A kind of real-time remote monitoring embedded terminal which is combined with mobile communication technology and GPS localization technology, has been developed. The results of preliminary experiments show that the terminal can transmit ECG signals and localization information in real time and continuously, supply a real-time monitoring of out-of-hospital cardiac patients and trace the patients.
Electrocardiography, Ambulatory ; instrumentation ; Telemedicine ; instrumentation ; Telemetry ; instrumentation

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

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

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 detect alterations of microsatellite loci [transforming growth factor beta receptor II (TGF-betaRII)(A)(10), TGF-betaRII(GT)(3), hMSH3(A)(8), hMSH6(C)(8), Bax(G)(8), IGFIIR(G)(8), IGFIIR(CT)(3)] and point mutations of TGF-betaRII (TGF-betaRII 452/454, TGF-betaRII 533).

METHODSPCR-SSLP, microdissection-PCR-SSLP, PCR-SSCP, clone sequencing and immunohistochemistry were used.

RESULTSThe mutation rate of TGF-betaRII(A)(10) in RER+ (replication error positive) colorectal carcinomas was 33% (3/9). Similar mutations were also observed in adenomas with severe dysplasia. No mutations at other microsatellite loci were found. RER+ colorectal cancers mainly occurred in male patients at a young age and were more common in the colon than in the rectum (P < 0.05).

CONCLUSIONSRER+ colorectal cancers were found in young males and commonly located in the colon. A one third mutation rate in TGF-betaRII(A)(10) in these patients is lower than that observed in western populations, which may imply diverse pathways of carcinogenesis of RER+ colorectal carcinoma. TGF-betaRII(A)(10) mutation may play a role in the transforming process from an adenoma with severe dysplasia to a full blown carcinoma.

Adult ; Aged ; Colorectal Neoplasms ; genetics ; Female ; Humans ; Male ; Microsatellite Repeats ; Middle Aged ; Point Mutation ; Polymerase Chain Reaction ; Protein-Serine-Threonine Kinases ; Receptors, Transforming Growth Factor beta ; genetics

OBJECTIVETo further study the mechanism of the inhibitory effect of interferon beta-1a (IFN beta-1a) on the activation of human hepatic stellate cell (HSC) LX-2, and to analyze the differences on the protein expression in LX-2 induced by I IFN beta-1a.

METHODSCultured LX-2 cells were treated with 2000 U/ml IFN beta-1a for 48 h. Two-dimensional gel electrophoresis (2-DE) was performed to compare protein patterns of the control (untreated) and IFN beta-1a treated LX-2 and for quantitative and qualitative analyses of protein expression. A rat liver fibrosis model was established and the rats were sacrificed and their various tissues were obtained for the same analyses. Western blotting and RT-PCR were used to validate the expression of the changed proteins after treatment of IFN beta-1a in LX-2 cells and of various tissues of the rats.

RESULTS708 +/- 25 spots were detected in control LX-2 cells and 804 +/- 32 spots in IFN beta-1a-treated LX-2 cells. A match rate of 73%-82% was achieved. The results also showed that 31 protein spots displayed quantitative changes in expression after IFN beta-1a treatment. Of the 31 spots, 21 proteins were identified, of which, one was newly found, two were enhanced in abundance and 18 showed lower expressions. The newly found protein was glia maturation factor beta (GMF beta). The treatment of LX-2 with IFN beta-1a increased the production of GMF beta(GMF beta) protein in comparison with the untreated cells (t=1.81, P < 0.01). The expression of GMF beta protein (1.81 vs 0.10) and mRNA (0.85 vs 0.12) were more in the normal liver tissues than in the cirrhotic liver tissues (t=2.53, 2.13 respectively, P < 0.01). The expressions of GMF beta protein and mRNA were weak in rat heart and lung tissues, however, they were strong in rat liver, kidney, spleen and brain tissues (t=1.91, 1.94 respectively, P < 0.01).

CONCLUSIONThere is a significant difference of protein expression levels between IFN beta-1a untreated and treated LX-2 cells. These proteins, especially GMF beta, may be involved in an inhibition process of IFN beta-1a on activation and apoptosis of LX-2 cells. This proteome study may be useful in further studies of the relationship of IFN beta-1a treatment and human liver diseases.

