RU2492244C2 - Method of assessment of degree of cell proliferation using marker genes by method of polymerase chain reaction in real time - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method comprises adding to the part of the wells with lymphocytes of mitogens or test substances, incubation of the contents of the wells, selection of the contents of the wells, cytolysis and isolation of nucleic acids. The reaction of reverse transcription (RT) is carried out with primers for genes tpa and cdc2: primer for RT with mRNA of gene tpa 5'-CATCTTCATCTCACTCTTC-3', primer for RT with mRNA of gene cdc2 5'-CTGGAGTTGAGTAACGAG-3'. Real-time PCR is carried out with the obtained cDNA on the specific primers and with use of the labeled oligonucleotides for genes tpa and cdc2: for gene tpa the forward primer is 5'-TGTCGTGTCAGACCTTGAAGC-3', the reverse primer is 5'-CCTTGGATTTCTTGCTTGTGAC-3', the probe (FAM)-5'-TGTACCACTGTCTCAAGCCCTCCTGC-3'-(BHQ1), for cdc2 gene the forward primer is 5'-CTTCACTTGTTAAGAGTTATTTATAC-3', the reverse primer is 5'-CCAGAGTGTTACTACCTCATGTG-3', the probe (FAM)-5'-TGCCTTGCCAGAGC(FdT)TTTGGAATAC-3'-(BHQ1). The degree of proliferation of lymphocytes is expressed in proliferation index which is the number indicating how many times the amount of mRNA of gene tpa and/or cdc2 increases during culturing lymphocytes with/without addition of PHA or the test substance as compared with the amount of mRNA of the genes in lymphocytes freshly isolated from the blood of healthy donor.

EFFECT: invention enables to monitor directly the number of dividing cells, assessing the degree of expression of the genes involved and regulating the cell division.

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Description

The proposed method relates to the field of medicine, biology and biotechnology and can be used to assess the degree of cell proliferation by mitosis. In particular, this method can be used to assess the functional activity of lymphocytes in vitro using the blast transformation of lymphocytes by polymerase chain reaction (PCR) with the registration of the accumulation of reaction products "in real time".

The classic model for assessing the functional activity of lymphocytes in vitro is the reaction of blast transformation of lymphocytes (RBTL). The principle of the method is based on the mitogenic effect on lymphocytes of certain substances of plant and bacterial origin, as well as various substances that affect the functioning of the immune system (cytokines, chemokines, CpG, etc.). Phytohemaglutinin (PHA), obtained from bean seeds, has a mitogenic effect mainly on T-lymphocytes. In the classical methodology of the blast transformation reaction, the incubation of peripheral blood mononuclear cells in the presence of PHA is carried out for 72 hours, the reaction results are evaluated by the inclusion of tritiated ³ H-thymidine 6-18 hours after incubation [1].

Polymerase chain reaction (PCR) is widely used for amplification of nucleic acids and allows, in particular, to detect the presence and quantity of nucleic acids with a specific nucleotide sequence in a DNA or RNA sample. The results of the polymerase chain reaction are analyzed either at the end of the reaction or directly during the reaction. To record the results of PCR during the process, it is carried out in a detecting amplifier in the presence of either fluorescent intercalating dyes or fluorescently labeled oligonucleotide primers or samples [2].

Attempts have already been made to evaluate the degree of proliferative response of cells using marker genes, but other methods have been used for this assessment. For example, a method is known for evaluating a proliferative response by determining bromodeoxyuridine (BrdU) incorporated in DNA using an ELISA method [3]. However, this method does not have a sufficiently high signal-to-noise ratio (about 10: 1) and its sensitivity is limited by the limits of determining the optical density at 450 nm (from 0.0 to 3.0).

Using the Northern blot method, it was shown that the mRNA level of the topoisomerase II alpha gene (tpa) and the cell cycle controller 2 (cdc2) gene changes during the cell cycle, and the mRNA level of these genes increases either directly (cdc2) [5], or immediately after mitosis (tpa) [6]. Thus, it is possible to determine the degree of proliferative response of cells using the Northern blot method, but this method requires hybridization with a radiolabeled sample. In addition, the level of mRNA expression of the topoisomerase II alpha gene was evaluated in dividing and non-dividing cells using the ribonuclease protection assay [4]. Using this method, it is also possible to assess the degree of cell proliferation, however, it also requires hybridization with a radioactively labeled sample, and the sensitivity of such methods loses the sensitivity of the real-time PCR method.

