KR102013060B1 - Method for stimulating chondrogenesis by using KLF-4 - Google Patents

Abstract

The present invention relates to a method of promoting the differentiation of chondrocytes by inducing overexpression of KLF-4 (Kruppel-like factor 4) in chondrocytes. In addition, the present invention relates to a method for promoting differentiation of chondrocytes by inducing overexpression of KLF-4 (Kruppel-like factor 4) in chondrocytes treated with simvastatin.
The method of promoting chondrocyte differentiation, induces KLF-4 (Krupple-like factor 4) overexpression in chondrocytes and expresses the expression of SOX-9, type II collagen or sulfated prothioglycan in chondrocytes. It can increase the differentiation of chondrocytes.

Description

Method for promoting chondrocyte differentiation using KLF-4 {Method for stimulating chondrogenesis by using KLF-4}

The present invention relates to a method of promoting the differentiation of chondrocytes by inducing overexpression of KLF-4 (Kruppel-like factor 4) in chondrocytes.

In addition, the present invention relates to a method for promoting differentiation of chondrocytes by inducing overexpression of KLF-4 (Kruppel-like factor 4) in chondrocytes treated with simvastatin.

Osteoarthritis (OA) is the most common disease of the joint, affecting nearly half of the elderly population, and characterized by progressive degeneration of articular cartilage. Osteoarthritis significantly affects the psychosocial status of patients, their families and occupations. Major clinical symptoms include symptoms of chronic knee pain, stiffness, knee edema and limited physical activity. OA causes severe pain and exercise problems at intermediate or advanced levels and represents a leading socio-economic burden in the developed world.

Statins include 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors and are a major therapeutic agent for the treatment of hypercholesterolemia because they significantly reduce the low density lipoprotein cholesterol (LDL) cholesterol circulation level. Statins are commonly used in the treatment of hyperlipidemic cardiovascular disease, reducing the likelihood of illness and death. The value of statin therapy in recent years has not been based solely on the reduction of lipids, but rather has a direct vascular effect. Another study showed a prophylactic anti-inflammatory effect because statins can act directly as an inhibitor of immune regulation of inflammatory cytokines or MMP expression in chondrocytes. As recently reported, simvastatin (SVT) can induce differentiation of chondrocytes, the structural formula of which is represented by the following [Formula 1].

Kruppel-like factor 4 (KLF-4) is a zinc finger-type transcription factor expressed in the gastrointestinal tract, skin, testicles, bone and other tissues. KLF-4 has three tandem C ₂ H ₂ zinc finger motifs at the C terminus that bind to the 5'-gagaggctgct-3 'or 5'-caccc-3' DNA sequence. This protein is considered a pleiotropic factor because it can activate or inhibit transcription.

During skeletal development, KLF-4 is upregulated in immature osteoblasts and gradually decreases as osteoblasts mature. It has also been shown to neutralize the carcinogenic effects of Notch signaling, which is closely related to skeletal formation. In addition, expression in fibroblasts is highest in growth arrest cells and lowest in the rapid proliferative phase. This suggests that physiological regulation of KLF-4 expression in various cell types is critical to development.

However, the role of KLF-4 in chondrocytes has not been studied or reported to date. Accordingly, the present inventors have made intensive efforts to provide a composition capable of promoting chondrocyte differentiation. As a result, KLF-4 promoted the differentiation of simvastatin-treated chondrocytes and completed the present invention.

The present inventors have completed the present invention by developing a method for promoting the differentiation of chondrocytes using KLF-4 (Kruppel-like factor 4) and SVT.

Accordingly, an object of the present invention provides a method for promoting chondrocyte differentiation, comprising: inducing KLF-4 (Kruppel-like factor 4) overexpression in chondrocytes.

Still another object of the present invention is to provide a method for promoting chondrocyte differentiation, comprising: inducing overexpression of KLF-4 (Krupple-like factor 4) in chondrocytes treated with simvastatin.

