CN113637633B - A method to promote the differentiation of mesenchymal stem cells into osteoblasts - Google Patents

Abstract

The invention provides a method for promoting mesenchymal stem cells to differentiate into osteoblasts, which comprises the following steps: constructing an expression vector of an ALX1 gene, and transferring the expression vector of the ALX1 gene into the mesenchymal stem cells to obtain the mesenchymal stem cells for efficiently expressing exogenous ALX 1; and taking the mesenchymal stem cells for efficiently expressing the exogenous ALX1, and carrying out directional induction culture by using osteoblast induction liquid to obtain the osteoblast. The mesenchymal stem cells which are constructed by the technology and express ALX1 exogenously are in an osteoblast induced differentiation system, so that the need of external cytokines or special equipment in a culture system is eliminated, and the culture cost of the mesenchymal stem cells for directional induced differentiation into osteoblasts is reduced.

Description

A method to promote the differentiation of mesenchymal stem cells into osteoblasts

(1) Technical field

The present invention relates to a method for promoting the differentiation of mesenchymal stem cells into osteoblasts through the ALX1-lncRNA-IGF2 pathway.

(2) Background technology

Human bone tissue contains stem cells with certain differentiation potential. Relevant research has shown that mesenchymal stem cells (MSCs) from bone marrow can differentiate into osteoblasts, chondrocytes, adipocytes, cardiomyocytes, etc., especially the research on the differentiation of mesenchymal stem cells into osteoblasts has become a stem cell-based research. It is an important foundation for bone damage repair and the development of bone tissue engineering technology. It will have great application value in osteoblast transplantation and bone tissue engineering in medicine.

At present, in the technology of directional differentiation of mesenchymal stem cells into osteoblasts, the main methods used are to add relevant cytokines or apply mechanical effects to promote the differentiation of mesenchymal stem cells into osteoblasts. The method of adding cytokines is based on the molecular mechanisms related to bone differentiation and development, and adding bone morphogenetic protein (BMP) and insulin-like growth factor (IGF2) and other cytokines to the in vitro mesenchymal stem cell directed induction differentiation culture medium to regulate bone differentiation. The activity of cell signaling pathways related to development induces osteoblast differentiation. The method of mechanical action is to flow the culture fluid through the adherent mesenchymal stem cells in a specific device (such as a fluid shearing plate or a perfusion device), thereby generating a certain fluid shear force on the mesenchymal stem cells. This fluid shear force acts on the extracellular matrix and receptors of cells, thereby converting extracellular mechanical signals into intracellular chemical signals, and then regulates the activity of cell signaling pathways related to bone cell differentiation and development, ultimately promoting mesenchymal replenishment. Stem cells differentiate into osteoblasts. However, these two technical methods have certain limitations in practical application, especially in clinical applications and preparation of seed cells for bone tissue engineering. The cytokine method not only increases the cost, but also has low bone cell differentiation efficiency. It is not convenient for clinical in vivo application; the fluid shear force method requires special equipment and is not suitable for in vivo bone damage repair treatment.

Therefore, if new key molecular targets related to bone differentiation are found through a large number of gene expression group analyses, and new technologies are used to promote osteoblast differentiation based on the new targets, it will not only open up new avenues for the directed differentiation of stem cells into osteoblasts. The research ideas of bone cells can also greatly reduce the cost of preparing osteoblasts in vitro, improve the efficiency of osteoblast differentiation, and also promote clinical application research on bone damage repair.

(3) Contents of the invention

In order to effectively improve the ability of mesenchymal stem cells to differentiate into osteoblasts, the present invention is based on the discovery of a new bone differentiation-specific long non-coding RNA (lncRNA), namely lncRNAAC132217.4, based on RNA-Seq analysis. The high expression vector of its upstream transcription factor type box 1 (ALX1) promotes the efficient expression of lncRNA AC132217.4 in mesenchymal stem cells and improves the activity of the lncRNA-IGF2 signaling pathway. The purpose is to provide an effective method to promote the directional differentiation of mesenchymal stem cells into osteogenesis. Cell methods.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The invention provides a method for promoting the differentiation of mesenchymal stem cells into osteoblasts. The method is:

(1) Construct an expression vector of the ALX1 gene, and transfer the expression vector of the ALX1 gene into the mesenchymal stem cells to obtain mesenchymal stem cells that efficiently express exogenous ALX1;

(2) Take the mesenchymal stem cells that highly express exogenous ALX1 prepared in step (1) and conduct targeted induction culture with an osteoblast induction solution to obtain the osteoblasts.

