The mechanisms of general anesthesia, which was introduced about 170 years ago, remain poorly under- stood. Even less well understood are the effects of general anesthesia on the human body. Recently we identified 18 G-protein coupled receptor (GPCR) genes of Daphnia pulex, an invertebrate model organism. Phylogenetic analysis identified these genes to be the homologs of the human γ-aminobutyric acid, type B (GABAB) receptor, metabotropic glutamate receptors (mGluR), adrenergic receptor, serotonin (5-HT) receptor, dopamine receptor and muscarinic acetylcholine receptor (mAChR). Using reverse transcription and quantitative PCR techniques, we systematically measured the effects of propofol, etomidate and ethanol on these 18 GPCR mRNA expressions in Daphnia pulex.
Animals ; Daphnia ; drug effects ; metabolism ; Ethanol ; pharmacology ; Etomidate ; pharmacology ; Phylogeny ; Propofol ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Receptors, GABA-B ; genetics ; metabolism

BACKGROUND: Precartilaginous stem cells (PSCs) have strong proliferation ability and differentiation potential,but they are instable and prone to differentiate.Importing exogenous gene could immortalize them and leave phenotype character unchanged.OBJECTIVE: To establish immortalized precartilaginous stem cells (PSCs) from neonatal SD rats in vitro for the further related research about the differentiation mechanism and clinical application of precartilaginous stem cells.DESIGN,TIME AND SETTING: Single sample observation.The study was carried out in the Department of Orthopedics.Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from October 2005 to September 2006.MATERIALS: Neonatal SD rats,irrespective of gender,24-hour old,were used for prepare PSCs.METHODS: By using LipofectamineTM 2000,a gene transfection reagent,plasmid pCMVSV40T/PUR containing the simian virus 40 large T antigene gene (SV40Tag) was transfected into the primary cultured PSCs isolated by immuniomagnetic beads coasted with the second antibody.Colonies were isolated by puromycin selection and expanded by many passages.MAIN OUTCOME MEASURES: Biological character of PSCs; plasmid identification; biological character of transfected cells and identification; RT-PCR; growth curve.RESULTS: Immunomagnetic beads separation system obtains PSCs,which was confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive PSCs.Double restriction enzyme was cut,electrophoresis confirmed pCMV was 3 kb,SV40T was 2.3 kb.A particular anti-puromycin cell clone was acquired,which was confirmed as FGFR-3 positive PSCs.The total RNA was isolated from the positive cell clones,and a 588 bp fragment,which was specific for the SV40T antigene gene,was amplified.The transfected cells were expanded to immortalized cell strain,named as immortalized precartilaginous stem cells (IPSCs).Thepopulation doubling time of IPSCs was (22.98±2.77) hours,no significant effect of subculture,freezing and recovering had been found.CONCLUSION: Precartilaginous stem cells could be isolated from neonatal SD rats,cultured in vitro,and immortalized through the transfection of pCMVSV40T/PUR.

Objective To retrospectively describe the technique and review the surgical results of OsteoMed M3 titanium plate and screws used to secure the posterior elements in the open position after expansive unilateral opendoor laminoplasty.Methods Twenty-six patients with multilevel cervical disc herniation and canal stenosis were treated with an expansive unilateral open-door laminoplasty with OsteoMed M3 plate and screws.The follow-up period was over 1 year.The improvement of spinal function after surgeries under JOA was evaluated to analyse the effects and releated factors.Results All of 26 cases' follow-up period was over 1 year.The mean JOA score increased significantly from 9.40 ± 1.658 ( range,5 to 13 ) points before surgery to 13.80 ± 1.958 ( range,7 to 16) points at final follow- up ( t =- 21.137,P =0.000 ).Mean recovery rate was 57.9％.Postoperative radiography,magnetic resonance imaging and computed tomography scan demonstrated significantly increased sagittal diameter and canal expansion.Two cases without relief of nurological symptoms underwent an additional anterior multilevel corpectomy.One case with ossification of the posterior longitudinal ligament had not good enough neurologic improvement after surgery.No neurologic deterioration owing to hinge reclosure or major surgery-related complications were observed.It would reduce the recovery for those with old age,long history,worse symptoms,cervical kyphosis and abnormal signal in MR imaging.It was good for patients to do early active cervical exercises after surgery.Conclusion Unilateral open- door laminoplasty with OsteoMed M3 titanium plate and screws fixation effectively maintains expansion of the spinal canal and resists closure while preserving alignment and stability.This modified technique is easy to perform with a low complication,is and economic,and is good for clinical application.

