CN113648417A - POSTN (polyhedral oligomeric silsesquioxane) as marker for regulating and controlling mesenchymal stem cell differentiation capacity and application thereof - Google Patents

Abstract

The invention belongs to the technical field of stem cells, and particularly relates to POSTN (polyhedral oligomeric silsesquioxane) as a marker for regulating and controlling the differentiation capacity of mesenchymal stem cells and application thereof. The invention discloses application of POSTN gene or protein as a marker for regulating differentiation capacity of umbilical cord mesenchymal stem cells. Furthermore, the invention also discloses a preparation for regulating and controlling the differentiation capacity of the mesenchymal stem cells. The research discovers that the POSTN gene can regulate the differentiation functions of adipogenic cells and osteoblastic cells of the human umbilical cord derived mesenchymal stem cells for the first time, discovers that POSTN is an important regulation and control gene of the osteogenic differentiation of the umbilical cord mesenchymal stem cells, and provides a new research strategy for the umbilical cord mesenchymal stem cells in the regenerative medicine field such as bone injury repair and osteoporosis treatment.

Description

POSTN (polyhedral oligomeric silsesquioxane) as marker for regulating and controlling mesenchymal stem cell differentiation capacity and application thereof

Technical Field

The invention belongs to the technical field of stem cells, and particularly relates to POSTN (polyhedral oligomeric silsesquioxane) as a marker for regulating and controlling the differentiation capacity of mesenchymal stem cells and application thereof.

Background

Mesenchymal Stem Cells (MSCs) are a class of adult stem cells with low immunogenicity, and have self-renewal and multidirectional differentiation potential, and MSCs are present in the interstitium of various tissues and organs throughout the body, are most abundant in bone marrow tissue, and can also be found in various tissues such as placenta, umbilical cord, liver, fat, muscle, skin, and the like. The MSC plays an important role in the process of maintaining balance of bone formation and bone resorption, can be differentiated into chondrocytes and osteoblasts under the induction of a specific microenvironment, wherein the osteoblasts can directly regulate bone formation and play an important role in repairing and regenerating bone defects and treating osteopathia such as osteoporosis, so the MSC has important clinical application value.

POSTN is a 90kDa extracellular matrix protein first found in the mouse osteoblast cell line, containing 1 canonical signal sequence, 4 homology repeat domains (FAS domains) and a C-terminal domain, lacking the transmembrane domain. It was initially discovered that POSTN promotes the aggregate differentiation of osteoblasts and osteogenic precursor cells in the periost; further research results show that POSTN can also be expressed in connective tissues, bone tissues and fibroblasts, can play a functional role in the regeneration process of tissues such as bones and hearts, and can promote wound healing. It has been shown that POSTN is an important regulator of cell growth and differentiation by binding to cell surface receptors of the integrin family FAK/AKT, transducing intracellular signals, and promoting the development or maintenance of various tissues or organs. POSTN also has a regulatory role in various cell proliferation, migration, angiogenesis, cytokine production and differentiation, etc. through multiple signaling pathways such as PI3K/AKT or Ras/p38 MAPK/CREB. However, whether POSTN regulates the osteogenic differentiation of hUC-MSC (umbilical cord mesenchymal stem cells) is currently unclear.

Disclosure of Invention

One of the purposes of the invention is to provide a target POSTN for regulating and controlling mesenchymal stem cell osteogenic and adipogenic differentiation and application thereof in preparation of mesenchymal stem cell osteogenic and adipogenic differentiation products.

The other purpose of the invention is to provide a POSTN inhibitor or accelerant and application thereof in bone-related diseases such as repair and regeneration of bone defects, osteoporosis and the like.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

the invention discloses that POSTN can regulate and control the differentiation of adipogenic cells and osteoblastic cells of umbilical cord mesenchymal stem cells. The POSTN knocked-down umbilical cord mesenchymal stem cell has obviously reduced osteogenic differentiation capacity and influences the application of the POSTN knocked-down umbilical cord mesenchymal stem cell in the fields of bone injury repair and the like, so that the inventor proves that POSTN is an important regulation molecule for osteogenic differentiation of umbilical cord mesenchymal stem cells and can research and develop the specificity of the POSTN over-expressed umbilical cord mesenchymal stem cell for treating diseases such as bone injury repair and the like.

