WO2013035209A1 - Etfbの細胞異常増殖への適用方法及び異常増殖抑制剤 - Google Patents
Etfbの細胞異常増殖への適用方法及び異常増殖抑制剤 Download PDFInfo
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Definitions
- the present invention relates to application of electron transfer flavonoprotein beta subunit (ETFB) to cell growth, an inhibitor thereof, and a therapeutic agent for abnormal proliferation of cells comprising the inhibitor.
- ETFB electron transfer flavonoprotein beta subunit
- TGF- ⁇ Abnormal growth of fibroblasts is associated with various diseases such as hypertrophic scars resulting from scars, fibrosis in the lungs, cirrhosis and nephrosclerosis. It is said that TGF- ⁇ is involved in the process of inducing abnormal growth (see, for example, Patent Document 1, Patent Document 2, and Patent Document 3), but TGF- ⁇ is a multitasking biological substance. Therefore, there are not a few aspects that cannot be dealt with by simple suppression, and the absence of an effective TGF- ⁇ inhibitor with few side effects is thought to be due to this.
- ETFB is a protein related to electron transfer (see, for example, Patent Document 4, Patent Document 5, Patent Document 6, and Patent Document 7), and is sometimes used as a biomarker. Is not well known.
- the present invention has been made under such circumstances, and an object of the present invention is to provide a means for distinguishing abnormal proliferation of fibroblasts at an early stage and suppressing it.
- the present inventors have identified fibroblast abnormal growth at an early stage, and have sought to find a means for suppressing this, and as a result of intensive research efforts, the fibroblasts are placed in the presence of tension.
- the expressed protein when cultured in collagen gel and when cultured in collagen gel in the absence of tension, it was found that the expression of ETFB was enhanced under the preconditions. Ascertaining that it is a factor of abnormal growth, the inventors have completed the invention.
- the present invention is as follows. ⁇ 1> A method for differentiating the proliferation state of fibroblasts using the degree of expression of ETFB (electron transfer flavonoprotein beta subunit) as an index, When the degree of expression of the ETFB is high, there is a high probability that abnormal proliferation of fibroblasts has occurred, A method for distinguishing the proliferative state of fibroblasts, in which, when the degree of expression of ETFB is low, it is determined that there is a high probability that the proliferation of fibroblasts is maintained normally.
- ETFB electron transfer flavonoprotein beta subunit
- ⁇ 2> The case where the expression level of the ETFB is high is a case where the expression level in the fibroblast of the test sample is higher than the expression level in the control fibroblast,
- ⁇ 3> The differentiation method according to ⁇ 1>, wherein the abnormal proliferation of fibroblasts is fibrosis.
- ⁇ 4> The differentiation method according to ⁇ 3>, wherein the fibrosis is caused by differentiation of fibroblasts into myofibroblasts.
- ⁇ 5> The differentiation method according to ⁇ 1>, wherein the abnormal proliferation of fibroblasts is hypertrophic scar formation.
- ⁇ 6> A method for differentiating fibrosis in an organ using the degree of ETFB expression in fibroblasts of an organ suspected of fibrosis as an index, When the expression level of ETFB is high, it is determined that there is a high probability that fibrosis has occurred in the organ, A method for distinguishing fibrosis in an organ, in which, when no increase in ETFB is observed, the probability of fibrosis occurring is low.
- ⁇ 7> The case where the expression level of the ETFB is high is a case where the expression level in the organ of the test sample is higher than the expression level in the control
- ⁇ 8> The method for distinguishing fibrosis in an organ according to ⁇ 7>, wherein the organ is skin.
- Fibroblasts are cultured in a collagen gel in the presence of tension in the presence and absence of a test substance, When cultured in the presence of a test substance, if the expression level of ETFB is lower than when cultured in the absence, the test substance is identified as a fibrosis inhibitor. .
- the differentiation method according to any one of ⁇ 1> to ⁇ 11>, wherein the expression of ETFB is protein expression.
- ⁇ 13> The differentiation method according to any one of ⁇ 1> to ⁇ 11>, wherein the ETFB expression is RNA expression.
- ⁇ 14> The differentiation method according to ⁇ 13>, wherein the degree of ETFB expression is measured by a polymerization chain reaction (PCR).
- PCR polymerization chain reaction
- Primers used in the PCR are as follows: The oligonucleotide described in SEQ ID NO: 1 or the oligonucleotide having this as a partial sequence and capable of amplifying the base sequence encoding ETFB, and The differentiation method according to ⁇ 14>, which is an oligonucleotide having the oligonucleotide described in SEQ ID NO: 2 or an oligonucleotide having this as a partial sequence and capable of amplifying a base sequence encoding ETFB.
- Fibroblasts are cultured in a collagen gel in the presence of tension in the presence and absence of the test substance.
- the fibroblasts When cultured in the presence of the test substance, the fibroblasts are cultured in the absence of the expression level of ETFB.
- a fibrosis inhibitor consisting of a test substance that is lower than sometimes.
- a hypertrophic scar treatment drug comprising the fibrosis inhibitor according to ⁇ 16> as an active ingredient.
- the hypertrophic scar treatment drug according to ⁇ 18> which is used for treatment of the resulting hypertrophic scar.
- the hypertrophic scar treatment drug according to ⁇ 18> which is used for prevention to prevent the resulting hypertrophic scar from deteriorating.
- the hypertrophic scar treatment drug according to ⁇ 18> which is used for preventing the occurrence of scars having a high probability of occurrence.
- FIG. 3 shows the results of culture in collagen gel of Example 1. It is the figure which showed the influence which it has on the proliferation of the fibroblast of ETFB of Example 1.
- FIG. 2 is a diagram (photograph) showing the effect of ETFB siRNA on the formation of stress fibers induced by TGF- ⁇ in Example 1.
- TGF- ⁇ 11 ng / ml
- ETFB or negative control (negative control) siRNA had been introduced, and cultured for 3 days, followed by Phalloidin staining.
- FIG. ETFB or NegativeETFcontrolNsiRNA was introduced, collagen gel culture was performed, RNA was collected every day of culture, and mRNA expression of collagen 1A1 (COL1A1) (B) and SMA (C) was detected by qRT-PCR confirmed.
- A shows the expression level of ETFB in the attachment state and free state under the introduction of Negative control siRNA
- B shows the expression amount of COL1A1 in the attachment state and free state under the introduction of Negative control siRNA
- C shows the expression amount of COL1A1 in the attachment state and free state.
- the expression level of SMA in an attached state and a released state is shown. It is a figure which shows the influence which the ETFB siRNA of Example 1 has on collagen production and SMA production.
- ETFB or Negative control siRNA was introduced and cultured in collagen gel with or without TGF- ⁇ (300ng / ml). After 1 day of culture, RNA was collected and COL1A1 (A), SMA (B ) And ETFB (C) mRNA expression was confirmed by qRT-PCR.
- A shows the change in the expression level of COL1A1 by the introduction of NegativeNcontrol siRNA or ETFB siRNA in the presence or absence of TGF- ⁇
- B shows the expression of SMA by the introduction of Negative control siRNA or ETFB siRNA in the presence or absence of TGF- ⁇
- C shows the change in the expression level of ETFB due to the introduction of NegativeETFcontrol siRNA or ETFB siRNA in the presence or absence of TGF- ⁇ .
- ETFB ETFB which is the main element of the present invention, is also referred to as an electron transfer flavonoprotein beta subunit, and its detailed base sequence and amino acid sequence have already been registered in gene data banks such as Gene Bank.
- a human-type ETFB is registered in Genebank as CAG33108.1. This was identified as a growth factor for fibroblasts through the following procedure.
- fibroblasts In culture under the tension loading condition in collagen gel used for identification of growth factors of fibroblasts, fibroblasts undergo cell proliferation, collagen expression increase, etc. In this proliferation, cell morphology, liquidity Factor expression approximates the state of fibrosis.
