OBJECTIVETo investigate the significance of cytokine interleukin-12p40 (IL-12p40) and interferon-gamma (IFN-gamma) in tissues formation and development of human oral lichen planus (OLP).

METHODSThe tissues of 11 cases of normal oral epithelium and 43 cases of OLP were investigated for the expression of IL-12p40 and IFN-gamma proteins by using Envision two-step immunohistochemistry. The correlations between the expressions of these two cytokines, and their clinical and pathological significance in OLP were analyzed.

RESULTS1) IL-12p40 and IFN-gamma proteins were up-regulated in OLP comparing with that in normal oral mucosa and there was statistical significance between their difference (P < 0.05). 2) The percentage of positive IL-12p40 staining in OLP of IFN-gamma positive group was higher than IFN-gamma negative group and there was statistical significance between their difference (Chi2 = 5.828, P = 0.016). A positive correlation was found between IL-12p40 and IFN-gamma proteins in OLP (Spearman r = 0.357, P = 0.019). 3) The percentage of positive IL-12p40 staining in OLP with short course (< 6 months) was higher than that in OLP with long course (> 6 months; Chi2 = 7.935, P = 0.005), and a significant association was found between IFN-gamma over expression and the degeneration of base cells in OLP lesions (Chi2 = 9.070, P = 0.011).

CONCLUSIONThese results indicate that at the primary phase of OLP, IL-12 may drive the pathological destruction in OLP lesions by elevating IFN-gamma protein locally. IFN-gamma may play an important role for the pathological destruction in OLP lesions.

Adult ; Female ; Humans ; Interferon-gamma ; Interleukins ; Lichen Planus, Oral ; Middle Aged ; Mouth Mucosa

Soil and Uncaria rhynchophylla in different functional areas were selected for the study,the content of heavy metals such as As, Cd, Cu, Cr, Pb, and Hg in soil and U. rhynchophylla was discussed, the characteristics of their accumulation in the U.rhynchophylla was analyzed, the contamination levels of heavy metals in soil in different functional areas was evaluated. The results showed that content of Cu, As, Pb and Cr in soil was being cropland>woodland>wasteland, content of Cd was being woodland>cropland>wasteland, content of Hg was being cropland>woodland>wasteland. According to quality standard of soil environment, soil Cd in woodland, cropland and wasteland all exceeded the state-level standards, soil Cd in woodland exceeded the secondary standard, soil Hg in cropland and wasteland all exceeded the state-level standards. According to technical conditions of green food producing area, soil Cd in woodland exceeded the limit value of standard. According to Green Trade Standards of Importing Exporting Medicinal Plants Preparations,the content of heavy metals of U.rhynchophylla in cropland,woodland and wasteland were correspond to the specification. From the single factor pollution index, the soil in woodland was polluted by Cd. From the comprehensive pollution index, the soils in different functional areas were not contaminated by heavy metals. The enrichment coefficient of heavy metals such as As, Cu, Cr, and Pb in hook of U.rhynchophylla was being wasteland>woodland>cropland, the enrichment coefficient of Cu in hook of U. rhynchophylla in wasteland was more than 1. Except Cu, the enrichment coefficient of other heavy metals was low.

The study aims at evaluating genetic diversity and medicinal quality of cultivated germplasm in Rehmannia glutinosa, and providing theoretical guidance for screening excellent germplasm. The genetic diversity of 21 species of R. glutinosa were analyzed by SRAP molecular markers, and the catalpol and verbascoside was determined by HPLC. The mass fraction of catalpol and verbascoside in R. glutinosa germplasm were respectively in the range of 2.393%-6.519% and 0.063%-0.478%, the germplasm 14, 16, 15 and 20 germplasm, witch catalpol and verbascoside content was higher. A total of 57 bands were produced by 10 primer, among which 40 polymorphic bands were polymorphic bands, and the percentage of polymorphic loci was 8.77%-54.39%, the Nei's genetic diversity index (H) was 0.374 1, Shannon's polymorphism information index (I) was 0.546 6. Gst and gene flow Nm were 0.608 8 and 0.321 3, respectively. Based on the genetic uniformity, 21 species of germplasm were grouped into 2 categories. The genetic diversity level of R. glutinosa was medium low. The comprehensive consideration of the genetic diversity and the content inculde catalpol and verbascoside, germplasm 7 and germplasm 18 could be used as the preferred materials for the cultivation of reticulum. Germplasm 15 and 16 can be used as the preservation and breeding object of rhubarb germplasm.
Animals ; Gene Flow ; Genetic Variation ; Phylogeny ; Plant Breeding ; Plants, Medicinal ; genetics ; Rehmannia ; genetics

