In vitro analysis of the proliferative capacity and cytotoxic effects of ex vivo induced natural killer cells, cytokine-induced killer cells, and gamma-delta T cells
Niu et al. BMC Immunology
In vitro analysis of the proliferative capacity and cytotoxic effects of ex vivo induced natural killer cells, cytokine-induced killer cells, and gamma-delta T cells
Chao Niu 0
Haofan Jin 0
0 Equal contributors Cancer Center, the First Hospital of Jilin University , 71 Xinmin Street, Changchun 130021 , China
Background: Recent studies have focused on the significant cytotoxicity of natural killer (NK) cells, cytokine-induced killer (CIK) cells, and gamma-delta (γδ) T cells in tumor cells. Nevertheless, the therapeutic features of these cell types have not been compared in the literature. The aim of this study was to evaluate the feasibility of activation and expansion of NK, CIK, and γδ T cells from cancer patients in vitro, and to clarify the differences in their antitumor capacities. Methods: NK, CIK, and γδ T cells were induced from the peripheral blood mononuclear cells of 20 cancer patients by using specific cytokines. Expression of CD69, NKG2D, CD16, granzyme B, perforin, IFN-γ, and IL-2 was measured by flow cytometry. Cytokine production and cytotoxicity were analyzed by enzyme-linked immunosorbent assay and Calcein-AM methods. Results: NK cell proliferation was superior to that of CIK cells, but lower than that of γδ T cells. NK cells had a much stronger ability to secrete perforin, granzyme B, IFN-γ, and IL-2 than did CIK and γδ T cells, and imparted significantly higher overall cytotoxicity. Conclusions: Expanded NK cells from cancer patients are the most effective immune cells in the context of cytokine secretion and anti-tumor cytotoxicity in comparison to CIK and γδ T cells, making them an optimal candidate for adoptive cellular immunotherapy.
Natural killer cells; Cytokine-induced killer cells; Gamma-delta T cells; Cytotoxicity
Cellular immunity plays an essential role in anti-tumor
activity. Immunocyte activity is often compromised in
tumor patients, due to the inhibitory tumor
environment. Therefore, numerous preliminary studies have
demonstrated the safety and efficacy of adoptive
cellular immunotherapy (ACI)—consisting of ex vivo
expansion and activation of patient immunocytes followed by
reinfusion . ACI has become a promising, innovative
strategy for personalized cancer therapy . Due to the
complexity of immune escape—such as antigen loss,
MHC class I down-regulation, and the expression of
inhibitory molecules—specific immune cells such as
cytotoxic T lymphocytes (CTL) have limited utility in cancer
therapeutics . Non-specific immune effector cells such as
natural killer (NK) cells, cytokine-induced killer (CIK) cells,
and gamma-delta (γδ) T cells have better potential, since they
have no MHC restriction, contribute to front-line anti-tumor
surveillance, and bridge the gap between innate and adaptive
immunity [4, 5]. These cell types share many common
mechanisms, including NKG2D and perforin-mediated
cytotoxicity, and cytokine secretion [6–8]. However, each of them
has unique features, and their therapeutic effects have been
shown in ex vivo studies and clinical trials.
NK cells, which are large, granular CD3−CD56+
lymphocytes, can be rapidly activated to spontaneously attack certain
abnormal cells in the body, particularly cancerous or
virusinfected cells [9, 10]. Individuals who have low NK cell activity
are at increased risk of developing cancer [11, 12]. Several
clinical trials have confirmed the therapeutic benefit and safety of
NK cell adoptive infusion [13, 14]. CIK cells are
heterogeneous. The main effector cells are CD3+CD56+ T cells, which
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exhibit both the powerful anti-tumor effect of T cells
and the non-MHC restriction of NK cells . Recent
clinical studies have indicated that CIK cells can
significantly improve progression-free survival, overall survival,
and effective clinical response in cancer patients . In
addition to NK and CIK cells, γδ T cells may display the
same cytotoxicity as NK and CIK cells, and act as
For the past decade, ACI with NK, CIK, and γδ T cells
has been a primary focus in cancer therapy, especially
for hematological malignancies such as leukemia,
lymphoma, and multiple myeloma [17–20]. However, there has
been no direct comparison of these cell types. In this
study, we evaluated the feasibility of in vitro expansion
of each immune cell type and compared their antitumor
activity against various forms of hematological cancers
with the aim of providing in vitro evidence for their use
in ACI cancer therapies.
