Endocrine
DOI 10.1007/s12020-013-9984-0
ORIGINAL ARTICLE
Effect of combined therapy of human Wharton’s jelly-derived
mesenchymal stem cells from umbilical cord with sitagliptin
in type 2 diabetic rats
Jianxia Hu • Fang Wang • Ruixia Sun • Zhongchao Wang
Xiaolong Yu • Li Wang • Hong Gao • Wenjuan Zhao •
Shengli Yan • Yangang Wang
Received: 6 February 2013 / Accepted: 7 May 2013
Ó Springer Science+Business Media New York 2013
Abstract Type 2 diabetes mellitus is the most common
endocrine disease all over the world, while existing ther-
apies can only ameliorate hyperglycemia or temporarily
improve the response to insulin in target tissues, they
cannot retard or improve the progressive b-cell dysfunction
persistently. Combined therapy of stem cells and sitagliptin
might resolve this problem, we verified this hypothesis in a
diabetic rat model. Except ten Wistar rats in normal control
group, diabetic rats were divided into diabetic control
group, WJ-MSCs group, sitagliptin group and WJ-
MSCs ? sitagliptin group and received homologous ther-
apy. Ten weeks after therapy, diabetic symptoms, FPG and
GHbA1c in WJ-MSCs group, sitagliptin group and WJ-
MSCs ? sitagliptin group were significantly less than
those in diabetic control group (P \ 0.05), while fasting
C-peptide and number of b cells in WJ-MSCs group and
WJ-MSCs ? sitagliptin group was significantly higher
than those in diabetic control and sitagliptin group
(P \ 0.01). Glucagon and number of a cells in sitagliptin
group and WJ-MSCs ? sitagliptin group were significantly
lower than those in WJ-MSCs group and diabetic control
group (P \ 0.01). No symptoms of rejection and toxic
effect were observed. Combined therapy of WJ-MSCs and
Jianxia Hu and Fang Wang contributed equally to this work.
J. Hu
L. Wang
H. Gao
Y. Wang (&)
Stem Cell Research Center, The Affiliated Hospital of Medical
College, Qingdao University, No. 16, Jiangsu Road,
Qingdao 266003, China
e-mail: qdyxylxy@126.com
F. Wang
R. Sun
Z. Wang
X. Yu
W. Zhao
S. Yan
Endocrinology Department, The Affiliated Hospital of Medical
College, Qingdao University, No. 16, Jiangsu Road,
Qingdao 266003, China
•
sitagliptin can effectively ameliorate hyperglycemia, pro-
mote regeneration of islet b cells and suppress generation
of islet a cells in diabetic rats, presenting a new therapy for
type 2 diabetes although the exact mechanisms are unclear.
Keywords Human umbilical cord mesenchymal stem
cells
Sitagliptin
Type 2 diabetes
Islet b cells
Introduction
Diabetes has become one of the most serious puzzles to
global public health, the total number of people with dia-
betes is projected to increase to 366 million in 2030 [1].
Type 2 diabetes mellitus (T2DM) is the most common
form of diabetes and characterized by a combination of
insulin resistance and pancreatic beta-cell dysfunction. The
mainstream therapies for T2DM include insulin sensitizers
and exogenous supply of insulin, but these drugs can only
ameliorate hyperglycemia or temporarily improve the
response to insulin in target tissues, they cannot retard or
improve the progressive b-cell dysfunction [2]. Recent
reports suggest that stem cells may assist in improving
metabolic control in subjects with T2DM [3].
A number of studies and clinical trials have revealed
that mesenchymal stem cells (MSCs) are capable of
reducing glucose levels in animals or subjects with type 1
and type 2 diabetes, but the exact mechanisms need to be
elucidated [4–6]. Based on current knowledge, it was
considered that the possible mechanism of the therapeutic
effect of MSCs on hyperglycemia might be its paracrine
effect on pancreatic islet, including inflammation sup-
pression and islet cells regeneration [7]. However, some
controversial studies have suggested that the limited
number of neogenetic b cells in vivo and the small amount
123

of insulin produced by these cells seemed to be inadequate
to maintain euglycemia [8, 9]. Our preliminary animal
study revealed that the intervenous infusion of umibilical
cord MSCs can stimulate the regeneration of both a and b
cells in mouse pancreatic islet, this can induce a increased
level of both insulin and glucagon and decreased blood
glucose, however, the exact mechanisms are not clear.
