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Objective To explore the management strategy and indications for revisionary internal fixation after percutaneous kyphoplasty/percutaneous vertebroplasty (PKP/PVP) in cancellous vertebral fractures.Methods A retrospective analysis was made of the 676 cases of single-segment PKP/PVP at Department of Orthopaedics,The Affiliated Hospital to Qingdao University from January 2008 to January 2019.They were subjected to 4 different managements after their primary PKP/PVP:rehabilitation without any treatment in 637 cases,conservative treatment in 19 cases (including 3 ones who refused any revision),KP/VP revision in 12 cases and internal fixation revision in 8 cases.The rate of volume reduction after bone cement dispersion (Vx) was calculated using software Mimics 17.0 on the basis of primary CT data of all the patients.The correlation regression analysis was made between the revision rate and the approximate quantization value of Vx.The Glasgow Coma Score (GCS) of conscious state was used to evaluate the 39 patients after failure of their primary surgery before the surgical strategy for revision was worked out.The cobb angle,pelvic incidence angle (PI),pelvic inclination angle (PT),sacral inclination angle (SS),sagittal deviation (SVA),pain visual analogue scale (VAS) were measured and recorded before operation and at the last follow-up for the KP/VP revision group and internal fixation revision group,indicated as △cobb,△PI,△PT,△SS,△SVA and △VAS,respectively.The indexes were compared between the 2 groups.Results The incidence of osteoporotic vertebral fractures treated with internal fixation revision was 1.18％ (8/676).The correlation between Vx and revision rate was y =0.53 + 0.04x (P ＜ 0.05).The regression analysis showed that Vx was positively correlated with the revision rate (r2 =0.860,P =0.001) and the fitting curve was correlated (r2 =0.916,P =0.001).The GSC scores revealed 31 normal,6 mild disturbance and 2 moderate disturbance cases.There were no significant differences in gender,age or VAS scores between the KP/VP revision group and the internal fixation revision group (P ＞ 0.05).There was a significant difference in △cobb between the 2 revision groups (6.3° ± 7.5° versus 19.2° ± 14.8°) (P ＜0.05),but there were no significant differences between the 2 groups in △PI (4.1°±5.2° versus 3.3°±6.7°),△PT (0.7°±4.6° versus 0.4° ± 3.2°),△SS (3.7° ± 6.2° versus 3.1° ± 5.3°) or △SVA (-3.2 ± 11.9 mm versus-7.9 ± 9.5 mm) (P ＞ 0.05).Conclusions The outcomes of primary PKP/PVP have a great impact on the decision-making of internal fixation revision.The mode and extent of diffusion after initial vertebral cement perfusion are particularly related to the revision rate.The revision plan should depend on clinical symptoms.The internal fixation revision should be individualized to ensure the quality of life of the patients in line with the principles of "resolving symptoms" and "moderate correction".
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BACKGROUND:Inhibiting the apoptosis of intervertebral disc cel s can postpone the degenerative process of intervertebral disc. Survivin has a strong function of regulating cel proliferation and anti-apoptosis. OBJECTIVE:To construct and identify the lentiviral vector encoding survivin gene of human. METHODS:The survivin gene of human (BIRC5) was synthesized through the gene synthesis technology, amplified by PCR and analyzed by electrophoresis. The target gene was cloned into lentiviral expression plasmid to obtain the recombinant lentiviral vector Lenti-BIRC5. After transformation into competent E. coli cel s, the candidate clones were identified by PCR firstly. The positive clones were identified by gene sequencing. The lentivirus plasmid containing target gene was transfected into 293T cel s, and the expression of recombinant lentiviral vector Flag-Survivin fusion protein was detected through western blot analysis. RESULTS AND CONCLUSION:The PCR results of electrophoresis and DNA sequencing showed that lentiviral vector containing human survivin gene was constructed successful y. Western blot analysis results showed that the target gene was transfected successful y and over-expressed in cultured cel s. The lentiviral expression vector of human survivin gene Lenti-BIRC5 was constructed successful y, which lays a foundation for the study addressing the anti-apoptotic effects of survivin on human nucleus pulposus cel s.
