人视网膜母细胞瘤肿瘤干细胞的分离鉴定及相关表面标志的初步研究
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摘要
视网膜母细胞瘤(retinoblastoma, RB)是婴幼儿最常见的眼内恶性肿瘤,目前国际上针对RB开展了以化疗为主结合局部治疗的综合疗法,极大改善了患儿的生命预后,然而化疗存在最大的问题就是肿瘤的复发和转移。肿瘤化疗失败后复发的主要原因是肿瘤细胞对化疗药物产生耐药性,而耐药性的产生机制相当复杂,主要包括多耐药性蛋白的功能表达、静息期细胞对化疗不敏感以及肿瘤细胞凋亡耐受等,虽然在这些方面的研取得了一定的进展,但在阐释耐药性机制及指导临床应用上并未取得根本性突破。
肿瘤干细胞(Cancer stem cells, CSCs)是一类极少的具有自我更新、不定潜能性并促使肿瘤形成的细胞,对肿瘤的生长、形成及转移起到关键作用’,更为重要的是这部分肿瘤起始性细胞对化疗药物的敏感性较其它非致瘤性肿瘤细胞明显降低。目前,临床上化疗很大程度上是针对大的非致瘤性肿瘤细胞组成的肿瘤块,而不能有效杀灭或抑制肿瘤干细胞,导致了化疗后肿瘤的复发和转移。虽然不是所有恶性肿瘤的生物学特点都符合肿瘤干细胞理论,但CSCs假说为理解RB化疗耐药性提出一种更为合理的细胞水平的理论解释。通过研究肿瘤干细胞,将有利于进一步理解RB发生、发展及转移的机制,可能为减少RB化疗后复发和转移提供新的治疗策略。目前在多种实体瘤中均已证实有肿瘤干细胞存在,包括乳腺癌、脑肿瘤、胰腺癌、结肠癌、黑色素瘤、前列腺癌和卵巢癌等。然而和其他肿瘤研究相比,作为眼内肿瘤的RB由于新鲜标本获取的困难性以及样本的变异性使得其肿瘤干细胞的研究相对滞后。
本实验通过对人RB肿瘤细胞体外采用无血清限定培养基长期培养,分离得到了以肿瘤球方式生长的细胞系,并可以在连续传代的同时保持了其自我更新、增殖及分化能力,同时通过化疗耐药性试验及小鼠致瘤试验评价其对化疗药物的反应性及异体致瘤能力。同时文章第三部分还测定了表面标志物ABCG2(ATP-binding cassette superfamily G member 2, ATP结合膜转运蛋白2)、CD133和CD44在该群细胞中的表达情况,为进一步研究视网膜母细胞瘤肿瘤干细胞(retinoblastoma cancer stem-like cell, RCSC)及其鉴定所需的表面标志奠定了基础。
Retinoblastoma (RB) is the most common malignant tumor of the retina in children. Chemoreduction using systemic chemotherapy combined with local therapy (photoablation, cryotherapy or thermotherapy) has become a mainstay of therapy for retinoblastoma, which has improved prognosis and preserved the eye of patients. Nonetheless, chemotherapeutic drug resistance is common in retinoblastoma, resulting in the increased incidences of unsuccessful treatments. Previous studies showed that P-glycoprotein and multidrug resistance proteins (MRP) may contribute to drug resistance. However, the mechanism for resistance and recurrence is still not clear and improved targeted therapies are essential for alleviating this devastating malignancy.
In past years, one emerging hypothesis postulates that the development of drug-resistant tumors is sustained by a self-renewing subpopulation termed as putative cancer stem cells (CSCs). Current therapies target rapidly dividing cells that comprise the bulk of the tumor while failing to eradicate the CSCs which subsequently re-initiate the entire malignancy. It is likely that these residual CSCs are able to survive in a dormant state for many years after remission due to their marked resistant ability. Although not all types of cancers follow the CSC theory, it provides a possible cellular mechanism to account for the metastasis and chemoresistance of RB. CSCs have been identified in many solid tumors including brain, breast, pancreas, prostate, melanoma, colon and ovarian cancers. However, compared to other solid tumors, cancer stem cell research is impeded in this intraocular cancer because of the shortage of fresh samples and difficulty of isolation from varying RB lesions, a mixture of tumor cells with necrosis and calcification.
