KR102159630B1 - Pharmaceutical composition for preventing or treating cholestatic liver injury or hyperlipidemia comprising placenta-derived mesenchymal stem cells - Google Patents

Abstract

The present invention provides a pharmaceutical composition for the prevention or treatment of cholestatic liver disease or hyperlipidemia, comprising placenta-derived mesenchymal stem cells as an active ingredient.

Description

A pharmaceutical composition for preventing or treating cholestatic liver injury or hyperlipidemia comprising placenta-derived mesenchymal stem cells}

The present invention relates to a pharmaceutical composition for the prevention or treatment of cholestatic liver disease or hyperlipidemia comprising placenta-derived mesenchymal stem cells.

The liver is one of the major living organs of humans and is responsible for protein synthesis and metabolism, especially lipid metabolism. Fatty liver caused by alcohol or metabolic syndrome causes overall body abnormalities. Diet therapy is being used as a treatment for alleviating the symptoms of lipid accumulation. However, until now, a method for fundamentally treating lipid accumulation has not been developed.

Cholestatic liver injury is a disease caused by the accumulation of bile acids and lipids. The liver regulates central lipid metabolism processes including fatty acid synthesis, mitochondrial β-oxidation, and phospholipid transport. Impaired bile secretion caused by biliary obstruction or liver damage interferes with cholesterol and phospholipid metabolism. In the bile duct ligation (BDL) rat model, the liver lipid concentration did not change, whereas the concentration of very low-density lipoprotein (VLDL) cholesterol and low-density lipoprotein (LDL) cholesterol. Serum levels are markedly increased (T. Kamisako, et al., Hepatology Research, vol. 25, no. 2, pp. 99-104, 2003).

In addition, hyperlipidemia refers to a disease in which lipids and/or lipoproteins in blood are elevated to abnormal levels. Hyperlipidemia is also classified into hypercholesterolemia, hypertriglyceridemia, and combined hyperlipidemia according to the type of elevated lipids. Triglycerides are known to be one of the independent risk factors for atherosclerosis. Although conclusive evidence for the association between hypertriglyceridemia and cardiovascular disease, including atherosclerosis is not clear, it is known that hypertriglyceridemia increases the risk of developing atherosclerosis (Cullen P. Evidence that triglycerides are an independent coronary heart). disease risk factor Am J Cardiol 2000; 86:... 943-9; Le NA, Walter MF The role of hypertriglyceridemia in atherosclerosis Curr Atheroscler Rep 2007; 9:110-5; Stalenhoef AF, de Graaf J. Association of fasting and nonfasting serum triglycerides with cardiovascular disease and the role of remnant-like lipoproteins and small dense LDL. Curr Opin Lipidol 2008; 19:355-61).

On the other hand, the placenta is involved in the synthesis and secretion of cytokines and proteins, which have many kinds of physiological activity, as well as the role of a transport medium for substances such as nutrients, waste products, and oxygen, which are essential for fetal development during pregnancy. It is an organ. The present inventors have developed an improved manufacturing method of placenta extract that can avoid the safety problem of the use of animal-derived protein sources such as fetal bovine serum, and minimize protein denaturation through an aqueous extraction method. (Korean Patent Publication No. 10-2010-0067789; Shin et al., Cell Proliferation, 2010). In addition, placenta-derived mesenchymal stem cells are stem cells separated and extracted from placenta excised after childbirth, and have no ethical problem, and are capable of recovering a large amount of stem cells more easily than other stem cell fields. The present inventors have also developed a method for separating placental chorionic plate membrane-derived mesenchymal stem cells with high purity (Korean Patent Registration No. 10-0900309).

The present inventors were studying the possibility of various applications of placenta-derived mesenchymal stem cells, while placenta-derived mesenchymal stem cells not only improved inflammation in a bile duct ligation (BDL) rat model of chronic cholestasis liver disease. In addition, by effectively reducing total cholesterol, LDL cholesterol and triglycerides, reducing the expression of lipid-generating genes, and reducing lipid accumulation, it can be usefully applied in the prevention and treatment of cholestasis liver disease and/or hyperlipidemia. I found it.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cholestasis liver disease or hyperlipidemia comprising placenta-derived mesenchymal stem cells.

According to an aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cholestasis liver disease or hyperlipidemia, comprising placenta-derived mesenchymal stem cells as an active ingredient.

The placenta-derived mesenchymal stem cells may include mesenchymal stem cells derived from the chorionic plate of the placenta; Alternatively, it may be a stem cell obtained by transducing the PRL-1 gene into mesenchymal stem cells derived from the chorionic plaque of the placenta. More preferably, the placenta-derived mesenchymal stem cells are mesenchymal stem cells derived from the chorionic plate membrane of the placenta; Alternatively, it may be a stem cell obtained by transducing the PRL-1 gene into mesenchymal stem cells derived from the chorionic valve of the placenta.

In one embodiment, the mesenchymal stem cells derived from the chorionic valve of the placenta may include: (a) obtaining a chorionic valve from the placenta separated after childbirth from the mother's mother; (b) scraping the cells inside the chorionic valve obtained in step (a) to collect mesenchymal stem cells; (c) adding a solution containing trypsin and ethylenediaminetetraacetate to the cells obtained in step (b), performing an enzymatic reaction at 20 to 30°C, and adding fetal calf serum to stop the enzyme reaction; And (d) culturing the cells recovered by centrifuging the reaction solution obtained in step (c) in a medium to which fetal calf serum and antibiotics are added.

In another embodiment, the pharmaceutical composition may be in the form of a tissue-implantable injection or intravenous injection.

According to the present invention, placenta-derived mesenchymal stem cells, specifically placental chorionic valve-derived mesenchymal stem cells, not only improve inflammation, but also effectively reduce total cholesterol, LDL cholesterol and triglycerides, and It has been found to decrease expression and also reduce lipid accumulation. In addition, when the PRL-1 gene is overexpressed in placental-derived mesenchymal stem cells, specifically, placental chorionic valve-derived mesenchymal stem cells, it has been shown that the expression of lipid-generating genes and lipid accumulation can be further significantly reduced. It was revealed by the invention. Accordingly, the pharmaceutical composition of the present invention can be usefully used in the prevention and treatment of cholestasis liver disease and/or hyperlipidemia.