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ObjectiveTo investigate the clinical features and treatment of ascending paralysis after thoracolumbar fracture.MethodsThree male patients with 2 fracture levels at T12 and one at L1 were retrospectively studied.Their mean age was 41.3 years(range,39-42 years).All 3 cases were undertaken open decompression,reduction and internal fixation.Paralysis level began to ascend at 2-5 days after injury,with 2 cases up to C2,3 and 1 case up to T7.Two patients suffered irritating pain over the paralysis level before onset of ascending.Postoperative MRI images demonstrated well reduction and no compression of spinal cord.In the early phase after ascending,MRI obviously showed swelling in spinal cord and long T1 and long T2 signals shaped patchy and stripy distribution in the central area.One patient's MRI displayed that the spinal cord shrinked 16 days after trauma with abnormal high signal in the central area.ResultsTwo cases died of respiratory muscle paralysis and 1 case suffered paraplegia with no recovery 5 years after surgery.ConclusionAscending paralysis after thoracolumbar fracture is a rare complication with very poor prognosis.MRI is available for evaluating operational effects and affected level.The exact mechanism and effective treatment are still unclear and need further investigated.
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Objective To investigate the risk factors and preventive measures for acute epidural hematoma after posterior thoracic spine surgery.Methods A retrospective study of 14 patients who developed acute epidural hematoma after thoracic spine surgery from May 2002 to May 2012 was conducted.There were 6 males and 8 females,aged from 41 to 69 years (average,61.2 years).There were 10 cases of thoracic spinal canal stenosis,3 cases of thoracic spinal meningioma,and 1 case of thoracic metastasis.About 3-14 h (average,6.6 h) after posterior thoracic spine surgery,the neurological deterioration was found,and according to the American Spinal Injury Association (ASIA) classification,there were 5 cases of grade A and 9 cases of grade B.The neurological function before evacuation of hematoma was compared with that after evacuation of hematoma and that at final follow-up.The correlations between hematoma compression time,neurological improvement rate and neurological function before evacuation of hematoma were statistically analyzed.Results After evacuation of hematoma,the ASIA classification of 14 patients was as follows:grade B in 1 case,grade C in 2 cases,grade D in 4 cases,and grade E in 7 cases.The hematoma compression time of 3 patients with grade B or C was more than 10 hours.Obvious difference of neurological function was found before and after evacuation of hematoma.The neurological improvement rate was 63.7％±23.3％ after evacuation of hematoma,which was negatively correlated with hematoma compression time and positively correlated with preoperative neurological function.The neurological function before evacuation of hematoma was significantly different from that at final follow-up.The neurological improvement rate was 86.97％±17.58％ at final follow-up,which was negatively correlated with hematoma compression time and positively correlated with preoperative neurological function.Conclusion The acute epidural hematoma after thoracic spine surgery could cause severe neurological deterioration.The neurological improvement was negatively correlated with hematoma compression time.Evacuation of hematoma must be done as soon as possible once progressive neurological deterioration is found.
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Objective 1) To verify the the potential of the adenoassociated viral vector as a strategy for intradiscal gene transfer in degenerative rabbit intervertebral discs. 2) To investigate the gene transduction efficacy and to quantify the biologic effects on the matrix synthesis after single gene transfer and combined gene transfer. Methods Rabbit models of disc degeneration were established by injecting the N-terminal 30×103 fibronectin fragment (Fn-f), 4 weeks later, saline with or without virus was injected directly into 144 lumbar discs of 36 skeletally mature New Zealand white rabbits. Group A (n =8) received the rAAV2-hTGFβ1; Group B (n=6) received rAAV2-hTGFβ3;Group C (n=6) recived rAAV2-hTGFβ1 and rAAV2-hTGFβ3; Group D (n=8) recived rAAV2-EGFP as the experimental control. Group E (n=8) recived PBS as the blank control. Two rabbits of the group A and group E were sacrefied 1 week after injection, immunohistochemical staining for hTGF-β1 was performed on the slices of nucleus pulposus (NP) tissues. On 4,8 and 12 weeks after gene transferring, NP tissues were cultured or decomposed to quantify the biochemical changes of the matrix using 35S-sulfate incorporation assay and western blot detection. The expression of EGFP was observed 12 weeks after injection. Results Discs in group A exhibited extensive and intense positive immunostaining for hTGF-β1 than the control discs in group E 1 week after gene transferring. The nucleus pulposus tissues in group A, B and C exhibited a 1.28-2.06 fold increase in proteoglycan synthesis and a 1.25-1.73 fold increase in collagen type Ⅱ production over those in group E (P＜0.05 or P＜0.01).Combination of two gene transfer in group C makes a significantly increased level of proteoglycan (1.195-1.290 fold)and collagen type Ⅱ (1.152-1.219 fold) than single gene transfer in group A and B(P＜0.05 or P＜0.01).No statistic differences shows between A group and B group. The difference of the matrix synthesis between group D and group E was also not statistically significant (P＞0.05). Extensive and intensive green fluorescence was observed on the slice of nucleus pulposus tissues received rAAV2-EGFP 12 weeks after gene delivery. The expression of EGFP kept for more than 12 weeks. Conclusion Findings showed that the disc tissue injected with rAAV2 mediated genes highly expressed the therapeutic proteins from 1 week to more than 12 weeks after delivery. It is suggested that adenoassociated virus be an valid vector for the transfer of the exogenous genes in the degenerative disc. The therapeutic factors hTGF-β1 and hTGF-β3 could efficiently increase the synthesis of proteoglycan and collagen type Ⅱ in the degenerative NP cells and combined transfer of two genes was more effective than single gene transfer. The two factors have an positive synergistic effects.
