Objective: Although benzene is a confirmed environmental carcinogen, the mechanism of its carcinogenicity remains largely unclear. The suggested oncogene, miR-221, is elevated and plays important roles in various tumors, but its role in benzene-induced carcinogenesis remains unknown. Methods: In the present study, we constructed hydroquinone (HQ, a representative metabolite of benzene with biological activity)-transformed malignant cell line (16HBE-t) and analyzed the level of miR-221 in it with qRT-PCR. Exosomes from 16HBE-t cells incubated with or without an miR-221 inhibitor were isolated by ultracentrifugation, characterized by transmission electron microscopy and laser scanning confocal microscope, and then transfected into 16HBE cells. The effects of exosomal miR-221 on apoptosis induced by HQ in recipient cells were determined using flow cytometry. Results: The amount of miR-221 in 16HBE-t was significantly increased compared with controls. When recipient cells ingested exosomes derived from 16HBE-t, miR-221 was increased, and apoptosis induced by HQ was inhibited. Blocking miR-221 in 16HBE-t using an inhibitor did not significantly alter miR-221 or apoptosis in recipient cells. Conclusion Exosomal miR-221 secreted by 16HBE-t inhibits apoptosis induced by HQ in normal recipient cells.
Apoptosis ; Bronchi/cytology* ; Cell Line, Transformed ; Epithelial Cells ; Exosomes ; Humans ; Hydroquinones ; MicroRNAs

Autophagy is an evolutionarily-conserved self-degradative process that maintains cellular homeostasis by eliminating protein aggregates and damaged organelles. Recently, vesicle-associated membrane protein-associated protein B (VAPB), which is associated with the familial form of amyotrophic lateral sclerosis, has been shown to regulate autophagy. In the present study, we demonstrated that knockdown of VAPB induced the up-regulation of beclin 1 expression, which promoted LC3 (microtubule-associated protein light chain 3) conversion and the formation of LC3 puncta, whereas overexpression of VAPB inhibited these processes. The regulation of beclin 1 by VAPB was at the transcriptional level. Moreover, knockdown of VAPB increased autophagic flux, which promoted the degradation of the autophagy substrate p62 and neurodegenerative disease proteins. Our study provides evidence that the regulation of autophagy by VAPB is associated with the autophagy-initiating factor beclin 1.
Autophagy ; drug effects ; physiology ; Beclin-1 ; genetics ; metabolism ; Cell Line, Transformed ; Gene Expression Regulation ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microtubule-Associated Proteins ; genetics ; metabolism ; R-SNARE Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; RNA-Binding Proteins ; genetics ; metabolism ; Transfection

To discover the nephroprotective substances from Huangkui capsule.The components of Huangkui capsule were isolated by preparative liquid chromatography, and the active components were screened by LC/MS and identified. The adriamycine-injured HK-2 cells were treated with various active components with different concentrations, and the malonaldehyde (MDA) content, adenosine triphosphate (ATP) level and mitochondrial oxygen consumption rate were measured to verify the protective activity of the compounds.Four active components in Huangkui capsule were identified to exert nephroprotective effects. Fifteen flavanoids from these four components were tentatively identified by LC/MS, and hyperin, myricetin, quercetin, rutin and isoquercetin were confirmed. Hyperin, myricetin quercetin and rutin showed dose-dependent protective effects on injured HK-2 cells. Espacially, hyperin significantly reduced MDA content, quercetin and rutin significantly increased ATP level, and myricetin significantly increased mitochondrial oxygen consumption rate.Hyperin, myricetin, querctein and rutin might be the potential nephroprotective compounds in Huangkui capsule, their effects may be related to the inhibition of lipid peroxidation and the alleviation of mitochondrial damage.
Abelmoschus ; chemistry ; drug effects ; Adenosine Triphosphate ; metabolism ; Cell Line, Transformed ; Chromatography, Liquid ; Doxorubicin ; Drugs, Chinese Herbal ; Epithelial Cells ; drug effects ; Flavonoids ; pharmacology ; Kidney Diseases ; chemically induced ; drug therapy ; prevention & control ; Kidney Tubules, Proximal ; drug effects ; Lipid Peroxidation ; drug effects ; Malondialdehyde ; metabolism ; Mass Spectrometry ; Mitochondria ; drug effects ; Oxygen Consumption ; drug effects ; Protective Agents ; chemistry ; pharmacology ; Quercetin ; analogs & derivatives ; pharmacology ; Rutin ; pharmacology

