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Objective:To study the effects of fluoride exposure on skeletal development of zebrafish larvae and its possible molecular mechanisms.Methods:Six hours post fertilization(6 hpf) wild-type zebrafish embryos were selected and exposed to sodium fluoride [NaF, control group (0 mg/L NaF), low fluoride group (25 mg/L NaF) and high fluoride group (100 mg/L NaF)] for 9 days. Fluorine ion selective electrode was used to detect the overall fluorine content of zebrafish larvaes, and the death and development of zebrafish larvaes were observed and counted. Bone mineralization and chondrogenesis of the zebrafish larvaes were analyzed by alizarin red staining and alcin blue staining, respectively. The expression levels of sry-related-high-mobilty-group box 9a (Sox9a), osteprotegerin (OPG) and receptor-activator of nuclear factor kappa beta ligant (RANKL) were analyzed by real-time quantitative PCR.Results:Compared with control group [(0.12 ± 0.01) μg/140 larvaes], the overall fluorine contents of zebrafish larvaes in low fluoride group [(0.28 ± 0.03) μg/140 larvaes] and high fluoride group [(0.64 ± 0.10) μg/140 larvaes] were significantly higher, and the differences were statistically significant ( P < 0.05). Compared with control group, zebrafish larvaes in high fluoride group had shorter body length, higher swim bladder loss rate and higher spinal curvature rate ( P < 0.05). The alizarin red staining area, integrated optical density (IOD) and the number of mineralized vertebrae were higher in low fluoride group, while the alcin blue staining area of cartilage formation was lower ( P < 0.05). In the high fluoride group, alizarin red staining area, IOD and the number of mineralized vertebrae were lower, while the alcin blue staining area of cartilage formation was higher ( P < 0.05). Compared with control group, the expression levels of OPG mRNA and OPG/RANKL mRNA in low fluoride group were higher ( P < 0.05); the expression level of RANKL mRNA was higher in high fluoride group, while the expression level of OPG/RANKL mRNA was lower ( P < 0.05). Conclusion:A short period of fluoride exposure from zebrafish embryo to zebrafish larvae can cause abnormal bone development of zebrafish larvae, which may be related to endochondral osteogenesis and OPG/RANKL pathway.
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OBJECTIVE@#: To observe the expression of gene in the early development stage of wild zebrafish embryos.@*METHODS@#: The collinearity of gene and the sequence similarity of G6pd protein were analyzed with gene database and BLAST software, respectively. Expression of gene in different development stages of zebrafish embryos was detected by hybridization. The -EGFP-pCS recombinant plasmids were microinjected into zebrafish embryos, and fluorescence was observed under a fluorescence microscope. The expression of G6pd protein at 24, 48 and 72 hour post fertilization (hpf) zebrafish embryos was detected by Western blotting; the enzyme activity of G6pd at 24, 48 and 72 hpf zebrafish embryos was detected by modified G6pd quantitative ratio method.@*RESULTS@#: The G6pd protein similarity of zebrafish and human was 88%, and that of zebrafish and mouse was 87%. The results of hybridization showed that the gene was mainly expressed in the hematopoietic tissues of zebrafish; the results observed after microinjection of -EGFP-pCS recombinant plasmid were consistent with the results of hybridization. At 24, 48 and 72 hpf, the relative expression levels of G6pd protein in zebrafish embryos were 1.44±0.03, 1.47±0.05, and 1.54±0.02, respectively(>0.05); the G6pd enzyme activity levels were 1.74±0.17, 1.75±0.12, 1.71±0.22, respectively (>0.05).@*CONCLUSIONS@#: The study has observed the expression of gene and G6pd protein, and G6pd enzyme activity in zebrafish embryos at different development phases, which provides a reference for the establishment of a zebrafish G6PD deficiency model.
