C-Reactive Protein ; analysis ; Calcitonin ; blood ; Calcitonin Gene-Related Peptide ; Cerebrospinal Fluid Proteins ; analysis ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Leukocyte Count ; Male ; Meningitis, Bacterial ; diagnosis ; Meningitis, Viral ; diagnosis ; Protein Precursors ; blood

Objective To repair segmental mandibular defects with autogenous bone marrow stromal cells(BMSCs)and?-triealcium phosphate.Methods Isolated BMSCs were in vitro expand- ed.A 3 cm-long segmental mandibular defect was created at right mandible in 12 canines,of which de- fects in six canines were repaired with BMSCs and?-tricalcium phosphate(?-TCP)and that in other six cases repaired with?-TCP,which was used as control.The engineered bone was evaluated by X-ray, CT,DXA,gross and histological examination,immunohistochemistry and biomechanical test 4,12,26,32 weeks after operation respectively.Results In induced BMSCs,histochemistry showed AKP activity. Oral X-ray showed obvious callus formation 4-26 weeks after operation in experimental group but minimal bone formation in control group.At 32 weeks after operation,gross observation,X-ray and CT demonstra- ted well bony-union in experimental group but bony-nonunion in control group.DXA indicated that the bone density of experimental group was significantly higher than that of control group.Biomechanical test revealed no statistical difference upon mechanical strength of mandibula between experimental group and normal group.Conclusions Canine segmental mandibular defects can be well repaired with the tissue- engineered bone generated by autogenous osteogenic BMSCs and?-TCP scaffold.

Mammalian cells are prone to apoptosis when cultured in large scale for production of biopharmaceuticals. And this will reduce production duration and result in high cost of production. Apoptosis is triggered by various factors, and delicately regulated by a set of genes. Bcl-2, a component integrated in mitochondria membrane, is an important member of these genes. By maintaining the integrity of mitochondria membrane, Bcl-2 keeps cytochrome C from releasing into cytoplasm, and thus blocks the activation of caspases, and subsequent onset of apoptosis. Over-expression of Bcl-2 has proven to be useful in blocking apoptosis in various cell lines, including CHO, hybridoma, myeloma, lymphoma and insect cells. Ammonia, a metabolite of cultured cells, however, showed apparent pro-apoptosis activity. In living cells, ammonia can be utilized by glutamine synthetase (GS) to synthesize glutamine, and thus lower the concentration of ammonia in medium, and its negative effects. Glutamine is essential to living cells. If not added into medium, glutamine can only be synthesized by GS, which makes GS a qualified selection marker. This marker can be used for gene amplification by adding into medium increased concentration of MSX, an inhibitor of GS. In this study, we over-expressed Bcl-2 using GS amplification in a recombinant CHO cell line stably expressing human interferon-beta. The modified cell line, with higher expression of Bcl-2 and lower production of ammonia, exhibited good anti-apoptosis quality and higher interferon-beta production in continuous culture.
Animals ; Apoptosis ; genetics ; physiology ; Biopharmaceutics ; CHO Cells ; cytology ; metabolism ; Cricetinae ; Cricetulus ; Glutamate-Ammonia Ligase ; genetics ; metabolism ; Interferon-beta ; metabolism ; Models, Genetic ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism

