[Objective] To investigate the correlation between human leukocyte antigen (HLA) gene polymorphism and hepatitis B virus (HBV) infection. [Methods] Venous blood samples were collected from 501 healthy individuals undergoing physical examinations at Yan’an Hospital in Kunming, Yunnan Province. Enzyme linked immunosorbent assay (ELISA) was used to detect HBV halves. Based on the results of HBV half detection, the patients were divided into three groups: HBV carrier group, previous infection group, and healthy control group. The HLA-A antigen genotype was detected using polymerase chain reaction with sequence specific primers (PCR-SSP) genotyping technology, and the distribution frequency of HLA-A gene polymorphism was compared between HBV carrier group and healthy control group, as well as between previous infection group and healthy control group. SPSS17.0 software was used for data statistical analysis. [Results] In the healthy control group, the HLA-A2 positivity rate was 47.49%, and the allele frequency was 31.29%.The overall frequency of gene distribution in the healthy control group was consistent with the HLA-A allele table commonly and confirmed in China published by the Chinese Bone Marrow Bank. The HLA-A2 positivity rate and allele frequency in the HBV carrier group were 63.04% and 42.23%, respectively; The difference in HLA-A2 positivity rate and allele frequency among carriers was statistically significant (P<0.05). the HLA-A2 positivity rate and allele frequency in the HBV previous infection group were 56.14% and 35.97%, respectively, which did not significantly differ from those in the healthy control group (P>0.05). [Conclusion] HLA-A2 gene may be a susceptibility gene for chronic hepatitis B HBV carriers.

Objective: Trimethylamine oxide (TMAO) is a metabolite of intestinal flora and is known to promote the progression of atherosclerotic plaques. However, how TMAO works, including its effect on vascular endothelial cells, is not fully understood. This study aims to explore the biological role of TMAO in human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. Methods: Cell pyroptosis and the loss of plasma membrane integrity were induced under TMAO stimulation in HUVECs. The plasma membrane integrity of the cells was measured by Hoechst 33342/propidium iodide (PI) staining and lactate dehydrogenase leakage assay, and the changes in cell morphology were observed by atomic force microscope. The expression of proteins related to pyroptosis was determined by Western blotting or immunofluorescence. Mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) activity in HUVECs was measured by the ALDH2 activity assay kit, and the level of reactive oxygen species (ROS) was detected by fluorescent probe DCFH-DA. Results: TMAO induced pyroptotic cell death, manifesting by the presence of propidium iodide-positive cells, the leakage of lactate dehydrogenase, the production of N-terminal gasdermin D (GSDMD-N), and the formation of plasma membrane pores. Moreover, TMAO induced elevated expression of inflammasome components, nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3),apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1 in cells. TMAO significantly inhibited ALDH2 activity and increased intracellular ROS production. However, the activation of ALDH2 by pharmacological manipulation attenuated TMAO-induced inflammasome activation and GSDMD-N production.Conclusion: TMAO induces pyroptosis of vascular endothelial cells through the ALDH2/ROS/NLRP3/GSDMD signaling pathway, which may be a potential therapeutic target for improving the treatment of atherosclerosis.

The study was designed to explore the drug-drug interactions mechanisms mediated by OATP1B1 between traditional Chinese medicine Danshensu and rosuvastatin. First, the changes of rosuvastatin pharmacokinetics were investigated in presence of Danshensu in rats. Then, the primary rat hepatocytes model was established to explore the effects of Danshensu on the uptake of rosuvastatin by hepatocytes. Finally, HEK293T cells with overexpression of OATP1B1*a and OATP1B1*5 were established using a lentiviral delivery system to explore the effects of Danshensu on the uptake of rosuvastatin. Rosuvastatin pharmacokinetic parameters of C(max0, AUCO(0-t), AUC(0-∞) were increased about 123%, 194% and 195%, by Danshensu in rats, while the CL z/F value was decreased by 60%. Uptake of rosuvastatin in the primary rat hepatocytes was decreased by 3.13%, 41.15% and 74.62%, respectively in the presence of 20, 40 and 80 μmol x L(-1) Danshensu. The IC50 parameters was (53.04 ± 2.43) μmol x L(-1). The inhibitory effect of Danshensu on OATP1B1 mediated transport of rosuvastatin was related to the OATP1B1 gene type. In OATP1B1*5-HEK293T mutant cells, transport of rosuvastatin were reduced by (39.11 ± 4.94)% and (63.61 ± 3.94)%, respectively, by Danshensu at 1 and 10 μmol x L(-1). While transport of rosuvastatin was reduced by (8.22 ± 2.40)% and (11.56 ± 3.04)% and in OATP1B1*1a cells, respectively. Danshensu significantly altered the pharmacokinetics of rosuvastatin in rats, which was related to competitive inhibition of transport by OATPJBI. Danshensu exhibited a significant activity in the inhibition of rosuvastatin transport by OATP1B1*5-HEK293T, but not by OATP1B1*1a, suggesting a dependence on OATP1B1 sequence.

