CN113637633B - Method for promoting differentiation of mesenchymal stem cells to osteoblasts - Google Patents
Method for promoting differentiation of mesenchymal stem cells to osteoblasts Download PDFInfo
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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
Field of the art
The present invention relates to a method for promoting differentiation of mesenchymal stem cells into osteoblasts through the ALX1-lncRNA-IGF2 pathway.
(II) background art
Human bone tissue has stem cells with certain differentiation potential. Related researches have shown that Mesenchymal Stem Cells (MSCs) from bone marrow can differentiate into osteoblasts, chondrocytes, adipocytes, cardiac myocytes and the like, and especially the research on the differentiation of mesenchymal stem cells into osteoblasts becomes an important foundation for the repair of bone injury based on stem cells and the development of bone tissue engineering technology, and has great application value in medicine for osteoblast transplantation and bone tissue engineering.
In the aspect of the directional differentiation of mesenchymal stem cells into osteoblasts at present, the main adoption is to add relevant cytokines or to apply mechanical action to promote the differentiation of mesenchymal stem cells into osteoblasts. The method for adding the cytokines is to add the cytokines such as Bone Morphogenetic Protein (BMP) and insulin-like growth factor (IGF 2) into the in vitro mesenchymal stem cell directional induction differentiation culture solution according to the molecular mechanism related to bone differentiation and development to regulate the activities of cell signal paths related to bone differentiation and development so as to perform osteoblast differentiation induction. In the mechanical action method, culture solution flows through mesenchymal stem cells attached to the wall in a specific device (such as a fluid shear plate or a perfusion device), so that a certain fluid shear force is generated on the mesenchymal stem cells, the fluid shear force acts on extracellular matrixes and receptors of the cells, so that extracellular mechanical signals are converted into intracellular chemical signals, and then the differentiation of the mesenchymal stem cells into osteoblasts is finally promoted by regulating the activity of cell signal paths related to the differentiation and development of the osteoblasts. However, these two techniques have certain limitations in practical application, especially in clinical application and preparation of bone tissue engineering seed cells, which are inconvenient: the cytokine method not only increases the cost, but also has lower induced differentiation efficiency of bone cells, and is not convenient for clinical in vivo application; the fluid shear method requires special equipment and is also not suitable for in vivo bone injury repair treatment.
Therefore, if a large number of genome analysis is adopted to find out a new key molecular target spot related to bone differentiation, and a new technology is adopted to promote the differentiation of osteoblasts aiming at the new target spot, a new research thought for the directional differentiation of stem cells into osteoblasts can be developed, meanwhile, the preparation cost of osteoblasts in vitro can be greatly reduced, the differentiation efficiency of osteoblasts is improved, and the clinical application research on the repair of bone injury can be promoted.
(III) summary of the invention
In order to effectively improve the capacity of the mesenchymal stem cells for directional differentiation into the bone cells, the invention discovers a novel long-chain non-coding RNA (lncRNA) specifically related to bone differentiation based on RNA-Seq analysis, namely lncRNA AC132217.4, constructs a high expression vector of an upstream transcription factor type box 1 (ALX 1), promotes the mesenchymal stem cells to efficiently express lncRNA AC132217.4, improves the activity of a lncRNA-IGF2 signal pathway, and aims to provide a method for effectively promoting the mesenchymal stem cells for directional differentiation into the bone cells.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a method for promoting mesenchymal stem cells to differentiate into osteoblasts, which comprises the following steps:
(1) 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;
(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.
The gene high expression technology adopts a lentivirus over-expression vector (Planti-CMV-Puro), and the target gene ALX1 amplification primer sequence is as follows: primers CGC GTC GAC ATG GAG TTT CTG AGC GAG A and CAT GGC CCA TGA AAT ATT GGC TAG CTA GC. The expression vector is transfected into 293T cells through lentiviral packaging plasmids PSPAX2 and PMD2G to carry out virus packaging, and then the 3 rd generation mesenchymal stem cells are infected.
Further, the mesenchymal stem cells may be derived from one of the following tissues: bone marrow, fat, neonatal umbilical cord, placenta, etc. The mesenchymal stem cells used in the invention are human bone marrow mesenchymal stem cells.
Further, the expression vector of the ALX1 gene is constructed by ligating the ALX1 gene to a lentiviral overexpression vector.
