AIM: To analyze the upregulated CD133 expression in tumorigenesis of primary colon cancer cells. METHODS: Upregulated CD133 expression in tumorigenesis of colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 an...AIM: To analyze the upregulated CD133 expression in tumorigenesis of primary colon cancer cells. METHODS: Upregulated CD133 expression in tumorigenesis of colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) was analyzed by flow cytometry. Human colon cancer tissue samples were stained with anti-human CD133. SW620 cells were sorted according to the CD133 expression level measured by fluorescence-activated cell sorting. Spheroids of colorectal cancer cells were cultured with the hanging drop. Expression of CD133 and Lgr5 in spheroids of colorectal cancer cells and monolayer culture was detected by RT-qPCR. Spheroids of colorectal cancer cells were analyzed using anti-human CD133 with immunohistochemical staining. RESULTS: CD133 antigen was expressed in colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) as well as in primary and metastatic human colon cancer tissues. However, the CD133 was differently expressed in these cell lines and tissues. The expression levels of CD133 and Lgr5 were significantly higher in spheroids of parental, CD133hi and CD133-cells than in their monolayer culture at the mRNA level (P < 0.05). Immunohistochemical staining of spheroids of CD133-cells showed that CD133 was highly expressed in colorectal cancer cell lines. CONCLUSION: Upregulated CD133 expression plays a role in tumorigenesis colorectal cancer cells, which may promote the expression of other critical genes that can drive tumorigenesis.展开更多
Objective:To explore the inhihitive and apoptosis inductive effect of IL-24 genes on CD133^+laryngeal cancer cells in Hep-2 line.Methods:Human peripheral blood monocytes were isolated.The total RNA was extracted by us...Objective:To explore the inhihitive and apoptosis inductive effect of IL-24 genes on CD133^+laryngeal cancer cells in Hep-2 line.Methods:Human peripheral blood monocytes were isolated.The total RNA was extracted by using Trizol method and reverse transcripted into cDNA using RT-PCR method.Primers P1 and P2 was designed for the amplification of human IL-24 genes.After confirmation of agarose gel electrophoresis tests,TA was cloned into pMD19-T simple vector.Nhe Ⅰ and Xho Ⅰ double digesting human IL-24 and pIRES2-ZsGreen1 and eukaryotic expression vector were used to establish the pIRES2-ZsGreen1-hIL-24 vector,and detected by enzyme digestion and gene sequencing methods.Flow cytometry(FCM) was used to isolate CD133^+ cells from Hep-2 cells.CD133^+ cells were transfected with pIRES2-ZsGreen1-hIL-24 through liposome 2000.After detection,MTT and FCM were used to observe the effect of IL-24 gene on CD133^+ laryngeal cancer Hep-2 cells.Results:Lipotin mediated transfection of recombinant pIRES2-ZsGreen1-hIL-24 plasmid into CD133^+ Hep-2 could expressed IL-24 gene in cells stably.MTT results showed that IL-24 transfected group was significantly suppressed compared to empty vector group and control group(P<0.05);FCM results showed that the apoptosis rate of experimental group increased significantly compared to empty vector group and control group(P<0.05).Conclusions:IL-24 gene expressions can inhibit proliferation of CD133^+laryngeal cells in Hep-2 line and promote their apoptosis.展开更多
AIM: To understand CD133 promoter hypermethyl-ation and expression in 32 colorectal cancer cell lines. METHODS: Nucleic acid was isolated from 32 colorectal cancer cell lines and CD133 expression levels were measured ...AIM: To understand CD133 promoter hypermethyl-ation and expression in 32 colorectal cancer cell lines. METHODS: Nucleic acid was isolated from 32 colorectal cancer cell lines and CD133 expression levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Promoter methylation status of the CD133 gene was analyzed with a methylation-specific PCR after sodium-bisulfi te modification and by clonal sequencing analysis. The correlation between expression and promoter methylation of CD133 gene was