WO2018147504A1 - Mesenchymal stem cell line useful for developing fibrosis treatment agent - Google Patents

Abstract

The present invention relates to a mesenchymal stem cell line useful for developing a fibrosis treatment agent and, particularly, to hepatic stellate cell-derived mesenchymal stem cell line ONGHEPA1(KCTC 13086BP) which is characterized by inducing fibrosis by way of TGF-β or PDGF treatment, and to a method for screening a fibrosis treatment agent by using the mesenchymal stem cell line. The mesenchymal stem cell line according to the presentinvention can be very useful not only as a fibrosis model of a cell or tissue, but also as a cell for developing a new fibrosis treatment agent. Further, the screening method according to the present invention can accurately and efficiently screen a fibrosis treatment agent from a variety of candidate substances.

Description

Mesenchymal stem cell line useful for the development of fibrotic agents

The present invention relates to mesenchymal stem cell lines useful for the development of therapeutic agents for fibrosis, specifically, hepatic mesenchymal cell-derived mesenchymal stem cell line ONGHEPA1 (KCTC13086BP), characterized in that fibrosis is induced by treatment with TGF-β or PDGF and fibrosis using the same It relates to a method of screening a therapeutic agent.

Fibrosis is a disease in which excessive fibrous connective tissue is formed in organs or tissues during regeneration or development, which is in contrast to the formation of normal fibrous tissue. If the fibrous connective tissue is excessively formed in the organ or tissue, the tissue is hardened and the influx of body fluids is reduced, and thus the original function cannot be sufficiently performed in vivo. Causes include injury, inflammation, burns, radiation, chemotherapy, lymphedema. The problem caused by fibrosis depends on the location of the fibrous connective tissue is formed, mainly liver, secretory organs, lungs, etc. are damaged. Fibrosis is typically idiopathic pulmonary fibrosis (IPF), myelo fibrosis, liver fibrosis and kidney fibrosis.

Idiopathic pulmonary fibrosis is a chronically progressive interstitial pulmonary disease with poor disease progression and yet no proven treatment. Diagnosis is made when a biopsy of the lung reveals a honeycomb or irregular shape. Slow respiratory distress and death with hypoxia or myocardial infarction. To date, no clear cause has been proven, but various factors such as environment, virus, genetics, and toxic compounds cause inflammation in the lungs and fibrosis in the lungs due to excessive proliferation of fibroblasts in the process of healing the inflammation. It is thought to be.

Myelofibrosis is a disease in which the fibers of bone marrow tissue are over-developed, which lowers the function of making blood and changes in the number of red blood cells and white blood cells, and their actions. It is divided into idiopathic and secondary. Dual idiopathic symptoms include severe fibrotic hyperplasia and hypertrophy of systemic bone marrow, with nucleated red blood cells and fragile granulocytes in peripheral blood. The cause is uncertain, and bone marrow poisoning and inflammation are thought to be the cause. Secondary manifestations occur during the course of leukemia, malignant lymphoma, cancer metastasis, and chemical poisoning. Effective therapeutics have not yet been developed.

Liver fibrosis is also called cirrhosis. Chronic inflammation causes normal liver tissue to turn into fibrotic tissue, such as regenerative nodules, resulting in decreased liver function. Chronic hepatitis B or hepatitis C, heavy drinking, hepatotoxic substances, etc. due to the inflammatory state of the liver mainly occurs. Treatment is aimed at delaying the progression of symptoms as much as possible. Depending on the cause, antiviral drugs are used, but if the causes are different, the effect is unknown.

Renal fibrosis is a progressive disease in which extracellular matrix accumulates and fibrosis occurs in the kidney. It is characterized by glomerulosclerosis and tubular interstitial fibrosis. Fibrosis of the kidneys causes side effects in kidney function. Causes include trauma, infection, surgery, environmental factors, chemicals, radiation exposure, and symptoms such as pain, urination problems, nausea and vomiting. Drugs or kidney transplants can be used to manage symptoms, but they also require the development of effective therapies.

On the other hand, Grande et al. Recently suggested that Twist or Snail, a major regulator of epithelial mesenchymal transition (EMT) (hereinafter referred to as 'EMT'), plays an important role in renal fibrosis. Lovisa et al. Also suggested that EMT plays an important role in renal fibrosis. EMT refers to a phenomenon in which normal cells are genetically reprogrammed in the form of mesenchymal cells that are likely to move due to an intermediate cytoskeletal change as they progress to tumor cells. Therefore, it is thought that inhibiting the expression of EMT-related proteins can inhibit the metastasis and proliferation of tumors. Therefore, various researchers are conducting studies related to these EMTs to develop tumor therapeutics. Hundreds of the known factors such as Twist, Snail, Slug, E-cadherin, vimentin, collagen11 a1 are known.

Suzuki et al. Reported that DEC2 (BHLHE41) is a transcription inhibitor of Twist, a regulator of EMT, and inhibiting its expression activates EMT and converts it into malignant tumors. Sato et al. Also reported that DEC2 inhibits the expression of Slug, a regulator of EMT, and thus suppresses malignant tumoration induced by TGF-β. In addition, various researchers have reported that modulators of EMT, such as DEC2, are associated with cancer metastasis.

As such, studies on EMT and its modulators have mostly been conducted only with respect to cancer or tumors. However, the present inventors paid attention to the relationship between EMT and fibrosis based on the results of some previous studies, and expected to be able to prevent and treat fibrosis if EMT can be controlled.

The present inventors have noted the association between EMT and fibrosis and the association between DEC2 and EMT, and the results of studying the possibility of eupatini as a therapeutic agent for fibrosis by observing that eupatini upregulates DEC2 mRNA of macrophages differentiated from bone marrow cells. In fact, it has been revealed in cell models and animal models that eupatillin can inhibit EMT, and it has been shown that eupatin can effectively inhibit fibrosis of organs or tissues by activation of EMT.

In this process, the present inventors have greatly felt the need for a method that can more accurately and efficiently identify the fibrosis treatment effect of a fibrosis treatment agent such as eupatillin. Accordingly, the present inventors have developed a method for screening an accurate and efficient fibrosis treatment agent and a cell model necessary for this. Was intended.

Grande MT, Sanchez-Laorden B, Lopez-Blau C, De Frutos CA, Boutet A, Arevalo M, Rowe RG, Weiss SJ, Lopez-Novoa JM, Nieto MA. Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease. Nat Med. 2015 Sep; 21 (9): 989-97.

Lovisa S, LeBleu VS, Tampe B, Sugimoto H, Vadnagara K, Carstens JL, Wu CC, Hagos Y, Burckhardt BC, Pentcheva-Hoang T, Nischal H, Allison JP, Zeisberg M, Kalluri R. Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis. Nat Med. 2015 Sep; 21 (9): 998-1009.

Suzuki M, Sato F, Bhawal UK. The basic helix-loop-helix (bHLH) transcription factor DEC2 negatively regulates Twist1 through an E-box element. Biochem Biophys Res Commun. 2014 Dec 12; 455 (3-4): 390-5.

Sato F, Kawamura H, Wu Y, Sato H, Jin D, Bhawal UK, Kawamoto T, Fujimoto K, Noshiro M, Seino H, Morohashi S, Kato Y, Kijima H. The basic helix-loop-helix transcription factor DEC2 inhibits TGF-β-induced tumor progression in human pancreatic cancer BxPC-3 cells. Int J Mol Med. 2012 Sep; 30 (3): 495-501.

Dong Y, Geng Y, Li L, Li X, Yan X, Fang Y, Li X, Dong S, Liu X, Li X, Yang X, Zheng X, Xie T, Liang J, Dai H, Liu X, Yin Z , Noble PW, Jiang D, Ning W. Blocking follistatin-like 1 attenuates bleomycin-induced pulmonary fibrosis in mice. J Exp Med. 2015 Feb 9; 212 (2): 235-52.