Serum used widely in mammalian cell culture is also a potential source of bacterial, mycoplasmal and viral contaminations. In addition, the complex biological components in serum make harder the subsequent product recovery process. High cost, high batch variation and potential source limitation are among the other shortcomings. So serum-free or even protein-free medium are preferable for recombinant protein production. However, without serum to provide essential components such as hormones, growth factors and binding proteins, cells are easy to die. In this study, CHO-dhfr- cells were genetically engineered to make them adapted to IMEM, a protein-free medium, and resistant to apoptosis. The genes in choice are insulin-like factor (Igf-1), Bcl-2 and cyclin E. Bcl-2 is a mitochondrial membrane-integrated protein. It can block the release of cytochrome c by maintaining the integrity of mitochondrial membrane, and thus inhibit apoptosis. Igf-1 is similar both in structure and function to insulin, a growth factor added to serum-free medium to promote cell growth and is the only protein component in many currently used serum-free media. cyclin E is a cell cycle protein expressed continuously in G1 phase. When cyclin E accumulates to certain amount, cell cycle was driven to S phase. So cyclin E is a proliferation-promoting protein. By co-express Igf-1/Bcl-2 or Bcl-2/ cyclin E in CHO-dhfr- cells with a dicistronic expression vector, we constructed two cell lines: CHO-IB and CHO-BC. The high expression of each protein was confirmed by Western blot and flow cytometry. Apoptosis was analyzed by flow cytometry and DNA ladder detection, and the two cell lines were both found much more resistant to apoptosis induced by withdrawal of serum or addition of actinomycin D than the CHO-dhfr- parent cell. Cell proliferation assay by MTT method showed that the two cell lines proliferated much faster than CHO-dhfr- in IMDM medium without serum. Continuously culture assay proved that the two cell lines grow very well in IMEM protein-free medium supplemented with fibronectin and vitronectin to ease adherence. When compared to CHO-dhfr-, the two cell lines exhibited much more viable cell numbers and faster growth rate.
Animals ; Apoptosis ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Culture Media ; Cyclin E ; genetics ; Genes, bcl-2 ; Insulin-Like Growth Factor I ; genetics

Without serum to provide adherent factors, CHO-dhfr- cells grow in suspension when cultured in serum-free medium. Although this offers advantages in some applications, in most production systems adherent cell growth is preferable. Gene transfection, clonal selection and amplification can be easier for adherent cells; the density of immobilized cells is often higher than those in suspension culture, which results in a higher protein productivity; washout of cells by perfused medium during continuous fermentation can be avoided; for high-throughput microplate assays, adherent cells are preferred to facilitate medium changes and cell washing. It has been proved that purified vitronectin alone was able to mediate attachment and spreading of CHO cells in serum-free medium. So we constructed a tricistronic expression vector expressing Igf-1, Vitronectin and Bel-2 at the same time. The vector was transfected into CHO-dhfr- cells and one clone, namely CHO-IVB2, expressing high level of the three proteins was screened out by Western blot. The cell line showed similar apoptosis-resistant and serum-independent properties to CHO-IB, an engineered cell line constructed before. When cultured in IMEM protein-free medium without any components supplemented, CHO-IVB can grow adherently. The viable cell numbers and growth rate of CHO-IVB were much higher than CHO-IB, making CHO-IVB an apoptosis-resistant host for production of recombinant proteins which can grow adherently in protein-free medium.
Animals ; Apoptosis ; CHO Cells ; physiology ; Cell Adhesion ; Cell Line ; Cell Proliferation ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Flow Cytometry ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; Vitronectin ; genetics

OBJECTIVETo investigate the feasibility of multiple displacement amplification (MDA) to apply in the non-invasive prenatal genetic diagnosis of Duchenne muscular dystrophy (DMD).

METHODSMaternal blood was obtained from 20 pregnant women at 7 to 25 weeks of gestation. After the discontinuous density gradient centrifugation with Percoll, the fetal nucleated red blood cells (NRBCs) were stained with Kleihauer test. All positive NRBCs were collected by micromanipulator and then performed with MDA. Sex and short tandern repeat (STR) analysis were determind from a small aliquot of the reaction. The origin of NRBCs was verified and prenatal diagnosis of DMD was made at the same time.

RESULTSThe product length of MDA was >15 kb, while primer extension preamplification (PEP) is only about 1 kb. We completed non-invasive prenatal genetic diagnosis of 6 fetus at high risk of DMD using MDA. The results were all coincident with amniotic fluid control.

CONCLUSIONThe MDA method which provides a highly uniform representation across the genome, representing the entire genome with minimal amplification bias, shows good application prospects.

