Objective To observe the activation of rat hepatic stellate cells (HSC-T6) and the changes of methyltransferase (DNMT)1,DNMT3a mRNA expression with different doses of sodium arsenite stimulation.Methods HSC-T6 cells were exposed to a final concentration of 0 (control),5 (low dose),15 (medium dose) and 25 (high dose) μmol/L sodium arsenite in culture medium for 24,48 and 72 h,cells and cell culture supernatant were harvested.Enzyme-linked immunosorbent assay (ELISA) kit was used to measure fibrosis factors contents of type Ⅰ collagen (COL-1),type 11Ⅲ collagen (COL-3) and alpha smooth muscle actin (α-SMA) and quantitative real-time PCR was used to detect the expression levels of DNMT1 and DNMT3a mRNA.Results Different arsenic exposure time (24,48,72 h) had a significant effect on COL-1,COL-3 and α-SMA contents in HSC-T6 cells (F =249.574,328.493,3 157.436,all P ＜ 0.01);Different arsenic exposure content (low,medium,high dose groups) had a significant effect on COL-1,COL-3 and α-SMA contents in HSC-T6 cells (F =3 946.521,1 006.399,13 025.770,all P ＜ 0.01).After arsenic exposure for 24 and 48 h,the expression levels of DNMT1 mRNA in high dose group (4.33 ± 0.24,2.34 ± 0.43) were higher than those of control group (1.00 ± 0.00,1.00 ± 0.00,all P ＜ 0.05).At the same arsenic exposure levels (low,medium or high dose),the expression level of DNMT1 mRNA was declined with prolongation of sodium arsenite stimulation time (all P ＜ 0.05).After arsenic exposure for 48 and 72 h,the expression levels of DNMT3a mRNA in high dose group (2.23 ± 0.50,5.02 ± 0.23) were higher than those of control group (1.00 ± 0.00,1.00 ± 0.00,all P ＜ 0.05).The expression levels of DNMT3a mRNA in medium and high dose groups at 72 h (3.80 ± 0.14,5.02 ± 0.23) were higher than those of 24 h (3.03 ± 0.12,0.42 ± 0.15,all P ＜ 0.05).Conclusion HSC-T6 cells are obviously activated with pro-fibrotic effect;the expression levels of DNMT1 and DNMT3a mRNA are both up regulated in HSC-T6 cells after being exposed to sodium arsenite.

