OBJECTIVE To explore the mechanism of action of epigallocatechin-3-gallate(EGCG) in inhibiting laryngeal cancer cells. METHODS The expression of epidermal growth factor receptor(EGFR) in laryngeal cancer cell lines AMC-HN-8, TU686 and TU212 was detected by Western blotting, and the inhibitory effects of cetuximab and EGCG on three laryngeal cancer cells were detected by CCK-8 assay. A lentiviral vector containing EGFR promoter and Luc reporter gene was constructed to generate a TU686-EGFR-Luc cell line that could steadily express Luc activity. Luciferase assay was performed to evaluate the effect of EGCG on the transcription activity of EGFR promoter. Cell cycle and apoptosis of EGCG-treated laryngeal carcinoma cells were analyzed by flow cytometry, and changes of the levels of EGFR and downstream ERK1/2, cell cycle-associated proteins P53 and P27, apoptosis-associated proteins BCL2 and PART, and autophagy marker LC3A/B were further examined. RESULTS The laryngeal carcinoma cell lines were insensitive to cetuximab but could be effectively suppressed by EGCG. EGCG effectively inhibited the transcription activity of EGFR promoter. Treatment of TU686 cells at sub-IC50 dose EGCG resulted in significant cell cycle arrest at S phase with partial apoptosis. Significant inhibition of expression and activation of EGFR and downstream signaling pathway were observed. CONCLUSION EGCG can effectively downregulate EGFR and suppress laryngeal carcinoma cells, further investigation on in vivo effect and mechanisms are anticipated.

ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation, invasion and drug resistance, and it serves as a therapeutic target. However, only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present. Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor. The recombinant shuttle plasmid pDC316-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc. The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged, characterized and amplified using AdMax^TM adenovirus packaging system. The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells. Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated. The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc, leading to significant inhibition of EGFR/ERK signaling pathway. Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.
Adenoviridae/genetics* ; Cell Proliferation ; Estrogen Receptor alpha/metabolism* ; Recombinant Proteins ; Transfection

Objective:To observe the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in regulating apoptosis during malignant transformation of human bronchial epithelial cells (HBE cells) induced by sodium arsenite (NaAsO 2). Methods:HBE cells were treated with 0.0 and 1.0 μmol/L NaAsO 2, which were control group and arsenic exposed group respectively. HBE cells were treated with 1.0 μmol/L NaAsO 2 for 43 passages to establish a malignant transformation model. The dynamic changes of indexes in different passages (0, 1st, 8th, 15th, 22nd, 29th, 36th, and 43rd) after exposure to NaAsO 2 were monitored, including the apoptosis rate detected by flow cytometry and apoptosis-related proteins and Nrf2 protein detected by Western blotting. Nrf2 siRNA was transfected into malignant transformed HBE cells (T-HBE cells) to silence Nrf2. The silencing effect of Nrf2 protein was verified. And, the apoptosis rate and apoptosis-related proteins were detected. Results:With the increase of arsenic exposure, the apoptosis rates of HBE cells decreased (0, 1, 8, 15, 22, 29, 36 and 43 passages were 0.370 ± 0.029, 0.443 ± 0.069, 0.357 ± 0.046, 0.330 ± 0.016, 0.273 ± 0.050, 0.160 ± 0.024, 0.110 ± 0.022, 0.097 ± 0.012, respectively, Ftrend = 22.981, P < 0.05). Compared with the 0 passage cells, the apoptosis rates of the 22nd, 29th, 36th and 43rd passages in the arsenite group were lower. The differences between them were statistically significant ( P < 0.05). With the increase of arsenic exposure, the expressions of pro-apoptotic proteins caspase-3, cleaved-caspase-3, C/EBP-homologous protein (CHOP) and B-cell lymphoma-2 (Bcl-2) associated X protein (Bax) showed downward trends ( Ftrend = 22.356, 3.738, 6.130, 8.061, P < 0.05), while the anti-apoptotic proteins myeloid cell leukemia 1 protein (Mcl-1) and Bcl-2 showed upward trends ( Ftrend = 58.201, 7.691, P < 0.05). Compared with the 0 passage and the control group of the same passage, from the 22nd passage of caspase-3, cleaved-caspase-3, from the 15th passage of CHOP, Mcl-1, and Bcl-2, from the 29th passage of Bax in the arsenite group, the differences of protein were statistically significant ( P < 0.05). However, there were no significant differences in caspase-8, cleaved-caspase-8, caspase-12 and cleaved-caspase-12 protein expressions in the arsenic group ( P > 0.05). Compared with the 0 passage and the control group of the same passage, from the 8th passage of Nrf2 proteins in the arsenite group, the differences of expressions were statistically significant ( P < 0.05). Compared with T-HBE cells transfected with Con siRNA (control), the apoptosis rate of T-HBE cells transfected with Nrf2 siRNA was higher ( P < 0.05). Compared with T-HBE cells transfected with Con siRNA, the expression levels of Nrf2, Bcl-2 and Mcl-1 in T-HBE cells transfected with Nrf2 siRNA were lower ( P < 0.05), while the expression levels of cleaved-caspase-3/caspase-3, caspase-3, cleaved-caspase-3, CHOP, and Bax were higher ( P < 0.05). Conclusion:Nrf2 may regulate mitochondrial apoptotic pathway through Bcl-2, Mcl-1 and Bax, and endoplasmic reticulum apoptotic pathway through CHOP, so as to inhibit the apoptosis of HBE cells and participate in the process of malignant transformation of HBE cells induced by NaAsO 2.

