Expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines
Aim: It has been demonstrated that the endogenous matrix metalloproteinases (MMPs) inhibitor reversion inducing cysteine rich protein with Kazal motifs (RECK) is a reliable prognostic marker for detecting several types of tumors. However, the RECK expressions in most of the normal and neoplastic tis...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | Expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines / S. Takagi, Y. Hoshino, T. Osaki, M. Okumura, T. Fuginaga // Experimental Oncology. — 2007. — Т. 29, № 1. — С. 30–34. — Бібліогр.: 31 назв. — англ. |
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irk-123456789-1385672018-06-20T03:05:19Z Expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines Takagi, S. Hoshino, Y. Osaki, T. Okumura, M. Fuginaga, T. Original contributions Aim: It has been demonstrated that the endogenous matrix metalloproteinases (MMPs) inhibitor reversion inducing cysteine rich protein with Kazal motifs (RECK) is a reliable prognostic marker for detecting several types of tumors. However, the RECK expressions in most of the normal and neoplastic tissues were extremely low, and to measure its expression is quite complicated. The purpose of the present study is to establish an easy method to quantify murine RECK mRNA expression for use in future experimental studies. Subsequently, in order to verify the reliability of the established quantification technique, we examined the change in RECK expression and gelatinase secretion in tumor cells when stimulated by the extracellular matrix. Methods: Several murine tumor cells were used in the present study. The real-time polymerase chain reaction (PCR) method and measurement conditions for murine RECK mRNA were studied using these tumor cells. Gelatinase activities were also examined by gelatin zymography. Results: Murine RECK mRNA expression was accurately quantified using real-time PCR. Among the tumor cells used in the study, osteosarcoma cells showed significantly higher RECK mRNA expression than the others. The RECK expression in the osteosarcoma cells was down-regulated by contact with matrigel-coated culture flasks due to increased secretion of gelatinases. Conclusion: The real-time PCR method employed in our study is useful to quantify RECK expression. Показано, что эндогенный ингибитор матриксных протеиназ (MMП) RECK может служить надежным прогностическим маркером для некоторых типов опухолей, однако его экспрессия в большинстве нормальных и неопластических тканей крайне низкая, поэтому возникают сложности, связанные с детекцией таковой. Цель работы — разработка количественного метода определения экспрессии мРНК для использования в экспериментальных исследованиях. Для дальнейшего подтверждения надежности разработанного метода исследованы изменения экспрессии RECK и секреции желатиназ в опухолевых клетках при стимуляции внеклеточным матриксом. Методы: в работе использовали несколько линий опухолевых клеток мыши, в которых экспрессию мРНК RECK анализировали методом ПЦР в режиме реального времени, активность желатиназ — методом зимографии. Результаты: экспрессию мРНК RECK количественно оценили методом ПЦР в режиме реального времени, причем среди исследованных клеточных линий наиболее высокий уровень экспрессии RECK выявили в клетках остеосаркомы. Экспрессия RECK в клетках остеосаркомы подавлялась при контакте с культуральным пластиком, обработанным матригелем, вследствие повышения секреции желатиназ. Выводы: для количественной оценки экспрессии мРНК RECK может быть использован метод ПЦР в режиме реального времени. 2007 Article Expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines / S. Takagi, Y. Hoshino, T. Osaki, M. Okumura, T. Fuginaga // Experimental Oncology. — 2007. — Т. 29, № 1. — С. 30–34. — Бібліогр.: 31 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138567 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Takagi, S. Hoshino, Y. Osaki, T. Okumura, M. Fuginaga, T. Expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines Experimental Oncology |
| description |
