Objective To study the changes in the expression of GABA and its receptors in the dorsal horn of rat spinal cord induced by hind paw formalin injection. Methods Twelve healthy male SD rats weighing 300-325 g were randomly divided into two groups: (A) control group (n=6) and (B) peripheral inflammatory hyperalgesia group in which 5% formalin 50?l was injected subcutaneously into the plantar region of right hind paw to produce persistent pain(n=6). The animals were killed 24h after formalin injection. L_(4-6) segment of the spinal cord was removed. The number of GABA immuno-reactive cells was examined by immuno-cytochemistry technique and the exprpession of GABA_(A?3) and GABA_(B1) receptor mRNA in the spinal dorsal horn were determined by in situ hybridization. Results In control group(A), GABA immuno-reactive cells and GABA_(A?3) and GABA_(B1) receptor mRNA were observed throughout the lumbar spinal dorsal horn. The density of GABA immuno-reactive cells and GABA_(B1) receptor mRNA was highest in the superficial laminae Ⅰ-Ⅲ; while the GABA_(A?3) receptor mRNA was evenly distributed in the spinal dorsal horn. In group B, 24h after formalin injection there was a significant increase in the GABA immuno-reactive cells in the ipsilateral spinal dorsal horn. The expression of GABA_(A?3) and GABA_(B1) receptor mRNA were significantly increased in bilateral dorsal horn compared to group A. The three parameters were positively correlated and their locations in the spinal dorsal horn were not significantly different from those in group A. Conchusion GABA and its receptors may play an important role in nociceptive modulation.

85%).From the in vitro release experiment,the rhBMP-2 was controlled to release from the chitosan microsphere over 30 days.[Conclusion]The rhBMP-2 loaded chitosan microsphere prepared by emulsion cross-linking method shows a good controlled release property.Such a novel type of controlled release system has the potential of being applied for controlled cytokine delivery and the bone tissue engineering.

[Objective]To construct the recombinant adenovirus vector co-expressing human vascular endothelial growth factor 165(VEGF165) and angiogenin-1(Ang-1),and to observe the expression of target gene after transfection.[Method]Molecular biologic techniques were used to clone the genes of VEGF165 and Ang-1,and PCR was used to amplify the DNA sequence of IRES(internal ribozyme entry site) from pIRES2-EGFP.The genes of VEGF165,IRES and Ang-1 were subcloned to pTrack-CMV one by one to get the plasmid named pTrack-CMV-VIA,which contained all the three genes.The linearized shuttle plasmid was cotransformed into BJ5183 bacteria with backbone vector AdEasy-1.Further the recombinant plasmid was packaged and amplified in QBI-293A cells after Pac I digestion to get adenovirus vector pAd-VIA.The expression of the gene of interests was evaluated by fluorescence microscopic analysis of GFP expression and enzyme linked immunosorbent assay(ELISA).[Result]The recombinant adenovirus plasmid was consistent with that shown by sequencing and restriction endonucleases digestion.The recombinant pAd-VIA vector was packaged and amplified successfully in QBI-293A cells.The titer was 2?1010 PFU/ml after amplifying.High positive GFP was expressed in the field through fluorescence microscopy after being transfected by recombinant pAd-VIA.ELISA indicated the expression of the target genes.The difference between the tansfected and non-transfected group was significant(P

