AIM:To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori(H.pylori) infection.METHODS:Aiming to overcome this limitation,clones of the ...AIM:To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori(H.pylori) infection.METHODS:Aiming to overcome this limitation,clones of the heterogenic cancer-derived NCI-N87 cell line were isolated,by stably-transducing it with the human telomerase reverse-transcriptase(h TERT) catalytic subunit gene.The clones were first characterized regarding their cell growth pattern and phenotype.For that we measured the clones' adherence properties,expression of cell-cell junctions' markers(ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance.The gastric properties of the clones,concerning expression of mucins,zymogens and glycan contents,were then evaluated by haematoxylin and eosin staining,Periodic acid Schiff(PAS) and PAS/Alcian Blue-staining,immunocytochemistry and Western blot.In addition,we assessed the usefulness of the h TERT-expressing gastric cell line for H.pylori research,by performing co-culture assays and measuring the IL-8 secretion,by ELISA,upon infection with two H.pylori strains differing in virulence.RESULTS:Compared with the parental cell line,themost promising NCI-hT ERT-derived clones(CL5 and CL6) were composed of cells with homogenous phenotype,presented higher relative telomerase activities,better adhesion properties,ability to be maintained in culture for longer periods after confluency,and were more efficient in PAS-reactive mucins secretion.Both clones were shown to produce high amounts of MUC1,MUC2 and MUC13.NCI-hT ERT-CL5 mucins were shown to be decorated with blood group H type 2(BG-H),Lewis-x(Lex),Ley and Lea and,in a less extent,with BG-A antigens,but the former two antigens were not detected in the NCI-h TERT-CL6.None of the clones exhibited detectable levels of MUC6 nor sialylated Lex and Lea glycans.Entailing good gastric properties,both NCIhT ERT-clones were found to produce pepsinogen-5 and human gastric lipase.The progenitor-like phenotype of NCI-hT ERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5,supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions.CONCLUSION:These traits,in addition to resistance to microaerobic conditions and good responsiveness to H.pylori co-culture,in a strain virulence-dependent manner,make the NCI-hT ERT-CL6 a promising model for future in vitro studies.展开更多
The expression of CH50 polypoptide in bladder cancer cell line BIU-87 and the effects on the invasion ability of BIU-87 were investigated. The eukaryotic expressing vector pCH510 of polypeptide CH50 was introduced int...The expression of CH50 polypoptide in bladder cancer cell line BIU-87 and the effects on the invasion ability of BIU-87 were investigated. The eukaryotic expressing vector pCH510 of polypeptide CH50 was introduced into BIU-87 cells by gene transfection in vitro. The expression of CH50 polypeptide was detected by using immunohistochemical S-P method. The expression of the transfected gene was identified by RT-PCR. Cell invasion assay kit was applied to detect the effect of CH50 polypeptide on the invasion ability of BIU-87. The results showed that the BIU-87 cells transfected with pCH510 could express the CH50 polypeptide, while in the control group, no CH50 polypeptide was detectable. In the transfection group, the invasion ability of BIU-87 in vitro was lower than in control group (P<0.05). It was concluded that CH50 polypeptide was successfully expressed in BIU-87 cells by gene transfection, by which the in vitro invasion ability of BIU-87 was inhibited.展开更多
目的对经过短串联重复序列(short tandem repeats,STR)分型检测技术鉴定后的两株U87胶质瘤细胞系[(美国典型培养物保藏中心(American Type Culture Collection,ATCC)数据库中U-87 MG Glioblastoma-Astrocytoma Human和德国微生物菌种保...目的对经过短串联重复序列(short tandem repeats,STR)分型检测技术鉴定后的两株U87胶质瘤细胞系[(美国典型培养物保藏中心(American Type Culture Collection,ATCC)数据库中U-87 MG Glioblastoma-Astrocytoma Human和德国微生物菌种保藏中心(Deutsche Sammlung von Mikroorganismen und Zellkulturen,DSMZ)数据库中U-87 MG]进行细胞生物学特性测定,对两株细胞用于后续研究工作的可能性进行评价。方法分别采用CCK-8法、划痕实验、Transwell实验等方法,并结合显微镜下细胞形态学特征,比较两株肿瘤细胞系的增生、迁移及侵袭能力等生物学特性。结果 (1)CCK-8法:细胞生长曲线提示细胞增生能力差异无统计学意义(P>0.05)。(2)划痕实验:两细胞系迁移能力差异无统计学意义(P>0.05)。(3)Transwell实验:两细胞系迁移能力差异无统计学意义(P>0.05),DSMZ数据库中U-87 MG细胞侵袭能力高于ATCC数据库中U-87 MG GlioblastomaAstrocytoma Human细胞,差异有统计学意义(P<0.05)。结论两株U87细胞系均可反映脑胶质瘤的生物学特性,可用于后续科研实验。两细胞系的增生、迁移能力差异无统计学意义,侵袭能力差异具有统计学意义,可根据实验目的选择合适的细胞系。展开更多