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Objective To retrospectively evaluate and analyze the clinical effect of posterior pedicle subtraction osteotomy in treating chronic, posttraumatic thoracolumbar kyphosis. Methods Nineteen patients (11 males and 8 females) with chronic, posttraumatic thoracolumbar kyphosis were corrected surgically. The patients were at age range of 29-61 years (mean 42 years). Preoperative kyphosis Cobb angle ranged from 31° to 63° (mean 47°) and trauma history ranged from 8 months to 63 months (mean 29 months). All patients were treated with pedicle subtraction osteotomy according to the size of Cobb angle, extent of spinal stenosis and source of compression. Results Sagittal alignment was improved to average 40.2°, with a correction rate of 85.8%. Two patients developed postoperative leakage of cerebrospinal fluid. Among them, one was combined with encephalic infection and cured with active treatment, and the other developed postoperative wound infection, which were treated conservatively with antibiotics and local wound care. There were no other severe complications. The average follow-up period was 15 months (range 6-41 months). At the last follow-up, clinical symptoms and neurological function were improved significantly. Neither loss of correction nor failure of internal fixators was observed. X-ray and dynamic X-ray films showed a 100% fusion in all patients. Conclusions The single-stage posterior pedicle subtraction osteotomy is a safe and effective procedure for correction of posttraumatic thoracolumbar kyphosis. It is possible and safe to obtain a correction within 55° on single segment by this technique.
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Objective To investigate the clinical outcomes of trans-facet joints approach to treat thoracic degenerative disease with anterior compression.Methods From January 2003 to December 2009,22 patients with thoracic myelopathy caused by anterior compression were studied retrospectively.The patients included 16 males and 6 females,aged from 36 to 72 years(average 54.2 years).There were thoracic ossification of posterior longitudinal ligament(OPLL)in 11 cases,thoracic disc protrusion with ossification in 8 cases,thoracic vertebra posterior osteophytes in 2 cases,ankylosing spondylitis with thoracic pseudoarthrosis in 1 cases.Preoperative Japanese Orthopaedic Association(JOA)score was 5.2(range,2-9).The characteristic of thoracic degeneration was analyzed by CT and MRI examination.Posterior decompressive laminectomies were performed by the technique of "cap uncovering".The facet joints were removed bilaterally.Anterior ossified compressions were cut via posterior-lateral approach,and then intervertebral bone graft and bilateral pedicle screws were implanted.Results All patients were followed up for 8 to 38 months.According to the revised Epstein standard,there were excellent in 7 patients,good in 9,fair in 4,and poor in 2.The total effective rate was 90.9%(20/22).The excellent and good rate was 72.7%(16/22).The mean postoperative JOA score was 8.7(range,2-11).Surgical complications included dural laceration in 1 patient,pleura injury in 1 patient,epidural hematoma in 2 patients.There were no cases of spinal instability or deep infection.Conclusion The anterior compression can be solved completely via trans-facet joints approach in thoracic degenerative disease patients.
