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ABSTRACT OBJECTIVE：To study the effects of Calpeptin inhibitor Calpeptin on the transformation and stemness markers
expression induced by estradiol （E2 ），and to investigate its mechanism. METHODS：Taking human mammary epithelial cells
MCF-10A as research object，transformed cells were induced by E2 treatment. Cells were divided into control group（0.1％DMSO），
E2-transformed group（50 nmol/L），E2-transformed+Calpeptin group（50 nmol/L E2+1 μmol/L Calpeptin），then continuously treated
with corresponding drug-containing culture medium for 15 generations. Then，MTT assay was used to determine the proliferation
rate of cells（24，48 h）；plate colony test was used to detect the Clone formation rate of cells；the number of sphere-forming cells
was measured by suspension spheroidization test；mRNA expressions of stemness marker（CD44，Nanog，OCT4）and extracellular
sigal-regulated kinase（ERK）were detected by RT-qPCR，and protein expressions of CD44，Nanog，OCT4 ，ERK and p-ERK
were detected by Western blotting assay. Another E2-transformed cells were divided into control group（0.1％DMSO）and U0126
（ERK inhibitor）group（10 μmol/L）. Clone formation rate，the number of sphere-forming，protein expressions of CD44，Nanog，
OCT4，ERK and p-ERK were determined with above methods，and to validate the relationship of ERK inhibition with transformed
cell behavior and the expression of stemness markers. RESULTS：Compared with control group，proliferation rate and clone
formation rate of E2 transformed group were increased significantly（P＜0.01），and the number of sphere-forming was increased
significantly（P＜0.01）；mRNA expression levels of CD44，Nanog，OCT4，ERK and protein expression levels of CD44，Nanog，
OCT4 and p-ERK in cells were increased significantly（P＜0.01）. Compared with E2-transformed group，proliferation rate（24，48
h） and clone formation rate of E2-transformed + Calpeptin group were decreased significantly （P＜0.01），and the number of
sphere-forming was decreased significantly （P＜0.05）；mRNA expression levels of CD44，Nanog，OCT4 ，ERK and protein
expression levels of CD44，Nanog，OCT4，p-ERK in cells were decreased significantly（P＜0.05 or P＜0.01）. After treated with
ERK inhibitor U0126，clone formation rate of E2-transformed cells，the number of sphere-forming，protein expression levels of
CD44，Nanog，OCT4 and p-ERK were increased significantly（P＜0.05 or P＜0.01）. CONCLUSIONS：Calpeptin can inhibit the
transformation and the expression of stemness markers of human mammary epithelial cells MCF-10A，and the mechanism of it may
be associated with inhibiting the activation of Calpain-ERK signaling pathway.
KEYWORDS Calpeptin； Estradiol； Human mammary epithelial cells MCF-10A； Cell transformation； Stemness marker；
Extracellular sigal-regulated kinase；Mechanism

雌二醇（E2）是诱导乳腺癌发生的主要风险因素 ， 导了上皮细胞转化以及癌细胞的迁移、增殖 [10-12] 。但有
[1]
其可通过激活雌激素受体（ER）或代谢产生有毒代谢产 关Calpain是否介导了E2诱导的乳腺上皮细胞MCF-10A
物，从而诱导乳腺上皮细胞转化以及癌变发生 [2-3] 。相关 干性特征增强，尚未见文献报道。另有研究显示，细胞
研究显示，乳腺肿瘤干细胞（BCSCs）在乳腺癌的发生、 外信号调节激酶（ERK）信号通路的激活与细胞的恶性
[13]
维持、转移、治疗抵抗和复发中起着重要作用，而 E2与 转化密切相关 。但 Calpeptin 是否能通过 ERK 通路干
BCSCs 的产生密切相关 。人乳腺上皮细胞 MCF-10A 预乳腺上皮细胞 MCF-10A 转化以及干性特征增强，也
[4]
是一种非致瘤性乳腺上皮细胞，正常情况下其呈不规则 同样尚未见文献报道。鉴于此，本研究旨在通过探讨
多边形贴壁生长，且增殖缓慢；但经 E2 长期诱导后， Calpain 抑 制 剂 Calpeptin 对 E2 诱 导 乳 腺 上 皮 细 胞
MCF-10A 细胞将失去上皮细胞相关特性，胞体变大，呈 MCF-10A 转化及干性标志物（CD44、OCT4、Nanog）表
长梭形贴壁生长，增殖能力增强，且具有一定的间质特 达的影响，并通过探究 Calpain-ERK 信号通路在其中的
[5]
征 。近期有研究发现，E2 能诱导人乳腺上皮细胞 介导作用，为阐明Calpeptin抑制E2诱导乳腺癌发生的作
MCF-10A 的自我更新、多潜能分化等干性特征增强，促 用机制提供参考。
进该细胞向BCSCs转化 。 1 材料
[6]
钙激活中性蛋白酶（Calcium-activated neutral prote- 1.1 仪器
ase，缩写为“Calpain”）是一种 Ca 依赖型半胱氨酸蛋白 ND2000 型超微量紫外分光光度计、2001HY-6003
2+
酶，主要成员有 Calpain-1 和 Calpain-2，可参与调控乳腺 型CO2细胞培养箱（美国Thermo Fisher Scientific公司）；
癌细胞的多种恶性生物学行为 [7-8] 。Calpeptin 是一种具 HH-W21-Cr600 型电热恒温水温箱、DW-86L486 型立式
有细胞穿透性的 Calpain 抑制剂，据相关研究报道，Cal- 超低温冰箱（青岛海尔特种电器有限公司）；SW-CJ-2D
peptin可通过抑制ER阳性乳腺癌细胞MCF-7中Calpain 型超净工作台（苏州净化设备有限公司）；FA2204N型电
的活性，从而抑制细胞的迁移和侵袭 。本课题组前期 子天平（上海菁海仪器有限公司）；ZHWY-103D 型恒温
[9]
研究发现，在 E2诱导人乳腺上皮细胞 MCF-10A 转化的 培 养 振 荡 器（上 海 智 城 分 析 仪 器 制 造 有 限 公 司）；
[10]
过程中，通常伴随着 Calpain 活性的增强 。而 Calpain DYY-7C 型电泳仪（北京市六一仪器厂）；Epoch 型全波
活性增强在癌症的发生发展中具有重要作用，其参与介 长酶标仪（美国Bio-Tek公司）；CKX41型倒置显微镜（日

·1550 · China Pharmacy 2020 Vol. 31 No. 13 中国药房 2020年第31卷第13期