KR20090025989A - Human mesenchymal stem cells improved tissue regeneration and production methods thereof - Google Patents

Abstract

A mesenchyme stem cell is provided to improve reproductive ability and the damaged tissue regeneration ability of mesenchyme stem cell. A mesenchyme stem cell is manufactured by being processed by antisense RNA of lzts2 gene coding amino acid sequence of a sequence number 1. An expression of the lzts 2 gene of the mesenchyme stem cell is held back. The antisense RNA is siRNA having base sequence selected from a group consisting of sequence number 2, 4, 6, 8, 10. A double-strand siRNA oligonucleotide of the lzts2 gene has a base sequence of a sequence number 2.

Description

Mesenchymal stem cells improved tissue regeneration and production methods

The present invention relates to mesenchymal stem cells with improved tissue regeneration ability, and more particularly, to mesenchymal stem cells treated with antisense RNA of lzts2 gene and suppressed the expression of lzts 2 gene, and a method for producing the same.

Ischemia is a condition in which the blood vessels that supply blood to the organs are blocked and the blood supply is impaired, causing local necrosis. Ischemia causes ischemic heart disease such as angina, myocardial infarction, and peripheral artery disease such as cerebral infarction and foot ulceration of the leg. Treatment of these ischemic diseases is performed by drug therapy, coronary angioplasty using a stent to dilate narrow blood vessels, and arterial bypass surgery in the heart. However, this method of treatment does not serve as a proper treatment in the case of problems with blood vessels. Therefore, new methods are needed to overcome the limitations of the existing treatment methods, and recently, studies on using stem cells to regenerate damaged tissues of ischemic diseases have been actively conducted.

Stem cells have the property of continuously producing the same cells as themselves for a certain period of time in an undifferentiated state and differentiating into specific cells under suitable conditions. Stem cells can be divided into embryonic stem cells and adult stem cells, depending on their origin. Human embryonic stem cells have many ethical problems because they are made from embryos that can occur in human life, but they are known to have superior cell proliferation and differentiation capacity as adult stem cells. Adult stem cells can be obtained from bone marrow, blood, brain, skin, etc., so there are few ethical problems, but they have limited multipotency compared to embryonic stem cells. The most researched adult stem cells are hematopoietic stem cells, and recent studies on mesenchymal stem cells are also invigorating.

The first bone marrow stromal cells found among mesenchymal stem cells were first identified by a study by Fiedenstein et al. (1968) (Friedenstein et al, 1968). And various connective tissues such as adipose tissue (Owen, 1988). Experiments have been attempted to differentiate bone marrow stromal cells into cardiomyocytes (Makino et al, 1999), hepatocytes (Oh et al, 2001) and neurons (Woodbury et al, 2000; Deng et al, 2001) in vitro. Mesenchymal stem cells, including myeloid matrix cells, can be used for a variety of purposes, including cell therapy of various organs of injury, gene transfer, and regulation of immune responses (Tocci & Forte, 2003).

  Administration of bone marrow cells, including endothelial progenitor cells, has been reported in recent clinical trials (Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, Breidenbach C, Fichtner S). , Korte T, Hornig B, Messinger D, Arseniev L, Hertenstein B, Ganser A, Drexler H. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial.Lancet. 2004 364 (9429): 141- Peripheral vascular disease (Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, Amano K, Kishimoto Y, Yoshimoto K, Akashi H, Shimada K, Iwasaka T, Imaizumi T; Therapeutic Angiogenesis using Cell Transplantation (TACT) Study Investigators.Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial.Lancet. 2002 360 (9331): 427-435) This was reported.

In experimental animals, Kaka C, Isner JM, Asahara T, et al. (2000) Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization.PNAS. 97, 3422-3427; Madeddu P, Emanueli C, Pelosi E , Salis MB, Cerio AM, Bonanno G, Patti M, Stassi G, Condorelli G, Peschle C. Transplantation of low dose CD34 + KDR + cells promotes vascular and muscular regeneration in ischemic limbs.FASEB J. 2004 18 (14): 1737- 1739), cardiac ischemia model (Botta R, Gao E, Stassi G, Bonci D, Pelosi E, Zwas D, Patti M, Colonna L, Baiocchi M, Coppola S, Ma X, Condorelli G, Peschle C. Heart infarct in NOD -SCID mice: therapeutic vasculogenesis by transplantation of human CD34 + cells and low dose CD34 + KDR + cells.FASEB J. 2004 18 (12): 1392-1394) and pulmonary arterial hypertension models (Takahashi M, Nakamura T, Toba T, Kajiwara N, Kato H, Shimizu Y. Transplantation of endothelial progenitor cells into the lung to alleviate pulmonary hypertension in dogs.Tissue Eng. 2004 10 (5-6): 771-7 79) reported the possibility of revascularization using endothelial progenitor cells.