Sitagliptin is a dipeptidyl-peptidase IV (DPP4) inhibitor
shown to increase insulin release and decrease glucagon
levels by impacting on the a and b cells in pancreatic islet
to prevent the inactivation of the incretin hormones glu-
cagon-like peptide-1 (GLP-1) and glucose-dependent in-
sulinotropic polypeptide (GIP) [10, 11]. The clinical trials
reviewed have demonstrated that sitagliptin, either alone or
in combination with metformin or thiazolidinediones, is
effective in reducing HbA1c values, fasting plasma glucose
(FPG) levels, and 2-h postprandial plasma glucose (PPG)
levels in patients with type 2 diabetes [12, 13].
Combination of MSCs and sitagliptin might promote the
regeneration of islet b cells and decrease the mass of islet a
cells, so effectively increase insulin release and decrease
glucagon levels. We explored this hypothesis by investi-
gating the effect of combined therapy of intravenous
infusion of Wharton’s jelly-derived mesenchymal stem
cells (WJ-MSCs) from human umbilical cord and intra-
gastric administration of sitagliptin in a diabetic rat model.
Materials and methods
Animals
This study was approved by the Institutional Animal Eth-
ical Committee, Qingdao and Ethics Committee of the
Affiliated Hospital of Medical College, Qingdao university.
Sixty male Wistar rats with 7–8 weeks old, weighing
180–220 g, were purchased from Slac Laboratory Animal
Centor, Shanghai and housed in cages, fed on regular rat
chow and maintained under optimal ambience of temper-
ature (22–23 °C), light (12 h dark/light cycles), oxygen,
humidity (60 %) and ventilation till sacrificed. A high-fat
diet and low-dose streptozotocin (STZ) intraperitoneal
injection was administered to 50 rats to induce type 2
diabetes as previously reported [14]. The remaining 10 rats
fed with normal diet served as normal controls. A high-fat
diet consisting of 22 % fat, 48 % carbohydrate, and 20 %
protein, with a total calorific value 44.3 kJ/kg and a normal
pellet diet consisting of 5 % fat, 53 % carbohydrate, 23 %
protein, with total calorific value 25 kJ/kg, was ordered
from the Stoyer Center of Slac Laboratory Animal Centor,
Shanghai. After consuming the diet for 6 weeks, rats in the
diabetes mellitus (DM) group were intraperitoneally
injected with 30 mg/kg of STZ. Ten days later, FPG of
123
Endocrine
the rats in the DM group were measured, the rats with
FPG C11.1 mmol/L twice were considered as diabetic. The
diabetic rats were allowed to continue to feed on a high-fat
diet until the end of the study.
Experimental plan
Diabetic rats were randomly divided into four groups
(n = 10), including diabetic control group, WJ-MSCs
group, sitagliptin group and WJ-MSCs ? sitagliptin group.
Rats in diabetic control group were as diabetic controls; rats
in WJ-MSCs group were treated with intravenous infusion of
WJ-MSCs by vena caudalis at the first week and fifth week
after diagnosed as diabetic rats, the cell number was
1 9 106/rat; rats in sitagliptin group were treated with
intragastric administration of sitagliptin (Merck Sharp and
Dohme Italia SPA) for 10 weeks, the dose of sitagliptin was
10 mg/kg/day; rats in WJ-MSCs ? sitagliptin group were
treated with combination of WJ-MSCs plus sitagliptin as
described above (showed in Fig. 1).
Stem cell preparation
WJ-MSCs were provided by Human Umbilical Cord
Mesenchymal Stem Cell Bank, Shandong Province, China.
Umbilical cord was obtained from a healthy mother, age
27, born a healthy term fetus and no genetic family history,
no cancer history, no hepatitis B virus (HBV), hepatitis C
virus (HCV), human immunodeficiency virus (HIV),
Epstein-Barr virus (EBV), cytomegalovirus (CMV), and
syphilis in serum. Umbilical cord collection was approved
by the Institutional Medical Research Ethics Committee of
the local maternity hospitals. Fully informed consent was
obtained several weeks prior to delivery from the mother.
