肝癌细胞边缘群和非边缘群中甲胎蛋白和细胞角蛋白19的表达差异分析
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摘要
肿瘤的发生机制是当前国内外研究的热点领域，目前,在学术界就其形成过程尚未获得统一的认识。近年来，有学者提出肿瘤细胞和干细胞都具有幼稚的细胞表型等多种相似性，并由此提出肿瘤的干细胞理论。有研究发现急性髓细胞样白血病细胞中能分离出与骨髓造血干细胞具有相同干细胞表面标志的癌细胞，这些细胞能在免疫缺陷小鼠中诱导急性髓细胞样白血病的发生；这表明肿瘤组织中可能存在既具有干细胞特征，又具有肿瘤特征的肿瘤干细胞。目前，有文献报道，在多种实体瘤中发现有肿瘤干细胞的存在，例如乳腺癌、脑肿瘤等。随着研究的深入，已有多种方法可应用于肿瘤组织中具有干细胞样特性的肿瘤细胞的分离纯化。利用干细胞对多种染料及异己物质具有排斥性的特异性的功能标志，结合使用紫外光源激发的流式细胞荧光激活细胞分选法(FACS)，可以分选得到低荧光并表现为边缘性聚集的边缘群细胞（side population;简称SP细胞）。经过较深入的研究，该亚群细胞已被初步证实富集有具有干细胞样特性的细胞。目前，这种分离富集干细胞样细胞的方法被成功应用在多种组织的成体干细胞的分离过程中，此方法较原有的磁性活化分选和荧光活化分选更为高效。肿瘤细胞和干细胞在无限增殖，多向分化及自我更新机制等方面具有相似性。肿瘤异质性理论也被广泛证实。原发性肝细胞癌是我国常见恶性肿瘤之一，由于预后差，故死亡率较高。对肝癌细胞中特殊亚群细胞的分离研究，可能是研究肝癌的发生、复发、转移、耐药形成的最佳途径。
甲胎蛋白（AFP）属胚胎类抗原，作为一种胚胎时期或病理状态的基因表达产物，它伴随着胚胎发育和细胞分裂旺盛的过程。此外，AFP还具有多种功能并在机体内发挥着多种重要的生理病理作用。CK19的表达有组织和分化特异性,是目前筛选出的肝干细胞高度表达且特异性较好的标志物之一，是胆管上皮细胞的特异性标志物。CK19在正常成体肝脏中仅表达于胆管上皮细胞而不表达于肝细胞。CK19表达阳性的肝细胞被认为是来源于肝干细胞，并位于分化的早期。有研究发现，当肝细胞发生癌变时，部分癌细胞可表达CK19,且其表达程度与细胞的分化程度呈负相关。
本实验研究拟通过紫外光激发的流式细胞荧光激活分选法获得肝癌细胞中的两个亚群细胞--边缘群细胞（SP）和非边缘群细胞（Non-SP），并用间接免疫荧光方法了解在肝癌细胞亚群中AFP和CK19的表达是否存在明显差异，通过对两个亚群细胞cDNA进行基因扩增，了解SP细胞中AFP和CK19mRNA转录水平是否与Non-SP细胞中有所不同。以此进一步了解SP细胞中是否富含的克隆扩增、形成肿瘤及自我更新能力强的，更为原始的干细胞样肿瘤细胞，并进一步揭示干细胞样肝癌细胞与肝癌的发生、复发、转移及耐药的关系。最后，利用免疫组化方法观察肝癌组织内AFP和CK19表达情况，揭示其二者与术后早期复发等临床现象之间的关系。
目的：
应用紫外光源激发的流式细胞荧光激活分选法分选肝癌细胞系MHCC97中边缘群及非边缘群两个亚群的细胞，采用间接免疫荧光方法观察蛋白质水平AFP和CK19的表达变化及含量，采用RT-PCR方法半定量分析AFP和CK19mRNA的转录水平，探讨在MHCC97细胞系不同亚群中AFP和CK19差异表达的意义。采用免疫组化方法观察肝癌组织中AFP和CK19的表达水平。
方法：
1对人类肝癌细胞系MHCC97中的边缘群细胞进行分选及边缘群细胞的分化能力的检测；
1）常规细胞培养；
2）0.25%胰酶温和消化细胞，常规离心并以HBSS液洗涤细胞；
3）SP细胞及Non-SP细胞的检测及分选
制备Hoechst33342染色的肝癌单细胞悬液，对照组在此基础上加入钙离子通道拮抗剂阻断ABC转运蛋白家族ABCG2分子的主动外排作用。流式细胞仪检测肝癌细胞中Hoechst33342荧光，紫外激发光源，通过双色性反射滤镜和不同的组合滤片将肝癌细胞中荧光信号分为两个部分，以HoechstRed为X轴，Hoechst Blue为Y轴作二维散点图，将低Hoechst Red及低Hoechst Blue且对照组缺失的区域设定为SP细胞的“门”，分选出SP细胞及Non-SP细胞。
4）根据Genebank中提供的甲胎蛋白（AFP）和细胞角蛋白19（CK19）的基因序列，使用Primer5引物设计软件分别对两条基因进行半定量引物的设计，并送公司合成。分别以边缘群细胞和非边缘群细胞两种细胞群的cDNA为模板，行半定量检测。
5）将两群细胞接种盖玻片，分别行AFP和CK19双色免疫荧光检测。利用Image-Pro Plus5.0.2软件分别检测SP细胞和non-SP细胞爬片的平均积分光密度，比较两组细胞中AFP和CK19表达量。用SPSS 11.0统计软件包采用配对t检验对数据进行分析。
2购买肝癌组织芯片，分别行AFP和CK19免疫组化染色。比较两组标本组织中细胞的AFP和CK19表达量。
结果：
1流式细胞仪的双波长荧光分析结果显示在二维散点图上有边缘群细胞存在。MHCC97细胞系中边缘群细胞的比例接近0.25%。加入钙通道拮抗剂后该群细胞明显减少，因为低荧光的特征消失而进入到了非边缘群细胞群中，这表明MHCC97细胞系中SP细胞表型与细胞膜表面主动转运泵分子ABCG2的主动外排功能有关；
2 SP细胞及Non-SP细胞AFP和CK19基因半定量RT-PCR分析利用琼脂糖凝胶电泳条带，对AFP和CK19 mRNA光密度值与G3PDH mRNA光密度值的比值进行统计分析表明SP细胞组AFP和CK19 mRNA转录水平明显高于Non-SP细胞组。
3 SP细胞及Non-SP细胞AFP和CK19双色免疫荧光检测SP细胞及Non-SP细胞分别行细胞爬片培养，4小时后观察均伸展贴壁，分别行AFP和CK19双色免疫荧光检测，激光共聚焦镜下观察，SP细胞及Non-SP细胞胞浆内均可见表达AFP蛋白的红色荧光颗粒，CK19蛋白的绿色荧光颗粒。应用Image-Pro Plus5.0.2软件分别检测SP细胞和non-SP细胞爬片的平均积分光密度，比较两组细胞中AFP和CK19表达量。用SPSS11.0统计软件包采用配对t检验对数据进行分析。表明MHCC97细胞系边缘群和非边缘群细胞间AFP和CK19的表达在蛋白质水平有显著性差异。
4肝癌组织芯片中AFP和CK19蛋白表达的免疫组化检测对组织芯片中肝癌组织分别行AFP和CK19染色，荧光显微镜下可见部分组织细胞胞浆内棕黄色颗粒，比较两组标本组织中细胞的AFP和CK19表达量。结果绝大多数肝癌组织表达AFP，部分肝癌组织表达CK19，少数肝癌组织既表达AFP，又表达CK19。
结论：
1利用Hoechst33342染料通过流式细胞仪技术确认人类肝癌细胞系MHCC97中存在边缘群细胞。
2 MHCC97来源的边群细胞共同表达AFP和CK19，这提示了边缘群细胞确实具有一定的双向分化能力。这说明边缘群细胞具有一定的原始特征。进一步阐明SP细胞富含了表型幼稚，克隆扩增、形成肿瘤及自我更新能力强的，更为原始的干细胞样肿瘤细胞，并提示其与肝癌的发生、复发、转移及耐药形成的关系。
3绝大多数肝癌组织表达AFP，部分肝癌组织表达CK19，少数肝癌组织既表达AFP，又表达CK19。由于正常肝细胞不表达CK19，提示表达CK19的肝癌细胞可能来源于肝癌组织中某些祖细胞。
