ORIGINAL ARTICLE
Expression of KGF-1 and KGF-2 in Skin Wounds and Its
Application in Forensic Pathology
Lin Sheng Yu, PhD,*†‡ Xing Biao Li, BM,*†‡ Yan Yan Fan, PhD,*†‡ Guang Hua Ye, MM,*†‡
Jun-Li Li, MM,*†‡ Tong-Tong Fu, BM,*†‡ Yi Liao, MM,*†‡ and Feng Li, MD, JD, PhD§||
Abstract: The expression of keratinocyte growth factor-1 (KGF-1) and
keratinocyte growth factor-2 (KGF-2) in skin wounds in mice was studied
using multiple methods. The dynamic expression of KGF-1 and KGF-2 for
antemortem and postmortem injuries as well as the examination of ante-
mortem injuries after death under different temperature and over varying
time periods was studied. It demonstrates that skin KGF-1 resulting from
an antemortem injury starts to rise at 6 hours, reaches its peak at 1 day,
and starts to drop at 5 days. The expression of skin KGF-2 resulting from
an antemortem injury starts to rise at 12 hours, reaches its peak at 7 days,
and begins to drop at 10 days. Skin KGF-1 and skin KGF-2 after death sta-
bilize within 7 days at 4°C and −20°C, within 5 days at 20°C, and within
1 day at 30°C. The application of KGF-1 and KGF-2 indicators in skin
wound age determination is both feasible and reliable.
Key Words: forensic sciences, antemortem injury, skin wounds, mice,
KGF-1, KGF-2
(Am J Forensic Med Pathol 2017;00: 00–00)
S kin is the largest tissue and organ in the human body. It is dis-
tributed over the entire body surface and is very vulnerable to
violent injuries. Accurate wound age determination provides clues
for determining the nature and detection of a crime and offers a
scientific basis for a conclusion and, therefore, becomes a very im-
portant topic in the field of forensic pathology.1–5 For this reason,
identifying and adjusting the damage repair processes and indica-
tors specifically related to injury time has been one of the major
tasks in forensic pathology and one of the hot topics of forensic
studies at home and abroad. Recently a number of scholars have
been using immunohistochemistry, in situ hybridization, Western
blot, and real-time polymerase chain reaction (PCR) technology in
studying the immediate expression of single indicator within dif-
ferent time periods after skin injury6–10; however, the application
of these tests in forensic pathology is very limited. While keeping
in mind the demands of forensic practice and taking into consider-
ation the findings of previous studies,11–13 this study plans to es-
tablish an animal model based on skin incised wounds in mice.
The study will utilize immunohistochemistry, Western blot, and
real-time PCR technology to study the dynamic distribution and
expression features of keratinocyte growth factor-1 (KGF-1) and
Manuscript received October 7, 2016; accepted February 27, 2017.
From the *Department of Forensic Medicine, †Forensic Center, and ‡Institute
of Forensic Science, Wenzhou Medical University, Wenzhou, Zhejiang
Province, China; §Forensic Medical Management Services, Nashville, TN;
and ||Shanghai Key Lab of Forensic Medicine, Shanghai City West,
Shanghai, China.
The present study was supported by the Shanghai Key Lab of Forensic
Medicine (grant no. KF1403).
Reprints: Feng Li, MD, JD, PhD, Shanghai Key Lab of Forensic Medicine,
Shanghai City West, 1347 Guangfu Rd, Shanghai 200063, China.
E-mail: fli@forensicmed.com.
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ISSN: 0195-7910/17/0000–0000
DOI: 10.1097/PAF.0000000000000315
Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017
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keratinocyte growth factor-2 (KGF-2) for antemortem and post-
mortem injuries, and the behavior of antemortem injuries after
death. The study will investigate different temperatures and differ-
ent time periods. Finally, the study will determine the feasibility of
combining the aforementioned indicators for determining the age
of the skin wound.
Skin wound healing is a basic but complicated biological
response, and it is generally composed of 3 phases: inflamma-
tion, proliferation, and maturation. Multiple substances, includ-
ing growth factors, cytokines, and adhesion molecules, are
known to be closely related in each phase of the skin wound
healing process, and some of them have been applied in the
wound age determination. Fibroblast growth factors (FGFs) be-
long to a large family related to protein structure. Currently, 23
members from the family, which are FGF1 to FGF23, have been
identified. Each member has certain sequence homology and
structural similarity. Keratinocyte growth factors belong to the
FGFs family and are considered to be polypeptide growth fac-
tors that may be combined with heparin. Currently, they have
2 members, which are KGF-1 (also known as FGF-7) and
KGF-2 (also known as FGF-10). Produced by mesenchymal
cells, they specifically stimulate the epithelial cell multiplication
through autocrine or paracrine and are closely related to organ de-
velopment and body homeostasis. To all kinds of epithelial cells,
KGF is somewhat effective, but special mitogen may also protect
cells against any damage and play an important role in repairing
damaged epithelial cells. Research shows that KGF-1 is mainly
produced in the early wound healing period and in the remodeling
process of the late wound healing period.14 However, KGF-2 has a
notable curative effect over the prevention of catarrh caused by ra-
diotherapy, ulceration, and enteritis.15 During the skin wound
healing period, the expression of both KGF-1 and KGF-2 in-
creases significantly, which indicates that they might also be par-
ticipating in the skin wound healing process.
MATERIALS AND METHODS
Preparation of Skin-Incised Wound Model and
Experimental Samples
Intraperitoneal anaesthesia with 2% pentobarbital sodium
(30 mg/kg) was applied, and a 2-cm longitudinal full-thickness
skin incision was made in the middle of the back of the Institute
