大鼠原发性肝癌肿瘤细胞与骨髓造血干细胞在肝癌个体化治疗应用中的实验研究
|
摘要
背景：
以肝切除为代表的外科治疗仍是原发性肝癌的首选治疗方法。但由于原发性肝癌的早期发现存在困难，肝癌切除术后的5年复发率高达60％以上，切除术后的综合治疗在原发性肝癌的治疗中仍然占有重要的地位。化疗是综合治疗的主要手段之一，而各种针对原发性肝癌的临床化疗总体有效率仅为18.2％～47％，并且进展缓慢。提供切实可行的个体化化疗方案成为临床工作者面对原发性肝癌治疗上的迫切需要。
随着近年来肿瘤干细胞(TSC)理论的确立，对于实体肿瘤的治疗有了新的认识：TSC是肿瘤发生、浸润、转移和复发等恶性生物学行为的核心，是肿瘤细胞成瘤能力的决定因素。只要存在肿瘤干细胞，肿瘤就难以治愈。本实验室的前期研究中在大鼠原发性肝癌已经初步鉴定了具备TSC特征(极强的致瘤能力和自我更新并多向分化)的肿瘤细胞亚群。由此如果能够建立一种针对原发性肝癌肿瘤干细胞的个体化化疗方案，靶向杀伤肿瘤干细胞，将有希望在原发性肝癌的化疗进展上取得突破。
目前对于包括肝癌在内的绝大多数实体瘤来说，缺乏鉴定TSC的统一标准，而分离出纯度高的TSC，直接进行药敏实验的技术难度、成本都比较大。较多研究表明，肝干细胞是肝细胞癌和胆管癌的祖细胞。来源于骨髓干细胞的肝卵圆细胞(Hepatic oval cell，HOC)具备肝脏干细胞性质，是肝癌干细胞的对应成体干细胞。卵圆细胞不仅具有同骨髓干细胞相似的表面标志，如CD34、Thy-1(CD90)、c-kit(CD117)，并且在耐药机制上也类似干细胞。骨髓干细胞分为造血干细胞和间充质干细胞，相比较TSC而言易于分离鉴定，药敏实验的可行性较高。基于上述理由，本课题利用肿瘤干细胞与骨髓干细胞的关系，重点研究骨髓干细胞在针对肝癌干细胞个体化化疗的药物敏感实验中的应用。
目的：研究大鼠骨髓造血干细胞与大鼠原发性肝癌细胞药敏与肝癌细胞成瘤能力的相关性；探讨利用造血干细胞，进行针对肝癌干细胞药物敏感实验的可能性。并研究了药物对骨髓间充质干细胞、肝癌细胞和HepG2细胞的药敏差异。
方法：30只雄性清洁级SD大鼠应用0.025％二乙基亚硝胺水溶液饲喂24周建立大鼠原发性肝癌模型，选取诱癌成功大鼠8只，用组织块法分离培养原代肝癌细胞；用免疫磁珠法分离骨髓造血干细胞，流式细胞仪检测传代细胞表面标志CD34。筛选五种不同作用机制抗肿瘤药物表阿霉素、5-氟尿嘧啶(5-FU)、健择、环磷酰胺、顺铂作用24小时后，SRB法分别检测化疗药物对造血干细胞与肝癌细胞的抑制率。同时药物作用后的肝癌细胞接种至裸鼠，6周后测量移植瘤重量观察其成瘤实验的差异，对模型大鼠造血干细胞、肿瘤细胞抑制率与化疗药物作用后肿瘤细胞移植裸鼠后的成瘤重量分别进行相关性分析。另取原代培养肿瘤细胞与间充质干细胞，同时与HepG2细胞系细胞，SRB法进行药物敏感实验，观察药物敏感性的差异。
结果：(1)表阿霉素、5-氟尿嘧啶、健择、环磷酰胺、顺铂五种药物对肝癌大鼠造血干细胞抑制率分别为26.44±3.14、29.72±5.05、25.98±3.34、26.91±2.70、27.21±3.93；对肝癌细胞抑制率分别为：44.43±5.80、56.53±6.50、41.93±7.31、45.53±4.65、54.07±5.02。表阿霉素、5-氟尿嘧啶、健择、环磷酰胺、顺铂五种药物对肝癌大鼠造血干细胞抑制率与对肝癌细胞抑制率比较有显著性差异，t值与P值分别为：t=7.051(P=0.000)；t=9.513(P=0.000)；t=7.239(P=0.000)；t=12.023(P=0.000)与t=10.091(P=0.000)。(2)表阿霉素、5-氟尿嘧啶、健择、环磷酰胺、顺铂五种药物对肝癌细胞抑制率与其作用肝癌细胞后移植瘤重量间进行相关分析，具体相关系数为：表阿霉素r=0.065(P=0.878)；5-氟尿嘧啶，r=0.033(P=0.939)；健择r=-0.539(P=0.168)；环磷酰胺r=0.026(P=0.950)；顺铂r=0.600(P=0.116)，均无相关性。而化疗药物对肝癌大鼠造血干细胞抑制率与对应药物作用后肝癌细胞移植瘤重量进行相关分析，具体相关系数为：表阿霉素r=-0.727(P=0.041)；5-氟尿嘧啶，r=-0.927(P=0.001)；健择r=-0.901(P=0.002)；环磷酰胺r=-0.774(P=0.024)；顺铂r=-0.783(P=0.022)，化疗药物对造血干细胞抑制率与肝癌细胞增殖侵袭能力有负相关性，并且相关有统计学意义。对阿霉素、顺铂、洛拉曲克的敏感度从高到低依次为HepG2细胞，肝癌细胞，间充质细胞。而丝裂霉素药敏显示，3种细胞药物敏感度从高到低依次为HepG2，间充质干细胞，肝癌细胞。
结论：(1)选取的五种临床常用化疗药物中，其作用机制互不相同。实验结果，同一个体的肝癌细胞和造血干细胞对不同的药物敏感性有差异。化疗药物对肝癌大鼠造血干细胞抑制率小于对肝癌细胞抑制率，肿瘤干细胞可能类似造血干细胞的耐药机制。(2)化疗药物对肝癌细胞抑制率不能完全反映肝癌细胞增殖侵袭能力。化疗药物对造血干细胞抑制率相对于肝癌细胞可以较好反映肿瘤细胞增殖侵袭能力。(3)肿瘤细胞药物敏感性与细胞系细胞和间充质细胞存在一定异同。
Background:
Surgery operation like Hepatectomy is the capital method to heal Primary hepatic carcinoma (PHC) in clinical practice. But the patients with PHC are very difficult to be diagnosed in earlier period. Moreover, we discovered that after Hepatectomy there is still a high recurrence rate. Therefore, combined therapies after Hepatectomy play an important role in PHC therapy. However, as a more important way among combined therapies, the effects of chemotherapy on PHC were very little and hard to improve. So, feasible individualization chemotherapy will be useful for the clinical workers.