1 shows the effect of inflammatory response and placental chorionic valve-derived mesenchymal stem cell (CP-MSC) transplantation induced by chronic cholestasis. Histological analysis using hematoxylin and eosin staining was performed (scale bar = 50 μm; original magnification, × 200). CTL: control group; NTx (non transplantation): non-transplant group; Tx (transplantation): transplant group.
2 is a change in serum lipid profile after BDL and/or CP-MSC transplantation, showing the serum levels of total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides. * P <0.05 (compared to non-grafted group). CTL: control group; NTx (non transplantation): non-transplant group; Tx (transplantation): transplant group.
3 shows the results of analyzing the production process of the placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene using the puncture method and the characteristics of the obtained cells.
4 shows the results of analyzing the expression of a lipid-generating gene according to the transplantation of placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene using qRT-PCR.
5 shows the results of histological analysis of lipid accumulation in liver tissue following transplantation of placental chorionic valve-derived mesenchymal stem cells transduced with PRL-1 gene.

In the present specification, the term'cholestatic liver injury or cholestatic liver disease' refers to a condition in which bile does not flow from the liver to the duodenum, and refers to cholestasis caused by the accumulation of bile acids and lipids.

In addition, in the present specification,'hyperlipidemia' refers to a disease in which lipids and/or lipoproteins in the blood are elevated to abnormal levels, and hypercholesterolemia, hypertriglyceridemia, and complex Includes combined hyperlipidemia.

In cholestatic liver disease, impaired bile secretion interferes with lipid homeostasis. In the bile duct ligation (BDL) rat model transplanted with mesenchymal stem cells (CP-MSCs) derived from the chorionic plate membrane of the placenta, in histological analysis, fat accumulation and inflammatory reaction in liver tissue were observed. Although observed, a tendency to decrease was observed in the CP-MSCs transplant group. In addition, the increased serum cholesterol level after BDL was significantly reduced by CP-MSC transplantation. These results indicate that CP-MSCs have therapeutic potential for cholestatic liver disease and hyperlipidemia.

The present invention provides a pharmaceutical composition for the prevention or treatment of cholestatic liver disease or hyperlipidemia, comprising placenta-derived mesenchymal stem cells as an active ingredient.

The placenta-derived mesenchymal stem cells may be derived from various tissues constituting the placenta, such as amniotic epithelial cells, amniotic membranes, trophoblasts, and chorionic membranes. Preferably, the placenta-derived mesenchymal stem cells may be mesenchymal stem cells derived from the chorionic plate of the placenta, more preferably mesenchymal stem cells derived from the chorionic plate membrane. I can. Placenta-derived mesenchymal stem cells can be separated using a known method, and more preferably, placental chorionic membrane obtained by the separation method developed by the present inventors (Separation method according to Korean Patent Registration No. 10-0900309). Valve-derived mesenchymal stem cells can be used. Accordingly, in the pharmaceutical composition of the present invention, the placenta-derived mesenchymal stem cells include (a) obtaining a chorionic plate membrane from the placenta separated after childbirth from the mother's mother; (b) scraping the cells inside the chorionic valve obtained in step (a) to collect mesenchymal stem cells; (c) adding a solution containing trypsin and ethylenediaminetetraacetate to the cells obtained in step (b) to perform an enzymatic reaction at 20 to 30°C, and adding fetal bovine serum to stop the enzyme reaction. ; And (d) culturing the cells recovered by centrifuging the reaction solution obtained in step (c) in a medium to which fetal calf serum and antibiotics are added. A more specific method may be performed according to the method disclosed in Korean Patent Registration No. 10-0900309, and the document is incorporated herein as a whole.

The present inventors have found that when the PRL-1 gene is overexpressed in placenta-derived mesenchymal stem cells, the expression and lipid accumulation of the lipid-generating gene can be more significantly reduced. Accordingly, the placenta-derived mesenchymal stem cells may be cells obtained by transducing the PRL-1 gene (the gene of SEQ ID NO: 1). PRL-1 (Phosphatase of regenerating liver-1, Protein Tyrosine Phosphatase Type IV A, Member 1; PTP4A1) is a gene encoded, including Prenylated Protein Tyrosine Phosphatase (PTPs). It has been reported that the PRL-1 gene stimulates the S phase in the G1 phase during mitosis to promote cell growth and proliferation, thereby activating the invasion or migration ability. This has a correlation with some cancer occurrence and metastasis. Recently, it has been reported that it is initially overexpressed during liver regeneration to prevent cirrhosis and aid liver regeneration (PMID 16508789). In addition, it has been reported that PRL-1, expressed in photoreceptor cells of the primate retina, regulates free radicals through the glutathione system (PMID 17673310). However, it has not yet been reported how transduction of the PRL-1 gene affects the expression of lipid-generating genes and lipid accumulation.

Therefore, the placenta-derived mesenchymal stem cells may be stem cells transduced with the PRL-1 gene, preferably stem cells obtained by transducing the PRL-1 gene into mesenchymal stem cells derived from the chorionic plaque of the placenta. It may be a cell, or more preferably a stem cell obtained by transducing the PRL-1 gene into a mesenchymal stem cell derived from the chorionic valve of the placenta.

The pharmaceutical composition of the present invention comprises, as an active ingredient, placenta-derived mesenchymal stem cells or placenta-derived mesenchymal stem cells transduced with the PRL-1 gene as described above, and a pharmaceutically acceptable carrier, that is, a cell therapeutic agent ( cell therapy) may include a carrier commonly used in the field. The pharmaceutical composition of the present invention may be formulated in the form of an intra-tissue-transplant injection, an intravenous injection, a lyophilized preparation for injection, and the like according to a conventional pharmaceutical method, and preferably, an intra-transplant injection or an intravenous injection. It can be formulated in a form. The pharmaceutically acceptable carrier may include a sterilized aqueous solution (eg, physiological saline, etc.), a non-aqueous solvent, and the like. In addition, if necessary, suitable stabilizers, adjuvants (eg, Freund's complete adjuvant, Freund's incomplete adjuvant, etc.), isotonic agents, preservatives, and the like may be included.