Preferably, the expression vector of the 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 lentiviral 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 a sticky end and a linear vector, and the target gene with the sticky end and the linear vector are connected by a ligase to obtain the expression vector of the ALX1 gene (the sequence of the expression vector of the ALX1 gene is shown as SEQ ID NO: 2).
Further, the cDNA sequence of the human ALX1 gene of the present invention is obtained by the following method: and (3) separating and extracting total RNA of the human bone marrow mesenchymal stem cells by using Trizol reagent (Invitrogen, carlsbad, calif.), and then carrying out first-strand cDNA synthesis by using a first-strand cDNA synthesis kit (Fermentas Co.) 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 by means of Lip8000 transduction kit (peyunnan biology ltd).
Specifically, the mesenchymal stem cells for efficiently expressing exogenous ALX1 are obtained by the following method: after the expression vector of the ALX1 gene is subjected to virus packaging through a slow virus packaging plasmid psPAX2 and a PMD2G transfected 293T cell, the mesenchymal stem cell for continuously culturing for 12 hours under the assistance of 8 mug/mL of 1, 5-dimethyl-1, 5-diazaundecene polymethylbromide (Polybrene, sigma, st. Louis, MO) to obtain the mesenchymal stem cell for efficiently expressing exogenous ALX 1.
Further, each liter of the osteoblast induction 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.
Further, the conditions of the directional induction culture are as follows: 37℃for 7 days.
Compared with the prior art, the invention has the beneficial effects that:
(1) In the technology provided by the invention, the mesenchymal stem cells are transduced by adopting the long-chain non-coding lncRNA AC132217.4 upstream specific transcription factor ALX1 gene high expression vector, so that the expression of the long-chain non-coding lncRNA AC132217.4 in the mesenchymal stem cells can be effectively improved, and the activation of the IGF2 cell signal path with specific osteoblast differentiation is promoted.
(2) 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.
(3) Compared with the traditional osteoblast directional induction differentiation, the mesenchymal stem cell directional induction differentiation osteoblast culture system produced by the technology of the invention effectively improves the efficiency of the mesenchymal stem cell directional differentiation osteoblast.
The key point of the invention is to carry out ALX1 expression vector construction and mesenchymal stem cell transfection according to the verification of novel bone formation specific long-chain non-coding lncRNA (lncRNA AC 132217.4) and upstream specific transcription factor ALX1 thereof, prepare the mesenchymal stem cells with high-efficiency expression ALX1 and promote the directional differentiation of the mesenchymal stem cells into bone cells. Expression vector construction, mesenchymal stem cell transfection, and other parameters during the process of osteoblast directional differentiation can be performed by conventional means in the art.
(IV) description of the drawings
FIG. 1.lncRNA AC132217.4 shows that the expression level increases with increasing osteoblastic differentiation (A) LncRNAs differentially expressed in osteoblastic differentiation of human bone marrow mesenchymal stem cells. (B-C) AC132217.4 increased expression with osteogenic differentiation. Data analysis was performed according to mean±sd; n is more than or equal to 3; * p <0.05, < p <0.01, < p <0.001.
FIG. 2 analysis of infection efficiency of ALX1 Virus by Green fluorescent protein
FIG. 3. AC132117.4 promotes osteogenic differentiation by modulating the stability of IGF2 gene mRNA (A) Starnase database examines potentially targeted mRNA downstream of lncRNA AC 132217.4. (B) Quantitative PCR (polymerase chain reaction) detection of the influence of lncRNAAC132117.4 knockdown on the stability of IGF2 gene mRNA; (C-D) effects of IGF2 knockdown on osteogenic differentiation-related proteins and genes; (E-F) effect of IGF2 knockdown on osteogenic differentiation ALP activity and alizarin red; (G-H) effects of overexpression of IGF2 on osteogenic differentiation-related proteins and genes in lncRNA AC132217.4 knockdown cells; (I-J) the effect of overexpression of IGF2 on osteogenic differentiation ALP activity and alizarin red in cells knockdown by this osteogenic differentiation lncRNA AC 132217.4. ACH: human bone marrow mesenchymal stem cells overexpressed by AC 132217.4; CTR: overexpressing empty human bone marrow mesenchymal stem cells; IGF2 shRNA, IGF2 knockdown human bone marrow mesenchymal stem cells; CTR shRNA: human bone marrow mesenchymal stem cells infected with empty plasmid are disturbed. Ach+igf2 shRNA: human bone marrow mesenchymal stem cells with IGF2 knockdown while AC132217.4 is overexpressed. The scale is 50um, data analysis is according to mean+ -SD; n is more than or equal to 3; * p <0.05, < p <0.01, < p <0.001.
