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Objective@#Fine particulate matter (PM 2.5) is an air pollutant that has become of great concern in recent years. Numerous studies have found that PM 2.5 may contribute to lung cancer, but the pathogenesis has not yet been fully elucidated. In this study, we explored the roles of exosomes from bronchial epithelial cells in PM 2.5-promoted lung cancer metastasis.@*Methods@#Exosomes were isolated from cell supernatants. An animal model of lung metastasis (established by tail vein injection of A549-luc) and in vitro studies with lung cancer cell lines were used to investigate the effects of exosomes derived from PM 2.5-treated human bronchial epithelial cells (PHBE-exo).@*Results@#The animal experiments revealed that PHBE-exo-treated mice showed stronger luciferase activity and a larger relative metastatic region in the lungs, thus indicating that PHBE-exo promoted the metastatic potential of lung cancer. Additionally, PHBE-exo promoted the migration, invasion and epithelial-to-mesenchymal transition of lung cancer cells, in a manner mediated by activation of c-Jun N-terminal kinase.@*Conclusion@#These results implied that PM 2.5 may promote the development of lung cancer through exosomes derived from bronchial epithelial cells, thus providing a potential interventional target for lung cancer. These findings broadened our understanding of cancer-promoting mechanisms of environmental pollutants.
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Animals , Humans , Mice , Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition , Exosomes/metabolism , Lung Neoplasms/metabolism , Particulate Matter/toxicityABSTRACT
Objective To construct the eukaryotic expression vector plasmid enhanced green fluorescent protein (pEGFP)-N1-CPNE3, and identify the expression and localization of Copine-3 protein in cells. Methods The Copine-3 coding sequences (CPNE3) was amplified by RT-PCR from human bronchial epithelial (HBE) cells and inserted into eukaryotic expression vector pEGFP-Nl. The recombinant plasmid pEGFP-Nl-CPNE3 was confirmed by endonuclease digestion and sequencing before it was transfected into 293T and H1299 cells. Cellular localization of Copine-3-EGFP fusion protein was detected by con-focal laser scanning. Expression of Copine-3 in 293T and H1299 cells was detected by Western blotting analysis. Localization of Copine-3 in clinical samples of the lung adenocarcinoma patients was detected by immunohistochemistry. Results CPNE3 was successfully constructed into the eukaryotic expression vector pEGFP-Nl and expressed in 293T and H1299 cells. Furthermore, the location of Copine-3 protein in cytoplasm and nucleus was determined by immunofluorescence staining, immuno Western blotting and immunohistochemistry in those cells and clinical samples. Conclusion The eukaryotic expression vector pEGFP-Nl-CPNE3 is constructed successfully, and Copine-3 protein is localized in cytoplasm and nucleus.
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Objective To study the molecular mechanism of interleukin 25 (IL-25)expression in the lung of asthmatic rats.Methods The expressions of IL-25 mRNA and protein in the lungs were detected by Real-time PCR and ELISA,respectively.The levels of IL-25 mRNA and protein were detected by ovalbumin (OVA)in human bronchial epithelial cells.And the transcription factors that regulate IL-2 5 expression were explored through site prediction.Results The expressions of IL-25 mRNA and protein in the lung of OVA-induced asthma rats were significantly increased during animal experiments.Cell experiments showed that OVA could increase the expression of IL-2 5 in human bronchial epithelial cells in a dose-dependent manner,and OVA could upregulate the expression of transcription factor AP1.AP1 was found in the promoter region of IL-25 by site prediction.The AP1 inhibitor (T5224)significantly reduced the expression of IL-25 in OVA-induced human bronchial epithelial cells. Conclusion The molecular mechanism of IL-25 expression induced by OVA in asthma is related to the increase of transcription factor AP1 .