Therefore, a proper in vitro model is required to study retinoblastoma cancer stem-like cells and to design future therapeutic approaches. Here, we report that a long-term culture of sphere-forming cells from human retinoblastoma was established; these cells maintain their cancer stem-like cell properties including the ability of self-renewal, proliferation, differentiation, tumorigenicity and chemoresistance. Moreover, the expression of cell surface markers, such as ABCG2 (ATP-binding cassette superfamily G member 2), CD 133, CD44, has been studied on these cultured cells. Further research on surface marker for identifying retinoblastoma cancer stem-like cells (RCSC) may be carried out on these results.
肿瘤干细胞(Cancer stem cells, CSCs)是一类极少的具有自我更新、不定潜能性并促使肿瘤形成的细胞,对肿瘤的生长、形成及转移起到关键作用’,更为重要的是这部分肿瘤起始性细胞对化疗药物的敏感性较其它非致瘤性肿瘤细胞明显降低。目前,临床上化疗很大程度上是针对大的非致瘤性肿瘤细胞组成的肿瘤块,而不能有效杀灭或抑制肿瘤干细胞,导致了化疗后肿瘤的复发和转移。虽然不是所有恶性肿瘤的生物学特点都符合肿瘤干细胞理论,但CSCs假说为理解RB化疗耐药性提出一种更为合理的细胞水平的理论解释。通过研究肿瘤干细胞,将有利于进一步理解RB发生、发展及转移的机制,可能为减少RB化疗后复发和转移提供新的治疗策略。目前在多种实体瘤中均已证实有肿瘤干细胞存在,包括乳腺癌、脑肿瘤、胰腺癌、结肠癌、黑色素瘤、前列腺癌和卵巢癌等。然而和其他肿瘤研究相比,作为眼内肿瘤的RB由于新鲜标本获取的困难性以及样本的变异性使得其肿瘤干细胞的研究相对滞后。
本实验通过对人RB肿瘤细胞体外采用无血清限定培养基长期培养,分离得到了以肿瘤球方式生长的细胞系,并可以在连续传代的同时保持了其自我更新、增殖及分化能力,同时通过化疗耐药性试验及小鼠致瘤试验评价其对化疗药物的反应性及异体致瘤能力。同时文章第三部分还测定了表面标志物ABCG2(ATP-binding cassette superfamily G member 2, ATP结合膜转运蛋白2)、CD133和CD44在该群细胞中的表达情况,为进一步研究视网膜母细胞瘤肿瘤干细胞(retinoblastoma cancer stem-like cell, RCSC)及其鉴定所需的表面标志奠定了基础。
Retinoblastoma (RB) is the most common malignant tumor of the retina in children. Chemoreduction using systemic chemotherapy combined with local therapy (photoablation, cryotherapy or thermotherapy) has become a mainstay of therapy for retinoblastoma, which has improved prognosis and preserved the eye of patients. Nonetheless, chemotherapeutic drug resistance is common in retinoblastoma, resulting in the increased incidences of unsuccessful treatments. Previous studies showed that P-glycoprotein and multidrug resistance proteins (MRP) may contribute to drug resistance. However, the mechanism for resistance and recurrence is still not clear and improved targeted therapies are essential for alleviating this devastating malignancy.
In past years, one emerging hypothesis postulates that the development of drug-resistant tumors is sustained by a self-renewing subpopulation termed as putative cancer stem cells (CSCs). Current therapies target rapidly dividing cells that comprise the bulk of the tumor while failing to eradicate the CSCs which subsequently re-initiate the entire malignancy. It is likely that these residual CSCs are able to survive in a dormant state for many years after remission due to their marked resistant ability. Although not all types of cancers follow the CSC theory, it provides a possible cellular mechanism to account for the metastasis and chemoresistance of RB. CSCs have been identified in many solid tumors including brain, breast, pancreas, prostate, melanoma, colon and ovarian cancers. However, compared to other solid tumors, cancer stem cell research is impeded in this intraocular cancer because of the shortage of fresh samples and difficulty of isolation from varying RB lesions, a mixture of tumor cells with necrosis and calcification.
Therefore, a proper in vitro model is required to study retinoblastoma cancer stem-like cells and to design future therapeutic approaches. Here, we report that a long-term culture of sphere-forming cells from human retinoblastoma was established; these cells maintain their cancer stem-like cell properties including the ability of self-renewal, proliferation, differentiation, tumorigenicity and chemoresistance. Moreover, the expression of cell surface markers, such as ABCG2 (ATP-binding cassette superfamily G member 2), CD 133, CD44, has been studied on these cultured cells. Further research on surface marker for identifying retinoblastoma cancer stem-like cells (RCSC) may be carried out on these results.
引文
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