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BACKGROUND:Ginsenoside can promote wisdom,prevent aging,protect cortical motor neurons,resist cell apoptosis,but the mechanisms are unclear.OBJECTIVE:To observe the effect of Ginsenoside Rb1 and Rg1 on nerve growth factor expression in Schwann cells.DESIGN,TIME AND SETTING:The in vitro cytological study was performed at the Second People's Hospital of Shenzhen City from March to June 2004.MATERIALS:Fresh adult ex vivo nerve was obtained from limbs that were dissociated by trauma and could not be reimplanted at the Second People's Hospital of Shenzhen City.Ginsenoside Rb1 and Rg1 was supplied by the Norman Bethune University of Medical Sciences.METHODS:Epineurium was removed and cut into 1.0-2.0 mm blocks.Schwann cells were isolated by enzyme digestion.Following removing fibroblasts by double 30-minute differential attachment,Schwann cells with above 95% purity rate were harvested,and then incubated on a 96-well culture plate coated with polylysine (105 cells/well).Schwann cells in the Ginsenoside Rb1 group were subjected to 20 uL of Ginsenoside Rb1 at 10,20,40,60,80 ug.Schwann cells in the Ginsenoside Rg1 group underwent 20 uL of Ginsenoside Rg1 at 10,20,40,60,80 ug.Schwann cells in the control group were treated with 20 uL of phosphate buffered saline.MAIN OUTCOME MEASURES:Nerve growth factor expression rate was determined in Schwann cells by using flow cytometry.RESULTS:Nerve growth factor expression rate in Schwann cells was significantly increased in the Ginsenoside Rb1 and Ginsenoside Rg1 groups compared with the control group at 48 hours following incubation (P ＜ 0.05),in a dose-dependent fashion.Nerve growth factor expression rate peaked when Ginsenoside Rb1 and Ginsenoside Rg1 were 60 mg/L.No significant difference in nerve growth factor expression rate was detected between the Ginsenoside Rb1 and Ginsenoside Rg1 groups (P ＞0.05).CONCLUSION:Ginsenoside Rb1 and Rg1 has potential of promoting the recovery of damaged peripheral nerve by increasing Schwann cell producing and secreting nerve growth factor.
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BACKGROUND: Glial cell-derived neurotrophic factor (GDNF) and transforming growth factor-beta 1 (TGF-β1)co-subordinate to TGF-β family. Both of them play very important roles in the development and differentiation of central and peripheral nervous system, and regulation of cell cycle in mammals.OBJECTIVE: To observe the differentiation of spinal cord-derived neural stem cells(NSCs) induced by GDNF combined with TGF-β1, and make a comparison of differentiation results with GDNF or TGF-β1 culture fluid.DESIGN: Controlled observation.SETTING: Central Laboratory, Shenzhen Hospital Affiliated to Southern Medical University.MATERIALS: Ten SD rats of clean grade, which were at conception for 16 days, were provided by the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technplogy. Main reagents and materials:DMEM/F12,B27(GIBCO); basic fibroblast growth factor (bFGF), GDNF; TGF-β1(PeproTech);fetal bovine serum (FBS,Hyclone); nestin multiple antibody (Boster, Wuhan); glial fibrillary acidic protein (GFAP) multiple antibody; neurofilament protein (NF-200) monoclonal antibody (Sigma).METHODS: This experiment was carried out in the Central Laboratory, Shenzhen Hospital Affiliated to Southern Medcial University between October 2005 and September 2006. Under the aseptic condition, rat fetus was isolated for isolation and culture of spinal cord-derived neural stem cells. In this study, five groups were divided: basal medium group, control group, bFGF group, TGF-β1 group, GDNF+ TGF-β1 group. In the basal medium group, DMEM/F12 containing penicillin,streptomycin, amphotericin (AMPH) B and 0.02 volume fraction of B27 annex solution. At 1 week after primary culture, rat spinal cord-derived NSC clones proliferated in vitro stably were harvested. In the control group, 0.1 volume fraction of FBS was added into basal medium. In the later three groups, induced medium was exchanged, i.e. 20 μg/L bFGF, 2 μg/L TGF-β1, and 10 μg/L GDNF+2 μg/L TGF-β1 were added into the basal medium, respectively. ①The differentiation of spinal cord-derived NSCs induced by different factors were observed under the optical microscope. ②The expressions of neurons and astrocytes were detected by immunocytochemical staining labeling. ③ The differentiated cells were counted by sorting technique by means of fluorescence excitation flow cytometer, and the percentage of NSCs differentiating