Using the Western blot method, the expression level of topoisomerase II alpha and beta protein was determined during the cell cycle [7], which also allows us to study the degree of cell proliferation, but the sensitivity of this method is low.

There is a method for assessing the degree of stimulation of lymphocytes by flow cytometry by measuring the expression level of specific marker molecules on the cell surface. For example, upon stimulation of certain CD markers with monoclonal antibodies, the expression of CD38 on the surface of lymphocytes increases, and the increase in the number of CD38 was determined by flow cytometry [3]. However, in this method, an increase in the expression of marker molecules on the surface of cells does not always coincide with their proliferation, and often depends on the age and state of health of the body.

The technical result achieved by the implementation of the proposed invention is to reliably determine the degree of cell proliferation in response to the studied stimuli or during spontaneous proliferation.

The problem solved by the proposed invention is to use the method of real-time PCR to assess the degree of proliferative response of cells using mRNA marker genes, the activity of products of which increases during mitosis. In particular, the following genes can be used: topoisomerase II alpha (topoisomerase II alpha, tpa, OMIM 126430) and the cell cycle controller 2 gene (cell division cycle 2, cdc2, OMIM 116940).

The proposed method for assessing the proliferative response of cells of the immune system in the blood to achieve the specified technical result provides:

- taking venous blood into a wakutainer with sodium heparin (BD Vacutainer, sodium heparin) or using any other material (tissues);

- isolation of mononuclear fraction cells on a density gradient of ficoll (ficoll or ficoll-verographin with a density of 1.077 g / ml) as described in the standard method [1] within 12 hours after blood sampling *;

- counting cells stained with trypan blue in a Goryaev’s chamber on an inverted microscope or in a hemacytometer;

- dilution of cells with a complete nutrient growth medium to a concentration of 1 million / ml (for example, RPMI-1640 medium, Sigma # R0883, with the addition of 10% Standart Fetal Bovine Serum serum, HyClone # SH30088.03, 2 mM PanEco glutamine # Ф032, 20 mM HEPES- Buffer PanEco # F134);

- digging out with an automatic pipette with a sterile tip into the wells of a 96-well plate, 1-2x10 ⁵ received cell suspension in a volume of 100-200 μl of medium;

- the addition of mitogens or test substances to the wells in the required concentration (in the case of the use of the phytohemagglutinin mitogen, its concentration is 2.5-5.0 μg / ml of medium with cells; in the case of studying the influence of various other substances, the experimenter selects the concentration). Under the test substance should be understood a substance whose influence on cell proliferation is investigated;

- incubation of the tablet during the test time in a CO ₂ incubator at 37 ° C, 5% CO ₂ in an atmosphere of saturated water vapor;

- selection of the contents of the wells after incubation with a frequency determined by the researcher, based on the goals and objectives of the experiment (in our work, the selection of the contents of the holes was carried out once every 12 hours for 7 days);

- lysis of cells and isolation of nucleic acids using the Proba-NK kit (NPF DNA-Technology CJSC) or another ready-made kit for RNA isolation and, if necessary, DNA or another method according to the established procedure;

- formulation of the reverse transcription reaction from specific primers for the tpa and cdc2 genes in order to obtain DNA complementary to the RNA of the studied genes;

- real-time PCR staging with the obtained complementary DNA on specific primers and using labeled oligonucleotides on the tpa and cdc2 genes;

- PCR delivery (when taking a different number of cells in test tubes) with isolated DNA on specific primers and using labeled oligonucleotide on the human growth hormone receptor (ghr) gene to normalize the results;

- the expression of the degree of proliferation of cells of the immune system in the proliferation index, which is a number that shows how many times the amount of tpa and / or cdc2 mRNA increases during lymphocyte cultivation with / without the addition of PHA or the test substance compared to the amount of mRNA of these genes in lymphocytes freshly isolated from blood or any other material from a healthy donor.

Thus, when analyzing the results of real-time PCR, it is determined how many times the amount of mRNA of the studied tpa and cdc2 genes increases during the cultivation of lymphocytes with the addition of mitogens or test substances (or without the addition of stimulants) compared with the amount of mRNA of these genes in freshly isolated lymphocytes and in the first hours of cultivation.

