Objective:To analyze whether the OAZI-1 (ornithine decarboxylase antizyme inhibitor-1) protein complex isolated from tumor cells could induce specific antitumor effects in the experiment mice .Methods:OAZI-1 protein complexes were isolated from B16-F1 melanoma cells by immune magnetic beads coated with OAZI-1 antibody and used as the vaccine to immune the C 57BL/6 mice.After immunization,the mice were inoculated subcutaneously with live B 16-F1 cells and then tumor formation and growth were ob-served.ELISA was used to determine the level of cytokine IFN-γin the serum of immunized mice.Lactate dehydrogenase assay (LDH) was performed to evaluate killing effect of spleen lymphocytes on B 16-F1 cells.The mice immunized by purified OAZI-1 from prokaryotic expression and PBS were used as controls in the animal experiment .Results: Compared with the control mice ,the spleen lymphocytes ( effector cells ) from the mice inoculated with OAZI-1 protein complexes had stronger killing ability on B 16-F1 cells (target cells).At three different effector:target ratio (10:1,50:1,100:1),the killing ability of these spleen lymphocytes were 46.2%, 59.5%and 92.5% respectively,which was significantly higher than the spleen lymphocytes from the mice inoculated with purified AZIN-1 protein (36.1%,26.8% and 45.9%) or inoculated with PBS (24.6%,24.0% and 27.2%).In addition,the content of serum anti-tumor cytokine IFN-γwas also significantly higher in the mice inoculated with OAZI-1 protein complexes (538.3 pg/ml) than the mice inoculated with purified AZIN-1 ( 256.2 pg/ml ) or with PBS ( 131.0 pg/ml ) .When B16-F1 live cells were subcutaneously inoculated into the immunized mice described above ,the tumor formation rate was only 40%in the mice immunized with OAZI-1 protein complex ,but 100%in the mice immunized with PBS or purified OAZI-1.The growth of inoculated tumors in the mice immunized with OAZI-1 protein complex was also much slower than the control mice .Conclusion:The results in this study suggest that the OAZI-1 protein complex isolated from B 16-F1 tumor cells could contain some tumor antigens .When used as tumor vaccine to inoculate mice ,this complex can induce anti-tumor immune killing activity in experimental animals .

ObjectiveTo explore the effects of ischemic preconditioning on the level of serum testosterone (T) and apoptosis of spermatogenic cells in rabbits with testicular ischemia-reperfusion injury induced by testicular torsion.

METHODSA total of 15 New Zealand male rabbits were randomly divided into groups A (control), B (ischemia-reperfusion), and C (ischemic preconditioning). The animals of group A were subjected to exposure of the right spermatic cord without ischemia, those of group B received 60-minute non-invasive occlusion of the right spermatic cord followed by 3 days of reperfusion, and those of group C underwent 5-minute occlusion plus 5-minute reperfusion of the right spermatic cord followed by the same procedure as that for group B. Then the rabbits were narcotized with 3% barbital sodium, the whole blood collected for examination of the serum T content and the testis tissues obtained from both the ischemic and healthy sides for HE and TUNEL staining.

RESULTSAfter operation, the body weight was significantly increased as compared with the baseline in groups A (［2.65±0.07］ vs ［2.45±0.07］ kg, P<0.05) and C (［3.03±0.11］ vs ［2.92±0.07］ kg, P<0.05), but not in group B (［3.05±0.07］ vs ［3.05±0.07］ kg, P>0.05). The serum T level showed no statistically significant difference in group A before and after operation (［139.59±9.39］ vs ［140.19±9.47］ ng/L, P>0.05), but was remarkably lower after operation than the baseline in groups B ［148.06±3.31］ vs ［74.12±4.00］ ng/L, P<0.01) and C (［133.75±6.48］ vs［94.76±3.13］ ng/L, P<0.01) as well as than the postoperative index in group A (P<0.01). In comparison with group A and the healthy side of group B, the testis tissue of the ischemic side in group B exhibited structural damage of most of the seminiferous tubules with disappearance of spermatogenic cell structures, apoptosis of spermatogenic cells, and exudation of light-eosin edema fluid in the mesenchyme and lumen, with a markedly increased apoptosis index (P<0.01) and a significantly decreased Johsen's score (P<0.01). Compared with ischemic side of group B, The testis tissue of the ischemic side in group C was restored to normal as compared with that in group B, with a dramatically decreased apoptosis index (P<0.01) and a remarkably increased Johnsen's score (P<0.01).

