脑肿瘤干细胞体外分化的形态、标志物及细胞增殖动力学特征

目的探讨脑肿瘤干细胞(BTSC)在体外分化过程中的细胞形态、分化相关标志物表达和增殖动力学变化,为进一步研究BTSC分化走向提供实验依据。方法取同一病例初发和复发的间变性室管膜瘤患者的肿瘤手术标本,用CD133免疫磁珠分离出CD133+细胞,加血清分化,相差显微镜下观察其形态,在未分化和分化后第3、7、10、21天收集细胞,流式细胞术检测细胞CD133+、神经上皮干细胞蛋白(Nestin)、胶质纤维酸性蛋白(GFAP)、β-微管蛋白(Tubulinβ-)Ⅲ的表达。在未分化和分化第7天做增殖周期测定。取正常神经干细胞(NSC)作为对照。结果(1)形态:BTSC由圆形到短梭形和多角形、再到长梭形及少量星形,第7天以后细胞形态往短梭形和圆形回复,出现细胞聚集成球重新飘浮在培养基中;NSC按固有规律分化。(2)标志物:BTSC和NSC未分化时CD133+和Nestin均高表达,BTSC分化后CD133+和Nestin全程表达,且先降后升,第7天表达率分别为(3·65±0·17)%和(28·99±1·26)%,第21天分别为(14·63±1·16)%和(45·46±1·27)%;GFAP和β-TubulinⅢ表达量一直偏低,但GFAP阳性表达率相对高于β-TubulinⅢ;NSC在分化第10天时失去了CD133+和Nestin的表达,GFAP和β-TubulinⅢ表达量明显增加,分别为(88·94±1·23)%和(11·94±0·36)%。(3)增殖周期及倍体:BTSC分化前为亚二倍体,分化后有少量二倍体和大量亚二倍体及超二倍体,处于G2-M期和S期的细胞比例大于NSC,复发BTSC分化后细胞组成比原发BTSC复杂;神经干细胞分化前后都为整二倍体,主要处于G0-G1期。结论细胞形态、标志物的表达、增殖周期及倍体变化反映出脑肿瘤干细胞分化走向和神经干细胞不同,前者存在明显的分化障碍。

Characteristics of morphology, differentiation related marker, and proliferation dynamics of differentiated brain tumor stem cells in vitro

OBJECTIVE: To pursue the changes of cell morphology, expression of differentiation related markers, and proliferation cycles of brain tumor stem cells (BTSCs) after differentiation in vitro. METHODS: Tumor stem cells of the line CD133(+) were obtained from two specimens from one clinical case with anaplasia ependymocytoma during operation, one specimen being obtained during the first operation and then second specimen being obtained during the second operation 6 months later on the recurrent tumor. CD133(+) cells were acquired by using magnetic sorting and then cultured to differentiate in medium containing 10% fetal bovine serum. The morphology of the cells was observed under phase contrast microscope. Cells were collected respectively before differentiation and 3, 7, 10, and 21 days after the differentiation. The cell surface markers such as CD133, nestin, glial fibrillary acidic protein (GFAP), and beta-tubulin III were detected with flow cytometry. Proliferation cycles were examined before differentiation and in the 7th day after differentiation. Normal neural stem cells (NSCs) obtained from fetal brain tissues were used as controls. RESULTS: (1) The BTSCs were round shape at the beginning, then changed to short fusiform, polygon and long fusiform. Seven days later the cells reversed to short fusiform and round shape. The cells accumulated into cell spheres and floated in the culture medium again. While the NSCs differentiated along their routine rules. (2) Both the undifferentiated BTSCs and NSCs showed high level expression of CD133 and nestin. After differentiation the BTSCs expressed CD133 and nestin, the expression levels decreased first and then increased. The expression rates of CD133 and nestin were (3.65 +/- 0.17)% and (28.99 +/- 1.26)% in the 7th day, (14.63 +/- 1.16)% and (45.46 +/- 1.27)% in the 21st day. While the positive expression rate of GFAP was higher than that of beta-tubulin III. In the 10th day the NSCs under differentiation lost the expression of CD133 and nestin. The percentage of GFAP positive cells and beta-tubulin III positive cells were (88.94 +/- 1.23)% and (11.94 +/- 0.36)% respectively. (3) All undifferentiated BTSCs were hypodiploid. After differentiation majority of the BTSCs were hypodiploid or hyperdiploid, The percentages of S phase and G(2)-M phase cells in the BTSCs were higher than that in the NSCs. The cell composition of recrudescent BTSCs was more complex than that of the primary BTSCs. All NSCs were diploid whether differentiated or not. Most of the NSCs were G(0)-G(1) phase cells. CONCLUSION: The differentiation direction of BTSCs is quietly different from that of the NSCs. There is an obvious dysdifferentiation in BTSCs.