KR101676609B1 - Methods for inhibiting senescence and dedifferentiation of chondrocytes using Rheb gene - Google Patents

Abstract

The present invention provides a method for inhibiting senescence or de-differentiation of chondrocytes, comprising the step of transforming chondrocytes with a gene encoding Rheb protein; And an aging or dedifferentiation-suppressed chondrocyte transformed with a gene encoding a Rheb protein. The method for inhibiting senescence or de-differentiation according to the present invention can inhibit aging and de-differentiation occurring in monolayer culture of chondrocytes, thereby allowing a large amount of chondrocytes having normal proliferative activity and phenotype to be cultured. In addition, the method according to the present invention can improve the low recovery rate of cartilage cells in autologous cartilage tissue, and thus can be usefully used for manufacturing cartilage cells for cell therapy.

Description

Methods for inhibiting senescence and dedifferentiation of chondrocytes using Rheb gene (Methods for inhibiting senescence and dedifferentiation of chondrocytes using Rheb gene)

The present invention relates to a method for inhibiting senescence and de-differentiation of chondrocytes, comprising the step of transforming chondrocytes with a gene encoding Rheb protein. The present invention also relates to chondrocyte cells which are transformed with a gene encoding a Rheb protein and whose aging and de-differentiation are suppressed.

Articular cartilage is a cartilage covering the tip of the bone that forms the joint, and it plays an important role in protecting the joint from mechanical stress by preventing friction between the bones. About 50 to 70% of the joint matrix is composed of type II collagen and about 15 to 30% is composed of proteoglycans. These matrix components provide strong tensile strength to articular cartilage. As the age increases, the percentage of cells in the articular cartilage decreases, and the non-collagen protein increases, resulting in a change in the substrate composition, which can not overcome the mechanical load on the joints and lead to joint arthritis (degenerative arthritis) (Bobacz K et al, Ann Rheum Dis, 63 (12), pp. 1618-1622, 2004). In addition, the articular cartilage is a tissue that is difficult to regenerate once it is damaged or degenerated because it is not distributed in blood vessels, lymphatic vessels and nerves (Brittberg M et al, New England Journal of Medicine, 331, pp. 889-895, 1994). Currently, efforts are being made to treat the articular cartilage disease by various engineering approaches, but there are still many limitations in normal cartilage tissue regeneration. So far, regeneration of cartilage tissue by autologous chondrocyte transplantation has been known as a clinically very stable and effective method. However, these autologous chondrocytes can not be used for a wide range of damage to the cartilage tissue due to the impossibility of securing a large amount of normal cartilage cells in the treatment of the cartilage cell, and there is a limit according to the location of the cartilage damage and the age of the patient.

Chondrocytes are already differentiated cells, and cell division is not strong in the tissues. However, when separated from the cells in vitro, proliferation is promoted and a sufficient number of chondrocytes can be obtained. However, when monolayer culture is performed for a long time in order to obtain a sufficient number of cells required, the aging phenomenon in which the cell proliferation ability is remarkably decreased and the demineralization phenomenon in which the phenotype of the chondrocyte cell is decreased (Yudoh K et al., Arthritis Res Ther. 19 (4), pp 325-38, 2009), fibrotic cartilage and calcified tissues were formed during in vivo transplantation (Fischer J et al. Arthritis & Rheumatism, 62 (9), pp. 2696-2706, 2010). In the case of degenerative arthritis, when the cells obtained from the cartilage tissue are already aged and normal cartilage cells are recovered from the normal cartilage tissue, the aging and de-differentiation of the normal cartilage cells rapidly occurs by the extracorporeal monolayer culture, Securing it is technically very difficult. In order to overcome this problem, a culture method using various techniques such as addition of various cell growth factors (for example, TGF-?), Three-dimensional culture in the form of pellets, and introduction of chondrogenic differentiation-related genes into stem cells has been proposed And it is very difficult to obtain a sufficient number of normal chondrocytes.

The present inventors have conducted various studies to develop a method capable of providing chondrocytes having normal proliferative potential and phenotype, that is, chondrocyte inhibiting aging and de-differentiation, which can be used for regeneration of cartilage tissue by chondrocyte transplantation. The present inventors have found that when the chondrocyte transformed with the Rheb gene, known as a cell growth regulating gene, maintains the proliferative capacity and phenotype of the chondrocyte even in continuous passaging, aging and dedifferentiation are effectively inhibited.

