Objective To investigate the application value of tuberculosis infection of T cells spot test in the diagnosis for pulmonary tuberculosis in elderly patients with chronic obstructive pulmonary disease(COPD).Methods According to test different ways,64 elderly COPD patients with pulmonary tuberculosis were divided into the two groups.The control group(n =32)was checked by tuberculosis antibody test,and the study group(n =32)received T cells infected with tuberculosis spot test.The two sets of results were observed and compared.Results The study group by T cell spot test TB infection,29 cases were detected positive(90.63%),3 cases of negative(9.37%);tuberculosis antibody detection by the control group,23 cases were detected positive(71.88%),9 negative patients (28.12%).Compared the two groups,the study group had higher positive rate(χ2 =3.954 7,P <0.05),and nega-tive rate was lower(χ2 =3.914 7,P <0.05).Conclusion Tuberculosis infection T cell spot test in the diagnosis of pulmonary tuberculosis in patients with COPD in the elderly has greater value,more accurate diagnosis,reliability and worthy of promotion.

Objective To compare the therapeutic effects of continuous veno-venous hemofiltration (CVVH) versus repeated intermittent veno-venous hemofiltration (RIVVH) on patients with severe acute pancreatitis (SAP).Methods Fifty-six patients with SAP were randomly divided into the CVVH group (n =28) and the RIVVH group (n =28).The clinical symptoms and signs,the APACHE Ⅱ and MODS scores,the result of biochemistry including amylase and lipase,and the plasma levels of TNF-α,IL-6,IL8 before and after treatment,the duration of mechanical ventilation,boosting drug application time,the length of stay in ICU,the surgical intervention rate and the mortality were compared between the two groups.Results The clinical symptoms improved in the two groups after treatment (P ＜ 0.05).The APACHE Ⅱ and MODS scores were all reduced in the two groups after treatment (P ＜ 0.05).When compared with the RIVVH group,the result of biochemistry including amylase and lipase,and the plasma levels of TNF-α,IL6,IL-8 were significantly decreased (P ＜ 0.05).The duration of mechanical ventilation,the length of stay in ICU and the mortality were also significantly decreased in the CVVH group (P ＜ 0.05).Conclusions CVVH was more efficacious than RIVVH in the treatment of SAP.

Objective:To investigate the stiffness characteristic of triple-negative human breast cancer at different size in a implantation nude female mice model using shear wave elastography(SWE) and to compare the clinical pathologic features of tumors with elasticity variables.Methods:Human breast cancer MDA-MB-231 cells were injected into 30 nude female mice and 27 transplanted tumors were successfully found in nude female mice. Ultrasound and SWE were longitudinally performed on maximum diameter plane of 21 tumours in 21 nude mice. The elastic parameters of maximal elasticity(Emax), mean elasticity (Emean) and standard deviation of elasticity(Esd) were recorded. The mice were divided into 3 groups according to the tumor size. They were group A with tumor size less than or equal to 5 mm, group B with tumor size greater than 5 mm and less than or equal to 10 mm, group C with tumor size larger than 10 mm and smaller than or equal to 15 mm. Compared with pathology, the relationships between Ki67 of transplanted tumor and elastic parameters were analyzed.Results:As the transplanted tumors increased, the values of Emax, Esd, Ki67 all increased. The lesions maximal size, Emax, Esd, Ki67 were significant higher in group B ( P<0.001, P=0.006, P=0.002, P=0.026) and group C ( P<0.001, P<0.001, P<0.001, P=0.028) than group A. The other parameters were not significantly different among the groups(all P>0.05). The size of transplanted tumors was significantly and positively correlated with Emax ( rs=0.673, P=0.001), Esd ( rs=0.661, P=0.001), and Ki67 ( rs=0.509, P=0.018). Conclusions:SWE Emax and Esd can reflect the tumor tissue stiffness change and biological activity during the tumor growth.