Firstly, the invention provides application of POSTN gene or protein as a marker for regulating and controlling the differentiation capacity of mesenchymal stem cells.

Preferably, the mesenchymal stem cell is a human umbilical cord mesenchymal stem cell.

Preferably, the differentiation potency is osteogenic differentiation potency and/or adipogenic differentiation potency.

The invention provides application of a POSTN gene or protein in preparation of mesenchymal stem cell osteogenesis and/or adipogenesis induced differentiation products.

Preferably, the product comprises an inhibitor or promoter of POSTN gene or protein expression.

The inhibitor or the accelerator for expressing the POSTN gene or the protein enables the expression of osteogenic and/or adipogenic differentiation markers to be obviously changed by knocking down or accelerating the POSTN gene, thereby influencing the osteogenic differentiation capacity of the MSC.

Preferably, the osteogenic differentiation markers include an early marker ALP for osteoblast differentiation maturation and a late marker OPN for osteoblast differentiation maturation; the adipogenic differentiation markers include ADI and PPAR γ.

The invention also provides a preparation for regulating and controlling the differentiation capacity of the mesenchymal stem cells, and the preparation takes an inhibitor of POSTN or an accelerant of POSTN as an active ingredient.

Preferably, the differentiation potency is osteogenic differentiation potency and/or adipogenic differentiation potency.

Preferably, the inhibitor or the accelerant of POSTN knocks down or promotes the expression of POSTN genes or proteins, so that the expression of osteogenic differentiation markers is obviously changed, and the osteogenic adipogenic differentiation capacity of the MSC is influenced.

Preferably, the POSTN accelerator is an agent that promotes the expression level of POSTN;

preferably, the promoter is a vector that overexpresses POSTN.

Preferably, the POSTN inhibitor comprises a POSTN shRNA;

preferably, the target sequence of the POSTN shRNA is the nucleotide sequence shown as SEQ ID NO. 1.

Further, the invention provides an application of the mesenchymal stem cell osteogenic differentiation preparation in preparing a medicine for treating bone defect repair and regeneration and osteoporosis by adjusting mesenchymal stem cell osteogenic differentiation.

Based on the technical scheme, the invention has the following beneficial effects:

the research discovers for the first time that the POSTN gene can regulate the differentiation function of adipogenic cells and osteoblastic cells of the human umbilical cord derived mesenchymal stem cells, discovers that POSTN is an important regulation and control gene for the osteogenic differentiation of the umbilical cord mesenchymal stem cells, provides a new research strategy for the umbilical cord mesenchymal stem cells in the regeneration medical field such as bone injury repair and osteoporosis treatment, and develops a POSTN over-expressed specificity which can be used for treating bone tissue regeneration and the like.

Drawings

FIG. 1 validation of the efficiency of lentivirus infection of hUC-MSC; wherein, the observation results of the fluorescence microscope in the figure 1A-F, the detection results of the flow cytometry in the figure 1G and the detection results of the POSTN gene knock-down effect in the figure 1H q-PCR.

FIG. 2 amplification and identification of sh-POSTN-MSC with stably knocked-down POSTN; FIG. 2A-F Giemsa staining observations, FIG. 2G flow cytometry detection results.

FIG. 3 identification of the induced differentiation capacity of sh-POSTN-MSC in vitro, comparing the induced group with the self-differentiated group; FIGS. 3A-D oil Red O staining test results; FIG. 3E-F q-PCR results for detecting adipogenic differentiation key transcription factors ADI and PPAR γ expression, where E is sh-NC-MSC, and FIG. 3G-J ALP staining for detecting cell ALP activity; FIG. 3K-L q-PCR examination of the expression results of ALP and OPN, which are key markers for osteogenic differentiation.

