CN113637633A - Method for promoting differentiation of mesenchymal stem cells into osteoblasts - Google Patents
Method for promoting differentiation of mesenchymal stem cells into osteoblasts Download PDFInfo
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- CN113637633A CN113637633A CN202110935409.2A CN202110935409A CN113637633A CN 113637633 A CN113637633 A CN 113637633A CN 202110935409 A CN202110935409 A CN 202110935409A CN 113637633 A CN113637633 A CN 113637633A
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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 (3) carrying out directional induction culture on the mesenchymal stem cells for efficiently expressing the exogenous ALX1 by using osteoblast induction liquid to obtain the osteoblasts. The mesenchymal stem cells exogenously expressing ALX1 constructed by the technology of the invention are in an osteoblast induced differentiation system, thus the need of adding cell factors or special equipment in a culture system is saved, and the culture cost of the mesenchymal stem cells for directionally inducing and differentiating the osteoblasts is reduced.
Description
(I) technical field
The invention relates to a method for promoting mesenchymal stem cells to differentiate into osteoblasts through an ALX1-lncRNA-IGF2 pathway.
(II) background of the invention
Stem cells having a certain differentiation potential exist in the bone tissue of the human body. Relevant researches show that Mesenchymal Stem Cells (MSCs) from bone marrow can be differentiated into osteoblasts, chondrocytes, adipocytes, cardiomyocytes and the like, and particularly, the research on the differentiation of the MSCs into the osteoblasts becomes an important basis for the bone injury repair and the development of bone tissue engineering technology based on the stem cells, so that the MSCs have important application values of osteoblast transplantation and bone tissue engineering in medicine.
At present, in the aspect of mesenchymal stem cell directional differentiation osteoblast technology, relevant cytokines are mainly added, or mechanical action is exerted to promote the differentiation of mesenchymal stem cells to osteoblasts. The method for adding the cell factors is that according to the molecular mechanism related to the bone differentiation and development, the cell factors such as Bone Morphogenetic Protein (BMP) and insulin-like growth factor (IGF2) and the like are added into the culture solution for the directional induction and differentiation of the mesenchymal stem cells in vitro to regulate the activity of cell signaling pathways related to the bone differentiation and development so as to carry out differentiation induction of the osteoblasts. The method for mechanical action is that a culture solution flows through adherent mesenchymal stem cells in a specific device (such as a fluid shear plate or a perfusion device) so as to generate certain fluid shear force on the mesenchymal stem cells, the fluid shear force acts on extracellular matrix and receptors of the cells so as to convert extracellular mechanical signals into intracellular chemical signals, and then the differentiation of the mesenchymal stem cells into osteoblasts is finally promoted by regulating cell signal pathway activity related to the differentiation and development of the osteoblasts. However, these two techniques have certain limitations in practical application, and especially have many inconveniences in clinical application and preparation of seed cells for bone tissue engineering: the cytokine method not only increases the cost, but also has low bone cell induced differentiation efficiency and is not convenient for clinical in vivo application; the fluid shear force method requires special equipment and is not suitable for repair treatment of bone injury in vivo.
Therefore, if a large number of gene expression group analyses are carried out to find a new key molecular target spot related to bone differentiation and a new technology is adopted to promote osteoblast differentiation aiming at the new target spot, a new research idea of directional differentiation of stem cells into osteoblasts can be developed, the preparation cost of in vitro osteoblasts can be greatly reduced, the differentiation efficiency of the osteoblasts can be improved, and the clinical application research of bone injury repair can be promoted.
Disclosure of the invention
In order to effectively improve the capability of the mesenchymal stem cells for directional differentiation into the osteoblasts, the invention discloses a novel long-chain non-coding RNA (lncRNA) specifically related to the bone differentiation based on RNA-Seq analysis, namely lncRNA AC132217.4, constructs a high-expression vector of an upstream transcription factor type box 1(ALX1), promotes the mesenchymal stem cells to efficiently express lncRNA AC132217.4, improves the activity of an lncRNA-IGF2 signal channel, and aims to provide a method for effectively promoting the mesenchymal stem cells to directionally differentiate into the osteoblasts.
