Objective To study the time effect of the gene expression of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes so as to lay a theoretical foundation for gene therapy of osteonecrosis. Methods The best multipl icity of infection (MOI) of BMSCs transfected with rAAV was detected by fluorescent cell counting. The 3rd generation rabbit bone mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group), respectively. The expression of GFP was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by RT-PCR assay and Western blot assay in vitro. The transfected cells in 2 groups were prepared into suspension with 5 × 106 cells/mL, and injected into the rabbit thigh muscles of experimental group 1 (n=9) and control group 1 (n=9), respectively. The muscle injected with rAAV-IRES-GFP was sl iced by frozen section method and the expression of GFP protein was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by Western blot assay and ELISA assay in vivo. Results The best MOI of BMSCs transfected with rAAV was 5 × 104 v.g/cell. In vitro, the expressions of GFP, hVEGF165, and hBMP-7 genes started at 1 day after transfection, the expressions obviously increased at 14 days after transfection, and the expression maintained the b level at 28 days after transfection. In vivo, the expressions of GFP, hVEGF165, and hBMP-7 genes could be detected at 2 weeks after injection, and b expressions were shown at 6 to 8 weeks after injection. The values of hVEGF165 and hBMP-7 were (248.67 ± 75.58) pg/mL and (4.80 ± 0.61) ng/mL respectively in experimental group 1, and were (32.28 ± 8.42) pg/mL and (0.64 ± 0.42) ng/mL respectively in control group 1; showing significant differences between 2 groups (P lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has efficient gene expression ability.
Objective To investigate the effect difference between the Solis fixation fusion and the titanium plate fixation by the cervical anterior approach after decompression and bone graft implantation. Methods Of the 104 patients with cervical disease from September 2001 to March 2004, 36 were treated with the Solis implantation after decompression by the cervical anterior approach, and 68 were treated with the titanium plate fixation after decompression and bone graft implantation. The recovery of the neurological function in all the patients were assessed with the JOA Scoring at 6 weeks,3,6,12,24 and 36 months. The fragment fusion and its stability as well as the changes in the intervertebral height were assessed with X-ray examination. Results According to the JOA Scoring, the excellent and good outcomes accounted for 94.4% in the Solis group and 94.1% in the titanium plate group. In allthe patients, the fragment fusion was achieved in 3 months. The change in the Cobb angle of the fused fragment was less than 5° at the flexionextension posture, 3.6±0.8° in the Solis group, 2.4±0.7° in the titanium plate group. There was significant differences between the two groups(P＜0.05). The intervertebral height of the operation fragment in the Solis group increased 1.6±0.7mm, which was higher than that in the titanium plate group(P＜0.05). Conclusion Clinical effects of the two internalfixation operations are good; however, the Solis fixation has more advantages because of its simpler performance,less trauma, and fewer complications.
Objective To determine whether the number of distal locking bolts have an impact on the biomechanical feature of locking intramedullary nails. Methods Twenty locking nails tested were divided into two groups randomly. One distal locking screw was used in first group (single bolt group); and two were used in the other group (double bolts group). After being fixed in the model, compressive and torsional strength of the interlocking nail were measured in each group. Results The average maximum strength of double bolts group and single bolt group was 2 160 N and 1 880 N respectively in compression tests(P＜0.05). In torsion tests, the average maximum torsional moment of double bolts group and single bolt group was 55.8 Nm and 55.5 Nm respectively(P＞0.05), the average maximum torsional angle indouble bolts group and single bolt group was 58.3° and 58.0° respectively(P＞0.05). Conclusion Single distal bolt used in interlocking nail system can meet clinical request, though the whole biomechanical behavior isnot better than that of double bolts. One distal bolt is enough for the stable fracture types and double bolts should be used in the serious fracture types.