The dosage of the placenta-derived mesenchymal stem cells or placenta-derived mesenchymal stem cells transduced with the PRL-1 gene contained as an active ingredient in the pharmaceutical composition of the present invention is the patient's condition and weight, the degree of disease, and administration. It depends on the route and duration, but can be appropriately selected by those skilled in the art. For example, the placenta-derived mesenchymal stem cells or placenta-derived mesenchymal stem cells transduced with the PRL-1 gene are at least 2 X 10 ⁶ cells/kg or more, preferably ⁶ X 10 as a daily dose. It can be administered at a dose of ⁶ to 8 X 10 ⁶ cells/kg, and the above administration may be administered once or several times a day. Accordingly, in the pharmaceutical composition of the present invention, the content of the placenta-derived mesenchymal stem cells or placenta-derived mesenchymal stem cells transduced with the PRL-1 gene per unit dosage form of the pharmaceutical composition is at least 2 X 10 ⁶ cells/kg or more, preferably 6 X 10 ⁶ to 8 X 10 ⁶ cells/kg in an amount suitable for administration (e.g., for a patient of 60 kg, 1.2 X 10 ⁸ cells or more, preferably 3.6 X 10 ⁸ ∼ 5.0 X 10 ⁹ cells). Of course, the content range can be appropriately changed as needed.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Placenta-derived stem cells (hereinafter referred to as "CP-MSCs") used in the following tests were obtained from the chorionic plate membrane of the placenta obtained according to Korean Patent Registration No. 10-0900309. The derived mesenchymal stem cells were used.

1. Materials and test methods

(1) cell culture

The collection of placenta samples for research purposes was approved by the Institutional Review Board of Gangnam Cha Hospital. All participants provided clear consent prior to sample collection. Placenta was obtained from a woman who delivered at 38±2 weeks as a woman without any medical, obstetric or surgical complications. CP-MSCs were isolated according to the reported method (MJ Lee et al., Journal of Cellular Biochemistry, vol. 111, no. 6, pp. 1453-1463, 2010), and 10% fetal bovine serum, FBS; Sigma-Aldrich), 1% penicillin/streptomycin (Sigma-Aldrich), 1 μg/mL heparin (Sigma-Aldrich), and 25 ng/mL hFGF-4 (human fibroblast growth factor-4; Peprotech Inc., Rocky Hill, NJ, USA) supplemented with DMEM/F12 medium (Dulbecco's modified Eagle medium/Ham's F-12 medium; Sigma-Aldrich, St. Louis, MO, USA) containing 20% O ₂ at 37°C. % CO ₂ incubated in an incubator.

(2) BDL rat model and transplantation of CP-MSCs

Seven-week-old male Sprague-Dawley rats (Orient Bio Inc., Seongnam, Korea) were maintained in an air-conditioned animal facility. Previously reported methods (MF Mahmoud, et al, European Journal of Pharmacology, vol. 742, pp. 118-124, 2014; and JH Jun, et al., Clinical and Molecular Hepatology, vol. 22, no. 3, pp. 372-381, 2016), the common bile duct was ligated under general anesthesia using Avertin (2,2,2-tribromoethanol, Sigma-Aldrich). One week after bile duct ligation (BDL), CP-MSCs (2×10 ⁶ cells, passages 8-10) were injected intravenously through the tail vein of the transplant group. CP-MSC numbers were determined by conventional dose-determining tests (MJ Lee et al., Journal of Cellular Biochemistry, vol. 111, no. 6, pp. 1453-1463, 2010; J. Jung, et al., International Journal of Stem Cells, vol. 8, no. 1, pp. 79-89, 2015). Liver tissue and blood samples were collected at 1, 2, 3, and 5 weeks after transplantation in the transplant group, and at 1, 2, 3, and 5 weeks after BDL in the non-transplant group. The experimental protocol was approved by the Institutional Animal Care and Use Committee of CHA University.

(3) Histological analysis

A liver tissue sample was fixed in 10% formalin, embedded in paraffin, and cut to a thickness of 5 μm. The obtained sections were stained with hematoxylin/eosin and observed with an optical microscope (Axioskop2, Carl Zeiss Micro-Imaging, Oberkochen, Germany) at 200x magnification.

(4) blood chemistry

Serum concentrations of total cholesterol, high-density lipoprotein (HDL) cholesterol, LDL cholesterol, triglycerides, albumin, and total bilirubin were measured enzymatically with an automatic analyzer (Hitachi 747, Hitachi, Tokyo, Japan). I did.

(5) Quantitative real-time polymerase chain reaction

Rat liver tissue was homogenized and lysed, and total RNA was isolated using TRIzol reagent (Invitrogen). Reverse transcription was performed using 500 ng of total RNA and Superscript III reverse transcriptase (Invitrogen). Real-time polymerase chain reaction (PCR) was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Then, cDNA was amplified by PCR using the following conditions: 40 cycles at 95°C for 5 minutes, 95°C for 5 minutes and 60°C for 30 minutes. The primer sequence, the reaction solution composition, and the amplification method are shown in Tables 1, 2, and 3, respectively. GAPDH or β-actin was used as an internal control for normalization.

