AIM:Neural stem cells can be induced to differentiate into various types of neural cells such as neurons and neuroglia cells,but the technique of depuration and cultivation does not consummate.This article determines the optimal culture technique of neural stem cells by different culture concentrations and passage methods. METHODS:Experiments were conducted from May to December 2006 at Laboratory of Transplantation Immunity of Sichuan University.①Clean pregnant female rats(embryonic age range from 12-16 days)and the disposition of animal met ethical standard.②The cerebral cortex of rat embryos were collected,and digested with trypsin and ethylenediamine tetraacetic acid mixture to obtain signal cell suspension.They were cultured in serum-free medium (DMEM/F12 medium containing B27,basic fibroblast growth factor and epidermal growth factor).The 3~(rd)passage cells were collected,and incubated at 1?10~7 L~(-1),1?10~8 L~(-1),1?10~9 L~(-1),1?10~(10)L~(-1),respectively.In addition,neural stem cells were collected 7-10 days after primary culture to harvest formative cell masses.Mechanical blow refers to soft blowing with haustorial tube from thick to thin after centrifugation,or sterile syringe with No.5 pinhead blowing cells when the blow was 5 times.Bubble production was avoided during the operation.Trypsin aspiration combined with mechanical blow refers to trypsin was added after centrifugation,at 37℃for 10 minutes,the neural stem cells were lightly blown with haustorial tube polished with flame or blown with sterile syringe with No.5 pinhead,and then fetal bovine serum was added to stop digestion.③The growth characteristic of the 3~(rd)passage cells at different culture concentration was observed and proliferation was measured at days 1,3,5 and 7 by MTT assay.The neural clone spheres of subcultured was counted to determine the optimal passage way.Immunofluorescence was carried out to detect nestin(special marker to neural stem cells),BrdU,neurone specific enolase,glial fibrillary acidic protein. RESULTS:①Growth characteristics and identification of rat embryonic neural stem cells in vitro:The dissociated neural stem cells from the cerebral cortex of rat embryos were continuously harvested and purified by suspension cultures to get the daughter cell clone.Nestin positive cells could be found in the neurospheres and after attachment they could differentiate into neurone specific enolase and glial fibrillary acidic protein positive cells,and immunofluorescence showed a great of BrdU-positive cells.②The effect of different incubated number of neural stem cells on proliferation:When the neural stem cells planted at the concentration of 1?10~9 L~(-1),the growth rate of the cells was the highest of all concentrations.The number of clone spheres exceeded others at concentration 1?10~7 L~(-1),1?10~8 L~(-1)and 1?10~10 L~(-1)(P

Objective To observe the effect of intravenous administration of bone marrow stromal cells (BMSCs) on the functional recovery after spinal cord injury in rats.Methods BMSCs harvested from 10 donor adult Sprague-Dawley rats were isolated,cultured,purified,amplified and labeled with bromodeoxyuridine (BrdU ).A total of 66 adult Sprague-Dawley rats were subjected to weight-drop impact causing complete paraplegia and randomly divided into three groups seven days after injury.Group A (22 rats) was treated with 2 × 106 BMSCs cultured in 1 ml phosphate-buffered saline by tail vein,Group B (22 rats) was treated with 1 ml Dulbecco' s modified Eagle medium and Group C (22 rats) was set as blank control group.The distribution and differentiation of donor cells in spinal cord were observed in recipient rats by using immunohistochemical staining and expressions of growth associated protein-43 ( GAP-43),neurofilament 200 (NF200) and nestin in the contused spinal cord measured at 2,3 and 6 weeks respectively after injection.The motor function of three groups was evaluated by the Basso-Beattie-Bresnahan ( BBB) scores at 1,2,3,4,5 and 6 weeks respectively after injection.Results BrdU-reactive cells were mainly distributed through the contused and near regions of spinal cord at 2,3 and 6 weeks after injection.The survived cells in contused spinal cord accounted for 4.9% ,4.4% and 2.6% of the total injection cells at 2,3 and 6 weeks respectively after injection.Two weeks after injection,BMSCs were mainly round or ellipse in shape,12.6% BrdU-reactive cells expressed glial fibrillary acidic protein (GFAP) and 5.4% expressed the neuronal nuclear antigen (NeuN).The expressions of GAP-43,NF200 and nestin were detected at 2,3 and 6 weeks after injection,with significant higher level in Group A than that in Groups B and C (P<0.05).BBB score in Group A was significantly higher than that in Groups B and C 3-6 weeks after transplantation (P<0.05).Conclusions With intravenous administration after spinal cord injury,BMSCs migrate and survive into contused spinal cord,exhibit site-dependent differentiation,up-regulate the expressions of GAP-43,NF200 and nestin,ameliorate nerve function and can be used for treatment of spinal cord injury.

