【Objective】 To evaluate the effectiveness of random quality control sampling in blood sample detetion by ELISA. 【Methods】 Blood samples of 5 mL specification of blood donors from our blood station from May to July 2022 were selected for routine operation on a fully automated sampler. J standard substances(3 mL specification) as daily samples were added to A1 well, H12 well and random wells of HBsAg, anti-HCV, anti-HIV, and -TP, and then placed in a fully automated enzyme immunoassay analyzer for testing. With random well quality control as the internal quality control judgment standard, 20 consecutive tests were conducted and were divided into A1 (well) group, H12 (well) group and random (well) group according to different well positions. Quality control maps were drawn using Levey-Jennings quality control chart with random group as the framework, and were compared with the quality control map of A1 well and H12 well results in the same day. 【Results】 The mean quality control levels of infectious indicators of blood transfusion in blood donors by ELISA were: HBsAg 3.87±0.28, anti-HCV 3.79±0.38, anti-HIV 3.64±0.30 and anti-TP 4.53±0.51. 【Comparison】 of HBsAg, anti-HCV, anti-HIV and anti-TP, between random group, A1 group and H12 group were HBsAg 3.87± 0.28 vs 4.09±0.30 vs 3.64±0.26, anti-HCV 3.78±0.37 vs 3.96±0.38 vs 3.63±0.38, anti-HIV 3.63±0.31 vs 3.82±0.32 vs 3.48±0.28 and anti-TP 4.51±0.51 vs 4.71±0.52 vs 4.36±0.51, The S/CO value of each indicator were H12 group0.05) . Using random group as the quality control framework standard, 5 points in group A1 fell outside of +2s, and 1 point in group H12 fell outside of -2s, resulting in a total of 6 alarms. With the quality control substance placed in A1 well of the ELISA plate, the judgment of detection results of the entire ELISA plate could be inevitably affected, especially the last row of low concentration virus marker samples on the ELISA plate. 【Conclusion】 The application of random quality control sampling method in donor blood by ELISA is scientific and reasonable, which can reduce the systematic error caused by artificial setting of ELISA plate fixed well positions and can also discover edge effects that affect the detection results.

Objective To investigate the effects of fluoride at different concentrations on proliferation and apoptosis of primary rat ameloblast in vitro.Methods Ameloblasts were isolated from tooth germ of 4 days SD rat maxillomandibular molar and cultured in vitro.Cells were treated with NaF at 0.0 (control group),0.4,0.8,1.6,3.2 and 6.4 mmol/L for 24,48 and 72 h,respectively.Inverted microscope was used to observe cell morphology;immunochemistry method was used to identify ameloblasts;3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazolium bromide (MTT) assay was applied to measure cell viability at each time point.The cells were treated with 1.6 mmol/L NaF for 24 and 48 h,or after 50 mol/L caspase pan-inhibitor Z-VAD-FMK pretreatment 1 h,1.6 mmol/L NaF treatment for 48 h.Cell apoptosis was then tested by flow cytometry.In addition,activation of caspase-3 and poly (ADP-ribose) polymerase (PARP) were assessed by Western blotting to explore potential involvement of caspase activation in NaF-induced apoptosis.All data analysis was performed using SigmaStat V 3.5 software.Results ①Primary rat ameloblasts were in polygonal shape at low density and appeared like paving stone at high density with obvious nucleus,showing typical morphological characteristics of cells with epithelial origin.②The results of immunochemistry assay indicated that the cultured cells were positive in cytokeratin 14 (CK14) and ameloblastin (AMBN) staining,in accordance with the immunocytochemical characteristics of ameloblasts.③The effects of NaF on ameloblast proliferation were in a dose-and time-dependent manner.For low dose NaF (0.4 and 0.8 mmol/L) groups,cells treated for 24 h had significantly higher cell proliferation rates than that of the control group (0.0 mmol/L,P ＜0.05),the proliferation indexes for 0.0,0.4 and 0.8 mmol/L groups were 1.00 ± 0.00,1.38 ± 0.11 and 1.29 ± 0.13,respectively;the same doses of NaF had no obvious influence on cell proliferation at 48 h (1.00 ± 0.00,1.16 ± 0.14 and 0.94 ± 0.07,P ＞ 0.05);cell proliferation indexes at 72 h were significantly lower than that of the control group (0.87 ± 0.03 and 0.80 ± 0.04,P ＜ 0.05).Medium dose of NaF (1.6 mmol/L) did not cause obvious alterations in cell proliferation at 24 h (0.90 ± 0.08,P ＞ 0.05);while cell proliferation indexes at 48 and 72 h were obviously reduced than that of the control group (0.38 ± 0.03 and 0.26 ± 0.04,P ＜ 0.01).For high NaF concentration (3.2 and 6.4 mmo]/L) groups,cell proliferation indexes were significantly decreased at all time points compared with control cells,the rates for 3.2 mmol/L groups were 0.57 ± 0.14,0.08 ± 0.03 and 0.00 ± 0.00,respectively,and the rates for 6.4 mmol/L groups were 0.11 ± 0.04,0.00 ± 0.00 and 0.00 ± 0.00,respectively (P ＜ 0.01).④Flow cytometry was used to detect apoptosis.The results showed that treatment with 1.6 mmol/L NaF resulted in significantly increased apoptosis in ameloblasts at both 24 h [(5.80 ± 2.03)％] and 48 h [(17.45 ± 4.97)％] compared to the control group [(2.59 ± 0.95)％,P ＜ 0.05].In cells pre-treated with pan-caspase inhibitor Z-VAD-FMK,NaF-induced apoptosis was significantly lower than that of cells treated with only 1.6 mmol/L NaF [(9.43 ± 3.79)％ vs (18.26 ± 3.39)％,P ＜ 0.05].⑤Cleavage of caspase-3 and PARP was detected in ameloblasts treated with 1.6 mmol/L NaF for 48 h.Conclusion Overdose fluoride could inhibit proliferation and induce apoptosis via activation of caspase cascade in primary cultured rat ameloblasts.

