Primary porcine tracheal epithelial cells(PTECs) are an appropriate model for studying the molecular mechanism of various porcine respiratory diseases, including swine-origin mycoplasmas, which are isolated from respi...Primary porcine tracheal epithelial cells(PTECs) are an appropriate model for studying the molecular mechanism of various porcine respiratory diseases, including swine-origin mycoplasmas, which are isolated from respiratory tract of pigs and mainly found on the mucosal surface surrounding swine trachea. However, the short proliferation ability of primary PTECs greatly limits their lifespan. In this study, primary PTECs were carefully isolated and cultured, and immortal PTECs were constructed by transfecting primary PTECs with the recombinant constructed plasmid pEGFPhTERT containing human telomerase reverse transcriptase(hTERT). Immortal PTECs(hTERT-PTECs) maintained both the morphological and functional characteristics of primary PTECs, as indicated by the expression of cytokeratin 18, cellcycle analysis, proliferation assay, Western blotting, telomerase activity assay, karyotype analysis and quantitative RTPCR. Compared to primary PTECs, hTERT-PTECs had an extended replicative lifespan, higher telomerase activity, and enhanced proliferative activity. In addition, this cell line resulted in a lack of transformed and grown tumors in nude mice, suggesting that it could be safely applied in further studies. Moreover, hTERT-PTECs were vulnerable to all swineorigin mycoplasmas through quantitative analysis as indicated by 50% color changing unit(CCU_(50)) calculation, and no significant differences of adhesion ability between primary and immortal PTECs were observed. For the representative swine mycoplasma Mycoplasma hyopneumoniae(Mhp), except for DNA copies quantitative real-time PCR assay, indirect immunofluorescence assay and Western blotting analysis also depicted that hTERT-PTECs was able to adhere to different Mhp strains of different virulence. In summary, like primary PTECs, hTERT-PTECs could be widely used as an adhesion cell model for swine-origin mycoplasmas and in infection studies of various porcine respiratory pathogens.展开更多
AIM: To analyze the localization of erythropoietin receptor on gastric specimens and characterize the effects of erythropoietin on the normal gastric epithelial proliferation using a porcine gastric epithelial cell c...AIM: To analyze the localization of erythropoietin receptor on gastric specimens and characterize the effects of erythropoietin on the normal gastric epithelial proliferation using a porcine gastric epithelial cell culture model. METHODS: Erythropoietin receptor was detected by RT-PCR, Western blotting and immunohistochermistry. Growth stimulation effects of erythropoietin on cultured gastric mucosal cells were determined by ELISA using bromodeoxyuridine (BrdU). RESULTS: Erythropoietin receptor was detected on cultured porcine gastric mucosal epithelial cells. Erythropoietin receptor was also detected histochemically at the base of gastric mucosal epithelium. BrdU assay demonstrated a dose-dependent increase in growth potential of cultured porcine gastric mucosal epithelial cells by administration of erythropoietin, as well as these effects were inhibited by administration of antierythropoietin antibody (P〈 0.01). CONCLUSION: These findings indicate that erythropoietin has a potential to proliferate gastric mucosal epithelium via erythropoietin receptor.展开更多
As the first line of defence against pathogens and endotoxins crossing the intestine-blood barrier,the intestinal epithelial barrier plays a determinant role in pigs’health and growth.4-Phenylbutyric acid(4-PBA),an a...As the first line of defence against pathogens and endotoxins crossing the intestine-blood barrier,the intestinal epithelial barrier plays a determinant role in pigs’health and growth.4-Phenylbutyric acid(4-PBA),an aromatic fatty acid,was reported to benefit homeostasis of endoplasmic reticulum and protein synthesis.However,whether 4-PBA affects intestinal epithelial barrier function in pigs is unknown.This study aimed to explore the effects of 4-PBA on the intestinal barrier function,using in vitro models of well-differentiated intestinal porcine epithelial cell(IPEC-J2)monolayers in the transwell plates.Cell monolayers with or without 4-PBA(1.0 mmol/L)treatment were challenged with physical scratch,deoxynivalenol(DON,2.0μg/mL,48 h),and lipopolysaccharide(LPS,5.0μg/mL,48 h),respectively.Transepithelial electrical resistance(TEER)and fluorescein isothiocyanate-dextran(FD-4)permeability were measured to indicate barrier integrity and permeability.Real-time PCR and Western blot were conducted to determine relative gene and protein expressions of tight junction proteins.As expected,physical scratch,DON,and LPS challenges decreased TEER and increased FD-4 permeability.4-PBA treatment accelerated cell mitigation and rehabilitation of the physical scratch-damaged intestinal epithelial barrier but did not alleviate DON or LPS induced barrier damage.However,once 48-h DON and LPS challenges were removed,rehabilitation of the epithelial barrier function of IPEC-J2 monolayer was accelerated by the 4-PBA treatment.Also,the relative gene and protein expressions of zonula occludens-1(ZO-1),occludin,and claudin-1 were further upregulated by the 4-PBA treatment during the barrier rehabilitation.Taken together,4-PBA accelerated the IPEC-J2 cell monolayer barrier recovering from physical scratch,DON-,and LPS-induced damage,via enhancing cell mitigation and expressions of tight junction proteins.展开更多
