OBJECTIVETo compare the tissue engineered cartilage constructed with chondrocytes derived from auricular and articular cartilage.

METHODSChondrocytes were isolated from porcine auricular and articular cartilage, and BMSCs were obtained from bone marrow aspirate and cultured. Each kind of chondrocytes were resuspended alone or mixed with BMSCs at a ratio of 1:1, and seeded onto PGA/PLA scaffolds to construct tissue engineered cartilage (n = 4). The constructs were cultured for 8 weeks in vitro and then subcutaneously implanted into nude mice for 6 weeks. The differences between chondrocytes monoculture from articular and auricular cartilage or between each of them co-cultured with BMSCs were evaluated by gross view, measurement of thickness and wet weight, histological examinations including H&E, Safranin O, type II collagen, and Ponceau's & Victoria blue staining, and gene expression analysis of cartilage related genes.

RESULTSNo obvious differences were found histologically among the complexes constructed in vitro 8 weeks except for few elastic fibers secreted in the auricular chondrocytes + BMSCs co-culture group. Neo-cartilage is thicker in the groups of articular chondrocytes (38. 1% than the group of auricular chondrocytes, P < 0.05) and articular chondrocytes + BMSCs co-culture (19.3% than the group of auricular chondrocytes + BMSCs, P < 0.05). However, after 6 weeks in vivo the elastic fibers were found positive in the complexes constructed by auricular chondrocytes, and its staining was even stronger and more homogenous in the group of auricular chondrocytes + BMSCs co-culture. The tissues generated by articular chondrocytes alone and co-cultured with BMSCs both formed the characteristic features of three-layer structure of hyaline cartilage and ossified in vivo with significant up-regulation of COL10A1 and MMP-13. To summarize, auricular chondrocytes formed the elastic cartilage while articular chondrocytes formed the hyaline cartilage during the development of tissue engineered cartilage either by monoculture or the co-culture with BMSCs.

CONCLUSIONSThe chondrogenic response of chondrocytes from different cartilage origins demonstrates that an initial chondrocyte and cartilage type recapitulates the same in later tissue-engineered development.

Animals ; Bone Marrow Cells ; cytology ; Cartilage, Articular ; cytology ; Cells, Cultured ; Chondrocytes ; Coculture Techniques ; Ear Auricle ; cytology ; Mesenchymal Stromal Cells ; cytology ; Mice, Nude ; Swine ; Tissue Engineering ; methods ; Tissue Scaffolds

Objective To observe the effectiveness of introducing Diazepam into combined aesthesia of Sumianxin and ketamine hydrochloride .Method A total of 80 rabbits of both genders for operation were randomly divided into A , B and C groups .The A group was injected with Sumianxin intramuscularly ( 0.3 mL/kg by weight ) .The B group was injected with Sumianxin and ketamine hydrochloride intramuscularly ( 0.3 mL/kg by weight ) .The C group was injected with Diazepam intravenously ( 1.5 mL/kg by weight ) combined with Sumianxin and ketamine hydrochloride injected intramuscularly (0.3 mL/kg by weight).The aesthetic effects, induction time, anesthesia maintaining time, total anaesthetic dose and operation time were observed , recorded and compared .Result The induction time of the C group was significantly shorter than A and B groups (P<0.01).The initial anesthesia maintaining time of the C group was the longest among the three (P<0.01) with least total anaesthetic dose (P<0.01).The operation time of the C group was the least with best aesthetic effects (P<0.01).Conclusion Introducing Diazepam into combined aesthesia of Sumianxin and ketamine hydrochloride can improve the aesthetic effects .Therefore , this is an optional aesthetic method for time-consuming animal operation or sensitive surgical sites of rabbits .

OBJECTIVETo investigate the effects of carnosic acid (CA) on reversal of the multidrug resistance (MDR) of human leukemia cell line K562/A02 and its mechanism.

METHODSMTT assay was used to determine the sensitivity of K562/A02 cells to adriamycin (ADM) pre-and post-treated with CA. Flow cytometry (FCM) and laser scanning confocal microscopy (LSCM) were used to measure intracellular fluorescence intensity and concentration of ADM respectively. The expression level of mdr1 was detected by semi-quantitative RT-PCR. P-glycoprotein (P-gp) expression was detected by FCM and Western blot.

