摘要
AIM:To characterize the regeneration-associated stem cell-related phenotype of hepatocyte-derived growth factor receptor(HGFR)-expressing cells in active ulcerative colitis(UC).METHODS:On the whole 38 peripheral blood samples and 38 colonic biopsy samples from 18 patients with histologically proven active UC and 20 healthy control subjects were collected.After preparing tissue microarrays and blood smears HGFR,caudal type homeobox 2(CDX2),prominin-1(CD133) and Musashi-1conventional and double fluorescent immunolabelings were performed.Immunostained samples were digitalized using high-resolution Mirax Desk instrument,and analyzed with the Mirax TMA Module software.For semiquantitative counting of immunopositive lamina propria(LP) cells 5 fields of view were counted at magnification x 200 in each sample core,then mean ± SD were determined.In case of peripheral blood smears,30 fields of view with 100 μm diameter were evaluated in every sample and the number of immunopositive cells(mean ± SD) was determined.Using 337 nm UVA Laser MicroDissection system at least 5000 subepithelial cells from the lamina propria were collected.Gene expression analysis of HGFR,CDX2,CD133,leucine-rich repeat-containing G-protein coupled receptor 5(Lgr5),Musashi-1 and cytokeratin20(CK20) were performed in both laser-microdisscted samples and blood samples by using real time reverse transcription polymerase chain reaction(RT-PCR).RESULTS:By performing conventional and double fluorescent immunolabelings confirmed by RT-PCR,higher number of HGFR(blood:6.7 ± 1.22 vs 38.5 ±3.18;LP:2.25 ± 0.85 vs 9.22 ± 0.65;P < 0.05),CDX2(blood:0 vs 0.94 ± 0.64;LP:0.75 ± 0.55 vs 2.11± 0.75;P < 0.05),CD133(blood:1.1 ± 0.72 vs 8.3± 1.08;LP:11.1 ± 0.85 vs 26.28 ± 1.71;P < 0.05)and Musashi-1(blood and LP:0 vs scattered) positive cells were detected in blood and lamina propria of UC samples as compared to controls.HGFR/CDX2(blood:0 vs 1± 0.59;LP:0.8 ± 0.69 vs 2.06 ± 0.72,P < 0.05)and Musashi-1/CDX2(blood and LP:0 vs scattered) coexpressions were found in blood and lamina propria of UC samples.HGFR/CD133 and CD133/CDX2 coexpressions appeared only in UC lamina propria samples.CDX2,Lgr5 and Musashi-1 expressions in UC blood samples were not accompanied by CK20 mRNA expression.CONCLUSION:In active UC,a portion of circulating HGFR-expressing cells are committed to the epithelial lineage,and may participate in mucosal regeneration by undergoing mesenchymal-to-epithelial transition.
AIM:To characterize the regeneration-associated stem cell-related phenotype of hepatocyte-derived growth factor receptor(HGFR)-expressing cells in active ulcerative colitis(UC).METHODS:On the whole 38 peripheral blood samples and 38 colonic biopsy samples from 18 patients with histologically proven active UC and 20 healthy control subjects were collected.After preparing tissue microarrays and blood smears HGFR,caudal type homeobox 2(CDX2),prominin-1(CD133) and Musashi-1conventional and double fluorescent immunolabelings were performed.Immunostained samples were digitalized using high-resolution Mirax Desk instrument,and analyzed with the Mirax TMA Module software.For semiquantitative counting of immunopositive lamina propria(LP) cells 5 fields of view were counted at magnification x 200 in each sample core,then mean ± SD were determined.In case of peripheral blood smears,30 fields of view with 100 μm diameter were evaluated in every sample and the number of immunopositive cells(mean ± SD) was determined.Using 337 nm UVA Laser MicroDissection system at least 5000 subepithelial cells from the lamina propria were collected.Gene expression analysis of HGFR,CDX2,CD133,leucine-rich repeat-containing G-protein coupled receptor 5(Lgr5),Musashi-1 and cytokeratin20(CK20) were performed in both laser-microdisscted samples and blood samples by using real time reverse transcription polymerase chain reaction(RT-PCR).RESULTS:By performing conventional and double fluorescent immunolabelings confirmed by RT-PCR,higher number of HGFR(blood:6.7 ± 1.22 vs 38.5 ±3.18;LP:2.25 ± 0.85 vs 9.22 ± 0.65;P < 0.05),CDX2(blood:0 vs 0.94 ± 0.64;LP:0.75 ± 0.55 vs 2.11± 0.75;P < 0.05),CD133(blood:1.1 ± 0.72 vs 8.3± 1.08;LP:11.1 ± 0.85 vs 26.28 ± 1.71;P < 0.05)and Musashi-1(blood and LP:0 vs scattered) positive cells were detected in blood and lamina propria of UC samples as compared to controls.HGFR/CDX2(blood:0 vs 1± 0.59;LP:0.8 ± 0.69 vs 2.06 ± 0.72,P < 0.05)and Musashi-1/CDX2(blood and LP:0 vs scattered) coexpressions were found in blood and lamina propria of UC samples.HGFR/CD133 and CD133/CDX2 coexpressions appeared only in UC lamina propria samples.CDX2,Lgr5 and Musashi-1 expressions in UC blood samples were not accompanied by CK20 mRNA expression.CONCLUSION:In active UC,a portion of circulating HGFR-expressing cells are committed to the epithelial lineage,and may participate in mucosal regeneration by undergoing mesenchymal-to-epithelial transition.
基金
Cell Analysis Laboratory, 2nd Department of Internal Medicine, and the 1st Department of Pathology and Experimental Oncology, Semmelweis University for their technical support