into neurons and astrocytes were detected under the different induction environments.MAIN OUTCOME MEASURES: ① Morphological feature of cell differentiation in each group. ② Immunohistochemical detection of NSCs in each group. ③ The percentage of NSCs differentiating into neurons and astrocytes in each group.RESULTS: ① Cell morphology during differentiation: At the early stage of differentiation, lots of cells creeped to all the directions, and one week later, most of the migrated cells adhered to the wall entirely. Neuron-like cells, astrocyte-like cells and oligodendrocyte-like cells could be identified in the low-density cell region. ②Immunohistochemical detection results: A lot of GFAP- positive astrocytes were found in the control group and TGF-β1 group; Many differentiated neurons and NF-200 staining positive were found in the bFGF group and GDNF+ TGF-β1 group. ③Percentage of stained neuron and astrocyte: at one week of induction, the percentage of stained neurons was higher in the GDNF+ TGF-β1 group than in the control group, bFGF group and TGF-β1 group (x2=24.15,19.56,25.32,P ＜ 0.05-0.01), and the percentage of stained astrocytes was lower in the GDNF+ TGF-β1 group than in the control group, bFGF group and TGF-β1 group (x2=24.45,23.79,P ＜ 0.01 ).CONCLUSION: The combined in vitro induction of GDNF and TGF-β1 contributes to the neuronal differentiation of spinal cord-derived NSCs, indicating that both of them have synergistic effect.
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BACKGROUND: Pathophysiological mechanism of local microenvironment is complex after central nerve injury; especially,both inflammatory reaction at an acute phase and formation of secondary glial scar have tremendous effects on effective regeneration of axon, regeneration and arrangement of local nerve cells, proliferation and migration of local stem cells;therefore, it becomes a basic reason for blocking nerve repair in an early period. Thus, how to effectively resist inflammatory factors in injured region at an acute phase and how to optimize microenvironment of neural regeneration are the most important strategies for repairing spinal cord injury in recent years.OBJECTIVE: To design, establish and screen the best expression of interleukin-6 receptor (IL-6R) α to inhibit shRNA adenovirus expression vector by using spinal cord injury models.DESIGN: Duplicative measurement study.SETTING: Department of Spine Surgery, the Second People's Hospital of Shenzhen.MATERIALS: A total of 40 healthy Wistar rats, either gender, 8-10 weeks old, were selected in this study. Rabbit-anti-rat glial fibrillary acidic protein (GFAP) antibody Ⅰ was provided by Santa Cruz Compan; siRNA eukaryon expression plasmid pGenesil (cohtaining green fluorescent expression system) was provided by Wuhan Jingsai Bioengineering Company.METHODS: The experiment was carried out in ImmuneOpening Laboratory, Basic Medical Faulty, Tongji Medical College, Huazhong University of Science and Technology, and Medica Laboratory Center, the Second People's Hospital of Shenzhen in November 2006. Three pairs of shRNA template which composed of 19 bp reverse repeated motif of IL-6 receptor (IL-6R) α target sequence with 9 bp spacer were designed and synthesized, then the recombinant adenovirus expression vectors with green fluorescence protein were constructed in vitro respectively. The acute spinal cord injury models were completed, and the adenovirus recombinants were regionally injected post 12 hours after spinal cord injury;in addition, the inhibitory effect of RNA interference (RNAi) on expression of IL-6R in local region after spinal cord injury were detected by using real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot so as to screen adenovirus expression vector which had the best inhibitory effect on expression of IL-6R.MAIN OUTCOME MEASURES: Inhibitory effect of RNAi on expressions of IL-6R RNA and protein in local region after spinal cord injury.RESULTS: Sequence analysis showed that IL-6R-shRNA recombinant adenovirus expression vector was successfully constructed, and optimal IL-6R-shRNA recombinant adenovirus expression vector was screened by using real-time fluorescence quantitative RT-PCR and Western blot. The IL-6R expressions were 49% and 56% at the levels of mRNA and protein, respectively.CONCLUSION: The IL-6R--shRNA recombinant adenovirus expression vector is successfully constructed and screened.The gene expression of IL-6R can be highly inhibited after acute spinal cord injury.