In order to achieve the above object, the present invention may provide a method for promoting chondrocyte differentiation, comprising: inducing KLF-4 (Kruppel-like factor 4) overexpression in chondrocytes.

According to a preferred embodiment of the present invention, overexpressing KLF-4 is induced by transfection of chondrocytes with a KLF-4 expression vector comprising a KLF-4 gene consisting of the nucleotide sequence of SEQ ID NO: 1. It may be.

According to another preferred embodiment of the present invention, overexpressing KLF-4 may be to directly transfect the gene sequence encoding KLF-4 in chondrocytes.

According to another preferred embodiment of the present invention, the differentiation-promoting chondrocytes may be an increase in the expression level of SOX-9, type II collagen or sulfated-proteeoglycan.

The present invention may also provide a method for promoting chondrocyte differentiation, comprising: inducing overexpression of KLF-4 (Krupple-like factor 4) in chondrocytes treated with simvastatin.

According to a preferred embodiment of the present invention, the concentration of simvastatin (Simvastatin) may be from 10 to 100μM.

According to a preferred embodiment of the present invention, the differentiation-promoting chondrocytes may be an increase in the expression level of SOX-9, type II collagen or sulfated prothioglycan (Sulfated-proteeoglycan).

Therefore, the present invention induces KLF-4 (Krupple-like factor 4) overexpression in chondrocytes and increases the expression of SOX-9, type II collagen or sulfated-proteeoglycan in chondrocytes. Since it promotes differentiation of chondrocytes, it provides a method for promoting chondrocyte differentiation.

Figure 1 confirmed the expression of KLF-4 in the chondrocyte differentiation process involving simvastatin (Simvastatin).
Figure 2 confirmed the effect of simvastatin (Simvastatin) on the interaction of KLF-4 and SOX-9.
Figure 3 confirmed the expression of KLF-4 induced by simvastatin (Simvastatin) after passage of chondrocytes in order to induce the differentiation of chondrocytes.
Figure 4 confirms the effect of amplifying KLF-4 using cDNA or knocking down KLF-4 using siRNA during chondrocyte differentiation involving simvastatin.
Figure 5 confirmed the location of KLF-4 and SOX-9 in the chondrocyte differentiation process involving simvastatin (Simvastatin).

Hereinafter, the present invention will be described in more detail.

As described above, studies on safe and effective method for promoting chondrocyte differentiation are required, but the role of KLF-4 in chondrocytes has not been studied or reported to date.

Cartilage differentiation promoting method comprising the step of inducing KLF-4 (Kruppel-like factor 4) overexpression in chondrocytes according to the present invention is SOX-9, type II collagen or sulfated prothioglycans in chondrocytes Since the expression of (Sulfated-proteeoglycan) is increased to promote the differentiation of chondrocytes, it is effective as a method of promoting chondrocyte differentiation.

Accordingly, the present invention provides a method for promoting chondrocyte differentiation, comprising: inducing KLF-4 (Kruppel-like factor 4) overexpression in chondrocytes.

Overexpressing the KLF-4 of the present invention is preferably induced by transfection of chondrocytes with a KLF-4 expression vector comprising a KLF-4 gene consisting of the nucleotide sequence of SEQ ID NO: 1, but not limited thereto. It doesn't work.

Overexpressing the KLF-4 of the present invention is preferably one that directly transfects the gene sequence encoding KLF-4 in chondrocytes, but is not limited thereto.

Preferably, the differentiation-promoting chondrocytes of the present invention show an increase in the expression level of SOX-9, type II collagen or sulfated-proteeoglycan, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors confirmed the expression of KLF-4 in the chondrocyte differentiation process involving simvastatin. Cartilage cells treated with SVT were found to increase the expression of KLF-4 and type II collagen and sulfated prodioglycan (FIG. 1).

