自体荧光技术筛选恶性胶质瘤干细胞的优势分析
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摘要
目的探讨利用自体荧光技术筛选胶质瘤干细胞的效果。方法利用胶质瘤干细胞嗜核黄素的特点,在体外普通胶质瘤干细胞中加入核黄素培养,利用流式细胞仪筛选具有自发荧光的胶质瘤干细胞。CCK-8检测其体外增殖能力。建立小鼠模型,观察小鼠生存能力,观察肿瘤体积、质量。检测肿瘤组织Ki67、PCNA和Nestin的表达水平。结果通过流式细胞仪筛选出具有自发荧光的胶质瘤干细胞; CCK-8结果显示荧光胶质瘤干细胞的增殖能力更强(P<0.01);体外荷瘤实验表明,荷有荧光干细胞的小鼠,其生存时间明显降低,肿瘤体积和质量明显增大(P<0.01)。肿瘤石蜡切片免疫组化显示,载有荧光干细胞肿瘤切片中Ki67、PCNA和Nestin含量明显增高(P<0.05)。荧光干细胞的增殖和恶性程度高于目前GL261肿瘤干细胞,同时也能筛选出部分CD133~-的具有高恶性程度高和高侵袭性的胶质瘤干细胞。结论自体荧光技术能较好地筛选出恶性程度高的胶质瘤干细胞。
Objective To determine the efficiency of autofluorescence technique in screening of glioma stem cells(GSCs). Methods Since GSCs favor riboflavin, the agent was added to the culture medium of normal glioma stem cells. Then flow cytometry was employed to screen glioma stem cells with autofluorescence. Subsequently, the proliferation of the obtained cells was detected by CCK-8 assay. Finally, a mouse model of xenograft was established to observe the survive and tumor size and weight. The expression of Ki67, PCNA and Nestin in the tumor mass was detected by immunohistochemical assay. Results Glioma stem cells with autofluorescence were successfully screened out by flow cytometry. CCK-8 assay indicated that the proliferation of autofluorescence glioma stem cells was significantly stronger(P<0.01). In vitro tumor-bearing experiments suggested that mice with xenograft of fluorescent stem cells had significantly reduced survival time and increased tumor volume and weight(P<0.01). Immunohistochemical assay showed that the expression levels of Ki67, PCNA and Nestin in the tumor mass were significantly increased(P<0.05). The proliferation and malignancy of the autofluorescence GSCs were stronger than glioma stem GL261 cells screened by CD133~+ labeling. The CD133~- GSCs with higher malignancy and invasion were also screened out with this method. Conclusion Autofluorescence technique can screen GSCs with high malignancy.
Objective To determine the efficiency of autofluorescence technique in screening of glioma stem cells(GSCs). Methods Since GSCs favor riboflavin, the agent was added to the culture medium of normal glioma stem cells. Then flow cytometry was employed to screen glioma stem cells with autofluorescence. Subsequently, the proliferation of the obtained cells was detected by CCK-8 assay. Finally, a mouse model of xenograft was established to observe the survive and tumor size and weight. The expression of Ki67, PCNA and Nestin in the tumor mass was detected by immunohistochemical assay. Results Glioma stem cells with autofluorescence were successfully screened out by flow cytometry. CCK-8 assay indicated that the proliferation of autofluorescence glioma stem cells was significantly stronger(P<0.01). In vitro tumor-bearing experiments suggested that mice with xenograft of fluorescent stem cells had significantly reduced survival time and increased tumor volume and weight(P<0.01). Immunohistochemical assay showed that the expression levels of Ki67, PCNA and Nestin in the tumor mass were significantly increased(P<0.05). The proliferation and malignancy of the autofluorescence GSCs were stronger than glioma stem GL261 cells screened by CD133~+ labeling. The CD133~- GSCs with higher malignancy and invasion were also screened out with this method. Conclusion Autofluorescence technique can screen GSCs with high malignancy.
引文
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[2] UNG N, YANG I. Nanotechnology to augment immunotherapy for the treatment of glioblastoma multiforme[J]. J Neurooncol, 2015, 123(3): 473-481. DOI:10.1007/s11060-015-1814-1.
[3] REYA T, MORRISON S J, CLARKE M F, et al. Stem cells, cancer, and cancer stem cells[J]. Nature, 2001, 414(6859): 105-111. DOI:10.1038/35102167.
[4] DONNENBERG V S, DONNENBERG A D. Multiple drug resistance in cancer revisited: the cancer stem cell hypothesis[J]. J Clin Pharmacol, 2005, 45(8): 872-877. DOI:10.1177/0091270005276905.
[5] SINGH S K, HAWKINS C, CLARKE I D, et al. Identification of human brain tumour initiating cells[J]. Nature, 2004, 432(7015): 396-401. DOI:10.1038/nature03128.
[6] SINGH S K, CLARKE I D, TERASAKI M, et al. Identification of a cancer stem cell in human brain tumors[J]. Cancer Res, 2003, 63(18): 5821-5828.
[7] CLéMENT V, DUTOIT V, MARINO D, et al. Limits of CD133 as a marker of glioma self-renewing cells[J]. Int J Cancer, 2009, 125(1): 244-248. DOI:10.1002/ijc.24352.
[8] HILL R P. Identifying cancer stem cells in solid tumors: case not proven[J]. Cancer Res, 2006, 66(4): 1891-1895. DOI:10.1158/0008-5472.CAN-05-3450.
[9] KERN S E, SHIBATA D. The fuzzy math of solid tumor stem cells: a perspective[J]. Cancer Res, 2007, 67(19): 8985-8988. DOI:10.1158/0008-5472.CAN-07-1971.
[10] MIRANDA-LORENZO I, DORADO J, LONARDO E, et al. Intracellular autofluorescence: a biomarker for epithelial cancer stem cells[J]. Nat Methods, 2014, 11(11): 1161-1169. DOI:10.1038/nmeth.3112.
[11] LATHIA J D, GALLAGHER J, HEDDLESTON J M, et al. Integrin alpha 6 regulates glioblastoma stem cells[J]. Cell Stem Cell, 2010, 6(5): 421-432. DOI:10.1016/j.stem.2010.02.018.
[12] SOEDA A, PARK M, LEE D, et al. Hypoxia promotes expansion of the CD133-positive glioma stem cells through activation of HIF-1alpha[J]. Oncogene, 2009, 28(45): 3949-3959. DOI:10.1038/onc.2009.252.
[13] CALABRESE C, POPPLETON H, KOCAK M, et al. A perivascular niche for brain tumor stem cells[J]. Cancer Cell, 2007, 11(1): 69-82. DOI:10.1016/j.ccr.2006.11.020.