In order to achieve the purpose, the 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 (2) directionally inducing and culturing the mesenchymal stem cells efficiently expressing the exogenous ALX1 prepared in the step (1) by using osteoblast induction liquid to obtain the osteoblasts.
The gene high expression technology adopts a lentivirus over-expression vector (Plenti-CMV-Puro), and the sequence of an amplification primer of a target gene ALX1 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 infects the 3 rd generation mesenchymal stem cell after being subjected to virus packaging by transfecting 293T cells by lentiviral packaging plasmids psPAX2 and PMD 2G.
Further, the mesenchymal stem cell may be from one of the following tissues: bone marrow, fat, neonatal umbilical cord, placenta, etc. The mesenchymal stem cells used in the invention are human mesenchymal stem cells.
Furthermore, the expression vector of the ALX1 gene is constructed by connecting the ALX1 gene to a lentivirus overexpression vector.
Preferably, the expression vector of the ALX1 gene is prepared by the following method: PCR amplification is carried out on a cDNA sequence of a human ALX1 gene 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, restriction enzymes SalI and NheI are used for respectively carrying out enzyme digestion on a lentivirus overexpression vector Plenti-CMV-Puro and the target gene 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 through ligase to obtain an 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 invention is obtained by the following method: the human bone marrow mesenchymal stem cell total RNA is separated and extracted by using Trizol reagent (Invitrogen, Carlsbad, CA), and then cDNA first strand synthesis is carried out by using a cDNA first strand synthesis kit (Fermentas company), so as to obtain the cDNA sequence of the human ALX1 gene.
Preferably, the expression vector of the ALX1 gene is transferred into the mesenchymal stem cell by Lip8000 transduction kit (bi yun tian bio ltd).
Specifically, the mesenchymal stem cell for efficiently expressing exogenous ALX1 is obtained by the following method: the expression vector of the ALX1 gene is transfected into 293T cells by lentivirus packaging plasmids psPAX2 and PMD2G for virus packaging, and then the mesenchymal stem cells are continuously cultured for 12 hours under the assistance of 8 mu g/mL of 1, 5-dimethyl-1, 5-dinitrogen undecamethylene polymethine bromide (Polybrene, Sigma, St.Louis, Mo), so that the mesenchymal stem cells efficiently expressing the exogenous ALX1 are obtained.
Further, each liter of the osteoblast inducing liquid consists of the following components: 900mL of DMEM-LG culture solution, 100mL of fetal bovine serum, 100U/mL of penicillin, 100U/mL of streptomycin, 10mM of beta-sodium glycerophosphate and 50 mu M of ascorbic acid.
Further, the conditions of the directional induction culture are as follows: 7 days at 37 ℃.
Compared with the prior art, the invention has the following beneficial effects:
(1) the technology provided by the invention adopts the osteoblast differentiation specific long-chain non-coding lncRNA AC132217.4 upstream specific transcription factor ALX1 gene high expression vector to transduce the mesenchymal stem cells, can effectively improve the expression of the long-chain non-coding lncRNA AC132217.4 in the mesenchymal stem cells, and promotes the activation of the osteoblast differentiation specific IGF2 cell signal channel.
(2) The mesenchymal stem cells exogenously expressing ALX1 constructed by the technology of the invention are in an osteoblast induced differentiation system, thus the need of adding cell factors or special equipment in a culture system is saved, and the culture cost of the mesenchymal stem cells for directionally inducing and differentiating the osteoblasts is reduced.
(3) Compared with the traditional osteoblast directional induced differentiation, the mesenchymal stem cell directional induced differentiation osteoblast culture system generated by the technology effectively improves the efficiency of the mesenchymal stem cell directional induced differentiation osteoblasts.
The key point of the invention is that according to the verification of a novel osteogenic specific long-chain non-coding lncRNA (lncRNA AC132217.4) and an upstream specific transcription factor ALX1 thereof, ALX1 expression vector construction and mesenchymal stem cell transfection are carried out, the mesenchymal stem cell for efficiently expressing ALX1 is prepared, and the capability of directionally differentiating the mesenchymal stem cell into the osteoblast is promoted. Other parameters in the processes of expression vector construction, mesenchymal stem cell transfection and osteoblast directional differentiation can be carried out by adopting conventional means in the field.