Therefore, the main object of the present invention is to provide a method capable of accurately and efficiently screening a fibrosis therapeutic agent.

Another object of the present invention to provide a cell line required for such a screening method.

According to an aspect of the present invention, the present invention is characterized in that the mesenchymal stem cell line derived from mesenchymal cell, characterized in that fibrosis is induced by treatment of transforming growth factor beta (TGF-β) or platelet-derived growth factor (PDGF) ONGHEPA1 (KCTC13086BP) is provided.

According to another aspect of the present invention, the present invention comprises the steps of treating the ONGHEPA1 one protein and candidate selected from TGF-β and PDGF; And comparing the degree of fibrosis of the ONGHEPA1 treated with the protein and the candidate with the control group not treated with the candidate with the fibrosis therapeutic agent.

The screening method of the present invention is used as a method for screening a therapeutic agent for fibrosis selected from the group consisting of idiopathic pulmonary fibrosis, myelofibrosis, liver fibrosis, and kidney fibrosis. It is preferable.

In the screening method of the present invention, the degree of fibrosis is preferably determined using a method of observing the morphology of cells.

In the screening method of the present invention, the method further comprises comparing the expression patterns of the ONGHEPA1 treated with the protein and the candidate and the control genes without the candidate.

In the screening method of the present invention, the gene is Actg2, Postn, Col11a1, Fn1, Thsd7a, Trabd2b, Col5a1, Slit3, Cemip, Inhba, Spta1, Exoc4, Adamts12, Efnb2, Figf, Eln, Hs6st2, Hspg2, Tbcd, Serpin , Tlcd2, Frem1, Cald1, Loxl2, Timp3, Col3a1, Pdia6, Ptn, Parm1, Dpysl3, Col12a1, Cryzl1, Calu, Fstl1, Vcl, Ccnd2, Adamts2, Dysf, Olfm2, Uba1, Lepre1, Psap, Ltbp1, Pbn , Fam53b, Cav1, Nisch, Fndc1, Tpm1, Dclk1, Actn4, Csf1, Tnc, Itsn1, Tacc2, Psd3, Ctbp2, Hsp90aa1, Sept2, Efemp2, Ehd2, Copg1, Mycn, Ligi1, Ilporap, Itbscr17, , Tnks2, Plod3, Btaf1, Dync1h1, Aurka, Wnk1, Col7a1, P4ha1, Syne2, Caprin1, Calr, Eng, Map4, Arhgef2, Ip6k1, TEAD1, Plod2, Fam175b, Asap1, Lama4, Serpine 1, Trrap It is preferably one or more genes selected from the group consisting of ERGIC1, Rnf145, Axl, Ltbp2, Ltbp4, Mcfd2 and Akt1.

The mesenchymal stem cell line of the present invention can be very useful not only as a fibrosis model of cells or tissues but also as a cell for the development of a new fibrosis therapeutic agent. According to the screening method of the present invention, a fibrosis therapeutic agent can be accurately and efficiently used from various candidate substances. Can be screened.

Figure 1 shows the results of the IHC analysis of ONGHEPA1 of the present invention, it can be seen that ONGHEPA1 is a cell of the Hepatic Stellate Cells (HSC) (hereinafter referred to as 'HSC') lineage. Left: IHC results using anti-GATA4 antibody response positive, right: IHC results using anti-CK-18 antibody response negative.

Figure 2 shows the results of FACS analysis of ONGHEPA1 of the present invention, it shows that ONGHEPA1 expresses the biofilm proteins CD29, CD44, CD71 and CD106 as mesenchymal stem cells.

Figure 3 is a photomicrograph of the morphology of the cells over time by treating the TGF-β in ONGHEPA1 of the present invention. None-treated: Control group in which ONGHEPA1 cells were cultured without any treatment. TGFβ: Experiment group treated with TGF-β at 5ng / ml.

Figure 4 is a photomicrograph of the morphology of cells over time by treating PDGF in ONGHEPA1 of the present invention. None-treated: Control group in which ONGHEPA1 cells were cultured without any treatment. PDGF: Experiment group treated with 5 ng / ml of PDGF.

Figure 5 is a graph showing the effect of increasing the DEC2 mRNA expression of eupatilli in macrophages differentiated from bone marrow cells.

Figure 6 shows the effect of inhibiting the fibrosis of eupatillin, after the recovery of three parts of the lung tissue from one mouse in each experimental group photographed by a microscope by masson's trichrome staining. Normal control group: normal mice bred by vehicle only without bleomycin and eupatylin, bleomycin group: bleomycin-induced pulmonary fibrosis, bleomycin + eupatin group: bleomycin Mice that induce pulmonary fibrosis by administration and administration of eupatillin (40 μg).

Figure 7 is treated with a fibrosis inducer (TGF-β or PDGF) and eupatillin in the ONGHEPA1 of the present invention and the photomicrograph of the shape of the cells (top of each A and B), and immunofluorescence staining and DAPI staining results (Bottom of each A and B). A) ONGHEPA1 was not treated separately (Non-treated) or treated with TGF-β (5 ng / ml) (TGFβ 24h) or TGF-β + Eufatlin (50 μM) (TGFβ + eupatilin 24h) Results for cells of B) ONGHEPA1 (Non-treated), PDGF (5ng / ml) (PDGF 24h) or PDGF + Eupatlin (50μM) (PDGF + eupatilin 24h) and 24 hours Result for later cells.

FIG. 8 is a microscopic photograph of the morphology of cells treated with fibrosis inducers (TGF-β or PDGF) and eupatillin in normal human lung fibroblasts (NHLF) (tops of A and B, respectively), and immunofluorescence and DAPI Dyeing result (bottom of A). A) No treatment to NHLF (Non-treated), TGF-β (5 ng / ml) (TGFβ 48h) or TGF-β + Eupatillin (50 μM) (TGFβ + eupatilin 48h) after 48 hours As a result, B) ONGHEPA1 was treated with non-treated (Non-treated), PDGF (5 ng / ml) (PDGF 48h) or PDGF + Eupatlin (50 μM) (PDGF + eupatilin 48h) 48 hours. Result for later cells.

FIG. 9 shows no treatment of ONGHEPA1 of the present invention (DMEM), only treatment with fibrosis inducers (TGF-β or PDGF) (5 ng / ml) (TGFβ 24h, PDGF 24h), fibrosis inducers and eupatini (50 μM). ) Together with (TGFβ + eupatilin 24h, PDGF + eupatilin 24h) or fibrosis inducer and ONGE200 (50 μM) together (TGFβ + ONGE200 24h, PDGF + ONGE200 24h) and microscopically capture the morphology of cells after 24 hours. One picture.

10 is a picture of the morphology of the cells after 24 hours after the treatment of the ONGHEPA1 of the present invention (DMEM) or only the fibrosis inducer (TGF-β) (5ng / ㎖) (TGFβ 24h) And after 24 hours of treatment with the fibrosis inducer, treatment with eupatillin (50 μM) or ONGE200 (50 μM) (TGFβ 24h + eupatilin 24h, TGFβ 24h + ONGE200 24h). .

11 is treated with only the fibrosis inducer (TGF-β or PDGF) (5 ng / ml) (TGF-b, PDGF) to the ONGHEPA1 of the present invention (Tup-b, PDGF), or the fibrosis inducer and eupatillin (50 μM) (Eup), fibrosis Induction agent and ONGE200 (50μM) is treated together (ONGE200) and the total RNA was purified by real-time PCR to analyze the expression pattern of each EMT related gene (Col11A1, slit3, Axl, Postn, Fn1 or Aurka). A) Results of experiment using TGF-β as a fibrosis inducer, B) Results of experiment using PDGF as a fibrosis inducer.

