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目的研究细胞密度是否会对从人卵巢透明细胞癌系ES-2和卵巢腺癌细胞A2780中分离的癌干细胞产生影响。方法将ES-2、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ES-2、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ES-2癌干细胞球生长速度和成瘤能力。结果 2×10~4 mL~(-1)密度下,ES-2细胞能在SFM中存活,但不能形成癌干细胞球;5×10~4、1×10~4 mL~(-1) ES-2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(1×10~5 mL~(-1)组、5×10~5 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5×10~4 mL~(-1)密度下,癌干细胞球成瘤能力更强。A2780细胞分别以1×10~4、3×10~4、5×10~4 mL~(-1)的密度接种于SFM中,10d后,1×10~4、3×10~4 mL~(-1)组中形成细胞团,但3×10~4 mL~(-1)组中形成的细胞团更大,透光性更强,细胞更致密,5×10~4 mL~(-1)组未形成细胞悬浮球。流式细胞术发现A2780细胞3×10~4 mL~(-1)组中细胞与1×10~4 mL~(-1)组相比,CD133~+细胞阳性率更高,差异有统计学意义(P<0.05)。3×10~4 mL~(-1)组的细胞生长速度最快。结论细胞密度5×10~4 mL~(-1)和3×10~4 mL~(-1)分别是诱导ES-2、A2780细胞株卵巢肿瘤干细胞球的合适条件。
Objective To investigate whether cell density affects cancer stem cells isolated from human ovarian clear cell carcinoma line ES-2 and ovarian adenocarcinoma cell line A2780. Methods ES-2 and A2780 cells were cultured in serum-free medium containing growth factor, bovine serum albumin and human insulin. The cancer stem cells were induced by continuous passage. Morphological changes of ES-2 and A2780 cells were observed under different cell densities. Flow cytometry was used to detect the changes of cell surface marker CD133 and CD44 before and after induction in serum-containing medium (SSM) and serum-free medium (SFM). Soft agar colony formation assay was used to detect the growth and tumorigenicity of ES-2 cancer stem cells under different induction conditions. Results Under the density of 2 × 10 ~ 4 mL ~ (-1), ES-2 cells could survive in SFM but could not form cancer stem cell spheres. The ES-2 cells could express 5 × 10 ~ 4 and 1 × 10 ~ 4 mL ~ -2 cells survive in SFM, proliferate and form cancer stem cell spheres. Compared with the adherent cells and the pre-induction, the expression of CD133 + and CD44 + cells in the cancer stem cells (1 × 10 -5 mL -1, 5 × 10 -5 mL -1) (P <0.05). After passage, the proliferation and clonogenic capacity of the cancer stem cells were higher than that of the control group (P <0.05). Under the density of 5 × 10 ~ 4 mL ~ (-1) A2780 cells were inoculated into SFM at a density of 1 × 10-4, 3 × 10-4 and 5 × 10-4 mL -1, respectively. After 10 days, the cells were treated with 1 × 10-4, 3 × 10-4 mL ~ (-1) group. However, the cell mass formed in 3 × 10 ~ 4 mL ~ (-1) group was larger, the translucency was stronger and the cells were denser. The volume of 5 × 10 ~ 4 mL ~ (- 1) The group did not form the cell suspension ball. Flow cytometry showed that the positive rate of CD133 ~ + cells in 3 × 10 ~ 4 mL ~ (-1) group was higher than that in 1 × 10 ~ 4 mL ~ (-1) group, the difference was statistically significant Significance (P <0.05). The cell growth rate of 3 × 10 ~ 4 mL ~ (-1) group was the fastest. Conclusion Cell density of 5 × 10 ~ 4 mL ~ (-1) and 3 × 10 ~ 4 mL ~ (-1), respectively, are suitable conditions for inducing ovarian tumor stem cell spheres of ES-2 and A2780 cell lines.