There is no unified opinion of tumorgenesis in academic circles.Recently,researchers proposed the theory of cancer stem cells because the sameimmature cell phenotype was included in the tumor cell and stem cell.Researchers have isolated leukaemia cells that have the markers of HSCs fromacute myeloid leukaemia (AML) It reveals that the cancer stem cells might existin many tumors, which have both capacities of stem cells and tumorigenicity.Now the cancer stem cells have been found in many solid tumors, for examplebreast cancer, human train tumor etc. With the development of research,manyapproaches have been used for the disassociation and purification of cancer stemcells. Recently, more and more studies showed that there is increased evidence offinding a small side population (SP) both from established cancer cell lines andprimary tumors.SP cells were identified of having stem like characteristics, thecapacity for self-renewal to allow the maintenance of an undifferentiated stemcell population and the ability to undergo differentiation.HCC was common inour country.It is possible for the research of the carcinogenesis, palindromia, metastasis and drug fast through the disassociation of SP.
AFP belongs to embryo antigen and plays important role in the body.CK19 iszighly expressed in the liver stem cell and regarded as the specific marker of bileduct.The expression of CK19 has negative correlation with the differentiation ofliver cancer cell.
Aim:
To investigate the significance of AFP and CK19 different expression indifferent subpopulations of MHCC97.
Methods:
1.To analyze the side population cells from human hepatocellular carcinomacell lines MHCC97, and to sort them in MHCC97. To assay the differentiationability of side population cells
1）Culture cells as usual;
2）0.25% trypsin digest cells, centrifuge them as usual and wash them by HBSS;
3）Detect and sort SP cell and Non-SP cell.
Aliquot the cells suspension into two labeled centrifuge tube. All two tubes ofcancer cells stain with the Hoechst dye, but one of tube add inhibitor of Ca2+channel to inhibit the exocytosis of ABCG2 in additionally. Both of two tubesincubate at 37℃on water bath. Then place Hoechst-stained cancer cells on thecytometer, excite the Hoechst dye at 355 nm and use dichroic mirror to separatethe emission wavelengths, collect Hoechst blue fluorescence with a 450nm bandpassfilter and Hoechst red fluorescence with a 675nm edge filter long-pass.Display the histogram of Hoechst red (x-axis) versus Hoechst blue (y-axis). Sidepopulation can be found on the histogram by establish gate. Compare the Hoechstfluorescence profile of two groups of cancer cells suspension
4) According to the sequences of AFP and CK19 from Genebank, we design the primers of two genes by Primer5 soft, and send them to biological company forcomposting. Then, using the cDNA from side population and non-side populationcells as template, we do the semi-quantity assay.
5) After being cultured on coverslips, we carry the immunofluorescence stainingassay of AFP, CK19 in two cells.
2. Tissue chip of liver cancer was purchased for the immunohistochemicalstain of AFP and CK19.
ResultResults:
1. The side population can be observed on the fluorescence profile when theemission of Hoechst analyzed by dual-wavelength. The percentage of SP cells inMHCC97 is about 0.25%.When the inhibitor of Ca2+ channel is used, the sidepopulation eliminate because of inhibition of efflux mediated by the ABCtransporter. The SP cells enter into the main population on the fluorescenceprofile because not have low fluorescence characteristics.
2. Reverse transcription–polymerase chain reaction (RT-PCR) for AFP andCK19 gene.
The results of AGE showed that the transcriptional level of AFP and CK19 insp cell group was significantly higher than that in the Non-SP cell group.
