2.Hypoxia-induced alterations of lipid metabolism in the normal human hepatic L02 cell line.
Wen-hui CHENG ; Wei SHEN ; Juan CHEN
Chinese Journal of Hepatology 2012;20(1):30-34
OBJECTIVETo investigate the effects of hypoxia on lipid metabolism in the normal human hepatic cell line L02 and to investigate the underlying molecular mechanisms.
METHODSL02 cells were exposed to hypoxic conditions (experimental groups: at 1% O2, 5% CO2, 94% N2 for 3, 6, 12, 24, or 48 hours) or normoxic conditions (control group: at 21% O2). Lipid droplet accumulation and triglyceride content were measured in each group by oil red O staining and biochemical assay, respectively. The mRNA expression levels of hypoxia-inducible factor (HIF)-2a and sterol regulatory element binding protein (SREBP)-1c were detected by reverse transcription-polymerase chain reaction. Protein expression levels of HIF-2a, adipose differentiation-related protein (ADRP), and Fas were tested by Western blot analysis.
RESULTSLipid droplet accumulation and the triglyceride content were significantly higher in the hypoxia group than the normoxia group. In addition, the hypoxia groups had significantly down-regulated mRNA expression of SREBP-1c (12h: 0.236+/-0.043, 24 h: 0.287+/-0.044, 48 h: 0.342+/-0.049 vs. normoxia: 0.503+/-0.037; F = 28.37, P less than 0.01) and FAS protein (12 h: 0.562+/-0.054, 24 h: 0.674+/-0.062, 48 h: 0.682+/-0.057 vs normoxia: 0.857+/-0.069; F = 16.08, P less than 0.01). In normoxic cells, little or no expression of HIF-2a protein was detected by Western blot. In hypoxic cells, HIF-2a protein expression peaked at 6h (0.973+/-0.067). ADRP protein expression was significantly higher in hypoxia groups than in the normoxia group (12 h: 0.319+/-0.043, 24 h: 0.732+/-0.056 and 48 h: 0.873+/-0.066 vs. 0.211+/-0.019; all, P less than 0.05.
CONCLUSIONExposure to hypoxic conditions might induce lipidosis in normal human hepatic cells by stimulating HIF-2a and ADRP expression.
Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Hypoxia ; Cell Line ; Down-Regulation ; Hepatocytes ; metabolism ; Humans ; Lipid Metabolism ; Membrane Proteins ; metabolism ; Oxygen ; metabolism ; Perilipin-2 ; Sterol Regulatory Element Binding Protein 1 ; metabolism
3.Effect of hepatitis B virus X protein on expression of lipid metabolism-related genes in HepG2 cells.
Juan CHEN ; Wei SHEN ; Wen-hui CHENG
Chinese Journal of Hepatology 2011;19(10):768-773
OBJECTIVETo investigate the effect of Hepatitis B Virus X Protein (HBx) on the expression of lipid metabolism-related genes and its role in pathogenesis of hepatocyte fatty degeneration.
METHODSHepatitis B Virus X gene eukaryon expression vector pIRES2-eGFP-HBx was transfected into HepG2 cells to establish HepG2/HBx cell model for HBx expression. HepG2 cells transfected with pIRES2-eGFP (HepG2/pIRES2 cell) and non-transfected were used as controls. At 24, 48 and 72 hours after transfection, the expression of green fluorescent protein (GFP) was observed by fluorescence microscope and the triglyceride(TG) content was detected. RT-PCR and Western blot were applied to detect the levels of sterol regulatory element binding protein-1 (SREBP-1), liver x receptor alpha (LXRalpha) mRNA and the levels of HBx, LXRalpha and fatty acid synthase (FAS) protein. At 24, 48 and 72 hours after transfection, the expression of GFP was found in HepG2/HBx and HepG2/pIRES2 cells, and increased gradually. The expression of HBx was detected only in HepG2/HBx cells, and was increased with time after transfection (F = 32.21, P less than 0.01). These suggested successful obtaining of HepG2-HBx cell model for HBx expression.
RESULTSAt 24h, 48h and 72h after transfection, the expression levels of LXRalpha mRNA (0.386+/-0.055, 0.505+/-0.071, 0.649+/-0.058 ) and SREBP-1 mRNA (0.395+/-0.055, 0.548+/-0.047, 0.795+/-0.058), as well as the levels of LXRalpha protein(0.178+/-0.036, 0.263+/-0.047, 0.347+/-0.058) and FAS protein(0.436+/-0.055, 0.608+/-0.053, 0.827+/-0.046) in HepG2-HBx group were dramatically higher than those in the controls at the same time points (all P less than 0.05/0.01), and were gradually increased with time (all P less than 0.05/0.01). A positive correlationship was observed between HBX protein level and the LXRalpha, SREbP-1 mRNA and LXRalpha, FAS protein levels. The difference of TG content between HepG2/HBx group and control groups was not statistically significant (P more than 0.05).