FIG. 4.ALX1 affects its transcription level by binding at the promoter of lncRNA AC132217.4 (A) transcription factors differentially expressed 7 days after osteoblast differentiation of human mesenchymal stem cells; (B) The PROMO database predicts the binding site of ALX1 within 3000bp upstream of the start site of lncRNA AC 132217.4; (C-D) effect of ALX1 knockdown on AC132217.4 and IGF2 expression; (E-F) construction of a reporter gene for the wild type and mutation of the AC132217.4 promoter, verifying the binding site of ALX1 to the AC132217.4 promoter. ALX1 shRNA1 is human bone marrow mesenchymal stem cells with ALX1 interference sequence 1 knocked down; ALX1 shRNA2, human bone marrow mesenchymal stem cells with ALX1 interference sequence 2 knocked down; shCTR: human bone marrow mesenchymal stem cells that interfere with empty plasmid infection; WT: a luciferase reporter gene of wild type AC132217.4 promoter sequence; MUT: a luciferase reporter gene of the ALX1 binding site mutated AC132217.4 promoter sequence; ALX1H: ALX1 overexpressing human bone marrow mesenchymal stem cells; CTR: overexpressing empty human bone marrow mesenchymal stem cells. Data analysis was performed according to mean±sd; n is more than or equal to 3; * p <0.05, < p <0.01, < p <0.001.
FIG. 5 ALX1 overexpression promotes expression of lncRNA AC132217.4 and IGF2 (A) quantitative PCR examined the effect of ALX1 overexpression on gene expression levels of lncRNA AC132217.4 and IGF 2. (B) Western detection of the effect of ALX1 overexpression on protein expression levels of ALX1 and IGF 2. ALX1H: ALX1 overexpressing human bone marrow mesenchymal stem cells; CTR: overexpressing empty human bone marrow mesenchymal stem cells. Data analysis was performed according to mean±sd; n is more than or equal to 3; * p <0.05, < p <0.01, < p <0.001.
FIG. 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: ALX1 overexpressing human bone marrow mesenchymal stem cells; CTR: overexpressing empty human bone marrow mesenchymal stem cells. The scale is 50um.
(fifth) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
the following cell cultures were cultured under the following conditions except for the specific descriptions: the culture was performed at 37℃in a 5% carbon dioxide cell incubator.
Example 1: analysis of Gene expression profiling
Collecting 3 rd generation human bone marrow mesenchymal stem cells, preparing into single cell suspension, and mixing with 2×10 4 The cells were inoculated onto a culture dish having a diameter of 30mM and subjected to an adherent culture in an alpha-MEM medium, and when the cells reached 80 to 90% of the confluence, the cells were induced in a specific manner by using an osteoblast induction solution (DMEM-LG culture solution, 900mL, 100mL of fetal bovine serum, 100U/mL of penicillin, 100U/mL of streptomycin, 10mM of beta-sodium glycerophosphate, 50. Mu.M of ascorbic acid) and the cells were subjected to a specific induction.
After 7 days of directional induction, cells after differentiation induction and cells without induced differentiation were taken 5X 10 each 5 Cell lysis was performed by adding 1mL of Trizol lysate, adding 1/5 volume of chloroform to the cell lysate, standing at room temperature for 5min, centrifuging at 13000rpm at 4℃for 15 min, collecting the uppermost aqueous phase, adding 5/3 volume of isopropanol, standing at room temperature for 10min, centrifuging at 13000rpm at 4℃for 10min, removing the supernatant, adding 1mL of 75% ethanol, centrifuging at 7500rpm at 4℃for 5min, removing the supernatant, removing 75% ethanol in vacuo, and dissolving with DEPC water (supplied by the detection side). The total concentration of RNA is measured to be more than 100 ng/. Mu.l, and the total concentration is more than 1. Mu.g, and then the RNA is sent to a detection party for RNA-Seq test analysis.
