膀胱移行细胞癌侧群细胞的分离与鉴定

Isolation and Identification of Side Population Cells from Human Bladder Transitional Cell Cancer

目的探讨膀胱移行细胞癌新鲜癌组织和一些细胞系中是否存在侧群(SP)细胞及其比例,并鉴定其功能。方法利用双波长FACS检测4例膀胱移行细胞癌新鲜癌组织和3个膀胱移行细胞癌细胞系EJ、UM-UC-3、T24中是否存在SP细胞及其比例,并从细胞增殖能力,克隆形成能力(CFE),细胞周期的分布,自我更新和长期分化能力,ABCG2和干性基因Notch-1、β-catenin、SMO的表达,对放化疗的抵抗等差异证实膀胱移行细胞癌中SP细胞是否有肿瘤干细胞的特点。结果 4例新鲜膀胱癌组织中SP的比例分别为1.1%、1.8%、4.1%、1.2%,与维拉帕米预孵30min后,SP的比例分别下降到0.8%、0.9%、2.4%、0.6%;3个膀胱癌细胞系中只有T24中存在SP细胞,比例为34.7%,EJ和UM-UC-3中不含SP细胞。与NSP细胞相比,T24中的SP细胞有更强的生长增殖能力和克隆形成能力(P<0.05);有更多的细胞处于G0/G1期(87.4%vs 63.3%,P<0.05),更少的细胞处于S期(31.1%vs 9.1%,P<0.05);分选后的SP和NSP细胞经过约10d的常规培养,SP细胞中NSP的比例占76.2%,而NSP细胞中SP的比例只占2.6%;RT-PCR的结果表明SP细胞中ABCG2和干性基因Notch-1、β-catenin、SMO的表达更高;SP细胞对放化疗有更强的抵抗能力,而且这种抵抗能力与ABCG2的高表达有关。结论膀胱移行细胞癌细胞中存在侧群细胞,而且T24中的侧群细胞有肿瘤干细胞的特点。

Objective To isolate and identify side population cells from human bladder transitional cell cancer. Methods Dual wavelength FACS was used to sort four fresh bladder transitional cell cancer tissues and three bladder cancer cell lines after Hoechst 33342 labeling and the potential stem cell characteristics of side population cells from T24 were compared according to proliferation, cell cycle distribution, self-renewal, differ- entiation, sensitivity to chemotherapy drugs and X-ray. Results Several bladder transitional cell cancer cell lines such as T24 and four fresh bladder transitional cell cancer tissues had side population cells of different proportions ,but EJ and UM-UC-3 had no side population. Moreover,T24 SP cells had stronger proliferative and colony formation abilities than non-SP cells. SP cells were also more resistant to chemotherapy and radiotherapy,which might be due to expression of the ATP-binding cassette half-transporter, sub-family G, member 2 protein. Cell cycle results suggested that more SP than NSP ceils were in the G0/G1 phase （87.4% vs 63.3% ,P 〈 0.05） and fewer SP than NSP cells were in S phase （31.1% vs 9.1% ,P 〈 0.05）. After about 10 days culture in normal condition,SP ceils still contained 23.8% of cells that stained poorly with Hoechst 33342. In contrast, NSP cells only contained 2.6% poorly staining ceils. RT-PCR results suggested SP cells expressed more AB- CG2, Notch-1, β-catenin and SMO than NSP cells. Conclusion Side population cells can be found in human bladder transitional cell cancer and side population cells from human bladder transitional cell cancers cell line T24 harbor stem-like cells.