CD133~+CD24~+肝癌干细胞的分离、鉴定及生物学特征的研究
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摘要
研究背景及目的：在世界范围内，原发性肝癌是最常见的恶性程度最高的肿瘤之一，为全球第5大癌症，死亡率位列第3位。据报道，每年有将近100万人被诊断为肝癌，60万人死于该病，且5年生存率低于5%，并且发病率还在逐年升高，因此肝癌严重威胁着人类的健康和生命。
手术治疗、肝移植、肝动脉化疗栓塞术（TACE）、射频消融治疗（RFA）为目前肝癌的主要治疗手段。手术治疗仍是肝癌治疗的首选方法，但是手术后复发仍然是至今无法解决的难题，并且复发率很高。对于合并有肝硬化的肝癌患者，肝移植是最有前景的治疗手段，但由于供体肝短缺，肝移植的开展受到较大限制。由于各治疗手段的局限性，导致肝癌患者的生存率低下，迫切需要结合肝癌的发病机制研究进一步开发新疗法。肿瘤干细胞假说认为在肿瘤组织中存在一种数量较少的肿瘤干细胞，被认为是肿瘤增殖、侵袭、转移及复发的根源。目前在白血病、乳腺癌、肺癌、脑肿瘤、结直肠癌、前列腺癌等多种肿瘤中均已成功分离出肿瘤干细胞。肝癌中存在肿瘤干细胞与其难治、复发及耐药等特点密切相关，而这些特点提示肝癌很可能是一种肿瘤干细胞疾病。随着研究地不断深入，越来越多的肝癌干细胞表面标志物被确立，并应用于干细胞的鉴别与分选，主要包括CD133、CD24、CD44、CD90、上皮细胞黏附分子（EpCAM）等。目前有报道，利用细胞表面分子分离出的肝癌干细胞在复发、耐药等方面发挥着重要作用。
方法：本研究通过CD133和CD24两个肿瘤干细胞标志物为筛选标记，从肝癌细胞系Huh-7细胞中分离出CD133+CD24+阳性细胞，在体外从自我更新能力、分化能力、克隆能力及耐药等方面进行了细胞学行为的鉴定，在体内从致瘤性、CD133和CD24蛋白的表达等方面进行了鉴定。
1．应用流式细胞术检测CD133、CD24等表面分子在肝癌细胞系的表达情况，并分选和鉴定CD133+CD24+细胞；
2．检测CD133+和CD24+细胞克隆形成能力及成球能力；
3．利用RT-PCR检测CD133和CD24表达水平；
4. Western blot法检检测分选出的细胞基因表达；
5．利用小鼠模型检测分选细胞的成瘤能力；
6．免疫组织及HE染色检测小鼠肿瘤组织中CD133及CD24的表达；
7．MTT检测细胞对洛铂的敏感性。
结果：本研究以CD133和CD24为肿瘤干细胞标志物，利用流式细胞分选法从Huh-7细胞中分选出CD133+CD24+细胞和CD133-CD24-细胞，并检测了其成球能力、克隆形成能力、CD133和CD24表达、在体内的成瘤能力及对洛铂的敏感性等，得到以下主要结果：
1．肿瘤干细胞标记物CD133和CD24在肝癌细胞系Huh-7呈共表达；
2．CD133+CD24+细胞具有很强的克隆形成能力及成球能力；
3．与对照组相比，CD133+CD24+细胞明显高表达CD133和CD24基因及蛋白；
4.分选出的CD133+CD24+细胞具有更强的致瘤性；
5.小鼠移植瘤模型分离出肝癌组织表达CD133和CD24蛋白；
6．CD133+CD24+细胞大多处于细胞周期静止期（G0/G1期）；
7. CD133+CD24+细胞对洛铂相对耐药，其IC50为9.9μg/mL；
结论：1.肝癌细胞系Huh-7同时表达肿瘤干细胞标记CD133和CD24
2. CD133+CD24+Huh-7细胞具有肿瘤干细胞的特性，包括具有自我更新及分化能力，更强克隆及成球能力，在免疫缺陷小鼠体内具有高致瘤性。
3.与CD133-CD24-Huh-7细胞相比，CD133+CD24+Huh-7细胞对洛铂更耐药。
总之，联合使用细胞表面分子CD133和CD24可以更好的分选、分离和鉴定肝癌干细胞，分选出的细胞具有高成球能力、高克隆形成能力、高致瘤性和高耐药性。CD133和CD24可以作为分离肝癌干细胞的表面标记物。
创新点：CD133和CD24可以作为分选肝癌干细胞的表面标记物，CD133和CD24联合应用能更好地从肝癌细胞系中分离出肝癌干细胞。
Background and objective：Hepatocellular carcinoma (HCC) is the fifth most prevalentmalignant cancer, and the third most common cause of cancer-related deaths worldwide. Itis reported that approximately one million cases of HCC are diagnosed annually, witharound600,000of these cases resulting in patient death. The incidence of HCC is expectedto increase globally, and with a5-year survival rate of below5%.
