人舌癌细胞株Tca8113中ABCG2蛋白表达的意义及其SP细胞群的分选
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摘要
目前肿瘤是威胁人类健康的重大疾病,肿瘤干细胞(tumor stem cells)学说认为肿瘤是一种干细胞疾病,是由具有成瘤能力的肿瘤细胞增殖发育而形成的异常组织。肿瘤细胞中仅有极少数的细胞具有成瘤潜能。现在肿瘤干细胞的研究已经成为当代研究的热点。
1996年Goodell[1]等首次提出在Hoechst33342染色的骨髓细胞中存在着一群染色很浅的细胞群,并将其命名为side population(SP细胞),在SP细胞里富含了大量的干细胞成分,不仅表型上大部分和干细胞表型一致,而且在功能验证中体现了很强的造血组织重建能力。ABCG2作为一种转运蛋白,在维持细胞自身稳定及机体正常生理功能等方面发挥着重要作用。随着干细胞研究的不断发展,发现它直接参与了干细胞特性——SP表型的形成。SP细胞较高表达ABCG2蛋白,可将荧光染料Hoechest33342泵出细胞外,与main population(MP)细胞比,SP细胞表现为较弱的Hoechst33342激发荧光特性。
本研究以人舌癌细胞株Tca8113为研究对象,通过流式细胞技术(FACS)分选人舌癌细胞株Tca8113中的侧群细胞(SP细胞)、非侧群细胞(MP细胞),检查ABCG2蛋白在SP细胞、非SP细胞、T(Tca8113)细胞中的表达情况,并探讨其意义。为今后进一步分选纯化类肿瘤干细胞及分离肿瘤干细胞提供有效手段。
研究方法及结果:
1.人舌癌中ABCG2蛋白表达的意义
方法:人舌癌细胞株Tca8113,用含15℅小牛血清的1640培养液培养,放于37℃,含5%CO2的培养箱中,等细胞贴壁生长达70%—80%时,用0.125%的胰蛋白酶消化传代,在盖玻片上爬片,置于6孔板中培养,观察细胞生长状态良好,把细胞爬片固定于载玻片上,用10﹪甲醛固定60～90 min。然后加入鼠抗人ABCG2一抗和FITC羊抗鼠二抗,双通道激光共聚焦显微镜下行细胞免疫荧光观察。另外收集生长状态良好的Tca8113细胞,200 uL PBS重悬细胞,加入鼠抗人ABCG2一抗和FITC羊抗鼠二抗,上流式细胞仪分析,检测ABCG2蛋白的表达情况。结果:绿色荧光为FITC染色的细胞,其中少量的绿色光点为FiTC染色的ABCG2蛋白。流式检测结果显示ABCG2在人舌癌的表达率为5﹪左右。实验结果表明:人舌癌细胞株Tca8113也象某些癌细胞一样含有SP细胞。
2.人舌癌细胞株Tca8113中SP细胞群的分离
方法:取25瓶已培养的人舌癌细胞株Tca8113细胞,细胞总量约为108,用0.01﹪PBS冲洗,适量的0.125﹪胰酶消化,中止消化后,HBSS洗涤,重悬细胞,加入Hoechest33342。对照组同时加入维拉帕米。碘化丙啶(PI)标记死细胞。流式细胞仪检测分选,发光偏弱的即为SP细胞。去除PI阳性死细胞,收集分选出来的SP细胞、非SP细胞(MP细胞)。结果: Hoechest33342染色后,分选出人舌癌细胞株Tca8113中SP细胞的含量约为0.8﹪。
3.人舌癌细胞株Tca8113细胞、MP细胞、SP细胞中ABCG2蛋白含量的检测及分析
方法:分别收集(1～3)×105数量级的SP细胞、MP细胞和T细胞,分别用200 uL PBS重悬细胞,加入鼠抗人ABCG2一抗和FITC羊抗鼠二抗,上流式细胞仪分析,检测ABCG2蛋白的表达情况。流式检测结果显示SP细胞中ABCG2的表达率为13.16﹪左右;未分选Tca8113细胞中ABCG2的表达率为3.29﹪左右;而MP细胞几乎没有ABCG2的表达。实验结果表明:人舌癌细胞株Tca8113中SP细胞较高表达ABCG2蛋白。
研究结果表明:人舌癌细胞株Tca8113中存在SP细胞,并且分离出了这一小群细胞。通过分选癌侧群细胞中的ABCG2+细胞,将有可能进一步分选纯化类肿瘤干细胞,为今后分离肿瘤干细胞提供了实验基础,同时也为以后靶向肿瘤干细胞的治疗提供了新方法和思路。
At present, tumor is a great disease of threatening mankind’s health. Tumor stem cells theory presumes that tumor is a stem cell disease and it is abnormal tissue of tumor cell which has the capacity of forming tumor proliferating. There is few cell having the capacity of forming tunor in tumor cell . Nowadays, the research of tumor stem cell has became a hot spot.