Aim To investigate the effect of TAL and MAPK signal transduction on alveolar macrophage (AM) apoptosis and activity of chronic bronchitis (CB) rats.Methods CB model was established by BCG+LPS method and the in vitro and in vivo experiments were used. MTT method was used to detect the AM activity,and the apoptosis of AM was observed by electron microscope.Results The number of AM in BALF of CB rats was increased than that of normal group (P<0.01).The activity of AM was increased in model group than in control group.The apoptotic rate of AM in CB group was much lower than that in the control group [(13.93±3.34)% vs (5.37±1.38)%] (P<0.01).ERK inhibitor PD98059 induced the apoptosis of cultured AM while JNK inhibitor Curcumin reduced the apoptosis.TAL could inhibit ERK MAPK phosphorylation in AM of CB rats.Further investigation showed that Bcl-2 protein expression was significantly increased while Bax evidently decreased in AM of CB rats.TAL could significantly decrease Bcl-2 expression and increase Bax protein expression, which might be the mechanism of its effect.Conclusions There is an increased activity and decreased apoptosis of AM in CB rats compared with normal rats. TAL can inhibit AM activity and increase apoptosis of AM in CB rats which may be related to the therapeutic effect of CB. ERK and JNK MAPK signal transduction participates in the apoptosis of AM. Regulation of Bcl-2/Bax imbalance and MAPK phosphorylation in AM of CB rats might be the mechanism of its effect.

Objective To investigate the incidence and pregnant outcome on vaginal birth after cesarean (VBAC). Methods From January 2005 to December 2015, clinical data of 507 cases with VBAC in West China Second Hospital were studied retrospectively. There were 370 cases of VBAC from January 2013 to December 2015 as study group (VBAC group), in contrast, 740 cases of elective repeat cesarean section (ERCS group) and 740 primiparas of vaginal delivery without history of cesarean section as control groups, the pregnancy outcome were analyzed between the study group and control groups respectively. Results (1) There were 76 547 total births from January 2005 to December 2015. Among these, 10 178 (13.296%, 10 178/76 547) patients had a single prior low transverse cesarean section, of which 4.981%(507/10 178) had VBAC. The incidence of VBAC was rising from 1.020%-3.704%during 2005-2012 to 6.028%-7.662% during 2013-2015. The rate of scared uterus during 2013-2015 was 18.269% (5 539/30 319), of which 9.26%(513/5 539) chose trial of labor after cesarean section (TOLAC). Successful VBAC occurred in 72.12%(370/513) of patients with TOLAC, and 27.88%(143/513) delivered by emergency cesarean. (2) The following parameters of the successful group and the unsuccessful VBAC group were compared, maternal age (29±4) versus (34±4) years, body mass index at prenatal visit (22.2±1.4) versus (22.6±1.4) kg/m2, gestational age (38.7±0.9) versus (39.6±1.3) weeks, birth weight (3 326±317) versus (3 404±285) g, and the rate of induction of labor 0 (0/370) and 6.29%(9/143), there were significant differences (all P<0.01). There was no statistical difference between two group for lower uterine segment (P=0.947). (3) The duration of labor of VBAC group and 740 primiparas of vaginal delivery without history of cesarean section was compared, (10.3± 1.8) versus (11.5 ± 2.0) hours, there was significant difference (P<0.01). There were no statistical difference between two groups for the following parameters, including postpartum hemorrhage, hospitalization duration, the ratio of 5-minute Apgar score≥8, neonatal admission rate (all P>0.05). (4) The postpartum hemorrhage and hospitalization duration in VBAC group incidence were respectively (194±34) ml and (2.32±0.49) days, and the indexes of the ERCS group were respectively (419 ± 57) ml and (4.14 ± 0.78) days, there were significant differences (all P<0.01). There were no statistical difference between two groups for the ratio of 5-minute Apgar score≥8 and neonatal admission rate (all P>0.05). Conclusions The majority of patients choose ERCS rather than TOLAC. It′s important to assess the indications and contraindications of patients for the successful VBAC, and to monitor maternal and fetal conditions during the delivery process. The premise of TOLAC is a comprehensive understanding of closely monitoring the progress of delivery. Compared with the ERCS, VBAC could reduce patients′ postpartum hemorrhage and hospitalization duration, improve the outcomes of pregnancy, and the cesarean section rate could be reduced.