Mesenchymal stem cells undergo cerebral infarction (Chen J, Zhang ZG, Li Y, Wang L, Xu YX, Gautam SC, Lu M, Zhu Z, Chopp M. Intravenous administration of human bone marrow stromal cells induces angiogenesis in the ischemic boundary zone after stroke in rats.Circ Res. 2003 92 (6): 692-699), myocardial infarction (Nagaya N, Fujii T, Iwase T, Ohgushi H, Itoh T, Uematsu M, Yamagishi M, Mori H, Kangawa K, Kitamura S Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis.Am J Physiol Heart Circ Physiol. 2004 287 (6): H2670-267) and chronic heart disease (Silva GV, Litovsky S, Assad. JA, Sousa AL, Martin BJ, Vela D, Coulter SC, Lin J, Ober J, Vaughn WK, Branco RV, Oliveira EM, He R, Geng YJ, Willerson JT, Perin EC.Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model.Circulation. 2005 111 (2): 150-156), It has been reported that it can promote sex and that cells isolated from adipose tissue can promote blood vessel regeneration and differentiate into endothelial cells (Planat-Benard V, Silvestre JS, Cousin B, Andre M, Nibbelink M, Tamarat R, Clergue M, Manneville C, Saillan-Barreau C, Duriez M, Tedgui A, Levy B, Penicaud L, Casteilla L. Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation. 2004 109 (5): 656-663).

However, stem cells transplanted into damaged tissues in the body should be differentiated into damaged tissue regeneration. The therapeutic effect using these stem cells alone is difficult to obtain excellent results. Therefore, it is essential to improve stem cell function by inducing genetic modification of stem cells to improve cell regeneration treatment.

  LZTS2 (lucine zipper tumor suppressor 2) has been cloned from human tumors chromosome 8p21-22 and is rarely expressed in human tumors. Previous studies have shown that overexpression of LZTS2 inhibits tumor cell proliferation. Cabeza-Arvelaiz, Y .; Thompson, TC; Sepulveda, JL; Chinault, AC: LAPSER1: a novel candidate tumor suppressor gene from 10q24.3.Oncogene 20: 6707-6717, 2001; Ishii, H., R. Baffa, SI Numata, Y. Murakumo, S. Rattan, H. Inoue, M. Mori, V. Fidanza, H. Alder, and CM Croce. 1999.The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors.Proc. Natl. Acad. Sci. USA 96: 3928-3933).

 Therefore, it was confirmed that mesenchymal stem cells that inhibited the lzts2 gene using small interfering RNA (siRNA) duplex oligo contributed to the promotion of damaged tissue cell regeneration in the ischemic animal model of immunodeficient mice. It will provide a basis for promising cell therapy in patients with Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior arts, and thus treated with double-stranded siRNA of the lzts2 gene to produce mesenchymal stem cells with suppressed expression of the lzts 2 gene, thereby contributing to promoting damaged tissue cell regeneration. It was confirmed to complete the present invention.

Therefore, the main object of the present invention is to provide a mesenchymal stem cell and a method for producing the same, which contribute to promoting damaged tissue cell regeneration. The main object of the present invention is to inhibit the lzts2 gene using Small interfering RNA (siRNA) duplex oligo to promote the proliferation of mesenchymal stem cells and to promote damaged tissue cell regeneration.

Another object of the present invention to provide a cell therapy for the treatment of ischemic diseases using mesenchymal stem cells with improved tissue regeneration and a method for producing the same.

According to an aspect of the present invention, the present invention provides mesenchymal stem cells which are treated with antisense RNA of the lzts2 gene encoding the amino acid sequence of SEQ ID NO: 1, and the expression of the lzts 2 gene is suppressed.

 In the present invention, the antisense RNA of the lzts2 gene is an RNA molecule having 15 to 30 nucleotides capable of causing RNA interference (abbreviated as "RNAi") by having a sequence complementary to the target site of the mRNA of the lzts2 gene. Means. Examples of the antisense RNA include siRNA (short interfering RNA) or shRNA (short hairpin RNA) using RNA interference of the lzts2 gene. RNA interference is a method of post-transcriptional gene regulation conserved in many eukaryotes. RNAi is induced by short double-stranded RNA ("dsRNA") molecules present in cells. These short dsRNA molecules, also called “siRNAs”, are separated into single strands and then bind to “RNA induced silencing (RISC)” to cleave targeted mRNAs or inhibit translation.

Therefore, the present invention provides an isolated siRNA comprising short double-stranded RNA consisting of about 17 nucleotides to about 25 nucleotides that target the mRNA of the lzts2 gene. The siRNA comprises antisense RNA strands complementary to the sense RNA strands, both of which are annealed to each other by standard Watson-Crick base pair interactions (hereinafter referred to as "base-paired"). ). The sense strand comprises a nucleic acid sequence identical to the target sequence in the target mRNA. The lzts2 mRNA target sequence used to prepare the siRNA of the present invention is an RNA sequence complementary to SEQ ID NO: 2,4,6,8,10.

