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该文主要探索从小鼠Lewis肺癌亲代细胞系(Lewis lung carcinoma parental cell line,LLC-Parental)中分离能够在体外长期培养的肿瘤干细胞(cancer stem cells,CSCs)的方法。在体外贴壁条件下培养LLC-Parental细胞,通过连续富集悬浮生长的细胞,经过8代后,成功富集出可悬浮成球的小鼠Lewis肺癌干细胞样细胞株(LLC-spheroid enrichment,LLC-SE)。为了验证其干细胞特性,我们使用荧光定量PCR法(Real-time PCR)检测表示干细胞特性的基因Bmi1(B-cell-specifi c moloney murine leukemia virus insertion site 1)、Cd133(cluster of differentiation-133)、Aldh1a1(aldehyde dehydrogenase family 1,subfamily A1)、Oct4(octamer-binding transcription factor 4)、Sox2(sexdetermining region Y box 2)、Nanog和Klf4(Kruppel-like factor 4)的表达。6孔板琼脂成球实验检测其增殖和悬浮成球能力。96孔板单细胞克隆形成实验检测单个细胞的克隆形成能力。裸鼠皮下移植这一干细胞特性检测的经典模型检测其体内致瘤能力以及C57同源小鼠左肺原位种植模型检测其肿瘤转移能力。Real-time PCR结果显示,LLC-SE细胞中Bmi1的表达水平显著高于LLC-Parental细胞。6孔板琼脂成球实验、96孔板单细胞克隆形成实验表明,LLC-SE细胞的悬浮成球能力和单细胞克隆能力显著高于LLC-Parental细胞。动物实验表明,LLC-SE细胞的体内致瘤能力、肿瘤转移能力强于LLC-Parental细胞。该文首次运用连续8次悬浮聚球法分离获得大量的可稳定传代的具有干细胞特性的LLC-SE细胞,为建立肿瘤干细胞细胞模型提供了新的方法,为进一步研究肿瘤干细胞的生物学特性奠定了基础。
This article mainly explores a method to isolate long-term cultured cancer stem cells (CSCs) from mouse Lewis lung carcinoma parental cell line (LLC-Parental). LLC-Parental cells were cultured in vitro and LLC-spheroid enrichment (LLC) cells were successfully enriched after eight generations by continuous enrichment of suspension-grown cells. -SE). In order to verify the characteristics of stem cells, we used real-time PCR to detect Bmi-1 cells, CD133 cluster of differentiation-133, Aldh1a1 (aldehyde dehydrogenase family 1, subfamily A1), Oct4 (octamer-binding transcription factor 4), Sox2 (sexdetermining region Y box 2), Nanog and Klf4 (Kruppel-like factor 4) 6-well agar into the ball test to test its proliferation and suspension into the ball ability. Single-cell Clonal Formation Experiments in 96-well Plates. The model of stem cell characterization was subcutaneously transplanted in nude mice to detect the tumorigenicity in vivo and to detect the metastasis ability of the C57 homologous mouse left lung in situ implantation model. Real-time PCR results showed that the expression level of Bmi1 in LLC-SE cells was significantly higher than that of LLC-Parental cells. 6-well plate agar into the ball experiments, 96-well single-cell cloning experiments showed that LLC-SE cells suspended into the ball ability and single cell clonal capacity was significantly higher than the LLC-Parental cells. Animal experiments show that LLC-SE cells in vivo tumorigenicity, tumor metastasis than LLC-Parental cells. In this paper, we firstly obtained a large number of stably-passaged LLC-SE cells with eight consecutive suspension spheres and provided a new method for establishing a tumor stem cell model, which laid the foundation for the further study of the biological characteristics of tumor stem cells The foundation.