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有证据表明肝癌含有一定数量的具有自我更新能力的癌干细胞亚群。然而,癌干细胞是如何被调控的还不清楚。肿瘤微环境中一些生物介质可以导致癌干细胞快速可逆性改变。本文旨在探讨,在肿瘤微环境中有较高含量,又是细胞关键的调控因子的亚硝酸盐是如何调控人肝癌干细胞样细胞表型的。用SMMC-7721细胞系无血清培养获得细胞球。流式细胞术检测细胞周期,体外克隆形成实验和球形成实验检测细胞自我更新能力,侵袭实验检测细胞侵袭能力,癌干细胞标志物检测采用流式细胞术和Westen blot方法,MTT检测细胞活力。皮下注射SMMC-7721球形成细胞建立裸鼠移植瘤模型,活性氧(ROS)检测试剂盒检测移植瘤组织内ROS水平,免疫荧光检测组织内缺氧有诱导因子-1α(HIF-1α)。结果显示,SMMC-7721细胞系来源的球细胞自我更新能力增强,干细胞标志物表达增加,耐药性增强,提示球形成细胞富集了较多的具有癌干细胞特征的细胞。用亚硝酸钠(150μmol·L~(-1))处理亲本细胞和球形成细胞后,G_0/G_1期细胞比例增加,肝癌干细胞标志物CD133、CD90和EpCAM表达增加,细胞耐药能力和侵袭能力增强。与亲本细胞相比,亚硝酸盐对球形成细胞的上述作用明显增强。体内实验结果也显示,亚硝酸盐促进球形成细胞移植瘤生长,组织内ROS水平下降,HIF-1α累积。结果表明,亚硝酸盐通过上调肝癌干细胞“干性”增强肝癌细胞侵袭能力。
There is evidence that liver cancer contains a certain number of self-renewing cancer stem cell subsets. However, it is unclear how cancer stem cells are regulated. Some biological mediators in the tumor microenvironment can cause rapid and reversible changes in cancer stem cells. This article aims to investigate how nitrite, which is a cell-critical regulatory factor in tumor microenvironments, regulates the phenotype of human hepatocellular carcinoma stem cell-like cells. Cell spheres were obtained by serum-free culture of SMMC-7721 cell line. Flow cytometry was used to detect cell cycle, in vitro clonogenicity assay and spherocytosis assay to detect cell self-renewal ability. Invasion assay was used to detect cell invasiveness. Flow cytometry and Westen blot were used to detect cancer stem cell markers. Cell viability was detected by MTT assay. The subcutaneous injection of SMMC-7721 cells into nude mice model of tumor xenografts, reactive oxygen species (ROS) detection kit detection of transplanted tumor tissue ROS levels, immunofluorescence detection of tissue hypoxia with induced factor-1α (HIF-1α). The results showed that the SMMC-7721 cell line derived from self-renewal ability of ball cells enhanced stem cell marker expression increased resistance increased, suggesting that the ball-forming cells are enriched with more features of cancer stem cells. After treated with 150μmol·L -1 sodium nitrite, the percentage of cells in G 0 / G 1 phase increased, the expression of CD133, CD90 and EpCAM in hepatocellular carcinoma cells increased, and the drug resistance and invasiveness of cells Enhanced. Compared with the parental cells, the above-mentioned effect of nitrite on the spherical cells is significantly enhanced. The results of in vivo experiments also showed that nitrite promoted the growth of the transplanted spherical tumor and the level of ROS in the tissue decreased and HIF-1α accumulated. The results show that nitrite can enhance the invasion ability of hepatocellular carcinoma cells by up-regulating the stem cells of stem cells.