The sense and antisense strands of the siRNA of the present invention comprise two complementary single-stranded RNA molecules, or two complementary portions form base pairs and are defined by a single "hairpin" region of the single strand. It may comprise a single molecule covalently bonded. The latter case is called shRNA (short hairpin RNA), and the shRNA has a single strand of about 50-70 nucleotides in length and forms a stem-loop structure in vivo. Long RNAs of 19-29 nucleotides complementary to both loop regions of 5-10 nucleotides form base pairs to form double stranded stems. shRNAs are generally synthesized in vivo by transcription of complementary DNA sequences from Pol III promoters. Pol-III-induced transcription begins at the well-defined start site and terminates at the second residue of the linear (-TTTT-) consisting of four or more thymidines to produce non-poly (A) transcripts. Pol III promoter is active in all cells and can express shRNA. After transcription, shRNAs are cleaved by Dicer and interact with RISC like siRNAs.

The siRNA of the present invention can be obtained using many techniques known to those of ordinary skill in the art. For example, siRNA can be chemically synthesized using a method known in the art, or produced by recombinant methods. Preferably, the siRNA of the invention can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and conventional DNA / RNA synthesizers. siRNA can be synthesized as two RNA molecules complementary and separated or as one RNA molecule with two complementary regions. Alternatively, siRNA can also be expressed from recombinant DNA plasmids using appropriate promoters. Suitable promoters for expressing siRNAs of the invention from plasmids include, for example, U6 or H1 RNA pol III promoter sequences and cytomegalovirus promoters. In addition, the recombinant plasmid of the present invention may include an inducible or controllable promoter for expressing siRNA in specific tissues or specific intracellular environments.

The siRNA of the invention can be expressed as two RNA molecules that are complementary and separated from the recombinant plasmid or as one RNA molecule having two complementary regions. Selection of a plasmid suitable for expressing siRNA of the present invention, a method for inserting a nucleic acid sequence for expressing siRNA into a plasmid, and a method for delivering a recombinant plasmid to a cell of interest are within the skill of the art. .

In the mesenchymal stem cells of the present invention, the antisense RNA includes any siRNA (short interfering RNA) or shRNA (short hairpin RNA) using RNA interference of the lzts2 gene, but SEQ ID NOs: 2, 4, 6, 8 , Is preferably any one siRNA selected from the group consisting of 10. The sequence numbers may each constitute a double stranded RNA with complementary sequences.

According to another aspect of the present invention, there is provided a cell therapy agent for the treatment of ischemia diseases comprising mesenchymal stem cells as an active ingredient. The ischemic disease includes any disease in which blood vessels supplying organs are blocked and blood supply is impaired and local necrosis occurs. Preferably, the ischemia, myocardial infarction, cerebral infarction, or foot ulcer are characterized. It is done.

In order to demonstrate the therapeutic effect of the ischemic disease of the present invention, in Example 5, after binding the femoral artery of the rat, the blood flow was blocked to induce lower limb ischemia. Were implanted respectively. As a result of measuring blood flow at 7 and 14 days after transplantation, it was confirmed that blood flow was increased in the group transplanted with cells that suppressed lzts2 gene expression than mesenchymal stem cells (Fig. 6). H & E) confirmed that histologically improved muscle injury recovery in the group transplanted with cells that suppressed lzts2 gene expression, rather than transplantation of mesenchymal stem cells alone.

The cell therapy of the present invention may be prepared in the form of a general cell therapy known in the art, for example, in the form of injections, which may be surgically implanted directly into an ischemic disease site or transferred to an ischemic disease site after intravenous administration. Can be.

The mesenchymal stem cells included in the composition of the present invention may vary depending on the type of disease, the route of administration, the age and sex of the patient, and the extent of the disease, but in the average adult, 10 ⁴ to 10 ⁸ cells are administered. can do.

According to another aspect of the present invention, there is provided a double-stranded siRNA oligonucleotide for the lzts2 gene having the nucleotide sequences of SEQ ID NOs: 2 and 3.

According to another aspect of the invention, preparing a mesenchymal stem cell; And it provides a method for producing mesenchymal stem cells with increased proliferative capacity and damaged tissue regeneration capacity comprising the step of inhibiting the expression of the lzts2 gene encoding the amino acid sequence of SEQ ID NO: 1 to the mesenchymal stem cells.

In the method for producing mesenchymal stem cells of the present invention, the step of inhibiting the expression of the lzts2 gene may cause RNA interference of the lzts2 gene using antisense RNA for the lzts2 gene. Examples of the antisense RNA may include short interfering RNA (siRNA) or short hairpin RNA (shRNA), and the siRNA may include a properly protected ribonucleoside phosphoramidites and a conventional DNA / RNA synthesizer. It can be synthesized chemically.