FIG. 4 is a graph showing the identification of osteogenic induced differentiation capacity of sh-POSTN-MSC in vitro, and the sh-NC-MSC induced group is compared with the sh-POSTN-MSC induced group; FIGS. 4A-B ALP staining to detect cellular ALP activity; FIG. 4C ALP stained area; FIG. 4D q-PCR detection of the key markers for osteogenic differentiation, ALP and OPN.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Part of the reagent configurations of the ALP staining solution experiments in embodiment 3 of the present invention are as follows:

fixing liquid: a pipettor takes 40 mul of citric acid, 2ml of deionized water and 3ml of acetone to be uniformly mixed in a penicillin bottle for later use;

dyeing liquid: a pipettor takes 62.5 mul of cobalt methyl blue, 125 mul of naphthol phosphate AS-MX-disodium salt and 3ml of deionized water to be mixed in a penicillin bottle for standby. The staining solution is prepared and used in dark place.

Part of reagent configurations of the oil red O staining solution experiment in embodiment 3 of the invention are as follows:

oil red O staining solution: measuring 100ml of isopropanol in a measuring cylinder, weighing 0.5g of oil red O powder by using a balance, putting the oil red O powder into the same beaker, putting the beaker into a magnetic stirrer, stirring for 1 hour at room temperature to fully dissolve and uniformly mix the oil red O powder, and storing the oil red O powder at 4 ℃ in a dark place. When in use, a mixed solution is prepared according to the proportion of 3:2 of the oil red O and the PBS buffer solution. After filtration through filter paper, staining was performed. It is used as it is.

Example 1 isolation and culture of hUC-MSC (human umbilical cord-derived mesenchymal Stem cells)

Taking umbilical cord tissue of sterile perinatal waste, placing the umbilical cord tissue in a sterile blue-covered bottle with an alpha-MEM culture medium, moving the umbilical cord tissue to a super clean bench, cleaning tissue blood with PBS, and separating the umbilical cord tissue; stripping 2 arterial blood vessels and 1 venous blood vessel of the umbilical cord tissue by using autoclave three-tooth forceps, and discarding the stripped blood vessels; cutting umbilical cord into 1cm pieces with sterile surgical scissors, soaking in sterile 70% ethanol for 5min, and placing in sterile culture dish containing PBS buffer solution; peeling amnion from the outer layer of 1cm umbilical cord segment with the above sterile three-tooth forceps, shredding the rest with forceps, and cutting into pieces of 1mm with scissors³Small tissue pieces, these small tissues were sealed in EP tubing with 0.1% collagenase and placed in 37 ℃ CO₂Culturing in an incubator, digesting for 40 min; the digested tissue was placed in a petri dish containing 10% FBS alpha-MEM in complete medium and 3day replaced with fresh complete medium; about 80% of long fusiform adherent cells from the tissue climb out, adding cold PBS buffer solution for washing for 2 times, digesting the cells with 0.25% of pancreatin for adherence, and carrying out passage according to the ratio of 1:3, wherein the cells after passage are the hUC-MSC of the P1 generation.

Example 2 lentivirus infection of hUC-MSC and identification of transfection efficiency

1. Lentivirus infection hUC-MSC

Constructing a lentivirus vector (simultaneously provided with green fluorescent protein and puromycin resistant gene sequences) containing an expression sh-POSTN gene to infect the hUC-MSC (sh-POSTN-MSC), and using the lentivirus containing the expression green fluorescent protein and puromycin resistant gene sequences to infect the hUC-MSC (sh-NC-MSC) as a reference.

