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BACKGROUND:In consideration of the food safety and ecological safety of transgenic plants,the retention of marker genes is the primary safety issue affecting transgenic plants. OBJECTIVE:Based on the principle of immune caries prevention,our research team successfully constructed the plant anti-caries vaccine fusion gene expression vector pCAMBIA-E8-APB-DOCK8 for these two caries causing virulence factors surface protein and glucosyltransferase,which provides a basis for the research and development of transgenic plant vaccine. METHODS:In this study,the selective marker genes Km and GUS in the plant caries vaccine fusion gene expression vector pCAMBIA-E8-APB-DOCK8 were removed by DNA recombination technology through a series of steps such as DNA fragment separation,connection,transformation,clone detection,and sequencing. RESULTS AND CONCLUSION:The efficiency of marker gene removal was 99%.This study has laid a good experimental foundation for the safe production of transgenic plant vaccine against dental caries,and also provided ideas for the construction of other plant vaccine vectors.
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BACKGROUND:Fluorosis is a disorder of enamel development caused by long-term intake of large amounts of fluoride during enamel development. OBJECTIVE:To further explore the molecular mechanism of dental fluorosis formation by screening the differentially expressed genes associated with calcium homeostasis in ameloblasts by transcriptome sequencing technology. METHODS:LS8 cells were treated with 0,0.4,0.8,1.6,3.2 and 6.4 mmol/L sodium fluoride(NaF)for 24,48 and 72 hours to observe the effects of different concentrations of NaF on the morphology,cell activity and intracellular Ca2+ concentration of LS8 cells.The differentially expressed genes were screened by transcriptome sequencing and validated. RESULTS AND CONCLUSION:After 24 hours of treatment,the cells treated with 0,0.4,and 0.8 mmol/L NaF were in good growth condition,with increased cell number and clear cell outline.When the NaF concentration was≥1.6 mmol/L,the cells were gradually shrunken and became smaller and the number of cells decreased with the increase of NaF concentration.After 48 and 72 hours of treatment,the number of cells increased in the 0,0.4 mmol/L NaF groups,while gradually decreased in the 0.8,1.6,3.2 mmol/L NaF groups,with rounded and smaller cell morphology.The cells in the 6.4 mmol/L NaF group were shrunken,rounded and suspended in the medium,with almost no adherent cells.When treated with the same concentration of NaF,LS8 cells were in optimal growth after 24 hours of treatment.Results from cell counting kit-8 assay showed that when treated with the same concentration of NaF,the cell activity decreased with the increase of treatment time;when the treatment time was the same,the cell activity decreased with the increase of NaF concentration.After 24 hours of treatment,the intracellular Ca2+ concentration increased with the increase of NaF concentration.Transcriptome sequencing analysis identified genes involved in the regulation of cellular calcium homeostasis:Hsp90b1,Canx,Calr,and Hspa5 that were significantly upregulated(P＜0.05)and Cacna1a that was significantly downregulated(P＜0.05).To conclude,the inhibitory effect of NaF on LS8 cell proliferation may be related to the abnormal increase in intracellular Ca2+ concentration,and the mechanism may be caused by the upregulation of the expression of protein processing and synthesis pathways Hsp90b1,Canx,Calr,and Hspa5 and the downregulation of the expression of calcium signaling pathway Cacna1a.
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Dental fluorosis is a manifestation of chronic oral fluorosis caused by excessive fluoride intake in childhood. At present, the molecular mechanism of dental fluorosis is still unclear. Ameloblasts are the most sensitive cells to fluoride in tooth tissue. Fluoride affects the proliferation and secretion of ameloblasts through the role of key molecules in the molecular signal pathways, leading to the formation of dental fluorosis. This paper reviews the relationship between the molecular signal pathways [mitogen-activated protein kinase (MAPK), transforming growth factor-β (TGF-β)/Smad, Wnt, Foxo1/Runx2], stress pathways (endoplasmic reticulum stress and oxidative stress) and the occurrence of dental fluorosis in recent years, in order to deeply understand the pathogenesis of dental fluorosis at the molecular level, and provide new ideas for the prevention and treatment of dental fluorosis.
