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Objective To explore the mechanism of the demethylase fat mass and obesity associatal(FTO)gene on the proliferation of human liver cancer cell line HepG2.Methods HepG2 cells were divided into the control group,FTO group(transfected with FTO over-expression plasmid),si-FTO group(transfected with si-FTO)and si-FTO+si-FoxO1 group(simultaneously transfected with si-FTO and si-FoxO1).The expression of FTO in cells was detected by RT-qPCR.Cell proliferation,invasion and apoptosis were examined using CCK-8 assay,Transwell chamber assay and flow cytometry,respectively.Dot blot assay was performed to measure m6 A methylation,and protein expression of FoxO1 in cells was detected by Western blot.Results Analysis of overall survival in liver cancer patients using The Cancer Genome Atlas(TCGA)showed higher expression of FTO associated with shorter survival(P＜0.05).Compared with normal liver cells HL7702,FTO relative expression was significantly increased in human liver cancer cells HepG2(P＜0.05).The relative expression of FTO in si-FTO group cells was lower than that in the control group,while the relative expression of FTO in FTO group was higher than that in the control group(P＜0.05).The relative expression of FTO in the si-FTO+si-FoxO1 group was higher than that in the si-FTO group(P＜0.05).Compared with the Control group,cell viability,count of invasive cells,relative level of m6 A were significantly decreased,while apoptosis rate and protein expression of FoxO1 were significantly increased in the si-FTO group(P＜0.05).Cell viability,count of invasive cells,and relative expression level of m6 A were sig-nificantly higher,while apoptosis rate and protein expression of FoxO1 were significantly lower in the FTO group compared to the Control group(P＜0.05).Compared with the si-FTO group,cell viability,invasive cells and rela-tive level of m6 A were significantly increased,while apoptosis rate and protein expression of FoxO1 were significant-ly decreased in the si-FTO+si-FoxO1 group(P＜0.05).Conclusion High expression of FTO is associated with poor clinical prognosis.Knockdown of the demethylase FTO gene inhibits proliferation and invasion of liver cancer cells and induces their apoptosis.The mechanism is potentially related to the regulation of FoxO1.
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Objective:To efficiently amplify NK cells and determine their cytotoxic activity against a variety of tumor cell lines in vitro,thereby providing evidence for potential clinical application.Methods: PBMCs were isolated from adult peripheral blood and co-cultured with K562 cells that were genetically modified to express 4-1BBL,IL-15 and IL-21 on the surface for 15 days to effectively amplify NK cells.The total cell number and Purity of CD3-CD56+ cells were measured.Granzyme B and perforin expression of the amplified NK cells were detected by flow cytometry and real-time PCR.The anti-tumor effect on different cancer cells was evaluated.Results: This method obtained a more than 1.1×1010 CD3-CD56+ NK cells with 95% purity over a 15 day amplification procedure.The expanded NK cells could efficiently release granzyme B and Perforin.The cytotoxicity against different tumor cells was followed the order from strong to weak:gastric,pancreatic,cervical,ovarian and renal cancer cells,with the highest activity against gastric cancer cell line A549 (90% at E∶T=10∶1) (P<0.05).A time-dependent killing effect of activated NK cells on cervical,liver and pancreatic cancer cells was observed.Conclusion: This amplification procedure can consistently generate large amounts of pure NK cells with effective cytotoxic function against a variety of tumor cells.
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Objective To investigate the status and correlation between fatigue and health locus of control in patients with chronic obstructive pulmonary disease ( COPD ) . Method The multidimensional fatigue symptom inventory-short form and health locus of control questionnaire were used to investigate the fatigue and health locus of control among 150 COPD patients. Results The score on fatigue was 78.82 ± 1.50, in the middle-high level. The two factors scored the higher were physical fatigue and activity. The total score on health locus of control was 61.76 ± 5.02, at a middle-low level. The dimensions of health locus of control, ranked in a descending way in scores, included external control by destiny and authorities and internal control. The scores on external control by destiny and authorities were positively related that of total score on fatigue (P<0.05 for both) and the score of internal control was negatively correlated (P<0.05). Conclusion The fatigue of COPD patients was at high level and the health locus of control was at middle level. In the clinical practice, nurses should pay attention to the enhancement of patients′health locus of control to help them take up health behaviors for the purpose of lowering their fatigue.
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<p><b>OBJECTIVE</b>To define the mechanism of acute hepatitis in non-human primates after liver directed gene therapy.</p><p><b>METHODS</b>Differences in immune response exhibited by 8 rhesus monkeys receiving adenovirus (Ad) or lipofectamine-mediated gene transfer by various routes, the time course, and the nature of the specific immune responses to both adenoviral vectors and transgene products were studied using HE staining (H&E) and immunohistochemical staining.</p><p><b>RESULTS</b>The monkeys developed mild to moderate acute hepatitis 1 to 3 weeks after intravenous or intrabiliary injection of first generation replication-defective adenoviruses carrying the Escherichia coli lacZ gene. This was accompanied by adenovirus-mediated T-cell proliferation and neutralizing antibodies to the adenovirus. Increased numbers of CD3(+), CD4(+) and CD8(+) T-lymphocytes were detected in the diseased livers, while B-lymphocytes were absent. Hepatocytes demonstrated increased expression of beta 2-microglobulins (beta 2-MG) and HLA-DR antigens in the plasma membranes. The development of acute hepatitis and the accompanying immune abnormalities were delayed in immunosuppressed monkeys until after the discontinuation of immunosuppressive therapy. The monkeys infused with Ad. CMVluc showed more significant and longer durations of hepatitis than the monkeys infused with adenoviruses carrying the lacZ gene. Lipofectamine-mediated gene transfer was inefficient. There was neither lacZ expression nor significant immune response in the liver of monkeys infused with lipofectamine via the portal vein or the common bile duct.</p><p><b>CONCLUSION</b>Immune response to the hepatocytes in liver directed gene therapy is MHC class I restricted and T-cell mediated. Both adenoviral vectors and foreign genes are related to the liver damage. Mild to moderate hepatic inflammation seen with the E-1 deleted vector is reversible. Immunosuppression regimens may prolong transgene expression and delay the development of acute adenoviral hepatitis.</p>