目的 观察评价盐酸氨基葡萄糖结合塞来昔布治疗膝骨关节炎（KOA）的疗效及安全性。 方法 2001年3月－2012年3月采用随机对照方法，将184例KOA患者随机分为对照组与试验组，各92例。对照组单独给予塞来昔布，试验组给予塞来昔布和盐酸氨基葡萄糖，共治疗8周，停药后继续观察4周。采用Lequesne指数作为疗效评分标准，观察服药前后的膝关节症状变化，包括休息痛、运动痛、压痛、肿胀、晨僵和行走能力的改善程度，纪录不良反应及实验室生化指标等。 结果 两组Lequesne指数在治疗前相比均明显下降，两组治疗8周后Lequesne总指数比较差异均有统计学意义（P＜0.05）。治疗8周后试验组总有效率明显优于对照组。安全性方面两组比较无差异。 结论 盐酸氨基葡萄糖结合塞来昔布治疗KOA，能明显改善患者的临床症状，疗效优于单纯的塞来昔布治疗，且不会增加药物不良反应，具有较好的临床价值。
Objective To compare the early rehabilitation effects of total hip arthroplasty (THA) with direct anterior approach (DAA) versus posterior approach (PA). Methods A retrospective analysis was made in the data of 83 patients with unilateral osteonecrosis of the femoral head between March 2014 and May 2017. Forty-eight patients were treated with THA via PA (PA group) and 35 patients were treated with THA via DAA (DAA group). There was no significant difference in gender, age, body mass index, stage of osteonecrosis of the femoral head, and disease duration between 2 groups (P>0.05). The length of incision, operation time, total amount of bleeding, the time of first postoperative walking with crutch, the time of first postoperative walking without crutch, the Harris scores, and the visual analogue scale (VAS) scores of 2 group were recorded and compared. Results All incisions healed primarily and no infection, dislocation, or fracture occurred. All patients were followed up 30.2 months on average (range, 6-44 months). The numbness symptom caused by the lateral femoral cutaneous nerve injury occurred in 2 patients of DAA group and released after symptomatic treatment. The length of incision, total amount of bleeding, the time of first postoperative walking with crutch, and the time of first postoperative walking without crutch of DAA group were significantly superior to those of PA group (P<0.05). The Harris scores of DAA group were significantly higher than those of PA group at 2 weeks and 1 month after operation (P<0.05), while no significant difference was found between 2 groups before operation and at 3 months and 6 months after operation (P>0.05). The VAS scores of DAA group were significantly lower than those of PA group at 1 day, 4 days, 1 week, and 2 weeks after operation (P<0.05), while no significant difference was found between 2 groups before operation (P>0.05). Conclusion DAA is meaningful to enhanced recovery after THA. But it should be chosen carefully because of the disadvantages, such as long learning curve, limited indications, and requirements of specific instruments.
ObjectiveTo investigate whether exosomes derived from miR-27a-overexpressing human umbilical vein endothelial cells (HUVECs)—exo (miR-27a) can promote bone regeneration and improve glucocorticoids (GC) induced osteonecrosis of femoral head (ONFH) (GC-ONFH).MethodsThe exo (miR-27a) were intended to be constructed and identified by transmission electron microscopy, nanoparticle tracking analysis, Western blot, and real-time fluorescent quantitative PCR (qRT-PCR). qRT-PCR was used to evaluate the effect of exo (miR-27a) in delivering miR-27a to osteoblasts (MC3T3-E1 cells). Alkaline phosphatase staining, alizarin red staining, and qRT-PCR were used to evaluate its effect on MC3T3-E1 cells osteogenesis. Dual-luciferase reporter (DLRTM) assay was used to verify whether miR-27a targeting Dickkopf WNT signaling pathway inhibitor 2 (DKK2) was a potential mechanism, and the mechanism was further verified by qRT-PCR, Western blot, and alizarin red staining in MC3T3-E1 cells. Finally, the protective effect of exo (miR-27a) on ONFH was verified by the GC-ONFH model in Sprague Dawley (SD) rats.ResultsTransmission electron microscopy, nanoparticle tracking analysis, Western blot, and qRT-PCR detection showed that exo (miR-27a) was successfully constructed. exo (miR-27a) could effectively deliver miR-27a to MC3T3-E1 cells and enhance their osteogenic capacity. The detection of DLRTM showed that miR-27a promoted bone formation by directly targeting DDK2. Micro-CT and HE staining results of animal experiments showed that tail vein injection of exo (miR-27a) improved the osteonecrosis of SD rat GC-ONFH model.Conclusionexo (miR-27a) can promote bone regeneration and protect against GC-ONFH to some extent.
