Objective To study the effect of Potentilla Discolour Bunge (PDB) on the NOS expression of the vascular endothelial cells (VEC) of DM rats. Methods The DM rat model was established by alloxan injected, and then the rats were treated with herb of PDB for 4 weeks continuously. The NOS expression of VEC were assayed by histochemistry method and image analysis system. Result NOS OD value of the PDB group was higher than that of model group and Glibenclamide group (P

Objective To discuss and analyze the CT appearances and differential diagnosis of pulmonary tuberculomas. Methods 40 cases of pulmonary tuberculomas proved by surgery and pathology were included in the study and compared to 40 cases of peripheral-type bronchogenic carcinomas, which were also surgico-pathologically proved. Results Tuberculomas were most found in the posterior segments of the lung and exhibited well-demarcated lesions of homogeneous density. Some lesions also possessed characteristics like rough spiculation, minute calcification within the lesion and marginal calcification. Thick-walled, thin-walled or stellate cavities were also found. On contrast enhanced scans, tuberculomas showed little or ring enhancement. In addition, satelite lesions or pleural thickening were often found near the tuberculomas. Hilar and mediastinal lymph-nodes were calcified but not enlarged. Anti-tuberbulosis therapy often resulted in little or even no absorption.Conclusion More accurate diagnosis of pulmonary tuberculomas could be made after careful analysis of all CT signs and the clinical data. CT-guided needle biopsy could be used when the dignosis could not be made clearly.

Objective To observe the immunological effect of tumor cell vaccines to mouse hepatoma A(HepA) treated by Haematoxylin. Methods HepA cell was treated by 0.1% Haematoxylin and made into three vaccines: HepA vaccine with complete Freund' s adjuvant, HepA vaccine with incomplete Freund' s adjuvant; and HepA vaccine without any adjuvants. Five times of immunization were given the grouped mice with the above three vaccines, then active HepA cells (1×105 for each mouse) were inoculated by intraperitoneal injection to attack them; reckoning the mean survival time (MST) of the grouped mice, comparing the immunoprotective action of the three vaccines to the tmnor-bearing mice. Those mice only receiving normal saline (equal volume to the vaccine) were as a control group. Results MST of control group was (23.30±1.24) day; MST of mice receiving H22 vaccine with complete adjuvant was (43.90±15.20) day (P<0.02); MST of mice receiving H22 vaccine with incomplete adjuvant was (39.60±13.77) day (P<0.05); and MST of mice receiving HepA vaccine without any adjuvant was (38.40±12.54) day (P<0.05); As compared with the control group, the three treated groups showed a life-lengthening rate (LLR) 88.41%, 69.96 % and 64.81% respectively. Conclusion The vaccines treated by Haematoxylin give a marked immunoprotective action to those tumor-bearing mice. The HepA vaccine' s immunological effect is increased by the Freund' s adjuvant (complete or incomplete).

Objective To investigate Nuclear factor-κB (NF-κB) expression in tumor associated inflammation in patients with adamantinomatous craniopharyngima..Methods Fifty-four patients (31 male and 23 female) with craniopharyngioma from 3 to 66 years of age were recruited from May 2004 to March 2006.NF-κB and Osteopontin (OPN) expression in human craniopharyngiomas were detected using immunohistochemical staining.High-sensitivity C-reactive protein (hs-CRP), a systemic marker of inflammation, was examined in patients' tumor hydatid fluid, cerebrospinal fluid and serum.Results NF-κB expression was significantly increased in the adamantinomatous craniopharyngioma.Spearman;s correlation analysis demonstrated that NF-κB expression was associated with OPN expression.The hs-CRP level was also increased in the tumor hydatid fluid (4.28±0.90 mg/mL), cerebrospinal fluid (0.035±0.006 mg/mL) and serum (1.72±0.54 mg/mL) in patients with adamantinomatous craniopharyngioma.Conclusions NF-kappa B is closely associated with tumor associated inflammation which further mediates adhesion of tumor to surrounding important structures in patients with adamantinomatous craniopharyngioma.

OBJECTIVETo observe the therapeutic efficacy of tetrandrine tablets combined with matrine injection in the treatment of silicosis.

METHODSSixty-three patients with silicosis were randomly divided into treatment group (n = 33) and control group (n = 30). Both groups received anti-inflammatory, cough-relieving, and anti-asthmatic treatment. Meanwhile, the treatment group was given tetrandrine tablets (100 mg bid) and matrine injection (150 mg qd). There were 4 courses of tetrandrine treatment (each course = 3 months), with one-month intervals among them. Matrine injection was used for 15 consecutive days in each month. There were 2 courses of matrine treatment (each course = 3 months), with a one-month interval in between. The clinical symptoms, pulmonary function, serum superoxide dismutase (SOD) activity, and chest X-ray images were observed before and after treatment.