Objective:To develop a method for the determination of palladium in bendamustine hydrochloride by GFAAS. Meth-ods:The sample was destroyed by heat, and the content of palladium was determined by GFAAS with the detection wavelength of 247. 6 nm. Results:The absorbance and the content of palladium showed a good linear relationship within the range of 20-60 ng· ml-1(r=0. 998 4). The average recovery of palladium was 102. 9%(RSD=1. 7%, n=9). Conclusion: The method is sensitive and simple, which can be used for the determination of palladium in bendamustine hydrochloride.

Objective To evaluate the clinical result and feasibility of anterior approach with posterior vertebral wall preserved in the treatment of thoracolumbar burst fracture with or without paraplegia. Methods From 2005 to 2010, 68 patients with thoracolumbar burst fracture were treated by corpectomy, strut graft and instrumentation with preserved posterior vertebral wall. There were 49 males and 19 females at average age of 39.8 years (16-62 years). Kyphotic Cobb' s angle and spinal stenotic rate was measured preoperatively and postoperatively. The neurological status was evaluated with Frankel impairment scale. Results All patients were successfully managed with this technique, with no neurological deteriorations, cerebrospinal fluid leakage or incision infections except for the pneumothorax in three patients who were then cured through expectant treatment. Screw distraction was performed for restoration of the disc height and kyphosis in 68 patients. After the fractured fragment became loose and was removed, the dura matter was exposed in 19 patients including the eerebrospinal fluid leakage in three patients. Retrograde lumbar myelography was applied in eight patients, of whom there found the blocked spinal canal in three patients and excision of the posterior vertebral wall was performed. Lack of vertebral distraction was found in two patients and extraction of the reversed bone fracture behind the posterior longitudinal ligament was performed in one. The preservation rate of the posterior vertebral wall was 95% (65/68). Fifty-two patients were followed up for mean 2.2 years (from 3 months to 4.5 years) ,which showed no lower back kyphosis. There showed 1-3 Frankel grades of improvement in spinal cord function except for five patients at Frankel grade A. The Cobb angle was average 18.2° in 68 patients preoperatively and was corrected to 9.7°in 52 patients at last follow-up. CT scan showed that the stenotic rate was 42% preoperatively and 9% at final follow up in 68 patients,with no breakage of the screw and plate. Conclusion In the management of thoracolumbar burst fractures,anterior approach is helpful for preservation of the posterior vertebral wall.

BACKGROUND: Hyperpolarization-activated cyclic nucleotide-gated (HCN) current plays an important role in regulating heart spontaneous pulsation.OBJECTIVE: To observe target gene expression and electrophysiological characteristics of pig bone marrow mesenchymal stem cells (BMSCs) transfected with hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) gene recombinant adenovirus.DESIGN, TIME AND SETTING: Cell-gene in vitro study was performed at the Laboratory of Thoracic and Cardiovascular Surgery,Second Military Medical University of Chinese PLA from July 2007 to March 2008.MATERIALS: Yorkshire pig was supplied by Animal Institute, Second Military Medical University of Chinese PLA. HCN2 plasmid was presented by Professor Dario DiFrancesco from Italy. Recombinant adenovirus Ad.HCN2 was constructed and stored using Ad5 in this laboratory.METHODS: Pig BMSCs were isolated with combination of gradient centrifugation of Percoll and adherent treatment in vitro.Ad.HCN2 was transfected at multiplicity of infection=50. We also set non-transfection and transfected Ad.Null groups.MAIN OUTCOME MEASURES: Expression of HCN2 mRNA was detected with RT-PCR, and expression of HCN2 channel protein was examined with immunofluorescent staining. Electrophysiology of HCN2 channel protein was measured with whole-cell patch clamp.RESULTS: No amplified fragments were found in the non-transfection and transfected Ad.Null groups, but amplified fragments were determined at 250-500 bp following Ad.HCN2 amplification, which was the same as plasmid carrying HCN2 gene. Staining strength of cell nuclei following transfection was significantly weaken compared with cell membrane and plasma, which showed identical distribution as HCN2 protein. No HCN2 protein was detected in the non-transfection and transfected Ad.Null groups.Pacemaker current could be recorded with a whole-cell patch clamp. It was fully activated around -140 mV with an activation threshold of -60 mV, presenting voltage dependence. CsCI (4 mmol/L) reversibly blocked the inward currents. No pacemaker current was detected in the non-transfection and transfected Ad.Null groups.CONCLUSION: The HCN2 recombinant adenovirus carrier was transferred into serial subcultivation porcine bone marrow mesenchymal stem cells. HCN2 channel protein has been expressing. Pacemaker current could be recorded with a whole-cell patch clamp.