Objective: To evaluate the aerosol concentration(PM2.5,PM10.0 and aerosol particle number) formation in non-contact "air-puff" tonometry and provide suggestions for medical workers to take appropriate daily protection during the prevalence of 2019-nCoV. Methods: A cross-sectional study was carried out in this study. Thirty healthy subjects were enrolled on February 22, 2020 at Eye Hospital of Wenzhou Medical University. The intraocular pressure (IOP) was measured by non-contact "air-puff" tonometer in the ophthalmic consulting room and the hall with or without masks. PM2.5, PM10.0 and aerosol particles were recorded by air quality detector. The cumulative effects of IOP measurement, PM2.5, PM10.0 and aerosol particle number were analyzed, and the aerosol density of subjects with and without masks was compared. Results: The PM2.5, PM10.0 and aerosol particles produced by the non-contact "air-puff" tonometry and increased with the increase of spray times. The IOP curves of 60 eyes of 30 subjects were measured respectively in two environments of medical consulting room and medical institution hall. It was found that PM2.5, pm10.0 and particle number fluctuated and increased with the increase of IOP measurement person times, showing cumulative effect, and the accumulation speed of aerosol density in hall was faster than that in consulting room. The density of PM2.5 and PM10.0 produced without gauze mask were (53.417±2.306) and (85.350± 3.488) μg/m³, which were higher than those of (50.567±0.862) and (80.617±1.463) μg/m³ with gauze mask. The differences were statistically significant (P=0.028, 0.019). Conclusions Aerosol can be produced by non-contact "air-puff" tonometer spraying, and it fluctuates with the increase of spraying times, showing a cumulative effect. The aerosol accumulation is higher in the hall with insufficient air circulation. And more aerosol can be produced without gauze mask.

