Objective To detect the gene expression of Pax6 in retinoblastoma (Rb).Design Experimental study.Participants Six cases of fresh Rb tumors and two established Rb cell lines.Six cases of retina tissues as normal control group.Methods Pax6 gene ex- pression was assessed in two established Rb cell lines,six primary tumors and six cases of retina tissues using the reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot.Main Outcome Measures Expression of Pax6 gene in fresh retinoblastoma tis- sue and cell lines.Results The mRNA of Pax6 gene was positive in all two established Rb cell lines and the six primary Rb tumors using RT-PCR.The protein of Pax6 gene was also positive in Western blot.Pax6 mRNA and protein expression in the Rb tumors were increased compared with normal control group (P

To construct a system of genetic transformation suitable for Rhizopus oryzae, we constructed a single-exchange vector pBS-hygro carrying hygromycin B resistance gene (hph) as its selective marker using gene splicing by overlap extension PCR (SOE PCR) technique. We introduced this recombinant vector into Rhizopus oryzae AS 3.819 by PEG/CaCl2-mediated transformation of protoplast, electroporation of protoplast and germinated spores; and we studied the effects of hydrolysis time, field strength and spore germination time on transformation frequency. We conducted quantitative real-time PCR (qPCR) assay to determine the gene copy number of ldhA integrated in the genome of R. oryzae transformants and its effect on the stability of transformants. We successfully achieved R. oryzae transformants integrated with pBS-hygro-ldhA vector. The optimal hydrolysis time for protoplast production was 140 min, and the optimal field strength of electroporation pulse for protoplast was 13 kV/cm. The optimal germination time of spores for electroporation was 2.5 h, and the optimal field strength of electroporation pulse was 14 kV/cm. The transformation frequency of method based on germinated spores was generally higher than the methods based on protoplast. The qPCR test results suggested that transformants with high copy number of integration in a certain range were relatively stable. Our results provided basis and support for metabolic regulation and genetic engineering breeding of R. oryzae.
DNA, Recombinant ; Electroporation ; Genetic Engineering ; Genetic Vectors ; Hygromycin B ; Protoplasts ; Real-Time Polymerase Chain Reaction ; Rhizopus ; genetics ; Transformation, Genetic

Antilymphocyte Serum ; HLA Antigens ; immunology ; Hematopoietic Stem Cell Transplantation ; Humans ; Isoantibodies ; Prognosis ; Transplantation, Homologous

Coix ; Epimedium ; Fatigue ; Humans ; Hypoxia ; Plant Leaves

OBJECTIVETo study the roles of allergen testing in vitro and impulse oscillometry for lung function measurements in preschool children with cough variant asthma (CVA).

METHODSethodsForty-four preschool children with acute asthma, 41 with chronic asthma, 46 with CVA, and 35 healthy preschool children as control were recruited in the study. Inhaled allergen, food allergen, and mite-specific IgE were determined by Pharmacia UniCAP System. Serum eosinophil cationic protein (ECP) and total IgE levels were measured. Lung function was assessed by impulse oscillometry.

RESULTSThe positive rates of inhaled allergen and food allergen, and total IgE levels in the CVA, acute asthma and chronic asthma groups were higher than those in the control group (P<0.01). However, no significant differences were found among the three case groups. The serum ECP levels in the CVA group were lower than those in the acute asthma group (P<0.01), but did not show differences when compared with the chronic asthma group. The impulse oscillometry demonstrated that the respiratory total impedance (Zrs), airway resistance at 5 Hz (R5), airway resistance at 20 Hz (R20), subtracting R5 from R20 (R5-R20) and resonant frequency (Fres) in the CVA, acute asthma and chronic asthma groups were higher than those in the control group (P<0.01). Zrs, R5, R20, R5-R20, and Fres in the CVA and chronic asthma groups were lower than those in the acute asthma group (P<0.01). Serum ECP levels were positively correlated with Zrs, R5, R5-R20 and Fres (P<0.05) in the CVA and chronic asthma groups.