- a proliferation phenomenon is not observed. That is, a protein that expresses or enhances expression only under a tension load condition by comparing the expressed protein or RNA when fibroblasts are cultured in collagen gel with tension load and without tension. If RNA is captured, there is a high probability that it is a factor of fibroblast proliferation and a factor of fibrosis.
- the protein or RNA thus identified is subjected to a knockdown experiment using siRNA or the like.
- RNA can be targeted, and a protein can also be targeted.
- proteome analysis by the DD method can be performed on the culture and / or culture product of fibroblasts. That is, development by two-dimensional electrophoresis, DD, and the obtained spot are identified by MALDI-TOF-MS. Inhibit the function of the identified protein and confirm whether the pathological condition improves or is possible. Then, the three-dimensional structure of the target protein is investigated, the active center is determined, and a compound that fits into the active center with high affinity is designed. It is a technique that is said to perform functional suppression technology with DD, siRNA, etc. in proteome analysis, structural analysis of target protein using computer, and study of active center by biological experiment.
- the fibroblast used for such analysis can be used without particular limitation as long as it is an established cell. However, since the final embodiment is a human fibroblast, a human fibroblast is used. I can do it.
- Human fibroblasts include CCD-1113Sk cells (ATCC No. CRL2439, normal human dermal fibroblasts, 39-year-old black female), CCD-1109Sk (ATCC No. CRL2361, human dermal normal fibroblasts, Suitable examples include 21-year-old Caucasian male) or CCD-1032Sk (ATCC No. CRL2439, human skin dermal normal fibroblasts, Caucasian newborn), etc. I can do it. These cultures can be cultured in an FBS-added DMEM medium or RPMI medium.
- RNA can be amplified by PCR.
- siRNA can be prepared and a knockout experiment can be performed. The knockout experiment can also verify whether the target protein actually contributes to fibroblast proliferation and fibrosis.
- the inventors of the present invention applied tension-loaded culture in a collagen gel of fibroblasts, specifically, under conditions where the cells were cultured while attached to the wall surface or bottom surface of the incubator. It was found that ETFB was produced specifically. That is, in the culture of fibroblasts in a collagen gel, it can be discriminated whether or not abnormal growth of fibroblasts such as fibrosis is promoted by using the production amount of ETFB as an index.
- one aspect of the present invention is a method for identifying a proliferative state using the degree of expression of ETFB (electron transfer flavonoprotein beta subunit) as an index in a method for identifying a proliferative state of fibroblasts,
- ETFB electron transfer flavonoprotein beta subunit
- the degree of expression of ETFB is high, there is a high probability that abnormal proliferation of fibroblasts has occurred
- the degree of ETFB expression is low, this is a method for distinguishing the proliferative state of fibroblasts, in which it is determined that there is a high probability that the proliferation of fibroblasts is maintained normally.
- “using the degree of ETFB expression as an index” can also be referred to as “measuring the degree of ETFB expression as a marker”.
- “High probability that abnormal proliferation of fibroblasts has occurred” can be said to be “high risk of abnormal proliferation of fibroblasts”.
- “High probability that the proliferation of fibroblasts is maintained normally” can be said to be “a low risk of abnormal fibroblast proliferation”.
- the “differentiation method of fibroblast proliferation state” can also be referred to as “a method of predicting the risk of abnormal proliferation of fibroblasts”.
- the case where the expression level of ETFB is high is a case where the expression level in the fibroblast of the test sample is higher than the expression level in the control fibroblast
- the case where the expression level of ETFB is low may be a case where the expression level of the test sample in the fibroblasts is the same or lower than the expression level in the control fibroblasts.
- the case where the expression level of ETFB is high is not limited, for example, it is expressed in the fibroblast of the test sample, but is not expressed in the control fibroblast, or the test sample
- the expression level in the fibroblasts may be 150% or more, 300% or more, 500% or more with respect to the expression level in the control fibroblasts,
- the case where the expression level of ETFB is low is not limited.
- the expression level of the test sample in the fibroblast is the same as the expression level in the control fibroblast, or 70% or less, 50% or less, It can be 10% or less.
- the fibroblast of the test sample is a target cell that needs to be differentiated from the proliferative state of the fibroblast, and is not particularly limited.
- the control fibroblast can be a normal fibroblast of the subject or a normal fibroblast of the same or similar species as the subject or an established normal fibroblast.
- the established normal fibroblasts the same established cells as described above can be used.
- the expression level of ETFB may be measured by ETFB protein expression or by measuring the expression of a gene such as RNA encoding ETFB.
- the method for measuring the gene encoding ETFB protein or ETFB in fibroblasts is not particularly limited.
- abnormal proliferation of fibroblasts may be a disease state due to abnormal proliferation of fibroblasts.
- the disease state due to abnormal proliferation of fibroblasts is not particularly limited.
- fibrosis specifically, hypertrophic scar, keloid, scleroderma, comedone scar, pulmonary fibrosis, liver fibrosis, etc.
- fibroblast This may be fibrosis caused by differentiation of cells into myofibroblasts.
- Another aspect of the present invention is a method for distinguishing fibrosis in an organ using the degree of expression of ETFB in fibroblasts of an organ suspected of fibrosis as an index, where the degree of expression of ETFB is high.
- a method of distinguishing fibrosis in an organ in which it is determined that there is a high probability that fibrosis has occurred in the organ, and if there is no increase in ETFB, the probability that fibrosis has occurred is low. is there.
- “the probability of fibrosis occurring in an organ is high” can be said to be “high risk of fibrosis occurring in an organ”.
- “The probability that fibrosis is occurring is low” can be said to be “the risk that fibrosis is occurring is low”.
- the “differentiation method of fibrosis in an organ” can also be referred to as “a method of predicting the risk of fibrosis in an organ”.
- the expression level of ETFB is high, the expression level in the organ of the test sample is higher than the expression level in the control, The case where no increase in ETFB is observed can be a case where the expression level in the organ of the test sample is the same or lower than the expression level in the control.
- the case where the degree of expression of ETFB is high is not limited, for example, it is expressed in the organ of the test sample, but is not expressed in the control, or the expression level in the organ of the test sample is 150% or more, 300% or more, 500% or more with respect to the expression level in the control,
- the case where the expression level of ETFB is low is not limited.
- the expression level in the organ of the test sample may be the same as that in the control or 70% or less, 50% or less, or 10% or less. .
- the organ of the test sample is a target organ that needs to be discriminated for fibrosis in the organ, and is not particularly limited, and mammals such as humans, pigs, monkeys, chimpanzees, dogs, cows, rabbits, rats, mice, etc. It can be an organ derived from the like. Note that cultured cells, cultured tissues, blood, and the like obtained from the target organ may be used as the test sample.
- the control can be a normal part of the same or another organ of the subject or an organ or cell of the same or similar species as the subject or an established normal cell. As the established normal cells, the same established cells as described above can be used.
- the “organ” as a target of the fibrosis differentiation method in the organ of the present invention is not particularly limited, and may be, for example, a viscera such as skin, lung, or liver.
- the expression level of ETFB may be measured by ETFB protein expression or by measuring the expression of a gene such as RNA encoding ETFB.
- the method for measuring ETFB protein or ETFB-encoding gene in organ fibroblasts is not particularly limited. For example, according to a conventional method, cells are disrupted to obtain a cell extract containing a gene encoding ETFB protein or ETFB. Thus, the ETFB protein or ETFB-encoded gene contained in the cell extract can be measured by the usual measurement method as described later.
- the abnormal growth may be a disease state due to abnormal growth of fibroblasts.
- the disease state due to abnormal proliferation of fibroblasts is not particularly limited.
