GLuc (Gaussia luciferase) is a secreted luciferase with high sensitivity. In this study, we primarily compared expression character of PTTR with that of PCMV, relied on easy secretion, high sensitivity and simple and fast detection of GLuc. We firstly constructed two plasmids pAAV2-neo-TTR-GLuc and pAAV2-neo-CMV-GLuc. Then, 4 cell lines were transfected with the two plasmids in aid of Lipofectamine 2000, including Huh7 and HepG2, which are derived from liver cells, as well as HEK293 and HeLaS3 cells, which are non-liver cell lines. We monitored the expression of GLuc in the supernatant of these cell cultures at different time points post-transfection. Furthermore, we injected the two plasmids with different doses into BALB/c mice by the means of hydrodynamic delivery and monitored the GLuc expression in vivo with 2.5 microl tail tip blood since 2 h post-injection. The cell assay results suggested that the expression of GLuc driven by CMV promoter was significantly higher than that of GLuc driven by TTR promoter. And, the luciferase activity of GLuc driven by CMV promoter was 50-300 times higher than that of GLuc driven by TTR promoter in HEK293 and HeLaS3 cell lines, but less than 10 times higher than that of GLuc driven by TTR promoter in the HepG2 and Huh7 cell lines, indicating the relative liver-specificity of TTR promoter. In the animal assay, the higher luciferase activity was determined in CMV promoter group than in TTR promoter group at different doses of the two plasmids. But the expression patterns for the two promoters differed obviously. The expression of GLuc driven by CMV promoter reached the maximum 10 hours post-injection and declined rapidly; while the expression of GLuc driven by TTR promoter reached the maximum 48 hours after delivery, and declined very slowly. These results implied that PTTR could keep expression of driven gene in a long time although its expression intensity is lower than PCMV's. Thus, it is more suitable for maintaining longer expression of target genes in liver.
Animals ; Cell Line ; Cytomegalovirus ; genetics ; metabolism ; Gene Expression ; Gene Transfer Techniques ; Genes, Reporter ; Humans ; Luciferases ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Prealbumin ; genetics ; metabolism ; Promoter Regions, Genetic

Objective To examine the activation and cytotoxieity of human peripheral blood T lymphocyte induced in vitro by human 4-1-BB ligand(4-1-BBL) gene transfected into tumor Tca8113 cells.Methods The eukaryotic expression vector pEGFP-h4-1-BBL was transfected into human oral carcinoma cell line Tca8113 by Lipofectamine<'TM>2000. The transfected cells were then selected in medium containing G-418, cloned by limited dilution and named as Tca8113-4-1-BBL Human 4-1-BBL mRNA and protein expression of transfected cells was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting respectively. The tumor cell vaccines (TCV) were obtained by treatment with mitomycin (MMC). Human peripheral blood mononuclenr cells (PBMC) were prepared from lymphoprep, and then stimulated with anti-CD-3 mAb and incubated with non-transfected or transfected TCV-TcaSI13 cells,respectively. The proliferation of T cells was evaluated by trypan blue exclusion; the CCK-8 was used to detect the cytotoxic effect of T lymphecytes. Meanwhile, the secretion of intefferom-γ (IFN-γ) and interleukin(IL)-2 in culture supernatant was detected by enzyme-linked immunosorbnent assay (ELISA).Results The Tca8113 cells trasfected by pEGFP-h4-1-BBL could express human 4-I-BBL efficiently. As compared with wild type Tca8113 cells, the transfected Tca8113 cells could markedly promote proliferation,IL-2 and IFN-γ production and cytotoxic activity of lymphocytos. Condusions The transfection of human 4-1-BBL gene in Tca8113 cells is effctive in enhancing its immunogenicity and inducing antitumor immune response in vitro.

Gastrodia elata Bl. is a famous and costful traditional Chinese medicine. Their genomic DNA fingerprints were investigated using a modified Randomly Amplified Polymorphic DNA method. DNA fragments common to all or to fine populations were identified and recovered. Five DNA fragments were proven not to be reported through DNA cloning, PCR identifying, nucleotide sequencing and bioinformatics analyses and were received in and recorded by NCBI GenBank. Gastrodine contents of the Gastrodia tuber samples were determined using high performance liquid chromatography technique. The distribution of the five DNA fragments in 9 Gastrodia elata Blue populations and the correlation with gastromedicine content were studied. The results show the distribution of these DNA sequences varied greatly among the populations whereby DNA Sequence 1 was the common and distinguishing molecular marker for all the populations studied and DNA Sequence 2 may relate to higher gastrodine content. In conclusion, these DNA marker sequences can be employed to identify genuine gastrodia tubers, better varieties and optimize their selection and cultivating.
Base Sequence ; Benzyl Alcohols ; analysis ; Cloning, Molecular ; Computational Biology ; DNA, Plant ; chemistry ; Gastrodia ; genetics ; Glucosides ; analysis ; Plant Tubers ; genetics

OBJECTIVETo investigate the clinical manifestation of patients with renal injury induced by chronic mercury intoxication and the application of the diagnostic criteria of occupational mercury poisoning.

