Objective To investigate the influence of Nogo extracellular peptide residues 1-40 (NEP1-40) gene modification on the survival and differentiation of the neural stem cells (NSCs) after transplantation. Methods NSCs were isolated from the cortex tissue of rat embryo at the age of 18 days and identified by Nestin immunofluorescence. The lentiviruses were transduced to NSCs to construct NEP1-40 gene modified NSCs. The spinal cords of 30 Sprague Dawley rats were hemisected at T9 level. The rats were randomly assigned to 3 groups: group B (spinal cord injury, SCI), group C (NSCs), and group D (NEP1-40 gene modified NSCs). Cell culture medium, NSCs, and NEP1-40 gene modified NSCs were transplanted into the lesion site in groups B, C, and D, respectively at 7 days after injury. An additional 10 rats served as sham-operation group (group A), which only received laminectomy. At 8 weeks of transplantation, the survival and differentiation of transplanted cells were detected with counting neurofilament 200 (NF-200), glial fibrillary acidic portein (GFAP), and myelin basic protein (MBP) positive cells via immunohistochemical method; the quantity of horseradish peroxidase (HRP) positive nerve fiber was detected via HRP neural tracer technology. Results At 8 weeks after transplantation, HRP nerve trace showed the number of HRP-positive nerve fibers of group A (85.17 ± 6.97) was significantly more than that of group D (59.25 ± 7.75), group C (33.58 ± 5.47), and group B (12.17 ± 2.79) (P lt; 0.01); the number of groups C and D were significantly higher than that of group B, and the number of group D was significantly higher than that of group C (P lt; 0.01). Immunofluorescent staining for Nestin showed no obvious fluorescence signal in group A, a few scattered fluorescent signal in group B, and b fluorescence signal in groups C and D. The number of NF-200-positive cells and MBP integral absorbance value from high to low can be arranged as an order of group A, group D, group C, and group B (P lt; 0.05); the order of GFAP-positive cells from high to low was group B, group D, group C, and group A (P lt; 0.05); no significant difference was found in the percentage of NF-200, MBP, and GFAP-positive cells between group C and group D (P gt; 0.05). Conclusion NEP1-40 gene modification can significantly improve the survival and differentiation of NSCs after transplantation, but has no induction on cell differentiation. It can provide a new idea and reliable experimental base for the study of NSCs transplantation for SCI.
【 Abstract】 Objective To construct a lentiviral expression vector carrying Nogo extra cellular peptide residues 1-40(NEP1-40) and to obtain NEP1-40 efficient and stable expression in mammalian cells. Methods The DNA fragment ofNEP1-40 coding sequence was ampl ified by PCR with designed primer from the cDNA l ibrary including NEP1-40 gene, and then subcloned into pGC-FU vector with in-fusion technique to generate the lentiviral expression vector, pGC-FU-NEP1-40. The positive clones were screened by PCR and the correct NEP1-40 was confirmed by sequencing. Recombinant lentiviruses were produced in 293T cells after the cotransfection of pGC-FU-NEP1-40， and packaging plasmids of pHelper 1.0 and pHelper 2.0. Green fluorescent protein (GFP) expression of infected 293T cells was observed to evaluate gene del ivery efficiency. NEP1-40 protein expression in 293T cells was detected by Western blot. Results The lentiviral expression vector carrying NEP1-40 was successfully constructed by GFP observation， and NEP1-40 protein expression was detected in 293T cells by Western blot. Conclusion The recombinant lentivirus pGC-FU-NEP1-40 is successfully constructed and it lays a foundation for further molecular function study of NEP1-40.