Marker gene Sequence number Primer sequence (5'-3') Junction temperature(℃) Stem cell Oct-4 2 F: AGT GAG AGG CAA CCT GGA GA 52 3 R; GTG AAG TGA GGG CTC CCATA Nanog 4 F: TTC TTG ACT GGG ACC TTG TC 52 5 R: GCT TGC CTT GCT TTG AAG CA Sox-2 6 F: AGA ACC CCA AGATGC ACA AC 52 7 R: GGG CAG CGT GTA CTT ATC CT HLA-G 8 F: GCG GCT ACTACA ACC AGA GC 58 9 R: GCA CAT GGC ACG TGT ATC TC TERT 10 F: GAGCTGACGTGGAAGATGAG 55 11 R: CTT CAA GTG CTG TCT GAT TCC AAT G Osteoblastic OC 12 F: CAC TCC TCG CCC TAT TGG C 58 13 R: CCC TCC TGC TTG GAC ACA AAG Col 1 14 F: AGA CAT CCC ACC AAT CAC CT 60 15 R: CGT CAT CGC ACA ACA CCT Lipid cellularity Adipsin 16 F: CAC GTA CCA TGA TGG GGC AA 55 17 R: TCG AGA TCC CCA CGT AAC CA PPARg 18 F: GAC AGA CCT CAG GCA GAT TG 55 19 R: GTC AGC GAC TGG GAC TTT TC Adiponectin 20 F: GAC TGC CAC TAA TTC AGA GC 55 21 R: CTC ATG GGG ATA ACA CTC AG LPL 22 F: ACA GGT GCA ATT CCA AGG AG 55 23 R: CTT TCA GCC ACT GTG CCA TA Leptin 24 F: ATC TAT GTG CAC CTG AGG GTA G 55 25 R: TCC TTT TCA CAA AGC CAC ACT AT FABP4 26 F: ACA TGA AAG AAG TGG GAG TTG GC 55 27 R: AAG TAC TCT CTG ACC GGA TGA CG - PRL-1 28 F: TAC TGC TCC ACC AAG AAG CC 60 29 R: AGG TTT ACC CCA TCC AGG TC Internal control human
GAPDH 30 F: GCA CCG TCA AGG CTG AGA AC 55 31 R: GTG GTG AAG ACG CCA GTG GA rat
GAPDH 32 F: TCC CTC AAG ATT GTC AGC AA 55 33 R: AGA TCC ACA ACG GAT ACA TT

PCR reaction solution volume cDNA 5 μl Forward primer 0.5 μl Reverse primer 0.5 μl Enzyme (FasStart universal SYBR Green master mix) 12.5 μl DEPC-treated water ^* 6.5 μl Total amount 25 μl

* Diethylpyrocarbonate (DEPC)-treated water

95 ℃ Tm ℃ 20 ℃ cycle denaturalization 10 minutes One Amplification 10 seconds 30 seconds (55 ℃) 40 Dissolution 1 second (60-94 ℃)
(melt curve 65-95℃, increasing 1℃) One final 1 min One

(6) Western blot analysis

Using RIPA containing a protease inhibitor cocktail (Roche, Branchburg, NJ, USA) and a phosphatase inhibitor (Sigma-Aldrich), rat liver tissues were homogenized and dissolved on ice. Protein lysates were separated by 8% to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis ((SDS-PAGE), and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). After that, blocking buffer (Tris-buffered saline (Tris-buffered saline (TBS)) 0.1% Tween 20 and 8% bovine serum albumin (BSA) for 1 hour). The membrane was blocked with Anti-PRL-1. A clonal antibody (1:1000; Abcam, Cambridge, UK) and an Anti-GAPDH polyclonal antibody (1:3000; Abfrontier, USA) were used to react for 16 hours at 4° C. After that, a washing solution (0.1%) Tween 20 (in TBS) was washed 3 times for 5 minutes, and then reacted for 1 hour at room temperature with a secondary antibody corresponding to the immunoglobulin of mice or rabbits conjugated with the enzyme (horseradish peroxidase). After washing three times for a minute, the protein was plated with chemiluminescence, and the light measured using an image densitometer (Bio-rad, USA) was analyzed using Image J analysis software. The optical density analysis for the hazardous protein band was based on the GAPDH optical density in the same gel.

(7) Preparation of placental chorionic valve-derived mesenchymal stem cells transduced with PRL-1 gene using electroporation method

Placental chorionic valve-derived mesenchymal stem cells (CP-MSCs) were treated with 10% Fetal Bovine Serum (FBS), 1% Penicillin-Streptomycin, 25 ng/ml Fibroblast Growth Factor-4. , FGF-4), 1 ug/ml Heparin was added in MEM-alpha medium and cultured in a CO ₂ incubator at 37°C. After the cells were washed with phosphate buffered saline (PBS), the cells were removed by treatment with trypsin for 2 minutes at 37°C, and then the removed cells were collected using PBS and centrifuged at 1200 rpm for 5 minutes. 1 ml of the culture solution was added to the obtained pellet and suspended, and then counted using a hemoglobin meter (hemocytometer). The recovered cells were centrifuged at 200 g for 10 minutes, Nucleofector was added to suspend the cells (5x10 ⁵ /100ul), and then 2 ug of DNA plasmid containing PRL-1 was added. The cells were transferred to a cuvette and placed in a Nucelofection machine to run the'U-23' program. Upon completion of the program, the culture dish containing the fresh medium was quickly stabilized in a 37°C, CO ₂ incubator. After 4 hours, the medium of the culture dish was removed using an inhaler, and the adhered cells were medium containing 1.5 mg/ml neomycin (10% Fetal Bovine Serum, FBS), 1% penicillin. Placental chorionic membrane transduced with PRL-1 gene by culture in MEM-alpha medium supplemented with streptomycin, 25 ng/ml fibroblast growth factor-4 (FGF-4), and 1 ug/ml Heparin Valve-derived mesenchymal stem cells were obtained.