CONCLUSIONSThe measurements of allergens, serum ECP and impulse oscillometry for lung function are helpful for the evaluation of airway inflammation and airway obstruction in preschool children with CVA.

Allergens ; immunology ; Asthma ; immunology ; physiopathology ; Child ; Child, Preschool ; Cough ; immunology ; physiopathology ; Eosinophil Cationic Protein ; blood ; Humans ; Immunoglobulin E ; blood ; Lung ; physiopathology ; Oscillometry ; methods

Objective To establish a method for content determination of total flavones and polysaccharides in Hedysari Radix in different producing areas in Gansu Province.Methods The contents of total flavones and polysaccharides in Hedysari Radix in different producing areas in Gansu Province were determined by ultraviolet spectroscopy with calycosin-7-glucoside and glucose as reference substance, and the wavelength was set at 260 nm and 484 nm.Results The contents were from 2.82 mg/g to 6.79 mg/g for total flavones and from 106.14 mg/g to 746.40 mg/g for total polysaccharides in Hedysari Radix in different producing areas in Gansu Province. The recoveries of total flavones and total polysaccharides were 97.96％ and 102.90％, respectively.Conclusion There was difference in contents of total flavones and polysaccharides of Hedysari Radix in different producing areas in Gansu Province, and the method of using ultraviolet spectroscopy is simple, reproducible, accurate and reliable, which can be preferably used as the method for content determination of total flavones and polysaccharides in Hedysari Radix.

OBJECTIVESTo investigate the effects of paclitaxel loaded ultrasound contrast agents on cell cycles and ultrastructural features of HepG2 cells.

METHODSHepG2 cells were cultured, and divided into a blank control group, a paclitaxel group, an ultrasound contrast agents group, and a paclitaxel loaded ultrasound contrast agents group. Cell cycles of the four groups were detected by flow cytometry, and the ultrastructural changes of the cells were observed under a transmission electron microscope.

RESULTSPaclitaxel loaded ultrasound contrast agents blocked the HepG2 cells at their G2/M phases, and it also induced more apoptosis of the HepG2 cells.

CONCLUSIONSPaclitaxel loaded ultrasound contrast agents can block HepG2 cells at the G2/M phase and induce apoptosis of the cells.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Contrast Media ; pharmacology ; Hep G2 Cells ; Humans ; Microscopy, Electron, Transmission ; Paclitaxel ; pharmacology

·AIM: To compare clinical effects and cost of panretinal photocoagulation (PRP) combined with Ranibizumab or triamcinolone acetonide (TA) for diabetic macular edema (DME). ·METHODS: Forty-eight patients (48 eyes) with DME and diabetic retinopathy ( DR) receiving PRP were randomly assigned to two groups, which were respectively intravitreally injected ranibizumab (0. 5mg) and TA (4mg). Ranibizumab (0.5mg) was intravitreal injected every 4wk for 3 times. The effects of injection for DME were evaluated using best-corrected visual acuity (BCVA ), central macular thickness ( CMT ) and intraocular pressure (IOP). During the follow-up, other injections were performed to eyes which had CMT greater than 400μ m. The medical costs were calculated at 12wk and 24wk.·RESULTS: BCVA and CMT between 2 groups were not significantly different (P>0.05); BCVA and CMT among different time points were significantly different(P<0.05);the treatments and the time points had significant interaction on BCVA (P<0.05). BCVA was improved in two groups at all the time after injection(P<0.05),except 1wk after injection of TA (P=0.33). There was significant difference between the two groups at 12wk and 16wk on BCVA and that injected with ranibizumab was better (P=0.03,0.045). CMT decreased in two groups at all the time after injection (P<0.05). There was significant difference only between the two groups at 1wk (P< 0. 01). All intraocular pressures were in the normal range, except one needed ocular hypotensive agents. The medical costs (yuan) of the ranibizumab group in 12wk and 24wk were 38 736 and 42 564,which of the TA group were 5 790 and 7 053,respectively. ·CONCLUSION:Both PRP combined with ranibizumab or TA for DME can effectively control disease progression in short time. Therapeutic effect is not significant between two methods, but PRP combined with TA is more economic.

