这周三跟大家分享的是来自Oncotarget 的Glioma stem cells-derived exosomes promote the angiogenic ability of endothelial cells through miR-21/VEGF signal。
摘要：
胶质瘤干细胞（
GSCs
）在胶质母细胞瘤预后中起重要作用。外泌体（
EXs
）可通过递送微小
RNA
（
miRs
）介导细胞通信。成胶质细胞瘤具有高水平的
miR-21
，后者可以上调血管内皮生长因子（
VEGF
）表达。我们假设
GSC-EX
可以通过
miR-21 / VEGF
信号通路促进内皮细胞（
ECs
）的血管生成能力。我们从
U-251
细胞株中分离出
GSC
，后者具有干细胞标记
CD133
。通过转染或不转染
scramble
或
miR-21 mimic
的方法，我们得到
GSC-EXs
con
，
GSC-EXs
sc
和
GSC-EXs
miR-21
。人脑内皮细胞（
EC
）与
vehicle
，
GSC-Xs
con
，
GSCEXs
sc
共同培养或
GSC-EXs
miR-21
加
VEGF siRNA
（
siRNA
VEGF
）。
24
小时后，我们评估了
ECs
的血管生成能力，并测定了
miR-21
，
VEGF
和
p-Flk1 /VEGFR2
的水平。结果表明：
1
）超过
90
％的纯化
GSCs
表达
CD133;2
）通过
miR-21 mimic
转染，
VEGF
和
miR-21
在
GSCs
和
GSC-Exs
中提高
; 3
）与
GSC-EXs
con
或
GSC-EXs
sc
相比，
GSC-EXs
miR-21
更有效提高
Ecs
中
miR-21
、
VEGF
的水平及
p-Flk1 /VEGFR2
的比例。
4
）
GSC-EXs
miR-21
在促进血管生成能力方面也比其他更有效，其效果亦可被
siRNA
VEGF
预处理后显着降低。结论
:GSC-EXs
可以通过激活
miR-21 / VEGF /VEGFR2
信号通路，促进
ECs
的血管生成能力。
背景
：肿瘤干细胞参与肿瘤生长、放化疗耐受。
GSCs
参与肿瘤血管形成及血清去除后细胞死亡耐受。
miR-21
（肿瘤
miR
）存在于多种肿瘤中，可促进细胞分裂及迁移。
VEGF
在星形胶质细胞瘤血管生成有重要作用。过表达
miR-21
可以增加前列腺细胞系的
VEGF
的表达。
GSCs
可以产生高水平
VEGF
进而促进肿瘤血管形成。
实验流程及结果：
1.分离
GSCs
（
anti-CD133 conjugated beads
及流式分选）
Figure1: MACS purification of GSCs from U-251 cells and characterization of GSCs by flow cytometry.
GSCs were defined as CD133
+
cells, which were separated by using MACS and assessed by flow cytometry for determining the efficiency of purification.(
A
) Representative flow plots showing the percentage of CD133
+
cells in U-251 cells and the purified GSCs; left curve: isotype control; right curve: antibody; (
B
)summarized data showing the percentage of CD133
+
cells before and after MACS; *
p
< 0.05, vs. U-251 cells. Data are expressed as mean ± SEM;
n
=3/group. MACS: magnetic activated cell sorting
2.通过转染改变GSCs的miR-21水平，并测定VEGF mRNA的水平
Figure 2: Up-regulation of miR-21 by miR-21 mimic transfection increased VEGF mRNAexpression in GSCs.
The purified GSCs were transfected with miR-21 for 48 hrs, and the levels of miR-21 and VEGF were determined by qRT-PCR. (
A
–
B
)the levels of miR-21 and VEGF in different types of GSCs; *
p
< 0.05,vs. GSCs
con
or GSCs
sc
; Data are expressed as mean ± SEM;
n
=4/group
3.检测GSC-EXsmiR-21中miR-21和VEGF水平
Figure 3: Up-regulation of miR-21 in GSCs by miR-21 mimic transfection increased the levels of miR-21 and VEGF mRNA expression in GSC-EXsmiR-21.
(
A
) representative NTAplots showing the same pattern of the size distribution of the three types ofGSC-EXs; (
B
) summarized data showing the concentration of EXs. (
C
)CD63 expression in the collected EXs. *
p
< 0.05, vs. GSCs; (
D
–
E
)summarized data showing the levels of miR-21 and VEGF in various types ofGSC-EXs. *
p
< 0.05, vs. GSC-EXs
con
or GSC-EXs
sc
; Data are expressed as mean ± SEM;
n
=4/group.
4.GSCs
来源外泌体被
Ecs
吞噬后物质含量变化
Figure 4: Incorporation of various GSC-EXs into ECs and GSC-EXsmiR-21 had better effects on increasing miR-21 level and VEGF secretion of ECs.
(
A
) left:Representative images showing the incorporation of GSC-EXs into ECs; bar: 100μm; red: PKH26 labeled GSC-EXs; blue: DAPI counterstained nucleus; right: summarized data showing the fluorescence intensity in ECs co-cultured with the three types of GSC-EXs; (
B
) summarized data showing the level of miR-21in ECs co-cultured with the three types of GSC-EXs; (
C
) summarized data showing the concentration of VEGF in the culture medium of ECs co-cultured withGSC-EXs. *
p
< 0.05, vs. vehicle;
+
p
< 0.05, vs. GSC-EXs
con
or GSC-EXs
sc
; Data are expressed as mean ±SEM;
n
= 4/group.
5.GSCs来源外泌体被Ecs吞噬功能变化