Cell quantification and expansion
NK, CIK, and γδ T cells were induced from the PBMCs
of 20 cancer patients. After 14 days induction,
immunocyte viability exceeded 85 % without contamination. All
cells were able to propagate in vitro: NK cells to a
median of 359-fold (250–669-fold, n = 20), CIK cells
to 253-fold (120–621-fold, n = 20), and γδ T to 550-fold
(159–879-fold, n = 20); γδ T cells had the strongest
amplification ability (Fig. 1).
After induction and expansion, the percentage of
expanded NK (r = 0.339, p = 0.001), CIK (r = 0.358, p = 0.003),
and γδ T (r = 0.344, p = 0.004) cells showed a slight positive
correlation with their percentage in the patients’ blood
(Spearman’s test). These data suggest the percentage of
Fig. 1 Fold expansion of NK, CIK, and γδ T cells. Fold expansion was
calculated by dividing the absolute output number of expanded NK,
CIK, and γδ T cells after 14 days of culture by the respective number
on day 0. Results are expressed as mean ± SD, n = 20; * p < 0.05,
** 0.001 < p < 0.01, *** p < 0.001
immune cells in the patients’ blood likely has little
effect on the ex vivo induction of these immune cell
types, further demonstrating the stability and broad
applicability of our methods.
Immunophenotyping of expanded immune cells
The percentages of NK (CD56+CD3−), γδ T (Vγ9+CD3+),
and CIK (CD56+CD3+) cells before and after induction
were 16.1 (1.7–29.7) vs. 79.7 (62.1–98.4), 4.4 (0.9–10.2)
vs. 65.9 (40.2–98.2) and 12.7 % (1.6–21.1 %) vs. 35.4 %
(16.3–55.6 %), respectively. A portion of γδ T cells
(10.2–45.9 %) was CD56-positive, and nearly no
expression of CD4 or CD8 was observed in the population.
However, the majority of CIK cells were CD8-positive
NK and CIK cells in patient blood rarely express
CD69, but γδ T cells exhibited high expression of this
marker. After induction and expansion, activated NK
and CIK cells expressed much higher levels of CD69.
However, there was only a slight increase in CD69
expression on γδ T cells before and after induction. After
NKG2D induction, only γδ T cells showed a very strong
increase (Fig. 3a). However, CD16 expression differed
between immune cell types. NK cells had the highest
CD16 expression at 78.7 % (50.2–98.2 %). In contrast,
some of the CD3+CD56+ CIK cells (30.7, 6.9–59.6) and
γδ T cells (11.7 %, 2.6–37.7 %) were also CD16-positive
(Fig. 3b). After induction, inhibitory KIR CD158a and
CD158b on NK cells were down-regulated. Representative
results from one of these patients are shown in Fig. 3c.
All three induced immune cells expressed perforin.
NK cells showed much higher perforin production than
the other two expanded immune cell types. γδ T cells
showed slightly higher perforin production than CIK
cells (Fig. 4a). Almost all NK (97.3 ± 2.2) and CIK
(94.8 % ± 5.2 %) cells and a majority of γδ T cells
(72.3 % ± 13.1 %) were granzyme B-positive. However,
NK cells yielded the strongest expression of granzyme B
Cytokine secretion of the expanded cells
Nearly all of the expanded NK cells and half of the CIK
and γδ T cells were IFN-γ-positive. About 40 % of the
NK cells were IL-2-positive, while only a small portion
of CIK and γδ T cells were positive for IL-2 (Fig. 5a). NK
cells were much more efficient secretors of IFN-γ and
IL-2 than were CIK and γδ T cells (Fig. 5b). Subsets of
NK cells or CIK and γδ T cells secreted a very high level
of IFN-γ, yet only a small amount of IL-2 was detected
in the supernatant of the CIK and γδ T cells. No
differences in INF-γ and IL-2 secretion were observed between
CIK and γδ T cells. Very low levels of IL-4, IL-6, and
IL10 were detected in the supernatants of NK, CIK, and γδ
T cells (Fig. 4b).