The preparation of WJ-MSCs were performed in the
laminar flow laboratory using a method previously
described [15], with some modifications. Briefly, the
umbilical cord was washed with phosphate-buffered saline
(PBS) twice and then dissected with scissors into pieces
approximately 1 cm3 in volume. These tissue pieces were
plated in a cell culture dish (Corning) in low-DMEM
medium supplemented with 5 % non-animal-derived
serum. Cell cultures were maintained in a humidified
atmosphere with 5 % CO2 at 37 °C. After 3 days of cul-
ture, the medium was replaced to remove the tissue and
non-adherent cells, and changed twice weekly thereafter.
Once 80 % confluence had been reached, the adherent cells
(passage 0) were detached with 0.125 % trypsin and pas-
saged in the cell culture dish. The WJ-MSCs were cultured
and expanded in laminar flow laboratory for four passages
to prepare final cell products which should be sterile and all
qualified for the examinations including aerobe, myco-
plasma, HBV, HCV, HIV, EBV, CMV, syphilis, and
Endocrine
Fig. 1 The experimental
procedure for this study
endotoxin testing. Also, cells were stained with a double
label and then analyzed by flow cytometry with a FACS
Calibur (BD). These cells expressed highly CD90, CD105,
CD73, and CD146 but not CD34, CD45, and HLA-DR.
Chromosomal karyotype of UC-MSC was normal.
Observation and assessment
All changes of rats, including fur, action, response, food
and drink, urine and body weight, were recorded carefully.
Body weight and glucometer tail-vein blood glucose levels
(OneTouch Horizon glucose measurement strips, Johnson
and Johnson Ltd, Milpitas, CA, USA) were measured bi-
weekly. Ten weeks after therapy, glycosylated hemoglobin
(GHbA1c), C-peptide, and glucagon were measured by
ELISA (Rat glycosylated hemoglobin/C-peptide/glucagon
Elisa kit, Mercodia, Sweden) as per the manufacturer’s
protocol.
Histologic examination
All rats were killed at the 10 weeks after therapy and tissue
of pancreas, heart, liver, and kidney were acquired. Tissue
specimens were fixed in 10 % paraformaldehyde for 24 h
and embedded in OCT. Six-micron-thick sections were
stained with H&E for light microscopy.
For immunohistochemistry, after deparaffinage and
hydration, tissue sections were soaked in 3 % H2O2 for
10 min, then sections were washed twice in distilled water
for 5 min, followed by incubation with antibody (anti-
insulin or glucagon, Sigma-Aldrich; 1:300) at 25 °C for
60 min. After washing with phosphate-buffered solution
(PBS), sections were incubated with secondary antibody
(Sigma-Aldrich; 1:300) at 25 °C for 25 min, then colora-
tion, counterstain with haematoxylin, dehydration with
gradient alcohol and mounting with neutral gum for light
microscopy.
Quantification of islet cells
Following immunofluorescence staining of the islets, the
numbers of different cell types (a, b) cells and total islet
cells in each islet were manually counted from the islet
microscopy images. For each cell type, 20–35 representa-
tive islets from 4 to 5 rats were counted. The islets con-
taining \30 cells were excluded.
Statistical analysis
All statistical analyses were performed using SPSSÒ ver-
sion 15.0 software (SPSS Inc., Chicago, USA). Results
were expressed as mean ± standard deviation (SD) unless
123

otherwise designated. Differences between the four dia-
betic groups at baseline were tested by one-way ANOVA
and post hoc analysis with Bonferroni correction for mul-
tiple comparisons. A two-way ANOVA was used to
determine the main effects of WJ-MSCs (plus or minus)
and sitagliptin (plus or minus) and their interaction. When
a significant interaction effect was identified, a one-way
ANOVA with Tukey’s multiple comparison post hoc test
was used to identify difference between groups. For
parameters where repeated measurements were taken over
time (i.e., FPG and GHbA1c), a two-way repeated-measure
ANOVA was performed. A P \ 0.05 was considered to be
statistically significant.
Results
Animal general characteristics
Success of fat-fed, STZ-induced T2DM rat model was con-
firmed by checking for blood glucose (FPG C11.1 mmol/L,
twice). Rats in normal group were good as before, the
hydroposia, cibation and body weight increased gradually.
Polydipsia, polyphagia, urorrhagia, nastiness, and hypoergy
were observed as expected among rats in diabetic groups,
and their body weight decreased as expected. There was no
significant difference in FPG and body weight of rats
between the four diabetic groups at the beginning of therapy.