Objective To study the relationship between socioeconomic status (SES) and esophageal squamous cell carcinoma(ESCC) in Taixing city,Jiangsu province.Methods A populationbased case-control study was carried out to collect data including demography,socioeconomic status indicators and possible risk factors.Unconditional logistic regression was used to compare different SES indicators and composite wealth scores constructed between cases and controls,using the principal component analysis methodology.Results Factors as:having received high school or higher education (OR=0.66,95％CI:0.46-0.96),living space over 67 m2 per-capita (OR=0.71,95％CI:0.54-0.94),drinking tap water longer than 5 years (OR=0.76,95％ CI:0.59-0.98) and wealth score ＞0.93 (OR=0.63,95％CI:0.48-0.83) were associated with increased risk of ESCC.Compared to those worked in farming,forestry,animal husbandry,fishery and water conservancy,the individuals who worked in business-service industry were at lower risk of ESCC (OR =0.65,95％CI:0.43-0.97).Conclusion An inverse association of low SES and ESCC were found in Taixing people that called for further explanation.

Objective To investigate the local immune response in condyloma acuminatum treated with aminolevulinic acid-photodynamic therapy (ALA-PDT).Methods In vitro and in vivo studies were performed.A previously established keratinocyte cell line human papilloma virus (HPV) 16E7/HaCaT which stably expresses HPV16E7 protein was used in this study.Peripheral blood mononuclear cells (PBMCs) were separated from 10 healthy volunteers.After pretreatment with ALA-PDT,HPV16E7/HaCaT cells were cocultured with the PBMCs for 3 hours in a Transwell chamber followed by the observation of chemotactic migration of PBMCs.Tissue samples were obtained from the lesions of 10 patients with condyloma acuminatum before,and at 1,2,3 and 48 hours after the first session of ALA-PDT.Immunohistochemistry was conducted to determine the number of CD4+ T cells,CD8+ T cells and CD68+ macrophages as well as CD4/CD8 T-cell ratio in the tissue samples.Results After 3-hour coculture with HPV16E7/HaCaT cells pretreated by ALA-PDT,PBMCs showed apparent chemotactic migration.Immunohistochemistry revealed a statistical increase in the number of CD4+ T cells,CD8+ T cells and CD4/CD8 T-cell ratio at 48 hours (all P ＜ 0.05),as well as in the number of CD68+ macrophages at 3 hours and 48 hours (both P ＜ 0.05) after the first session of ALA-PDT.Conclusion ALA-PDT may induce local antiviral immune response in condyloma acuminatum.

The medical records of all 12 patients diagnosed as chronic active Epstein-Barr virus (CAEBV) infection at our hospital were analyzed retrospectively.There were 7 males and 5 females with a median onset age of 28 years.CAEBV was characterized by fever,splenomegaly,hepatomegaly and lymphadenopathy,etc.The abnormalities of laboratory examination included liver dysfunction,thrombocytopenia,anemia and leucopenia.EBV-DNA detected by real-time polymerase chain reaction was (1.7 × 103-3.5 × 107) copies/μg DNA in peripheral blood mononuclear cell.Among them,the outcomes were death (n =5),lost to follow-up (n =2) and T cell lymphoma (n =1).It is necessary to improve our awareness of CAEBV infection because of its poor prognosis and high mortality.

Objective To investigate the protective effect of butyl flufenamate ointment against ultraviolet (UV)-induced skin damage,skin aging,and cutaneous squamous cell carcinoma (CSCC) in SKH-1 hairless mice.Methods A total of 128 mice were randomly and equally divided into four groups:UV group receiving UV irradiation only,butyl flufenamate ointment group and matrix cream group receiving UV irradiation after 30-minute pretreatment with topical butyl flufenamate ointment and matrix cream respectively,and blank control group receiving neither pretreatment nor irradiation.In the sunburn experiment (n =24),mice were exposed to single session of UV irradiation (1.5 minimal erythema doses (MEDs)),and 24 hours later,erythema and swelling response was observed,and skin tissue was obtained from the irradiated area on the back of mice followed by the determination of COX-2 expression using the streptavidin biotin peroxidase complex (SABC) method.To establish a photoaging (n =24) and CSCC (n =80) model,mice were exposed to four sessions of UV irradiation every week for 12 and 28 successive weeks respectively,with the irradiation dose starting at 0.9 MED and increasing gradually.After 12-week irradiation,skin tissue was resected from the back of photoaged mice and subjected to Masson staining for the evaluation of collagen changes as well as immunohistochemical analysis for the quantification of Bax,Bcl-2 and Caspase 3 expression.The initiation and progression of CSCC were observed in mice on a once-a-week basis from 12 to 28 weeks.SPSS 21.0 software was used for statistical analysis.One way analysis of variance was carried out for multiple-group comparisons of numerical data,Ridit analysis for the comparison of immunohistochemical staining intensity.Kaplan-Meier method and log-rank test were utilized for the comparison of tumor-free survival time.Results Both the degree of erythema and swelling response and expression level of COX-2 were significantly lower in the butyl flufenamate ointment group than in the other two UV-irradiated groups (all P ＜ 0.05).After 12-week irradiation,the butyl flufenamate ointment group showed milder degree of skin aging,together with higher density of collagen in dermis,weaker expression of Bcl-2 but stronger expression of Bax and Caspase 3,by comparison with the other two UV-irradiated groups (all P ＜ 0.05).During the 28 weeks of irradiation,the median tumor-free survival time was statistically longer in the butyl flufenamate ointment group than in the matrix cream group and UV group((25.0 ± 0.4) months vs.(24.0 ± 0.3) months and (23.0 ± 0.4) months,P ＜ 0.05 and 0.01 respectively).Conclusion Butyl flufenamate ointment has a certain photoprotective effect.