3. Immunocytochemistry
SP cells and Non-SP cells were grown on coverslips respectively for 4 h, fixedwith 4% paraformaldehyde , then cells were exposed to 0.3% H2O2 in absolutemethanol at room temperature for 15 minutes to inhibit endogenous peroxidaseactivity. Immunocytochemical staining was performed with commerciallyavailable primary antibody and other reagents completed according to themanufacturer's protocol.Under confocal microscopy，AFP-positive grains andCK19-positive grains could be detected both in SP cell and Non-SP cell. Lg IOD(integrated optical density)was used for measurement of the expression ofAFP and CK19.These results showed that there were obvious difference of AFPand CK19 between SP cell and Non-SP cell.
4. Immunocytochemistry of AFP and CK19 were performed in the Tissue chipof liver cancer.
Under fluorescence microscope，buffy grains were partly expressed in thecytoplasm.The results showed that AFP was expressed in most liver cancer,CK19 was expressed in partial liver cancer and only a few liver cancer expressedboth AFP and CK19.
ConclusiConclusion：
1. The side population cells existed in human hepatocelluar carcinoma cell linesMHCC97.
2. The side population cells from MHCC97 co-expressed AFP and CK19, it wasinferred that the side population cells had bilateral differentiation ability tosome extent. It suggested that side population cells had some primitivecharacteristics.
3. The liver cancer with the expression of CK19 has the high relapserate.Because of the absence of ck19 in normal liver,it suggested that ck19positive HCC origined from the progenitor cell.
甲胎蛋白（AFP）属胚胎类抗原，作为一种胚胎时期或病理状态的基因表达产物，它伴随着胚胎发育和细胞分裂旺盛的过程。此外，AFP还具有多种功能并在机体内发挥着多种重要的生理病理作用。CK19的表达有组织和分化特异性,是目前筛选出的肝干细胞高度表达且特异性较好的标志物之一，是胆管上皮细胞的特异性标志物。CK19在正常成体肝脏中仅表达于胆管上皮细胞而不表达于肝细胞。CK19表达阳性的肝细胞被认为是来源于肝干细胞，并位于分化的早期。有研究发现，当肝细胞发生癌变时，部分癌细胞可表达CK19,且其表达程度与细胞的分化程度呈负相关。
本实验研究拟通过紫外光激发的流式细胞荧光激活分选法获得肝癌细胞中的两个亚群细胞--边缘群细胞（SP）和非边缘群细胞（Non-SP），并用间接免疫荧光方法了解在肝癌细胞亚群中AFP和CK19的表达是否存在明显差异，通过对两个亚群细胞cDNA进行基因扩增，了解SP细胞中AFP和CK19mRNA转录水平是否与Non-SP细胞中有所不同。以此进一步了解SP细胞中是否富含的克隆扩增、形成肿瘤及自我更新能力强的，更为原始的干细胞样肿瘤细胞，并进一步揭示干细胞样肝癌细胞与肝癌的发生、复发、转移及耐药的关系。最后，利用免疫组化方法观察肝癌组织内AFP和CK19表达情况，揭示其二者与术后早期复发等临床现象之间的关系。
目的：
应用紫外光源激发的流式细胞荧光激活分选法分选肝癌细胞系MHCC97中边缘群及非边缘群两个亚群的细胞，采用间接免疫荧光方法观察蛋白质水平AFP和CK19的表达变化及含量，采用RT-PCR方法半定量分析AFP和CK19mRNA的转录水平，探讨在MHCC97细胞系不同亚群中AFP和CK19差异表达的意义。采用免疫组化方法观察肝癌组织中AFP和CK19的表达水平。
方法：
1对人类肝癌细胞系MHCC97中的边缘群细胞进行分选及边缘群细胞的分化能力的检测；
1）常规细胞培养；
2）0.25%胰酶温和消化细胞，常规离心并以HBSS液洗涤细胞；
3）SP细胞及Non-SP细胞的检测及分选
制备Hoechst33342染色的肝癌单细胞悬液，对照组在此基础上加入钙离子通道拮抗剂阻断ABC转运蛋白家族ABCG2分子的主动外排作用。流式细胞仪检测肝癌细胞中Hoechst33342荧光，紫外激发光源，通过双色性反射滤镜和不同的组合滤片将肝癌细胞中荧光信号分为两个部分，以HoechstRed为X轴，Hoechst Blue为Y轴作二维散点图，将低Hoechst Red及低Hoechst Blue且对照组缺失的区域设定为SP细胞的“门”，分选出SP细胞及Non-SP细胞。
4）根据Genebank中提供的甲胎蛋白（AFP）和细胞角蛋白19（CK19）的基因序列，使用Primer5引物设计软件分别对两条基因进行半定量引物的设计，并送公司合成。分别以边缘群细胞和非边缘群细胞两种细胞群的cDNA为模板，行半定量检测。
5）将两群细胞接种盖玻片，分别行AFP和CK19双色免疫荧光检测。利用Image-Pro Plus5.0.2软件分别检测SP细胞和non-SP细胞爬片的平均积分光密度，比较两组细胞中AFP和CK19表达量。用SPSS 11.0统计软件包采用配对t检验对数据进行分析。
2购买肝癌组织芯片，分别行AFP和CK19免疫组化染色。比较两组标本组织中细胞的AFP和CK19表达量。
结果：
1流式细胞仪的双波长荧光分析结果显示在二维散点图上有边缘群细胞存在。MHCC97细胞系中边缘群细胞的比例接近0.25%。加入钙通道拮抗剂后该群细胞明显减少，因为低荧光的特征消失而进入到了非边缘群细胞群中，这表明MHCC97细胞系中SP细胞表型与细胞膜表面主动转运泵分子ABCG2的主动外排功能有关；
2 SP细胞及Non-SP细胞AFP和CK19基因半定量RT-PCR分析利用琼脂糖凝胶电泳条带，对AFP和CK19 mRNA光密度值与G3PDH mRNA光密度值的比值进行统计分析表明SP细胞组AFP和CK19 mRNA转录水平明显高于Non-SP细胞组。
3 SP细胞及Non-SP细胞AFP和CK19双色免疫荧光检测SP细胞及Non-SP细胞分别行细胞爬片培养，4小时后观察均伸展贴壁，分别行AFP和CK19双色免疫荧光检测，激光共聚焦镜下观察，SP细胞及Non-SP细胞胞浆内均可见表达AFP蛋白的红色荧光颗粒，CK19蛋白的绿色荧光颗粒。应用Image-Pro Plus5.0.2软件分别检测SP细胞和non-SP细胞爬片的平均积分光密度，比较两组细胞中AFP和CK19表达量。用SPSS11.0统计软件包采用配对t检验对数据进行分析。表明MHCC97细胞系边缘群和非边缘群细胞间AFP和CK19的表达在蛋白质水平有显著性差异。
4肝癌组织芯片中AFP和CK19蛋白表达的免疫组化检测对组织芯片中肝癌组织分别行AFP和CK19染色，荧光显微镜下可见部分组织细胞胞浆内棕黄色颗粒，比较两组标本组织中细胞的AFP和CK19表达量。结果绝大多数肝癌组织表达AFP，部分肝癌组织表达CK19，少数肝癌组织既表达AFP，又表达CK19。
结论：
1利用Hoechst33342染料通过流式细胞仪技术确认人类肝癌细胞系MHCC97中存在边缘群细胞。
2 MHCC97来源的边群细胞共同表达AFP和CK19，这提示了边缘群细胞确实具有一定的双向分化能力。这说明边缘群细胞具有一定的原始特征。进一步阐明SP细胞富含了表型幼稚，克隆扩增、形成肿瘤及自我更新能力强的，更为原始的干细胞样肿瘤细胞，并提示其与肝癌的发生、复发、转移及耐药形成的关系。
3绝大多数肝癌组织表达AFP，部分肝癌组织表达CK19，少数肝癌组织既表达AFP，又表达CK19。由于正常肝细胞不表达CK19，提示表达CK19的肝癌细胞可能来源于肝癌组织中某些祖细胞。