Fig. 2 The percentage of NK, CIK, and γδ T cells before and after induction. a The percentage of NK cells (CD3−CD56+) before and after induction.
b The percentage of γδ T (CD3+Vγ9+) and CIK (CD3+CD56+) cells before and after induction. γδ T and CIK cells were immunostained with CD4 and
CD8. Representative results from a single patient are shown
Cytotoxicity of expanded immune cells against
hematologic malignancies cell lines
We compared the cytotoxicity of NK, CIK, and γδ T
cells against hematologic tumors, as bulk expanded
immune cells tested in the Calcein-AM assay showed
substantial cytotoxic capacity. NK cells exhibited the
most significant cytotoxicity in K562 (Fig. 6a), NB4
(Fig. 6b), HL-60 (Fig. 6d), and U266 (Fig. 6e) cells. Both
NK and γδ T cells produced stronger cytotoxic effects
against Jurkat cells than CIK cells (Fig. 6c). Although all
Fig. 3 (See legend on next page.)
(See figure on previous page.)
Fig. 3 Expression of CD69, NKG2D, and KIR on induced immune cells. a The expression of CD69 and NKG2D were analyzed by flow cytometry (gray
histogram). CD69 and NKG2D expression peaks are shifted to the right in expanded cells versus pre-induction cells (black histogram). b Expression of
CD16; gray histograms depict isotype controls. c Expression of KIR (CD158a and CD158b) on NK cells before and after induction. Gray histograms depict
isotype controls and the black histogram depicts the specific antibody. Data from one representative patient is shown
three expanded cell types exhibited limited cytotoxicity
against Raji cells compared to other target cells, NK cells
exerted the strongest antitumor activity (Fig. 6f ).
Interestingly, the addition of rituximab significantly enhanced
Raji lysis by all cell types; however, NK cells continued
to show a greater synergistic cytotoxicity in Raji cells
with rituximab in comparison to CIK and γδ T cells.
Nevertheless, there were no notable differences in
cytotoxicity against Raji cells between CIK and γδ T cells in
the presence of rituximab (Fig. 7).
Although many studies have shown the importance of
NK, CIK, and γδ T cells in the prevention of tumor
relapse and metastasis in mice and humans [8, 21, 22], few
studies have directly compared their cytotoxic effects to
identify which would be the optimal candidate for
clinical application. There are many issues to be considered
prior to clinical application, including the number of
introduced cells, the feasibility of in vitro expansion, and
their respective anti-tumor activities.
Obtaining an adequate number of immune cells is a
key challenge for ACI application in a clinical setting
. Studies indicate that sufficient numbers of CIK and
γδ T cells can be obtained by current methods [3, 20];
however, methods for the expansion of NK cells in vitro
have being investigated by using feeder cells, including
irradiated allogenic PBMCs , K562 cells , and
Epstein-Barr virus-transformed lymphoblastoid cell lines
. Although these NK cell infusions are well tolerated
and partially effective, current methods for NK cell
preparation involve complex separation protocols and often
require ethical approval that render them expensive and
sub-optimal or unfeasible for large-scale clinical studies
. To overcome these disadvantages, we examined
whether the use of CD16 antibody-coated flasks could
be used in place of feeder cells. Our studies demonstrate
that a substantial number of NK cells could be
generated, ranging from 250–669-fold expansions, after
14 days induction and culture. Importantly, this is
comparable to the yields of feeder-cell methods . In
addition to NK cells, we amplified CIK and γδ T cells in
Fig. 4 Perforin and granzyme B production in expanded NK, CIK, and γδ T cells. Three types of immune cells from twenty cancer patients were
harvested after 14 days induction in vitro. NK and CIK cells were stained with mAbs to CD3 and CD56, and γδ T cells were stained with CD3 and
Vγ9. After fixation and permeabilization, cells were stained for perforin and granzyme B using specific conjugated anti-human cytokine mAbs.
a Perforin-positive cells and mean fluorescence intensity (MFI) of NK, CIK, and γδ T cells. b Granzyme B-positive cells and MFI of NK, CIK, and γδ T
cells. Results are expressed as mean ± SD, n = 20. * p < 0.05, ** 0.001 < p < 0.01, *** p < 0.001, ns p > 0.05
Fig. 5 Cytokine production in NK, CIK, and γδ T cells. a Intracellular staining of IFN-γ and IL-2 in NK, CIK, and γδ T cells. Intracellular levels of IL-2
and IFN-γ were measured as described for perforin and granzyme B detection. b Extracellular cytokine production of NK, CIK, and γδ T cells. The
culture supernatants were collected and analyzed by ELISA for IFN-γ, IL-2, IL-4, IL-6, and IL-10. Results are expressed as the mean ± SD, n = 20. * p < 0.05,
** 0.001 < p < 0.01, *** p < 0.001, ns p > 0.05
vitro using conventionally optimized methods. Although
several studies have shown that CIK cells easily
proliferate in vitro , our investigation shows that the
proliferative ability of NK cells in our culture setup was
superior to that of CIK cells. We also found that the
ratios of NK, CIK, and γδ T cells in the blood of cancer
patients had no effect on their amplification efficiency.