RNA-Seq analysis data are stored in NCBI gene expression database (accessible via GEO series accession number GSE 114117) (https:// www.ncbi.nlm.nih.gov/GEO/query/acc. Cgi). Analysis found that the long-chain non-coding lncRNAAC132117.4 expression level was very variable among all genes with changes in expression level (FIGS. 1A, B) and was confirmed by qRT-PCR analysis (FIG. 1C).
Wherein fig. 1: (A) LncRNAs differentially expressed in osteoblastic differentiation of human bone marrow mesenchymal stem cells. (B-C) AC132217.4 increased expression with osteogenic differentiation. Data analysis was performed according to mean±sd; n is more than or equal to 3; * p <0.05, < p <0.01, < p <0.001.
Example 2: construction of lncRNA AC132217.4 knock-down vector
The lncRNA AC132217.4 knock-down vector is purchased from Ji Ma gene company, constructed by pGPU6/GFP/Neo vector, and constructed by homologous recombination. The short hairpin RNA (shRNA) lentiviral vector is constructed as shown in SEQ ID NO: 1.
Example 3: short hairpin RNA (shRNA) lentiviral vector transfection of lncRNA AC132217.4 mesenchymal stem cells
The preparation of recombinant viral vector suspension and the transduction of mesenchymal stem cells were performed using Lip8000 transduction kit (Biyundian biological Co., ltd.). Recombinant viral vector suspensions were prepared using the facultative packaging cell line 293T cells (ATCC, rockfiller, mass.) according to the kit instructions. 2.0X10 pairs in 10-cm Petri dishes (Nunc Co.) at 37℃in a cell incubator with the aid of 8. Mu.g/mL of 1, 5-dimethyl-1, 5-diazaundecene methylene polymethine bromide (Polybrene; sigma, st. Louis, MO) 5 Individual mesenchymal stem cells were transduced with recombinant viral vectors for 8 hours. Cell transduction efficiency was analyzed using enhanced Green Fluorescent Protein (GFP) (see fig. 2), which shows that transduced shRNA can be expressed in mesenchymal stem cells for long periods.
Example 4: analysis of specificity of lncRNA AC132217.4 for osteoblast differentiation
The potential targeting mRNA downstream of lncRNA AC132217.4 was examined by the Starbase database and the mRNA of insulin-like growth factor IGF2 was found to have the highest confidence score (silign score) with lncRNA AC132217.4 (fig. 3A), indicating that the mRNA of IGF2 gene may be the targeting mRNA downstream of lncRNA AC 132217.4. :
to verify that mRNA of IGF2 gene was targeted mRNA downstream of lncRNA AC132217.4, mesenchymal stem cells transfected with short hairpin RNA (shRNA) lentiviral vector of lncRNA AC132217.4 were cultured with osteoblast induction medium (DMEM-LG medium 900mL, 100mL fetal bovine serum, 100U/mL penicillin, 100U/mL streptomycin, 10mM beta-sodium glycerophosphate, 50 μm ascorbic acid) for 7 days and then treated with actinomycin D to block mRNA synthesis, whereby stability of IGF2 gene mRNA after lncRNA AC132217.4 knockdown was examined. The results showed that the stability of IGF2 gene mRNA was significantly reduced after lncRNA AC132217.4 knockdown (fig. 3B).
The osteoblast specific alkaline phosphatase (ALP), collagen 1 (COL 1A 1) and Runt related transcription factor 2 (RUNX 2) gene messenger RNA (mRNA) expression levels were analyzed by Real-time quantitative PCR (Real-time PCR) technology (reaction system of 1. Mu.l cDNA+1. Mu.l primer+8. Mu.l ultra pure water+10. Mu.l 2X SYRB quantitative PCR enzyme mix; reaction conditions: 40 cycles after 5 minutes of pre-denaturation at 95 ℃,10 s at 60 ℃ C. Each cycle). The amplification primers for ALP, COL1A1 and RUNX2 are shown in the following Table:
analysis of COL1A1 and Osteocalcin (OCN) activities by immunoblotting (western blot) technique, analysis of ALP activity and cell mineralization nodules by alkaline phosphatase activity detection technique and alizarin red staining technique, analysis of osteoblast directional differentiation ability of mesenchymal stem cells after knockdown of lncRNA AC132217.4 as shown in fig. 3C-J, shows that expression levels of osteoblast specific genes and transcription factors of mesenchymal stem cells are significantly reduced by knockdown of lncRNA AC132217.4, and restoration of the ability of mesenchymal stem cells to directionally differentiate into osteoblasts after overexpression of IGF2 in lncRNA AC132217.4 knockdown osteoblasts indicates that lncRNA AC132217.4 is improved by regulating stability of IGF2 gene mRNA.
