Biomarkers, Tumor ; Humans ; Lung Neoplasms ; Mesothelioma

Objective To prepare stable aqueous dispersions of chitosan/multi-walled carbon nanotubes (CS/MWCNTs) composites,and observe the effects of CS/MWCNTs on the growth of human umbilical vein endothelial cells (HUVEC).Methods CS/MWCNTs composites were prepared by electrostatic interactions between negatively charged MWCNTs and positively charged low-molecular-weight CS.The prepared CS/MWCNTs were characterized by transmission electron microscopy and Zetasizer nano-analyser.The cellular uptake of the fluorescently labeled CS/MWCNTs was observed by laser confocal microscopy after incubating with HUVEC for 24 h at different concentrations.In vitro cytotoxicity and cellular reactive oxygen were also detected.Results When the mass ratio of low-molecular-weight CS to MWCNTs was equal or greater than 10∶1,the CS/MWCNTs can be stabilized in solution.Cellular uptake experiments showed that the CS/MWCNTs could enter into the cells and locate mainly in the cytoplasm.Cytotoxicity study showed that the CS/MWCNTs composites was less toxic than MWCNTs alone at high concentration (10 and 20 μg/ml).However,there was no significant differencein the level of cellular reactive oxygen between the two groups (P＜0.05).Conclusions CS/MWCNTs composites showed low cytotoxicity and high stability,which would be a promising carrier for drug delivery.

Objective To investigate the impact of chitosan and alkylated chitosan DNA nanoparticles on the function of human naive CD4+T cells.Methods The secretion of cytokines (IL-4 and TNF-γ) was observed after the co-incubation of human naive CD4+T cells with nanoparticles 12 h,24 h and 48 h,respectively.ResultsNone of the nanoparticles induced the production of cytokines ( IL-4 and TNF-γ ).Conclusion Chitosan and alkylated chitosan DNA nanoparticles will not induce the differentiation of human naive CD4+ T cells into T1 or T2 and may be considered as a safe gene carrier.

Objective To generate recombinant Pichia pastoris for high-copy expression of human tissue factor pathway inhibitor (TFPI). Methods The cDNA encoding human TFPI was inserted into the expression vector pPIC9K and the constructed expression vector rhTFPI-pPIC9K was confirmed by restriction endonuclease analysis and DNA sequencing. The recombinant plasmids were subsequently transformed into Pichia pastoris GS115 cells, and the transformants were confirmed by PCR amplification of the genomic DNA.The recombinant Pichia pastoris with high copies of TFPI cDNA was screened by G418 selection. Western blot and TFPI ELISA Kit were employed to analyzing. The temperature, time and concentration of methanol for the induction of recombinant protein were optimized. Results PCR analysis and DNA sequencing confirmed the successful construction of the expression vector rhTFPI-pPIC9K. Real time quantitative PCR and Western blot analysis demonstrated the positive correlation between TFPI expression level and the copy number of TFPI cDNA in Pichia pastoris cells. Optimization of the induction condition significantly elevated the expression level and activity of TFPI (9.95±0.78 mg/L and 3.91±1.37 U/mL). Conclusion The Pichia pastoris strain with high copy of TFPI expression was successfully constructed, which lays a solid foundation for the further investigation on the function of TFPI and its application in the prevention and therapy of diseases.

ObjectiveTo isolate and identify endothelial progenitor cells (EPCs) from human umbilical cord,and to study the cell proliferation and gene transfection of green fluorescent protein plasmid in vitro.MethodsEPCs were isolated from human umbilical cord in enzyme digestion method.The biological characteristics of EPCs were identified by flow cytometry and laser confocal microscope.The enhanced green fluorescent protein (EGFP) gene transfection mediated by EPCs was investigated using Lipofectamine 2000 as transfection reagent.ResultsEndothelial progenitor cells isolated from umbilical cord formed typical endothelial cell colony 9 days later.These cellsdisplayed an improved positive expression of CD133 and kinase insert domain receptor (KDR).The endotheliallineage characteristics of expanded cells were confirmed by fluorescein isothiocyanate (FITC)-UEA-1 binding and DiI-ac-LDL uptake assay with the aid of laser confocal microscope.The transfection results demonstrated high expression of EGFP taking EPCs as host cell.ConclusionEndothelial progenitor cells isolated from umbilical cord can be propagated and induced to differentiate into endothelial cells in the appropriate culture conditions.EPCs demonstrated to be an ideal carrier for gene and cell therapy.