These results demonstrate that all three expanded
immunocyte types have the potential for in vitro
expansion to generate sufficient numbers for clinical
The antitumor capacity of effector cells is another
challenge in ACI. Our study assessed the phenotype,
cytokine production, and cytotoxicity of the expanded
cell types. Since NK, CIK, and γδ T cells have shown
significant efficacy in the treatment of hematological
Fig. 6 Anti-tumor cytotoxicity of induced immune cells in vitro. Cytolysis against hematological malignancy targets K562 (a), NB-4 (b), Jurkat
(c), HL-60 (d), U266 (e), and Raji (f) cells were compared at 10:1 effector-target ratios (E:T). NK cells showed the most significant cytotoxicity.
Results are expressed as the mean ± SD, n = 20. * p < 0.05, ** 0.001 < p < 0.01, *** p < 0.001, ns p > 0.05
Fig. 7 Antibody-dependent cellular cytotoxicity of induced NK, CIK,
and γδ T cells. Raji cells were pre-coated with rituximab (Genentech)
at 10 mg/mL for 30 min. The ADCC capacity at 10:1 effector-target
ratios (E:T) were measured by the calcein-AM method. Effector cells
were bulk samples of the cultured cells (i.e., no cell sorting was done).
Results are expressed as mean ± SD, n = 20. * p < 0.05, ** 0.001 <
p < 0.01, *** p < 0.001, ns p > 0.05
malignancies in vivo and in vitro, we selected leukemia
and multiple myeloma cell lines as target cells for our
There are two main NK cell subsets distinguished by
the level of CD56 expression in human blood, namely
CD56dim and CD56 bright . CD56dim cells are fully
matured and strongly express CD16, a low-affinity Fc
immunoglobulin G receptor that allows immune cells to
participate in ADCC . In contrast, CD56bright cells
are more immature and are primarily considered to be
cytokine producers with a limited role in cytolytic
responses . The NK cells expanded in this study had
very high CD16 (Fig. 3b) and CD56 expression, and may
represent a subset of NK cells undergoing
differentiation. NK cells also express inhibitory and stimulatory
receptors, such as killer immunoglobulin-like receptor
(KIR), CD16, and NKG2D. The cytotoxic reactivity of
NK cells results from the integration of signals from the
stimulatory and inhibitory receptors as they engage
ligands on the target cell surface. Our studies show that
NK cells propagated in CD16 antibody-coated flasks
exhibit increased expression of NKG2D and CD69, and
decreased expression of inhibitory KIR. Intracellular and
extracellular cytokine detection indicated that NK cells
were more potent producers of IFN-γ than CIK and γδ
T cells (Figs. 4a and 5e). IFN-γ enhances antigen
presentation by dendritic cells and stimulates production of the
CXC chemokines MIG (CXCL9) and IP-10 (CXCL-10),
which inhibit tumor angiogenesis and recruit
CXCR3bearing effector cells to tumor sites [29, 30]. Therefore, NK
cells producing high amounts of IFN-γ could contribute
greatly to tumor regression during cancer immunotherapy.
Since induced immunocytes are usually used for ACI
without purification, these cultured cells were directly
harvested for cytotoxicity detection. In comparison to
CIK and γδ T cells, the NK cells expanded in this study
were significantly more cytotoxic toward many kinds of
malignant cells lines including NK-resistant Raji cells
(Fig. 6f ). Raji cells normally elude NK cell recognition
due to their expression of inhibitory KIR ligands
(HLACw3 and HLA-Cw4) and a lack of NKG2D ligands
(MICA) [31, 32]. The increased cytotoxic effects
observed with our cultured NK cells likely result from their
down-regulation of inhibitory KIR–CD158a and CD158b
that engage HLA-Cw4 and HLA-Cw3, respectively.
