摘要
目的运用无血清细胞培养法培养出源于结肠癌细胞株SW620的细胞球,并实现其在体外的繁殖及传代,为深入研究结肠癌提供坚实的基础。方法逐步驯化人结肠癌细胞株SW620适应无血清培养条件,1周后,收集悬浮生长的细胞球。采用免疫荧光法检测胚胎干细胞标记物SSEA-4、TRA-1-60在SW620细胞及细胞球的表达情况;采用PI染色法检测2种细胞的细胞周期;采用脂肪诱导分化液对2种细胞进行体外诱导培养;采用裸鼠成瘤实验比较2种细胞的成瘤能力。结果 SW620细胞在无血清培养液中培养1周后,可生出悬浮生长的细胞球。胚胎干细胞标记物SSEA-4、TRA-1-61在所有细胞球细胞中均呈阳性表达;在SW620细胞中的表达率分别为(25.74±7.62)%、(27.74±4.31)%,两者相比,差异具有统计学意义(P<0.05)。细胞球细胞有(7.18±1.35)%处于分裂期(S期),(84.19±2.52)%处于分列前期(G0/G1期);SW620细胞有(20.89±3.84)%处于S期,(63.02±6.73)%处于G0/G1期,两者相比差异有统计学意义(P<0.05)。脂肪诱导液中培养7d后,油红特异性染色的脂肪颗粒在每100个细胞球细胞中为(583.80±77.69)个;在每100个SW620细胞中为(169.20±26.43)个,两者相比差异有统计学意义(P<0.05)。2×104个细胞球细胞即可在裸鼠皮下成瘤,移植瘤体积为(2 279.98±346.27)mm3;2×105个SW620细胞才能形成裸鼠皮下移植瘤,移植瘤体积为(889.75±78.79)mm3,两者相比差异有统计学意义(P<0.05)。结论人结肠癌细胞SW620经过无血清驯化,可培养出细胞球,并可进行体外的大量繁殖和稳定传代;细胞球表现出肿瘤干细胞样细胞的特性。
Objective To generate suspended spheres ,Human colon cancer cell line SW620 cells were acclimated gradually in the free serum culture medium ,1 week later ,a great quantity of spheres were obtained for the further research .Methods SW620 cells and spheres were incubated with the following Abs ：anti‐SSEA‐4 Ab and anti‐TRA‐1‐60 Ab and detected with confocal microscopy . SW620 cells and spheres were collected and stained by PI ,Cell cycle distribution was then acquired by flow cytometry .SW620 cells and spheres were transferred into Adipocyte Differentiation Medium and cultured for 7 d and then lipid droplets were counted . SW620 cells and sphere cells were injected subcutaneously into the lateral root of one posterior limb of a nude mouse ,tumor volume was calculated .Results Spheres formed when grown under serum‐free conditions in 7 days ,and the ability of SW620 cells to form spheres was sustained .A few of SW620 adherent cells be cultured in DF＋10% FBS clearly showed the expression of pluripotent stem cell markers Ssea‐4（25 .739 ± 7 .62）% ,Tra‐1‐60（27 .742 ± 4 .311）% ,but almost all spheres expressed the 2 markers and there was a significant difference between the two groups in the expression ratio （P〈0 .05） .It had been shown that the spheres exhibited a relatively high proportion of cells in G0/G1 phase（84 .19± 2 .52）% and low proportion in S phase（7 .18 ± 1 .35）% ;when com‐pared to SW620 cells ,G0/G1 phase（63 .02 ± 6 .73）% ,S phase（20 .89 ± 3 .84）% （P〈0 .05） .Adipocytes drops from spheres and ad‐herent cells were calculated under microscope after been cultured in adipocytes liquid for 7 d .The number of adipocytes drops in spheres was （583 .80 ± 77 .69） ,but（169 .20 ± 26 .43）in SW620 adherent（P〈0 .05） .2 × 104 sphere cells injected subcutaneously into the lateral root of one posterior limb of a nude mouse fromed transplantation tumor after 4 weeks ,and the tumor size was （2 279 .98 ± 346 .27） mm3 ;2 × 105 adherent cells
出处
《国际检验医学杂志》
CAS
2015年第15期2221-2223,共3页
International Journal of Laboratory Medicine
基金
2015年中山市人民医院科研基金项目(201516)
关键词
结肠癌
肿瘤干细胞
无血清培养
colorectal cancer
cancer stem cells
serum-free culture