(IV) description of the drawings
FIG. 1 shows that the expression level of lncRNA AC132217.4 increases with the increase of osteogenic differentiation, (A) LncRNAs differentially expressed in osteogenic differentiation of human mesenchymal stem cells. (B-C) AC132217.4 increased with osteogenic differentiation expression. Data analysis was 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 AC132217.4 promotes osteogenic differentiation by modulating the stability of IGF2 gene mRNA (A) Starbase database examination of potential targeting mRNA downstream of IncRNA AC 132217.4. (B) Quantitative PCR is used for detecting the influence of the LncRNAACC132217.4 knocked-down on the stability of IGF2 gene mRNA; (C-D) effects of IGF2 knockdown on osteogenic differentiation-related proteins and genes; (E-F) effects 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 AC 132217.4-knockdown cells; (I-J) Effect of overexpression of IGF2 in osteoblastic differentiated IncRNA AC132217.4 knockdown cells on osteoblastic differentiation ALP activity and alizarin Red. ACH is human bone marrow mesenchymal stem cells over-expressed by AC 132217.4; CTR: overexpresses unloaded human bone marrow mesenchymal stem cells; IGF2 shRNA IGF2 knockdown human mesenchymal stem cells; CTR shRNA: human mesenchymal stem cells interfering with unloaded plasmid infection. ACH + IGF2 shRNA: human bone marrow mesenchymal stem cells with overexpression of AC132217.4 and concomitant IGF2 knockdown. Scale 50um. data analysis was according to mean + -SD; n is more than or equal to 3; p <0.05, p <0.01, p < 0.001.
FIG. 4 ALX1 influences its transcription level by binding at the promoter of lncRNA AC132217.4 (A) a transcription factor differentially expressed after 7 days of osteogenic differentiation of human mesenchymal stem cells; (B) the PROMO database predicts a binding site for 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 AC132217.4 promoter wild type and mutant reporter gene, and verification of ALX1 and AC132217.4 promoter binding site. ALX1 shRNA1 ALX1 interference sequence 1 knockdown human mesenchymal stem cells; ALX1 shRNA2 ALX1 interference sequence 2 knockdown human mesenchymal stem cells; shCTR: human bone marrow mesenchymal stem cells interfering with unloaded plasmid infection; WT: a luciferase reporter gene of the wild-type AC132217.4 promoter sequence; MUT: a luciferase reporter gene comprising the AC132217.4 promoter sequence mutated at the ALX1 binding site; ALX 1H: ALX 1-overexpressed human bone marrow mesenchymal stem cells; CTR: overexpresses unloaded human bone marrow mesenchymal stem cells. Data analysis was 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 the expression of IncRNA AC132217.4 and IGF2 (A) quantitative PCR examination of the effect of ALX1 overexpression on the gene expression levels of IncRNA AC132217.4 and IGF 2. (B) Western analysis examined the effect of ALX1 overexpression on the protein expression levels of ALX1 and IGF 2. ALX 1H: ALX 1-overexpressed human bone marrow mesenchymal stem cells; CTR: overexpresses unloaded human bone marrow mesenchymal stem cells. Data analysis was 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 assay of ALX1 overexpression on osteogenic differentiation. (B) Alizarin red measures the effect of ALX1 overexpression on osteogenic calcification. ALX 1H: ALX 1-overexpressed human bone marrow mesenchymal stem cells; CTR: overexpresses unloaded human bone marrow mesenchymal stem cells. Scale bar 50um.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to 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 special instructions: the cells were cultured in a 5% carbon dioxide cell incubator at 37 ℃.