OBJECTIVEOmenn syndrome is a rare autosomal recessive hereditary severe combined immunodeficiency. The purpose of this study was to understand clinical characteristics and genetic mutation type of Omenn syndrome and to improve the recognition of Omenn syndrome among pediatric clinicians.

METHODOne suspected case of severe combined immunodeficiency was found to have pneumonia repeatedly, intractable diarrhea, poor antibiotic treatment effect, lymphadenopathy, hepatosplenomegaly and erythroderma. The patient was diagnosed as having Omenn syndrome by RT-PCR, and the expression of RAG1/RAG2 and gene analysis of RAG1/RAG2 were performed.

RESULTThe classification of lymphocyte was CD3(+) cells (35.3%), CD19(+) cells (0.4%), CD16(+) cells (57.6%). After stimulation with phytohemagglutinin (PHA), lymphocyte proliferation of the child was extremely low. Genetic studies showed RAG1 homozygous deletion mutation (2302 del T). He had detectable activated T-lymphocytes with low circulating B-lymphocytes and no evidence of maternal T-cell engrafment as indicated by the short tandem repeat (STR) analysis.

CONCLUSIONOmenn syndrome is a severe combined immunodeficiency disease caused by mutations in the RAG1/RAG2 gene. The disease has been reported rarely in China. The clinical manifestations of the disease is early postnatal repeated infections and erythroderma. Mutation analysis of RAG1/RAG2 gene may help to confirm the diagnosis and may be useful in early immune reconstitution and genetic counseling.

Amino Acid Sequence ; Biomarkers ; blood ; DNA Mutational Analysis ; DNA-Binding Proteins ; genetics ; Genotype ; Homeodomain Proteins ; genetics ; Humans ; Infant ; Lymphocytes ; immunology ; pathology ; Male ; Microsatellite Repeats ; Mutation ; Nuclear Proteins ; genetics ; Phenotype ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Severe Combined Immunodeficiency ; diagnosis ; genetics ; pathology

AIM: To study the expression and cancer-promoting mechanism of miR-130b in human retinoblastoma(RB).

METHODS: Detected the expression levels of miR-130b in human RB carcinoma tissues, adjacent tissues and human RB cell lines(HXO-Rb44 and Y79)by qRT-PCR; detected the expression levels of PTEN in HXO-Rb44 and Y79 cells by qRT-PCR, Western Blot and immunofluorescence; verified the target relationship between miR-130b and PTEN by dual-luciferase reporter gene test; the co-transfection test was used to investigate the relationship between PTEN and miR-130b on the expression of PI3K/Akt signaling pathway in RB cell line.

RESULTS: The expression level of miR-130b in cancer tissue of RB was significantly higher than that of paracancerous tissue(P<0.05). Compared with axben-181 cells, the expression level of miR-130b in HXO-Rb44 and Y79 cells was significantly increased(P<0.05). Compared with RB cancer tissue, the expression level of PTEN in its paracancerous tissue was significantly increased(P<0.05). The expression level of miR-130b was negatively correlated with the expression level of PTEN(P<0.001). The mRNA and protein expression levels of PTEN in HXO-Rb44 cells overexpressing miR-130b were significantly reduced, while the mRNA and protein expression levels of PTEN in Y79 cells after miR-130b interference were significantly increased(P<0.05). Compared with miR-130b mimics+PTEN-NC group, the luciferase activity of miR-130b mimics+wt-PTEN group was significantly reduced(P<0.05).In the HXO-Rb44 cells co-transfected with miR-130b mimics+PTEN-NC, the expression levels of p-Akt 308 and p-Akt 473 protein were significantly increased(P<0.05), while the expression levels of PTEN protein were significantly decreased(P<0.05). In the HXO-Rb44 cells co-transfected with miR-130b mimics+PTEN, no significant changes were observed in the above three proteins.