In 1996,Goodell at first proposed that there were a few colored fairly shallow cells in Hoechst33342 colored bone marrow cells. He named them side population cells which contained many stem cell components. Not only their phenotype was the same as stem cell ,but they had very fortis capacity of reestablishing hematopoietic tissue. ABCG2 is a transport protein . It plays important effect in keeping cell’s homeostasis and organism normal physiological functions .With the development of stem cell research, it is though that it participates the formation SP phenotype which is stem cell character.Side population cells expressed highly ABCG2 protein. They could pump Hoechst33342 out from cells. Compared with main population cells, side population cells showed fairly lepto Hoechst33342 stimulating fluorescence character.
We sorted SP(Side Population) cells and MP(Non-SP) cells from Human Tongue Cancer Cell Line Tca8113 through fluorescence-activated cell sorting(FACS)and investigated the expression rate and significance of ABCG2 in SP cells ,MP cells and T(Tca8113) cells. The research may be a useful method to purity alike Tumor Stem Cell(TSC) and isolate TSC in following research.
Research method and result
1. the expressed significance of ABCG2 in human tongue cancer
Method: cultivate the human tongue cancer cell line Tca8113 in the RPMI1640 (15%CS), put the culture bottle in the CO2(5%) ,37℃incubator. When the cells proliferated to 70%~80% of the culture bottle’s bottom, trypsinase(0.125%) digested and passaged. After creeped piece on the cover glass. cultivate Tca8113 in the six shadow mask.When seeing the cells growing well, we fixed cell creeping piece on the glass slide and used formaldehyde(10%) to fix it about 60~90 minutes. Then added ABCG2 antibody and FITC antibody, and detected ABCG2 expression of Tca8113 under immunofluorescence . In addition, collected the cells growing well , used 200 ul PBS to suspend the cells and added ABCG2 antibody and FITC antibody. Then detected ABCG2 expression of Tca8113 through FACS. Results: the cells which FITC colored emited virent fluorescent light and ABCG2 protein which FITC colored emited few virent light spot. The expression rate of ABCG2 in Tca8113 is about 5℅ by flow cytometry measuring. Experimental results indicated that human tongue cancer cell line Tca8113 contained SP cells like some cancer cells.
2. side population cells dissociated from human tongue cancer cell line Tca8113
Method: Collected 25 bottles of cultivated Tca8113 cells , Prepared about 108 cells for staining. Then used PBS(0.01%) to washout the cells and trypsinase(0.125%) digested them. After broke off diagesting, we used HBSS to washout them , suspended the cells and added Hoechest33342. At the same time, added Verapalmil in the control group.Used PI to mark dead cells. Detected and sorted SP cells which illuminated slightly through FACS. After removed PI+ dead cells, collected sorted SP, MP cells. Results: After colored by Hoechest33342, there were expressed only in 0.8% of the cells in human tongue cancer cell line Tca8113. Experimental results indicated that SP cells exited in human tongue cancer cell line Tca8113.
3. detection and analysis of ABCG2 in human tongue cancer cell line Tca8113,MP cells and SP cells
Method: Prepared about (1~3)×105 SP,MP,T cells for detecting.Used 200 ul PBS to suspend the cells and added ABCG2 antibody and FITC antibody. Then detected ABCG2 expression of Tca8113 through FACS. Results: The expression rate of ABCG2 in SP cells was about 13.16℅, and in T cells the expression rate was about 3.29℅, on the contrary ,there was nearly no expression in MP cells(1.07℅). Experimental results indicated that SP cells in human tongue cancer cell line Tca8113 overexpressed ABCG2.
Research results indicated that SP cells exited in human tongue cancer cell line Tca8113 and we sorted them. So isolation of ABCG2+ cell from SP cells may lay experimental basis for puriting alike Tumor Stem Cell(TSC) . At the same time, it could provide new method and thinking for targeting to Tumor Stem Cell therapy.