Example 1: analysis of Gene expression profiles
Collecting 3 rd generation human bone marrow mesenchymal stem cells, preparing into single cell suspension, and culturing at 2 × 104Inoculating each culture dish to a culture dish with the diameter of 30mM for alpha-MEM adherent culture, and performing osteoblast directional induction by using osteoblast induction liquid (900 mL of DMEM-LG culture liquid, 100mL of fetal calf serum, 100U/mL of penicillin, 100U/mL of streptomycin, 10mM of beta-sodium glycerophosphate and 50 mu M of ascorbic acid) when the cells reach 80-90% of the layer.
After 7 days of the directional induction, the differentiation-induced cells and the non-induced cells were each 5X 105The cells were lysed by adding 1mL of Trizol lysate to the lysate, adding 1/5 volumes of chloroform to the lysate, standing at room temperature for 5min, centrifuging at 13000rpm for 15 min at 4 ℃, then taking the uppermost aqueous phase, adding 5/3 volumes of isopropanol, standing at 13000rpm for 10min, centrifuging at 13000rpm for 10min at 4 ℃, removing the supernatant, adding 1mL of 75% ethanol, then centrifuging at 7500rpm at 4 ℃ for 5min, removing the supernatant, removing the residual 75% ethanol under vacuum, and then dissolving with DEPC water (provided by the detection method). The total concentration of RNA is determined, the concentration exceeds 100 ng/mu l, the total amount exceeds 1 mu g, and then the RNA is sent to a detection partyRNA-Seq test analysis was performed.
RNA-Seq analysis data was stored in the NCBI gene expression database (accessible via GEO series accession number GSE 114117) (https:// www.ncbi.nlm.nih.gov/GEO/query/acc. cgi). The analysis found that the expression level of long-chain non-coding lncrnaac132217.4 was significantly changed in all genes with changed expression level (fig. 1A, B), and was confirmed by the analysis of qRT-PCR method (fig. 1C).
Wherein, in the figure 1: (A) LncRNAs which are differentially expressed in osteogenic differentiation of human bone marrow mesenchymal stem cells. (B-C) AC132217.4 increased with osteogenic differentiation expression. Data analysis was 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 knockdown vector
The lncRNA AC132217.4 knock-down vector is purchased from Jima gene company, is constructed by pGPU6/GFP/Neo vector, and is constructed by a homologous recombination method. Constructing a short hairpin RNA (shRNA) lentiviral vector as shown in SEQ ID NO: 1 is shown.
Example 3: transfection of mesenchymal Stem cells with short hairpin RNA (shRNA) Lentiviral vector of IncRNA AC132217.4
Preparation of recombinant virus vector suspension and transduction of mesenchymal stem cells were performed using Lip8000 transduction kit (bi yun tian bio ltd). Recombinant viral vector suspensions were prepared using the compatible packaging cell line 293T cells (ATCC, Rockofeller, Mass.) according to kit instructions. 2.0X 10 pairs of 1, 5-dimethyl-1, 5-dinitrogen undecamethylene polymethine bromide (Polybrene; Sigma, St. Louis, Mo.) in 10-cm dishes (Nunc) at 37 ℃ in a cell incubator with the aid of 8. mu.g/mL of 1, 5-dimethyl-1, 5-dinitrogen undecamethylene polymethine bromide5The mesenchymal stem cells were transduced with the recombinant viral vector for 8 hours. Cell transduction efficiency was analyzed using enhanced Green Fluorescent Protein (GFP) (see fig. 2), and the results showed that the transduced shRNA could be expressed in mesenchymal stem cells for a long period of time.
Example 4: specific analysis of lncRNA AC132217.4 on osteoblast differentiation
Potential targeting mrnas downstream of lncRNA AC132217.4 were examined by the Starbase database and the mRNA for insulin-like growth factor IGF2 was found to have the highest confidence score (siiign score) with lncRNA AC132217.4 (fig. 3A), indicating that the mRNA for IGF2 gene is likely to be the targeting mRNA downstream of lncRNA AC 132217.4. :
to verify that the mRNA of IGF2 gene is the targeting mRNA downstream of lncRNA AC132217.4, mesenchymal stem cells transfected with lncRNA AC132217.4 short hairpin RNA (shRNA) lentiviral vector were taken, cultured for 7 days in osteoblast induction medium (DMEM-LG medium 900mL supplemented with 100mL fetal bovine serum, 100U/mL penicillin, 100U/mL streptomycin, 10mM sodium β -glycerophosphate, 50 μ M ascorbic acid) and then treated with actinomycin D to block mRNA synthesis, thereby investigating the stability of IGF2 gene mRNA after lncRNA AC132217.4 knockdown. The results showed that the stability of IGF2 gene mRNA was significantly decreased after lncRNA AC132217.4 knock-down (fig. 3B).