confirmed with treatment of 5-aza-2’-deoxycytidine. RESULTS: We measured CD133 expression levels in 32 colorectal cancer cell lines. RT-PCR analysis showed undetectable or low levels of CD133 expression in 34.4%of cell lines. To verify the relation between CD133 expression and methylation status of the CD133 gene promoter in colorectal carcinogenesis, CD133 gene promoter hypermethylation was analyzed in 32 cancer cell lines. Promoter hypermethylation was detected in 13 (40.6%) of the cell lines using methylation specificPCR and confirmed by bisulfite sequencing analysis. Treatment of 11 of the cell lines with the demethylation agent 5-aza-2’-deoxycytidine recovered CD133 expression in most of them. CONCLUSION: Transcriptional repression of CD133 is caused by promoter hypermethylation of the CD133 CpG islands in some of colorectal cancer cell lines. The study may contribute to the understanding of the role of CD133 inactivation in the progression of colorectal cancers.展开更多
Cellular senescence is a signal transduction process which maintained genomic stability and stopped mammalian cell growth. Furthermore, cellular senescence induces a protective response to a variety of DNA damage. How...Cellular senescence is a signal transduction process which maintained genomic stability and stopped mammalian cell growth. Furthermore, cellular senescence induces a protective response to a variety of DNA damage. However, this process is also associated with apoptosis, upregulated secretion of inflammatory cytokine, and promoted surrounding tissue damage. When cellular senescence accumulates to a certain extent, it triggers geriatric diseases, such as chronic inflammation, immune senescence-associated tumors and incontrollable infections. Cellular senescence gene SENEX, which was cloned in 2004, has been demonstrated to play a unique gatekeeper function in human endothelial cells when stress-induced pre-mature senescence and apoptosis occurr. The phenomenon that CD4+CD25+ Treg cells accumulated in the aged population has been well studied in recent years. Now Treg accumulation related to immune-pathology has attracted more interest. CD4+CD25+ Treg did not decline and age, but accumulated and suppressed immunoreaction. The enhanced Treg number and function may be associated with stress-induced premature senescence-mediated unique cellular senescence protection mechanisms, and SENEX may play a critical role in this process. In this article, we summarize the cellular senescence and SENEX gene in the accumulation and functional activity of CD4+CD25+ Treg in the elderly.展开更多
Angioarchitecture plays an important role in the malignant development of intracranial hemangiopericytoma. It remains poorly understood whether high frequency of hemorrhage during clinical surgery for intracranial hem...Angioarchitecture plays an important role in the malignant development of intracranial hemangiopericytoma. It remains poorly understood whether high frequency of hemorrhage during clinical surgery for intracranial hemangiopericytoma is associated with angioarchitecture. The present study utilized hematoxylin-eosin staining, and immunohistochemical staining with epithelial membrane antigen, vimentin, CD34, von Willebrand factor （vWF） and CD133 to observe characteristics of angioarchitecture. In addition, silver stains were used to demonstrate changes in reticular fibers in the wall of vessel channels in intracranial hemangiopericytoma and meningioma. Five patterns of angioarchitecture were identified in intracranial hemangiopericytoma, namely tumor cell islands, vasculogenic mimicry, mosaic blood vessels, sprouting angiogenesis, and intussusceptive angiogenesis. Several CD133＋ tumor cells were found to form tumor cell islands. A connection between vWF ^＋ and vWF channels was detected in the pattern of intussusceptive angiogenesis, and some vimentin^＋ tumor cells were embedded in the periodic acid-Schiff positive channel wall. Incomplete threads of reticular fibers formed the walls of larger pseudo-vascular channels and some tumor clumps or scattered tumor cells were detected ＂floating＂ in them. The angioarchitecture, specific markers and reticular fibers of intracranial hemangiopericytoma were significantly different from meningioma. Angioarchitecture provides a functional vascular network for vascular evolution in intracranial hemangiopericytoma and contributes to significant intra-operative bleeding.展开更多