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BACKGROUND: It is demonstrated that bone marrow stem cells (BMSCs) can generate neurosphere structures, which is similar to cloning sphere of neuron-specific enolase (NSE), in a specially induced system in vitro; therefore, BMSCs draw more and more attention as seed cells to repair central nerve injury.OBJECTIVE: To investigate the differentiation from BMSCs into neuron-like calls induced by fetal spinal cord tissue.DESIGN: Observational study.SETTING: Department of Spine Surgery, Second People's Hospital of Shenzhen.MATERIALS: SD rats (16 pregnant days old and 2 months old) were provided by Animal Center, Tongji Medical College,Huazhong University of Science and Technology. Single antibody of NSE, multi-antibody of glial fibrillary acidic protein (GFAP) and single antibody of neurofilament (NF200) were provided by Wuhan Boster Company.METHODS: The experiment was carried out in Immune Opening Laboratory and Central Laboratory, Basic Medical Department, Tongji Medical College, Huazhong University of Science and Technology in September 2006. Bones of lower extremities of rats were collected to centrifuge BMSCs. Fetal spinal cord tissue homogenate was extracted from 16 pregnant days old rats to make inducing solution and induce differentiation of BMSCs. Otherwise, embryo muscle tissue was used to make muscle tissue homogenate as the same way so as to regard as the controls.MAIN OUTCOME MEASURES: BMSCs underwent morphological observation after induction; in addition, anti-NSE, NF200 and anti-GFAP were used to label neurons and astrocytes, respectively. Ten non-overlapping sights were randomly selected from positive reactive-induced cells after immunohistochemical staining under optic microscope to calculate ratio of positive cells of NSE and NF200 counting for total numbers of cells.RESULTS: ① Morphological changes of BMSCs after induction: During early induction, optic microscope indicated that soma of partial cells rebounded; whose apophysis was long and thin; apophysis of differentiated cells grew gradually and intercrossed each other. It was similar to nerve cells and some branches were similar to dendrite branches. However,morphological changes of cells in the control group were not obvious. ② Expression of relevant antigens differentiated from BMSCs into neuron-like cells at one week after induction: Most cells in spinal cord homogenate group expressed as positive NSE and NF200; a few of cells expressed as GFAP. While, positive staining of nerve cell antibody was not observed in the control group; meanwhile, positive reaction of nerve cell antigen was not observed in the control group,too. Immunohistochemistry examination demonstrated that positive rates of NSE and NF200 expressions were (68±1.7)%and (76.2±2.9)%, respectively.CONCLUSION: Fetal spinal cord tissue homogenate can induce differentiation from BMSCs into neuron-like cells.
