Construction and identification of the recombinant adenovirus vector carrying a small interfering RNA targeting the peroxisome proliferator-activated receptor-γ / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Steroid-induced osteonecrosis of the femoral head (ONFH) is a common clinical disease, with a high disability rate. At present, efficient prevention and treatment of steroid-induced ONFH is still lacking. The peroxisome proliferator-activated receptor-γ (PPARγ) is recognized as an important pathogenic gene for the development of steroid-induced ONFH. RNA interference (RNAi) is a tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target proteins. Therefore, down-regulation of PPARγ expression by RNAi may prevent the incidence of steroid-induced ONFH.</p><p><b>METHODS</b>According to the principles of siRNA design, three duplex siRNA sequences (971 - 989, 1253 - 1271 and 1367 - 1385) derived from the PPARγ gene (NM_001082148) were synthesized. These duplexes were annealed, purified and ligated into 1.0-cytomegalovirus (CMV) shuttle vector. The shuttle vector was transfected into HEK293 cells. The HEK293 generated recombinant adenovirus vector carrying PPARγ siRNA sequences was purified and the titer of recombinant adenovirus was determined.</p><p><b>RESULTS</b>After the annealing of single-strand DNA oligo encoding short hairpin RNA (shRNA) sequences, products were identified by gel electrophoresis. These products were ligated into the 1.0-CMV shuttle vector and the recombinant shuttle vectors 1.0-CMV-971, 1.0-CMV-1253 and 1.0-CMV-1367 were constructed. These sequences of these recombinant vectors were confirmed. We then successfully constructed the recombinant adenovirus vector carrying siRNA targeting PPARγ. After purification, the virus titer was higher than 10(10) plaque forming unit (PFU)/ml.</p><p><b>CONCLUSION</b>In this study, three recombinant adenovirus shuttle vectors carrying siRNA targeting PPARγ, including shuttle vectors 1.0-CMV-971, 1.0-CMV-1253 and 1.0-CMV-1367, were successfully constructed and high titers of recombinant adenovirus were obtained.</p>

Osteogenic potential of human calcitonin gene-related peptide alpha gene-modified bone marrow mesenchymal stem cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Most of the basic and clinical studies of osteonecrosis of the femoral head (ONFH) are restricted to bone tissues only, whereas various systems are involved in the onset and development of ONFH, including nervous system. Peptidergic nerve participates in the neuronal regulation of bone metabolism and anabolism, and plays key roles in the growth, repair and reconstruction of bone. Calcitonin gene-related peptide (CGRP), which is secreted by peptidergic nerve, is the main mediator of bone metabolism. It dramatically promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Additionally, it enhances the osteoblast mass and the rate of osteoblast formation, and reduces the bone resorption by acting on osteoblasts and osteoclasts. Hence, we aimed to construct recombinant retrovirus vector pLNCX(2)-hCGRPα and to investigate the proliferation and osteogenic potential of hCGRPα-producing BMSCs (BMSCs/pLNCX(2)-hCGRPα) after virus infection.</p><p><b>METHODS</b>The constructed recombinant retrovirus vector pLNCX(2)-hCGRPα was transfected into PT67 packaging cells by lipofectamine 2000. Virus was collected for BMSCs infection. The mRNA and protein expression of hCGRPα was examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. The cell proliferation was determined by methyl thiazoleterazolium (MTT) assay. The osteogenic potential of BMSCs was evaluated by alkaline phosphatase (ALP) activity.</p><p><b>RESULTS</b>Both mRNA and protein expression of hCGRPα was detected in BMSCs/pLNCX(2)-hCGRPα cells. These cells exhibited significantly elevated proliferation and ALP value as compared with control BMSCs (P < 0.05).</p><p><b>CONCLUSION</b>BMSCs/pLNCX(2)-hCGRPα cells could stably express hCGRPα and showed promoted proliferation ability and osteogenic potential as compared with control BMSCs.</p>