CONCLUSION: miR-130b is highly expressed in RB tissues and cell lines. PTEN is the target gene of miR-130b, and miR-130b may negatively regulate PTEN to affect the expression of PI3K/Akt signaling pathway and ultimately play a role in promoting cancer.

OBJECTIVETo study the correlation between cyclin E protein overexpression and centrosome amplification in oral squamous cell carcinoma (OSCC).

METHODSFormalin-fixed, paraffin-embedded tissues from 12 normal oral epithelium cases and 46 cases of OSCC were studied. Their centrosome status was analyzed by indirect immunofluorescence double staining with antibodies to centrosome protein gamma-tubulin and cytokeratin. The expression of cyclin E protein was studied by immunohistochemical methods. The correlation between cyclin E protein expression and centrosome amplification in OSCC was statistically analyzed by SPSS 12.0.

RESULTSThirty-seven of the 46 OSCC cases (80.4%) studied showed evidence of centrosome amplification, as signified by enlargement and/or increase in number of centrosomes, while normal oral epithelium possessed centromeres of normal size and number. Positive staining for cyclin E protein was observed in 30 of the 46 OSCC cases (65.2%), while all the normal oral epithelium cases were cyclin E protein-negative. The percentage of centrosome amplification in OSCC with positive cyclin E protein staining (90.0%, 27/30) was higher than that in OSCC with negative cyclin E protein staining (62.5%, 10/16) (chi(2) = 5.014, P < 0.05). Centrosome amplification showed positive correlation with cyclin E protein overexpression (r = 0.330, P < 0.05).

CONCLUSIONUp-regulation of cyclin E protein may represent one of the possible mechanisms for centrosome amplification in OSCC.

Carcinoma, Squamous Cell ; metabolism ; pathology ; surgery ; Centrosome ; pathology ; ultrastructure ; Cyclin E ; metabolism ; Epithelium ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Microscopy, Confocal ; Mouth Mucosa ; metabolism ; pathology ; Mouth Neoplasms ; metabolism ; pathology ; surgery ; Up-Regulation

OBJECTIVETo explore the influence of arsenic pollution caused by coal-burning on methylation (promoter and exon 5) and mutation (exon 5) of human p53 gene, and to analyze the relationship between methylation, mutation and arsenism.

METHODSAccording to the diagnostic criteria of endemic arsenism, 112 patients with arsenism (including 38 mild cases, 43 moderate cases and 31 severe cases) were selected in the areas with endemic arsenism from Xingren, Guizhou province. Among the subjects, 43 cases were diagnosed by dermatopathological methods, and they were divided into non-cancerous group (24 cases) and cancerous group (19 cases). 90 controls were selected from the non-arsenic polluted areas. Under the principle of informed consent, blood samples were collected from individuals. The methylation of p53 gene in promoter region and exon 5 were detected by extinction enzyme-PCR, the mutation of p53 gene (exon 5) was detected by PCR-SSCP, PCR products cloning and sequencing technology.