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[ ABSTRACT] AIM:To investigate the role of canonical transient receptor potential channel 1 ( TRPC1 ) in the epithelial-mesenchymal transition ( EMT) of human bronchial epithelial ( HBE) cells induced by transforming growth fac-tor-β1 (TGF-β1).METHODS:EMT of 16HBE cells induced by TGF-β1 were identified by microscopy, immunofluores-cence and Western blotting.Immunofluorescence, real-time PCR and Western blotting were applied to detect the mRNA and the protein expression of TRPC1 in the 16HBE cells.The influence of SKF96365 (a TRPC1 blocker) and siRNA-me-diated silencing of TRPC1 on the EMT of the 16HBE cells were detected by microscopy and Western blotting.RESULTS:Treatment with TGF-β1 induced significant morphological changes of the 16HBE cells.Exposure to TGF-β1 decreased the expression of E-cadherin protein (P<0.01) and increased the expression of α-SMA protein (P<0.05) in the 16HBE cells.Immunofluorescence observation indicated that TRPC1 expression in the 16HBE cells was positive.The expression of TRPC1 at mRNA and protein levels was significantly increased in the 16HBE cells after stimulation with TGF-β1 ( P<0.05).The morphological changes of the 16HBE cells induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silen-cing compared with TGF-β1 group.The protein expression of E-cadherin andα-SMA induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silencing compared with TGF-β1 group (P<0.05).CONCLUSION:TGF-β1 induces EMT with the mechanism of up-regulating TRPC1 in human bronchial epithelial cells.
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Objective:To investigate YKL-40-mediated inflammation in human bronchial epithelial cells and analyzed the soluble factors secreted by bronchial epithelial cells exposed to YKL-40 that were responsible for increasing proliferation and migration of primary normal human bronchial smooth muscle cells (BSMCs).Methods:YKL-40-induced inflammation was assayed in two human bronchial epithelial cells (BEAS-2B cell line and primary human bronchial epithelial cells ,namely HBECs).In addition,we treated BEAS-2B cells and HBECs with YKL-40,and added the conditioned culture media ( YKL-40-BEAS-2B-CM) and ( YKL-40-HBECs-CM) to BSMCs.The proliferation and migration of BSMCs were determined by premixed WST-1 cell proliferation reagent and QCM chemotaxis migration assay ,respectively.Results: Bronchial epithelial cells treated with YKL-40 resulted in a significant increase of IL-8 production,but have no effect about RANTES ,Eotaxin and TNF-α.YKL-40-BEAS-2B-CM and YKL-40-HBECs-CM induced IL-8 was found to further stimulate proliferation and migration of BSMCs ,and the effects were inhibited after neutralizing IL-8.Conclusion:Through investigating the interaction of airway epithelium and smooth muscle ,our findings implicate that YKL-40 may be involved in the inflammation of asthma by induction of IL-8 from epithelium,subsequently contributing to BSMCs proliferation and migration.Moreover, inhibition of IL-8 signaling is a potential therapeutic target for YKL-40-induced inflammation and remodeling of asthma.
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Objective To explore whether TNF-α involves in the modulation of Cysteinyl leukotriene receptor 1 (CysLT1) expression in bronchial epithelial cells.Methods The bronchial epithelial cell lines 16HBE cells were stimulated with different concentration (0.00,0.05,0.50,5.00,20.00 μg/L) of TNF-α for 48 hours,and CysLT1 mRNA in 16HBE cells was measured by reverse transcription(RT)-PCR.CysLT1 expression was detected by immunohistochemistry.Results 16HBE cells did not express CysLT1,after the cells were treated with TNF-α,obvious expression of CysLT1 were detected by immunohistochemistry.The weak CysLT1 mRNA expression was observed by RT-PCR in 16HBE cells,and after the cells were treated with TNF-α for 48 hours,CysLT1 mRNA expression were upregulated.When the concentrations of TNF-α were 0.00,0.05,0.50,5.00,and 20.00 μg/L respectively,the relative intensities of CysLT1 mRNA/β-actin were 0.048,0.105,0.177,0.182,0.495,respectively.Conclusions TNF-α can upregulate CysLT1 mRNA expression in 16HBE ceils in a dose-dependent manner.When infected by virus,respiratory tract produces abundant TNF-α.The TNF-α can upregulate the expression of CysLT1 in epithelial cells,enhance inflammation reaction in respiratory tract.This may explain partially the mechanism of exacerbation of asthma induced by respiratory tract infection.