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Endemic fluorosis is a common biogeochemical disease. Although the etiology is clear, its molecular mechanism is not fully understood. So far, there is no specific method to effectively treat fluorosis, mainly to prevent. In recent years, a large number of studies have confirmed that a variety of macro elements such as calcium, magnesium, phosphorus, and trace elements such as selenium and boron play a positive regulatory role in the occurrence and development of fluorosis, and have different degrees of influence in the body's antagonism against fluorosis. High levels of trace elements such as arsenic, lead, aluminum and chromium are risk factors for fluorosis. In view of the importance of a variety of macro and trace elements in human nutrition and health, this article reviews the latest developments in multiple elements and fluorosis, especially skeletal fluorosis and dental fluorosis, in order to further understand the causes of fluoride-induced health damage, and provide theoretical reference for improving the prevention strategies of fluorosis in a targeted manner.
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Objective:To investigate the epidemic characteristics of human brucellosis in Inner Mongolia Autonomous Region from 2018 to 2020, and provide a reliable scientific basis for formulating brucellosis prevention and control strategies in Inner Mongolia Autonomous Region.Methods:A retrospective study was carried out to collect data of human brucellosis in Inner Mongolia Autonomous Region from 2018 to 2020 from the "China Disease Control and Prevention Information System", and the monitoring data and information of confirmed cases were collected from the annual summary data reported by the leagues (cities) of the Inner Mongolia Autonomous Region. Using descriptive epidemiological methods, the epidemic situation, three distributions (time, region and population distributions) of brucellosis, and the serological and pathogenic test results of active monitoring population were analyzed.Results:From 2018 to 2020, a total of 40 665 cases of brucellosis were reported in Inner Mongolia Autonomous Region, with an annual average incidence rate of 53.47/100 000. The number of annual incidence had increased from 10 111 in 2018 to 16 406 in 2020, and the annual incidence rate had increased from 39.99/100 000 in 2018 to 64.60/100 000 in 2020. The spring and summer was the peak incidence, mainly in March to August, accounting for 64.90% (26 390/40 665) . There were reports of brucellosis cases in 12 leagues (cities) of Inner Mongolia Autonomous Region, and the top 3 regions with the number of reported cases were Tongliao City (9 896 cases), Xing'an League (6 136 cases) and Chifeng City (4 934 cases). The age of onset of brucellosis cases was mainly 30 - < 65 years old(33 539 cases), and the sex ratio between men and women was 2.18 ∶ 1.00 (27 890 ∶ 12 775); the occupational distribution was mainly farmers, accounting for 79.23% (32 221/40 665). From 2018 to 2020, 704 085 people were actively monitored in the region, of which 391 941 were serologically tested, and the infection rate was 4.57% (17 920/391 941); and there were 9 539 new cases in the active monitoring population. In 3 years, 19 strains of Brucella sheep type 3 and 11 strains of Brucella sheep type 1 were isolated. Conclusions:From 2018 to 2020, the incidence rate of brucellosis in Inner Mongolia Autonomous Region is increasing year by year. There are many new cases in the active monitoring population, and more underreporting cases. It is recommended to expand the scope of monitoring, strengthen pathogen monitoring among humans and animals, and joint prevention and control of various departments to improve the self-protection awareness of the masses.
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Objective:To explore the molecular mechanism of fluoride toxicity to ameloblasts.Methods:Mouse ameloblast cell line (LS8 cells) was taken and divided into control group [0.0 mmol/L sodium fluoride (NaF)] and fluoride exposed group (1.6 mmol/L NaF) according to the final concentration of NaF. Transcriptome sequencing was performed to screen differentially expressed genes (DEGs), and gene ontology (GO) analysis and gene set enrichment analysis (GSEA) were performed on DEGs. The STRING database was used to construct the protein-protein interaction (PPI) network of DEGs, and Cytoscape 3.8.0 software was used to visualize the PPI network to screen key modules and key genes. At the same time, real-time fluorescence quantitative PCR was used to detect the mRNA expression level of key genes, and the key genes were verified by gene expression database (GEO database).Results:Compared with the control group, there were 709 DEGs in the fluoride exposed group, including 223 up-regulated genes and 486 down-regulated genes. The GO analysis of DEGs mainly involved molecular functions such as receptor-ligand activity, cell adhesion molecule binding, structural components of extracellular matrix, cellular components such as collagen of extracellular matrix, receptor complex, membrane raft, biological processes such as external packaging structure organization, extracellular structure organization, and extracellular matrix organization. The GSEA of the whole gene set found that the interleukin-17 (IL-17) signaling pathway, ribosome biogenesis in eukaryotes, and the nuclear factor kappa-B (NF-κB) signaling pathway were activated, while fatty acid degradation, pyruvate metabolism and fatty acid metabolism were inhibited. After constructing PPI network, three key modules and four key genes [typeⅠcollagen α1 (Col1a1), typeⅠcollagen α2 (Col1a2), typeⅤcollagen α1 (Col5a1) and fibrinogen 1 (Fbn1)] were obtained. Compared with the control group, the mRNA expression levels of Col1a1, Col1a2, Col5a1 and Fbn1 in LS8 cells of the fluoride exposed group were significantly decreased ( P < 0.05), which was consistent with the change trend of gene expression in the GEO database. Conclusion:Key genes such as Col1a1, Col1a2, Col5a1, Fbn1, and signaling pathways such as IL-17 and NF-κB, which are screened by bioinformatics method, may be closely related to the toxic effects of fluoride on ameloblasts.
