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 develop a novel scaffolding method for the copolymers poly lactide-co-glycolide acid (PLGA) to construct a three-dimensional (3-D) scaffold and explore its biocompatibility through culturing Schwann cells (SCs) on it.

METHODSThe 3-D scaffolds were made by means of melt spinning, extension and weaving. The queueing discipline of the micro-channels were observed under a scanning electronic microscope (SEM).The sizes of the micropores and the factors of porosity were also measured. Sciatic nerves were harvested from 3-day-old Sprague Dawley (SD) rats for culture of SCs. SCs were separated, purified, and then implanted on PLGA scaffolds, gelatin sponge and poly-L-lysine (PLL)-coated tissue culture polystyrene (TCPS) were used as biomaterial and cell-supportive controls, respectively. The effect of PLGA on the adherence, proliferation and apoptosis of SCs were examined in vitro in comparison with gelatin sponge and TCPS.

RESULTSThe micro-channels arrayed in parallel manners, and the pore sizes of the channels were uniform. No significant difference was found in the activity of Schwann cells cultured on PLGA and those on TCPS (P larger than 0.05), and the DNA of PLGA scaffolds was not damaged.

CONCLUSIONThe 3-D scaffolds developed in this study have excellent structure and biocompatibility, which may be taken as a novel scaffold candidate for nerve-tissue engineering.

Animals ; Biocompatible Materials ; Cell Adhesion ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Lactic Acid ; Microscopy, Electron, Scanning ; Polyglycolic Acid ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; Tissue Engineering ; methods ; Tissue Scaffolds

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

AIMTo study the synthesis and antitumour activities of some aryl-substituted pteridines.

METHODSA series of aryl-substituted pteridines were synthesized from 4, 6-diamino-5-nitrosopyrimidines by cyclization with 4-aminophenylacetonitriles. The antitumour activities were tested by MTT method.

RESULTSNine new compounds (I-III) were synthesized and their structures were characterized by EA, IR, 1HNMR and MS spectra. Compounds I-III showed antitumour activities in vitro.

CONCLUSIONCompounds I-III showed remarkable antitumour activities in vitro. No interaction was determined between the title compounds and calf thymus DNA. It indicated that these compounds possibly inhibit dihydrofolate reductase (DHFR) or other enzymes on which folic acid depends.

Adenocarcinoma ; pathology ; Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; drug effects ; Humans ; KB Cells ; drug effects ; Lung Neoplasms ; pathology ; Molecular Structure ; Pteridines ; chemical synthesis ; chemistry ; pharmacology

OBJECTIVETo establish two-dimensional electrophoresis profiles from human laryngeal squamous cell carcinoma tissue and paired normal tumor-adjacent mucosa epithelia tissue, and to identify differential expression proteins.

METHODSThe total proteins of human laryngeal squamous carcinoma tissue and paired normal tumor-adjacent mucosa epithelia tissue were separated by immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE). The differential expression proteins were analyzed using image analysis software, then identified using mass spectrometry and database searching.

RESULTSWell-resolved, reproducible 2-DE patterns of laryngeal squamous cell carcinoma and adjacent normal mucosa epithelial were obtained. Differential protein spots were defined as spots in 2-DE gels. Thirteen proteins were preliminarily identified, naming which 10 proteins were upregulated in laryngeal cancer tissue. Such as cofilin-1, nuclear body protein SP140, GRP94, HSP 90, GSTP1-1, superoxide dismutase [Mn], cyclophilin A, proteasome activator complex subunit 2, apolipoprotein A-I precursor, CaM-like protein and so on. There were 3 proteins downregulated in laryngeal cancer tissue, which were fatty acid-binding protein, calgranulin A and calgranulin B.

CONCLUSIONSThirteen proteins which are associated with the tumorigenesis of the laryngeal squamous cell carcinoma were characterized. These extensive protein variations indicate that multiple protein molecules should be simultaneously targeted as an effective strategy to counter the disease. It is better for understanding of the oncogenesis and pathogenesis in a global way, which in turn is a basis-for the rational designs of diagnostic and therapeutic methods.