After homologous therapy, symptoms described above
among rats in WJ-MSCs group, sitagliptin group and
WJ-MSCs ? sitagliptin group were improved, so as body
weight. However, at the end of therapy, body weight of rats
in WJ-MSCs group, sitagliptin group and WJ-MSCs ? si-
tagliptin group were still less than that in normal group
(P \ 0.05, showed in Fig. 2).
FPG and GHbA1c
There was an effect of WJ-MSCs (P = 0.001) and sitag-
liptin (P = 0.001) and their interaction (P = 0.001) for
repeated FPG and GHbA1c measurement. After therapy,
FPG in WJ-MSCs group, sitagliptin group and WJ-
MSCs ? sitagliptin group began to decrease. Two weeks
later, FPG reached the lowest level, after that, FPG in WJ-
MSCs and sitagliptin group showed a little fluctuation
while FPG in WJ-MSCs ? sitagliptin group continued as a
low level. Ten weeks after therapy, FPG and GHbA1c of
rats in WJ-MSCs group, sitagliptin group and WJ-
MSCs ? sitagliptin group were statistically improved
compared with those in diabetic control group (P \ 0.05),
especially for WJ-MSCs ? sitagliptin group (P = 0.004,
0.021), as showed in Fig. 3. FPG and GHbA1c in WJ-
MSCs ? sitagliptin group was a little higher than those in
123
Endocrine
450 Body Weight
400
body weight (g)
350
300
250
200
0 2w 4w 6w 8w 10w
normal control diabetic control
WJ-MSCs sitagliptin
WJ-MSCs + sitagliptin
Fig. 2 Difference in body weight between groups. Body weight were
measured bi-weekly. Compared with that in diabetic control group,
median body weight of rats in WJ-MSCs group, sitagliptin group and
WJ-MSCs ? sitagliptin group were improved after homologous
therapy. Time 0 was the day as the beginning of experiment. Median
body weight of rats in WJ-MSCs group, sitagliptin group and WJ-
MSCs ? sitagliptin group was significantly more than that in diabetic
control group but less than that in normal group after 4 weeks using
repeated measures ANOVA (*P \ 0.05)
normal group, but there was no statistical difference
(P = 0.342, 0.097).
Glucagon
Glucagon in plasma was affected by WJ-MSCs (P = 0.007)
and sitagliptin (P = 0.001). The interaction between WJ-
MSCs and sitagliptin was significant for glucagon
(P = 0.005), wherein sitagliptin alone had no greater role on
glucagon compared with WJ-MSCs ? sitagliptin (P =
0.059). Ten weeks after therapy, glucagon of rats in WJ-
MSCs group was significantly higher than that in other four
groups (P \ 0.05), while in sitagliptin group and WJ-
MSCs ? sitagliptin group, glucagon was obviously lower
than that in WJ-MSCs group and control group (P \ 0.01,
showed in Fig. 4). There was no significant difference
between sitagliptin group and WJ-MSCs ? sitagliptin group
(P = 0.087).
C-peptide
Fasting C-peptide in plasma was affected by WJ-MSCs
(P = 0.001) and sitagliptin (P = 0.014) and their interac-
tion (P = 0.003). Ten weeks after therapy, fasting C-pep-
tide of rats in WJ-MSCs group and WJ-MSCs ? sitagliptin
group was significantly higher than that in diabetic control
group (P = 0.001, 0.001) and sitagliptin group (P = 0.009,
0.004), but still lower than normal control group (P =
0.005, 0.008, showed in Fig. 4). There was no statistical
difference between WJ-MSCs group and WJ-MSCs ?
sitagliptin group (P = 0.074).