To observe the effect of uniform and shift rotation culture on the formation and activity of the alginate-chitosan (AC) microencapsulated HepLL immortalized human hepatocytes and HepG2 cells aggregates.AC microcapsulated HepG2 and HepLL cells were randomly divided into two groups. Each group was divided into 3 subgroups according to uniform and shift rotation culture.The size and number of aggregates were observed and measured under laser confocal microscopy and inverted microscope dynamically. The amount of albumin synthesis was detected by ELISA, the clearance of ammonia was detected by colorimetry, and diazepam conversion function was detected by high performance liquid chromatography (HPLC).On day 6, 8, 10, 12, 14 and 16, the number and size of the aggregates, albumin synthesis, diazepam clearance and ammonium clearance increased significantly in shift rotation culture group than in uniform group (all<0.01). The albumin synthesis, diazepam clearance, and ammonium clearance in the microencapsulated HepLL groups were significantly higher than those of HepG2 cells at any time (all<0.01).Shift rotation culture can significantly promote the formation and increase the activity of AC microencapsulated HepLL and HepG2 aggregates, and HepLL cells may be more suitable for bioartificial liver than HepG2.
Albumins ; biosynthesis ; metabolism ; Alginates ; Ammonia ; metabolism ; Animals ; Cell Aggregation ; physiology ; Cell Culture Techniques ; methods ; Cell Line, Transformed ; physiology ; Chitosan ; Diazepam ; metabolism ; Glucuronic Acid ; Hep G2 Cells ; cytology ; physiology ; Hepatocytes ; cytology ; physiology ; Hexuronic Acids ; Humans ; Liver, Artificial ; Rotation

Liver fibrosis is an important health problem that can further progress into cirrhosis or liver cancer, and result in significant morbidity and mortality. Inhibiting proliferation and inducing apoptosis of hepatic stellate cells (HSCs) may be the key point to reverse liver fibrosis. At present, anti-fibrosis drugs are rare. Kinetin is a type of plant-derived cytokinin which has been reported to control differentiation and induce apoptosis of human cells. In this study, the HSCs were incubated with different concentrations of kinetin. The proliferation of rat HSCs was measured by MTT assay, cell cycle and apoptosis were analyzed by flow cytometry, and the apoptosis was examined by TUNEL method. The expression of Bcl-2 and Bax proteins was detected by immunocytochemistry staining. It was found that kinetin could markedly inhibit proliferation of HSCs. In a concentration range of 2 to 8 μg/mL, the inhibitory effects of kinetin on proliferation of HSCs were increased with the increased concentration and the extension of time (P < 0.01). Flow cytometry indicated that kinetin could inhibit the DNA synthesis from G0/G1 to S phase in a dose-dependent manner (P < 0.01). The apoptosis rates of the HSCs treated with 8, 4 and 2 μg/mL kinetin (25.62% ± 2.21%, 15.31% ± 1.9% and 6.18% ± 1.23%, respectively) were increased significantly compared with the control group (3.81% ± 0.93%) (P < 0.01). All the DNA frequency histogram in kinetin-treated groups showed obvious hypodiploid peak (sub-G1 peak), and with the increase of kinetin concentrations, the apoptosis rate of HSCs also showed a trend of increase. It was also found that kinetin could down-regulate the expression of Bcl-2, and up-regulate the expression of Bax, leading to the decreased ratio of Bcl-2/Bax significantly. The kinetin-induced apoptosis of HSCs was positively correlated with the expression of Bax, and negatively with the expression of Bcl-2. It was concluded that kinetin can inhibit activation and proliferation of HSCs by interrupting the cell cycle at G1/S restriction point and inducing apoptosis of HSCs via reducing the ratio of Bcl-2/Bax.
Animals ; Apoptosis ; drug effects ; Cell Line, Transformed ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; G1 Phase Cell Cycle Checkpoints ; drug effects ; genetics ; Gene Expression Regulation ; Growth Inhibitors ; pharmacology ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Kinetin ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; genetics ; metabolism ; Rats ; Signal Transduction ; bcl-2-Associated X Protein ; agonists ; genetics ; metabolism