The shRNA sequences are shown in Table 1:

TABLE 1shRNA sequences

Gene	Target Seq
		POSTN	AAGCAGAAGATGACCTTTCAT，SEQ ID NO:1
NC	TTCTCCGAACGTGTCACGT，SEQ ID NO:2

The specific method comprises the following steps: culturing P3 generation hUC-MSC at 75cm²After the cell fusion degree is about 60%, the fresh alpha-MEM complete culture medium is replaced, and simultaneously the infection enhancing solution HitransG A and the lentivirus suspension are added, the virus infection index MOI is 10, and the virus suspension volume is (MOI multiplied by the number of viruses/virus titer). After 24h, puromycin (4X 10) is replaced^-6mol/L) of alpha-MEM complete medium, 4d later, replacement of puromycin-containing (2X 10)^-6mol/L) of the complete medium.

2. Characterization of transfection efficiency

When the cell fusion degree reaches about 80%, observing the expression of Green Fluorescent Protein (GFP) after the lentivirus vector infects the hUC-MSC by a fluorescence microscope; the GFP fluorescence expression was measured by flow cytometry.

The method for detecting the expression of the GFP protein by flow cytometry is as follows:

after digestion and centrifugation of the cells (direct centrifugation of the suspended cells), the cells were resuspended in PBS buffer and the total cell count was determined. Removing a plurality of 1.5ml EP tubes, adding cells into a corresponding number of EP tubes according to experimental purpose, wherein the cells in each EP tube are at 5X 10⁵-1×10⁶In the meantime. Centrifuging at 4 deg.C for 10 min at 3000 rpm in a low-temperature high-speed centrifuge. After centrifugation, the supernatant was discarded, and the lower cell pellet was resuspended in 200. mu.l of PBS buffer, and then thoroughly mixed with a pipette and tested on the machine.

A lot of green fluorescence was observed in sh-NC-MSC and sh-POSTN-MSC cells under a fluorescence microscope (FIGS. 1A-F). The detection of GFP protein expression by flow cytometry revealed that virus-free hUC-MSC group was (3.19. + -. 0.9)%, sh-NC-MSC group was (97.5. + -. 2.6)%, and sh-POSTN-MSC group was (97.8. + -. 1.7)% (FIG. 1G).

The experiments show that the lentiviral vector can efficiently infect the hUC-MSC, and the carried gene is integrated into the hUC-MSC genome and is normally expressed.

q-PCR detection of POSTN Gene mRNA level expression

Each group of hUC-MSC was continued with puromycin (1X 10)^-6mol/L) of the total culture medium, collecting partial cells, extracting total RNA, detecting the mRNA level expression condition of POSTN by q-PCR, and amplifying the rest cells until P4 generation for freezing and storing for later use.

The q-PCR expression method for detecting the POSTN gene mRNA level is as follows:

selecting required cDNA samples and primers according to experimental requirements, and preparing a qPCR system in a MicroTM Optical 8-Tube Strip: mu.l of cDNA, 10. mu.l of 2q-PCR Master Mix, 1. mu.l of primers (10. mu.M), 8. mu.l of double distilled water, and 20. mu.l of total volume. The primers are shown in table 2. Detection was performed on a QuantStudio1 PCR instrument after transient centrifugation, procedure: 50 ℃, 2 minutes, 95 ℃, 10 minutes; at 95 deg.C, 5s, 60 deg.C, 1 min, 95 deg.C, 15 s, 40 cycles; 60 ℃, 1 minute, 95 ℃, 30s, 60 ℃, 15 seconds. The data obtained, expressed as x. + -.s, were analyzed using QuantStaudio Design & Analysis software for q-PCR results, SPSS 18.0 software for data, and GraphPad prism7.00 for plots. And (3) analyzing the difference among the groups by adopting a t test, wherein the statistical significance is obtained when the P value is less than 0.05.