RESULTSCA decreased IC(50) of ADM in K562/A02 cells from 16.31 µg/mL to 1.35 µg/mL, being a 12.08-fold decrease. The intracellular ADM fluorescence intensity of K562/A02 was increased from 17.05 to 60.53 after treated with CA (P < 0.01). In living K562/A02 cells, after treated with CA, the diffuse distribution of intracellular ADM was recovered in both nuclear and cytoplasm, and the concentration of intracellular ADM increased from 4.93µg/mL to 15.43µg/mL. RT-PCR assay showed that CA inhibited the expressions of mdr1 mRNA in K562/A02 cells (P < 0.01). Mean fluorescence intensity of P-gp detected by FCM in CA-treated K562/A02 was decreased to 22.80 as compared with that in untreated K562/A02 cells (44.40, P < 0.05).

CONCLUSIONCA can reverse the MDR of K562/A02 cells in vitro. The mechanism may be associated with down-regulation of mdr1 and inhibition of P-gp function.

ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Humans ; K562 Cells

The purpose of this study was to evaluate the safety of cryopreserved and thawed peripheral blood stem cell (PBSC) fractionated return infusions in children. 35 children patients with malignant tumors (13 acute leukaemias, 15 neuroblastomas and 7 malignant lymphomas) received fractionated return infusions of cryopreserved stem cells after undergoing high-dose chemotherapy without or with total body irradiation. The toxicities of 70 return infusions were evaluated. All patients were mobilized by chemotherapy plus recombination human granulocyte colony-stimulating factor (rhG-CSF), and then PBSCs were collected by a separator CS-3000 plus or COBE spectra-4. The grafts were cryopreserved in 10% dimethyl sulfoxide (DMSD) and stored in liquid nitrogen. There were totally 70 PBSC transfusions. The total volume of PBSCs transfused: 190 - 420 ml (265 +/- 73 ml or 13.7 +/- 4.2 ml/kg) with a mean of (4.43 +/- 1.91) x 10(8)/kg of PBSCs, and 0.94 +/- 0.18 g/kg of DMSO. The single dose: 90 - 300 ml (132 +/- 37 ml or 6.6 +/- 5.2 ml/kg) with a mean of 0.68 +/- 0.12 g/kg of DMSO. Symptoms occurring during the infusions were recorded. All patients were monitored for 24 hours after infusion. Pulse, blood pressure, body temperature, and respiratory rate were recorded every 15 minutes. At four hours before and 8 hours after infusion, urinalysis was performed. Serum potassium, sodium, creatinine, total bilirubin, aspartate amino transferase (AST), and alanine amino transferase (ALT) levels were examined within 24 hours before and after the first infusion. The results showed that the toxicities observed included hemoglobinuria in 54 return infusions (77.1%), headache in 28 (40.0%), nausea in 24 (34.3%), vomiting in 17 (24.3%), and abdominal pain in 8 (11.4%). Patients who received a graft > 200 ml tended to have a higher frequency of hemoglobinuria, headache, nausea, vomiting, or abdominal pain (P<0.01), and they disappeared quickly, too. Total bilirubin increased after the first return infusion (P<0.01), and there was a significant correlation between the volume of infusion and the degree of total bilirubin increase (r=0.8977, P<0.01). No renal failure or shock occurred. It is concluded that transient hemoglobinuria, headache, nausea, vomiting, and abdominal pain are common toxicities associated with PBSC autograft, and these toxicities are related with a single volume of PBSCs transfused. Total bilirubin increase is correlated with the volume of infusion. In a word, the toxicity is less frequent and lower severe in children with fractionated infusions of cryopreserved peripheral blood stem cell.
Acute Disease ; Adolescent ; Child ; Child, Preschool ; Cryopreservation ; Female ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Headache ; etiology ; Hematopoietic Stem Cell Mobilization ; methods ; Hemoglobinuria ; etiology ; Humans ; Leukemia ; therapy ; Lymphoma ; therapy ; Male ; Nausea ; etiology ; Neoplasms ; therapy ; Neuroblastoma ; therapy ; Peripheral Blood Stem Cell Transplantation ; adverse effects ; methods ; Recombinant Proteins

OBJECTIVETo investigate the synergistic effects of carnosic acid (CA) with arsenic trioxide (As₂O₃) on proliferation and apoptosis in HL-60 human myeloid leukemia cells, and the major cellular signaling pathway involved in these effects.