RESULTSThe positive rates of methylation of p53 gene in promoter region were 13.16% (5/38), 27.91% (12/43) and 45.16% (14/31) respectively among mild, moderate and severe arsenism group, which were obviously higher than the rates in the control group (1.11% (1/90), χ² values were 8.679, 23.690, 41.199, respectively, both P values < 0.017). The positive rates of methylation of p53 gene were 25.00% (6/24) and 63.16% (12/19) in non-cancerous and cancerous group respectively, which were obviously higher than those in the control group (1.11% (1/90), χ² values were 18.762, 57.497, respectively, both P values < 0.025). The positive rates of methylation of p53 gene (exon 5) were 55.26% (21/38), 51.16% (22/43) and 48.39% (15/31) respectively among mild, moderate and severe arsenism group, which were obviously lower than the rates in the control group (88.88% (80/90), χ² values were 18.151, 23.168, 22.420, respectively, both P values < 0.017). The positive rates of methylation of p53 gene (exon 5) were 54.17% (13/24) and 42.11% (8/19) in non-cancerous and cancerous group respectively, which were obviously lower than those in the control group (88.88% (80/90), χ² values were 15.201, 22.075, respectively, both P values < 0.025). The mutation rates of p53 gene (exon 5) were respectively 5.26% (2/38), 16.28% (7/43) and 25.81% (8/31) among mild, moderate and severe arsenism group; while the results in moderate and severe arsenism group were obviously higher than in the control group (0.00%, χ² values were 15.465, 24.870, respectively, both P values < 0.017). The positive rate of mutation of p53 gene (exon 5) were respectively 16.67% (4/24) and 31.58% (6/19) in non-cancerous and cancerous group, which were obviously higher than it in the control group (0.00%, χ² values were 15.545, 30.077, both P values < 0.025). The hypermethylation of p53 gene in promoter region was related with the mutation of p53 gene (exon 5) (coefficient of association was 0.294, P value < 0.05); and the hypomethylation of p53 gene (exon 5) was related with the its mutation (coefficient of association was 0.410, P value < 0.05).

CONCLUSIONArsenic pollution caused by coal-burning can cause the hypermethylation of p53 gene in promoter region, hypomethylation and mutation of p53 gene (exon 5), and the changes of methylation of p53 gene are related with its mutation and might be one of the important etiological factors of arsenic pathogenicity or carcinogenesis.

Adult ; Arsenic Poisoning ; etiology ; genetics ; Case-Control Studies ; Coal ; adverse effects ; DNA Methylation ; Environmental Pollution ; adverse effects ; Female ; Genes, p53 ; Humans ; Male ; Middle Aged ; Mutation ; Promoter Regions, Genetic ; Tumor Suppressor Protein p53 ; genetics

OBJECTIVETo test expression of carbonic anhydrase II (Ca II) mRNA in osteoclasts which were applied with fluid shear stress.

METHODSThe bone marrow cells of Sprague-Dawley rats were cultured with the presence of 1,25-(OH)2D3 and dexamethasone. The osteoclast-like cells were identified by tartrate-resistant acid phosphatase(TRAP) staining and scanning electron microscope (SEM) observation, then purified with trypsin/ethylenediamine tetraacetic acid (EDTA). Different values and lasting time of steady fluid shear stress were exerted on the osteoclasts with parallel plate flow system. The Ca II expression of osteoclasts were detected by real time reverse transcription-polymerase chain reaction(RT-PCR) and nested polymerase chain reaction(PCR).

RESULTSThe levels of Ca II mRNA were down-regulated correspondingly with the increase of stress and time (P < 0.05).

CONCLUSIONIt's indicated that steady fluid shear stress within a certain range may down-regulate the expression of Ca II in osteoclasts.

Animals ; Bone Marrow Cells ; Carbonic Anhydrase II ; Cells, Cultured ; Osteoclasts ; Polymerase Chain Reaction ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Stress, Mechanical