CONCLUSIONSIschemic preconditioning can raise the decreased serum T level and reduce the apoptosis of spermatogenic cells in rabbits with testicular ischemia-reperfusion injury, which could be applied as a potential option for the clinical treatment of testicular ischemia-reperfusion injury.

Animals ; Apoptosis ; Germ Cells ; In Situ Nick-End Labeling ; Ischemia ; physiopathology ; Ischemic Preconditioning ; Male ; Rabbits ; Random Allocation ; Reperfusion Injury ; blood ; physiopathology ; Spermatic Cord Torsion ; Testis ; physiopathology ; Testosterone ; blood

Objective To evaluate the efifcacy and safety of hemostatics of injected gelatin matrix (HIGM) under the guidance of contrast-enhanced ultrasound (CEUS) for treating splenic trauma in canine model. Methods A total of 24 commercial hybrid dogs underwent celiotomy with creation of uniformly blunt splenic trauma lesion of 4.0 cm×4.0 cm×2.5 cm (length, width and depth, respectively) by hemostatic clamp. Subjects were prospectively randomized into two groups. The treatment group was treated with HIGM under the guidance of CEUS and the positive control group received thrombin solution. Conventional ultrasound and CEUS were performed to record the ascites and the splenic lesion areas at 1st, 3rd, 7th, 14th and 21st day. The ifne needle biopsy and splenectomy were performed for histopathologic examination. The weight, free intraperitoneal lfuid and injury site were compared with t test between HIGM and postive group. Results All animals in two groups survived. All dogs stopped hemorrhage after injection of HIGM under CEUS guidance. The area of injury site was (12.91±0.89) cm2, (4.45±0.75) cm2 and (1.38±0.23) cm2 at 1st, 3rd and 7th day and splenic lesions were not found at 14th and 21st day in all dogs (n=12) of HIGM group. The splenic lesion was (16.74±0.91) cm2, (11.26±0.99) cm2, (8.02±0.82) cm2 and (1.58±0.36) cm2 in the postive group at 1st, 3rd, 7th and 14th day and splenic lesions were not found at 21st day in all dogs (n=12). At 7th and 14th day post-injection, lesion areas were statistically significant between two groups (t=27.162, P=0.008;t=15.129, P=0.001). Free intraperitoneal lfuid was (0.91±0.05) cm at 1st day detected by conventional ultrasound and free intraperitoneal fluid was not found at 3rd, 7th, 14th and 21st day in all dogs (n=12) of HIGM group. The free intraperitoneal fluid in thepositive group was (1.96±0.17) cm, (1.30±0.11) cm and (0.81±0.12) cm at 1st, 3rd and 7th day and free intraperitoneal lfuid was not found at 14th and 21st day in all dogs (n=12). At 1st, 3rd and 7th day post-injection, free intraperatitoneal lfuid was statistically significant between two groups (t=20.934, P=0.003; t=41.310, P=0.000; t=22.520, P=0.000). Histopathological examination showed that there was no foreign body and foreign body granuloma and the structure of red pulp was recovered at 7th, 14th and 21st day. Gross anatomy showed that the splenic injury site was recovered completely without complications. Conclusion This study explored the value of HIGM for splenic trauma and provided a preliminary experimental evidence for clinical treatment.

OBJECTIVETo investigate the mRNA expression profiles of megakaryocytes (MKs) from human cord blood CD34+ cells ex vivo expanded using Solexa technique.

METHODSCD34+ Cells were isolated using density gradient centrifugation and magnetic activated cell sorting. Cultures were stimulated with recombinant human thrombopoietin (100 ng/ml). After 12 days, the MKs fraction was separated from the non-MKs fraction using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The mRNA expression of MKs and non-MKs was detected by Solexa sequencing.

RESULTSWe obtained 3 773 147 and 3 533 805 Tags from MKs and non-MKs, respectively. The amounts of unambiguous tags were 3 291 132 and 2 967 947 and those of distinct tags were 197 769 and 245 318. The expression of 1161 genes was up-regulated and that of 902 genes down-regulated. The expression of 2717 tags was up-regulated and that of 1519 tags down-regulated.

CONCLUSIONSMKs and non-MKs have remarkably different mRNA expression profiles. The differential gene-encoded products may be involved in cellular development, adhesion, apoptosis metabolism, intra- and intercellular signal transduction, and immune response. Further studies on this topic may clarify the expression mechanism, signal transduction, and regulation mechanisms.