for Cancer Research mouse. The wound was allowed to stop
bleeding naturally without binding or applying medication. The
mouse was euthanized with dislocation of the cervical vertebra
during the experiment. A 1.5 cm  1 cm tissue block was selected
from the skin wound of the mouse and divided into 3 portions.
One portion was provided with immunohistochemistry after 4%
paraformaldehyde fixation, the second portion was preserved for
protein extraction and real-time fluorescent quantitation PCR, and
the third portion was 0.1 cm3 precooled and prefixed with 2.5% glu-
taraldehyde for transmission via electron microscopic examination.
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Yu et al
FIGURE 1. Skin wound bleeding at 0.5 hours and 1 hour post injury.
Inflammatory cell infiltration occasionally occurs (HE 100).
Laboratory Animal Grouping
Antemortem Injury Group and Control Group
The mice were randomly divided into 12 groups, including
11 antemortem injury groups (wound samples are collected at
0.5 h, 1 h, 3 h, 6 h, 12 h, 1 d, 3 d, 5 d, 7 d, 10 d, and 14 d after in-
jury) and 1 normal control group. The researcher sheared the hair
and prepared the skin of the mouse without wounding them, and
then collected the skin tissue with the same size at the same place
after euthanizing the mouse. Each group contains 5 mice.
Postmortem Injury Group and Control Group
The mice were randomly divided into 6 groups, including 5
postmortem injury groups. The skin incision samples were col-
lected at 0.5 hour, 1 hour, 2 hours, 4 hours, and 6 hours after death.
Wound sample was taken 0.5 hour after injury. For the 1 control
group, the researcher sheared the hair and prepared the skin of
the mouse without wounding them, and then collected the skin tis-
sue with the same size at the same place after euthanizing the
mouse. Again, each group is comprised of 5 mice.
FIGURE 2. Skin wound bleeding at 0.5 hours and 1 hour post injury.
Inflammatory cell infiltration occasionally occurs (HE 100).
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Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017
FIGURE 3. Skin wound inflammatory cell infiltration at 3 hours and
6 hours post injury (HE 100).
Postmortem Stability Group and Control Group
The mice survived for a while after the skin incision and
then were put to death. Next, they were placed under various
temperatures, −20°C, 4°C, 20°C, and 30°C, respectively. Sam-
ples were taken at 6 hours, 12 hours, 1 day, 3 days, 5 days, and
7 days. The control group was 1 day antemortem injury. Each
group is comprised of 5 mice.
Immunohistochemical Determination
(SABC Method)
For regular skin tissue embedding and sectioning with a
thickness of 5 μm, the instructions on the diagnostic kit for details
was referred to.
1. Three percent H2O2 was used to incubate inactivated endog-
enous peroxidase and flushed thoroughly with phosphate-
buffered saline (PBS) solution.
2. One to 2 drops of goat serum was added, incubated at 37°C for
20 minutes, and then released.
FIGURE 4. Skin wound inflammatory cell infiltration at 3 hours and
6 hours post injury (HE 100).
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Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017
FIGURE 5. Skin wound inflammatory cell infiltration heavy at
12 hours and 1 day post injury (HE 100).
3. Diluted primary antibodies was added and flushed with
PBS overnight.
4. Secondary antibodies were added, incubated at 37°C for
3 minutes, and flushed thoroughly with PBS solution.
5. Streptavidin-biotin complex (SABC) working solution was
added, incubated at 37°C for 30 minutes, and flushed thor-
oughly with PBS solution.
6. Skin tissue was placed under the microscope, and the results
were observed.
7. Skin tissue was leached with distilled water, and developing
was stopped. The sheet was sealed after counterstaining with
hematoxylin, and the results were observed.
8. For the control group, PBS was used in substitution of primary
antibodies as a negative control (with the exception of this step,
all remaining steps are exactly the same as the experimental
groups). Next, an image analysis system was used and the num-
ber of positive cells was counted.
Western Blot
The spare skin tissue was removed (weight, 100 mg) and then
cut and homogenated; 1 mL was extracted, and 2 mL of protein
FIGURE 6. Skin wound inflammatory cell infiltration heavy at
12 hours and 1 day post injury (HE 100).
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Expression of KGF-1 and KGF-2 in Skin Wounds
FIGURE 7. Skin wound granulation tissue growth at 3 days and
5 days post injury (HE 100).
lysis buffer was added. Next, the supernatant was removed by
centrifugation and 3% sodium dodecyl sulfate (SDS) was
added; it was sealed and added to a boiling water for modification.
Protein quantification was performed, and SDS polyacrylamide
gel electrophoresis (PAGE) gel was prepared for electrophoresis.
Membrane was transferred for 1 hour (100 V, 350 mA), antibodies
were added onto the transferred cellulose acetate membrane at
4°C, and left overnight. The next day, it was rinsed 3 times
(5 min per rinse) with Tris-buffered saline with Tween 20 (TBST).
Diluted KGF (1:3000) was added and incubated for 2 hours,
then rinsed 3 times (5 min per rinse) with TBST, glowed, and
developed (model of the fully automatic imaging system, Kodak
Image station 440). Relative content is expressed in integrated
optical density.
RNA Extraction and Analysis
RNA Extraction
The specimen was removed from the liquid nitrogen and
2 mL of Trisol was added. The tissues were pyrolyzed with
an electric homogenizer. The lysate was transferred into the
centrifuge tube at room temperature and was allowed to sit for
FIGURE 8. Skin wound granulation tissue growth at 3 days and
5 days post injury (HE 100).
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Yu et al Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017