BACKGROUND: The increasing prevalence of diabetes mellitus has been become one of the diseases which threaten the heath of human being in the 21st century. Islet transplantation is considered to be the most effective approach to cure type Ⅰ diabetes mellitus. However, lack of donor tissue limits the application of this therapy. However, recent progress of stem cell research shows that stem cell therapy may be a potential means to solve this problem.OBJECTIVE: To take activin A and all-trans retinoic acid (AR) in inducing the differentiation of bone marrow mesenchymal stem cells (MSCs) and explore its possibility DESIGN: A randomized controlled experiment.SETTING: Institute of Biotechnology, Academy of Military Medical SciencesMATERIALS: This experiment was conducted at the Institute of Biotechnology, Academy of Military Medical Sciences from November 2004to June 2005. Six male Sprague-Dawley rats, with body mass of 150-160g, were provided by the Experimental Animal Center of Academy of Military Medical Sciences.METHODS: Femoral bone marrow of the rats was extracted under aseptic condition. Bone marrow mesenchymal stem cells (MSCs) were isolated with density gradient centrifugation. Passaged MSCs were randomly divided into 4 groups: high concentration of glucose (HG), AR, beta-mercaptoethanol (ME) and negative control groups. MSCs were induced to differentiate into IPCs with conditional medium containing high concentration glucose, activin A, RA and ME etc. After induction, phenotypes of differentiated cells were examined by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR).MAIN OUTCOME MEASURES: Expression of insulin and glucagon of differentiated cells were examined by immunocytochemistry. Insulin-1 mR-NA expression of differentiated cells was detected by RT-PCR.RESULTS: After bone marrow mesenchymal stem cells were induced,there were scattered insulin-and glucagon-positive cells in the HG group,many insulin-and glucagon-positive cells in the AR and ME groups, and these cells formed insulin-like structure. The expression of insulin-1mRNA could be observed in the HG, AR and ME groups. Insulin-and glucagonpositive cells and the expression of insulin-1mRNA were not observed in the negative control group.CONCLUSION: We adopt an induction scheme based on AR and other matured factors, and successfully make bone marrow mesench.ymal stem cells induce and differentiate into insulin positive reaction cells and form insulin-like structure, but its induction efficiency needs further improvement.

Objective To investigate the role of additional post core biopsy ultrasound in clinically node negative breast cancer.Methods Axillary ultrasound was performed before and after breast cancer was diagnosed on core biopsy samples.Post core biopsy ultrasound were performed by radiologists of this department of ultrasound at random.Post-diagnosis ultrasounds were performed by a radiologist with over 20 years of experience for the diagnosis of breast cancer with axi[lary disease.Results were compared to the final axillary pathological result.Results Of the 96 patients,17 were pathology lymph node positive.Post biopsy ultrasound identified 8 of the 17 positive nodes,with a sensitivity 47.1％,specificity 88.6％,positive predictive value of 47.1％,negative predictive value of 88.6％,accuracy of 81.3％.While the diagnosis index of pre-biopsy ultrasound were 47.1 ％,88.6％,47.1％,88.6％,81.3％,respectively.Conclusions Post-biopsy ultrasounds had an increased sensitivity for identifying positive axillary nodes,at the same times,specificity decreased.

Hepatitis B surface antigen (HBsAg) carrying preS sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. Here we report the success in achieving efficient and stable expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells. The HMRCHEF53u/Neo-S/preS1 expression vector carrying S/preS1 gene was constructed and transfected into CHO-S cells. A stable and high-expression CHO cell line, named 10G6, was selected by ELISA and limiting dilution analysis. Western blotting analysis showed S/preS1 expressed from 10G6 cells possessed both S and preS1 antigenicity. 10G6 cells displayed characters of favorable growth and stable S/preS1 expression in repeated batch cultures as evaluated by viable cell density, viability and S/preS1 concentration. And cultivation of 10G6 cells in fed-batch mode resulted in S/preS1 production at 17-20 mg/L with viable cell density at 7 x 10(6)-10 x 10(6) cells/mL.
Animals ; CHO Cells ; Cricetulus ; Epitopes ; biosynthesis ; genetics ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B Vaccines ; biosynthesis ; genetics ; Hepatitis B virus ; Protein Precursors ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