Brief Description of the Drawings

Figure 1 shows graphs for determining the amplification efficiency of E (%) (see formula 1). To carry out the calculations, we determined the amplification efficiency of each gene (E) and the values of the difference of the threshold cycles when diluting the sample 10 times (slope), for which we made dilutions of the samples 3.2 and 10 times (0.5 lg step), constructed calibration lines and determined the parameters necessary for calculations according to formula 1 and the readings of the device. Differences in the slope angles of the lines are caused by the inhomogeneity of the level of the recorded fluorescence intensity (for example, due to the heterogeneity of the temperature of the fuser, the inhomogeneities of the optical path of the device, the dispersion of the optical parameters of the reaction tubes, errors in the preparation of the reaction mixture, etc.).

Figure 2 shows the kinetics of changes in the levels of mRNA expression of tpa and cdc2 genes in peripheral blood mononuclear cells taken from two healthy donors and stimulated with PHA. CDC-1 — level of cdc2 gene mRNA expression in the donor 1 cell population; CDC-2 - level of cdc2 gene mRNA expression in the cell population of donor 2; TPA-1 - level of expression of tpa gene mRNA in the cell population of donor 1; TPA-2 - level of expression of tpa gene mRNA in the cell population of donor 2.

Figure 3 presents the kinetics of changes in the levels of mRNA expression of the tpa and cdc2 genes in peripheral blood mononuclear cells taken from two healthy donors and cultured without the addition of PHA. CDC is the level of cdc2 gene mRNA expression in the cell population, the average of two donors; TPA is the level of tpa gene mRNA expression in the cell population, the average of two donors.

Figure 4 presents the kinetics of changes in expression levels of tpa and cdc2 genes in the first hours of cultivation of peripheral blood mononuclear cells stimulated by PHA. CDC-1 — level of cdc2 gene mRNA expression in the donor 1 cell population; CDC-2 - level of cdc2 gene mRNA expression in the cell population of donor 2; TPA-1 - level of expression of tpa gene mRNA in the cell population of donor 1; TPA-2 - level of expression of tpa gene mRNA in the cell population of donor 2.

The implementation of the invention

1. The reaction of blast transformation

Blood should be taken on an empty stomach in vakuteyner with heparin, the selection of lymphocytes from the PC should be carried out no later than 24 hours after taking the blood. The selection of the mononuclear cell fraction is carried out as described in [1] with slight changes. Briefly, 7 ml of fresh blood taken in a heparin vacutainer is diluted 1: 2 by volume with 199 medium with Hanks salts and glutamine, layered on 3-4 ml of ficoll solution (density 1.077 g / ml), centrifuged at 1600 rpm, 15 ° C, 15 min. The interphase is carefully transferred to clean tubes and 5-8 ml of 199 medium are washed three times in the same centrifuge at 1200 rpm, 15 ° C, 10 min. After washing, the supernatant is drained, the obtained cells are resuspended on a vortex in 1 ml of complete nutrient medium RPMI-1640, the number of isolated mononuclear cells in the hemocytometer is determined under a microscope. Add the required amount of complete nutrient medium RPMI-1640 to a concentration of 10 ⁶ cells / ml medium.

In a sterile 96-well plate, 10 ⁵ isolated cells per well are digested in a volume of 100 μl, 10 μl of a PHA stock solution is added (final concentration 2.5 μg / ml). No stimulants are added to the wells with cells to control spontaneous proliferation. The tablet is placed in a CO ₂ incubator saturated with water vapor for 7-8 days at 37 ° C, 5% CO ₂ . After a certain number of hours, cell samples are taken.

2. Isolation of RNA and DNA

To isolate nucleic acids, the Proba-NK kit (DNA-Technology # 05-017) or any other ready-made kit or method for isolating RNA and DNA according to the procedure is used.

3. Reverse transcription reaction

The reverse transcription reaction is carried out in a volume of 20 μl (16.5 μl of the obtained ribonucleic acid preparation is taken in the reaction). Specific oligonucleotides are used as primers for reverse transcription. The reaction is carried out at a temperature of 40 ° C for 1 hour, followed by inactivation of reverse transcriptase at 95 ° C for 15 minutes.