Objective:Glioma is a highly invasive tumor,frequently disposed in essential areas of the brain,which makes its surgical excision extremely difficult;meanwhile adjuvant therapy remains quite ineffective.Methods:In the...Objective:Glioma is a highly invasive tumor,frequently disposed in essential areas of the brain,which makes its surgical excision extremely difficult;meanwhile adjuvant therapy remains quite ineffective.Methods:In the current report,a new therapeutic approach in curing malignant neoplasms has been performed on the U87 human glioblastoma model.This approach,termed"Karanahan",is aimed at the eradication of cancer stem cells(CSCs),which were recently shown to be capable of internalizing fragments of extracellular double-stranded DNA.After being internalized,these fragments interfere in the process of repairing interstrand cross-links caused by exposure to appropriate cytostatics,and such an interference results either in elimination of CSCs or in the loss of their tumorigenic potency.Implementation of the approach requires a scheduled administration of cytostatic and complex composite double-stranded DNA preparation.Results:U87 cells treated in vitro in accordance with the Karanahan approach completely lost their tumorigenicity and produced no grafts upon intracerebral transplantation into immunodeficient mice.In SCID mice with developed subcutaneous grafts,the treatment resulted in reliable slowing down of tumor growth rate(P<0.05).In the experiment with intracerebral transplantation of U87 cells followed by surgical excision of the developed graft and subsequent therapeutic treatment,the Karanahan approach was shown to reliably slow down the tumor growth rate and increase the median survival of the mice twofold relative to the control.Conclusions:The effectiveness of the Karanahan approach has been demonstrated both in vitro and in vivo in treating developed subcutaneous grafts as well as orthotopic grafts after surgical excision of the tumor.展开更多
基金Supported by Grants from the Fundao para a Ciência e a Tecnologia(FCT,Portugal),No.PPCDT/SAL-IMI/57297/2004 and No.PTDC/BIM-MEC/1051/2012The Swedish Cancer foundation+2 种基金The Swedish Research Council,No.K2010-79X-21372-01-3Forska utan djurfrsk,Animal Free ResearchResearch fellowship 2011 from the Sociedade Portuguesa de Gastrenterologia(Portugal)
文摘AIM:To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori(H.pylori) infection.METHODS:Aiming to overcome this limitation,clones of the heterogenic cancer-derived NCI-N87 cell line were isolated,by stably-transducing it with the human telomerase reverse-transcriptase(h TERT) catalytic subunit gene.The clones were first characterized regarding their cell growth pattern and phenotype.For that we measured the clones' adherence properties,expression of cell-cell junctions' markers(ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance.The gastric properties of the clones,concerning expression of mucins,zymogens and glycan contents,were then evaluated by haematoxylin and eosin staining,Periodic acid Schiff(PAS) and PAS/Alcian Blue-staining,immunocytochemistry and Western blot.In addition,we assessed the usefulness of the h TERT-expressing gastric cell line for H.pylori research,by performing co-culture assays and measuring the IL-8 secretion,by ELISA,upon infection with two H.pylori strains differing in virulence.RESULTS:Compared with the parental cell line,themost promising NCI-hT ERT-derived clones(CL5 and CL6) were composed of cells with homogenous phenotype,presented higher relative telomerase activities,better adhesion properties,ability to be maintained in culture for longer periods after confluency,and were more efficient in PAS-reactive mucins secretion.Both clones were shown to produce high amounts of MUC1,MUC2 and MUC13.NCI-hT ERT-CL5 mucins were shown to be decorated with blood group H type 2(BG-H),Lewis-x(Lex),Ley and Lea and,in a less extent,with BG-A antigens,but the former two antigens were not detected in the NCI-h TERT-CL6.None of the clones exhibited detectable levels of MUC6 nor sialylated Lex and Lea glycans.Entailing good gastric properties,both NCIhT ERT-clones were found to produce pepsinogen-5 and human gastric lipase.The progenitor-like phenotype of NCI-hT ERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5,supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions.CONCLUSION:These traits,in addition to resistance to microaerobic conditions and good responsiveness to H.pylori co-culture,in a strain virulence-dependent manner,make the NCI-hT ERT-CL6 a promising model for future in vitro studies.