Various treatments can be employed for HCC management according to the clinicalliver cancer staging system. Hepatic resection is a common therapy for HCC, particularlyfor noncirrhotic patients. It is also a first-line treatment for patients with early HCC. But thetumor recurrence is still a problem after resection, and the recurrence rate remains high afteroperation, indicating the incomplete eradication of tumor cells in patients. Orthotopic livertransplantation (OLT) has become a promising therapeutic option for cirrhotic patients withunresectable HCC, but these treatments are limited by the advanced stage when the diseaseis usually diagnosed and the shortage of liver grafts available. Local ablative strategy is abetter treatment of choice for the management of early-stage HCC with small tumor. Inspite of technological advances in treatment interventions, the survival outcome of HCCpatients at present is still unsatisfactory. Chemotherapy either by TACE or systemictreatment is also available, due to the highly chemotherapy-resistant nature of the disease,however, response rates are low.
The exact mechanism of tumor recurrence remains vague due to a limitedunderstanding of hepatocarcinogenesis. It is suspected that tumor relapse is likely to becaused by cancer stem cells (CSCs) that are resistant to current treatments. Based on theCSC theory, it is believed that CSCs only represent a minority number of the entire tumor mass (generally less than5%of the total tumor). However, the percentage of CSCs isolatedfrom hepatocellular cancer cell lines or tissue is more than50%. So it is not likely that allthe separated from HCC cell lines are CSCs by CSC markers-CD133. And CSCs in othercancer types have also been characterized by more than one marker. For this reason, tobetter characterize the CSC population in HCC, the identification of other markers,expressed along with CD133, is much required and was also our purpose for this study.Methods:
1．Fluorescence activated cell sorting was used to assess cancer stem cell markerexpression in hepatocellular cancer cell lines, and identify and isolate CD133+CD24+cells;
2．Detection of clonogenic ability and tumorsphere formation ability of sorted cells;
3．CD133and CD24expression was analyzed by RT-PCR;
4．CD133and CD24expression was analyzed by Western blot;
5. Tumor formation ability of the sorted cell was determined in Balb/C mice in vivo;
6．CD133and CD24expression was detection in HCC tissue specimens by HE andIHC stainning;
7．Cell viability was analysed by MTT array.Results: We identified and isolated a small population hepatic cancer cells co-expressionCD133and CD24surface markers from human liver cell lines Huh-7. The sorted cellspossessed property as follows:
1. CD133and CD24were co-expressed in hepatocellular cancer cell line
2. Increased spheroid formation ability and higher clonogenic ability inCD133+CD24+cells
3. Compared to CD133-CD24-cells, and CD24were preferentially co-expressed inCD133+CD24+cells, RT-PCR and western blot analyses revealed that the CD133and CD24gene expression of sorted cells was in line with their phenotype in RNA lever and proteinlevel.
4. The sorted CD133+CD24+cells from Huh-7cells had a stronger tumorigenicity.
5. Significant expression of CD133and CD24in HCC tissue specimens fromxenograft mice model and the tumor was very similar with the primary cancer.
6. The percentage of G0and G1phrase was more higher in CD133+CD24+cells.
7. CD133+CD24+cells from Huh-7cells were highly resistant to chemotherapeuticagent lobaplatin, and the IC50was9.9μg/mL.
In a word, CD133+CD24+cells possessed a greater colony-forming efficiency, higherproliferative output, and greater ability to form tumor in vivo, which was identical to theoriginal tumor, and had features of hepatic cancer stem cells. CD133and CD24double-positive cells were more resistant to chemotherapeutic agents.
Conclusions:
1. Hepatocellular cancer cell line Huh-7expressed cancer stem cell marker CD133and CD24.
2. CD133+CD24+Huh-7cells possessed cancer stem cell propersity, includingself-renewal, differentiation, increased spheroid formation ability, higher clonogenic ability,and a stronger tumorigenicity in SCID mice.
3. Compared to CD133-CD24-Huh-7cells，CD133+CD24+Huh-7cells werehighly resistant to chemotherapeutic agent lobaplatin。
Taken together, Our results suggest that hepatic cancer stem cell could be preciselydefined by co-expression of CD133and CD24cell surface markers. The identification oftumorigenic liver CSCs could provide new insight into the HCC tumorigenic process andpossibly bear great therapeutic implications.