Accordingly, it is an object of the present invention to provide a method for inhibiting senescence and / or dedifferentiation of cartilage cells, which comprises transforming cartilage cells with a gene encoding Rheb protein.

It is another object of the present invention to provide a chondrocyte transformed with a gene encoding a Rheb protein, which is suppressed in aging and / or de-differentiation.

According to one aspect of the present invention, there is provided a method for producing a chondrocytic cell, comprising the step of transforming chondrocytes with a gene encoding a Rheb protein having the amino acid sequence of SEQ ID NO: 1, preferably a gene having the nucleotide sequence of SEQ ID NO: Methods of inhibiting senescence or dedifferentiation are provided.

According to another aspect of the present invention, there is provided an aged or undifferentiated chondrocyte transformed with a gene encoding a Rheb protein having the amino acid sequence of SEQ ID NO: 1, preferably a gene having the nucleotide sequence of SEQ ID NO: 2 do.

It has been found by the present invention that aging and de-differentiation are effectively inhibited by maintaining the proliferative capacity and phenotype of chondrocyte cells even when the chondrocytes transformed with the Rheb gene are continuously cultured. Therefore, the method according to the present invention can inhibit the aging and demineralization in the monolayer culture of existing cartilage cells, thereby allowing a large amount of cartilage cells having normal proliferative activity and phenotype to be cultured. In addition, the method according to the present invention can improve the low recovery rate of cartilage cells in autologous cartilage tissue, and thus can be usefully used for manufacturing cartilage cells for cell therapy.

FIG. 1 shows the results of analysis of cell markers (SA-β-Gal) expression (A), cell size analysis (B), cartilage cell marker genes (Col II and Col X) and Rheb gene expression analysis (C) and analysis of expression of Rheb protein (D) by Western blotting.
Figure 2 is a cleavage map of the pEGFP vector used in cloning of the Rheb plasmid DNA vector.
FIG. 3 shows flow cytometric analyzes (A) and (B) for cholesteric cells of the third passage (P3-Mock) into which an empty vector was introduced and a third passage of cartilage cells (P3-Rheb) transformed with a Rheb plasmid DNA vector RT-PCR analysis (B).
FIG. 4 shows the results of the in vitro culture of the 3rd passage chondrocytes transfected with the empty vector and the 3rd passage chondrocytes transformed with the Rheb plasmid DNA vector up to the 6th passage (P6-Mock / P6-Rheb ), Cell marker (SA-β-Gal) (A), cell size (B) and cell proliferative capacity (C).
FIG. 5 is a graph showing the results of the third passage of cartilage cells (P3-Mock) into which an empty vector was introduced and the third passage of cartilage cells (P3-Rheb) transformed with a Rheb plasmid DNA vector, The results of RT-PCR analysis (A) and real-time PCR analysis (B) on cultured chondrocytes (P6-Mock / P6-Rheb) are shown.

As used herein, the term "chondrocytes" refers to chondrocytes that are excreted in vitro from the cartilage tissue of an animal including a human; Refers to chondrocytes obtained by differentiating from stem cells (for example, embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs), etc. by known methods). The chondrocytes are preferably human The chondrocyte may be a chondrocyte that is excreted from the cartilage tissue of the human, and more preferably, a chondrocyte that is excreted from the human normal vitrified cartilage (connective tissue) to the outside of the human body. The chondrocyte may be treated with an enzyme treatment (for example, a type of chondroitin sulfate, etc.) from a cartilage tissue isolated from a human body for the purpose of treatment or the like, according to a known method II collagenase treatment), centrifugation, etc. The isolated chondrocyte may be cultured in a culture medium for normal chondrocyte culture, for example, 1% penicillin May be incubated in a streptomycin and 10% fetal bovine serum (FBS) is added to a Dulbecco's modified Eagle's medium (Dulbecco's modified Eagle medium (DMEM), Gibco BRL, Gaithersburg, MD)].

In addition, the term " senescence "of chondrocytes refers to changes in the genetic or morphological phenotype of chondrocytes due to changes in the proliferation of chondrocytes by continuous monolayer culture and other causes, It means a change that loses its characteristics. The aging phenotype or biomarker of chondrocytes includes senescence associated beta-galactosidase (SA-beta-Gal). Therefore, aging of chondrocytes can be defined as a significant increase in the expression of aging biomarkers such as SA-β-Gal.