To observe the effect of pIgR on Salmonella Enteritidis induced inflammation in jejunum and ileum in Chicken,7 day Hyline chickens were taken orally with Salmonella Enteritidis (SE) and killed after 1 d,3 d,7 d and 14 d.The mRNA expression of Toll-like receptor 4(TLR4),MyD88,TRAF6,NF-κB and pIgR was detected by real-time RT-PCR and pIgR protein level was detected by Western blot.The results showed that TLR4 signaling pathway was activated and mR-NA level of the pIgR in jejunum and ileum was enhanced (P＜0.01) and protein level of the pIgR in jejunum and ileum was up-regulated by SE.The study proved that TLR4 signal pathway on mucosal cell surface of jejunum and ileum was activated and expression of pIgR was up-regulated and gut mucosal immunity of chicken was strenghtened.

BACKGROUND:Studies have shown that placement, needling point, angle and length of pedicle screws for thoracic vertebral fractures have been unified. Many studies concerned axial pul-out force and biomechanical behavior of a single screw, but few reports addressed the biomechanical behavior after the replacement with three kinds of screw connecting to rods. OBJECTIVE:To further observe and compare biomechanical behavior after three kinds of thoracic screw placement connecting rod using biomechanical testing, to understand the strength of thoracic pedicle screw replacement connecting rods, and the effects of its puling out on the stress of the bone surrounding the placement site. METHODS:The fresh frozen specimens of thoracic vertebra were divided into three groups at random: the pedicle fixation group, the transverse process-vertebral body fixation group, and modified rib transverse process fixation group. Thoracic pedicle screw system was inserted by the standard placement method in each group. The experiment was measured on the mechanical equipment. During puling out, the strength that bone bore was described with dynamic curve, folowed by statistical analysis. The difference in the gripping force on the thoracic vertebra among three kinds of screw placement method was compared. RESULTS AND CONCLUSION: Among three kinds of screw placement methods and among three kinds of screw placement methods on the injured vertebral body, the gripping force was significantly bigger in the pedicle fixation group than in the transverse process-vertebral body fixation group and modified rib transverse process fixation group (P < 0.01). No significant difference in the gripping force was detected between the transverse process-vertebral body fixation group and modified rib transverse process fixation group. Results verified that the stability of loading ability was apparently better in the pedicle fixation group compared with the transverse process-vertebral body fixation group and modified rib transverse process fixation group.

Objective To evaluate the effects of focal vibration on the physical properties and surface electroyograms of persons with neck-shoulder pain.Methods Thirty-four patients with neck-shoulder pain received 3 to 5 applications of vibratory stimulation of the inner side of the trapezius and scapula for 30 s each time.They reported their pain level using a visual analogue scale.Muscle tone and the elasticity and stiffness of the trapezius muscle were measured bilaterally using a MyotonPRO Muscular Function Evaluation System.Surface electromyography (sEMG) was performed bilaterally during arm extension while standing.The testing was repeated four times:before treatment (baseline),immediately after treatment (IAT),2 hours after treatment (2AT) and 6 hours after treatment (6AT).Results There was a significant reduction in reported pain at IAT and 2AT compared to baseline,but no significant difference was found between the perceived pain at baseline and at 6AT.The values of oscillation frequency,logarithmic decrement and dynamic stiffness had decreased significantly at IAT and 2AT compared to baseline.The average AEMG during upper-arm flexion of the trapezius on the painful side had increased significantly at IAT comparcd to that before the treatment,while the average IEMG and MF were both significantly higher than before the treatment.At 2AT the average AEMG and IEMG were both still significantly higher than at the baseline,but there was no longer a significant difference in the average MF.By 6AT none of the three indicators differed significantly from those before the treatment.Conclusions A single session of focal vibration can relieve pain,increase muscle activation as well as decrease muscle stiffness for people with neck-shoulder pain within 2 hours.