Effect of growth hormone on the immune function of dendritic cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Dendritic cells (DCs) are one of the most important antigen presenting cells in the human body, and DCs at various stages of maturation possess different or even opposite functions. The aim of this study was to investigate the influence of growth hormones on the functional status of cord blood-derived DCs encompassing immunophenotype, ability to excrete interleukin (IL)-12 and provoke autologous leukomonocyte.</p><p><b>METHODS</b>Mononuclear cells were isolated from fresh cord blood, with IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) used to induce and stimulate the mononuclear cells. Growth hormone at different concentrations was used to modify DCs, and then DCs morphology, number and growth status were observed. The immunophenotype of DCs was detected with a flow cytometer. The concentration of IL-12 in the DCs supernatant was determined by enzyme linked immunosorbent assay (ELISA) and DCs functional status was evaluated by autologous mixed lymphocyte reactions.</p><p><b>RESULTS</b>Mononuclear cells from cord blood can be differentiated into DCs by cytokine induction and growth hormone modification. With the increase in growth hormone concentrations (5 - 100 microg/L), the expression of DCs HLA-DR, CD1alpha, CD80 and CD83 were significantly increased (P < 0.05). The ability of DCs to secrete IL-12 was significantly improved (P < 0.05), and the ability of DCs to activate autologous lymphocytes was significantly enhanced (P < 0.05). Pegvisomant was able to ablate the effects of growth hormone on DCs.</p><p><b>CONCLUSIONS</b>Growth hormone may facilitate DCs induction and maturation, and improve the reproductive activity of autologous lymphocytes in a dose-dependent manner. Growth hormone may serve as a factor of modifying DCs to achieving maturity.</p>

A modified technique of bone grafting pedicled with femoral quadratus for alcohol-induced osteonecrosis of the femoral head / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Quadratus femoris pedicled bone grafting has yielded satisfactory long-term clinical outcome for osteonecrosis of the femoral head (ONFH) in pre-collapse ONFH without extensive lesion. However, for pre-collapse ONFH with extensive necrotic area, it is still challenging to preserve the femoral head. The current study aimed to introduce a new technique of deliquesce strut with titanium mesh containing bone grafting pedicled with the femoral quadratus and to evaluate its short-term outcomes.</p><p><b>METHODS</b>From January 2008 to December 2008, 10 ONFH patients (12 hips) underwent operations by a new technique of deliquesce strut with titanium mesh containing bone grafting pedicled with the femoral quadratus (group A). According to the ARCO classification system, there were two hips in stage II B and 10 hips in stage II C. Also in the same period, 12 ONFH patients (16 hips) underwent operations by the conventional procedure of quadratus femoris pedicled bone grafting (group B). There were 6 hips in stage II B and 10 hips in stage II C. All patients were males and suffered from alcohol induced ONFH. For the new technique, the necrotic area was evaluated, and a titanium mesh piece of the same size (range from 2.5 cm × 2.8 cm to 2.8 cm × 3.4 cm) was obtained and shaped to match the contour of the head. The cancellous bone was first placed underneath the subchondral bone and was densely impacted (about 1 to 2 mm thick). Then the titanium mesh piece was inserted. The length of the decompressive trough was measured. A titanium cylinder mesh cage with a diameter of 1.6 cm of the same length was obtained, with a "U" shaped window in the wall being created to make room for the muscle pedicle. The muscle pedicle bone was inserted into the titanium mesh cage to form a bone graft-titanium cage complex and, then the complex was inserted. The hundred percent score method was used for outcome evaluation. Clinical and radiographic outcomes were compared between group A and group B.</p><p><b>RESULTS</b>The average operative time was 150 minutes (130 to 185 minutes) in group A, with an average of 130 minutes (120 to 180 minutes) in group B. The mean blood loss was 400 ml (300 to 500 ml) in group A and 350 ml (250 to 500 ml) in group B. Group A patients were followed up for an average of 19.2 months (14 to 24 months), with an average of 18.5 months (12 to 24 months) for Group B. Full weight bearing was allowed 5 to 7 months postoperatively. Pain and function were obviously improved. For group A, pain score improved from a mean of 9.8 points preoperatively to an average of 24.6 points postoperatively, and function score improved from a mean of 9.0 points preoperatively to an average of 17.4 points postoperatively. In group B, pain score improved from a mean of 9.5 points preoperatively to an average of 24.2 points postoperatively and function score improved from a mean of 9.2 points preoperatively to an average of 17.2 points postoperatively. The range of motion changed the least, with score improvement from a preoperative mean of 13.9 points to postoperative 16.8 points for group A and from a preoperative mean of 13.7 points to postoperative 16.5 points for group B. Radiographic score improved from preoperative 31 points to postoperative 38 points for group A, in comparison with an improvement from preoperative 31 points to postoperative 37 points for group B. At the latest follow up, 11 hips were rated as excellent and 1 hip was better for group A, with 14 hips being rated as excellent and 2 hips being better in group B. There was no statistically significant difference between groups A and B in clinical and radiographic outcomes.</p><p><b>CONCLUSION</b>For ONFH in stage ARCO IIC, satisfactory clinical outcome can be achieved by the new technique in the short-term period while the long-term clinical outcome has yet to be determined.</p>

