Objective: To explore the effect of three-dimensional echocardiography (RT-3DE) for evaluating right atrial function in patients after atrial septal defect (ASD) closure.
Methods: Our research included 2 group: ASD group,n=50 patients with successful ASD closure and Control group, n=30 normal subjects from physical examination. RT-3DE was conducted at pre-operation and at 3 days, 1 month, 3 months post-operation; the volume of right atrium before contraction (Vpre), minimum volume of the right atrium (Vmin), maximum volume of the right atrium (Vmax), total emptying volume of right atrium (Vt), total emptying volume fraction of right atrium (Ft), active emptying volume fraction (Fa) and passive emptying volume fraction (Fp) were measured and compared between 2 groups at pre- and post-operation respectively.
Results: At pre-operation, compared with Control group, ASD group had increased Vmax, Vmin, Vpre, Vt, and Fa, while decreased Ft and Fp, allP<0.05. At 3 days post-operation, compared with Control group, ASD group had increased Vmax, Vmin, Vpre, Vt, and Fa, while decreased Ft and Fp, allP<0.05. Compared with pre-operation, ASD group had decreased post-operative Vmax, Vmin, Vpre, Vt and Fa while increased Ft and Fp at 3 time points, allP<0.05. At 3 months post-operation, the above 7 indexes were similar between ADS group and Control group,P>0.05.
Conclusion: ADS patients had abnormal volume and function in right atrium, the abnormalities could be improved at 3 days after ASD closure and could be recovered to normal level at 3 months after operation. RT-3DE has the important role for evaluating right atrial volume and function in patients after ASD closure.

OBJECTIVETo investigate the expression of cytokeratin 17 (CK17) in oral squamous cell carcinoma (OSCC) as well as its clinical significance.

METHODSDetection of the mRNA level and protein expression of CK17 in the in vitro cellular carcinogenesis model of OSCC, some OSCC cell lines and tissue specimens from 30 primary OSCC patients were performed using real-time polymerase chain reaction (PCR), Western blot and immunohistochemistry, respectively.

RESULTSIncreased CK17 mRNA level was observed in the HB56 and OSC cell lines compared with the HIOEC using real-time PCR technique. Western blot showed increased CK17 protein expression in all the cell lines compared with the HIOEC. Increased CK17 mRNA and immunoreaction levels were also observed in the cancerous tissue specimens from OSCC patients compared with normal adjacent tissues (P<0.01).

CONCLUSIONThe significantly increased CK17 gene may be associated with the tumorigenesis and development of OSCC.

Adult ; Blotting, Western ; Carcinoma, Squamous Cell ; Cell Line ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Keratin-17 ; Male ; Middle Aged ; Mouth Neoplasms ; RNA, Messenger