Aim: It has been demonstrated that the endogenous matrix metalloproteinases (MMPs) inhibitor reversion inducing cysteine rich protein with Kazal motifs (RECK) is a reliable prognostic marker for detecting several types of tumors. However, the RECK expressions in most of the normal and neoplastic tissues were extremely low, and to measure its expression is quite complicated. The purpose of the present study is to establish an easy method to quantify murine RECK mRNA expression for use in future experimental studies. Subsequently, in order to verify the reliability of the established quantification technique, we examined the change in RECK expression and gelatinase secretion in tumor cells when stimulated by the extracellular matrix. Methods: Several murine tumor cells were used in the present study. The real-time polymerase chain reaction (PCR) method and measurement conditions for murine RECK mRNA were studied using these tumor cells. Gelatinase activities were also examined by gelatin zymography. Results: Murine RECK mRNA expression was accurately quantified using real-time PCR. Among the tumor cells used in the study, osteosarcoma cells showed significantly higher RECK mRNA expression than the others. The RECK expression in the osteosarcoma cells was down-regulated by contact with matrigel-coated culture flasks due to increased secretion of gelatinases. Conclusion: The real-time PCR method employed in our study is useful to quantify RECK expression. |
| format |
Article |
| author |
Takagi, S. Hoshino, Y. Osaki, T. Okumura, M. Fuginaga, T. |
| author_facet |
Takagi, S. Hoshino, Y. Osaki, T. Okumura, M. Fuginaga, T. |
| author_sort |
Takagi, S. |
| title |
Expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines |
| title_short |
Expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines |
| title_full |
Expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines |
| title_fullStr |
Expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines |
| title_full_unstemmed |
Expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines |
| title_sort |
expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2007 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138567 |
| citation_txt |
Expression of membrane-anchored matrix metalloproteinase inhibitor reversion inducing cysteine rich protein with kazal motifs in murine cell lines / S. Takagi, Y. Hoshino, T. Osaki, M. Okumura, T. Fuginaga // Experimental Oncology. — 2007. — Т. 29, № 1. — С. 30–34. — Бібліогр.: 31 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-10T06:04:30Z |
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2025-07-10T06:04:30Z |
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| fulltext |
30 Experimental Oncology 29, 30–34, 2007 (March)
Matrix metalloproteinases (MMPs) play impor-
tant roles in the mechanism of tumor invasion and
metastasis by selectively degrading the extracellular
matrix. Among the various MMPs, gelatinases (type-IV
collagenases), particularly MMP-2 and -9, are closely
linked with tumor malignancy [11, 20], and the increa-
sed levels of these enzymes in tumor patients are
strongly related to poor prognosis [7, 13, 14].
A novel endogenous specific MMP inhibitor, which
was designated as “reversion inducing cysteine rich
protein with Kazal motifs” (RECK), has been recently
isolated [28]. RECK inhibits tumor invasion and tumor
angiogenesis by negatively regulating MMP-2, -9, and
membrane-type 1-MMP (MT1-MMP) [19, 28]. It has
been suggested that this membrane-anchored gly-
coprotein can be considered as one of the reliable pro-
gnostic factors in several tumor patients [10, 18, 24].
In the majority of studies, the RECK mRNA expres-
sion levels were quantified by Northern blotting [10, 18,
28]. However, the RECK mRNA expression levels were
reported to be considerably low in most of the normal
human tissues and tumor cells [28]. Therefore, to detect
the low expression levels of the RECK mRNA by Northern
blotting, a large volume of specimens was required to
purify sufficient amounts of the mRNA. In view of this, the
quantitative real-time polymerase chain reaction (PCR)
analysis is advantageous because it allows detection of
the mRNA even in small amounts of specimens, and it
is suitable for treating a large number of samples. Real
time PCR had been used to quantify human and canine
RECK mRNA expression, and the amplification condition
was established [24, 26, 27]. However, this technique
has not been established for murine RECK.
The purpose of the present study was to investigate
the RECK expression levels in several murine tumor cells
by employing the quantitative PCR. Subsequently, in order
to confirm the reliability of the established quantification
technique and correlation of gelatinase activity, the change
in RECK expression and gelatinase secretion was studied
in tumor cells attached to the extracellular matrix.