FIG. 12 shows no treatment with NHLF (Non treated control), PDGF (5 ng / ml) treatment only (PDGF_48h, PDGF_72h), or PDGF and eupatillin (50 μM) together (Eup_48h, Eup_72h) total RNA This is a graph analyzing the expression of each EMT-related gene (Col11A1, vimentin or periostin) by real-time PCR.

Figure 13 shows the results of the analysis of the transcriptome of the control ONGHEPA1, ONGHEPA1 induced fibrosis, and ONGHEPA1 induced fibrosis and eupatyline treated. Control group: ONGHEPA1, TGFβ: control group in which TGF-β was added to the culture medium and cultured with ONGHEPA1 for 24 hours, and TGFβ + Eup: experimental group in which TGF-β and eupatillin were added to the culture solution and cultured for ONGHEPA1 for 24 hours.

FIG. 14 is a volcano plot showing gene induction fold and p-value of genes whose expression is changed in normal ONGHEPA1, fibrosis-induced ONGHEPA1, and fibrosis-induced and opathyin-treated ONGHEPA1.

FIG. 15 shows the interactionome of genes whose expression is significantly changed in normal ONGHEPA1, fibrosis-induced ONGHEPA1, and induction of fibrosis and UFHEPA1 treated in ubiastine in an unbiased manner.

The mesenchymal stem cell line ONGHEPA1 of the present invention has been deposited with the accession number KCTC13086BP in the Biological Resource Center (KCTC) of the Korea Research Institute of Bioscience and Biotechnology.

ONGHEPA1 of the present invention is a mesenchymal stem cell (MSC) of the Hepatic Stellate Cells (HSC) family of mice (hereinafter, referred to as 'MSC'), and is a cell capable of infinite growth.

Fibrosis can be induced by a simple method of treating transforming growth factor beta (TGF-β) or platelet-derived growth factor (PDGF), which is very useful not only as a cell fibrosis model but also as a cell for screening of fibrotic therapies. In particular, ONGHEPA1 is a liver-derived cell, which can be used as a model for liver fibrosis. Since there is no suitable liver fibrosis model, its value is considered very high.

ONGHEPA1 of the present invention can be cultured in DMEM medium, but is not limited thereto. It can be cultured with or without FBS (fetal bovine serum), and it is preferable to include antibiotics such as penicillin and streptomycin to prevent contamination. The culture temperature is preferably about 37 ℃, it is preferable to incubate at about 5% CO ₂ state.

The present invention provides a method for screening a fibrosis therapeutic agent using ONGHEPA1 as described above, the screening method comprising the steps of treating ONGHEPA1 with a protein and a candidate selected from TGF-β and PDGF; And comparing the degree of fibrosis of the ONGHEPA1 treated with the protein and the candidate and the control group not treated with the candidate.

As a method for treating TGF-β, PDGF, or a candidate substance, a method of contacting ONGHEPA1 by suspending the candidate substance in the culture medium of ONGHEPA1 or suspending the candidate substance in the buffer solution may be used. Each concentration may be appropriately selected depending on the case, but according to the present invention, the TGF-β or PDGF for inducing fibrosis is preferably 2 to 10 ng / ml, and more preferably 5 ng / ml. .

Treatment of the fibrosis inducer TGF-β or PDGF and the candidate may be performed simultaneously or at different times. For example, a method of simultaneously treating a fibrosis inducing agent and a candidate material, a method of treating a fibrosis inducing agent first, and then treating the candidate after a predetermined time, a method of treating a candidate material first, and then treating a fibrosis inducing agent after a certain time. Can be used. If the fibrosis inducing agent is treated first, the effect of the candidate substance occurs after a certain degree of fibrosis, and thus the therapeutic effect can be more clearly investigated. If the candidate substance is treated first, the prevention effect can be more clearly investigated. have.

There is no restriction | limiting in the candidate substance applicable to the screening method of this invention. That is, both natural and artificial synthetic compounds can be applied, and it is considered that the present invention can be applied without limitation to the form of purified compounds or extracts.

The screening method of the present invention compares the degree of fibrosis of ONGHEPA1 treated with one of TGF-β and PDGF, that is, a fibrosis inducer and a candidate, with a control without the candidate. Judgment can be made using an observation method. The morphology of the cells can be observed through a microscope. When fibrosis is induced, ONGHEPA1 differentiates into myofibroblasts and secretes extracellular matrix protein (ECM) between cells, slowing cell movement and overexpressing αSMactin (alpha smooth muscle actinin), resulting in an overexpression of the cytoskeleton. Cures. Thus, the cell morphology is clear and elongated compared to the fibrosis inducer. The degree of fibrosis can be determined by comparing the morphology of these cells. If the degree of fibrosis is less than that of the control group without the candidate material, or close to the cell type of ONGHEPA1 not treated with both the fibrosis inducing agent and the candidate material, then the candidate material may have a therapeutic effect on fibrosis. If eupatitiline, which is effective in treating fibrosis, is used as a positive control group, the fibrosis therapeutic agent can be screened more accurately and the activity level of the candidate substance can be determined.

In addition to comparing the degree of fibrosis as described above, using a method of comparing the expression patterns of genes can more accurately and effectively screen for a fibrosis therapeutic agent. Expression of genes can be confirmed using methods such as real-time PCR, but is not limited thereto. According to the present invention, the expression of several genes is regulated by eupatyline having an excellent fibrotic effect in fibrosis-induced ONGHEPA1, and among these genes, genes related to Epithelial Mesenchymal Transition (EMT) were found. Experiments with eupatillin have demonstrated that EMT plays a very important role in fibrosis, and thus, the fibrotic therapeutic agent can be screened more accurately and effectively by comparing the expression patterns of these EMT-related genes according to the treatment of candidates. Among these EMT-related genes, in particular Actg2, Postn, Col11a1, Fn1, Thsd7a, Trabd2b, Col5a1, Slit3, Cemip, Inhba, Spta1, Exoc4, Adamts12, Efnb2, Figf, Eln, Hs6st2, Hspg2, Tbcd, Serpinf1, Tlc1, Tlc1 , Cald1, Loxl2, Timp3, Col3a1, Pdia6, Ptn, Parm1, Dpysl3, Col12a1, Cryzl1, Calu, Fstl1, Vcl, Ccnd2, Adamts2, Dysf, Olfm2, Uba1, Lepre1, Psap, Ltbp1, Sptbn1, Palld Cbd1, 53 , Nisch, Fndc1, Tpm1, Dclk1, Actn4, Csf1, Tnc, Itsn1, Tacc2, Psd3, Ctbp2, Hsp90aa1, Sept2, Efemp2, Ehd2, Copg1, Mycn, Ligi1, Il18rap, Wbscr17, Col1a5, Synlo3gb , Btaf1, Dync1h1, Aurka, Wnk1, Col7a1, P4ha1, Syne2, Caprin1, Calr, Eng, Map4, Arhgef2, Ip6k1, TEAD1, Plod2, Fam175b, Asap1, Lama4, Serpine 1, Trrap, SURF1, RGbpl9, Osbpl9 It is preferable to compare the expression patterns of one or more genes selected from the group consisting of Axl, Ltbp2, Ltbp4, Mcfd2 and Akt1. These genes are genes whose expression is upregulated when fibrosis is induced in ONGHEPA1, and that expression is significantly downregulated when eufatlin is treated.