(8) Characteristic analysis of placental chorionic valve-derived mesenchymal stem cells transduced with PRL-1 gene

Quantitative RT-PCR, RT-PCR, and Western blotting were performed to confirm whether the PRL-1 gene was overexpressed and stem cell ability of the placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene. Before performing quantitative RT-PCR, lysis cells with Trizol and synthesize cDNA using reverse transcriptase, gene-specific sequence and FastStart universal SYBR Green master mix and polymerization The qRT-PCR analysis was performed based on the PCR amplification step using an enzyme (Polymerase) and the numerical results of the amplified PCR products. The primer sequence used for the amplification of each gene, the composition of the PCR reaction, and the reaction conditions are shown in Tables 1 to 3, respectively. The cells overexpressing the PRL-1 gene were selectively recovered using an antibiotic medium containing 1.5 mg/ml neomycin using neomycin resistance included in the PRL-1 clone. The recovered PRL-1 overexpressed cells were centrifuged (12000rpm, 15min. 4°C) using RIPA Lysis buffer (SIGMA, USA) containing a protease inhibitor, and then proteins were separated. Protein (20ug) was transferred to a semipermeable membrane of polyvinylidinedifluoride (PVDF) by immunoblotting after SDS-PAGE. The semipermeable membrane was reacted in a blocking solution (5% bovine serum albumin, Bovine Serum Albumin, BSA) for 1 hour at room temperature, and then analyzed in the same manner as the Western blot method.

In order to detect the surface antigen of placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene, flow cytometry analysis was performed. Native placental chorionic valve-derived mesenchymal stem cells and placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene were converted to a specific concentration of fluorescent isothiocyanate (FITC)- or phycoethrin. (phycoerythrin, PE)-containing antibody was reacted and measured by flow cytometry (FACS). Information on each antibody is shown in Table 4 below.

Marker company Bell CD34-PE BD mouse CD13-PE BD mouse CD90-PE BD mouse CD105-PE BD mouse HLA-ABC-FITC BD mouse HLA-DR-FITC BD mouse HLA-G-FITC Abcam mouse

In addition, tumor formation experiments were conducted to confirm the safety of placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene. Four immune deficient NOD/SCID mice (Non Obese Diabetic-Severe Combined Immunodeficiency mice, NOD/SCID mice) (9 weeks old, male) (Orient Bio, Korea) were used. The experimental animals were grouped into a non-grafted group (control group, Con) and a placental chorionic valve-derived mesenchymal stem cell transplant group (direct transplantation into testicular tissue, Tx) transduced with the PRL-1 gene into testicular tissue. I did. Placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene were directly transplanted to 5x10 ⁵ cells/20ul diluted by adding 20ul phosphate buffered saline (PBS) using a 24 gage catheter. The control group was administered the same amount of phosphate buffered saline. After 14 weeks of breeding at 22~26℃ and 55~60% relative humidity, the mice were sacrificed to obtain testicular tissue, and then genomicDNA extraction and hematoxylin&eosin (H&E) staining were performed.

To confirm the differentiation ability of stem cells, placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene were induced to differentiate into adipocytes and bone cells. Placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene were cultured at a density of 5×10 ³ cells/cm ² , and then exchanged with adipocytes and bone cell differentiation induction medium (Gibco, USA). Adipocyte differentiation and bone cell differentiation maintenance medium was changed once every 2 days. After about 21 days, the cells were fixed in 4% PFA to accumulate lipid vesicles and calcium through Oil Red O (Sigma-Aldrich) and Von Kossa (Sigma-Aldrich) chemical staining. Was confirmed. Adipocytes and osteoblasts Placental chorionic valve-derived mesenchymal stem cells transduced with the differentiated PRL-1 gene were harvested, and expression of specific genes for adipocytes and bone cells was confirmed by qRT-PCR. gene The primer sequences used for amplification of each gene, the composition of the PCR reaction, and reaction conditions are shown in Tables 1 to 3, respectively.

(9) Analysis of expression of genes related to lipid production and expression regulation at the RNA level

Genes related to lipid production and expression regulation after transplantation of natural placental chorionic valve-derived mesenchymal stem cells (native) and placental chorionic valve-derived mesenchymal stem cells (PRL-1+) transduced with the PRL-1 gene into BDL rats Expression was subjected to quantitative RT-PCR (quantitative RT-PCR, qRT-PCR) analysis. After BDL was performed, liver tissues of the group transplanted with natural placental chorionic valve-derived mesenchymal stem cells or the placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene were collected and used with a mortar. The tissue was finely polished. After mixing the tissues for each group, lysis the tissue with Trizol, and synthesize cDNA using reverse transcriptase, gene-specific sequence and FastStart universal SYBR Green master mix The qRT-PCR analysis was performed based on the PCR amplification step using and the numerical result (CT value) of the amplified PCR product. The primer sequences used for amplification of each gene, the composition of the PCR reaction, and reaction conditions are shown in Tables 1 to 3, respectively.

(10) Analysis of lipid accumulation at the histological level of liver

In (3) above, the liver was removed and the liver was sectioned into 8 μM. The sectioned liver tissue was fixed with formaldehyde and washed with distilled water. Thereafter, 100% propylene glycol was reacted for 5 minutes, and then Oil Red O staining was reacted at room temperature for 1 hour. Thereafter, the reaction was carried out in 85% propylene glycol for 3 minutes for 3 minutes, followed by washing with water, followed by nuclear staining with hematoxylin. After washing with water, the slide was sealed and observed using an optical microscope.

(11) Statistical analysis

All tests were performed twice or three times. Data are expressed as mean±standard deviation. Student's t-tests were performed for comparison between groups, and P <0.05 was measured as a significance level by multiple comparison. Statistical analysis was performed using PASW version 22.0 (SPSS Inc., Chicago, IL, USA).

2. Test result

(1) CP-MSC transplant improves inflammation in rat liver.

To evaluate the effect of CP-MSCs transplantation on cholestatic liver injury, BDL rats were divided into 2 groups, rats of the transplanted group were injected with CP-MSCs, and the rats of the non-transplanted group were medium ( culture medium) was injected. As shown in FIG. 1, after 1 week of BDL, invasion of inflammatory cells around the bile duct and proliferation of bile ducts at the distal site were observed in both the non-grafted group and the transplant group. After 2 weeks of BDL, in the non-grafted group, the distal region was expanded due to extensive bile duct proliferation and concentric periductal fibrosis, and normal lobular structures were destroyed. Compared to the non-transplant group, bile duct proliferation was less pronounced in the transplant group, and the lobular pattern was preserved (FIG. 1). Fatty liver was not observed in the control group, the non-transplant group, or the transplant group.