TABLE 2 primer sequences

The results show that the mRNA expression level of POSTN in sh-POSTN-MSC is significantly reduced compared with hUC-MSC and sh-NC-MSC (FIG. 1H). Compared with the hUC-MSC, the expression quantity of POSTN gene mRNA in the sh-POSTN-MSC is reduced by 86.3%, and compared with the sh-NC-MSC, the expression quantity of POSTN gene mRNA in the sh-POSTN-MSC is reduced by 86.0%.

The results prove that both the lentivirus with the POSTN knockdown shRNA sequence and the lentivirus of a control group can efficiently infect the hUC-MSC, and can effectively inhibit the expression of the POSTN gene in the hUC-MSC.

Example 3 amplification and characterization of sh-POSTN-MSC with stably knocked-down POSTN

Resuscitating the sh-NC-MSC and sh-POSTN-MSC of P4, adding puromycin (1 × 10)^-6mol/L) of alpha-MEM and subcultured. When the cells are cultured to the P5 generation or the P6 generation, the complete culture medium without puromycin is replaced for culture, and the cell morphology and the phenotype are detected when the cell fusion degree reaches 70 to 80 percent.

1. Giemsa staining detection of cell morphology

When the confluency of the cells is 70-80%, discarding cell supernatant, washing the cells with PBS buffer solution for 2 times, adding 70% ethanol for fixation for 15 minutes, discarding ethanol, washing the cells with PBS buffer solution for 2 times, taking a proper amount of 20 XGiemsa staining solution according to the number of the cells, diluting the staining solution with PBS buffer solution to 1 XGiemsa staining solution, adding the staining solution into a culture container of the cells, and staining for 1 hour. And after dyeing is finished, removing the dyeing solution, washing the cells for 2 times by using PBS buffer solution, adding a proper amount of PBS buffer solution to prevent the sample from being dried, and observing the dyed cells under an inverted microscope.

2. Flow cytometry detection of cell phenotype

After digestion and centrifugation of the cells examined (direct centrifugation of the suspended cells), they were resuspended in PBS buffer and the total cell mass was counted. Several 1.5ml EP tubes were removed and cells were added to the corresponding number of EP tubes for experimental purposes, the cells in each EP tube being between 5X 105 and 1X 106. Centrifuging at 4 deg.C for 10 min at 3000 rpm in a low-temperature high-speed centrifuge. After centrifugation, the supernatant was discarded, and the lower cell pellet was resuspended in 200. mu.l of PBS buffer, at which time the corresponding flow antibody, 1. mu.l of each antibody, was added, the antibodies were thoroughly mixed using a pipette, and incubated for 30 minutes at 4 ℃ in the dark. After that, the cell pellet was obtained by further centrifugation and washed 2 times with PBS buffer, and the temperature, the centrifugation time and the rotation speed of the centrifuge were the same as those described above. Finally, the cells were resuspended in 200. mu.l PBS buffer and tested on the machine. All manipulations were performed in the dark during and after the addition of the flow antibody. And (3) observing the sh-NC-MSC and the sh-POSTN-MSC infected with the lentivirus for 7 days by an inverted microscope, wherein the cells are seen to grow in a fiber-like long fusiform shape and a vortex-shaped adherent manner.

3. Results

Giemsa staining observation (FIGS. 2A-F), the cultured sh-NC-MSC and sh-POSTN-MSC still grow adherent in fiber-like long fusiform and vortex when being transferred to P6; the flow cytometric analysis of the flow phenotypes of sh-NC-MSC and sh-POSTN-MSC showed that (FIG. 2G) the phenotype of sh-POSTN-MSC was not changed.

The results show that the morphology and phenotype of the cells of the sh-NC-MSC and the sh-POSTN-MSC are not obviously changed compared with those of the cells before the lentivirus infection.

Example 4 identification and comparison of differentiation-inducing Capacity of sh-POSTN-MSC in vitro

1. Identification of fat-forming induced differentiation capability of in vitro sh-POSTN-MSC

P3 MSC generation was inoculated to 6-well plate for adipogenic induced differentiation assay.

