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Lung cancer stem cells play an important role in chemoresistance and invasion and metastasis of lung cancer. Increasing evidence sustains that microRNAs(miRNAs)play a major role in regulating tumor angiogenesis,drug resistance and metastasis. Cancer stem cells are considered to be one of the reasons leading to cancer recurrence,metasta-sis and resistance to chemotherapy,therefore better understanding how miRNAs regulate gene expression in lung cancer stem cells will help identify cancer biomarkers and new therapeutic targets,and will help to develop better treatment strate-gies for lung cancer.
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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.
ABSTRACT
Notch signaling pathway is involved in the abnormal differentiation and self-renewal of lung cancer stem cells.The further studies for the roles of Notch signaling pathway in the regulation of lung cancer stem cells are expected to find new targets in the diagnosis and treatment of lung cancer.Inhibitors of the Notch signaling pathway may be effective in the treatment of lung cancer.Lung cancer stem cells are thought to be a major cause of recurrence of lung cancer,therefore,targeted therapy for lung cancer stem cells may be more effective than treatment for the entire tumor.
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Objective To determine the factors contributing to the development of deep vein thrombosis (DVT) in the lower extremity in patients after hip or knee arthroplasty and hip fracture internal fixation.Methods One hundred and forty-seven consecutive patients receiving hip or knee arthroplasty and hip fracture internal fixation from 2004 to 2005 were included in this study. Their age ranged from 33 to 92 years. Duplex color ultrasonic inspection was performed on veins of the bilateral lower extremities before operation and 2 weeks after operation for detection of DVT. The patients were divided into a DVT group and a DVT-free group based on the development of DVT after operation. Detailed perioperative clinical information about the patients, surgery and anesthesia was collected.Results Lower extremity DVT was found in 42.2% of the patients after operation, while the incidence of proximal DVT was 2.7%. Compared with the DVT-free group, the usage rate and dosage of ephedrine increased significantly, the duration of anesthesia was significantly longer, and the white blood cell count (WBC) on the 1st postoperative day and the highest WBC count were significantly higher in the DVT group(P＜0.05). Logistic regression analysis indicated that the above factors were closely related to DVT.Conclusion Duration of anesthesia ＞ 3 h, ephedrine administration and a marked increase in WBC count after operation are the risk factors for DVT in the lower extremities in patients after hip or knee arthroplasty and hip fracture internal fixation.
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Objective This research attempted at prevention of steam contamination and protection of medical staff in periodontal scaling. Methods Thirty sample patients without oral cavity or upper res-piratory tract infection, chronic disease, infectious disease or other contraindication were randomly classified into the experimental group and the control group.The former gargled with chlorhexidine in the process of periodontal scaling while the latter rinsing only. Oral cavity bacteria of both groups were sampled before and after operation;air samples were got at five periods of time(before operation, 10 minutes in operation, 20 minutes in operation, 30 minutes in operation, and end of operation) and germicnltured.The entire data were analyzed.Results Air samphng in periodental scaling and oral sample germiculture after the scaling in the experimental group were lower than those of the control group, P<0.01. Conclusions With effective disinfection and sta-ble characteristics, chlorbexidine can be popularly utilized in skin mucous membrane disinfection. Gargling with chlorhexidine in periodontal scaling eliminates bacteria and microorganism in oral cavity and resulting steam,thus reducing environmental pollution and protecting the health of medical staff.
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Objective To compare the effects of different dose aprotinin on cardiopulmonary bypass induced inflammation and myocardial injury Methods Eighteen adult patients undergoing cardiopulmonary bypass (CPB) were divided into three groups (n=6): control,low dose aprotinin and full dose aprotinin Plasma levels of tumor necrosis factor ?(TNF?), interleukin 6(IL 6), interleukin 10(IL 10), intercellular adhesion molecule 1(ICAM 1), troponin T(TnT) and neutrophil integrin cluster of differentiation 11b(CD11b) were measured at following points: before CPB(T1);1 h of CPB (T2) ;30 min after weaning from CPB (T3); 4 h after weaning from CPB (T4) ; and 24 h after weaning from CPB(T5) Results TNF?: the values in two aprotinin using groups were lower than the control levels at T2, T3 and T4 , furthermore, in full dose group was lower than in low dose group at T4 , and lower than two other groups at T5 (P
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Objective To observe the changes of calcitonin gene related peptide (CGRP) in rat spinal motoneuron after upper motoneuron injury. Methods Thirty six adult male Wistar rats were selected. Spinal cord transection was made at the lower thoracic segment. The lumbar regions of spinal cord were removed immediately, 1, 2, 4, 7 and 14 days after surgery to be divided into dorsal and ventral sections. The content of CGRP in ventral section was detected using radioimmunoassay method.Results The level of CGRP in spinal motoneuron of the lumbar region was decreased significantly after transection of spinal cord, reached the lowest level on the 2nd day after surgery and then maintained at the low level.Conclusions The upper motoneuron injury can result in the decrease of CGRP content in spinal motoneuron.
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Objective To determine the plasma concentrations and pharmacodynamics of fentanyl and nerphine used in postoperative epidural or intravenous patient-controlled analgesia (PCEA,PCIA) .Methods Sixty ASA Ⅰ - Ⅱ patients (36 male, 24 female), aged 18-25 yr undergoing elective major operation were randomly divided into 3 groups : group Ⅰ received PCEA with 0.13 % bupivacaine + fentanyl 2?g?ml-1 (n = 20) ; groupⅡ received PCEA with 0.13 % bupivacaine + morphine 0.08 mg? ml-1 ( n = 20); group Ⅲ received PCIA with morphine 0.5 mg ? ml-1 ( n = 20). In group Ⅰ and Ⅱ the background infusion rate was 4 ml ? h-1 , PCA bolus dose 2 ml and lock-out interval 20 min, while in group Ⅲ the back ground infusion rate was 1 ml?h-1 , PCA bolus dose 2 ml and lock-out interval 6 min. PCA was maintained for 48 h in all three groups. The vital signs, analgesic effect (VAS, VRS) and side-effects were recorded and venous blood samples were taken for determination of plasma fentanyl and morphine concentrations at 4 h, 24 h and 48 h after PCA was commenced. Results The demographic data were comparable among the three groups. There was no significant difference in MAP, HR and RR during PCA among the three groups. The analgesia was satisfactory in all three groups and no other analgesic was used during PCA. The rate of excellent analgesia ranged between 80%-85 % . The incidences of side-effects were higher in group Ⅱ and Ⅲ as compared with those in group Ⅰ . The volume of epidural PCA solution administered in 48 h was significantly larger in group Ⅰ than that in group Ⅱ( P