(2) CP-MSC transplantation reduces BDL-induced hypercholesterolemia, but does not affect fatty acid uptake.

Inhibition of bile secretion induced by BDL causes an elevation of biliary phospholipids in circulating blood (SR De Vriese, et al., Annals of Nutrition & Metabolism, vol. 45, no. 5, pp. 209 -216, 2001). Therefore, the effect of transplantation of CP-MSCs on cholesterol metabolism was evaluated by measuring the cholesterol concentration in serum. Compared to the control group, the total cholesterol was significantly increased after 2 weeks of BDL in the non-transplanted group, whereas the total cholesterol in the transplanted group was significantly decreased compared to the non-transplanted group ( P <0.05; FIG. 2). Results similar to those for total cholesterol were also found in serum LDL cholesterol and triglycerides (FIG. 2 ). These results indicate that BDL-induced cholestasis and hypercholesterolemia are improved by CP-MSC transplantation.

(3) Characterization of placental chorionic valve-derived mesenchymal stem cells transduced with PRL-1 gene

For transduction of the PRL-1 gene into natural placental chorionic valve-derived mesenchymal stem cells, placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene were prepared by electroporation. The PRL-1 plasmid contains resistance to Neomycin antibiotics, and cells containing Green Fluorescent Protein (GFP) were selectively cultured using this. As a result of qRT-PCR and Western blotting analysis, PRL-1 expression was statistically significantly increased in placental chorionic valve-derived mesenchymal stem cells transduced with the PRL-1 gene compared to natural placental chorionic valve-derived mesenchymal stem cells. . In addition, as a result of analyzing the expression of stem cell ability (Oct4, Nanog, Sox2), immune tolerance (HLA-G), and proliferative ability (TERT) through RT-PCR, placental chorionic valve transduced with PRL-1 gene- It was confirmed that the derived mesenchymal stem cells maintained as much as the natural placental chorionic valve-derived mesenchymal stem cells. As a result of Flow Cytometrey, hepatopoietic markers (CD34, CD45 and HLA-DR) were negative, and non-hematopoietic markers (CD90, CD105, HLA-ABC and HLA-G) were positive. As a result of tumor analysis through placental-chorionic valve-derived mesenchymal stem cell transplantation transduced with the PRL-1 gene, tumor formation was not observed in the group (Tx) transplanted compared to the control group in the testis. In addition, as a result of confirming the differentiation ability of stem cells, lipid vesicles after differentiation into adipocytes and bone cells were detected by Oil Red O staining, and accumulation of calcium was detected by Bonkosa staining. As a result of quantitative RT-PCR analysis, it was observed that adipocyte-specific gene expression (Adipsin, PPAR gamma) and bone cell-specific gene expression (OC, Collagen Type 1) were statistically significantly increased in differentiated cells compared to undifferentiated cells. On the other hand, undifferentiated gene expression (Oct4) in differentiated cells was decreased compared to undifferentiated (FIG. 3 ).

(4) Analysis of gene expression related to lipid production and expression regulation

Expression of lipid production-related genes, that is, genes related to lipid production and maturation in the liver in the BDL model, Adipsin, Adiponectin, PPAR gamma, Leptin, LPL, and FABP4 were analyzed through qRT-PCR (FIG. 4). In the group transplanted with natural placental chorionic valve-derived mesenchymal stem cells, a significant difference was observed in that the expression of each adipogenesis, expression, and maturation-related gene decreased significantly up to about 3 weeks compared to the NTx group. In particular, the PRL-1 gene The transfected placental chorionic valve-derived mesenchymal stem cells transplanted group (PRL-1+) significantly decreased the expression of lipid production and maturation-related genes compared to the cholangioarthrosis group. In addition, gene expression was statistically significantly decreased in the placental chorionic valve-derived mesenchymal stem cell transplantation group transduced with the PRL-1 gene than in the natural placental chorionic valve-derived mesenchymal stem cell transplantation group. In particular, it was observed that the expression of PPAR gamma and Leptin was significantly reduced.

(5) Analysis of lipid accumulation after transplantation of placental chorionic valve-derived mesenchymal stem cells transduced with PRL-1 gene

After transplantation of natural placental chorionic valve-derived mesenchymal stem cells or placental chorionic valve-derived mesenchymal stem cells transduced with PRL-1 gene, lipid accumulation in the BDL model was analyzed by Oil Red O staining. Use 200 ul of phosphate buffered saline (PBS) to dilute natural placental chorionic valve-derived mesenchymal stem cells and placental chorionic valve-derived mesenchymal stem cells transduced with PRL-1 gene to 2x10 ⁶ cells/200 ul, and then diluted to 24 gage It was injected through the tail vein using a catheter of. The control group was administered the same amount of phosphate buffered saline. As can be seen in FIG. 5, it was observed that the accumulation of lipids decreased in the group administered with placental chorionic valve-derived mesenchymal stem cells compared to the BDL model. In particular, it was observed that the accumulation of lipids was significantly reduced in the placental chorionic valve-derived mesenchymal stem cell transplant group transduced with the PRL-1 gene compared to the natural placental chorionic valve-derived mesenchymal stem cells, and the result was also PRL- In the placental chorionic valve-derived mesenchymal stem cell transplant group transduced with 1 gene, lipids were significantly reduced (FIG. 5).