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Objective To study the influence of parental generation coal-burning-borne fluorosis on tooth development of their offspring.Methods High fluoride air model was established on the basis of burning coal habit of the epidemic areas.Fluoride feed was made of coal drying corn from the epidemic areas.Totally 48 SD rats were randomly divided into 4 groups with male to female ratio of 1:1 by random number table method.Rats in high,middle and low fluoride groups were put in the high fluoride air room and feed food with 40,25 and 10 mg/kg fluorine,and the control group was put in normal air room and feed normal food.After 8 weeks,rats were mating and parturition.Tooth eruption time of offspring rat was observed;and dental fluorosis incidence,the tooth length and fluorine content were observed at 21 d.Results In high and middle fluoride groups [(6.83 ± 0.94),(6.25 ± 1.06) d],tooth eruption time of offspring rat was later than that of control group [(5.34 ± 0.89) d,all P ＜ 0.01].At 21 d,dental fluorosis was observed in the lower incisors of the high and middle fluorine groups;compared with control group [(5.21 ± 0.19) mm,(223.00 ± 14.08) μg/kg],the tooth length was decreased [(4.83 ± 0.22),(4.96 ± 0.25) mm,P ＜ 0.01or ＜ 0.05],and tooth fluoride content was increased [(362.64 ± 20.35),(289.79 ± 19.18) μg/kg,all P ＜ 0.01].Dental fluorosis incidence of offspring rats was positively correlated with the fluorine dose (r =0.704,P ＜ 0.01).Conclusion Parental generation rats’ intaking excessive fluoride can affect offspring rats tooth development and dental fluorosis,which is related to the fluorine dose.
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Objective SD rats were immuned with the transgenic tomatoes which carried fused gene of a region of PAc Strep-tococcus mutants and cholera toxin B subunit.The immunogenicity was tested to explore secure and economic edible vaccines a-gainst dental caries.Methods A total of 18 eighteen-day-old female SD rats were subdivided randomly into three groups:the exper-imental group which were fed with transgenic tomato juice containing chimaera protein PAcP/CTB;the positive control group which were treated with deactivated S.mutans;the negative control group which were not treated with transgenic tomato juice.Rats were immuned once per week for four weeks.Blood and saliva were collected at one day before the first immunity and one week after each immunization.IgG of blood serum and SIgA of saliva were detected using the enzyme-linked immunosorbent assay (indirect ELISA) testing.On day 70,rats were terminated.The maxillary and mandibular bones were subsequently taken out to count dental caries′scores.Results Post immunization,the experimental group and the positive control group had statistical significant levels of speci-ficity IgG in serum and SIgA in saliva compared to the negative control group (P <0.05).There was a significance difference be-tween the experimental group and the negative control group except in Dx levels of caries loss (P <0.05).Conclusion The targeted protein expressed on the transgenic tomatoes is immunogenic,which can effectively induce mucous membrane immune response and the systematical immunoreaction to suppress the occurrence of the dental caries.
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Objective:To survey the expression of MMP-1,MMP-2 of human periodontal ligament cells(HPDLCs)treated by tea polyphenols(TP)and lipopolysaccharide(LPS).Methods:HPDLCs were in vitro cultured in vitro and treated by TP(200 μg/ml) and /or LPS(100 μg/ml)for 24,48 and 72 h respectively,the secretion of MMP-1 and MMP-2 were examined by ELISA,MMP-1 and MMP-2 mRNA expression was examined by real-time PCR.Results:The secression and mRNA expression of MMP-1 and MMP-2 of HPDLCs increased by LPS treatment and significantly inhibited by TP at the different times.Conclusion:TP can inhibit the col-lagen degradation of HPDLCs mediated by LPS.