Objective: To explore the effects of decline of pH value on cardiomyocyte viability of rats, and to analyze the possible mechanism. Methods: Hearts of five newborn Sprague-Dawley rats were isolated, and then primary cardiomyocytes were cultured and used in the following experiments. (1) The primary cardiomyocytes were divided into pH 7.4+ 6 h, pH 7.0+ 6 h, pH 6.5+ 6 h, pH 6.0+ 6 h, pH 6.5+ 1 h, and pH 6.5+ 3 h groups according to the random number table, with 4 wells in each group. After being routinely cultured for 48 h (similarly hereinafter), cells in pH 7.4+ 6 h, pH 7.0+ 6 h, pH 6.5+ 6 h, and pH 6.0+ 6 h groups were cultured with pH 7.4, pH 7.0, pH 6.5, and pH 6.0 DMEM-F12 medium (similarly hereinafter), respectively, and then they were cultured for 6 h. Cells in pH 6.5+ 1 h and pH 6.5+ 3 h groups were cultured with pH 6.5 medium, and then they were cultured for 1 h and 3 h, respectively. Viability of cells was detected by methyl-thiazolyl-tetrazolium (MTT) method. (2) The primary cardiomyocytes were divided into pH 7.4, pH 6.5, and pH 6.5+ taxol groups according to the random number table, with 2 wells in each group. Cells in pH 7.4 group were cultured with pH 7.4 medium, while cells in pH 6.5 and pH 6.5+ taxol groups were cultured with pH 6.5 medium. Cells in pH 6.5+ taxol group were added with taxol of a final molarity of 0.2 μmol/L in addition, and then they were cultured for 6 h. Morphology and density of microtubule of cells was detected by immunofluorescence assay. (3) The primary cardiomyocytes were grouped and treated as in experiment (2), with 2 wells in each group. The expressions of polymerized microtubulin and free microtubulin were determined with Western blotting. (4) The primary cardiomyocytes were grouped and treated as in experiment (2), with 4 wells in each group. Viability of cells after treated with taxol was detected by MTT method. Data were processed with one-way analysis of variance and LSD-t test. Results: (1) The viability of cells in pH 7.4+ 6 h, pH 7.0+ 6 h, pH 6.5+ 6 h, pH 6.0+ 6 h, pH 6.5+ 1 h, and pH 6.5+ 3 h groups were 1.00±0.08, 0.90±0.08, 0.85±0.06, 0.83±0.04, 0.91±0.10, and 0.89±0.10, respectively. Compared with that in pH 7.4+ 6 h group, viability of cells in pH 7.0+ 6 h, pH 6.5+ 6 h, pH 6.0+ 6 h, pH 6.5+ 1 h, and pH 6.5+ 3 h groups were all decreased in different degrees (t=2.476, 4.002, 4.996, 2.168, 2.400, P<0.05). (2) Microtubules of cells in pH 7.4 group were radially distributed around the nucleus with clear tubular structure. Compared with that in pH 7.4 group, the skeleton of microtubules of cells in pH 6.5 group was obviously damaged, with broken structure of microtubule and reduced density. Compared with that in pH 6.5 group, the damage degree of microtubules of cells in pH 6.5+ taxol group was obviously alleviated, and the structure of microtubules basically returned to normal. (3) Compared with that in pH 7.4 group, the expression of free microtubulin of cells in pH 6.5 group was significantly increased (t=3.030, P<0.05), while the expression of polymerized microtubulin of cells was significantly decreased (t=8.604, P<0.05). Compared with that in pH 6.5 group, the expression of free microtubulin of cells in pH 6.5+ taxol group was significantly decreased (t=4.559, P<0.05), while the expression of polymerized microtubulin of cells was significantly increased (t=5.472, P<0.05). (4) Viability of cells in pH 7.4, pH 6.5, and pH 6.5+ taxol groups were 1.00±0.10, 0.83±0.04, and 0.93±0.10, respectively. Compared with that in pH 7.4 group, the viability of cells in pH 6.5 group was obviously declined (t=4.412, P<0.05). Compared with that in pH 6.5 group, the viability of cells in pH 6.5+ taxol group was obviously increased (t=2.461, P<0.05). Conclusions The decline of pH value reduces the viability of cardiomyocytes of rats through destroying the skeleton of microtubule. Stabilizing microtubule skeleton can significantly reduce acidic treatment-induced damage and ameliorate cardiomyocyte viability.