By the establishment of tumor stem cell theory, the tumor stem cells theory gave us a new viewpoint in solid tumor therapy. From the theory, we were told that tumor stem cells were source of tumorigenesis、infiltration and metaptosis and the cure of the tumor might be impossible if the tumor stem cells were not eradicated thoroughly. So tumor stem cells should be the target in the therapy of solid tumor. And the subgroup of hepatocarcinoma cells characterized by TSC was separated in earlier study in our laboratory. If we can establish a therapy way to aim and targeted kill TSC, the effect of chemotherapy on PHC will be enhanced greatly.
But by now there is no unified sign on TSC in solid tumor including PHC, and it is difficult and costly to separate TSC. Many research support the point that liver stem cell are the ancestral cell of hepatocellular carcinoma and cholangiocarcinoma. Hepatic oval cell (HOC) which comes from bone marrow stem cell is a sort of liver stem cell corresponding TSC in PHC. They have same surface markers like CD34、Thy-1 (CD90)、c-kit(CD117) and same mechanisms of multidrug resistance. Besides, bone marrow stem cell is easy to be separated and identified. In view of above, we will study on the correlation of chemosensitivity between hepatocarcinoma cells and marrow stem cells by SRB way and find out its application for individualization chemotherapy.
Objective: To study the correlation of anticancer drugs sensitivity between hepatocarcinoma cells and marrow hemopoietic stem cell (HSC) in rats; and study whether HSC can be used to take the place of tumor stem cells in PHC for anticancer drug sensitiveness evaluation in vitro. Study the differences in chemosensitivity to 4 anticancer drugs among hepatocarcinoma cells, MSCs (marrow mesenchymal stem cells) and HepG2 cells by SRB assay.
Methods: The primary liver carcinoma was induced by 0.025% diethylnitrosamine in rats. Tumor cells and HSC were separated from 8 hepatocarcinoma rat. The cell surface marker CD34 was tested by flow cytometry. The inhibition ratio of Epirubicin、5-Fliorouracil、Gemcitabine、Cyclophosphamide and Cisplatin to hepatocarcinoma cells and HSC were measured by SRB way after 24 hours. The weight of the tumor in nude mice, which were injected rats' hepatocarcinoma cells treated by anticancer drugs, was measured after 6 weeks. Then the differences of drugsensitivity between hepatocarcinoma cells and HSC and the correlation of the inhibition ratios of anticancer drug to the weight of the tumor in nude mice were analyzed. The inhibition rates of hepatocarcinoma cells, MSCs and HepG2 cells were measured and analyzed by SRB assay.