In addition, in the method for producing mesenchymal stem cells of the present invention, the antisense RNA includes any antisense RNA capable of inducing the inhibition of the expression of the lzts2 gene by binding complementarily with the mRNA of the lzts2 gene, preferably SEQ ID NO: 2 It is characterized in that the siRNA having any one base sequence selected from the group consisting of, 4,6,8,10.

In the method for producing mesenchymal stem cells of the present invention, the step of inhibiting the expression of the lzts2 gene may be carried out by transfection with a plasmid overexpressing the NF-κB gene, such as pCMVp65NF-κB (see Example 1).

As described above, according to the present invention, cell proliferation was improved by inhibiting the expression of lzts2 gene in human mesenchymal stem cells derived from adipose tissue. It can be seen that it promotes tissue cell regeneration. Therefore, based on the mechanism regulating the expression of lzts2 gene in mesenchymal stem cells, it can be used for the development of mesenchymal stem cell therapy useful for promoting damaged tissue regeneration.

Example  One. NF - kB  By inhibitors and overexpression Mesenchyme  In stem cells lzts2  Gene expression regulation

Mesenchymal stem cells used in the present invention used mesenchymal stem cells prepared by the method of inducing differentiation of human stem cells into animal-free serum culture medium composition and hepatocytes (Korea Patent 10-06276950000). Mesenchymal stem cells derived from adipose tissue were SN50 (50 or 100 mg / ml, inhibitor of NF-κB nuclear translocation), NF-kB decoy oligo deoxynucleotide, mutant NF-kB decoy oligo Deoxynucleotides were injected to each other. Also, 48 hours after transfection of p65 plasmid (pCMVp65 NF-κB) for NF-kB overexpression in mesenchymal stem cells, RNA expression was confirmed by RT-PCR and Real-Time PCR. . NF-kB Decoy is a promising inhibitor with high selectivity for the transcription factor NF-kB. NF-kB Decoy; synthetic double-stranded phosphothiorate oligonucleotides containing NF-κB sequence for sequence-specific inhibition of NF-κB a NF-κB consensus sequence) was used. NF-kB Decoy inhibits NF-kB activity by acting as a decoy (decoy), a cis element that binds to transcription factors in vivo. example In the present invention, double-stranded phosphorothioate oligonucleotides synthesized by NF-κB decoy and mutated control were used, respectively. ')to be.

 In the RT-PCR experiment, 5'-CTGTGTCCTGGAAGGAAAGC-3 'was used as the forward primer and 5'-CTCCCACTTGGTCTCCTCAA-3' was used as the forward primer for the lzts2 gene. In the GAPDH gene used as a control, 5'-TCCATGACAACTTTGGTATCG-3 'was used as the forward primer and 5'-TGTAGCCAAATTCGTTGTCA-3' was used as the reverse primer. As a result, it was confirmed that lzts2 gene expression was increased upon treatment of NF-kB inhibitor SN50 and NF-kB decoy, and lzts2 gene expression was reduced upon p65 overexpression (FIGS. 1-A and B).

Example  2. NF - kB  Β- by inhibitors and overexpression catenin Of Tcf signaling  control

To determine how β-catenin / Tcf signaling is regulated by NF-kB inhibitors and overexpression in adipose tissue and bone marrow-derived mesenchymal stem cells, luciferase constructs plasmid (pTOP-flash) containing Tcf promoter in cells. ) And mutant plasmid (pFOP-flash), and then N50-50B / 50 (50 or 100 mg / ml, inhibitor of NF-κB nuclear translocation), NF-kB decoy (10 mM, Sequence-) Specific inhibition of NF-kB, 5'-AGTTGAGGGGACTTTCCCAGGC-3 ') and mutant decoy (10 mM, 5'AGTTGAGGCGACTTTCCCAGGC-3') and p65 plasmid (pCMVp65 NF-κB) for NF-kB overexpression After luciferase assay was performed. All transient transtection experiments used Lipofectamine Plus Reagent sold by Invitrogen. The pTOP-flash plasmid consists of the Tcf / Lef1 binding DNA enhancer, the Thymidine kinase promoter, and the firefly Luciferase reporter gene. In the case of the pFOP-flash plasmid, the Tcf / Lef1 binding DNA sequence was mutated. pNF-κB-Luc plasmid (Clontech Laboratories, Inc. CA) is a plasmid with DNA sequence (Enhancer) that binds NF-κB. p65 plasmid (pCMVp65 NF-κB) is a plasmid used for NF-kB overexpression. As shown in FIG. 2, after 48 hours of transformation under each condition, 0.25 M Tris, 2 mM DTT, 2 Mm 1,2-diaminocyclohexane-N, N, N ', N'-tetraacetic acid, 10% glycerol and 1 Luciferase enzyme activity was measured using Luciferase Assay System (Promega corporation, Madison, WI) by treating cells in% Triton X-100 buffer (lysis buffer). Reduction of luciferase activity in SN50 and NF-kB decoy-treated cells inhibited β-catenin / Tcf signaling (Figs. 2-A, B). -catenin / Tcf signaling increased (Fig. 2-B).

