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BACKGROUND:Intervertebral disc degeneration is the pathological basis of degenerative spinal diseases. Studies on the influentialfactors of intervertebral disc degeneration contribute to the prevention and treatment of degenerative spinal disease. OBJECTIVE:To observe the growth and proliferation of nucleus pulposus cels isolated by trypsin plus type II colagenase digestion in complete medium with different glucose concentrations, exploring the optimal glucose concentration for growth of nucleus pulposus cels. METHODS:Nucleus pulposus cels isolated and cultured by trypsin plus type II colagenase digestion method were observed under an inverted microscope, and thecelnumber was counted. Morphology of nucleus pulposus cels was observed afterhematoxylin-eosinstaining and toluidine blue staining. Colagen type II immunoreactivity was detected by immunohistochemical staining combined with immunofluorescent staining.Nucleus pulposuscels were incubated in complete medium containing various glucose concentrations (0, 6.25, 12.5, 17.5, and 25 mmol/L) for 24 hours, and then cel proliferation and apoptosis were determined. RESULTS AND CONCLUSION:The stained nucleus pulposus cels showed polygonal and short spindle, with one or two nuclei. Celularpseudopod appeared gradualy and then became slim with increased passage numbers. The isolated and cultured nucleus pulposus cels positively expressed colagen type II and aggrecanProliferative activity of nucleus pulposus cels cultured in medium with 17.5 mmol/L glucose was significantly higher than that in medium with 0 and 25 mmol/L glucose (P< 0.05 orP< 0.01). There wasno significant differencein cel apoptosis between these groups except for 0 mmol/L glucose (P<0.05). These results confirm that a large number of nucleus pulposus celscan beharvested by trypsin plustype II colagenase digestion and the optimal glucose concentration is 17.5 mmol/L.
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BACKGROUND: A number of studies have suggested that lycopene is an antioxidant which is used to decrease the risk of age-related chronic diseases.OBJECTIVE: To establish a rat model of postmenopausal osteoporosis, and to investigate the effects of lycopene in preventing bone loss in ovatiectomized rat models of osteoporosis.METHODS: The 6-month-old female Wistar rats, SPF grade, which had never given birth, were divided into sham and ovatiectomized groups. The ovatiectomized rats were further sub-divided into four groups which were administered with corn oil orally as model group, lycopene (10 mg/kg, 20 mg/kg, daily) or treated by intraperitoneal injection with estradiol benzoate (25 mg/kg, twice).RESULTS AND CONCLUSION: After 12-week administration, as compared with the model group, the uterus weight of high-dose and low-dose lycopene groups significantly increased (P < 0.05); the Ca, P, superoxide dismutase levels, percent trabecular area,and trabecular number were obviously higher, and serum alkaline phosphatase, urine deoxypyridinoline, malondialdehyde,trabecular separation and number of osteoclasts were obviously lower in the lycopene groups than the model group (P < 0.05). The bone mass index, bone bio-mechanics and bone histomorphometry were better in the lycopene groups than the model group (P < 0.05). These findings indicate that lycopene has a definite antiosteoporotic effect on ovatiectomized rats.
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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 investigate the relationship between a functional single nucleotide polymorphisms(SNP) in the core promoter region of GDF5 gene(+104T＞c ;rs143383) and spinal fusion in Qingdao Han people. Methods This study included 201 patients who needed to be treated with spinal fusion and 200 healthy controls. They were all out of tuberculosis, tumor, infection, and long time of related medicine using. Their operation regions were similar, containing L4.5, C4.5, C5.6, and C6.7. The SNP was defined in all people, using PCR-restriction fragment length polymorphism (RFLP) and gene sequencing. The patients were followed-up 3,6, 12 months after operation. The conditions of bone graft fusion were carried out into different grades according to imaging. According to the common used fusion criterion, to analyze the relationship between this SNP and spinal fusion. Results The results obtained from PCR-RFLP were confirmed by gene sequencing. The patients and the healthy control all showed the SNP in this site. There were no relationship between the spinal fusion patients and the healthy control in the SNP (x2=0.304, P=0.859). But it showed correlation (x2=4.752, P=0.023) with fusing or not in the spine and the speed of fusion (x2=9.864, P=0.007)in Qingdao Han people. In the fusion group, the site rs143383 showed more C allele than the non-fusion group. T allele may affect the transcription of GDF5, thus reduce the expression of GDF5. Patients with the genotype TC+CC showed larger proportion stable fusion and faster speed than the patients with TT. Conclusion SNP in the core promoter region of GDF5 (+104T＞C) is associated with spinal fusion in Qingdao Han people. The allele C may be an important factor to promote spinal fusion. Detect the TT genotype early and intervene, the spinal fusion effect may be improved. Or, the genotype may be changed by gene technology,making the efficient fusion.
