BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase ( TERT ) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT . However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16 -specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION P16 methylation drives TERT -mediated immortalization and transformation of normal human cells that may contribute to cancer development.
Humans ; Telomerase/genetics* ; DNA Methylation/physiology* ; Fibroblasts/cytology* ; Cyclin-Dependent Kinase Inhibitor p16/metabolism* ; Cell Line ; Cell Transformation, Neoplastic/genetics*

Objective To explore the effects of TRAF6 inhibition on autophagy,myocardial inflammation and cardiac function in septic mice.Methods Twenty-four male Kunming mice were randomly divided into 4 groups:sham,sham + C25-140(sham+C),cecal ligation and puncture(CLP),and cecal ligation and puncture+C25-140(CLP+C)group.Sham+C group and CLP+C group were intraperitoneally injected with C25-140 after operation.LVEF and LVFS were evaluated by ultrasound 24 hours after operation.Serum TNF-α and IL1-β were measured by ELISA.HE staining was used to evaluate myocardial inflammatory response.Autophagosomes and mitochondrial microstructure of cardiomyocytes were observed by transmission electron microscopy.TRAF6 mRNA in myocardial tissue was detected by qPCR.The expression of TRAF6,P62,Beclin-1 and LC3B protein was detected by W-B.The effect of C25-140 on myocardial injury in the septic mice was observed by inhibiting autophagy with 3-MA.Results Compared with the sham group,the levels of TRAF6 mRNA and TRAF6 in the myocardial tissue in the CLP group were significantly increased(P<0.05)and the serum TNF-α and IL1-β concentrations were signifi-cantly increased(P<0.05).Meanwhile,the myocardial tissue HE staining showed inflammatory cell infiltration and the LVEF and LVFS levels were significantly decreased in the CLP group(P<0.05).Compared with CLP group,the CLP+C group showed that the expression of TRAF6 mRNA and TRAF6 protein decreased(P<0.05),serum TNF-α and IL1-β decreased(P<0.05),myocardial histopathological myocardial inflammatory cell infiltration decreased,the LVEF and LVFS levels increased(P<0.05).Electron microscopy showed that the mitochondrial swelling decreased,autophagosomes increased,expression of Beclin-1 and LC3Ⅱ/Ⅰ increased,and P62 expression decreased(P<0.05).As compared with CLP+C group,the CLP+C+3-MA group showed that obvious inflamma-tory cell infiltration in the myocardial pathology and the LVEF and LVFS levels decreased after 3-MA inhibited autophagy(P<0.05).Conclusion Inhibition of TRAF6 can not only ameliorate myocardial inflammatory injury and cardiac dysfunction in septic mice,but promote the involvment of cardiomyocyte autophagy in provention from sepsis-induced myocardial injury.

Objective:To investigate the clinical characteristics of infantile neuroblastoma (NB) and the factors that affect prognosis.Methods:We retrospectively analyzed the clinical data collected from 44 cases of NB who received treatment in The Fourth Affiliated Hospital of China Medical University from March 2008 to March 2017 to summarize the clinical characteristics of NB and the factors that affect prognosis.Results:Among the 44 cases, 21 were male and 23 were female, with a median age of 5.5 months (range, 0 days-12 months). Four cases had stage I NB, seven cases stage II NB, five cases stage III NB, 15 cases stage IV NB, and 13 cases stage IVs NB. The tumors were located in the adrenal glands and retroperitoneum ( n = 26, 59.1%), posterior mediastinum ( n = 15, 34.1%), pelvis ( n = 2, 4.5%), and neck ( n = 1, 2.3%). The median follow-up time was 90 months (range, 2-144 months). The 3-year and 5-year overall survival rates were 93.2% and 90.9%, respectively. Among 35 cases who survived more than 5 years, 30 cases survived healthily, 5 cases survived with tumor, and 4 cases died. Bone marrow metastasis, bone metastasis, and the extent of tumor resection greatly affect the prognosis of NB ( χ2 = 6.92, 12.19, 4.70, all P < 0.05). Conclusion:The overall prognosis of NB is good in infants. NB mainly occurs in the abdomen. The survival rate of infants with stage IVs NB is lower than that of infants with stages I, II, and III NB. The prognosis of NB occurring in the abdomen is poorer than that occurring in other regions. Bone marrow metastasis, bone metastasis, and the extent of tumor resection are adverse factors affecting the prognosis.