Objective: To investigate eye care behaviors among primary students in Wenzhou during Novel Coronavirus epidemic(COVID-19),and to provide a basis for eye care education and myopia prevention strategy. Methods: A total of 1 127 students from grade one to grade six of six primary schools in Wenzhou were selected to participate in an on-line invistigation regarding class attendance and eye care behaviors during the epidemic, March 6-9, 2020. Results: During the epidemic period, the primary school students in Wenzhou mainly took classes on the Internet (936, 83.1%), and the main learning tools were computers (391, 34.7%) and mobile phones (344, 30.5%), with an average of 3.00 h of online class. On average, students needed 2.00 h to complete homework and 1.00 h of extracurricular reading every day. In addition to learning, the daily use of mobile phone or ipad, computer and TV was 1.00,0.50,1.00 h respectively. Limited by the epidemic situation, the average daily exercise time of students was 0.81 h, including 0.00 h of outdoor activities; the average daily sleep was 9.00 h . During the epidemic period, 553 pupils (49.1%) reported eye discomfort, of which the most common was dry eyes (379, 47.4%). Multivariate Logistic regression analysis showed that the main learning tools of non-electronic products, reading distance > 30 cm or not reading, exercise time >0.5 h and outdoor activity time > 0.5 h were the protective factors of ocular discomfort (P<0.05). More than 2 h homework and recreational screen time higher than 0.5 h were risk factors for ocular discomfort(P＜0.05). Conclusion During the epidemic period, online learning increasedeye strain.Time of electronic devices usage,correct posture for reading,moderate level of ourdan physical activity,as well as prevention and treatment of eye strain should be strictly monitored.

Drugs targeting immune checkpoint are used for cancer treatment, but resistance to single drug may occur. Combination therapy blocking multiple checkpoints simultaneously can improve clinical outcome. Therefore, we designed a recombinant protein rPC to block multiple targets, which consists of extracellular domains of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The coding sequence was inserted into expression vector and stably transfected into HEK293 cells. The culture supernatant was collected and rPC was affinity-purified. Real-time quantitative PCR was used to evaluate the expression levels of ligands for PD-1 and CTLA-4 in several human cancer cell lines. The binding of rPC with cancer cells was examined by immunofluorescence cell staining, the influence of rPC on cancer cell growth was assayed by CCK-8. The results showed that rPC could be expressed and secreted by stably transfected HEK293 cells, the purified rPC could bind to lung cancer NCI-H226 cells which have high levels of ligands for PD-1 and CTLA-4, no direct impact on cancer cell growth could be observed by rPC treatment. The recombinant protein rPC can be functionally assayed further for developing novel immunotherapeutic drugs for cancer.
Animals ; CTLA-4 Antigen ; genetics ; Cell Proliferation ; HEK293 Cells ; Humans ; Lung Neoplasms ; metabolism ; Programmed Cell Death 1 Receptor ; genetics ; Protein Binding ; Protein Domains ; genetics ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism

To construct a eukaryotic expression plasmid containing the luciferase reporter gene (Fluc) to quickly detect apoptosis. Four amino acids, Asp-Glu-Val-Asp (DEVD), the recognize motif of Caspase-3, were introduced into the middle of the Fluc-C and N fragment. Meanwhile, four amino acids, Asp-Glu-Val-Gly (DEVG), were selected as a negative control. Subsequently, the recombinant gene was cloned into the N and C terminal end of the split intein, and named as pFluc-DEVD and pFluc-DEVG. Then the plasmids were transfected into cells and renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. Then the apoptosis level was detected by the double luciferase reporter gene and the Western blotting analysis. The results showed that when apoptosis occurred, the content of firefly luciferase expressed in the pFluc-DEVD plasmid transfected group was about 3 times higher than pFluc-DEVG plasmid transfected group. Furthermore, Western blotting detection indicated that the Fluc level was significantly increased in pFluc-DEVD transfected group when pre-treated by apoptosis stimulants. The activation degree of Caspase-3 was closely related to the expression of Fluc, and had a significant statistical difference. These results confirmed that firefly luciferase protein expressed by pFluc-DEVD plasmid can be cleaved by the intracellular Caspase-3 enzyme, and this plasmid can accurately reflect the cell apoptosis level, which provides a useful method for quantitative detection of apoptosis.
Apoptosis ; Genes, Reporter ; Luciferases, Firefly ; Transfection