There is no unified opinion of tumorgenesis in academic circles.Recently,researchers proposed the theory of cancer stem cells because the sameimmature cell phenotype was included in the tumor cell and stem cell.Researchers have isolated leukaemia cells that have the markers of HSCs fromacute myeloid leukaemia (AML) It reveals that the cancer stem cells might existin many tumors, which have both capacities of stem cells and tumorigenicity.Now the cancer stem cells have been found in many solid tumors, for examplebreast cancer, human train tumor etc. With the development of research,manyapproaches have been used for the disassociation and purification of cancer stemcells. Recently, more and more studies showed that there is increased evidence offinding a small side population (SP) both from established cancer cell lines andprimary tumors.SP cells were identified of having stem like characteristics, thecapacity for self-renewal to allow the maintenance of an undifferentiated stemcell population and the ability to undergo differentiation.HCC was common inour country.It is possible for the research of the carcinogenesis, palindromia, metastasis and drug fast through the disassociation of SP.
AFP belongs to embryo antigen and plays important role in the body.CK19 iszighly expressed in the liver stem cell and regarded as the specific marker of bileduct.The expression of CK19 has negative correlation with the differentiation ofliver cancer cell.
Aim:
To investigate the significance of AFP and CK19 different expression indifferent subpopulations of MHCC97.
Methods:
1.To analyze the side population cells from human hepatocellular carcinomacell lines MHCC97, and to sort them in MHCC97. To assay the differentiationability of side population cells
1）Culture cells as usual;
2）0.25% trypsin digest cells, centrifuge them as usual and wash them by HBSS;
3）Detect and sort SP cell and Non-SP cell.
Aliquot the cells suspension into two labeled centrifuge tube. All two tubes ofcancer cells stain with the Hoechst dye, but one of tube add inhibitor of Ca2+channel to inhibit the exocytosis of ABCG2 in additionally. Both of two tubesincubate at 37℃on water bath. Then place Hoechst-stained cancer cells on thecytometer, excite the Hoechst dye at 355 nm and use dichroic mirror to separatethe emission wavelengths, collect Hoechst blue fluorescence with a 450nm bandpassfilter and Hoechst red fluorescence with a 675nm edge filter long-pass.Display the histogram of Hoechst red (x-axis) versus Hoechst blue (y-axis). Sidepopulation can be found on the histogram by establish gate. Compare the Hoechstfluorescence profile of two groups of cancer cells suspension
4) According to the sequences of AFP and CK19 from Genebank, we design the primers of two genes by Primer5 soft, and send them to biological company forcomposting. Then, using the cDNA from side population and non-side populationcells as template, we do the semi-quantity assay.
5) After being cultured on coverslips, we carry the immunofluorescence stainingassay of AFP, CK19 in two cells.
2. Tissue chip of liver cancer was purchased for the immunohistochemicalstain of AFP and CK19.
ResultResults:
1. The side population can be observed on the fluorescence profile when theemission of Hoechst analyzed by dual-wavelength. The percentage of SP cells inMHCC97 is about 0.25%.When the inhibitor of Ca2+ channel is used, the sidepopulation eliminate because of inhibition of efflux mediated by the ABCtransporter. The SP cells enter into the main population on the fluorescenceprofile because not have low fluorescence characteristics.
2. Reverse transcription–polymerase chain reaction (RT-PCR) for AFP andCK19 gene.
The results of AGE showed that the transcriptional level of AFP and CK19 insp cell group was significantly higher than that in the Non-SP cell group.
3. Immunocytochemistry
SP cells and Non-SP cells were grown on coverslips respectively for 4 h, fixedwith 4% paraformaldehyde , then cells were exposed to 0.3% H2O2 in absolutemethanol at room temperature for 15 minutes to inhibit endogenous peroxidaseactivity. Immunocytochemical staining was performed with commerciallyavailable primary antibody and other reagents completed according to themanufacturer's protocol.Under confocal microscopy，AFP-positive grains andCK19-positive grains could be detected both in SP cell and Non-SP cell. Lg IOD(integrated optical density)was used for measurement of the expression ofAFP and CK19.These results showed that there were obvious difference of AFPand CK19 between SP cell and Non-SP cell.