Objective:We recombine the suicide gene CD,UPRT into plasmid pTRKH2 and clone the recombinant dual suicide gene therapy system into tumor-hypoxia-targeting vector Bifidobacterium infantis and characterize its function...Objective:We recombine the suicide gene CD,UPRT into plasmid pTRKH2 and clone the recombinant dual suicide gene therapy system into tumor-hypoxia-targeting vector Bifidobacterium infantis and characterize its function.Methods:CD gene,UPRT gene and lactic acid bacteria expression plasmid pTRKH2 were digested by restriction endonuclease BamH I and Sal I,and constructed recombinant plasmids pTRKH2/CD and pTRKH2/UPRT in E.coli.The recombinant plasmids were then transfected into Bifidobacterium Infantis by electroporation.Identification of pTRKH2/CD and pTRKH2/UPRT was processed by dual restriction endonuclease digesting and sequencing.RT-PCR and SDS-PAGE were used to examine the expression of CD and UPRT genes at RNA and protein levels.The killing effects on Melanoma B16-F10 cells by pTRKH2/CD and pTRKH2/UPRT suicide gene therapy system with 5-FC were examined by MTT assay.Results:The CD gene and UPRT gene was successfully recombined into lactic acid bacteria expression plasmid pTRKH2.After dual endonuclease digestion of plasmid purified from the positively transfected E.coli,two fragments of 6.9 Kb and 1.3 Kb were found for CD gene and two fragments of 6.9 Kb and 620 bp were found for UPRT gene.The sequencing of CD gene and UPRT gene proved consistent sequences with Genebank published data.A fragment of 1.3 Kb for CD gene and fragment of 620 bp for UPRT gene was found in recombinant Bifidobacterium by RT-PCR.A 52 KDa protein for CD gene was identified in whole-cell protein of recombinant Bifidobacterium and a 26 KDa protein for UPRT gene was identified in supernatant fluid of recombinant Bifidobacterium.The survival rate of tumor cells treated by extracts from culture of recombinant Bifidobacterium with 5-FC showed a strong killing effects of pTRKH2/CD and pTRKH2/UPRT dual suicide gene therapy system on Melanoma B16-F10 cells.Conclusion:CD gene and UPRT gene are successfully inserted into pTRKH2 and transfected into tumor-hypoxia-targeting vector Bifidobacterium Infantis.This dual suicide gene therapy system shows a high efficiency for tumor cells killing.展开更多
文摘AIM: To analyze the upregulated CD133 expression in tumorigenesis of primary colon cancer cells. METHODS: Upregulated CD133 expression in tumorigenesis of colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) was analyzed by flow cytometry. Human colon cancer tissue samples were stained with anti-human CD133. SW620 cells were sorted according to the CD133 expression level measured by fluorescence-activated cell sorting. Spheroids of colorectal cancer cells were cultured with the hanging drop. Expression of CD133 and Lgr5 in spheroids of colorectal cancer cells and monolayer culture was detected by RT-qPCR. Spheroids of colorectal cancer cells were analyzed using anti-human CD133 with immunohistochemical staining. RESULTS: CD133 antigen was expressed in colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) as well as in primary and metastatic human colon cancer tissues. However, the CD133 was differently expressed in these cell lines and tissues. The expression levels of CD133 and Lgr5 were significantly higher in spheroids of parental, CD133hi and CD133-cells than in their monolayer culture at the mRNA level (P < 0.05). Immunohistochemical staining of spheroids of CD133-cells showed that CD133 was highly expressed in colorectal cancer cell lines. CONCLUSION: Upregulated CD133 expression plays a role in tumorigenesis colorectal cancer cells, which may promote the expression of other critical genes that can drive tumorigenesis.