In another specific embodiment of the present invention, the inventors confirmed the effect of simvastatin on the interaction of KLF-4 and SOX-9. Expression of KLF-4 and SOX-9 increased and coexisted in the nucleus when SVT was treated, whereas type II collagen was present in the cytoplasm (FIG. 2).

In another specific embodiment of the present invention, the inventors confirmed the expression of KLF-4 induced by simvastatin after passage of chondrocytes in order to induce dedifferentiation of chondrocytes. In the case of dedifferentiated chondrocytes, the expression levels of type II collagen and sulfated prothioglycan were increased, and it was confirmed that the treatment of SVT increased the expression level of KLF-4 (FIG. 3).

In another specific embodiment of the present invention. Simulating the cartilage cell differentiation involving simvastatin (Simvastatin) amplified KLF-4 using the cDNA of SEQ ID NO: 1, or knockdown KLF-4 using siRNA was confirmed (knockdown) effect. Irrespective of the KLF-4 transfection method, expression of collagen II was elevated when overexpressed in chondrocytes, and expression of collagen II was significantly decreased when KLF-4 was knocked down (FIG. 4).

In another specific embodiment of the present invention, the location of KLF-4 and SOX-9 was identified during chondrocyte differentiation involving simvastatin. Expression of KLF-4 increased the expression of SOX-9 and confirmed that it also coexisted in the nucleus (FIG. 5).

Therefore, the method of promoting chondrocyte differentiation, comprising inducing KLF-4 (Kruppel-like factor 4) overexpression in chondrocytes, is characterized in that SOX-9, type II collagen or sulfated prothioglycan (Sulfated) in chondrocytes. increased expression of proteeoglycans can promote chondrocyte differentiation.

In addition, the present invention provides a method for promoting chondrocyte differentiation, comprising: inducing overexpression of KLF-4 (Krupple-like factor 4) in chondrocytes treated with simvastatin.

The concentration of simvastatin of the present invention (Simvastatin) is preferably 10 to 100μM, but is not limited thereto.

Preferably, the differentiation-promoting chondrocytes of the present invention show an increase in the expression level of SOX-9, type II collagen or sulfated-proteeoglycan, but is not limited thereto.

Therefore, the method of promoting chondrocyte differentiation, comprising inducing KLF-4 (Kruppel-like factor 4) overexpression in SVT-treated chondrocytes, is characterized in that SOX-9, type II collagen or sulfated prothiocation in chondrocytes. Increased expression of sulfated-proteeoglycan can promote the differentiation of chondrocytes.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Sample and Processing

<1-1> Sample

SVT was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Cells were treated with 50 μM SVT or vehicle (dimethyl sulfoxide, DMSO) for 24 hours. Sodium nitroprussade (SNP; Sigma Chemicals, St. Louis, MO, USA) was used for the differentiation of chondrocytes. KLF-4 cDNA (GFP-tag) and pCMV-AC6 vectors were purchased from Origene technologies (Rockville, MD, USA). KLF-4 siRNA was obtained from Bioneer (Daejeon, Korea). Transfection of cDNA and siRNA was performed using TurpFect transfection reagent (TurboFect transfection; Fisher Thermo Scientific, Waltham, Mass., USA) according to the manufacturer's instructions.

<1-2> Isolation and Monolayer Culture of Rabbit Joint Cartilage Cells

New Zealand white rabbits (2 weeks old) were purchased from Kotec (Pyeongtaek, South Korea). Animals were sacrificed with ether anesthesia, then disinfected with 75% ethanol and rinsed with PBS containing penicillin (100 units / mL) and streptomycin (100 μg / mL). Chondrocytes are cut into small pieces of 1 to 3 mm ² , digested with DMEM containing 0.2% collagenase II (Sigma-Aldrich, St. Louis, MO, USA) for 7 hours at 37 ° C. and 1000 Centrifugation was performed for 10 minutes at rpm. After centrifugation, cells (2 × 10 ⁴ cells / dish) were resuspended in DMEM supplemented with 10% (v / v) fetal bovine serum, penicillin (50 units / mL), and streptomycin (50 μg mL). , Monolayer culture in 5% CO ₂ incubator at 37 ℃. After incubation for one day, chondrocytes were attached to the wall of the culture dish and the medium was changed every two days. Stretched cells showed irregular spindles or polygonal shapes. Subsequent experiments were performed at least three times independently. The present invention protocol was approved by Gongju University Ethics Committee.