- fibrosis specifically, hypertrophic scar, keloid, comedone scar, scleroderma, pulmonary fibrosis, liver fibrosis, etc.
- fibroblast This may be fibrosis caused by differentiation of cells into myofibroblasts.
- fibroblasts are cultured in a collagen gel in the presence of tension in the presence and absence of a test substance,
- the test substance When cultured in the presence of a test substance, if the expression level of ETFB is lower than when cultured in the absence, the test substance is identified as a fibrosis inhibitor. It is.
- the case where the expression level of ETFB is lower than when cultured in the absence is not limited. For example, it is not expressed in the presence of the test substance or the test substance
- the expression level in the presence of can be 70% or less, 50% or less, 10% or less with respect to the expression level in the absence of ETFB.
- the culture conditions may be such as, but not limited to, for example, the culture can be performed by culturing at 30 to 40 ° C. for 12 to 72 hours in a gel having a collagen concentration of 0.05 to 0.5 wt%.
- the “differentiation method of fibrosis inhibitor” can also be referred to as “screening method of fibrosis inhibitor”.
- the test substance may be either a natural product or a synthetic product, and may be a drug or other candidate substance, and specifically, extracts derived from plants and microorganisms and purified products thereof, Examples include low-molecular synthetic compounds, antibodies, peptides, aptamers, siRNA, nucleic acids used in gene therapy, modified products and derivatives thereof, and the like.
- ETFB can be quantified as a protein, or can be quantified as a gene such as RNA encoding ETFB.
- a protein or gene quantification method a conventional method can be used. For example, it can be measured by an immunoassay method such as gel electrophoresis, Western blotting, ELISA or the like, immunohistochemical analysis, Northern blotting, PCR or the like. Since the amplification method by PCR can be applied to quantification of genes such as RNA, it is particularly preferable.
- a reagent capable of staining the protein in the gel or an antibody that specifically recognizes ETFB may be used.
- the antibody may be either a monoclonal antibody or a polyclonal antibody. These antibodies can be produced by a known method.
- the antibody may be labeled with a fluorescent dye, a radioisotope, an enzyme or the like, if necessary.
- a pair of nucleic acid primers comprising a nucleic acid primer that can specifically hybridize with ETFB mRNA and a nucleic acid primer that can specifically hybridize with cDNA synthesized using the mRNA as a template may be used. .
- the primer can be designed based on the sequence information of the gene to be measured.
- the oligonucleotide shown in SEQ ID NO: 1 in the sequence listing or SEQ ID NO: 2 in the sequence listing can be used. Oligonucleotides containing such a sequence as a partial sequence and capable of amplifying a base sequence encoding ETFB and exhibiting similar effects belong to the technical scope of the present invention.
- the length of the oligonucleotide is not limited, and is, for example, about 10 to 50 bases and about 15 to 30 bases.
- a test substance that has been evaluated and has an inhibitory effect on ETFB expression can be identified as a fibroblast abnormal growth inhibitor.
- Such an agent for suppressing abnormal growth of fibroblasts can be used to prevent diseases caused by abnormal growth of fibroblasts such as scars, particularly hypertrophic scars, by processing the same into an external preparation for skin or a pharmaceutical composition using the inhibitor as an active ingredient. Or it can be set as the pharmaceutical for a treatment.
- a preventive agent it is pre-administered before hypertrophic scars occur to prevent the formation of hypertrophic scars at sites with high probability of scarring, especially hypertrophic scars, for example, around surgical resection sites.
- the pharmaceutical preparation includes the above-mentioned fibroblast abnormal growth inhibitor and optional ingredients for preparation, such as excipients, disintegrants, binders, coloring agents, flavoring agents, flavoring agents, dispersants, surfactants.
- fibroblast abnormal growth inhibitor such as excipients, disintegrants, binders, coloring agents, flavoring agents, flavoring agents, dispersants, surfactants.
- the pharmaceutical preparation can be processed into a dosage form such as an internal medicine, an external preparation, and an injection, but it is particularly preferable to process it into an external preparation.
- surroundings of the said sample can be known by confirming how much ETFB is expressed in the said sample by PCR etc. .
- CCD-1113Sk cells from 39-year-old black women
- KELFIB keloid cell line
- Fibroblasts were incubated at 37 ° C and CO 2 concentration of 5% in DMEM (D-6046, Sigma) with FBS 10% (tissue culture flask (353024, BD FALCON) with added medium). -3158, Forma Scientific, Sanyo). At the time of subculture, the cells were washed with PBS, 0.05% trypsin solution (T4049, Sigma) was added, and the cells were incubated for 2-3 minutes in an incubator, and then the cells were collected, subcultured and used. Passaging was carried out every 3 to 4 days, and in this case, passage numbers from 7 to 12 were used.
- TUNEL staining After digesting the collagen in the collagen gel and recovering the cells, 1 mL of DMEM medium and cell fixative (Collection Fluid, 6676315, Thermo Shandon) were added and mixed by inversion. The cell suspension was centrifuged with Cytospin (Thermo scientific) and attached to a slide glass to prepare a specimen. The prepared specimens were subjected to TUNEL staining using an in situ apoptosis detection kit (MK500, Takara) according to the instruction manual. The microscopic examination was performed with a fluorescence microscope (ECLIPSE E600, Nikon) at a magnification of 100 to 400 times, and the appearance of positive cells under the adhesion and free conditions was observed.
- ECLIPSE E600 Fluorescence microscope
- the digested cells were collected in an Eppendorf tube by centrifugation and washed twice with PBS. The work during this period was performed under ice cooling as much as possible. At that time, the wet cell weight was 100 to 200 mg.
- Membrane fractions were extracted using ProteoPrep Membrane Extraction Kit (Sigma) according to the instruction manual. When thawing the cells, protease (Protease Inhibitor Cocktail, P2714, Sigma) and phosphatase inhibitor (Phosphatase Inhibitor Cocktail I & II, P2850 & P5726, Sigma) were added according to the instruction manual.
- the amount of the extracted protein was measured with Coomassie
- the extraction rate in one extraction was about 1/300.
- Two-dimensional electrophoresis was performed using a two-dimensional electrophoresis apparatus manufactured by Pharmacia.
- a two-dimensional electrophoresis apparatus manufactured by Pharmacia.
- isoelectric focusing an 18 cm gel (Immobile Dry Strip, Amersham) was used, and pI ranges of 3 to 10 NL (non-linear), 5.5 to 6.7, and 6 to 9 were examined.
- the amount of protein applied was 100 to 300 ⁇ g.
- Isoelectric focusing was developed by the cup loading method according to the instruction manual.
- the electrophoresis apparatus was Ettan IPGphor IEF System (Amersham), and the development conditions were as shown below. 50 ⁇ A / Strip, 20 °C
- S1 Gradient 500V 1min.
- S2 Gradient 4000V 4h
- S3 Step-n-hold 8000V 10h
- S4 Step-n-hold up to 6000V recovery
- SDS-PAGE was performed with Multiphor II II electrophoresis unit (18-1018-06, Amersham) and Electrophoresis Power supply (19-3500-01, Amersham).
- the gel was a precast gel (Excel-Gel SDS, 80-1255-53, Amersham, 245 ⁇ 110mm, 0.5mm thin, Gradient 8-18%), and electrophoresis conditions were S1: 600V, 400mA, 13W, 30min., S2 : 600V, 400mA, 30W, 16h.
- the developed gel is silver-stained using Silver stain MS kit (299-58901, Wako Pure Chemicals) with an automatic staining device (Hoefer Processor Plus, Amersham), and the staining conditions are the same for each gel. Staining was carried out so that The development time was 6 minutes. By repeating the above operation, 5 gels were prepared for each of the attached state, the released state, and the pI range. Until MALDI-TOF-MS analysis, it was stored in 8.7% Glycerol solution at 4 ° C.