基金supported by Jilin Province Natural Science Foundation of China.No:20130101151JCChinese Ministry of Education Projects of Doctoral New Teachers.No:20120061120092
文摘Objective:To explore the inhihitive and apoptosis inductive effect of IL-24 genes on CD133^+laryngeal cancer cells in Hep-2 line.Methods:Human peripheral blood monocytes were isolated.The total RNA was extracted by using Trizol method and reverse transcripted into cDNA using RT-PCR method.Primers P1 and P2 was designed for the amplification of human IL-24 genes.After confirmation of agarose gel electrophoresis tests,TA was cloned into pMD19-T simple vector.Nhe Ⅰ and Xho Ⅰ double digesting human IL-24 and pIRES2-ZsGreen1 and eukaryotic expression vector were used to establish the pIRES2-ZsGreen1-hIL-24 vector,and detected by enzyme digestion and gene sequencing methods.Flow cytometry(FCM) was used to isolate CD133^+ cells from Hep-2 cells.CD133^+ cells were transfected with pIRES2-ZsGreen1-hIL-24 through liposome 2000.After detection,MTT and FCM were used to observe the effect of IL-24 gene on CD133^+ laryngeal cancer Hep-2 cells.Results:Lipotin mediated transfection of recombinant pIRES2-ZsGreen1-hIL-24 plasmid into CD133^+ Hep-2 could expressed IL-24 gene in cells stably.MTT results showed that IL-24 transfected group was significantly suppressed compared to empty vector group and control group(P<0.05);FCM results showed that the apoptosis rate of experimental group increased significantly compared to empty vector group and control group(P<0.05).Conclusions:IL-24 gene expressions can inhibit proliferation of CD133^+laryngeal cells in Hep-2 line and promote their apoptosis.
基金Supported by (in part) The Korea Science and Engineering Foundation (KOSEF) funded by the Korean government (MEST R01-2008-000-20108-0)
文摘AIM: To understand CD133 promoter hypermethyl-ation and expression in 32 colorectal cancer cell lines. METHODS: Nucleic acid was isolated from 32 colorectal cancer cell lines and CD133 expression levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Promoter methylation status of the CD133 gene was analyzed with a methylation-specific PCR after sodium-bisulfi te modification and by clonal sequencing analysis. The correlation between expression and promoter methylation of CD133 gene was confirmed with treatment of 5-aza-2’-deoxycytidine. RESULTS: We measured CD133 expression levels in 32 colorectal cancer cell lines. RT-PCR analysis showed undetectable or low levels of CD133 expression in 34.4%of cell lines. To verify the relation between CD133 expression and methylation status of the CD133 gene promoter in colorectal carcinogenesis, CD133 gene promoter hypermethylation was analyzed in 32 cancer cell lines. Promoter hypermethylation was detected in 13 (40.6%) of the cell lines using methylation specificPCR and confirmed by bisulfite sequencing analysis. Treatment of 11 of the cell lines with the demethylation agent 5-aza-2’-deoxycytidine recovered CD133 expression in most of them. CONCLUSION: Transcriptional repression of CD133 is caused by promoter hypermethylation of the CD133 CpG islands in some of colorectal cancer cell lines. The study may contribute to the understanding of the role of CD133 inactivation in the progression of colorectal cancers.
文摘Cellular senescence is a signal transduction process which maintained genomic stability and stopped mammalian cell growth. Furthermore, cellular senescence induces a protective response to a variety of DNA damage. However, this process is also associated with apoptosis, upregulated secretion of inflammatory cytokine, and promoted surrounding tissue damage. When cellular senescence accumulates to a certain extent, it triggers geriatric diseases, such as chronic inflammation, immune senescence-associated tumors and incontrollable infections. Cellular senescence gene SENEX, which was cloned in 2004, has been demonstrated to play a unique gatekeeper function in human endothelial cells when stress-induced pre-mature senescence and apoptosis occurr. The phenomenon that CD4+CD25+ Treg cells accumulated in the aged population has been well studied in recent years. Now Treg accumulation related to immune-pathology has attracted more interest. CD4+CD25+ Treg did not decline and age, but accumulated and suppressed immunoreaction. The enhanced Treg number and function may be associated with stress-induced premature senescence-mediated unique cellular senescence protection mechanisms, and SENEX may play a critical role in this process. In this article, we summarize the cellular senescence and SENEX gene in the accumulation and functional activity of CD4+CD25+ Treg in the elderly.