The term " dedifferentiation "in the present specification refers to the genetic or morphological expression of chondrocytes due to a comprehensive change in the synthetic profile of chondrocytes due to continuous monolayer culture and other causes And the change of the chondrocytes is lost. Here, the term "synthetic profile" refers to the synthesis of gene expression and related proteins. The phenotype of the cartilage cell, that is, the biomarker, includes Col II, and the phenotype of the demineralized cartilage cell, that is, the biomarker, includes Col X. Thus, demyelination of chondrocytes may also be defined as a significant decrease in the expression of normal chondrocyte biomarkers of Col II and a significant increase in the expression of demineralized biomarkers such as Col X.

The present invention provides a method for inhibiting aging or de-differentiation of chondrocytes, comprising the step of transforming chondrocytes with a gene encoding a Rheb protein having the amino acid sequence of SEQ ID NO: 1. The present invention also provides a chondrocyte cell transformed with a gene encoding a Rheb protein having the amino acid sequence of SEQ ID NO: 1, wherein the chondrocyte inhibited senescence or de-differentiation.

According to the present invention, it has been revealed by the present invention that the aging and dedifferentiation are effectively inhibited by maintaining the proliferative capacity and phenotype of chondrocyte cells even when the chondrocytes transformed with the Rheb gene are continuously cultured. Therefore, the method according to the present invention can inhibit the aging and demineralization in the monolayer culture of existing cartilage cells, thereby allowing a large amount of cartilage cells having normal proliferative activity and phenotype to be cultured. In addition, the method according to the present invention can improve the low recovery rate of cartilage cells in autologous cartilage tissue, and thus can be usefully used for manufacturing cartilage cells for cell therapy.

The gene encoding the Rheb protein may be composed of the nucleotide sequence of SEQ ID NO: 2. The method of inhibiting senescence or dedifferentiation according to the present invention is preferably applied to chondrocytes subcultured in the first to sixth passages, more preferably chondrocytes subcultured in the third passages. Therefore, the chondrocyte inhibited by senescence or de-differentiation according to the present invention is preferably a chondrocyte cell subcultured in a first passage to a sixth passage, more preferably a chondrocyte subcultured in a third passage, Lt; RTI ID = 0.0 > transformed < / RTI > with a coding gene.

Transformation of the chondrocytes can be performed by conventional genetic engineering methods. For example, the transformation can be carried out using the Micoroporator method, a non-viral infection method. The microporous method is a method for stimulating cells (that is, cartilage cells) at a high voltage to instantaneously change the composition of a cell membrane to allow a gene (that is, a gene encoding a Rheb protein) . ≪ / RTI > For example, the transformation can be carried out by cloning a gene encoding a Rheb protein into a plasmid DNA vector (for example, pEGFP vector (Enzynomics, Korea) etc.) according to a conventional method to obtain a recombinant Rheb plasmid DNA vector, And introducing it into chondrocytes by a porator method. The microporous method can be performed by mixing the Rheb plasmid DNA vector and chondrocytes in a Resuspension solution (Invitrogen, USA), and then reacting at about 1700 voltage, about 20 ms, and about 1 pulse, (For example, a DMEM culture medium without antibiotics).

In addition, the above-mentioned transformation can use a virus infection method. For example, the transformation can be accomplished by cloning a gene encoding a Rheb protein in a viral vector, for example, a retroviral vector or a lentiviral vector, and infecting the host cell with the resulting vector, And then infecting the cartilage cells with the virus.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1. Cultivation and aging of human chondrocytes Depletion  evaluation

(1) Isolation of human chondrocytes

Under the approval of the Ethics Committee of the University of Science and Technology of Chungcheong University, the patient received consent from the patient and removed cartilage tissue that was removed during knee surgery in orthopedic surgery. In order to remove contaminated blood, the obtained cartilage tissue was washed three times with phosphate buffered saline (PBS) (Hyclone, USA) and cut with an operating knife to facilitate the enzyme treatment. The cartilage tissue was then digested with PBS containing 0.05 w / v% bovine serum albumin and 2 mg / mL Type II collagenase (Sigma) for 15 hours at 37 ° C. After filtration through a 70 mu m filter, the filtrate was centrifuged and the floating fat cells were removed. Isolated chondrocytes were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) (IHyclone, USA) BRL, Gaithersburg, Md.) At 37 ° C in a 5% CO ₂ incubator to isolate human chondrocytes. Once subcultured cells were used in the following tests.