Subject(s)
Animals , Humans , Mice , Embryo, Nonmammalian , Gene Expression Regulation, Developmental , Glucosephosphate Dehydrogenase , Genetics , In Situ Hybridization , Plasmids , Genetics , Zebrafish , Embryology , GeneticsABSTRACT
Objective To investigate the effects of Danusertib on the changes in Aurora kinase B (Aurora B)/ribosomal protein p70S6 kinase (p70S6K)/ribosomal protein 15 (RPL15) signaling pathway and autophagy in human leukemia cells and its mechanism.Methods Myeloid leukemia cell lines THP-1 and K562 were selected as the research subjects.The experiment was divided into 2 phases.Phase 1:each cell line was treated with the concentration of Danusertib in 0.1 p mol/L,1.0 p mol/L and 5.0 μ mol/L.In control group,2 mL/L dimethyl sulfoxide (DMSO)was given.All the treated cells were cultured for 24 hours.The viability of each cell line was examined by methyhhiazoletrazolium assay and the autophagy was assessed by flow cytometry.In addition,the protein levels of p70S6K,AuroraB,phosphatidylinositol 3-kinase (PI3K),AKT(phosphorylated protein kinase B),mammalian target of rapamycin (mTOR),microtubule-associated protein (LC3),Beclin1 and RPL15 were determined by using Western blot.Part 2:Aurora B and RPL15 were down-regulated in THP-1 and K562 cells,respectively.DMSO was used to dissolve Danusertib(5.0 μ mol/L).The grouping was designed as following:DMSO group (blank control group),Danusertib treated group,empty plasmid group,small interfering RNA(siRNA) group,empty plasmid + Danusertib-treated group and siRNA + Danusertib treated group.The protein levels of Aurora B,p70S6K,RPL15,Beclinl and LC3 were detected by using Western blot.Results (1) Danusertib decreased the viability of THP-1 and K562 cells and the half maximal inhibitory concentration values were 26.9 pmol/L and 30.2 μmol/L for THP-1 and K562 cells,respectively.(2)The protein levels of p-Aurora B/Aurora B,p-p70S6K/p70S6K,RPL15,p-mTOR/mTOR and p-AKT/AKT decreased compared with control cells after being treated with 0.1 μmol/L,1.0 μ mol/L and 5.0 pmol/L of Danusertib in THP-1 and K562 cells,and the differences were statistically significant (all P ＜ 0.05).(3) In THP-1 cells,compared with the empty plasmid group,the protein levels of p70S6K and RPL15 decreased by 22.1％,61.3％ (F =18.1,P =0.001) and 55.4％,56.1％ (F =19.4,P =0.001) in siRNA group and siRNA + Danusertib-treated group after knockdown of Aurora B.In contrast,the protein levels of LC3 increased by 13.6％ and 17.1％ (F =15.4,P =0.001)compared with the empty plasmid group.In addition,the protein levels of Beclin1 and LC3 increased by 39.5％,92.3％ (F=25.2,P=0.001) and 40.2％,58.3％ (F=23.9,P=0.001) in siRNA group and siRNA + Danusertib treated group,compared with the empty plasmid group after down-regulation of RPL15.In K562 cells,compared with the empty plasmid group,the protein levels of p70S6K and RPL15 decreased by 24.2％,62.7％ (F =20.4,P=0.001) and 57.2％,60.1％ (F =23.9,P =0.001) in siRNA group and siRNA + Danusertib treated group after downregulation of Aurora B.But the protein levels of LC3 increased by 17.9％ and 56.7％ (F =20.9,P =0.001)compared with the empty plasmid group.Moreover,the protein levels of Beclin1 and LC3 were increased by 20.6％,98.4％ (F=22.4,P =0.001) and 41.5％,70.1％ (F=26.2,P =0.001) in siRNA group and siRNA + Danusertib treated group,compared with the empty plasmid group after downregulation of RPL15.Conclusion Danusertib can induce autophagy via inhibition of the PI3K/AKT/mTOR signaling pathway and can negatively regulate Aurora B/p70S6K/RPL15 axis in THP-1 and K562 cells.In addition,RPL15 may be a key target of Aurora B/p70S6K/RPL15signaling pathway in the inhibition of tumor proliferation.