SVT  Type II collagen KLF Whether to promote the expression of -4

We examined the differentiation of chondrocytes to determine the correlation between KLF-4 and chondrocyte differentiation. The differentiation state of chondrocytes was confirmed by examining the expression of type II collagen and sulfated proteoglycan synthesis.

Specifically, rabbit articular chondrocytes were treated with SVT in primary culture and KLF-4 and type II collagen expression was confirmed by Western blot analysis and Alcian blue staining.

Western blot analysis showed that total protein was harvested from chondrocytes and the same amount of protein (20-40 μg) was separated by 8% sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) and Transferred to nitro cellulose membrane. The membranes were blocked for 1 hour at room temperature with 5% (w / v) skim milk powder in Tris buffered saline-0.01% Tween 20 (TBST, pH 8.0) buffer and then incubated with primary antibody for one day. Antibodies used were as follows: goat anti-collagen type II monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, SC-7764, 0.2 μg / mL); Rabbit anti-KLF-4 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, SC-20691, 0.2 μg / mL); Mouse anti-GAPDH (Santa Cruz Biotechnology; SC-166545; 0.2 μg / ml); Anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA; A0545; 80 ng / mL); Anti goat IgG (Chemicon International, Billerica, MA, USA, AP106P, 40 ng / mL); And anti mouse IgG (Enzo Life Sciences International, Farmingdale, NY, USA, ADI-SAB-100, 80 ng / mL). After washing three times with TBST for 10 minutes, the membrane was incubated with secondary mustard peroxidase-binding antibody for 2 hours. Reactive bands were visualized using an improved chemiluminescent reagent (Dogen, Seoul, Republic of Korea) and quantified using the LAS4000 camera system (Fuji Film, Tokyo, Japan).

For alcianblue staining, cells (P0) were fixed for 15 min with 3.5% paraformaldehyde in PBS, stained with 1% alcianblue in 3% acetic acid for 1 day and washed three times with 0.1N HCl. Stained cells were washed three times with distilled water and 6M guanidine HCl was added for 6 hours. The production of sulfated-proteoglycans was measured at 595 nm. The data represents the results of at least four independent experiments.

As a result, as shown in FIG. 1, SVT-treated chondrocytes showed type II collagen expression consistent with upregulation of KLF-4 in a dose- and time-dependent manner (FIGS. 1A and B). Consistent with the expression of type II collagen, SVT treated chondrocytes showed enhanced alcian blue staining for sulfated proteoglycans (FIG. 1C). These results show that KLF-4 expression is increased in SVT treated primary cultured rabbit articular chondrocytes, suggesting a relationship between KLF-4 expression and chondrocyte differentiation.

SVT KLF The impact of SO-4 on SOX-9 interaction

<3-1> RT- PCR  inspection

To determine the association between KLF-4 expression and differentiation of the present invention, chondrocytes were treated with SVT and the expression of chondrocyte markers type II collagen, SOX-9 and KLF-4 were examined by RT-PCR.