Comparison of surgical treatment in single thoracolumbar-lumbar adolescent idiopathic scoliosis: anterior versus posterior surgery / 中华外科杂志

<p><b>OBJECTIVE</b>To retrospectively compare the clinical outcomes of anterior and posterior surgical treatment in single thoracolumbar-lumbar adolescent idiopathic scoliosis.</p><p><b>METHODS</b>Between January 2004 and August 2008, 22 female patients, averaged 14.5 years old (12 to 18 years), of thoracolumbar-lumbar adolescent idiopathic scoliosis were corrected by anterior correction and fusion. At the same time, 20 female patients, average 14.8 years old (11 to 19 years), were corrected by posterior segmental pedicle screw correction and fusion. Operation time, SRS-24 score, intraoperative blood loss, and coronal and sagittal plane correction were compared between the two groups.</p><p><b>RESULTS</b>All patients were followed up for 12 to 63 months, the mean follow-up time was 28.3 months. Operation time was (334 + or - 36) min in anterior group and (292 + or - 17) min in posterior group; intraoperative blood loose was (940 + or - 207) ml in anterior group and (596 + or - 227) ml in posterior group; fusion levels were (5.2 + or - 0.8) in anterior group and (6.7 + or - 1.2) in posterior group. There were statistically significant difference in operation time, intraoperative blood loss and fusion levels (P < 0.05). Coronal correction was (93 + or - 5)% in anterior group and (88 + or - 5)% in posterior group. SRS-24 scores averaged 98 in anterior group and averaged 94 in posterior group. There was no statistical difference in coronal correction or SRS-24 scores (P > 0.05).</p><p><b>CONCLUSIONS</b>Posterior surgery has the same correction results compared with anterior surgery in treating thoracolumbar-lumbar adolescent idiopathic scoliosis. Posterior surgery takes less operation time, brings less trauma but has longer fusion levels.</p>

Application of lumbar-pelvic fixation in lumbosacral reconstruction after resection of sacral tumors / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the short-term clinical results of a new approach of lumbar-pelvic fixation for lumbosacral reconstruction after resection of sacral tumors.</p><p><b>METHODS</b>Fifteen patients with sacral tumors underwent lumbar-pelvic fixation using TSRH-3D, CDH-M8 or ISOLA with iliac screws. The lumbosacral stability was evaluated according to the X-ray result to assess the feasibility and therapeutic effect of this approach.</p><p><b>RESULTS</b>X-ray showed that high lumbosacral stability was achieved in all the 15 cases after the operation, and satisfactory therapeutic effect was obtained.</p><p><b>CONCLUSION</b>Lumbar-pelvic fixation with iliac screw is feasible for lumbosacral reconstruction after resection of the sacral tumors, which provides strong internal fixation and produce good clinical outcomes.</p>

