OBJECTIVETo investigate the effects of botulinum toxin type A (BTXA) on the expression of alpha smooth muscle actin(alpha-SMA) and myosin-II of fibroblasts in scars. Methods Fibroblasts were isolated from tissue specimens of scars contracture. Cells from passages 3-5 were randomly divided into 3 groups (control group, low BTXA group (1 U/10(6) Cells), and high BTXA group (2.5 U/ 10(6)Cells)). Growth condition of fibroblasts was observed at 1 , 4, 7 day after BTXA treated. Changes of alpha-SMA and myosin-II in fibroblasts were detected by Western blot.

RESULTSFibroblasts grew well in control group. The proliferation was decreased 4 days later in BTXA groups. Lots of apoptotic cells were seen in high BTXA group at 7th day. Proteins of alpha-SMA and myosin-II in fibroblasts were statistically different between BTXA group and control groups at 4th day (P < 0.05). The expression of alpha-SMA and myosin-II in low BTXA group was higher than that in high BTXA group at 7th day (P < 0.05).

CONCLUSIONSBTXA could induce the apoptosis of fibroblasts and decrease the expression of alpha-SMA and myosin-II in fibroblasts. The inhibitory effect was strengthened with BTXA concentration increase within a certain range.

Actins ; metabolism ; Botulinum Toxins, Type A ; pharmacology ; Cicatrix ; Fibroblasts ; drug effects ; metabolism ; Humans ; Muscle, Smooth ; metabolism ; Myosin Type II ; metabolism ; Random Allocation

Objective To evaluate the validity of botulinum toxin type A (BTXA) injections for the treatment of scar contracture.Methods 26 patients with scar contracture were randomly assigned into BTXA group and triamcinolone acetonide (TAC) group.Pinpoint tattooing was performed on each side of each scar in the plane of its longest axis.A template was used to ensure consistent length.These two tattoo points were measured to assess scar contraction at baseline,at every month for a total of 6 months.Histological analysis was conducted to study the physiological environment and immunohistochemistry to detect the expression of α-SMA and myosin-Ⅱ at different groups.Results Scar contraction was more relaxed in BTXA group than that in TAC group after 1 month (P＜0.05),especially in the 6th month (the D value in BTXA group and TAC group was (1.23±0.42) cm,and (0.56±0.33) cm respectively).For immunohistochemistry,the expression of α-SMA and myosin-Ⅱ also decreased in BTXA group (P＜0.05).Conclusions The treatment of scar contracture by suitable BTXA injections is safe and effective.

INTRODUCTIONThe purpose of this study was to explore whether diabetes mellitus (DM) correlates with the risk of kidney cancer in Taiwan.

MATERIALS AND METHODSWe designed a population-based case-control study from the Taiwan National Health Insurance Database, which consisted of 116 patients with newly diagnosed kidney cancer as cases and 464 subjects without kidney cancer as controls in 2000 to 2009. Both cases and controls were aged ≥20 years. Baseline comorbidities were compared between kidney cancer cases and controls.

RESULTSMultivariable analysis showed no association was detected between DM and kidney cancer (OR 1.06, 95% CI, 0.58 to 1.94). Hypertension (OR 2.05, 95% CI, 1.23 to 3.42), chronic kidney diseases (OR 2.57, 95% CI, 1.23 to 5.37), cystic kidney diseases (OR 18.6, 95% CI, 1.84 to 187.6) and kidney stones (OR 4.02, 95% CI, 2.43 to 6.66) were significant comorbidities associated with increased risk of kidney cancer. Use of alpha-glucosidase inhibitor was associated with increased risk of kidney cancer (OR 4.31, 95% CI, 1.07 to 17.3).

CONCLUSIONDM does not correlate with the risk of kidney cancer. Hypertension, chronic kidney diseases, cystic kidney diseases, kidney stones and use of alpha-glucosidase inhibitors are associated with kidney cancer.

Carcinoma, Renal Cell ; etiology ; Case-Control Studies ; Diabetes Complications ; Female ; Humans ; Hypoglycemic Agents ; therapeutic use ; Kidney Neoplasms ; etiology ; Male ; Middle Aged ; Risk Factors

OBJECTIVETo study the content of mineral elements and the principal components in Gastrodia elata.