Objective: To investigate the effect of human antigen R on lysosomal acidification during autophagy in mouse cardiomyocytes cultured in vitro. Methods: The hearts of 20 C57BL/6 mice aged 1-2 days no matter male or female were isolated to culture primary cardiomyocytes which were used in the following experiments. (1) The cells were divided into 5 groups according to the random number table (the same grouping method below), i. e., normal control group and sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups. The cells in normal control group were routinely cultured for 54.0 h with Dulbecco′s modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), and the cells in sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups were firstly regularly cultured for 53.5, 53.0, 51.0, 48.0 h and then cultured with replaced sugar-free serum-free medium for 0.5, 1.0, 3.0, and 6.0 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 Ⅱ (LC3Ⅱ), autophagy-related protein 5, and adenosine triphosphatase V1 region E1 subunit (ATP6V1E1) were detected by Western blotting. (2) The cells were divided into normal control group and sugar-free serum-free 3.0 h group. The cells in corresponding groups were treated the same as those in experiment (1), and the cell lysosomal acidification level was observed and detected under a laser scanning confocal microscope. (3) Two batches of cells were grouped and treated the same as those in experiment (1). The protein expression of human antigen R in the whole protein of cells of one batch and its protein expression in the cytoplasm and nucleus protein of cells of the other batch were detected by Western blotting. (4) The cells were divided into normal control group, simple control small interfering RNA (siRNA) group, simple human antigen R-siRNA1 (HuR-siRNA1) group, simple HuR-siRNA2 group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group. After 48 hours of regular culture, the cells in simple control siRNA group and sugar-free serum-free+ control siRNA group were transfected with negative control siRNA for 6 h, the cells in simple HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA1 group were transfected with HuR-siRNA1 for 6 h, and the cells in simple HuR-siRNA2 group and sugar-free serum-free+ HuR-siRNA2 group were transfected with HuR-siRNA2 for 6 h. Hereafter, the cells in these 8 groups were continuously cultured for 48 h with regular conditon, and then the cells in normal control group and each simple siRNA-treated group were replaced with DMEM/F12 medium, the cells in the other groups were replaced with sugar-free serum-free medium, and they were cultured for 3 h. The protein expression of human antigen R in the whole protein of cells was detected by Western blotting. (5) Two batches of cells were divided into sugar-free serum-free+ control siRNA group and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The distribution and expression of human antigen R in the cells of one batch were observed and detected by immunofluorescence method, and the lysosomal acidification level in the cells of the other batch was observed and detected under a laser scanning confocal microscope. (6) Three batches of cells were divided into sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group, and the cells in corresponding groups were treated the same as those in experiment (4). The protein expressions of cathepsin D in the whole protein of cells of one batch, human antigen R in the cytoplasm protein of cells of one batch, and ATP6V1E1 in the whole protein of cells of the other batch were detected by Western blotting. (7) The cells were divided into normal control group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The mRNA expression of ATP6V1E1 in cells was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The sample number of each experiment was 3. Data were processed with independent data t test, one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: (1) Compared with those of normal control group, the protein expressions of LC3Ⅱ and ATP6V1E1 in the whole protein of cells of sugar-free serum-free 1.0, 3.0, and 6.0 h groups were significantly increased (t=12.16, 4.05, 4.82, 11.64, 3.29, 8.37, P<0.05 or P<0.01). Compared with that of normal control group, the protein expression of autophagy-related protein 5 in the whole protein of cells of sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups was significantly increased (t=6.88, 10.56, 5.76, 9.91, P<0.05 or P<0.01). (2) Compared with 1.03±0.08 of normal control group, the lysosomal acidification level in the cells of sugar-free serum-free 3.0 group (2.92±0.30) was significantly increased (t=6.01, P<0.01). (3) There was no statistically significant difference in the overall comparison of protein expression of human antigen R in the whole protein of cells among the 5 groups (F=1.09, P>0.05). Compared with that of normal control group, the protein expression of human antigen R in the cytoplasm protein of cells was significantly increased in sugar-free serum-free 1.0, 3.0, and 6.0 h groups (t=43.05, 11.07, 5.39, P<0.05 or P<0.01), while the protein expression of human antigen R in the nucleus protein of cells was significantly decreased in sugar-free serum-free 3.0 and 6.0 h groups (t=11.18, 12.71, P<0.01). (4) Compared with that of simple control siRNA group, the protein expression of human antigen R in the whole protein of cells of simple HuR-siRNA1 group and simple HuR-siRNA2 group was significantly decreased (t=4.82, 4.44, P<0.05). Compared with that of sugar-free serum-free+ control siRNA group, the protein expression of human antigen R in the whole protein of cells of sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group was significantly decreased (t=4.39, 6.27, P<0.05). (5) Compared with those of sugar-free serum-free+ control siRNA group, the distribution of human antigen R in the cytoplasm of cells and its expression level were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group (t=10.13, P<0.01). Compared with 1.00±0.06 of sugar-free serum-free+ control siRNA group, the lysosomal acidification level (0.73±0.06) in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased (t=3.28, P<0.01). (6) Compared with those of sugar-free serum-free+ control siRNA group, the protein expressions of cathepsin D in the whole protein of cells, human antigen R in the cytoplasm protein of cells, and ATP6V1E1 in the whole protein of cells were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group (t=4.16, 3.99, 4.81, 5.07, 11.68, 12.97, P<0.05 or P<0.01). (7) Compared with that of normal control group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free 3.0 h group was significantly increased (t=5.51, P<0.05). Compared with that of sugar-free serum-free+ control siRNA group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased (t=5.97, P<0.05). Conclusions After sugar-free serum-free treatment in vitro, the autophagy in mouse primary cardiomyocytes is activated, the lysosomal acidification is enhanced, and the expression of human antigen R in cytoplasm is increased. Human antigen R function is activated and involved in maintaining lysosomal acidification during autophagy in mouse cardiomyocytes.