Cementum is critical for anchoring the insertion of periodontal ligament fibers to the tooth root. Several aspects of cementogenesis remain unclear, including differences between acellular cementum and cellular cementum, and between cementum and bone. Biomineralization is regulated by the ratio of inorganic phosphate (Pi) to mineral inhibitor pyrophosphate (PPi), where local Pi and PPi concentrations are controlled by phosphatases including tissue-nonspecific alkaline phosphatase (TNAP) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). The focus of this study was to define the roles of these phosphatases in cementogenesis. TNAP was associated with earliest cementoblasts near forming acellular and cellular cementum. With loss of TNAP in the Alpl null mouse, acellular cementum was inhibited, while cellular cementum production increased, albeit as hypomineralized cementoid. In contrast, NPP1 was detected in cementoblasts after acellular cementum formation, and at low levels around cellular cementum. Loss of NPP1 in the Enpp1 null mouse increased acellular cementum, with little effect on cellular cementum. Developmental patterns were recapitulated in a mouse model for acellular cementum regeneration, with early TNAP expression and later NPP1 expression. In vitro, cementoblasts expressed Alpl gene/protein early, whereas Enpp1 gene/protein expression was significantly induced only under mineralization conditions. These patterns were confirmed in human teeth, including widespread TNAP, and NPP1 restricted to cementoblasts lining acellular cementum. These studies suggest that early TNAP expression creates a low PPi environment promoting acellular cementum initiation, while later NPP1 expression increases PPi, restricting acellular cementum apposition. Alterations in PPi have little effect on cellular cementum formation, though matrix mineralization is affected.
Alkaline Phosphatase ; metabolism ; Animals ; Cell Line, Transformed ; Dental Cementum ; cytology ; metabolism ; physiology ; Gene Expression Profiling ; Humans ; Mice ; Models, Animal ; Phosphoric Diester Hydrolases ; metabolism ; Pyrophosphatases ; metabolism ; Tooth Root ; metabolism ; physiology ; X-Ray Microtomography

This study was aimed to explore the method for induction and expansion of EB virus specific cytotoxic T lymphocytes (EBV-CTL) in vitro, and to detect their killing effect. Peripheral blood mononuclear cells (PBMNC) were collected from 6 EBV seropositive healthy donors, and EBV-transformed B lymphoblastoid cells (BLCL)were used as the antigen-presenting cells and antigen stimulant which was irradiated by 40 Gy (60)Co irradiator. The autologous PBMNC and irradiated BLCL were cultured to induce and expand the EBV-CTL, and the immunophenotype was identified by the flow cytometry. The killing effect of the EBV-CTL against the autologous BLCL (autoBLCL), the autologous PHA cultured B lymphoblastoid cells( PHA-BLCL), the allogeneic BLCL (alloBLCL) and the K562 cells were measured with LDH release assay under different effector-to-target ratio. The results showed that the 6 cell lines of EBV-CTL were induced and expanded from the EBV seropositive healthy donors, the overall increase in cell numbers varied from 18.6 to 55.0 times. After 10 stimulations, the specific killing efficiency of the EBV-CTL for the autoBLCL were 59.4%, 43.2% and 29.0% under the effector-to-target ratio of 20: 1, 10: 1 and 5: 1. The nonspecific killing efficiency for the PHA-blast, alloBLCL and K562 cells were 7.1%, 9.4% and 10.3% (P < 0.05) under the 20: 1 ratio; 6.6%, 8.3% and 8.1% (P < 0.05) under 10: 1; 5.4%, 7.3% and 6.3% (P < 0.05) under 5: 1, respectively. It is concluded that the EBV-CTL can be successfully induced and expanded ex vivo for specific killing of HLA matched BLCL and may become a potential treatment for EBV related post-transplant lymphoproliferative disorders.
B-Lymphocytes ; immunology ; Cell Line, Transformed ; Herpesvirus 4, Human ; immunology ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; immunology ; virology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; virology