1996年Goodell[1]等首次提出在Hoechst33342染色的骨髓细胞中存在着一群染色很浅的细胞群,并将其命名为side population(SP细胞),在SP细胞里富含了大量的干细胞成分,不仅表型上大部分和干细胞表型一致,而且在功能验证中体现了很强的造血组织重建能力。ABCG2作为一种转运蛋白,在维持细胞自身稳定及机体正常生理功能等方面发挥着重要作用。随着干细胞研究的不断发展,发现它直接参与了干细胞特性——SP表型的形成。SP细胞较高表达ABCG2蛋白,可将荧光染料Hoechest33342泵出细胞外,与main population(MP)细胞比,SP细胞表现为较弱的Hoechst33342激发荧光特性。
本研究以人舌癌细胞株Tca8113为研究对象,通过流式细胞技术(FACS)分选人舌癌细胞株Tca8113中的侧群细胞(SP细胞)、非侧群细胞(MP细胞),检查ABCG2蛋白在SP细胞、非SP细胞、T(Tca8113)细胞中的表达情况,并探讨其意义。为今后进一步分选纯化类肿瘤干细胞及分离肿瘤干细胞提供有效手段。
研究方法及结果:
1.人舌癌中ABCG2蛋白表达的意义
方法:人舌癌细胞株Tca8113,用含15℅小牛血清的1640培养液培养,放于37℃,含5%CO2的培养箱中,等细胞贴壁生长达70%—80%时,用0.125%的胰蛋白酶消化传代,在盖玻片上爬片,置于6孔板中培养,观察细胞生长状态良好,把细胞爬片固定于载玻片上,用10﹪甲醛固定60～90 min。然后加入鼠抗人ABCG2一抗和FITC羊抗鼠二抗,双通道激光共聚焦显微镜下行细胞免疫荧光观察。另外收集生长状态良好的Tca8113细胞,200 uL PBS重悬细胞,加入鼠抗人ABCG2一抗和FITC羊抗鼠二抗,上流式细胞仪分析,检测ABCG2蛋白的表达情况。结果:绿色荧光为FITC染色的细胞,其中少量的绿色光点为FiTC染色的ABCG2蛋白。流式检测结果显示ABCG2在人舌癌的表达率为5﹪左右。实验结果表明:人舌癌细胞株Tca8113也象某些癌细胞一样含有SP细胞。
2.人舌癌细胞株Tca8113中SP细胞群的分离
方法:取25瓶已培养的人舌癌细胞株Tca8113细胞,细胞总量约为108,用0.01﹪PBS冲洗,适量的0.125﹪胰酶消化,中止消化后,HBSS洗涤,重悬细胞,加入Hoechest33342。对照组同时加入维拉帕米。碘化丙啶(PI)标记死细胞。流式细胞仪检测分选,发光偏弱的即为SP细胞。去除PI阳性死细胞,收集分选出来的SP细胞、非SP细胞(MP细胞)。结果: Hoechest33342染色后,分选出人舌癌细胞株Tca8113中SP细胞的含量约为0.8﹪。
3.人舌癌细胞株Tca8113细胞、MP细胞、SP细胞中ABCG2蛋白含量的检测及分析
方法:分别收集(1～3)×105数量级的SP细胞、MP细胞和T细胞,分别用200 uL PBS重悬细胞,加入鼠抗人ABCG2一抗和FITC羊抗鼠二抗,上流式细胞仪分析,检测ABCG2蛋白的表达情况。流式检测结果显示SP细胞中ABCG2的表达率为13.16﹪左右;未分选Tca8113细胞中ABCG2的表达率为3.29﹪左右;而MP细胞几乎没有ABCG2的表达。实验结果表明:人舌癌细胞株Tca8113中SP细胞较高表达ABCG2蛋白。
研究结果表明:人舌癌细胞株Tca8113中存在SP细胞,并且分离出了这一小群细胞。通过分选癌侧群细胞中的ABCG2+细胞,将有可能进一步分选纯化类肿瘤干细胞,为今后分离肿瘤干细胞提供了实验基础,同时也为以后靶向肿瘤干细胞的治疗提供了新方法和思路。
At present, tumor is a great disease of threatening mankind’s health. Tumor stem cells theory presumes that tumor is a stem cell disease and it is abnormal tissue of tumor cell which has the capacity of forming tumor proliferating. There is few cell having the capacity of forming tunor in tumor cell . Nowadays, the research of tumor stem cell has became a hot spot.