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Objective To investigate retrospectively the long-term results of discectomy for patients with lumbar intervertebral disc herniation. Methods From July 1988 to May 2003, 273 cases of 1040 patients with lumbar intervertebral disc herniation undergone surgical treatment in our hospital were followed up. All patients were divided three groups according the time of follow-up. The follow-up time was three years as middle follow-up group (Ⅰ), five years as longer follow-up group(Ⅱ) and ten years and more as sup-longer follow-up group (Ⅲ). Sixty-eight cases(24.91%) were in group Ⅰ, including 42 males and 26 females, with the average age of 43.7 years (14-63 years). The group Ⅱ included 141 cases (51.65%), 92 males and 49 females, with the average age of 46.1 years (18-76 years). As group ⅡⅢ, 64 cases (23.44%) were included 46 males and 18 females, with the average age of 43.5 years (20-63 years). The standards Scoring System of Chinese Spinal Association (CSA) and Japan Orthopaedic Association (JOA) were used for investigation. Results According to CSA system, the total good and excellent rate of surgical treatment for lumbar intervertebral disc herniation was 89.0%. The percentage of the satisfactory of the group Ⅰ, Ⅱ, Ⅲ were 92.6%, 91.5% and 79.7% respectively. There was significant difference between group Ⅰ and group Ⅱ, Ⅲ. The score of JOA were 24.75±5.08, 22.43±6.55, 21.64±7.18 postoperatively, with significant difference between group Ⅰ and group Ⅱ, Ⅲ. Conclusion The mid-term results of surgery for patients with lumbar iutervertebral disc herniation is good, and the good and excellent rate decreases gradually with the follow-up time. The results were similar to each other for evaluation between the standard of CSA and JOA.
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BACKGROUND: There are no reports about adult degenerative disc cell culture model currently. This establishment of disc cell culture is the cellular basis of intervertebral disc tissue engineering. OBJECTIVE: To establish cell culture models from human degenerative intervertebral disc, and to settle the cytology base of disc tissue engineering. DESIGN AND SETTING: The controlled observational experiment with regard to cytology was performed in Shandong Institute of Orthopaedics and Traumatology. MATERIALS: Five degenerative intervertebral disc patients were recruited from Department of Orthopaedics, Affiliated Hospital of Qingdao University Medical College, including one case in L2-3, three cases in L4-5 and one case in L5-S1. METHODS: Five samples were taken from the operational disectomy for lumbar disc herniation, and these samples were cultured using enzymatic digestion cell culture and tissue cell culture method differently. All the samples were cultured with HAMF12 medium with the addition of 10% and 20% fetal bovine serum. The morphology of the cultured cell was observed with Giernsa staining and transmission electron microscopy respectively. MAIN OUTCOME MEASURES: ①Cellular growth; ②Morphology of the cultured cells;③Ultrastructure Of the cultured cells. RESULTS: By means of enzymatic digestion cell culture and tissue cell culture method, numerous degenerative lumbar disc cells were obtained and successfully subcultured. The cell secretions around the cultured cells may influence the growth of these cells. Destroying the secretions, the cultured cells could be highly proliferated. The degenerative intervertebral disc cells were proliferated vigorously in HAMF12 medium with 20% fetal bovine serum compared with that with 10% fetal bovine serum. The notochordal cell was observed in all the specimens. CONCLUSION: The adult degenerative disc can be used to obtain the required cell for tissue engineering.
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@#Objective To investigate the expression of DR5 and DcR2 protein and mRNA in lumbar intervertebral disc (LID) of patients with prolapse of LID and normal controls.Methods The expression and distribution of DR5 and DcR2 protein were immunostained in prolapsed LIDs of 60 patients and 22 normal LIDs of 8 normal controls. In parallel, mRNA of DR5 and DcR2 was quantified by real time fluorescent reverse transcriptase-polymerase chain reaction (RT-PCR) in prolapsed LIDs of 30 patients and 9 normal LIDs of 3 normal controls.Results The positive rates of DR5 protein in prolapsed IVDs and normal IVDs were 41.60% and 26.09% respectively. There were a significant difference between the two groups ( P=0.001). Further similar evidences were obtained by quantification of DR5 mRNA ( P=0.025). There was no difference in the expression of DcR2 protein and mRNA between the two groups.Conclusion The apoptosis pathway induced by DR5/tumor necrotic factor-related apoptosis-inducing ligand (TRAIL) maybe exists in LID tissues.