CONCLUSIONSHBx-LXRalpha-SREBP-1/FAS pathway suggested regulating transcription and expression of lipid metabolism-related genes, which might be one of the important molecular mechanism causing hepatocyte fatty degeneration.
Carcinoma, Hepatocellular ; metabolism ; Fatty Acid Synthase, Type I ; metabolism ; Hep G2 Cells ; Humans ; Lipid Metabolism ; genetics ; Liver Neoplasms ; metabolism ; Liver X Receptors ; Orphan Nuclear Receptors ; metabolism ; Sterol Regulatory Element Binding Protein 1 ; metabolism ; Trans-Activators ; genetics ; Transfection
4.The concurrent control study of elective nodal prophylactic irradiation with intensity modulated radiotherapy for esophageal carcinoma
Shuchai ZHU ; Hui DONG ; Wenbin SHEN ; Zhikun LIU ; Juan LI ; Jingwei SU
Chinese Journal of Radiological Medicine and Protection 2014;34(10):758-762
Objective To explore the value of elective nodal prophylactic irradiation with intensity modulated radiotherapy(IMRT) for esophageal carcinoma.Screening patients who are suitable for elective nodal prophylactic irradiation (ENI),in order to improve locoregional control and overall survival.Methods The concurrent control study was conducted to esophageal cancer patients who were treated by definitive radiotherapy.A total of 148 patients finished treatment were identified.Seventy-four patients received ENI,while the other seventy-four patients received involved-field irradiation (IFI).Kaplan-Meier method was used for calculation of locoregional control rates and overall survival rates.The univariate and multivariate analysis of prognostic factors were also tested.Results The 1-,3-,and 5-year locoregional control rates of ENI group and IFI group were 72.5%,52.8%,50.6% and 58.4%,35.8%,21.9% (x2 =7.881,P <0.05),respectively.The 1,3,and 5 years survival rates of the ENI group and IFI group were 74.3%,44.2%,24.5% and 68.9%,27.6%,15.9% (x2 =1.903,P < 0.05),respectively.In Cox multivariate analysis,clinical T stage,tumor location,different radiotherapy region were independent factors for the locoregional control of all patients,and clinical T,N stage,the length of esophageal barium meal and chemotherapy were independent factors for the overall survival of all patients.Conclusions Esophageal carcinoma patients treated with ENI could achieve better locoregional control than those treated with IFI.Esophageal carcinoma patients with early stage or middle thoracic lesion could benefit from ENI for local control and overall survival.
5.Construction of lung cancer cell model overexpressing human MutS homologue 2(hMSH2)
Bichao LU ; Qingli SHEN ; Wenli LI ; Juan LI ; Hui CHEN ; Yumei DAI ; Wei HE
Basic & Clinical Medicine 2017;37(7):929-934
Objective To construct human lung cancer cell model with human MutS homologous protein 2 (hMSH2) overexpression for exploring the effect of hMSH2 molecule in the cytotoxicity of γδ T cell against lung cancer cells.Methods hMSH2 coding sequence was cloned by PCR for construction of recombinant vector which over expressed hMSH2-EGFP fusion protein using homologous recombination.The recombinant vector was transfected to lung cancer cell line NCI-H520 to construct human lung cancer cell model overexpressing hMSH2 molecule.The expression of hMSH2 molecule in NCI-H520 was detected by Western blot.The expression of hMSH2 on the cell membrane was measured by flow cytometry.Cytotoxicity of expanded γδ T cells from peripheral blood mononuclear cells against NCI-H520 cells was detected by LDH release assay in vitro.Results hMSH2 coding sequence (2805 bp) was cloned and the result of restriction endonuclease digestion of Fugw-hMSH2 recombinant vector was accordance with the anticipated objective strip size.Exogenous hMSH2-EGFP fusion protein was expressed in NCIH520 cells.The level of hMSH2 molecule on the surface of NCI-H520 cells with overexpression of hMSH2 was significantly increased (P<0.001).Cytotoxicity of γδ T cells against NCI-H520 cells with overexpression of hMSH2 was significantly increased compared to the wild type NCI-H520 cells (P<0.05).Conclusions Lung cancer cell model that overexpresses hMSH2 molecule is successfully constructed,hMSH2 molecule on the cell membrane is increased and the cytotoxicity of γδ T cells against lung cancer cells is enhanced.
