Objective: To determine the role of semaphorin 4D (Sema4D) in osteolytic bone destruction of bone metastasis in lung cancer, and to explore its mechanism. Methods: Immunohistochemistry was used to detect the expression of Sema4D in bone metastases of lung cancer and normal bone tissues. The expression levels of Sema4D protein and mRNA in four kinds of lung cancer cell lines were detected by Western blotting and real-time fluorescent quantitative PCR, respectively. The conditioned medium of lung cancer cells was collected, and ELISA was used to detect the secretion level of Sema4D in lung cancer conditioned medium. The conditioned medium from lung cancer cells with Sema4D high expression (PC9 cells) or low expression (A549 cells) was collected and co-cultured with osteoblastic precursor MC3T3-E1 cells, then the osteoblast differentiation ability was detected by alkaline phosphatase staining, the mineralization ability of osteoblasts was detected by alizarin red staining, the expression levels of osteoblast differentiation genes alkaline phosphatase, liver/bone/kidney (ALPL), Oxterix and collagen type alpha 1 (Col1α1) were detected by real-time fluorescent quantitative PCR. Sema4D shRNA was transfected into lung cancer PC9 cells, then the expression and secretion levels of Sema4D in lung cancer cells were detected by Western blotting, real-time fluorescent quantitative PCR and ELISA, respectively. The conditioned medium of lung cancer PC9 cells transfected with Sema4D shRNA was collected and co-cultured with preosteoblasts, and the effect of the conditioned medium from lung cancer cells with Sema4D interference on the osteoblast differentiation was determined by alkaline phosphatase staining, alizarin red staining and real-time fluorescent quantitative PCR, respectively. Results: Compared with the normal bone tissues, Sema4D was highly expressed in osteolytic bone metastases of lung cancer. Sema4D was expressed and secreted in different lung cancer cell lines in different degrees. The expression and secretion levels of Sema4D were the highest in lung cancer PC9 cells, but lowest in lung cancer A549 cells (P < 0.05). The conditioned medium of lung cancer cells significantly inhibited the osteogenic differentiation of osteoblasts induced by osteogenic inducer, which was characterized by the reduction of relative staining area of alkaline phosphatase staining and alizarin red staining (both P < 0.05), and the decreased expression levels of osteogenic differentiation genes ALPL, Oxterix and Col1α1 (all P < 0.05). The conditioned medium of PC9 cells with Sema4D high expression exhibited more aggressive inhibitory effect on osteoblast differentiation than the conditioned medium of A549 cells with Sema4D low expression (P < 0.05). shRNA transfection significantly reduced the expression and secretion of Sema4D in lung cancer cells (both P < 0.05), and significantly attenuated the inhibitory effect of lung cancer conditioned medium on osteoblast differentiation (all P < 0.05). Conclusion: Lung cancer-derived Sema4D plays an important role in osteolytic bone destruction by inhibiting osteoblast differentiation, suggesting that it is expected to become a new therapeutic target for bone metastasis of lung cancer.

Objective:To study the role of Jagged1 and Notch signaling pathway played in the differentiation and proliferation of RAW 264.7 cells.Methods: RAW 264.7 cells were divided into three groups to culture:The control group:RAW 264.7 cells were threated with culture and RANKL.The Jagged1 group:RAW 264.7 cells were threated with recombinant protein Jagged 1 besides the control group.The DAPT group:RAW 264.7 cells were threated with DAPT besides the Jagged 1 group.The mRNA expression of osteoclast markers(TRAP,CK,CTR) and Notch key target genes (HES-1 and HEY-1) were measured by real-time PCR.The formation of osteoclast , bone resorption , Notch expression and proliferation of RAW 264.7 cells were detected by TRAP staining , scanning electron microscope ,immunofluorescence and cell counting kit-8 ( CCK-8 ).Results: TRAP, CK, CTR , HES-1 and HEY-1 mRNA expression were significantly higher than the control group and DAPT group in Jadded 1 group ( P<0.05 ).TRAP+cell count ,osteolytic area was significantly increased in Jagged 1 group compared with control and DAPT group , and no significant difference observed between the last two groups.Immunofluorescence results showed high expression of N ICD in cell membrane and cytoplasm in all groups and additionally expressed in nucleus in Jadded 1 group.Cell proliferation was inhibited in Jagged 1 group also ( P<0.05 ).Conclusion:Jagged1 promotes RAW264.7 cells osteoclast differentiation and inhibits proliferation by activating Notch signaling pathway .

