Objective To immortalize rat astrocytes which could be used as cell carriers for transgenic cellular analgesia. Methods Astrocytes were isolated from cerebral cortex of newborn SD rats by trypsin digestion and cultured according to the method of differential cell adhesiveness and transfected with plasmid pCMVSV40T/ PUR containing the simian virus 40 large tumor antigen (SV40Tag) gene. The positive colonies were isolated by puromycin selection and expanded by many passages. The integration and expression of large T antigen gene were detected and the immuno-reactivity of glial fibrillary acidic protein (GFAP) was determined by PCR, RT-PCR and immuno-cytochemistry. Results Rat astrocytes were successfully cultured in vitro and positively stained for the astrocytic marker GFAP. The positive colonies were isolated and subcultured for 50 passages. PCR and RT-PCR products were analyzed using 1.5% agarose gel electrophoresis. The size of amplifacation product of target gene was identical to that of the positive control (558 bp) . There was no PCR and RT-PCR product from non-transfected cells. DNA sequencing and BLAST showed that 558 bp nucleotides were identical to the SV40Tag gene of the Genbank (100%). The transfected cells were positively stained for the SV40Tag and GFAP. Conclusion Immortalized rat astrocyte strain with SV40 tag gene is constructed successfully.

Objective To study the biological character of rat astrocyte strain immortalized by simian virus 40 large T antigen gene(SV40Tag) and explore the feasibility of using it as cell vehicle for transgenic cellular analgesia.Methods Rat cerebral cortical astrocytes (AST) and immortalized rat astrocyte strain (IAST) were cultured in vitro. Morphology and growth features of AST and IAST were examined and compared after subculture, freezing and recovery. The ultrastructure of the cells was observed by transmission electron microscopy. The glial fibrillary acidic protein (GFAP) in these cells was detected by immuno-cytochemistry. The cell proliferation rate and cell cycle were determined by bromodeoxyuridine labelling and flow cytometry. AST and IAST were cultured in soft agar and inoculated in nude mice to investigate the tumorigenesis of IAST. Results LAST could be subcultured successively. The cells remained monolayer, anchorage dependent and the growth was attachment-inhibited. When AST was subcultured for only 10 passages, replicative senescense began. Subculture, freezing and recovery did not influence the shape and proliferation of IAST (94%?5%) , but decreased the vitality rate of AST (54%?4% ) (P

Objective To investigate the changes of histamine receptors in bladder before and after the treatment by sodium hyaluronate in rats of interstial cystitis (IC). Methods Twenty IC model rats were randomly divided into experimental group and control group, with 10 for each group.The bladders in experimental group were filled with sodium hyaluronate, while the rats in control group were executed at once. The bladders were dyed with HE staining, special staining and immunohistochemistry staining to count the number of mononuclear cells and mast cells and observe the changes of histamine receptors. Results In experimental group,the counts of mononuclear cells and mast cells were 12.20±2.48 and 2.90±0.87 respectively;the numbers of average optical of histamine receptor H1, H2, H3, H4 were 0.015±0.007, 0.006±0.001, 0.007±0.004, 0.061±0.026 , respectively. In control group, the counts of mononuclear cells and mast cells were 23.90±3.07 and 7.08±1.23;the numbers of average optical of histamine receptor H1, H2, H3, H4 were 0.055±0.033, 0.031±0.023, 0.033±0.017, 0.091±0.059, respectively. The differences of mononuclear cells and mast cells were significant between the 2 groups(P＜0.01). The differences of average optical of histamine receptor H1, H2, H3 between the 2 group were significant (P＜0.05), while the difference of histamine receptor H4 was not significant(P＞0.05). Conclusion Histamine receptor H1, H2, H3 take part in the development of IC, the antagons may be used for the treament of IC.

e combina-tion of protamine sulfate and potassium chloride to establish interstitial cystitis animal model is reliable and feasible. Researchers can choose the right time of irrigation based on the intent of the experiment.