Animals ; Cell Line ; Female ; Glia Maturation Factor ; metabolism ; Hepatic Stellate Cells ; metabolism ; Humans ; Interferon beta-1a ; Interferon-beta ; pharmacology ; Liver ; cytology ; Liver Cirrhosis ; metabolism ; Proteome ; Rats ; Rats, Sprague-Dawley

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

3a and 7a are nonstructural proteins of SARSCoV, which are encoded separately by ORF 3a and ORF 7a in SARSCoV genome. The expression of 3a has been founded in cells infected by virus in vivo or in vitro. Firstly, the pGL3Control vector was reconstructed , the pGL3Enhancer vector deletious of SV40 promoter gene was obtained . Then the IFN? promoter gene was cloned into the pGL3Enhancer vector and pGLIP21, the Luciferase reporter plasmid with IFN? promoter was established. The availability of pGLIP21 was verified by NDV ,the inductor of IFN?, the Luciferase activity was assayed in cells transfected with pGLIP21 by Luminometer. In order to see the function of 3a and 7a protein of SARSCoV,CHO cells expressing 3a or 7a protein were transfected with pGLIP21, the intensity of luciferase activity was analyzed . By analysis, in vitro, 3a and 7a protein of SARSCoV had the similar ability in triggering the expression of Luceferase gene, i.e 3a and 7a protein of SARSCoV could effectively activate the promoter fragment of IFN? gene. This result will help studying the function of 3a and 7a protein and provide a method to study the nosogenesis mechanism of SARSCoV.

OBJECTIVETo explore the etiology and clinical characters of hepatitis caused by non-hepatotropic virus.

METHODS68 non-hepatotrophic viral hepatitis patients with negative anti-HAV-anti-HEV were diagnosed by detecting antibodies of anti-HSV IgM, anti-EBV IgM, anti-CMV IgM, anti-CSV IgM and anti-ANA, anti-mitochondrion antibody. Their clinical symptoms and signs were compared with that of acute viral hepatitis patients at the same time.

RESULTSAmong the 68 patients, 9 were infected by HSV, 12 by EBV, 8 by CMV, 14 by CSV, and the other 13 patients and 12 patients were positive for anti-ANA and anti-mitochondrion antibody, respectively. 35 of 43 non-hepatotrophic viral hepatitis patients were infected in winter and spring season. Their clinical symptoms and signs were milder than that of acute viral hepatitis patients.

CONCLUSIONLiver damage and dysfunction may be the prominent phenomenon during HSV, EBV, CMV and CSV infection, just like that of acute viral hepatitis but with milder clinical symptom and signs.

Adult ; Antibodies, Viral ; blood ; Cytomegalovirus ; immunology ; Female ; Hepatitis, Viral, Human ; etiology ; virology ; Herpesvirus 4, Human ; immunology ; Humans ; Immunoglobulin M ; blood ; Male ; Simplexvirus ; immunology

OBJECTIVETo improve the diagnosis and treatment level of spontaneous bacterial peritonitis (SBP) in the patients with advanced liver disease, get better curative effect and prognosis.

METHODSRegistered the body temperature, symptoms and signs in the abdomen, and blood routine test, the polymorphonuclear (PMN) cell count, and ascites culture in the patients with cirrhosis and fulminant hepatitis. These patients were given supporting therapies including use plasma and albumin as well as antibiotics treatment according to drug sensitivity or empiric. Changes of the body temperature, symptoms and signs were used to evaluate the effect of therapy.

RESULTS186 of 275 inward patients with end-stage liver disease during this period were considered as SBP by ascites culture or clinical experience with various degree symptoms and signs such as pain, distention, higher tension and touch pain in the abdomen. Infective rate was 67.6%. Among them 138 patients had abnormal body temperature more than 37.4 degrees C. 106 patients with leukocyte count in the peripheral blood more than 10 x 10(9)/L; 137 patients with PMN more than 80% in differential cell count; 103 patients with PMN more than 250/mm(3) in ascites. Only 29 patients were culture positive. 82 patients were cured, 17 patients with improvement, 18 patients with inefficacy or deterioration. 42 patients died of hepatic-renal failure and 27 patients died because of upper alimentary tract bleeding, respectively.

CONCLUSIONSigns and symptoms of SBP were atypical in the patients with end-stage liver disease. Ascites culture positive rate was not high. Early diagnosis and proper use antibiotics according to culture and empirics were important to increase effect and improve prognosis

Adolescent ; Adult ; Aged ; Anti-Bacterial Agents ; therapeutic use ; Bacterial Infections ; diagnosis ; microbiology ; therapy ; Female ; Humans ; Liver Diseases ; complications ; Male ; Middle Aged ; Peritonitis ; diagnosis ; microbiology ; therapy ; Prognosis