Animals ; Area Postrema ; physiology ; Blood Pressure ; Electric Stimulation ; Electrocardiography ; Male ; Neurotransmitter Agents ; Peroneal Nerve ; physiology ; Rats ; Rats, Sprague-Dawley

Method of isolation and purification of recombinant hepcidin was described, and the bioactivity of the protein was assayed in this paper.The oxidation of his-hepcidin was carried out in the cysteine-cystine system, and the multimers were removed through gel filtration under denaturation condition.Then the protein was refolded by continuous dilution and digested by enterokinase.The total yield of his-hepcidin before enterokinase cleavage is 50%, and the purity is above 95%.Through agar diffusion assay, the recombinant hepcidin displayed obvious antibacterial activity against B.subtilis.The LC-ESI-MS analysis of recombinant hepcidin showed that the measured molecular weight accorded with the calculated molecular weight, and the CD spectrum indicated that the secondary structure of recombinant hepcidin is similar with native hepcidin.

OBJECTIVETo establish a method to determine the content of Curcumol in Rhizoma Curcumae.

METHODThe samples were determined by GC on a HP-5 column (0.32 mm x 30 m, 0.25 micron), Inlet temperature 200 degrees C, FID 250 degrees C, flow 1.0 mL x min(-1), splitless. Temperature programming started at 60 degrees C, holding for 4 min, then increased to 210 degrees C at a rate of 3 degrees C x min(-1).

RESULTThe calibration curve of curcumol is linear over the range of 40.0-2,000 microg x mL(-1), r = 0.9997. The high and low additive recovery were 95.01% (RSD 2.52%), 96.46% (RSD 2.86%).

CONCLUSIONThis method was accurate and reliable with a good reproducibility, and the procedure of samples pretreatment is simple.

Chromatography, Gas ; Curcuma ; chemistry ; Plants, Medicinal ; chemistry ; Reproducibility of Results ; Rhizome ; chemistry ; Sesquiterpenes ; analysis

Objective:The recombinant human retinal pigment epithelium-derived factor(PEDF)protein to be obtained and the angiogenesis of the rPEDF to be identified.Methods: PEDF gene gene was amplified by PCR and cloned into pET32a,rPEDF protein was expressed in E.coli BL21 and confirmed by SDS-PAGE and Western blot.The rPEDF was purified by Ni-NTA on denature condition.The concentration of the rPEDF was determined by Bradford method.The angiogenesis of the rPEDF was determined by chick chorioallantoic membrane(CAM) method.Results: The expression plasmid pET32a-PEDF was constructed successfully.The rPEDF was expressed with stable efficiency in E.coli BL21.The results of the CAM experiment showed that the rPEDF had notable angiogenesis effect in the concentration 0.4、0.04 ng/ml,but had no effect in 4 ng/ml.Conclusion:The PEDF gene was cloned and expressed efficiently,the angiogenesis of the rPEDF to be identified and the activity was worked in certain range.The results can facilitate studying its function and spreading its application.

OBJECTIVETo establish an animal model of acute blunt scrotal trauma (BST) and evaluate the types of lesion by conventional ultrasonography (CUS) and contrast-enhanced ultrasonography (CEUS).

METHODSWe made acute BST models in 21 healthy male New Zealand rabbits by striking 3 - 12 times the unilateral testes randomly selected with a 0. 5 kg iron ball falling freely from a 30 cm height. Then we evaluated the lesion types in the models by CUS and CEUS and verified our evaluation against pathological results.

RESULTSAcute BST models were successfully established in all the 21 animals, including contusion in 10, hematoma in 6, and rupture in 5, all confirmed by pathology. CUS clearly manifested the morphology, internal echoes, and blood flow of the testes, but had a low rate of accurate diagnosis in testicular contusion for over 6 hours as well as in complex lesions. CEUS revealed an earlier perfusion of the contrast agent and shorter arriving time (AT) and time to peak intensity ( TP) in testicular contusion than in the control testes (P <0.05) , but showed no statistically significant difference between the two groups in the half time of descending peak intensity (P>0.05). For testicular hematoma, contrast agent clearly presented its outline and a delayed low enhancement in the surrounding tissue, with significant differences from the control in AT and TTP. In severe testis rupture, occasional outflow but no perfusion of contrast agent was observed.

CONCLUSIONBST models can be established in rabbits by repeated strikes of the unilateral testes lesion of contrast agent was observed. with a freely falling iron ball. Simple contusion injury can be induced by less than 6 strikes, while complex injuries can be inflicted by more than 10. Combined application of CUS and CEUS can improve the accuracy of diagnosis of different types of lesion.

Acute Disease ; Animals ; Disease Models, Animal ; Male ; Rabbits ; Scrotum ; diagnostic imaging ; injuries ; Ultrasonography ; Wounds, Nonpenetrating ; diagnostic imaging

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

OBJECTIVETo observe the fetus protection effects of Zhixue Baotai Decoction (ZBD) on women of early threatened abortion with dark area surrounding pregnancy sac.

METHODSThe 105 patients with early threatened abortion, in whom vaginal bleeding was shown already, were randomly assigned to the treatment group and the control group, who were treated respectively with ZBD and progesterone to protect fetus. The efficacy of treatment was evaluated by dynamic monitoring of serum hormone and B-ultrasonic examination.

RESULTSAmong the 54 cases in the treatment group the fetus was protected successfully, showing a fetus protecting rate of 81.5%; while among the 51 cases in the control group, the protection was effective in 22 cases (43.1%), the success rate in the former was better (P<0.01). The dark area was absorbed in 16 out of 19 cases (84.2%) in the treatment group, while in the control group absorption occurred only in 6 out of 17 (35.3%).