In 1996,Goodell at first proposed that there were a few colored fairly shallow cells in Hoechst33342 colored bone marrow cells. He named them side population cells which contained many stem cell components. Not only their phenotype was the same as stem cell ,but they had very fortis capacity of reestablishing hematopoietic tissue. ABCG2 is a transport protein . It plays important effect in keeping cell’s homeostasis and organism normal physiological functions .With the development of stem cell research, it is though that it participates the formation SP phenotype which is stem cell character.Side population cells expressed highly ABCG2 protein. They could pump Hoechst33342 out from cells. Compared with main population cells, side population cells showed fairly lepto Hoechst33342 stimulating fluorescence character.
We sorted SP(Side Population) cells and MP(Non-SP) cells from Human Tongue Cancer Cell Line Tca8113 through fluorescence-activated cell sorting(FACS)and investigated the expression rate and significance of ABCG2 in SP cells ,MP cells and T(Tca8113) cells. The research may be a useful method to purity alike Tumor Stem Cell(TSC) and isolate TSC in following research.
Research method and result
1. the expressed significance of ABCG2 in human tongue cancer
Method: cultivate the human tongue cancer cell line Tca8113 in the RPMI1640 (15%CS), put the culture bottle in the CO2(5%) ,37℃incubator. When the cells proliferated to 70%~80% of the culture bottle’s bottom, trypsinase(0.125%) digested and passaged. After creeped piece on the cover glass. cultivate Tca8113 in the six shadow mask.When seeing the cells growing well, we fixed cell creeping piece on the glass slide and used formaldehyde(10%) to fix it about 60~90 minutes. Then added ABCG2 antibody and FITC antibody, and detected ABCG2 expression of Tca8113 under immunofluorescence . In addition, collected the cells growing well , used 200 ul PBS to suspend the cells and added ABCG2 antibody and FITC antibody. Then detected ABCG2 expression of Tca8113 through FACS. Results: the cells which FITC colored emited virent fluorescent light and ABCG2 protein which FITC colored emited few virent light spot. The expression rate of ABCG2 in Tca8113 is about 5℅ by flow cytometry measuring. Experimental results indicated that human tongue cancer cell line Tca8113 contained SP cells like some cancer cells.
2. side population cells dissociated from human tongue cancer cell line Tca8113
Method: Collected 25 bottles of cultivated Tca8113 cells , Prepared about 108 cells for staining. Then used PBS(0.01%) to washout the cells and trypsinase(0.125%) digested them. After broke off diagesting, we used HBSS to washout them , suspended the cells and added Hoechest33342. At the same time, added Verapalmil in the control group.Used PI to mark dead cells. Detected and sorted SP cells which illuminated slightly through FACS. After removed PI+ dead cells, collected sorted SP, MP cells. Results: After colored by Hoechest33342, there were expressed only in 0.8% of the cells in human tongue cancer cell line Tca8113. Experimental results indicated that SP cells exited in human tongue cancer cell line Tca8113.
3. detection and analysis of ABCG2 in human tongue cancer cell line Tca8113,MP cells and SP cells
Method: Prepared about (1~3)×105 SP,MP,T cells for detecting.Used 200 ul PBS to suspend the cells and added ABCG2 antibody and FITC antibody. Then detected ABCG2 expression of Tca8113 through FACS. Results: The expression rate of ABCG2 in SP cells was about 13.16℅, and in T cells the expression rate was about 3.29℅, on the contrary ,there was nearly no expression in MP cells(1.07℅). Experimental results indicated that SP cells in human tongue cancer cell line Tca8113 overexpressed ABCG2.
Research results indicated that SP cells exited in human tongue cancer cell line Tca8113 and we sorted them. So isolation of ABCG2+ cell from SP cells may lay experimental basis for puriting alike Tumor Stem Cell(TSC) . At the same time, it could provide new method and thinking for targeting to Tumor Stem Cell therapy.
引文
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[3] Han B,Zhang J.Muhidrug resistance in cancer chemotherapy and xenobiotic protection mediated by the haft ATP·binding cassette transporter ABCG2[J].Cur Med Chem Anti·Cane Agents,2004,4(1):31-42.