- ⁇ 4> DD of cultured cells in attached and free state Using the 2D Master Database, a t-test with a rejection rate of 10% was performed on the matched spots, and spots with differences in spot intensity were extracted. Thereafter, the calculated spot was visually confirmed, and it was confirmed whether there was a difference visually. At the same time, the entire spot was reconfirmed visually, and spots that were not detected by computer processing and that differed in concentration between the attached state and the released state were added.
- spots that were found to have different expression levels by the above procedure were estimated by MALDI-TOF-MS. From the 3-4 gels judged to be equivalent by analysis, the spot of the group with the higher expression level was cut out. Since it was expected that the amount of protein was small, these gel fragments were collected and used for the test as one sample. Spot desilvering and trypsin digestion were performed using a 96-well trypsin digester and kit (Montage In-GelDigest Kit, Millipore) according to the instructions.
- Trypsin-digested protein is spotted on an anchor chip (74115, Bruker Daltonics) with a matrix (CHCAmatrix; 10 mg / mL ⁇ -CHCA in acetone-Ethanol 1: 1 v / v), dried, and MALDI-TOF-MS ( Peptide® Mass® Fingerprinting (PMF) analysis was performed using AutoFlex® II and Bruker Daltonics. PMF analysis is a method for estimating a protein from the mass pattern of peptide fragments jumping out of a matrix. Spots with sufficient protein content were identified by pseudo-MS / MS analysis and post-source-decay (PSD) analysis that could analyze amino acid sequences.
- PDD post-source-decay
- LF2000 Lipofectamine 2000
- OPTI-MEM 31985062, Invitrogen
- LF2000 is a cationic liposome, forms a complex with an anionic siRNA, and is taken into cells via endosomes or lysosomes.
- the siRNA described in Table 3 was used, and other than Galectin-3 was purchased.
- Galectin-3 was designed using Takara's HP siRNA Design Support System (http://www.takara-bio.co.jp/rnai/intro.htm). In use, suppression of mRNA expression was confirmed by QuantiGene using CCD-1113sk cells cultured for 1 day after transfection in monolayer culture. In addition, in order to confirm the specificity of the siRNA used, BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) is used to search the sequence on which the siRNA acts, and the gene has a similar sequence. MRNA was measured from the same sample using the QuantiGene probe against, and siRNA that was not found to have an inhibitory effect was used. Silencer Negative control siRNA # 1 (4611, Ambion) was used as a negative control.
- siRNA suppression effect and specificity confirmation test 2.5 ⁇ 10 4 CCD-1113sk cells were cultured the day before in a 12-well plate, and siRNA was introduced using LF2000 according to the instruction manual.
- the amount of LF2000 used was 4 ⁇ L / well at the final concentration, 33 nM for siRNA, and 2 mL of medium.
- the measurement was carried out using a plate reader (ARVO.SX, Wallac, PerkinElmer) and chemiluminescence was measured at 45 ° C.
- the suppression rate was calculated by the following formula 1, and the suppression rate of the target gene was 20% or less, and the specificity confirmation gene was not suppressed.
- CCD-1113sk cells were seeded in 1.5 ⁇ 10 5 cells / 10 cm dish (353003, FALCON), and after overnight culture, LF2000 was used according to the instruction manual and transfected with siRNA.
- n 2 per time point gene in the same manner as described above.
- the number of cells in the gel was counted every day from the start of the culture in the collagen gel to 4 days after the culture. The number of cells was measured by the method described above.
- the cells used for the measurement on the start date of culture and 3-4 days after the culture were collected from each group, 100 ⁇ L of Lysis Buffer was added, and mRNA was measured with QuantiGene. QuantiGene purchased and used probes shown in Table 4 below.
- fibroblast abnormal growth inhibitory protein was identified by siRNA transfection. That is, siRNA of each protein was purchased, cultured for 24 hours in a 10 cm 2 petri dish, then subjected to siRNA transfection treatment, and then cultured for 24 hours after that in collagen gel. The results are shown in FIG. The black bar graph shows the result of the attached state, and the white bar graph shows the result of the free state. Factors that showed inhibition of cell proliferation in the attached state were GAL3, EHD1 and ETFB. DnaJB5 also showed increased cell number proliferation. On the other hand, GAL3 and DnaJB5 showed a decrease in cell number under free conditions, suggesting cytotoxicity. MO25 and CS showed enhanced cell proliferation.
- GAL3 showed cell growth inhibition in the attached state, it was excluded from the candidates because it was not possible to rule out this cell growth inhibition due to cytotoxicity because a decrease in the number of cells was observed in the free state.
- EHD1 and ETFB showed no significant decrease in the number of cells even in the free state, and showed cell growth suppression in the attached state. Therefore, in order to confirm the reproducibility of the two factors, a more detailed test was conducted. As a result, EHD1 was not reproducible.
- ETFB was more suppressed in cell growth than negative control in the attached state, and the number of cells did not decrease significantly in the attached state (see FIG. 2), and the same tendency was observed, and reproducibility was obtained.
- a knockdown experiment using siRNA was performed. That is, CCD-1113sk cells (ATCC No. CRL2439, human skin dermal normal fibroblasts, derived from a black 39-year-old woman) were used and maintained in the same manner as described above. The siRNA and introduction reagent were the same as described above. Silencer Negative control siRNA # 1 (4611, Ambion) or AllStars Negative Control siRNA (Cat. No. 1027281, QIAGEN) was used as a negative control. The introduction method was the same as that described above.
- the mRNA expression level was measured by real-time PCR machine (ABI PRISM 6700, Applied Biosystem Inc.) using QuantiTect SYBR GreenPCR Kit (204145, QIAGEN) for 5 ⁇ L of cDNA. The measurement was triplicate and simultaneously the expression level of GAPD was measured. The following primers listed in Table 5 were used. Further, collagen gel culture was performed, and a 24-well plate was photographed using a scanner (Multiscanner III, Amersham) from day 0 of the culture. After photographing, the area of the gel in the well was measured using NIH Image. The percentage of the measured area was calculated by setting the bottom area of the well as 100%.
- Excessive collagen expression is caused by the differentiation of fibroblasts into myofibroblasts, and an increase in the expression of SMA ( ⁇ -smooth muscle actin) has been reported as a myofibroblast differentiation marker in fibroblasts. Moreover, the increase in SMA expression is caused by stimulation of TGF- ⁇ . If suppression of ETFB function suppresses collagen and SMA expression, this factor increases the expectation as a therapeutic agent for hypertrophic scar in addition to the possibility of suppressing hyperplasia by suppressing excessive cell number proliferation of fibroblasts . Therefore, the behavior of these factors was confirmed.
- SMA smooth muscle actin
- ETFB is a factor of abnormal growth of fibroblasts. It can also be seen that by monitoring this trend, the probability of abnormal growth of fibroblasts such as scars can be determined.
- the abnormal growth of fibroblasts is suppressed by discriminating how the trend of ETFB in the presence and absence of tension changes depending on the test substance.
- Ingredients to be screened can also be screened.
- the component which suppresses the fibroblast abnormal proliferation screened on such conditions can suppress an increase in SMA expression, ie, myofibroblast differentiation, in addition to suppressing fibroblast proliferation.
- the same component suppresses fibroblast proliferation, and further suppresses excessive collagen expression caused by fibroblast differentiation into myofibroblast, thereby causing a disease involving such a phenomenon, that is, It can be seen that it can be used as a therapeutic and prophylactic agent for fibrosis and hypertrophic scarring.
- the present invention can be applied to the development of a medicine that suppresses abnormal proliferation of fibroblasts.