Example  3. Mesenchyme  In stem cells lzts2  By gene expression inhibition NF -κB and  β- catenin Of Tcf  signaling regulation

In order to suppress the expression of lzts2 gene in adipose tissue-derived mesenchymal stem cells, small interfering RNA (siRNA) duplex oligo containing lzts2 gene was prepared, and then siRNA duplex oligo and oligofectamine (reagent, invitrogen) Treated. SiRNA duplex oligo used in the present invention is a pair of double-stranded siRNA having a complementary base sequence selected from the group consisting of SEQ ID NO: 2 and 3, 4 and 5, 6 and 7, 8 and 9, and 10 and 11 Used. Double-stranded siRNA consisting of double SEQ ID NO: 2 and 3 is a novel production and use for the present invention, double stranded siRNA consisting of SEQ ID NO: 4 to 11 was purchased from Dharmacon Co. (on-TARGET plus SMART pool , Dharmacon, Inc. CO). In addition, non-targeting duplex oligo (on-TARGET plus siCONTROL, Dharmacon, Inc. CO; DharmaFECT Transfection Reagent) was used as a negative control for verification of the invention.

The lzts2 gene expression was measured by RT-PCR and Real-Time PCR. In the RT-PCR experiment, the primer sequence was 5'-CTGTGTCCTGGAAGGAAAGC-3 'and 5'-CTCCCACTTGGTCTCCTCAA-3' as the forward primer for the lzts2 gene. Real-Time PCR experiment using SYBR Green I (primers β-actin, 5'-CTGGTGCCTGGGGCG-3 ', 5-AGCCTCGCCTTTGCCGA-3', lzts2, 5'-AGAAGCGGCAATTGCAGGAC-3 ', 5', respectively) -CTCGCCTGATTTCTGGCACA-3 '). As a result, it was confirmed that lzts2 gene expression was inhibited by RT-PCR and Real-Time PCR (FIGS. 3-A and B). In addition, to confirm the regulation of NF-κB and β-catenin / Tcf signals by inhibition of lzts2 gene expression, pTOP-flash for identifying β-catenin / Tcf signals in mesenchymal stem cells (slzts2) that inhibited lzts2 gene expression with siRNA. To determine the plasmid, pFOP-flash, and NF-κB signal, luciferase activity was measured 48 hours after transfection with pNF-κB-Luc. As a result, it was confirmed that lzts2 gene expression inhibition increased both NF-κB and β-catenin / Tcf signals (FIG. 3 -C). To confirm the above results, Western blot confirmed the translocation of β-catenin and p65 protein to the nucleus 48 hours after lzts2 gene expression was inhibited in adipose tissue-derived mesenchymal stem cells. Inhibit lzts2 gene expression in adipose tissue-derived mesenchymal stem cells, and after 48 hours, trypsin-treated, wash in PBS solution at 5 ° C, and then pellet the ice-cold lysis buffer (210 mM mannitol, 70 mM sucrose 5). treated with mM Tris, pH 7.5, 1 mM EDTA, protease inhibitors) and stored on ice for 15 minutes. The nucleus was then separated by centrifuge (10 minutes, 4 ° C, 14,000rpm) and the cytosolic portion was boiled in the sample buffer. Cell pellets containing nuclei were washed with PBS, resuspensioned for 5 minutes in RIPA buffer (Santa Cruz Biotechnology, Inc. CA), centrifuged again (10 minutes, 4 ° C, 14,000 rpm), and then supernatant (nuclear). fractions) were boiled in the sample buffer. Nucleic and cytosolic proteins were separated by 10% SDS polyacrylamide gels, electrotransferred to nitrocellulose membranes (Hybond-ECL, Amersham Pharmacia Biotech, Piscataway, NJ) and monoclonal antibodies (β-catenin, and actin; BD Biosciences Clontech, CA, NF-κBp65 : Santa Cruz Biotechnology, Inc. CA). Subsequently, the lzts2 gene was developed by treatment with secondary antibodies (anti-mouse and anti-rabbit peroxidase-conjugated secondary antibodies; Amersham Pharmacia Biotech, Piscataway, NJ) and enhanced chemiluminescence (ECL detection kit, Amersham Pharmacia Biotech, Piscataway, NJ). After inhibition of expression, it was confirmed that translocation was increased to the nucleus of β-catenin and p65 protein present in the cells (Fig. 3-D). β-TrCP, which is regulated by the β-catenin / Tcf pathway, is known to induce NF-κB activation by IκB degradation. Therefore, the effect of β-catenin regulated by the inhibition of lzts2 gene expression affects β-TrCP and IκB degradation in real-time PCR (primary sequence of β-TrCP, 5'-TAGCAGAGCAGTCCAACCCAGA-3 ', 5'-CTGTTGGTGAACAACTGTGTGGAG-3 ) And western blot (IκB: Santa Cruz Biotechnology, Inc. CA). As a result, it was confirmed that lzts2 gene expression inhibition induced β-TrCP expression (Fig. 4-A) and IκB degradation (Fig. 4-B).