Results: (1) The inhibition ratio of Epirubicin、5-Fluorouracil、Gemcitabine、Cyclophosphamide and Cisplatin to HSC were 26.44±3.14, 29.72±5.05 and 25.98±3.34, 26.91±2.70, 27.21±3.93, which were different from that of the hepatocarcinoma cells, which were 44.43±5.80(t=7.051, P=0.000), 56.53±6.50(t=9.513, P=0.000), 41.93±7.31(t=7.239, P=0.000), 45.53±4.65(t=12.023, P=0.000) and 54.07±5.02 (t=10.091, P=0.000). (2)No correlation was found between the inhibition ratio of 5 anticancer drugs on hepatocarcinoma cells and the tumor weight of nude mice. The correlation coefficient were: r=0.065 (Epirubicin, P=0.878), r=0.033 (Fluorouracil, P=939), r=-0.539 (Gemcitabine, P=0.168), r=0.026 (Cyclophosphamide, P=0.950) and r=0.600 (Cisplatin, P=0.116). But a significant negative correlation was found between the inhibition ratio of 3 anticancer drugs on MSCs and the tumor weight of nude mice. The correlation coefficient were: r=-0.727 (Epirubicin, P=0.041), r=-0.927 (Fluorouracil, P=0.001), r=-0.901 (Gemcitabine, P=0.002), r=-0.774 (Cyclophosphamide, P=0.024) and r=-0.783 (Cisplatin, P=0.022). The chemosensitivity of hepatocarcinoma cells and HepG2 cells to adriamycin, cisplatin and nolatrexed dihydrochloride were higher than that of MSCs, while the chemosensitivity of HepG2 cells and MSCs to MCC was higher than that of hepatocarcinoma.
Conclusions: (1) We used 5 different mechanisms of action chemotherapeutics which were commonly used in clinic. The result shows the hepatocarcinoma cells from the same rat have different drug sensitivity to HSC. The inhibition ratio of anticancer drugs to HSC was lower than that it to hepatocarcinoma cells. It suggested that the HSC has a distinct chemoresistance mechanism like the tumor stem cells. (2) The inhibition ratio of anticancer drugs on hepatocarcinoma cells could not correctly appraise the invasion potential of hepatocarcinoma cells. However the inhibition ratio of anticancer drugs on HSC can correctly appraise the invasion potential of hepatocarcinoma cells. (3) The MSCs have a distinct chemoresistance mechanism like the tumor stem cells.
以肝切除为代表的外科治疗仍是原发性肝癌的首选治疗方法。但由于原发性肝癌的早期发现存在困难，肝癌切除术后的5年复发率高达60％以上，切除术后的综合治疗在原发性肝癌的治疗中仍然占有重要的地位。化疗是综合治疗的主要手段之一，而各种针对原发性肝癌的临床化疗总体有效率仅为18.2％～47％，并且进展缓慢。提供切实可行的个体化化疗方案成为临床工作者面对原发性肝癌治疗上的迫切需要。
随着近年来肿瘤干细胞(TSC)理论的确立，对于实体肿瘤的治疗有了新的认识：TSC是肿瘤发生、浸润、转移和复发等恶性生物学行为的核心，是肿瘤细胞成瘤能力的决定因素。只要存在肿瘤干细胞，肿瘤就难以治愈。本实验室的前期研究中在大鼠原发性肝癌已经初步鉴定了具备TSC特征(极强的致瘤能力和自我更新并多向分化)的肿瘤细胞亚群。由此如果能够建立一种针对原发性肝癌肿瘤干细胞的个体化化疗方案，靶向杀伤肿瘤干细胞，将有希望在原发性肝癌的化疗进展上取得突破。
目前对于包括肝癌在内的绝大多数实体瘤来说，缺乏鉴定TSC的统一标准，而分离出纯度高的TSC，直接进行药敏实验的技术难度、成本都比较大。较多研究表明，肝干细胞是肝细胞癌和胆管癌的祖细胞。来源于骨髓干细胞的肝卵圆细胞(Hepatic oval cell，HOC)具备肝脏干细胞性质，是肝癌干细胞的对应成体干细胞。卵圆细胞不仅具有同骨髓干细胞相似的表面标志，如CD34、Thy-1(CD90)、c-kit(CD117)，并且在耐药机制上也类似干细胞。骨髓干细胞分为造血干细胞和间充质干细胞，相比较TSC而言易于分离鉴定，药敏实验的可行性较高。基于上述理由，本课题利用肿瘤干细胞与骨髓干细胞的关系，重点研究骨髓干细胞在针对肝癌干细胞个体化化疗的药物敏感实验中的应用。