Objective: To study the impact of aging on pancreatic atrophy, fibrosis and exocrine hypofunction in patients with post-ERCP pancreatitis (PEP) and its severity. Methods: A retrospective study was conducted on 786 patients who underwent ERCP at the Affiliated Zhongshan Hospital of Dalian University from June 2011 to April 2018. Patients who were aged over 75 years were grouped into the elderly group while those aged less than 75 years were grouped into the younger group. The incidences and severity of post-ERCP pancreatitis in the two groups were analyzed. Results: In the elderly group, there were 308 patients. The average age was (81.8±4.8) years. In the younger group, there were 478 patients. The average age was (57.7±12.0) years. The average operation time for the elderly group was (52.5±14.1) minutes, and that for the younger group was (50.7±14.9) minutes. There were no significant differences in operation time and in the related factors between the two groups (P>0.05). There was no significant difference in the rates of hyperamylasemia between the two groups (29.9% vs 30.1%, P>0.05). The overall rate of PEP was 11.3% (89/786). In the elderly group, the rate of PEP was 6.5% (20/308), which was significantly lower than that in the younger group (χ²=11.765, P<0.05). The rates of mild, moderate and severe PEP in the elderly group was significantly lower than those in the younger group (all P<0.05). Hyperamylasemia and pancreatitis in the 2 groups were alleviated after conservative treatment. Conclusions Aging (≥75 years) resulted in pancreatic atrophy, fibrosis, exocrine hypofunction which had a protective effect on PEP.

Non-small-cell lung cancer (NSCLC), the most common type of lung cancer accounting for 85% of the cases, is often diagnosed at advanced stages owing to the lack of efficient early diagnostic tools. 5-Hydroxymethylcytosine (5hmC) signatures in circulating cell-free DNA (cfDNA) that carries the cancer-specific epigenetic patterns may represent the valuable biomarkers for discriminating tumor and healthy individuals, and thus could be potentially useful for NSCLC diagnosis. Here, we employed a sensitive and reliable method to map genome-wide 5hmC in the cfDNA of Chinese NSCLC patients and detected a significant 5hmC gain in both the gene bodies and promoter regions in the blood samples from tumor patients compared with healthy controls. Specifically, we identified six potential biomarkers from 66 patients and 67 healthy controls (mean decrease accuracy >3.2, P < 3.68E-19) using machine-learning-based tumor classifiers with high accuracy. Thus, the unique signature of 5hmC in tumor patient's cfDNA identified in our study may provide valuable information in facilitating the development of new diagnostic and therapeutic modalities for NSCLC.
5-Methylcytosine ; analogs & derivatives ; blood ; Biomarkers, Tumor ; blood ; genetics ; Carcinoma, Non-Small-Cell Lung ; blood ; diagnosis ; genetics ; Case-Control Studies ; Circulating Tumor DNA ; blood ; DNA Methylation ; Epigenomics ; Female ; Humans ; Lung Neoplasms ; blood ; diagnosis ; genetics ; Male ; Middle Aged

Objective To determine the risk factors for postoperative hyperactive-type delirium (PHTD) in elderly patients undergoing orthopedic surgery.Methods A total of 7 171 elderly patients of both sexes,aged more than or equal to 65 yr,of American Society of Anesthesiologists physical status Ⅰ-Ⅳ,who underwent orthopedic surgery from January 2008 to December 2012 in Second Affiliated Hospital of Wenzhou Medical University,were retrospectively analyzed.Data such as gender,age,preoperative electrolytes,blood glucose,hemoglobin,albumin,senile dementia and use of benzodiazepines,type of operation,anesthesia methods,operation time,intraoperative use of anticholinergic agents and benzodiazepines and hypotension (decrease more than 20％ of the baseline),and postoperative electrolyte,hemoglobin,albumin and hypotension were collected.The patients were divided into postoperative PHTD group (group PHTD) and postoperative non-PHTD group (group non-PHTD) according to whether PHTD developed within 7 days after operation.The risk factors of which P values were less than 0.05 would enter the multivariate logistic regression to stratify the risk factors for postoperative PHTD.Results Ninety-nine patients developed PHTD,and the incidence was 1.38％.The results of logistic regression analysis showed that age more than or equal to 80 yr,hip surgery and preoperative anemia were independent risk factors for postoperative PHTD (P＜0.05).Conclusion Age more than or equal to 80 yr,hip surgery and preoperative anemia are independent risk factors for postoperative PHTD in elderly patients undergoing orthopedic surgery.