Objective To study the effects of ghrelin on action potential (AP) and transient outside K+ current (Ito) in ventricular myocytes of diabetic cardiomyopathy (DCM) rats.Methods A rat model of DCM was established by single intraperitoneal injection of streptozotocin (STZ, 60mg/kg), and whole cell patch-clamp technique was used to record the effects of 10-7mmol/L ghrelin on AP and Ito current density in the DCM rat ventricular myocytes.Results Compared to DCM group, Ghrelin of 10-7mmol/L could significantly prolong APD50 [(12.49±2.32)％;n=7, P=0.037] and APD90 [(26.29±5.13)％;n=7, P=0.006] and decrease Ito current peak value [(23.14±3.07)％;n=9, P=0.021] in DCM rat ventricular myocytes.Conclusion Exogenous ghrelin is involved in the electrophysiological reconstruction of the heart of diabetic rats by decreasing Ito current and prolonging APD in STZ-induced DCM rat ventricular myocytes.

Background The advantages of endothelium keratoplasty(EK)include without corneal surface incision and sutures,remaining normal corneal topography,minimizing astigmatism and providing a healthy donor endothelial cell count and function.Using anterior segment optical coherence tomography(AS-OCT) to examine the morphology of the corneal graft after EK is helpful for the evaluation of the outcome of EK.ObjectiveThis study applies AS-OCT to observe the relationship of recipient cornea and donor graft and offer a basis for the evaluation of operative outcome.MethodsThe clinical data from 44 eyes of 42 patient who received DSAEK in Peking University Eye Center from September 2007 through April 2009 was reviewed retrospectively.These patients included 30 male and 12 female with the age from 15 to 83 years.Structure of cornea and relationship between recipient cornea and graft disc after surgery were clinically examined and recorded by slit-lamp digital camera and AS-OCT.This study was approved by the Ethics Committee of Peking University.Written informed consent was obtained from all of the patients before the operation.ResultsThe donor graft attached to posterior central surface of recipient in 28 eyes under the slit-lamp microscopy and AS-OCT.The thickness of donor graft was decreased,and graft sticked more closely to recipient cornea and was more transparent with the lapse of time.Complete dislocation of the donor graft in early stage was found in 5 eyes(11.36%) and partial dislocation in 2 eyes(4.55%).Curliness of graft edge occurred in 1 eye(2.27%) and eccentricity of donor graft in 8 eyes(18.18%).ConclusionAS-OCT can display the morphology and structure of cornea as well as the relationship between donor graft and recipient after DSAEK.It is helpful for evaluating the clinical effectiveness of DSAEK.