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0.05)between left AF(238,36.62%)and right AF(220,33.85%).More HIZs(446,68.62%)were located in inferior AF than that of middle or superior AF.The motion segments from L3、4 to L5S1 were the region that the HIZ occurred frequently and it could present in single segment or multi-segment.In anterior AF,HIZs often occurred at L2、3 and/or L3、4 discs.Whereas,they usually developed at L4、5 and/or L5S1 in posterior AF. Conclusion The incidence rate of HIZ in lumbar disc is higher.Posterior and inferior AF of discs and lower motive segments have more risk of HIZs.It could develop in single motive segment or multi-segments at one time.
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BACKGROUND: Human transforming growth factor-β1 gene can be used for gene therapy of the degeneration of intervertebral discs, but the key to the experiment is to construct its effective vector.OBJECTIVE: To determine whether or not adult degenerated intervertebrai disc cells cultured in vitro after transfected by eukaryotic expression vector can express the product of human transforming factor-βl, and to provide the experimental basis of gene therapy for intervertebral disc degeneration.DESIGN: Single sample experiment. SETTING: Traumatic Orthopedic Institute of Shandong Province and the Orthopedic Department of Weihai Municipal Hospital.MATERIALS: The experiment was conducted at the laboratory of Traumatic Orthopedic Institute of Shandong Province between October 1999and January 2001. Intervertebral disc samples were from the operated patients with protrusion of irtervertebral disc after the patients were informed.Sample 1 was intervertebral disc at L4/5 from a 30-year-old woman; sample 2 was intervertebral disc at L5/S1from a 30-year-old woman.METHODS: ① Culture of adult degenerated intervertebral disc cells:Samples ex vivo were taken back to the laboratory within 30 minutes; fibrous ring cells and myelin nucleus cells cultured primarily were collected.② Transfection: Cells were put in the 24-well culture plate with 5.5×105cells in each well. Constructed PCI-hTGF-β1 eukaryotic expression vector was used to perform transfection, then transfected PCI group and nontransfected group were set. ③ The expression product of cells transfected for 48 hours was determined with immunohistochemical staining method.MAIN OUTCOME MEASURES: Comparison of absorbance of the positive cell product of eukaryotic expression vector PCI-hTGF-β1 in the primary fibrous ring cells and myelin nucleus cells in each group.RESULTS: ① Sample 1: The absorbance of positive cell product of eukary otic expression vector PCI-hTGF-β1 in the primary fibrous ring cells and myelin nucleus cells was 3.49 and 3.69 times that in PCI group, and 3.55times that in non-transfected group. ② Sample 2: The absorbance of positive cell product of eukaryotic expression vector PCI-hTGF-31 in the primary fibrous ring cells and myelin nucleus cells was 3.56 and 3.46 times that in PCI group, and 3.43 times and 3.33 times that in non-transfected group.CONCLUSION: PCI-hTGF-31, as the effective eukaryotic expression vector in the transfection of transforming growth factor-31 gene to culture degenerated intervertebral disc cells in vitro, can transfect adult degenerated intervertebral disc cells cultured in vitro and obtain the high expression of human transforming growth factorβ1 gene.