【Abstract】 Objective To construct a recombinant adeno-associated virus (AAV) shuttle vector expressing nervegrowth factor β (NGF-β) gene. Methods By PCR amplification, the structural element of pAAV-multi ple cloning site(MCS) and the functional element of pGenesil-1.1 were obtained and cloned into T-easy vector, respectively; the recombinant T-easy vectors were digested by restriction enzyme, then the target fragments were reclaimed and connected by DNA l igase, so the recombinant AAV shuttle vector pAAV-U6/CMV-enhanced green fluorescent protein (EGFP) containing U6 promoter and CMV promoter was obtained. The vector was transfected into 293 cells. The human Miapaca-2 cell l ine was cultured, and total RNA was extracted, then human NGF-β gene was obtained by RT-PCR. T-easy-NGF-β vector was constructed by cloning human NGF-β gene into T-easy vector and identified by RT-PCR, digestion, and DNA sequencing. As NGF-β gene was cloned into pAAV-U6/CMV-EGFP vector, the recombinant AAV shuttle vector expressing NGF-β gene was obtained and identified by RT-PCR, digestion, and DNA sequencing. Results The bands of 800 bp and 4 250 bp were detected when pAAV-U6/CMVEGFP was digested. The GFP was detected when pAAV-U6/CMV-EGFP was transfected into 293 cells. The bands of 736 bp and 3 015 bp were detected when T-easy-NGF-β was digested; DNA sequencing result of T-easy-NGF-β was fully consistent. The bands of 736 bp and 4 250 bp were detected when pAAV-U6/CMV-NGF-β was digested. DNA sequencing result of pAAV-U6/ CMV-NGF-β showed that sequences were completely correct. Conclusion The AAV shuttle vector pAAV-U6 /CMV-NGF-β is successfully constructed, providing experimental basis for investigation of the repair of spinal cord injury.
Objective To analyze the biomechanical changes of the adjacent cervical facet joints when the angled cervical prosthesis is replaced. Methods A total of 400 northwestern people were involved, with an age of 40 years or older.The cervical vertebra lateral X-ray films were taken, and the cervical angles were measured by professional computer aided design software, then the cervical intervertebral disc prosthesis with 10° angle was designed. The finite element models of C4，5and C4-6 segments with intact cervical discs were developed; the C4，5 disc was replaced by the cervical prosthesis with 0° and 10° angle respectively; and then all models were subjected to axial loading, flexion/extension, lateral bending, and torsion loading conditions; the stress effects on adjacent facet joints after replacement were observed by comparing with that of the intact model. Results The cervical angles were (9.97 ± 3.64)° in C3，4, (9.95 ± 4.34)° in C4，5, (8.59 ± 3.75)° in C5，6, and (8.49 ± 3.39)° in C6，7, showing no significant difference between C3，4 and C4，5, C5，6 and C6，7 (P gt; 0.05) and showing significant differences between the other cervical angles (P lt; 0.05). When C4，5 model was axially loaded, no significant difference in equivalent shearing stress were observed in intact, 0°, and 10° groups; at flexion/extension loading, the stress was biggest in intact group, and was smallest in 10° group; at lateral bending, the stress got the high rank in intact group, and was minimum in 10° group; at torsion loading, the stress state of 10° group approached to the intact one condition. When C4-6 model was loaded, the facet joint stress of the replaced segment (C4，5) decreased significantly at axial loading, flexion/extension, and lateral bending; while no obvious decrease was observed at torsion loading; the stress of the adjacent inferior disc (C5，6) decreased significantly at axial loadingand lateral bending condition, while less decrease was observed at torsion loading, no significant change at flexion/extension condition, it approached to that of the intact one. Conclusion The finite element analysis reveals that the biomechanical properties of 10° designed prosthesis is approximate to that of the intact cervical disc, thus the 10° designed prosthesis can meet the requirements of biomechanical function reconstruction of the cervical spine.