Objective To evaluate therapeutic effects of simvastatin on serum expressions of cytokines and synovial tissue aspartic protease-3 (Caspase-3) in collagen induced arthritis (CIA) in rats, and the mechanism thereof. Methods The rat model of CIA was established by injecting bovine Ⅱ collagen. Sixteen model rats were randomly divided into two groups:CIA model group (sterile water 5 mL·kg-1·d-1 by gavage) and simvastatin group (2.0 mg·kg-1·d-1 by gavage). Seven normal rats were included in control group (sterile water 5 mL·kg-1·d-1 by gavage). The arthritis index (AI) and hind paw vol-umes were recorded once a week. The serum levels of cytokine, tumor necrosis factor (TNF)-αand interleukin (IL)-6 were measured by ELISA 42 days after the initial immunization. The expression of Caspase-3 in ankle synovial tissue was detect-ed by immunohistochemical method, and pathological results of HE staining in rat ankle were compared between three groups. Results Values of AI were decreased on the 24-d of the initial immunization in simvastatin group and CIA model group, which was significantly decreased on the 35-d of the initial immunization in simvastatin group than that of CIA model group (P＜0.05). The values of hind paw volumes were decreased on the 14-d of the initial immunization in simvastatin group and CIA model group, which was still significantly higher than those of control group (P＜0.05). The values of hind paw volumes were decreased on the 35-d and 42-d of the initial immunization in simvastatin group than those of CIA model group (P＜0.05). The serum levels of TNF-αand IL-6 on the 42-d of the initial immunization were significantly lower in simvastatin group than those of CIA model group, but which were significantly higher than those of control group ( P＜0.05). There were more synovial hyperplasia in simvastatin group than those of CIA model group. Only a small amount of inflamma-tory cell infiltration was found in simvastatin group. The expression of Caspase-3 was significantly higher in simvastatin group than that of CIA model group. Conclusion Simvastatin can significantly inhibit the serum levels of TNF-αand IL-6 in CIA model rats, and can up-regulate the expression of Caspase-3 in ankle of model rats.

Objective To investigate the changes of 4 histamine receptors (H1R, H2R, H2R and H4 R)in interstitial cystitis on animal experimental models. Methods Thirty female SD rats (250-300 g) were randomly divided into 2 groups as follows: 20 in experimental group and 10 in control group. The experimental group was filled with protamine sulfate+ potassium chloride to create interstitial cystitis model, the control group was sacrificed directly. At the end of the experiment, the bladders of all these SD rats were studied by the immunohistochemistry staining and the value of their mean absorbance (-A) was calculated by IPP4.5 image analysis software. The SPSS 11.5 was used to analyze the differences between the groups. Results Four kinds of histamine receptors mainly expressed in the bladder epithelium. In the experimental group, the (-A) of H1 R was 0. 054±0.031, the of H2R was 0.032±0.021, the (-A) of H3R was 0.047±0.033 and the (-A) of H4R was 0. 149±0. 191,respectively. In the control group, the A of H1R was 0. 017±0. 011, the (-A) of H2R was 0. 018±0. 015, the (-A) of H3R was 0. 014±0. 011, the (-A) of H4R was 0. 060±0.039, respectively. In contrast,the A of H1 R, H2 R and H3R in experimental group was increased significantly(P＜0.05); there was no significant change in (-A) of H4R expression(P＞0.05). Conclusions H1, H2 and H3 receptors in rat model interstitial cystitis bladder epithelium have increased and it indicates H1 R, H2R and H3R may be related to interstitial cystitis. H3 R may be a new treatment target of interstitial cystitis.

