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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.
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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 investigate whether metabolic pathway-related gene polymorphisms are associated with arterial plaque stability and their gene-gene interactions increase the risk of cerebral infarctions.Methods Totally 294 patients with atherothrombosis stroke admitted to the Department of Neurology, the Third Affiliated Hospital of Wenzhou Medical University from September 2010 to December 2012 were divided into a carotid vulnerable plaque group ( n=69 ) and a stable plaque group ( n=225 ) according to the results of carotid B-mode ultrasonography.A total of 282 healthy volunteers excluded carotid plaque and stroke were enrolled as well.Genetic polymorphisms of ALOX5AP and CYP3A5, CYP2C9*2, CYP2C9*3 and EPHX2 were genotyped using polymerase chain reaction and mass spectrometry analysis.The SPSS16.0 software was used to compare genotype frequencies and the generalized multifactor dimensionality reduction ( GMDR ) method was applied for gene-gene interaction analyses.Results The results showed that EPHX2 GG genotype might protect against stroke ( OR =0.520, 95% CI 0.288 -0.940, P=0.030).The distribution of CYP3A5 genotypes showed statistically significant differences (χ2 =7.284, P=0.026) between the vulnerable plaque ( AA: 5 cases, AG: 36 cases, GG: 28 cases) and stable plaque ( AA: 26 cases, AG: 77 cases, GG: 122 cases ) groups.Multivariate Logistic regression analysis showed that the GG genotype of CYP3A5 was protective factor for unstable plaques ( OR=0.405, 95%CI 0.178 -0.920, P =0.031 ).Differences in other SNPs did not reach statistical significance between the two groups.The GMDR analysis showed a significant gene-gene interaction between SG13S114 and A6986G, with scores of 10 for cross-validation consistency and 9 for the sign test (P=0.011).The best model for ischemic stroke was found to be SG13S114 AA and A6986G AA.Adjusting for age, hypertension and diabetes, the certain gene-gene interaction predicted a significantly higher risk of cerebral infarction (OR=1.804, 95%CI 1.180-2.759, P=0.006).Conclusions The EPHX2 G860A gene might be linked with the incidence of cerebral infarctions.Only a CYP3A5 gene polymorphism might be associated with carotid plaque instability in patients with stroke.The gene-gene interaction predicts a significantly higher risk of cerebral infarction.There is a 1.804-fold increased risk for ischemic stroke in individuals with these combined genetic factors.
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Objective:To explore the relationship between tissue factor (TF),tissue factor pathway inhibitor (TFPI) and the severity in patients with diabetic cerebral infarction.Methods: 226 patients with diabetic cerebral infarction were included into the study,National Institute of Health Stroke Scale ( NIHSS) was used to evaluate the severity of CIS.The single factor analysis and multiple regression analysis were used to explore the relationship between TF , TFPI and the severity.Results: The concentrations of TF,TFPI,TF/TFPI,cholesterol and triglyceride in the NIHSS≤12 group were significantly lower than that in the NIHSS>12 group ( P<0.05);the NIHSS was significantly positive correlate with TF (r=0.354,P=0.012),TFPI (r=0.302,P=0.027),TF/TFPI (r=0.410,P=0.000),cholesterol (r=0.364,P=0.006) and triglycerides (r=0.334,P=0.018);Multiple linear regression analysis showed that the TF , TFPI, TF/TFPI, cholesterol were independent risk factors of the severity in patients with diabetic cerebral infarction.Conclusion:The level of TF and TFPI could reflect the severity in patients with diabetic cerebral infarction according to the NIHSS.
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Objective To explore the influencing factors of gastrointestinal bleeding(GB) in secondary prevention patients with cerebral ischemic stroke(CIS).Methods A total of 616 patients were divided into bleeding group and control group according to the status (yes,no) of suffering GB during the 2years follow-up.Single factor analysis and Logistic regression analysis was used to explore the influencing factors of GB in CIS patients.Results The proportion of age≥65,a history of GB,gastric disease,renal insufficiency,sudden onset,NIHSS ≥12 and CIS ≥2 in the bleeding group was significantly higher than that in the control group (P ＜ 0.05) ; The proportion of combined with statins,proton pump inhibitors and gastric mucosal protective agent in the bleeding group was significantly lower than that in the control group (P ＜0.05) ; The Logistic regression analysis showed that age≥65,a history of GB,gastric disease,renal insufficiency,sudden onset,NIHSS≥12 and the times of CIS≥2 were risk factors of GB; however,combined with statins,proton pump inhibitors were protective factors.Conclusions Aging,a history of GB,gastric disease,sudden onset,higher NIHSS score and the times of CIS ≥ 2 were the risk factors of GB,combined with statins and proton pump inhibitors could reduce the risk of GB.
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Objective To explore the characteristics of operation,curative effects and operative management in elderly patients of age older than 70 years with coronary heart disease receiving coronary artery bypass grafting.Methods 108 elderly patients of age older than 70 years with coronary heart disease were divided into two groups:OPCAB group(n = 76) and CCABG group (n = 32) The clinical curative effects, early postoperative mortality and complications of the two groups were compared and analyzed respectively. Results OPCAB group was better than CCABG group in these series(P ＜ 0.05): The early postoperative mortality (5.8%, 11.2%)、 myocardial infarction (2.9%, 10.6%), respiration failure(8.7%, 17.5%), pulmonary complications: (11.8%, 31.5%) 、complication of CNS:(1.8% ,9.8%) 、acute renal failure(1.8% ,6.2%) ,the time of intubation: (9.3 ±4.5), (25.4 ±7.5) h,ICU stay(3.1 ± 1.8) ,(7.1 ±2.9) d,hospital stay(15.5 ±8.6) ,(26.4 ±8.6)d. Conclusion OPCAB could reduce operative mortality and complication, it should be the first option for the surgery of elder patients with coronary heart disease;surgical skills and correct perioperative management were the key factors to assure surgical outcome.