<110> CHA University Industry-Academic Cooperation Foundation SUNGKWANG MEDICAL FOUNDATION <120> Pharmaceutical composition for preventing or treating cholestatic liver injury or hyperlipidemia comprising placenta-derived mesenchymal stem cells <130> PN0868 <160> 33 <170> KoPatentIn 3.0 <210> 1 <211> 5093 <212> DNA <213> Homo sapiens <400> 1 aaagatgacc gtggttaagg cagtagaaag caggctcgac actgagacgc ggttccagcc 60 ctagaaggat tgcattttac gattcaggca aacttcgagt ctcctcatgt gacatgtgtg 120 cactcctgga ccgttttaat tatgagaaac gtggtttgca gcatgattcc ttccagtcga 180 taaatcggaa tctctctcgc tcccacccct tcttaacttc aggcttcctg catcccggag 240 cactcccggc agccccttcc ctcccccgcc ccggggatgc tccgactcgg cgcttagcca 300 ttcatcaacc ggttcacacc ggcggcggcc gccgcggagt gacgtccgga gggggcgggc 360 ctccgccccc gcctgtcggc tcctggcccg cggttccagg ccgcgattgg tggctggagg 420 gttgcacgtc gcgccggcta taaaggggag ggcttgtgac gcaagggcgc ctcggcgcgt 480 gtattggctc cttcggctgc gggccggctc ggctacgcgc tctgctccga gccgctcact 540 gcatggtaga gtctggtgcc cccgccgccg cctgcatcgc cgccaccgcc gctccgccac 600 gaccaccgcc gcctcctgcc ctgcagccac cgccaccgcc tgtgtcgccg ccgcctcggg 660 accggctgta tgattaggcc acaatcttca atgagtaaac atattcctca attctgtggt 720 gttcttggtc acacatttat ggagtttctg aagggcagtg gagattactg ccaggcacag 780 cacgacctct atgcagacaa gtgaactgta gaaactgatt actgctccac caagaagccc 840 ccataagagt ggttatcctg gacacagaag tgttgaattg aaatccacag agcattttac 900 aagagttctg acctggatgg ggtaaacctc agtgcacttc ttttctgttg gcctcagtat 960 tactggattg aagaattgct gcttcttgtt aggaggttca tttcacttat cattacttac 1020 aacttcatac tcaaagcact gagaatttca agtggagtat attgaagtag acttcagttt 1080 ctttgcatca tttctgtatt caattttttt aattatttca taaccctatt gagtgttttt 1140 taactaaatt aacatggctc gaatgaaccg cccagctcct gtggaagtca catacaagaa 1200 catgagattt cttattacac acaatccaac caatgcgacc ttaaacaaat ttatagagga 1260 acttaagaag tatggagtta ccacaatagt aagagtatgt gaagcaactt atgacactac 1320 tcttgtggag aaagaaggta tccatgttct tgattggcct tttgatgatg gtgcaccacc 1380 atccaaccag attgttgatg actggttaag tcttgtgaaa attaagtttc gtgaagaacc 1440 tggttgttgt attgctgttc attgcgttgc aggccttggg agagctccag tacttgttgc 1500 cctagcatta attgaaggtg gaatgaaata cgaagatgca gtacaattca taagacaaaa 1560 gcggcgtgga gcttttaaca gcaagcaact tctgtatttg gagaagtatc gtcctaaaat 1620 gcggctgcgt ttcaaagatt ccaacggtca tagaaacaac tgttgcattc aataaaattg 1680 gggtgcctaa tgctactgga agtggaactt gagatagggc ctaatttgtt atacatatta 1740 gccaacatgt tggcttagta agtctaatga agcttccata ggagtattga aaggcagttt 1800 taccaggcct caagctagac agatttggca acctctgtat ttgggttaca gtcaacctat 1860 ttggatactt ggcaaaagat tcttgctgtc agcatataaa atgtgcttgt catttgtatc 1920 aattgacctt tccccaaatc atgcagtatt gagttatgac ttgttaaatc tattcccatg 1980 ccagaatctt atcaatacat aagaaattta ggaagattag gtgccaaaat acccagcaca 2040 atacttgtat atttttagta ccatacagaa gtaaaatccc aggaactatg aacactagac 2100 cttatgtggt ttattccttc aatcatttca aacattgaaa gtagggccta catggttatt 2160 tgcctgctca ctttatgttt acatctccca cattcatacc aatatacgtc aggtttgctt 2220 aaccattgat tttttttttt ttttaccaag tcttacagtg attattttac gtgtttccat 2280 gtatctcact ttgtgctgta ttaaaaaaac ctccattttg aaaatctacg ttgtacagaa 2340 gcacatgtct ttaatgtctt cagacaaaaa agccttacat taatttaatg tttgcactct 2400 gaggtgcaac ttaacaggga gggcctgaga aaagaatggg agggggctat taattatttt 2460 tagcaaaatg ttgcctttgt cttgtgcaaa catgtagaat atgctcttta atttagtaaa 2520 atattttttt aaaaggtaga gatgctttgt tattgtaatc ataaacttcc tgaaattctt 2580 gtaatttttt tcccatactt atcagaagtg tgtttaccaa cttatttttg tttgaaagtg 2640 tgattttttt tttccttccc aacctctctt gcaaaaaaag aaatgggttt ctgctaatga 2700 attgagcaga catctaatat tttatatgcc ttttgagctg tgtaacttaa tatttggata 2760 cttgacaatt tgttttatta tgtaattgat aaaatggtga tgtgtattaa tgttagttca 2820 accatatatt tatactgtct ggggatgtgt ggttatagtt ctgtgggaga aataattttg 2880 tcagtgttca ccagcttgta aaaacttagt gcgagagctg aaacatctaa ataaataatg 2940 acatgcattt atcatcattg agattggttt gcttaaaatt aacttatttt gtagaagaca 3000 aaatgaattg cacttcactt aatgtgtgtc ctcatctttt tacaaataaa tgaaggatta 3060 taaatgatgt cagcatttta gtaaactttt agacaaaatt tgttagggtc attcatgaaa 3120 actttaatac taaaagcact ttccattata tactttttaa aggtctagat aattttgaac 3180 caatttatta ttgtgtactg aggagaaata atgtatagta gaggacagcc ttggtttgta 3240 aagctcagtt ccactagttc atggttttgt gcaacttctg agcctcagtt ttctcctttg 3300 caaattaata attacatacc tttatagatt ttgaaattaa tttaaatatt agtatttggt 3360 acatgaaggc ttaatgttaa gtttccttta atgatccaca ataatccctt tgatcacgtt 3420 aatctaaatc tagatgtctt tgtctaattt tttttgaata gcagttataa atgtaaagga 3480 ctcaaagttt aagtaaaaag tgatactcca ccttgtgttt caaagaattt agttccacct 3540 cttcatacca gtttaacact taatatattt cattggattt tagacagggc aaaaggaaga 3600 acaggggcct ctggaggccc ttggttattt aaatcttgga ttatttgtga tagtaatcac 3660 aaatttttgg ctaattttta acctgaggtt ttgttttttt tttaaaggaa atgcagccta 3720 gtcttgagaa cataatttta tataatcaat tactaaatgt taaactatta ccacacagcc 3780 cataaaacag catttgcgtt tattgagaga gaggatgtgc catcatgatt aatgaaaact 3840 atcttttgag tttgaaaaga aattaatttg cagtgtttgg attgtatata tggtgctaaa 3900 aataaattaa tttactttat aaaccttatc tgtacattat acgatgtgat gaaatttgct 3960 ttttatccaa atattttgta tcttgtaaat atggctaatt ataggaatgc ctataataca 4020 tcttagattc cttatatcta ataagagttc aaagagttat gagttgaagt cttgaatgca 4080 ggaaactatc tgatagtgtt ctaaaatttg gttacttggg tttggatacc cttagtggga 4140 tgatgtaaat agaggctagc tacctaggct tgtctatagc aaccataatg ttgatgtaag 4200 taatgcggtt actgaatcat aagaaaatgc catctctttt tagttgaagg aaaactctgg 4260 aagtaggtgc cattggtcat tctgcagtgc actgcaacca ttgtttcccc tagtgccctc 4320 ttttccctag ggcattgctc tcctattccc acgccttaac acagctctat acctagaagc 4380 agccagccca ggcatgcagt cacatttaat cacatccccc ttctagagtg cttcaaaatg 4440 atgtagtccc tcaacttggc taaagaatct caatctcttg aaatttattt ttttaatgtc 4500 atattcatct ggtaaatatc tactgtttgc caggcattta agaatatggc aaagaacata 4560 aaagatggtg tcaccagatt ttggtcacca atgagtaccc gacccgttgc catgattaag 4620 agagaatgct ttctattgga gtttcaggaa atataatttg agaatacttt aaagggaagt 4680 ggaagtataa gtgaatgata tttttctttt acatgtaaac aatgaagtta tttcaaagtt 4740 aagttttaaa caaaatacat gaagtagtgt ctgccataca tgttaatatt ctacattctt 4800 gcttccttaa attaatatgt ttgtgtgtat atatgtgcct cacacctgaa ttgaaaatta 4860 aagactggtt taaaagtggt ttaaaagtga catttaatgt ttctccatta cgtttggggt 4920 aaccagccta agtggaatct tggaaggaaa gtaagggaaa aacttgtatt tgccttcaat 4980 gaattaaacc agtgatatgt gttaacgtat gaatgaaagg attgatggtg attttataat 5040 tatatatatt gccgcagtaa ccagttaata aattgatagc taccatttaa aaa 5093 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 agtgagaggc aacctggaga 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gtgaagtgag ggctcccata 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 ttcttgactg ggaccttgtc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gcttgccttg ctttgaagca 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 agaaccccaa gatgcacaac 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 gggcagcgtg tacttatcct 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 gcggctacta caaccagagc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 gcacatggca cgtgtatctc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 gagctgacgt ggaagatgag 20 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 cttcaagtgc tgtctgattc caatg 25 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 cactcctcgc cctattggc 19 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 ccctcctgct tggacacaaa g 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 agacatccca ccaatcacct 20 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 15 cgtcatcgca caacacct 18 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 16 cacgtaccat gatggggcaa 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 17 tcgagatccc cacgtaacca 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 18 gacagacctc aggcagattg 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 19 gtcagcgact gggacttttc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 20 gactgccact aattcagagc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 21 ctcatgggga taacactcag 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 22 acaggtgcaa ttccaaggag 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 23 ctttcagcca ctgtgccata 20 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 24 atctatgtgc acctgagggt ag 22 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 25 tccttttcac aaagccacac tat 23 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 26 acatgaaaga agtgggagtt ggc 23 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 27 aagtactctc tgaccggatg acg 23 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 28 tactgctcca ccaagaagcc 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 29 aggtttaccc catccaggtc 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 30 gcaccgtcaa ggctgagaac 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 31 gtggtgaaga cgccagtgga 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 32 tccctcaaga ttgtcagcaa 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 33 agatccacaa cggatacatt 20

