Preparation and characterization of Ad-ERα-36-Fc-GFP / 生物工程学报

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 AdMaxTM 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.

Role of Nrf2 regulating apoptosis in arsenite induced malignant transformation of HBE cells / 中华地方病学杂志

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.

Aerosol formation during non-contact "air-puff" tonometry and its significance for prevention of COVID-19 / 中华实验眼科杂志

Objective:To evaluate the aerosol and concentration (PM2.5, PM10 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 in 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.Particulate matter (PM)2.5, PM10 and aerosol particles were recorded by air quality detector.The cumulative effects of IOP measurement, PM2.5, PM10 and aerosol particle number were analyzed, and the aerosol density of subjects with and without masks was compared.Results:The PM2.5, PM10 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 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 produced without gauze mask were (53.417±2.306) and (85.350± 3.488) μg/m 3, which were higher than those of (50.567±0.862) and (80.617±1.463) μg/m 3 with gauze mask.The differences were statistically significant (P=0.028, 0.019), and significant difference was found in aerosol density between with and without gauze mask, although more aerosol was produced without gauze mask in comparison to gauze mask (P=0.123). 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 and particulate matter accumulation is higher in the hall with insufficient air circulation, and more aerosol and PM can be produced without gauze mask.

Construction, expression and purification of a mammalian secretory recombinant fusion protein rPC / 生物工程学报

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.

Aerosol formation during non-contact "air-puff" tonometry and its significance for prevention of COVID-19 / 中华实验眼科杂志

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.

Long term exposure to low doses of sodium arsenite on effect of apoptosis in immortalized human skin cutin forming cells / 中华地方病学杂志

Objective To explore the mechanism of cell apoptosis of immortalized human keratinocytes (HaCaT cells) and protein expression related to this process after long term exposure to sodium arsenite (NaAsO2,1.0 μmol/L).Methods Malignant transformation model was set up through long-term exposure of HaCaT cells to 1.0 μmol/L NaAsO2.Cell passage for 0,1,7,14,21,28 and 35 generations in the process of malignant transformation were collected for measurement of cell apoptosis rate by flow cytometry,and apoptosis related proteins by Western blotting,including activation of cysteine protease 3,8 (cleaved-caspase-3,8),C/EBP homologous protein (CHOP),B-cell leukemia/lymphoma 2 (Bcl-2),and Bcl-2 associated X protein (Bax).Results Along with the arsenite treatment,the apoptosis levels were significantly decreased (F =26.770,all P ＜ 0.05),the apoptosis levels (0.307 ± 0.049,0.213 ± 0.055,0.163 ± 0.057,0.147 ± 0.035,0.053 ± 0.012) of the 7th,14th,21st,28th and 35thgenerations of cells after arsenite treatment were lower than that of control group of the 0th generation (0.393 ±0.021,all P ＜ 0.05).Compared between generations,there were statistical differences of the protein expression levels of cleaved-caspase-3,Chop,Bax and Bcl-2 in arsenite group (cleaved-caspase-3:1.000 ± 0.000,1.030 ± 0.027,1.104 ± 0.069,1.016 ± 0.087,0.838 ± 0.075,0.753 ± 0.082,0.677 ± 0.073;Chop:1.000 ± 0.000,1.059 ± 0.018,0.934 ± 0.095,0.976 ± 0.216,0.793 ± 0.136,0.651 ± 0.042,0.564 ± 0.056;Bax:1.000 ± 0.000,1.069 ± 0.037,1.028 ± 0.042,0.954 ± 0.118,0.641 ± 0.135,0.531 ± 0.132,0.429 ± 0.085;Bcl-2:1.000 ± 0.000,1.072 ± 0.023,1.249 ± 0.134,1.334 ± 0.143,1.633 ± 0.221,1.507 ± 0.152,1.461 ± 0.145,F =7.730,7.355,27.802,12.438,all P ＜ 0.05),compared with control group of the 0th generation (1.000 ± 0.000) and the same generation control group (1.000 ± 0.000),after the 21st generation,the differences were statistically significant (all P ＜ 0.05),while there was no difference of the protein expression levels of cleaved-caspase-8 (F =0.832,P ＞ 0.05).Conclusion In the process of malignant transformation,the apoptosis levels of HaCaT cells are inhibited after long term sodium arsenite exposure through mitochondria and endoplasmic reticulum (ER) stress signaling pathways.