Endocrine
Fig. 3 Difference in level of FPG and GHbA1c between groups. FPG
were measured bi-weekly from beginning of therapy, and GHbA1c
were examined 10 weeks after therapy. Time 0 was the day as the
beginning of therapy. Median FPG and GHbA1c of rats in sitagliptin
group was significantly lower than that in diabetic control group using
repeated measures ANOVA (*P = 0.035, 0.029). Median FPG and
GHbA1c of rats in WJ-MSCs group and WJ-MSCs ? sitagliptin
group was significantly lower than that in diabetic control group using
repeated measures ANOVA (#P \ 0.01). There was no significant
difference in FPG between WJ-MSCs ? sitagliptin group and normal
group (P = 0.342, 0.097)
Histologic examination
Regeneration of islet b cells in WJ-MSCs group and WJ-
MSCs ? sitagliptin group was observed as expected when
compared with diabetic control group, moreover the b cells
regeneration in WJ-MSCs ? sitagliptin group was a little
more than that in WJ-MSCs group. Regeneration of islet a
cells in WJ-MSCs group was observed as expected com-
pared to diabetic control group, while the a cells regener-
ation in WJ-MSCs ? sitagliptin group was not obvious
(showed in Fig. 5).
Also, we quantified a and b cells in each islet. There was
an effect of WJ-MSCs for pancreatic b cells (P = 0.001).
The number of b cells in WJ-MSCs group and WJ-
MSCs ? sitagliptin group was obviously more than that in
Fig. 4 Difference in level of glucagon and fasting C-peptide between
groups. C-peptide and glucagon were measured 10 weeks after
therapy. Glucagon in WJ-MSCs group was higher than that in diabetic
control group (*P = 0.037). Glucagon in sitagliptin group and WJ-
MSCs ? sitagliptin group was lower than that in diabetic control
group (#P = 0.003, 0.009) and WJ-MSCs group (łP = 0.001, 0.001).
Fasting C-peptide in WJ-MSCs group and WJ-MSCs ? sitagliptin
group was higher than that in diabetic control group (#P = 0.001,
0.001) and sitagliptin group (§P = 0.009, 0.004), and lower than
normal control group (#P = 0.005, 0.008)
sitagliptin group (P = 0.001, 0.001) and diabetic control
group (P = 0.001, 0.001, showed in Fig. 6). b cells in WJ-
MSCs ? sitagliptin group was a little more than that in WJ-
MSCs group, but it was not significant (P = 0.086). Pan-
creatic a cells were affected by sitagliptin (P = 0.001) and
WJ-MSCs (P = 0.003) and their interaction (P = 0.001).
The number of a cells in WJ-MSCs group was more than that
in diabetic control group (P = 0.029), while number of a
cells in sitagliptin group and WJ-MSCs ? sitagliptin group
was significantly less than diabetic control group
(P = 0.003, 0.002) and WJ-MSCs group (P = 0.002,
0.001). There was no statistical difference in a cells between
sitagliptin group and WJ-MSCs ? sitagliptin group
(P = 0.053).
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Fig. 5 Difference in insulin and glucagon between groups by
immunohistochemistry (9200). Ten weeks after therapy, tissue of
pancreas were acquired to do histologic examination. Pancreas were
For heart, liver, kidney with HE stain, there was no
conspicuous difference in lymphocyte infiltration and his-
tomorphology between rats in normal control group, dia-
betic control group, and WJ-MSCs group (showed in
Fig. 7).
Side effect
There were no symptoms of rejection and toxic effect
observed by macroscopic observation, laboratory and his-
tological examination.
123
Endocrine
stained with H&E for light microscopy. Insulin and glucagon were
examined by immunohistochemistry
Discussion
Type 2 diabetes mellitus (T2DM) is the most common
endocrine disease all over the world, affecting more than
200 million people [16]. Characteristics of this disease
involve abnormalities in glucose and lipid metabolism,
including inadequate insulin secretion from pancreatic islet
b cells and resistance to insulin activity. The denominator
of beta-cell dysfunction in T2DM is inflammation leading
to loss of functional pancreatic endocrine mass and a need
for exogenous insulin therapy [17, 18]. However, so far,
Endocrine
Fig. 6 Difference in number of a and b cells in islet between groups.
Following immunofluorescence staining of the islets, the numbers of
different cell types (a, b) cells and total islet cells in each islet were
manually counted from the islet microscopy images. The number of b
cells in WJ-MSCs group and WJ-MSCs ? sitagliptin group was
higher than that in sitagliptin group (§P = 0.001, 0.001) and diabetic
control group (#P = 0.001, 0.001). There was no significant differ-
ence between WJ-MSCs group and WJ-MSCs ? sitagliptin group
(P = 0.086). The number of a cells in sitagliptin group and WJ-
MSCs ? sitagliptin group was lower than that in WJ-MSCs group
(łP = 0.002, 0.001) and diabetic control group (#P = 0.003, 0.002).