70 patients including 60 cases under acupuncture anesthesia and 10 cases under drug anesthesia were observed. Before and 20 minutes after acupuncture the blood samples were taken from the patient ear lobes respectively, and in some patients taken once again 24 hrs after acupuncture. The electroacupuncture point Hegu or Zusanli was mainly adopted. As the method for detection of cell-mediated immunity(CMI) in vitro the improved microtechnique of whole blood for E-rosette (active and nonactive) and lymphocyte transformation tests was used. In performance of active rosetting the total leucocyte count and the differential lymphocyte count were done for calculation of absolute number of active rosette forming cells (RFC). The mean value of increase of active RFC was 12.7?1.43, the decrease was 6.8?1.77 after acupuncture. The increment of the absolute number of active RFC was 175?63.59. However no marked effect on the drug anesthesia group was found. In the lymphocyte transformation assay the increase was 12.7?1.49, the decrease was 7.0?2.19, and the enhancement effect still exhibited 24 hrs after acupuncture. In these tests an increase was mostly found in those with a lower or a usual CMI level; a decrease often found in those with a higher CMI level prior to acupuncture. The increase or decrease level in the results of three kinds of test (active, nonactive RFC and lymphocyte transformation) was similar, the increase range was 12～13%, the decrease range 6～7%. As the former compared with the latter, the promotion was prominent by all means.

ObjectiveTo investigate the biomechanical characteristics of different types of fixation with bioactive cervical fusion cage made of hydroxyapatite and poly L-lactic acid in cervical spinal fusion.MethodsIliac crest bone,bioactive cervical fusion cage and bioactive cervical fusion cage with plate fixation were used for anterior interbody implants after anterior discectomy across C5-6 in six fresh human cervical spine specimens respectively,and the range of motion of the cervical vertebrae interbody fusion were measured through the motional stability test.Results After discectomy,Bioactive Cervical Fusion Cage with plate fixation exhibited a significant increase in stability and a decrease of range of motion in angular motion than others in all motional directions ( P ＜ 0.005 ). Bioactive cervical fusion cage exhibited a decrease in stability and an increase of range of motion (6.25 ± 0.29) in angular motion than the intact spine (5.76 ± 0.40) in extension,but the difference was not significantly ( P ＞ 0.05 ).Bioactive cervical fusion cage exhibited a decrease in angular motion than iliac crest bone and a significant increase in stability in all motional directions except extension (P ＜ 0.005).ConclusionsBioactive cervical fusion cage' s biomechanical performance was excellent and bioactive cervical fusion cage with plate fixation was excellent in stability in all motional direction,and could remain initial stability of cervical vertebrae.

Objective: Our purpose was to explore the change regularity of β-glucuronidase (β-G) in body of patients with Non-Hodgkin malignant lymphoma. Methods: β-G was examined synchronously both in the serum and in the tumor tissue of 13 cases patient with Non-Hodgkin malignant lymphoma by using the method of enzymlinked immunsorbent assay (ELISA) and immunohistochemistry separately. Among them, 3 cases were studied by using the immuno electron microscopic technique. Results: β-G was highly expressed both in the serum and tumorous tissue in patients with non-Hodgkin malignant lymphoma and there was obviously difference as compared with the control group (P<0.01). Conclusion: The combined detection with functional and morphological methods to β-G, it may be assistant target to early discovery and early diagnosis of Non-Hodskin malignant lymphoma.

By culturing bone marrow mesenchymal stem cells of rabbits with fibrin glue in vitro, the biocompatibility of fibrin glue was investigated to study whether this material can be used as scaffolds in bone tissue engineering. After 2-months old New Zealand rabbits had been anesthetized, about 4-6 ml of bone marrow were aspirated from rabbit femoral trochanter. The monocytes suspension was aspirated after bone marrow was centrifuged with lymphocyte separating medium and cultured primarily. Then the cells were divided into two groups: one was cultured with complete medium and the other with induced medium. The cells of the two groups were collected and inoculated to the culture plate containing fibrin glue. In the control group, cells were inoculated without fibrin glue. The implanted cells and materials were observed at different stages under a phase-contrast microscope and scanning electron microscope. MTT and alkaline phosphatase (ALP) were measured. Bone marrow mesenchymal stem cells grew on the surface of fibrin glue and adhered to it gradually. Cells light absorption value (A value) and the ALP content showed no significant difference. Fibrin glue had no inhibitory effect on cell morphology, growth, proliferation and differentiation. It has good biocompatibility and can be used as scaffold materials for bone marrow mesenchymal stem cells in bone tissue engineering.
Biocompatible Materials/*pharmacology ; Bone Marrow Cells/*cytology ; Cells, Cultured ; Coculture Techniques ; Fibrin Tissue Adhesive/*pharmacology ; Mesenchymal Stem Cells/*cytology ; Tissue Engineering