4. Immunocytochemistry of AFP and CK19 were performed in the Tissue chipof liver cancer.
Under fluorescence microscope，buffy grains were partly expressed in thecytoplasm.The results showed that AFP was expressed in most liver cancer,CK19 was expressed in partial liver cancer and only a few liver cancer expressedboth AFP and CK19.
ConclusiConclusion：
1. The side population cells existed in human hepatocelluar carcinoma cell linesMHCC97.
2. The side population cells from MHCC97 co-expressed AFP and CK19, it wasinferred that the side population cells had bilateral differentiation ability tosome extent. It suggested that side population cells had some primitivecharacteristics.
3. The liver cancer with the expression of CK19 has the high relapserate.Because of the absence of ck19 in normal liver,it suggested that ck19positive HCC origined from the progenitor cell.
引文
1. Al-Hajj M., Wicha M.S., Benito-Hernandez A., et al.Prospective identification of tumorigenic breast cancercells[ J ], Proc Natl Acad Sci U S A. 2003;100(7): 3983-3988.
2. Ingo R, Katrin B, Ronny L,et al. Stem Cell Fate AnalysisRevisited: Interpretation of Individual Clone Dynamics in theLight of a New Paradigm of Stem Cell Organization[ J ], JBiomed Biotechnol. 2007;4(3): 255-260.
3. Reya T., Morrison S.J., Clarke M.F., et al. Stem cells, cancer,and cancer stem cells[ J ], Nature. 2001;414 (6859): 105-111.
4. Tannishyha Reya, Sean J, Morrison, et al. Stem cells, cancer,and cancer stem cells[ J ]. Nature,2001,414(6859):534-537.
5. Larderet G., Fortunel N.O., Vaigot P., et al. Human sidepopulation keratinocytes exhibit long-term proliferativepotential and a specific gene expression profile and can forma pluristratified epidermis[ J ], Stem Cells. 2006;24(4):965–974.
6. Decraene C., Benchaouir R., Dillies M.A., et al. Globaltranscriptional characterization of SP and MP cells from themyogenic C2C12 cell line: effect of FGF6[ J ], PhysiolGenomics. 2005;23(2): 132–149.
7. Challen G.A., Little M.H., et al. A side order of stem cells:the SP phenotype[ J ], Stem Cells. 2006;24(1): 3–12.
8. Triel C., Vestergaard M.E., Bolund L., et al. Side populationcells in human and mouse epidermis lack stem cellcharacteristics[ J ], Exp Cell Res. 2004;295(1): 79–90.
9. Goodell M.A., Brose K., Paradis G., et al. Isolation andfunctional properties of murine hematopoietic stem cells thatare replicating in vivo[ J ], J Exp Med. 1996;183(4):1797–1806.
10. Zhou S., Schuetz J.D., Bunting K.D., et al. The ABCtransporter Bcrp1/ABCG2 is expressed in a wide variety of stemcells and is a molecular determinant of the side-populationphenotype[ J ], Nat Med. 2001;7(9): 1028–1034.
11. Behbod F., Xian W., Shaw C.A., et al. Transcriptionalprofiling of mammary gland side population cells[ J ], StemCells. 2006;24(4): 1065–1074.
12. Majka S.M., Beutz M.A., Hagen M., et al. Identification ofnovel resident pulmonary stem cells: form and function of thelung side population[ J ], Stem Cells. 2005;23(8): 1073–1081.
13. Meeson A.P., Hawke T.J., Graham S., et al. Cellular andmolecular regulation of skeletal muscle side populationcells[ J ], Stem Cells. 2004;22(7): 1305–1320.
14. Shimano K., Satake M., Okaya A., et al. Hepatic oval cellshave the side population phenotype defined by expression ofATP-binding cassette transporter ABCG2/ BCRP1[ J ], Am JPathol. 2003;163(1): 3–9.
15. Kim M., Morshead C.M., et al.Distinct populations of forebrainneural stem and progenitor cells can be isolated using sidepopulationanalysis[ J ], J Neurosci. 2003;23(33):10703–10709.
16. Hirschmann-Jax C., Foster A.E., Wulf G.G., et al.A distinct“side population” of cells with high drug efflux capacity inhuman tumor cells[ J ], Proc Natl Acad Sci USA. 2004;101(39):14228–14233.
17. Grichnik J.M., Burch J.A., Schulteis R.D., et al.Melanoma, atumor based on a mutant stem cell[ J ], J Invest Dermatol.2006;126 (1): 142–153.
18. Kondo T., Setoguchi T., Taga T., et al. Persistence of a smallsubpopulation of cancer stem-like cells in the C6 glioma cellline[ J ], Proc Natl Acad Sci USA. 2004;101(3): 781–786.
19. Morita Y., Ema H., Yamazaki S., et al. Non-side-populationhematopoietic stem cells in mouse bone marrow[ J ], Blood2006;108(8): 2850-2856.
20. Shackleton M., Vaillant F., Simpson K.J., et al. Generation ofa functional mammary gland from a single stem cell[ J ],Nature. 2006;439 (7072): 84–88.
21. Ponti D., Costa A., Zaffaroni N., et al. Isolation and invitro propagation of tumorigenic breast cancer cells withstem/progenitor cell properties[ J ], Cancer Res. 2005;65(13):5506–5511.
22. Benchaouir R., Rameau P., Decraene C., et al. Evidence for aresident subset of cells with SP phenotype in the C2C12myogenic line: a tool to explore muscle stem cell biology[ J ],Exp Cell Res. 2004;294(1): 254–268.
23. Haraguchi N., Utsunomiya T., Inoue H., et al. Characterizationof a side population of cancer cells from humangastrointestinal system[ J ], Stem Cells. 2006;24(3): 506–513.
24. Parmar K., Sauk-Schubert C., Burdick D., et al.Sca+CD34?murine side population cells are highly enriched for primitivestem cells[ J ], Exp Hematol. 2003;31(3): 244–250.
25. de Paiva C.S., Pflugfelder S.C., Li D.Q., et al. Cell sizecorrelates with phenotype and proliferative capacity in humancorneal epithelial cells[ J ], Stem Cells. 2006;24(2):368–375.
