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 effect of ketamine on spatial learning and memory in a rat model of acute pain produced by an incision in plantar area. Methods Seventy-two 3-month old male SD rats weighing 200-250g were randomly divided into 3 groups: (1) control group (C) received no plantar incision (n = 24); (2) acute pain group (M) received an incision in the sole of the hindpaw according to the method described by Brennan (n = 24) and (3) ketamine group (K) received plantar incision and intraperitoneal ketamine 10 mg?kg-1 every day for 7 days starting from the day when plantar incision was made ( n = 24). In group C and M normal saline (NS) was given i.p. instead of ketamine. The three groups were further divided into 2 subgroups according to the time when Morris water maze (MWM) testing was started-1 week (C1, M1,K1) or 3 weeks (C3, M3, K3) after plantar incision. MWM test was performed 4 times a day for 6 consecutive days. The latent periods and swimming distances were recorded automatically by MWM monitoring system. Six days after the last MWM testing the animals were anesthetized and killed. The hippocampus was removed for microscopic examination. Results (1) MWM test: From the 1st to the 6th day of testing, the latent period and swimming distance in group K1 were significantly longer than those in group C1 and M1 (P

Objective To study the effects of profound hemodilution with 6% HES (200/0.5) on coagulation.Methods Ten male new Zealand long-ear rabbits weighing (2.43 ?0.19) kg were anesthetized with intravenous thiopentone 8 mg-1 and tracheotomized and mechanically ventilated (VT = 15 ml-1 , RR = 24 bpm) . Anesthesia was maintained with intravenous infusion of thiopentone and succinylcholine. Femoral artery and vein were cannulated for BP and CVP monitoring. Hemodilution was performed in 6 steps at 30 min intervals. Blood was withdrawn from artery and simultaneously replaced by intravenous infusion of equal volume of 6% HES until Hct was 5 % -8 % . Blood samples were taken before hemodilution and 30 min after each step of hemodilution for determination of coagulation function curve using Sonoclot coagulation and platelet function analyzer. Results When Hct was 25%-30% there was no significant difference in coagulation function before and after hemodilution. When Hct was 15%-20% there was significant difference in ACT, TTP, clot rate and MCS before and after hemodilution but the normal coagulation process was not affected. When Hct

Objective To construct the recombinant adeno-associated virus vector with human preproenkephalin gene (rAAV-hPPE). Methods The human preproenkephalin (hPPE) gene was cloned into the adeno-associated virus (AAV) vector plasmid pSNAV which contained neo expression box. The recombinant pSNAV-hPPE was then transfected into BHK cells using lipofectamine?2000. The G418-resistant cells, BHK / SH1, were obtained. The BHK / SH1 cells were infected with HSV1-rc /△UL2 which has the function of packaging the recombinant AAV(rAAV) . After purification, the construction of rAAV-hPPE was achieved.Results The construction of pSNAV-hPPE was confirmed by digestion with restriction enzyme. Southeon bolting was used to detect the virus liters (2.5 ? 1212 v.g /ml) .Conclusion This rAAV-hPPE virus vector with high liter and strong infectivity can be used in transgenic analgesic research.

0.05). Conclusion:The stress reaction to human injury stimulation are inhibited effectively by propofol - fentanyl intraveous anesthesia

Objective To establish immortalized neural progenitor cell strain and provide stable cell resource for cell-transplantation and gene therapies. Methods Plasmid pCMVSV40T/PUR containing the simian virus 40 large T antigen gene (SV40Tag) were transfected into the primary cultured neural progenitor cells (NPCs) of newborn rat using lipofectin transfection method. Colonies were isolated by puromycin selection and expanded by many passages. Anti-nestin antibodies were used to identify the cultured cells. The specific molecular marker of neurons and astrocytes were detected using immunocytochemistry method to investigate the capability of differentiation of the transfected cells. The expression of SV40Tag in expanded cell lines was identified by RT-PCR, Southern blot and immunocytochemistry method.Results One anti-puromycin cell clone was obtained, which was microtubule-associated protein-2 (MAP-2) positive cells with the capability of proliferation and could differentiate into MAP-2 or glial fibrillary acidic protein positive cells. The existence of SV40Tag cDNA and the expression of mRNA and protein of SV40Tag were confirmed in transfected cells. The transfected cells were expanded to immortalized cell strain maintained for more than 50 passages, named as immortalized neural progenitor cell (INPC) . INPCs were elliptical or triangular cells with two or three short axons. The population doubling time of INPC was (22.9?2.7)h, subculture, freezing and recovering had no effect on cellular shape and proliferation of INPC. Conclusion Immortalized neural progenitor cell strain was established successfully. It may provide stable cell resource for the basic researches and cell-transplantation therapies with NPC.