FIGURE 9. Skin wound scar formation, epidermal cells of FIGURE 11. Healing of the skin wound scar at 14 days post injury
regeneration covering the wound at 7 days and 10 days post (HE 100).
injury (HE 100).

5 minutes. The tube was placed into a centrifuge at 12,000g at 4°C
for 10 minutes. The supernatant was absorbed carefully, and the
contents were placed into a new centrifuge tube. Two hundred
microliters of chloroform was added, shaken vigorously at room
temperature for 5 minutes at 4°C, and centrifuged at 12,000g for
15 minutes. Next, the supernatant was absorbed, 500 μL of iso-
propyl alcohol was added, mixed evenly and placed under room
temperature for 10 minutes at 4°C, and then centrifuged at
12,000g for 10 minutes. The supernatant was discarded and
rinsed with 1 mL 70% precipitation ethyl alcohol; the superna-
tant was discarded and dried naturally under room temperature
for 10 to 20 minutes; 20 μL diethylpyrocarbonate (DEPC)-
treated water was added to dissolve RNA as reverse transcription
template. One microliter of RNA sample was taken and applied
in 3-(N-morpholino)propanesulfonic acid (MOPS) agarose gel
electrophoresis, and the molecular level of the extracted RNA
was preliminarily inspected.
Real-Time Fluorescence PCR
1. All primer sequence is synthesized by Shanghai Sunny
Biotechnology Co. Ltd. The upstream primer sequence for
KGF-1 is 5′-CGTGCTTCCACCTCGTCT-3′, the downstream
primer sequence is 5′-TCTCCTGGGTCCTTTCA-3′, and the
length is 222 bp; the upstream primer sequence for KGF-2
is 5′-CGGAGTTGTTGCCGCAAAG-3′, the downstream
primer sequence is 5′-GCCGTTGTGCTGCCAGTTA-3′,
and the length is 166 bp; the upstream primer sequence for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is 5′-
GGCACAGTCAAGCTGAGATTG-3′, the downstream primer
sequence is 5′-ATGGTGGTGAAGACGCCAGTA-3′, and the
length is 143 bp.
2. Reverse transcription reaction system includes the fol-
lowing: RNA (5 μL), 5 reaction buffer (4 μL), Oligo dT (1 μL),
RNase inhibitor (1 μL), reverse transcriptase (1 μL), and distilled
water (8 μL).