目的：研究大鼠骨髓造血干细胞与大鼠原发性肝癌细胞药敏与肝癌细胞成瘤能力的相关性；探讨利用造血干细胞，进行针对肝癌干细胞药物敏感实验的可能性。并研究了药物对骨髓间充质干细胞、肝癌细胞和HepG2细胞的药敏差异。
方法：30只雄性清洁级SD大鼠应用0.025％二乙基亚硝胺水溶液饲喂24周建立大鼠原发性肝癌模型，选取诱癌成功大鼠8只，用组织块法分离培养原代肝癌细胞；用免疫磁珠法分离骨髓造血干细胞，流式细胞仪检测传代细胞表面标志CD34。筛选五种不同作用机制抗肿瘤药物表阿霉素、5-氟尿嘧啶(5-FU)、健择、环磷酰胺、顺铂作用24小时后，SRB法分别检测化疗药物对造血干细胞与肝癌细胞的抑制率。同时药物作用后的肝癌细胞接种至裸鼠，6周后测量移植瘤重量观察其成瘤实验的差异，对模型大鼠造血干细胞、肿瘤细胞抑制率与化疗药物作用后肿瘤细胞移植裸鼠后的成瘤重量分别进行相关性分析。另取原代培养肿瘤细胞与间充质干细胞，同时与HepG2细胞系细胞，SRB法进行药物敏感实验，观察药物敏感性的差异。
结果：(1)表阿霉素、5-氟尿嘧啶、健择、环磷酰胺、顺铂五种药物对肝癌大鼠造血干细胞抑制率分别为26.44±3.14、29.72±5.05、25.98±3.34、26.91±2.70、27.21±3.93；对肝癌细胞抑制率分别为：44.43±5.80、56.53±6.50、41.93±7.31、45.53±4.65、54.07±5.02。表阿霉素、5-氟尿嘧啶、健择、环磷酰胺、顺铂五种药物对肝癌大鼠造血干细胞抑制率与对肝癌细胞抑制率比较有显著性差异，t值与P值分别为：t=7.051(P=0.000)；t=9.513(P=0.000)；t=7.239(P=0.000)；t=12.023(P=0.000)与t=10.091(P=0.000)。(2)表阿霉素、5-氟尿嘧啶、健择、环磷酰胺、顺铂五种药物对肝癌细胞抑制率与其作用肝癌细胞后移植瘤重量间进行相关分析，具体相关系数为：表阿霉素r=0.065(P=0.878)；5-氟尿嘧啶，r=0.033(P=0.939)；健择r=-0.539(P=0.168)；环磷酰胺r=0.026(P=0.950)；顺铂r=0.600(P=0.116)，均无相关性。而化疗药物对肝癌大鼠造血干细胞抑制率与对应药物作用后肝癌细胞移植瘤重量进行相关分析，具体相关系数为：表阿霉素r=-0.727(P=0.041)；5-氟尿嘧啶，r=-0.927(P=0.001)；健择r=-0.901(P=0.002)；环磷酰胺r=-0.774(P=0.024)；顺铂r=-0.783(P=0.022)，化疗药物对造血干细胞抑制率与肝癌细胞增殖侵袭能力有负相关性，并且相关有统计学意义。对阿霉素、顺铂、洛拉曲克的敏感度从高到低依次为HepG2细胞，肝癌细胞，间充质细胞。而丝裂霉素药敏显示，3种细胞药物敏感度从高到低依次为HepG2，间充质干细胞，肝癌细胞。
结论：(1)选取的五种临床常用化疗药物中，其作用机制互不相同。实验结果，同一个体的肝癌细胞和造血干细胞对不同的药物敏感性有差异。化疗药物对肝癌大鼠造血干细胞抑制率小于对肝癌细胞抑制率，肿瘤干细胞可能类似造血干细胞的耐药机制。(2)化疗药物对肝癌细胞抑制率不能完全反映肝癌细胞增殖侵袭能力。化疗药物对造血干细胞抑制率相对于肝癌细胞可以较好反映肿瘤细胞增殖侵袭能力。(3)肿瘤细胞药物敏感性与细胞系细胞和间充质细胞存在一定异同。
Background:
Surgery operation like Hepatectomy is the capital method to heal Primary hepatic carcinoma (PHC) in clinical practice. But the patients with PHC are very difficult to be diagnosed in earlier period. Moreover, we discovered that after Hepatectomy there is still a high recurrence rate. Therefore, combined therapies after Hepatectomy play an important role in PHC therapy. However, as a more important way among combined therapies, the effects of chemotherapy on PHC were very little and hard to improve. So, feasible individualization chemotherapy will be useful for the clinical workers.
By the establishment of tumor stem cell theory, the tumor stem cells theory gave us a new viewpoint in solid tumor therapy. From the theory, we were told that tumor stem cells were source of tumorigenesis、infiltration and metaptosis and the cure of the tumor might be impossible if the tumor stem cells were not eradicated thoroughly. So tumor stem cells should be the target in the therapy of solid tumor. And the subgroup of hepatocarcinoma cells characterized by TSC was separated in earlier study in our laboratory. If we can establish a therapy way to aim and targeted kill TSC, the effect of chemotherapy on PHC will be enhanced greatly.