Objective To assess the image quality (IQ) of an iterative reconstruction (IR) technique (iDose4) from prospective electrocardiography (ECG)-triggered coronary CTA on a 256 MSCT scanner and determine the optimal dose reduction using IR that can provide IQ comparable to filtered back projection (FBP).Methods Prospectively ECG gated CCTA were performed on 120 patients [76 men,44 women; age:(53 ± 10)y] using a 256-slice MSCT (Brilliance iCT,Philips Healthcare).The control group (Group A,n =30) were scanned using the conventional tube output (120 kVp,210 mAs) and reconstructed using FBP.The other 3 groups were scanned with the same kVp but successively reduced tube output as follows:B (n =30):105 mAs,C (n =30):84 mAs:D (n =30):65 mAs and reconstructed using IR levels of L4 to L6,respectively.All images were reconstructed using the same kernel (XCB).Two radiologists graded IQ in a blinded fashion on a 4-point scale (4-excellent,3-good,2-fair and 1-poor).Quantitative measurements of CT values,image noise,Signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were obtained in each group.Analysis of variance (ANOVA) was used for comparisons of objective evaluation indices (noise,CNR) and radiation dose (CTDIvol,DLP,ED) between the four groups.The Kruskal-Wallis test was used for comparisons of demographic data and for detection of differences in subjective evaluation of IQ among groups.A level of P ＜ 0.05 was considered statistically significant.A ROC analysis was performed to determine a radiation reduction threshold up to which excellent IQ was maintained.Results There was no significant differences in objective noise among Groups A (37.4 ±7.9) HU,B(33.2±7.1) HU,C(35.7±9.8) HU,and D(36.0±6.8) HU (F=1.48,P=0.22).There was no significant differences in CNR among Groups A(15.0 ±2.3),B(16.5 ±3.6),C(16.3 ±3.5),and D(15.3±2.8) (F=1.70,P =0.17).Group B and C had good and excellent scores of the subjective IQ (≥3),and there was no significant differences in the scores of the subjective IQ between Group A,and Groups B,C (P =0.30-1.00).Significant differences in image sharpness and study acceptability were observed between groups A and D (P ＜ 0.01).Using the criterion of excellent IQ (score 4),the ROC curve of dose levels and IQ acceptability established a reduction of 60％ of tube output (Group C) as optimum cutoff point (AUC:0.76,95％ CI:0.65-0.87).The effective dose (ED) of Group C was 61％ lower than that of Group A,(1.2 ± 0.1) mSv vs.(3.1 ± 0.6) mSv.Conclusion Iterative reconstruction techniques can provide 61％ ED reduction in prospectively-triggered coronary CTA using 256-slice MSCT while maintaining excellent image quality.

Objective To study the imaging characteristics of localized myositis ossificans for improving its diagnosis and differen-tial diagnosis ability.Methods The Clinical and radiographic data of 45 cases with localized myositis ossificans proved by pathology or clinical follow up were analyzed retrospectively,summarize its imaging features.Results Of the 45 cases,1 7 cases occurred in the elbow joint,1 5 in the hip joint,13 in the other parts of the body.X-ray showed lining or lamellar high-density ossification in soft tis-sues in 34 cases,of those 12 cases with “shell”ossification.Compared with the X-ray,CT showed more clear ossification.MRI showed the soft tissue mass with peripheral edema in 18 cases;3 cases obvious ossification,no edema around.SPECT/CT showed abnormal imaging agent concentration in soft tissue within 12 cases,of those 5 cases concentration range greater than the range of ossification,4 cases concentration range less than the range of ossification.Conclusion Localized myositis ossificans have certain im-aging characteristics.Integrated application of a variety of imaging combined with the clinical can fully display the evolution of the disease,and improve its diagnosis rate.

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