Materials and Methods
Murine cell lines and cell culture. The following
mouse tumor cell lines were cultured in Dulbecco’s
Modified Eagle’s Medium (DMEM) containing
1,000 U/ml of penicillin, 0.1 mg/ml streptomycin, and
10% fetal bovine serum (FBS) at 37 °C and 5% CO2
condition: B16 (melanoma) [12], B16F1 (pulmonary
metastatic melanoma derived from B16) [23], P815
(mast cell tumor) [8], YAC-1 (lymphoma) [15], KLN205
(squamous cell carcinoma) [22], S-180 (sarcoma)
[31], Dunn (osteosarcoma) [21], LM8 (pulmonary
metastatic melanoma derived from Dunn) [1].
RNA extraction and reverse transcription. Each
tumor cell line was washed twice and incubated for 24 h
in a culture medium lacking FBS. Then, total RNA was
extracted by the guanidine isothiocyanate method [5].
The total RNA (2 µg) was denatured at 70 °C for 10 min,
immediately cooled, and added to a solution containing
200 units of M-MLV reverse transcriptase (Invitrogen,
USA), 50 nmol dithiothreitol, 10 pmol poly (dT) primer,
and 20 nmol dNTPs in a total volume of 20 µl. After the
poly (dT) primer was annealed at 20 °C for 10 min, cDNA
synthesis was performed at 37 °C for 1 h.
expression of MeMbrane-anchored Matrix
Metalloproteinase inhibitor ReveRsioN iNduciNg cysteiNe
Rich pRoteiN with KAzAl Motifs in Murine cell lines
S. Takagi*, Y. Hoshino, T. Osaki, M. Okumura, T. Fuginaga
Laboratory of Veterinary Surgery, Department of Veterinary Clinical Sciences, Graduate School of
Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan
Aim: It has been demonstrated that the endogenous matrix metalloproteinases (MMPs) inhibitor reversion inducing cysteine rich
protein with Kazal motifs (RECK) is a reliable prognostic marker for detecting several types of tumors. However, the RECK ex-
pressions in most of the normal and neoplastic tissues were extremely low, and to measure its expression is quite complicated. The
purpose of the present study is to establish an easy method to quantify murine RECK mRNA expression for use in future experi-
mental studies. Subsequently, in order to verify the reliability of the established quantification technique, we examined the change
in RECK expression and gelatinase secretion in tumor cells when stimulated by the extracellular matrix. Methods: Several murine
tumor cells were used in the present study. The real-time polymerase chain reaction (PCR) method and measurement conditions
for murine RECK mRNA were studied using these tumor cells. Gelatinase activities were also examined by gelatin zymography.
Results: Murine RECK mRNA expression was accurately quantified using real-time PCR. Among the tumor cells used in the study,
osteosarcoma cells showed significantly higher RECK mRNA expression than the others. The RECK expression in the osteosarcoma
cells was down-regulated by contact with matrigel-coated culture flasks due to increased secretion of gelatinases. Conclusion: The
real-time PCR method employed in our study is useful to quantify RECK expression.
Key Words: RECK, gelatinase, MMP inhibitor, real-time PCR, osteosarcoma.
Received: December 28, 2006.
*Correspondence: E-mail: staka@vetmed.hokudai.ac.jp
Fax: +81-11-706-5229
Abbreviations used: MMPs — matrix metalloproteinases; mRNA —
messenger ribonucleic acid; PCR — polymerase chain reaction;
RECK — reversion-inducing-cysteine-rich protein with Kazal motifs.
Exp Oncol 2007
29, 1, 30–34
Experimental Oncology 29, 30–34, 2007 (March) 3129, 30–34, 2007 (March) 31March) 31) 31 31
Quantitative pcR analysis of RecK gene ex-
pression in mouse tumor cell lines. All PCR reac-
tions were performed using a detection kit (Lightcy-
cler-FirstStart DNA Master SYBR Green kit, Roche
Molecular Biochemicals, Mannheim, Germany). The
primer pairs used for amplifying RECK mRNA were
as follows: sense, 5ʹ-GCA TGC AAG CAG GCA TCT
TC-3ʹ and antisense, 5ʹ-CTG TGG ACT GAT AGA GGC
AC-3ʹ. To ensure correctness of mRNA extraction and
reverse-transcription and standardize the samples,
all the samples were subjected to PCR amplifica-
tion using oligonucleotide primers specific for the
constitutively expressed gene glyceraldehydes-3-
phoshate-dehydrogenase (GAPDH). The primer pairs
for amplifying GAPDH mRNA were as follows: sense,
5ʹ-GAA GGT CGG TGT GAA CGG ATT-3ʹ and antisense,
5ʹ-GAA GAC ACC AGT AGA CTC CAC GAC ATA-3ʹ.