METHODMineral elements were determined by ICP and the data was analyzed by SPSS.

RESULTK element has the highest content-and the average content was 15.31 g x kg(-1). The average content of N element was 8.99 g x kg(-1), followed by K element. The coefficient of variation of K and N was small, but the Mn was the biggest with 51.39%. The highly significant positive correlation was found among N, P and K . Three principal components were selected by principal components analysis to evaluate the quality of G. elata. P, B, N, K, Cu, Mn, Fe and Mg were the characteristic elements of G. elata.

CONCLUSIONThe content of K and N elements was higher and relatively stable. The variation of Mn content was biggest. The quality of G. elata in Guizhou and Yunnan was better from the perspective of mineral elements.

Drugs, Chinese Herbal ; analysis ; Gastrodia ; chemistry ; Minerals ; analysis ; Principal Component Analysis

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

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

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 evaluate whether the clustering of risk factors, both environmental and genetic, increases the risk of essential hypertension (EH) and the accumulation of risk factors influences the blood pressure level in normotensives.

METHODSOn the basis of a prevalence survey, 501 subjects of Mongolian ethnicity (243 hypertensives and 258 normotensives) who were not related to each other were selected to conduct a case-control study. All subjects were interviewed with questionnaires and their blood specimens were collected. Renin gene insertion/deletion (I/D) polymorphism, a new genetic marker, was genotyped with PCR and polyacrylamide gel electrophoresis.

RESULTSOverweight, alcohol consumption, and renin gene I/D polymorphism were significant risk factors of EH (P<0.05). The odds ratios (OR) for the number of risk factors were 2.39 (95%CI: 0.98-6.74) for one risk factor, 5.03 (95%CI: 2.06-14.18) for two, and 6.09 (95%CI: 1.85-22.38) for three respectively after adjusting for age and sex. In normotensives, age- and sex-adjusted mean blood pressures increased with more accumulation of risk factors. However, there were no significant differences among the different blood pressure levels according to the number of risk factors (P>0.05).

CONCLUSIONOverweight, alcohol consumption, and renin gene I/D polymorphism are risk factors of EH in the Mongolian ethnic population of China. The accumulation of the risk factors causes a sharp increase of the risk of EH.

Adult ; China ; ethnology ; Cluster Analysis ; Female ; Humans ; Hypertension ; epidemiology ; Male ; Middle Aged ; Mongolia ; epidemiology ; ethnology ; Odds Ratio ; Risk Factors

PURPOSE: To distinguish lupus flare-up from infection in systemic lupus erythematosus (SLE), we analyze the expression of circulating CD27(high) plasma cells in SLE patients with and without infection, in comparison to non-SLE patients with infection. MATERIALS AND METHODS: The percentage of circulating CD27(high) plasma cells was measured by flow cytometry in the following four groups: 36 SLE patients without infection, 23 SLE patients with infection, eight non-SLE patients with infection, and 26 healthy controls. RESULTS: The frequency of CD27(high) plasma cells had a correlation with the SLE disease activity index (SLEDAI) (r = 0.866, p < 0.05), level of anti-dsDNA (r = 0.886, p < 0.05), C3 (r = - 0.392, p < 0.05), and C4 (r = - 0.337, p < 0.05) in SLE patients without infection, but there was no correlation with disease activity in SLE patients with infection. Among three groups in particular-SLE without infection, SLE with infection, and non-SLE with infection-the percentages of CD27(high) plasma cells were elevated. The percentage of CD27(high) plasma cells was higher in SLE patients with infection, when compared to SLE patients without infection. CONCLUSION: The percentage of CD27(high) plasma cells is a biomarker for disease activity of SLE without infection, under correlation with SLEDAI, anti-dsDNA, and C3 and C4 level. However, when the SLE patients have an infection, the percentage of CD27(high) plasma cells is not an adequate biomarker for the survey of disease activity. The percentage of CD27(high) plasma cells may serve as a potential parameter to distinguish a lupus flare-up from infection.
Adult ; Antigens, CD27/*biosynthesis ; Bacterial Infections/complications ; Biological Markers/metabolism ; Case-Control Studies ; Female ; Flow Cytometry/methods ; Humans ; Lupus Erythematosus, Systemic/*blood/immunology ; Male ; Plasma Cells/cytology/*immunology ; Virus Diseases/complications