METHODSHL-60 cellular proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Cell cycle distribution and apoptosis were monitored by flow cytometry. The activation of casepase-9, Bcl-2-associated agonist of cell death (BAD), p-BAD, p27, phosphatase and tensin homolog deleted on chromosome ten (PTEN), Akt, p-Akt was assessed by Western blot analysis. The expression of PTEN mRNA was tested by reverse transcription polymerase chain reaction (RT-PCR) analysis.

RESULTSCA reduced HL-60 cell viability in a dose- and time-dependent manner, and induced G1 arrest and apoptosis. Moreover, CA upregulated PTEN expression, blocked the Akt signaling pathway, subsequently inhibited phosphorylation of BAD, reactivated caspase-9, and elevated levels of p27. CA also augmented these effects of As₂O₃.

CONCLUSIONCA might be a novel candidate of the combination therapy for leukemia treatment; these effects were apparently associated with the modulation of PTEN/Akt signaling pathway.

Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Base Sequence ; Blotting, Western ; Cell Cycle ; drug effects ; DNA Primers ; Diterpenes, Abietane ; pharmacology ; Drug Synergism ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; metabolism ; pathology ; Oxides ; pharmacology ; PTEN Phosphohydrolase ; metabolism ; Plant Extracts ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects

Infectious bursal disease virus (IBDV), the causative agent of a highly contagious disease in chickens, carries a small nonstructural protein (NS). In this study, vvIBDV Gx-VP5 genes were cloned into plasmid pET30a( + ) and expressed in E. coli with IPTG inducing. BALB/c mice were immunized with the purified recombinant fusion protein. SP2/0 myeloma cells and spleen cells of BALB/c mice were fused by PEG(MW1500), three hybridoma cell lines were examined by indirect ELISA and clone for three times by limited dilution, and were named as 4B4, 6D12, 3E8. The subtype of the monoclonal antibodies were IgG1 with a subtype identified ELISA kit, and light chains were kappa. The ascites titers of monoclonal antibodies were 5 x 10(4), 3.5 x 10(4), 3 x 10(4) by indirect ELISA, respectively. Indirect ELISA and Western blot results showed that the monoclonal antibodies only acted with VP5 protein, IF analysis indicated that three monoclonal antibodies acted with IBDV Gt. There were specific fluorescence in detected Vero E6 cells which transient expressed VP5 protein by IFA. Therefore, monoclonal antibodies specific to IBDV VP5 proteins are specific method for detected VP5 proteins, and base on establish stabilize expressed VP5 protein Vero cell lines to research IBDV VP5 protein function.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Chickens ; Escherichia coli ; genetics ; metabolism ; Female ; Hybridomas ; secretion ; Immunization ; Infectious bursal disease virus ; immunology ; Mice ; Mice, Inbred BALB C ; Viral Nonstructural Proteins ; immunology

OBJECTIVETo find out association mapping of loci related to schizophrenia on chromosome 1 with microsatellite markers in DNA pooling samples from schizophrenic cases and normal controls in Shandong peninsula.

METHODSA total of 31 microsatellite markers on chromosome 1 spaced at approximately 10 cM were scanned to two separated DNA pooling samples consisting of 119 schizophrenic cases and 119 normal controls respectively. Statistic analysis was performed by Chi-square test method to compare the difference between the ratio of each allele between the two pooling samples.

RESULTSSignificant statistic difference was found at D1S2878 between cases and controls, and P< 0.01 at this loci.

CONCLUSIOND1S2878 locus on chromosome 1 associates with schizophrenia in Shandong peninsula. Fine mapping and searching for candidate genes are warranted in this region.

