摘要目的:探讨腺性肥大乳房乳腺上皮细胞(hMEC-GAHB)的原代培养方法，观察该原代细胞的形态学及生物学特点。方法:采用0.25%胰蛋白酶和Ⅰ型胶原酶混合液，结合长时间4 ℃和短时间37 ℃消化，配合低转速离心的方法，从2010年4月至2011年4月华中科技大学同济医学院附属协和医院整形外科手术治疗的10例腺性乳房肥大症患者(M组，实验组)和10例正常体积乳房患者(N组，对照组)的乳腺组织中消化获取乳腺上皮细胞并进行原代培养，评价获取原代细胞的效果，观察各组细胞的形态学特点，采用多抗体免疫细胞化学法(MICC)鉴定原代细胞种属，并采用碘化丙锭(PI)染色法、四甲基偶氮唑盐(MTT)法和流式细胞术等方法对各组细胞的细胞周期和体外增殖及凋亡等生物学特点等进行分析。结果:腺性肥大乳房每克乳腺组织可获取细胞数为5×10 6个，细胞存活率为70%，接种后48 h细胞贴壁率为60%，贴壁细胞活性良好，接种后约72 h胞质展开，约120 h相互融合。细胞增殖明显，未见明显凋亡。hMEC-GAHB与正常体积乳房乳腺上皮细胞在细胞形态学上差异无统计学意义，且正常表达典型的上皮细胞骨架蛋白[CK8、CK14、CK18、α-平滑肌肌动蛋白(α-SMA)等]。在含有相同浓度的17β-雌二醇的体外环境中，处于同一时期细胞对数生长期时，hMEC-GAHB组的群体倍增率(PDR)为0.548、1.634、2.062、3.946、5.210、8.700，正常体积乳房组的群体倍增率(PDR)为0.394、1.455、1.820、3.822、4.467、5.213，两者间差异有统计学意义( P<0.05)；hMEC-GAHB组和正常体积乳房组的G 2/M期与G 0/G 1期细胞数比值分别为0.45±0.01和0.34±0.01，两者间差异有统计学意义( P<0.05)。 结论:采用改良的酶消化法可获得较满意的细胞数和细胞活性。hMEC-GAHB具有典型上皮细胞的形态学及生物学特点，在含有雌激素的体外环境中，hMEC-GAHB比较正常体积乳房乳腺上皮细胞具有更强的增殖活力，并具有正常的细胞衰退期。

abstractsObjective:To investigate the proper methods of primary cell culture of human mammary epithelial cells of gland-associated hypertrophic breast (hMEC-GAHB), and find out its morphological and biological characteristics in vitro. Methods:Harvesting the primary mammary epithelial cells from 10 cases of gland-associated hypertrophic breasts and 10 cases of breasts for normal volume, using 0.25% trypsin and typeⅠcollagenase, which interact with the mammary tissue under 4 ℃ for a long time and then under 37 ℃ for a while, companying the employment of low-speed centrifugation, and then culture the cells in vitro. evaluating the result of harvest of primary cells, observing their morphological characteristics. identifying the primary cell species by multiantibody immunocytochemistry. analyzing their biological characteristics in cell cycle and proliferation and apoptosis in vitro by propidium iodide (PI) staining method and methyl thiazolyl tetrazolium (MTT) method and flow cytometry. Results:About 5×10 6 cells could be obtained from 1g mammary tissue of gland-associated hypertrophic breast, the livability of the cells was about 70%, and the adherence rate after 48 h was about 60%. The adherent cells showed good activity, and cytoplast stretched after 72 h and started to connect each other after 120 h. Obvious proliferation could be observed, without apparent apoptosis. hMEC-GAHB seemed to have no distinct difference with normal volume breast group in cell morphology, and it expressed normally typical epithelial skeleton protein [CK8, CK14, CK18, α-smooth muscle actin (α-SMA)]. Under the environment containing proper 17b-estradiol in vitro and in logarithmic growth phase, the population doubling rate of hMEC-GAHB were 0.548, 1.634, 2.062, 3.946, 5.210, 8.700, while the data of normal volume breast group were 0.394, 1.455, 1.820, 3.822, 4.467, 5.213 with statistical difference ( P<0.05); the cell number ratio of G 2/M and G 0/G 1 phase of hMEC-GAHB and normal volume breast group were respectively 0.45±0.01 and 0.34±0.01 with statistical difference ( P<0.05). Conclusion:Satisfactory cell number and activity could be obtained by the improved methods of enzyme digestion. hMEC-GAHB possessed typical biological and morphological characteristics of epithelial cells. Compared with hMEC-NVB, it showed relative great proliferative activity in the environment containing estrogen in vitro, and had normal senescence phase.