Objective To investigate the efficacy of using Xinjiang wild Artemisia rupestris L. crude polysaccharides ( WARCP) as an immunologic adjuvant for influenza virus vaccine( IVV) .Methods ICR mice were subcutaneously immunized with 0.3 μg of IVV and 1.5 μg of IVV alone or co-administered with 200 μg of WARCP on 0 d and 14 d.Antibody levels in serum samples were detected by using indirect ELISA.MTT method was used to measure the proliferation of splenocytes.The growth conditions of mice were observed as well.Results No significant differences in the body weight were observed between mice from different groups (P>0.05).The levels of influenza virus-specific IgG, IgG1 and IgG2a were signifi-cantly increased in mice injected with WARCP adjuvant (P<0.05).The levels of IgG antibody in mice im-munized with low-dose of IVV and WARCP were significantly higher than those in mice immunized with high-dose of IVV alone (P<0.05), indicating at least 80% reduction in vaccine dosage by adding WARCP as adjuvant.Moreover, WARCP significantly promoted the proliferation of lymphocytes (P<0.05).Conclu-sion Adding WARCP to IVV enhanced the efficacy of IVV by boosting humoral and cellular immunity re-sponses with the advantages of high safety and dose-sparing.This study suggested the possibility of using WARCP as a novel immunologic adjuvant for influenza virus vaccine.

Objective:To study the mechanism of ginsenoside Rh2 inhibiting HepG2 cells migration.Methods:HepG2 cells in logarithmic growth phase were cultured in 96-well plates,which were induced by different concentration Rh2,respectively for 24,48,72 hours.The cell inhibition was detected by Cell Counting Kit.Transwell chambers was used to checked HepG2 cell migration ability;luciferase was tested by Luciferase Reporter Assay system reagent;The expressions of P-ERK,ERK,P-P38,P-38,P-JUK,JUK,MMP3 proteins were detect by Western blot;the expression of AP1,MMP3 gene were detected by Quantitative PCR;The expression of AP1, MMP3 fluorescence protein were observed by fluorescence microscopy.Results:Administrated with different concentration of Rh2 after 24 ,48 ,72 h,the proliferation of HepG2 cells were inhibited ( P<0.05) ,and in dose-and time-dependent manner.Transwell assay showed Rh2 could significantly inhibited migration of HepG2 cells.The expressions of P-ERK , MMP3 proteins were significantly decreased,the expressions of P-JUK, P-P38 proteins were significantly increased, expression levels of ERK, P-38, JUK were no significant difference.Expression of AP1,MMP3 gene were significantly decreased,the expressions of AP1,MMP3 fluorescence proteins were significantly decreased.Conclusion:Ginsenoside Rh2 can activate MAPK pathway to inhibit the migration of HepG2 cells.