METHODSThe clinical data of 8 patients with chronic occupational mercury intoxication were analysed and evaluated.

RESULTSAll the observed clinical signs of chronic mercury intoxication correspond with the items of the diagnostic criteria of occupational mercury poisoning. The increasing beta2-MG was one of the clinical manifestations of renal injury induced by chronical mercury intoxication. The renal injury obviously was dose-dependent and reversible.

CONCLUSIONSThe national diagnostic criteria of occupational mercury poisoning is practically valuable. The renal injury induced by chronic mercury intoxication should not be neglected.

Adult ; Female ; Humans ; Male ; Mercury Poisoning ; diagnosis ; Middle Aged ; Occupational Diseases ; diagnosis ; Reference Standards

Objective To explore the clinical features,diagnoses,differential diagnoses and treatments of superior cerebellar artery aneurysms.Methods The clinical data of 16 patients with superior cerebellar artery aneurysms,admitted to our hospital from January 2013 to March 2018,were retrospectively collected.Their clinical manifestations,imaging features,surgical effects and related problems in the process of diagnoses and treatments were analyzed.Results Among the 16 patients,11 were caused by aneurysm rupture;8 had subarachnoid hemorrhage alone,and three had subarachnoid hemorrhage accompanied by ventricular hemorrhage;CT and CTA confirmed that 8 were superior cerebellar artery aneurysms,two were posterior cerebral artery aneurysms,and one was with unclear diagnosis.In the other 5 patients,three had eyelid ptosis and two had abducent nerve palsy;CT,CTA or MR imaging showed that two were considered as ventral brainstem occupying lesions,and three did not have clear diagnosis.Finally,all patients were diagnosed as having superior cerebellar artery aneurysms by three-dimensional DSA.Five patients were treated with interventional embolization first,and one was treated with surgical clipping because of vertebral artery stenosis and difficulty of catheter access;two patients were transferred to our department for surgical clipping due to aneurysm rupture after embolization treatment in other hospitals;and 9 patients were treated by surgical clipping directly.After treatments,one patient was in bed for a long time due to cerebellar infarction and systemic complications,and the other 15 patients recovered well;two of them underwent ventricular peritoneal shunt due to hydrocephalus.Conclusions Superior cerebellar artery aneurysm has onset of subarachnoid hemorrhage mostly,and oculomotor and abductor nerve paralysis,and space occupying manifestation around the brainstem sometimes.For patients with suspicious posterior circulation aneurysms whose diagnosis or location are unclear,three-dimensional DSA examination should be performed early to confirm the diagnosis.Treatment should be taken as soon as possible once the superior cerebellar artery aneurysm is defined.Interventional embolization may be the first choice,but it is necessary to master the methods of surgical clipping in order to treat the disease timely.

Objective To investigate the effect of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) as adjuvant on immune response in adults of non-and hyporesponders to hepatitis B vaccine. Methods Those who were once immunized with recombined yeast gene hepatitis B vaccine more than one standard scheme in two years and negative for hepatitis B markers were randomly sorted as group A and group B. 33 adults of group A were given hepatitis B vaccine 10 μg each time. The immune procedure was O, 1 and 6month. 34 adults of group B were given rhGM-CSF 300 μg for the first day, then 10 μg each time for routine immune. The blood samples were collected before the first injection and in 1, 2 and 8 months (T1, T2, TS)following the first injection to test Anti-HBs. Results Anti-HBs positive conversion rates of group A and B at T8 was 39.39% and 64.71% respectively(P=0.038). Anti-HBs levels of group B at TI, T2, T8 were(113.85±198.56) mIU/ml, (312.40±349.44) mIU/ml, (427.74±411. 58) mIU/ml (P=0.001). There was significant difference between group A and B in T8 Anti-HBs levels(P=0.010). Conclusion Better immune response was found in the group of rhGM-CSF with hepatitis B vaccine. So rhGM-CSF can induce the immune respond to hepatitis B vaccine.