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Abstract
Description
一方、ETFBは電子伝達に係わる蛋白質であり(例えば、特許文献4、特許文献5、特許文献6、特許文献7を参照)、時として、バイオマーカーに使用されることもあるが、その実態についてはあまり知られていない。脂質貯留性筋疾患においては、ETFBの変異が見られるとの報告(例えば、非特許文献1を参照)、II型糖尿病の要因の一つであるとの示唆(例えば、非特許文献2を参照)、マルチプル・アシルコエンザイムAデヒドロゲナーゼ・デフィシェンシー(Multiple acyl-CoA dehydrogenase deficiency;MADD)の一要素であるとの示唆(例えば、非特許文献3を参照)などが知られているが、皮膚などとの関連も、線維芽細胞との関係も全く知られていない。また、ETFBとTGF-βとの関連も全く知られていない。
<1> ETFB(エレクトロントランスファー フラボノプロテイン ベータ サブユニット)の発現の度合いを指標とする、線維芽細胞の増殖状態の鑑別法であって、
当該ETFBの発現の度合いが高い場合には、線維芽細胞の異常増殖が生じている蓋然性が高く、
ETFBの発現の度合いが低い場合には、線維芽細胞の増殖が正常に保たれている蓋然性が高い、と判別する、線維芽細胞の増殖状態の鑑別法。
<2> 前記ETFBの発現の度合いが高い場合が、被検試料の線維芽細胞における発現量が、対照の線維芽細胞における発現量に対して高い場合であり、
前記ETFBの発現の度合いが低い場合が、被検試料の線維芽細胞における発現量が、対照の線維芽細胞における発現量に対して同じ又は低い場合である、<1>に記載の鑑別法。
<3> 前記線維芽細胞の異常増殖が、繊維化である、<1>に記載の鑑別法。
<4> 前記線維化が、線維芽細胞のマイオファイブロブラストへの分化に起因しているものである、<3>に記載の鑑別法。
<5> 線維芽細胞の異常増殖が、肥厚性瘢痕の形成である、<1>に記載の鑑別法。
<6> 線維化を疑われる臓器の線維芽細胞における、ETFBの発現の度合いを指標とする、臓器における線維化の鑑別法であって、
ETFBの発現の度合いが高い場合には、当該臓器において線維化が起こっている蓋然性が高いと判別し、
ETFBの亢進が認められない場合には、線維化が起こっている蓋然性は低いと判別する、臓器における線維化の鑑別法。
<7> 前記ETFBの発現の度合いが高い場合が、被検試料の臓器における発現量が、対照における発現量に対して高い場合であり、
ETFBの亢進が認められない場合が、被検試料の臓器における発現量が、対照における発現量に対して同じ又は低い場合である、<6>に記載の臓器における線維化の鑑別法。
<8> 前記臓器が皮膚である、<7>に記載の臓器における線維化の鑑別法。
<9> 線維芽細胞を、張力の存在下コラーゲンゲル中で、被検物質の存在下及び非存在下培養し、
被検物質の存在下培養した場合、ETFBの発現量が非存在下培養したときよりも低い場合には、当該被検物質が、線維化抑制剤であるとする、線維化抑制剤の鑑別法。
<10> 前記線維化は、肥厚性瘢痕の形成である、<9>に記載の異常線維化抑制剤の鑑別法。
<11> 前記線維化が皮膚における肥厚性瘢痕である、<10>に記載の線維化抑制剤の鑑別法。
<12> 前記ETFBの発現がタンパク質の発現である、<1>~<11>の何れかに記載の鑑別法。
<13> 前記ETFBの発現がRNAの発現である、<1>~<11>の何れかに記載の鑑別法。
<14> ETFBの発現の度合いをポリメリゼーションチェインリアクション(PCR)で測定する、<13>に記載の鑑別法。
<15> 前記PCRに用いるプライマーは、
配列番号1に記載のオリゴヌクレオチド又はこれを部分配列に持ち、ETFBをコードする塩基配列を増幅することができるオリゴヌクレオチド、及び、
配列番号2に記載のオリゴヌクレオチド又はこれを部分配列に持ち、ETFBをコードする塩基配列を増幅することができるオリゴヌクレオチドである、<14>に記載の鑑別法。
<16> 線維芽細胞を、張力の存在下コラーゲンゲル中で、被検物質の存在下及び非存在下培養し、被検物質の存在下培養した場合、ETFBの発現量が非存在下培養したときよりも低い被検物質からなる、線維化抑制剤。
<17> 前記線維化は、肥厚性瘢痕の形成である、<16>に記載の線維化抑制剤。
<18> <16>に記載の線維化抑制剤を有効成分とする、肥厚性瘢痕処置薬。
<19> 生じた肥厚性瘢痕の治療用である、<18>に記載の肥厚性瘢痕処置薬。
<20> 生じた肥厚性瘢痕が悪化をしないための予防用である、<18>に記載の肥厚性瘢痕処置薬。
<21> 生じる蓋然性が高い瘢痕が生じることを防ぐ予防用である、<18>に記載の肥厚性瘢痕処置薬。
<22> ETFBをコードする塩基配列を増幅することができる、配列番号1又は2に表されるオリゴヌクレオチドと実質同一なオリゴヌクレオチド。
本発明の主要素となる、ETFBは、エレクトロントランスファー フラボノプロテイン ベータ サブユニットとも称され、その詳細な塩基配列、アミノ酸配列は既にジーンバンクなどの遺伝子データバンクに登録されている。例えば、ヒト型のETFBであれば、ジーンバンクにCAG33108.1で登録されている。このものは、以下の手続を経て、線維芽細胞の増殖因子として同定された。
したがって、本発明では、RNA等の遺伝子をターゲットとすることも出来るし、蛋白質をターゲットとすることも出来る。
この様な過程を経て、本願発明者らは、線維芽細胞のコラーゲンゲル中での張力負荷培養、具体的には、細胞が培養器の壁面乃至は底面に付着した状態で培養される条件において、ETFBが特異的に産生されることを見出した。即ち、線維芽細胞のコラーゲンゲル中での培養において、ETFBの産生量を指標に線維化等の線維芽細胞の異常増殖が促進されているか否かを鑑別することが出来る。
すなわち、本発明の一形態は、線維芽細胞の増殖状態の鑑別法において、ETFB(エレクトロントランスファー フラボノプロテイン ベータ サブユニット)の発現の度合いを指標とする、増殖状態の鑑別法であって、
ETFBの発現の度合いが高い場合には、線維芽細胞の異常増殖が生じている蓋然性が高く、
ETFBの発現の度合いが低い場合には、線維芽細胞の増殖が正常に保たれている蓋然性が高い、と判別する、線維芽細胞の増殖状態の鑑別法である。
ここで、「ETFBの発現の度合いを指標とする」とは、「ETFBの発現の度合いをマーカーとして測定する」と言うこともできる。
「線維芽細胞の異常増殖が生じている蓋然性が高い」とは、「線維芽細胞の異常増殖が生じているリスクが高い」と言うこともできる。「線維芽細胞の増殖が正常に保たれている蓋然性が高い」とは、「線維芽細胞の異常増殖が生じているリスクが低い」と言うこともできる。「線維芽細胞の増殖状態の鑑別法」は、「線維芽細胞の異常増殖のリスクの予測方法」と言うこともできる。
ETFBの発現の度合いが高い場合とは、被検試料の線維芽細胞における発現量が、対照の線維芽細胞における発現量に対して高い場合であり、
ETFBの発現の度合いが低い場合とは、被検試料の線維芽細胞における発現量が、対照の線維芽細胞における発現量に対して同じ又は低い場合であり得る。
ETFBの発現の度合いが高い場合とは、限定されないが、例えば、被検試料の線維芽細胞においては発現しているが、対照の線維芽細胞においては発現していないか、又は被検試料の線維芽細胞における発現量が、対照の線維芽細胞における発現量に対して150%以上、300%以上、500%以上であり得、
ETFBの発現の度合いが低い場合とは、限定されないが、例えば、被検試料の線維芽細胞における発現量が、対照の線維芽細胞における発現量に対して同じ又は70%以下、50%以下、10%以下であり得る。
被検試料の線維芽細胞は、線維芽細胞の増殖状態を鑑別される必要のある対象の細胞であり、特に限定されず、ヒト、ブタ、サル、チンパンジー、イヌ、ウシ、ウサギ、ラット、マウス等の哺乳動物等由来の細胞であり得る。
