CN113846064A - FGF18 gene modified mesenchymal stem cell and preparation method and application thereof - Google Patents
FGF18 gene modified mesenchymal stem cell and preparation method and application thereof Download PDFInfo
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- CN113846064A CN113846064A CN202111098403.0A CN202111098403A CN113846064A CN 113846064 A CN113846064 A CN 113846064A CN 202111098403 A CN202111098403 A CN 202111098403A CN 113846064 A CN113846064 A CN 113846064A
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to FGF18 gene modified mesenchymal stem cells, a preparation method thereof and application thereof in treating osteoarthritis. The invention discloses a FGF18 gene modified mesenchymal stem cell, which is an umbilical cord mesenchymal stem cell over-expressing FGF18 without influencing the phenotype and differentiation capacity of MSC. By using the modified mesenchymal stem cells to over-express FGF18 gene and using a variant or autologous local joint cavity injection mode to treat osteoarthritis, the cartilage defect of arthritis patients can be fundamentally improved, the curative effect is improved, and the pain and the side effect of the patients in the treatment process are reduced. The invention provides technical support for preparing the medicament for promoting the osteoarthritis repair.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to FGF18 gene modified mesenchymal stem cells, a preparation method thereof and application thereof in treating osteoarthritis.
Background
Osteoarthritis (OA) is a degenerative disease caused by age, metabolic abnormality or congenital, and the main cause is degenerative damage of joint cartilage. The clinical manifestations include joint pain and joint deformity. Currently, there are over 4 billion osteoarthritis patients worldwide. And there is no effective drug on the market to treat or block the progression of osteoarthritis.
Currently, the conventional treatments for osteoarthritis patients include oral administration of anti-inflammatory analgesic and glucosamine drugs, intra-articular injection of sodium hyaluronate, physical therapy, surgery, and the like. The osteoarthritis treatment drug which is only recommended by the American orthopedists institute of America (AAOS) guidelines is a non-steroidal anti-inflammatory analgesic drug which can delay the course of disease and improve the symptoms of patients to a certain extent, but can not reverse the pathological process, and most of the drugs have obvious side effects. When conventional therapies are ineffective, patients with joint deformity and joint dysfunction need surgical treatment. Surgical treatment is mainly through arthroscopy (speculum) or open surgery, and although temporary pain relief is achieved, the long-term results are not ideal.
In regenerative medicine and clinical treatment, Mesenchymal Stem Cells (MSCs) have the ability of self-renewal, multi-lineage differentiation and low immunogenicity, so that they have a wide clinical application prospect in various alternative treatments. Fibroblast growth factor 18(FGF18) was shown to be effective in stimulating cartilage regeneration, significantly increasing the thickness of cartilage at the knee joint of a patient, preventing further cartilage degradation. Given that the currently available treatments for osteoarthritis are limited by several drawbacks, it is imperative to find a new, safe, and fundamental treatment for osteoarthritis.
Disclosure of Invention
In view of the problems of the prior art in methods for treating osteoarthritis, the invention aims to provide the FGF18 gene modified mesenchymal stem cell, a preparation method thereof and application in treating osteoarthritis. The FGF18 is used for modifying the mesenchymal stem cells, so that the modified mesenchymal stem cells specifically and stably express FGF18 at a high level. Experiments prove that the FGF18 gene modified mesenchymal stem cells achieve the effect of treating osteoarthritis through local joint cavity injection. The mesenchymal stem cells modified by FGF18 gene can fundamentally improve the cartilage defect of arthritis patients, improve the curative effect and reduce the pain and side effect of the patients in the treatment process. Not only contributes to the wide application of the mesenchymal stem cells in clinical treatment, but also contributes to the promotion of the industrialization of the mesenchymal stem cells.
In order to achieve the purpose, the invention adopts the following technical scheme.
An FGF18 gene modified mesenchymal stem cell, wherein the FGF18 gene modified mesenchymal stem cell is a mesenchymal stem cell over-expressing FGF 18.
