Acta Physiologica Sinica, April 25, 2009, 61 (2): 185-193
Received 2008-12-03 Accepted 2009-02-19
This work was supported by Natural Science Foundation of Education Department of Jiangsu Province, China (No. 05KJD310235).
**These authors contributed equally to this work.
*Corresponding author. Tel: +86-516-85802033; E-mail: email@example.com
Mobilization of bone marrow-derived Nkx2-5+ cardiac progenitor cells under
condition of acute myocardial ischemia
DONG Hong-Yan1,**, XU Zhi-Wei2,**, ZHANG Zhong-Ming2,*, YU Hong-Li1, XU Xia-Hong1
1Center of Neurobiological Research; 2Department of Cardiothoracic Surgery of Affiliated Hospital, Xuzhou Medical College, Xuzhou
Abstract: The present study aimed to observe the morphological distribution of bone marrow (BM)-derived Nkx2-5+ cardiac progeni-
tor cells (CPCs) in bone marrow niche and evaluate the effect of acute myocardial ischemia (AMI) on the mobilizion of BM-derived
Nkx2-5+ CPCs. Animal models of BALB/c mouse AMI, cerebral and hind-limb ischemia were established. Nanogold labeling method,
immunofluorescence and Western blot were used to identify the distribution of BM-derived Nkx2-5+ CPCs and the expressions of
Nkx2-5 protein in peripheral blood and BM after AMI. Meanwhile, in different ischemia organ models and after AMD3100 (SDF-1/
CXCR4 antagonist) pretreatment in AMI model, Nkx2-5 protein expressions in peripheral blood were also assayed. Nkx2-5+ CPCs
were found to locate in cavitas medullaris. The percentage of Nkx2-5+ CPCs in blood increased immediately after AMI. Nkx2-5 protein
expression in peripheral blood was also upregulated at the timepoint of 24 h post-AMI (P<0.01) and kept stable without further
enhancement from day 1 to day 7 post-AMI. In BM, Nkx2-5 protein expression was upregulated immediately after AMI and
downregulated afterwards (P<0.01). After AMD3100 pretreatment in AMI group, Nkx2-5 protein expression was significantly
inhibited in peripheral blood (P<0.05). In cerebral and hind-limb ischemia models, Nkx2-5 protein expressions were significantly lower
than that in AMI group (P<0.01), but with no significant difference to control group. These results suggest that Nkx2-5+ CPCs are
physiologically resident in BM and AMI initiates mobilization of BM-derived Nkx2-5+ CPCs in a predominant organ-specific manner.
In the procedure of mobilization, SDF-1 may play a critical role in a chemoattracted manner.
Key words: cardiac progenitor cells; bone marrow; Nkx2-5; mobilization; myocardial ischemia
急性心肌缺血对骨髓急性心肌缺血对骨髓 Nkx2-5+心脏祖细胞的动员作用 心脏祖细胞的动员作用
摘摘 要： 要：骨髓源性Nkx2-5+心脏祖细胞(bone marrow-derived Nkx2-5+ cardiac progenitor cells)具有高度特异性分化为心肌细胞的潜
员作用，探讨其细胞动员的可能机制。分别建立小鼠急性心肌缺血及脑、后肢急性缺血动物模型。采用纳米金 - 银免疫标
祖细胞的动员作用；应用SDF-1/CXCR4 通路特异性阻断剂AMD3100，分析SDF-1 在急性心肌缺血后对Nkx2-5+心脏祖细胞
心脏祖细胞比例显著增加(P<0.01)。心肌缺血早期(1 d)，外周血Nkx2-5 蛋白表达显著增加(P<0.01)，并可持续7 d；而此间，
骨髓中Nkx2-5 蛋白表达立即升高，随后则降低。应用AMD3100 阻断剂后，心肌缺血组外周血Nkx2-5 蛋白表达受到明显抑
Acta Physiologica Sinica, April 25, 2009, 61 (2): 185-193
关键词： 关键词：心脏祖细胞；骨髓；Nk x2 -5 ；动员；心肌缺血
The regenerative capacity of cardiomyocyte is poor so that
the heart itself can not repair serious injury and recover
function by self-proliferation. Numerous basic studies were
performed using a variety of cell types with the hope of
improving myocardial performance. The studies on adult
mammalian bone marrow (BM) mesenchymal stem cells
(MSCs) have given rise to the upsurge of experiments and
early-stage clinical trials to regenerate infarcted myocar-
dium[1-5]. It is undoubted that BM could be a convenient
source of autogeneic cell seeds without immunological
rejection and available in large quantities for transplantation,
thus, this solution represents a promising method.