The study aimed to establish an UPLC-MS/MS method for the determination of baicalin in rat plasma,in order to study the effect of PEG400 on pharmacokinetics of baicalin and baicalein in normal and gut microbiotadysbiosis rats. Plasma was precipitated with ethyl acetate and determined by UPLC-MS/MS method,with genistein as an internal standard. In terms of specificity,linearity,range,accuracy,precision and stability,the method was suitable for the determination of baicalin in plasma. The gut microbiotadysbiosis rat model was induced through the oral administration with lincomycin hydrochloride(5 g·kg-1·d-1) for one week. Samples of plasma of rats were obtained at different time points,after the rats were administrated with baicalin,baicalin and PEG400. Baicalin in rats were detected by UPLC-MS/MS method,and pharmacokinetic parameters were calculated by DAS 3. 2. 2 software. The results showed that the β-glucosidase activity and the number of colonies in the feces of gut microbiotadysbiosis rats induced by lincomycin hydrochloride were significantly reduced. The Cmaxand AUC0-tof the baicalinand PEG400 group in the intestinal flora were significantly lower than those in the normal rat baicalin and PEG400 group. There was no significant difference in Cmaxand AUC0-tbetween the baicalin group and the baicalin+PEG400 group of gut microbiotadysbiosis rats. The Cmaxand AUC0-tof the normal rats baicalin group were significantly higher than those of the gut microbiotadysbiosis rats baicalin group and the baicalin + PEG400 group. There was no significant difference in Cmaxand AUC0-tbetween the normal rat baicalein and PEG400 group and the baicalein group. The Cmaxand AUC0-tof the baicalein group in the gut microbiotadysbiosis rats were lower than those in the normal baicalein group,but significantly higher than those in the baicalein and PEG400 group. PEG400 could increase the absorption of baicalin in normal rats,but is ineffective in gut microbiotadysbiosis rats,with no impact on the absorption of baicalein in rats.
Animals ; Chromatography, Liquid ; Dysbiosis ; drug therapy ; Flavanones ; pharmacokinetics ; Flavonoids ; pharmacokinetics ; Gastrointestinal Microbiome ; drug effects ; Polyethylene Glycols ; Rats ; Tandem Mass Spectrometry

Objective:To investigate the effects of Da Jianzhongtang on substance P （SP）， mast cells （MC）， Toll like receptor 2 （TLR2）， TLR4 on MC model and nuclear transcription factor （NF）-κB p65 in visceral pain rats with irritable bowel syndrome （IBS）， and explore its mechanism of action on IBS visceral pain. Method:Forty-eight 3-day-old SD rats were randomly divided into 6 groups： the control group （control）， irritable bowel syndrome group （IBS）， ketotifen group （Ketotifen，0.18 mg·kg^-1）， Da Jianzhongtang low， medium and high dose groups （DJZT-L， DJZT-M， DJZT-H，2.16，1.08，0.54 g·kg^-1）， with 8 rats in each group. Intragastric administration lasted for 2 weeks. Maternal separation method was used to establish the IBS visceral pain model in rats. The visceral sensitivity of rats was evaluated at 60， 40 and 20 mmHg （1 mmHg≈0.133 kPa） with Abdominal wall withdrawal response （AWR） scale. SP and NF-κB p65 protein expression levels in colon tissue were detected with Western blotting technique. TLR2 and TLR4 proteins on mast cell membrane were detected by immunofluorescence staining. The degranulation rate of mast cells in colon tissue was detected by toluidine blue staining. Result:Compared with normal rats， AWR scores of model rats significantly increased at 60， 40， and 20 mmHg pressure （P<0.05，P<0.01）， the degranulation rate of mast cells in colon tissue and SP protein expression in colon tissue significantly increased （P<0.01）， TLR2， TLR4， and nuclear NF-κB p65 expression on mast cell membrane significantly increased （P<0.01）. Compared with model rats， the AWR scores of DJZT-H group （pressure of 40， 20 mmHg） and DJZT-M group （pressure of 60， 40， 20 mmHg） significantly decreased. Meanwhile， the degranulation rate of colon mast cells， and the SP， TLR2， TLR4， and NF-κB p65 expression also significantly decreased （P<0.05，P<0.01）. Conclusion:Da Jianzhongtang can affect mast cell activity and finally decrease visceral pain of IBS rats by down-regulating SP in colon tissue.