TABLE 1. Detection of KGF-1 Protein Expression levels in Skin

Tissues of the Various Groups by SABC Immunohistochemistry
(OD values; n = 5)

Time After Optical Density Standard

Injury Control Values Deviation P
Control 0
0.5 h 0
1h 0
3h 16.5 1.8
6h 20.8 2.7 *
12 h 31.5 2.2 *
1d 55.3 2.6. *
3d 48.5 2.1 *
5d 51.4 1.3 *
7d 50.1 2.6 *
10 d 46.2 2.9 *
14 d 36.7 2.3 *
FIGURE 10. Skin wound scar formation, epidermal cells of All values are expressed as mean ± SD (n = 5).
regeneration covering the wound at 7 days and 10 days post *P < 0.05 (vs control group).
injury (HE 100).

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Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017 Expression of KGF-1 and KGF-2 in Skin Wounds

TABLE 2. Detection of KGF-2 Protein Expression Levels in Skin TABLE 3. Relative Intensity of KGF-1 and KGF-2 at Different
Tissues of the Various Groups by SABC Immunohistochemistry Posttraumatic Intervals
(OD values; n = 5)
Time After KGF-1 KGF-1 KGF-2 KGF-2
Time After Optical Density Standard Injury Mean SD Mean SD
Injury Control Values Deviation P
Control 0.58 0.03 0.63 0.03
Control 0 3h 0.61 0.04 0.66 0.04
0.5 h 0 6h 0.8 0.03 0.69 0.04
1h 0 12 h 1.36 0.07 1 0.05
3h 15.3 1.5 1d 1.59 0.09 1.2 0.06
6h 15.8 2.4 3d 1.26 0.06 1.22 0.06
12 h 20.6 2.1 * 5d 1.42 0.08 1.36 0.08
1d 28.5 2.6 * 7d 1.05 0.05 1.43 0.07
3d 32.1 1.9 * 10 d 0.83 0.04 1.19 0.05
5d 40.6 2.1 * 14 d 0.71 0.04 0.84 0.04
7d 51.5 2.2 *
All values are expressed as mean ± SD (n = 5).
10 d 47.8 2.7 *
14 d 45.9 2.4 *
All values are expressed as mean ± SD (n = 5).
*P < 0.05 (vs control group).

FIGURE 12. Graph (A) is the analysis of KGF-1, KGF-2, and GAPDH protein by Western blotting at different posttraumatic intervals. The control
(C) column represents the result of the control skin. Representative results from 5 individual animals are shown. Graph (B) shows the relative
intensity of KGF-1 to GAPDH. Graph (C) shows the relative intensity of KGF-2 to GAPDH. All values are expressed as mean ± SD (n = 5).
*P < 0.05 (vs control group); **P < 0.05 (vs control group or preceding posttraumatic group).

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Yu et al Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017

3. Quantitative polymerase chain reaction system includes
the following: RNA (5 μL), Master Mix (12.5 μL), Primer F′
(stock, 10 μM) (0.5 μL), Primer R′ (stock, 10 μM) (0.5 μL),
50 SYBR Green Solution (0.5 μL), and, lastly, DEPC-treated
distilled water till 25 μL.
4. Each sample expands the target gene and internal refer-
ence; repeat after every 3 of them.
5. Polymerase chain reaction condition setting: reaction
system continually reacts at 42°C (30 min), repeats at 95°C
(20 s) → 62°C (30 s) → 7°C 2 (30 s), and repeats 40 times. The
reaction system is maintained for 1 minute at 95°C and reacts
for 1 minute at 55°C. Lastly, starting from 55°C, the temperature
increases 0.5°C after each 5 seconds until it reaches 95°C.
Light Microscopic Observation
Regular paraffin embedding was used and cut into slices.
Hematoxylin-eosin (HE) stain was applied, the morphological
changes of the skin tissue were observed under a lighted micro-
scope, and photographs were taken.
Data Processing
SPSS11.0 statistical software was used. The data are shown
in the form of (±s). Statistical analysis is done by using intergroup
one-way analysis of variance as well as compared in pairs and
inspected with q. It shall be deemed as significantly different at
P value less than 0.05.
RESULTS
Light Microscope Observation Results of Skin
Tissue Morphology
Skin wound bleeding and inflammatory cell infiltration
occasionally occur at 1 hour after injury. Skin wound inflam-
matory cell infiltration occurs at 6 hours after injury. Skin wound