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BACKGROUND: Schwann cell-derived neurotrophic factor is a bioactive protein isolated and purified from the kytoplasm of Schwann cell. It can obviously maintain the survival of spinal cord anterior horn motor neuron and promote the regeneration of peripheral nerve.OBJECTIVE: To observe the protective effect of Schwann cell-derived neurotrophic factor on the high injury of peripheral nerve-induced apoptosis of sensory neurons in spinal dorsal root ganglia.DESIGN: Randomized and controlled animal experiment.SETTING: Shenzhen Second People's Hospital.MATERIALS: Totally 30 3-week-old SD infant rats, of clean grade and either gender, were used in this experiment. They were randomly divided into neurotrophic factor group and control group with 15 rats in each one.Left sides of the animals in both two groups were set as normal sides and right sides as injured sides.METHODS: This experiment was carried out at the Experimental Animal Center, Medical College of Sun Yat-sen University from May 2003 to July 2003. ① L4.5 nerve root high-mutilation animal models were developed on the rats in two groups. Proximal nerve stump was connected with silicone tube. According to grouping, 60 mg/L Schwann cell-derived neurotrophic factors and 20 μL normal saline were injected into the silicone tubes respectively. Two ends of silicone tube were enveloped with vaseline.② Sample collecting was conducted at postoperative 4 weeks, survival rate and morphological change of sensory neurons in dorsal root ganglia of injured nerve was observed.MAIN OUTCOME MEASURES: ① Gross observation of sciatic nerve regeneration at injured side of the rats in two groups ② Survival of sensory neurons in dorsal root ganglia ③ Morphological change of sensory neurons in dorsal root ganglia.RESULTS: All the 30 rats entered the stage of result analysis. ① Gross observation of sciatic nerve regeneration: In the neurotrophic factor group,nerve new born axon grew along silicone tube, with 1cm in length; there were few and thin newborn axons in control group with 0.8 cm in length.② Survival of neuron in dorsal root ganglia of the rats in two groups: There was little fibrous tissue proliferation in the dorsal root ganglion in neurotrophic factor group. The loss of neurons was not obvious and the survival rate was 91.8%. Obvious fibrous tissue proliferation appeared in the dorsal root ganglia in control group, and a great many neurons were lost with the survival rate of 58.6%. Survival rate of neurons was 33.2% higher in neurotrophic factor group than in control group (P ＜ 0.01 ). ③ Morphological change of neurons in dorsal root ganglia: The diameter and area of neurons in dorsal root ganglia were significantly lower in control group than in neu rotrophic factor group and normal side [(21.8±1.4) μm,(373.1±50.9) μm2 vs (24.8±1.1) μm, (482.8±42.2) μm2 and (24.5±1.3) μm, (471.5±51.4) μm2,P ＜ 0.01], while there were no significant difference in diameter and area of neurons between neurotrophic factor group and normal side(P ＞ 0.05).CONCLUSION: Schwann cell-derived neurotrophic factors have obvious neurotrophic bioactivity for sensory neurons in the injured dorsal root ganglia.
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BACKGROUND: Schwann cell derived neurotrophic factor, which is isolated and purified from the kytoplasm of Schwann cell with the relative molecular mass of 58000, is a kind of neurotrophic substance possessing obvious neurotrophic activity. It can be against neurovirulent substance of nitrogen monoxidum.OBJECTIVE:To create root avulsion animal models and observe the protective effects of Schwann cell derived neurotrophic factor (SDNF) on motoneurons of spinal anterior horn from spinal root avulsion induced cell death.DESIGN: Repeated observation and measure.SETTING: Third Department of Orthopaedics, Second People's Hospital of Shenzhen; Department of Micro-surgery , First Hospital Affiliated to Sun Yat-sen University.MATERIALS: This experiment was conducted at the Experimental Animal Center of Medical College of Sun Yat-sen University from March to May 2003. Twenty Sprague-Dawley rats with the age of 3-4 months, of clean degree, were selected and divided randomly into experimental group of Schwann cell derived neurotrophic factor and control group of normal saline with 10 rats in each group. The right side was injured, and the left side was intact served as normal control side.METHODS : ①A rat model of C6,7 spinal root avulsion induced motoneuron degeneration was established. ② A small piece of gelfoam presoaked in 40 μL SDNF solutions (1 g/L) was placed in contact with the injured spinal cord in the animals of the experimental group. Normal saline was added as the same way as above in the animals of the control group. ③ A silica pipe was put on the surface of gleform, one end of the silica was sutured to the glefoam , and the other end wasfixed subcutaneously with vaselinum. Local intramuscular injection of penicillinum was performed on the wound following closing the incision. All rats received an injection (20 μL) of either SDNF or normal saline solution at the lesion site through the silica pipe sutured to the glefoam once a week after the surgery. All the animals were killed by the end of the third weeks. ④The spinal region of C6,7 level was dissected out for observing