Objective To investigate the pathological changes of spinal cord tissues after spinal cord injury and their significance for clinical diagnosis and treatment of complete spinal cord injury.Methods Patients with complete spinal cord injury were selected.Mter rigorous ethical review and patient informed consent,damaged section of the spinal cord and necrotic tissue were removed and transplanted with the function biological material with autologous stem cell.The specimen of injured spinal cord collected during the operation were divided into three phases according to injury time:3 cases of acute phase (≤ 3 days),3 cases of subacute phase (4-14 days),and 1 case of chronic phase (＞ 14 days).The pathological changes of injured spinal cord tissue were observed through HE staining,and the expression of hypoxia inducible factor 1α (HIF-1α),tumor necrosis factor (TNF-α),microtubule-associated protein(MAP2) were detected by immunofluorescence.Results (1) HE staining showed that the acute injury mainly manifested as diffuse hyperemia and liquefaction,subacute injury infiltration of inflammatory cells and spinal cord liquefaction,and chronic injury mainly scar repair.(2)The fluorescence intensity (24.67 ±0.51) of HIF-1o in chronic injury was higher than acute (3.17 ± 0.40) and subacute injury (4.62 ± 0.48) (P ＜ 0.05),and the fluorescence intensity of subacute injury was higher than that of acute injury (P ＜ 0.05).(3) The fluorescence intensity (17.60 ± 1.17) of TNF-α in subacute injury was higher than that of acute injury (5.35 ± 0.33) and chronic injury (1.81 ± 0.17) (P ＜0.05);(4) The fluorescence intensity of MAP2 was 9.46 ±0.41 in acute injury,higher than 3.25 ± 0.42 in subacute injury and 1.16 ± 0.08 in chronic injury (P ＜ 0.05).Conclusions There are hypoxia,inflammation,neuronal apoptosis and repair in the spinal cord tissues after complete spinal cord injury,such as hyperemia,liquefaction,necrosis and gradual scar repair.The severity of inflammation and hypoxia significantly differs among different injury phases.The inflarmnatory cytokines are mainly active in the microenvironment during subacute injury.Hypoxia is mainly involved in the pathological changes of chronic injury.This can provide a new theoretical basis for the clinical treatment of complete spinal cord injury and the timing of surgical treatment.

Objective:To investigate the feasibility and clinical outcome of single-stage posterior total en bloc spondylectomy via posterior approach for lowerlumbar spinal malignant tumors.Methods:The clinical data of 23 patients with metastatic tumors of the lower lumbar spine who underwent single-stage posterior total En bloc spondylectomy in our hospital from January 2012 to June 2018 were analyzed retrospectively. There were 14 males and 9 females, age 57.9±10.8 years old (range, 37-74 years old). All patients were treated with single-stage posterior total en blocspondylectomy, titanium mesh implantation and posterior pedicle screw fixation. Observation items included operation time, intraoperative blood loss, postoperativehospital stays,the visual analogue scale (VAS) and the Eastern Cooperative Oncology Group (ECOG) physical condition score of the patients before operation,1 month after operationand 6 months after operation, the American spinal injury association (ASIA) spinal cord injury grade pre-operation andpostoperation, perioperative complications, local recurrence and survival state.Results:The median fellow-up time of this group was 20 months (range 6-56 months). At the end of the last follow-up, there were 3 patients who survived, the average follow-up time of the three patients who survived to the last follow-up was 37.3±11.7 months. One of them had local recurrence, but survived with tumor. The operative time was 155-510 min, with an average of 258±96 min, the intraoperative blood loss was 750-2 500 ml, with an average of 1 258.7±528.6 ml, and the postoperative hospital stay was 10-30 d, with an average of 18.4±4.6 d. VAS score decreased from 7.4±0.8 before operation to 2.6±0.6 1 month after operation, and ECOG score decreased from 1.6±0.9 before operation to 0.9±0.76 months after operation, showing statistically significant differences ( P<0.05). 6 patients presented with postoperative acute nerve root stimulation, 3 patients presented with postoperative cerebrospinal fluid leakage, 3 patients presented with postoperative surgical site infection, 1 with pulmonary infection, and 3 patients presented with titanium mesh displacement. Conclusion:Single-stage posterior total En bloc spondylectomy is feasible for the treatment of metastatic tumors of the lower lumbar spine. Although the operation is quite challenging due to its special anatomical structure and biomechanical characteristics,the long-term follow-up effect is satisfactory.