The high gene expression technology uses lentiviral overexpression vector (Plenti-CMV-Puro), and the target gene ALX1 amplification primer sequence is: primer CGC GTC GAC ATG GAG TTT CTG AGC GAG A and CAT GGC CCA TGA AATATT GGC TAG CTA GC. The expression vector was used to transfect 293T cells with lentiviral packaging plasmids psPAX2 and PMD2G for viral packaging and then infect third-generation mesenchymal stem cells.

Furthermore, the mesenchymal stem cells can come from one of the following tissues: bone marrow, fat, newborn umbilical cord, placenta, etc. The mesenchymal stem cells used in the present invention are human bone marrow mesenchymal stem cells.

Furthermore, the expression vector of the ALX1 gene is constructed by connecting the ALX1 gene to a lentiviral overexpression vector.

Preferably, the expression vector of the ALX1 gene is prepared as follows: PCR amplification of the cDNA sequence of the human ALX1 gene is performed with primers CGC GTC GAC ATGGAG TTT CTG AGC GAG A and CAT GGC CCA TGA AAT ATT GGC TAG CTA GC to obtain For the target gene, use restriction endonucleases SalI and NheI to digest the lentiviral overexpression vector Plenti-CMV-Puro and the target gene respectively to obtain the target gene and linear vector with sticky ends. Use ligase to cleave the target gene. The target gene with sticky ends is connected to the linear vector to obtain the expression vector of the ALX1 gene (the sequence of the expression vector of the ALX1 gene is shown in SEQ ID NO: 2).

Further, the cDNA sequence of the human ALX1 gene of the present invention is obtained by the following method: using Trizol reagent (Invitrogen, Carlsbad, CA) to isolate and extract total RNA of human bone marrow mesenchymal stem cells, and then using a cDNA first strand synthesis kit (Fermentas Company) performed first-strand cDNA synthesis to obtain the cDNA sequence of the human ALX1 gene.

Preferably, the expression vector of the ALX1 gene is transferred into the mesenchymal stem cells through Lip8000 transduction kit (Beyotime Biotechnology Co., Ltd.).

Specifically, the mesenchymal stem cells that highly express exogenous ALX1 were obtained as follows: the expression vector of the ALX1 gene was transfected into 293T cells with lentiviral packaging plasmids psPAX2 and PMD2G for virus packaging, and then incubated in 1 , the mesenchymal stem cells were continued to be cultured for 12 hours with the assistance of 5-dimethyl-1,5-dizundemethyl bromide (Polybrene, Sigma, St. Louis, MO), and the above results were obtained. Mesenchymal stem cells that highly express exogenous ALX1.

Further, each liter of the osteoblast induction medium consists of the following components: 900 mL DMEM-LG culture medium, 100 mL fetal calf serum, 100 U/ml penicillin, 100 U/ml streptomycin, 10 mM β-glycerophosphate sodium, and 50 μM ascorbic acid.

Further, the conditions for the directional induction culture are: 37°C, 7 days.

Compared with the existing technology, the beneficial effects of the present invention are mainly reflected in:

(1) In the technology provided by the present invention, the long-chain non-coding lncRNA AC132217.4 upstream specific transcription factor ALX1 gene high-expression vector is used to transduce mesenchymal stem cells, which can effectively improve the long-chain expression of mesenchymal stem cells. The expression of non-coding lncRNA AC132217.4 promotes the activation of the osteoblast differentiation-specific IGF2 cell signaling pathway.

(2) The mesenchymal stem cells expressing exogenous ALX1 constructed by the technology of the present invention are used in the osteoblast induction differentiation system, eliminating the need for external cytokines or special equipment in the culture system, and reducing the orientation of mesenchymal stem cells. Cost of culturing osteoblasts induced to differentiate.

(3) Compared with the traditional directional induction and differentiation of osteoblasts, the mesenchymal stem cell directional induction and differentiation into osteoblast culture system produced by the technology of the present invention effectively improves the efficiency of the directional differentiation of mesenchymal stem cells into osteoblasts.

The key to the present invention is to construct an ALX1 expression vector and transfect mesenchymal stem cells based on the verification of a new osteogenic-specific long non-coding lncRNA (lncRNA AC132217.4) and its upstream specific transcription factor ALX1, to prepare a highly efficient expression of ALX1 of mesenchymal stem cells, and promote the ability of mesenchymal stem cells to differentiate into bone cells. The expression vector construction, mesenchymal stem cell transfection, and other parameters in the osteoblast directional differentiation process can all be carried out by conventional means in this field.