26. Zhou S., Morris J.J., Barnes Y., et al. Bcrp1 gene expressionis required for normal numbers of side population stem cellsin mice, and confers relative protection to mitoxantrone inhematopoietic cells in vivo[ J ], Proc Natl Acad Sci USA.2002;99 (19): 12339–12344.
27. Yano S., Ito Y., Fujimoto M., et al. Characterization andlocalization of side population cells in mouse skin[ J ], StemCells. 2005;23 (6): 834–841.
28. Summer R., Kotton D.N., Sun X., et al. Side population cellsand Bcrp1 expression in lung[ J ], Am J Physiol Lung Cell MolPhysiol. 2003;285 (1): L97–L104.
29. Tsinkalovsky O., Rosenlund B., Laerum O.D., et al.Clock geneexpression in purified mouse hematopoietic stem cells[ J ],Exp Hematol. 2005;33(1): 100–107.
30. Chen J., Hersmus N., Van D., et al. The adult pituitarycontains a cell population displaying stem/progenitor cell andearly embryonic characteristics[ J ], Endocrinology.2005;146(9): 3985–3998.
31. Patrawala L., Calhoun T., Schneider-Broussard R., et al.Sidepopulation is enriched in tumorigenic, stem-like cancer cells,whereas ABCG2+ and A[ J ], Cancer Res. 2005;65(14): 6207–6219.
32. Liu B.Y., McDermott S.P., Khwaja S.S., et al. The transformingactivity of Wnt effectors correlates with their ability toinduce the accumulation of mammary progenitor cells[ J ], ProcNatl Acad Sci USA. 2004;101(12): 4158–4163.
33. Uchida N., Fujisaki T., Eaves A.C., et al. Transplantablehematopoietic stem cells in human fetal liver have a CD34(+)side population (SP) phenotype[ J ], J Clin Invest.2001;108(7): 1071–1077.
34. Lechner A., Leech C.A., Abraham E.J., et al. Nestin-positiveprogenitor cells derived from adult human pancreatic islets ofLangerhans contain side population (SP) cells defined byexpression of the ABCG2 (BCRP1) ATP-binding cassettetransporter[ J ], Biochem. Biophys. Res. Commun. 2002;293 (2):670–674.
35. Montanaro F., Liadaki K., Schienda J., et al. Demystifying SPcell purification: viability, yield, and phenotype are definedby isolation parameters[ J ], Exp. Cell Res. 2004;298 (1):144–154.
36. Al Hajj M., Becker M.W., Wicha M., et al. Therapeuticimplications of cancer stem cells[ J ], Curr. Opin. Genet. Dev.2004;14 (1): 43–47.
37. Soltysova A., Altanerova V., Altaner C., et al. Cancer stemcells[ J ], Neoplasma 2005;52 (6): 435–440.
38. Al Hajj M., Wicha M.S., Benito-Hernandez A., et al.Prospectiveidentification of tumorigenic breast cancer cells[ J ], Proc.Natl. Acad. Sci. U. S. A. 2003;100 (7): 3983–3988.
39. Marx J., et al. Cancer research. Mutant stem cells may seedcancer[ J ], Science 2003;301 (5638): 1308–1310.
40. Woodward W.A., Chen M.S., Behbod F., et al. On mammary stemcells[ J ], J. Cell Sci. 2005;118 (Pt16): 3585–3594.
41. Raaijmakers M.H., de Grouw E.P., Heuver L.H., et al. Breastcancer resistance protein in drug resistance of primitiveCD34+38? cells in acute myeloid leukemia[ J ], Clin. CancerRes. 2005;11 (6): 2436–2444.
42. Hu Y, Wang JS, Jia XS. et al. Progress of stem cells research.Progress Japanese Medicine[ J ], 2002, 23(12):570-573.
43. Bonnet D, Dick JE. et al. Human acute myeloid leukemia isorganized as ahierarchy that originates from a primitivehematopoietic cell[ J ]. Nat Med, 1997,3(7):730-737.
44. Webb T. et al. Work on breast cancer stem cells raisesquestions about treatment strategies[ J ]. J CancerIns,2003,95:774-775.
45. Guo Y, Follo M, Geiger K, Lubbert M, et al. Side populationcells from different precursor compartments［J］. HematotherStem Cell Res, 2007,12(1): 71-82.
46. Preffer FI, Dombkowski D, Sykes M, et al. Lineage-negativeside population cells with restricted hematopoietic capacitycirculate in normal human adult blood: immunophenotypic andfunctional characterization［J］. Stem cells, 2002, 20(5):417-427.identification of tumorigenic breast cancer cells[ J ], Proc.Natl. Acad. Sci. U. S. A. 2003;100 (7): 3983–3988.
39. Marx J., et al. Cancer research. Mutant stem cells may seedcancer[ J ], Science 2003;301 (5638): 1308–1310.
40. Woodward W.A., Chen M.S., Behbod F., et al. On mammary stemcells[ J ], J. Cell Sci. 2005;118 (Pt16): 3585–3594.
41. Raaijmakers M.H., de Grouw E.P., Heuver L.H., et al. Breastcancer resistance protein in drug resistance of primitiveCD34+38? cells in acute myeloid leukemia[ J ], Clin. CancerRes. 2005;11 (6): 2436–2444.
42. Hu Y, Wang JS, Jia XS. et al. Progress of stem cells research.Progress Japanese Medicine[ J ], 2002, 23(12):570-573.
43. Bonnet D, Dick JE. et al. Human acute myeloid leukemia isorganized as ahierarchy that originates from a primitivehematopoietic cell[ J ]. Nat Med, 1997,3(7):730-737.
44. Webb T. et al. Work on breast cancer stem cells raisesquestions about treatment strategies[ J ]. J CancerIns,2003,95:774-775.
45. Guo Y, Follo M, Geiger K, Lubbert M, et al. Side populationcells from different precursor compartments［J］. HematotherStem Cell Res, 2007,12(1): 71-82.