6.Clinical study of combined detection of troponin T,myoglobin and NT-ProBNP in diagnosis of myocardia linfarction
Fei SHEN ; Chengfang ZHANG ; Juan ZHAO ; Guangxin CHEN ; Hui LI ; Xiaoming LIAO
International Journal of Laboratory Medicine 2017;38(9):1220-1221,1224
Objective To observe the clinical application of combined detection of troponin T(cTnT),myoglobin(Myo) and NT-ProBNP in the diagnosis of myocardial infarction(MI).Methods A total of 913 cases of acute chest pain complaints to our hospital for examination and treatment from January 2012 to December 2015 were selected and divided into the acute MI group and non-acute MI group according to the final diagnostic results.The cTnT,Myo and NT proBNP levels were detected in all subjects.The differences of cTnT,Myo and NT-proBNP levels were observed in the two groups.The sensitivity and specificity of single indicator detection,2-indicator combined detection and 3-indicator combined detection for diagnosing MI were observed.Results The levels of cTnT,Myo and NT-proBNP in the acute MI group were significantly higher than those in the non-acute MI group,the difference was statistically significant(P<0.05).The sensitivity and specificity of the two-indicator combined detection were higher than those of cTnT,Myo and NT-proBNP single detection,the sensitivity and specificity of the 3-indicator combined detection were 95.18% and 89.86% respectively,which were significantly higher than those of the single indicator test and the two-indicator combined detection.Conclusion The combined examination of cTnT,Myo and NT-proBNP has important significance in the diagnosis of MI,can improve the specificity and sensitivity of MI diagnosis and provides a reliable basis for clinical treatment.
7.Investigation of water defluoridation projects and the concentration of water fluoride
Hao, WANG ; Chun-an, SHEN ; Fu-juan, LENG ; Zhi-bao, ZHANG ; Guang-Shun, DUAN ; Hui-jie, CAO
Chinese Journal of Endemiology 2012;31(2):202-204
ObjectiveTo investigate the concentration of water fluoride and the application of water defluoridation projects in Suizhou,and to provide a basis for water improvement and prevention of the disease.MethodsCross-sectional study was carried out to investigate completely the water defluoridation projects built between 1986 and 2009 in the area under Suizhou's jurisdiction; 3 source water and terminal water samples of the projects in use were collected,respectively; 1 water sample was collected,respectively in the 3 projects with out-ofcommission or discarded water sources that used to be major water supply,for detection of fluoride,chloride,total hardness,heavy metals and other indicators.ResultsA total of 21 projects were investigated,14 projects were in normal use (66.7%),7 projects were out of order and abandoned (33.3%).Of the 14 projects normally used,13projects with water fluoride concentration ≤ 1.0 mg/L(92.9%)and 1 project with water fluoride concentration higher than 1.0 mg/L(7.1%),the concentration of water fluoride in the 7 abandoned projects was higher than 1.0 mg/L(100%).Arsenic and lead levels of all the water samples were normal.There were two water samples with iron content exceeded the standard,and one total hardness of water samples exceeded the standard in the normally used 14 peripheral water sources.One chloride content exceeded the standard,one manganese content exceeded the standard,two iron content exceeded the standard and three total hardness exceeded the standard in the seven abandoned projects.ConclusionsSome defluoridation projects are stopped using and abandoned in Suizhou,and the water fluoride exceeds the standard.
8.Relationship between hearing threshold and peripheral neuropathy in type 2 diabetic patients
Juan SHEN ; Fang LIU ; Hui ZENG ; Jie YU ; Qiag LI ; Yuqian BAO ; Weipiag JIA
Chinese Journal of Endocrinology and Metabolism 2011;27(8):644-648
Objective In order to investigate the relationship between hearing loss and diabetic peripheral neuropathy( DPN ) via comparing the pure tone thresholds and vibration perception threshold(VPT) in type 2 diabetic patients and control person without diabetes. Methods 173 subjects including 138 type 2 diabetic patients(DM)and 35 non-diabetes controls were examined for VPT and hearing threshold. Nerve conducting velocity ( NCV )including sensory nerve conducting velocity( SCV )and motorial nerve conducting velocity( MCV )of diabetic patients were determined. The participants were divided into three groups: control group (n = 35 ), DM group without peripheral neuropathy( non-PN group, n = 74 ), and DM group complicated with peripheral neuropathy (PN group,n = 64 ). The clinical characteristics, biochemical parameters , the incidence of sensorineural hearing loss ( SNHL),pure tone threshold, and VPT were compared among three groups. At last, the relationship between hearing thresholds and NCV were analyzed. Results The incidence of hearing impairment of sensorineural type was 29.69% in PN group, which was significantly higher than that of non-PN group( 17.57% )and control group( 17. 14% ). There was significant differences in age, duration of diabetes, glycolated hemoglobin (HbA1c), glycolated serum albumin ( GA), Fasting blood glucose( FPG), 2h postprandial blood glucose( PPG), VPT, and hearing threshold among the three groups( all P<0. 05 ). The value of hearing threshold increased significantly( all P<0. 05 ) in 3 VPT subgroups with VPT≤ 15 V, VPT 16-25 V, and VPT >25 V. The Spearman correlation analysis showed median NCV was negatively correlated with hearing threshold on 1.00, 2.00, 4. 00, and 8. 00 kHz ( All P < 0. 05 ). The logistic regression analysis indicated that the age( regression coefficient =0. 088, P<0. 01 ) was the independent risk factor of SNHL, median nerve MCV ( regression coefficient = -0. 135, P = 0. 046 ) was the important influencing factor of SNHL. Conclusion Diabetic patients are more likely to suffer from impaired middle-frequency and high-frequency hearing, DPN in patients is often complicated with hearing impairment. Age and median nerve MCV were major risk factors of SNHL in diabetic patients.