BACKGROUND:The structure of tissue engineering carrier affects the bio-action of cells greatly.OBJECTIVE: To investigate the biological characteristics of bone marrow stem cells (MSCs) in different concentrations of alginate combined with de-antigen bone xenograft (DBX).DESIGN: Observational trial.SETTING: PLA Institute of Trauma and Orthopedics, the Fourth Military Medical University of Chinese PLA.MATERIALS: Alginate, calcium chloride, MSCs, bone xenograft.METHODS: Bovine cancellous bone was out into cubes, which were degreased, deproteinized and then lyophilized.Cubes in pore size within 300-500 μm were selected for use after ethylene oxide sterilization. The purified sodium alginate was dissolved in DMEM cell culture medium of concentrations as different as 0.5%, 2%, 8% and 16%; 1×1012 L-1 induced MSCs were blended with isopyknic alginate-DMEM and compounded with DBX at a status of 0.5 Mpa negative pressure for 5 minutes in order to make a cell suspension fully fill into the pores of the cancellous bone. Then alginate was crosslinked with 50 g/L calcium gluconic acid for 30 seconds. The complex was put into a CO2 incubator and cultured for 4 days. The gel compound and cell growth in the pores of the complex were grossly observed with an inverted microscope. Status of cell growth in the complex with different concentrations of alginate was observed with scanning electron microscopeMAIN OUTCOME MEASURES: Compound status of alginate and bone xenograft, cell growth status and matrix secretion in compound carries.RESULTS: When the concentration of alginate was 0.25% or 1%, alginate was equally combined in DBX, while that of 4% and 8% only combined on the surface of cancellous bone. After in vitro cultured for 4 days, alginate of 0.25% were broken off from DBX surface. But alginate of 1% was equally combined with DBX pores with cells secreting well in alginate. Development of cells in alginate of 4% was restricted and no cells were seen in alginate of 8%.CONCLUSION: Alginate of 1% is suitable for constructing the carrier of bone tissue engineering with bone xenograft.

Objective To observe the long-term efficacy of anti-infective reconstituted bone xenograft (ARBX) combined with external fixation of posttraumatic long bone infection in lower extremities.Methods This retrospective case series study included 36 patients with posttraumatic long bone infection in lower extremities followed up for more than 18 months after receiving one-stage ARBX bone grafting combined with ring external fixation from January 2004 to December 2013.There were 21 male and 15 female patients,at the age of 19-72 years (mean,35.8 years).Multiple fractures were seen in 24 patients and single fractures in 12 patients.Bone and functional results were evaluated using the association for the study and application of the method of Ilizarov (ASAMI) classification.Results Follow-up ranged from 18 to 72 months (mean,38 months).Bone union was seen in 33 cases in a mean period of 5.2 months,and infection was completely cured.Length of limbs in all patients reached the expected extension length with the bone extension length of 3-10 cm (mean,5.2 cm).All the extended areas showed bone healing.According to the ASAMI classification,bone result was excellent in 20 patients,good in 13 and fair in 3,with the excellent and good rate of 92％,and limb function recovery was excellent in 18 patients,good in 11 and fair in 7,with the excellent and good rate of 81％.Conclusion ARBX adjuvant external fixator treatment of posttraumatic long bone infection in lower extremities improves bone healing rate and limb function recovery rate and shortens bone healing time at one stage.