OBJECTIVE: To explore the changes of mTOR signal pathway in HeLa cells under different nutritional conditions infected with Coxsackie virus B3 (CVB3). METHODS: The HeLa cells were cultured with two methods: the conventional culture method cultured HeLa cells with medium with 10% fetal bovine serum for 24 h and changed the medium next day, and then infected with CVB3; the serum starvation method cultured HeLa cells with medium without fetal bovine serum for 24 h, and then infected with CVB3. The expression of the coat protein of CVB3, mTOR, p70S6K mRNA was detected with RT-PCR at different time points. RESULTS: The virus group showed the expressions of mTOR and p70S6K mRNA were significantly higher than those in the control group at 12 h and 24 h (P<0.05) in the conventional culture. The virus group showed the expressions of mTOR and p70S6K mRNA were lower than those in the control group (all P<0.05) in the starvation serum. The expression of mTOR mRNA in the starvation serum virus group was higher than that in the conventional culture virus group (all P<0.05) and the control group. The expression of p70S6K mRNA was not significantly different in the two groups (P>0.05). CONCLUSION CVB3 can down-regulate the expressions of mTOR and p70S6K mRNA. The mTOR expression in the starvation serum is higher than that in the conventional culture.
Cell Culture Techniques ; Down-Regulation ; Enterovirus B, Human ; pathogenicity ; HeLa Cells ; Humans ; RNA, Messenger ; genetics ; metabolism ; Signal Transduction ; physiology ; TOR Serine-Threonine Kinases ; genetics ; metabolism

Objective To establish a model for cutaneous squamous cell carcinoma by irradiation of SKH-1 hairless mice with solar-simulated ultraviolet (solar UV), and to explore the biological characteristics of the model. Methods A total of 91 SKH-1 hairless mice were randomly divided into seven experimental groups (n = 10) and seven control groups (n = 3). The mice in experimental groups were irradiated with minimal erythema dose of solar UV 4 times per week for various durations (4, 8, 12, 16, 20, 24, 28 weeks), while the control mice received no irradiation. The general status and skin appearance of mice were observed during the treatment process. Mice were killed immediately after the last irradiation at different time points and pathological examination was carried out to observe the histological changes of skin lesions. Results Papules measuring equal to or more than 1 mm in diameter began to develop in some mice in experimental group 10 weeks after the first irradiation; tumors began to appear in 39.3% (11/28) of the remaining mice in experimental group on week 20, and in 100% (10/10) of the remaining mice on week 28. The cumulative dose approximated to 26.99 J/cm2 for UVB and 242.91 J/cm2 for UVA after 28-week irradiation. No tumor was observed in the control mice. Pathological examination revealed characteristic changes of squamous cell carcinoma in 30% of the mice on week 12, 33.3% on week 16, 60% on week 20, 87% on week 24, and 100% on week 28. Conclusions Ultraviolet could induce the hyperplasia of skin in SKH-1 hairless mice, and even cause the development of cutaneous squamous cell carcinoma after prolonged irradiation.

Objective To establish a human keratinocyte cell line (HaCaT) stably expressing HPV16E7 protein. Methods HPV16E7 gene was amplified from CaSki cells using PCR and inserted into the eukaryotic expression plasmid pcDNA3.1. Then, the recombinant expression plasmid pcDNA3.1-HPV16E7 was transfected into HaCaT cells followed by G418 selection and identification by RT-PCR and Western blot. Results The recombinant eukaryotic expression plasmid pcDNA3.1-HPV16E7 was successfully identified by restriction enzyme digestion pattern and sequence analysis. Agarose gel electrophoresis of RT-PCR products detected the 297-bp fragment of HPV16E7 cDNA, and Western blot confirmed the stable expression of HPV16E7 protein. Conclusion A human keratinocyte cell line (HaCaT) stably expressing HPV16E7 protein is successfully established.