RESULTSAfter treatment, chest distress, chest pain, shortness of breath, and other respiratory symptoms were relieved significantly (P < 0.05). The treatment group showed significantly higher SOD activity than before treatment and the control group (P < 0.05) and significantly higher forced vital capacity and forced expiratory volume in one second than before treatment and the control group (P < 0.05). After treatment, 5 patients (4 stage II cases and 1 stage III case, all in rapidly progressive forms) in the treatment group showed smaller, lighter, and clearer shadows with decreased overall intensity on chest X-ray; 12 patients showed significantly fewer and clearer lung markings on chest X-ray.

CONCLUSIONTetrandrine tablets combined with matrine injection have some therapeutic effect on silicosis.

Aged ; Alkaloids ; administration & dosage ; therapeutic use ; Benzylisoquinolines ; administration & dosage ; therapeutic use ; Humans ; Injections ; Male ; Middle Aged ; Quinolizines ; administration & dosage ; therapeutic use ; Silicosis ; drug therapy ; Tablets ; Treatment Outcome

BACKGROUND: Inhibiting the degradation of extracellular matrix in the intervertebral disc can delay the degenerative process of intervertebral disc. Matrix metalloproteinases-3 (MMP3) is considered as a key enzyme for degradation of extracelular matrix components such as type II collagen and aggrecan.
OBJECTIVE:To construct the short hairpin RNA lentiviral vector targeting human MMP3 gene and to detect its efficiency of gene silence by infecting human degenerative nucleus pulposus cells.
METHODS:According to the human MMP3 mRNA (NM_002422.4) sequence, four groups of the short hairpin RNA gene sequences targeting MMP3 were designed, synthesized and annealed to form double stranded DNA fragments, which were connected with the LV3 vectors digested by BamHI andEcoRI enzymes, and then transfected into the competent cels. The positive clones were identified by PCR, and analyzed by sequencing. The packaging and titer of lentivirus were determined after transfecting 293T cells. Human degenerative nucleus pulposus cels were infected with lentivirus vector, and the transfection efficiency of each group was observed under inverted fluorescence microscope. The interfering efficiency was detected by real time-PCR and western blot at 72 and 96 hours.
RESULTS AND CONCLUSION:The ds-oligo DNA was successfully inserted into the lentiviral vector as confirmed by electrophoresis and sequence analysis. The recombinant lentivirus was harvested from 293T cels with a viral titer of 1-5 ×108 TU/mL. RNA interference targeting the GCC AGG CTT TCC CAA GCA AAT sequences with the highest interfering efficiency in MMP3 gene at 72 and 96 hours resulted in suppression of MMP3 mRNA expression by 98% and 72%, respectively; and at 96 hours, the interfering efficiency of protein expression was 57.2%. The recombinant lentivirus vector containing RNA interference targeting MMP3 gene is successfuly constructed, which lays a foundation for further studies on the MMP3 function and gene therapy.

Objective To observe the effect of Puerarin on the level of tau phosphorylation in the olfactory bulb of Alzheimer's disease rat brain, and explore the underlying molecular mechanism.Methods ① Twenty-two male SD rats were randomly divided into the normal control group, model control group and Puerain-treated group.The levels of tau-1, PS396 and tau-5 in the olfactory bulb were detected by Western blotting.② Twenty male SD rats were randomly divided into model control group, low-dose Puerarin (40 mg·kg-1·d-1), medium-dose puerarin (80 mg·kg-1·d-1) and high-dose puerarin (160 mg·kg-1·d-1) groups.The levels of tau-1 and PS396 phosphorylation in the olfactory bulb were detected by Western blotting.③ The level of GSK-3β phosphorylation in the olfactory bulb of the normal control group, model control group A and puerain-treated group was detected by Western blotting.Results ① It was shown by Western blotting that the relative expression of tau-1 was significantly decreased in the olfactory bulb of the model group A(0.49±0.07)rat brain compared with the normal control group(0.85±0.03)(P<0.01), and the level of tau-1 was obviously higher in the puerarin-treated group(0.58±0.03)compared with that of the model group A(P<0.05).The differences of the levels of tau-5 and PS396 in the olfactory bulb were insignificant among the 3 groups.②Compared with the model group B, the expression of tau-1 in the olfactory bulb was significantly enhanced in the low-, medium-and high-dose of puerarin group: (0.39±0.09)vs(0.69±0.11),(0.55±0.11),(0.70±0.04);and the level of PS396 was significantly decreased in the olfactory bulb of low-dose puerarin group(0.36±0.07) compared with the model group B(0.55±0.05)(P<0.01).③Compared with the normal control group(0.96±0.07), the ratio of pS9-GSK-3β/tGSK-3β was obviously decreased in the olfactory bulb of the model group A(0.51±0.12),while that was significantly increased in the puerarin group(0.62±0.03) compared with the model group A(P<0.01).Conclusion Puerarin can attenuate AD-like tau hyperphosphorylation in the olfactory bulb of Alzheimer's disease rat brains, and decreased activity of GSK-3β might be involved in the effects of puerarin on tau hyperphosphorylation.