Specifically, total RNA was extracted from cells using TRIzol reagent (Life Technologies, Gaithersburg, MD, USA) to perform RT-PCR. Reverse transcription and cDNA synthesis were performed using the Maxime ™ RT premix kit (iNtRON Biotechnology, Seongnam), and RNA (0.2 μg) was reverse transcribed into cDNA according to the manufacturer's instructions. Primers used are shown in Table 1 below, wherein the annealing temperature and the number of cycles were 52 ° C and 27 cycles for type II collagen, 62 ° C and 27 cycles for SOX-9, and 52 cycles for KLF-4. ° C and 27 cycles, GAPDH is 56 ° C and 25 cycles.

object Length direction order SEQ ID NO: Type II collagen
370 bp
Forward direction 5'-GAC CCC ATG CAG TAC ATG CG-3 '' 2 Reverse 5'-AGC CGC CAT TGA TGG TCT CC-3 '' 3 SOX-9
386 bp
Forward direction 5'-GAC CCC ATG CAG TAC ATG CG-3 '' 4 Reverse 5'-AGC CGC CAT TGA TGG TCT CC-3 ' 5 KLF-4
253 bp Forward direction 5'-AGC TCA TGC CAC CGG GTT C-3 ' 6 Reverse 5'-GTG TGT TTG CGG TAG TGC CTG-3 ' 7 GAPDH
299 bp
Forward direction 5'-TCA CCA TCT TCC AGG AGC GA-3 '' 8 Reverse 5'-CAC AAT GCC GAA GTG GTC GT-3 ' 9

The amplified DNA product was isolated on 1% agarose gel and stained with EcodyeTM nucleic acid staining solution (BioFact, Daejeon, Korea). The mRNA of the target gene was normalized based on the expression level of GAPDH and then quantitatively calculated.

As a result, as shown in [FIG. 2A], when the cells were treated with SVT, type II collagen, SOX-9, and KLF-4 expression increased.

<3-2> IP Analysis

The inventors of the present invention wished to establish a functional link between KLF-4 and SOX-9 and to investigate how KLF-4 may play a role in the differentiation of chondrocytes. Collected and performed IP analysis.

Specifically, for IP analysis, cell extracts were first prepared with IP buffer (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA) and then lysed. Antisox-9 antibody and protein G beads (GenDEPOT, Barker, TX, USA) were added to the supernatant and incubated for one day. The antibodies used were as follows: Goat anti SOX-9 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, SC-17340, 0.2 μg / mL). IP fractions were analyzed using the anti-KLF-4 antibody in the same manner as the Western blot assay described in Example 2 above. Since SNP is a nitric oxide donor known to reduce SOX-9 expression and regulate chondrocyte differentiation, SNP treated chondrocytes were used as a negative control for SOX-9 expression.

As a result, as shown in [FIG. 2B], it was found that the interaction between KLF-4 and SOX-9 was significantly induced by SVT treatment in chondrocytes.

<3-3> Immunofluorescence  dyeing

The present inventors performed an immunofluorescent staining method to confirm the expression sites of KLF-4 and type II collagen.

Specifically, to perform immunofluorescence staining, chondrocytes were grafted at a concentration of 2 × 10 ⁴ cells in a 35 mm dish with a pre-set sterile cover slip. After attaching the cells, the cells were washed with PBS and fixed in 3.5% paraformaldehyde for 30 minutes. After washing three times with PBS, cells were blocked with 5% BSA at 37 ° C. for 1 hour. Samples were incubated with primary antibodies (Mouse Anti-COL2A1 / II collagen, MAB8887; 0.2 μg / mL) for 1 day at 4 ° C. Cells were then incubated with peroxidase-conjugated secondary antibody for 1 hour at 37 ° C. and then 4 ', 6--diamino-2phenylindole (4', 6- for 5 minutes at room temperature). Cells were counterstained with diamidino-2-phenylindole, DAPI; Invitrogen, Burlington, ON, Canada). Immunofluorescence was observed using a BX51 fluorescence microscope (Olympus, Tokyo, Japan). Since the cartilage matrix is rich in type II collagen, the synthesis and secretion of its components can be used as specific markers to confirm the differentiation phenotype of cartilage cells.

