CN117448379A - Construction method and application of iPSC-derived IL-10 protein over-expression MSC cell strain - Google Patents
Construction method and application of iPSC-derived IL-10 protein over-expression MSC cell strain Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology and cell technology, and provides a construction method of an IL-10 protein over-expression MSC cell strain from iPSC and application of the cell strain in cell medicines for treating osteoarthritis. The construction method comprises the steps of introducing a plasmid containing Cas 9 protein and targeted AAVS1 safety site sgRNA into cells by using a CRISPR system, inserting a donor template plasmid encoding an IL-10 protein sequence to obtain an iPSC cell strain with the IL-10 gene inserted at fixed points, and inducing differentiation of the iPSC cell strain to obtain the MSC capable of over-expressing the IL-10 protein. The MSC over-expressing the IL-10 protein can efficiently regulate the intra-articular immune environment, and has better treatment effect compared with the MSC of natural sources.
Description
Technical Field
The invention belongs to the field of molecular biology and cell technology, and in particular relates to a construction method of an IL-10 protein over-expression MSC cell strain and application thereof in cell medicines for treating osteoarthritis.
Background
Osteoarthritis is a common degenerative joint disease, and in the early stages of the onset, inflammatory properties of varying severity already exist. At present, the role of various inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis is still under investigation, wherein IL-10 is a cytokine thought to exhibit chondroprotective effects during the pathogenesis of osteoarthritis, and IL-10 has been shown to be involved in stimulating the synthesis of type II collagen and agrin, and after administration of IL-10 protein under in vitro conditions, proteoglycan synthesis and its percentage increase in extracellular matrix are shown in articular cartilage in healthy and osteoarthritis mice.
The combination of gene expression control techniques with human ipscs allows for accurate disease modeling and engineering in pluripotent cells and subsequently derived specialized cell types.
Disclosure of Invention
Aiming at the problems of limited immunoregulation capability and the like of natural MSC from tissue sources in osteoarthritis treatment, the invention inserts exogenous human IL-10 genes into iPSC at fixed points by using a CRISPR system, and then induces and differentiates iPSC cell lines to obtain MSC capable of over-expressing IL-10 protein, and has better treatment effect compared with the MSC from natural sources. The invention develops an IL-10 protein over-expression MSC cell strain from an iPSC, can express an anti-inflammatory factor IL-10 protein at a high level, can better protect articular cartilage, relieve the pathogenesis of osteoarthritis and repair joint injury.
In order to achieve the above object, one embodiment of the present invention provides a construction method of an iPSC-derived IL-10 protein over-expressed MSC cell line, which is characterized in that a plasmid containing Cas 9 protein and targeted genomic safety site sgRNA is introduced into a cell using a CRISPR system, and a donor template plasmid encoding an IL-10 protein sequence is inserted to obtain an iPSC cell line into which an exogenous IL-10 gene is inserted at a fixed point, and then induced to differentiate the iPSC cell line, thereby obtaining an MSC over-expressing the IL-10 protein.
The construction method of the IL-10 protein over-expression MSC cell strain derived from the iPSC is characterized in that the genome safety site is a site in the middle of an intron No. 1 of the AAVS1 safety site.
The construction method of the IL-10 protein over-expression MSC cell strain from the iPSC is characterized in that the CACCG sequence is added at the front end of a designed sgRNA sequence to serve as an F primer, and the AAAC sequence is added at the front end of a reverse complementary sequence of the sgRNA to serve as an R primer.
The construction method of the IL-10 protein over-expression MSC cell strain from the iPSC is characterized in that a CRISPR gene editing vector selects pX330 obtained from Addgene, and the sequence between two Bbs I sites, namely 245 sites and 267 sites, is replaced by a corresponding sgRNA sequence through a conventional molecular cloning method to obtain the sgRNA vector, wherein the vector sequence is shown as SEQ ID NO: shown at 9.
The construction method of the IL-10 protein over-expression MSC cell strain from the iPSC is characterized in that the donor template plasmid mainly comprises a left homologous arm, an IL-10 protein expression sequence and a right homologous arm.
The construction method of the IL-10 protein over-expression MSC cell strain derived from the iPSC is characterized in that the IL-10 protein expression sequence mainly comprises a promoter, an IL-10 protein CDS region sequence and a terminator, wherein the promoter is a CMV promoter sequence, and the terminator is a bGH Poly (A) signal sequence.