The composition of the reaction:

- 16.5 μl of the RNA preparation;

- 2 μl of 10x buffer * for reverse transcription;

- 0.01 μl of primer for reverse transcription at a concentration of 100 pM (or of each primer of 0.01 μl, if you plan to put PCR on several systems of specific primers);

- 0.24 μl of a mixture of four types of deoxyribonucleotide triphosphates at a concentration of 25 mM of each deoxyribonucleotide triphosphate;

- 0.5 μl of the enzyme reverse transcriptase (200 units / μl);

- water mQ treated with DEPC, up to 20 μl.

* - the composition of the buffer for reverse transcription: 500 mm Tris-HCl pH 8.3 at 25 ° C; 500 mM KCl; 40 mM MgCl ₂ ; 100 mM DTT

4. Real-time PCR

For each sample, PCR with specific primers for the tpa and cdc2 genes is performed. The ghr gene (human growth hormone receptor receptor gene for DNA normalization) is used as the normalization gene. The hprt1 gene (the hypoxanthine phosphoribosyltransferase 1 gene, which is often chosen for normalization by RNA) cannot be used in this case, since the degree of its expression changes upon stimulation by some mitogens.

Primers and probes for PCR are selected taking into account the structures of exons and introns in such a way as to exclude annealing on the matrix of genomic DNA on the genes tpa and cdc2. This allows not to use the additional step of processing nucleic acids with DNase. The absence of amplification on the genomic DNA matrix from specific primers for tpa and cdc2 genes was tested experimentally on samples that did not undergo a reverse transcription reaction. All probes contain the Fam label (dT). Accordingly, amplification reactions from specific primers to tpa, cdc2, and ghr are performed in different tubes. To increase the sensitivity and specificity of PCR, a “hot start” is used, which is achieved by separating the reaction components with paraffin. Amplification is carried out in real-time mode in a volume of 35 μl according to the following program:

80 ° С 30 sec, 94 ° С 1 min 1 cycle

94 ° C 10 s, 64 ° C 20 s 50 cycles.

For real-time PCR, use the DT-322 device (NPF DNA-Technology) or any other device that allows for real-time PCR. The measurement of the fluorescence level is carried out on each cycle at a temperature of 64 ° C.

Additional control over the progress of the reaction is carried out by electrophoresis of PCR products in a 2% agarose gel.

To determine the mRNA content of the studied genes, the following primers and probes were developed:

tpa gene mRNA: reverse transcription primer 5'-CATCTTCATCTGACTCTTC-3 ', forward primer 5'-TGTCGTGTCAGACCTTGAAGC-3', reverse primer 5'-CCTTGGATTTCTTGCTTGGG-3 ', probe -CTGCT-CCTAG-TAG-TAG BHQ1)

cdc2 gene mRNA: primer for reverse transcription 5'-CTGGAGTTGAGTAACGAG-3 ', direct primer 5'-CTTCACTTGTTAAGAGTTATTTATAC-3', reverse primer 5'-CCAGAGTGTTACTACCTCATGTG-3 ', probe (TGTAG-TAG-TAG) BHQ1)

Ghr gene DNA: direct primer 5'-CATTCCCATCATTGAGTGTGGAGTGAG-3 ', reverse primer 5'-CTGGGGATCAGGTGTTTATGGACCA-3', probe (BHQ1) -5'-CCTTCTGCCTGGCTTGCTT 3CTT-3CTT-3CTT

The composition of the reaction mixture in vitro:

bidistilled water - 25.37 μl

- reaction buffer ** - 3.5 μl

- a mixture of four types of deoxyribonucleotide triphosphates at a concentration of 25 mM of each deoxyribonucleotide triphosphate - 0.24 μl

- direct primer at a concentration of 100 μm - 0.125 μl

- reverse primer at a concentration of 100 μm - 0.125 μl

- detecting sample at a concentration of 50 μm - 0.07 μl

- Taq - polymerase at a concentration of 5 international units of activity - 0.5 μl

- test DNA sample - 5 μl

** - composition of the reaction buffer: 100 mM Tris-HCl (pH 8.8 at 25 ° С), 500 mM KCl, 0.8% P40, 20 mM MgCl ₂

5. Parameters used to describe real-time PCR

1. Wed - the value of the threshold cycle, automatically determined by the device.

2. Slope - the difference in the values of Cp (Δ Cp) when diluting the sample 10 times.

3. The effectiveness of amplification is estimated by the formula:

E = 10 ^{- (1 / slope)} ; E (%) = (E - 1) x100% (formula 1)