ObjectiveTo investigate the value of small incision approach in the anterior surgery of thoracic and lumbar spinal tuberculosis.MethodsA clinical data of 65 patients with thoracic or lumbar spinal tuberculosis treated with posterior-anterior surgery between January 2015 and January 2018 was retrospectively analyzed. The patients were divided into small incision group (group A, 29 patients) and traditional incision group (group B, 36 patients) according to the length of anterior incision. There was no significant difference in general data such as gender, age, disease duration, segment of lesion, American Spinal Cord Injury Association (ASIA) grading, preoperative pain visual analogue scale (VAS) score, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and Cobb angle of spinal kyphosis between 2 groups (P>0.05). The length of anterior incision, operation time, intraoperative blood loss, postoperative complications, postoperative hospitalization time, ESR, and CRP were recorded and compared. The VAS score was used to evaluate the pain after operation. The Cobb angles in patients with spinal kyphosis were measured and the loss of angle and correction rate of angle were calculated. The result of bone graft fusion was assessed according to the Bridwell standard.ResultsThe length of anterior incision, operation time, intraoperative blood loss, and hospitalization time of group A were all significantly less than those of group B (P<0.05). All patients in both groups were followed up 12-29 months (mean, 20 months). There were 4 cases (13.8%) and 14 cases (38.9%) of postoperative complications in groups A and B respectively, showing significant difference (χ2=5.050, P=0.025). The ESR and CRP in 2 groups all returned to normal at 6 months after operation, and there was no significant difference in ESR and CRP between 2 groups at 3 months, 6 months, and last follow-up (P>0.05). At last follow-up, the neurological function of patient with neurological symptoms was significantly better than that before operation, and there was no significant difference between 2 groups (Z=0.167, P=0.868). The VAS scores of 2 groups at each time point after operation were significantly lower than those before operation (P<0.05); the VAS score in group A was significantly lower than that in group B (t=−2.317, P=0.024) at 1 day after operation, but there was no significant difference between 2 groups (t=−0.862, P=0.392) at last follow-up. Among the patients with kyphosis, the Cobb angle was significantly decreased at 1 day after operation and last follow-up when compared with preoperative angle (P<0.05); but there was no significant difference between 1 day after operation and last follow-up (P>0.05). There was no significant difference in Cobb angle, loss of angle, and correction rate between 2 groups after operation (P>0.05). The bone graft healed well at last follow-up in 2 groups. There was no significant difference in bone graft fusion rate between 2 groups at 6 months after operation, 1 year after operation, and last follow-up (P>0.05). At last follow-up, all patients cured, and no recurrence occurred.ConclusionIn the anterior surgery of thoracic and lumbar tuberculosis, the application of small incision approach can achieve the similar effectiveness as traditional incision surgery with the advantages of minimally invasive, less complications, and quick recovery.
ObjectiveTo investigate the efficacy of transcranial combined with peripheral repetitive magnetic stimulation on motor dysfunction after stroke.MethodsA total of 40 patients after stroke who were hospitalized in the Department of Rehabilitation Medicine, the Second Affiliated Hospital of Xi’an Jiaotong University between January and December 2019 were selected. The patients were divided into the trial group and the control group by random number table method, with 20 cases in each group. Both groups received conventional rehabilitation and medicine treatment, on that basis, the trial group received repetitive transcranial magnetic stimulation (rTMS) combined with repetitive peripheral magnetic stimulation (rPMS), while the control group received rTMS combined with fake rPMS, both lasted for 2 weeks. Before treatment and 2, 4, 12 weeks after the initiation of treatment, the Fugl-Meyer Assessment (FMA) [including FMA-Upper Limb (FMA-UL), FMA-Lower Limb (FMA-LL)], National Institute of Health Stroke Scale (NIHSS), and Modified Barthel Index (MBI) were used to evaluate the efficacy of rTMS combined with rPMS.ResultsFive patients fell off, and 35 patients were finally included, including 18 in the trial group and 17 in the control group. No adverse reaction occurred during the study. Before treatment, there was no significant difference in FMA, FMA-UL, FMA-LL, NIHSS or MBI scores between the two groups (P>0.05). After treatment, the FMA score of the trial group changed from 36.44±28.59 to 75.56±19.94, and that of the control group changed from 39.05±29.85 to 54.64±23.25; the between-group difference was statistically significant at the end of the 4th and 12th weeks (P<0.05). The FMA-UL score of the trial group changed from 21.39±22.14 to 46.94±15.84, and that of the control group changed from 20.82±20.47 to 31.29±16.98; the between-group difference was statistically significant at the end of the 4th and 12th weeks (P<0.05). The FMA-LL score of the trial group changed from 15.06±9.10 to 28.61±5.69, and that of the control group changed from 18.23±10.33 to 23.35±8.20; the between-group difference was statistically significant at the end of the 12th week (P>0.05). The NIHSS score of the trial group changed from 6.83±4.54 to 2.78±2.05, and that of the control group changed from 6.35±3.67 to 3.94±2.56; the MBI score of the trial group changed from 53.33±17.90 to 83.06±12.50, and that of the control group changed from 60.88±25.45 to 78.82±15.67; there was no statistically significant difference in NIHSS or MBI between the two groups at any timepoint (P>0.05). Except for the FMA-LL of the control group, the other outcome indicators in each group were significantly different after treatment compared with those before treatment (P<0.05).ConclusionsBoth rTMS and rTMS combined with rPMS can improve the limb motor function and activities of daily living of stroke patients. The treatment mode of rTMS combined with rPMS has better effect on motor dysfunction after stroke, which is of great significance for improving the overall rehabilitation effect.