OBJECTIVETo investigate the roles of Kruppel-like factor 6 (KLF6) and its splice variant KLF6V on suppressing growth and inducing differentiation of human hepatocellular carcinoma hepG2 cells.

METHODKLF6V cDNA was amplificated by RT-PCR from human hepatocellular carcinoma (HCC) tissue and then sequenced. The recombinant vectors expressing KLF6 variant (KLF6V) were constructed using molecular clone technology based on established plasmid pcDNA3.1A(-)/wtKLF6. KLF6V or KLF6-transfected HepG2 cells were established after being screened with G418. Growth activity of HepG2/KLF6 or HepG2/KLF6V cells was detected by in vitro MTT assay. Expression of p21WAF1 or cyclin D1 protein was detected by Western blot, and expressions of AFP or ALB protein were measured by radioimmunoassay.

RESULTSA novel alternatively spliced transcript of the human KLF6 gene was found and its sequencing revealed that the variant form of KLF6 lacked 126nt and its encoded protein products had a deletion of 42 aa near the COOH-terminal amino acid in comparison with full-length KLF6. Although KLF6 alternative splicing was present in both normal and cancerous tissues, expression of the KLF6 splice variants seemed to be up-regulated in HCCs tissues. The isoform of KLF6 proteins antagonized the ability of wild-type KLF6 to up-regulate p21 expression or down-regulate cyclin D1 expression and suppress HepG2 cell proliferation. KLF6 gene increased albumin production and decreased alpha fetoprotein production of the cells.

CONCLUSIONThe isoform of KLF6 protein, present in HCC tissue, antagonizes the ability of wild-type KLF6 to suppress cell proliferation and induce cellular differentiation.

Cell Differentiation ; Cell Proliferation ; DNA, Complementary ; Gene Expression Regulation, Neoplastic ; Hep G2 Cells ; Humans ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; Protein Isoforms ; genetics ; Proto-Oncogene Proteins ; genetics ; Transfection

Objective To analysis the clinical efficacy of alteplase com-bined with butylphthalide sodium chloride injection on patients with acute cerebral infarction and its effect on the expression of serum stress factors. Methods A total of 142 patients with acute cerebral infarction were ran-domly divided into treatment group and control group, with 71 cases each.Patients in two groups were all treated with butylphthalide sodium chloride injection100 mL each time, twice a day, with over 6 h interval, injection time was 50-70 min.Alteplase was added in treatment group on the basis of control group, 5 mg alteplase was dissolved in 10 mL 0.9%NaCl for intravenous injection within 10 seconds.The remaining 45 mg was dissolved 0.9% NaCl in 100 mL for intravenous injection within 60 min, the injections were proceeded once a day.The course was 2 weeks of the two groups.The clinical efficacy and the changes of neu-rological function and expression of stress factor of the 2 groups were compared.Results The total effective rate in treatment group was 95.8%, and 83.1% in control group ( P<0.05 ) .Cerebral infarction volume and US National institutes of health stroke scale ( NHISS ) significantly reduced at 1 and 2 weeks after treatment in both groups, Barthel index ( BI) significantly increased ( P<0.05).The improvement in the treatment group was more obvious (P<0.05).Interleukin -6 (IL-6), IL-8, IL-10, C-reaction protein significantly decreased (P<0.05) after 1 and 2 weeks treatment in both groups, treat-ment group obviously decreased( P<0.05).Conclusion Alteplase combined with butylphthalide sodium chloride in-jection could reduce the expression of serum stress factor in patients with acute cerebral infarction, and could control the volume of cerebral infarction and improve the neural function.