Innovations: We demonstrated that CD133and CD24could served as hepatic cancer
stem markers, we can better characterize the CSC population in HCC by combination
CD133with CD24.
手术治疗、肝移植、肝动脉化疗栓塞术（TACE）、射频消融治疗（RFA）为目前肝癌的主要治疗手段。手术治疗仍是肝癌治疗的首选方法，但是手术后复发仍然是至今无法解决的难题，并且复发率很高。对于合并有肝硬化的肝癌患者，肝移植是最有前景的治疗手段，但由于供体肝短缺，肝移植的开展受到较大限制。由于各治疗手段的局限性，导致肝癌患者的生存率低下，迫切需要结合肝癌的发病机制研究进一步开发新疗法。肿瘤干细胞假说认为在肿瘤组织中存在一种数量较少的肿瘤干细胞，被认为是肿瘤增殖、侵袭、转移及复发的根源。目前在白血病、乳腺癌、肺癌、脑肿瘤、结直肠癌、前列腺癌等多种肿瘤中均已成功分离出肿瘤干细胞。肝癌中存在肿瘤干细胞与其难治、复发及耐药等特点密切相关，而这些特点提示肝癌很可能是一种肿瘤干细胞疾病。随着研究地不断深入，越来越多的肝癌干细胞表面标志物被确立，并应用于干细胞的鉴别与分选，主要包括CD133、CD24、CD44、CD90、上皮细胞黏附分子（EpCAM）等。目前有报道，利用细胞表面分子分离出的肝癌干细胞在复发、耐药等方面发挥着重要作用。
方法：本研究通过CD133和CD24两个肿瘤干细胞标志物为筛选标记，从肝癌细胞系Huh-7细胞中分离出CD133+CD24+阳性细胞，在体外从自我更新能力、分化能力、克隆能力及耐药等方面进行了细胞学行为的鉴定，在体内从致瘤性、CD133和CD24蛋白的表达等方面进行了鉴定。
1．应用流式细胞术检测CD133、CD24等表面分子在肝癌细胞系的表达情况，并分选和鉴定CD133+CD24+细胞；
2．检测CD133+和CD24+细胞克隆形成能力及成球能力；
3．利用RT-PCR检测CD133和CD24表达水平；
4. Western blot法检检测分选出的细胞基因表达；
5．利用小鼠模型检测分选细胞的成瘤能力；
6．免疫组织及HE染色检测小鼠肿瘤组织中CD133及CD24的表达；
7．MTT检测细胞对洛铂的敏感性。
结果：本研究以CD133和CD24为肿瘤干细胞标志物，利用流式细胞分选法从Huh-7细胞中分选出CD133+CD24+细胞和CD133-CD24-细胞，并检测了其成球能力、克隆形成能力、CD133和CD24表达、在体内的成瘤能力及对洛铂的敏感性等，得到以下主要结果：
1．肿瘤干细胞标记物CD133和CD24在肝癌细胞系Huh-7呈共表达；
2．CD133+CD24+细胞具有很强的克隆形成能力及成球能力；
3．与对照组相比，CD133+CD24+细胞明显高表达CD133和CD24基因及蛋白；
4.分选出的CD133+CD24+细胞具有更强的致瘤性；
5.小鼠移植瘤模型分离出肝癌组织表达CD133和CD24蛋白；
6．CD133+CD24+细胞大多处于细胞周期静止期（G0/G1期）；
7. CD133+CD24+细胞对洛铂相对耐药，其IC50为9.9μg/mL；
结论：1.肝癌细胞系Huh-7同时表达肿瘤干细胞标记CD133和CD24
2. CD133+CD24+Huh-7细胞具有肿瘤干细胞的特性，包括具有自我更新及分化能力，更强克隆及成球能力，在免疫缺陷小鼠体内具有高致瘤性。
3.与CD133-CD24-Huh-7细胞相比，CD133+CD24+Huh-7细胞对洛铂更耐药。
总之，联合使用细胞表面分子CD133和CD24可以更好的分选、分离和鉴定肝癌干细胞，分选出的细胞具有高成球能力、高克隆形成能力、高致瘤性和高耐药性。CD133和CD24可以作为分离肝癌干细胞的表面标记物。
创新点：CD133和CD24可以作为分选肝癌干细胞的表面标记物，CD133和CD24联合应用能更好地从肝癌细胞系中分离出肝癌干细胞。
Background and objective：Hepatocellular carcinoma (HCC) is the fifth most prevalentmalignant cancer, and the third most common cause of cancer-related deaths worldwide. Itis reported that approximately one million cases of HCC are diagnosed annually, witharound600,000of these cases resulting in patient death. The incidence of HCC is expectedto increase globally, and with a5-year survival rate of below5%.