(4) Description of drawings

Figure 1. The expression of lncRNA AC132217.4 increases with the increase of osteogenic differentiation: (A) Differentially expressed LncRNAs in the osteogenic differentiation of human bone marrow mesenchymal stem cells. (B-C) AC132217.4 expression increases with osteogenic differentiation. Data analysis is based on mean±SD; N≥3; *p<0.05, **p<0.01, **p<0.001.

Figure 2. Green fluorescent protein analysis of infection efficiency of ALX1 virus

Figure 3. AC132217.4 promotes osteogenic differentiation by regulating the stability of IGF2 gene mRNA: (A) Starbase database checks potential targeting mRNA downstream of lncRNA AC132217.4. (B) Quantitative PCR detects the effect of lncRNAAC132217.4 knockdown on the stability of IGF2 gene mRNA; (C-D) The effect of IGF2 knockdown on osteogenic differentiation-related proteins and genes; (E-F) IGF2 knockdown on osteogenic differentiation Effects of ALP activity and alizarin red; (G-H) Effects of overexpression of IGF2 in cells with lncRNA AC132217.4 knockdown on osteogenic differentiation and differentiation-related proteins and genes; (I-J) This osteogenic differentiation of lncRNA AC132217.4 knockdown cells Effects of overexpression of IGF2 on ALP activity and alizarin red in osteogenic differentiation. ACH: human bone marrow mesenchymal stem cells overexpressing AC132217.4; CTR: human bone marrow mesenchymal stem cells overexpressing empty vector; IGF2shRNA: human bone marrow mesenchymal stem cells knocking down IGF2; CTR shRNA: interfering with empty vector plasmid infection of human bone marrow mesenchymal stem cells. ACH+IGF2 shRNA: Human bone marrow mesenchymal stem cells with AC132217.4 overexpression and IGF2 knockdown. Scale bar: 50um. Data analysis is based on mean±SD; N≥3; *p<0.05, **p<0.01, **p<0.001.

Figure 4. ALX1 affects the transcription level of lncRNA AC132217.4 by binding to its promoter: (A) Differentially expressed transcription factors after 7 days of osteogenic differentiation of human mesenchymal stem cells; (B) PROMO database predicts the onset of lncRNA AC132217.4 The binding site of ALX1 within 3000bp upstream of the start site; (C-D) The effect of ALX1 knockdown on the expression of AC132217.4 and IGF2; (E-F) Construction of AC132217.4 promoter wild-type and mutant reporter genes to verify the relationship between ALX1 and Binding site of AC132217.4 promoter. ALX1 shRNA1: human bone marrow mesenchymal stem cells with knockdown of ALX1 interference sequence 1; ALX1 shRNA2: human bone marrow mesenchymal stem cells with knockdown of ALX1 interference sequence 2; shCTR: human bone marrow mesenchymal stem cells infected with interference empty plasmid; WT : Luciferase reporter gene of wild-type AC132217.4 promoter sequence; MUT: Luciferase reporter gene of AC132217.4 promoter sequence with ALX1 binding site mutation; ALX1H: ALX1 overexpressing human bone marrow mesenchymal stem cells ;CTR: overexpressing empty human bone marrow mesenchymal stem cells. Data analysis is based on mean±SD; N≥3; *p<0.05, **p<0.01, **p<0.001.

Figure 5. ALX1 overexpression promotes the expression of lncRNA AC132217.4 and IGF2: (A) Quantitative PCR examines the effect of ALX1 overexpression on the gene expression levels of lncRNA AC132217.4 and IGF2. (B) Western detection of the effect of ALX1 overexpression on the protein expression levels of ALX1 and IGF2. ALX1H: human bone marrow mesenchymal stem cells overexpressing ALX1; CTR: human bone marrow mesenchymal stem cells overexpressing null. Data analysis is based on mean±SD; N≥3; *p<0.05, **p<0.01, **p<0.001.

Figure 6. Effect of ALX1 overexpression on osteogenic differentiation: (A) Alkaline phosphatase detection of the effect of ALX1 overexpression on osteogenic differentiation. (B) Alizarin red detects the effect of ALX1 overexpression on osteogenic calcification. ALX1H: human bone marrow mesenchymal stem cells overexpressing ALX1; CTR: human bone marrow mesenchymal stem cells overexpressing null. Scale: 50um.