4. Determination of the expression level of tpa mRNA relative to ghr gene DNA.

Determination of the expression level of tpa mRNA relative to the ghr gene DNA was carried out according to the formula:

[tpa] / [ghr] = E _ghr ^Cp1 / E _ghr ^Cp2 (formula 2),

where E is the amplification efficiency,

Сp1 - value of the threshold cycle in the sample for ghr

Сp2 is the value of the threshold cycle in the sample for tpa.

5. The proliferation index was used as the main characteristic of the experiment — the ratio (R) of the expression level of reaction markers to the background expression value of these markers obtained in the first minutes / hours of lymphocyte culture.

Two methods were used to determine R:

CT delta-delta method (ΔΔ Ср) using normalization genes. R was considered as the ratio of the normalized level of expression at a certain point in time [tpa _i ] / [ghr _i ] to the background value [tpa _f ] / [ghr _f ]

R = [tpa _i ] / [ghr1 _i ] x [hprt1 _f ] / [ghr _f ] (formula 3);

method without the use of normalization genes

R = 10 ^{(Cpf-Cpi) / slope} (formula 4),

where Cpi is the value of the threshold cycle at a certain point in time RBTL,

Cpf is the background value of the threshold cycle in the first hours of RBTL.

A brief summary of the steps in implementing the method is as follows.

Taking cells, stimulating them with selected substances, then isolating RNA from them, turning it into DNA (because PCR can be done with DNA, not RNA).

PCR with labeled oligonucleotides to determine the amount of DNA taken into the reaction and, therefore, the amount of the original RNA. The earlier we detect the fluorescence signal due to the luminescence of labeled oligonucleotides, the more DNA (therefore, the original RNA) will be.

To exclude the situation when the RNA of the cdc2 and tpa genes may be larger due to the fact that different numbers of cells were taken in different tubes, we use the data normalization.

Together with RNA, we isolate total DNA from cells. This DNA is then directly used in PCR without reverse transcription. In DNA, we selected the ghr growth hormone receptor gene, which exists in a certain number of copies in all cells of higher eukaryotes.

Thus, in the PCR reaction, we simultaneously obtain two signals: one from labeled oligonucleotides to the cdc2 or tpa gene, and the second from labeled oligonucleotides to the ghr gene.

When processing the results, we divide the value of the received signal from cdc2 or tpa by the value of the signal from the ghr gene. Thus, we get normalized values for our genes: the value of the signal from the DNA (therefore, mRNA) of the cdc2 or tpa genes per one ghr gene molecule.

Now you can directly compare the fluorescence values. If fluorescence increases, then this means that indeed there are more mRNA molecules of the cdc2 or tpa genes, and there is no suspicion that more cells were taken into the reaction.

Next, we divide the obtained fluorescence value of cdc2 or tpa for cells stimulated by the selected substance by the fluorescence value of cdc2 or tpa for unstimulated cells, and we obtain the proliferation index. This index shows how many times the level of mRNA expression of cdc2 or tpa genes increased when the analyte was added at the time when the cells were removed and placed in a lysis solution.

If you carefully count the cells and take the standard amount into the reaction, you can not determine the fluorescence from the ghr gene, but you can use the fluorescence data from the cdc2 and tp genes directly, without normalization.

The principal difference of the proposed method is that the degree of cell proliferation in response to the studied stimuli or without the addition of stimulants is determined using real-time PCR using specific primers and probes for mpa of the tpa and cdc2 genes, whose activity increases during mitosis. The proposed method is highly specific and has a high sensitivity; the mRNA content of selected marker genes during proliferation increases several hundred times compared with the mRNA level in non-dividing cells. This method allows you to directly monitor the number of dividing cells, evaluating the degree of expression of genes involved in proliferation and regulating cell division, moreover, regardless of age, the presence of diseases, the state of the body and the cells being studied.