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<p><b>OBJECTIVE</b>To investigate the roles of Rho/Rock signaling pathway in silica-induced Epithelial-mesenchymal transition (EMT) in human bronchial epithelial cells (BEC) in vitro.</p><p><b>METHODS</b>Human BEC were incubated with silica with various concentrations for indicated times. Cell viability was assayed by MTT test. Morphologic Changes were observed by microscope. Mesenchymal marker α-smooth muscle actin (α-SMA), vimentin (Vim), and epithelial marker E-cadherin (E-cad) were analyzed by Western Blot. The pull-down assay was used to measure Rho activity. In the prevention experiments, the specific inhibitor for Rho effector ROCK (Y27632) was used to inhibit the activity of Rho.</p><p><b>RESULTS</b>Human BEC stimulated with silica were converted from a "cobblestone" epithelial structure into an elongated fibroblast-like shape structure. Incubation of human BEC with silica induced de novo expression of α-SMA and Vim, and loss of E-cad. Also, silica treatment resulted in Rho activation in human BEC. Y27632 up-regulated the E-cad expression but attenuated α-SMA and Vim expression in silica-stimulated cells.</p><p><b>CONCLUSION</b>The activation of Rho/ROCK signaling pathways is most likely involved in Silica-induced EMT in human bronchial epithelial cells.</p>
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Humans , Actins , Metabolism , Bronchi , Cell Biology , Cadherins , Metabolism , Cells, Cultured , Epithelial Cells , Metabolism , Epithelial-Mesenchymal Transition , Quartz , Toxicity , Signal Transduction , Vimentin , Metabolism , rho-Associated Kinases , MetabolismABSTRACT
Objective To investigate the mechanism of malignant transformation in human bronchial epithelial cell line BEP2D exposed to α-particles.Methods The levels of intracellular ROS and malonaldehyde (MDA) in BEP2D,RH22 (passage 22 of α-particle-irradiated BEP2D cells) and BERP35T-1 cells (derived from nude mice bearing malignant transformed cells generated from the passage 35 of α-particle-irradiated BEP2D cells) were assayed with DCFH-DA and MDA kit,respectively.The expressions of 8-OH-dG and γ-H2AX in BEP2D,RH23 (passage 23 of α-particle-irradiated BEP2D cells)and BERP35T-1 cells were also measured with immunocytochemistry and immunofluorescence staining.Results Compared to BEP2D cells,the levels of ROS ( t =4.30 and 3.94,P ＜ 0.05 ) and MDA ( t =4.89 and 15.10,P ＜0.05) increased in RH22 and BERP35T-1 cells.The expressions of 8-OH-dG (t =3.80 and 2.92,P ＜ 0.05 ) and γ-H2AX ( t =7.61 and 12.67,P ＜ 0.05 ) in RH23 and BERP35T-1 cells were also higher than those in BEP2D cells.Conclusions Oxidative stress induces lipid peroxidation and DNA damage leading to genomic instability,which could contribute to cellular malignant transforming process in the human bronchial epithelial cell line BEP2D with α-particle exposure.
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Objective To identify the changes of DNA methylation profile in the process of malignant transformation of BEP2D cell induced by α particles.Methods The genomic DNAs were isolated from the malignant transformation BERP35T4 cells and immortalized human bronchial epithelial cell line BEP2D.Genomic DNAs were digested by MseI and ligated of PCR linkers.Methylated DNAs were digested by BstUI and amplified by PCR.The methylated DNA probes were prepared by labeling with Cy3 and Cy5 fluorescence dyes individually and hybridized to the methylation CpG-Island microarray.The hybridization results were scanned and analyzed.Intensity values were quality controlled and normalized.The normalized data were used to identify the differentially expressed genes based on a 1.5 fold difference of the expression level.Results There were 16 genes which showed changes of methylation level in malignant transformation BERP35T4 cells, 9 of them were hypermethylation and 7 were hypomethylation.These genes were including the SKIP gene, PPP3CC gene, MAP2K6 gene, KIR2DL1 gene, KIR2DL4 gene, KIR3DP1 gene, ZNF493 gene, ZNF100 gene, NKX2-5 gene, TFAP2D gene, DR1 gene, KCNJ16 gene, CCDC18 gene, FNBP1L gene, IRX4 gene, EPB41L3 gene, TCP10 gene and so on.Conclusions The DNA methylation might have effects on ionizing radiation drived tumorigenesis.