Example  4. lzts2  By gene expression inhibition Mesenchyme  Stem cell Proliferative capacity

      In order to confirm the proliferative capacity of lzts2 expression inhibition, 20,000 cells were placed on 6well plate and mesenchymal stem cells derived from adipose tissue and lzts2 expression suppressed cells (slzts2), respectively, were placed on the NF-κB inhibitors SN50 and NF-κB. After incubation with decoy, the cells were recovered after 48 hours and 72 hours, respectively, and the number of cells was measured by haemacytometer under a microscope. As a result, it was confirmed that the proliferative capacity was improved in mesenchymal stem cells derived from adipose tissue in which lzts2 expression was suppressed (Fig. 5-A). The control cells treated with SN50 and NF-κB decoy were found to inhibit proliferative activity, but the inhibition of proliferative activity by SN50 and NF-κB decoy was reduced in cells with lzts2 expression inhibition (FIGS. 5-A and B). .

In order to confirm the effect of increasing proliferation, TTts2 expression was inhibited in mesenchymal stem cells derived from adipose tissue and bone marrow, and then MTT [3- (4,5-dimethylthiazol-2-yl) -2 after SN50 48 hours treatment. , 5-diphenyltetrazolium bromide] assay, Cell Cycle analysis by FACS and Cyclin D1 expression were measured by Real Time PCR (forword primer 5'-TGATGCTGCGCACTTCATCTG, revers primer 5'-TCCAATCATCCCGAATGAGAGTC-3 '). Washing 10 ⁵ -10 ⁶ cells with phosphate-buffered saline and immobilizing with 70% ethanol containing 4 ° C 0.5% Tween 20; Washing and re-dissolving with propidium iodide solution containing RNase A (10 mg / ml) and propidium iodide (50 μg / ml); Incubate at 37 ° C. for 15 minutes or at room temperature for 30 minutes; The cell cycle was analyzed by performing flow analysis (FACsort Becton Dickinson, CA). Human adipose tissue-derived mesnechymal stem cells (hASCs) and human bone marrow stromal cells (hBMSCs) from adipose tissue and bone marrow are also plated on 6-well plates (density of 2,000 / cm2). step; Staining with 0.4% trypan blue (Sigma, St Louis, MO, USA) dye after trypsin treatment after 48 or 72 hours; The proliferation rate of the cells was analyzed by performing a step of measuring the number of cells with a hemocytometer. Cell proliferation was evaluated by MATT assay, and the cultured cells containing 0.5 mg / ml MTT were added to each well, incubated at 37 ° C for 2 hours, and then supernatant was removed. Formazan crystals formed of living cells were dissolved in 110 μl of dimethyl sulfoxide solvent.

A 110 μl aliquots were transferred to 96-well plates and absorbed at 550 nm with an ELISA Reader (Bio-Tek instrument, Winooski, VT, USA). As a result, MTT was increased in adipose tissue and bone marrow-derived mesenchymal stem cells that inhibited lzts2 expression, and S- and G2 / M-phase cells of the cell cycle were observed predominantly. It could be confirmed (Fig. 5-CE). The control cells treated with SN50 had decreased MTT, S and G2 / M cells, and Cyclin D1 expression, but the effect of SN50 was inhibited in cells that inhibited lzts2 expression (Fig. 5-C-E).

  Inhibits lzts2 expression in adipose tissue and bone marrow-derived mesenchymal stem cells, and then controls β-catenin / Tcf signal by administering SN50 (50 or 100 mg / ml, inhibitor of NF-κB nuclear translocation), an NF-kB inhibitor I checked. As a result, inhibition of lzts2 expression increased β-catenin / Tcf signal, and it was confirmed that β-catenin / Tcf signal was inhibited in control cells treated with SN50, but β-catenin / Tcf signal inhibition by SN50 was inhibited in cells with lzts2 expression inhibited. It could be confirmed that it is reduced (Fig. 5-FI).

Example  5. Ischemia  On animal models Mesenchyme  Stem Cell Transplantation

  One day after induction of lower limb ischemia by blocking blood flow by binding the femoral artery to immunodeficient mice, PBS, mesenchymal stem cells and cells which inhibited lzts2 gene expression were transplanted. As a result of measuring blood flow at 7 and 14 days after transplantation, it was confirmed that blood flow was increased in the group transplanted with cells that suppressed lzts2 gene expression than mesenchymal stem cells (Fig. 6). H & E) showed that histologically improved muscle injury recovery in the group transplanted with the cells that suppressed the expression of lzts2 gene rather than the transplantation of mesenchymal stem cells alone (Fig. 7-A, B).