(5) Specific implementation methods

The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

Unless otherwise specified, the following cell culture conditions are: culture in a 37°C, 5% carbon dioxide cell incubator.

Example 1: Gene expression profiling analysis

Collect the third generation human bone marrow mesenchymal stem cells, prepare them into single cell suspension, and inoculate them into a 30 mm diameter culture dish at 2×10 ⁴ cells/culture dish for adherent culture in α-MEM medium until the cells reach a layer of 80 ~90%, use osteoblast induction medium (DMEM-LG culture medium 900mL, add 100mL fetal bovine serum, 100U/ml penicillin, 100U/ml streptomycin, 10mM β-glycerophosphate sodium, 50μM ascorbic acid) for osteoblast induction Directional induction.

After 7 days of directional induction, take 5 × 10 ⁵ cells each of cells after induction of differentiation and cells without induction of differentiation, and add 1 mL of Trizol lysis solution for cell lysis. Add 1/5 volume of chloroform to the cell lysis solution. , after standing at room temperature for 5 minutes, centrifuge at 13000 rpm and 4°C for 15 minutes, take the uppermost water phase, add 5/3 volume of isopropyl alcohol, let stand at room temperature for 10 minutes, centrifuge at 13000 rpm and 4°C for 10 minutes, remove the supernatant, and add 1 mL 75% ethanol, then centrifuge at 7500 rpm for 5 minutes at 4°C, remove the supernatant, vacuum remove the remaining 75% ethanol, and dissolve it in DEPC water (provided by the tester). After the total RNA concentration is determined, if the concentration exceeds 100ng/μl and the total amount exceeds 1μg, it will be sent to the detection party for RNA-Seq test analysis.

RNA-Seq analysis data are stored in the NCBI gene expression database (accessible through GEO series accession number GSE114117) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). The analysis found that among all genes whose expression levels changed, the expression level of long non-coding lncRNA AC132217.4 changed extremely significantly (Figure 1A, B), which was confirmed by qRT-PCR analysis (Figure 1C).

Figure 1: (A) Differentially expressed LncRNAs in osteogenic differentiation of human bone marrow mesenchymal stem cells. (B-C) AC132217.4 expression increases with osteogenic differentiation. Data analysis is based on mean±SD; N≥3; *p<0.05, **p<0.01, **p<0.001.

Example 2: Construction of lncRNA AC132217.4 knockdown vector

The lncRNA AC132217.4 knockdown vector was purchased from Jima Gene Company and constructed using the pGPU6/GFP/Neo vector. The vector was constructed using homologous recombination. The short hairpin RNA (shRNA) lentiviral vector is constructed as shown in SEQ ID NO: 1.

Example 3: Transfection of mesenchymal stem cells with short hairpin RNA (shRNA) lentiviral vector of lncRNA AC132217.4

Lip8000 transduction kit (Beyotime Biotechnology Co., Ltd.) was used to prepare the recombinant viral vector suspension and transduce mesenchymal stem cells. According to the instructions of the kit, the facultative packaging cell line 293T cells (ATCC, Rockefeller, MA) were used to prepare the recombinant viral vector suspension. With the assistance of 8 μg/mL of 1,5-dimethyl-1,5-diazoundecanepolymethyl bromide (Polybrene; Sigma, St. Louis, MO), in a 37°C cell culture incubator, 2.0 × 10 ⁵ mesenchymal stem cells were transduced with the recombinant viral vector in 10-cm culture dishes (Nunc Corp.) for 8 hours. Cell transduction efficiency was analyzed using enhanced green fluorescent protein (GFP) (Figure 2). The results showed that the transduced shRNA could be expressed in mesenchymal stem cells for a long time.

Example 4: Specific analysis of osteoblast differentiation by lncRNA AC132217.4

Checking the potential targeting mRNA downstream of lncRNA AC132217.4 through the Starbase database, it was found that the mRNA of insulin-like growth factor IGF2 and lncRNA AC132217.4 have the highest silign score (Figure 3A), indicating that the mRNA of the IGF2 gene may be It is the targeting mRNA downstream of lncRNA AC132217.4. :

In order to verify that the mRNA of the IGF2 gene is the targeting mRNA downstream of lncRNA AC132217.4, mesenchymal stem cells transfected with the short hairpin RNA (shRNA) lentiviral vector of lncRNA AC132217.4 were taken and cultured using osteoblast induction medium (DMEM). - 900mL of LG culture medium, add 100mL fetal bovine serum, 100U/ml penicillin, 100U/ml streptomycin, 10mM β-glycerophosphate sodium, 50μM ascorbic acid) and culture for 7 days, then treat with actinomycin D to block the synthesis of mRNA, This was used to examine the stability of IGF2 gene mRNA after knockdown of lncRNAAC132217.4. The results showed that the stability of IGF2 gene mRNA was significantly reduced after knockdown of lncRNA AC132217.4 (Figure 3B).