(2) Phenotypic analysis of chondrocytes in the 2nd, 4th, 6th and 8th passages

The chondrocytes obtained in the above (1) were inoculated at a density of ¹ × ^{10 4} cells / cm ² and cultured for 4 to 5 days in a culture medium containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) (Invitrogen) supplemented with DMEM (Gibco)] and subcultured to passage 8 with the same medium. The incubation period of each subculture was the same as 4 to 5 days.

Cell senescence was assessed by analyzing senescence associated beta-galactosidase (SA-β-Gal), a marker of cell senescence, on chondrocytes obtained from the 2nd, 4th, 6th and 8th passages (P2, Respectively. That is, the cells obtained from each passage were washed once with PBS, and fixed with a fixation solution at room temperature for 15 minutes at room temperature. After washing twice with PBS, β-galactosidase staining solution (Cell signaling) was added, and incubated in a 37 ° C incubator for 24 hours. The stained cells were observed through an inverted microscope and the results are shown in Fig. 1 (A). From FIG. 1 (A), it was observed that SA-β-Gal was expressed only in some cells in the second and fourth passage chondrocytes, whereas in the sixth and eighth passage chondrocytes, Lt; RTI ID = 0.0 > SA-beta-Gal < / RTI >

The size of chondrocytes in each passage (2nd, 4th, 6th and 8th passages) was also measured. That is, the cells were photographed through an inverted microscope (Olimpus) and the size of the cells was quantified using the Image J (National Institutes of Health) program. The results are shown in Fig. 1 (B). As can be seen from the results of FIG. 1 (B), the chondrocytes of the sixth and eighth passages were statistically significantly larger than those of the second and fourth chondrocytes (*, p < 0.05).

As can be seen from the results of FIGS. 1 (A) and 1 (B), the aging of cartilage cells increases as the number of passages (passages 6 and 8) increases.

The expression of the marker gene was evaluated for the chondrocytes of each passage (2nd, 4th, 6th and 8th passages) by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Expression of Col II was measured as a chondrocyte-associated marker gene, and expression of Col X as a dedifferentiation marker gene was measured. Expression of Rheb gene was also measured and analyzed using GAPDH as a housekeeping gene.

The RT-PCR was performed as follows. That is, cells obtained from each passage (2nd, 4th, 6th and 8th passages) were washed three times with PBS, and then cells were recovered with trypsin-EDTA and treated with triazole (TRIzol, Life Technologies, Inc. Grand Island, NY). 2 μg of the extracted RNA was subjected to RT-PCR using an RT-PCR kit (Bioneer, Seoul, Korea), and then the change in gene expression was measured by electrophoresis using 1.2% agarose gel . The primer sets used for the RT-PCR and the respective differentiation markers are shown in Table 1 below.

Marker order Source ^* Col II SEQ ID NO: 3 sense 5'-TTC AGC TAT GGA GAT GAC AAT C-3 ' NM_013227 SEQ ID NO: 4 Antisense 5'-AGA GTC CTA GAG TGA CTG AG-3 ' Col X SEQ ID NO: 5 sense 5'-AGG GTT ACC AGG TCC AAA AG -3 ' NM_00493.3 SEQ ID NO: 6 Antisense 5'-TTC CAG TCC TTG GGT CAT AA-3 ' Rheb SEQ ID NO: 7 sense 5'-ATG CCG CAG TCC AAG TCC-3 ' NM_005614 SEQ ID NO: 8 Antisense 5'-CAC ATC ACC GAG CAT GAA G-3 ' GAPDH SEQ ID NO: 9 sense 5'-CGG ATT TGG TCG TAT TGG GC-3 ' NM_002046 SEQ ID NO: 10 Antisense 5'-CAG GGA TGA TGT TCT GGA GA-3 '

* Origin: NCBI accession number

The results of RT-PCR as described above are shown in FIG. 1 (C). 1 (C), it can be seen that the expression of Col II, a chondrocyte-related marker, in the second and fourth chondrocytes is higher than that of the sixth and eighth chondrocytes. On the other hand, Col X, a chondrocyte cell dehiscence marker, showed low expression in the second and fourth passage cartilage cells, but high expression was observed in the cartilage cells of passage number 8. Therefore, it can be seen that the dehisceration of chondrocyte cells increases as the number of passages increases. In addition, the expression of Rheb gene, which is a cell proliferation related marker, was observed to be remarkably reduced in chondrocytes of the 6th and 8th decideways.