Lumbar interspinous non-fusion techniques: comparison between Coflex™ and Wallis / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare the short-term clinical outcome of non-fusion techniques using interspinous implantation Coflex(TM) and Wallis treatment in patients with lumbar spine degenerative diseases.</p><p><b>METHODS</b>Forty-one cases of lumbar stenosis, 18 of lumbar disc herniation, and 34 of lumbar stenosis with lumbar disc herniation were evaluated. Among the 43 cases receiving Coflex(TM) implantation, 41 had operations in one segment and 2 in 2 segments. In the other 50 cases with Wallis implantation, 47 had fixation of 1 segment and 3 had 2 segments fixed. JOA Score, Oswestry Disable Index (ODI) and VAS were used to evaluate the short-term clinical results.</p><p><b>RESULTS</b>The average operating time was 64.55 min in Coflex(TM) implantation with an average blood loss of 81.82 ml. The average operating time was 82.71 min in Wallis implantation, which caused an average blood loss of 89.66 ml. Significant improvements in the JOA Score, ODI and VAS were noted after the operations.</p><p><b>CONCLUSION</b>The two interspinous non-fusion techniques, Coflex and Wallis, produce good short-term clinical outcome in the treatment of lumbar spine degenerative diseases.</p>

A comparative study on maintenance of disc height by different anterior cervical fusion methods / 中华外科杂志

<p><b>OBJECTIVE</b>To compare the effect of autograft, cage and autograft plus internal fixation with plate on maintenance of disc height and recovery of spinal cord function.</p><p><b>METHODS</b>From March 1998 to June 2004, 117 cervical spondylotic patients who received anterior fusion surgery were reviewed. There were 65 males and 52 females with an average age of 57 years (range from 31 to 72 years). One level was involved in 91 patients, and two levels in 26. Fusion methods included autogenous tri-cortical iliac bone grafting alone in 31 patients, cage with autograft bone in 38 and autogenous tri-cortical iliac bone grafting combined with self-locking plate in 49. The measured fusion length was determined as the distance from the midpoint between the anterior and posterior aspects of the superior endplate of the top level of the fusion to the midpoint of the inferior endplate of the bottom level of the bottom level of the fusion. JOA rating criteria was used for cord function evaluation. One-way variation analysis was used to compare the disc height loss and cord function among the 3 groups.</p><p><b>RESULTS</b>All patients were followed-up for an average period of 23 months (range, 13 to 59 months). At final follow- up, disc height loss averaged 1.94 mm in autogenous tri-cortical iliac bone grafting group, greater than that in the other two groups (1.48 mm in cage group and 1.25 mm in instrumented group) with statistical significance. Recovery of spinal cord function was satisfactory in all groups at one year postoperatively with no statistical difference. Three implants failure occurred in self-locking plate group and was salvaged with uneventful recovery.</p><p><b>CONCLUSIONS</b>In anterior cervical fusion surgery, maintenance of disc height could be achieved by proper application of cage or internal fixation with plate.</p>

Anterior fusion for unstable lower cervical spine / 中华外科杂志

<p><b>OBJECTIVE</b>To assess the clinical outcome of anterior fusion for unstable lower cervical spine.</p><p><b>METHODS</b>From October 2000 to October 2004, anterior fusion were performed for 67 cases with unstable lower cervical spine with corresponding clinical manifestations. There were 38 males and 29 females with an average age of 33 years. Instability of lower cervical spine was radiographically determined by sagittal plane displacement between 2 cervical vertebrae of more than 3.5 mm or relative sagittal plane angulation greater than 11 degrees on maximal lateral flexion and extension films. Anterior interbody fusion methods included bone grafting with plate fixation or cage. JOA rating system was used for spine cord function assessment.</p><p><b>RESULTS</b>All patients were followed up for an average period of 14 months. Solid fusion was achieved for all operated levels. The majority of the patients got a satisfactory relief of preoperative symptoms. JOA scores for cord function improved from preoperative 10.15 to postoperative 14.95 with statistical difference (P < 0.05). Complications included screws loosing and backout in 2 cases. Revision surgery were performed with uneventful recovery.</p><p><b>CONCLUSION</b>Satisfactory clinical outcome can be achieved by proper selection of anterior fusion methods for patients with unstable lower cervical spine.</p>