Through real-time quantitative PCR (Real-time PCR) technology (the reaction system of real-time quantitative PCR is: 1μl cDNA + 1μl primer + 8μl ultrapure water + 10μl 2x SYRB quantitative PCR enzyme mix; the reaction conditions are: 95°C for 5 minutes after pre-denaturation 40 cycles, each cycle 95℃10s, 60℃40s) Analysis of osteoblast-specific alkaline phosphatase (ALP), collagen 1 (COL1A1) and Runt-related transcription factor 2 (RUNX2) gene messenger RNA (mRNA) expression level. The amplification primers for ALP, COL1A1 and RUNX2 are shown in the table below:

Western blot technology was used to analyze COL1A1 and osteocalcin (OCN) activities, alkaline phosphatase activity detection technology and alizarin red staining technology were used to analyze ALP activity and cell mineralization nodules, and lncRNA AC132217.4 knockdown was used to analyze the The osteoblast-directed differentiation ability of mesenchymal stem cells is shown in Figure 3C-J, which shows that knockdown of lncRNA AC132217.4 leads to a significant decrease in the expression levels of osteoblast-specific genes and transcription factors of mesenchymal stem cells. The ability of mesenchymal stem cells to differentiate into osteoblasts was restored after overexpression of IGF2 in osteoblasts, indicating that lncRNA AC132217.4 improved the stability of IGF2 gene mRNA.

In Figure 3: (A) Starbase database checks potential target mRNA downstream of lncRNA AC132217.4. (B) Quantitative PCR detects the effect of lncRNA AC132217.4 knockdown on the stability of IGF2 gene mRNA; (C-D) The effect of IGF2 knockdown on osteogenic differentiation-related proteins and genes. (E-F) Effects of IGF2 knockdown on ALP activity and alizarin red on osteogenic differentiation. (G-H) Effects of overexpression of IGF2 in lncRNA AC132217.4 knockdown cells on osteogenic differentiation-related proteins and genes. (I-J) Effects of overexpression of IGF2 on osteogenic differentiation ALP activity and alizarin red in cells knockdown of this osteogenic differentiation lncRNA AC132217.4. ACH: Human bone marrow mesenchymal stem cells overexpressing AC132217.4; CTR: Human bone marrow mesenchymal stem cells overexpressing the empty vector; IGF2 shRNA: Human bone marrow mesenchymal stem cells knocking down IGF2; CTR shRNA: Interfering empty plasmid Infected human bone marrow mesenchymal stem cells. ACH+IGF2 shRNA: Human bone marrow mesenchymal stem cells with AC132217.4 overexpression and IGF2 knockdown. Scale bar: 50um. Data analysis is based on mean±SD; N≥3; *p<0.05, **p<0.01, **p<0.001.

Example 5: Analysis of lncRNA AC132217.4 upstream specific transcription factor ALX1

In order to increase the transcriptional expression level of lncRNA AC132217.4 during the directional differentiation of mesenchymal osteoblasts, we looked for potential specific transcription factors upstream of AC132217.4 based on the RNA-Seq technical analysis results in Example 1, and found that there are 12 17 up-regulated and 17 down-regulated transcription factors (Figure 4A). We then used the PROMO database to analyze the transcription factor binding sites in the promoter region of lncRNA AC132217.4 (the genomic region about 3000 bp upstream of lncRNA AC132217.4) and found 8 high-level binding sites for ALX1 (Figure 4B), indicating that ALX1 may It is a transcription factor that specifically regulates the expression of lncRNAAC132217.4.