But by now there is no unified sign on TSC in solid tumor including PHC, and it is difficult and costly to separate TSC. Many research support the point that liver stem cell are the ancestral cell of hepatocellular carcinoma and cholangiocarcinoma. Hepatic oval cell (HOC) which comes from bone marrow stem cell is a sort of liver stem cell corresponding TSC in PHC. They have same surface markers like CD34、Thy-1 (CD90)、c-kit(CD117) and same mechanisms of multidrug resistance. Besides, bone marrow stem cell is easy to be separated and identified. In view of above, we will study on the correlation of chemosensitivity between hepatocarcinoma cells and marrow stem cells by SRB way and find out its application for individualization chemotherapy.
Objective: To study the correlation of anticancer drugs sensitivity between hepatocarcinoma cells and marrow hemopoietic stem cell (HSC) in rats; and study whether HSC can be used to take the place of tumor stem cells in PHC for anticancer drug sensitiveness evaluation in vitro. Study the differences in chemosensitivity to 4 anticancer drugs among hepatocarcinoma cells, MSCs (marrow mesenchymal stem cells) and HepG2 cells by SRB assay.
Methods: The primary liver carcinoma was induced by 0.025% diethylnitrosamine in rats. Tumor cells and HSC were separated from 8 hepatocarcinoma rat. The cell surface marker CD34 was tested by flow cytometry. The inhibition ratio of Epirubicin、5-Fliorouracil、Gemcitabine、Cyclophosphamide and Cisplatin to hepatocarcinoma cells and HSC were measured by SRB way after 24 hours. The weight of the tumor in nude mice, which were injected rats' hepatocarcinoma cells treated by anticancer drugs, was measured after 6 weeks. Then the differences of drugsensitivity between hepatocarcinoma cells and HSC and the correlation of the inhibition ratios of anticancer drug to the weight of the tumor in nude mice were analyzed. The inhibition rates of hepatocarcinoma cells, MSCs and HepG2 cells were measured and analyzed by SRB assay.
Results: (1) The inhibition ratio of Epirubicin、5-Fluorouracil、Gemcitabine、Cyclophosphamide and Cisplatin to HSC were 26.44±3.14, 29.72±5.05 and 25.98±3.34, 26.91±2.70, 27.21±3.93, which were different from that of the hepatocarcinoma cells, which were 44.43±5.80(t=7.051, P=0.000), 56.53±6.50(t=9.513, P=0.000), 41.93±7.31(t=7.239, P=0.000), 45.53±4.65(t=12.023, P=0.000) and 54.07±5.02 (t=10.091, P=0.000). (2)No correlation was found between the inhibition ratio of 5 anticancer drugs on hepatocarcinoma cells and the tumor weight of nude mice. The correlation coefficient were: r=0.065 (Epirubicin, P=0.878), r=0.033 (Fluorouracil, P=939), r=-0.539 (Gemcitabine, P=0.168), r=0.026 (Cyclophosphamide, P=0.950) and r=0.600 (Cisplatin, P=0.116). But a significant negative correlation was found between the inhibition ratio of 3 anticancer drugs on MSCs and the tumor weight of nude mice. The correlation coefficient were: r=-0.727 (Epirubicin, P=0.041), r=-0.927 (Fluorouracil, P=0.001), r=-0.901 (Gemcitabine, P=0.002), r=-0.774 (Cyclophosphamide, P=0.024) and r=-0.783 (Cisplatin, P=0.022). The chemosensitivity of hepatocarcinoma cells and HepG2 cells to adriamycin, cisplatin and nolatrexed dihydrochloride were higher than that of MSCs, while the chemosensitivity of HepG2 cells and MSCs to MCC was higher than that of hepatocarcinoma.
Conclusions: (1) We used 5 different mechanisms of action chemotherapeutics which were commonly used in clinic. The result shows the hepatocarcinoma cells from the same rat have different drug sensitivity to HSC. The inhibition ratio of anticancer drugs to HSC was lower than that it to hepatocarcinoma cells. It suggested that the HSC has a distinct chemoresistance mechanism like the tumor stem cells. (2) The inhibition ratio of anticancer drugs on hepatocarcinoma cells could not correctly appraise the invasion potential of hepatocarcinoma cells. However the inhibition ratio of anticancer drugs on HSC can correctly appraise the invasion potential of hepatocarcinoma cells. (3) The MSCs have a distinct chemoresistance mechanism like the tumor stem cells.
引文
[1] 张思维，李连第，鲁风珠，等．中国1990-1992年原发性肝癌死亡调查分析[J]．中华肿瘤杂志，1999，21：254．
[2] Fujiyama S ,Shibata J ,Maeda S ,et all .Phase Ⅰ clinical study of a novel lipophilic platinum complex (SM-11355) in patients with hepatocellular carcinoma refractory to cisplatin/lipiodol [J]. Br J Cancer, 2003, 89(9):1614-1619.
[3] Taieb J, Bonyhay L, Golli L, et all Gemcitabine plus oxaliplatin for patients with advanced hepatocellular carcinoma using two different schedules [J]. Cancer, 2003, 98 (12):2664-2670.
[4] Seki S, Yamada T, Kawkakita N, et al1. A new chemotherapeutic regimen for advanced unresectable hepatocellular carcinoma [J]. Hepato gastroenterology, 2003, 50(53):1598-1602.