survival rate and morphological change of motoneurons of spinal anterior horn as well as the expression of nitricoxide synthase(NOS).MAIN OUTCOME MEASURES: ① Survival and morphological change of spinal motor neurons. ②Change of nitricoxide synthase expression of spinal motor neurons.RESULTS: Totally 20 rats were enrolled in the experiment, and all of them entered the stage of result analysis. ① Survival and morphological changeof spinal motor neurons: 68.6% motoneurons of injured side of the control group died at 3 weeks after surgery. The survival rate was 31.4%,which was significantly lower than that of the intact side (P ＜ 0.01), and the survived neurons was shrinked significantly; the death rate of spinal motor neurons of injured side of experimental group was decreased by 35%as compared with control group (P＞ 0.05). The survival rate was 66.4%,and the survived neuron body was increased, similar to the intact side (P ＞ 0.05). ② Change of nitricoxide synthase expression of spinal motor neurons: In normal spinal cord, NOS positive neurons were shown in dorsal horn, surrounding the central canal and in the intermediolateral column.NOS was not seen in the anterior horn motonurons. At the end of the third week after C6,7 spinal root avulsion, increased NOS expression was not found at the injured side in the Schwann cell derived neurotrophic factor group and the intact side in the control side, while the significantly increased NOS expression of spinal motoneurons was found at the injured side of the control group.CONCLUSION: Degeneration of spinal motoneuron and increased expression of NOS can be induced by spinal root avulsion. SDNF has a significant effect in protecting spinal motoneurons from spinal root avulsion induced cell death and inhibiting the expression of NOS. These results suggest that the effects .of SDNF on motoneuron survival may be achieved by modifying the expression of certain cellular molecule such as NOS.
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[Objective]To explore the main points,skills,features and indication of orthophoria microscope cervical anterior discectomy(OMD)to treat cervical spinal intervertebral disk herniation,through its clinical application.[Method]Retrospective analysis of the clicnical data of 36 cases of cervical disc herniction treated with OMD were made.[Result]All the patients of the group were discharged in 7 days after operation.After an average followed-up 9.4 months.The rate of fineness was 90%.No serious complication.In the 36 cases,the X ray of the operation appears that the reasult satisfied.[Conclusion]Orthophoria microscope discectomy(OMD)is the best way now to take diseectomy.
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BACKGROUND: The free border of meniscus is avascular portion, for which, it is not susceptible for the meniscus to be cured naturally after injury. Therefore, it is necessary to induce fibrous tissue healing probably under certain situation.OBJECTIVE: To adopt tissue engineered cartilage and fibrin adhesive to treat meniscus injury in avascular portion and compare the results.DESIGN: Randomized group division and blank control experiment was designed.SETTING: Animal Laboratory of a Shenzhen Second People's Hospital.green-purplish-blue adult rabbits were selected, randomized into 3 groups,12 rabbits in each, named blank control, fibrin adhesive group(FA group)and tissue engineered cartilage group(TE-C group).METHODS: The experiment was performed in Animal Laboratory of Shenzhen Second People's Hospital from September 2003 to March 2004.Ten baby rabbits borne in 3 to 5 days were sacrificed to collect fibrochondrocytes for culture so as to prepare tissue engineered cartilage containing 12 × 108 L-1chondrocytes. Thirty-six adult rabbits were prepared into the injured model in avascular portion of meniscus (0. 7 × 0. 3) cm with full-thickness laceration. In blank control, no any filler was applied for management; in FA group, fibrin adhesive was infused in laceration; and in TE-C group, tissue engineered cartilage was infused in laceration. Four animals of each of 3 groups were sacrificed in the 2nd, 6th and 12th weeks after operation. Eight menisci were collected in each group each time for gross morphological observation and histological examination.MAIN OUTCOME MEASURES: Gross morphological observation and histological examination in injured meniscus model of rabbit.logical observation in injured meniscus model of rabbit: In blank control, the splits in meniscus were not been healed and tissue filler was not apparent. In FA and TE-C groups, the splits had been filled up with tissue fillers comblank control, 2 to 12 weeks after operation, there was chondrocyte proliferation presented on the border of splits. In FA group, 12 weeks after operation, on the defect border, there were many fibroblastic cells that closely adhered to adjacent tissue, resulting in scar tissue healing. In TE-C group,12 weeks after operation, cartilage cavities and capsule were apparent in the defect and chondrocftes were in cell condensation.CONCLUSION: Tissue engineered cartilage is survived in the acceptors, resulting in fibrocartilaginous healing and specific biological label of chondrocytes. But the remarkable difference presents in collagen arrangement among the repaired tissue, adjacent normal meniscus tissue and normal cartilage.