Further, the FGF18 gene modified mesenchymal stem cell has the sequence of the FGF18 gene as shown in SEQ ID NO: 1, or SEQ ID NO: 1 by replacing one or more nucleotides.
Further, the gene modification is carried out by virus transfection, lipofection, electrotransfer, gene editing or mRNA transfection.
Preferably, the genetic modification is viral transfection technology.
More preferably, the genetic modification is a lentiviral transfection technique.
Further, the FGF18 gene is derived from an FGF18 lentiviral vector or an FGF18 plasmid vector.
Further, the mesenchymal stem cells are bone marrow tissue, adipose tissue, umbilical cord tissue or placenta tissue-derived mesenchymal stem cells.
Preferably, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells (hUC-MSCs).
Further, the FGF18 gene modified mesenchymal stem cells are used for detecting the transfection efficiency of the FGF18 gene by using GFP green fluorescence or Cherry red fluorescence as a reporter gene.
Further, the preparation method of the FGF18 gene modified mesenchymal stem cell comprises the following steps.
Step 1, obtaining primary human mesenchymal stem cells.
And 2, subculturing the primary human mesenchymal stem cells.
And 3, constructing the FGF18 overexpression lentivirus.
And 4, transfecting the mesenchymal stem cells by the FGF18 overexpression lentiviruses, and screening a stable transfer cell line to obtain the FGF18 gene modified mesenchymal stem cells.
A composition comprising FGF18 genetically modified mesenchymal stem cells, wherein the FGF18 genetically modified mesenchymal stem cells are mesenchymal stem cells overexpressing FGF 18.
The FGF18 gene modified mesenchymal stem cell, the FGF18 gene modified mesenchymal stem cell prepared by the preparation method or the composition are applied to preparation of a medicament for treating osteoarthritis.
Further, the dosage form of the medicine is injection.
Further, the FGF18 gene modified mesenchymal stem cells are administrated by local joint cavity allogenic injection to achieve the effect of treating osteoarthritis.
The FGF18 gene modified mesenchymal stem cells adopt a co-culture mode to detect the influence of FGF18 modified mesenchymal stem cells on the proliferation of chondrocytes in vitro. And detecting the expression condition of the target gene FGF18 by using a protein immunoblotting method.
The FGF18 gene modified mesenchymal stem cells are subjected to local injection by establishing an osteoarthritis model, and the influence of the FGF18 modified mesenchymal stem cells on cartilage regeneration in vivo is detected.
The specific process of subculturing the primary human mesenchymal stem cells comprises the following steps: the obtained primary human umbilical cord mesenchymal stem cells are subcultured in a culture dish, and the human umbilical cord mesenchymal stem cells are obtained by amplification in the culture dish after culture and are first generation human umbilical cord mesenchymal stem cells, namely P1 generation; and performing secondary subculture on the obtained P1 generation human umbilical cord mesenchymal stem cells to obtain P2 generation human umbilical cord mesenchymal stem cells and P3 generation human umbilical cord mesenchymal stem cells.
The FGF18 described in the present invention belongs to the FGF family, members of which are involved in numerous cellular activities and life processes. FGF18, as a member of this group, has been shown to be effective in stimulating cartilage regeneration, significantly increasing the thickness of cartilage in the knee joint of a patient, and preventing further cartilage degradation.
The mesenchymal stem cell is an important member of a stem cell family, is derived from a mesoderm at the early development stage, is a stem cell with differentiation pluripotency, can perform foreign body feedback due to low immunological rejection reaction, and achieves a treatment effect, and at present, the application of FGF18 gene in modification of the mesenchymal stem cell for treating osteoarthritis is not reported.
The FGF18 gene in the invention is derived from an FGF18 lentiviral vector or an FGF18 plasmid vector (i.e., a lentiviral vector or a plasmid vector containing the coding sequence of FGF18 gene).