ADCC is another mechanism by which cells convey
antitumor activity, which is dependent on immunocyte
CD16 expression. NK cells showed the highest
expression of CD16 and the strongest ADCC efficacy in the
presence of rituximab (Fig. 7). This implies that
largescale NK cell expansion can promote the development
of an effective combination therapy for cancer. While few
differences were observed in ADCC function between
CIK and γδ T cells, a significant difference in CD16
expression was detected between CD3+CD56+ CIK and γδ T
cells. Interestingly, CIK cells displayed a stronger cytotoxic
effect toward K562 and HL-60 cells than γδ T cells, in
contrast to our observations in Jurkat cells.
In this study, we established a cytokine-based expansion
system for NK cells with the aim of developing them for
clinical application. NK cells not only have a higher
expansion capacity, but also much stronger antitumor
activity in cytokine production, director cytotoxicity, and
ADCC effect. Our analyses found that NK cells exhibit
stronger cytotoxicity against lymphoma, leukemia, and
myeloma cells than expanded CIK and γδ T cells.
Therefore, our study provides ex vivo evidence for the direct
comparison of NK, CIK, and γδ T cells. We also provide
experimental evidence for their clinical application in
Cells and culture
Human leukemia (K562, HL-60, NB4, and Jurkat) cells,
multiple myeloma (U266) cells, and lymphoma (Raji)
cells were cultured in RPMI-1640 medium (Gibco, USA)
supplemented with 10 % heat-inactivated fetal bovine
serum (Gibco), 100 U/mL penicillin, and 100 mg/mL
streptomycin (Invitrogen, USA) at 37 °C in a humidified
5 % CO2 incubator.
Isolation of peripheral blood mononuclear cells
Heparinized peripheral blood samples were obtained
from 20 volunteer cancer patients. Blood samples were
centrifuged at 1800 × g for 10 min and plasma was
transferred to new tubes. Peripheral blood mononuclear cells
(PBMCs) were isolated by density gradient
centrifugation using Ficoll (Nycomed Pharma AS, Norway) at
800 × g for 30 min.
Expansion of NK, CIK, and γδ T cells
NK cells were expanded as described . Briefly, PBMCs
were resuspended in AIM-V (Invitrogen) medium with
5 % auto-plasma, 500 U/mL IL-2, 2 ng/mL IL-15 (both
from Miltenyi Biotec, Germany), and 1 μg/mL OK432
(Shandong Luya Pharmaceutical Co., China) at a
concentration of 1 × 106 cells/mL. PBMCs were cultured in flasks
coated with anti-CD16 (Beckman, USA) for 24 h at 39 °C
in a humidified 5 % CO2 atmosphere. The cells were
cultured in AIM-V medium supplemented with 5 %
autoplasma, 1000 U/mL IL-2, and 2 ng/mL IL-15 at 37 °C for
the next 13 days.
To generate CIK cells, PBMCs were cultured in AIM-V
medium with 5 % auto-plasma at 37 °C with 1000 U/mL
IFN-γ (Miltenyi Biotec). After 24 h, 100 ng/mL mouse
anti-human CD3 monoclonal antibody (Peprotech, USA),
1000 U/mL IL-2, and 1000 U/mL IL-1α (Miltenyi Biotec)
were added. Fresh complete medium and IL-2 supplement
(1000 U/mL) were added every three days.
To amplify γδ T cells, PBMCs were cultured in
complete medium with 1 μM zoledronate (Zoledronic
Acid, Jilin Province Xidian Pharmaceutical Sci-Tech
Development Co., China) and 400 U/mL human IL-2.
Fresh complete medium and IL-2 supplement (400 U/mL)
were added every 2 or 3 days.
Cell expansion was expressed as “fold expansion,” which
was calculated by dividing the absolute output number
of NK, CIK, and γδ T cells after 14 days of culture by
their number on day 0. Absolute output numbers of
these three immune cells were calculated by multiplying
(ADCC) assays, Raji cells were pre-coated with
rituximab (Roche, Switzerland) at 10 mg/mL for 30 min.
Effector cells were bulk samples of the cultured cells,
i.e., no cell sorting was done. Lysis of the target cells
by effector cells was assessed by Calcein release assay
at a 10:1 effector-target ratio (E:T). After 4 h incubation,
calcein release in the supernatant was assessed on a
BioTek Synergy HT Microplate Reader (BioTek Instruments,
USA). The percentage Calcein release was calculated
according to the formula: % specific release = [(experimental
release – spontaneous release)/(maximum release –
spontaneous release)] × 100 %.