The expression levels of osteoblast specific alkaline phosphatase (ALP), collagen 1(COL1A1) and Runt-related transcription factor 2(RUNX2) gene messenger RNA (mRNA) were analyzed by Real-time quantitative PCR (Real-time PCR) technique (reaction system of Real-time quantitative PCR: 1. mu.l cDNA + 1. mu.l primer + 8. mu.l ultrapure water + 10. mu.l 2X SYRB quantitative PCR enzyme mix; reaction conditions: 40 cycles after 5 minutes of pre-denaturation at 95 ℃,95 ℃ and 40 cycles of 95 ℃ for 40 seconds and 40 seconds at 60 ℃ for each cycle). Amplification primers for ALP, COL1A1 and RUNX2 are shown in the following Table:
the activity of COL1A1 and Osteocalcin (OCN) is analyzed by an immunoblotting (western blot) technology, the activity of ALP and cell mineralized nodules are analyzed by an alkaline phosphatase activity detection technology and an alizarin red staining technology, the osteoblast directional differentiation capacity of the mesenchymal stem cells after the deletion of lncRNA AC132217.4 is analyzed as shown in FIGS. 3C-J, the fact that the deletion of lncRNA AC132217.4 causes the significant reduction of the expression level of osteoblast specific genes and transcription factors of the mesenchymal stem cells, and the restoration of the capacity of the mesenchymal stem cells after the overexpression of IGF2 in the osteoblasts subjected to the deletion of lncRNA AC132217.4 to directionally differentiate into the osteoblasts shows that lncRNA AC132217.4 is improved by regulating the stability of IGF2 gene mRNA.
In fig. 3: (A) the Starbase database examines potential targeting mRNAs downstream of lncRNA AC 132217.4. (B) Quantitative PCR is used for detecting the influence of the IncRNA AC132217.4 on the stability of IGF2 gene mRNA after being knocked down; (C-D) Effect 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) Effect of overexpression of IGF2 in (G-H) lncRNA AC 132217.4-knocked-down cells on osteogenic differentiation-related proteins and genes. (I-J) Effect of overexpression of IGF2 in osteoblastic differentiated IncRNA AC132217.4 knockdown cells on osteoblastic differentiation ALP activity and alizarin Red. ACH is human bone marrow mesenchymal stem cells over-expressed by AC 132217.4; CTR: overexpresses unloaded human bone marrow mesenchymal stem cells; IGF2 shRNA IGF2 knockdown human mesenchymal stem cells; CTR shRNA: human mesenchymal stem cells interfering with unloaded plasmid infection. ACH + IGF2 shRNA: human bone marrow mesenchymal stem cells with overexpression of AC132217.4 and concomitant IGF2 knockdown. Scale 50um. data analysis was according to mean + -SD; n is more than or equal to 3; p <0.05, p <0.01, p < 0.001.
Example 5: lncrRNA AC132217.4 upstream specific transcription factor ALX1 analysis
In order to increase the transcriptional expression level of lncRNA AC132217.4 during mesenchymal committed differentiation into osteoblasts, potential specific transcription factors upstream of AC132217.4 were searched according to the RNA-Seq technical analysis result in example 1, and 12 up-regulated and 17 down-regulated transcription factors were found (FIG. 4A). Then, we analyzed the transcription factor binding site of lncRNA AC132217.4 promoter region (genomic region about 3000bp upstream of lncRNA AC132217.4) by using the PROMO database, and found 8 ALX1 high binding sites (fig. 4B), indicating that ALX1 may be a transcription factor specifically regulating the expression of lncRNA AC 132217.4.