Adult ; Case-Control Studies ; China ; Chromosome Mapping ; Chromosomes, Human, Pair 1 ; genetics ; DNA ; genetics ; Female ; Genomics ; Humans ; Male ; Microsatellite Repeats ; genetics ; Schizophrenia ; genetics ; pathology

Objective To investigate the presentationand significance of circulating autoantibodies to erythropoietin receptor (EPOR) in sera from patients with systemic lupus erythematosus (SLE). Methods One hundred and twenty-four consecutive patients with SLE, seven with autoimmune hemolytic anemia (AIHA), 19 patients with iron deficiency anemia (IDA) and 45 normal individuals were involved in this study. In all patients with SLE, the disease activity was evaluated using the European consensus Lupus Activity Measurement scale. Antibodies to EPOR were detected by enzyme-linked immunosorbent assay (ELISA). All data were tested with Chi-squared or Student's t tests by SPSS software. Results A higher frequency of antibodies to EPOR were detected in SLE patients than healthy controls (20.2% vs 2.2%, P=0.004), however, they could not be detected in AIHA and IDA patients. Moreover, anti-EPOR antibodies were detected in 17 (33.3%) of 51 SLE patients with anemia, compared with that in 8 (11.0%, P=0.002) of 73 patients without anemia. Furthermore, patients with antibodies to EPOR had more severe anemia and often presented as microcytic anemia (P =0.005) than those without anti-EPOR antibodies. Finally, anti-EPOR antibodies seemed to be more likely to occur in patients with skin rash (P=0.014), low levels of C3 component of complement (P=0.01), positive anti-dsDNA antibodies (P=0.000) and higher disease activity scores (P= 0.024). Conclusion The higher incidence of antibodies to EPOR in SLE patients with anemia suggest that anti-EPOR antibodies might play a vital role in the development of anemia in SLE patients. Thus, detecting anti-EPOR antibodies in SLE patients with anemia may be helpful.

Objective:To measure the parameters of median nerve under ultrasound in patients with diabetes mellitus to explore the relationship between ultrasonic parameters and nerve conduction study. Methods:From June to November, 2019, 79 diabetic patients were divided into diabetic peripheral neuropathy (DPN) group (n = 37) and non-DPN group (n = 42) according to the diagnosis. Their right median nerve were measured cross-sectional area (CSA) and the proportion of hypoechoic areas (HA) 5 cm and 10 cm proximal to the wrist (CSA-5, CSA-10, HA-5 and HA-10, respectively) with sonography; while the conductive latency and velocity of right median nerves were measured with nerve conductive study. Results:CSA-5, CSA-10 and HA-10 were more in DPN group than in non-DPN group (|Z| > 2.282, P < 0.05). HA-5 correlated with the motor conductive latency (r = 0.264, P < 0.05) and velocity (r = -0.270, P < 0.05); HA-10 correlated with the abnormal sensory conductive latency (r = 0.234, P < 0.05) and velocity (r = 0.363, P < 0.01), and the motor conductive latency (r = 0.235, P < 0.05) and velocity (r = -0.255, P < 0.05). Conclusion:DPN can be found under sonography, some of them are related with the nerve electrophysiology, which may be a useful tool to screen DPN.

Objective To investigate the effect of CoCl2-induced hypoxia on the expressions of hypoxia-inducible factor-1α(HIF-lα),glucocorticoid receptor(GR)and opiomelanocortin(POMC)in AtT-20 cells.Methods AtT-20 cells were exposed to different concentrations(25,50,100,and 200 μmol/L)of CoCl2 to induce hypoxia.MTT assay was employed to assess the changes in the cell proliferation after the exposure,and real-time PCR and Western blotting were performed to detect the expressions of HIF-1α,GR and POMC genes at both the mRNA and protein levels.Results Compared with the cells in normoxic conditions,the AtT-20 cells exposed to CoCl2 treatment at 25,50,and 100 μmol/L for 24 and 48 h showed obviously enhanced cell proliferation.CoCl2 significantly increased the mRNA and protein expressions of HIF-1α,GR and POMC genes in a dose-dependent manner(P＜0.05).Conclusion CoCl2-induced hypoxia up-regulates the mRNA and protein expressions of HIF-1α,GR and POMC genes in AtT-20 cells.