Objective To construct,express,purify and identify the gene encodi ng major outer membrane protein of Neisseria gonorrhoeae (Porin I, or PI). Metho ds The gene encoding for PI of N.gonorrhoeae was amplified by PCR and cloned int o expression plasmid pGEX-4T-2 to form pGEX-4T-2/PI recombinants. A high lev el expression of GST-PI fusion protein was obtained in GST gene fusion system (GST:glutathione S transferase). The analysis indicated that the expressed pr otein was present predominantly in the insoluble form. Therefore, the induced pr otein was purified by SDS-PAGE, and bands corresponding to polypeptides of GST-PI fusion protein were excised and subjected to electroelution. A dot immunoch romatographic assay was employed to demonstrate whether the purified protein was gonococcal PI specific. Results The pGEX-4T-2/PI expression recombinants were constructed,expressed,purified and identified successfully. SDS-PAGE analysis and dot immunochromatographic assay suggested that the recombinant GST-PI fusio n protein was a 60 000 molecular weight protein andidentical in size to native PI and reacted with anti-PI monoclonal antibody. Conclusion Our results may lead to a potentiality for further study of diagnosti c kits and vaccine for Neisseria gonorrhoeae.

Objective To construct four expression plasmids, pcDNA3.1-GFP/HPV6bE6, pcDNA3.1-GFP/HPV6bE7, pcDNA3.1-GFP/HPV11E6, pcDNA3.1-GFP/HPV11E7 and their transfected murine cell lines. Methods The Four recombinant expression plasmids comprising HPV6bE6,HPV6bE7,HPV11E6 and HPV11E7 linked with GFP, respectively, were constructed and transfected to B16 cells by lipofectamine kit. Positive clones were selected by G418 and observed by fluorescent microscopy and identified by RT-PCR. Results The four constructed recombinant plasmids were authenticated by restriction enzyme digestion and DNA sequencing. Under the fluorescent microscope, the green fluorescence could be observed in cytoplasm and nucleus of four transfected B16 cell lines. The RNA extracted from positively transfected clones resistant to G418 were analyzed by RT-PCR, which demonstrated the presence of four expected fragments. Conclusions The transfected murine cell lines B16 can express HPV6bE6,HPV6bE7,HPV11E6 and HPV11E7 gene. These transfected cell lines can be further transplanted to mice in order to investigate the biological properties and immunological mechanisms of these genes in vivo.

BACKGROUND: As the preliminary experiment for gene therapy in intervertebral disc degeneration, this study aims to construct a recombinant plasmid containing fluorescent pAAV-hSOX9-IRES-tdTomato for adeno-associated virus packaging, in a broader attempt to lay the foundation for late experiments in vitro and in vivo.OBJECTIVE: To construct human SOX9 gene overexpressing adeno-associated virus, pAAV-hSOX9-IRES-tdTomato, packaging. METHODS: The plasmid pAAV-IRES-tdTomato and plasmid pUC57-hSOX9 were connected into pAAV-hSOX9-IRES-tdTomato by enzyme digestion method. The adeno-associated virus was packaged with plasmid co-transfections method. The recombinant pAAV-hSOX9-IRES-tdTomato was transfected into 293AAV cell by calcium phosphate transfection. The purification and drop of adeno-associated virus was tested by determination of biological titer. RESULTS AND CONCLUSION: The results of BLAST sequence comparison analysis showed that, pAAV-hSOX9-IRES-tdTomato exactly matched the synthetic gene sequence hSOX9. The titer is 1×107 TU/mL. Human gene SOX9 recombinant adenoviruses, pAAV-hSOX9-IRES-tdTomato, have been constructed successfully.