OBJECTIVETo investigate the effects of RCCS on cell seeding onto 3-D scaffold and cell-scaffold composite culture in vitro.

METHODSRabbit skin fibroblasts of passage 2 were seeded at 2 x 10(6) cell per cm(3) onto/into polyglycolic acid (PGA) foams by static seeding (dropping a cell suspension onto foams) or dynamic seeding (rotating PGA foams and a cell suspension in RCCS). Attachment of cells in foams was observed by cell-counting after trypsin digestion. The effects of culture condition were next studied by culturing cell-PGA complexes in RCCS versus static culture condition. Distribution and proliferation of cells in foams were investigated with MTT, stereomicroscope and scan electron microscope.

RESULTSNumbers of cells adhering to polymers increased gradually during an initial period of 24 hours. Eight, 12 and 24 hours after seeding, the rates of adhering cells were significantly higher in the dynamic seeding group than in the static seeding group (46.70% + 2.16% vs. 31.50% +/- 3.54%; 56.36% +/- 3.18% vs. 34.28% +/- 3.16%; 66.32% +/- 4.60% vs. 37.38% +/- 4.66%; P < 0.01). The dynamic culture method as compared to the static method resulted in new tissues with a higher cellularity and more uniform cell distribution during a 3 period of weeks.

CONCLUSIONSRCCS has advantages of promoting cell attachment, uniform migration and proliferation in polymer scaffolds and can be used for construction of 3-D cell-polymer tissues in vitro.

Animals ; Cell Adhesion ; Cell Culture Techniques ; methods ; Cell Division ; Cell Movement ; Fibroblasts ; cytology ; Polyglycolic Acid ; pharmacology ; Rabbits ; Time Factors

To obtain high-yield avilamycin-producing strains,low energy N~+ ion implantation technology and screening of streptomycin-re- sistant mutants are used in the study on breeding mutation.The results show that,“saddle”region,which range is from 3?10~(15) to 5?10~(15) ions/cm~2,has got better induced mutation action.It also means that the strain's resistant mutation and yield mutation closely correlate to each other,and the method of streptomycin resistant screening is feasible.We have isolated a high-yield strain SVT-45 which the productivi- ty is 195% higher than the original strain's in the rotation-flask experiments.These results showed that the ion implantation was an effective method for microbe mutagensis.

OBJECTIVETo investigate the ability of human dendritic cells (DCs) inducing specific T lymphocyte response and inhibit the expression of HBeAg and HBsAg in 2.2.15 cell culture supernatant.

METHODSDCs were prepared from peripheral blood mononuclear cells induced with granulocyte macrophage colony-stimulating factor(GM-CSF) and interleukin 4. DCs was impulsed with pure HBsAg before DCs maturation and cocultured with self-blood T lymphocyte, while DCs without pure HBsAg stimulated group, T lymphocyte group and only T lymphocyte group were prepared as control group. The culture supernatant of 2.2.15 cell with stimulated T lymphocytes was collected on day 1, day 3, day 5 and day 7, respectively. The expressed levels of HBeAg and HBsAg were detected by ELISA method.

RESULTSDCs after antigen stimulation had a strong ability to present antigen and induce immune activation, DCs after loading with antigen in normal control and chronic hepatitis patients group had stronger stimulative ability for T lymphocytes proliferation than that of DCs without loading with antigen and only T lymphocyte group(P less than 0.01). The stimulating ability of DCs had a positive correlation to the dosage of loaded antigen; CTLs produced as a result of DCs stimulation had a specific inhibitive effect on the expression of HBeAg in 2.2.15 cell supernatant,but not on the expression of HbsAg.

CONCLUSIONHuman dendritic cells stimulated with HBsAg in vitro can efficiently present antigen and stimulate the production of specific CTLs to HBV, which can efficiently inhibit the expression of HBeAg in 2.2.15 cell supernatant- DC vaccine may become an antiviral therapy strategy for chronic hepatitis B virus infected patients in future.

Adolescent ; Adult ; Antigen Presentation ; Cells, Cultured ; Child ; Dendritic Cells ; drug effects ; immunology ; Female ; Hepatitis B Surface Antigens ; pharmacology ; Hepatitis B, Chronic ; immunology ; Humans ; Lymphocyte Activation ; Male ; Middle Aged

Aged ; Biopsy, Needle ; methods ; Coal Mining ; Humans ; Lung ; pathology ; Lung Neoplasms ; complications ; diagnosis ; Middle Aged ; Pneumoconiosis ; complications

Animals ; Apoptosis ; Concanavalin A ; toxicity ; Liver ; drug effects ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; metabolism ; Nitric Oxide ; biosynthesis ; Tumor Necrosis Factor-alpha ; biosynthesis

OBJECTIVETo identify a novel HLA-A allele in a Chinese Han individual.

METHODSOne mismatch was observed in HLA-A locus in HLA typing for CMDP donors using bi-allelic SBT kit. A confirmatory test for novel HLA allele was performed with mono-allelic SBT kit.

RESULTSThe DNA sequence was confirmed to be a novel HLA-A allele. There was 1 nucleotide differed from the closest matching HLA-A*11:01:01 at position 393(G→A), which resulting a change from GGG to GGA at codon 107, led to a silent mutation, conserving the amino acid Gly.

CONCLUSIONA novel HLA-A allele was confirmed and officially named HLA-A*11:01:37 (Genbank accession number, JN209962) by the WHO Nomenclature Committee for Factors of the HLA System in January 2012.

