细胞密度对卵巢癌干细胞悬浮球诱导的影响

目的 研究细胞密度是否会对从人卵巢透明细胞癌系ES2 和卵巢腺癌细胞A2780中分离的癌干细胞产生影响。方法 将ES2、A2780细胞置于含生长因子、牛血清白蛋白和人胰岛素的无血清培养基中培养，采用连续传代诱导培养癌干细胞。观察不同细胞密度下ES2、 A2780细胞形态变化，流式细胞术分别检测含血清培养基（SSM）组、无血清培养基（SFM）组细胞诱导前后细胞表面标志CD133、CD44表达的改变。软琼脂克隆形成实验检测不同诱导条件下ES2癌干细胞球生长速度和成瘤能力。结果 2×104 mL-1密度下，ES2细胞能在SFM中存活，但不能形成癌干细胞球；5×104、1×104 mL-1 ES2细胞能在SFM中存活、增殖并形成癌干细胞球。与贴壁的细胞及诱导前相比，诱导后形成的癌干细胞球（1×105 mL-1组、5×105 mL-1组）高表达CD133+、CD44+ （ P<0.05）,传代后具有更高的增殖能力和克隆形成能力，且5×104 mL-1密度下，癌干细胞球成瘤能力更强。A2780细胞分别以1×104、3×104、5×104 mL-1的密度接种于SFM中，10 d后，1×104、3×104 mL-1组中形成细胞团，但3×104 mL-1组中形成的细胞团更大，透光性更强，细胞更致密，5×104 mL-1组未形成细胞悬浮球。流式细胞术发现A2780细胞3×104 mL-1组中细胞与1×104 mL-1组相比，CD133+细胞阳性率更高，差异有统计学意义( P<0.05)。3×104 mL-1组的细胞生长速度最快。结论 细胞密度5×104 mL-1和3×104 mL-1分别是诱导ES2、 A2780细胞株卵巢肿瘤干细胞球的合适条件。

Effects of Cellular Density on the Induction of Suspension Globe of Ovarian Cancer Stem Cells

【Abstracts】 Objective To determine the effect of cellular density on the separation and identification of cancer stem cells from human ovarian clear cell carcinoma cell line ES-2 and adenocarcinoma cell line A2780. Methods ES-2 and A2780 cells were cultured with human recombinant epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and bovine serum albumin and insulin in serum free medium. The cancer stem cells were obtained through serial passages. Changes in cell morphology, expressions of surface marker CD133 and CD44, and soft AGAR clone forming in the stem cells were examined under different cell density, either in serum-supplemented medium (SSM group) or in serum free medium (SFM group). Results Under the density of 2×104 mL-1, ES-2 cells survived in SFM, but did not form stem cells. When the density increased to 5×104 mL-1 or 1×105 mL-1, ES-2 cells survived in SFM, proliferated and formed stem cells. Compared with adherent cells, the suspension globe of stem cells expressed high levels of CD133 and CD44 ( P<0.05), with proliferation and clone forming ability after serial passages. The stem cell balls under the density of 5×104 mL-1 had stronger ability of tumor formation. A2780 cells formed suspension globe under the density of 1×104 mL-1 and 3×104 mL-1, but larger and more transparent balls were observed under the density of 3×104 mL-1 density. No suspension globe was formed under the density of 5×104 mL-1. More CD133+/CD44+cells were detected by flow cytometry under the density of 3×104 mL-1, compared with that under the density of 1×104 mL-1 ( P<0.05). Tumor stem cells grew faster under the density of 3×104 mL-1. Conclusion The optimal density for identifying stem cells from human ovarian cancer is 5×104 mL-1 for ES-2 and 3×104 mL-1 for A2780, respectively.