CAJ | 학술논문

目的研究全反式维甲酸(ATRA)对脑肿瘤干细胞(BTSCs)增殖和分化的影响.方法采用有限稀释法从人脑胶质母细胞瘤新鲜标本中分离、克隆筛选BTSCs;在无血清条件下培养BTSCs.根据培养基中的成分不同分为对照组、ATRA组、ATRA/生长因子组、生长因子组,用MTT法检测BTSCs的增殖效应;在含血清条件下诱导分化BTSCs.根据血清培养基中的成分不同分为ATRA组、对照组.于诱导分化第10天用免疫荧光技术检测BTSCs子代分化细胞CD133和胶质纤维酸性蛋白(GFAP)的表达率;将BTSCs子代分化细胞更换培养条件,观察其在无血清培养基中增殖逆转再次形成脑肿瘤干细胞球(BTS)的比例和形成时间.结果在无血清条件下,ATRA组BTSCs的增殖速度较对照组明显加快,但低于生长因子组和ATRA/生长因子组,形成的BTS亦小于后两者:在含血清条件下,ATRA组BTSCs子代分化细胞CD133、GFAP的表达率分别为2.29%±0.27%和75.60%±4.03%,对照组为7.05%±0.49%和12.51%±0.77%,前者的分化程度明显高于后者,差异有统计学意义(P<0.05),但前者仍然存在CD133的表达;BTSCs子代分化细胞在回复无血清条件下能够再次增殖形成BTS,ATRA组再形成BTS的比例为4.84%±0.32%,形成时间为(10.07+1.03)d.对照组分别为17.71%±0.78%和(4.08±0.35)d,前者再形成BTS的比例较后者更低、时间更长,差异有统计学意义(P<0.05).结论 ATRA能促进BTSCs增殖并诱导BTSCs子代细胞走向分化,但分化不彻底,细胞不能达到终末分化,可再次形成BTS.
목적 연구전반식유갑산(ATRA)대뇌종류간세포(BTSCs)증식화분화적영향.방법 채용유한희석법종인뇌효질모세포류신선표본중분리、극륭사선BTSCs;재무혈청조건하배양BTSCs.근거배양기중적성분불동분위대조조、ATRA조、ATRA/생장인자조、생장인자조,용MTT법검측BTSCs적증식효응;재함혈청조건하유도분화BTSCs.근거혈청배양기중적성분불동분위ATRA조、대조조.우유도분화제10천용면역형광기술검측BTSCs자대분화세포CD133화효질섬유산성단백(GFAP)적표체솔;장BTSCs자대분화세포경환배양조건,관찰기재무혈청배양기중증식역전재차형성뇌종류간세포구(BTS)적비례화형성시간.결과 재무혈청조건하,ATRA조BTSCs적증식속도교대조조명현가쾌,단저우생장인자조화ATRA/생장인자조,형성적BTS역소우후량자:재함혈청조건하,ATRA조BTSCs자대분화세포CD133、GFAP적표체솔분별위2.29%±0.27%화75.60%±4.03%,대조조위7.05%±0.49%화12.51%±0.77%,전자적분화정도명현고우후자,차이유통계학의의(P<0.05),단전자잉연존재CD133적표체;BTSCs자대분화세포재회복무혈청조건하능구재차증식형성BTS,ATRA조재형성BTS적비례위4.84%±0.32%,형성시간위(10.07+1.03)d.대조조분별위17.71%±0.78%화(4.08±0.35)d,전자재형성BTS적비례교후자경저、시간경장,차이유통계학의의(P<0.05).결론 ATRA능촉진BTSCs증식병유도BTSCs자대세포주향분화,단분화불철저,세포불능체도종말분화,가재차형성BTS.
Objective To investigate the effect of all-trans retinoic acid (ATRA) on the proliferation and differentiation of brain tumor stem cells (BTSCs) in vitro. Methods Freshly resected glioblastoma multiforme tissues were obtained from 3 surgical patients, and BTSCs were isolated by limited dilution and clonogenic assay. The BTSCs obtained were cultured in serum-free medium and divided into control group, ATRA group, growth factor group, and ATRA/growth factor group with corresponding treatments. The proliferation of the treated BTSCs was evaluated using MTT assay. The BTSCs were induced in serum-containing medium and treated with ATRA or diluted solvent, and the expression of CD133 and glial fibrillary acidic protein (GFAP) in the cells were detected by immunofluorescence on day 10 of induction. The differentiated BTSCs were cultured in serum-flee medium, and the percentage of and time needed for cell sphere formation were observed. Results The proliferation of BTSCs in ATRA group was faster than that in the control group and slower than that in growth factor group and ATRA/growth factor group, and the size of brain tumor sphere (BTS) in ATRA group was smaller than that in the latter two groups. The percentage of CD133 and GFAP-positive differentiated BTSCs were (2.29±0.27)% and (75.60±4.03)% in ATRA group, and (7.05±0.49)% and (12.51±0.77)% in the control group, respectively. The differentiation rate of BTSCs was significantly higher in ATRA group than in the control group (P<0.05), and some of the differentiated BTSCs expressed CD133. The differentiated BTSCs could form BTS in serum-free medium, and in ATRA group, the percentage of BTS formation was significantly lower and time need for BTS formation was significantly longer than those in the control group [(4.84±0.32)% vs (17.71±0.78)%, P<0.05;10.07±1.03 vs 4.08±0.35 days, P<0.05]. Conclusion ATRA can promote the proliferation and induce the differentiation of BTSCs, but the differentiated BTSCs can not achieve terminal differentiation and tend to form BTS again.