The PCR reaction mixture containing the following
reaction components was prepared to the indicated
end concentration: 13.4 µl water, 2.4 µl MgCl2 (4 mM),
0.1 µl forward primer (100 µM), 0.1 µl reverse primer
(100 µM), and 2.0 µl LightCycler Fast Start DNA Master
SYBR Green I (Roche Molecular Biochemicals). As a
PCR template, 18 µl of master-mix and 2 µl of cDNA as
a PCR template were added to glass capillaries.
The cycling conditions were as follows: initial dena-
turation at 95 °C for 30 s, followed by 35 cycles (RECK)
or 30 cycles (GAPDH) at 95 °C for touchdown (0 s), 60 °C
for 5 s, and 72 °C for 10 s. In order to improve the SYBR
Green I quantification, the temperature of the fluores-
cence measurement point was set at 84 °C (RECK) and
83 °C (GAPDH). The expression level of each mRNA in
the tissue samples was determined relative to the stan-
dard curve by using the LightCycler computer software
(Roche Molecular Biochemicals). The identity of each
PCR product was confirmed by electrophoresis.
stimulation by extracellular matrix. Matrigel-
coated plates were used to stimulate Dunn and LM8
cells by direct contact with the extracellular matrix.
Matrigel (BD Biosciences, USA) is composed of the
extracellular matrix that is secreted by Engelbreth-
Holm-Swarm mouse sarcoma cells. Matrigel was
diluted to 0.1 mg/ml with phosphate-buffered saline
(PBS) on ice. Subsequently, 1 ml of the solution was
poured into each well of the 6-well cell culture plates.
These solutions were dried by leaving plates overnight
on a clean bench under an air flow. Cell culture medium
containing 10% FBS (2 ml) was poured 2 h before
seeding it with the cell suspension solution (2 ml).
The concentration of cells in the culture medium was
adjusted to 1.0 x 106 cells/ml. After 24 h of incubation,
cells were washed twice with serum free medium.
The collection of RNA from each cell line and culture
medium was performed after 24 h.
gelatin zymography. Gelatin zymography was
employed to investigate the correlation between murine
RECK mRNA expression and gelatinize activity. By using
the previously reported method, gelatin zymography
was performed [2, 29]. Briefly, 20 µl of the supernatant
of each conditioned medium was mixed with an equal
volume of 2 x SDS-PAGE sample buffer without boiling.
The samples were fractionated by electrophoresis on a
10% polyacrylamide gel containing gelatin (0.5 mg/mL).
In order to remove the SDS, the gels were soaked in
2.5% Triton X-100 containing 10 mM Tris (pH 8.0) for
30 min at room temperature and incubated in a diges-
tion buffer (50 mM Tris [pH 8.0], 0.5 mM CaCl2, and
1 µM ZnCl2) at 37 °C for 30 h to enable the proteinase
digestion of the substrate. The gels were stained with
0.25% Coomassie brilliant blue R-250 and destained
with 10% isopropanol and 5% acetic acid. The gelati-
nolytic activities were observed due to the appearance
of clear bands of digested gelatin against a dark blue
background of stained gelatin.
statistical analysis. For the comparison of the mu-
rine RECK expression levels in tumor cells, analysis of va-
riance followed by Scheffe’s multiple comparison as the
post-hoc test was performed. For the comparison of con-
trol and stimulated cells, the Student’s t-test was used.
Differences were considered significant when p < 0.05.