In addition, proteins were extracted and subjected to Western blotting to confirm Rheb gene expression and protein expression. The western blotting was carried out as follows. The cell lysate was centrifuged at 13000 rpm for 10 minutes (Micro 17R, Hanil Science Industrial, Seoul, Korea), and the cell lysate was centrifuged for 10 minutes at 13000 rpm. Korea). The supernatant containing the protein was denatured at 95 ° C for 5 minutes in a SDS-PAGE sample buffer (SDS-PAGE sample buffer). The proteins in the gel were transferred to PVDF (Millipore, Billerica, MS) and incubated with anti-α-tubulin antibody (ABM, Canada), anti-Rheb antibody (abcam, USA) (Horseradish peroxidase, HRP), followed by enhanced chemiluminescence (ECL). The results of the Western blotting are shown in FIG. 1 (D). As can be seen from FIG. 1 (D), the expression pattern of the Rheb gene, which is a cell proliferation related marker, was observed to be remarkably reduced in the sixth and seventh passage chondrocytes where the depolymerization proceeded.

Example  2. Rheb  Gene introduction and identification

According to a known method (Bulavin, DV et al., Nature genetics 31, 210-215 (2002) and Bulavin, DV et al., Nature genetics 36, 343-350 (2004), NheI and HindIII were used as restriction enzymes The Rheb gene of SEQ ID NO: 2 was inserted into the pEGFP-NI vector to prepare a Rheb plasmid DNA vector. The cleavage map of the pEGFP vector is shown in FIG.

Using the Rheb plasmid DNA vector, gene introduction was performed using a microporator (Microporator, Invitrogen), a non-viral gene introduction system. That is, 5 μg of the Rheb plasmid DNA vector and 1 × 10 ⁶ cells of chondrocytes were mixed with 100 Resuspension solution (Invitrogen, USA) according to the manufacturer's instructions, and the mixture was reacted at about 1700 voltage, about 20 ms, Respectively. The cells thus obtained were transformed into a third passage of chondrocytes by culturing in a conventional culture medium (for example, a DMEM culture medium without antibiotics). For comparison, the third line of chondrocytes was transformed in the same manner using an empty vector without the Rheb gene, that is, a pEGFP-N1 vector.

The chondrocytes obtained by performing the gene introduction were cultured for 2 days in medium (DMEM (Gibco) supplemented with 1% Penicillin-Streptomycin and 10% fetal bovine serum (FBS) (Hyclone) Flow cytometry analysis (FACS) was used to observe the expression pattern of Rheb protein. The flow cytometry analysis was performed as follows. The third passage chondrocytes (P3-Mock), into which an empty vector was introduced, and the third passage cartilage cells (P3-Rheb) transformed with a Rheb plasmid DNA vector, were washed twice with PBS and then treated with trypsin- Cells were harvested with trypsin-EDTA and suspended in PBS at a concentration of 1 × 10 ⁶ cells / ml. Cell viability was measured using Accuri C6 (BD Biosciences) and Accuri CSampler software.

The chondrocytes obtained by gene introduction were cultured for 3 days in medium (DMEM (Gibco) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) (Hyclone) And RNA was extracted and subjected to RT-PCR in the same manner as in Example 1 (2).

The flow cytometry and RT-PCR results are shown in FIGS. 3A and 3B, respectively. As can be seen from FIGS. 3A and 3B, the transfection efficiency of the Rheb plasmid DNA vector into the chondrocyte was 32%, and the transfection efficiency of the third transfection line transformed with the Rheb plasmid DNA vector It was confirmed that the Rheb gene in the cartilage cell (P3-Rheb) was markedly increased as compared with the third passage cartilage (P3-Mock) into which the empty vector was introduced.

Example  3. Aging of cartilage cells and Depletion  Inhibition assessment

(P3-Mock) into which an empty vector was introduced and a third passage of chondrocytes (P3-Rheb) transformed with a Rheb plasmid DNA vector were obtained in the same manner as in Example 2, respectively. In addition, aging and depletion of chondrocytes (P6-Mock / P6-Rheb) obtained by subculturing each cell to passage 6 were evaluated.