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Interferon beta (IFN-beta) and TNF-related apoptosis-inducing ligand (TRAIL) are effective anticancer agents. Adeno-associated virus (AAV) is one of the current most promising gene delivery vectors. Previously, we constructed tumor-targeting AAV-hTERT-IFN-beta and AAV-hTERT-TRAIL by inserting IFN-beta or TRAIL gene into AAV controlled by hTERT promoter. The studies showed that either single IFN-beta or TRAIL gene therapy exhibited a certain extent anticancer effect. Here, we report their inhibitory effects on A549 lung cancer cell growth in vitro and in vivo by combined AAV-hTERT-IFN-beta and AAV-hTERT-TRAIL. Expression of secreted IFN-beta in lung cancer A549 cells infected by AAV-hTERT-IFN-beta was detected by enzyme-linked immunosorbent assay (ELISA). The growth-suppressing effect of AAV-hTERT-IFN-beta in combination with AAV-hTERT-TRAIL on several cancer cell lines was assessed by MTT assay. Apoptosis of A549 cancer cells infected by AAV-hTERT-IFN-beta alone, AAV-hTERT-TRAIL alone, and their combination was evaluated by apoptotic cell staining and flow cytometry (FCM), respectively. The antitumor effect of the combination of AAV-hTERT-IFN-beta with AAV-hTERT-TRAIL in vivo was further evaluated through A549 lung cancer xenograft in nude mice. The results showed that the combinational treatment was superior to any alone and presented intensified tumor cytotoxic and apoptotic effect on A549 cancer cells. Most importantly, the combination of AAV-hTERT-IFN-beta with AAV-hTERT-TRAIL exhibited significant antitumor effect and eliminated all tumor masses in nude mice, which lay a foundation for exploring the molecular mechanisms of combined IFN-beta and TRAIL anti-tumor activity.
Subject(s)
Animals , Humans , Mice , Antineoplastic Agents , Metabolism , Pharmacology , Cell Line, Tumor , Dependovirus , Genetics , Metabolism , Genetic Therapy , Genetic Vectors , Genetics , Interferon-beta , Genetics , Pharmacology , Lung Neoplasms , Pathology , Therapeutics , Mice, Nude , Neoplasm Transplantation , Recombinant Fusion Proteins , Genetics , Pharmacology , TNF-Related Apoptosis-Inducing Ligand , Genetics , PharmacologyABSTRACT
Based on the requirement of culture conditions for hematopoietic stem and progenitor cells (HSPCs) ex vive expansion, we developed a new-type bioreactor by combining superiorities of static and stirred culture models. Stem cell factor (SCF), thrombopoietin (TPO), FLT-3 ligand(Flt-3) were used as the cytokines cocktails. The effects of the static and cyclic culture on the expansion characteristics of CD34+ selected cells were compared in the new-type bioreactor. After 7 d cultures, the expansion of total cells in the static culture was 13.86 +/- 4.26 fold, higher than that in the cyclic culture (7.23 +/- 2.67 fold). The analysis of the fold expansion and the proportion of CD34+ cells showed that there was no marked difference between the static culture and the cyclic culture. However, the fold expansion and the proportion of CD34+CD38- cells were higher in the cyclic culture than those in the static culture (3.90 +/- 0.85 versus 1.82 +/- 0.58), which reflected more primary CD34+CD38- cells were obtained in the cyclic culture. The above results demonstrated that both the static culture and the cyclic culture could be used in ex vive expansion of CD34+ cells with the new-type bioreactor. In static culture hematopoietic stem cells differentiated into progenitor cells, whilst the cyclic culture favored the expansion of primary HSPCs.
Subject(s)
Humans , Antigens, CD34 , Metabolism , Bioreactors , Cell Culture Techniques , Methods , Cell Differentiation , Physiology , Cell Proliferation , Cytokines , Pharmacology , Fetal Blood , Cell Biology , Hematopoietic Stem Cells , Cell Biology , Allergy and Immunology , Stem Cell Factor , PharmacologyABSTRACT
The hematopoietic repopulating ability of fresh and cultured CD34+ cells and CD34- cells derived from cord blood were compared by nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. Fresh CD34+ cells and CD34- cells were isolated from fresh cord blood. Cultured CD34+ cells and CD34- cells were separated from cultured mononuclear cells (MNC). We transplanted these cells into sublethally irradiated NOD/SCID mice via the tail vein and sacrificed surviving mice after 6 weeks. The peripheral blood, spleen and bone marrow from each mouse were harvested for flow cytometry, colony-forming cells and human Alu sequences analyses. The proportions of CD45+ cells and human multilineage hematopoietic cells in NOD/SCID mice received CD34+ cells were close to that in the mice received both CD34+ cells and CD34- cells, while it was significantly higher than that in the mice received CD34- cells. Six weeks after transplantation, all the mice injected with cultured CD34- cells dead. The survival rate of mice injected with cultured CD34+ cells was 66.7%. All of the mice injected with both cultured CD34- and CD34+ cells survived. Moreover, CD45+ cells could be detected in all surviving mice, and human CD34, CD3, CD19, CD33 and CD71 antigen also could be detected on these CD45+ cells. The results showed that both fresh and cultured CD34+ cells had the capability of engraftment and hematopoiesis reconstitution, but CD34- cells hadn't the ability. However, CD34- cells had assistant effect on the hematopoietic repopulating ability of CD34+ cells.