List of references

1. Current Protocols in Immunology. Ed. Coligan J.E., Kruisbeek A.M., Margulies D.H., Shevach E.M., Strober W. John Wiley &Sons;2003; ISBN: 0471522767

2. US2002123062, 2002-09-05.

3. Tsehaynesh Messele, Marijke T. L. Roos, Dorte Hamann, Maarten Koot, Arnaud L. Fontanet, Frank Miedema, Peter T. A. Schellekens, and Tobias F. Rinke de Wit. Nonradioactive Techniques for Measurement of In Vitro T-Cell Proliferation: Alternatives to the [3H] Thymidine Incorporation Assay. Clin Diagn Lab Immunol. 2000 July; 7 (4): 687-692.

4. Isaacs RJ, Harris AL, Hickson ID. Regulation of the human topoisomerase II alpha gene promoter in confluence-arrested cells. J Biol Chem. 1996 Jul 12; 271 (28): 16741-16747.

5. Welch PJ, Wang JY. Coordinated synthesis and degradation of cdc2 in the mammalian cell cycle. Proc Natl Acad Sci USA. 1992 Apr 1; 89 (7): 3093-3097.

6. Goswami PC, Roti Roti JL, Hunt CR. The cell cycle-coupled expression of topoisomerase IIalpha during S phase is regulated by mRNA stability and is disrupted by heat shock or ionizing radiation. Mol Cell Biol. 1996 Apr; 16 (4): 1500-1508.

7. Christensen MO, Larsen MK, Barthelmes HU, Hock R, Andersen CL, Kjeldsen E, Knudsen BR, Westergaard O, Boege F, Mielke C. Dynamics of human DNA topoisomerases II alpha and II beta in living cells. J Cell Biol. 2002 Apr 1; 157 (1): 31-44.

Claims (1)

A method for assessing the degree of proliferation of lymphocytes using marker genes by the method of polymerase chain reaction in real time, including the procedure for taking blood,
isolation and cultivation of lymphocytes with their subsequent counting,
cultivation of cells in a complete nutrient medium,
digging into the wells of the tablet obtained cell suspension,
the addition of mitogens or test substances to part of the wells,
incubation of the contents of the wells of the tablet,
selection of the contents of the wells after incubation for a certain time,
cell lysis and nucleic acid excretion,
formulation of the reverse transcription reaction using specific primers for the tpa and cdc2 genes of the following composition: primer for reverse transcription from mRNA of the tpa 5'-CATCTTCATCTGACTCTTC-3 'gene, primer for reverse transcription from mdNA of the cdc2 5'-CTGGAGTTGAGTAAC mRNA gene
“real-time” PCR setup using the obtained cDNA on specific primers and using labeled oligonucleotides for the tpa and cdc2 genes of the following composition: to determine the amount of tpa mRNA, direct primer 5'-TGTCGTGTCAGACCTTGAAGC-3 ', reverse primer 5'-CCTTTGTGTGTGTGTGTGTGTGT ', probe (FAM) -5'-TGTACCACTGTCTCAAGCCCTCCTGC-3' - (BHQ1), for determining the amount of cdc2 gene mRNA direct primer 5'-CTTCACTTGTTAAGAGTTATTTACAC-3 ', reverse primer 5'-CCAGAGTGTTTC-3TC-TCTTC-3 5'-TGCCTTGCCAGAGC (FdT) TTTGGAATAC-3 '- (BHQ1),
if necessary, in case of difficulties with the isolation of RNA from cells, PCR should be performed with the extracted DNA on specific primers and using labeled oligonucleotide on the human growth hormone gene (human growth hormone, ghr) to normalize the results, using primers of the following composition: direct primer 5 '-CATTCCCATCATTGAGTGTGGAGTGAG-3', reverse primer 5'-CTGGGGATCAGGTGTTTATGGACCA-3 'and probe (FAM) -5'-CCTTCTGCCTGGCTTGCTTTCCC-3' - (BHQ1),
expression of the degree of lymphocyte proliferation in the proliferation index, which is a number showing how many times the amount of tpa and / or cdc2 mRNA increases during lymphocyte culture with / without the addition of PHA or an analyte compared to the amount of mRNA of these genes freshly isolated from blood healthy donor lymphocytes; in the case of difficulties in RNA isolation, the proliferation index can be determined by dividing the signal value from the tpa and cdc2 genes by the signal received from the DNA of the growth hormone receptor gene ghr.

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