FIGURE 13. Graph (A) is an analysis of KGF-1, KGF-2, and GAPDH protein by Western blotting at different postmortem intervals. The control
(C) column represents the results of the control skin. Representative results from 5 individual animals are shown. Graph (B) demonstrates the
relative intensity of KGF-1 to GAPDH. Graph (C) shows the relative intensity of KGF-2 to GAPDH. All values are expressed as mean ± SD (n = 5).
There were significant differences in each group.

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Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017 Expression of KGF-1 and KGF-2 in Skin Wounds

inflammatory cell infiltration is heavy between 12 hours and 1 day
post injury. Skin wound granulation tissue growth occurs between
3 and 5 days after injury. Skin wound scar formation occurs be-
tween 7 and 10 days after injury. A photo of the healing of skin
wound scar at 14 days post injury is found in Figures 1 to 11.
SABC Immunohistochemical Analysis of KGF-1 and
KGF-2 Expression Levels
The focus of this study is to investigate the protein expres-
sion levels of KGF-1 and KGF-2 in the skin tissue samples
from the various groups using the SABC immunohistochemi-
cal method. In addition, optical density (OD) values of the
KGF-1 and KGF-2 staining were quantified and compared
(Tables 1 and 2). Keratinocyte growth factor-1 protein expression
levels were increased in the skin at 6 hours after injury, reached
the peak at 1 day after injury, and began to decline at 5 days
after injury. Keratinocyte growth factor-2 protein expression
levels were increased in the skin at 12 hours after injury, reached
the peak at 7 days after injury, and began to decline at 10 days
after injury.

FIGURE 14. Graph (A) is an analysis of KGF-1, KGF-2, and GAPDH protein by Western blotting at different postmortem intervals at 4°C.
Column (c) KGF-1 represents the result of the wound 1 day post injury. Graph (c) of KGF-2 represents the results of the wound at 1 day after
injury. Representative results from 5 individual animals are shown. Graph (B) is the relative intensity of KGF-1 to GAPDH. Graph (C) is the
relative intensity of KGF-2 to GAPDH. All values are expressed as mean ± SD (n = 5). *P < 0.05 (vs control group); **P < 0.05 (vs control group
or preceding posttraumatic group).

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FIGURE 15. Graph (A) is an analysis of KGF-1, KGF-2, and GAPDH protein by Western blotting at different postmortem intervals at 20°C.
Column (c) of KGF-1 represents the result of the wound at 1 day after injury. Column C (c) of KGF-2 represents the result of the wound at
1 day after injury. Representative results from 5 individual animals are shown. Graph (B) shows the relative intensity of KGF-1 to GAPDH. Graph
(C) shows the relative intensity of KGF-2 to GAPDH. All values are expressed as mean ± SD (n = 5). *P < 0.05 (vs control group); **P < 0.05
(vs control group or preceding posttraumatic group).

Western Blot Analysis of the KGF-1 and KGF-2
Protein Expression
Western blot analysis was conducted to further investigate al-
terations in the protein expression levels of KGF-1 and KGF-2 in
the skin tissues derived from the various groups. Results were sim-
ilar in the immunohistochemistry (Fig. 12, Table 3).
KGF-1 and KGF-2 Stability Analysis Degradation
After Death
Stability influences the estimation of the skin damage
time. Using the Western blot method, skin KGF-2 and KGF-1
were tested at 4°C and −20°C within 7 days of stability, at
20°C within 5 days of stability, and at 30°C within 1 day of stability
(Figs. 13–17, Tables 4–8).
KGF-1 and KGF-2 Gene Expression Analysis
The mRNA expression levels of KGF-1 and KGF-2 in the
various groups were detected by reverse transcription quantitative
polymerase chain (RT-qPCR). The mRNA expression levels of
KGF-1 were increased in the skin between 6 hours and 1 day after
injury. The mRNA expression levels of KGF-2 were increased in
the skin at 12 hours to 3 days after injury. These results were

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Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017 Expression of KGF-1 and KGF-2 in Skin Wounds

