不同周期新辅助化疗后乳腺癌干细胞的分离培养和体外增殖特性

doi:10.3969/j.issn.2095-4344.2015.36.012

摘要/Abstract

摘要：

背景：接受不同周期新辅助化疗的乳腺癌组织中是否都存在乳腺癌化疗微球体以及其对乳腺癌干细胞分离培养的影响尚不清楚。

目的：探讨不同周期新辅助化疗后乳腺癌组织乳腺癌干细胞的体外增殖特性。

方法：从不同周期新辅助化疗后的乳腺癌组织中分离微球体，绘制细胞生长曲线，免疫细胞化学检测乳腺癌干细胞标记物ALDH1表达情况。

结果与结论：从新辅助化疗1个周期的标本中无法获得微球体，化疗2，3，4个周期的部分标本中可以培养出微球体。在培养的前3 d，化疗3个周期和化疗2个周期细胞数差异无显著性意义，但在 3 d 之后，化疗3个周期的细胞增殖速度显著快于化疗2个周期，6 d 之后，化疗2个周期的细胞生长曲线呈现出平缓的状态，化疗3个周期则呈现出迅速上升的状态。化疗 3 个周期标本的乳腺癌干细胞微球体中ALDH1阳性细胞比例大于化疗 2 个周期。结果表明化疗2、3个周期的乳腺癌干细胞均具有一定的增殖和分化能力，从化疗后的乳腺癌组织中分离乳腺癌干细胞宜选取化疗3个周期及以上的标本。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 干细胞, 肿瘤干细胞, 乳腺癌, 新辅助化疗, 乳腺癌干细胞, 细胞分离, 细胞培养, 增殖

Abstract:

BACKGROUND: Whether there are breast cancer stem cell microspheres in the breast cancer tissues and whether these microspheres have an impact on isolation and culture of breast cancer stem cells after different cycles of neoadjuvant chemotherapy are still unclear.

OBJECTIVE: To explore the proliferation and differentiation of breast cancer stem cells in breast cancer tissues after different cycles of neoadjuvant chemotherapy.

METHODS: Breast cancer stem cell microspheres were isolated from the breast cancer tissues after different cycles of neoadjuvant chemotherapy to drawn a cell growth curve. Immunocytochemical method was used to detect ALDH1 expression.

RESULTS AND CONCLUSION: Microspheres could be obtained from the specimens of neoadjuvant chemotherapy for two, three and four cycles rather than one cycle. At 3 days prior to culture, there was no difference in the number of cells isolated after two- and three-cycle neoadjuvant chemotherapy; but after 3 days, the cells from the three-cycle neoadjuvant chemotherapy proliferated faster than those from the two-cycle neoadjuvant chemotherapy; after 6 days, the cell growth curve of two-cycle neoadjuvant chemotherapy was in the plateau stage, and the proliferation of cells from the three-cycle neoadjuvant chemotherapy showed a rapid increase trend. The positive expression of ALDH1 in the microspheres from the three-cycle neoadjuvant chemotherapy was higher than that from the two-cycle neoadjuvant chemotherapy. These findings indicate that breast cancer stem cells from the specimens of two- and three-cycle neoadjuvant chemotherapy both have proliferation and differentiation potentials, and the specimens of three-cycle neoadjuvant chemotherapy or above are preferred.

Key words: Breast Neoplasms, Antineoplastic Combined Chemotherapy Protocols, Cell Proliferation

中图分类号:

R394.2

刘伟，魏冬冬，韩立杰. 不同周期新辅助化疗后乳腺癌干细胞的分离培养和体外增殖特性[J]. 中国组织工程研究, 2015, 19(36): 5806-5810.

Liu Wei, Wei Dong-dong, Han Li-jie. Isolation, culture and in vitro proliferation of breast cancer stem cells after different cycles of neoadjuvant chemotherapy [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(36): 5806-5810.

图/表（结果） 2

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引言

材料方法

讨论

目前，临床对乳腺癌患者进行治疗过程中，新辅助化疗是一种常用的方法^[27-30]。本实验对化疗1个周期之后的乳腺癌标本进行体外分离和培养，未得到微球体，分析出现这一结果的原因可能是在新辅助化疗过程中，化疗药物可以对相应的乳腺癌细胞进行有效的杀伤^[31]，同时也会对微球体造成一定的损伤^[32]，这一阶段进行微球体分离和培养，相当于对细胞予以二次损伤，微球体无法适应新生环境，导致死亡^[33]。在化疗2个周期之后，部分微球体对化疗药物的敏感性较高，在杀灭乳腺癌细胞的同时，可以有效清除微球体^[31]，而另一部分微球体对化疗的药物敏感性不高，在化疗之后可以幸存，受到化疗时间相对较短这一因素的影响，细胞无法在体内进行大规模的扩增^[32-33]，在对其进行体外培养的时候，需要一定的时间方可形成微球体。在新辅助化疗达到3个周期以上之后，大多数癌细胞已经杀灭^[34-37]，在化疗药物的影响下，对化疗不敏感的微球体会进行自我更新，使其拥有充足的时间在体内进行增殖，并通过扩增形成与微球体十分相似的细胞团，这一结果表明，微球体对化疗药物的反应存在一定的不同之处，属于具有异质性的群体^[38-39]。本实验培养分离的微球体在经过消化成为单细胞之后，在无血清培养基中，均可以重建产生新的微球体^[40-42]。利用细胞计数法对无血清悬浮培养的原代细胞生长情况进行测定，发现细胞处于持续生长状态。在培养的前3 d，细胞增殖不明显，化疗3个周期和化疗2个周期细胞数差异无显著性意义，但在3 d之后，化疗3个周期的细胞增殖速度显著快于化疗2个周期，6 d之后，化疗2个周期的细胞生长曲线呈现出平缓的状态，化疗3个周期则呈现出迅速上升的状态，说明受到化疗药物的影响，微球体的自我更新以及多向分化潜能均得到有效的促进^[43-46]。研究表明，ALDH1表型乳腺癌干细胞比CD44⁺CD24^-表型的乳腺癌干细胞在乳腺癌组织中分布更广泛。经免疫细胞化学实验检测还得出在微球体中均含有ALDH1阳性细胞，化疗3个周期标本的微球体中ALDH1阳性细细胞比例大于化疗2个周期标本，这一结果也表明，在微球体中普遍存在ALDH1表型细胞，随着新辅助化疗周期的不断增加，ALDH1表型微球体所占比例也会出现不断上升^[47-50]。综上所述，新辅助化疗2、3个周期的微球体，均具有一定的增殖和分化能力，说明从化疗后的乳腺癌组织中分离乳腺癌干细胞宜选取化疗3个周期及以上的标本。随着微球体分离、培养以及鉴定等水平的不断提高，其可成为研究乳腺癌干细胞，提高临床治疗水平的有力工具^[51-54]。中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程