The pGenesil-1-Beclin1 eukaryotic expression vectors were constructed to establish an SH-SY5Y cell line stably expressing shRNA-Beclin1. The shRNA was connected to pGenesil-1 to construct the recombinant plasmid pGenesil-1-Beclin1, which was transformed into JM109 E.coli. Positive clones were identified by digestion with restriction endonuclease and DNA sequencing. SH-SY5Y cells were cultured by the conventional method. The pGenesil-1-Beclin1 and pGenesil-1 plasmids were transfected into SH-SY5Ycells, and the cells were screened by G418 until the stable G418-resistant monoclonal cells were acquired. Beclin1 mRNA and Beclin1 protein were detected by RT-PCR and Western blot analysis respectively. The results of restriction endonuclease analysis and DNA sequencing confirmed the correct construction of the eukaryotic expression vector pGenesil-1-Beclin1. Two SH-SY5Y transfected cell lines were successfully selected. Compared with the control group, RT-PCR and Western blot showed that the expression of Beclin1 mRNA and protein were down regulated 71.28%±1.45%(P<0.05)and 75.50%±2.63%(P<0.05), respectively. The results indicated that the eukaryotic expression vector pGenesil-1-Beclin1 was successfully constructed and the SH-SY5Y cell lines with inhibited Beclin1 expression were established. It provides a useful cell model for studying the biological function of Beclin1.
ObjectiveTo investigate the effect of cyclic stretch stress on the osteogenic differentiation of human cartilage endplate-derived stem cells (CESCs). MethodsCESCs were isolated from the endplate cartilage tissues by the method of agarose suspension culture system. The endplate cartilage tissue was harvested for immunohistochemical staining. Flexercell-4000TM Tension Plus system was used to apply cyclic stretch on CESCs at a frequency of 1 Hz and at a stretch rate of 10% for 1, 6, 12, or 24 hours (experimental group). No stretch stress was performed on CESCs in the same culture condition (control group). After mechanical loading, the protein expression of bone morphogenetic protein 2 (BMP-2) was measured by Western blot, and gene expressions of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and SOX9 were detected by real-time fluorescent quantitative PCR. ResultsImmunohistochemical staining showed BMP-2 protein expression in chondrocytes. The continuous cyclic stretch stress of 10% can increase the expression of BMP-2 protein in CESCs. Significant differences were observed in the expressions of BMP-2 protein (P<0.05) between 2 groups at the other time points except at 1 hour (P>0.05), in a time-dependent manner. The real-time fluorescent quantitative PCR indicated that the gene expressions of Runx2 and ALP showed an increasing tendency with time in the experimental group when compared with the control group, but there was down-regulated expression of SOX9. Significant difference was found in mRNA expressions of Runx2 and ALP at 12 and 24 hours and in mRNA expressions of SOX9 at 6, 12, and 24 hours between 2 groups (P<0.05), in a time-dependent manner. ConclusionCyclic stretch stress may induce osteogenic differentiation of CESCs by regulating the expressions of some genes related osteogenesis in CESCs.