[4] Qingcheng Mao , Jashvant D , Unadkat . Role of the cancer resistence protein(ABCG2) in drug transport[J].The AAPs Journal,2005,7(1):l18-134.
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[8] Krishnamurthy P.Schuetz JD.Role of ABCG2/ BCRP inBiology[J].Annu Rev Pharmacol Toxicol,2006,46:381-410.
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[12] Loo TW,Clarke DM.Mutations to amino acids located in predicted transmembrane segment 6 (TM6) modulate the activity and substrate specificity of human P-glycoprotein[J].Biochemistry.1994.33(47):14049-l4057.
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[15] Janvilisri r,Venter H,Shahi S,et a1.Steml transport by the human breast cancer resistance protein(ABCG2)expressed in Lactococcus lactis [J].J Biol Chem,2003,278(23):20645-20651.
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[1] Zhang J T.Use of arrays to investigate the contribution of ATP binding cassette transporters to drug resistance in cancer chemo therapy and prediction of chemosensitivity[J].Cell Res,2007,17(4):311-323.
[2] Dean M,Rzhetsky A,Allikmets R.Human and Drosophila ABC Proteins/Holland 1B,Cole SP,Kuchler K,et a1.ABC Proteins,from Bacteria to Man[M].Amsterdam:Academic Press,2003:47-61.
[3] Han B,Zhang J.Muhidrug resistance in cancer chemotherapy and xenobiotic protection mediated by the haft ATP·binding cassette transporter ABCG2[J].Cur Med Chem Anti·Cane Agents,2004,4(1):31-42.
[4] Qingcheng Mao , Jashvant D , Unadkat . Role of the cancer resistence protein(ABCG2) in drug transport[J].The AAPs Journal,2005,7(1):l18-134.
[5] Doyle L A ,Yang W ,Abruzzo L V,et a1. A multidrug resistance transporter from human MCF-7 breast cancer cells[J].Proc Natl Acad Sci USA,1998,95(26):15665-15670.
[6] Allikmets R,Sehriml L M ,Hutchinson A ,et a1.A human placenta specific ATP—binding cassette gene(ABCP)on chromosome4q22 that is involved in muhidrug resistance[J].Cancer Res1998,58(23):5337-5339.
[7] Miyake K,Mickley L,Litman T,et a1.Molecular cloning of cDNAs which are highly overexpressed in nlitoxantrone resistant cells:demonstration of homology to ABC transport genes[J].Cancer Res,1999,59(1):8-13.
[8] Krishnamurthy P.Schuetz JD.Role of ABCG2/ BCRP inBiology[J].Annu Rev Pharmacol Toxicol,2006,46:381-410.
[9] Zhang W,Mojsilovic-Petrovic J,Andrade MF, et a1.The expression and functional characterization of ABCG2 in brain endothelial cells and vessels[J].FASEB J, 2003.17(14):2O85-2087.
[10] Robey RW,Honjo Y,Morisaki K,et a1.Mutations at aminoacid 482 in the ABCG2 gene afect substrate an d antagonist specificity [J].Br J Cancer,2003.89(10):1971-1978.
[11] Graf G A,Li W P,Gerard R D,et a1.Coexpression ofATP.Binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface[J].J Clin Invest,2002,110(5):594-569.
[12] Loo TW,Clarke DM.Mutations to amino acids located in predicted transmembrane segment 6 (TM6) modulate the activity and substrate specificity of human P-glycoprotein[J].Biochemistry.1994.33(47):14049-l4057.
[13] Ozvegy C,Litman T,Szakacs G,et a1.Functional characterization of the human muhidrug transporter, ABCG2,expressed in insect cells [J].Biochem Biophys Res Commun,2001,285(1):111-ll7.
[14] Ozvegy C,Varadi A,Sarkadi B.Characterization of drug transport,ATP hydrolysis , and nucleotide trapping by the human ABCG2 muhidrug transporter. Modulation of substrate specificity by a point mutation [J].J Biol Chem,2002,277(50):47980-47990.
[15] Janvilisri r,Venter H,Shahi S,et a1.Steml transport by the human breast cancer resistance protein(ABCG2)expressed in Lactococcus lactis [J].J Biol Chem,2003,278(23):20645-20651.
[16] Zhou S,Morris JJ,Barnes Y,et a1.Bcrpl gene expression is required for nomal numbers of side population stem cel in mice,and confers relative protectionto mi toxantrone in hematopoietic cels in vivo[J].PNAS,2002,99(19):12339-12344.