The pharmaceutical composition of the present invention may be in any suitable dosage form. Preferred are injections, suspensions, emulsifiers and the like. The pharmaceutical composition of the present invention can be administered into the body by known means. For example, local joint space autologous or allogeneic injections to achieve therapeutic effects in osteoarthritis can also be administered by other delivery methods. Such administration may be via a single dose or multiple doses.
It will be understood by those skilled in the art that the actual dosage to be administered herein may vary widely depending upon a variety of factors, such as the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.
Compared with the prior art, the invention has the following beneficial effects.
The FGF18 gene modified mesenchymal stem cell provided by the invention is a mesenchymal stem cell over-expressing FGF18, and does not influence the phenotype and differentiation capability of the MSC. Osteoarthritis is treated by means of local joint cavity injection of variant by using modified mesenchymal stem cells to over-express FGF18 gene. Aggrecanan is an ideal marker for representing the state of a cartilage matrix, and immunohistochemical staining results show that after FGF18 is injected into a joint cavity to overexpress modified hUC-MSCs, the expression of Aggrecan is obviously increased and basically returns to a normal level, and is obviously superior to an untreated group and an untreated group treated by hUC-MSCs. Meanwhile, the TUNEL staining result shows that a large amount of apoptosis occurs in chondrocytes in articular cartilage of a model untreated group (PBS), and the number of apoptotic chondrocytes is remarkably reduced after FGF18 is injected into articular cavities to over-express modified hUC-MSCs. The invention can fundamentally improve the cartilage defect of arthritis patients, improve the curative effect and simultaneously reduce the pain and side effect of the patients in the treatment process. The invention provides technical support for preparing the medicament for promoting the osteoarthritis repair.
Drawings
Figure 1 is a morphological feature (x 100) of a cultured primary umbilical cord mesenchymal stem cell.
Fig. 2 is a morphological feature (x 100) of umbilical cord mesenchymal stem cells after passage expansion to passage 2 (P2).
FIG. 3 shows the expression of Green Fluorescent Protein (GFP) in cells after transfection of lentivirus by fluorescence microscopy.
FIG. 4 shows measurement of FGF18 expression level of cells by Western blotting (Western Blot).
FIG. 5 shows that FGF18 gene overexpression modified human umbilical cord mesenchymal stem cells promote rat knee osteoarthritis cartilage repair.
FIG. 6 shows that FGF18 gene overexpression modified human umbilical cord mesenchymal stem cells restore rat knee osteoarthritis proteoglycan expression.
FIG. 7 shows that the human umbilical cord mesenchymal stem cells modified by FGF18 gene overexpression inhibit rat knee osteoarthritis chondrocyte apoptosis.
Detailed Description
In order to facilitate the understanding of the technical scheme of the present invention, the mesenchymal stem cell modified by FGF18 gene, its preparation method and its application in treating osteoarthritis are further described in the following with reference to the accompanying drawings and specific examples. The source of mesenchymal stem cells used in the present invention is illustrated: the human tissues used in the invention are clinical waste or isolated human tissues. For example, the umbilical cord in the examples was obtained from the gynecological ward of the gynecology and obstetrics hospital in Shenyang city. The technical scheme of the invention does not relate to the specific operation of the process of acquiring the human tissue. Unless otherwise specified, all biochemical reagents used in the examples are commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 preparation of FGF18 gene-modified mesenchymal stem cells and measurement of FGF18 expression amount.
The experimental equipment and reagents were as follows.
1.5ml EP tube (Bio, China), 15ml or 50ml centrifuge tube (Corning, America), desk top centrifuge (Eppendorf, German), 10cm petri dish (Corning, America), constant temperature shaker (Linbel, China), inverted microscope (leica, German), CO2 incubator (Eppendorf, America), vertical electrophoresis apparatus (Beijing hexakis, China), fluorescence microscope (Nikon, Japan), PCR apparatus (Eppendorf, America), gel imaging system (Bio-rad, America), endotoxin-free plasmid extraction kit (Tiangen, China), trypsin (Gibco, America), Gibco, America, DMEM (Gibco, America), double antibody (Gibco, America), glutamine (Gibco, America) (Mill, America), PVDF (Abli, Japan antibody), Japan antibody (Ulva), Japan antibody (Umber, America).