Objective To observe the changes of serum iron in patients with bronchopneumonia (bacterial pneumonia,mycoplasmal pneumonia) before and after treatment,and to discuss whether it can be used as an indicator of infection.Methods Forty cases with bacterial pneumonia and 41 cases with mycoplasmal pneumonia were recruited.The serum iron levels before and after treatment in bacterial pneumonia group and mycoplasmal pneumonia group were compared and analyzed.The correlation between white blood cell count,percentage of neutrophils,serum C-reactive protein (CRP) levels and the serum iron levels before treatment,and changes of the serum iron before and after treatment were analyzed.Results The level of serum iron after treatment in the bacterial pneumonia group [(16.28 ±5.81) μmol/L] was significantly higher than that before treatment [(4.83 ± 2.12) μ mol/L] (t =-11.19,P＜0.001).The level of serum iron after treatment in the mycoplasmal pneumonia group [(15.17 ±5.31) μmoL/L] was also significantly higher than that before treatment [(4.77 ± 1.99) μmol/L] (t =-11.29,P ＜0.001).The serum iron levels between the two groups before and after treatment had no statistically significant differences (t =0.135,0.898,P =0.893,0.373).There was no correlation between white blood cell count,percentage of neutrophils,CRP and serum iron levels before treatment (bacterial pneumonia group:r =-1.87,-0.219,-0.152;mycoplasmal pneumonia group:r =-0.032,-0.302,-0.274) and changes of the serum iron before and after treatment (bacterial pneumonia group:r =0.098,0.062,0.205;mycoplasmal pneumonia group:r =0.01 1,0.171,-0.105,P ＞ 0.05).Conclusion The serum iron level is significantly decreased in children with pulmonary infectious diseases and increased to normal level after anti-infection treatment.Serum iron can not be used as an indicator of infection in children.

This experiment was aimed to create A20 gene site-specific zinc finger DNA-binding protein. The sequence of A20 gene promoter was analyzed with bioinformatics means and submitted to ZF Tools Server at TSRI. Using the database of the web site, we determined the A20 gene valid target sites and designed the amino acid sequence of zinc finger protein predicted to be bound to the target site. And then, the structure of the protein sequence was analyzed and homology was modeled with various bioinformatics means. Based on the characteristic of this protein, the prokaryotic expression vector pTYB11-ZFP was constructed and expressed. Thus, the artificial zinc finger protein that recognized A20 specific sequence was designed, and expressed in Escherichia coli. The results indicate that it is feasible to design engineered artificial Zinc finger proteins by means of bioinformatics.
Amino Acid Sequence ; Base Sequence ; DNA-Binding Proteins ; chemistry ; genetics ; Humans ; Molecular Sequence Data ; Protein Binding ; Protein Engineering ; methods ; Transcription Factors ; chemistry ; genetics ; Zinc Fingers ; genetics

To investigate the biomechanical behavior of human intestines. The tensile test human intestine was performed with the electronic tension machine in this paper. The results indicate that the exponential relationship for the stress-strain of the human intestine was obtained, and the exponential coefficient a of each segment of the intestine is almost the same although the constant C is different. It also shows that the relative rate of stretch length of each segment intestines is different in longitudinal and circumferential directions. And the incremental elastic modulus of colon is less than those of small intestine. It is considered that the colon can be more easily deformed. The experimental results provide the theoretic basis for research on intestinal endoscopic microrobot.
Biomechanical Phenomena ; Colon, Transverse ; physiology ; Elasticity ; Humans ; In Vitro Techniques ; Intestine, Small ; physiology ; Stress, Mechanical ; Tensile Strength

