Objective To investigate the effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells.Methods Lung cancer A549 cells and SPC-A-1 cells were selected and divided into control group,Nc-shRNA group and Notch1-shRNA group.The Nc-shRNA group was a negative control RNAi lentivirus group,and the Notch1-shRNA group was a Notch1 inhibitory RNAi lentivirus group.The lentiviral-mediated shRNA interference technology was used to target the silencing of Notch1.The silencing effect of Notch1 gene was verified by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting.Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and sarcosphere formation assay.Apoptosis was detected by Annexin V/7-AAD double staining.Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA),B-cell lymphoma-2 (Bcl-2) and Notch1 downstream gene Hes-1.Results The results of qRT-PCR showed that the relative expression levels of Notch1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were 1.000 ± 0.000,0.937 ± 0.025,0.490 ± 0.036 and 1.000 ± 0.000,1.077 ± 0.070,0.373± 0.038,with statistically significant differences (F =359.707,P ＜0.001;F =210.455,P ＜0.001),further paired comparison,the relative expression of Notch1 in Notch1-shRNA group was significantly lower than that in Nc-shRNA group (all P ＜ 0.05).Western blotting showed that the expressions of Notch1 protein in A549 cells and SPC-A-1 cells were consistent with the mRNA results.MTT assay showed that the 24 h A values of A549 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.209 ± 0.005,0.219 ± 0.009,0.159 ±0.006,48 h A values were 0.293 ± 0.004,0.302 ± 0.004,0.205 ± 0.005,72 h A values were 0.450 ± 0.003,0.430 ± 0.012,0.348 ± 0.017,with statistically significant differences (F =79.487,P＜0.001;F =508.664,P ＜0.001;F =57.156,P ＜0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 24,48,72 h (all P ＜ 0.05).The 48 h A values of SPC-A-1 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.438 ±0.022,0.412 ± 0.015,0.364 ± 0.010,72h A values were 0.540 ± 0.016,0.519 ± 0.009,0.438 ± 0.019,with statistically significant differences (F =15.667,P =0.004;F =37.299,P ＜ 0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 48 h and 72 h (all P ＜ 0.05).The sphere sizes of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were (149.667 ± 6.506) μm,(136.667 ± 7.095) μm,(86.676 ± 7.638) μm,with statistically significant difference (F =65.940,P ＜ 0.001).The sphere sizes of the three groups in SPC-A-1 cells were (118.667 ± 6.658) μm,(128.000 ± 7.000) μm,(60.675 ± 4.509) μm,with statistically significant difference (F =105.372,P ＜0.001).Further paired comparison,the sphere size of Notch1shRNA group was significandy smaller than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).The apoptosis rates of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1cells were (0.489 ± 0.014)％,(0.633 ± 0.021)％,(1.683 ± 0.221)％ and (1.323 ± 0.194)％,(1.690 ± 0.188) ％,(3.017 ± 0.356) ％,with statistically significant differences (F =77.660,P ＜ 0.001;F=32.200,P =0.001),further paired comparison,the apoptosis rate of Notch1-shRNA group was significantly higher than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).Western blotting showed that the expressions of PCNA,Bcl-2 and Hes-1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were statistically significant (F =155.343,P ＜ 0.001;F =22.576,P =0.002;F =70.108,P＜0.001),and the expressions of PCNA,Bcl-2 and Hes-1 in the three groups in SPC-A-1 cells were statistically significant (F =49.419,P ＜0.001;F =28.090,P =0.001;F =12.040,P =0.007).Further paired comparison,the expressions of PCNA,Bcl-2 and Hes-1 in Notch1-shRNA group were significantly lower than those in Nc-shRNA group in the two kinds of cells,and the differences were statistically significant (all P ＜0.05).Conclusion Targeted silencing of Notch1 can reduce the proliferation activity of lung cancer stem cells and promote apoptosis,which may be related to the down-regulation of its downstream gene Hes-1.

Objective To analyze the perioperative risk factors of postoperative complications after posterior lumbar fusion operation.Methods The clinical data of 654 patients with posterior lumbar fusion during 2010 and 2014 were retrospectively analyzed.Using x2 test and one-way ANOVA,the predicted risk factors were screened for further Logistic regression.Results The total complication rate was 11.6％ among all 654 patients.The major complications included cardiac infarction,deep infection,sepsis,neurological impairment,and secondary operation.And the minor complications included wound dehiscence,urinary tract infection,pulmonary infection,gastrointestinal bleeding,CSF leakage and others.According to x2 test and one-way ANOVA,renal function insufficiency,preoperative neurological injury,ASA higher than Ⅲ level,intraoperative blood loss,long operation length,and usage of autogenous bone were screened as risk factors of complications.Renal function insufficiency,preoperative neurological injury,intraoperative blood loss,and long operation length were screened as risk factors of minor complications.And male,renal function insufficiency,preoperative neurological injury,intervertebral fusion,and posteriolateral fusion were screened as risk factors of major complications.However,according to Logistic regression,the independent risk factor of complications were preoperative neurological injury and long operation length;independent risk factors of minor complications were renal function insufficiency,preoperative neurological injury and long operation length;and independent risk factor of major complications was preoperative neurological injury.Conclusion Preoperative neurological injury,renal function insufficiency and long operation length are proved to be the risk factors of postoperative complication in lumbar fusion surgery.

OBJECTIVETo evaluate the clinical outcomes of total en-bloc spondylectomy (TES) in recurrence spinal tumor.

METHODSThe study was a retrospective study of recurrence spinal tumor from January 2010 to October 2013. A total of 6 patients with recurrent spinal tumor underwent TES procedures, with 5 cases located in thoracic spine and 1 case located in L1. There were 3 male and 3 female patients, with a mean age of 33.2 years. Pathological diagnosis included giant cell tumor of bone in 3 cases, breast cancer, lung cancer and nasopharyngeal carcinoma with 1 case in each. The operation time, bleeding loss, resected segments, cutting edge, spinal cord function and complications was evaluated.

RESULTSSingle segment resected in 1 case, 2 segments resected in 2 cases and 3 segments resected in 3 cases. The average operation time was 8.9 hours (7.5 to 12.0 hours). The average blood loss was 3 116 ml (2 500 to 4 500 ml). The average follow-up period was 23.2 months (12 to 47 months) without recurrence. There was no spinal cord injury during operation. The neurologic function was significantly improved in 2 cases (American Spinal Injury Association (ASIA) grade C to grade D), unchanged in 1 cases (ASIA grade B) and no deteriorated case in 3 cases (ASIA grade E). There was no perioperative deaths case. Complications included 2 cases pleural rupture, 1 case dural tear and 1 case massive haemothorax. No peri-operation death case.

CONCLUSIONSome of the recurrent spinal tumors are still suitable for en-bloc resection and TES procedure with the extent of its applicability under strict control.

Adult ; Female ; Humans ; Lung Neoplasms ; Male ; Neoplasm Recurrence, Local ; Retrospective Studies ; Spinal Cord Neoplasms ; Spinal Neoplasms ; surgery ; Spine