As a result, as shown in FIG. 2C, KLF-4 was localized around the nucleus, but type II collagen was mainly present in the cytoplasm. In contrast, as shown in [FIG. 2D], SOX-9 coexisted with KLF-4 in the nucleus.

SVT  In continuously differentiated chondrocytes KLF Up to -4? Check whether

Continuous experiments in monolayer cultures are known to result in the loss of differentiated phenotype in chondrocytes. We identified the effect of this marker on dedifferentiated chondrocytes through prolonged steps in monolayer cultures to determine if KLF-4 positively regulates type II collagen expression induced by SVT.

Specifically, Western blot analysis method and Alsian blue staining method of [Example 2] were used.

As a result, as shown in FIG. 3, SVT increased sulfated prothioglycan synthesis associated with type II collagen expression compared to the differentiated chondrocytes of the control group. Western blot analysis showed that SVT mediates the gradual increase of KLF-4 during the differentiation of chondrocytes. These results indicate that KLF-4 may delay dedifferentiation to maintain differentiation of chondrocytes.

Simvastatin ( SVT A) mediated chondrocyte differentiation KLF Effect of Overexpression or Knockdown of -4

<5-1> KLF Through transfection of -4 cDNA KLF Overexpression of -4

In order to confirm the effect of overexpression of KLF-4 on chondrocyte differentiation, we transfected cDNA (SEQ ID NO: 1) of KLF-4.

Specifically, Western blot analysis method and Alsian blue staining method of [Example 2] were used.

As a result, as shown in [FIG. 4A], type II collagen upregulation was induced as compared to expression in SVT treated control cells. In the absence of SVT, there was no significant difference in cell viability between chondrocytes transfected with pCMV6 vector and chondrocytes transfected with KLF-4 cDNA. In this context, as shown in FIG. 4B, RT-PCR showed that ectopic expression of KLF-4 mRNA in chondrocytes increased type II collagen expression.

<5-2> siRNA  through KLF -4 knockdown

In order to further investigate the results of Example <5-1>, the inventors performed a complementary experiment in which KLF-4 siRNA was introduced into chondrocytes to lower protein levels.

Specifically, expression of collagen II, KLF-4 and GAPDH was confirmed by Western blot analysis method of [Example 2] and RT-PCR method of [Example 3]. The synthesis of antioxidant prothioglycans was confirmed by the Alcian blue staining method of [Example 2].

As a result, as shown in [FIG. 4C], type II collagen induced by SVT in chondrocytes was significantly reduced due to KLF-4 knocked down with siRNA. Alcian blue staining also showed that KLF-4 treated with SVT after KLF-4 cDNA transfection increased 24% staining compared to SVT alone. In contrast, as shown in [FIG. 4D], knockdown of KLF-4 completely prevented the synthesis of SVT induced sulfated prothioglycans. These results indicate that upregulation of KLF-4 is required for the differentiation of SVT-induced chondrocytes and that exogenous KLF-4 may increase this process.

<5-3> Simvastatin ( SVT ) -In mediated chondrocyte differentiation KLF Location of -4 and SOX-9

Specifically, the position where KLF-4 and SOX-9 are expressed using the immunofluorescent staining method of Example <3-3> was confirmed.

As a result, it was confirmed that KLF-4 and SOX-9 are present in the nucleus of the SVT-treated chondrocytes as shown in FIG. 5. Ectopic KLF-4 expression through transfection of KLF-4 cDNA allowed SOX-9 to be present in the nucleus compared to SVT treated cells. This result shows an association between KLF-4 and SOX-9 in SVT mediated differentiation.