The construction method of the IL-10 protein over-expression MSC cell strain from the iPSC is characterized in that the donor template plasmid skeleton is a pUC57 vector obtained from Addgene, a left homology arm is synthesized, 1000bp is synthesized, an IL-10 protein insertion sequence is synthesized, a right homology arm is synthesized, 500bp is synthesized, the three sequences are sequentially inserted between EcoR I sites and Hindlll sites of the pUC57 vector through a molecular cloning method, and a donor template plasmid carrying a left homology arm, a right homology arm and an IL-10 homology repair template is obtained, wherein the template plasmid sequence is shown as SEQ ID NO: shown at 10.
The construction method of the IL-10 protein over-expression MSC cell strain from the iPSC is characterized in that the induction differentiation is in vitro induction differentiation, and comprises the following steps: culturing iPSC cell strain, inducing to form neural crest cell line, amplifying, culturing and storing neural crest cell, and differentiating neural crest cell into MSC to obtain MSC capable of over-expressing IL-10 protein.
The construction method of the IL-10 protein over-expression MSC cell strain derived from the iPSC is characterized in that the PCR detection of the obtained iPSC cell comprises the following steps: extracting genome DNA of each hole cell, PCR amplification and sequencing, and selecting cell clone with homozygous insertion sequence. The corresponding cross homology arm primer sequence is shown as SEQ ID NO:11 to SEQ ID NO: shown at 16.
The invention also provides an application of the MSC cell strain obtained by the construction method of the IL-10 protein over-expression MSC cell strain from the iPSC in the cell medicine for treating the osteoarthritis disease.
The invention has the following advantages:
1. the cell strain of the invention is a known safe site for inserting exogenous genes into human genome, can overexpress IL-10 protein and does not influence normal physiological functions of cells.
2. The invention provides the MSC capable of expressing the IL-10 protein at a high level, which is beneficial to joint protection and injury repair of osteoarthritis patients.
Drawings
FIG. 1 is a schematic diagram of a map of a No. 1 sgRNA plasmid targeting the AAVS1 site;
FIG. 2 is a schematic diagram of a homologous repair template plasmid map targeting the AAVS1 site;
FIG. 3 is a schematic diagram of PCR sequencing of AAVS1 site insertion of exogenous human IL-10 gene;
FIG. 4 is a morphology diagram of differentiation of positive clone iPSC cell lines targeting AAVS1 site toward MSC;
FIG. 5 is a graph of MSC cell differentiation potential;
FIG. 6 is a graph showing the relative expression level of IL-10 gene in MSC;
FIG. 7 is a WB map of MSC cell line IL-10 protein;
FIG. 8 is a graph showing histological index evaluation of a mouse osteoarthritis model after injection of MSC over-expressing IL-10.
Detailed Description
The present invention will be further described with reference to examples and drawings, but the present invention is not limited to the examples.
Example 1: design of targeting AAVS1 site sgRNA
1) The human genome AAVS1 gene in NCBI website is used as a template, the introns between the exons 1 and 2 are selected, the DNA sequence positioned in the middle of the introns is used as a target, and the gRNA is utilized to design the website on linehttp:// crispor.tefor.net/Sgrnas were designed.
2) Adding CACCG sequence as F primer to the front end of the selected sgRNA sequence, adding AAAC as R primer to the front end of the reverse complementary sequence of the sgRNA, and the related primer sequence is shown as SEQ ID NO in the sequence table: 1 to SEQ ID NO: shown at 6.
Example 2: construction of targeting AAVS1 locus sgRNA vector and detection of cleavage efficiency
1) Plasmids expressing both Cas 9 and sgrnas were selected from pX330 of Addgene.
2) The sequence between the two Bbs I sites (245 site and 267 site) of the pX330 plasmid is replaced by the corresponding sgRNA sequence by a conventional molecular cloning method, and the sgRNA vector is obtained.
3) And (3) respectively transfecting the vector successfully constructed in the previous step into HEK293T cells with the same cell quantity by using a Lip 3000 liposome transfection method, and collecting the cells after 48 hours of transfection to extract genome DNA.
4) Amplifying target fragments by using PCR, wherein corresponding primers are shown as SEQ ID NO:7 and SEQ ID NO: shown at 8. After recovery and purification, the efficiency of NHEJ generation after target site cleavage is detected by using a T7E1 enzyme digestion detection mode, and according to gray level analysis of a band after agarose electrophoresis, a No. 1 sgRNA with higher cleavage efficiency is selected for subsequent experiments, and the corresponding vector sequence is shown as SEQ ID NO:9, the plasmid map is shown in FIG. 1.
Example 3: construction of IL-10 donor template plasmid
1) Among the IL-10 protein insertion sequences, the promoter was selected from CMV promoters with higher transcription levels, and the terminator sequence was selected from the bGH poly (A) signal sequence.
2) IL-10 gene homologous repair template vector pUC57 vector obtained from Addgene was selected as backbone.