1. And (3) separating and culturing the umbilical cord-derived mesenchymal stem cells.
The specific steps of the separation and culture of the umbilical cord-derived mesenchymal stem cells are as follows.
(1) The umbilical cord tissue is cut off and washed in a sterile cup with Phosphate Buffer Saline (PBS) until clear (at which time the wash should be clear).
(2) The umbilical cord tissue was soaked in 75% alcohol for 3 minutes and washed twice with PBS containing 10% double antibody to remove alcohol residues. 2-3cm of umbilical cord tissue is cut off by scissors and put into a 10cm sterile culture dish.
(3) Removing vein wall, two arteries and amnion with hemostatic forceps, and peeling off Wharton's jelly. Peeling off 2g of Wharton's jelly, transferring into a 50ml centrifuge tube, and shearing.
(4) 800g, centrifuge for 5min, discard the supernatant and spread the tissue mass evenly.
(5) After 24 hours, 10ml of 10% FBS complete medium was added and incubation at 37 ℃ in a 5% CO2 incubator was continued.
(6) The primary cells grow for about 15-20 days, and as shown in figure 1, fusiform adherent cells grow out around the tissue block and can be subcultured.
(7) The culture medium was aspirated, washed twice with PBS to remove the remaining medium, 3ml of pancreatin was added, and 5ml of stop solution was added after the cells were in suspension.
(8) The cell suspension was transferred to a 50ml centrifuge tube and the flask was rinsed with saline. Centrifuge at 1000rpm for 5min, discard the supernatant and repeat the washing once.
(9) Resuspend cells were blown with complete medium and filtered through a 100 micron screen into a single cell suspension while allowing for tissue mass. The 1-pass 2 or 1-pass 1 culture was continued in a 5% CO2 incubator at 37 ℃.
(10) After that, the medium was replaced with fresh DMEM medium containing 10% fetal bovine serum every 3 days, and the cells were observed under a microscope as shown in FIG. 2.
The extraction, culture, cell bank establishment and other work of the umbilical cord mesenchymal stem cells are all finished in a GMP production workshop of the company. Carrying out detection on exogenous microorganisms, viruses, endotoxins and the like after the cells are separated and cultured and are subcultured and amplified to the 2 nd generation (P2); and simultaneously detecting the immunophenotype, differentiation capacity, cell biological efficacy and the like of the cells. And (3) taking the qualified cells as seed bank cells, and storing the seed bank cells in a liquid nitrogen tank at the temperature of-196 ℃.
2. And (4) cell transfection.
The umbilical cord mesenchymal stem cells are inoculated on a culture dish and are randomly divided into an overexpression control group and an overexpression group. The overexpression control group used unloaded lentiviruses and the overexpression group used lentiviruses containing the FGF18 gene. The transfection method was performed according to the lentivirus transfection instructions. After 2-3 days of lentivirus transfection, cells were observed for Green Fluorescent Protein (GFP) expression using a fluorescence microscope, as shown in FIG. 3. When Green Fluorescent Protein (GFP) expression was the strongest, cells that were not successfully transfected with lentivirus were killed by selection in complete medium containing puromycin and no virus. Successfully transfected cells can be further subcultured.
3. The expression level of FGF18 in cells was determined by Western blotting (Western Blot).
After transfection treatment, each group of cells was collected, lysed with protein lysate (RIPA), centrifuged at 12000rpm for 20min, and the supernatant was collected and assayed for protein content using BCA kit. The protein was loaded at 30. mu.g and subjected to SDS-PAGE. Transferring the protein to the PVDF membrane by a wet transfer method; sealing with 5% skimmed milk powder solution at room temperature for 1h, adding FGF18 and beta-actin primary anti-dilution solution, and incubating overnight at 4 deg.C in a shaking table; PBST is washed, and secondary antibody is added to incubate for 1h in a shaking table at room temperature; PBST was washed thoroughly, reacted with a chemiluminescent substrate and photographed by a chemiluminescent analyzer as shown in FIG. 4.