Claims (5)

A pharmaceutical composition for the prevention or treatment of cholestasis liver disease or hyperlipidemia, comprising placenta-derived mesenchymal stem cells overexpressing the PRL-1 gene as an active ingredient. The method of claim 1, wherein the placenta-derived mesenchymal stem cells overexpressing the PRL-1 gene are stem cells obtained by transducing the PRL-1 gene into mesenchymal stem cells derived from the chorionic plaque of the placenta. Pharmaceutical composition. The method of claim 1, wherein the placenta-derived mesenchymal stem cells overexpressing the PRL-1 gene are stem cells obtained by transducing the PRL-1 gene into mesenchymal stem cells derived from the chorionic valve of the placenta. Pharmaceutical composition. The method of claim 3, wherein the mesenchymal stem cells derived from the chorionic valve of the placenta are obtained by: (a) obtaining a chorionic valve from the placenta separated after childbirth from the mother's mother; (b) scraping the cells inside the chorionic valve obtained in step (a) to collect mesenchymal stem cells; (c) adding a solution containing trypsin and ethylenediaminetetraacetate to the cells obtained in step (b), performing an enzymatic reaction at 20 to 30°C, and adding fetal calf serum to stop the enzyme reaction; And (d) culturing the recovered cells by centrifuging the reaction solution obtained in step (c) in a medium to which fetal calf serum and antibiotics are added. A pharmaceutical composition, characterized in that it is obtained by a separation method. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is in the form of an intra-tissue-transplant injection or an intravenous injection.

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