脑微血管内皮细胞和脑肿瘤干细胞共培养的体外实验研究

目的 研究体外培养条件下,脑微血管内皮细胞(brain microvascular endothelial cells,BMECs)对脑肿瘤干细胞(braintumor stem cells,BTSCs)增殖、自我更新及分化功能的影响.方法 应用Transwell小室建立BMECs与BTSCs共培养模型,并建立对照组,共培养14 d后,测量肿瘤球(brain tumor sphere,BTS)直径大小,检测BTSCs增殖曲线,测定单细胞克隆形成率.在含血清的培养基中培养10 d后,行CD133、Nestin、GFAP、DAPI免疫荧光染色,计数两组的CD133、Nestin、GFAP染色的细胞数及同一视野的DAPI染色细胞核数,观察两组之间的差异.结果 共培养组BTS直径是对照组的5.05倍,增殖快,共培养组有61.4%能形成下一代BTSCs,而对照组为39.0%.在含血清的培养基中培养10 d后,共培养组的GFAP阳性率较对照组低,而CD133、Nestin阳性率较对照组高.结论 BMECs能够促进BTSCs增殖、自我更新能力,保持BTSCs未分化状态.

Effect of brain microvascular endothelial cells on brain tumor stem cells in co-culture system in vitro

Objective To investigate the effect of the brain microvascular endothelial cells (BMECs) on proliferation, self - renewal and differentiation of the brain tumor stem cells (BTSCs) in vitro. Method To establish a co - cultural system between BMECs and BTSCs on Transwell inserts in vitro. While the control group was preformed. The co - culture was maintained for 14 days. Then the size of brain tumor spheres (BTS) and the growth curves were determined. The single - colone formed rates of two groups were determined. The BTSCs of two group were cultured in DMEM/F12 containing 10% fetal calf groups were determined.The BTSCs of two group were cultured in DMEM/F12 containing 10% fetal calf serum for 10 days. Then CD133,Nestin,GFAP and DAPI immunofluorescence staining were made. CD133, Nestin,GFAP positive rate of two groups were calculated separately. The differences between the two groups were observed. Results BTS that were co - cultured with BMECs increased in size up to 5. 05 times larger than those grown in control group. There were 61. 4% BTSCs of the co - cultural group generate next tumor spheres. But that of the control group was 39. 0%. After BTSCs were cultured in the culture medium with 10% fetal bovine serum for 10 days,we found that GFAP - positive rate of the co - cultural group was lower than that of the control group. While CD 133 -positive and Nestin - positive rate of the co-cultural group was higher than that of the control group. Conclusions Brain microvascular endothelial cells could promote the proliferation and self - renewal and maintain the undifferentiated state of brain tumor stem cells.