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Objective To investigate the antioxidant ability and radiosensitivity in the malignant transformed human bronchial epithelial cell line BEP2D induced by α-particle exposure.Methods Glutathione Peroxidase (GPX),Catalase (CAT) and Glutathione (GSH) assay kits were employed to detect GPX and CAT enzyme abilities and the levels of GSH in BEP2D,RH21 ( passage 21 of α-particle-irradiated BEP2D cells),and BERP35T-1 cells (derived from nude mice bearing malignant transformed cells generated from cells of passage 35 of α-particle-irradiated BEP2D cells).MTT assay were used to test the growth rate of BEP2D,RH21 and BERP35T-1 cells treated with 0,30,60,90,120,and 150 μmoL/L H2O2.Colony-forming test and MTT assay were used to examine the cell survival fraction and the growth rate of BEP2D,RH21 and BERP35T-1 cells exposed to 0,2,4,and 8 Gy of γ-rays,respectively.Results GPX and CAT enzyme activities in RH21 and BERP35T-1 cells were obviously lower than in BEP2D( t = 5.75-67.92 ,P ＜ 0.05 ).CAT enzyme activity in BERP35T-1 was lower than that in RH21 cells (t =22.25 ,P ＜0.01 ).Compared to BEP2D cells,decreased level of GSH was detected in BERP35T-1 cells(t = 7.76,P ＜ 0.05 ),but there was no change in RH21.After treatment with 30,60,90,120,and 150 μmol/L H2O2,the growth rates of BEP2D were all higher than those of BERP35T-1 cells(t = 10.37-58.36,P ＜0.01 ).Meanwhile,the growth rates of BEP2D were all higher than those of RH21 cells after treatment with 60,90,120,and 150 μ mol/L H2O2 (t = 29.90-84.68,P ＜ 0.01 ).In addition,compared to BEP2D cells,decreased cell survival fraction and growth rate of BERP35T-1 cells were observed after irradiation with 2,4,and 8 Gy of y-rays ( t = 5.87-34.17,P ＜ 0.05 ).The cell survival fraction and growth rate of RH21 were all lower than those of BEP2D cells at 4 and 8 Gy post-irradition( t =6.33- 45.00,P ＜ 0.05 ).Conclusion The function of antioxidant system decreased in the α-particleinduced transformed cells,which could contribute to the acceleration of cellular malignant transforming process and radiosensitivity.
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Objective To investigate the role of Rho in SiO2 induced α-SMA expression in human bronchial epithelial cells (HBECs). Methods HBECs were cultured and stimulated with SiO2. Immunocytochemistry and Western blot were used to detect the expression of α-SMA. The activity of Rho was determined by GST pull down assay. In the prevention experiment,SiO2-stimulated HBECs were incubated with Rho inhibitor Y27632,and the expression of α-SMA was examined by Western blot. Results With SiO2 (0-300 μg/mL) treatment,the expression of α-SMA increased gradually,and 200 μg/mL of SiO2 led to the highest expression of α-SMA which was (5.09±1.98) times of the expression of α-SMA in the control group(P＜0.01). HBECs treated with SiO2 (200 μg/mL) for indicated time (1,2,6,12,and 24 h)showed an obvious increase of Rho activity(P＜0.01). Y27632 inhibited SiO2-induced α-SMA expression significantly,and the inhibition rate of 20 and 30 μmol/L Y27632 was 68% and 75%,respectively (P＜0.01).Conclusion Rho signaling pathway may mediate SiO2 induced α-SMA expression in HBECs.
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Objective: To investigate the effects of Aspergillus fumigatus extract (AFE) on the expression of GM-CSF in human bronchial epithelial cells and its possible mechanism. Methods: Human bronchial epithelial cells (16HBE-14o) were cultured in vitro and were exposed to different concentrations of AFE (0, 8, 16 and 20 mg/L) for different peroids (12, 24, 48, 72 h). Moreover, heat-treated AFE, serine protease inhibitors (aprotinin), protease-activated receptor-2 (PAR 2) agonist (SLIGLV-NH2) and PAR-2 antagonist (FSLLRY-NH2) were also used to treat 16HBE-14o cells. The production and release of GM-CSF in different supernatants was determined by ELISA. The expression of GM-CSF mRNA was measured by RT-PCR. Results: 16HBE-14o cells in the normal control group only had slight expression of GM CSF; the expression was significantly increased after AFE treatment(P<0.01); and the effect of AFE was in a time- and dose-dependent manner. PAR 2 agonist also promoted release of GM-CSF. Aprotinin and PAR-2 antagonist (FSLLRY-NH2) almost totally inhibited the effect of AFE on GM-CSF production by 16HBE-14o cells. Heat-treated AFE, which lost protease activities, exerted no effect on GM-CSF production. Conclusion: AFE, with its protease activity, can activate PAR 2, and subsequently causes GM-CSF synthesis and release by airway epithelial cells, which may contribute to the development and deterioration of asthma.