According to the screening method of the present invention, a therapeutic agent for fibrosis selected from the group consisting of idiopathic pulmonary fibrosis, myelofibrosis, liver fibrosis, and kidney fibrosis can be screened. Although ONGHEPA1 is a cell derived from the liver, most of the genes whose expression is upregulated when fibrosis is induced and the expression is significantly downregulated when fibrosis is inhibited are genes involved in EMT. Experiments have demonstrated that this EMT is also involved in the fibrosis of other tissues or cells, and according to the screening method of the present invention, in addition to hepatic fibrosis, the treatment for such other fibrosis can also be screened accurately and effectively.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Production of ONGHEPA1

After removing the liver tissue of C57BL / 6 mice, a single cell suspension was prepared to obtain infinitely proliferating cells through continuous culture, and reverse transcription polymerase chain reaction (RT-PCR), immunoohistochemistry (IHC), and FACS ( HSC-like MSC cell lines were selected by fluorescence-activated cell sorting.

Since HSC is different from normal hepatocytes, cells that are albumin-negative are selected by RT-PCR, and GATA4 and CK-18, endo / ectodermal markers, by IHC analysis using anti-GATA4 and anti-CK-18 antibodies. HSC was selected by selecting cells expressing.

In addition, MSC markers were selected by FACS analysis using antibodies against CD29, CD44, CD71, and CD106 by selecting cells in which the MSC markers are expressed on the surface of cells.

As a result, it was possible to select a cell line that satisfies all the above conditions (see FIGS. 1 and 2), named this cell line ONGHEPA1 and deposited in the Korea Institute of Bioscience and Biotechnology (KCTC) Accession No. (KCTC13086BP) Was given.

Example 2. Fibrosis Characterization of ONGHEPA1

The most important causative cell of liver fibrosis is HSC. HSCs differentiate into several cells, one of which is myofibroblast. HSCs also secrete large amounts of extracellular matrix protein (ECM) between cells, slowing their movement. Collagen α1 is the most abundant ECM protein. At the same time, αSMactinin is overexpressed in fibroblasts to harden the cytoskeleton.

Using the properties of the HSC as described above to determine the fiberization properties of ONGHEPA1. After treatment with TGF-β (5 ng / ml) or PDGF (5 ng / ml) to induce fibrosis and observing the morphology of cells according to the elapsed time, 6 to 8 hours after TGF-β or PDGF treatment Signs of fibrosis were seen from the time point, a significant fibrosis progressed at the point of 24 hours elapsed, it was confirmed that further fibrosis progressed at the point of 48 hours (see FIGS. 3 and 4).

Example 3 Validation of ONGHEPA1 in Fibrosis Therapeutic Screening

3-1. The Effect of Eupatillin on Fibrosis Treatment for Validation of ONGHEPA1

The fibrotic therapeutic effect of eupatillin used as a fibrosis therapeutic agent to verify the availability of ONGHEPA1 in the screening of a fibrosis therapeutic agent was demonstrated through experiments as in Examples 3-1-1 to 3-1-2. The chemical structure of eupatillin is represented by the following Chemical Formula 1.

[Formula 1]

3-1-1. Increased DEC2 Expression by Eupatillin

DEC2 is known as a transcription inhibitor of Twist and Slug, which are regulators of EMT. Therefore, if the expression of DEC2 is increased, transcription of EMT regulators such as Twist and Slug is inhibited, and thus, tissue fiber can be suppressed because EMT is inhibited. Therefore, it was confirmed whether the expression of DEC2 was increased by eupatini using mouse bone marrow cells (MBMC).

MBMC treated with macrophage-colony stimulating factor (M-CSF) and receptor activator of NFκB (RANKL) ligand to activate it, followed by incubation at a concentration of 50 μM for 4 days, followed by culture of DEC2. It was confirmed.

As a result, as shown in Figure 5, eupatillin appeared to act as a DEC2 inducer (inducer) to increase the mRNA expression of DEC2 7-8 times.

3-1-2. Effect of Eupatillin on Fibrosis of Bleomycin-Induced Lung Tissue

A real animal model was used to determine whether eupatini could actually inhibit tissue fibrosis. Five-week-old male C57BL / 6J mice (18.2-20.5 g in weight) (KOATECH, Korea) were used as experimental animals. The experimental animals were divided into five groups in each group as shown in Table 1 below.

Experimental group	Bleomycin Administration	Eupatillin Administration
Normal control	-	-
Bleomycin group	40 µg / head	vehicle
Bleomycin + Eupatillin 1 ㎍ administration group	40 µg / head	1 µg / 20 µl
5 g of bleomycin + eupatin	40 µg / head	5 µg / 20 µl
Bleomycin + Eupatillin 10 ㎍ administration group	40 µg / head	10 µg / 20 µl
20 µg administration group of bleomycin + eupatin	40 µg / head	20 µg / 20 µl
Bleomycin + Euphatiline 40 ㎍ administration group	40 µg / head	40 µg / 20 µl

The experimental animals used polysulfone material, 369L x 156W x 132H (mm) (EU, USA, UK GL compliance) standard breeding box in SPF (Specific Pathogen Free), BSL (Bio Safety Level) Level 2 facility. Breeding was done. The number of animals per breeding box was 2 to 3 in the quarantine / purifying period and 2 to 3 in the testing period.The temperature was 22 ± 2 ℃, the relative humidity was 50.0 ± 15.0%, the ventilation frequency was 10-20 times / hour, and the contrast was Cycle (lighting time) 12 hours / day (07:00 ~ 19:00), was bred under the conditions of the illumination of 150 ~ 300 Lux.

In order to induce pulmonary fibrosis, a bleomycin solution was directly injected into the lungs through trachea according to the method of intratracheal instillation (IT) by Kremer et al., Laxer et al. And Berkman et al. That is, while inhalation of C57BL / 6J mice with 70% N ₂ O, 30% O ₂ gas and 1.5% isoflurane, the skin was cut in the foreground, the muscles were cleaned, the organs were exposed, and the organs were removed with ophthalmic surgical scissors. A little incision was made. Using a 1 ml syringe equipped with a round 19 gauge needle, 50 μl of bleomycin-dissolved distilled water was injected directly into the lungs through the incision. Immediately after injection, the incision skin was sutured, awakened from anesthesia, and housed in a normal breeding cage. The administration of bleomycin was performed using a video instillobot, and 12-day pulmonary fibrotic disease induction period was set by one administration of 40 µg / 50 µl of bleomycin-HCl.

Eupatillin was dissolved in DPBS buffer (containing 1% DMSO), and the dosage of eupatillin was 1 ml / kg, and the individual dose was calculated based on recent weight measurement. Twelve days after bleomycin administration, the group was forced nasal administration once a day (five times a week) for one week using a micropipette. Toxicity symptoms and mortality were observed for two to three days after eupatillin administration, but no abnormal symptoms were observed after administration of bleomycin and eupatillin.

Three animals were selected for each experimental group to separate lung tissue. Microscopic observation of isolated lung tissue by masson's trichrome staining revealed that pulmonary fibrosis was induced by bleomycin treatment, and pulmonary fibrosis was inhibited by the administration of eupatillin in the 20 and 40 µg administration groups. . In particular, more effective inhibition of pulmonary fibrosis was confirmed in the 40 μg administration group of upatillin (see FIG. 6).

3-2. Validation of the availability of ONGHEPA1 using eupatini

As described above, the availability of ONGHEPA1 in fibrosis therapy screening was verified using the eupatylin whose fibrosis treatment effect was confirmed. If fibrosis is inhibited by eupatillin in conditions in which fibrosis of ONGHEPA1 is induced, it means that ONGHEPA1 can be used as a cell for screening for fibrosis therapeutics.