Sox9 gene was cloned from immortalized precartilaginous stem cells and its eukaryotic expression vector constructed in order to explore the possibility of bone marrow-derived stromal cells differentiation into precartilaginous stem cells induced by Sox9. A full-length fragment of Sox9 was obtained by RT-PCR, inserted into pGEM-T Easy clone vector, and ligated with pEGFP-IRES2 expression vector by double digestion after sequencing. The compound plasmid was transfected into born marrow-derived stromal cells by Lipofectamine 2000, and the transfection efficacy and the expression of Sox9 and FGFR-3 were observed. Flow cytometry was used to identify the cell phenotype, and MTT was employed to assay proliferative viability of cells. Sequencing, restrictive endonuclease identification and RT-PCR confirmed that the expansion of Sox9 and construction of Sox9 expression vector were successful. After transfection of the recombinant vector into bone marrow-derived stromal cells, the expression of Sox9 and FGFR-3 was detected, and proliferative viability was not different from that of precartilaginous stem cells. It was concluded that Sox9 gene eukaryotic expression vector was successfully constructed, and the transfected bone marrow-derived stromal cells differentiated into the precartilaginous stem cells.
Base Sequence ; Bone Marrow Cells/*cytology ; Cartilage/*cytology ; Cell Differentiation/genetics ; Cells, Cultured ; Cloning, Molecular ; Genetic Vectors/genetics ; Molecular Sequence Data ; Receptor, Fibroblast Growth Factor, Type 3/metabolism ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; SOX9 Transcription Factor/biosynthesis ; SOX9 Transcription Factor/*genetics ; Stem Cells/*cytology ; Stromal Cells/*cytology ; Transfection

BACKGROUND: Bone marrow stromal cells(BMSCs) are the ideal gene target cells and will have a bright future in the gene therapy of spinal cord injury.OBJECTIVE :To detect the expression of glial cell line - derived neurotrophic factor(GDNF) gene after BMSCs were infected by adenovirus-medialed GDNF (Adv-GDNF) in vitro and to explore its biological activity.DESIGN: A randomized controlled trial study.SETTING: Laboratory of Orthopedic DepartmentMATERIALS: The experiment was completed in the Laboratory of Orthopedic Department, Affiliated Tongji Hospital of Tong ji Meidcal College,Huazhong University of Science and Technology. Twenty-four SD rats of either gender, weighing (180 ± 20) g.INTERVENTIONS: BMSCs were infected by Adv-GDNF in vitro and then cocultured with spinal cord dorsal root ganglion. The three methods, immunofluorescent chemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay(ELISA) were used to evaluate GDNF expression in the BMSCs. The biological activity of GDNF was observed by a phase contrast microscope.MAIN OUTCOME MEASURES:Primary outcomes:①RT-PCR;②results of immunofluorescent chemical examination;③biological activity of GDNF in vitro. Secondary outcomes:①culturing and identification of BMSCs②time-effect relationship of GDNF expression revealed by ELISA.RESULTS: Immunofluorescence displayed expression of GDNF in BMSCs 48hours after Adv-GNDF infection. RT-PCR analysis demonstrated expression of GDNF mRNA 24 hours after Adv-GNDF infection. ELISA confirmed the presence of GDNF in the liquid supernatant of BMSCs 24 hours after Adv-GDNF infectionn and showed that GDNF was secreted. The supernatant can promote the neurite outgrowth in the rat dorsal root ganglion(DRG).CONCLUSION: It is demonstrated that BMSCs infected by Adv-GDNF can express GDNF steadily and the expressed GDNF has the activity of promoting neurite outgrowth, which lays a foundation of the GDNF gene therapy for spinal cord injury.

By culturing bone marrow mesenchymal stem cells of rabbits with fibrin glue in vitro, the biocompatibility of fibrin glue was investigated to study whether this material can be used as scaffolds in bone tissue engineering. After 2-months old New Zealand rabbits had been anesthetized, about 4-6 ml of bone marrow were aspirated from rabbit femoral trochanter. The monocytes suspension was aspirated after bone marrow was centrifuged with lymphocyte separating medium and cultured primarily. Then the cells were divided into two groups: one was cultured with complete medium and the other with induced medium. The cells of the two groups were collected and inoculated to the culture plate containing fibrin glue. In the control group, cells were inoculated without fibrin glue. The implanted cells and materials were observed at different stages under a phase-contrast microscope and scanning electron microscope. MTT and alkaline phosphatase (ALP) were measured. Bone marrow mesenchymal stem cells grew on the surface of fibrin glue and adhered to it gradually. Cells light absorption value (A value) and the ALP content showed no significant difference. Fibrin glue had no inhibitory effect on cell morphology, growth, proliferation and differentiation. It has good biocompatibility and can be used as scaffold materials for bone marrow mesenchymal stem cells in bone tissue engineering.
Animals ; Biocompatible Materials ; pharmacology ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Coculture Techniques ; Fibrin Tissue Adhesive ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; Rabbits ; Tissue Engineering