Objective To investigate influence of nicotinic acid adenine dinucleotide phosphate (NAADP) on autophagy in hypoxic cardiomyocytes of rats and its mechanism.Methods Five neonatal Sprague-Dawley rats were collected and sacrificed to harvest the hearts,and primary cardiomyocytes were separated for the following experiments.(1) Primary cardiomyocytes were collected and divided into normoxia group,hypoxia 9 h group,and hypoxia 9 h + NAADP group according to random number table,with 5 wells in each group.Cells in normoxia group were cultured routinely in the constant temperature incubator at 37 ℃ for 9 hours.Cells in hypoxia 9 h group and hypoxia 9 h + NAADP group were cultured in hypoxic incubator with volume fraction 94％ nitrogen,5％ carbon dioxide,and 1％ oxygen for 9 hours.Before hypoxia,cells in hypoxia 9 h + NAADP group were dealt with final amount-of-substance concentration 10 μmol/L NAADP.Cell counting kit 8 was used to measure cell viability.(2) Primary cardiomyocytes were collected and divided into normoxia group,hypoxia 9 h group,hypoxia 9 h + NAADP group,hypoxia 9 h + tran-Ned-19 group,and hypoxia 9 h + trans-Ned-19 + NAADP group according to the random number table,with 2 wells in each group.Cells in normoxia group were cultured routinely in the constant temperature incubator at 37 ℃ for 9 hours.And cells in the other 4 groups were cultured in hypoxic incubator as that in experiment (1) Before hypoxia,cells in hypoxia 9 h + NAADP group were dealt with amount-of-substance concentration 10 μmol/L NAADP,cells in hypoxia 9 h + tran-Ned-19 group were dealt with amount-of-substance concentration 1 μmol/L trans-Ned-19,and cells in hypoxia 9 h + trans-Ned-19 + NAADP group were dealt with amount-of-substance concentration 10 μmol/L NAADP and 1 μmol/L trans-Ned-19.Protein expressions of microtubule associated protein 1 light chain 3-Ⅱ and P62 were detected by Western blotting.(3) Primary cardiomyocytes were collected and grouped as those in experiment (1).The lysosomal acidity was determined by immunofluorescence method.Data were processed with one-way analysis of variance and least-significant difference test.Results (1) The cell viability in normoxia group was 1.114 ± 0.024,which was significantly higher than 0.685 ± 0.079 of cells in hypoxia 9 h group (P ＜ 0.01).The cell viability of hypoxia 9 h + NAADP group was 0.886 ± 0.061,which was obviously higher than that of cells in hypoxia 9 h group (P ＜0.05).(2) Expressions of microtubule-associated protein 1 light chain 3-Ⅱ and P62 of cells in hypoxia 9 h group were significantly higher than those of cells in normoxia group (P ＜ 0.0l).Compared with those in hypoxia 9 h group,expression of P62 in hypoxia 9 h + NAADP group was significantly decreased (P ＜ 0.01),while expression of microtubule-associated protein 1 light chain 3-Ⅱ did not change significantly (P ＞ 0.05).There were no significantly statistical difference in expressions of microtubule-associated protein 1 light chain 3-Ⅱ and P62 between hypoxia 9 h group and hypoxia 9 h + trans-Ned-19 group (P ＞ 0.05).Compared with those of cells in hypoxia 9 h + NAADP group,expression of P62 of cells in hypoxia 9 h + trans-Ned-19 + NAADP group was obviously increased (P ＜ 0.01),while expression of microtubule-associated protein 1 light chain 3-Ⅱ did not change significantly (P ＞ 0.05).(3) The intensity of green fluorescence of cells in normoxia group was strong and co-localized well with red fluorescence,and internal environment of lysosome was with stronger acidity.The intensity of green fluorescence in cells of hypoxia 9 h group was significantly lower than that of cells in normoxia group,and acidity of internal environment of lysosome was weakened.The intensity of green fluorescence and acidity of internal environment of lysosome in hypoxia 9 h + NAADP were significantly stronger than those of cells in hypoxia 9 h group,but significantly lower than those of cells in normoxia group.Conclusions NAADP can improve myocardial cell viability through acidifying internal environment of lysosome of cardiomyocyte after hypoxia,promoting degradation of autophagosomes,reducing autophagic lysosomal accumulation,and repairing damaged autophagie flow.