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BACKGROUND:Basic research demonstrated that type Ⅰ collagen exhibited prominent effect on osteogenesis,bone mass and bone fracture,which also participated in the bone fusion.However,few reports concerning the polymorphism of type Ⅰ collagen gene and spinal fusion.OBJECTIVE:To investigate the polymorphism of type Ⅰ collagen and to explore its relationship with the spinal fusion rate following metal implant or autogenous bone transplantation.METHODS:A total of 200 volunteers who need to receive spinal fusion in the Affiliated Hospital of Qingdao University Medical College were selected,including 102 cases received anterior cervical subcorpectomy combined with lilac bone implantation fusion following decompression,and 98 cases received posterior laminectomy for decompression combined with intertransverse process fusion.Meantime,223 normal adults were served as the control group.The peripheral blood was drawn-off and genomic DNA was extracted from white blood cells.The specific fragment which includes the objective gene was amplified by polymerase chain reaction (PCR),with length of 293 bp.The genotypes of Pcol2 site in type Ⅰ collagen were detected by PCR-restriction fragment length polymorphism (PCR-RFLP) method.The PCR product was digested with restriction endonuclease Eco311 and the result was observed by agarose gel electrophoresis.The G gene represented for the presence of the restriction endonuclease site,while the T gene for the absence of the restriction endonuclease site.The fusion rate of the bone graft was evaluated by x-ray film prior to and at months 3,6 and 12 after operation,and the results were compared by stages including quick (＜3 months),middle (3-6 months) and slow (6-12 months).RESULT AND CONCLUSION:There were the-1997G/T polymorphisms of the type Ⅰ collagen gene in 423 cases,including 166cases with GG,232 cases with GT,and 25 cases with TT,in addition,there was some correlation between the GG genotype and the lilac bone implantation fusion (P =0.004).The GG genotype accounted for 50% in the fast group,which was obviously greater than that of the middle and slow groups (33.3% and 16.7%,respectively).However,the-1997G/T polymorphisms had no correlation with the bone graft fusions inter transverse process of lumbar vertebra (P=0.831).The GG genotype in the-1997G/T polymophsim of the type Ⅰ collagen gene may be the essential factor which can promote the C-spine auto-ilium graft fusion.
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Objective To investigate the association of Mycoplasma pneumoniae(MP) infection with disease activity of ankylosing spondylitis. Methods A total of 158 subjects in our hospital were enrolled in this study, including patients with ankylosing spondylitis(AS, n=66), rheumatoid arthritis (RA, n=31),osteoarthritis(OA, n=25) and normal controls(NC, n=36). MP infection was defined as anti-MP IgM antibody positive. Anti-MP IgM antibodies were determined by a mycoplasma pneumoniae(Mac strain)membrane-based agglutination test. AS patients were divided into two groups: MP infection group and non-MP infection group. T-test was used for statistical analysis of age, blood white cells, ESR, CRP, immunoglobulin, BASDAI index, global assessment on VAS scale, Schober test and chest expansion reflecting spinal mobility.χ2-test was used to compare the positive rate of MP infection in different groups. Gender difference and prevalence of clinical infection in past four weeks between MP infection and MP-free group in AS patients was also compared. Ridit analysis was used to analyze the association of MP infection with degree of sacroiliac damage on CT. Results The prevalence of MP infection in AS (52%, 34/66) was much higher than that in rheumatoid arthritis (RA, 6%, P＜0.01 ), osteoarthritis(OA, 4%, P＜0.01 ) and normal controls (NC, 11%, P＜0.01) . Compared with the non-MP infection group, the MP infection group had more active disease in term of BASDAI(4.0±1.1 vs 3.0±1.9, P=0.017), ESR[(44±32) mm/1h vs (28±23) mm/1h, P=0.029], CRP [(40±38) mg/L vs (22±21) mg/L, P=0.025] serum total IgG level [(18±3) g/L vs (16±5) g/L, P=0.027],but not in serum total IgA and IgM. Regarding to the sacroiliac joint and spinal mobility, MP infection group did not exhibit any association with the sacroiliac grading on CT, Schober test and expansion. In AS patients with MP infection, only 44.1%(15/34) was complicated by clinical manifestations of upper respiratory tract in the past 4 weeks. However, a higher prevalence of MP infection was found in AS patients with clinical manifestation of upper respiratory tract, compared with those with negative clinical manifestation(71% vs 42%,P=0.027). Conclusion Mycoplasma pneumoniae is the most common reported pathogen in ankylosing spondylitis and relates to the disease activity of AS. MP infection is probably a principal triggering factor in the pathogenesis of AS.