[5] Tanioka H, Tsuji A, Morita S, et al1.Combination chemotherapy with continuous 5-fluorouracil and low dose cisplatin infusion for advanced hepatocellular carcinoma [J]. Anticancer Res ,2003,23 (2C):1891-1897.
[6] Pathak S, Multani AS. Aneuploidy, stem cells and cancer[J]. EXS. 2006;(96):49-64.
[7] Zhang M, Rosen JM. Stem cells in the etiology and treatment of cancer[J]. Curr Opin Genet Dev. 2006, 16(1):60-64.
[8] Al Hajj M, Wicha M S, Benito-Hernandez, A, et al. Prospective identification of tumorigenic breast cancer cells [J]. Proc. Natl. Acad. Sci. USA. 2003,100(7): 3983-3988.
[9] Nakano J, Hernmati H D, Komblum H J. Cancer stem cells in pediatric brain tumors[J]. No Shinkei Geka, 2004, 32(8):827-834.
[10] Huntly B J, Gilliland D G. Leukaemia stem cells and the evolution of cancer-stem-cell research[J]. Nat Rev Cancer, 2005,5(4):311-321.
[11] Singh SK, Clarke ID, Terasaki M,et al.Identification of a cancer stem cell in human brain tumors[J]. Cancer Research .2003, 63(18):5821-5828.
[12] Houman D, Hemmati I,chiro Nakano et al. Cancerous stem cells can arise from pediatric brain tumors. Proc. Natl. Acad. Sci. USA..2003, 100(25): 15178-15183.
[12] Taipale J,Beachy PA. The Hedgehog and Wnt signalling pathways in cancer [J].Nature, 2001, 411(6835):349-354.
[13] Guzman M L, Swiderski C F, Howard D S,et al.Preferential induction of apoptosis for primary human leukemic stem cells[J]. Proc. Natl. Acad. Sci. USA.2002, 99(25):16220-16225.
[14] Blair, A., Hogge, D. E., Ailles, L. E.,et al. Lack of expression of Thy-1 (CD90) on acute myeloid leukemia cells with long-term proliferative ability in vitro and in vivo[J].Blood.1997,89(9):3104-3112.
[15] Bonnet, D, Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell [J]. Nat Med. 1997, 3(7): 730-737.
[16] T Megha, F Ferrari, A Benvenuto,et al. p53 mutation in breast cancar, correlation with cell kinetics and cell of origin[J]. J Clin Pathol, 2002,55:461-466.
[17]] Zheng YW, Taniguchi H. Diversity of hepatic stem cells in the fetaland adult liver. Semin Liver Dis, 2003,23(4): 337—348.
[18] Paku S, Schnur J, Nagy P, et al. Origin and structural evolution ofthe early proliferating oval cells in rat liver. Am J Pathol, 2001,158 (4): 1313—1323.
[19] Marchand H, Morosan S, Charneau P, et al. Bipotentialmouse embryonic liver stem cell lines contribute to liver regenerationand differentiate as bile ducts and hepatocytes. Proc Natl Acad Sci USA, 2004,101 (22): 8360—8365.
[20] 龚加庆，方驰华，李雅等．卵圆细胞参与实验性肝癌形成过程的研究．中华外科杂志．2004：42：291-295．
[21] Germain L, Noel M, Gourdeau H,Marceau N. Promotion of growth and differentiation of rat ductular oval cells in primary culture. Cancer Research. 1998;48:368-378.
[22] Lowes KN,Brennan BA, Yeoh GC,et al.Oval cell numbers in human chronic liver disease are directly related to disease severity[J].Am J Pathol,1999,154(2):537.
[23] 邱德凯，马雄，彭延中，等．慢性肝病患者肝脏卵圆细胞的定位和定量研究—29例慢性肝病患者肝组织病理学分析[J]．中华消化杂志，2000，20(5)：301．
[24] Kang XQ, Zang WJ, Bao LJ, et al. Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes. World J Gastroenterol. 2005, 11(47): 7461-7465.
[25] Petersen BE ,Bowen WC, Patrene KD, et al1Bone marrow as a potentialsource of hepatic oval cells [J]. Scienc, 1999, 284 (5 417):1 168-1 170.
[26] Lagasse E, Connors H, Dhalimy M, et al. Purified hematopoi2etic stem cells can differentiate into hepatocytes in vivo. Nat Med, 2000, 6(11): 1229-1234.
[27] Oh SH ,Miyazaki M,Kouchi H ,et allHepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro[J ] .Biochem Biophys Res Commun ,2000,279(2) :500-504.
[28] Miyazaki M,Akiyama I ,Sakaguchi M,et all. Improved conditions to induce hepatocytes from rat bone marrow cells in culture [J]. Biochem BiophysRes Commun, 2002, 298 (1):24-30.