Objective To construct and screen neurite outgrowth inhibitory 66-samll interfering RNA (nogo66-siRNA) eukaryotic expression vectors of effective interference, so as to lay a foundation for further reconstruction of related viral vector. Methods The nogo66-siRNA fragments were designed and cloned into pGenesil-1.1, 4 plasmids of pGenesil-nogo66-siRNA-1, pGenesil-nogo66-siRNA-2, pGenesil-nogo66-siRNA-hk, and pGenesil-nogo66-siRNA-kb were obtained, sequenced and identified, then were transfected into C6 cell l ine. The transfection efficiency was measured by fluorescence microscope. RT-PCR and Western blot were used to detect the expression of nogo gene and select the plasmid of effective interference. Results DNA sequencing results showed interference sequences were correct. The bands of 800 bp and 4.3 kb were detected when pGenesil-nogo66-siRNAs were digested by Kpn I /Xho I. The expression of green fluorescent protein could be detected under fluorescence microscope, and the transfection efficiency was about 73%. RT-PCR and Western blot results showed that compared to non-transfected cells, the transfection of pGenesil-nogo66-siRNA-1 made the expression of nogo gene decl ine 22% and the expression of nogo protein decl ine 73%; the transfection of pGenesil-nogo66-siRNA-2 made the expression of nogo gene decl ine 28% and the expression of nogo protein decl ine 78%; the differences were significant (P lt; 0.05); and the transfection of pGenesil-nogo66-siRNA-hk and pGenesil-nogo66- siRNA-kb did not make the expressions of nogo gene and nogo protein decrease significantly (P gt; 0.05). Conclusion Nogo66-siRNA eukaryotic expression vector is successfully constructed, it lays an experimental foundation for repair of spinal cord injury.
Objective To study the effect of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes on bone regeneration and angiopoiesis in vivo so as to provide a theoretical basis for the gene therapy of avascular necrosis of thefemoral head (ANFH). Methods Twenty-four male adult New Zealand rabbits were made the ischemic hind l imb model and divided into 4 groups (n=6). The 3rd generation rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with the following 4 virus and were administered intramuscularly into the ischemic thigh muscle of 4 groups, respectively: rAAVhVEGF165- internal ribosome entry site (IRES)-hBMP-7 (group A), rAAV-hVEGF165-green fluorescent protein (GFP) (group B), rAAV-hBMP-7-GFP (group C), and rAAV-IRES-GFP (group D). At 8 weeks after injection, the blood flow of anterior tibial artery in the rabbit hind l imb was detected by ultrasonographic image. Immunohistochemical staining for CD34 was performed to identify the prol iferation of capillary. Another 24 male adult New Zealand rabbits were made the femur muscle pouch model and divided into 4 groups (n=6). The above 4 BMSCs transfected with rAAV were administered intramuscularly into the muscle pouch. At 8 weeks after injection, X-ray radiography was used to assess orthotopic bone formation, and von Kossa staining to show mineral ization. Results No symptoms of local or systemic toxicity were observed after rAAV injection. At 8 weeks after injection, the ratio of ischemic to normal blood flow and the number of capillaries in group A were the highest among 4 groups (P lt; 0.05). The ratio of ischemic to normal blood flow and the number of capillaries in group B were significantly higher than those in group C and group D (P lt; 0.05). However, there was no significant difference between group C and group D (P gt; 0.05). At 8 weeks after injection, orthotopic ossification and mineral ization were evidently detected in group A and group C, and group A was ber than group C. No obvious evidence of orthotopic ossification and mineral ization were observed in group B and group D. Conclusion rAAV-hVEGF165-IRES-hBMP-7 vector has the biological activities of inductive bone regeneration and angiopoiesis in vivo.