Objective To study the mechanism of liver fibrosis in rats caused by chronic exposure through drinking water containing sodium arsenite,to identify the differential proteins via proteomics technique.Methods Totally 40 healthy 8-week-old male Sprague Dawley (SD) rats of specific pathogen free (SPF) grade were randomly divided into 4 groups,which were control group (deionized water),0.68,1.36 and 2.73 mg/kg sodium arsenite (iAs3+) treated groups,respectively.The rats were fed with iAs-treated drinking water freely for 24 consecutive weeks.Twenty-four hour urine sample,blood and liver samples were collected.Hepatic fibrosis indices,specifically,type Ⅲ precollagen (PC Ⅲ),type Ⅳ collagen (Ⅳ-C),hyaluronic acid (HA) and laminin (LN) were detected by enzymelinked immunoassay (ELISA).Based on the isobaric tags for relative and absolute quantitation (iTRAQ) reagent 8-plex experiment,combined with 2DLC-MS/MS,the proteins in rats liver tissue of the medium dose group and the high dose group were compared with the those of control groups.Results ①The serum HA contents in the C (control) group,the L (low dose) group,the M (medium dose) group and the H (high dose) group were (198.51 ± 16.64),(218.39 ± 34.98),(261.72 ± 30.56) and (297.31 ± 35.72) ng/L;the serum PCⅢ contents in C,L,M and H groups were (15.32 ± 2.15),(16.78 ± 2.64),(19.51 ± 0.85) and (21.42 ± 1.63) μg/L;the serum LN contents in C,L,M and H groups were (734.57 ± 86.00),(792.65 ± 94.15),(916.83 ± 84.40) and (1 008.09 ± 64.17) μg/L;the serum Ⅳ-C contents in C,L,M and H groups were (52.34 ± 14.65),(59.72 ± 12.84),(74.38 ± 4.83) and (78.46 ± 4.30) μ.g/L,respectively.The differences in serological indices of liver fibrosis between-groups were statistically significant (F =21.136,19.957,22.007,14.288,all P ＜ 0.05).In multiple comparison of serum HA,PCⅢ and LN,there were no statistical significant differences between L group and C group.M and H groups were higher than L group and C group,significant statistical difference was found between H group and M group (all P ＜ 0.05).②Combining iTRAQ with 2DLC-MS/MS,based on the confidence threshold of protein (unused protScore) ＞ 1.3 and at least 1 matched peptides within the 95％ confidence interval,2 948 proteins were identified.Totally 2 162 proteins were detected in three groups compared with Venn diagram,after removing significant different proteins in C group,687 up-regulated proteins and 548 down-regulated proteins were identified in M group;633 up-regulated proteins and 519 downregulated were found in H group;the differences of protein expression between M and H groups were not statistically significant (P ＞ 0.05).③Up-regulated proteins related to the metabolism including AS3MT,MAT,SHMT,CHDH,CTH,CSAD and BHMT in M and H groups;of the two kinds of proteins of MTR,METK1 was up-regulated and F1LRB8 was down-regulated.Proteins associated with GSH including Gsta1,Gsta4,Gsta5,Gstt1,Gstt2,Gstk1,Gstp1,Gstm1,Gstm2,Gstm3,Gss,Gpx1,Gpx4,Esd,Hagh,Glo1,Mgst1 and B6DYQ5 which were all up-regulated.Proteins associated with liver fibrosis were Hic-5,Gss and six kinds of Tpm,and six kinds of Tpm subunits including two kinds of Tpm1,three kinds of Tpm2 and one kind of Tpm3 which were all up-regulated.Conclusions There is liver accumulation of arsenic after chronic arsenic exposure and resulting in liver fibrosis and decline of liver function.Expressions of AS3MT,MTR,MAT,SHMT,BHMT,CHDH,CTH and CSAD are up-regulated;arsenic meta bolism methionine cycle,folic acid cycle and sulfur transfer pathways are closely related.GSH plays an important role in arsenic metabolism and liver fibrosis,Hic-5,GSS and TPM may be associated with the occurrence of liver fibrosis.

Asingleparticle-inductivelycoupledplasmamassspectrometric(SP-ICP-MS)methodwas established to detect the size distribution and number concentrations of silver nanoparticle ( AgNPs) in dilute aqueous solution. The optimal dwell time was 3 ms to reduce possibility of two or more particles entering into detector simultaneously. An iterative algorithm was applied to distinguish AgNPs as outliers from baseline and dissolved metal ion signal if the measured intensity was beyond five time standard deviation of whole data. Size distribution and number concentration of three commercial silver nanoparticle dispersions ( nominal diameters of 30, 50, 100 nm) were determined using SP-ICP-MS. The result of SP-ICP-MS is accurately similar to the transmission electron microscopy ( TEM) , indicating that SP-ICP-MS is able to size silver nanoparticles. The particle size detection limit is 25 nm and the limit of number concentration is 8 × 104 particles/L in dilute solution. Tap water added with silver nanoparticle was tested to obtain a similar size distribution and number concentration. This method is simple, fast and highly sensitive, which can be used to investigate risk assessment of silver nanoparticle in aqueous environment and monitor silver nanoparticle in drinking water.