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Objective To investigate the effects of gefitinib on early embryonic development and expression of abcb4 tumor resistance genes in zebrafish.Methods Zebrafish were adopted as experimental animals and divided into gefitinib group,mixture of doxorubicin and gefitinib group and blank group.Zebrafish embryos of 0.5-1.5 hours after fertilization(0.5-1.5 hpf) were exposed to different concentrations of gefitinib,and then embryo development of zebrafish in 24-120 hpf was observed and the number of death,hatch and malformation was recorded.The embryo mortality was calculated under different concentrations of gefitinib at different time points,and the numerical value of IC50 was calculated;Hatching rates of zebrafish embryo in 48 hpf and 72 hpf and malformation rates of zebrafish embryo in 120 hpf were calculated.The zebrafish embryos exposed to different concentrations of gefitinib in different groups were collected,and the expressionof abcb4 gene in zebrafish embryos was detected by real-time quantitative PCR.Results Gefitinib IC50 in zebrafish embryos was 16.18 μmol/L.Compared with the control group,higher dosage (20 μmol/L) of gefitinib in other two groups significantly decreased hatching rates of embryos,and had obvious embryonic lethal effects and teratogenic effects.Moreover,the mRNA levels of the abcb4 gene in the zebrafish embryos of gefitinib group were not significantly changed,whereas the mRNA levels of the abcb4 gene in mixture of doxorubicin and gefitinib group were significantly different(P＜0.05).Conclusion Gefitinib has no significant effects on the expression of abcb4 tumor resistance gene in early development of zebrafish embryos(P＞0.05),but it can reverse the drug resistant effects of doxorubicin,suggesting that zebrafish can construct tumor resistance model.
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Objective To explore the effects of vinblastine on abcb4 tumor resistance gene expression in early development zebrafish and lay an important foundation for multi-drug resistant antineoplastic screening in zebrafish model.Methods Zebrafish embryos of 0.5~1.5 hours post-fertilization were exposured to different concentrations of vinblastine, and then calculated the IC50 of vinblastine.Zebrafish embryos of 0.5~1.5 hours post-fertilization were treated with different concentrations of vinblastine and embryo culture medium respectively, and then observed zebrafish embryo development in 24~120 hours post-fertilization and recorded the number of death, hatch and malformation.Evaluating the impact of vinblastine on abcb4 gene expression in zebrafish with quantitative real-time PCR and whole-mount in situ hybridization by collecting zebrafish embryos exposed to different concentrationsof vinblastine.Results Vinblastine IC50 in zebrafish embryos was 3.08 μmol/L.The mRNA level of abcb4 gene in vinblastine treated embryos was significantly increased compared to blank control group.Moreover, abcb4 gene positive hybridization signals were found in the small intestine of zebrafish embryos after 120 hours post-fertilization and also found in the brain and heart of zebrafish embryos by the method of whole-mount in situ hybridization.Conclusions Vinblastine can significantly increase the expression level of abcb4 tumor resistancegene in early development zebrafish embryos, which indicates that zebrafish can be used as a tumor resistant drug screening model.
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Objective To choose zebrafish as the experimental animal model for studying the spatiotemporal expression rule of rap1 gen in zebrafish embryo early development process .Methods The Rap1 gene fragment was cloned from the zebrafish emby‐oscDNA ,then the Rap1 gene fragment and pCS2+ plasmid were performed the in vitro connetion and recombination was extracted , the combinant plasmid was correct after the double enzyme digestion ,colony PCR and sequencing identification .T3 RNA polymer‐ase in vitro transcription system was used to obtain the digoxin (DIG)‐labeled anti‐sense mRNA probe of Rap1 gene .The whole mount in situ hybridization method was adopted to detect the Rap1 expression in zebrafish embryo early development process . Results The positive hybridization signal of Rap1 gene was detected at the cell division junction region of 0 .75 hpf ,animal pole of 3 .70 hpf and 6 .00 hpf ,and notochord of 12 .00 -72 .00 hpf .Conclusion Rap1 gene might be involved in the early development process of notochord nervous system in zebrafish .