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Objective To explore the effect of BPDE on the expression of N-Ras in the human bronchial epithelial cell line. Methods The levels of mRNA and protein expression in BPDE transformed 16HBE cells(16HBE-T) and untransformed control 16HBE cells(16HBE-N) were examined by using RT-PCR and Western blot. Locations and expression levels of protein in both kinds of cells were analyzed by immunocytochemical method. Results Compared with 16HBE-N, the levels of mRNA and protein of N-Ras significantly increased to 3.616 and 1.600 times in 16HBE-T. Immunocytochemical method showed N-Ras protein in 16HBE-T and 16HBE-N expressed in the cytomembrane and cytoplasm, but the expression level of protein in 16HBE-T was significantly higher than that in 16HBE-N. Conclusion The up-regulated expression of N-Ras oncogene may play an important role in the malignant transformation of 16HBE induced by BPDE
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PURPOSE: Respiratory syncytial virus (RSV) which is the most common cause of bronchiolitis in infants is an important triggering factor of asthma exacerbation in children. This virus stimulates chemokine such as regulated on activation normal T-cell expressed and secreted (RANTES) production by human airway epithelial cells in vitro. Corticosteroids are effective medications for prevention of asthma exacerbation. We examined whether the inhibitory effect of corticosteroid on RANTES production in human bronchial epithelial cells (BEAS-2B) infected with RSV varies according to the kinds of corticosteroids and the timing of corticosteroid treatment in vitro. METHODS: In the pretreatment group, BEAS-2B cells were pretreated with budesonide (BUD), fluticasone propionate (FP), or methylprednisolone (MP), and then the cells were infected with RSV. The supernatants of the cell culture were collected after virus infection. In the posttreatment group, BEAS-2B cells were infected with RSV, and then treated with corticosteroid, and the supernatants of the cell culture were collected. RANTES levels in each of the collected supernatant were determined. RESULTS: In the pretreatment group, the strength of each corticosteroid's inhibitory effect on RANTES product was not significantly different. In the posttreatment group, the strength of the inhibitory effect on the production of RANTES was in the order of BUD> MP=FP. BUD have more inhibitory effect on RANTES production at posttreatment group. CONCLUSION: The inhibitory effect of corticosteroid on the production of RANTES in BEAS-2B cells infected with RSV varied widely according to the kinds of corticosteroids and the timing of the corticosteroid treatment.
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Child , Humans , Infant , Adrenal Cortex Hormones , Asthma , Bronchiolitis , Budesonide , Cell Culture Techniques , Chemokine CCL5 , Diethylpropion , Epithelial Cells , Methylprednisolone , Respiratory Syncytial Viruses , T-Lymphocytes , FluticasoneABSTRACT
Objective To explore the mechanisms of chemical carcinogenesis and the relationship between malignant transformation and genomic/chromosomal instability of human bronchial epithelial cell line by formaldehyde.Methods BEAS-2B cell line was toxicated by formaldehyde and the LC_(50) of formaldehyde was tested by MTT assay.BEAS-2B cells were induced by formaldehyde at 20% LC_(50),and the transformation of BEAS-2B cells was identified and induced clones were selected.By G-banding technique,the metaphase modal chromosome number,karyotypes and nonrandom structural change of induced cell strains were analyzed.Results The growth rate of BEAS-2B cells induced by formaldehyde was significantly increased.The induced cells showed a trend of infinite proliferation and the anchorage independent growth was seen in 15~(th) generation cells.As compared with the relatively stable pseudodiploid karyotype of BEAS-2B cells,most of induced cells were triploid and/or tetraploid karyotypes and their chromosome 14 had lost two copies of chromosome replaced by the other two.They also had high frequency of unstable aberration(39%),including chromosome loss,endoduplication,translocation,breakage,two and/or three centromeres etc..Conclusion The formaldehyde could lead to progression of malignant transformation and result in genomic instability of human bronchial epithelial cells in vitro.