Objective: To investigate the inhibitory effect of Rh2 on HepG2 cells and explore the underlying mechanism.Methods: We used lentivirus carrying β-catenin to infect HepG2 cell, and detected expression of β-catenin using fluorescence microscopy.The effect of Rh2 on proliferation of HepG2-β-catenin and HepG2 cells was measured by CKK-8 assay,and flow cytometry was used to detect cell cycle and apoptosis.The activity of Gsk-3βwas checked by ELISA kit.The expression of Gsk-3β,β-catenin,Bax,Bcl2,CyclinD1,MMP3 genes were measured by qRT-PCR.In order to checked the relationship between β-catenin and TCF4,CHIP assay kit was used,the expression of Bax,Bcl2,CyclinD1,MMP3 genes were measured by PCR.The expressions of Gsk-3β,β-catenin,Bax,Bcl2,CyclinD1,MMP3 proteins were examined by Western blot.Results:HepG2 cells were successfully infected by pLOV-EF1a-MCS-3FLAG-β-catenin lentivirus,named HepG2-β-catenin.CCK-8 showed that ginsenoside Rh2 could effectively inhibit the proliferation of HepG2 and HepG2-β-catenin cells in vitro,which exhibits a dose-dependent manner at range of 10-160 μmol/L Rh2.The IC50 of Rh2 exposure on HepG2 cell for 48,72 h were 100 μmol/L and 58.12 μmol/L,but the IC50 of Rh2 exposure on HepG2-β-catenin for 48,72 h were 129.2 μmol/L,83.33 μmol/L,respectively.The IC50 of Rh2 exposure on HepG2-β-catenin cell was higher than HepG2 cell, compared with HepG2 group the differences was statistically significant ( P<0.01 ).Flow cytometry indicated that Rh2 could arrest HepG2 and HepG2-β-catenin cells in G0/G1 phase;the cell population in G0/G1 phase of HepG2+Rh2 group was(64.57±0.65)%,HepG2-β-catenin+Rh2 group was(58.61±2.01)%.Flow cytometry indicated that Rh2 could induced early apoptosis in HepG2 and HepG2-β-catenin cells.The apoptosis rate of HepG2 +Rh2 group was (17.27 ±2.77)%,HepG2-β-catenin +Rh2 group(9.02 ±1.76)%.The ELISA results indicated that HepG2 cells was induced by Rh2 for 12,24,48,72 h,the activity of Gsk-3βgradually increased,peak in 48 h,then decreased.Compared with control group,Rh2 induced HepG2 and HepG2-β-catenin cells for 48 hours, Gsk-3βactivity were increased, and their activity reduced after adding Bio, there were no significant differences between HepG2+Rh2 and HepG2-β-catenin+Rh2 groups.The PCR,CHIP and WB results showed that the expression of Gsk-3β,Bax gene and proteins increased,while theβ-catenin,CyclinD1,Bcl2,MMP3 gene and proteins down-regulation in HepG2 and HepG2-β-catenin cell induced by Rh2.Compared with HepG2-β-catenin +Rh2 group, the expression of other gene and proteins changed significantly,however,Gsk-3βwas no significant difference.Conclusion:Over-expression of β-catenin may weaken the phar-macological effects of ginsenoside Rh2 on HepG2 cells.The activity of Gsk-3βwas increased by ginsenoside Rh2 to degradeβ-catenin, affecting the expression of downstream genes,promoting apoptosis of liver cancer cells and inhibiting metastasis.

OBJECTIVETo investigate the inhibitory effect of trichostatin A (TSA) on the proliferation of HepG2 cells and explore the underlying mechanism.

METHODSHepG2 cells exposed to different concentrations of TSA for 24, 48, or 72 h were examined for cell growth inhibition using a cell counting kit, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under inverted microscope. The expressions of beta-catenin, HDAC1, HDAC3, H3K9, cyclinD1 and Bax proteins in the exposed cells were detected by Western blotting, and the expressions of HDAC1 and HDAC3 mRNAs by quantitative fluorescent PCR.

RESULTSExposure to TSA caused significant dose- and time-dependent inhibition of HepG2 cell proliferation (P<0.05) and resulted in increased cell percentage in G0/G1 and G2/M phases and decreased cell percentage in S phase. The apoptotic index in the control group was (6.22 ± 0.25)%, which increased to (7.17 ± 0.20)% and (18.14 ± 0.42)% after exposure to 250 and 500 nmol/L TSA, respectively. Exposure to 250 and 500 nmol/L TSA also caused cell morphology changes with numerous floating cells. The expressions of beta-catenin, H3K9 and Bax proteins were significantly increased and CyclinD1, HDAC1, and HDAC3 protein expressions decreased in TSA-treated cells, but the expressions of HDAC1 and HDAC3 mRNAs showed no significant changes.

CONCLUSIONSTSA can inhibit the proliferation of HepG2 cells and induce cell cycle arrest and apoptosis by inhibiting HDAC activity, promoting histone acetylation, and activating Wnt/beta-catenin signaling pathway.

Acetylation ; Apoptosis ; Cell Cycle Checkpoints ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Hep G2 Cells ; drug effects ; Histone Deacetylase 1 ; metabolism ; Histone Deacetylases ; metabolism ; Histones ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; Wnt Signaling Pathway ; bcl-2-Associated X Protein ; metabolism ; beta Catenin ; metabolism