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BACKGROUND: It has been successful to repair articular cartilage defects by using solid carrier as cytoskeleton. We tried to transplant liquid or gel carrier materials combined cells into the body of animals, and investigated its feasibility.OBJECTIVE: To investigate the feasibility of homo-transplatation with liquid or gel carrier materials of Pluronic F-127-recombinant human bone morphogenetic protein-2 (rhBMP-2) engineered chondrocytes for the repair of full-thickness rabbit articular cartilage defect.DESIGN: A controlled experiment.SETTINGS: Department of Orthopaedics, Weihai Municipal Hospital;Shandong Institute of Orthopaedics and Traumaology.MATERIALS: The experiments were carried out in the laboratory of Shandong Institute of Orthopaedics and Traumaology from November 2001 to September 2003. Thirty-six healthy adult New Zealand rabbits of 2.5-4.5 kg, either male or female, were divided into four groups according to the method of random number table: Pluronic F-127-rhBMP-2 engineered chondrocytes group, Pluronic F-127-rhBMP group, Pluronic F127 engineered chondrocytes group and blank control group, with 9 rabbits in each group.METHODS: After grouping, the 36 rabbits were made into models of articular cartilage defects. Pluronic F-127-rhBMP-2 was used as a vector of chondrocytes which were obtained from New Zealand rabbits after cultured and amplified in vitro. The mixture of Pluronic F-127, Pluronic F-127-rhBMP-2 and cultured chondrocytes was transplanted into the defects of articular cartilage that had been made previously with φb3.5 mm drill.There was not any treatment in the blank control group. At 4, 8 and 12 weeks postoperatively, the repairing conditions of the defects were evaluated with gross observation and histological observation under light microscope and under electron microscope. The repaire quality was assessed blindly according to the Wakitani scoring standard.MAIN OUTCOME MEASURES: ① Healing of cartilage defects; ② Property and morphology of the chondrocytes, characteristics, number and arrangement of collagens in matrix.RESULTS: ① In the Pluronic F-127-rhBMP-2 engineered chondrocytes group, the transplanted chondrocytes could grow better than those in other groups, the defected areas were completely filled at 4 weeks. The regenerated tissues at 8 and 12 weeks had similar appearance with the surrounding normal cartilage tissue, but vague. Delimitation. The histological examination showed that transparent cartilages formed, and the defects were healed. ② Under electron microscope at 8 and 12 weeks, there were mature transparent cartilages in the repaired tissues, and there were irregularly arranged slight, even and non-periodical collagen Ⅱ in surrounding. In the blank control group, only fibrous repair was observed, the regenerated tissue lacked elasticity with rough surface. ③ Repairing quality score: The scores at each time point in the Pluronic F-127-rhBMP-2 engineered chondrocytes group were significantly different from those in the other groups.Those in the Pluronic F-127-rhBMP-2 engineered chondrocytes group and Pluronic F-127-rhBMP-2 group and Pluronic F-127 engineered chondrocytes group were significantly different from those in the blank control group [4 weeks: (3.93±1.91), (4.56±1.07), (4.78±1.09), (8.44±1.13) points:8 weeks: (2.80±1.45), (3.24±1.00), (3.33±1.00), (8.44±1.13) points; 12 weeks (2.22±1.10), (3.01±0.69), (3.00±0.71), (9.00±0.87) points, P ＜ 0.001],but there were no significant differences between the two groups (P ＞ 0.05).CONCLUSION: The mixture of Pluronic F-127-rhBMP-2 and cultured chondrocytes can repair successfully the cartilage defects of femoral condyle of rabbit knees by means of hyaline cartilage than simple application of Pluronic F-127-rhBMP-2 or Pluronic F-127 engineered chondrocytes.
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<p><b>OBJECTIVE</b>To determine whether the synthesis of proteoglycan, collagen and associated ultrastructure are related to the adenovirus-mediated gene transferred to adult degenerative cells.</p><p><b>METHODS</b>Adenovirus/cytomegalovirus human transforming growth factor-beta 1 (Ad/CMV-hTGF-beta 1) was used to transfect degenerative cells. Antonopulos method, Miamine method and transmission electron microscopy were conducted to study the synthesis of proteoglycan, collagen, and ultrastructure, respectively. Cell cultures were established from the nucleus pulpous and annulus fibrosus tissues, which were taken from surgery.</p><p><b>RESULTS</b>Nucleus pulpous and annulus fibrosus cells were efficiently transduced by the adenoviral vector carrying hTGF-beta 1 gene. The synthesis of proteoglycan and collagen increased compared with the control group (P < 0.05). The metabolism of cells was slightly improved. No significant toxic effects were found.</p><p><b>CONCLUSIONS</b>Expression of hTGF-beta 1 gene is efficient to accelerates proteoglycan synthesis and thus accelerates the improvement of collagen. The function and structure of degenerative cells are improved. Ad/CMV-hTGF-beta 1 may be suitable for treating disc degeneration.</p>