Objective To evaluate the efficacy of pedicle screw fixation combined with bucking bar technique in treatment of C2 pedicle fractures.Methods A retrospective case series study was conducted for 26 cases of C2 pedicle fractures undergone posterior pedicle screw fixation after putting the bucking bar on the anterior surface of C2 via oral cavity from April 2004 to April 2014.There were 19 males and 7 females,with a mean age of 42 years (range,27-71 years).Bilateral C2 pedicle screw placement status,operation time and intraoperative blood loss were recorded.American Spinal Injury Association (ASIA) impairment scale and Visual Analogue Scale (VAS) were used for outcome evaluation.Fracture reduction and symptoms such as sore throat,expectoration,hoarse voice,dysphagia and condition of posterior pharyngeal wall were detected after operation.Results Pedicle fixation of C2 pedicle fractures was completed successfully in 21 cases,repeat C2 pedicle fixation took place in three,and C1-3 fixation was conducted for two.Operation time was (83.2 ± 16.6) min and blood loss was (171.2 ± 115.5)ml.One of the cases experiencing repeat manipulation for screw insertion had transient body balance disorder caused by cerebellar ischemia and recovered within 2 days.X-ray and CT showed complete reduction in 17 cases and incomplete reduction in nine cases.Bony fusion was obtained 6 months after operation.Twenty cases were followed up for 3-46 months (mean,26 months).Final follow-up showed anatomical reduction and bony healing in 19 cases,while malunion in other three cases.ASIA grade improved from C to D in one case,whereas the rest showed a complete neurological recovery (ASIA grade E).VAS improved from preoperative 3-8 points [(5.2 ± 2.5) points] to 0-4 points [(1.6 ±1.5) points] at the final follow-up (P ＜ 0.05).Mild swollen sore throat occurred in four cases after operation.Laryngoscopy showed injury to the posterior pharyngeal wall mucosa (Grade Ⅰ in six cases and Grade Ⅱ in two cases),which were healed within 5-7 days after operation.Conclusion For C2 pedicle fractures,pedicle screw fixation combined with bucking bar technique via oral cavity benefits fracture reduction and stability without apparent impairment to the soft tissue of pharyngeal mucosa.

BACKGROUND: Current commonly used bone graft materials include cancellous bone or bone blocks from ilium, tibia, fibula and others. There is no report of bone graft and fusion in cervical spine from manubrium sterni. OBJECTIVE: To measure lateral X-ray of cervical vertebrae with no degeneration and manubrium sterni samples to explore feasibility of autologous cancellous bone graft of manubrium sterni with pyramesh in anterior cervical spinal fusion surgery, so as to develop a new source of autogenous bone graft.METHODS: A total of 40 manubrium sterni specimens were selected and the length, maximum width and minimum width, thickness, anterior and posterior cortex thickness of manubrium sterni were measured. Manubrium sterni area was calculated: the length, maximum width and minimum width were 4 mm reduced from those of manubrium sterni, its thickness was 2 mm reduced from that of manubrium sterni. The formula of measuring the manubrium sterni volume is (maximum width of manubrium sterni + the minimum width of manubrium sterni)×length of manubrium sterni × thickness of manubrium sterni×1/2. Intervertebral height and vertebral height from C2 to C7 were measured from lateral cervical radiograph of 106 patients with cervical radiolopathy, and the length of pyramesh needed in anterior cervical spinal surgery for one, two, three discs resection, one vertebra plus two discs resection and two vertebrae plus three discs resection was calculated. RESULTS AND CONCLUSION: The manubrium sterni volume was (17 735.51±5 234.92) mm3 and the volume of bone-grafting area was (8 982.83±2 427.76) mm3. The length of pyramesh and volume of bone used in operation were minimal for one disc resection, and maximal for two vertebrae plus three discs resection. The volume of bone graft in the pyramesh was significantly less than the donor area in any anterior cervical spinal fusion operation. Results show that autologous cancellous graft of manubrium sterni combined with pyramesh supporting can be used in anterior cervical spinal fusion surgery. Compared with autologous iliac graft, this method is simpler and does not influence early functional exercise or walking bearing weight.