摘要：
Objective To evaluate the feasibility of modified serum-free medium for adherent cell culture,the biological phenotype of adherent tumor and normal cells was observed in vitro.Methods Tumor and normal cell lines of human and rat were cultured in serum-free medium (DOBA) or serum-containing medium (D10).DOBA medium is basically composed of equal volumes of dulbecco's modified eagle medium (DMEM) high glucose and reduced serum (Opti-MEM) medium containing 2％ B27 and 1％ non-essential amino acids.Serum-containing medium is basically composed of DMEM medium containing 10％ fetal bovine serum (FBS).Cell growth and proliferation were assessed by cell counting kit-8 (CCK-8).20 ng/ml epidermal growth factor (EGF) and 25 μmol/L β-catenin-responsive transcription inhibitor (iCRT3) were used to detect the drug sensitivity.Apoptosis was determined by Annexin Ⅴ/propidium iodide (PI) double staining.Colony formation experiment was utilized to observe long-term cell culture.Results Cells cultured in DOBA grew and expanded following time lapse.After cultured 72 h,relative proliferation rate of A172 and C6 cells was (2.49 ± 0.08) ％ vs.(2.10 ± 0.07) ％,(6.91 ±0.40) ％ vs.(2.97 ± 0.27) ％,resulting in a decrease of cell proliferation capacity (P ＜ 0.01).Relative proliferation rate of Hela and HREC cells was (4.59 ±0.70)％ vs.(6.01 ±0.31)％,(2.84 ±0.33)％ vs.(3.95 ± 0.20) ％,resulting in an increase of cell proliferation capacity (P ＜ 0.01).SW480 and U87 cells showed no significant differences in DOBA culturing (P ＞ 0.05).After stimulated by EGF for 48 h,the proliferation rate of U251 and HUNEC was (1.29 ±0.03) times and (1.18 ±0.08) times higher than that of D10 group.While stimulated by iCRT3 for 48 h,the proliferation rate of HCT116 and SW480 was (90± 2)％ and (78 ±10)％ lower than that of D10 group.Thus,the cells in D0BA were more sensitive to EGF and iCRT3 than those in serum-containing medium (P ＜ 0.05,P ＜ 0.01).Flow assay showed that serum-free medium did not increase cell apoptosis (P ＞ 0.05).Colony-forming experiment showed that cells in DOBA formed clones,but clone number and cell number of each clone were both less than cells in serum-containing medium.After cultured 10-14 d,clone numbers of Hela、SW480 and U251 were (368 ± 29) cells vs.(300 ± 6) cells,(396 ± 13) cells vs.(324 ± 13) cells,(372 ± 9) cells vs.(251 ± 12) cells (P＜ 0.05,P＜ 0.01).Conclusion DOBA medium maintains self-renewal,long-term culture and subculture of adherent cells,increases the sensitivity of cells to drug stimulation,and does not induce the apoptosis rate.%目的 观察改良的无血清培养基对体外贴壁培养的肿瘤细胞和正常细胞生物学表型的影响,探讨以无血清培养基为基础的培养方案用于贴壁细胞培养的可行性.方法 培养人和大鼠多种肿瘤细胞和正常细胞株,分为无血清培养基组(DOBA)和有血清培养基组(D10),无血清培养基以等体积杜尔伯科改良伊格尔(DMEM)高糖和减血清(Opti-MEM)培养基为基础,加入2％B-27和1％非必需氨基酸,有血清培养基用DMEM培养基添加10％胎牛血清.通过细胞计数试剂盒(CCK-8)检测不同培养基对细胞正常生长和增殖的影响,或评估细胞对20 ng/ml表皮生长因子(EGF)和25 μmol/L Wnt通路小分子抑制剂(iCRT3)的敏感性,膜联蛋白Ⅴ(Annexin Ⅴ)/碘化丙锭(PI)双染流式细胞术检测对细胞凋亡的影响,克隆形成实验观察对细胞长期培养的影响.结果 无血清组细胞均能正常生长和扩增,培养72 h后,A172、C6细胞的相对增殖率分别为(2.49±0.08)％比(2.10±0.07)％、(6.91±0.40)％比(2.97±0.27)％,DOBA组细胞增殖明显减弱(P＜0.01),Hela和HREC细胞相对增殖率分别为(4.59±0.70)％比(6.01±0.31)％、(2.84±0.33)％比(3.95±0.20)％,DOBA组细胞增殖能力明显增强(P＜0.01),SW480和U87细胞增殖差异无统计学意义(P＞0.05);EGF刺激48 h后,U251和HUNEC增殖率是D10组的(1.29±0.03)倍和(1.18 ±0.08)倍,iCRT3刺激48 h后,HCT116和SW480细胞增殖率为D10组的(90±2)％和(78±10)％,所以DOBA组细胞对EGF或iCRT3刺激的敏感性高于有血清组(P ＜0.05,P＜0.01);与有血清培养基比较,DOBA不影响细胞凋亡(P＞0.05),长期培养能形成细胞克隆集落,但克隆数和单个克隆的细胞数均较有血清组少,培养10～14 d后,Hela、SW480和U251细胞克隆数分别为(368 ±29)个比(300 ±6)个、(396±13)个比(324±13)个以及(372±9)个比(251±12)个(P＜0.05,P＜0.01).结论 DOBA无血清培养基可以维持贴壁培养的肿瘤和正常细胞的自我更新,可实现长时间培养和传代而不增加细胞凋亡比例,并且增加细胞对药物的敏感性.