Aged ; Carcinoma, Squamous Cell ; metabolism ; Humans ; Laryngeal Neoplasms ; metabolism ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Proteomics ; methods

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

The paper aimed to elucidate the specific impact of supramolecular chemistry on the Chinese medicine theories (CMT) in their modernization, after had summarized up the research status of supramolecular chemistry and analyzed the possible supramolecular forms of Chinese medicine (CM), as well as considered the problems in modernization of CM theories. On comparison of the classical chemistry that delt with chemical bonds among atoms, the supramolecular chemistry was rather concerned with varietes of weak noncovalent bonds intermolecules, and reflected the macro-apparent chemical properties of each molecules, and was the most appropriate chemical theories to explain the CMT and microcosmic materials. The molecules in the human body and Chinese material medica (CMM) formed supramolecules by way of self-assembly, self-organization, self-recognition and self-replication, with themselves or with complexation, composition, chelation, inclusion, neutralization etc. Meridian and Zang-fu viscera in CMT might be a space channel structure continuously consisted of unique molecules cavity that was imprinted with the supramolecularly template inside and outside of cells, through which the molecules in CMM interacted with the meridian and Zang-fu viscera. When small molecules in human body imprinted with macromolecules in meridian and Zang-fu viscera, in other words, they migrated along within imprinting channels of meridian and Zang-fu viscera on behavior of "Qi chromatography" impulsed by the heart beat, finally showed up on macroscopic the anisotropy of tissue and organ, as described namely as visceral manifestation in Chinese medical science. When small molecules in CMM interacted with imprinting channel on meridian and Zang-fu viscera, the natural properties and efficacy regularities of CMM was reflected on macroscopic. Therefore, the special representation forms of basic CMT is based on the macroscopic expression of "Qi chromatography" abided by imprinting effect regularities, and on whether the imprinted template of small molecules matched with cavity template of macromolecules in meridian and Zang-fu viscera, only is the adequate representation of supramolecular chemistry for them. The CMM materials is the mixture including single molecules and supramolecules. The compatibility for CM prescriptions can significantly change the function rules. Therefore in the study of basic CMT, we should pay special attention to the laws of supramolecular chemistry. It is the most essential differences of the CMT from the modern medicine which established by the laws of single molecular theories.
Chemistry, Pharmaceutical ; Drug Therapy ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Humans ; Meridians ; Nanotechnology

Objective To assess the neointimal coverage after the implantation of various drug eluting stents (DES) by optical coherence tomography ( OCT). Methods The study comprised of 62 patients implanted DES for ( 15. 3 ± 5.7 ) months. Patients were divided into three groups according to the type of implanted stent: Cypher group ( patient = 26, stent = 57 ), Endeavor group ( patient = 17, stent = 23 )and Firebird group (patient = 19, stent = 32). OCT images of the stent were analyzed by software equipped by Light Lab system. Intimal thickness of 64 μm, 168 μm and 366 μm represents 10%, 25% and 50%lumen area loss, respectively. Neointimal coverage was thin with intimal thickness≤64 μm, satisfactory with intimal thickness between 65 μm and 366 μm and hyperplaisa and restenosis with intimal thickness ＞ 366μm. Results The percent of complete neointimal coverage was similar among groups ( P ＞ 0. 05 ). The thickness of neointimal coverage in Cypher and Endeavor and Firebird group was (178.7 ± 11.9)μm,(228.7 ± 17. 1 ) μm and ( 170. 3 ± 13.3 ) μm, respectively( all P ＜ 0. 05 ). The symmetry of Cypher stent was better than Firebird stent, and the symmetry of Firebird stent was better than Endeavoe stent. Conclusion There was significant difference on neointimal coverage after various types of DES implantation, and OCT can be used to evaluate the symmetry of neointimal coverage post implantation of various DES.