BACKGROUND: Bone morphogenetic protein(BMP) is one of the most important cytokines that induce and promote seed cells to be transformed into osteocytes. Insoluble natural BMP can hardly affect the life of cultured seed cells. The expensive soluble recombinant BMP is also hard to work on the seed cells at the appropriate time and dose. Therefore, gene therapy technique provides us with a brand new idea of using gene-modified seed cells.OBJECTIVE: To transfect exogenous BMP-3 gene into the fibroblasts and screen the positive fibroblast clones that can express BMP-3 stably.DESIGN: Simple sample study.SETTING: Orthopaedic Research Institute, Xijing Hospital, Fourth Military Medical University of Chinese PLA.MATERIALS: The fibroblasts(NIH3T3) were kindly presented by Professor Situ Zhen-qiang of the Stomatological College of Fourth Military Medical University of Chinese PLA.METHODS: This experiment was conducted in the Key Laboratory of Chinese PLA, which belongs to the Orthopaedic Research Institute of Fourth Military Medical University. BMP-3 gene was transfected into the fibroblasts through lipofectamin. The transfected cells were screened by G418. The separated cloned cells were identified through immunohistochemistry. The positively stained cells were the clones of BMP-3 expressing fibroblasts.MAIT OUTCOME MEASURES: The screening concentration of NIH3T3 cells, screening of positive transfected cells, and expression of BMP-3 in screened cells.RESULTS: BMP-3 gene was successfully transfected into the fibroblasts. BMP-3 expressing fibroblast clones were creened and identified through immunohistochemistry. Fibroblast strains with stable BMP-3 expression were obtained.CONCLUSION: The transfection of BMP-3 gene eukaryonic expression vector into the fibroblasts and obtaining of fibroblast strains with BMP-3 expression have laid foundation for the usage of gene-modified seed cells in future research of bone tissue engineering.

Objective To validate the effect of anti-infective reconstituted bone xenograft (ARBX) in treating posttraumatic osteomyelitis by one-stage grafting in the adults.Methods With clinical application approval of Medical Command,Logistics Ministry of PLA,ARBX was used to treat 27 adult patients (29 lesions) with posttraumatic osteomyelitis by one-stage grafting after debridement since September 2001.The study analyzed 27 patients (29 grafts) who were followed up for average 26 months (12-63 months).Results The follow-up for average 26 months (12-63 months) in 27 patients showed that infection of 22 patients (24 lesions) was controlled and cured,except for three with failure to control the infection or with recurrence of infection,two with controlled infection but with postoperative nonunion.The infection control rate was 89.7% (26/29) and the cure rate was 82.8% (24/29) ,which were better than the results of traditional therapy.Conclusions ARBX has high osteoinductive activity and enhanced anti-infective capability,which enables it to be used as one-stage grafting to treat posttraumatic osteomyelitis in the adults.

Objective To evaluate the effect of dexmedetomidine on necroptosis during liver injury in septic rats.Methods Eighteen SPF adult male Sprague-Dawley rats,weighing 200-220 g,were divided into 3 groups (n=6 each) using a random number table:sham operation group (group SH),sepsis group (group SEP) and dexmedetomidine group (group DEX).Sepsis was induced by cecal ligation and puncture in chloral hydrate-anesthetized rats in SEP and DEX groups.Dexmedetomidine 5 μg/kg was injected via the caudal vein at 1 h before operation in group DEX.Blood samples were collected from the caudal vein at 6 h after operation for determination of serum aspartate amino-transferase (AST) and alanine aminotransferase (ALT) concentrations.The rats were then sacrificed and livers were removed for determination of the level of reactive oxygen species (ROS) in liver tissues (using chemiluminescence assay) and expression of receptor-interacting protein 1 (RIP1),RIP3,mixed lineage kinase domain-like (MLKL),high-mobility group box 1 protein (HMGB1) and dynamin-related protein 1 (Drpl) in liver tissues (by Western blot).Results Compared with group SH,the serum AST and ALT concentrations were significantly increased,the expression of RIP1,RIP3,MLKL,HMGB1 and Drpl in liver tissues was up-regulated,and the level of ROS in liver tissues was increased in SEP and DEX groups (P＜0.05).Compared with group SEP,the serum AST and ALT concentrations were significantly decreased,the expression of RIP1,RIP3,MLKL,HMGB1 and Drp1 in liver tissues was down-regulated,and the level of ROS in liver tissues was decreased in group DEX (P＜0.05).Conclusion The mechanism by which dexmedetomidine attenuates liver injury may be related to inhibition of necroptosis in septic rats.