OBJECTIVETo evaluate the effect of FLAMIGEL (hydrogel dressing) on the repair of residual burn wound.

METHODSSixty burn patients with residual wounds hospitalized in 6 burn units from November 2011 to May 2012 were enrolled in the multi-center, randomized, and self-control clinical trial. Two residual wounds of each patient were divided into groups T (treated with FLAMIGEL) and C (treated with iodophor gauze) according to the random number table. On post treatment day (PTD) 7 and 14, wound healing rate was calculated, with the number of completely healed wound counted. The degree of pain patient felt during dressing change was evaluated using the visual analogue scale (VAS). The mean numbers of wounds with score equal to zero, more than zero and less than or equal to 3, more than 3 and less than or equal to 6, more than 6 and less than or equal to 10 were recorded respectively. Wound secretion or exudate samples were collected for bacterial culture, and the side effect was observed. Data were processed with repeated measure analysis of variance, t test, chi-square test, and nonparametric rank sum test.

RESULTSWound healing rate of groups T, C on PTD 7 was respectively (67 ± 24)%, (45 ± 25)%, and it was respectively (92 ± 16)%, (72 ± 23)% on PTD 14. There was statistically significant difference in wound healing rate on PTD 7, 14 between group T and group C (F = 32.388, P < 0.01). Ten wounds in group T and four wounds in group C were healed completely on PTD 7, with no significant difference between them (χ(2) = 0, P > 0.05). Forty-two wounds in group T and seven wounds in group C healed completely on PTD 14, with statistically significant difference between them (χ(2) = 42.254, P < 0.01). Patients in group T felt mild pain during dressing change for 37 wounds, with VAS score higher than zero and lower than or equal to 3. Evident pain was observed in patients of group C during dressing change for 43 wounds, and it scored higher than 3 and less than or equal to 6 by VAS evaluation. There was statistically significant difference in mean number of wounds with different grade of VAS score between group T and group C (Z = -4.638, P < 0.01). Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, E. coli, Baumanii, and Staphylococcus epidermidis were all detected in both groups, but there was no statistical difference between group T and group C (χ(2) = 0.051, P > 0.05). No side effect was observed in either of the two groups during the whole trial.

CONCLUSIONSFLAMIGEL can accelerate the healing of residual burn wounds and obviously relieve painful sensation during dressing change.

Adolescent ; Adult ; Aged ; Bandages ; Burns ; therapy ; Female ; Humans ; Hydrogels ; Male ; Middle Aged ; Young Adult

Objective: To investigate the effect of 2, 2', 4, 4'-tetrabromodiphenyl ether (PBDE-47) on the mitochondrial mass in rat adrenal pheochromocytoma (PC12) cells and the potential mechanisms. Methods: Highly differentiated PC12 cells were divided into control, 1, 10 or 20 μmol/L PBDE-47-treated groups and cultured for 24 h. Transmission electron microscopy was employed to observe the changes in mitochondrial morphology and quantity in PC12 cells. Flow cytometry was used to measure the fluorescence intensity of Nonyl Acridine Orange (NAO) , a fluorescent indicator of mitochondrial membrane cardiolipin, to reflect mitochondria mass. Western blotting was used to determine the expression levels of Mitofusion 1 (Mfn1) and Fission 1 (Fis1) proteins. To further explore the role of abnormal mitochondrial fusion and fission in PBDE-47-induced mitochondrial mass changes, PC12 cells were divided into control group, 5 μmol/L M1 treatment group, 20 μmol/L PBDE-47 treatment group and 5 μmol/L M1+20 μmol/L PBDE-47 combined treatment group and cultured for 24 h, then the fluorescence intensity of NAO and expression levels of Mfn1 and Fis1 proteins were detected. Results: The control group showed numerous mitochondria with normal morphology, while the number of mitochondria decreased after PBDE-47 treatment. Especially, the disappeared cristae, swelling and vacuoles of mitochondria and decreased fluorescence intensity of NAO (P<0.05) were observed in 10 and 20 μmol/L PBDE-47-treated groups. Meanwhile, the expression levels of Mfn1 and Fis1 proteins in the 10 and 20 μmol/L PBDE-47-treated groups were significantly decreased compared with control group (P<0.05) . However, 5 μmol/L M1 co-treatment with 20 μmol/L PBDE-47 significantly increased the levels of Mfn1 and Fis1 proteins and fluorescence intensity of NAO compared with the 20 μmol/L PBDE-47 group (P<0.05) . Conclusion PBDE-47 can inhibit the mitochondrial fusion and fission process, thus leading to damage of mitochondria mass in PC12 cells.