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Objective:To explore the DNA methylation patterns and methylation differential genes of patients with coal-burning-borne endemic fluorosis, and to provide a basis for study of the pathogenesis of fluoride-induced body injury.Methods:A case-control study was conducted in Shuicheng County, Liupanshui, Guizhou Province, ten patients with severe fluorosis were selected as the fluorosis group in Douqing Township, where people burning high fluorine coal in open range all year round; and ten people without fluorosis phenotype were selected as the control group in Huaga Township, where firewood was the main fuel. Peripheral blood samples were collected from the two groups of people. Reduced representation bisulfite sequencing (RRBS) technique was used to detect the whole genome DNA methylation pattern ( n = 4) and DNA differentially methylated region (DMR), the DMR differential degree (log 2Ratio) and KEGG pathway enrichment analysis were used to screen the methylation differential genes, and real-time PCR was used to verify the mRNA expression levels of the candidate methylation differential genes( n = 10). Results:The methylation pattern analysis results showed that the methylation levels of all C bases in the genome DNA of the fluorosis group and the control group were (61.53 ± 0.59)% and (62.48 ± 1.53)%, respectively; among them, the methylated levels at CG sites were (63.75 ± 0.65)%, (64.36 ± 1.01)%, at CHG sites were (13.79 ± 0.72)%, (16.69 ± 4.06)%, and at CHH sites were (25.12 ± 1.72)%, (29.77 ± 3.97)%. Compared with the control group, patients in the fluorosis group had 1 000 DMR distributed on different autosomes; and the chromosome 19 was the most with 104 segments. There were 978 DMR-related genes, including 265 hypermethylation genes and 713 hypomethylation genes; KEGG pathway enrichment analysis showed that methylation differential genes were mainly involved in cell metabolism, cancers, and phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) and other signaling pathways; combined with the differential degree of DMR, the hypermethylated succinate dehydrogenase complex flavoprotein subunit A pseudogene 3 (SDHAP3, log 2Ratio = 3.487) and hypomethylated nuclear factor κB inhibitor kinase regulatory subunit γ (IKBKG, log 2Ratio =-4.436) were selected as the candidate genes. There were statistically significant differences in the mRNA expression levels of SDHAP3 (0.54 ± 0.08, 1.00 ± 0.00) and IKBKG (1.32 ± 0.39, 1.00 ± 0.00) between fluorosis group and control group ( F = 22.94, 15.09, P < 0.01 or < 0.05). Conclusion:There are a large number of methylation differential genes in the genomes of patients with coal-burning-borne endemic fluorosis and controls, the hypermethylated SDHAP3 and hypomethylated IKBKG may be involved in fluoride induced body injury.
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Objective:To understand the current situation of children's fluorosis in the coal-burning-borne endemic fluorosis areas (abbreviated as coal-burning fluorosis) in Suojia Miao, Yi and Hui Township (Suojia Township for short) in Liuzhi Tequ, Guizhou Province, and to provide scientific basis for formulating prevention and control strategies and measures.Methods:In 2019, the cluster sampling method was adopted to select children aged 8-12 years old from 6 primary schools in Suojia Township, Liuzhi Tequ, Guizhou Province to conduct a questionnaire survey to collect basic information, and perform dental fluorosis examination and indexing in accordance with the "Diagnosis of Dental Fluorosis" standards. Immediate urine samples were collected from children in April and October, and urinary fluoride content was determined by ion selective electrode method.Results:A total of 1 381 children aged 8-12 years old were investigated, aged (9.84 ± 1.38) years old, including 679 boys and 702 girls. A total of 625 children with dental fluorosis were detected, and the detection rate was 45.26%; the dental fluorosis index was 1.00, and the prevalence intensity was moderate; the main score of dental fluorosis was extremely mild, accounting for 37.00% (511/1 381). The detection rates of dental fluorosis in children aged 8 to 12 years old were 35.10% (106/302), 35.83% (115/321), 47.96% (129/269), 55.23% (153/277), and 57.55% (122/212), respectively; the difference between different ages was statistically significant (χ 2 = 48.949, P < 0.01), and the detection rate of dental fluorosis in children increased with age(χ 2trend = 45.254, P < 0.01).The detection rates of dental fluorosis in boys and girls were 43.59% (296/679) and 46.87% (329/702), respectively, and there was no significant difference between different genders (χ 2 = 1.492, P > 0.05). In April and October, 123 and 107 urine samples of children aged 8-12 years old were tested. The geometric mean of urinary fluoride was 1.55 and 0.47 mg/L, respectively. The urinary fluoride level in April was higher than the normal range (< 1.40 mg/L). Conclusions:Suojia Township in Liuzhi Tequ of Guizhou Province is still a fluorosis area, and there is a big difference in urinary fluorine level in different months, which indicates that the residents in this area may have intermittent high fluorine intake, and prevention and control of endemic fluorosis should be further strengthened.