<110> KONGJU NATIONAL UNIVERSITY INDUSTRY-UNIVERSITY COOPERATION FOUNDATION <120> Method for stimulating chondrogenesis by using KLF-4 <130> 1063544 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 1437 <212> DNA <213> Artificial Sequence <220> <223> KLF-4_cDNA <400> 1 atgaggcagc cacctggcga gtctgacatg gctgtcagcg acgcgctgct cccatctttc 60 tccacgttcg cgtctggccc ggcgggaagg gagaagacac tgcgtcaagc aggtgccccg 120 aataaccgct ggcgggagga gctctcccac atgaagcgac ttcccccagt gcttcccggc 180 cgcccctatg acctggcggc ggcgaccgtg gccacagacc tggagagcgg cggagccggt 240 gcggcttgcg gcggtagcaa cctggcgccc ctacctcgga gagagaccga ggagttcaac 300 gatctcctgg acctggactt tattctctcc aattcgctga cccatcctcc ggagtcagtg 360 gccgccaccg tgtcctcgtc agcgtcagcc tcctcttcgt cgtcgccgtc gagcagcggc 420 cctgccagcg cgccctccac ctgcagcttc acctatccga tccgggccgg gaacgacccg 480 ggcgtggcgc cgggcggcac gggcggaggc ctcctctatg gcagggagtc cgctccccct 540 ccgacggctc ccttcaacct ggcggacatc aacgacgtga gcccctcggg cggcttcgtg 600 gccgagctcc tgcggccaga attggacccg gtgtacattc cgccgcagca gccgcagccg 660 ccaggtggcg ggctgatggg caagttcgtg ctgaaggcgt cgctgagcgc ccctggcagc 720 gagtacggca gcccgtcggt catcagcgtc agcaaaggca gccctgacgg cagccacccg 780 gtggtggtgg cgccctacaa cggcgggccg ccgcgcacgt gccccaagat caagcaggag 840 gcggtctctt cgtgcaccca cttgggcgct ggaccccctc tcagcaatgg ccaccggccg 900 gctgcacacg acttccccct ggggcggcag ctccccagca ggactacccc gaccctgggt 960 cttgaggaag tgctgagcag cagggactgt caccctgccc tgccgcttcc tcccggcttc 1020 catccccacc cggggcccaa ttacccatcc ttcctgcccg atcagatgca gccgcaagtc 1080 ccgccgctcc attaccaaga gctcatgcca cccggttcct gcatgccaga ggagcccaag 1140 ccaaagaggg gaagacgatc gtggccccgg aaaaggaccg ccacccacac ttgtgattac 1200 gcgggctgcg gcaaaaccta cacaaagagt tcccatctca aggcacacct gcgaacccac 1260 acaggtgaga aaccttacca ctgtgactgg gacggctgtg gatggaaatt cgcccgctca 1320 gatgaactga ccaggcacta ccgtaaacac acggggcacc gcccgttcca gtgccaaaaa 1380 tgcgaccgag cattttccag gtcggaccac ctcgccttac acatgaagag gcatttt 1437 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> type2_collagen_primer_F <400> 2 gaccccatgc agtacatgcg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> type2_collagen_primer_R <400> 3 agccgccatt gatggtctcc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX9_primer_F <400> 4 gaccccatgc agtacatgcg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX9_primer_R <400> 5 agccgccatt gatggtctcc 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> KLF-4_primer_F <400> 6 agctcatgcc accgggttc 19 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KLF-4_primer_R <400> 7 gtgtgtttgc ggtagtgcct g 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_primer_F <400> 8 tcaccatctt ccaggagcga 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_primer_R <400> 9 cacaatgccg aagtggtcgt 20

Claims (7)

i) directly transfecting the gene sequence encoding KLF-4 in chondrocytes in vitro; And
ii) inducing overexpression of KLF-4 (Kruppel-like factor 4) by treating simulstatin at a concentration of 10-100 μM to the chondrocytes of step i); Containing
A method of increasing the expression level of SOX-9, type II collagen or sulfated-proteeoglycan in chondrocytes.
According to claim 1, wherein the transfection of step i) is characterized in that by inducing the transfection (transfection) to the chondrocytes KLF-4 expression vector comprising a KLF-4 gene consisting of the nucleotide sequence of SEQ ID NO: How to.


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