Thus, while ONGHEPA1 was incubated, TGF-β (5 ng / ml) or PDGF (5 ng / ml) was treated to induce fibrosis, but eupatini was added to the culture at a concentration of 50 μM. As a result, in the experimental group treated with eupatillin, unlike the fibrosis experimental group treated with TGF-β or PDGF, HSC was differentiated into myofibroblast after 24 hours, and no fibrosis was observed (see FIG. 7).

In order to verify the above experiment, the experiment was performed in the same manner using NHLF (normal human lung fibroblast), not ONGHEPA1. NHLF is known to cause idiopathic fibrosis. As a result, as shown in Figure 8 NHLF also showed the same trend as in ONGHEPA1.

Example 4. Maintenance and Management of ONGHEPA1

4-1. Equipment and Supplies

4-1-1. equipment

Biosafety cabinet (Ex. BL2, SterilGard Hood, The Baker Company), Pipet-aid (Drummond), Centrifuge (Ependorf), 37 ° C water bath, CO ₂ incubator (Thermo), Hemocytometer, Phase-contrast microscopy (Olympus CKX31) , Freezing container (cryobox, eg Nalgene), cryogenic freezer (Thermo -80 ℃), Nitrogen tank (eg MVE500)

4-1-2. Expendables

Polypropylene Conical Tube (e.g. BD FalconTM), Spray with 70% Ethanol, Serological Pipet, Cryovial (e.g. Nunc cryo freezing vial cat no. 377267), Label Markers and Labels, Dry Ice and Styrofoam Boxes

4-1-3. Medium and general reagent

DMEM high glucose (Hyclone, US), Penicillin streptomycin (Hyclone, US), Fetal Bovine Serum (FBS) (Hyclone, US), Dulbesco phosphate Buffered Saline, Trypan blue solution 0.4% (in normal saline) (Gibco, Cat No. 15250-061) (used for cell counting), DMSO (Sigma-aldrich, Cat no. D2650) (used for freezing), Trypsin / EDTA (Gibco, Cat no. 25300) (used for cell separation) )

Example) Preparation of culture solution (based on 500ml): DMEM high glucose (500ml) + FBS (50ml) + 100U / ml penicillin / streptocmycin (5.5ml)

4-2. Thawing of Frozen ONGHEPA1

The thawing of frozen ONGHEPA1 in Cryovial is as follows.

Preliminary Preparation: Prepare the cell dilution solution in advance (e.g., put about 35ml of cooled culture solution in a 50ml conical tube) and preheat the bath.

(1) Transfer the cell cryovial from the liquid nitrogen tank to the styrofoam box containing dry ice.

(2) Grab the cap of the Cryovial and immerse it within about 20 ~ 25 seconds so that the lid is not locked in the 37 ℃ thermostatic water bath. Floating racks may be used to treat multiple cryovials, but limit the number of cryovials that can be processed before the iceballs that form inside the vial melt and thaw. Do not shake or flip the partially thawed vial.

(3) Sprinkle 70% ethanol spray on the outside of the partially thawed vial quickly and clean it with a clean paper towel.

(4) Open the cryovial cap inside the cleanbench, add 0.5-1 ml of culture with a 2 ml selogical pipet or 1 ml blue tip pipette, and transfer to a 50 ml conical tube containing the prepared media. Fill up to 50 ml with culture (about 0.5-1 ml of culture can be added to recover the cells remaining in the cryovial).

(5) Centrifuge at room temperature for 5 minutes at a speed of 250 ~ 300xg (about 1200 ~ 1300rpm).

(6) Remove the supernatant by vacuum suction or directly (be careful not to lose the cell pallet when using vacuum suction).

(7) Add 5-20 ml of culture (concentration of approximately 2x10 ⁶ cells per ml) to cell precipitation and gently float with pipette.

(8) Measure cell number by using hematocytometer.

(9) Inoculate the cells in T-75 flask or dish for cell culture containing warm culture.

(10) After 24 hours incubation in a 5% CO ₂ incubator at 37 ℃, the culture medium was replaced to remove the non-adherent cells and cell debris.

(11) Replace the broth every 3-4 days.

4-3. Subculture of ONGHEPA1

(1) On the 7th day of culture, when the density of the cells was increased by 80% on the culture plate under a microscope, the culture medium was discarded, washed twice with DPBS (5ml) and removed by vacuum suction. .

(2) Add 2 ml of 0.05% trypsin / EDTA (Gibco) and incubate at 37 ° C for 3-5 minutes.

(3) Check the isolation state of the cells, put the same amount of trypsin / EDTA and the same amount of the culture solution, collect the cells using a pipette, put them in a conical tube and centrifuge for 3 minutes at a speed of 250 ~ 300xg (about 1200 ~ 1300rpm). .

(4) Carefully remove the supernatant using vacuum suction, and add 8 ml of warm culture solution to the conical tube to suspend the cell pallet using a pipette.

(5) Add warm culture solution (18ml) to four 75T-flasks, and add 2ml of the cell suspension prepared in (4) to the flask. (Cell passage ratio is about 1: 4 ~ 5).

(6) Incubate in a 5% CO ₂ incubator at 37 ° C.

4-4. Cryopreservation of ONGHEPA1

Preparation: Prepare Cryovial (Nunc cryo freezing vial cat no. 377267), label markers and labels, Freezing container (cryobox (eg Nalgene) and cryopreservation solution (FBS: DMSO = 9: 1 with complement-inactivation).

(1) Discard the culture medium, wash twice with DPBS (5 ml) and remove using vacuum suction.

(2) Add 2 ml of 0.05% trypsin / EDTA (Gibco) and incubate at 37 ° C for 3-5 minutes.

(3) Check the separation state of the cells, put the same amount of trypsin / EDTA and the same culture medium, and collect the cells using a pipette, and then put the cells in a conical tube and centrifuge for 3 minutes at a speed of 250 ~ 300xg (about 1200 ~ 1300rpm).

(4) Carefully remove the supernatant using vacuum suction, add 1 ml of the cryopreservation solution (preservation can be increased depending on the number of cells) at 37 ° C into the conical tube and resuspend the cells using a pipette. (Usually suspended 5-10x10 ⁶ cells per ml of medium).

(5) Immediately after suspension, the cells suspended in a pipet are dispensed into pre-labeled cryovials, then placed in a cyrobox filled with isopropanol and stored in a -80 ° C freezer.

(6) Cells stored within a minimum of three hours or one week in a -80 ° C freezer are transferred to the vapor phase of the liquefied nitrogen reservoir for long-term preservation.

Example 5. Screening with ONGHEPA1

5-1. Way

Preparation: DMEM high glucose without Serum, DMEM high glucose without Serum, Recombinant human TGFb-1 (10㎍, Peprotech), Eupatillin (5mg, Adipogen), DPBS, 24 well plate or 6 well plate , DMSO

Final concentration of drug used in this example

Concentration of hTGFβ-1 to induce fibrosis: 5ng / ml

Concentration of Eupatillin and Screening Compounds for Fibrosis Inhibition: 50 μM / mL

Dissolution of hTGFβ-1 using culture medium without serum

Dissolution of Eupatilin and Screening Compounds Using DMSO

5-1-1. Serum condition

(1) When using a 24 well plate, seed 4x10 ⁴ cells in DMEM medium containing 10% FBS, 1% penicillin streptomycin, and incubate in 37 ° C and 5% CO ₂ incubator.

(2) When about 50 ~ 60% of the cells are filled in the culture plate (2 days after initial seeding), start the experiment (if there is a serum, the cells proliferate steadily over time, which is a good result due to overproliferation. Cannot be obtained).

(3) Remove the culture by vacuum suction and wash it 2 ~ 3 times using DPBS.

(4) Put 1 ml of culture solution (including serum) in which eupatini or screening compound is dissolved into each well.