To verify the above speculation, 2 independent ALX1 shrnas were used to reduce expression of ALX 1. The ALX1 shRNA interference plasmid was constructed on the basis of pRNAi-U6.2/Lenti Vector empty Vector. The ALX1 shRNA primer sequence is synthesized by Huada biology company, and the two ends of the synthesized upstream and downstream primers are annealed after phosphorylation by phosphorylase to form a double-chain structure (the phosphorylation and annealing system is that 1ul of the upstream primer +1ul of the downstream primer +1ul of the T4 PNK enzyme +7ul of ultrapure water, the program is 30min at 37 ℃, and is reduced to 25 ℃ at the speed of 5 ℃/min after 5min at 95 ℃), and then the double-chain structure is connected with the ALX1 shRNA sequence through the HpaI and XhoI enzyme digestion vector. 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 ALX 1-knocked down cells compared to non-ALX 1-knocked down cells (fig. 4C-D), and the luciferase reporter assay method 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 osteogenic differentiation of human mesenchymal stem cells; (B) the PROMO database predicts a binding site for 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 AC132217.4 promoter wild type and mutant reporter gene, and verification of ALX1 and AC132217.4 promoter binding site. ALX1 shRNA1 ALX1 interference sequence 1 knockdown human mesenchymal stem cells; ALX1 shRNA2 ALX1 interference sequence 2 knockdown human mesenchymal stem cells; shCTR: human bone marrow mesenchymal stem cells interfering with unloaded plasmid infection; WT: a luciferase reporter gene of the wild-type AC132217.4 promoter sequence; MUT: a luciferase reporter gene comprising the AC132217.4 promoter sequence mutated at the ALX1 binding site; ALX 1H: ALX 1-overexpressed human bone marrow mesenchymal stem cells; CTR: overexpresses unloaded human bone marrow mesenchymal stem cells. Data analysis was 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 mesenchymal stem cells, then adopts a 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 the ALX1 gene sequence. The sequence of the ALX1 gene amplification primer is as follows: CGC GTC GAC ATG GAG TTT CTG AGC GAG A, and CAT GGC CCA TGA AAT ATT GGC TAG CTA GC. The PCR reaction of ALX1 was performed in a total volume of 25. mu.l, at 95 ℃ for 5 minutes followed by 30 cycles of pre-denaturation, each cycle consisting of 94 ℃ for 60 seconds, 55 ℃ for 45 seconds, and 72 ℃ for 60 seconds. The length of the amplified fragment is 981 bp.
The PCR reaction system (20. mu.l) was: mu.cDNA + 1. mu.l primer + 8. mu.l ultrapure water + 10. mu.l 2 XPCR enzyme mix.
The PCR-amplified fragment of ALX1 was ligated to a lentiviral over-expression vector (Plenti-CMV-Puro) (Jinan Vivi organism Co., Ltd.) via the cleavage sites of SalI and NheI (TaKaRa) using ligase (TaKaRa) to construct an ALX1 lentiviral over-expression vector such as SEQ ID NO: 2, respectively.
The constructed ALX1 lentivirus overexpression vector is used for preparing a recombinant virus vector suspension and transducing mesenchymal stem cells by a Lip8000 transduction kit (Biyuntian biology, Ltd.). Recombinant viral vector suspensions were prepared using the packaging cell line 293T (ATCC, Rockofeller, Mass.) according to kit instructions. (Virus-packaging protocol: 15ug of total plasmid (ALX1 overexpression plasmid: psPAX 2: PMD 2G:. about.4: 3: 1) was mixed with 750ul of Opti-MEM medium, 24ul of Lip8000 transfection reagent (Biyunyinan Bio Inc.) was added and mixed, the transfection solution was added to 293T cells in 10cm dishes at 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, which was the virus stock solution, was collected as a virus stock solution in 8. mu.g/mL of 1, 5-dimethyl-1, 5-diaza-undecylmethylene polybromide (Polybrene; Sigma, St. Louis, Mo.) and mixed in 6-cm dishes (Nunc Corp.) at 2.0X 105The mesenchymal stem cells were transduced with the recombinant viral vector for 12 hours. Western blotting verifies the overexpression efficiency, and the result shows that the transduced overexpression plasmid can be expressed in the mesenchymal stem cells for a long time.