文摘The expression of CH50 polypoptide in bladder cancer cell line BIU-87 and the effects on the invasion ability of BIU-87 were investigated. The eukaryotic expressing vector pCH510 of polypeptide CH50 was introduced into BIU-87 cells by gene transfection in vitro. The expression of CH50 polypeptide was detected by using immunohistochemical S-P method. The expression of the transfected gene was identified by RT-PCR. Cell invasion assay kit was applied to detect the effect of CH50 polypeptide on the invasion ability of BIU-87. The results showed that the BIU-87 cells transfected with pCH510 could express the CH50 polypeptide, while in the control group, no CH50 polypeptide was detectable. In the transfection group, the invasion ability of BIU-87 in vitro was lower than in control group (P<0.05). It was concluded that CH50 polypeptide was successfully expressed in BIU-87 cells by gene transfection, by which the in vitro invasion ability of BIU-87 was inhibited.
文摘目的对经过短串联重复序列(short tandem repeats,STR)分型检测技术鉴定后的两株U87胶质瘤细胞系[(美国典型培养物保藏中心(American Type Culture Collection,ATCC)数据库中U-87 MG Glioblastoma-Astrocytoma Human和德国微生物菌种保藏中心(Deutsche Sammlung von Mikroorganismen und Zellkulturen,DSMZ)数据库中U-87 MG]进行细胞生物学特性测定,对两株细胞用于后续研究工作的可能性进行评价。方法分别采用CCK-8法、划痕实验、Transwell实验等方法,并结合显微镜下细胞形态学特征,比较两株肿瘤细胞系的增生、迁移及侵袭能力等生物学特性。结果 (1)CCK-8法:细胞生长曲线提示细胞增生能力差异无统计学意义(P>0.05)。(2)划痕实验:两细胞系迁移能力差异无统计学意义(P>0.05)。(3)Transwell实验:两细胞系迁移能力差异无统计学意义(P>0.05),DSMZ数据库中U-87 MG细胞侵袭能力高于ATCC数据库中U-87 MG GlioblastomaAstrocytoma Human细胞,差异有统计学意义(P<0.05)。结论两株U87细胞系均可反映脑胶质瘤的生物学特性,可用于后续科研实验。两细胞系的增生、迁移能力差异无统计学意义,侵袭能力差异具有统计学意义,可根据实验目的选择合适的细胞系。
基金supported by the Russian Ministry of Science and High Education,State Budgeted Project 0259-2021-0013 for the Institute of Cytology and Genetics,Novosibirskthe Russian Foundation for Basic Research(Grant No.18-34-20016)+1 种基金Microscopic examination of bone marrow cells was supported by the Russian Ministry of Science and High EducationState Budgeted Project 0246-2021-0017 for the Institute of Molecular and Cellular Biology,Novosibirsk。
文摘Objective:Glioma is a highly invasive tumor,frequently disposed in essential areas of the brain,which makes its surgical excision extremely difficult;meanwhile adjuvant therapy remains quite ineffective.Methods:In the current report,a new therapeutic approach in curing malignant neoplasms has been performed on the U87 human glioblastoma model.This approach,termed"Karanahan",is aimed at the eradication of cancer stem cells(CSCs),which were recently shown to be capable of internalizing fragments of extracellular double-stranded DNA.After being internalized,these fragments interfere in the process of repairing interstrand cross-links caused by exposure to appropriate cytostatics,and such an interference results either in elimination of CSCs or in the loss of their tumorigenic potency.Implementation of the approach requires a scheduled administration of cytostatic and complex composite double-stranded DNA preparation.Results:U87 cells treated in vitro in accordance with the Karanahan approach completely lost their tumorigenicity and produced no grafts upon intracerebral transplantation into immunodeficient mice.In SCID mice with developed subcutaneous grafts,the treatment resulted in reliable slowing down of tumor growth rate(P<0.05).In the experiment with intracerebral transplantation of U87 cells followed by surgical excision of the developed graft and subsequent therapeutic treatment,the Karanahan approach was shown to reliably slow down the tumor growth rate and increase the median survival of the mice twofold relative to the control.Conclusions:The effectiveness of the Karanahan approach has been demonstrated both in vitro and in vivo in treating developed subcutaneous grafts as well as orthotopic grafts after surgical excision of the tumor.