Objective:To investigate the predictive value of early thyroid function changes on the efficacy of patients with Graves′ disease (GD) after 131I therapy. Methods:Data of patients with GD (59 males, 214 females; age (37.4±11.4) years) who underwent single therapy of 131I in Tianjin Medical University General Hospital from November 2017 to January 2019 were retrospectively analyzed. Symptoms, signs and laboratory tests (serum free triiodothyronine (FT 3) and serum free thyroxine (FT 4)) of patients were observed to assess the efficacy of 131I treatment. Efficacy was divided into complete remission (CR), partial remission (PR), non-remission (NR) or relapse. The changes of thyroid function (ΔFT 3=FT 3 before treatment-FT 3 after treatment)/FT 3 before treatment×100%; ΔFT 4=FT 4 before treatment-FT 4 after treatment)/FT 4 before treatment×100%) 1 month after 131I therapy in each efficacy group and differences among them were compared by using independent-sample t test, χ2 test, one-way analysis of variance and the least significant difference t test. ROC curves were drawn to analyze the predictive values of early thyroid function changes on the efficacy of 131I treatment for GD. Logistic regression analyses were performed to identify the influencing factors for the efficacy of 131I therapy. Results:CR rate and total effective rate of 273 GD patients after single therapy of 131I were 67.03%(183/273) and 92.67%(253/273), respectively. After 1 month, CR rate of euthyroidism group ( n=95) was significantly higher than that of hyperthyroidism group ( n=178; 81.05%(77/95) vs 59.55%(106/178); χ2=4.60, P=0.032). ΔFT 3 and ΔFT 4 at the first month were statistically significant and decreased sequentially in the CR group ( n=183), PR group ( n=70), NR or relapse groups ( n=20; F values: 15.40, 12.54, both P<0.001). ROC curve analysis showed that patients with ΔFT 3≥73.64% and (or) ΔFT 4≥59.03% had a higher probability of achieving CR, with sensitivities of 84.3% and 86.7%, and specificities of 62.6% and 62.6%, respectively. Logistic regression analysis showed that 24 h radioactive iodine uptake (odds ratio ( OR)=1.095, 95% CI: 1.031-1.139), dose of 131I given per gram of thyroid tissue ( OR=1.562, 95% CI: 1.321-1.694), ΔFT 3 ( OR=1.354, 95% CI: 1.295-1.482), ΔFT 4 ( OR=1.498, 95% CI: 1.384-1.608) were factors affecting the outcome of patients with GD treated with 131I treatment (all P<0.05). Conclusion:Effects of 131I treatment can be predicted based on the change of the thyroid function at the first month after 131I treatment in patients with GD.

Objective: To analyze the impact of metabolic risk factors on the epidemiological characteristics of the reactivation of inactive HBsAg carriers (IHC) and provide effective intervention measures to standardize the management of chronic hepatitis B infections. Methods: Based on the chronic hepatitis B infection cohort established in 2010 in Jiangsu province, six follow-up visits from 2012 to 2020 were conducted to analyze the characteristics and influencing factors of the hepatitis B reactivation of IHC and the impact of metabolic risk factors, including obesity, high blood pressure, diabetes and hyperglycemia. Results: From 2012 to 2020, 2 527 IHC and 17 730 person-years were observed during a median follow-up period of 7.0 person-years. Ninety-eight cases of hepatitis B reactivation, with a cumulative reaction rate, was 3.9%, and the incidence density was 5.53/1 000 person-years. Multivariate Cox proportional risk regression analysis showed that age and baseline HBV DNA were independent risk factors of HBV reactivation. Compared with the patients ≥60 years, 40-49 age group (aHR=2.16, 95%CI:1.20-3.90) and 20-29 age group (aHR=5.48, 95%CI:2.07-14.48) were significantly associated with hepatitis B reactivation. Compared with the HBV DNA negative patients at baseline, the risk of hepatitis B reactivation was higher in the group with low HBV DNA level 100-1 999 IU/ml (aHR=1.67, 95%CI:1.11-2.52). Stratification analysis results showed that compared with those without metabolic risk factors, in the ≥50 age group, patients with ≥2 metabolic risk factors showed adjusted HR of 2.73 (95%CI:1.08-6.96). Conclusions: The risk of hepatitis B being reactive is the persistent existence of IHC in communities in Jiangsu province, especially young adults, low-level HBV DNA carriers, and IHC with ≥2 metabolic risk factors. Follow-up for these IHC should be strengthened to reduce the risk of disease progression by antiviral treatment at the right time.
Cohort Studies ; DNA, Viral ; Hepatitis B/epidemiology* ; Hepatitis B Surface Antigens ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/epidemiology* ; Humans ; Risk Factors ; Young Adult