46. Preffer FI, Dombkowski D, Sykes M, et al. Lineage-negativeside population cells with restricted hematopoietic capacitycirculate in normal human adult blood: immunophenotypic andfunctional characterization［J］. Stem cells, 2002, 20(5):417-427.apoptosis in tumor cells via a pathway independent of CD，TNFR1and TNFR2 through activation of easpase-3-likeproteases[J]．Eur J Biochem，1999，266(3)：750-761．
56. Um S H．Mulhall C．Alisa A．et a1．Alpha-fetoprotein impairs APCfunction and induces their apoptosis[J]．J lmmuno，2004，173(3)：1772-1778．
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2. Ingo R, Katrin B, Ronny L,et al. Stem Cell Fate AnalysisRevisited: Interpretation of Individual Clone Dynamics in theLight of a New Paradigm of Stem Cell Organization[ J ], JBiomed Biotechnol. 2007;4(3): 255-260.
3. Reya T., Morrison S.J., Clarke M.F., et al. Stem cells, cancer,and cancer stem cells[ J ], Nature. 2001;414 (6859): 105-111.
4. Tannishyha Reya, Sean J, Morrison, et al. Stem cells, cancer,and cancer stem cells[ J ]. Nature,2001,414(6859):534-537.
5. Larderet G., Fortunel N.O., Vaigot P., et al. Human sidepopulation keratinocytes exhibit long-term proliferativepotential and a specific gene expression profile and can forma pluristratified epidermis[ J ], Stem Cells. 2006;24(4):965–974.
6. Decraene C., Benchaouir R., Dillies M.A., et al. Globaltranscriptional characterization of SP and MP cells from themyogenic C2C12 cell line: effect of FGF6[ J ], PhysiolGenomics. 2005;23(2): 132–149.
7. Challen G.A., Little M.H., et al. A side order of stem cells:the SP phenotype[ J ], Stem Cells. 2006;24(1): 3–12.
8. Triel C., Vestergaard M.E., Bolund L., et al. Side populationcells in human and mouse epidermis lack stem cellcharacteristics[ J ], Exp Cell Res. 2004;295(1): 79–90.
9. Goodell M.A., Brose K., Paradis G., et al. Isolation andfunctional properties of murine hematopoietic stem cells thatare replicating in vivo[ J ], J Exp Med. 1996;183(4):1797–1806.
10. Zhou S., Schuetz J.D., Bunting K.D., et al. The ABCtransporter Bcrp1/ABCG2 is expressed in a wide variety of stemcells and is a molecular determinant of the side-populationphenotype[ J ], Nat Med. 2001;7(9): 1028–1034.
11. Behbod F., Xian W., Shaw C.A., et al. Transcriptionalprofiling of mammary gland side population cells[ J ], StemCells. 2006;24(4): 1065–1074.
12. Majka S.M., Beutz M.A., Hagen M., et al. Identification ofnovel resident pulmonary stem cells: form and function of thelung side population[ J ], Stem Cells. 2005;23(8): 1073–1081.
13. Meeson A.P., Hawke T.J., Graham S., et al. Cellular andmolecular regulation of skeletal muscle side populationcells[ J ], Stem Cells. 2004;22(7): 1305–1320.
14. Shimano K., Satake M., Okaya A., et al. Hepatic oval cellshave the side population phenotype defined by expression ofATP-binding cassette transporter ABCG2/ BCRP1[ J ], Am JPathol. 2003;163(1): 3–9.
15. Kim M., Morshead C.M., et al.Distinct populations of forebrainneural stem and progenitor cells can be isolated using sidepopulationanalysis[ J ], J Neurosci. 2003;23(33):10703–10709.
16. Hirschmann-Jax C., Foster A.E., Wulf G.G., et al.A distinct“side population” of cells with high drug efflux capacity inhuman tumor cells[ J ], Proc Natl Acad Sci USA. 2004;101(39):14228–14233.
17. Grichnik J.M., Burch J.A., Schulteis R.D., et al.Melanoma, atumor based on a mutant stem cell[ J ], J Invest Dermatol.2006;126 (1): 142–153.
18. Kondo T., Setoguchi T., Taga T., et al. Persistence of a smallsubpopulation of cancer stem-like cells in the C6 glioma cellline[ J ], Proc Natl Acad Sci USA. 2004;101(3): 781–786.
19. Morita Y., Ema H., Yamazaki S., et al. Non-side-populationhematopoietic stem cells in mouse bone marrow[ J ], Blood2006;108(8): 2850-2856.
20. Shackleton M., Vaillant F., Simpson K.J., et al. Generation ofa functional mammary gland from a single stem cell[ J ],Nature. 2006;439 (7072): 84–88.
21. Ponti D., Costa A., Zaffaroni N., et al. Isolation and invitro propagation of tumorigenic breast cancer cells withstem/progenitor cell properties[ J ], Cancer Res. 2005;65(13):5506–5511.
22. Benchaouir R., Rameau P., Decraene C., et al. Evidence for aresident subset of cells with SP phenotype in the C2C12myogenic line: a tool to explore muscle stem cell biology[ J ],Exp Cell Res. 2004;294(1): 254–268.
23. Haraguchi N., Utsunomiya T., Inoue H., et al. Characterizationof a side population of cancer cells from humangastrointestinal system[ J ], Stem Cells. 2006;24(3): 506–513.
24. Parmar K., Sauk-Schubert C., Burdick D., et al.Sca+CD34?murine side population cells are highly enriched for primitivestem cells[ J ], Exp Hematol. 2003;31(3): 244–250.
25. de Paiva C.S., Pflugfelder S.C., Li D.Q., et al. Cell sizecorrelates with phenotype and proliferative capacity in humancorneal epithelial cells[ J ], Stem Cells. 2006;24(2):368–375.
26. Zhou S., Morris J.J., Barnes Y., et al. Bcrp1 gene expressionis required for normal numbers of side population stem cellsin mice, and confers relative protection to mitoxantrone inhematopoietic cells in vivo[ J ], Proc Natl Acad Sci USA.2002;99 (19): 12339–12344.
