目的

通过比较观察激素敏感的MCF-7 细胞在体外干细胞培养和常规培养条件下雌激素受体(ER)变化及对内分泌治疗药物的敏感性变化,初步探讨肿瘤干细胞与内分泌耐药的关系。

方法

分别于常规培养及干细胞培养条件下(悬浮球培养)培养激素敏感的MCF-7 细胞株,流式细胞仪检测分子表型CD44⁺CD24^-/low与CD44⁺CD24⁺亚群细胞比例变化,免疫细胞化学法测定ERα 和ERβ 的表达变化,MTT 法检测细胞对他莫西芬的敏感程度。 分别以t 检验、卡方检验、方差分析进行统计分析。

结果

干细胞培养条件下CD44⁺CD24^-/low亚群细胞的比例为(1.60±0.08)%,比常规培养条件下的(0.27±0.08)%显著增加(t=-12.10,P=0.00),而CD44⁺CD24⁺亚群细胞比例由(5.59±0.88)%增至(30.63±4.40)%(t=-5.58,P=0.00)。 干细胞培养条件培养下ERα 和ERβ 表达率较常规培养下调,分别由85.27% 和90.53% 降至69.43% 和73.20%,差异有统计学意义(χ²=214.64,P=0.00;χ²=303.58,P=0.00),且对他莫西芬的敏感性降低,IC₅₀值由(9.82±0.31)μmol/L 升至(16.46±0.50)μmol/L,再次诱导分化后ER 并未出现上调,对他莫西芬的敏感性仍旧降低(F=113.63, P=0.00)。

结论

与常规培养相比较,体外干细胞培养条件下可以培养出含高比例具有干细胞特性的CD44⁺CD24^-/low与CD44⁺CD24⁺亚群细胞的微球囊,其ER 仍为阳性表达,但对他莫昔芬治疗敏感性减低,推测其可能是乳腺癌内分泌耐药的原因。

Objective

To explore estrogen receptor (ER) expression and endocrine resistance of hormone-sensitive MCF-7 cells in normal culture or in stem cell culture in vitro.

Methods The MCF-7 cells were cultured in normal culture or in stem cell culture in vitro(suspension sphere). The proportion of CD44⁺ CD24^-/low phenotype and CD44⁺ CD24⁺phenotype cells were determined using flow cytometry (FCM). The ERa and ERβ expression was detected using immunohistochemical method. The susceptibility to tamoxifen was determined using MTT essay. The data were processed with t-test, Chi-square test and analysis of variance, respectively.

Results

In suspension culture as stem cells, the proportions of the CD44⁺CD24^-/low and CD44⁺CD24⁺ phenotype cells were (1.60±0.08)%and (30.63±4.40)% respectively, significantly higher than (0.27±0.08)% and (5.59±0.88)% in normal medium (t=-12.10,P=0.00; t=-5.58,P=0.00). The positive expressions of ERα and ERβ were 85.27% and 90.53% in normal medium, 69.43% and 73.20% in suspension culture, respectively,and the differences were statistically significant(χ²=214.64,P=0.00;χ²=303.58,P=0.00). With regards to tamoxifen sensitivity, IC₅₀ was(9.82±0.31)μmol/L in normal medium, and (16.46±0.50) μmol/L in suspension culture. After the second induced differentiation, the positive expression of ER was not upregulated and the susceptibility to tamoxifen remained low (F = 113.63, P = 0.00).

Conclusion

Suspension sphere with a high ratio of CD44⁺CD24^-/low cells and CD44⁺CD24⁺cells can be incubated in stem cell culture in vitro, with higher positive expression of ER and lower sensitivity to tamoxifen, which may be attributed to endocrine resistance.