Quantitative PCR detection of mesenchymal stem cells infected with ALX1 overexpression plasmid was used to analyze the gene expression levels of IncRNA AC132217.4 and IGF2 (FIGS. 5A-B), and western and Elisa were used to detect the protein expression level of IGF2, and as a result, it was found that overexpression of ALX1 promoted the expression levels of AC132217.4 and IGF 2.
In fig. 5: (A) quantitative PCR examined the effect of ALX1 overexpression on the gene expression levels of lncRNA AC132217.4 and IGF 2. (B) Western analysis examined the effect of ALX1 overexpression on the protein expression levels of ALX1 and IGF 2. ALX 1H: ALX 1-overexpressed human bone marrow mesenchymal stem cells; CTR: overexpresses unloaded human bone marrow mesenchymal stem cells. Data analysis was 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 capability of transduction ALX1 high-expression vector for promoting directional differentiation of mesenchymal stem cells into osteoblasts
Digesting the mesenchymal stem cells of the untransfected ALX1 high expression vector and the mesenchymal stem cells of the transfected ALX1 high expression vector into single cell suspension at the ratio of 3 x 104The cells are inoculated in a 16-well culture plate at a density of one cell per milliliter, after the cells are fused to 80-90 percent, osteoblast induction liquid (900 mL of DMEM-LG culture liquid, 100mL of fetal calf serum, 100U/mL of penicillin, 100U/mL of streptomycin, 0.1 mu mol of dexamethasone, 10mM of beta-sodium glycerophosphate and 50 mu M of ascorbic acid) is added for directional induction of osteoblasts for 7 days. The solution was changed every 2 days.
After termination of induction, differentiation effect was observed by alkaline phosphatase staining: the cells obtained were washed 3 times with PBS (pH 7.2), 1 minute each time, fixed with 1% paraformaldehyde containing 7.5% sucrose at room temperature for 10-20 minutes, and then stained with an alkaline phosphatase kit according to the manufacturer's instructions, first equilibrated with a substrate buffer, stained in a freshly prepared staining solution of 5-bromo-4-chloro-3-indacenaphthyl phosphate/nitro blue tetrazolium (BCIP/NBT) in the dark at room temperature for more than 30 minutes, and washed with water. The differentiated cells were observed under a 100-fold inverted microscope as shown in FIG. 6A.
After termination of induction, alizarin red S staining was used to observe the differentiation effect: the differentiated cells were washed 3 times with PBS (pH 7.2) for 1 minute each, the specimens were fixed with 4% paraformaldehyde, incubated with 2% alizarin red S stain at 37 ℃ for 5 minutes, rinsed with distilled water and observed (FIG. 6B).
In fig. 6: (A) alkaline phosphatase measures the effect of ALX1 overexpression on osteogenic differentiation. (B) Alizarin red measures the effect of ALX1 overexpression on osteogenic calcification. ALX 1H: ALX 1-overexpressed human bone marrow mesenchymal stem cells; CTR: overexpresses unloaded human bone marrow mesenchymal stem cells. Scale bar 50um.
To sum up:
(1) the technology of the invention effectively improves the capability of directional differentiation of mesenchymal stem cells into osteoblasts:
by constructing the ALX1 high expression vector and transfecting the mesenchymal stem cell, the expression level of long-chain non-coding lncRNA AC132217.4 in the cell can be effectively improved, the stability of mRNA of IGF2 gene is enhanced, the activity of IGF2 signal channel is improved, the efficiency of the mesenchymal stem cell for directional differentiation into osteocyte is remarkably promoted, and compared with the mesenchymal stem cell which is not transfected with the ALX1 high expression vector, the directional differentiation efficiency is improved by 68.2%.