(5) Photographing and RNA collection for cell morphology 12 hours and 24 hours after eupatini or screening compound treatment (fiberization rate in serum conditions is faster than serum free and needs to be checked for 12 hours)

5-1-2. Serum free condition

(1) In case of using a 24 well plate, seed 1x10 ⁵ cells in DMEM medium containing 10% FBS and 1% penicillin streptomycin, and then incubate in 37 ° C and 5% CO ₂ incubator.

(2) After about 2 days, about 80% of the cells will be filled in the culture plate, and at this point, the test will be performed. (If 4x10 ⁴ cells are seeded, 80% of the cells will be filled after 3-4 days. In the case of experiments, it is better to increase the seeding cells early)

(3) Remove the medium using vacuum suction and wash it three times with DPBS so that no residual FBS remains. (If only a small amount of FBS remains, this may affect the effect of the drug.)

(4) Inject 1 ml of serum free medium in which upatillin or screening compound is dissolved into each well.

(5) Photographing and RNA collection to confirm cell morphology 6, 12, 24 and 48 hours after eufatlin or screening compound treatment

5-2. result

The compound having an antifibrotic effect was screened using the method of Example 5-1. As a result, ONGE200 [5,7-dihydroxy-2- (4-hydroxyphenyl) -6-methoxy-chromone], including eufatlin, was found to have excellent antifibrotic effect among the various compounds (Fig. 9 and 10). The chemical structure of ONGE200 is shown in the following formula (2).

[Formula 2]

Example 6 Analysis of Expression Patterns of EMT-Related Genes

To investigate the expression patterns of EMT-related genes under conditions in which fibrosis is induced in ONGHEPA1 and in the condition that fibrosis is inhibited by eupatin or ONGE200, ONGHEPA1 (control) and TGF- treated with TGF-β or PDGF EMT-related gene expression patterns were analyzed by real-time PCR by purifying total RNA from ONGHEPA1 (experimental group) (fibrosis inhibition), which was treated with β or PDGF and eupatin or ONGE200. As a result, as shown in FIG. 11, the expression of EMT-related genes was inhibited by eupatini or ONGE200.

In order to verify the above experiment, the experiment was conducted in the same manner using NHLF, not ONGHEPA1. As a result, as shown in Figure 12 NHLF also showed the same trend as in ONGHEPA1.

The above experimental results indicate that ONGHEPA1 is very effective for screening compounds exhibiting antifibrotic effect through the regulation of EMT-related genes.

Example 7. Global Gene Expression Pattern Analysis

To investigate the expression of various genes under the conditions of fibrosis induction and inhibition of fibrosis of ONGHEPA1, ONGHEPA1 (control) and TGF-β treated with DMSO were treated with fibrosis-induced ONGHEPA1, TGF-β, and eupatiliin. Global gene expression patterns of ONGHEPA1 treated together were examined.

After 24 hours of treatment with DMSO, TGF-β or eupatillin, total RNA was isolated to prepare a library, and the Illumina High-seq sequencer analyzed approximately 10,000 expressed mRNAs of 30 Giga total transcriptome.

As a result, the following results were obtained (see FIG. 13).

1) Number of genes commonly expressed in three experimental groups: 9,033

2) Genes expressing specifically in the control ONGHEPA1 cell line: 628

3) Gene expressing specifically when TGF-β is treated: 169

4) Genes that are specifically expressed when TGF-β and Eupatillin are treated: 150

5) Commonly expressed genes in TGF-β treated group and TGF-β + eupatillin treated group: 657

6) Commonly expressed genes in control and TGF-β treatment groups: 171

7) Commonly expressed genes in control group and TGF-β + eupatin treatment group: 188

Accordingly, the treatment of TGF-β in the ONGHEPA1 cell line induces the expression of 826 (= 169 + 657) genes, which in turn activates the EMT program, and the treatment of euphatilin results in the expression of 328 (= 150 + 188) genes. It can be seen that trans-differentiation occurs again, inverting the EMT program to its original state.

The gene induction fold and p-value of the gene whose expression is changed in the TGF-β treatment group and the TGF-β + eupatillin treatment group are shown in a volcano plot as shown in FIG. 14.

EMT is a cellular program involved in tumor development, stem cell differentiation and fibrosis, which has been known to involve hundreds of genes. Col11a1 (Collagen type XI alpha 1), Postn (Periostin), Slit3 (Slit homolog 3), Fn1 (Fibronectin 1), Axl (AXL receptor tyrosine kinase), and Aurka (Aurora kinase A) are EMT markers. In Example 6, it was shown that eupatillin inhibits the expression of Col11a1, Postn, Slit3, Fn1, and Aurka genes by TGF-β, and RNA-Seq results show that 976 genes were treated by TGF-β or eupatin treatment. The expression of 9,033 genes commonly found in all treatments was also found to be microscopically increased and decreased, and both of them would directly or indirectly affect EMT. Therefore, based on big data that selects all of these 10,000 genes whose expression is regulated to p <0.05 or less by treatment with TGF-β + Eupatillin and integrates all the functions of the protein encoded by each of the selected genes. Gene interactionomes were analyzed in an unbiased manner to investigate the networks between them (see FIG. 15).

Surprisingly, there are eight hubs of these gene networks, forming one network framework through several nodes, and the eight hubs are mostly composed of genes related to EMT. The cytoskeleton protein hub (I) and collagen protein hub (II) are networked by integrin α1 (Itga1) nodes, and cell cycle protein hub-1 (III), including Cyclin B1, a key factor in EMT, is protein kinase. It is shown that the integrin beta 3 (Itbg3) node, together with the three protein hubs, is connected to the C alpha (PKCA) node to form a robust network. The second cell cycle hub-2 (IV) is the transcription factor Lymphoid Enhancer Binding Factor 1 (Lef1) node, which is linked by Cyclin D1 (Ccdn1), and the Adenylate Cyclase 9 (Adcy9) signaling hub, which is an important chemokine for EMT, is CXCL16. EMT factors were networked with C-Fos, CCL2 and Junb. Here, Adcy9 signaling herb is thought to be a new EMT regulator that is influenced by eupatillin. The CD44 herb (V), known to have a profound effect on cancer cell EMT, has been linked to secreted phosphoprotein 1 (Spp1 / Osteopontin) nodes involved in cell migration and invasion. Spp1 (osteopontin) is also known as an important EMT factor. The Spp1 node was networked with Mmp3, an ECM protease. Hub (VI), which is an important protein of the ECM matrix and major EMT factors, Fibrillin and Elastin, is linked to the central node, integrin b3 (Itgb3). The EMT-causing cytokine transforming growth factor b2 protein hub (VII) contains serpine1 and Figf (= VegfD) and has been shown to network with kinase insert domain receptor (Kdr) nodes. The semaphorin and cholesterol receptor (Vldr) hubs (VIII), known as important receptors for EMT, contain plexin D, semaphoring 3E, neurophilin, and NGF, and the semaphorin receptor hub is networked with Kdr nodes. The Vldr receptor hub is connected to insulin receptor substrate 2I (rs2) via nerve growth factor (Ngf), indicating that the signaling axis is formed. Snail2 (= Slug) -Ecadherin (= Cadh1) nodes, the major transcription factors of EMT, establish a network with cell cycle protein hub (II), and Ecadherin is an important EMT factor, Mmp3, caviolin (Cav), Tenascin C (Tnc) and It has been shown to be associated with PKCa. PKCa was networked with cytoskeleton and collagen protein hubs by integrin b4. Tenascin C was directly associated with integrin B3.

