Down-regulation of
rpl15 gene expression inhibits development of early hematopoiesis in zebrafish- Journal of Third Military Medical University Vol. 43, Issue 17, Pages: 1611-1618(2021)
- 作者机构：
- 作者简介：
- 基金信息：the Regional Program of National Natural Science Foundation of China(31860325);the Project of Science and Technology Plan of Guizhou Province(黔科合基础〔2020〕1Y092)
DOI：10.16016/j.1000-5404.202103070
CLC：Received：07 March 2021，
Published：15 September 2021
- 稿件说明：
移动端阅览
Weijun ZHOU, Dongzhen ZHOU, Tao PENG, et al. Down-regulation of
rpl15 gene expression inhibits development of early hematopoiesis in zebrafish[J]. Journal of Third Military Medical University, 2021, 43(17): 1611-1618.Weijun ZHOU, Dongzhen ZHOU, Tao PENG, et al. Down-regulation of
rpl15 gene expression inhibits development of early hematopoiesis in zebrafish[J]. Journal of Third Military Medical University, 2021, 43(17): 1611-1618. DOI： 10.16016/j.1000-5404.202103070.
摘要
目的
2
探究核糖体蛋白L15（ribosomal protein l15
rpl15
）基因表达下调对斑马鱼早期造血发育的影响。
方法
2
设计并合成
rpl15
吗啉代反义寡核苷酸（morphilino antisense oligo
MO），通过显微注射到单细胞期斑马鱼（0~0.75 hpf）的受精卵中来下调
rpl15
的表达，通过qRT-PCR和Western blot技术验证下调
rpl15
基因表达的有效性。使用体式显微镜分别观察注射
rpl15
MO（MO）组与野生型（WT）组的发育情况。O-dianisidine染色检测血红蛋白表达的情况，qRT-PCR检测红系特异性转录因子
hbae3
、
hbbe1
的表达变化。全胚原位杂交和qRT-PCR检测红系特异性转录因子
gata1
、髓系转录因子
pu.1
、粒系转录因子
mpo
、定向造血标志转录因子
runx1
和
c-myb
、原始祖细胞造血标志物
scl
和
lmo2
等早期造血发育过程的关键转录因子的表达变化。TUNEL实验检测造血组细胞的凋亡变化。
结果
2
显微注射
rpl15
MO能显著下调斑马鱼
rpl15
的表达
与WT组相比，MO组表现出体节弯曲、发育迟缓、心包水肿等异常表型。MO组血红蛋白合成量与
hbae3
、
hbbe1
的表达量相较于WT组明显减少（
P
<
0.05）。原位杂交及qRT-PCR结果表明，
gata1
的表达明显减少（
P
<
0.05），
pu.1
、
mpo
、
runx1
、
c-myb
的表达无明显变化，
scl
的表达明显减少（
P
<
0.01），
lmo2
的表达无明显变化。TUNEL实验显示造血祖细胞的凋亡无明显变化。
结论
2
下调
rpl15
的表达抑制了斑马鱼早期的造血发育，并可能与
gata1
和
scl
表达明显降低有关。
Abstract
Objective
2
To investigate the effect of down-regulation of ribosomal protein l15 (
rpl15
) gene on early hematopoietic development in zebrafish.
Methods
2
The
rpl15
morphilino antisense oligo (MO) was designed and synthesized to down-regulate the expression of
rpl15
by microinjection into embryo of zebrafish (0~0.75 hpf) at the unicellular stage. qRT-PCR and Western blotting were used to verify the effectiveness of down-regulating
rpl15
gene. The development of the injected (MO) and wild-type (WT) groups were observed separately using a somatic microscope. O-dianisidine staining was used to detect the expression of hemoglobin. qRT-PCR was used to detect the changes in the expression of the red lineage-specific transcription factors
hbae3
and
hbbe1
. Whole embryo in situ hybridization and qRT-PCR were performed to detect the changes in the expression of key transcription factors of early hematopoietic development such as red lineage-specific transcription factor
gata1
myeloid transcription factor
pu.1
granulocyte marker
mpo
targeted he matopoietic marker transcription factors
runx1
and
c-myb
and primitive group cell hematopoietic markers
scl
and
lmo2
. TUNEL assay was used to detect the changes of apoptosis in hematopoietic group.
Results
2
Microinjection of
rpl15
MO successfully down-regulated the expression of
rpl15
in zebrafish
and the MO-injected group exh
ibited abnormal phenotypes such as body segment curvature
developmental delay
and pericardial edema when compared with the control group. Hemoglobin synthesis was significantly reduced in the MO group than the control group. The results of in situ hybridization indicated that the expression of
gata1
was significantly reduced (
P
<
0.05)
but the mRNA levels of
pu.1
mpo
runx1
c-myb
and
lmo2
were not affected
while that of
scl
was obviously reduced (
P
<
0.05). TUNEL assay showed no significant change in apoptosis of hematopoietic progenitor cells.
Conclusion
2
Down-regulation of
rpl15
inhibits the early hematopoietic development of zebrafish
and it may be related to the significantly reduced expression of
gata1
and
scl
.
关键词
Keywords
references
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