基金the Science and Technology Projects in Liaoning Province, No. 2010225036
文摘Angioarchitecture plays an important role in the malignant development of intracranial hemangiopericytoma. It remains poorly understood whether high frequency of hemorrhage during clinical surgery for intracranial hemangiopericytoma is associated with angioarchitecture. The present study utilized hematoxylin-eosin staining, and immunohistochemical staining with epithelial membrane antigen, vimentin, CD34, von Willebrand factor （vWF） and CD133 to observe characteristics of angioarchitecture. In addition, silver stains were used to demonstrate changes in reticular fibers in the wall of vessel channels in intracranial hemangiopericytoma and meningioma. Five patterns of angioarchitecture were identified in intracranial hemangiopericytoma, namely tumor cell islands, vasculogenic mimicry, mosaic blood vessels, sprouting angiogenesis, and intussusceptive angiogenesis. Several CD133＋ tumor cells were found to form tumor cell islands. A connection between vWF ^＋ and vWF channels was detected in the pattern of intussusceptive angiogenesis, and some vimentin^＋ tumor cells were embedded in the periodic acid-Schiff positive channel wall. Incomplete threads of reticular fibers formed the walls of larger pseudo-vascular channels and some tumor clumps or scattered tumor cells were detected ＂floating＂ in them. The angioarchitecture, specific markers and reticular fibers of intracranial hemangiopericytoma were significantly different from meningioma. Angioarchitecture provides a functional vascular network for vascular evolution in intracranial hemangiopericytoma and contributes to significant intra-operative bleeding.
基金Supported by a grant from the Science and Technology Bureau of Sichuan Province (No. 04JY029-020-2)
文摘Objective:We recombine the suicide gene CD,UPRT into plasmid pTRKH2 and clone the recombinant dual suicide gene therapy system into tumor-hypoxia-targeting vector Bifidobacterium infantis and characterize its function.Methods:CD gene,UPRT gene and lactic acid bacteria expression plasmid pTRKH2 were digested by restriction endonuclease BamH I and Sal I,and constructed recombinant plasmids pTRKH2/CD and pTRKH2/UPRT in E.coli.The recombinant plasmids were then transfected into Bifidobacterium Infantis by electroporation.Identification of pTRKH2/CD and pTRKH2/UPRT was processed by dual restriction endonuclease digesting and sequencing.RT-PCR and SDS-PAGE were used to examine the expression of CD and UPRT genes at RNA and protein levels.The killing effects on Melanoma B16-F10 cells by pTRKH2/CD and pTRKH2/UPRT suicide gene therapy system with 5-FC were examined by MTT assay.Results:The CD gene and UPRT gene was successfully recombined into lactic acid bacteria expression plasmid pTRKH2.After dual endonuclease digestion of plasmid purified from the positively transfected E.coli,two fragments of 6.9 Kb and 1.3 Kb were found for CD gene and two fragments of 6.9 Kb and 620 bp were found for UPRT gene.The sequencing of CD gene and UPRT gene proved consistent sequences with Genebank published data.A fragment of 1.3 Kb for CD gene and fragment of 620 bp for UPRT gene was found in recombinant Bifidobacterium by RT-PCR.A 52 KDa protein for CD gene was identified in whole-cell protein of recombinant Bifidobacterium and a 26 KDa protein for UPRT gene was identified in supernatant fluid of recombinant Bifidobacterium.The survival rate of tumor cells treated by extracts from culture of recombinant Bifidobacterium with 5-FC showed a strong killing effects of pTRKH2/CD and pTRKH2/UPRT dual suicide gene therapy system on Melanoma B16-F10 cells.Conclusion:CD gene and UPRT gene are successfully inserted into pTRKH2 and transfected into tumor-hypoxia-targeting vector Bifidobacterium Infantis.This dual suicide gene therapy system shows a high efficiency for tumor cells killing.
