CAJ | 학술논문

背景与目的:肿瘤干细胞学说的提出为肿瘤治疗提供了新的靶点和方向,但肿瘤干细胞的分离纯化一直是个难题.本研究拟从人小细胞肺癌细胞株H446中分离并鉴定出具有干细胞特性的侧群(SP)细胞,研究其生物学特征,为肿瘤干细胞的分离纯化奠定基础.方法:采用荧光激活细胞分选(FACS)技术分选得到H446细胞中SP细胞和非侧群(NSP)细胞,并检测纯度.观察形成悬浮肿瘤细胞球的能力,采用逆转录-聚合酶链反应(RT-PCR)及荧光定量PCR检测这两种细胞亚群中CD133、ABCG2、Nucleostemin mRNA水平.MTT法比较SP细胞、NSP细胞及未分选细胞体外增殖能力及耐药性差异,流式细胞仪检测体外分化能力,裸鼠成瘤实验检测体内成瘤能力.结果:荧光显微镜下H4-46细胞中Hoechst33342阴性细胞约为(5.1±0.2)%.流式细胞分选结果显示,H446中SP细胞比例为(6.3±0.1)%.SP细胞在无血清培养基中形成悬浮肿瘤细胞球的能力强于NSP细胞.CD133、ABCG2在SP细胞中的表达是NSP细胞的(21.60±0.26)倍、(7.10±0.14)倍,差异有统计学意义(P＜0.01);Nucleostemin在SP细胞中的表达是非sP细胞的(1.02±0.08)倍,差异无统计学意义(P＞0.05).SP细胞体外增殖能力及耐药存活能力均明显强于NSP细胞及未经分选的细胞(P＜0.01);SP细胞在体外可分化为NSF,细胞,但NSP细胞在体外不可分化为SP细胞;SP细胞在裸鼠体内具有较强的致瘤性.结论:人小细胞肺癌细胞株H446中存在具有肿瘤干细胞特性的SP细胞,CD133、ABCG2可能是人小细胞肺癌千细胞的分子标志物.
배경여목적:종류간세포학설적제출위종류치료제공료신적파점화방향,단종류간세포적분리순화일직시개난제.본연구의종인소세포폐암세포주H446중분리병감정출구유간세포특성적측군(SP)세포,연구기생물학특정,위종류간세포적분리순화전정기출.방법:채용형광격활세포분선(FACS)기술분선득도H446세포중SP세포화비측군(NSP)세포,병검측순도.관찰형성현부종류세포구적능력,채용역전록-취합매련반응(RT-PCR)급형광정량PCR검측저량충세포아군중CD133、ABCG2、Nucleostemin mRNA수평.MTT법비교SP세포、NSP세포급미분선세포체외증식능력급내약성차이,류식세포의검측체외분화능력,라서성류실험검측체내성류능력.결과:형광현미경하H4-46세포중Hoechst33342음성세포약위(5.1±0.2)%.류식세포분선결과현시,H446중SP세포비례위(6.3±0.1)%.SP세포재무혈청배양기중형성현부종류세포구적능력강우NSP세포.CD133、ABCG2재SP세포중적표체시NSP세포적(21.60±0.26)배、(7.10±0.14)배,차이유통계학의의(P＜0.01);Nucleostemin재SP세포중적표체시비sP세포적(1.02±0.08)배,차이무통계학의의(P＞0.05).SP세포체외증식능력급내약존활능력균명현강우NSP세포급미경분선적세포(P＜0.01);SP세포재체외가분화위NSF,세포,단NSP세포재체외불가분화위SP세포;SP세포재라서체내구유교강적치류성.결론:인소세포폐암세포주H446중존재구유종류간세포특성적SP세포,CD133、ABCG2가능시인소세포폐암천세포적분자표지물.
Background and Objective:Recently,the theory of cancer stem cells(CSCs)has presented new targets and orientations for tumor therapy.The major difficulties in researching CSCs include their isolation and purification.The aim of this study is to identify and characterize the side population(SP)cells in small cell lung cancer(SCLC)cell line H446,which Iays the foundation for the isolation and purification of CSCs.Methods:Fluorescence-activated cell sorting(FACS)was used to sort SP and non-SP (NSP)cells from H446,Both subgroups were cultivated to survey the capacity to form into suspended tumor cell spheres.Reverse transcriptionpolymerase chain reaction(RT-PCR)and real-time PCR were used to evaluate the expression levels of the mRNA of CD133,ABCG2,and nucleostemin in both subgroups.The capacity of proliferation and the differences in drug resistance of both subgroups and unso rted cells were tested by the MTT method.The differentiation ability of both subgroups was determined by FACS.Proliferation was determined by subcutaneous tumor formation in nude mice.Results:The percent of Hoechst 33342 negative cells was about(5.1±0.2)%in H446 by fluorescence microscopy.The percent of SP cells was(6.3±0.1)%by flow cytometry.SP cells had a stronger capability of fOrming into tumOr spheres than NSP cells.The mRNA expression Ievels of ABCG2,CD133,and nucleostemin in SP cells were 21.60±0.26,7.10±0.14,and 1.02±0.08 folds higher than that in NSP cells(P＜0.01,P＜0.01,and P＞0.05,respectively).In vivo,SP cells showed better proliferative ability and tougher viability when treated with drugs.SP cells can differentiate into NSP cells,but NSP cells cannot differentiate into SP cells.SP cells had a greater ability to form tumors.Conclusions:The H446 cell line contained some SP cells with stem cell properties.CD133 and ABCG2 may be cancer stem celI markers of SCLC.