ObjectiveConjugation of fluorescent dye onto plasmid DNA was investigated in order to monitor delivery process of plasmid DNA.MethodsPlasmid was activated with bromine,stored for different timeintervals at 4 ℃ or room temperature,and subsequently coupled with 1,10-diaminodecane to prepare aminemodified plasmid DNA.Amine-modified plasmid was then reacted with isothiocyanate (FITC) for fluorescent labeling,and the labeling ratio was calculated after purification.The effect of storage conditions (time/temperature) of bromine-actived plasmid (BP) on fluorescent labeling efficacy was estimated,and the cell transfection efficiency of fluorescent plasmid-lipofectamine complex was observed.The fluorescent plasmid delivered by lipofectamine 2000 in A10 cells was observed by laser scanning confocal microscope (LSCM) and flow cytometry.ResultsThe experimental data showed that prolonged storage time of bromine-activated DNA had a negative effect on the labeling ratio,and lower storage temperature had a positive effect on the labeling ratio.It also demonstrated that FITC modification had no effect on the transfection efficiency of plasmid-lipofectamine complex as compared with that of unlabeled plasmid-lipofectamine complex,and FITC modified plasmid had enough fluorescent intensity to monitor cell uptake with flow cytometer and sub-cellular distribution with LSCM.ConclusionA facile method for conjugating fluorescent dye onto plasmid was established in the study,and could be utilized to trace the plasmid delivery for investigating the transfection mechanism.

Objective: To investigate the effect of chrysotile exposure on ribosomal DNA (rDNA) copy number and DNA damage response, so as to provide insights into the mechanism of asbestos-induced carcinogenesis. Methods: Human pleural mesothelial MeT-5A cells were treated with chrysotile suspensions at doses of 1.25, 2.5 and 5 μg/cm2 (low-, medium-, high-dose group), while PBS served as controls. MeT-5A cells were harvested 6, 24, 48 and 72 h post-treatment, and the rDNA copy numbers and the BIRC5, HRAS, GINS4 and RRM2 mRNA expression were determined using a quantitative real-time PCR (qPCR) assay. The apoptosis of MeT-5A cells and DNA damage were detected using Muse cell analyzer. The rDNA copy numbers, DNA damage responses and BIRC5, HRAS, GINS4 and RRM2 mRNA expression were compared in MeT-5A cells treated with different doses of chrysotile suspensions. Results: There were significant differences in 45S rDNA copy numbers among low-, medium-, high-dose groups and the control groups 6, 48 and 72 h post-treatment with chrysotile suspensions, and significantly lower 45S rDNA copy numbers were measured in low-, medium- and high-dose groups than in the control group 6 h post-treatment, while significantly higher 45S rDNA copy numbers were found in the high-dose group than in low- and medium-dose groups 48 and 72 h post-treatment (all P<0.05). There were significant differences in 5S rDNA copy numbers among low-, medium-, high-dose groups and the control groups 24, 48 and 72 h post-treatment with chrysotile suspensions, and significantly lower 5S rDNA copy numbers were measured in medium- and high-dose groups than in the control group 24 and 48 h post-treatment, while significantly lower 5S rDNA copy numbers were found in medium- and high-dose groups than in the low-dose group 24, 72 h post-treatment (all P<0.05). There were significant differences in the overall apoptotic rate of MeT-5A cells among groups at different time points, and the overall apoptotic rate of MeT-5A cells were significantly higher in medium- and high-dose groups than in the control group (all P<0.05), with late-stage apoptosis predominantly detected. There were significant differences in the rates of ATM activation and DNA double-strand break in MeT-5A cells among groups 72 h post-treatment, and higher rates of ATM activation and DNA double-strand break were measured in medium- and high-dose groups than in the control group (all P<0.05). In addition, there were significant differences in the relative mRNA expression of BIRC5, HRAS, GINS4 and RRM2 genes among groups 24 and 48 h post-treatment, and significantly lower BIRC5, HRAS, GINS4 and RRM2 mRNA expression was quantified in medium- and high-dose groups than in the control group (all P<0.05). Conclusion Exposure to chrysotile may induce rDNA copy number variations and altered expression of nucleolar proteins in human pleural mesothelial cells, which may be involved in the regulation of DNA damage responses.