Objective To study the alteration of Th17 cells and related molecules in patients with Hashimoto's thyroiditis (HT).Methods Th17 cells were determined by flow cytometry.Real-time PCR method was applied to detect the expression of the orphan nuclear receptor RORγt and interleukin(IL) -17.Serum IL-6 and IL-23 were detected by ELISA method.Results ( 1 ) Compared with healthy controls,the frequency of Th17 cells [ (0.75± 0.79) ％ vs ( 0.28 ± 0.23 )％ ] and expression of RORγt ( 0.30 ± 0.38 vs 0.04 ± 0.02,both P ＜ 0.05 ) were significantly increased in HT patients.( 2 ) The levels of IL-6 and IL23 in HT patients were higher than those in healthy controls ( 3.66 ± 4.70 vs 0.47 ± 1.11,154.7 ± 75.81 vs 80.65 ± 61.41,both P＜0.05 ).( 3 ) A positive correlation between Th17 cells and serum TgAb was revealed in HT patients ( r =0.848 4,P =0.007 7 ).(4) The results of PCR showed that IL-17 and RORγt expressed in thyroid tissues of patients HT.Conclusion An increased frequency of Thl7 cells was found in HT patients,implying that this cell subset may play an important role in the pathogenesis of autoimmune thyroid disease.

Objective To investigate the effect and mechanism of decorin (DCN) on invasion of colorectal cancer cell line HCT116 in vitro.Methods Transwell assay was employed to detect the invasion of HCT116 cells;Real-time PCR was used to detect the expression of CD133 and TIMP-2 mRNA of HCT116 cells;Western blot method was used to detect the expression of HIF-1α, CD133 and TIMP-2 protein of HCT116 cells.Results 1) When the concentrations of DCN was 0, 1 and 3 mg/L, under the conditions of normal oxygen and hypoxia, the numbers of invasive cells were (241±46), (168±46), (51±17) fields in each well (P<0.01) and (207±61), (213±64), (156±54), (44±17) fields in each well (P<0.01).2) Under the normoxic conditions, the TIMP-2 mRNA and protein in HCT116 cells were increased by DCN (3 mg/L) (P<0.01), but that of CD133 were not affected.3) DCN (3 mg/L) significantly decreased the expression of HIF-1α/CD133/TIMP-2 protein in HCT116 cells under hypoxia (P<0.01), but had no significant effect on the expression of CD133 mRNA.ConclusionsUnder the conditions of hypoxia and normal oxygen, DCN may function through different mechanisms to inhibit the invasion of colorectal adenocarcinoma cell line HCT116 in vitro.

Objective:Through determination of urinary arsenic metabolites in high water arsenic exposed areas of Jilin and Shanxi provinces, to explore the mode and possible influencing factors of arsenic metabolism in different populations.Methods:From October 2018 to August 2019, a cluster sampling was carried out in villages (arsenic in drinking water ≥0.05 mg/L) of some townships (towns) in Lyuliang City, Shanxi Province and Baicheng City, Jilin Province for epidemiological investigation and general health examination. The residents over 35 years old drinking water from local centralized water supply and small well water sources were selected as arsenic exposure group, and people (nearby low-arsenic water source areas) with the same diet and living habits and similar economic conditions were selected as control group. Urine samples were collected. Liquid chromatography-atomic fluorescence spectrometry(LC-AFS) technology was used to separate and detect 4 species of arsenic compounds, including trivalent inorganic arsenic (iAs Ⅲ), pentavalent inorganic arsenic (iAs Ⅴ), methylated arsine (MMA), and dimethylated arsine (DMA). Total arsenic (tAs), inorganic arsenic percentage (iAs%), MMA percentage (MMA%), DMA percentage (DMA%), primary methylation index (PMI) and the secondary methylation index (SMI) were calculated. The influencing factors of arsenic metabolism were analyzed by multiple linear regression. Results:A total of 1 415 villagers were investigated, including 1 256 in arsenic exposure group and 159 in control group. Compared with the control group, there were no significant differences in age, gender ratio and occupation distribution between arsenic exposure group and control group ( P > 0.05), but there were significant differences in smoking, drinking, body mass index (BMI) and education level distribution ( P < 0.05). The median of urinary tAs, iAs%, MMA%, DMA%, PMI and SMI in control group and arsenic exposure group were 12.86 μg/L, 15.03, 5.23, 76.35, 84.97, 93.68 and 69.68 μg/L, 10.24, 8.37, 79.31, 89.76, 90.65, respectively, the levels of urinary tAs, DMA% and PMI in arsenic exposed group were higher than those in control group, while iAs% and SMI were lower than those in control group, the differences were statistically significant ( U=- 13.87, - 4.30, - 6.64, - 6.64, - 1.99, P < 0.05). After analysis of the factors influencing urinary arsenic metabolism in the population, we found that age and BMI had an impact on iAs% ( β=- 0.08, - 0.08, P < 0.05); gender, drinking, BMI and education level were influencing factors of MMA% ( β =- 0.11, - 0.09, - 0.07, 0.08, P < 0.05); DMA% was mainly affected by age, gender, BMI and education level ( β = 0.06, 0.09, 0.10, - 0.09, P < 0.05); PMI was mainly affected by age and BMI ( β = 0.08, 0.08, P < 0.05); while SMI was affected by gender, drinking, BMI and education level ( β=0.09, 0.08, 0.08, - 0.09, P < 0.05). Conclusions:The urinary arsenic metabolism models of different arsenic exposed groups are different. Age, gender, smoking, drinking, BMI and education level may be influencing factors of different arsenic metabolism models.