Objective To investigate the effect ot ionizing radiation (IR) on the expressions of HMGB1 in the radiation-sensitive and radiation-resistant human cervical cancer cells and to analysis the role of HMGB1 in the regulation of radiosensitivity.Methods Human cervical cancer cells HeLa and its radioresistant strain HeLaR cells were irradiated with different doses of X-rays.The cells were collected at different time points after irradiation.The expressions of protein and mRNA of HMGB1 were detected by Western blot and real-time quantitative PCR.Results At the protein level,the expression of HMGB1 in HeLaR cells was significantly reduced at 6-36 h after 2,5 and 10 Gy X-ray irradiation (t =3.574-9.754,P ＜0.05),and then it was recovered to the control level at 48 h after IR.On the contrary,the expression of HMGB1 in HeLa cells was significantly increased at 6,12,48 h after 2 Gy IR (t =3.945-4.864,P＜0.05),at 6,36,48hafter5 GyIR (t=-2.875-3.295,P＜0.05),and at 36,48 h after 10 Gy IR (t =-4.480,-4.517,P ＜ 0.05).At mRNA level,the trend of HMGB1 expression alteration was consistent with that of protein expression.Conclusions The changes of HMGB1 expression can be differently induced by X-rays in the human cervical cancer radiation-sensitivity cells and radiation-resistant cells.HMGB1 may be involved in the radioresistance of human cervical cancer.

To construct an immortalized rat astrocyte strain genetically modified by rat preprogalanin gene (IAST/GAL) and detect its galanin (GAL) expression and secretion, a cDNA fragment of rat GAL in plasmid of pBS KS(+)-GAL was inserted into eukaryotic expression vector pcDNA3.1 (+) by DNA recombinant technology, then the restriction enzyme digestion and DNA sequencing were carried out to evaluate the recombinant. The pcDNA3.1 (+)-GAL and pcDNA3.1 (+) construct were transfected into immortalized rat astrocyte strain (IAST) by lipofectamine and the population of cells which stably integrated the construct was selected with 600 microg/mL G418. Individual clones were screened and expanded into clonal cell strains. Detection of Neo gene was used to validate the success of the transfection. Immunocytochemical staining, RT-PCR and radioimmunoassay were used to detect the expression and secretion level of GAL. The recombinant had been successfully constructed by restriction enzyme digestion and DNA sequencing. Detection of Neo gene showed that the pcDNA3.1 (+)-GAL and pcDNA3.1 (+) have been successfully transfected into IAST. After selection by using G418, IAST/GAL and IAST/Neo cell strains were obtained. IAST/GAL, IAST/Neo and IAST were immunostained positively for GAL, but the GAL average optical density of IAST/GAL was significantly higher than that of IAST/Neo and IAST (P< 0.01). The level of GAL mRNA expression and the supernatant concentration of GAL in cultured IAST/GAL were significantly higher than those of IAST and IAST/Neo (P<0.01), but no significant differences were found between the IAST and IAST/Neo (P>0.05). It was concluded that IAST/GAL strain was constructed successfully and it might provide a basis for the further study of pain therapy.
Astrocytes/cytology ; Astrocytes/*metabolism ; Cell Line, Transformed ; Cells, Cultured ; Galanin/*biosynthesis ; Galanin/genetics ; Genetic Vectors ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Transfection

The stability of melittin in different solvents (water, deoxygenated water, physiological saline, PBS, 50% ethanol, ethanol, glycerol)was studied and the results showed that the stability of melittin is not influenced by light, temperature and pH in 50% ethanol, which melittin can be completed dissolved when compared with ethanol and glycerol, in such, 50% ethanol was chosen as solvent storage when measured content of melittin. Then the effect of different concentrations of PBS, the pH of PBS and rat skin ho- mogenates were tested, and the results showed that melittin was degraded rapidly at low concentration solution and low ionic strength. Increasing pH of PBS and rat skin homogenate can accelerate the degradation of melittin. These researches provide an experimental ba- sis for further study of melittin.
Animals ; Drug Stability ; Ethanol ; chemistry ; Hydrogen-Ion Concentration ; Melitten ; chemistry ; Rats ; Skin ; drug effects ; Solvents ; chemistry ; Temperature

Adult ; Chondrosarcoma ; metabolism ; pathology ; Collagen Type II ; metabolism ; Diagnosis, Differential ; Femoral Neoplasms ; metabolism ; pathology ; Giant Cells ; pathology ; Humans ; Male ; Osteoclasts ; pathology ; Osteosarcoma ; metabolism ; pathology ; S100 Proteins ; metabolism ; Vimentin ; metabolism