To verify the above speculation, two independent ALX1 shRNAs were used to reduce the expression of ALX1. ALX1 shRNA interference plasmid was constructed based on pRNAi-U6.2/Lenti Vector empty vector. The ALX1 shRNA primer sequence was synthesized by BGI. Both ends of the synthesized upstream and downstream primers are phosphorylated by phosphorylase and then annealed to form a double-stranded structure (the phosphorylation and annealing system is: 1ul upstream primer + 1ul downstream primer) + 1ul of T4 PNK enzyme + 7ul of ultrapure water, the program is 37℃ for 30min, 95℃ for 5min, then lowered to 25℃ at a rate of 5℃/min), and then digest the vector and ALX1 shRNA sequence by HpaI and XhoI Make a connection. The ALX1 shRNA primer sequence is as follows:

The results showed that compared with cells without ALX1 knockdown, the expression level of lncRNA AC132217.4 in ALX1 knockdown cells was significantly reduced (Figure 4C-D). The luciferase reporter assay method verified that ALX1 can bind to the lncRNA AC132217.4 gene. Startup zone (Figure 4E-F).

In Figure 4: (A) Differentially expressed transcription factors after 7 days of osteogenic differentiation of human mesenchymal stem cells; (B) PROMO database predicts the binding site of ALX1 within 3000 bp upstream of the start site of lncRNA AC132217.4; (C-D) Effect of ALX1 knockdown on the expression of AC132217.4 and IGF2; (E-F) Construct wild-type and mutant AC132217.4 promoter reporter genes to verify the binding site of ALX1 and AC132217.4 promoter. ALX1 shRNA1: human bone marrow mesenchymal stem cells with knockdown of ALX1 interference sequence 1; ALX1 shRNA2: human bone marrow mesenchymal stem cells with knockdown of ALX1 interference sequence 2; shCTR: human bone marrow mesenchymal stem cells infected with interference empty plasmid; WT : Luciferase reporter gene of wild-type AC132217.4 promoter sequence; MUT: Luciferase reporter gene of AC132217.4 promoter sequence with ALX1 binding site mutation; ALX1H: ALX1 overexpressing human bone marrow mesenchymal stem cells ;CTR: overexpressing empty human bone marrow mesenchymal stem cells. Data analysis is based on mean±SD; N≥3; *p<0.05, **p<0.01, **p<0.001.

Example 6: Construction of ALX1 high expression vector and transfection of mesenchymal stem cells

The technology of the present invention uses Trizol reagent (Invitrogen, Carlsbad, CA) to isolate and extract total RNA of human bone marrow mesenchymal stem cells, and then uses a cDNA first strand synthesis kit (Fermentas Company) to synthesize the first strand of cDNA, and then use the first strand of cDNA to Use it as a PCR amplification template to amplify the ALX1 gene sequence. The amplification primer sequences of the ALX1 gene are: CGC GTCGAC ATG GAG TTT CTG AGC GAG A and CAT GGC CCA TGA AAT ATT GGC TAG CTA GC. The total volume of the ALX1 PCR reaction was 25 μl, and 30 cycles were performed after pre-denaturation at 95°C for 5 minutes. Each cycle included 94°C for 60 seconds, 55°C for 45 seconds, and 72°C for 60 seconds. The length of the amplified fragment is 981bp.

The PCR reaction system (20 μl) is: 1 μl cDNA + 1 μl primer + 8 μl ultrapure water + 10 μl 2x PCR enzyme mix.

The PCR amplified fragment of ALX1 was connected to the lentiviral overexpression vector (Plenti-CMV-Puro) (Jinan Weizhen Biological Co., Ltd.) (Jinan Weizhen Biological Co., Ltd.) through SalI and NheI (TaKaRa Company) restriction sites and ligase (TaKaRa Company). The ALX1 lentiviral overexpression vector is shown in SEQ ID NO: 2.

The constructed ALX1 lentiviral overexpression vector was used to prepare the recombinant viral vector suspension and transduce mesenchymal stem cells using the Lip8000 transduction kit (Beyotime Biotechnology Co., Ltd.). According to the instructions of the kit, the packaging cell line 293T (ATCC, Rockefeller, MA) was used to prepare the recombinant viral vector suspension. (Virus packaging scheme: Mix 15ug of total plasmid (ALX1 overexpression plasmid: psPAX2:PMD2G=4:3:1) with 750ul of Opti-MEM medium, then add 24ul of Lip8000 transfection reagent (Beyotime Biotechnology Co., Ltd. Company), mix well, add the transfection solution to 293T cells in a 10cm dish with a density of 70-90%, replace the fresh 293T cell culture medium (DMEM high sugar culture medium + 10% FBS) for 6 hours, and collect the supernatant after 72 hours. The supernatant is the virus stock solution), with the assistance of 8 μg/mL 1,5-dimethyl-1,5-diaz undecanemethylene polymethyl bromide (Polybrene; Sigma, St. Louis, MO), 2.0 × 10 ⁵ mesenchymal stem cells were transduced with the recombinant viral vector in a 6-cm culture dish (Nunc Corporation) for 12 hours. Western blotting verified its overexpression efficiency, and the results showed that the transduced overexpression plasmid could be expressed in mesenchymal stem cells for a long time.