Erythroblasts ; metabolism ; Feasibility Studies ; Female ; Fetal Diseases ; blood ; diagnosis ; genetics ; Humans ; Muscular Dystrophy, Duchenne ; blood ; diagnosis ; genetics ; Polymerase Chain Reaction ; methods ; Pregnancy ; Prenatal Diagnosis ; methods

OBJECTIVETo investigate the association between the expression of turnor necrosis factor alpha (TNF-alpha) mRNA in fat tissue of intrauterine growth retarded (IUGR) rats and insulin resistance, and the long-term effects of early different nutritional diet.

METHODSThe IUGR rat model was established by food restriction of pregnant rats. A total of 32 newborn IUGR rats were randomly divided into 4 groups: IUGR model (S/N) group, IUGR high caloric diet (A) group, IUGR high caloric and high protein diet (B) group, IUGR high protein diet (C) group. Only the mother rats were given those different diets individually, and all IUGR newborn pups were lactated for 3 weeks. From the beginning of the 4(th) week, all IUGR pups were weaned and fed with normal diet till the end of the experiment. Eight normal birth weight newborn rats were used as the control group fed with the normal diet. Weight, perirenal fat weight, fasting glucose and insulin concentration and quantified TNF-alpha mRNA expression in adipose cell were measured at the 48(th) week. The insulin sensitive index (ISI) and the relation index between TNF-alpha mRNA and fat weight, fat weight/body weight (fw/bw) ratio and ISI were calculated.

RESULTSISI of IUGR model group, IUGR A and B groups was lower than normal control group, while perirenal fat weight, fw/bw and the expression of TNF-alpha mRNA in adipose cells were all significantly higher (P < 0.05 or 0.01). There were no significant differences in these indexes between IUGR C group and normal control groups (P > 0.05). A positive correlation was found between TNF-alpha mRNA and fat weight and fw/bw (r(1) = 0.755, r(2) = 0.782, P = 0.000). Significant inverse associations between ISI and TNF-alpha mRNA (r = -0.556, P = 0.000) and fw/bw (r = -0.513, P = 0.02) were also found.

CONCLUSIONThe occurrence of insulin resistance in IUGR rats is possibly associated with central obesity and accumulation of the abdominal fat and adipose cell over-expression of TNF-alpha. The adipose TNF-alpha may be an important pathogenic factor of insulin resistance of IUGR. High protein diet is a reasonable nutritional intervention. Because it promotes the skeleton muscle catch-up growth but not fat catch-up growth, it can avoid the occurrence of central obesity and insulin resistance in IUGR rats.

Adipose Tissue ; metabolism ; Animals ; Diet ; Female ; Fetal Growth Retardation ; Insulin Resistance ; Nutritional Status ; Pregnancy ; Random Allocation ; Rats ; Tumor Necrosis Factor-alpha ; metabolism

Chinese hamster ovary cells (CHO) are preferable to prokaryotic, yeast or insect cells as hosts for biopharmaceutical production due to the products are more similar to their natural conformation. However, CHO cells confront tremendous difficulties when cultured in large scale such as mal-adaptation to serum-free medium, apoptosis and over-growth without limitation. So in addition to optimizing CHO system in respect of medium, environment and expression vector, modification of CHO cells themselves has drawn more and more attention. Here the main progress in CHO-modification is reviewed.
Animals ; Apoptosis ; genetics ; CHO Cells ; drug effects ; metabolism ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; drug effects ; Cell Division ; drug effects ; Cricetinae ; Genetic Vectors ; genetics ; Transfection

OBJECTIVETo investigate the clinical indications of asthma control test (ACT).

METHODSA total of 120 asthmatic patients with a diagnosis in line with the American Thoracic Society criteria and treated for over a month were enrolled in this study. The patients were asked to complete a survey to assess their symptoms and asthma attacks, and ACT evaluation was conducted by physicians familiar with ACT evaluation. The patients were classified into two groups based on the pulmonary function test (positive for bronchodilator test and provocation test) or based on disease severity (mild and moderate-to-severe asthma groups). The effect of ACT evaluation was graded as good (no less than 4 item available for evaluation), fair (2-3 items available) and poor (no more than 1 item). To further analyze the ACT sensitivity in relation to different disease severity, 29 asthmatic patients with an initial diagnosis and BDT positivity were included, and the ACT score of the patients with mild, moderate and severe asthma based on FEV1% were compared.