1 is a result showing that lzts2 gene expression is regulated by NF-κB inhibitor and overexpression in mesenchymal stem cells derived from adipose tissue.

Figure 2 is a result showing the regulation of β-catenin / Tcf signaling by NF-κB inhibitor and overexpression in mesenchymal stem cells.

Figure 3 shows that NF-κB and β-catenin / Tcf signaling is regulated by the inhibition of lzts2 gene expression using Small interfering RNA (siRNA) duplex oligo in mesenchymal stem cells.

Figure 4 shows the results of controlling β-TrCP expression and IκB degradation by inhibiting lzts2 gene expression using Small interfering RNA (siRNA) duplex oligo in adipose tissue-derived mesenchymal stem cells.

Figure 5 shows that the proliferation is promoted when the lzts2 gene is inhibited by using small interfering RNA (siRNA) duplex oligo in mesenchymal stem cells.

Figure 6 is a result of measuring the blood flow by transplanting the mesenchymal stem cells suppressed lzts2 gene using small interfering RNA (siRNA) duplex oligo in the ischemic animal model of the immunodeficient mice.

Figure 7 is a result showing the tissue regeneration ability in the lower limb ischemia of the immunodeficient mice when transplanted mesenchymal stem cells lzts2 function is suppressed.

<110> Pusan Nationanl University The Industry-University Cooperation Foundation <120> human mesenchymal stem cells improved tissue regeneration and          production methods <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 668 <212> PRT <213> Homo sapiens <400> 1 Met Ala Ile Val Gln Thr Leu Pro Val Pro Leu Glu Pro Ala Pro Glu   1 5 10 15 Ala Ala Thr Ala Pro Gln Ala Pro Val Met Gly Ser Val Ser Ser Leu              20 25 30 Ile Ser Gly Arg Pro Cys Pro Gly Gly Pro Ala Pro Pro Arg His His          35 40 45 Gly Pro Pro Gly Pro Thr Phe Phe Arg Gln Gln Asp Gly Leu Leu Arg      50 55 60 Gly Gly Tyr Glu Ala Gln Glu Pro Leu Cys Pro Ala Val Pro Pro Arg  65 70 75 80 Lys Ala Val Pro Val Thr Ser Phe Thr Tyr Ile Asn Glu Asp Phe Arg                  85 90 95 Thr Glu Ser Pro Pro Ser Pro Ser Ser Asp Val Glu Asp Ala Arg Glu             100 105 110 Gln Arg Ala His Asn Ala His Leu Arg Gly Pro Pro Pro Lys Leu Ile         115 120 125 Pro Val Ser Gly Lys Leu Glu Lys Asn Met Glu Lys Ile Leu Ile Arg     130 135 140 Pro Thr Ala Phe Lys Pro Val Leu Pro Lys Pro Arg Gly Ala Pro Ser 145 150 155 160 Leu Pro Ser Phe Met Gly Pro Arg Ala Thr Gly Leu Ser Gly Ser Gln                 165 170 175 Gly Ser Leu Thr Gln Leu Phe Gly Gly Pro Ala Ser Ser Ser Ser Ser             180 185 190 Ser Ser Ser Ser Ser Ala Ala Asp Lys Pro Leu Ala Phe Ser Gly Trp         195 200 205 Ala Ser Gly Cys Pro Ser Gly Thr Leu Ser Asp Ser Gly Arg Asn Ser     210 215 220 Leu Ser Ser Leu Pro Thr Tyr Ser Thr Gly Gly Ala Glu Pro Thr Thr 225 230 235 240 Ser Ser Pro Gly Gly His Leu Pro Ser His Gly Ser Gly Arg Gly Ala                 245 250 255 Leu Pro Gly Pro Ala Arg Gly Val Pro Thr Gly Pro Ser His Ser Asp             260 265 270 Ser Gly Arg Ser Ser Ser Ser Lys Ser Thr Gly Ser Leu Gly Gly Arg         275 280 285 Val Ala Gly Gly Leu Leu Gly Ser Gly Thr Arg Ala Ser Pro Asp Ser     290 295 300 Ser Ser Cys Gly Glu Arg Ser Pro Pro Pro Pro Pro Pro Ser 305 310 315 320 Asp Glu Ala Leu Leu His Cys Val Leu Glu Gly Lys Leu Arg Asp Arg                 325 330 335 Glu Ala Glu Leu Gln Gln Leu Arg Asp Ser Leu Asp Glu Asn Glu Ala             340 345 350 Thr Met Cys Gln Ala Tyr Glu Glu Arg Gln Arg His Trp Gln Arg Glu         355 360 365 Arg Glu Ala Leu Arg Glu Asp Cys Ala Ala Gln Ala Gln Arg Ala Gln     370 375 380 Arg Ala Gln Gln Leu Leu Gln Leu Val Phe Gln Leu Gln Gln Glu Lys 385 390 395 400 Arg Gln Leu Gln Asp Asp Phe Ala Gln Leu Leu Gln Glu Arg Glu Gln                 405 410 415 Leu Glu Arg Arg Cys Ala Thr Leu Glu Arg Glu Gln Arg Glu Leu Gly             420 425 430 Pro Arg Leu Glu Glu Thr Lys Trp Glu