Herb I. Skeletal Protein Herbs

Troponin I1 & Troponin I2, Tropomyosin 2, Transgelin, α2 smooth muscle actin, Myosin heavy chain 9 & 11, Leiomodin 1, γ2 smooth muscle acitin, Laminin subunit α4

Herb II. Collagen Proteomic Herbs>

Collagen 4 α5 & α6, Collagen 5 α1 & α3, Collagen 6 α3, Collagen 8 α1 & α5, Collagen 11 α1, Collagen 12 α1, Collagen 15 α1

Herb III. Cell cycle Protein Herb-1>

Cyclin B1, Gadd45a, Cyclin F, ASPM, NIMA-related kinase (Nek2), Optineurin

Herb IV. Cell cycle Proteomic Herbs-2>

Cyclin D1, Cdk14, C-Fos, Junb, CCL2, CCL7

Herb V. CD44 Associated Protein Hub

Cd44, Hypoxia Up-Regulated 1 (Hyou1), Ncam, Calreticulin, Immunity-Related GTPase M (Irgm1), Parp4, Parp9, Pdia4 & Pdia6

Herb VI. Fibrillin Protein Herbs>

Efemp2 (EGF Containing Fibulin-Like Extracellular Matrix Protein 2), Fibrillin 5 (Fbn5), Fibrillin 2 (Fbn2), Elastin (Eln), Fibrillin 1 (Fbn1)

Herb VII. Transforming Growth Factor Beta 2 Protein Herbs>

RAR Related Orphan Receptor A (Rora), Neuronal PAS Domain Protein 2 (Npas2), Serpine1, Transforming Growth Factor Beta 2 (Tgfb2), Vascular Endothelial Growth Factor D (Figf)

Herb VIII. Semaphorin & Vldr Receptor Protein Herbs>

Plexin D1, Semaphorin 3E, Semaphorin 3A, Neuropilin 1, Very Low Density Lipoprotein Receptor, Nerve Growth Factor (Ngf)

Major Network Nodes of the TGF-β-Eupatilin Interactome

Integrin α1, Integrin β3, Integrin β4, Protein kinase Cα, Lef1, Slug, Cadherin1 (= E-Cadherin), Adenylate cyclase 9, Spp1 (= Osteopointin), Fibrilin1, Dedicator of cytokinesis 1 (Dock1), Syk2, Notch4, etc.

These results indicate that the EMT program induced by TGF-β treatment in ONGHEPA1 is reversed by eupatini, and the mechanism is based on eight EMT protein hubs, namely cytoskeleton protein hub (I) and collagen protein hub (II). , Cell cycle protein hub-1 (III), Cell cycle protein hub-2 (IV), CD44 associated protein hub (V), Fibrillin protein hub (VI), TGF-β2 protein hub (VII), and Semaphorin and Vldr receptors Protein hub (VIII) is produced and destroyed, and integrin α1, intergrin β3, protein kinase Cα, Snali2, Kdr, Ecadherin and adenylate cyclase 9 are important nodes.

Example 8. Target Gene Analysis

On the basis of the results of the global gene expression profile investigation as in Example 7, expression was induced by TGF-β treatment in ONGHEPA1 cells, and the gene was regressed when treated with eufatlin, ie, TGF-β treatment group. Among the genes with difference in expression between the and eupatini treatment groups, the genes were selected so that the expression was greatly increased by the TGF-β treatment and the expression was hardly achieved by the treatment with the eupatini.

As a result, 103 genes as shown in Tables 2 and 5 were selected.

No.	Description (Abbreviation)	Log2fc	p-value
One	Actin, gamma2 (Actg2)	-5.83	0.00005
2	Periostin (postn)	-4.92	0.00005
3	Collagen, type XI, alpha 1 (Col11a1)	-3.11	0.00005
4	Fibronectin 1 (Fn1)	-infinite	0.0002
5	Thrombospondin, type I domain containing 7A (Thsd7a)	-5.06	0.0002
6	TraB domain containing 2B (Trabd2b)	-2.75	0.0003
7	Collagen type XV, alpha 1 (Col5a1)	-2.26	0.00055
8	Slit homolog 3 (Slit3)	-3.65	0.00065
9	Cell migration inducing protein, hyaluronan biding (Cemip)	-2.75	0.00095
10	Inhibition beta-A (Inhba)	-2.19	0.0015
11	Spectrin alpha, erythrocytic 1 (Spta1)	-4.01	0.00165
12	Exocyst complex component 4 (Exoc4)	-2.9	0.0019
13	A disintegrin-like and metallopeptidase with thrombospondin type 1 motif 12 (Adamts12)	-2.09	0.0025
14	Ephrin B2 (Efnb2)	-1.92	0.0038
15	c-fos induced growth factor (Figf)	-2.49	0.0044
16	Elastin (Eln)	-3.28	0.00555
17	Heparan sulfate 6-O-sulfotransferase 2 (Hs6st2)	-3.26	0.0056
18	Perlecan (Heparan sulfate proteoglycan2) (Hspg2	-infinite	0.0057
19	Tubulin-specific chaperone d (Tbcd)	-2.11	0.00595
20	Natriuretic peptide receptor 3 (Npr3)	-2.82	0.00675
21	Serin (or cysteine) peptidase inhibitor, clade F, member 1 (Serpinf1)	-1.99	0.00685
22	TLC domain-containing protein 2 (Tlcd2)	-infinite	0.0074
23	Fras 1 related extracellular matrix protein 1 (Frem1)	-2.36	0.0075
24	Caldesmon 1 (cald1)	-infinite	0.0074
25	Lysyl oxidase-like 2 (Loxl2)	-1.93	0.0078
26	Tissue inhibitor of metalloproteinase 3 (Timp3)	-infinite	0.00785
27	Collagen, type III, alpha 1 (Col3a1)	-infinite	0.0083
28	Protein disulfide isomerase associated 6 (Pdia6)	-1.85	0.00835
29	Pleiotrophin (Ptn)	-2.06	0.00875
30	Prostate androgen-regulated mucin-like protein 1 (Parm1)	-1.57	0.01225

No.	Description (Abbreviation)	Log2fc	p-value
31	Dihydropyrimidinase-like 3 (Dpysl3)	-infinite	0.0138
32	Collagen, type XII, alpha 1 (Col12a1)	-2.02	0.01435
33	Crystallin, zeta (quinone reductase) -like 1 (Cryzl1)	-infinite	0.01475
34	Calumenin (calu)	-infinite	0.015
35	Follistatin-like 1 (Fstl1)	-infinite	0.0156
36	Vinculin (Vcl)	-infinite	0.01575
37	Cyclin D2 (Ccnd2)	-2.29	0.01585
38	A disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif 2 (Adamts2)	-2.08	0.1685
39	Dysferlin (dysf)	-2.06	0.01765
40	Olfactomedin 2 (Olfm2)	-1.9	0.01845
41	Ubiquitin-like modifier activating enzyme 1 (Uba1)	-infinite	0.01855
42	Leprecan 1 (Lepre1)	-1.75	0.01865
43	Prosaposin (Psap)	-infinite	0.01875
44	Latent transforming growth factor beta binding protein 1 (Ltbp1)	-3.32	0.01985
45	Spectrin beta, non-erythrocytic 1 (Sptbn1)	-infinite	0.02
46	Palladin, cytoskeletal associated protein (Palld)	-infinite	0.02005
47	Protein FAM 53B (Fam53b)	-infinite	0.02015
48	Caveolin 1, Caveolae protein (Cav1)	-1.76	0.02025
49	Nischarin (Nisch)	-infinite	0.02075
50	Fibronectin type III domain containing 1 (Fndc1)	-1.75	0.02105
51	Tropomyosin 1, alpha (Tpm1)	-infinite	0.02145
52	Doublecortin-like kinase 1 (Dclk1)	-1.54	0.023
53	Actin alpha 4 (Actn4)	-infinite	0.0241
54	Colony stimulating factor 1 (macrophage) (Csf1)	-2.15	0.02535
55	Tenascin C (Tnc)	-5.1	0.02575
56	Intersectin 1 (SH3 domain protein 1A) (Itsn1)	-infinite	0.0263
57	Transforming, acidic coiled-coil containing protein 2 (Tacc2)	-infinite	0.0267
58	Pleckstrin and sec7 domain containing 3 (Psd3)	-1.43	0.0275
59	C-terminal-binding protein 2 (Ctbp2)	-infinite	0.0277
60	Heat shock protein 90, alpha (cytosolic), class A member 1 (Hsp90aa1)	-infinite	0.029