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BACKGROUND: Besides being a structural barrier for infectious pathogens, bronchial epithelium also participates actively in the modulation of airway inflammatory reactions through the synthesis of a variety of proinflammatory mediators such as RANTES (regulated on activation, normal T cell expressed & secreted) and IL-8 (interleukin 8) which are important chemokines for chemotaxis of eosinophils and neutrophils and their activation. Respiratory syncytial virus (RSV) which is the most common cause of bronchiolitis in infants is an important trigger of asthma exacerbation and stimulates chemokine production by human airway epithelial cells in vitro. OBJECTIVES: The objectives of this study are to determine the effects of dexamethasone (DX) on RSV induced RANTES and IL-8 gene expression and their production by BEAS-2B cell, a human bronchial epithelial cell line. METHODS: Then, total RNA of RANTES was collected at 6, 12, 24 and 48 hour intervals after inoculation with RSV and total RNA of IL-8 was collected at 2, 4 and 6 hour intervals after inoculation with RSV. Gene expressions of RANTES and IL-8 were determined by RT-PCR semi- quantitatively from total RNA. DX at varying concentrations (10-9, 10-8, 10-7, 10-6M) on RSV (MOI=1, MOI=10) induced chemokine production. RESULTS: Gene expressions of RANTES and IL-8 were higher in RSV infected group compared to the non-infected group at each time and at each MOI. Gene expressions of RANTES and IL-8 were decreased in DX-treated RSV infected groups compared to the non-treated RSV infected group and this tendency was prominent in higher concentrations of DX. CONCLUSION: DX may modulate the inflammatory response of the airway in RSV induced bronchiolitis and asthma exacerbation through the inhibition of virus-induced chemokine production by bronchial epithelial cells. Bronchial epithelium also participates actively in the modulation of airway inflammatory reactions through the synthesis of proinflammatory mediators such as RANTES and IL-8.
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Humans , Infant , Asthma , Bronchiolitis , Chemokine CCL5 , Chemokines , Chemotaxis , Dexamethasone , Eosinophils , Epithelial Cells , Epithelium , Gene Expression , Interleukin-8 , Neutrophils , Respiratory Syncytial Viruses , RNA , RNA, MessengerABSTRACT
Purpose:To detect the abnormalities of WWOX(WW domain containing oxidoreductase) gene in human lung adenocarcinoma cell line A549. Methods:Deletion of WWOX exons 6-8 transcript was analyzed by reverse transcriptase-PCR technology; loss of heterozygosity (LOH) of WWOX gene was analyzed by PCR-based assays for dinucleotide repeat polymorphisms technology. Aberrant expression of WWOX protein was analyzed by western blot. Results:A549 cells samples showed loss of WWOX exons 6-8 transcript.This deletion was not detected in normal primary cultured human bronchial epithelial cells samples.Three microsatellites(D16S3029、D16S3096、D16S504)did not have LOH in the normal primary cultured human bronchial epithelial cells samples, but D16S2029 and D16S3096 were all found to have LOH in A549 Cells samples. We further observed that expression of WWOX protein was significantly lower in A549 cell samples compared to the normal primary cultured human bronchial epithelial cells samples. Conclusions:WWOX gene may be important during tumorigenesis in lung adenocarcinoma cancer.Deletion of exons 6-8,LOH and aberrant expression of protein are all modes of WWOX gene inactivity in lung adenocarcinoma cancer.
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Objective To explore the effect of sodium fluoride(NaF) on the tumor markers of human embryonic bronchial epithelium (HEBE) cells.Methods HEBE cells were collected from the human abortive fetues.The cells were exposed to NaF of several concentrations for 36 h.After rinsed,the cells were incubated for 36 h again.The NaF toxicity to HEBE cells was detected using MTT method.The supernatant was collected and the lung tumor markers were detected using ELISA method.Results NaF was toxic to the HEBE cells,and the toxicity was increased with the NaF doses.There were few survival HEBE cells at 6 mmol/L NaF.The tumor markers in both of the control and experiment groups were very low,and no significant difference had been seen between them.Conclusion NaF may damage HEBE cells,but may not influence the tumor markers of HEBE cells.