27. Yano S., Ito Y., Fujimoto M., et al. Characterization andlocalization of side population cells in mouse skin[ J ], StemCells. 2005;23 (6): 834–841.
28. Summer R., Kotton D.N., Sun X., et al. Side population cellsand Bcrp1 expression in lung[ J ], Am J Physiol Lung Cell MolPhysiol. 2003;285 (1): L97–L104.
29. Tsinkalovsky O., Rosenlund B., Laerum O.D., et al.Clock geneexpression in purified mouse hematopoietic stem cells[ J ],Exp Hematol. 2005;33(1): 100–107.
30. Chen J., Hersmus N., Van D., et al. The adult pituitarycontains a cell population displaying stem/progenitor cell andearly embryonic characteristics[ J ], Endocrinology.2005;146(9): 3985–3998.
31. Patrawala L., Calhoun T., Schneider-Broussard R., et al.Sidepopulation is enriched in tumorigenic, stem-like cancer cells,whereas ABCG2+ and A[ J ], Cancer Res. 2005;65(14): 6207–6219.
32. Liu B.Y., McDermott S.P., Khwaja S.S., et al. The transformingactivity of Wnt effectors correlates with their ability toinduce the accumulation of mammary progenitor cells[ J ], ProcNatl Acad Sci USA. 2004;101(12): 4158–4163.
33. Uchida N., Fujisaki T., Eaves A.C., et al. Transplantablehematopoietic stem cells in human fetal liver have a CD34(+)side population (SP) phenotype[ J ], J Clin Invest.2001;108(7): 1071–1077.
34. Lechner A., Leech C.A., Abraham E.J., et al. Nestin-positiveprogenitor cells derived from adult human pancreatic islets ofLangerhans contain side population (SP) cells defined byexpression of the ABCG2 (BCRP1) ATP-binding cassettetransporter[ J ], Biochem. Biophys. Res. Commun. 2002;293 (2):670–674.
35. Montanaro F., Liadaki K., Schienda J., et al. Demystifying SPcell purification: viability, yield, and phenotype are definedby isolation parameters[ J ], Exp. Cell Res. 2004;298 (1):144–154.
36. Al Hajj M., Becker M.W., Wicha M., et al. Therapeuticimplications of cancer stem cells[ J ], Curr. Opin. Genet. Dev.2004;14 (1): 43–47.
37. Soltysova A., Altanerova V., Altaner C., et al. Cancer stemcells[ J ], Neoplasma 2005;52 (6): 435–440.
38. Al Hajj M., Wicha M.S., Benito-Hernandez A., et al.Prospectiveidentification of tumorigenic breast cancer cells[ J ], Proc.Natl. Acad. Sci. U. S. A. 2003;100 (7): 3983–3988.
39. Marx J., et al. Cancer research. Mutant stem cells may seedcancer[ J ], Science 2003;301 (5638): 1308–1310.
40. Woodward W.A., Chen M.S., Behbod F., et al. On mammary stemcells[ J ], J. Cell Sci. 2005;118 (Pt16): 3585–3594.
41. Raaijmakers M.H., de Grouw E.P., Heuver L.H., et al. Breastcancer resistance protein in drug resistance of primitiveCD34+38? cells in acute myeloid leukemia[ J ], Clin. CancerRes. 2005;11 (6): 2436–2444.
42. Hu Y, Wang JS, Jia XS. et al. Progress of stem cells research.Progress Japanese Medicine[ J ], 2002, 23(12):570-573.
43. Bonnet D, Dick JE. et al. Human acute myeloid leukemia isorganized as ahierarchy that originates from a primitivehematopoietic cell[ J ]. Nat Med, 1997,3(7):730-737.
44. Webb T. et al. Work on breast cancer stem cells raisesquestions about treatment strategies[ J ]. J CancerIns,2003,95:774-775.
45. Guo Y, Follo M, Geiger K, Lubbert M, et al. Side populationcells from different precursor compartments［J］. HematotherStem Cell Res, 2007,12(1): 71-82.
46. Preffer FI, Dombkowski D, Sykes M, et al. Lineage-negativeside population cells with restricted hematopoietic capacitycirculate in normal human adult blood: immunophenotypic andfunctional characterization［J］. Stem cells, 2002, 20(5):417-427.identification of tumorigenic breast cancer cells[ J ], Proc.Natl. Acad. Sci. U. S. A. 2003;100 (7): 3983–3988.
39. Marx J., et al. Cancer research. Mutant stem cells may seedcancer[ J ], Science 2003;301 (5638): 1308–1310.
40. Woodward W.A., Chen M.S., Behbod F., et al. On mammary stemcells[ J ], J. Cell Sci. 2005;118 (Pt16): 3585–3594.
41. Raaijmakers M.H., de Grouw E.P., Heuver L.H., et al. Breastcancer resistance protein in drug resistance of primitiveCD34+38? cells in acute myeloid leukemia[ J ], Clin. CancerRes. 2005;11 (6): 2436–2444.
42. Hu Y, Wang JS, Jia XS. et al. Progress of stem cells research.Progress Japanese Medicine[ J ], 2002, 23(12):570-573.
43. Bonnet D, Dick JE. et al. Human acute myeloid leukemia isorganized as ahierarchy that originates from a primitivehematopoietic cell[ J ]. Nat Med, 1997,3(7):730-737.
44. Webb T. et al. Work on breast cancer stem cells raisesquestions about treatment strategies[ J ]. J CancerIns,2003,95:774-775.
45. Guo Y, Follo M, Geiger K, Lubbert M, et al. Side populationcells from different precursor compartments［J］. HematotherStem Cell Res, 2007,12(1): 71-82.
46. Preffer FI, Dombkowski D, Sykes M, et al. Lineage-negativeside population cells with restricted hematopoietic capacitycirculate in normal human adult blood: immunophenotypic andfunctional characterization［J］. Stem cells, 2002, 20(5):417-427.apoptosis in tumor cells via a pathway independent of CD，TNFR1and TNFR2 through activation of easpase-3-likeproteases[J]．Eur J Biochem，1999，266(3)：750-761．
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