Objective To investigate the influence of knocking down Ezrin expression in combination with HSP70-induced immune killing on the apoptosis and proliferation of mouse osteosarcoma cells.Methods Human osteosarcoma cell line MG63 was cultured,the HSP70 and Ezrin-shRNA DNA fragments were cloned into the expression vector pGFP-V-RS containing CMV and pU6 promoters,and constructed the expression vector pGFP-V-RS-shRNA and pGFP-V-RS-shRNA-HSP70.The vectors were transfected into MG63 cell line,respectively.The status of transfected MG63 cells was observed by fluorescent microscope.The expressions of Ezrin and HSP70 were examined by real time RT-PCR and Western blot.Flow cytometry 、MTS test was used to detect the changes of cell apoptosis and proliferation,and changes of the expression of apoptosis and cell cycle related proteins was detected by Western blot.The specific cytotoxic T lymphocytes (CTLs) were induced by HSP70,and its killing effect on target MG63 tumor cells was analyzed by MTT assay.Results The specific vector simultaneously knocking down Ezrin and overexpressing HSP70 was constructed for the first time in China and confirmed by fluorescence microscope and gene/protein expression analysis.Compared to Ezrin knock-down alone,simultaneous HSP70 overexpression partially recovered the promoted cellular apoptosis and proliferation suppression by Ezrin knock-down,however,when compared to the normal control,the apoptosis rate of LM 8 cells was still significantly increased from 11.01％±0.22％ to 24.28％±0.50％,while the proliferation rate decreased from 395.14％±2.24％ to 310％± 2.83％.In addition,Western detected that Ezrin-shRNA could promote the expression of Bax.However,the expression of Ezrin-shRNA could reduce the Bcl-2 and Cyclin D1.Ezrin-shRNA/HSP70 also has the same effect.MTT assays revealed that the CTL cytotoxic effect on target MG63 tumor cells at all concentrations were significantly higher in CT+IL-2+HSP70 group compared with that in CT+ IL-2 group,with a cytotoxicity as high as 56.33％± 1.95％.Conclusion Simultaneous knocking down Ezrin and overexpressing HSP70 promotes the apoptosis and inhibits the proliferation of osteosarcoma cell.And the HSP70 can induce CTL which enhances the killing effect on tumor cell.And the HSP70 can induce CTL which enhance the killing effect on tumor cell.

Objective To evaluate the operation methods and effects of transforaminal wedge osteotomy (TWO) combined with intervertebral grafting bone for correcting the rigid thoracolumbar kyphosis.Methods From January 2003 to June 2013, treatment of 52 cases of the rigid thoracolumbar kyphosis by TWO combined with intervertebral bone graft were studied (including 33 males and 19 females, and the age range was from 15 to 72 with average age of 42.3 years old).In these 52 cases, there were 17 cases of ankylosing spondylitis, 25 cases of obsolete thoracolumbarvertebral fractures, 7 cases of chronic thoracolumbar tuberculosis and 3 cases of congenital vertebral malformations.52 cases presented as lumbar kyphosis, low back pain aggravated,with an average VAS score of 8.6;24 cases were accompanied with different extent of neurological dysfunction which obviously influenced daily life or work.Osteotomy was located at thoracolumbar intervertebral disc.Pedicle screws internal fixation system combined with intervertebral grafting bone was used to compress the vertebra and fuse after TWO for correction.Preoperative and postoperative low back pain was evaluated by visual analog score (VAS), Frankel score and Oswestry disability index(ODI) in patients with neurological dysfunction.The above indexes were compared with the PSO osteotomy group.Results Preoperative mean kyphosis Cobb angle was 65.6°(31°-137°).The average post-operative kyphosis Cobb angle was 19.2° (8°-35°), and the average correction rate was 68.4％.The mean operation time was 3.8 h (2.2-5.3 h), and the mean intraoperative blood loss was 1220ml (500-2 900 ml).Preoperative VAS score was 8.6± 1.2 while the last follow-up was 1.8±0.5;preoperative Oswestry dysfunction index was 75.6±8.2 while the last follow-up was 18.4±8.1.The results were statistically significant.The spinal function was 4 cases of Frankel C and 20 cases of Frankel D before operation, all of which dropped to E level after operation.52 patients were followed up with the average time of 41 months (from 6 to 60 months).The bony fusion rate was 94.23％ at 6 months after operation.All of the patients were fused in 3 years after operation.The average loss of kyphosis Cobb angle was 5.5°(4°-12°).Compared with PSO, the average operation time, the amount of bleeding and the improvement of neurological function after surgery all had some advantages.Conclusion Transforaminal wedge osteotomy has many advantages, such as less bleeding wounds, less nerve interference and limited multi-segmental osteotomy, with little trauma and high bony fusion rate.