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BACKGROUND:Apoptosis,which result in various reasons,plays an important role in intervertebral disc degeneration. One of important reasons for apoptosis is oxidative stress. OBJECTIVE:To investigate effects and mechanism of hydrogen-peroxide(H2O2) on nucleus pulposus cell injury induced by oxidative stress at intracellular signal transduction level in rats. DESIGN,TIME AND SETTING:A single sample observation was performed at the institute of Traumatology and Orthopaedics of Shandong Province from March to October in 2008. MATERIALS:The specific p38 mitogen-activated protein kinase(p38 MAPK) inhibitor SB203580,the specific JNK inhibitor SP600125 were purchased from Beyotime Institute of Biotechnology;Two 24-hour-old SD rats of SPF degree were purchased from Qingdao Institute for Drug Control. METHODS:The primary cultured nucleus pulposus cells were divided into four groups:the H2O2 group,stimulated by H2O2 with concentrations of 0,50,100,200,400,and 800 ?mol/L;Control group;SB203580+H2O2 group:cells were preincubated with SB203580,and then were treated by stimulated by H2O2;SP600125+H2O2 group,cells were preincubated with SP600125,followed by stimulated by H2O2. MAIN OUTCOME MEASURES:The expression and cellular localization of P-p38MAPK and P-JNK was detected by immunohistochemistry,and the expression of total and phosphorylated SAPK/JNK,p38MAPK were measured by Western blotting. RESULTS:H2O2 could activate the activity of P38 and JNK. The expression of P38MAPK was significantly inhibited with the pretreatment of SB203580;however,the SP600125 could inhibit the expression of JNK. Immunofluorescence analysis demonstrated that P-p38MAPK and P-JNK were expressed and distributed mainly in cytoplasmic and nuclear exception of the control group. CONCLUSIONS:Apoptosis of rat nucleus pulposus cells are induced by oxidative stress via p38MAPK and JNK signal pathway.
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[Objective]To evaluate the accuracy and influenceing factors of somatosensory evoked potential in spinal cord monitoring during cervical and thoracic spinal surgery and intraoperative nerve root monitoring in lumbar surgery.[Method]The somatosensory evoked potential(SEP) were used during arvical and thoracic spinal surgery and evaluated the accuracy of SEP according to the record of different stages and spinal cord function after surgery.The EMG were used to monitor the nerve root function in lumber operation to estimate whether nerve root being stimulated or tensioned.In addition,affected fators of SEP and EMG during operation were observed.[Result]Of 128 cases of cervical and thoracic surgery,116 cases did not reach the warning standards(amplitude decreasing 50% or diappearing) and showed no postoperative enhancement of symptom of nerve roots injury.12 cases reached the warning standards intraoperatively and the surgeon were warned to take some steps to finish the operations,only in one case incompletely transient paralysis occurred due to the time of amplitude decreasing of intraoperative SEP more than 10 minutes.Effect of other factors such as anaesthesia and low blood pressure did not reach the warning standards.There were 3 artifical negative cases.Only 1 was artifical positive case.of 40 cases of lumbar surgery,12 cases were found myoelectic responses,which warning the surgeon at any time to avoid nerve roots injury,no nerve roots injury were found after operation.[Conclusion]During cervical and thoracic spinal operation,the somatosensory evoked potential can reflect the physiological and pathological conditions of spinal cord after ruling out the interfering factors.Intraoperative spontaneous electromyography can reflect the nerve roots function promptly and accurately and assure the safety of lumbar surgery.
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[Objective]To study the pathogenesis,clinical manifestation and surgical treatment of the posterior margin separation of lumbar vertebral epiphysis.[Method]Sixteen patients suffering from the posterior margin separation of lumbar vertebral epiphysis were followed up.The clinical manifestation and radiologic examination were analyzed and results of surgical treatment were evaluated.[Result]The patients were usually young and manifested with the sign and symptom of lumbar disc herniation and/or lumbar stenosis.CT was helpful for the accurate diagnosis of this disease.The different surgical measures were taken for the treatment according to the type and range of protrusion.[Conclusion]The posterior margin separation of lumbar vertebral epiphysis were divided into three types:end plate separation and moving into posterior margin,Schmorl node and avulsion fracture.The good results can be obtained with surgery.
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Sleep and memory are the basic function of the brain. A large number of studies from both humans and animals experiments have offered a substantive body of evidence supporting that sleep contributes crucially to memory consolidation. The processes of memory consolidation in hippocampus and cortex during sleep was reviewed and the primary cellular and molecular mechanism were briefly introduced.
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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 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.