CONCLUSIONThe effect of ZBD is superior to that of progesterone in treating women of early threatened abortion with dark area surrounding pregnancy sac.

Abortion, Threatened ; diagnostic imaging ; drug therapy ; Adult ; Drugs, Chinese Herbal ; therapeutic use ; Extraembryonic Membranes ; diagnostic imaging ; Female ; Humans ; Phytotherapy ; Pregnancy ; Pregnancy Trimester, First ; Treatment Outcome ; Ultrasonography ; Young Adult

OBJECTIVETo investigate the origin of childhood B-cell acute lymphoblastic leukemia (B-ALL) and its epitopes recognized by cytotoxic T lymphocytes (CTL) in immunoglobulin heavy chain variable region (IgHV).

METHODSSeven IgHV gene families were respectively amplified by PCR and directly sequenced in 108 childhood ALL. The amino acid sequences were deducted from sequenced nucleotides. Bioinformatics was applied to analyses of recombination patterns, somatic mutations and germline gene segments usage, and to prediction of epitopes recognized by CTL.

RESULTSIgHV gene rearrangements were identified in 66% of the cases, including 37 (52.1%) monoallelic rearrangements, 26 (36.6%) biallelic rearrangements and 8 (11.3%) oligoclonal rearrangements. Among the obtained 40 B-ALL IgHV gene sequences, 8 (20.0%) were in frame rearrangements without stop codons. V(H3) (11/40), V(H4) (11/40) and V(H1) (8/40) amounted to 75% rearranged V(H) families. V(H)(4-59) and V(H)(4-34) were the most frequently rearranged V(H)(4) family gene segments. Usage of D2 and D3 families was most prominent (35.9% and 28.2%, respectively). Increased frequency of D7-27 (15.4%) was found as compared to that of normal peripheral B lymphocytes (P = 0.02). J(H)(6) was found in 47.5% rearrangements followed by J(H)(4) (27.5%). 8/40 (20.0%) DJ(H) junctions lacked N nucleotides, which was higher than that reported for normal peripheral B lymphocytes (P = 0.02). 17.5% B-ALL IgHV contained scattered replacement mutations with replacement (R) to silent (S) substitution ratio (R/S ratio)

CONCLUSIONB-ALL originated from progenitor or precursor B lymphocytes. B-ALL IgHV genes are of germline characteristics. Potential T cell epitopes were derived from framework regions 1 and 3 of immunoglobulin heavy chain in B-ALL.

Adolescent ; Child ; Child, Preschool ; Female ; Gene Rearrangement ; Genes, Immunoglobulin ; HLA-A Antigens ; genetics ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Infant ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology

OBJECTIVETo induce anti-B-cell acute lymphoblastic leukemia (B-ALL) cytotoxic T lymphocyte response against immunoglobulin heavy chain frame-derived nonapeptide.

METHODSThe peptide, QLVQSGAEV, containing IgHV1 frame region 3rd-11th amino acids (IgHV1(3-11)), was synthesized. IgHV1(3-11)-T2 binding tests were performed. HLA-A * 0201-positive normal peripheral blood mononuclear cells (PBMNC) were stimulated by IgHV1(3-11)-loaded antigen presenting cells three times at weekly intervals. HLA-A * 0201/IgHV1(3-11) tetramers were used to detect the proliferation of IgHV1(3-11)-specific CD8(+) T cells in the culture. Seven IgHV gene families of B-ALL patients were respectively amplified by PCR and the PCR products were sequenced to select IgHV1 and IgHV3 family monoallelic functional rearrangements. Among them, HLA-A * 0201 positive individuals were subsequently identified. Cytotoxicity of IgHV1(3-11)-specific CD8(+) T cells against HLA-A * 0201-positive IgHV1/IgHV3 family B-ALL cells was measured by MTT assay.

RESULTSThe synthesized IgHV1(3-11) up-regulated HLA-A * 0201 expression on T2 cell surface by 1.63-folds. The percentage of IgHV1(3-11)-specific CD8(+) T cells in HLA-A * 0201-positive normal PBMNC was increased from 1.64% after second stimulation to 82.57% after third stimulation. At effector: target ratio of 20:1, the killing rate of IgHV1(3-11)-specific CD8(+) T cells against IgHV1 family B-ALL cells was 18.24%, being 1.8-folds as that against IgHV3 family B-ALL cells (P = 0.01).

CONCLUSIONCytolytic T lymphocytes generated in vitro against immunoglobulin heavy chain frame-derived nonapeptides can kill B-ALL cells expressing these peptides.

Cells, Cultured ; Humans ; Immunoglobulin Heavy Chains ; immunology ; pharmacology ; Leukemia, B-Cell ; immunology ; pathology ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology

Bone marrow transplantation (BMT) is an effective therapy for a variety of hematologic and immunodeficient diseases. Two possible situations of chimerism can be encountered when assessing the hematological status in recipient post-allo-BMT: 1. complete chimerism, where the marrow and circulating blood cells all are donor origin; 2. mixed chimerism, the cell components in recipient are both donor and recipient origins. Formation of the different kinds of chimerism are influenced by some factors, included the amount of infused hematopoietic stem cells, the ratio of T cells from donor and recipient, conditioning regimen and patient's condition. The clinical significance was different for the forms and development of chimerism. There are several detecting methods for chimerism, such as microsatellite assay, karyotyping of chromosome, conversion of blood group, red blood cell antigen, restriction fragment length polymorphism, variable number of tandem repeat, and PCR. Above-mentioned aspects of chimerism research were reviewed.
Bone Marrow Transplantation ; Hematopoietic Stem Cells ; cytology ; Humans ; Research ; trends ; Research Design ; T-Lymphocytes ; cytology ; Transplantation Chimera ; blood ; Transplantation, Homologous