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Objective Type A lamins are encoded by LMNA and a major component of the nuclear lamina, which have been suggested to play important roles in chromatin organization, transcription, DNA replication, and cell apoptosis. The aim of this study was to analyze the bioinformation of zebrafish lamins. Methods A phylogeny analysis was figured out with protein sequences of different species by Clustal X and MEGA 4?0 software. Then we compared the lamin protein sequences of different species with that of zebrafish by BLAST tool from NCBI. A figure of synteny analysis results was done with lamin sequence information of humans, murine and zebrafish cited from UCSC, Vega and Ensemble. Results The a?nalysis results showed that lmna, lmnb1, and lmnb2 genes of zebrafish are highly conservative and they may be homology of human LMNA, LMNB1 and LMNB2 genes. Conclusions Zebrafish lamins and human lamins have homologous sequence similarity, indicating that these two genes are orthologous genes.
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Objective Lamins are the major components of nuclear lamina underneath the inner nuclear membrane (INM).Lamins express in most cells and are involved in the whole process of growth, also play a major role in cell stability and embryonic development.Mutant in human LMNA gene may lead to a series of disorders, which are similar to progeria or other aging-associate syndrome.In this study, we report a new lmna knockdown animal model generated in our laboratory in order to provide a useful tool for studying laminopathies.Methods Two plasmids tagged to zebrafish lmna gene were designed based on morpholino oligonucleotides technology.Co-microinjected the plasmids into zebrafish embryos to knockdown lmna gene.Imagining and western blot detection were used to identify the mutants.Results Two different proteins, Lamin A/C, were expressed in the zebrafish embryos.Two plasmids lmna-MO and lmna-EGFP-pCS 2 + were generated and co-microinjected into embryos.The results of imagining and western blot showed that the expression of lmna gene was downregulated in the zebrafish embryos.Conclusions Lamin A/C are expressed in zebrafish.lmna gene can be knocked down by the injection of lmna-MO and lmna-EGFP-pCS 2 +.This new animal model may be a powerful tool for study on laminopathies.
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To investigate the effect ofgene down-regulation on early hematopoietic development of zebrafish.Phosphorodiamidate morpholino oligomer (PMO) technology was used to downregulategene expression in Zebrafish. Zebrafish embryos injected phosphorodiamidate morpholino antisense oligonucleotide ofgene mRNA by microinjection at unicellular stage were taken as the experimental group, and those injected meaningless phosphorodiamidate morpholino antisense oligonucleotide were taken as the control. The embryos were collected at 18, 24, 30 and 36 hpf after the fertilization. The real-time fluorescent quantitative PCR (RT-PCR) and whole embryohybridization methods were used to detect the expression of myeloid hematopoietic transcription factorand erythroid hematopoietic transcription factorin zebrafish.RT-PCR showed that the expressions ofanddecreased in the experimental group compared with the control group (all<0.05). Whole embryohybridization showed that the blue-black positive hybridization signals ofandin experimental group were shallow than those in the control group.Myeloid hematopoietic and erythroid hematopoietic of zebrafish are blocked with the downregulation ofgene.