However, many recent reports that are mainly concerned
about the efficacy and safety of MSCs-based myocardial
regeneration hold skeptical views to open an academic
debate[6,7]. Because the detailed mechanisms that MSCs
participate in myocardial regeneration remain highly
controversial, altered interest to identify specific BM-
derived cellular subset(s) for cardiomyocyte regeneration
has been evoked extensively.
A recent study has revealed that BM contains not only
haemopoietic stem cells (HSCs) and MSCs, but also a
crowd of differentiated tissue-committed stem cells
(TCSCs). TCSCs population maintains a high regenera-
tive capacity of terminally differentiated organs both under
basal condition and following local tissue injury. For
instance, after the onset of myocardial ischemia, one of
the TCSCs marked with Nkx2-5, GATA-4 and MEF-2C,
also known as BM-derived cardiac progenitor cells (BM-
derived CPCs), will be thereby mobilized into the periph-
eral blood circulation, via terminal homing to the ischemic
myocardia, to initiate endogenous cardiomyocyte regen-
Nevertheless, the morphological evidence of Nkx2-5+
CPCs residing in BM niche and the detailed mechanism of
BM-derived CPCs mobilization, specially, the dynamic
variation of Nkx2-5+ CPCs in BM after acute myocardial
ischemia (AMI), remain uncertain.
In this study, we set Nkx2-5, one of the pivotal factors
of cardiomyogenesis, as the key marker, firstly, to observe
the morphological distribution of BM-derived Nkx2-5+ CPCs
in BM niche, and secondly, to evaluate the effects of AMI
on the mobilizion of BM-derived Nkx2-5+ CPCs.
Meanwhile, we intended to reveal the impacts of ischemia
in different organs, besides the heart, on mobilizing BM-de-
rived Nkx2-5+ CPCs.
1 MATERIALS AND METHODS
BALB/c mice, 4 to 6 weeks old and weighing 16-25 g,
were obtained from Experimental Animal Centre of Xuzhou
Medical College. The animal studies were approved by the
Animal Care and Use Committee of the Xuzhou Medical
College. All animals received humane care in compliance
with the Guideline for Care and Use of Laboratory Animals
published by Jiangsu Province, China.
1.2 Animal models
Myocardial ischemia models were established by using
sequential Isoprenaline (ISO) (Shanghai Harvest Pharma-
ceutical Co., Ltd. China) injection. Briefly, high dose of
ISO (10 mg/kg) was intraperitoneally injected once daily
for 3 d with a 24-hour interval. As a negative control, ISO
was replaced by 0.9% normal saline. An individual electro-
cardiogram was recorded once daily after ISO
administration, and an ST-segment-elevation was consid-
ered as qualified model for further study. After total three
days of ISO administration, under basal anesthesia induced
by intraperitoneal injection of 2% Carbrital (40 mg/kg),
animal hearts were harvested, embedded in paraffin, sliced
into 5 μm sections and stained with Hematoxylin-Eosin for
To establish BALB/c mouse cerebral ischemia model,
the animal anesthesia was initially induced by intraperito-
neal injection of 2% Carbrital (40 mg/kg), and maintained
by inhalation of 1.5% Halothane in 100% O2 via face masks.
The animal spontaneous breath was kept. With an inserted
rectal probe, the body temperature was strictly controlled
at 37 ºC during the procedure until the animals were placed
in incubators. A midline cervical incision was made, taking
care not to damage the vagus nerve, bilateral common ca-
rotid arteries were exposed and isolated using 4/0 silk su-
ture line and the animals were allowed to stabilize for 5
min. The common carotid arteries were occluded using
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