対照の線維芽細胞は、対象の正常線維芽細胞又は対象と同一又は類似の生物種の正常線維芽細胞又は確立された正常線維芽細胞であり得る。確立された正常線維芽細胞としては、上記と同様の確立された細胞を用いることが出来る。
ETFBの発現の度合いは、後記のとおり、ETFB蛋白質の発現を測定してもよく、ETFBをコードしたRNA等の遺伝子の発現を測定してもよい。線維芽細胞におけるETFB蛋白質又はETFBをコードした遺伝子の測定方法は、特に限定されず、例えば、常法に従って、細胞を破砕し、ETFB蛋白質又はETFBをコードした遺伝子を含む細胞抽出液を得て、細胞抽出液に含まれるETFB蛋白質又はETFBをコードした遺伝子を、後記のような通常の測定方法により測定することにより行うことが出来る。
本発明において「線維芽細胞の異常増殖」とは、線維芽細胞の異常増殖による疾患状態であり得る。線維芽細胞の異常増殖による疾患状態は、特に限定されず、例えば線維化(具体的には、肥厚性瘢痕、ケロイド、強皮症、面皰瘢痕、肺線維症、肝線維症等)、線維芽細胞のマイオファイブロブラストへの分化に起因している線維化等であり得る。
ここで、「臓器において線維化が起こっている蓋然性が高い」とは、「臓器において線維化が起こっているリスクが高い」と言うこともできる。「線維化が起こっている蓋然性が低い」とは、「線維化が起こっているリスクが低い」と言うこともできる。「臓器における線維化の鑑別法」は、「臓器における線維化のリスクの予測方法」と言うこともできる。
ETFBの発現の度合いが高い場合とは、被検試料の臓器における発現量が、対照における発現量に対して高い場合であり、
ETFBの亢進が認められない場合とは、被検試料の臓器における発現量が、対照における発現量に対して同じ又は低い場合であり得る。
ETFBの発現の度合いが高い場合とは、限定されないが、例えば、被検試料の臓器においては発現しているが、対照においては発現していないか、又は被検試料の臓器における発現量が、対照における発現量に対して150%以上、300%以上、500%以上であり得、
ETFBの発現の度合いが低い場合とは、限定されないが、例えば、被検試料の臓器における発現量が、対照における発現量に対して同じ又は70%以下、50%以下、10%以下であり得る。
被検試料の臓器は、臓器における線維化を鑑別される必要のある対象の臓器であり、特に限定されず、ヒト、ブタ、サル、チンパンジー、イヌ、ウシ、ウサギ、ラット、マウス等の哺乳動物等由来の臓器であり得る。なお、対象の臓器から得られる培養細胞、培養組織、血液等を被検試料として用いてもよい。
対照は、対象の同じ又は別の臓器の正常部分又は対象と同一又は類似の生物種の臓器又は細胞あるいは確立された正常細胞であり得る。確立された正常細胞としては、上記と同様の確立された細胞を用いることが出来る。
本発明の臓器における線維化の鑑別法の対象となる「臓器」とは、特に限定されず、例えば皮膚、肺或いは肝臓などの内臓等であり得る。
ETFBの発現の度合いは、後記のとおり、ETFB蛋白質の発現を測定してもよく、ETFBをコードしたRNA等の遺伝子の発現を測定してもよい。臓器の線維芽細胞におけるETFB蛋白質又はETFBをコードした遺伝子の測定方法は、特に限定されず、例えば、常法に従って、細胞を破砕し、ETFB蛋白質又はETFBをコードした遺伝子を含む細胞抽出液を得て、細胞抽出液に含まれるETFB蛋白質又はETFBをコードした遺伝子を、後記のような通常の測定方法により測定することにより行うことが出来る。
本発明の線維芽細胞の増殖状態の鑑別法を応用すれば、被検物質の存在下でETFBの産生量が高まるか、抑制されるかで当該被検物質が異常増殖促進剤であるか、異常増殖抑制剤であるかが鑑別される。
ここで、異常増殖とは、線維芽細胞の異常増殖による疾患状態であり得る。線維芽細胞の異常増殖による疾患状態は、特に限定されず、例えば線維化(具体的には、肥厚性瘢痕、ケロイド、面皰瘢痕、強皮症、肺線維症、肝線維症等)、線維芽細胞のマイオファイブロブラストへの分化に起因している線維化等であり得る。
本発明の一形態は、線維芽細胞を、張力の存在下コラーゲンゲル中で、被検物質の存在下及び非存在下培養し、
被検物質の存在下培養した場合、ETFBの発現量が非存在下培養したときよりも低い場合には、当該被検物質が、線維化抑制剤であるとする、線維化抑制剤の鑑別法である。
被検物質の存在下培養した場合、ETFBの発現量が非存在下培養したときよりも低い場合とは、限定されないが、例えば、被検物質の存在下において発現していないか、被検物質の存在下における発現量が、ETFBの発現量が非存在下における発現量に対して70%以下、50%以下、10%以下であり得る。
コラーゲンゲル中での被検物質の存在下及び非存在下の培養は、陰性対照の細胞の増殖が良好であり、被検物質の存在下及び非存在下で細胞の増殖の差異が明確となるような培養条件であってよく、限定されないが、例えば、コラーゲン濃度0.05~0.5wt%ゲル中で、30~40℃、12~72時間の培養により、行うことが出来る。
「線維化抑制剤の鑑別法」は、「線維化抑制剤のスクリーニング法」と言うこともできる。
被検物質としては、天然物又は合成品のいずれであってもよく、通常医薬等の候補物質となり得るものであってよく、具体的には、植物および微生物由来の抽出物およびその精製品、低分子合成化合物、抗体、ペプチド、アプタマー、siRNA、遺伝子治療に用いられる核酸、その修飾体や誘導体などを挙げることができる。
PCR反応を行うためのプライマーとしては、例えば、配列表の配列番号1或いは配列表の配列番号2に示すオリゴヌクレオチドを用いることが出来る。かかる配列を部分配列として含み、ETFBをコードする塩基配列を増幅することができる、同様の効果を示すオリゴヌクレオチドは、本発明の技術範囲に属する。同オリゴヌクレオチドの長さとしては、限定されないが、例えば、10~50塩基、15~30塩基程度である。
斯くの如くに、被検物質を評価し、ETFB発現の抑制作用を認めたものについては、線維芽細胞異常増殖抑制剤とし判別することが出来る。かかる線維芽細胞異常増殖抑制剤は、同抑制剤を有効成分として皮膚外用剤や、医薬組成物に加工することにより、瘢痕、特に肥厚性瘢痕などの線維芽細胞異常増殖に起因する疾病の予防又は治療用の医薬とすることが出来る。予防薬としては、瘢痕、特に肥厚性瘢痕などが生じる蓋然性の高い部位、例えば、手術における切除箇所の周辺などに、肥厚性瘢痕が生じる前に予め投与し肥厚性瘢痕の生成を防ぐ、また、生じた肥厚性瘢痕が悪化しない様に予め投与する様な使用が好ましく開示でき、治療においては、既に生じてしまった肥厚性瘢痕等の瘢痕に投与し、当該瘢痕を縮小させる使用が好ましく例示できる。当該医薬の製剤化は、前記線維芽異常増殖抑制剤と製剤化のための任意成分、例えば、賦形剤、崩壊剤、結合剤、着色剤、矯味剤、矯臭剤、分散剤、界面活性剤等を加えて、常法に従って処理することにより、医薬製剤に加工することが出来る。医薬製剤としては、内服薬、外用剤、注射剤などの剤形に加工可能であるが、外用剤に加工することが特に好ましい。
ヒト皮膚正常線維芽細胞の種類は、ATCCより入手可能な細胞が多数存在するが、ケロイド細胞株である(KELFIB)のプロファイルに最も近いCCD-1113Sk細胞(39歳黒人女性由来)を優先的に用いた。
コラーゲンゲル中での培養はVarediらの方法の変法を用いた。即ち、粉末培地より通常の5倍に濃縮したDMEM(5xDMEM、D5523、Sigma)を作製し、以下に示す混合比で最終的にDMEM培地と同様な組成となるようにコラーゲン溶液を作製した。作製したコラーゲン溶液は恒温槽にて12℃にて使用まで保存した。コラーゲン原液の必要量(1mg/mLの際は7mL)、5xDMEM 7mL、200mM HEPES 3.5mL、0.03% NaH2CO3 3.5mL、0.01% NaOH 1.5mL、FBS必要量(10%の際は3.5mL)、H2Oにて総量31.5mLとした。Basal layerを作製する場合には、あらかじめ24穴または6穴組織培養プレート(24穴;353047、6穴;353046、BD FALCON)に24穴の場合は250μL、6穴の場合には1mLのコラーゲン溶液を添加し、インキュベーターにて15分以上静置しコラーゲンを固化させた。