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Animals , Down-Regulation , Genetics , Embryo, Nonmammalian , GATA1 Transcription Factor , Genetics , Metabolism , Gene Knockdown Techniques , Hematopoiesis , In Situ Hybridization , Lamin Type A , Genetics , Physiology , Proto-Oncogene Proteins , Genetics , Metabolism , Trans-Activators , Genetics , Metabolism , Zebrafish , Embryology , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To assess the impact of work-related musculoskeletal disorders (WRMDs) on work ability among workers.</p><p><b>METHODS</b>A total of 1686 workers in various occupations, such as administration and education, were enrolled as subjects using the random cluster sampling method. The WRMDs and work ability of all subjects were evaluated using standardized Nordic questionnaires for the analysis of musculoskeletal symptoms and the Work Ability Index (WAI) scale, respectively. Comparison of work ability and its classification between the disease group and the non-disease group was performed by paired t test, RxC table χ2 test, and the Wilcoxon rank-sum test. The relationship between work duration and work ability was analyzed by the Spearman correlation test and a multi-level model.</p><p><b>RESULTS</b>(1). The work ability of workers in the disease group was significantly lower than that in the non-disease group (P<0.0 1). (2) There were significant differences in work ability between workers with different work durations (<10 years, 10-20 years, and ≥20 years) (F=22.124, P< 0.01). With the increase in work duration, the work ability of workers declined in both groups, and the work ability of workers in the disease group (Spearman coefficient rs=-0. 172, P<0.01) had a more significant decline than that in the non-disease group (Spearman coefficient rs=-0.104, P<0.01). WRMDs were important risk factors for the decrease in work ability among workers. (3) There were significant differences in constituent ratios and levels of work ability classification between the disease group and the non-disease group (χ2=121.097, P<0.01; Z=-10.699, P<0.01). The proportions of workers with poor and medium work ability in the disease group were significantly higher than those in the non-disease group, while the proportion of works with excellent work ability in the disease group was significantly lower than that in the non-disease group. The similar characteristics in constituent ratios and levels of work ability classification could be found between the disease group and the non- disease group in various occupations (P<0.01).</p><p><b>CONCLUSION</b>WRMDs have a harmful effect on the work ability of workers, and the work ability of workers substantially declines with the increase in exposure time (work duration).</p>
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Humans , Musculoskeletal Diseases , Occupational Health , Occupations , Risk Factors , Surveys and Questionnaires , Work PerformanceABSTRACT
<p><b>OBJECTIVE</b>To characterize the distribution of work-related musculoskeletal disorders (WRMD) among the occupational population.</p><p><b>METHODS</b>A total of 1686 people of various occupations were recruited with random cluster sampling. Standardized Nordic questionnaires for the analysis of musculoskeletal systems were used to evaluate WRMD at the neck, shoulder, or lower back in the past one year. The annual prevalence of WRMD was determined. Difference analysis was performed with t-test, ANOVA, or chi-square test. The relationship between personal characteristics and WRMD was analyzed by unconditional logistic regression.</p><p><b>RESULTS</b>(1) WRMD were most frequently observed at the neck, followed by the lower back, and was least observed at the shoulder (P < 0.05). The prevalence of WRMD among mental workers was significantly higher than those among physical workers and mental-physical workers (P < 0.01). The prevalence of WRMD among female workers was significantly higher than that among male workers (P < 0.05). (2) In general, the prevalence of WRMD significantly rose with the increases in age (<30, 30∼, 40∼, and ≥ 50 years) or working years (<10, 10∼, and ≥ 20 years) (P < 0.05). (3) In the face of sickness or injury, physical workers and mental workers showed a relatively high absence rate but a relatively low medical visiting rate (13.05%). (4) Unconditional logistic regression analysis showed that mental work, gender, and working year were the main influential factors for WRMD among workers.</p><p><b>CONCLUSION</b>Workers of different types of occupation, genders, ages, and working years have different risks of WRMD at the neck, shoulder, and lower back.</p>
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Adult , Female , Humans , Male , Middle Aged , Logistic Models , Musculoskeletal Diseases , Epidemiology , Occupational Diseases , EpidemiologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the polymorphism of mitochondrial DNA region V in Bouyei people and Miao people living in Guizhou province of China.</p><p><b>METHODS</b>Polymerase chain reaction and direct sequencing were used in the study.</p><p><b>RESULTS</b>Only two kinds of polymorphism were found in Bouyei and Miao people. One was standard pattern, the other short pattern. And the frequencies of short pattern(9 bp deletion) in Bouyei and Miao people were 31.1% and 33.3% respectively.</p><p><b>CONCLUSION</b>The polymorphisms of mitochondrial DNA region V in Bouyei people and Miao people living in Guizhou province of China are similar, but they are different from those in other people.</p>