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To construct TeI3c/4c-based and temperature-inducible gene inactivation system (Thermotargetron) and to apply it to gene inactivation of mesophilic bacteria. The subunit of flagellum (fliC) and C4 dicarboxylate orotate:H⁺ symporter (dctA) genes were chosen as targets in the genome of Escherichia coli HMS174 (DE3) strain. According to recognition roles of TeI3c/4c intron, the fliC489a, fliC828s, fliC1038s and dctA2a sites were chosen as target sites. Gene-targeting plasmids were constructed based on pHK-TT1A by using overlap PCR method and transformed into HMS174 cells. An aliquot mid-log phase cultures of the transformants were shocked at 48 °C and plated on LB plate (containing chloramphenicol). Afterwards, gene mutants were screened by using colony PCR and DNA sequencing. After the mutants were obtained, the phenotypes of ΔfliC and ΔdctA gene mutants were characterized by using agar puncture and carbon metabolism experiments. Colony PCR and sequencing results show that TeI3c/4c intron was inserted in the designed sites of fliC and dctA genes. The gene-targeting efficiency of Thermotargetron system was 100%. Phenotype verification experiments of the mutants demonstrated that the cell motility of all ΔfliC mutants was damaged and the malate assimilation ability of ΔdctA mutant was deprived comparing to wild-type HMS174 strain. In our study, a temperature-inducible and high-efficiency gene inactivation system was established for mesophilic bacteria. This system could achieve high efficiency and precise gene inactivation by modulation of the incubation duration of the transformants at 48 °C.
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Objective To investigate whether microRNA-34a (miR-34a) participates in lipopolysaccharide (LPS) mediated sepsis related renal function impairment via Kruppel-like factor 4 (KLF4). Methods Thirty healthy male Sprague-Dawley (SD) rats, weighing 180-200 g, were randomly divided into two groups: control group and model group, with 15 rats in each group. The SD rats from model group were injected with LPS 7.5 mg/kg to induce sepsis related renal function impairment model, the SD rats from control group were injected with normal saline. The serum creatinine concentration (SCr) and blood urine nitrogen (BUN) content was detected by multifunction biochemical analyzer; the morphological changes of renal tissue were observed by hematoxylin and eosin stain (HE) staining; the expression of miR-34a and KLF4 gene in plasma and renal tissue were detected by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR); the protein expression of KLF4 in renal tissue was detected by Western Blot; the target gene of miR-34a was verified by double luciferase reporter gene analysis. Results Compared with control group, inflammatory cell infiltration in renal tissue was increased in model group, the SCr and BUN were significantly increased [SCr (μmol/L): 142.5±10.6 vs. 46.4±5.6, BUN (mmol/L): 31.6±6.2 vs. 8.5±1.2, both P < 0.01], the gene expression of miR-34 in plasma and renal tissue were significantly increased (2 -ΔΔCt: 2.26±0.11 vs. 1.14±0.05 in plasma, 4.23±0.12 vs. 1.12±0.04 in renal tissue, both P < 0.01), the gene and protein expressions of KLF4 were significantly decreased [KLF4 gene (2 -ΔΔCt): 0.52±0.03 vs. 1.21±0.06, KLF4 protein (A value): 0.72±0.03 vs. 1.05±0.04, both P < 0.01], which indicated that kidney injury occurred in rats. Pearson correlation analysis showed that plasma miR-34a was positively correlated with SCr and BUN (r value were 0.678, 0.721, respectively, both P < 0.05). Double luciferase reporter assay confirmed that KLF4 was the target gene of miR-34a. Conclusion The miR-34a participates in LPS mediated sepsis related renal function impairment via KLF4.