Alleles ; Base Sequence ; Blood Donors ; HLA-A11 Antigen ; genetics ; Humans ; Sequence Analysis, DNA

Objective To study the screening of trace amount mutation of BRAF V 600E gene for avoiding the appearance of ineffective treatment in cancer patients .Methods The internal competitive amplification fragments were used to improve the inhibition of wild-type blocking (WTB) probe on wile-type BRAF V600E gene to increase the detection efficiency of BRAF V600E genotype of trace amount mutation occurrence .Re-sults When the template DNA concentration was 50 -200 ng/μL ,the constructed trace amount gene muta-tion real time fluorescence quantitative detection method could completely block the amplification of the wild-type BRAF V600E gene .The sensitivity of this assay reached as high as 0 .1％ ,which was in line with the sen-sitivity requirement for the gene trace amount mutation detection technique .In the colorectal biopsy tissues from 50 cases of suspected colorectal cancer ,8 cases (16 .0％ ) of BRAF V600E gene trace amount mutation were detected by using this constructed method ,which had higher detection rate .Conclusion The constructed gene trace amount mutation detection method can make the rapid ,simple and low cost quantitative analysis for BRAF V600E gene trace amount mutation in clinical samples .

Objective To analyze and summarize the clinical manifestations,diagnosis and treatment of pure spinal epidural cavernous hemangiomas.Methods We presented a case series of 6 patients with pure spinal epidural cavernous hemangiomas in our hospital from April 2012 to November 2016 with previous articles discussing their clinical presentation,radiological characteristics,surgical technique,pathological features,and functional outcome.Results All patients were diagnosed as pure spinal epidural cavernous hemangiomas by pathology.The cavernous hemangiomas were totally cut and patients recovered well after operations.All patients gradually improved neurologically and achieved a good outcome with no recurrence during the follow-up for 13 to 68 months.Conclusion Pure spinal epidural cavernous hemangioma is rare.It is easily confused with other spinal diseases.MRI is an important imaging examination method.Pathology is the method of diagnosis.Surgical resection is the main method of treatment,and the prognosis is good.

This study examined the expressions of miR-22 and miR-135a in rats with acute edematous pancreatitis (AEP) and their target genes in order to shed light on the involvement of miR-22 and miR-135a in the pathogenesis of acute pancreatitis (AP). The in vivo model of AEP was established by introperitoneal injection of L-arginine (150 mg/kg) in rats. The miRNA microarray analysis was used to detect the differential expression of miRNAs in pancreatic tissue in AEP and normal rats. The in vitro AEP model was established by inducing the rat pancreatic acinar cell line (AR42J) with 50 ng/mL recombinant rat TNF-α. Real-time quantitative RT-PCR was employed to detect the expression of miR-22 and miR-135a in AR42J cells. Lentiviruses carrying the miRNA mimic and anti-miRNA oligonucleotide (AMO) of miR-22 and miR-135a were transfected into the AR42J cells. The AR42J cells transfected with vehicle served as control. Western blotting was used to measure the expression of activated caspase3 and flow cytometry analysis to detect the apoptosis of AR42J cells. Targets of miR-22 and miR-135a were predicted by using TargetScan, miRanda, and TarBase. Luciferase reporter assay and quantitative real-time RT-PCR were performed to confirm whether ErbB3 and Ptk2 were the target gene of miR-22 and miR-135a, respectively. The results showed that the expression levels of miR-22 and miR-135a were obviously increased in AEP group compared with the control group in in-vivo and in-vitro models. The expression levels of miR-22 and miR-135a were elevated conspicuously and the expression levels of their target genes were reduced significantly in AR42J cells transfected with lentiviruses carrying the miRNA mimic. The apoptosis rate was much higher in the TNF-α-induced cells than in non-treated cells. The AR42J cells transfected with miRNA AMOs expressed lower level of miR-22 and miR-135a and had lower apoptosis rate, but the expression levels of ErbB3 and Ptk2 were increased obviously. It was concluded that the expression levels of miR-22 and miR-135a were elevated in AEP. Up-regulating the expression of miR-22 and miR-135a may promote the apoptosis of pancreatic acinar cells by repressing ErbB3 and Ptk2 expression in AEP.

OBJECTIVETo identify a novel human leukocyte antigen (HLA) allele in Chinese and investigate its inheritance in the family.

METHODSExceptional reaction pattern was detected in HLA-B locus in HLA typing using Luminex DNA polymerase chain reaction with sequence specific oligonucleotide probe hybridization (PCR-SSOP) assay. A confirmatory test for the novel HLA allele was performed by DNA sequencing based typing of the proband's family.

RESULTSThe DNA sequence was confirmed to be a novel HLA B allele. There were 7 nucleotides which differed from the closest matching HLA B*40:06:01 at positions 302(G to A), 309(G to C), 311(A to C), 313(C to G), 314(T to C), 317(G to T), and 319(G to C) in exon 2, which resulted in 5 amino acid changes at codon 101 (Ser to Asn), 104 (Asn to Thr), 105 (Leu to Ala), 106 (Arg to Leu), and 107 (Gly to Arg), respectively. Family investigation indicated that the novel allele was transmitted from the proband's father.

CONCLUSIONA novel HLA B allele was identified and officially named as HLA-B*40:96 (GenBank accession No. FJ374890) by the WHO Nomenclature Committee for Factors of the HLA System.

Alleles ; Base Sequence ; Female ; HLA-B Antigens ; genetics ; Haplotypes ; Histocompatibility Testing ; Humans ; Molecular Sequence Data ; Pedigree ; Sequence Alignment