Flow cytometry data were collected before and after
induction and analyzed for correlation using Spearman’s
test. Other data were analyzed by Wilcoxon’s rank-sum
test in SPSS13.0 software. Results were considered
significant at p ≤ 0.05.
Informed consent/patient enrollment
In this study, 20 tumor patients were enrolled.
Informed consent in accordance with the Declaration of
Helsinki was obtained from all patients, and approval
was obtained from the Ethics Committee of the the
First Hospital of Jilin University (protocol #2010–019).
Table 1 Clinicla data of the patients in the study
the total number of viable cells by the percentages of
these three immune cells as determined by flow
cytometry. Total viable numbers of NK, CIK, and γδ
T cells were determined by the CASY cell counter
The cultures were collected, washed, incubated for 15 min
with mouse mAbs against human CD3-PerCP,
CD56FITC, or PE, CD69-APC, CD16-PE (BD Biosciences,
USA), and NKG2D-PE (BioLegend, USA). NK cells were
incubated with CD158a-PE and CD158b-PE (BD
Pharmingen, USA), CIK cells were incubated with CD4-PE
and CD8-APC (BD Biosciences) and γδ T cells were
incubated with Vγ9-FITC (BD Pharmingen), CD4-PE, and
CD8-APC. Isotype-matched antibodies were used as
controls. Perforin and granzyme B detection was performed
according to the BD Cytofix/Cytoperm™ Kit manual (BD
Biosciences). Briefly, NK, CIK, and γδ T cells were
harvested and adjusted to 1 × 106 cells/mL in RPMI-1640
medium containing 10 % fetal calf serum, and incubated
0.1 % GolgiStop (BD Biosciences) for 4 h. After
preincubation with 10 % normal human serum, cells were
stained with mAbs to identify NK (CD3−CD56+), CIK
(CD3+CD56+), and γδ T cells (CD3+Vγ9+), followed by
intracellular staining for perforin-PE and granzyme B-PE
(BD Pharmingen), and the corresponding isotype
antibodies to determine intracellular cytokine levels.
Flow cytometry data acquisition was performed on a
BD FACS Calibur (BD Biosciences) with Cell Quest Pro
software. Analysis was performed with FlowJo software
(Tree Star, USA).
Cytokine secretion analysis
NK, CIK, and γδ T cells were collected and suspended
(1 × 106 cells/mL) in AIM-V medium and incubated at
37 °C for 24 h in a humidified atmosphere of 5 % CO2.
Supernatants were collected for detection of IFN-γ, IL-2,
IL-4, IL-6, and IL-10. Cytokine secretion was quantified
using commercially available enzyme-linked
immunosorbent assay (ELISA) kits. Intracellular cytokine levels
of IL-2 and IFN-γ were measured as described above for
perforin and granzyme B.
NK, CIK, and γδ T cells were used as the effectors and
leukemia cells (K562, HL-60, NB-4, and Jurkat),
lymphoma cells (Raji), and multiple myeloma cells (U266) were
used as targets. Briefly, target cells were collected,
washed once with PBS, and suspended in PBS at 1 × 106
cells/mL. Calcein-AM was added to a final
concentration of 1 μM. Cells were incubated in a humidified
atmosphere of 5 % CO2 for 30 min and then washed twice
with PBS. For antibody-dependent cellular cytotoxicity
Inclusion criteria were no radiation or chemotherapy
for at least one month before blood collection. Only
patients with solid tumors were included. Patient
characteristics are listed in the Table 1.
CN and HFJ contributed equally to this manuscript. CN conceived, designed,
and carried out the studies and drafted the manuscript. HFJ and WL
participated in the design of the study. JWC conceived and designed the
study and revised and edited the manuscript. ML, JTX, and DSX carried out
part of the studies. JFH revised and edited the manuscript. HH performed
statistical analyses. All authors read and approved the final manuscript.
This work was supported in part by grants from National Science Foundation
for Young Scholars (30901702), National Natural Science Foundation of China
(31470798), Ministry of Education Key Project of Science and Technology
(311015), National Major Scientific and Technological Special Project for
Significant New Drugs Development during the Twelfth Five-year Plan Period
(2013ZX09102032), Provincial Science Fund of Jilin Provincial Department of
Finance (3D5116523428, 20150204027YY), and Norman Bethune Program of
Jilin University (2012202).
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