RESULTSIn patients positive for bronchodilator test, good, fair and poor evaluation effects were found in 48, 15, and 5 cases, as compared to 10, 15, and 27 in those positive for provocation test, respectively, showing significant differences between the two groups (P < 0.001). In mild asthma group, good, fair and poor evaluation effects were found in 12, 15, and 18 cases, respectively, significantly different from those in moderate-to- severe asthma group (50, 21, and 4 cases, P < 0.001). ACT scores showed a positive correlation to FEV1% in 29 patients with positive BDT (r = 0.55, P = 0.003). ACT scores had no significant difference between mild and moderate asthma groups (P > 0.05), but showed significant differences between mild and severe groups (P = 0.009) and between moderate and severe groups (P = 0.008).

CONCLUSIONACT is more suitable for evaluating patients positive for bronchodilator test or with moderate to severe asthma.

Adolescent ; Adult ; Aged ; Asthma ; diagnosis ; physiopathology ; Female ; Humans ; Male ; Mass Screening ; Middle Aged ; Predictive Value of Tests ; Respiratory Function Tests ; Sensitivity and Specificity ; Severity of Illness Index ; Surveys and Questionnaires ; Young Adult

BACKGROUND:In recent years, the application of stem cel s to treat autoimmune diseases has become a hot spot. But, studies on umbilical cord blood mesenchymal stem cel s transplantation for the treatment of polymyositis/dermatomyositis are rarely reported. OBJECTIVE:To explore the immunologic mechanism of Th cytokines on the occurrence and development of polymyositis/dermatomyositis by observing the changes in serum interferon-γ, interleukin-4 and interleukin-17 in patients after umbilical cord blood mesenchymal stem cel s transplantation. METHODS:Eighty-one polymyositis/dermatomyositis patients were selected and divided into conventional therapy group (n=44) undergoing glucocorticoid and immunosuppressants therapy and cel transplantation group (n=37) undergoing intravenous infusion of umbilical cord blood mesenchymal stem cel s at a density of (3.5-5.2 )×107 . Dosing regimen was same in the two groups. After fol ow-up of 1, 3, 6 months, the changes of creatine kinase and myodynamia were evaluated;after fol ow-up of 3 and 6 months, lung imaging was evaluated;in the cel transplantation group, interferon-γ, interleukin-4 and interleukin-17 levels were detected before treatment and at 3 and 6 months after treatment. RESULTS AND CONCLUSION:At 1, 3, 6 months after treatment, the creatine kinase level was significantly decreased, and the muscle force grade was significantly increased in both groups (both P<0.001). Compared with the conventional therapy group, the creatine kinase level was lower and the muscle force grade was higher in the cel transplantation group (both P<0.001). Results from lung imaging test showed a remarkable improvement after cel transplantation, and it indicated that umbilical cord blood mesenchymal stem cel s transplantation had good stability. At 6 months after transplantation, the level of interferon-γwas significantly increased, while the interleukin-4 level was decreased significantly (both P<0.01);at 3, 6 months after cel transplantation, the levels of interleukin-17 were significantly decreased (P<0.01). Levels of interleukin-4 and interleukin-17 were positively correlated with the level of creatine kinase at 6 months after cel transplantation (r=0.467, 0.488, both P<0.05), but there was no obvious correlation between the levels of interferon-γand creatine kinase (r=0.213, P>0.05). These findings indicate umbilical cord blood mesenchymal stem cel s transplantation combined with glucocorticoid and immunosuppressants therapy can adjust immune network effects and improve the immune tolerance in polymyositis/dermatomyositis patients, which is safe and effective.