Migraine is a complex disorder of brain function. The hypothalamus has been identified to play a crucial role in attack generation and secretes various neuropeptides. The orexinergic system plays a role in energy metabolism, arousal, sleep, stress and pain modulation. These disorders are closely related to clinical symptoms of migraine. Therefore, the study of the mechanism of hypothalamic orexinergic system in migraine may provide a new perspective for treatment. This article discusses the relationship between hypothalamic orexin system and migraine premonitory symptoms, and briefly summarizes the possible role and mechanism of hypothalamic orexin system in migraine.

Objective To observe the expression dynamics of lens-related transcription factors in human embryonic stem cell (hESC) differentiated into lentoid body(LB).Methods A "three-stage" protocol was used for LB directional differentiation from hESC in vitro.The hESC (D0) and three differentiation stages cells were collected to analyze the expression dynamics of lens development-associated transcription factors by high throughput RNA sequencing technology in hESC-induced LB.Western blot and cell immunofluorescence were used to observe the involved genes at protein level.Results During D0-D6,cells became more round and compact.And during D7-D18,cells morphology gradually changed to spindle.At the end of D35,three-dimensional and transparent structurelentoid body (LB) was obtained.RT-PCR results showed that the stem cell related genes reduced and the lens specific genes increased significantly,and the LB was characterized by the expression of crystallins.According to clustering analysis of high throughput sequencing,a distinct difference in transcription factors gene expression was observed between D0 and D32.Meanwhile,the difference between D6 and D18 was minimum.The expressions of preplacodal genes,including DLX3,DLX5,DLX6,HES1,HES4,OTX2 and EYA1 increased remarkably at the first induction stage and then decreased.Lens-specification gene SOX2 declined gradually and then increased.In addition,the expression of PAX6 increased during all three induction stages.Furthermore,lens-differentiation genes including MAB21L1,CMAF,PROXI and PITX3 had no significant change in the early induction stage,but increased significantly at the third induction stage.Conclusions The expression dynamics of lens development-associated transcription factors in the hESC induced LB corresponded to those in vivo,which indicate that this induction system can recapitulate early lens development well and lay the foundation of studying lens embryonic development andtranscription factor associated congenital lens diseases.