4. And (5) experimental results.
1) Lentivirus transfection results.
The western blot results showed that the expression of FGF18 was significantly up-regulated in the over-expressed group. This indicates that the cell model of over-expressing FGF18 is successfully established, and the purpose of increasing FGF18 expression is achieved.
2) Proliferation and differentiation of mesenchymal stem cells over-expressed with FGF 18.
According to a proliferation curve obtained by a real-time monitoring system and a statistical analysis result, after FGF18 is over-expressed, the proliferation rate of umbilical cord mesenchymal stem cells is obviously accelerated, and the population doubling time is obviously reduced.
The gene modification method of the invention not only can adopt an overexpression FGF18 lentiviral vector, but also can adopt an overexpression FGF18 plasmid vector. Meanwhile, the source of the mesenchymal stem cells can also be placenta, adipose tissue or peripheral blood. The detailed description of these alternatives is not repeated here in view of the skilled person in the art's knowledge of the corresponding technical measures.
Example 2 study experiment of FGF18 gene-modified mesenchymal stem cells for treatment of osteoarthritis.
The experimental equipment and reagents were as follows.
Horizontal shaker (wonder, china), full-automatic tissue hydroextractor (Leika, German), full-automatic rotary microtome (Leika, German), pathological tissue rinsing and baking apparatus (zhongwei, china), tissue embedding machine (Leika, German), fluorescence microscope (Olympus, Japan), glass slide, cover slip (shitai, china), hemostat, surgical scissors, scalpel, forceps, suture (shitai, china), hematoxylin powder (solibao, china), eosin powder (beisoxol, china), safranin O powder (Sigma, America), toluidine blue staining solution (solibao, china), trypsin digestion solution (solibao, china), universal SP detection kit (zhongshan bridge, DAB), chinese chromogenic kit (zhongshan bridge, china), anti-agregadan antibody (Abcam, Britain), phosphate buffer solution (maixin, PBS).
1. Preparation of FGF18 gene modified mesenchymal stem cell preparation.
Taking 1 × 10 human umbilical cord mesenchymal stem cells modified by FGF18 gene overexpression prepared in example 16Mixing completely to obtain injection.
2. An osteoarthritis model building method.
The instruments needed by the operation are sterilized in advance for standby. Injecting sodium pentobarbital (45 mg/kg) into abdominal cavities of all rats, and shaving the hairs at the knee joints by using electric clippers after the rats are successfully anesthetized for several minutes; fixing the rat on an operating table in a supine posture, bending the knee joint at the right side of the hind limb by 90 degrees, and wiping the iodophor for sterilization; longitudinally cutting an incision of about 4 cm from the inner side of the patella by using an operation blade, tearing a fascia, retracting a patellar ligament to the side, exposing an anterior cruciate ligament, transecting the anterior cruciate ligament by using an operation blade, observing that no obvious bleeding exists, and flushing a joint cavity by using physiological saline; resetting the patella, and verifying whether the molding is successful by a front drawer test; after the success of the model building is confirmed, fascia and skin are sutured by using a special surgical needle and a suture line, the weak rat after the operation is placed on an electric blanket, and the rat returns to a cage after the rat is recovered, and the rat can freely move and eat. Thereafter, the rats were observed 2-3 times a week in the animal room and their health status was recorded.
3. And (5) grouping experiments.
In the experiment, 43 male SD rats of 12 weeks of age were used. After 1 month of building an animal arthritis model, the animal knee osteoarthritis model was set into 3 groups: one group injected 50 μ l of 10-containing solution into knee joint cavity of animal6(ii) individual untreated human umbilical cord mesenchymal stem cells; another group was injected into the knee joint cavity of rat with 50. mu.l of 106The FGF18 overexpression modified human umbilical cord mesenchymal stem cells are obtained, and the rest solution except the cells is PBS; the negative control group was a knee joint injection of knee osteoarthritis model animals with an equal amount of PBS solution. The positive control group was normal SD rats without arthritis molding).