3) Synthesizing an AAVS1 left homology arm, 1000bp, synthesizing an IL-10 protein insertion sequence shown in 1), synthesizing an AAVS1 right homology arm, 500bp, and sequentially inserting the three sequences between EcoR I sites and Hindlll sites of a pUC57 vector by a conventional molecular cloning method to obtain a donor plasmid carrying the left and right homology arms and a homologous recombination template, wherein the corresponding vector sequences are shown as SEQ ID NO:10, and the plasmid map is shown in FIG. 2.
Example 4: co-transduction of sgRNA plasmids and donor template plasmids
1) ipscs were cultured to 90% confluence using standard methods, cells were digested and counted.
2) After cell counting, 1X 10 was removed 6 - 1 × 10 7 And centrifuging the individual cells at a low speed, taking out the centrifuge tube into a biosafety cabinet, opening a tube cover, and sucking and discarding the supernatant.
3) The sgRNA plasmid and the homologous donor template plasmid are respectively absorbed by 0.5 ug-5 ug according to different proportions, and specifically 0.5 ug, 0.6 ug, 0.7 ug, 0.8 ug, 0.9 ug, 1 ug, 1.1 ug, 1.2 ug, 1.3 ug and the like can be designed and added into an electrotransfer buffer.
4) After being blown and evenly mixed for several times by a liquid-transferring gun, all liquid is sucked out and added into the obtained cell sediment, after the cells are resuspended, the cell sediment is transferred into an electrotransfer cup, then the electrotransfer cup is placed into a transfection chamber of an electrotransfer instrument for electrotransfer operation, after the electrotransfer operation is finished, the electrotransfer cup is taken out and placed into a biological safety cabinet, and the cells are transferred to a culture dish.
Example 5: clone isolation and identification
1) The cells after electrotransformation were changed daily to medium, after two days of culture, the cells were digested, counted and 5000 cells were inoculated in 10 cm dishes.
2) Culturing for 7 days, picking up the monoclonal in a laminar flow workbench, inoculating the monoclonal into 24-hole plates respectively, culturing for 7 days, and verifying positive clones.
3) And (3) carrying out PCR detection on the obtained iPSC cells, extracting genome DNA of each hole cell by using a DNA extraction kit, carrying out PCR amplification and sequencing, and selecting cell clones with homozygous insertion sequences. The corresponding cross homology arm primer sequence is shown as SEQ ID NO:11 to SEQ ID NO:16, the sequencing result of the positive clone is shown in FIG. 3, which shows that the expression sequence of IL-10 is correctly inserted into the target site.
Example 6: differentiation of iPSC into neural crest cells
1) Performing induced differentiation experiment when the above gene is inserted positive and cultured to 70% -80% confluency by detected iPSC clone, sucking the maintenance medium, washing off residual medium with PBS solution, adding Accutase or TrypLE digestive juice, standing at 37deg.C, and 5% CO 2 Incubate in an incubator at concentration and saturation for 3-8 minutes, gently shake to detach the cells from the bottom of the plate.
2) The cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 1000 revolutions per minute for 5 minutes.
3) Sucking supernatant, adding 1 mL fresh neural crest cell induction culture medium to resuspend cells, gently beating for 1-2 times, counting cells, inoculating into a fibroblast/iMatrix coated orifice plate with an inoculation density of 0.1-110 4 cells/cm 2 。
4) Continuously culturing for 10 days, and changing fresh culture medium daily with culture medium usage amount of 0.2-0.4 mL/cm 2 。
Example 7: differentiation of neural crest cell lines into MSC and MSC identification
1) Replacing fresh MSC differentiation medium with the cells with neural crest cell line morphology, and continuing at 37 ℃ and 5% CO 2 Culturing in an incubator with saturated humidity. Fresh medium is replaced every day, and the usage amount of the medium is changedIs 0.2-0.4 mL/cm 2 After the continuous culture for 6 days, the cells were observed and photographed, and the cell morphology was the MSC cell morphology as shown in FIG. 4.
2) The differentiation of MSCs was induced using bone, cartilage and adipocyte differentiation medium, and the obtained cells were demonstrated to have the ability to differentiate into osteogenic, chondrogenic and adipogenic cells, and the results are shown in fig. 5, which show that positive clones have the ability to differentiate into three lines.
3) qPCR measurement is carried out on the cells meeting the MSC identification standards in 1) and 2), and after total RNA is extracted from partial cells and cDNA is obtained through reverse transcription, qPCR measurement is carried out on the target gene expression quantity. The quantitative primer is shown as SEQ ID NO:17 to SEQ ID NO:20, and the quantitative results are shown in FIG. 6, which shows that the target RNA transcription level is high.