[29] Miyazaki M, Kataoka K, Medina R, et al1Differentiation of bone marrowcells in culture and in vivo [J]. International Congress Series, 2003, 1252:461-464.
[30] 陈涛，褚忠华，刘建平，等．体外化疗药物敏感实验对指导原发性肝癌个体化疗的临床意义[J]．癌症，2005，24(8)：1018-1022．
[31] 施新猷,李六金,顾为望,等．现代医学实验动物学[M]．人民军医出版社．2000：436．
[1] 周思朗，李鹏，曹漫明，等．大鼠肝癌干细胞生物学行为研究[J]．中华肝脏疾病杂志，2006，14(5)：364-366．
[2] 陈涛，褚忠华，刘建平，等．体外化疗药物敏感实验对指导原发性肝癌个体化疗的临床意义[J]．癌症，2005，24(8)：1018-1022．
[3] Liu S, Peng Z, Low J, et al. An evaluation of adenosine triphosphate bioluminescence assay for tumor in Vitro) chernosensitivity testing. Huaxi Yike Daxue Xuebao,2002; 31(3):330-333.
[4] Lwadate Y, Fujimoto S, Yamauia A. Differential—chemosensitivity in human intracerebral gliomas measured by flow cytometric DNA and analysis[J].Int J Mol Med, 2002 ;10(2):187-192.
[5] Behbod F, Rosen JM.Will. Cancer stem cells provide new therapeutic targets? [J].Carcinogenesis. 2005, 26(4):703-711.
[6] Behbod F, Rosen JM.Will cancer stem cells provide new therapeutic targets? [J].Carcinogenesis. 2005, 26(4):703-711.
[7] Nakano I, Hemmati HD, Kornblum HI.Cancer stem cells in pediatric brain tumors [J]. No Shinkei Geka. 2004, 32(8):827-834.
[8] Knutsen T ,Rao VK, Ried T, et al. Amplification of 4q21-q22and the MXR gene in independently derived mitoxantrone-resistant cell lines [J]. Genes Chromosomes Cancer, 2000,27(1):110-116.
[9] Filip S, Mokry J, English D.Stem cell plasticity and carcinogenesis. Neoplasma. 2006; 53(2):87-91.
[2] Fujiyama S ,Shibata J ,Maeda S ,et all .Phase Ⅰ clinical study of a novel lipophilic platinum complex (SM-11355) in patients with hepatocellular carcinoma refractory to cisplatin/lipiodol [J]. Br J Cancer, 2003, 89(9):1614-1619.
[3] Taieb J, Bonyhay L, Golli L, et all Gemcitabine plus oxaliplatin for patients with advanced hepatocellular carcinoma using two different schedules [J]. Cancer, 2003, 98 (12):2664-2670.
[4] Seki S, Yamada T, Kawkakita N, et al1. A new chemotherapeutic regimen for advanced unresectable hepatocellular carcinoma [J]. Hepato gastroenterology, 2003, 50(53):1598-1602.
[5] Tanioka H, Tsuji A, Morita S, et al1.Combination chemotherapy with continuous 5-fluorouracil and low dose cisplatin infusion for advanced hepatocellular carcinoma [J]. Anticancer Res ,2003,23 (2C):1891-1897.
[6] Pathak S, Multani AS. Aneuploidy, stem cells and cancer[J]. EXS. 2006;(96):49-64.
[7] Zhang M, Rosen JM. Stem cells in the etiology and treatment of cancer[J]. Curr Opin Genet Dev. 2006, 16(1):60-64.
[8] Al Hajj M, Wicha M S, Benito-Hernandez, A, et al. Prospective identification of tumorigenic breast cancer cells [J]. Proc. Natl. Acad. Sci. USA. 2003,100(7): 3983-3988.
[9] Nakano J, Hernmati H D, Komblum H J. Cancer stem cells in pediatric brain tumors[J]. No Shinkei Geka, 2004, 32(8):827-834.
[10] Huntly B J, Gilliland D G. Leukaemia stem cells and the evolution of cancer-stem-cell research[J]. Nat Rev Cancer, 2005,5(4):311-321.
[11] Singh SK, Clarke ID, Terasaki M,et al.Identification of a cancer stem cell in human brain tumors[J]. Cancer Research .2003, 63(18):5821-5828.
[12] Houman D, Hemmati I,chiro Nakano et al. Cancerous stem cells can arise from pediatric brain tumors. Proc. Natl. Acad. Sci. USA..2003, 100(25): 15178-15183.
[12] Taipale J,Beachy PA. The Hedgehog and Wnt signalling pathways in cancer [J].Nature, 2001, 411(6835):349-354.
[13] Guzman M L, Swiderski C F, Howard D S,et al.Preferential induction of apoptosis for primary human leukemic stem cells[J]. Proc. Natl. Acad. Sci. USA.2002, 99(25):16220-16225.
[14] Blair, A., Hogge, D. E., Ailles, L. E.,et al. Lack of expression of Thy-1 (CD90) on acute myeloid leukemia cells with long-term proliferative ability in vitro and in vivo[J].Blood.1997,89(9):3104-3112.