No.	Description (Abbreviation)	Log2fc	p-value
61	Septin2 (Sept2)	-infinite	0.02975
62	Epidermal growth factor-containing fibulin-like extracellular matrix protein 2 (Efemp2)	-1.66	0.03005
63	EH-domain containing 2 (Ehd2)	-infinite	0.03025
64	Coatomer protein complex, subunit gamma 1 (Copg1)	-infinite	0.03045
65	v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (Mycn)	-2.05	0.031
66	Lethal giant larvae homolog 1 (Ligi1)	-infinite	0.0331
67	Interleukin 18 receptor accessory protein (Il18rap)	-1.69	0.0332
68	Willians-Beuren syndrome chromosome reion 17 homolog (Wbscr17)	-2.83	0.03325
69	Collagen type 1 alpha 1 (Col1a1)	-1.64	0.0334
70	Synaptopodin (Synpo)	-infinite	0.03375
71	Integrin beta 5 (Itgb5)	-infinite	0.0342
72	Tankyrase, TRF1-interacting ankyrin-related ADP-rebose polymerase 2 (Tnks2)	-infinite	0.0349
73	Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (Plod3)	-2.01	0.0355
74	BTAF1 RNA polymerase II, B-TFIID transcription factor-associated (Btaf1)	-infinite	0.0356
75	Dynein cytoplasmic 1 heavy chain 1 (Dync1h1)	-infinite	0.03565
76	Aurora kinase A (Aurka)	-15.3	0.03595
77	WNK lysine deficient protein kinase 1 (Wnk1)	-infinite	0.03685
78	Collagen type VII alpha1 (Col7a1)	-15.2	0.03715
79	Procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha 1 polypeptide (P4ha1)	-1.81	0.0375
80	Spectrin repeat containing nuclear envelope 2 (Syne2)	-infinite	0.0376
81	Cell cycle associated protein 1 (Caprin1)	-infinite	0.0382
82	Calreticlin (cal)	-1.74	0.03845
83	Endoglin (Eng)	-infinite	0.03895
84	Microtubule-associated protein 4 (Map4)	-infinite	0.039
85	rho / rac guanine nucleotide exchange factor (GEF) 2 (Arhgef2)	-infinite	0.03915
86	Inositol hexakisphosphate kinase 1 (Ip6k1)	-infinite	0.03985
87	TEA Domain Transcription factor 1 (TEAD1)	-infinite	0.04005
88	Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 (Plod2)	-1.48	0.0402
89	Family with sequence similarity 175, member B (Fam175b)	-infinite	0.0413
90	ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (Asap1)	-infinite	0.0414

No.	Description (Abbreviation)	Log2fc	p-value
91	Laminin, alpha 4 (Lama4)	-1.29	0.04145
92	Serine (or cysteine) peptidase inhibitor, clade E, member 1 (Serpine 1)	-1.78	0.0419
93	Importin-4 (Ipo4)	-infinite	0.04235
94	Transformation / transcription domain-associated protein (Trrap)	-infinite	0.043
95	Surfeit 1 (SURF1)	-infinite	0.044
96	Oxysterol binding protein-like 9 (Osbpl9)	-infinite	0.0458
97	Endoplasmic reticulum-golgi intermediate compartment 1 (ERGIC1)	-1.26	0.0465
98	Ring finger protein 145 (Rnf145)	-infinite	0.04665
99	AXL receptor tyrosine kinase (Axl)	-infinite	0.048
100	Latent transforming growth factor beta binding protein 2 (Ltbp2)	-infinite	0.04845
101	Latent transforming growth factor beta binding protein 4 (Ltbp4)	-infinite	0.0492
102	Multiple coagulation factor deficiency 2 (Mcfd2)	-1.28	0.04935
103	Thymoma viral proto-oncogene 1 (Akt1)	-infinite	0.04985

As a result of examining the functions of the 103 genes and related studies, most of them were factors related to EMT. Especially in the case of the Follistatin-like 1 (Fstl1) gene, when knocked out, the lung was induced with bleomycin, There was a study showing that little fiberization (Dong et al., 2015).

Therefore, if the expression patterns of the 103 genes described above are applied to the screening of ONGHEPA1 fibrosis therapeutic agent, it will be possible to more efficiently screen the compounds that can inhibit fibrosis through the regulation of EMT.

The mesenchymal stem cell line and the screening method of the present invention can be very useful for the development of new and effective fibrotic therapeutic agents from various candidates.

[Accession number]

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13086BP

Trust Date: 20160825

Claims (6)

1. Hepatic mesenchymal cell-derived mesenchymal stem cell line ONGHEPA1 (KCTC13086BP), characterized in that fibrosis is induced by treatment of transforming growth factor beta (TGF-β) or platelet-derived growth factor (PDGF).
2. Treating ONGHEPA1 of claim 1 with one protein and a candidate selected from TGF-β and PDGF; And

   Comparing the degree of fibrosis of the ONGHEPA1 treated with the protein and the candidate material and the control group not treated with the candidate material; Screening method for a fibrosis treatment comprising a.
3. The method of claim 2,

   The screening method is a method for screening a therapeutic agent for fibrosis selected from the group consisting of idiopathic pulmonary fibrosis, myelofibrosis, liver fibrosis and kidney fibrosis. Screening method.
4. The method of claim 2,

   The degree of fibrosis is screened by the method of observing the morphology of the cells.
5. The method of claim 2,

   Comparing the expression pattern of the ONGHEPA1 treated with the protein and the candidate material and the gene of the control group not treated with the candidate material; Screening method further comprising.
6. The method of claim 5,

   The genes are Actg2, Postn, Col11a1, Fn1, Thsd7a, Trabd2b, Col5a1, Slit3, Cemip, Inhba, Spta1, Exoc4, Adamts12, Efnb2, Figf, Eln, Hs6st2, Hspg2, Tbcd, Serpinf1, Tlcd2, Frem1, , Timp3, Col3a1, Pdia6, Ptn, Parm1, Dpysl3, Col12a1, Cryzl1, Calu, Fstl1, Vcl, Ccnd2, Adamts2, Dysf, Olfm2, Uba1, Lepre1, Psap, Ltbp1, Sptbn1, Palld, Fam1b, Cav1 Ni , Tpm1, Dclk1, Actn4, Csf1, Tnc, Itsn1, Tacc2, Psd3, Ctbp2, Hsp90aa1, Sept2, Efemp2, Ehd2, Copg1, Mycn, Ligi1, Il18rap, Wbscr17, Col1a1, Synpo, Itgb1, Tnk3c Bc , Aurka, Wnk1, Col7a1, P4ha1, Syne2, Caprin1, Calr, Eng, Map4, Arhgef2, Ip6k1, TEAD1, Plod2, Fam175b, Asap1, Lama4, Serpine 1, Trrap, SURF1, Osbpl9, ERGIC1, Rnft, 2xl Ltbp4, Mcfd2 and Akt1 screening method characterized in that one or more genes selected from the group consisting of.

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