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Objective To adopt the three-dimensional speckle tracking technology to analyze the left ventricular three-dimensional strain in patients with hypertension of normal geometric patterns to provide a basis for early finding the hypertensive cardiac function change.Methods Fifty patients with hypertension of normal geometric patterns and age-matched individuals undergoing physical examination were selected.The routine cardiac parameters were measured with cardiac echocardiography,furthermore the three-dimensional speckle tracking technology was adopted to detect the global longitudinal strain(GLS),global circumferential strain(GCS),global area strain(GAS),global peak radial strain(GRS) and spherical index(SPI).All parameters were compared between the two groups.Results The left ventricular internal diameter,posterior wall of left ventricle,interventricular septum,relative wall thickness(RWT),left ventricular mass index(LVMI),left ventrieular ejection fraetion(EF),SPI and GCS had no statistical differences between the two groups(P＞0.05);GLS,GAS and GRS in the hypertension with normal geometric patterns group were significantly lower than those in the control group(P＜0.05).Conclusion Three-dimensional speckle tracking technology displays the decrease of left ventricular systolic function in the patients with hypertension of normal geometric patterns,its sensitivity is superior to EF,which is a good method to detect early changes of left ventricular systolic function in hypertension.
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OBJECTIVE:To observe effect and safety of continuous renal replacement therapy (CRRT) combined with differ ent doses of xuebijing sepsis patients with acute kidney injury (AKI).METHODS:One hundred and four patients diagnosed as sepsis complicated with AKI were collected from ICU of our hospital during Jun.2012 to Aug.2015.They were divided into small-dose group,medium-dose group,large-dose group and control group according to random number table,with 26 cases in each group.Four groups received routine treatment.Small-dose group was given Xuebijing injection 50 mL added into 0.9％ Sodium chlo ride injection (NS) 100 mL,ivgtt,bid;medium-dose group was given Xuebijing injection 100 mL added into NS 100 mL,ivgtt,bid;large-dose group was given Xuebijing injection 100 mL added into NS 100 mL,ivgtt,qid.All patients were treated for 1 week.The levels of IL-6,TNF-α and hs-CRP were observed in 4 groups before and after treatment;renal function indexes as BUN,SCr and CysC,blood coagulation function indexes as PT,APTT and Fib were also observed as well as APACHE Ⅱ score and Marshall score.The occurrence of ADR were recorded during treatment.RESULTS:Ninety-five cases meeting the criteria were finally included in the study after 9 cases withdrew from the study,and there were 23,24,25,23 cases in small-dose group,medium-dose group,large-dose group and control group,respectively.Before treatment,there was no statistical significance in above indexes among 4 groups (P＞0.05).After treatment,the serum levels of IL-6,TNF-α,CRP,PT and APTT,APACHE Ⅱ score and Marshall score in 4 groups were all decreased significantly,while the serum levels of BUN,SCr,CysC and Fib were increased significantly;the improvement of above indexes in small-dose group,medium-dose group and large-dose group were all better than in control group,and the large-dose group was significantly better than the small-dose group and medium-dose group,with statistical significance (P＜0.05).There was no statistical signifi cance in the incidence of ADR among 4 groups (P＞ 0.05).CONCLUSIONS:Large-dose xuebijing combined with CRRT can effectively inhibit inflammatory reaction in sepsis patients with AKI,improves renal function and coagulation function,and dose not increase the risk of ADR.