Antigens, CD34 ; Cells, Cultured ; Fetal Blood ; cytology ; Humans ; Megakaryocytes ; cytology ; metabolism ; RNA, Messenger ; genetics ; Transcriptome

BACKGROUNDThe non-operation treatment of intra-abdominal trauma guided contrast enhanced ultrasound (CEUS) is one of the hottest research topic. Gelatin/thrombin/calcium (GTC) was developed as a novel haemostatic agent for non-operable intra-abdominal trauma. We hypothesized that GTC can achieve haemostasis (without the use of pressure) within a short time in a large wound model by percutaneous injection under CEUS guidance.

METHODSForty Wister rats received large liver injuries by haemostatic clamp and were randomly divided into four groups, according to the haemostatic agent used. These included normal saline (NS) group A, lyophilising thrombin powder (LTP) group B, GTC group C, and absorbable α-cyanoacrylate (ACNA) group D. Each injury site was treated with one of the above materials and total bleeding time was recorded. All liver wounds were evaluated using CEUS at three periods: pre-injury, injury and post-treatment. The liver wounds were also evaluated by histology 3, 6, and 9 days after injury and the extents of abdominal adhesions were recorded.

RESULTSThe sensitivity of CEUS (100%) in detecting blunt traumatic liver lesions was significantly higher than conventional ultrasound (42.5%). Bleeding times at the injury site in the GTC group C ((129.3 ± 14.0) seconds) and ACNA group D ((5.2 ± 1.0) seconds) were significantly shorter than those in the NS group A ((369.5 ± 48.8) seconds, P < 0.01) and LTP group B ((324.7 ± 52.22) seconds, P < 0.01). The LTP group B showed no significant difference compared with the NS group A. Gross examination of liver tissue revealed that there were fewer intra-abdominal adhesions in the GTC group C (10%) than in the ACNA group D (100%). Histopathologic examination showed that GTC was completely absorbed after nine days.

CONCLUSIONSGTC, delivered by percutaneous injection under CEUS, may achieve haemostasis (without the use of pressure) within a short time in a large wound model. GTC is absorbable and may prevent intra-abdominal adhesions. Therefore, it may be the optimal choice for first aid treatment of large abdominal wounds in the setting of blunt trauma.

Animals ; Calcium ; administration & dosage ; therapeutic use ; Gelatin ; administration & dosage ; therapeutic use ; Hemorrhage ; diagnostic imaging ; drug therapy ; Hemostatics ; administration & dosage ; therapeutic use ; Injections ; Liver ; diagnostic imaging ; injuries ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Thrombin ; administration & dosage ; therapeutic use ; Ultrasonography

OBJECTIVETo study the effect of the traditional Chinese herbal drug Ciwujia in inducing the differentiation of marrow stromal cells (MSCs) into neuron-like cells.

METHODSRat MSCs isolated from the whole bone marrow were amplified by adherent culture in vitro and induced to differentiate into neuron-like cells using serum-free DMEM/F12 containing Ciwujia. The protein and mRNA expressions of nestin, beta-Tubulin III and glial fibrillary acidic protein (GFAP) in the differentiated cells were detected by indirect immunofluorescence method, Western blotting and RT-PCR.

RESULTSThe third-passage MSCs showed positive expression rates for CD44 and CD54 beyond 90% with decreased CD14 expression rate to 2.37%. Induction by Ciwujia of the MSCs resulted in cell body shrinkage and protrusion of the cell processes resembling those of neurons. The differentiated cells were positive for nestin and beta-Tubulin III expression and negative for GFAP as shown by immunofluorescence assay, Western blotting and RT-PCR.

CONCLUSIONCiwujia can induce the differentiation of rat MSCs into neuron-like cells in vitro.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Eleutherococcus ; Female ; Glial Fibrillary Acidic Protein ; metabolism ; Intermediate Filament Proteins ; metabolism ; Male ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurons ; cytology ; Plant Extracts ; pharmacology ; Rats ; Rats, Wistar ; Stromal Cells ; cytology ; drug effects ; Tubulin ; metabolism

OBJECTIVETo study the role of contrast-enhanced ultrasound (CEUS) in the management of splenic trauma

METHODSForty patients with splenic trauma underwent CEUS examination and then different management were provided according to the examination results and the general status of patients: conservative treatment (group I, n = 19); CEUS-guided injective therapy (group II, n = 6); and surgery (group III, n = 15).