4) The cell with higher qPCR detection result content in 3) is further verified by a WB method, whether the protein expression amount of the target gene IL-10 is increased is detected, and the specific result is shown in figure 7, wherein the IL-10 protein expression amount is obviously increased relative to the wild type MSC.
Example 8: model mice are used for verifying the therapeutic effect of IL-10 over-expression MSC cell strain on osteoarthritis
1) The sgrnas targeting the mouse AAVS1 site and the corresponding homologous repair template plasmids inserted into the exogenous mouse IL-10 gene were designed separately according to the methods described in examples 1-3 above.
2) Mouse MSC cells were cultured and then plasmid transfected, and the success of the exogenous IL-10 gene transfer into mouse MSC was confirmed by detecting the total level of mRNA expression.
3) A model of medial meniscus instability (DMM) in mice was established surgically, a mouse model commonly used in osteoarthritis studies. Respectively injecting Phosphate Buffer (PBS) into joint cavity, wild type MSC and MSC over-expressing IL-10 twice a week for 8 weeks, and by histologically analyzing several aspects of OARSI score, chondrocyte number, proteoglycan loss and cartilage thickness of osteoarthritis, we confirmed that the MSC group over-expressing IL-10 protein can significantly protect articular cartilage structure and inhibit the development of osteoarthritis more effectively.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (10)
- The construction method of the IL-10 protein over-expression MSC cell strain from the iPSC is characterized in that a CRISPR system is used for introducing plasmids containing Cas 9 protein and target genome safety site sgRNA into cells, inserting donor template plasmids encoding IL-10 protein sequences to obtain the iPSC cell strain with the exogenous IL-10 gene inserted at fixed points, and inducing differentiation of the iPSC cell strain to obtain the MSC over-expressing the IL-10 protein.
- 2. The method for constructing an iPSC-derived IL-10 protein over-expressed MSC cell line according to claim 1, wherein the genomic safety site is a site in the middle of intron No. 1 of AAVS1 safety site.
- 3. The method for constructing an iPSC-derived IL-10 protein overexpressed MSC cell line according to claim 1, wherein a CACCG sequence is added as an F primer to the front end of the designed sgRNA sequence, and an AAAC sequence is added as an R primer to the front end of the reverse complement of the sgRNA sequence.
- 4. The method for constructing an iPSC-derived IL-10 protein over-expressed MSC cell line according to claim 1, wherein the CRISPR gene editing vector selects pX330 obtained from Addgene, and the sequence between two bbsi sites, namely 245 and 267 sites, is replaced with the corresponding sgRNA sequence by a conventional molecular cloning method, to obtain the sgRNA vector, the vector sequence of which is as shown in SEQ ID NO: shown at 9.
- 5. The method of claim 1, wherein the donor template plasmid comprises a left homology arm, an IL-10 protein expression sequence, and a right homology arm.
- 6. The method according to claim 5, wherein the IL-10 protein expression sequence mainly comprises a promoter, an IL-10 protein CDS region sequence and a terminator, wherein the promoter is a CMV promoter sequence and the terminator is a bGH Poly (A) signal sequence.
- 7. The construction method of an iPSC-derived IL-10 protein over-expression MSC cell strain according to claim 1, wherein the donor template plasmid skeleton is a pUC57 vector obtained from Addgene, a left homology arm is synthesized, 1000bp is synthesized, an IL-10 protein insertion sequence is synthesized, a right homology arm is synthesized, 500bp is synthesized, the three sequences are sequentially inserted between EcoR I sites and Hindlll sites of the pUC57 vector by a molecular cloning method, and a donor template plasmid carrying a left homology arm, a right homology arm and an IL-10 homology repair template is obtained, wherein the template plasmid sequence is shown as SEQ ID NO: shown at 10.
- 8. The method for constructing an iPSC-derived IL-10 protein over-expressed MSC cell line according to claim 1, wherein the induced differentiation is in vitro induced differentiation, comprising the steps of: culturing iPSC cell strain, inducing to form neural crest cell line, amplifying, culturing and storing neural crest cell, and differentiating neural crest cell into MSC to obtain MSC capable of over-expressing IL-10 protein.
- 9. The method for constructing an iPSC-derived IL-10 protein over-expressed MSC cell line according to claim 1, wherein the PCR detection is performed on the obtained iPSC cells, comprising the steps of: extracting genome DNA of each hole cell, performing PCR amplification and sequencing, selecting cell clone with homozygous insertion sequence, and corresponding cross homology arm primer sequence as shown in SEQ ID NO:11 to SEQ ID NO: shown at 16.
- 10. Use of an MSC cell line obtained by the construction method of an iPSC-derived IL-10 protein over-expressed MSC cell line according to any one of claims 1 to 9 in a cell medicament for the treatment of osteoarthritis disease.
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