悬浮培养法富集结肠癌肿瘤干细胞的研究

Enrichment of colon cancer stem cells by suspension cell culture

目的采用悬浮培养法从人结肠癌细胞HT29中富集结肠癌干细胞,为进一步研究奠定基础。方法 HT29细胞悬浮培养使用添加多种因子的无血清培养基,显微镜下观察微球规则后,胰酶消化,制备单细胞悬液进行传代;用双苯酰亚胺33342荧光染料检测HT29细胞中侧群细胞的比例;采用流式细胞术检测CD133的表达;免疫印迹法(Western blotting)检测ALDH1A1的表达;克隆形成试验检测HT29微球的克隆形成能力。结果 HT29细胞中侧群细胞含量为3.45%±0.05%,经过维拉帕米阻断后,HT29中侧群细胞比例明显减少,差异有统计学意义(P<0.01);具有CD133和ALDH1A1表达阳性的细胞,表明存在结肠癌干细胞亚群。HT29细胞悬浮培养24 h,单个细胞以出芽的方式分裂,72 h可见细胞微球体积逐渐增大,7 d细胞团致密微球形态规则,形成漂浮的球体。HT29细胞微球ALDH1A1蛋白表达水平(1.98±0.41)和CD133+表达(76.04%±4.73%)明显高于常规培养的HT29细胞(分别为1.09±0.32、45.38%±10.65%),差异均有统计学意义(P<0.05);HT29细胞微球的克隆形成率(66%±6%)明显高于常规培养的HT29细胞(18%±6%),差异有统计学意义(P<0.01),表明结肠癌干细胞富集率达70%左右。结论悬浮培养法富集结肠癌干细胞操作简便、相对成本较低、便于掌握,培养基组成和传代次数是影响富集效果的关键。

Objective To enrich the colon cancer stem cells （CSC） from the established human colon cancer cell HT29 with susp- ension culture method and to provide the base for further study. Methods The HT29 cells were cultured in serum-free condition medium containing growth factors. The trypsin was used to digest for preparation of single cell suspension to passage after microspheres were formed .Hoechst 33342 staining test was used to assess the proportion of side population （SP） cells in cultured HT29 cells. Flow cytometry was used to detect CD133 expression. Western blotting assay was utilized to measure ALDH1A1 expression and colony formation assay was used to observe the colony formation ability of HT29 tumor sphere cells. Results The percentage of SP cells in HT29 cells was 3.45%±0.05%, which decreased significantly after incubation with verapamil （P〈0.01 ）. The HT29 cells with CD133 ＋ and ALDH1A1 ＋indicated the subpopulation of colon CSC. After cultured for 24 hours, single cell was divided in the form of budding, after cultured for 72 hours, the size of cellular microspheres gradually increased, and after cultured for 7 days, the suspended cell spheres with regular morphology were formed. The ALDH1A1 protein expression level （1.98±0.41） and CD133^＋ expression level （76.04%±4.73%） in HT29 cellular microspheres were significantly higher than those （1.09±0.32 and 45.38%±10.65%） in routine cultures of HT29 cells （P〈0.05）. The clone formation rate （66%±6%） of HT29 sphere cells was significantly higher than that （18%±6%） in routine cultures of HT29 cells （P〈0.01）. The enrichment rate of CSC was 70%. Conclusion Suspension culture method used to enrich CSC is a simple, inexpensive and easy method for operation. Composition of culture medium and the number of passage are key issues for the enrichment effects.