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Adulto , Humanos , Adenoviridae , Células Cultivadas , Colágeno , Matriz Extracelular , Metabolismo , Vectores Genéticos , Disco Intervertebral , Proteoglicanos , Enfermedades de la Columna Vertebral , Metabolismo , Patología , Transfección , Factor de Crecimiento Transformador beta , Genética , Factor de Crecimiento Transformador beta1RESUMEN
<p><b>OBJECTIVE</b>To provide a highly efficient adenoviral vector Ad-CMV-hTGFbeta1 for the study of gene therapy for reversion of the intervertebral disc degeneration.</p><p><b>METHODS</b>A newly developed recombinant adenoviral vector construction system was used in the study. The cDNA of hTGFbeta1 was first subcloned into a shuttle plasmid pShuttle-CMV. The resultant plasmid was linearized by digesting with restriction endonuclease PmeI, and subsequently transformed into E.coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Finally, the recombinant plasmid linearized by PmeI was transfected into 293 cells. Recombinant adenoviruses were generated within 2 weeks.</p><p><b>RESULTS</b>The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8% agarose electrophoresis. The infected 293 cells showed evident cytopathic effect (CPE). The productions of PCR confirmed the presence of recombinant adenovirus. The expression of hTGFbeta1 was verified by immunohistochemical staining.</p><p><b>CONCLUSIONS</b>The successful generation of the adenoviral vector Ad-CMV-hTGFbeta1 and the confirmation of the interest gene expression make it possible for the experimental study of the reversion of the intervertebral disc degeneration by gene therapy.</p>
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Humanos , Adenoviridae , Genética , Secuencia de Bases , Células Cultivadas , Citomegalovirus , Genética , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Genética , Inmunohistoquímica , Disco Intervertebral , Biología Celular , Patología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Recombinación Genética , Sensibilidad y Especificidad , Factor de Crecimiento Transformador beta , Genética , Factor de Crecimiento Transformador beta1RESUMEN
Objective To determine sensitive indices for defining degenerative cervical spinal canal stenosis(DCSCS) with a new radiograghic measurement method. Methods One hundred normal lateral radiographs of the cervical spine were divided into two groups according to ages. The following indices were measured or calculated: sagittal diameter of spinal canal(a), sagittal diameter of cervical body(b), sagittal diameter of edge or degenerative cervical body(c), cervical spinal canal ratio[(a/b),CSCR] and effective cervical spinal canal ratio [(a+b-c)/c,ECSCR]. The comparisons of these indices between two groups were performed to select the most sensitive index for clinical reference. Results There was no significant difference between the two age groups in terms of a, b and a/b. The comparison of c and (a+b-c)/c between two age groups showed significant difference at the level of C4,C5 and C6 (P
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Objective To evaluate the reversion possibility of hVEGF165 and TGF?1 to intervertebral disc degeneration by gene method. Methods The hVEGF165cDNA obtained from plasmid pcDNA3(+)-hVEGF165 was subcloned into the packaging plasmid pSNAV of AAV by molecular clone ways. The recombinant plasmid pSNAV-hVEGF165 was identified by restriction enzymes analysis and sequencing analysis, and then transfered to the HEK293 cell and VEC by lipofectamine mediated gene transfer method. The protein hVEGF165 was detected by immunofluorescence for immunocytochemistry and explored the influence to the proliferation of vascular endothelial cell by MTT. Whereafter the AAV-hVEGF165 was packaged by Benyuan Zhengyang Company. AAV-hVEGF165 and AAV-TGF?1 were cotransfected into annulus fibrosus cell of intervertebral disc, then the expression of hVEGF165 and TGF?1, and the change of collagen Ⅰin annulus fibrosus cell were detected by Western blot. Results The recombinant pSNAV-hVEGF165 was completely constructed and confirmed by restriction enzymes analysis and sequencing analysis. The protein hVEGF165 was detected by immunofluorescence for immunocytochemistry in experimental group, and hVEGF165 could promote the proliferation of vascular endothelial cell. The bioactive AAV-hVEGF165 was successfully constructed. The expression of AAV-hVEGF165 and AAV-TGF?1 were manifested in degenerative annulus fibrosus cell by Western blot, and the expression of collagen Ⅰin annulus fibrosus cell cotransfected by AAV-hVEGF165 and AAV-TGF?1 was markedly more than that of the monogenic transfected cell. Conclusion hVEGF165 could cooperate with TGF?1 to promote the expression of collagen Ⅰ.
