinide and pravastatin sodium in rat plasma were separated by Agilent HC-C18 with the mobile phase consisting of methanol-0.1% formic acid (80 : 20). Detection and quantification were performed by using ESI-MS. The detector was operated in selected Reaction-monitoring mode at m/z 453.3-->230.1 for repaglinide, m/z 447.2-->327.4 for pravastatin sodium and m/z 285.1-->192.9 for diazepam as the internal standard. The calibration curve obtained was linear (R2>0.99) over the concentration range of 9.77-10,000 ng.mL-1 for repaglinide and 4.88-625 ng.mL-1 for pravastatin sodium. Compared with the single administration group, Cmax and AUC0-6h of repaglinide increased significantly (P<0.05) and tmax of pravastatin sodium prolonged (P<0.05) in co-administration group. The method is found to be simple, sensitive and accurate for determining the concentration of repaglinide and pravastatin sodium in rat plasma. There exists pharmacokinetic interactions in the co-administration of repaglinide and pravastatin sodium.