それぞれの実験によって決められた培養時間後、最終濃度310U/mLとなるようにDMEMを用いて調製したコラゲナーゼ溶液に、コラーゲンゲルをスパチュラでプレートよりはがして添加し、37℃にて20~40分振盪しコラーゲンを消化した。消化した細胞浮遊液を1500rpmにて3~5分遠心分離し細胞を回収した。回収した細胞に対し100μLの培地を添加し、トリパンブルー染色を施した後Burkel-Turk血球計算盤にて1/5の希釈率にて生細胞数を計測した。
コラーゲンゲルのコラーゲンを消化し、細胞を回収後、1mLのDMEM培地および細胞固定液(Collection Fluid、6768315、Thermo Shandon)を加え、転倒混和した。その細胞浮遊液をCytospin(Thermo scientific)にて分遠心し、スライドガラスに付着させ標本を作製した。作製した標本にin situ Apoptosis Detection Kit(MK500, Takara)にて取扱説明書にしたがってTUNEL染色を施した。検鏡は蛍光顕微鏡(ECLIPSE E600、Nikon)にて100~400倍の倍率で行い、付着条件下および遊離条件下の陽性細胞出現を観察した。
付着状態および遊離状態培養のコラーゲンゲルプレートをそれぞれ8~10枚作製し、培養後1日において前述の手法と同様にコラーゲンを消化した。ただし、コラゲナーゼによる非特異的なタンパク質分解を可能な限り抑制するため、1mMのBenzamidinehydrochrolide Hydrate(B6506、Sigma)および0.1mMのN-α-p-Tosyl-L-Lysine Chloromethylketone Hydrochloride(TLCK、T7254、Sigma)存在下にて実施した。
50μA/Strip、20℃
S1:Gradient 500V 1min.
S2:Gradient 4000V 4h
S3:Step-n-hold 8000V 10h
S4:Step-n-hold 6000V 回収まで
染色が終了したゲルは、スキャナー(ImageScanner III、28-9076-07、Amersham)を用いてコンピューターに取り込んだ。取り込んだ画像は画像解析ソフト(2D Master Elite Ver. 4.01、2D Master Database Ver. 4.00、Amersham)を用いて以下の手順にて解析を実施した。DDにおける全体的な流れは以下に示す。
(1)コンピューターによるスポットの自動検出、目視確認による重複スポットの修正
(2)スポットとして検出されていないゲルのバックグランドおよび染色されたスポット全体の染色強度のゲル間での標準化
(3)ゲル間で移動度から共通に存在すると認識されるスポットのナンバリング(マッチング、ここで検出される番号をMatch No.と呼ぶ)、ならびに同じサンプルの泳動像が真に同等であるかどうかの統計処理(本処理で同等と判断されたゲルをDDに供した)。
(4)t検定によるDD(棄却率10%)
また、同時に目視にてDDを実施し、コンピューター処理にて算出されたスポットが真に染色強度が異なるかどうかの判断、ならびにコンピューター処理では有意とはならなかったが、目視にて強度が異なるスポットの追加を実施した。
ImageScanner IIIにて取り込んだ画像をトリミングし、サイズを同等とした。2DMaster Eliteにて自動スポット検出を以下のパラメーターで実施した。
Peak Dilation Parameters:
Min. Peak Area 85
Max. Peak Area 1100
Min. Aspect Ratio 0.4
Max. Aspect Ratio 4.5
Min. Area Ratio 50
2D Master Eliteに搭載されているバックグランド除去設定およびスキャンされたゲル染色濃度を標準化する作業を取扱説明書に従い実施した。
Max. Number of Touching Peaks 20
Background Intensity 0
Step Size 1
Smoothing Size 1
Histogram Equalization なし
Tidy Edges あり
その後目視によりスポットを確認し、重複して認識されているスポットの修正を実施した。
スポット像が良好でスポット数の多い遊離状態のゲル像をReference gelに設定し、取扱説明書に従いスポットのマッチングを実施した。また、2D Master Databaseに搭載されているTest Hypothesis機能を用いてReference gelとのDunnett検定を実施し、5枚のゲルのうち、3枚以上同等と認められた群について<4>の作業を実施した。
2D Master Databaseを用い、マッチングしたスポットについて棄却率10%のt検定を実施し、スポット強度に違いのあるものを抽出した。その後算出されたスポットを目視にて確認し、目視でも違いがあるかを確認した。また同時にスポット全体について目視にて再確認し、コンピューター処理で検出されなかった、付着状態・遊離状態で濃度に違いがあるスポットを追加した。
細胞は前述と同様にCCD-1113sk細胞(ATCC No. CRL2439、ヒト皮膚真皮正常線維芽細胞、黒人39歳女性由来)を用い、前述の手法と同様に維持した。導入試薬としてリポフェクタミン2000(LF2000、11668019、Invitrogen)を取扱説明書に従い用いた。導入の際にはOPTI-MEM(31985062、Invitrogen)を培地として使用した。LF2000は陽イオン性のリポソームであり、陰イオン性のsiRNAとコンプレックスを形成し、エンドソームまたはリソソームを経由して細胞内に取り込まれる。siRNAは表3に記載したものを用い、Galectin-3以外は購入した。Galectin-3はTakaraのHPでのsiRNA Design Support System(http://www.takara-bio.co.jp/rnai/intro.htm)を用いて設計した。使用に際し、単層培養にてトランスフェクション後1日間培養したCCD-1113sk細胞を用いて、QuantiGeneによりmRNAの発現抑制を確認した。また、使用したsiRNAの特異性を確認するために、BLAST(http://blast.ncbi.nlm.nih.gov/Blast.cgi)によりsiRNAが作用する配列を検索し、その類似配列を持つ遺伝子に対するQuantiGeneプローブを用いて同様のサンプルよりmRNAを測定し、抑制効果が認められかったsiRNAを使用した。また、陰性対照としてSilencer Negative control siRNA #1(4611、Ambion)を用いた。
前述のETFBの機能を調べるために、siRNAによるノックダウン実験を行った。即ち、CCD-1113sk細胞(ATCC No. CRL2439、ヒト皮膚真皮正常線維芽細胞、黒人39歳女性由来)を用い、上述と同様に維持した。siRNAおよび導入試薬は上述と同様のものを用いた。また、陰性対照としてSilencer Negative control siRNA #1(4611、Ambion)、またはAllStars Negative Control siRNA(Cat. No. 1027281, QIAGEN)を用いた。導入方法は前述の方法と同様に実施した。
TGF-βを添加する場合は、コラーゲン溶液作製時にHuman rTGF-β1(100-B-010、R&D Systems)を添加し混合後、同様に播種した。