[15] Bonnet, D, Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell [J]. Nat Med. 1997, 3(7): 730-737.
[16] T Megha, F Ferrari, A Benvenuto,et al. p53 mutation in breast cancar, correlation with cell kinetics and cell of origin[J]. J Clin Pathol, 2002,55:461-466.
[17]] Zheng YW, Taniguchi H. Diversity of hepatic stem cells in the fetaland adult liver. Semin Liver Dis, 2003,23(4): 337—348.
[18] Paku S, Schnur J, Nagy P, et al. Origin and structural evolution ofthe early proliferating oval cells in rat liver. Am J Pathol, 2001,158 (4): 1313—1323.
[19] Marchand H, Morosan S, Charneau P, et al. Bipotentialmouse embryonic liver stem cell lines contribute to liver regenerationand differentiate as bile ducts and hepatocytes. Proc Natl Acad Sci USA, 2004,101 (22): 8360—8365.
[20] 龚加庆，方驰华，李雅等．卵圆细胞参与实验性肝癌形成过程的研究．中华外科杂志．2004：42：291-295．
[21] Germain L, Noel M, Gourdeau H,Marceau N. Promotion of growth and differentiation of rat ductular oval cells in primary culture. Cancer Research. 1998;48:368-378.
[22] Lowes KN,Brennan BA, Yeoh GC,et al.Oval cell numbers in human chronic liver disease are directly related to disease severity[J].Am J Pathol,1999,154(2):537.
[23] 邱德凯，马雄，彭延中，等．慢性肝病患者肝脏卵圆细胞的定位和定量研究—29例慢性肝病患者肝组织病理学分析[J]．中华消化杂志，2000，20(5)：301．
[24] Kang XQ, Zang WJ, Bao LJ, et al. Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes. World J Gastroenterol. 2005, 11(47): 7461-7465.
[25] Petersen BE ,Bowen WC, Patrene KD, et al1Bone marrow as a potentialsource of hepatic oval cells [J]. Scienc, 1999, 284 (5 417):1 168-1 170.
[26] Lagasse E, Connors H, Dhalimy M, et al. Purified hematopoi2etic stem cells can differentiate into hepatocytes in vivo. Nat Med, 2000, 6(11): 1229-1234.
[27] Oh SH ,Miyazaki M,Kouchi H ,et allHepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro[J ] .Biochem Biophys Res Commun ,2000,279(2) :500-504.
[28] Miyazaki M,Akiyama I ,Sakaguchi M,et all. Improved conditions to induce hepatocytes from rat bone marrow cells in culture [J]. Biochem BiophysRes Commun, 2002, 298 (1):24-30.
[29] Miyazaki M, Kataoka K, Medina R, et al1Differentiation of bone marrowcells in culture and in vivo [J]. International Congress Series, 2003, 1252:461-464.
[30] 陈涛，褚忠华，刘建平，等．体外化疗药物敏感实验对指导原发性肝癌个体化疗的临床意义[J]．癌症，2005，24(8)：1018-1022．
[31] 施新猷,李六金,顾为望,等．现代医学实验动物学[M]．人民军医出版社．2000：436．
[1] 周思朗，李鹏，曹漫明，等．大鼠肝癌干细胞生物学行为研究[J]．中华肝脏疾病杂志，2006，14(5)：364-366．
[2] 陈涛，褚忠华，刘建平，等．体外化疗药物敏感实验对指导原发性肝癌个体化疗的临床意义[J]．癌症，2005，24(8)：1018-1022．
[3] Liu S, Peng Z, Low J, et al. An evaluation of adenosine triphosphate bioluminescence assay for tumor in Vitro) chernosensitivity testing. Huaxi Yike Daxue Xuebao,2002; 31(3):330-333.
[4] Lwadate Y, Fujimoto S, Yamauia A. Differential—chemosensitivity in human intracerebral gliomas measured by flow cytometric DNA and analysis[J].Int J Mol Med, 2002 ;10(2):187-192.
[5] Behbod F, Rosen JM.Will. Cancer stem cells provide new therapeutic targets? [J].Carcinogenesis. 2005, 26(4):703-711.
[6] Behbod F, Rosen JM.Will cancer stem cells provide new therapeutic targets? [J].Carcinogenesis. 2005, 26(4):703-711.
[7] Nakano I, Hemmati HD, Kornblum HI.Cancer stem cells in pediatric brain tumors [J]. No Shinkei Geka. 2004, 32(8):827-834.
[8] Knutsen T ,Rao VK, Ried T, et al. Amplification of 4q21-q22and the MXR gene in independently derived mitoxantrone-resistant cell lines [J]. Genes Chromosomes Cancer, 2000,27(1):110-116.
[9] Filip S, Mokry J, English D.Stem cell plasticity and carcinogenesis. Neoplasma. 2006; 53(2):87-91.