In this study, we prepared the acellular bone matrix of the inbred-line Banna mini-pig by using tissue engineering method and evaluated its possible application in bone tissue engineering. Histological analysis, xenoantigen expression and biomechanical measurement were performed on the matrix. HE staining and scanning electron microscopy showed the cellular components were almost removed. Immunohischemical result demonstrated that the xenoantigen, alpha-gal,was also eliminated. There was no statistically significant difference between the acellular bone matrix group and control group. The acellular bone matrix can provide appropriate space structure and strength for grafts. In conclusion, our data suggest that acellular bone matrix is a new kind of ideal bone scaffold material.
Animals ; Antigens, Heterophile ; analysis ; Biomechanical Phenomena ; Bone Matrix ; immunology ; Female ; Male ; Stress, Mechanical ; Swine ; Swine, Miniature ; Tissue Engineering ; alpha-Galactosidase ; analysis

This study was aimed to examine the effectiveness of a gene transfer of human TGFbeta1 gene into endothelial cells and to determine whether TGFbeta1 increases ECM expression of endothelial cells. With the help of DOTAP, endothelial cells were transfected with pMAMneoTGFbeta1. The positive cell clones were selected with G418. The stable transfection and expression of TGFbeta1 in the endothelial cells were determined by immunofluorescence analysis. The expression levels of collagen I and fibronectin in the transfected and untransfected endothelial cells were determined by Western blot. The adhesion force between endothelial cells and matrix was determined by a micropipette technical system. The results showed abundant TGFbeta1 stable expression in the endothelial cells. TGFbeta1 gene was noted to increase collagen I and fibronectin expression and increase the adhesion between endothelial cells and matrix. These findings indicated that TGFbeta1 can be used in vascular tissue engineering for the enhancement of endothelial cells adhesion.
Cell Adhesion ; Cells, Cultured ; Collagen Type I ; biosynthesis ; genetics ; Endothelium, Vascular ; cytology ; physiology ; Extracellular Matrix ; metabolism ; physiology ; Fibronectins ; biosynthesis ; genetics ; Humans ; Tissue Engineering ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Umbilical Veins ; cytology

To explore the human smooth muscle cells seeding in blood vessel of minor pig after trypsin treatment and provide data for xenotransplantation and for using pig vessel in tissue engineering. HE and silver stain were used for checking the smooth muscle cells seeding in acellular blood vessel. The results showed that the smooth muscle cells seeding succeeded and the smooth muscle cells were in normal morphological distribution. These demonstrate that the pig aorta can be used for smooth muscle cells seeding, and hence for constructing new vascular grafts.
Animals ; Bioprosthesis ; Blood Vessel Prosthesis ; Blood Vessels ; cytology ; Cell Separation ; Cell Transplantation ; Female ; Humans ; Male ; Muscle, Smooth, Vascular ; cytology ; Swine ; Swine, Miniature ; Tissue Engineering ; methods ; Transplantation, Heterologous

This study sought to explore the change of the major histocompatibility complex (MHC) antigen expression and the endothelization of blood vessel in minor pig after trypsin treatment, and to provide data for xenotransplantation and pig vessel for use in tissue engineering. Western blot assays were conducted for detecting the expression of MHC xenoantigens. Scanning electron microscopy was used for checking the endothelization of decellularized blood vessel. The results showed that MHC antigen is not expressed after trypsin treatment. The endothelization is accomplished. The endothelial cells have normal morphological distribution. These demonstrate that the antigen of pig aorta is significantly decreased and it can be used for constructing new vascular grafts.
Animals ; Bioprosthesis ; Blood Vessel Prosthesis ; Cells, Cultured ; Endothelial Cells ; cytology ; Female ; Major Histocompatibility Complex ; physiology ; Male ; Swine ; Swine, Miniature ; Tissue Engineering ; methods ; Tissue Scaffolds