4. Hematoxylin-eosin staining.
Drying the slices in a rinsing and drying instrument for more than 30 min, and performing conventional dewaxing until the slices are soaked in xylene I and xylene II for 15 min respectively, fully dewaxing, then soaking in gradient ethanol of 100% -100% -95% -95% -75% for 2 min respectively, washing off the xylene, and lightly washing with tap water for 1 min; immersing the slices in hematoxylin staining solution for dip-staining for 8 min, wherein before staining, attention needs to be paid to whether the surface of the hematoxylin staining solution has a film forming phenomenon, if the surface of the hematoxylin staining solution has the film forming phenomenon, a surface film needs to be scraped off by newspaper, otherwise, the slices are stained or color blocks are accumulated when the slices are stuck on the slices; washing with tap water for 2 times, each for 1 min; soaking the slices in 1% hydrochloric acid alcohol for differentiation for 5 s; washing with tap water for 2 times, each for 1 min; returning blue for 5 s by 0.5 percent ammonia water; washing with tap water for 2 times, each for 1 min, and observing under the mirror whether blue is clear; immersing the slices into 1% eosin staining solution for dip-dyeing for 1 min; quickly washing with water for 2 times; 75% -95% -95% -100% -100% of upward ethanol dehydration, transparent xylene, sealing with neutral gum and observing under a mirror.
5. Safranin O staining.
Placing the slices in a rinsing and drying instrument, drying the slices for more than 30 min, and dewaxing to water by a conventional method; immersing into hematoxylin staining solution for dip-staining for 8 min to make cell nucleus appear dark blue; washing with tap water for 2 times, each for 1 min; soaking the slices in 1% hydrochloric acid alcohol for differentiation for 5 s; washing with tap water for 2 times, each for 1 min; returning blue for 5 s by 0.5 percent ammonia water; washing with tap water for 2 times, each for 1 min, and observing under the mirror whether blue is clear; immersing the slices into 0.1% safranin O staining solution for 30 s-1 min; washing with water quickly, and observing the dyeing condition under a mirror; 75% -95% -95% -100% -100% of upward ethanol dehydration, transparent xylene and sealing with neutral gum.
6. Toluidine blue stain.
Placing the slices in a floating drying instrument, drying the slices for more than 30 min, and dewaxing to water conventionally; toluidine blue staining solution is fast drop-dyed, which can cause too deep dyeing for a long time; washing with water quickly, and observing the dyeing condition under a mirror; ascending ethanol for dehydration, transparent xylene and neutral gum for sealing.
7. Immunohistochemical staining.
Placing the slices in a rinsing and drying instrument for drying the slices overnight, and dewaxing to water conventionally; the antigen repairing process comprises the following steps: wiping the periphery of the hydrated section, placing the hydrated section in a moisture preservation box, dropwise adding trypsin (0.1%) on the section, and incubating for 1h at room temperature; after the incubation, washing the cells on a horizontal shaking table for 3 times, each time for 5min, in a vertical dye vat containing 1 × PBS (pH 7.2-7.4); dropping endogenous peroxidase blocking agent according to the specification of the second antibody, dropping 1 drop of endogenous peroxidase blocking agent into each slice, and incubating for 10 min at room temperature; the sections were washed 3 times with 1 × PBS on a horizontal shaker for 5min each time; dropwise adding normal goat serum working solution for sealing, sealing at room temperature for 15 min, and removing serum without washing; primary anti-dilution: primary anti-agrrechan (ab 36861) was diluted 1:200 with 1 × PBS; dropwise adding a proper amount of diluted primary antibody, covering a cover of a moisturizing box, and incubating overnight in a refrigerator at 4 ℃; washing with 1 × PBS for 3 times in the next day, 5min each time, and spin-drying; dropwise adding a biotinylation general secondary antibody working solution, and incubating for 15 min at room temperature; washing with 1 × PBS on a horizontal shaker for 3 times, 5min each time; dropwise adding HRP-labeled streptavidin working solution, and incubating for 15 min at room temperature; washing with 1 × PBS for 3 times, 5min each time; preparing DAB color development working solution: adding 1 drop of DAB concentrated solution (reagent 1) into 1 mL of DAB substrate solution (reagent 2), and uniformly mixing to obtain DAB working solution; dropwise adding a freshly prepared DAB developing working solution, incubating at room temperature for 5-20 min for developing, and observing the developing condition under a mirror; the dyeing is stopped by washing with tap water, the gradient ethanol is dehydrated, the dimethylbenzene is transparent, and the neutral resin is encapsulated.