Objective To explore the expression profiles and their clinical significance of serum exosomal microRNA (miRNA ) in the different phases of natural history in chronic hepatitis B (CHB ) patients .Methods A total of 92 treatment-naive CHB patients in Shanghai Public Health Clinical Center between January 2014 and January 2017 were retrospectively studied .The cases in immune tolerant (IT) phase ,immune reactive (IR) phase ,inactive carrier (IC) phase and HBeAg-negative CHB (ENH) phase were 24 ,24 ,24 ,and 20 ,respectively .Exosomes were isolated by ExoQuick solution from 250μL serum . The expressions of the surface protein markers LAMP2 and TSG101 in serum exosomes were determined by Western blotting . The expressions of miRNA-122 , miRNA-125a , miRNA-29c , miRNA-200c in exosomes were detected by real-time fluorescent quantitative PCR .The Kruskal-Wallis H test and Mann-Whitney U test were used to determine intergroup differences . The correlation coeffcients ( r) were calculated using Spearman′s correlation .Receiver operating characteristic (ROC) and the area under the curve (AUC ) were used to calculate the diagnostic values . Results Western blotting showed that exosome-specific markers LAMP2 and TSG101 were positive .The levels of serum exosomal miRNA-122 , miRNA-125a ,miRNA-29c and miRNA-220c were significantly different in CHB patients in different phases (H=41 .06 ,29 .31 ,49 .14 and 31 .73 ,respectively ,all P<0 .05) .Based on the results of liver function test and liver biopsy , serum exosomal miRNA-122 , miRNA-29c and miRNA-200c in inflammation group were down-regulated (U = 804 ,317 and 574 ,respectively ,all P< 0 .05) ,while miRNA-125a was up-regulated (U=279 ,P<0 .01) compared with non-inflammation group .The level of exosomal miRNA-200c was negatively correlated with ALT (r= -0 .3932 ,P<0 .01) ,while the level of miRNA-125a was positively correlated with ALT (r=0 .5981 , P<0 .01) .AUC of the above exosomal miRNA were 0 .6193 ,0 .8396 ,0 .8243 and 0 .6883 ,respectively .The highest AUC was miRNA-125a with the sensitivity of 84 .62％ and the specificity of 74 .47％ .AUC of ALT discrimination for liver inflammation was 0 .7953 ,with the sensitivity of 81 .08％ and the specificity of 70 .97％ .Conclusions The serum exosomal miRNA levels are significantly different among the different phases of natural history in CHB patients .Inflammation-associated exosomal miRNA is expected to be the non-invasive diagnostic biomarkers of liver inflammation and is of great benefit for determining the best time for clinical medication of CHB patients .

Objective To evaluate a model for axillary lymph node involvement combining CK19 mRNA with contrast enhanced ultrasound sonography (CEUS) score in operable breast cancer.Methods Operable breast cancer patients planned for sentinel lymph node (SLN) biopsy were enrolled.Preoperative CK19mRNA expressions in peripheral blood and CEUS score of axillary lymph nodes were tested before surgery.In the training set,postoperative sentinel lymph node (SLN) and non-sentinel lymph node (nSLN) pathological results were taken as the gold standard,effective modeling variables were screened,logistic regression was used to establish the prediction model.Parallel control studies were conducted between the validation set and the MSKCC model to evaluate the prediction accuracy and prediction efficiency.Results From Oct 2015 to Nov 2016,359 cases (training set) were enrolled and mathematical formulas for predicting SLN and nSLN were established,respectively.The sensitivity,specificity and AUC of predicting SLN involvement were 91.36％,94.92％ and 0.979 respectively.The sensitivity,specificity and AUC of predicting nSLN metastasis were 91.04％,90.53％ and 0.932 respectively.From Dec 2016 to Jul 2017,219 cases (verification set) were included.The sensitivity of SLN metastasis predicted by the model was 91.84％,the specificity was 96.69％,and the AUC was 0.979,significantly superior to the MSKCC model (0.739).The sensitivity,specificity and AUC of predicting nSLN metastasis were 95.35％,92.73％ and 0.945 respectively,significantly superior to the MSKCC model (0.873).Concolusions Combined with peripheral blood CK19 mRNA and CEUS score,the prediction model for axillary lymph node involvement for operable breast cancer,SLN/nSLN involvement probability can be calculated and qualitative judgment can be made.The overall accuracy and AUC of this model are better than the prediction model of MSKCC.