Dexamethasone-induced adipogenesis in primary marrow stromal cell cultures: mechanism of steroid-induced osteonecrosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>In steroid-induced osteonecrosis, hypertrophy and hyperplasia of marrow fat cells and lipid deposition of osteocytes can be found in the femoral head. However, the precise reason is not clear yet. The aim of this study was to observe the effect of dexamethasone (Dex) on differentiation of marrow stromal cells (MSCs), and to investigate the pathobiological mechanism of steroid-induced osteonecrosis.</p><p><b>METHODS</b>MSCs in cultures were treated with increasing concentrations of Dex (0, 10(-9), 10(-8), 10(-7), and 10(-6) mol/L) continuously for 21 days. The cells, which were exposed to 0 mol/L (control) or 10(-7) mol/L Dex for 4 - 21 days, were then cultured for 21 days without Dex. MSCs were stained with Sudan III. Number of adipocytes was counted under a light microscope. The activity of alkaline phosphatase (ALP) of MSCs treated with 0, 10(-8), 10(-7), and 10(-6) mol/L Dex for 12 days, and that treated with 0 mol/L and 10(-7) mol/L Dex for 8, 10, or 12 days were determined. The levels of triglycerides, osteocalcin and cell proliferation of MSCs treated with 0 mol/L and 10(-7) mol/L Dex were detected. The mRNA expression levels of adipose-specific 422 (aP2) gene and osteogenic gene type I collagen in MSCs treated with 0 mol/L and 10(-7) mol/L Dex for 6 days were analyzed by whole-cell dot-blot hybridization. Statistical analysis was performed using Student's t test and analysis of variance. P values less than 0.05 were considered significant statistically.</p><p><b>RESULTS</b>The number of adipocytes in cultures increased with the duration of MSCs' exposure to Dex and the concentration of Dex. The level of ALP activity in the MSCs decreased with concentration of Dex. In the control group, it was 8.69 times of that in the 10(-7) mol/L Dex group on day 12 (t = 20.51, P < 0.001). The level of triglycerides in 10(-7) mol/L Dex group was 3.40 times of that in the control (t = 11.00, P < 0.001). The levels of cell proliferation and osteocalcin in the control were 1.54 and 2.42 times of that in the 10(-7) mol/L Dex group respectively. As compared to the control, the mRNA expression of adipose-specific 422 (aP2) gene in 10(-7) mol/L Dex group was significantly increased (t = 36.48, P < 0.001), and that of osteogenic gene type I collagen was decreased (t = 42.07, P < 0.001).</p><p><b>CONCLUSIONS</b>Dex can directly induce the differentiation of MSCs into a large number of adipocytes and inhibit their osteogenic differentiation, which provide a novel explanation for the pathologic changes of steroid-induced osteonecrosis.</p>

Estimation of Surgical Effect of Giant Cell Tumor of Bone in Children / 实用儿科临床杂志

Objective To estimate the effects of different surgical treatments of giant cell tumor of bone(GCTB) of long bone in children.Methods The effects of surgical treatment of 15 cases suffered from GCTB were observed.The tumors were rated according to the system of Enneking classification,stageⅠwas managed with a curettage combined with local adjuvant treatment and filled by allograft bone or with curettage and filled by cement of bone.StageⅡb was treated by en bloc resection followed by reconstruction with large segment bone of allograft.The follow-up was given in all patients.Results The follow-up period was 7 years(ranged 2-16 years).In all 15 children patients with GCTB,there were 13 children in stageⅠ,9 patients with curettage combined with local adjuvant treatment and allograft bone showed good bone knitting and rehabilitation without deformity and functional problem and locally recurred;4 patients with curettage and filled by cement of bone showed 2 cases locally recurred.Two children in stageⅡb treated by en bloc resection followed by reconstruction with large segment bone of allograft were perfectly incorporated without evidence of obvious immune rejection,collapse,fracture and locally recurred,but the limb-suffered was short about 2 cm.Conclusions Stage Ⅰtreated by a curettage combined with local adjuvant treatment and filled by allograft bone can get good effect,but only treated by a curettage and filled by cement of bone has 50% locally recurred.TheⅡb treated by en bloc resection followed by reconstruction with large segment bone of allograft might influence the limb growth,but without locally recurred.