FIGURE 16. Graph (A) is an analysis of KGF-1, KGF-2, and GAPDH protein by Western blotting at different postmortem intervals at 30°C.
Column (c) of KGF-1 represents the result of the wound at 1 day after injury. Column (c) of KGF-2 represents the result of the wound at 1 day
after injury. Representative results from 5 individual animals are shown. Graph (B) shows the relative intensity of KGF-1 to GAPDH. Graph
(C) shows the relative intensity of KGF-2 to GAPDH. All values are expressed as mean ± SD (n = 5). *P < 0.05 (vs control group); **P < 0.05
(vs control group or preceding posttraumatic group).

consistent with those of the SABC immunohistochemical and
Western blot analyses (Tables 9 and 10).
DISCUSSION
When a skin cell is irritated by injuries, it may release multi-
ple growth factors to stimulate the multiplication of similar cells
or, possibly, cells developed from the same germ layer to encourage
repair. Although a lot of chemical media may affect cell regenera-
tion and differentiation, the polypeptide growth factors are the most
important as they are not only able to stimulate cell multiplication
but also participate in the repair of damaged tissues. Some growth
factors may work on many types of cells, whereas others work on
specific target cells only. Key growth factors that may participate
in the damage repair include platelet-derived growth factor, FGF,
epidermal growth factor, transforming growth factor, vascular en-
dothelium growth factor, other cell factors that may stimulate
growth such as interleukins and tumor necrosis factor, and so
on. The regular changes and characteristics of aforementioned
growth factors during the repair process reflect their relationships
with the injury time and may be used as molecular biomarker to
determine the injury time.

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FIGURE 17. Graph (A) is an analysis of KGF-1, KGF-2, and GAPDH protein by Western blotting at different postmortem intervals at −20°C.
Column (c) of KGF-1 represents the result of the wound at 1 day after injury. Column (c) of KGF-2 represents the result of the wound at 1 day
after injury. Representative results from 5 individual animals are shown. Graph (B) is the relative intensity of KGF-1 to GAPDH. Graph (C) is the
relative intensity of KGF-2 to GAPDH. All values are expressed as mean ± SD (n = 5). There were significant differences in each group.

TABLE 5. Relative Intensity of KGF-1 and KGF-2 at Different

TABLE 4. Relative Intensity of KGF-1 and KGF-2 at Different Posttraumatic Intervals at 4°C
Posttraumatic Intervals
Posttraumatic KGF-1 KGF-1 KGF-2 KGF-2
Posttraumatic KGF-1 KGF-1 KGF-2 KGF-2 Intervals Mean SD Mean SD
Intervals Mean SD Mean SD
Control 1.6 0.07 1.45 0.06
Control 0.53 0.03 0.61 0.07 6h 1.57 0.08 1.43 0.06
0.5 h 0.55 0.04 0.59 0.04 12 h 1.58 0.07 1.39 0.07
1h 0.58 0.06 0.63 0.04 1d 1.56 0.09 1.4 0.1
2h 0.52 0.05 0.57 0.06 3d 1.6 0.1 1.39 0.08
4h 0.57 0.07 0.64 0.06 5d 1.5 0.08 1.35 0.09
6h 0.52 0.06 0.62 0.07 7d 1.37 0.09 1.19 0.07
All values are expressed as mean ± SD (n = 5). All values are expressed as mean ± SD (n = 5).

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Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017 Expression of KGF-1 and KGF-2 in Skin Wounds