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Objective To understand the ABO blood type distribution of Buyi and Shui ethnic populations in Libo county of Guizhou province .Methods Totally 726 Buyi and 163 Shui individuals who were unrelated within three generations were detected ABO blood stype with glass-clotted method .Results The ABO blood type distributions in the Buyi and Shui ethnic populations both were in line with the Hardy-Weinberg equilibrium .The Buyi ethnic population ABO phenotypic distribution and gene frequen-cies were O>B>A>AB and r>q>p (r=0 .676 9 ,q=0 .176 7 ,P=0 .146 4) and the national index was 0 .842;while the Shui eth-nic population ABO phenotypic distribution and gene frequencies were O>A>B>AB and r>p>q(r=0 .522 6 ,q=0 .104 0 ,P=0 .164 7) and the national index was 1 .529 .Conclusion The Buyi and Shui ethnic populations′ABO blood type distribution in Libo county of Guizhou province is basically consistent with that of the same ethnic populations in other regions of Guizhou Province of the same ethnic groups .The genetic distance analysis prompts that the regional and ethnic differences exist in ABO blood type dis-tribution .
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Objective To explore the correlation between myeloperoxidase (MPO) genetic variation and coal-burning endemic fluorosis, and to understand the influence of integrated intervention including stove changes and health education on people’s health in the area. Methods In 2007, coal-burning endemic fluorosis disease areas were selected in Bijie City, Guizhou Province. No stove changes in Yachi Town, 150 patients with dental fluorosis were selected as fluorosis non-intervention group, and the intervention group was 150 patients in Changchun Town where the stoves were changed 2 years ago. The population in control group was selected in an area with non-endemic fluorosis in Changshun County. The mRNA expressions of MPO in leukoxytes were detected by real-time PCR. HepG2 cells were cultured in vitro and divided into four groups: pGL3-A group, pGL3-G group, pGL3-Control group and pGL3-Basic group. pGL3-A and pGL3-G were recombinant plasmid, while pGL3-Basic as a blank control and pGL3-Control as a positive one. The internal reference plasmid pRL-TK co-transfected the HepG2 cells with pGL3-G, pGL3-A, pGL3-Basic and pGL3-Control, respectively. The influence of sudden change of MPO gene promoter on the gene transfection activity was evaluated by a dual luciferasereporter gene system. Results The expression level of MPO mRNA in peripheral blood leukocytes in non-intervention group(0.054 ± 0 . 003 ) were higher than control and intervention groups (0.019 ± 0.004,0.019 ± 0.003, all P0.05). After the MPO-463G/A locus genetic variation occured, the luciferase reporter gene expression level of the recombinant plasmid pGL3-G(0.753 4 ± 0.086 6) was higher than that of the pGL3-A(0.490 0 ± 0.022 3, P < 0.05). Conclusions The study on MPO gene promoter-463G/A locus has prompted that MPO gene allele may be a protective factor to coal-burning fluorosis. The integrated interventions have a role in the prevention and treatment of endemic fluorosis.
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Objective:To investigate the association between polymorphisms of FGFR2 and the susceptibility of breast cancer in Han population in Guizhou province.Methods:Genotyping was performed using PCR-sequence-specific primers(PCR-SSP)in 106 histologically confirmed breast cancer cases and 116 cancer-free controls.Results:The genotype frequencies of rs1219648 TT,TC,and CC were 50%,25.47%.and 24.53% in breast cancer cases and 29.31%,48.28%,and 22.41% in the controls.The gene frequencies of T in breast cancer cases and the controls were 62.74% and 53.45%.respectively.The gene frequencies of C were 37.26% and 46.55%.respectively.The distribution of allele and genotype frequencies of FGFR2 rs1219648 was statistically different between breast cancer cases and the controls(P<0.05).Conclusion:FGFR2 rs1219648 polymorphism influences the susceptibility of breast cancer.TT genotype might serve as a risk factor for breast cancer.