In fig. 3: (A) The Starbase database examined potentially targeted mRNA downstream of lncRNA AC 132217.4. (B) Quantitative PCR detects the influence of the knockdown of the lncRNA AC132217.4 on the stability of the IGF2 gene mRNA; (C-D) Effect of IGF2 knockdown on osteogenic differentiation-related proteins and genes. (E-F) Effect of IGF2 knockdown on ALP activity and alizarin red in osteogenic differentiation. (G-H) Effect of overexpression of IGF2 on osteogenic differentiation-related proteins and genes in lncRNA AC132217.4 knockdown cells. (I-J) the effect of overexpression of IGF2 on osteogenic differentiation ALP activity and alizarin red in cells knockdown by this osteogenic differentiation lncRNA AC 132217.4. ACH: human bone marrow mesenchymal stem cells overexpressed by AC 132217.4; CTR: overexpressing empty human bone marrow mesenchymal stem cells; IGF2 shRNA, IGF2 knockdown human bone marrow mesenchymal stem cells; CTR shRNA: human bone marrow mesenchymal stem cells infected with empty plasmid are disturbed. Ach+igf2 shRNA: human bone marrow mesenchymal stem cells with IGF2 knockdown while AC132217.4 is overexpressed. The scale is 50um, data analysis is according to mean+ -SD; n is more than or equal to 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 mesenchymal directed differentiation into osteoblasts, potential specific transcription factors upstream of AC132217.4 were found based on the RNA-Seq technical analysis results in example 1, resulting in 12 up-regulated and 17 down-regulated transcription factors (fig. 4A). We then analyzed the transcription factor binding site of the lncRNA AC132217.4 promoter region (genomic region about 3000bp upstream of lncRNA AC 132217.4) using the PROMO database, and found 8 ALX1 high binding sites (FIG. 4B), indicating that ALX1 may be a transcription factor that specifically modulates lncRNA AC132217.4 expression.
To verify the above hypothesis, 2 independent ALX1 shRNA were used to reduce ALX1 expression. ALX1 shRNA interference plasmid is constructed on the basis of pRNAi-U6.2/Lenti Vector empty Vector. ALX1 shRNA primer sequence is synthesized by Huada biological company, two ends of synthesized upstream and downstream primers are phosphorylated by phosphorylase and annealed to form a double-chain structure (a system of phosphorylating and annealing is that 1ul of upstream primer+1 ul of downstream primer+1 ul of T4 PNK enzyme+7 ul of ultrapure water, the procedure is that the temperature is 37 ℃ for 30min, the temperature is 95 ℃ for 5min, the temperature is reduced to 25 ℃ according to the speed of 5 ℃/min), and HpaI and XhoI enzyme are used for cutting the carrier and ALX1 shRNA sequence and then connection is carried out. The ALX1 shRNA primer sequences are shown in the following table:
the results showed that the expression level of lncRNA AC132217.4 was significantly reduced in ALX1 knockdown cells compared to non-knockdown ALX1 cells (fig. 4C-D), and that luciferase reporter assay methods demonstrated that ALX1 could bind to the promoter region of lncRNA AC132217.4 gene (fig. 4E-F).
In fig. 4: (A) A transcription factor differentially expressed 7 days after osteoblast differentiation of human mesenchymal stem cells; (B) The PROMO database predicts the binding site of ALX1 within 3000bp upstream of the start site of lncRNA AC 132217.4; (C-D) effect of ALX1 knockdown on AC132217.4 and IGF2 expression; (E-F) construction of a reporter gene for the wild type and mutation of the AC132217.4 promoter, verifying the binding site of ALX1 to the AC132217.4 promoter. ALX1 shRNA1 is human bone marrow mesenchymal stem cells with ALX1 interference sequence 1 knocked down; ALX1 shRNA2, human bone marrow mesenchymal stem cells with ALX1 interference sequence 2 knocked down; shCTR: human bone marrow mesenchymal stem cells that interfere with empty plasmid infection; WT: a luciferase reporter gene of wild type AC132217.4 promoter sequence; MUT: a luciferase reporter gene of the ALX1 binding site mutated AC132217.4 promoter sequence; ALX1H: ALX1 overexpressing human bone marrow mesenchymal stem cells; CTR: overexpressing empty human bone marrow mesenchymal stem cells. Data analysis was performed according to mean±sd; n is more than or equal to 3; * p <0.05, < p <0.01, < p <0.001.