P6-Mock chondrocytes and P6-Rheb chondrocytes were examined for SA-β-Gal expression in chondrocytes in the same manner as in Example 1 (2). The results are shown in FIG. As can be seen from the results of FIG. 4 (A), the number of cells stained with SA-β-Gal was significantly decreased in P6-Rheb chondrocytes as compared to P6-Mock chondrocytes. In addition, the sizes of P6-Mock chondrocytes and P6-Rheb chondrocytes were measured in the same manner as in Example 1 (2), and the results are shown in FIG. 4 (B). As shown in FIG. 4 (B), P6-Rheb chondrocyte cells showed a statistically significant decrease in cell size compared to P6-Mock chondrocytes (*, p <0.05).

Cell proliferation was measured using Cell counting kit-8 (CCK, Dojindo laboratories, Japan) on days 1, 3, 5, 7 and 9 of P6-Mock chondrocytes and P6-Rheb chondrocytes. Cells were cultured in a 24-well plate, CCK was added to each well, reacted in a 37 ° C incubator for 3 hours, and then the absorbance was measured with an ELISA reader at a wavelength of 450 nm to compare cell viability. The result is shown in Fig. 4 (C). As can be seen in FIG. 4 (C), the proliferative capacity of P6-Mock chondrocytes rapidly decreased from 7 days after in vitro culture, but P6-Rheb chondrocytes proliferated continuously at 7 and 9 days of culture (*, P < 0.05). Therefore, it is statistically significant that P6-Mock cartilage cells exhibit high proliferative activity in P6-Rheb chondrocytes.

In addition, RNA was extracted from P6-Mock chondrocytes and P6-Rheb chondrocytes in the same manner as in Example 1 (2), and RT-PCR was performed to determine the expression patterns of Col II, ColX and Rheb genes The results are shown in Fig. 5 (A). The left panel of FIG. 5 (A) is the result of analyzing the mRNA of the chondrocytes of the third passage, and the right panel is the result of analyzing the mRNA expression pattern of the chondrocytes of the sixth passage. As can be seen from FIG. 5 (A), high Col II expression was observed in P3-Rheb chondrocytes, whereas ColX expression was very low. When these cells were cultured up to passage 6, gene expression patterns were examined in the same manner. As a result, P6-Rheb chondrocytes showed a higher expression of Col II than P6-Mock, and the expression of ColX was remarkably decreased.

Real-time quantitative RT-PCR was performed to quantitatively confirm gene expression patterns. 1 μl of a cDNA template obtained by performing RT-PCR after extracting RNA in the same manner as in Example 1 (2), 1 μl each of a 10 pM Rheb gene sense and an antisense primer, 25 μl of a Power CYBR Green PCR master mix (Invitrogen) 25 And 22 μl of DW were added to prepare samples, which were then measured using a StepOnePlus (Invitrogen) instrument. First, the samples were reacted at 94 ° C for 10 minutes for initial denaturation, and then a total of 45 cycles were amplified. The time and temperature for each cycle were 10 seconds at 94 ° C, 10 seconds at 60 ° C, and 30 seconds at 25 ° C. Expression of Rheb 2 ^- were compared in a relative value with the quantification of GAPDH by ^{△△ Ct} method. As a primer for measuring Rheb gene expression, the sense and antisense primers are 5'-GAG TCC ACA AAT TGG CCT TC-3 '(SEQ ID NO: 11) and 5'-CAG TCC AAG TCC CGG AAG AT- No. 12) was used. The results of the RT-PCR are shown in FIG. 5 (B). The left panel of FIG. 5 (B) is the result of analyzing the mRNA of the chondrocytes of the third passage, and the right panel is the result of analyzing the mRNA expression pattern of the chondrocytes of the sixth passage. 5 (B), the amount of ColII and Rheb gene expression in P3-Rheb chondrocytes was 2.4 ± 0.2 and 1.7 ± 0.0 fold higher than that of P3-Mock chondrocytes, and ColX, a chondrocyte demolding marker, was 0.4 ± 0.1 times lower (*, p <0.05). In addition, the expression of ColII and Rheb gene in P6-Rheb chondrocytes was 3.6 ± 0.2 and 1.4 ± 0.0 fold higher than that of P6-Mock chondrocytes, respectively, and the degeneration marker ColX was decreased by 0.4 ± 0.1 fold (*, p <0.05 ). Therefore, the third line of chondrocytes transformed with the Rheb plasmid DNA vector exhibited a statistically significant increase in the expression of ColI even when the in vitro culture was increased to the sixth passage, and the expression of ColX, the depletion marker, .