The statistical analyses were performed using the Stat
View 5.0 software (SAS Institute, Cary, NC, USA).
results and discussion
RecK gene expression in murine tumor cells.
Quantitative real time PCR could accurately measure the
amount of RECK mRNA, and the amplified cDNA comp-
letely corresponded with a part of the reported murine
RECK gene (Fig. 1). Northern blotting was used in most
of the previous studies to quantify the expression level
of the RECK mRNA [10, 18, 28]. The findings of these
studies were that the RECK mRNA expression level in
normal human tissues and tumor cells was considerably
low. Therefore, to detect the low RECK mRNA expression
by Northern blotting, it was necessary to purify the poly
A (+) mRNA from the total RNA and obtain an adequate
number of samples. The quantitative PCR used in this
study indicated that this was an easy, useful, and sensitive
technique to quantify the mRNA levels of specific genes.
Moreover, the process did not involve a treatment step
for purifying the mRNA from total RNA.
fig. 1. Standard curve for quantification of murine reversion
inducing cysteine rich protein with Kazal motifs (RECK) mRNA.
All measurements of murine RECK was performed when the error
value was less than 0.2
The murine tumor cells showed variable levels of mu-
rine RECK gene expression (Fig. 2). Although most cell
lines showed extremely low RECK mRNA expression, two
osteosarcoma cell lines showed a clear expression and
32 Experimental Oncology 29, 30–34, 2007 (March)
the S-180 cell — line comparatively higher RECK mRNA
expression. The previous studies reported that there was
little RECK mRNA expression in the murine and human
tumor cells [28]. However, in canine osteosarcoma cell
line, the high RECK mRNA expression was confirmed
[26]. In the present study, murine osteosarcoma was
found to exhibit significantly higher levels of RECK mRNA
compared to other tumor cell lines. The highly metastatic
LM8 cells derived from the Dunn osteosarcoma cell line
showed significantly lower expression than parental cell
line. As it was reported [28], the more malignant tumor
cells exhibited lower RECK mRNA expression compared
to parental cell lines. In particular, B16F1 and LM8 cells,
more metastatic than the cell lines that they are derived
from (B16 and Dunn, respectively) [1, 23], are showed
to possess lower RECK mRNA expression.
fig. 2. Murine RECK gene expression in various types of tumor
cells. GAPDH: glyceraldehyde-3-phoshate-dehydrogenase
Regulation of RecK mRNA expression under
extracellular matrix stimulation. Osteosarcoma
cells that were cultured on matrigel-coated plates were
attached to the bottom of the plates in a manner similar
to that observed on standard culture plates. However,
the cells exhibited different morphology depending on
the extracellular matrix present in the culture plates.
The tumor cells cultured on matrigel-coated plates
were fibroblastic, although the cells appeared to be
osteoblastic when cultured on standard plates. The
RECK mRNA expression in the cells grown on matri-
gel was significantly lower (by 2/3 nad ½ in Dunn and
LM8 cells respectively) than that in the cells grown in
standard conditions (Fig. 3).
fig. 3. The RECK gene expression in the osteosarcoma cell lines
upon contact with matrigel. The RECK expression in Dunn cells
grown in the matrigel-coated flask was significantly lower than
that in control flasks (*p < 0.05). LM8 cells also showed changes
identical to that shown by Dunn
There was one report stating that type-1 downregula-
tion of the RECK expression in human glioma cells grown
on collagen-gel coated plate [6]. However, our data (un-
published) indicated that type-1 collagen did not cause
RECK downregulation in the murine osteosarcoma. In
the present report, RECK expression in Dunn and LM8
cells was downregulated due to direct contact with matri-
gel — an extracellular matrix secreted by the Engelbreth-
Holm-Swarm mouse sarcoma [16]. The requirement for
direct contact of the cells with an extracellular matrix to
induce RECK downregulation might be due to the fact
that RECK is a membrane-anchored protein.
gelatin zymography. In the present study, gelatin
zymography was performed to validate the correlation
between RECK downregulation and gelatinase activities
and secretion. The MMPs are secreted from several
types of cells as inactivated proenzymes that are sub-
sequently activated by other MMPs or serine proteina-
ses [9]. Gelatin zymography is a method that enables
visualizing the amount of gelatinases as clear bands.