Objective : To examine the effect of asbestos exposure on oxidative stress, so as to provide insights into the elucidation of pathogenesis and management of asbestos-related diseases. Methods : Totally 245 subjects were recruited from an asbestos manufacturing area in Zhejiang Province, and their gender, age and history of asbestos exposure were collected through a questionnaire survey. The serum levels of 8-hydroxy-2'deoxyguanosine ( 8-OHdG ), glutathione ( GSH ), malondialdehyde ( MDA ), superoxide dismutase ( SOD ) and total antioxidative capacity ( TAOC ) were measured using an enzyme-linked immunosorbent assay ( ELISA ), and the levels of catalase ( CAT ), peroxiredoxin 2 ( PRX2 ), SOD1, SOD2 and thioredoxin-1 ( TRX1 ) were detected in peripheral white blood cells ( WBCs ) using a liquid-chip assay. Multivariable linear regression analysis was performed to identify the association between asbestos exposure and oxidative stress parameters. Results : There were 50 subjects without a history of asbestos exposure (unexposed group), 102 subjects with asbestos exposure for less than 10 years ( AE<10-year group ) and 93 subjects with asbestos exposure for 10 years and more ( AE≥10-year group ). No significant differences were found among the three groups in terms of age, gender, proportion of smokers or proportion of alcohol consumers ( P>0.05 ). Significantly higher 8-OHdG and MDA in serum, and higher PRX2 in peripheral WBCs were detected in the AE≥10-year group than in the unexposed group ( P<0.05 ); lower GSH and TAOC in serum, and lower CAT in peripheral WBCs were detected in the AE≥10-year group than in the unexposed group ( P<0.05 ); higher 8-OHdG and MDA in serum, and higher PRX2 in peripheral WBCs were detected in the AE≥10-year group than in the AE<10-year group ( P<0.05 ). Multivariable linear regression analysis showed that asbestos exposure significantly correlated with 8-OHdG, MDA and TAOC in serum, and CAT and PRX2 in peripheral WBCs ( P<0.05 ). Conclusion Asbestos exposure may induce the oxidative stress damage, suggesting that oxidative stress may be involved in asbestos-related diseases.

Objective : To analyze the survival of patients with malignant mesothelioma, so as to provide insights into the management of malignant mesothelioma. Methods : Totally 36 patients with malignant mesothelioma admitted to Cixi Third People’s Hospital from October 2012 to January 2021 were enrolled, and the demographic features, exposure to asbestos, and diagnosis and treatment were retrospectively reviewed. The survival rate and median survival time were calculated with the life-table method, and the factors affecting the survival rate of malignant mesothelioma were identified using the Kaplan-Meier estimate and log-rank test. Results : The 36 patients with malignant mesothelioma included 6 men ( 16.67% ) and 30 women ( 83.33% ), and had a median age of 61 ( interquartile range, 14 ) years. There were 30 cases with pleural malignant mesothelioma ( 83.33% ) and 6 cases with peritoneal malignant mesothelioma ( 16.67% ), 32 cases ( 88.89% ) with a history of occupational exposure to asbestos, and 26 cases ( 72.22% ) receiving palliative treatment. The 1-, 2- and 3-year cumulative survival rates were 30%, 15% and 3%, respectively, and the median survival time was 0.71 years. In addition, there were no significant differences in the survival period among patients with malignant mesothelioma in terms of gender, age, route of asbestos exposure, duration of asbestos exposure, pathogenic site and treatment regimens ( P>0.05 ). Conclusion The 36 patients with malignant mesothelioma had a median survival period of 0.71 years, and no association was found between the survival period and asbestos exposure or pathogenic site.

Objective : To examine the diagnostic and prognostic value of long non-coding RNA (lncRNA) JPX in mesothelioma, so as to provide insights into diagnosis and prognosis of mesothelioma. Methods: Patients with clinically definitive diagnosis of mesothelioma from 2015 to 2019 that were sampled from asbestos processing plants in Zhejiang Province from 2015 to 2019 were recruited in the mesothelioma group, while healthy residents without asbestos exposure or asbestos-related diseases in the same area served as controls. Participants' demographics, pathologic diagnosis and imaging features were collected, and the expression of blood lncRNA JPX was detected using lncRNA microarrays. The diagnostic value of lncRNA JPX for mesothelioma was evaluated using the receiver operating characteristic (ROC) curve, and the correlation between lncRNA JPX expression and prognosis was examined among mesothelioma patients using survival analysis. Results: There were 17 subjects in the mesothelioma group, with a mean age of (65.71±8.36) years, and 34 subjects in the controls, with a mean age of (64.24±8.70) years. LncRNA microarray detected significantly high lncRNA JPX expression in mesothelioma patients, and higher blood lncRNA JPX expression was detected in the mesothelioma group than in the control group [median (interquartile range), 1.10 (1.31) vs. 0.89 (0.54); t'=-2.300, P=0.034]. The area under the ROC curve was 0.673 (95%CI: 0.507-0.839, P=0.046), and if the cutoff was 1.759, the sensitivity and specificity were 35.3% and 100.0%, respectively. Survival analysis showed no significant difference in the survival rate of mesothelioma patients between the high lncRNA JPX expression group and the low expression group (χ2=0.212, P=0.645). Conclusions LncRNA JPX overexpression is detected in the blood of patients with mesothelioma, and lncRNA JPX expression presents a diagnostic value for mesothelioma; however, it shows little prognostic value for mesothelioma.