Val Cys Gln Lys Ser Gly Glu         435 440 445 Ile Ser Leu Leu Lys Gln Gln Leu Lys Glu Ser Gln Ala Glu Leu Val     450 455 460 Gln Lys Gly Ser Glu Leu Val Ala Leu Arg Val Ala Leu Arg Glu Ala 465 470 475 480 Arg Ala Thr Leu Arg Val Ser Glu Gly Arg Ala Arg Gly Leu Gln Glu                 485 490 495 Ala Ala Arg Ala Arg Glu Leu Glu Leu Glu Ala Cys Ser Gln Glu Leu             500 505 510 Gln Arg His Arg Gln Glu Ala Glu Gln Leu Arg Glu Lys Ala Gly Gln         515 520 525 Leu Asp Ala Glu Ala Ala Gly Leu Arg Glu Pro Pro Val Pro Pro Ala     530 535 540 Thr Ala Asp Pro Phe Leu Leu Ala Glu Ser Asp Glu Ala Lys Val Gln 545 550 555 560 Arg Ala Ala Ala Gly Val Gly Gly Ser Leu Arg Ala Gln Val Glu Arg                 565 570 575 Leu Arg Val Glu Leu Gln Arg Glu Arg Arg Arg Gly Glu Glu Gln Arg             580 585 590 Asp Ser Phe Glu Gly Glu Arg Leu Ala Trp Gln Ala Glu Lys Glu Gln         595 600 605 Val Ile Arg Tyr Gln Lys Gln Leu Gln His Asn Tyr Ile Gln Met Tyr     610 615 620 Arg Arg Asn Arg Gln Leu Glu Gln Glu Leu Gln Gln Leu Ser Leu Glu 625 630 635 640 Leu Glu Ala Arg Glu Leu Ala Asp Leu Gly Leu Ala Glu Gln Ala Pro                 645 650 655 Cys Ile Cys Leu Glu Glu Ile Thr Ala Thr Glu Ile             660 665 <210> 2 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> sense sequence of siRNA duplex oligo <400> 2 uuuccuucca ggacacagug cagca 25 <210> 3 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> anti-sence of siRNA duplex oligo <400> 3 ugcugcacug uguccuggaa ggaaa 25 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> sence sequence of siRNA oligo <400> 4 gggacagucu ggacgagaau u 21 <210> 5 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> anti-sence of siRNA duplex oligo <400> 5 uucucgucca gacugucccu u 21 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> sence of siRNA duplex oligo <400> 6 ggcaauugca ggacgacuuu u 21 <210> 7 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> anti-sence of siRNA duplex oligo <400> 7 aagucguccu gcaauugccu u 21 <210> 8 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> sence of siRNA duplex oligo <400> 8 aggagcaggu gauccgcuau u 21 <210> 9 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> anti-sence of siRNA duplex oligo <400> 9 uagcggauca ccugcuccuu u 21 <210> 10 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> sence of siRNA duplex oligo <400> 10 aggcauacga ggagcggcau u 21 <210> 11 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> anti-sence of siRNA duplex oligo <400> 11 ugccgcuccu cguaugccuu u 21  

Claims (8)

Mesenchymal stem cells treated with antisense RNA of the lzts2 gene encoding the amino acid sequence of SEQ ID NO: 1 to suppress the expression of the lzts 2 gene. The mesenchymal stem cell according to claim 1, wherein the antisense RNA is siRNA having any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 2,4,6,8,10. Cell therapy for the treatment of ischemia diseases comprising the mesenchymal stem cells of claim 1 or 2 as an active ingredient. 4. The cell therapeutic agent according to claim 3, wherein the ischemic disease is any one selected from cardiac angina, myocardial infarction, cerebral infarction, or foot ulcer. Double-stranded siRNA oligonucleotide for the lzts2 gene, characterized in that it has the nucleotide sequence of SEQ ID NO: 2. Preparing mesenchymal stem cells; And inhibiting the expression of the lzts2 gene encoding the amino acid sequence of SEQ ID NO: 1 in the mesenchymal stem cells. The method of claim 6, wherein the inhibiting the expression of the lzts2 gene comprises treating antisense RNA of lzts2. The method of claim 6, wherein the antisense RNA is a method for producing mesenchymal stem cells, characterized in that the siRNA having any one base sequence selected from the group consisting of SEQ ID NO: 2,4,6,8,10.

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