1.1 Induction of adipogenesis

Number of cells seeded: self-differentiated group 2X 10⁴Well, Induction group 8X 10⁴A hole. The self-differentiated group was cultured in alpha-MEM complete medium, the induced group was cultured in adipogenic induction medium, and the total volume of each group was changed every 3 d. Adipogenic induction medium: in a clean bench, 50ml of α -MEM medium containing 10% FBS was placed in a disposable centrifuge tube, and the types and final concentrations of adipogenic inducer were as follows: dexamethasone 1X 10^-7mol/L, vitamin C phosphate 5X 10^-5mol/L and beta-glycerophosphate 1X 10^-2mol/L. Stored at 4 ℃ and used within one week.

1.2 adipogenic induced differentiation assay

When the cells are cultured for 16 days, the fat cells are subjected to oil red O staining to detect the distribution of fat droplets. Removing the culture medium from cells of the self-differentiation group and the adipogenic induction group in a 6-hole plate, rinsing with PBS (phosphate buffer solution) for 2 times, adding 1ml of 4% neutral formaldehyde buffer solution into each hole, fixing for 20 minutes, rinsing with PBS buffer solution for 2 times, adding 1ml of oil red O staining solution into each hole, staining for 20 minutes in a dark place at room temperature, adding PBS buffer solution, washing for 2 times, adding 1ml of PBS buffer solution into each hole, and observing the distribution condition of red lipid drops under an inverted microscope. And (6) taking a picture.

q-PCR was performed to detect the expression levels of mRNA of peroxisome proliferator-activated receptor (PPAR γ), which is an adipogenic marker, and lipase (ADI) in adipocytes, referring to the q-PCR detection method in example 2, and the primer sequences are shown in Table 3.

As shown in FIGS. 3A-D, compared with the self-differentiated group, the sh-NC-MSC and sh-POSTN-MSC cells showed a large number of lipid droplets after adipogenic induction differentiation, and the mRNA expression levels of adipogenic differentiation key transcription factors ADI and PPAR γ were significantly increased by q-PCR (FIGS. 3E-F, P < 0.001).

2. In vitro sh-POSTN-MSC osteogenic induced differentiation capacity identification

P3 generation MSC was inoculated to 6-well plate for osteogenic induced differentiation assay.

2.1 osteogenic Induction

Number of cells seeded: self-differentiated group 2X 10⁴Well, Induction group 2X 10⁴A hole. The self-differentiation group was cultured in alpha-MEM complete medium, and the induction group was cultured in osteogenic induction medium with a total change of medium every 3 d. Osteogenesis inducing medium 50ml of alpha-MEM medium containing 10% FBS was taken from a clean bench and placed in a disposable centrifuge tube, and the types and final concentrations of osteogenesis inducing agents were as follows: dexamethasone 1X 10^-6mol/L, insulin 1X 10^-5ng/L，IBMX 5×10^-4mol/L and indometacin 5X 10^-4mol/L. Stored at 4 ℃ and used within one week.

2.2 osteogenic induced differentiation assay

When the cells are cultured for 16 days, the osteoblasts are stained by Alkaline phosphatase (ALP) to detect the ALP activity in the cells, the cells of a control group and an osteogenesis inducing group in a 6-well plate are discarded, the culture medium is rinsed 2 times by PBS buffer, 1ml of acetone is added into each well for fixation for 30s, the cells are rinsed 2 times by PBS buffer, 1ml of ALP staining solution is added into each well for dark staining for 30min at room temperature, 1ml of PBS buffer is added into each well for washing 2 times by PBS buffer, and then 1ml of PBS buffer is added into each well to observe the ALP activity under an inverted microscope. And (6) taking a picture.

The relative mRNA expression amounts of Osteopontin (OPN) and ALP, which are osteogenic differentiation markers, were measured by q-PCR, which is referred to the q-PCR measurement method of example 2, and the primer sequences are shown in Table 3.