Example 6: ALX1 high expression vector construction and mesenchymal stem cell transfection
The technology of the invention adopts Trizol reagent (Invitrogen, carlsbad, CA) to separate and extract total RNA of human bone marrow mesenchymal stem cells, then adopts cDNA first-strand synthesis kit (Fermentas company) to carry out cDNA first-strand synthesis, and then uses the cDNA first-strand as a PCR amplification template to amplify ALX1 gene sequence. The amplification primer sequences of the ALX1 gene are as follows: CGC GTC GAC ATG GAG TTT CTG AGC GAG A and CAT GGC CCA TGA AAT ATT GGC TAG CTA GC. The total volume of the PCR reaction for ALX1 was 25. Mu.l, 30 cycles after 5 minutes of pre-denaturation at 95℃each comprising 94℃for 60 seconds, 55℃for 45 seconds and 72℃for 60 seconds. The amplified fragment length was 981bp.
The PCR reaction system (20. Mu.l) was: 1. Mu.cDNA+1. Mu.l primer+8. Mu.l ultra pure water+10. Mu.l 2X PCR enzyme mix.
The PCR amplified fragment of ALX1 was ligated to a lentiviral overexpression vector (Planti-CMV-Puro) (Jinan Vietnam Limited) by means of a ligase (TaKaRa) at the cleavage site of SalI and NheI (TaKaRa) to construct an ALX1 lentiviral overexpression vector as shown in SEQ ID NO: 2.
The constructed ALX1 lentivirus over-expression vector is used for preparing a recombinant viral vector suspension and transducing mesenchymal stem cells through a Lip8000 transduction kit (Biyun Tianshengmbh). Recombinant viral vector suspensions were prepared using the packaging cell line 293T (ATCC, rockfiller, mass.) according to the kit instructions. (Virus packaging protocol: 15ug of total plasmid (ALX 1 overexpressing plasmid: psPAX2: PMD2 G=4:3:1) was mixed with 750ul of Opti-MEM medium, then 24ul of Lip8000 transfection reagent (Biyun Biotechnology Co., ltd.) was added, the transfection solution was added to 293T cells of 10cm dishes with a density of 70-90%, fresh 293T cell medium (DMEM high sugar medium+10% FBS) was replaced for 6 hours, and after 72 hours the supernatant was collected as a virus stock solution) was purified in 8. Mu.g/mL of 1, 5-dimethyl-1, 5-diazaundecene polymethylene bromide (Polybrene; sigma, st. Louis, MO) with the aid of 2.0X10X 10 in 6-cm dishes (Nunc Co.) 5 Each mesenchymal stem cell was transduced with the recombinant viral vector for 12 hours. Western blotting verifies the over-expression efficiency, and the result shows that the transduced over-expression plasmid can be expressed in mesenchymal stem cells for a long time.
Quantitative PCR assay of mesenchymal Stem cells infected with ALX1 over-expression plasmid for gene expression levels of lncRNA AC132217.4 and IGF2 (FIGS. 5A-B), western and Elisa detected protein expression levels of IGF2, and as a result, it was found that over-expression of ALX1 promoted expression levels of AC132217.4 and IGF 2.
In fig. 5: (A) Quantitative PCR examined the effect of ALX1 overexpression on gene expression levels of lncRNA AC132217.4 and IGF 2. (B) Western detection of the effect of ALX1 overexpression on protein expression levels of ALX1 and IGF 2. ALX1H: ALX1 overexpressing human bone marrow mesenchymal stem cells; CTR: overexpressing empty human bone marrow mesenchymal stem cells. Data analysis was performed according to mean±sd; n is more than or equal to 3; * p <0.05, < p <0.01, < p <0.001.