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Objective To exoplore the causes in healing difficulties of osteoporotic fracture, and to study the feasible approaches and methods for clinical treatment of osteoporotic fracture. Methods Forty female Sprague-Dawley rats, used as donor animals, were divided randomly into ovariectomized (OVX) group and control (CON) group, with each group of 20 animals. OVX group was subjected to bilateral ovariectomy and CON group was subjected to sham operation. Four months after surgery, the rats were sacrificed, and long bones of the extremities were harvested respectively for preparation of bone matrix gelatin (BMG) in terms of Urist's method. BMG implants obtained from OVX and CON rats were implanted into left and right femoral muscle pouches respectively of 17 female SD rats. The host rats were killed after 14-day feeding, and tissue block of BMG implants were removed for histologic study, alkaline phosphatase (ALP) activity, and calcium content (CA) determination, respectively. Results ALP activity of BMG tissue block from OVX group was (7.22? 2.59)U/g, and that from CON group was (12.01?6.18)U/g. Significant difference was found between the two groups (P
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Objective To study the biological effects of pSNAV2-hTGF?1 and pSNAV2-hTGF?3 on the reversion of rabbit disc degeneration. Methods Rabbit nucleus pulpous and annulus fibrosus cells were isolated and cultured. The fluorescence labled pSNAV2 were used to detect the transfect rates of rabbit disc cells at first. Then, the pSNAV2-hTGF?1 and pSNAV2-hTGF?3 were transfected into the degenerated rabbit disc cells respectively. The biological effects of hTGF?1 and hTGF?3 on degenerated rabbit disc cells were detected with Western-bloting and 35S detection to analyze and compare the matrix synthesis of the tranfected cells. Results pSNAV2 could transfect degenerated disc cells effectively in the early stages. Both the pSNAV2-hTGF?1 and pSNAV2-hTGF?3 could stimulate the synthesis of collagen Ⅱ and proteoglycan of the rabbit disc cells. For the early stage of degenerated disc cells, the synthesis of collagen Ⅱ and proteoglycan were greater transfected with pSNAV2-hTGF?1 than transfected with pSNAV2-hTGF?3. The pSNAV2-hTGF?1 could promote the degenerated rabbit annulus fibrosus cells to synthesize collagen Ⅰ and pSNAV2-hTGF?3 could promote the degenerated nucleus pulpous cells of later stage to synthesize the collagen Ⅱ. Conclusion Both pSNAV2-hTGF?1 and pSNAV2-hTGF?3 can promote the degenerated rabbit disc cells of early stage to synthesize the matrix. pSNAV2-hTGF?3 can efficently promote the seriously degenerated nucleus pulpous cells to synthesize the collagen Ⅱ.
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Objective To define the role of melatonin in the pathogenesis of chickens scoliosis following pinealectomy and constant light irradiation. Methods Ten white leghorn chickens in the control group were kept in light-dark (12h:12h) cycle, 500 lx in daytime and 0-5 lx in nighttime after birth. Pinealectomy was performed in 20 white leghorn chickens when 3-day-old and then kept in light-dark cycle as the control group. Constant light (500 lx) irradiation was used to reduce the secretion of melatonin in 20 chickens after their births. Radiologic examinations were performed on all chicken spines for scoliosis monthly. When the chickens were 3-month-old, their mid-day and mid-night serum samples were collected and analyzed with ELISA kit for melatonin. Results There was no scoliosis in the control group and constant light group when the chickens were 3-month-old. In the pinealectomy group, 4 chickens had obvious scoliosis in the first month when X-ray examination was taken. The curved deformity progressed and became serious when the chickens grew up. There were 7 chickens with severe curved deformity in the second month. When the chickens were 3-month-old, there were totally 11 chickens with scoliosis, Cobb' angle 11?-85?, average 30.63?. The level of melatonin in control group was low in daytime (10.6 pg/ml) and high in nighttime (110.4 pg/ml) alternately. The melatonin level was much lower, daytime 8.4 pg/ml and nighttime 6.9 pg/ml in pinealectomy group and 10.8 pg/ml in constant light group. There was no statistical significance in the serum melatonin between the pinealectomy group and constant light group. Both groups remained low level of serum melatonin. Conclusion Pinealectomy can reduce the secretion of melatonin and induce scoliosis in chickens. Although constant light could suppress the secretion of melatonin in chicken serum, it did not induce scoliosis. The pathogenesis of chickens scoliosis might not be mediated by low-level melatonin.