TABLE 3 primer sequences

Similarly, after the sh-NC-MSC and the sh-POSTN-MSC are subjected to osteogenic induced differentiation, ALP staining is used for detecting the ALP activity of the cells, and the ALP staining area is analyzed by Image J, and the results show that compared with the self-differentiation group, the ALP activities of the sh-NC-MSC and the sh-POSTN-MSC in the induction group are obviously increased (FIGS. 3G-J); in the osteogenesis induction group, the intracellular ALP activity of the sh-NC-MSC is obviously higher than that of the sh-POSTN-MSC (shown in FIGS. 4A-B), and the result of analyzing ALP staining area by Image J shows that the sh-NC-MSC is (59.27 +/-15.1%) and the sh-POSTN-MSC is (26.49 +/-6.75%) (shown in FIG. 4C).

q-PCR detects mRNA expressions of osteogenic differentiation key markers ALP and OPN in the sh-NC-MSC and the sh-POSTN-MSC in the self-differentiation group and the induced differentiation group, and the results show that the mRNA expressions of the osteogenic markers ALP and OPN in the sh-NC-MSC induced group are both obviously increased compared with the sh-NC-MSC self-differentiation group (figure 3K, P is less than 0.001); OPN expression of sh-POSTN-MSC induced group was significantly increased compared to sh-POSTN-MSC self-differentiated group, but ALP expression was not statistically different from self-differentiated group (fig. 3L). Meanwhile, mRNA expression conditions of ALP and OPN in the sh-NC-MSC induction group and the sh-POSTN-MSC induction group are compared, and the results show that compared with the sh-NC-MSC induction group, mRNA expression of ALP and OPN in the sh-POSTN-MSC induction group is remarkably reduced (FIG. 4D).

The results show that the expression of ALP and OPN which are osteogenic differentiation markers can be inhibited after the knocking-down of POSTN. The results prove that the osteogenic differentiation capacity of hUC-MSC can be inhibited after the POSTN gene is knocked down.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> military medical research institute of military science institute of people's liberation force of China

<120> POSTN as marker for regulating and controlling mesenchymal stem cell differentiation capacity and application thereof

<130> P210043

<160> 14

<170> PatentIn version 3.5

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Claims (10)

1. An application of POSTN gene or protein as the marker for regulating the differentiation ability of umbilical cord mesenchymal stem cells.

2. The use of claim 1, wherein the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

3. Use according to claim 2, wherein said differentiation capacity is an osteogenic differentiation capacity and/or an adipogenic differentiation capacity.

4. A preparation for regulating and controlling the differentiation capability of mesenchymal stem cells is characterized in that the preparation takes an inhibitor of POSTN or an accelerant of POSTN as an active ingredient.

5. The formulation according to claim 4, wherein the differentiation capacity is osteogenic differentiation capacity and/or adipogenic differentiation capacity.

6. The preparation of claim 5, wherein the inhibitor or promoter of POSTN is capable of affecting the osteogenic and/or adipogenic differentiation capacity of MSC by knocking down or promoting the expression of POSTN gene or protein, so that the expression of osteogenic and/or adipogenic differentiation markers is significantly changed.

7. The preparation of claim 6, wherein the POSTN promoter is an agent that promotes the expression level of POSTN, and preferably, the promoter is a vector that overexpresses POSTN.

8. The preparation as claimed in claim 6, wherein the POSTN inhibitor comprises POSTN shRNA, preferably, the target sequence of the POSTN shRNA is the nucleotide sequence shown in SEQ ID NO. 1.

9. Use of a preparation of mesenchymal stem cell differentiation capacity according to any one of claims 4-8 for the preparation of a mesenchymal stem cell osteogenic and/or adipogenic induced differentiation product.

10. Use of a preparation of mesenchymal stem cell differentiation capacity according to any one of claims 4-8 for the preparation of a medicament for the treatment of bone defect repair and regeneration and osteoporosis by modulating mesenchymal stem cell osteogenic differentiation.

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