RESULTSEighteen patients were cured in group I and one patient experienced rehaemorrhagia. The haemostatic effect of CEUS-guided injective therapy was obvious in all six patients in group II. Among them, one patient experienced arteriovenous fistula, which was resolved after one week of injective therapy. Fifteen patients in Group III underwent surgery and were cured.

CONCLUSIONCEUS can provide reliable information for therapy mode selection in patients with splenic trauma and can be used to guide injective therapy.

Contrast Media ; Humans ; Spleen ; diagnostic imaging ; injuries ; Ultrasonography ; Wounds, Nonpenetrating ; diagnostic imaging ; therapy

The aim of study was to analyze natural killer immunoglobulin (Ig)-like receptor (KIR) gene content in HLA-identical sibling and to investigate the possibility of their KIR match. Samples were genotyped for HLA by Luminex method and polymerase chain reaction sequence based typing, the KIR gene was detected by polymerase chain reaction sequence-specific primers. The results showed that 17 KIR genes could be observed in the 27 pairs HLA-A, -B, -Cw and -DRB1 locus identical sibling samples. All individuals contained KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2.; 20 different KIR genotypes and 12 haplotypes have been found, the most common KIR genotypes was 2,2 with frequency 29.6% and KIR haplotype was 2 with frequency 53.0%. The A KIR haplotype was the most prevalent with frequency 67.2%; 12 pairs (44.4%) HLA identical sibling donor-recipients showed KIR match in genotype and haplotype, 13 pairs (48.1%) with one KIR haplotype mismatch and 2 pairs (7.4%) with two KIR haplotype mismatch; 1 pair was matched between donor KIR2DL1 and patient HLA-Cw (Lys80) ligand, 17 pairs were matched between KIR2DL2/KIR2DL3 and HLA-Cw (Asn80) ligand, 5 pairs were matched between KIR3DL1 and HLA-Bw4 ligand. It is suggested that the probability of KIR mismatch is high in HLA-identical sibling.
HLA Antigens ; genetics ; immunology ; Haplotypes ; Hematopoietic Stem Cell Transplantation ; methods ; Histocompatibility Testing ; Humans ; Multigene Family ; Receptors, KIR ; genetics ; Siblings

This study was aimed to investigate the molecular genetics basis of a novel allele HLA-A * 2459 in Chinese population. DNA was extracted from whole blood by PEL-FREEZ DNA extraction kit. The amplification of HLA-A exons 2 - 4 of the proband was preformed by allele specific primer PCR and the amplified product was sequenced bidirectionally with primers. The sequencing results showed HLA-A alleles of the proband as A * 1101 and the novel allele. The sequences of the novel allele have been submitted to GenBank (DQ313255, DQ313256, DQ313257). After Blast HLA analysis, the novel allele showed only one nucleotide differences with HLA-A * 24020101 at nucleotide position 527 T to C in exon 3. This results in an amino acid changes from Val to Ala at codon 152. In conclusion, this allele is a novel one and has been officially named HLA-A * 2459 by the WHO Nomenclature Committee.
Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Blood Donors ; China ; HLA-A Antigens ; genetics ; Humans ; Molecular Sequence Data ; Sequence Analysis, DNA

The study was purposed to investigate the molecular genetic basis for HLA novel allele HLA-B*5408N in Chinese population. DNA was extracted from whole blood by commercial DNA extraction kit, the HLA-B exons 2 - 4 of the proband was amplified by allele specific primers PCR and the amplified product was sequenced for exons 2, 3 and 4 bidirectionally. The sequencing results showed HLA-B alleles of the proband as B*1527 and the novel allele. The sequences of the novel allele have been submitted to Genbank (DQ295998, DQ295999, DQ296000). After blast analysis, the novel allele showed a single nucleotide mismatch with HLA-B*5401 in exon 3 at position 553 G-->T, which resulted in an amino acid changing from Glu to premature stop codon at position 161. No the HLA-B54 antigen specificity expression in the proband cells was found using HLA-AB serological Typing Trays. It is concluded that this allele is a novel null allele and has been officially named B*5408N by the WHO Nomenclature Committee.
Alleles ; China ; Exons ; genetics ; HLA-B Antigens ; genetics ; Humans ; Polymerase Chain Reaction ; Sequence Analysis, DNA