The number of a cells in WJ-MSCs group was higher than that in
normal control group and diabetic control group (*P = 0.001, 0.029)
there are no consummate therapeutic options able to effi-
ciently act not only on the tight glucose control but also on
the prevention of T2DM evolution and its complications.
Combination of MSCs and sitagliptin might present an
ideal option for T2DM. In our type 2 diabetic model, FPG
of rats in WJ-MSCs group was obviously decreased at
2 weeks after therapy, but after that, FPG increased slowly.
Previous studies in diabetic animal models have also
shown that MSCs are able to lower blood glucose levels
[19–22]. Nevertheless, the precise mechanisms underlying
these effects are still unclear. Very few MSCs could be
found to differentiate into functionally competent b cells
in vivo, it seems likely that there might be another mech-
anism underlying the therapeutic effect of MSCs in dia-
betes [20]. Recent studies have shown that MSCs can
produce a variety of trophic cytokines to improve the
microenvironment of the pancreas and promote expansion
of endogenous pancreatic stem cells [23–25]. Moreover,
the beneficial effect of a single infusion of MSCs in ame-
liorating hyperglycemia was maintained only for a limited
period. This rapid and relatively temporary effect of MSC
infusion in ameliorating hyperglycemia could not be ade-
quately explained by the partial restoration of islet function
[26]. Our results showed the levels of both C-peptide and
glucagon were increased after therapy in WJ-MSCs group,
also pancreatic histological examination showed the
regeneration of islet b cells and a cells. This indicated that
infusion of WJ-MSCs could efficiently ameliorated
hyperglycemia due to partly recovery of islet b cells, but it
did not last a longer time possibly due to the regeneration
of a cells or other unknown reasons.
Decreased FPG and obvious suppressed glucagon were
observed among rats in sitagliptin group in our study.
Histological results showed stationary b cells and depres-
sive a cells in islet. Sitagliptin, as an orally available DPP-
IV inhibitor, has been developed to be used as a once daily
treatment for T2DM with the ability of extending the
biological effect of incretin hormones through the inhibi-
tion of GLP-1 and GIP degradation [27–31]. In animal
models, continuous infusion of GLP-1 or injection of long-
acting GLP-1 mimetics has shown a remarkable glucose-
lowering efficacy, together with an ability to increase
b cells neogenesis and reduce a cells glucagon secretion
[32–34]. The results in our study confirmed this role of
sitagliptin.
As expected, rats in WJ-MSCs ? sitagliptin group
showed a combined effect of WJ-MSCs and sitagliptin.
FPG of rats in WJ-MSCs ? sitagliptin group was lower
than that in WJ-MSCs group and sitagliptin group, and the
fluctuation of glucose was very little, the increased
C-peptide and decreased glucagon were also observed.
Pancreatic histologic results of regeneration of islet b cells
and suppression of islet a cells in WJ-MSCs ? sitagliptin
group also confirmed the effect of combined therapy.
Moreover, there was not any side effect found among rats
in WJ-MSCs and sitagliptin treated group when compared
with normal and diabetic control group. All these results
indicated that combination of WJ-MSCs and sitagliptin
could be efficient and safe for type 2 diabetes.
In conclusion, combined therapy of WJ-MSCs and si-
tagliptin can efficiently ameliorate hyperglycemia, promote
123

Fig. 7 Histological difference in heart, liver, and kidney between
groups by HE stain (9200). Ten weeks after therapy, tissue of heart,
liver, and kidney were acquired to do histologic examination. There
regeneration of islet b cells and suppress generation of islet
a cells in diabetic rats, presenting a new therapy for type 2
diabetes although the exact mechanisms are unclear.
However, the clinical application of combined therapy of
WJ-MSCs and sitagliptin still need further studies and
clinical trials to elucidate the mechanisms and verify the
effect of this therapy.
Acknowledgments We appreciate all work done by Jiangsu Yu in
this study.
Conflict of interest The authors declare that they have no conflict
of interest.
Ethical standards The experiments comply with the current laws
of the country in which they were performed. This study was
approved by the Institutional Animal Ethical Committee, Qingdao
and Ethics Committee of the Affiliated Hospital of Medical College,
Qingdao University.
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