9.A comparative study of different irradiation ranges in radical radiotherapy for early-stage esophageal cancer
Shuchai ZHU ; Hui DONG ; Zhikun LIU ; Wenbin SHEN ; Juan LI ; Jingwei SU ; Jinrui XU
Chinese Journal of Radiation Oncology 2015;(6):615-618
Objective To compare the efficacy of elective nodal prophylactic irradiation ( ENI) and involved?field irradiation ( IFI) in radical radiotherapy for early?stage esophageal cancer and to determine the appropriate irradiation range for early?stage esophageal cancer. Methods The clinical data of 121 patients with early?stage esophageal cancer receiving radical radiotherapy in our hospital from January 2006 to December 2011 were collected and respectively analyzed. Sixty?one patients received ENI, and the other 60 patients received IFI. The Kaplan?Meier method was used to calculate local control ( LC) and overall survival ( OS) rates;the log?rank test was used for survival difference analysis and univariate prognostic analysis;the Cox regression model was used for multivariate prognostic analysis. Results The 1?, 3?, and 5?year LC rates in ENI group and IFI group were 81. 1%, 60. 1%, and 57. 5% vs. 64. 5%, 43. 9%, and 27. 2%, respectively ( P=0. 003 ) . The 1?, 3?, and 5?year OS rates in ENI group and IFI group were 86. 9%, 56. 8%, and 34. 8% vs. 86. 7%, 34. 3%, and 19. 1%, respectively ( P=0. 019) . The 1?, 3?,and 5?year overall failure rates in ENI group and IFI group were 22. 3%, 53. 8%, and 63. 2% vs. 43. 3%, 65. 8%, and 78. 8%, respectively ( P=0. 023) . Multivariate analysis showed that irradiation range was the influencing factor for LC and OS. Conclusions As for the radical radiotherapy for early?stage esophageal cancer, ENI can significantly increase LC and reduce locoregional failure, and therefore improve long?term OS.
10.A comparative study of failure patterns of definitive treatment of esophageal cancer with elective nodal irradiation and involved-field irradiation
Hui DONG ; Shuchai ZHU ; Jingwei SU ; Wenbin SHEN ; Zhikun LIU ; Juan LI
Chinese Journal of Radiation Oncology 2014;23(6):479-483
Objective To compare the failure pattern between esophageal cancer patients receiving definitive elective nodal irradiation (ENI) and involved-field irradiation (IFI) and to investigate the reasons and influential factors for locoregional recurrence and metastasis.Methods A retrospective analysis was performed on the clinical data of 245 patients with esophageal cancer who received definitive radiotherapy in our hospital from January 2006 to December 2012.One hundred and twenty-six patients received ENI,and the other 119 patients received IFI.Failure patterns were analyzed after treatment.Locoregional failures included local esophageal lesion uncontrol or recurrence and regional lymph node recurrence or metastasis.Distant metastases included distant organ metastasis and distant lymph node metastasis.Comparison of failure pattern between the two therapies was made by chi-square test.Results One hundred and sixty-three patients had failure after treatment.Locoregional failure was observed in 92 patients,distant metastasis in 36 patients,and locoregional failure plus distant metastasis in 35 patients.The 1-,3-,and 5-year overall failure rate for the ENI group were 35.4%,62.5%,and 69.0%,respectively,versus 46.5%,71.5%,and 81.5% for the IFI group (P =0.036).The 1-,3-,and 5-year locoregional failure rates for the ENI group were 29.9%,48.4%,and 50.0%,respectively,versus 39.6%,62.1%,and 71.4% for the IFI group (P =0.003).Conclusions For esophageal cancer patients receiving definitive radiotherapy,ENI can significantly reduce locoregional failures and increase locoregional control,thus improving the long-term survival.