Deoxynivalenol(DON)is considered to be the most harmful mycotoxin that affects the intestinal health of animals and humans.Phenethyl isothiocyanate(PEITC)in feedstuff is an anti-nutritional factor and impairs nutrient...Deoxynivalenol(DON)is considered to be the most harmful mycotoxin that affects the intestinal health of animals and humans.Phenethyl isothiocyanate(PEITC)in feedstuff is an anti-nutritional factor and impairs nutrient digestion and absorption in the animal intestinal.In the current study,we aimed to explore the effects of PEITC on DON-induced apoptosis,intestinal tight junction disorder,and its potential molecular mechanism in the porcine jejunum epithelial cell line(IPEC-J2).Our results indicated that PEITC treatment markedly alleviated DON-induced cytotoxicity,decreasing the apoptotic cell percentage and pro-apoptotic mRNA/protein levels,and increasing zonula occludens-1(ZO-1),occludin and claudin-1 mRNA/protein expression.Meanwhile,PEITC treatment ameliorated DON-induced an increase of the inducible nitric oxide synthase(iNOS)and cyclooxygenase 2(COX-2)mRNA levels and intracellular reactive oxygen species(ROS)level,and a decrease of glutathione peroxidase 1(GPx1),superoxide dismutase 2(SOD2),catalase(CAT)and heme oxygenase 1(HO-1)mRNA levels.Additionally,PEITC treatment significantly down-regulated autophagy-related protein 5(ATG5),beclin-1 and microtubuleassociated protein 1 light chain 3B(LC3-II)mRNA/protein levels,decreased the number of green fluorescent protein-microtubule-associated protein 1 light-chain 3(GFP-LC3)puncta and phosphatidylinositol 3 kinase(PI3K)protein expression,and up-regulated phospho-protein kinase B(p-Akt)and phospho-mammalian target of rapamycin(p-mTOR)protein expression against DON.However,the activation of autophagy by rapamycin,an autophagy agonist,abolished the protective effects of PEITC against DON-induced cytotoxicity,apoptosis and intestinal tight junction disorder.Collectively,PEITC could confer protection against DON-induced porcine intestinal epithelial cell injury by suppressing ROSmediated autophagy.展开更多
基金supported by the National Natural Science Foundation of China(31800161,31700157,31800160,31900159,and 31770193)the Natural Science Foundation of Jiangsu Province,China(BK20180297 and BK20170600)the Independent Research Project Program of Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base,Ministry of Science and Technology,China(2019sy004)。
文摘Primary porcine tracheal epithelial cells(PTECs) are an appropriate model for studying the molecular mechanism of various porcine respiratory diseases, including swine-origin mycoplasmas, which are isolated from respiratory tract of pigs and mainly found on the mucosal surface surrounding swine trachea. However, the short proliferation ability of primary PTECs greatly limits their lifespan. In this study, primary PTECs were carefully isolated and cultured, and immortal PTECs were constructed by transfecting primary PTECs with the recombinant constructed plasmid pEGFPhTERT containing human telomerase reverse transcriptase(hTERT). Immortal PTECs(hTERT-PTECs) maintained both the morphological and functional characteristics of primary PTECs, as indicated by the expression of cytokeratin 18, cellcycle analysis, proliferation assay, Western blotting, telomerase activity assay, karyotype analysis and quantitative RTPCR. Compared to primary PTECs, hTERT-PTECs had an extended replicative lifespan, higher telomerase activity, and enhanced proliferative activity. In addition, this cell line resulted in a lack of transformed and grown tumors in nude mice, suggesting that it could be safely applied in further studies. Moreover, hTERT-PTECs were vulnerable to all swineorigin mycoplasmas through quantitative analysis as indicated by 50% color changing unit(CCU_(50)) calculation, and no significant differences of adhesion ability between primary and immortal PTECs were observed. For the representative swine mycoplasma Mycoplasma hyopneumoniae(Mhp), except for DNA copies quantitative real-time PCR assay, indirect immunofluorescence assay and Western blotting analysis also depicted that hTERT-PTECs was able to adhere to different Mhp strains of different virulence. In summary, like primary PTECs, hTERT-PTECs could be widely used as an adhesion cell model for swine-origin mycoplasmas and in infection studies of various porcine respiratory pathogens.
基金Supported by the grants from National Defense Medical College.