mRNAの測定はqRT-PCRを実施した。すなわち、各実験にて回収した細胞よりRNeasy mini kit(74104、QIAGEN)を取扱説明書にしたがってtotal RNAを精製した。RNA0.1μgよりQuantiTect Reverse Transcription Kit(205311、QIAGEN)を用いて取扱説明書に従いcDNAを調製した。cDNA5μLに対しQuantiTect SYBR GreenPCR Kit(204145、QIAGEN)を用いて、real-time PCR machine(ABI PRISM 6700、Applied Biosystem Inc.)によりmRNA発現量を測定した。なお、測定はtriplicateとし、同時にGAPDの発現量測定を実施した。プライマーは表5に記載の以下のものを使用した。また、コラーゲンゲル培養を実施し、培養0日からスキャナー(Multiscanner III、アマシャム)を用いて24穴プレートを撮影した。撮影後、NIH Imageを用いてwell内のゲルの面積を測定した。Wellの底面積を100%とし、測定した面積のパーセントを算出した。
Claims (22)
- ETFB(エレクトロントランスファー フラボノプロテイン ベータ サブユニット)の発現の度合いを指標とする、線維芽細胞の増殖状態の鑑別法であって、
当該ETFBの発現の度合いが高い場合には、線維芽細胞の異常増殖が生じている蓋然性が高く、
ETFBの発現の度合いが低い場合には、線維芽細胞の増殖が正常に保たれている蓋然性が高い、と判別する、線維芽細胞の増殖状態の鑑別法。 - 前記ETFBの発現の度合いが高い場合が、被検試料の線維芽細胞における発現量が、対照の線維芽細胞における発現量に対して高い場合であり、
前記ETFBの発現の度合いが低い場合が、被検試料の線維芽細胞における発現量が、対照の線維芽細胞における発現量に対して同じ又は低い場合である、請求項1に記載の鑑別法。 - 前記線維芽細胞の異常増殖が、繊維化である、請求項1に記載の鑑別法。
- 前記線維化が、線維芽細胞のマイオファイブロブラストへの分化に起因しているものである、請求項3に記載の鑑別法。
- 線維芽細胞の異常増殖が、肥厚性瘢痕の形成である、請求項1に記載の鑑別法。
- 線維化を疑われる臓器の線維芽細胞における、ETFBの発現の度合いを指標とする、臓器における線維化の鑑別法であって、
ETFBの発現の度合いが高い場合には、当該臓器において線維化が起こっている蓋然性が高いと判別し、
ETFBの亢進が認められない場合には、線維化が起こっている蓋然性は低いと判別する、臓器における線維化の鑑別法。 - 前記ETFBの発現の度合いが高い場合が、被検試料の臓器における発現量が、対照における発現量に対して高い場合であり、
ETFBの亢進が認められない場合が、被検試料の臓器における発現量が、対照における発現量に対して同じ又は低い場合である、請求項6に記載の臓器における線維化の鑑別法。 - 前記臓器が皮膚である、請求項6に記載の臓器における線維化の鑑別法。
- 線維芽細胞を、張力の存在下コラーゲンゲル中で、被検物質の存在下及び非存在下培養し、
被検物質の存在下培養した場合、ETFBの発現量が非存在下培養したときよりも低い場合には、当該被検物質が、線維化抑制剤であるとする、線維化抑制剤の鑑別法。 - 前記線維化は、肥厚性瘢痕の形成である、請求項9に記載の異常線維化抑制剤の鑑別法。
- 前記線維化が皮膚における肥厚性瘢痕である、請求項10に記載の線維化抑制剤の鑑別法。
- 前記ETFBの発現がタンパク質の発現である、請求項1~11の何れか1項に記載の鑑別法。
- 前記ETFBの発現がRNAの発現である、請求項1~11の何れか1項に記載の鑑別法。
- ETFBの発現の度合いをポリメリゼーションチェインリアクション(PCR)で測定する、請求項13に記載の鑑別法。
- 前記PCRに用いるプライマーは、
配列番号1に記載のオリゴヌクレオチド又はこれを部分配列に持ち、ETFBをコードする塩基配列を増幅することができるオリゴヌクレオチド、及び、
配列番号2に記載のオリゴヌクレオチド又はこれを部分配列に持ち、ETFBをコードする塩基配列を増幅することができるオリゴヌクレオチドである、請求項14に記載の鑑別法。 - 線維芽細胞を、張力の存在下コラーゲンゲル中で、被検物質の存在下及び非存在下培養し、被検物質の存在下培養した場合、ETFBの発現量が非存在下培養したときよりも低い被検物質からなる、線維化抑制剤。
- 前記線維化は、肥厚性瘢痕の形成である、請求項16に記載の線維化抑制剤。
- 請求項16に記載の線維化抑制剤を有効成分とする、肥厚性瘢痕処置薬。
- 生じた肥厚性瘢痕の治療用である、請求項18に記載の肥厚性瘢痕処置薬。
- 生じた肥厚性瘢痕が悪化をしないための予防用である、請求項18に記載の肥厚性瘢痕処置薬。
- 生じる蓋然性が高い瘢痕が生じることを防ぐ予防用である、請求項18に記載の肥厚性瘢痕処置薬。
- ETFBをコードする塩基配列を増幅することができる、配列番号1又は2に表されるオリゴヌクレオチドと実質同一なオリゴヌクレオチド。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/342,774 US9562912B2 (en) | 2011-09-05 | 2011-12-27 | Method of identifying abnormal cells by expression levels of ETFB |
EP11872070.5A EP2754719B1 (en) | 2011-09-05 | 2011-12-27 | Method of using etfb as biomarker for identifying the growth state of fibroblasts |
CN201180074671.7A CN103917659A (zh) | 2011-09-05 | 2011-12-27 | Etfb在细胞异常增殖中的应用方法以及异常增殖抑制剂 |
KR1020147007797A KR101856114B1 (ko) | 2011-09-05 | 2011-12-27 | Etfb의 세포 이상증식에 대한 적용 방법 및 이상증식 억제제 |
JP2013532386A JP5963757B2 (ja) | 2011-09-05 | 2011-12-27 | Etfbの細胞異常増殖への適用方法及び異常増殖抑制剤 |
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- 2011-12-27 EP EP11872070.5A patent/EP2754719B1/en active Active
- 2011-12-27 CN CN201180074671.7A patent/CN103917659A/zh active Pending
- 2011-12-27 JP JP2013532386A patent/JP5963757B2/ja not_active Expired - Fee Related
- 2011-12-27 WO PCT/JP2011/080247 patent/WO2013035209A1/ja active Application Filing
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Publication number | Publication date |
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JPWO2013035209A1 (ja) | 2015-03-23 |
US9562912B2 (en) | 2017-02-07 |
KR101856114B1 (ko) | 2018-06-20 |
EP2754719B1 (en) | 2018-08-29 |
EP2754719A4 (en) | 2015-04-01 |
JP6189466B2 (ja) | 2017-08-30 |
US20140235491A1 (en) | 2014-08-21 |
JP5963757B2 (ja) | 2016-08-03 |
KR20140064913A (ko) | 2014-05-28 |
EP2754719A1 (en) | 2014-07-16 |
CN103917659A (zh) | 2014-07-09 |
JP2016116537A (ja) | 2016-06-30 |
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