(2) The technology of the invention has the advantages of reducing the experimental cost of directional induced differentiation of the mesenchymal stem cells into the osteoblasts:
the induction differentiation and the preparation of osteoblasts by the technology can avoid the requirement of adding related cell factors and special equipment, and effectively reduce the high cost caused by expensive cell factors and special equipment.
(3) The technology of the invention can be applied to the clinical bone injury repair and treatment and the research of bone tissue engineering technology:
the invention provides a method for directionally inducing and differentiating mesenchymal stem cells with high osteoblast differentiation efficiency, which can provide sufficient seed cells for bone tissue engineering research and application, and simultaneously, the high-expression technology of ALX1 and lncRNA AC132217.4 can be applied to the clinical bone injury repair and the gene therapy technical research of patients with nonunion.
Sequence listing
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ttctgagcga gaagtttgcc ctcaagagcc ctccgagtaa aaacagtgac ttttacatgg 240
gcgcaggagg tcctctggag cacgttatgg agacgctgga caatgagtcc ttttacagca 300
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 (10)
1. A method of promoting differentiation of mesenchymal stem cells into osteoblasts, the method comprising:
(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 (2) directionally inducing and culturing the mesenchymal stem cells efficiently expressing the exogenous ALX1 prepared in the step (1) by using osteoblast induction liquid to obtain the osteoblasts.
2. A method of promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 1, characterised in that the mesenchymal stem cells are from one of the following tissues: bone marrow, fat, neonatal umbilical cord, placenta.
3. The method of promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 2, wherein: the mesenchymal stem cell is human bone marrow mesenchymal stem cell.
4. The method of promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 1, wherein: the expression vector of the ALX1 gene is constructed by connecting the ALX1 gene to a lentivirus overexpression vector.
5. The method for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 4, wherein the expression vector for the ALX1 gene is prepared by: carrying out PCR amplification on a cDNA sequence of a human ALX1 gene by using 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, carrying out enzyme digestion on a lentivirus overexpression vector Plenti-CMV-Puro and the target gene by using restriction endonucleases SalI and NheI respectively to obtain the target gene with a sticky end and a linear vector, and connecting the target gene with the sticky end and the linear vector by using a ligase to obtain an expression vector of the ALX1 gene.
6. The method for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 5, wherein the cDNA sequence of the human ALX1 gene is obtained by: and (3) separating and extracting total RNA of the human mesenchymal stem cells by using a Trizol reagent, and then performing first strand cDNA synthesis by using a first strand cDNA synthesis kit to obtain a cDNA sequence of the human ALX1 gene.
7. The method of promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 1, wherein: the expression vector of the ALX1 gene is transferred into the mesenchymal stem cells through a Lip8000 transduction kit.
8. The method for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 7, wherein the mesenchymal stem cells highly expressing exogenous ALX1 are obtained by: the expression vector of the ALX1 gene is transfected into 293T cells by lentivirus packaging plasmids psPAX2 and PMD2G to carry out virus packaging, and then the mesenchymal stem cells are continuously cultured for 12 hours under the assistance of 8 mu g/mL 1, 5-dimethyl-1, 5-dinitrogen undecamethylene polymethine bromide, so that the mesenchymal stem cells for efficiently expressing the exogenous ALX1 are obtained.
9. The method for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 1, wherein said osteoblast inducing liquid is composed of, per liter: 900mL of the culture solution of the MEM-LG, 100mL of fetal bovine serum, 100U/mL of penicillin, 100U/mL of streptomycin, 10mM of beta-sodium glycerophosphate and 50 mu M of ascorbic acid.
10. The method for promoting differentiation of mesenchymal stem cells into osteoblasts according to claim 1, wherein the conditions for the directed induction culture are: 7 days at 37 ℃.
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| CN115851608A (en) * | 2022-12-23 | 2023-03-28 | 广州医科大学附属第二医院 | Gene editing mesenchymal stem cell and preparation method and application thereof |
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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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| CN115851608A (en) * | 2022-12-23 | 2023-03-28 | 广州医科大学附属第二医院 | Gene editing mesenchymal stem cell and preparation method and application thereof |
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