ObjectiveTo investigate the effect of Notch signaling pathway important target Hey1 expression on the differentiation and proliferation of C3H10T1/2 cells induced by bone morphogenetic protein 9 (BMP-9). MethodsHey1 lentivirus and Hey1 short hairpin RNA lentivirus were constructed and used to infect C3H10T1/2 cells to change the expression level of Hey1 in C3H10T1/2 cells. C3H10T1/2 cells infected with LV-Blank (empty plasmid) as control. The Hey1 expression levels of different groups were detected by fluorescence microscope, real-time fluorescence quantitative PCR, and Western blot. The C3H10T1/2 cells with different Hey1 expression level were induced by BMP-9 conditioned medium (BMP-9+C3H10T1/2 group, BMP-9+C3H10T1/2-Hey1 group, and BMP-9+C3H10T1/2-shHey1 group); the cells of control groups (C3H10T1/2 group and C3H10T1/2-Blank group) were cultured with normal medium. The mRNA and protein expression levels of osteogenesis related transcription factors (Runx2, osteopontin, and osteocalcin) were detected at 48 hours by real-time fluorescence quantitative PCR and Western blot assay. The cells proliferation and cycles were detected by MTT assay at 4, 5, 6, and 7 days and flow cytometry at 4, 5, and 10 days. The alkaline phosphatase (ALP) activity was analyzed by ELISA and observed by ALP staining at 4 and 7 days. ResultsC3H10T1/2 cell lines with different Hey1 expression levels were successfully established. In osteogenesis compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 enhanced the mRNA and protein expressions of transcription factors (Runx2, osteopontin, and osteocalcin), and the expression of osteogenic differentiation marker (ALP) (P < 0.05); however, inhibition of Hey1 expression significantly decreased the above indexes (P < 0.05). In cell proliferation activity compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 increased absorbance (A) value in MTT assay and pecentage of G2+S cells in cytometry assay, but inhibition of Hey1 expression significantly decreased the indexes (P < 0.05). ConclusionExpression of Hey1 is the important link in the osteogenic differentiation process of C3H10T1/2 cells induced by BMP-9, and plays an important role in the regulation of early cell proliferation.
ObjectiveTo study the effect of inhibitor of differentiation 1 (Id1) gene transfection on bone morphogenetic protein 2 (BMP-2) promoting the expressions of collagen type Ⅱ (COL Ⅱ) and aggrecan (ACAN) in intervertebral cartilage endplate cells (EPCs). MethodsEPCs were harvested from the New Zealand white rabbits, the 2nd generation EPCs were used for experiment. The transfection efficiency of green fluorescent protein blank lentivirus, high expression of Id1 lentivirus, RNA interference (RNAi) Id1 lentivirus transfection in the EPCs were observed by the fluorescence microscopy, real-time fluorescence quantitative PCR, and Western blot. Blank vector, single BMP-2 gene, BMP-2 and Id1 genes were transfected into EPCs, respectively. The cell morphology and the expressions of COL Ⅱ and ACAN in each group were observed. ResultsLentiviral transfection had no significant effect on the cell morphology. The EPCs were effectively transfected by the high expression Id1 lentivirus and RNAi Id1 lentivirus; the expression of Id1 mRNA was also significantly interfered. The expressions of COL Ⅱ and ACAN mRNA and synthesis of COL Ⅱ and ACAN protein were significantly higher in BMP-2 lentivirus and high expression Id1 lentivirus groups than control group (P<0.05). The expression of COL Ⅱ and ACAN protein were down regulated in the cartilage endplate cells when the expression of Id1 gene was decreased (P<0.05). ConclusionUp-regulation of Id1 gene expression can enhance the effects of BMP-2 on the synthesis of COL Ⅱ and ACAN in EPCs.
ObjectiveTo explore the effectiveness of intertransverse bone graft after debridement and fusion combined with posterior instrumentation in patients with single segmental thoracic tuberculosis. MethodsBetween March 2014 and May 2015, 17 cases of thoracic tuberculosis were treated by the surgery of intertransverse bone graft after debridement and fusion combined with posterior instrumentation. There were 10 males and 7 females with an average age of 48.5 years (range, 18-70 years), and with a mean disease duration of 4 months (range, 1-9 months). The affected segments included T4, 5 in 2 cases, T6, 7 in 5 cases, T7, 8 in 3 cases, T9, 10 in 2 cases, T10, 11 in 4 cases, and T11, 12 in 1 case. The operation time, intraoperative blood loss, and hospitalization time were recorded. Postoperative plain radiography was taken to assess the decompression and internal fixation, and the fusion effect was evaluated by X-ray or CT examination. The erythrocyte sedimentation rate (ESR), C reactive protein (CRP), visual analogue scale (VAS), Oswestry disability index (ODI), and Kyphosis angle were recorded and compared; the nerve function was evaluated by American Spinal Injury Association (ASIA). ResultsThe mean operation time, intraoperative blood loss, and hospitalization time were 184 minutes (range, 165-220 minutes), 231 mL (range, 150-800 mL), and 18 days (range, 12-26 days) respectively. No complication of hematoma or wound dehiscence was found. All patients were followed up 17.9 months on average (range, 9-22 months). No bone graft failure, internal fixation broken, pleural effusion, cerebrospinal fluid leakage, wound infection, fistula formation, and other complications occurred. Satisfactory intervertebral fusion was obtained in all patients at 3-8 months (mean, 5.3 months) after surgery. The ESR, CRP, VAS score, ODI score, and Kyphosis angle were significantly improved at immediate after operation and last follow-up when compared with preoperative ones (P < 0.05), and the ESR, CRP, VAS score and ODI score at last follow-up were significantly better than those at immediate after operation (P < 0.05). At last follow-up, the nerve function was recovered to ASIA grade E from grade C (1 case) and grade D (6 cases). ConclusionIntertransverse bone graft is a reliable, safe, and effective way of bone graft applied to the single segmental thoracic spinal tuberculosis.