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Objective To study the feasibility of applying nanometer ceramics artificial bone in clinical repair of bone defects. Methods The animal models of bone defect was made on the unilateral radius of 45 New Zealand white rabbits, which were divided into experimental group(repair with nanometer ceramics artificial bone), control group (repair with ceramics artificial bone) and blank group (unrepaired) randomly. The reconstructive effect in each group was evaluated by gross observation, alkaline phosphatase(ALP) detection of blood serum, histopathological observation, X-ray examination and SEM detection at 4th, 8th and 12th weeks postoperatively. Results In the experimental group there was more bone formation than in the control and the blank groups. The differences in reconstructive effect were statistically significant ( P
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Objective To discuss the strategies for diagnosis and treatment of severe pelvic fracture. Methods 38 patients with severe fracture of pelvis circle and different complications were studied in this paper from May, 1997 to June, 2005. 28 cases underwent emergency treatment for shock, 31 cases had internal fixation, and 14 cases received operative procedures for their complications. Results All the 38 cases survived. The integrity of the pelvic circle was restored in patients with unstable pelvic fracture. Most of the patients were back to their former work. Conclusions In treatment of severe pelvic fractures, the hemorrhagic shock and other complications endangering life should be promptly treated, and restoration of the integrity of the pelvic circle is the most important step.
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Objective To study the clinical outcome of thoracolumbar fractures treated by ante rior route decompression,bone-graft-fusion and internal fixation.Methods 51cases of thoracolumbar fractures from Jun1994to Mar 2002were treated by anterior route decompression,bone-graft-fusion and internal fixation,of w hich32cases by Kaneda system and 19cases by Z-Plate system.Their clinical data were analyzed.Results The patients were followed up from five months to eight years(average of 3.5years).45cases had a good anatomic reduction and maintain thoracic-lu mbar lordosis.48cases gained one to three Frankel grades of neurologic r e-covery as a consequence of operation.Conclusion Kaneda internal fixation has the characteristics of low cost,neurological decompression and reliable fixation;and Z-Plate systems has the advantages of simple manipul ation,intrinsic stability,less complications and good biocompatibility.
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Objective To report the clinic experience of tr eatment of severe pelvic fracture in order to improve the early diagnosis and operation of the injury.Methods We retrospectively studied the clin ic data of106patients admitted to our hospita l for severe pelvic fracture from Apr il 1994to May 2002.Results The main causes for pelvic fracture were traffic accident injury(69cases,65.1%)and falling accident injury(31cases,29.3%).87cases were in the survival group,and 19cases in the death group,with t he mortality of about 17.9%.In the death group,10di ed from hemorrhagic shock,4of severe cerebral injury,3from MOF,and 2from ARDS.32cases with pelvic f ractures were treated by opening red uction and internal fixation,91.7%of which achieved good results.Conclusion In the treatment of severe pelvic fra ctures,prehospital emergency care is very important.Complicated severe injuries should be treated pr omptly and pelvic fractures be fixed with internal fixation as soon as possibl e.