文摘AIM: To analyze the localization of erythropoietin receptor on gastric specimens and characterize the effects of erythropoietin on the normal gastric epithelial proliferation using a porcine gastric epithelial cell culture model. METHODS: Erythropoietin receptor was detected by RT-PCR, Western blotting and immunohistochermistry. Growth stimulation effects of erythropoietin on cultured gastric mucosal cells were determined by ELISA using bromodeoxyuridine (BrdU). RESULTS: Erythropoietin receptor was detected on cultured porcine gastric mucosal epithelial cells. Erythropoietin receptor was also detected histochemically at the base of gastric mucosal epithelium. BrdU assay demonstrated a dose-dependent increase in growth potential of cultured porcine gastric mucosal epithelial cells by administration of erythropoietin, as well as these effects were inhibited by administration of antierythropoietin antibody (P〈 0.01). CONCLUSION: These findings indicate that erythropoietin has a potential to proliferate gastric mucosal epithelium via erythropoietin receptor.
基金funded by the“Shennong Scholar funding of Hunan Agricultural University”,the“Changsha Municipal Natural Science Foundation(Grant No.kq2014068)”the“Open Project Program of Key Laboratory of Feed Biotechnology,the Ministry of Agriculture and Rural Affairs of the People’s Republic of China”。
文摘As the first line of defence against pathogens and endotoxins crossing the intestine-blood barrier,the intestinal epithelial barrier plays a determinant role in pigs’health and growth.4-Phenylbutyric acid(4-PBA),an aromatic fatty acid,was reported to benefit homeostasis of endoplasmic reticulum and protein synthesis.However,whether 4-PBA affects intestinal epithelial barrier function in pigs is unknown.This study aimed to explore the effects of 4-PBA on the intestinal barrier function,using in vitro models of well-differentiated intestinal porcine epithelial cell(IPEC-J2)monolayers in the transwell plates.Cell monolayers with or without 4-PBA(1.0 mmol/L)treatment were challenged with physical scratch,deoxynivalenol(DON,2.0μg/mL,48 h),and lipopolysaccharide(LPS,5.0μg/mL,48 h),respectively.Transepithelial electrical resistance(TEER)and fluorescein isothiocyanate-dextran(FD-4)permeability were measured to indicate barrier integrity and permeability.Real-time PCR and Western blot were conducted to determine relative gene and protein expressions of tight junction proteins.As expected,physical scratch,DON,and LPS challenges decreased TEER and increased FD-4 permeability.4-PBA treatment accelerated cell mitigation and rehabilitation of the physical scratch-damaged intestinal epithelial barrier but did not alleviate DON or LPS induced barrier damage.However,once 48-h DON and LPS challenges were removed,rehabilitation of the epithelial barrier function of IPEC-J2 monolayer was accelerated by the 4-PBA treatment.Also,the relative gene and protein expressions of zonula occludens-1(ZO-1),occludin,and claudin-1 were further upregulated by the 4-PBA treatment during the barrier rehabilitation.Taken together,4-PBA accelerated the IPEC-J2 cell monolayer barrier recovering from physical scratch,DON-,and LPS-induced damage,via enhancing cell mitigation and expressions of tight junction proteins.
基金This study was financially supported by the National Natural Science Foundation of China(32072926,31772811)the Priority Academic Program Development of Jiangsu Higher Education Institutions(Jiangsu,China).
文摘Deoxynivalenol(DON)is considered to be the most harmful mycotoxin that affects the intestinal health of animals and humans.Phenethyl isothiocyanate(PEITC)in feedstuff is an anti-nutritional factor and impairs nutrient digestion and absorption in the animal intestinal.In the current study,we aimed to explore the effects of PEITC on DON-induced apoptosis,intestinal tight junction disorder,and its potential molecular mechanism in the porcine jejunum epithelial cell line(IPEC-J2).Our results indicated that PEITC treatment markedly alleviated DON-induced cytotoxicity,decreasing the apoptotic cell percentage and pro-apoptotic mRNA/protein levels,and increasing zonula occludens-1(ZO-1),occludin and claudin-1 mRNA/protein expression.Meanwhile,PEITC treatment ameliorated DON-induced an increase of the inducible nitric oxide synthase(iNOS)and cyclooxygenase 2(COX-2)mRNA levels and intracellular reactive oxygen species(ROS)level,and a decrease of glutathione peroxidase 1(GPx1),superoxide dismutase 2(SOD2),catalase(CAT)and heme oxygenase 1(HO-1)mRNA levels.Additionally,PEITC treatment significantly down-regulated autophagy-related protein 5(ATG5),beclin-1 and microtubuleassociated protein 1 light chain 3B(LC3-II)mRNA/protein levels,decreased the number of green fluorescent protein-microtubule-associated protein 1 light-chain 3(GFP-LC3)puncta and phosphatidylinositol 3 kinase(PI3K)protein expression,and up-regulated phospho-protein kinase B(p-Akt)and phospho-mammalian target of rapamycin(p-mTOR)protein expression against DON.However,the activation of autophagy by rapamycin,an autophagy agonist,abolished the protective effects of PEITC against DON-induced cytotoxicity,apoptosis and intestinal tight junction disorder.Collectively,PEITC could confer protection against DON-induced porcine intestinal epithelial cell injury by suppressing ROSmediated autophagy.