Quantitative PCR detection and analysis of the gene expression levels of lncRNA AC132217.4 and IGF2 in mesenchymal stem cells infected with ALX1 overexpression plasmid (Figure 5A-B). The protein expression level of IGF2 was detected by western and Elisa, and the results showed that the overexpression of ALX1 Promoted the expression levels of AC132217.4 and IGF2.

In Figure 5: (A) Quantitative PCR examines the effect of ALX1 overexpression on the gene expression levels of lncRNA AC132217.4 and IGF2. (B) Western detection of the effect of ALX1 overexpression on the protein expression levels of ALX1 and IGF2. ALX1H: human bone marrow mesenchymal stem cells overexpressing ALX1; CTR: human bone marrow mesenchymal stem cells overexpressing null. Data analysis is based on mean±SD; N≥3; *p<0.05, **p<0.01, **p<0.001.

Example 7: Analysis of the ability of transduction of ALX1 high expression vector to promote directional differentiation of mesenchymal stem cells into osteoblasts

The mesenchymal stem cells that have not been transfected with the ALX1 high expression vector and the mesenchymal stem cells that have been transfected with the ALX1 high expression vector are digested into a single cell suspension and seeded in a 16-well culture plate at a density of 3×10 ⁴ cells/ml. , after the cells reach 80%-90% confluence, add 900mL of osteoblast induction medium (DMEM-LG culture medium, add 100mL fetal bovine serum, 100U/ml penicillin, 100U/ml streptomycin, 0.1μmol dexamethasone , 10mM β-sodium glycerophosphate, 50μM ascorbic acid) for targeted induction of osteoblasts for 7 days. Change the medium every 2 days.

After terminating the induction, alkaline phosphatase staining was performed to observe the differentiation effect: the resulting cells were washed three times with PBS (pH 7.2) for 1 minute each time, and fixed with 1% paraformaldehyde containing 7.5% sucrose at room temperature for 10 to 20 minutes. Then use an alkaline phosphatase kit for staining. According to the manufacturer's instructions, first balance with substrate buffer and stain with freshly prepared 5-bromo-4-chloro-3-indophosphonate/nitroblue tetrazolium (BCIP/NBT). Dye in the liquid at room temperature and protected from light for more than 30 minutes, then wash with water. Observe differentiated cells under a 100x inverted microscope as shown in Figure 6A.

After termination of induction, Alizarin Red S staining was used to observe the differentiation effect: Wash the differentiated cells 3 times with PBS (pH 7.2), 1 minute each time, fix the specimen with 4% paraformaldehyde, and incubate with 2% Alizarin Red S staining solution at 37°C for 5 minutes, rinse with distilled water and observe (Figure 6B).

In Figure 6: (A) Alkaline phosphatase detects the effect of ALX1 overexpression on osteogenic differentiation. (B) Alizarin red detects the effect of ALX1 overexpression on osteogenic calcification. ALX1H: human bone marrow mesenchymal stem cells overexpressing ALX1; CTR: human bone marrow mesenchymal stem cells overexpressing null. Scale: 50um.

To sum up:

(1) The technology of the present invention effectively improves the ability of mesenchymal stem cells to differentiate into osteoblasts:

The ALX1 high expression vector was constructed and transfected into mesenchymal stem cells, which can effectively increase the expression level of long non-coding lncRNA AC132217.4 in the cells, thereby enhancing the stability of IGF2 gene mRNA, thus improving the IGF2 signaling pathway. The activity significantly promoted the efficiency of directional differentiation of mesenchymal stem cells into osteoblasts. Compared with mesenchymal stem cells that were not transfected with the ALX1 high expression vector, the directional differentiation efficiency increased by 68.2%.

(2) The technology of the present invention has the advantage of reducing the experimental cost of directional induction and differentiation of mesenchymal stem cells into osteoblasts:

Inducing differentiation and preparing osteoblasts through this technology can eliminate the need to add related cytokines and special equipment, effectively reducing the high costs caused by expensive cytokines and special equipment.