[17] Xu J,Liu Y,Yang Y,et a1.Characterization of oligomeric human half -ABC transporter ATP-binding cassette G2[J].J Biol Chem 2004,279(19):19781-19789.
[18] McDevitt C A,Collins R F,Conway M,et a1.Purification and 3D structural analysis of oligomeric human muhidrug transporter ABCG2 [J].Structure,2006,14(11):1623-1632.
[19] Bhatia A,SchMer H J,Hryeyna C A.Oligomerization of the human ABC transporter ABCG2 : evaluation of the native protein and chimeric dimers[J].Biochemistry,2005,4(32):10893-10904.
[20] Henriksen U,Fog J U,Litman T,et a1.Identification of intra and intemmlecular disulfide bridges in the muhidrug resistance trans -porter ABCG2[J].J Biol Chem,2005,280(4):36926-36934.
[21] Fetsch PA,Abati A.Htman T。et a1.Localization of the ABCG2 mi -toxantrone resistance-associated protein in normal tissues [J] .Cancer Lett.2006,235(1):84-92.
[22] Xu J,Peng H,Chen Q,et a1.Oligomerization domain of the multidrug resistance-associated transporter ABCG2 and its dominant inhibitory activity[J].Cancer Res,2007,67(9):4373-4381.
[23] Honjo Y,Hrycyna C A,Yan Q w,et a1.Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP overexpressing cells[J].Cancer Res,2001,61(18):6635-6639.
[24] Alqawi O,Bates S,Georges E in the breast cancer resistance Argininc482 to threonine mutation protein ABCG2 inhibits rhodamine 123 transport while increasing binding[J].Biochem J,2004,382(Pt 2):711-716.
[25] Patrawala L,Calhoun T,Sehneider-Broussard R.et a1.Sidepopulation is enriched in tumorigenic, stem-like cancer cells,whereas ABCG2+ and ABCG2- cancer cels are similarly tumorigenic[J].Cancer Res,2005 ,65(14):6207-6220.
[26] Honjo Y , Morisaki K , ftuf L M , et a1 . Single—nucteotide polymorphism(SNP)analysis in the ABC half·transporter ABCG2(MXR /BCRPABCP1)[J].Cancer Biol Ther,2002,1(6):696-702.
[27] Backstrom G,Taipalensuu J,Melhus H,et a1.Genetic variation in the ATP binding cassette transporter genc ABCG2 (BCRP)in a Swedish population[J].Eur J Pharm Sci,2003,18(5):359-364.
[28] Bosch T M,Kjellberg I M,Bouwers A,et a1.Detection of single nucleotide polymorphisms in the ABCG2 gene in a Dutch population[J].Am J Phammcogenomics 2005,5(2):123-131.
[29] Yanase K,Tsukahara S,Mitsuhashi J,et a1.Functional SNPs of the breast cancer resistance protein-therapeutic effects and inhibitor development[J].Cancer Lett,2006,234(1):73-80.
[30] Tamura A,Watanabe M,Saito H,et a1.Functional validation of the geneticpolymorphisms of human ATP·binding cassette(ABC) transporter ABCG2 :identification of alleles that are defective in porphyrin transport[J].Mol Phammcol,2006,70(1):287-296.
[31] Zhou S,Schuetz J D,Bunting K D,et at.The ABC transporter Bcrpl/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side—population phenotype[J].NatMed,2001,7(9):1028-1034.
[32] Zhou S,Morris JIl,Barnes Y,et a1.Bcpl gene expression is required for normal numbers of side population stem cells in mice,and confers relative protection to mltoxantrone in hematopoietic cells in vivo [J].Pme Natl Aead Sei USA,2002,99(19):12339-l2344.
[33] Alvi A J,Clayton H,Joshi C.et a1.Functional and molecular charaeterisation of nlanlnlaly side population cells[J].Breast Cancer Res.2003,5(1):1-8.
[34] Hirschmann-Jax C,FosterA E,WulfG G,et a1.A distinct“side population”of cells with high drug efflux capacity in human tumor cells[J].Proc Natl Aead Sci USA,2004,101(39):14228-l4233.
[35] Raaijmakers M H,de Grouw E P,Heuver I H,et a1.Breast cancer resistance protein in drug resistance of primitive CD34+38- ce11s in aeute myeloid leukemia f J].Clin Cancer Res.2005,1 1(6):243-244.