To establish a gene regulation system compatible with biopharmaceutical industry and gene therapy, we constructed a fusion protein of biotin ligase from Bacillus subtilis (BS-BirA) and the trans-activation domain, and used its expression vector as the regulatory vector. Meanwhile, BS-BirA-specific operators were ligated upstream of attenuated CMV promoter to obtain the response vector. In this way, a novel eukaryotic gene regulation system responsive to biotin was established and named BS-Biotin-On system. BS-Biotin-On system was further investigated with the enhancing green fluorescent protein (EGFP) as the reporter gene. The results showed that our system was superior to the current similar regulation system in its higher induction ratio, and that the expression of interest gene could be tuned in a rapid and efficient manner by changing the biotin concentrations in the cultures, Our results show that the established system may provide a new alternative for the exogenous gene modulation.
Bacillus subtilis ; Biotin ; chemistry ; Eukaryotic Cells ; metabolism ; Gene Expression Regulation ; Genetic Vectors ; Promoter Regions, Genetic ; Trans-Activators

With suspension adapted recombinant Chinese hamster ovary (CHO) cell lines 11G-S expressing human pro-urokinase (pro-UK) as the object of study, a serum-free medium for the cultivation of recombinant CHO cells in suspension was formulated by using Plackett-Burman design and response surface methodology. The two-level Plackett-Burman design was used to evaluate the effect of 10 medium supplements on the growth of the 11G-S cells in suspension culture. Among the 10 medium supplements, insulin, transferrin, and putrescine were identified as the most significant factors (P < 0.05). The response surface methodology with three factors and three levels was used to determine the optimal levels of these factors. And a serum-free medium, SFM-CHO-S for recombinant CHO cells suspension culture was formulated. The maximum cell density of 11G-S cells in SFM-CHO-S in suspension batch culture reached 4.12 x 10(6) cells/mL with a maximum pro-UK activity at 5614 IU/mL, which was superior to the commercial serum-free medium for recombinant CHO cells.
Animals ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Genetic Engineering ; Insulin ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Transferrin ; pharmacology ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics

Taking a suspension adapted recombinant CHO cell line, 11G-S expressing human Pro-urokinase (Pro-UK) as the object of study, the impacts of different feeding nutrients, the start time of feeding and cell inoculation density on the growth and Pro-UK production of 11G-S cells in serum-free fed-batch culture were evaluated in 100 mL shacking flasks. The results indicated that amino acids, serum-free supplements and inorganic salts played important role in cell growth, cell viability and protein expression. And the effects of cells fed-batch culture was much better with the initial cell inoculation density at 3 x 10(5)-4 x 10(5) cells/mL and the start time of feeding set at 72 h, a maximum viable cells density of 7.8 x 10(6) cells/mL with a peak Pro-UK activity at 8570 IU/mL was achieved during 12 d fed-batch culture. Further, the mu of the 11G-S cells at the middle phase of the fed-batch culture, and both the q(glu) and q(gln) of the 11G-S cells at the middle and later phases of the fed-batch culture was higher than that of the 11G-S cells at the same phase of the batch culture, respectively.
Animals ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics ; metabolism

Currently, exogenous gene expression system based on retroviral vector has been widely used as efficient gene expression system in both gene therapeutic research and RNA interference. In this study, we evaluated the efficiency of exogenous gene expression mediated by the retroviral vector in mammalian cells. First, we constructed EGFP (enhanced green fluorescent protein) vector using pcDNA3.1(+) and retroviral vector pQCXIN as backbone vector respectively. Then, we transfected or infected HEK293 cells and CHO-K1 cells with above vector or corresponding retroviral virus, and measured the relative fluorescence intensity (RFI) of EGFP. The results showed that the RFI of the retroviral virus-infected cells was two times higher than that of the plasmid-transfected cells. Further experiments revealed repeated virus infection enhanced the expression of EGFP markedly, with RFI increasing twice after four rounds of virus infection. Furthermore, the EGFP expression in HEK293 cells mediated by the retroviral vector was more stable than transfected with plasmid pcDNA3.1(+). Finally, we further validated the efficiency of exogenous gene expression system based on the retroviral vector by expressing recombinant human activated protein C (rhAPC) in HEK293 cells. We obtained HEK293 cell lines with rhAPC expression between 10 and 15 microg/(10(6) cells d). In conclusion, the exogenous gene expression system based on the retroviral vector is an alternative method for the generation of stable and high-expressing mammalian cell lines.
Animals ; CHO Cells ; Cricetinae ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; HEK293 Cells ; Humans ; Protein C ; biosynthesis ; genetics ; RNA Interference ; Recombinant Proteins ; biosynthesis ; genetics ; Retroviridae ; genetics ; metabolism ; Transfection