This technique is distinct from Western blotting and en-
zyme-linked immunosorbent assay (ELISA) [3] because
it can measure not only activated gelatinases but also
simultaneously measure inactivated pro-MMPs [4, 30].
RECK is a membrane-anchored protein that negatively
regulates secretion and activities of gelatinases. Thus,
the data obtained by measuring gelatinase activity can
be used for evaluation of RECK expression.
Initially, zymographic analysis was carried out on the
same medium on which the Dunn osteosarcoma cells
were cultured without any stimulation. No visible band
indicating the secretion of pro-MMP-2 and pro-MMP-9
was observed (Fig. 4). LM8 cells secreted larger amounts
of pro-MMP-2 in their conditioned medium, and this
result was consistent to that of a previous report [1].
When these tumor cells are in contact with matrigel, they
exhibited increased gelatinases secretion. The contact
of the cells with matrigel induced secretion of MMP-9 in
Dunn cells and secretion of both the gelatinases MMP-2
and 9 in LM8 cells. These results might indicate a close
correlation between the increased gelatinase secretion
and downregulation of murine RECK mRNA.
fig. 4. Gelatinases activity observed in the two types of os-
teosarcoma cells stimulated by matrigel. Gelatinase activation
was induced due to direct contact with matrigel. MMP: Matrix
metalloproteinase
It is known that the level of gelatinase activity is
altered in tumor tissues [1, 10, 18, 25]. Several reports
have shown that gelatinases can be upregulated or
activated in osteosarcoma cells [17, 20]. In the present
study, we have shown that direct contact with matrigel
induced MMP secretion and RECK downregulation.
Experimental Oncology 29, 30–34, 2007 (March) 3329, 30–34, 2007 (March) 33March) 33) 33 33
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34 Experimental Oncology 29, 30–34, 2007 (March)
экспрессия ингибитора мембраносвязанных
матриксных металлопротеиназ rec�� в линияхrec�� в линиях в линиях
опухолевых клеток мыши
Показано, что эндогенный ингибитор матриксных протеиназ (MMП)MMП)П) RECK может служить надежным прогностическим
маркером для некоторых типов опухолей, однако его экспрессия в большинстве нормальных и неопластических тканей
крайне низкая, поэтому возникают сложности, связанные с детекцией таковой. Цель работы — разработка количествен-
ного метода определения экспрессии мРНК для использования в экспериментальных исследованиях. Для дальнейшего
подтверждения надежности разработанного метода исследованы изменения экспрессии RECK и секреции желатиназ в
опухолевых клетках при стимуляции внеклеточным матриксом. Методы: в работе использовали несколько линий опу-
холевых клеток мыши, в которых экспрессию мРНК RECK анализировали методом ПЦР в режиме реального времени,
активность желатиназ — методом зимографии. Результаты: экспрессию мРНК RECK количественно оценили методом
ПЦР в режиме реального времени, причем среди исследованных клеточных линий наиболее высокий уровень экспрессии
RECK выявили в клетках остеосаркомы. Экспрессия RECK в клетках остеосаркомы подавлялась при контакте с культу-
ральным пластиком, обработанным матригелем, вследствие повышения секреции желатиназ. Выводы: для количественной
оценки экспрессии мРНК RECK может быть использован метод ПЦР в режиме реального времени.
Ключевые слова: RECK, желатиназа, ингибитор MMП, метод ПЦР в режиме реального времени, остеосаркома.RECK, желатиназа, ингибитор MMП, метод ПЦР в режиме реального времени, остеосаркома., желатиназа, ингибитор MMП, метод ПЦР в режиме реального времени, остеосаркома.MMП, метод ПЦР в режиме реального времени, остеосаркома.П, метод ПЦР в режиме реального времени, остеосаркома.
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