(3) The technology of the present invention can be applied to clinical bone damage repair treatment and research on bone tissue engineering technology:

The present invention provides a method for directional induction and differentiation of mesenchymal stem cells into osteoblasts with high osteoblast differentiation efficiency, which can provide sufficient seed cells for bone tissue engineering research and application. At the same time, the high levels of ALX1 and lncRNAAC132217.4 Expression technology can be applied to clinical bone damage repair and gene therapy research in patients with nonunion.

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Hainan Disen Biotechnology Co., Ltd.

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aagcgtctgc aggcaaatgc gtgcaggcct tcggacccct gccccgcgcc gagcatcacg 360

tgcgcttgga gaggacctcg ccctgtcagg acagcagcgt gaactatggg atcactaaag 420

tagaaggaca gccccttcac accgaactga atagagctat ggacaactgt aacagtctcc 480

gaatgtctcc cgtgaaaggg atgcaagaga agggagagct ggatgaactt ggggataaat 540

gtgatagcaa tgtatccagc agtaagaaac ggaggcaccg aaccaccttc accagtttgc 600

agctagagga gctggagaaa gtctttcaga aaactcatta cccggatgtg tatgtcagag 660

aacagcttgc tctgaggaca gagctcactg aggccagggt ccaggtttgg tttcaaaatc 720

gaagggccaa atggagaaaa agggaacgtt atggccaaat acaacaagcg aaaagccatt 780

ttgctgccac ctatgatata tcagttttgc caaggactga cagctaccca cagattcaga 840

acaatttgtg ggcaggaaat gcaagtggtg gttctgtggt tacttcatgc atgt 894

Claims (8)

1. A method for promoting differentiation of mesenchymal stem cells into osteoblasts, the method comprising:

(1) Constructing an expression vector of a human ALX1 gene, and transferring the expression vector of the human ALX1 gene into the mesenchymal stem cells to obtain the mesenchymal stem cells for efficiently expressing exogenous ALX 1; the mesenchymal stem cells are human bone marrow mesenchymal stem cells;

(2) And (3) taking the mesenchymal stem cells which are prepared in the step (1) and efficiently express exogenous ALX1, and carrying out directional induction culture by using osteoblast induction liquid to obtain the osteoblast.

2. The method for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 1, wherein: the expression vector of the human ALX1 gene is constructed by connecting the human ALX1 gene to a lentivirus over-expression vector.

3. The method for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 2, wherein the expression vector of human ALX1 gene is prepared as follows: the cDNA sequence of the human ALX1 gene is amplified by PCR through primers CGC GTC GAC ATG GAG TTT CTG AGC GAG A and CAT GGC CCA TGA AAT ATT GGC TAG CTA GC to obtain a target gene, the lentivirus overexpression vector Plenti-CMV-Puro and the target gene are respectively subjected to enzyme digestion by restriction enzymes SalI and NheI to obtain a target gene with an adhesive end and a linear vector, and the target gene with the adhesive end and the linear vector are connected by using a ligase to obtain the expression vector of the human ALX1 gene.

4. The method for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 3, wherein the cDNA sequence of the human ALX1 gene is obtained by the following method: and (3) separating and extracting total RNA of the human bone marrow mesenchymal stem cells by adopting a Trizol reagent, and then adopting a cDNA first-strand synthesis kit to carry out cDNA first-strand synthesis to obtain the cDNA sequence of the human ALX1 gene.

5. The method for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 1, wherein: the expression vector of the human ALX1 gene is transferred into the mesenchymal stem cells through a Lip8000 transduction kit.

6. The method for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 5, wherein the mesenchymal stem cells highly expressing exogenous ALX1 are obtained by: after the expression vector of the human ALX1 gene is transfected into 293T cells through lentiviral packaging plasmid psPAX2 and PMD2G for virus packaging, the mesenchymal stem cells are continuously cultured for 12 hours under the assistance of 8 mug/mL of 1, 5-dimethyl-1, 5-diazaundecene polymethyl bromide, so that the mesenchymal stem cells for efficiently expressing exogenous ALX1 are obtained.

7. The method for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 1, wherein each liter of the osteoblast inducing liquid consists of the following components: 900mL DMEM-LG broth, 100mL fetal calf serum, 100U/mL penicillin, 100U/mL streptomycin, 10mM sodium beta-glycerophosphate, 50. Mu.M ascorbic acid.

8. The method for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 1, wherein the conditions of the directional induction culture are as follows: 37℃for 7 days.