Example 7: analysis of capacity of transduction ALX1 high expression vector to promote directional differentiation of mesenchymal stem cells into osteoblasts
Digesting the mesenchymal stem cells not transfected with the ALX1 high-expression vector and the mesenchymal stem cells transfected with the ALX1 high-expression vector into single fine cellsCell suspension at 3X 10 4 The cells were inoculated into 16-well culture plates at a density of 0.1. Mu. Mol/mL, and after 80% -90% of the cells were fused, osteoblast induction was performed for 7 days by adding an osteoblast induction solution (DMEM-LG culture solution 900mL, 100mL fetal bovine serum, 100U/mL penicillin, 100U/mL streptomycin, 0.1. Mu. Mol dexamethasone, 10mM sodium beta-glycerophosphate, 50. Mu. M ascorbic acid). The liquid is changed every 2 days.
After termination of induction, alkaline phosphatase staining was observed for differentiation effect: the resulting cells were washed 3 times with PBS (pH 7.2) for 1 minute each, fixed with 1% paraformaldehyde containing 7.5% sucrose at room temperature for 10 to 20 minutes, and then stained with alkaline phosphatase kit according to manufacturer's instructions, equilibrated with substrate buffer, stained with freshly prepared 5-bromo-4-chloro-3-indolephosphoric acid/azoblue tetrazolium (BCIP/NBT) staining solution for more than 30 minutes at room temperature in the absence of light, and washed with water. Differentiated cells were observed under a 100-fold inverted microscope as in fig. 6A.
After termination of induction, alizarin red S staining observed differentiation effect: differentiated cells were washed 3 times with PBS (pH 7.2) for 1 min each, 4% paraformaldehyde fixed specimens, 2% alizarin red S dye incubated at 37℃for 5min, and observed after rinsing with distilled water (FIG. 6B).
In fig. 6: (A) Alkaline phosphatase the effect of ALX1 overexpression on osteogenic differentiation was examined. (B) Alizarin red detects the effect of ALX1 overexpression on osteogenic calcification. ALX1H: ALX1 overexpressing human bone marrow mesenchymal stem cells; CTR: overexpressing empty human bone marrow mesenchymal stem cells. The scale is 50um.
To sum up:
(1) The technology effectively improves the capability of directional differentiation of mesenchymal stem cells into bone cells:
by constructing the ALX1 high expression vector and transfecting the mesenchymal stem cells, the expression level of long-chain non-coding lncRNA AC132217.4 in the cells can be effectively improved, the stability of IGF2 gene mRNA is enhanced, the activity of IGF2 signaling pathway is improved, the directional differentiation efficiency of the mesenchymal stem cells into bone cells is obviously promoted, and compared with the mesenchymal stem cells without the ALX1 high expression vector, the directional differentiation efficiency is improved by 68.2%.
(2) The technology of the invention has the advantage of reducing the experimental cost of directional induction and differentiation of mesenchymal stem cells into osteoblasts:
the induced differentiation and the preparation of the osteoblast by the technology can eliminate the need of adding related cytokines and special equipment, and effectively reduce the high cost caused by expensive cytokines and special equipment.
(3) The technology of the invention can be applied to the clinical bone injury repair treatment and the research of bone tissue engineering technology:
the invention provides a method for directional induction and differentiation of mesenchymal stem cells into osteoblasts with high-efficiency osteoblast differentiation, which can provide sufficient seed cells for bone tissue engineering research and application, and can be applied to clinical bone injury repair and gene therapy technology research of patients with bone nonunion by adopting high expression technology of ALX1 and lncRNA AC 132217.4.
Sequence listing
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Hainandison biotechnology Co.Ltd
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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.
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| CN1553951A (en) * | 2000-11-03 | 2004-12-08 | ���ﰲҽ�ƹɷ�����˾ | Human cord blood derived unrestricted somatic stem cells (USSC) |
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| CN1553951A (en) * | 2000-11-03 | 2004-12-08 | ���ﰲҽ�ƹɷ�����˾ | Human cord blood derived unrestricted somatic stem cells (USSC) |
| CN1818071A (en) * | 2005-07-04 | 2006-08-16 | 浙江大学 | Method for transferring bone marrow filling dry cell by TPO/FL |
| CN111269942A (en) * | 2020-02-28 | 2020-06-12 | 中国人民解放军军事科学院军事医学研究院 | Application of TGFBI as marker for regulating and controlling osteogenic differentiation of mesenchymal stem cells |
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