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This study examined the effects of flow shear stress on the bio-capacity of the endothelial cells' induced from mesenchymal stem cells (MSCs). After cultivating the SD rat mesenchymal stem cells in vitro, we exposed them under different intensity of flow shear stress and induced these cells to endothelial cells. The variations of total anti-oxidation competence (T-AOC) and quantity of nitrogen monoxide (NO) were tested. The results showed that shear stress has an enhanced effect on the T-AOC and NO of endothelial cells induced from MSCs in an intensity-dependent manner. Flow shear stress could provide a protective action on the in vitro induction of endothelial cells, thus formulating a theoretical foundation for the therapeutics of ischemic heart diseases and vascular tissue engineering.
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Animals , Female , Male , Rats , Antioxidants , Metabolism , Bone Marrow Cells , Cell Biology , Physiology , Cell Differentiation , Physiology , Cells, Cultured , Endothelial Cells , Cell Biology , Metabolism , Mesenchymal Stem Cells , Cell Biology , Physiology , Oxidation-Reduction , Rats, Sprague-Dawley , Stress, MechanicalABSTRACT
<p><b>OBJECTIVE</b>To study the genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) among the Han, Buyi and Miao populations in Guizhou and to provide genetic data for establishment of the genetic polymorphism bank of Guizhou Minorities.</p><p><b>METHODS</b>The technique of polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) was used to detect the genotype and allele frequencies at two mononucleotide sites (677 and 1298) of MTHFR among the Han population in Libo county, the Buyi population in Libo county and the Miao population in Leishan county.</p><p><b>RESULTS</b>At the site of 677, the T allele frequencies were found to be 22.8%, 16.1%, 10.6%, for the Han, Buyi, Miao populations respectively. At the site of 1298, the C allele frequencies were 28.9%, 39.1%, 48.7% for the Han, Buyi, Miao populations respectively. The frequencies for the combined heterozygote of 677CT/1298AC were 16.66%, 22.7%, 11.1% for the three populations respectively. Moreover, one case with combined homozygote of 677TT/1298CC was seen in the Miao population.</p><p><b>CONCLUSION</b>The polymorphisms of the two mononucleotide sites (677 and 1298) of MTHFR are diverse in different populations. The C allele frequencies at the site of MTHFR 1298 of the Miao population in Leishan county and the Buyi population in Libo county are high, and the C allele frequency in the Miao population is higher than those hitherto reported in literature.</p>
Subject(s)
Female , Humans , Male , Base Sequence , China , Gene Frequency , Genotype , Methylenetetrahydrofolate Reductase (NADPH2) , Genetics , Polymerase Chain Reaction , Polymorphism, Genetic , Genetics , Polymorphism, Restriction Fragment Length , Sequence Analysis, DNAABSTRACT
Objective The frequency of G6PD deficiency in the Tujia nationality in Jiangkou,Guizhou was investigated.Methods In Oct.2002,227 male subjects were selected randomly from the local people,and NBT qualitative and G6PD/6PGD quantitative methods were used to detect G6PD deficiency in them.Results Among the 227 subjects,17 cases of G6PD deficiency were found.The gene frequency of G6PD deficiency was 0.0749.Conclusion There is a high incidence of G6PD deficiency in Tujia nationality in Jiangkou,Guizhou.The investigation will illustrate the distribution of G6PD deficiency in Guizhou,and provide some useful data for the preventing G6PD deficiency,directing clinical management,improving minority population diathesis and studying the origin of Tujia nationality.
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Objective To explore the change of hippocampal neurons cholinergic receptor in rats with vascular dementia(VD).Methods VD rat models were established by employing improved method of Pulsinelli's four-vessel occlusion.1 month later,the abilities of learning and memory of VD models were tested by Morris water maze.The activities of acetylcholinesterase (AChE) in plasma and hippocampus were detected by the improved Ellman's colorimetric methods.The expressions of hippocampal nicotinic acetylcholine receptor (nAChR) subunits ?3,?4 and ?7 proteins and mRNA were detected respectinely by Western Blotting and RT-PCR,respectively.The results were compared with sham operation group.Results Compared to the sham operation group,the escape latency of Morris water maze test and the first time crossing platform in VD group were significantly prolonged,the number of crossing platform was decreased (all P