TABLE 6. Relative Intensity of KGF-1 and KGF-2 at Different TABLE 9. KGF-1 Gene Expression Analysis by RT-qPCR
Posttraumatic Intervals at 20°C
Time After Injury KGF-1 GAPDH 2−(ΔΔCt)
Posttraumatic KGF-1 KGF-1 KGF-2 KGF-2
Intervals Mean SD Mean SD Control 31.84418 23.58583 1
0.5 h 31.59597 23.92597 1.503528
Control 1.62 0.05 1.45 0.05 1h 32.95239 25.32073 1.464736
6h 1.58 0.07 1.43 0.06 3h 33.48205 24.7071 0.699016
12 h 1.57 0.06 1.41 0.08 6h 31.56207 24.93643 3.100965
1d 1.55 0.1 1.39 0.09 12 h 31.00159 23.87402 2.189764
3d 1.53 0.08 1.35 0.08 1d 33.56416 24.42312 5.295825
5d 1.28 0.09 1.09 0.11 3d 31.70304 22.88239 0.677224
7d 0.71 0.11 0.65 0.13 5d 31.19499 23.92403 1.98259
All values are expressed as mean ± SD (n = 5). 7d 34.30572 24.73463 0.402555
10 d 32.52349 24.7977 1.446496
14 d 33.21764 24.50425 0.729491
Recently, many researchers have used the immunohisto-
chemical stain method in situ hybridization, Western blot, and
real-time PCR technology to study the instant expression of skin sion of each indicator will go through a stage of gradually
injury across different time periods. Despite that, their application strengthening before it gradually decreases again. Therefore,
in forensic practice has been very limited. First, they have not identical expression values may appear at 2 different points in
taken into consideration the research finding of what occurs after time. Because the maximum expression and change pattern of
death. In a forensic practice, pathologists examine bodies that each indicator will vary after skin injury, it is very hard to fully
have been dead for varied periods of time and, therefore, may and objectively determine the time of the skin injury using only
overlook or ignore certain changes that may occur after death. 1 indicator. Therefore, it would be more scientifically accurate
The postmortem stability of the research finding may impact to combine a number of different indicators.
the accuracy of the determination of the time of tissue injury. In accordance with the forensic demands, this research stud-
Secondly, using only 1 indicator to determine the skin injury ied the dynamic distribution and expression features of KGF-1
time also has its limitations because, after skin injury, the expres- and KGF-2 for antemortem injury, postmortem injury, and the be-
havior of antemortem injuries after death under different tempera-
ture conditions and at different time periods. The research used an
TABLE 7. Relative Intensity of KGF-1 and KGF-2 at Different
Posttraumatic Intervals at 30°C established animal model of skin incised wounds in mice and uti-
lized immunohistochemistry, Western blot, and real-time PCR
Posttraumatic KGF-1 KGF-1 KGF-2 KGF-2 technology. This research shows that the antemortem injury of
Intervals Mean SD Mean SD skin KGF-1 (also known as FGF-7) starts to rise at 6 hours,
reaches its peak at 1 day, and starts to drop at 5 days. The antemor-
Control 1.61 0.06 1.47 0.05 tem injury of skin KGF-2 (also known as FGF-10) starts to rise at
6h 1.55 0.08 1.39 0.09 12 hours, reaches its peak at 7 days, and begins to drop at 10 days.
12 h 1.51 0.13 1.35 0.11 The postmortem stability of skin KGF-1 and skin KGF-2 stabi-
1d 1.25 0.11 1.05 0.11 lizes within 7 days at 4°C and −20°C, within 5 days at 20°C,
3d 0.32 0.19 0.13 0.07 and within 1 day at 30°C. Through the use of multiple indicators
5d 0.18 0.13 0.11 0.06 and different testing methods, researchers should be able to pro-
vide information that can be used in the determination of skin in-
7d 0.17 0.15 0.12 0.05
jury time in the practice of forensic medicine.
All values are expressed as mean ± SD (n = 5).

TABLE 10. KGF-2 Gene Expression Analysis by RT-qPCR

TABLE 8. Relative Intensity of KGF-1 and KGF-2 at Different Time After Injury KGF-2 GAPDH 2−(ΔΔCt)
Posttraumatic Intervals at −20°C
Control 36.26373 23.58583 1
Posttraumatic KGF-1 KGF-1 KGF-2 KGF-2 0.5 h 34.03217 23.92597 5.945084
Intervals Mean SD Mean SD 1h 34.04815 25.32073 5.406006
3h 34.63536 24.7071 6.725446
Control 1.62 0.07 1.43 0.05
6h 32.04025 24.93643 7.63908
6h 1.63 0.09 1.46 0.06
12 h 32.68709 23.87402 14.56894
12 h 1.65 0.07 1.45 0.07
1d 35.85558 24.42312 12.370908
1d 1.66 0.08 1.38 0.08
3d 31.76991 22.88239 19.83612
3d 1.59 0.11 1.41 0.05
5d 36.96203 23.92403 0.779105
5d 1.62 0.09 1.43 0.06
7d 34.70536 24.73463 6.530353
7d 1.58 0.1 1.37 0.09
10 d 35.49599 24.7977 3.94385
All values are expressed as mean ± SD (n = 5). 14 d 36.12836 24.50425 2.075967

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Yu et al
ACKNOWLEDGMENTS
The authors would like to thank James Farris, Ed. D., for his
assistance in reviewing the article.
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