ObjectiveTo investigate the changes and relationship of cervical spine sagittal alignment and other spinal-pelvic sagittal parameters in adolescent idiopathic scoliosis. MethodsBetween July 2011 and July 2014, 35 patients with idiopathic scoliosis who met the inclusion criteria underwent posterior pedicle screw instrumentation and fusion. There were 12 males and 23 females with a mean age of 16.2 years (range, 13-20 years), including 16 cases of Lenke type 1, 7 cases of Lenke type 2, 4 cases of Lenke type 3, 3 cases of Lenke type 4, 4 cases of Lenke type 5, and 1 case of Lenke type 6. The average follow-up time was 10.9 months (range, 5-36 months). The pelvic incidence (PI), pelvic tilt (PT), sacral slope (SS), lumbar lordosis (LL), thoracic kyphosis (TK), cervical lordosis (CL), T1 slope, C2 slope, C7 sagittal vertical axis (C7 SVA), C2-7 plumbline (cSVA) were measured on pre-and post-operative standing lateral X-ray film. Based on preoperative CL, the patients were divided into kyphosis group (CL>0°) and lordosis group (CL < 0°); after operation, the patients were divided into restored lordosis (group A), decreased kyphosis (group B), and increased lordosis (group C) after operation. All data about sagittal profile changes were analyzed. The relations between CL and other spinal-pelvic parameters in the kyphosis and lordosis groups before operation were determined via Spearman correlation coefficient. ResultsStatistically significant changes were observed in PT, PI, SS, and LL between at pre-and post-operation (P < 0.05), but no significant difference was found in the other parameters (P>0.05). There were 17 patients in lordosis group and 18 in kyphosis group before operation. Intra-group comparisons showed significant changes in PT, PI, SS, C2 slope, and C7 SVA in lordosis group, and in PT, PI, SS, LL, CL, TK, T1 slope, and C2 slope in kyphosis group (P < 0.05). Subgroup comparisons showed significant changes in CL, TK, C2 slope, C7 SVA, and T1 slope before operation (P < 0.05) and T1 slope at last follow-up between 2 groups (P < 0.05). In kyphosis group, 7 cases (group A) had restored lordosis, 7 cases (group B) had decreased kyphosis, and 4 cases had increased lordosis. In lordosis group, 9 cases (group C) had increased lordosis, 3 cases had decreased lordosis, and 5 cases had kyphotic cervical alignment after operation. Significant difference was found in LL, CL, T1 slope, C2 slope, and C7 SVA of group A, in TK and CL of group B, and in CL and cSVA of group C between pre-and post-operation (P < 0.05). There were significant differences in pre-and post-operative LL between groups A and B (P < 0.05). In lordosis group, there was a strong correlation between CL and C2 slope (P < 0.05) at pre-operation. CL had strong correlation with C2 slope and T1 slope (P < 0.05) at pre-operation in kyphosis group, and CL had moderate correlation with cSVA (P < 0.05). ConclusionCervical sagittal alignment plays an important role in the balance of the spine and pelvis. The change of cervical sagittal alignment has a certain correlation with the change of thoracic kyphosis. Attention to properly maintaining or restoring cervical sagittal lordosis alignment should be considered in preoperative evaluation of adolescent indiopathic scoliosis.
ObjectiveTo identify the prevalence of distal adding-on phenomenon after posterior selective fusion in type Lenke 1A idiopathic scoliosis, to analyze its risk factors so as to find the reasonable choice for lowest instrumented vertebra (LIV). MethodsA retrospective study was made on the clinical data of 43 patients with type Lenke 1A idiopathic scoliosis undergoing posterior selection fusion with pedicle screw instrumentation between July 2011 and December 2015. There were 15 males and 28 females, aged 12-18 years (mean, 16 years). The preoperative Cobb angle was (50.1±11.3)°. The anteroposterior and lateral radiographs were taken at preoperation, immediate after operation, and last follow-up to measure the radiographic parameters. Forty-three patients were divided into adding-on group and control group according to whether or not the occurrence of distal adding-on phenomenon on anteroposterior radiographs of the spine at last follow-up. All the factors that maybe cause distsal adding-on were evaluated by statistical analysis. ResultsThe patients were followed up 12-50 months (mean, 26.5 months). At last follow-up, distal adding-on was observed in 10 of 43 patients (23.3%), including 2 males (13.3%) and 8 females (28.6%). Univariate analyses showed following several factors associated with adding-on:preoperative lumbar flexibility, preoperative pelvic tilt in coronal plane, preoperative LIV+1 deviation from center sacral vertical line, L4 subtype, the difference between LIV and last touching vertebra (LTV) (LIV-LTV), the difference between LIV and lower end vertebra (LEV) (LIV-LEV), and the difference between LIV and stable vertebra (SV) (LIV-SV). The risk factors above were brought into Logistic regression model, the results showed that preoperative LIV+1 deviation (deviation>10 mm, odds ratio=10.812, P=0.026), LIV-LTV (LIV-LTV<1, odds ratio=9.017, P=0.04), and L4 subtype (1A-R, odds ratio=9.744, P=0.047) were significantly associated with adding-on. ConclusionPreoperative LIV+1 deviation of >10 mm, L4 subtype (1A-R) and LIV-LTV of <1 are independent predictive factors of adding-on after surgery. As high risk of adding-on was closely related with the incorrect fusion level, it plays an important role for surgeons to decide the level of the fusion before surgery thoroughly. The LIV should be extended at least to LTV+1 to avoid adding-on phenomenon.