ABSTRACT:OBJECTIVE To investigate the effect of peiminine on MEK1/2,ERK1/2 and the phosphorylatioin of bleomycin-induced pulmonary fibrosis in rats.METHODS Bleomycin of 5 mg/kg was instilled into the rats with a microliter injector to induce the acute lung injury.Sham-operated group and control group were given distilled water of 1 mL/100 g.Dexamethasone group was given equal volume of dexamethasone distilled water solution.Peiminine A and B group were given equal volume of peiminine distilled water solution of 5 mg/kg and 2.5 mg/kg,respectively.After consecutive daily gavage for 28 d,the rats were anesthetized and killed by carotid exsanguination.Lungs were fixed and embedded,and 4 μm sections were prepared. MEK1/2 and ERK1/2 in the lung tissues were detected by immunohistochemistry.The p-MEK1/2 and p-ERK1/2 were detec-ted by Western blot.RESULTS Peiminine significantly reduced the levels of ERK1/2 and MEK1/2 in lung tissues of rats with bleomycin-induced pulmonary fibrosis(P <0.01),and the effects was similar to dexamethasone (P > 0.05).No significant difference was observed between peimine A group and B group(P >0.05).Compared to the model group and dexamethasone group,peiminine significantly reduced the level of p-MEK1/2 and p-ERK1/2 in lung tissue of bleomycin-induced pulmonary fi-brosis rats(P <0.01).Compared to peimine A group,the B group displayed more significant efficacy(P <0.01).CONCLU-SION Peiminine attenuates the level of MEK1,ERK1 / 2 and the phosphorylation in lung tissues of bleomycin-induced pul-monary fibrosis rats,which may be one of the mechanism of anti-fibrosis.

Objective To evaluate the clinical effect and serum cytokines levels of mouse nerve growth factor during the treatment of senile dementia.Methods A total of 90 senile dementia patients were randomly divided into treatment group and control group,each group 45 cases.Control group was given conventional treatment.Treatment group was intravenous drip mouse nerve growth factor 20 μg,qd on the basis of control group.All patients were treated for 6 weeks.The cognitive function,dementia degree,daily life ability before treatment and 6,12 weeks after treatment in two groups were observed.The levels of serum interleukin-1β (IL-1β),interleukin-6 (IL-6),C-reactive protein(CRP),tumor necrosis factor-α (TNF-α) in two group were observed.Results The mental state examination(MMSE),activity of daily life (ADL),clinical dementia rating (CDR) in two group after 12 weeks treatment had significant difference (P ＜ 0.05).The IL-1 β in treatment group after 6,12 weeks treatment were (20.88 ± 5.83),(18.35 ±3.96) ng · L-1,and were (21.12 ±6.12),(20.76 ±4.83) ng· L-1 in control group.The IL-6 in treatment group after 6,12 weeks treatment were (24.33 ± 8.56),(18.85 ±5.83) ng · L-1,and were (24.84 ±9.13),(21.64 ± 6.68) ng · L-1 in control group.The CRP in treatment group after 6,12 weeks treatment were (6.43 ± 1.65),(5.27 ± 0.63)ng · L-1,and were (6.73 ± 1.73),(6.17 ± 0.95) ng · L-1 in control group.The TNF-α in treatment group after 6,12 weeks treatment were (40.52 ±8.33),(36.34 ±5.92) ng · L-1,and were (40.85 ±9.15),(39.46 ±6.44) ng · L-1 in control group.The IL-1β,IL-6,CRP,TNF-α in two group after 12 weeks treatment had significant difference (P ＜0.05).There was 1 case of mild headache in control group,the incidence of adverse drug reactions was 0.02％ (1/45 cases).There was no adverse drug reaction occurred in treatment group.Conclusion The clinical effect of nerve growth factor in the treatment of senile dementia is significant,and can improve the living ability,relieve the symptoms of dementia,reduce the level of serum cytokines.