Various treatments can be employed for HCC management according to the clinicalliver cancer staging system. Hepatic resection is a common therapy for HCC, particularlyfor noncirrhotic patients. It is also a first-line treatment for patients with early HCC. But thetumor recurrence is still a problem after resection, and the recurrence rate remains high afteroperation, indicating the incomplete eradication of tumor cells in patients. Orthotopic livertransplantation (OLT) has become a promising therapeutic option for cirrhotic patients withunresectable HCC, but these treatments are limited by the advanced stage when the diseaseis usually diagnosed and the shortage of liver grafts available. Local ablative strategy is abetter treatment of choice for the management of early-stage HCC with small tumor. Inspite of technological advances in treatment interventions, the survival outcome of HCCpatients at present is still unsatisfactory. Chemotherapy either by TACE or systemictreatment is also available, due to the highly chemotherapy-resistant nature of the disease,however, response rates are low.
The exact mechanism of tumor recurrence remains vague due to a limitedunderstanding of hepatocarcinogenesis. It is suspected that tumor relapse is likely to becaused by cancer stem cells (CSCs) that are resistant to current treatments. Based on theCSC theory, it is believed that CSCs only represent a minority number of the entire tumor mass (generally less than5%of the total tumor). However, the percentage of CSCs isolatedfrom hepatocellular cancer cell lines or tissue is more than50%. So it is not likely that allthe separated from HCC cell lines are CSCs by CSC markers-CD133. And CSCs in othercancer types have also been characterized by more than one marker. For this reason, tobetter characterize the CSC population in HCC, the identification of other markers,expressed along with CD133, is much required and was also our purpose for this study.Methods:
1．Fluorescence activated cell sorting was used to assess cancer stem cell markerexpression in hepatocellular cancer cell lines, and identify and isolate CD133+CD24+cells;
2．Detection of clonogenic ability and tumorsphere formation ability of sorted cells;
3．CD133and CD24expression was analyzed by RT-PCR;
4．CD133and CD24expression was analyzed by Western blot;
5. Tumor formation ability of the sorted cell was determined in Balb/C mice in vivo;
6．CD133and CD24expression was detection in HCC tissue specimens by HE andIHC stainning;
7．Cell viability was analysed by MTT array.Results: We identified and isolated a small population hepatic cancer cells co-expressionCD133and CD24surface markers from human liver cell lines Huh-7. The sorted cellspossessed property as follows:
1. CD133and CD24were co-expressed in hepatocellular cancer cell line
2. Increased spheroid formation ability and higher clonogenic ability inCD133+CD24+cells
3. Compared to CD133-CD24-cells, and CD24were preferentially co-expressed inCD133+CD24+cells, RT-PCR and western blot analyses revealed that the CD133and CD24gene expression of sorted cells was in line with their phenotype in RNA lever and proteinlevel.
4. The sorted CD133+CD24+cells from Huh-7cells had a stronger tumorigenicity.
5. Significant expression of CD133and CD24in HCC tissue specimens fromxenograft mice model and the tumor was very similar with the primary cancer.
6. The percentage of G0and G1phrase was more higher in CD133+CD24+cells.
7. CD133+CD24+cells from Huh-7cells were highly resistant to chemotherapeuticagent lobaplatin, and the IC50was9.9μg/mL.
In a word, CD133+CD24+cells possessed a greater colony-forming efficiency, higherproliferative output, and greater ability to form tumor in vivo, which was identical to theoriginal tumor, and had features of hepatic cancer stem cells. CD133and CD24double-positive cells were more resistant to chemotherapeutic agents.
Conclusions:
1. Hepatocellular cancer cell line Huh-7expressed cancer stem cell marker CD133and CD24.
2. CD133+CD24+Huh-7cells possessed cancer stem cell propersity, includingself-renewal, differentiation, increased spheroid formation ability, higher clonogenic ability,and a stronger tumorigenicity in SCID mice.
3. Compared to CD133-CD24-Huh-7cells，CD133+CD24+Huh-7cells werehighly resistant to chemotherapeutic agent lobaplatin。
Taken together, Our results suggest that hepatic cancer stem cell could be preciselydefined by co-expression of CD133and CD24cell surface markers. The identification oftumorigenic liver CSCs could provide new insight into the HCC tumorigenic process andpossibly bear great therapeutic implications.
Innovations: We demonstrated that CD133and CD24could served as hepatic cancer
stem markers, we can better characterize the CSC population in HCC by combination
CD133with CD24.
引文
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