To develop a stable cell line that could express the RSV NS1, the full-length RSV NS1 gene was generated by RT-PCR amplification from respiratory syncytial virus. NS1 gene was ligated with pBABE-puro to construct the recombinant retroviral expression plasmid pBABE-NS1, which was cotransfected into 293FT packaging cells with PIK packaging plasmid by calcium phosphate co-precipitation. The supernatant of 293FT was collected to infect HEp-2 cells, the resulting cell clones stably expressing NS1 were screened by puromycin. Using QPCR, CPE staining method and indirect immunofluorescence assay, the expression of NS1 at both gene and protein levels was identified. The recombinant plasmid pBABE-NS1 was identified by EcoRI and BamHI endonuclease digestion and the sequence analysis. QPCR results showed that the NS1 gene amplification in HEp-2-NS1 cells was 8483 fold higher than that in HEp-2 cells. Although the exogenous interferon was added, all cells were destroyed after 48 hours post infection using CPE staining method, showing that HEp-2-NS1 cells remained sensitive to the VSV virus. The results of RT-PCR and indirect immunofluorescence assay showed that the NS1 gene in HEp-2 cells could not only transcribe mRNA, but also express NS1 protein steadily. We had successfully established HEp-2-NS1 cell lines with stable expression of respiratory syncytial virus non-structural protein NS1.
Cell Line, Transformed ; HEK293 Cells ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; Respiratory Syncytial Viruses ; genetics ; Viral Nonstructural Proteins ; biosynthesis ; genetics

Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) is a novel phlebovirus, causing a life-threatening illness associated with the symptoms of severe fever and thrombocytopenia syndrome. The sequence and structure of the genome have already been illustrated in previous study. However, the characteristics and function of the structure and non-structure proteins is still unclear. In this study, we identified the density of the purified SFTSV virions as 1.135 g/mL in sucrose solution. Using RT-PCR method, we amplified the full coding sequence of RNA dependent RNA polymerase(RdRp), glycoprotein precursor (M), glycoprotein n (Gn), glycoprotein c (Gc), nuclear protein (NP) and non structural protein (NSs) of SFTSV (strain HB29). Respectively inserted the target genes into eukaryotic expression vector pcDNA5/FRT or VR1012, the target protein in 293T cell were successfully expressed. By analyzing the SFTSV virions in SDS-PAGE and using recombinant viral proteins with SFTS patients sera in Western blotting and Immunofluorescent assay, the molecule weight of structure and non-structure proteins of SFTSV were defined. The study provides the first step to understand the molecular characteristics of SFTSV.
Bunyaviridae Infections ; virology ; Cell Line, Transformed ; Fever ; virology ; HEK293 Cells ; Humans ; Orthobunyavirus ; genetics ; metabolism ; Thrombocytopenia ; virology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; Viral Structural Proteins ; biosynthesis ; genetics ; Virion ; genetics ; metabolism

OBJECTIVETo establish immortalized lymphoblastoid cell lines of a Miao core pedigree with Bardet-Biedl syndrome (BBS), in order to provide a long-term source of material for research.

METHODSWith Epstein-Barr virus transformation of B cells and addition of cyclosporine A to inhibit the activity of T cells, fresh anticoagulated blood samples with heparin were collected from 12 members of the core pedigree, and were used to establish the immortalized lymphoblastoid cell lines of B lymphocytes.

RESULTSTwelve immortalized lymphoblastoid cell lines of the core BBS pedigree were obtained successfully.

CONCLUSIONThe immortalized B lymphoblastoid cell lines of the Miao pedigree with BBS can preserve the whole genome information and provide long-term research materials for BBS study.

B-Lymphocytes ; cytology ; Bardet-Biedl Syndrome ; blood ; genetics ; Cell Line ; Cell Line, Transformed ; Cell Transformation, Viral ; China ; ethnology ; Ethnic Groups ; genetics ; Herpesvirus 4, Human ; Humans ; Pedigree