Therefore, from the results of FIGS. 4 and 5, that is, from SA-β-Gal, cell size measurement, and CCK, RT-PCR and Real-time PCR, the introduction of the Rheb gene effectively inhibited the aging and depolarization of chondrocytes , It turns out to be an excellent cartilage cell having a normal phenotype that can be used for regeneration of cartilage tissue.

<110> College of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> Methods for inhibiting senescence and dedifferentiation of          chondrocytes using Rheb gene <130> PN0710 <160> 12 <170> Kopatentin 1.71 <210> 1 <211> 184 <212> PRT <213> Homo sapiens <400> 1 Met Pro Gln Ser Lys Ser Arg Lys Ile Ala Ile Leu Gly Tyr Arg Ser   1 5 10 15 Val Gly Lys Ser Ser Leu Thr Ile Gln Phe Val Glu Gly Gln Phe Val              20 25 30 Asp Ser Tyr Asp Pro Thr Ile Glu Asn Thr Phe Thr Lys Leu Ile Thr          35 40 45 Val Asn Gly Gln Glu Tyr His Leu Gln Leu Val Asp Thr Ala Gly Gln      50 55 60 Asp Glu Tyr Ser Ile Phe Pro Gln Thr Tyr Ser Ile Asp Ile Asn Gly  65 70 75 80 Tyr Ile Leu Val Tyr Ser Val Thr Ser Ile Lys Ser Phe Glu Val Ile                  85 90 95 Lys Val Ile His Gly Lys Leu Leu Asp Met Val Gly Lys Val Gln Ile             100 105 110 Pro Ile Met Leu Val Gly Asn Lys Lys Asp Leu His Met Glu Arg Val         115 120 125 Ile Ser Tyr Glu Glu Gly Lys Ala Leu Ala Glu Ser Trp Asn Ala Ala     130 135 140 Phe Leu Glu Ser Ser Ala Lys Glu Asn Gln Thr Ala Val Asp Val Phe 145 150 155 160 Arg Arg Ile Ile Leu Glu Ala Glu Lys Met Asp Gly Ala Ala Ser Gln                 165 170 175 Gly Lys Ser Ser Cys Ser Val Met             180 <210> 2 <211> 552 <212> DNA <213> Homo sapiens <400> 2 atgccgcagt ccaagtcccg gaagatcgcg atcctgggct accggtctgt ggggaaatcc 60 tcattgacga ttcaatttgt tgaaggccaa tttgtggact cctacgatcc aaccatagaa 120 aacactttta caaagttgat cacagtaaat ggacaagaat atcatcttca acttgtagac 180 acagccgggc aagatgaata ttctatcttt cctcagacat actccataga tattaatggc 240 tatattcttg tgtattctgt tacatcaatc aaaagttttg aagtgattaa agttatccat 300 ggcaaattgt tggatatggt ggggaaagta caaataccta ttatgttggt tgggaataag 360 aaagacctgc atatggaaag ggtgatcagt tatgaagaag ggaaagcttt ggcagaatct 420 tggaatgcag cttttttgga atcttctgct aaagaaaatc agactgctgt ggatgttttt 480 cgaaggataa ttttggaggc agaaaaaatg gacggggcag cttcacaagg caagtcttca 540 tgctcggtga tg 552 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 ttcagctatg gagatgacaa tc 22 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 agagtcctag agtgactgag 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 agggttacca ggtccaaaag 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 ttccagtcct tgggtcataa 20 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 atgccgcagt ccaagtcc 18 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 cacatcaccg agcatgaag 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 cggatttggt cgtattgggc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 cagggatgat gttctggaga 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 gagtccacaa attggccttc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 cagtccaagt cccggaagat 20

Claims (6)

A method for inhibiting senescence or de-differentiation of chondrocytes, comprising the step of transforming chondrocytes subcultured in a first passage to a sixth passage with a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1. 2. The method according to claim 1, wherein the gene consists of the nucleotide sequence of SEQ ID NO: 2. delete A chondrocyte cell having an aged or undifferentiated state that is transformed with a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1. 5. The chondrocyte cell according to claim 4, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 2. The aged or undifferentiated cartilage cell according to claim 4 or 5, which is obtained by transforming chondrocytes subcultured in the first to sixth passages.

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