8. TUNEL staining.
Placing the slices in a rinsing and drying instrument, drying the slices for more than 2 hours, and dewaxing to water conventionally; wiping the water around the slices, dripping 0.1% trypsin, and pretreating the slices for 60 min at normal temperature; washing with PBS buffer solution twice, each for 3 min; preparation of TUNEL reaction mixture: component 1 (enzyme solution): the proportion of the component 2 (marking solution) is 1:9, the two are mixed uniformly, and the mixture is ready to use after being prepared and placed on ice temporarily; wiping the tissue periphery, and dripping TUNEL reaction mixed solution, wherein each slice is 25 mu L; cutting the PE gloves into a shape slightly larger than the slices, and covering the slices dropwise added with the reaction solution to ensure that the reaction solution can completely and uniformly cover tissues; placing the slices in a moisture preservation box, and incubating for 60 min in a dark place at room temperature; washing with PBS buffer solution for 3 times; and (4) dropwise adding an anti-fluorescence quenching agent, sealing, and photographing and observing under a fluorescence microscope.
9. Experimental results and analysis.
Knee joint samples were collected from each group of rats 3 months later for decalcification and paraffin embedding and staining of sections. The results are shown in fig. 5-7, and the staining shows that the thickness of the articular cartilage and the number of the cartilage cells in the model-making untreated group (PBS) are both significantly reduced, the articular cartilage matrix is seriously lost, the articular cartilage surfaces of the hUC-MSCs treated groups injected with untreated hUC-MSCs and the hUC-MSCs treated groups injected with FGF18 over-expression modification are smooth and flat, the cartilage matrix is increased to a certain extent, and the cartilage repair effect of the hUC-MSCs injected and modified with FGF18 over-expression is significantly better than that of the hUC-MSCs injected and untreated. Aggrecanan is an ideal marker for representing the state of a cartilage matrix, and immunohistochemical staining results show that after FGF18 is injected into a joint cavity to overexpress modified hUC-MSCs, the expression of Aggrecan is obviously increased and basically returns to a normal level, and is obviously superior to an untreated group and an untreated group treated by hUC-MSCs. Meanwhile, the TUNEL staining result shows that a large amount of apoptosis occurs in chondrocytes in articular cartilage of a model untreated group (PBS), and the number of apoptotic chondrocytes is remarkably reduced after FGF18 is injected into articular cavities to over-express modified hUC-MSCs.
The foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way and substantially, so that those skilled in the art may make various changes, modifications and equivalents without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and variations of the above embodiments according to the essential technology of the present invention are within the scope of the technical solution of the present invention.
Sequence listing
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Claims (11)
1. An FGF18 gene modified mesenchymal stem cell, wherein the FGF18 gene modified mesenchymal stem cell is a mesenchymal stem cell over-expressing FGF 18.
2. The FGF18 gene-modified mesenchymal stem cell of claim 1, wherein the sequence of the FGF18 gene has the sequence set forth in SEQ ID NO: 1, or SEQ ID NO: 1 by replacing one or more nucleotides.
3. The FGF18 gene-modified mesenchymal stem cell of claim 1 or 2, wherein the gene modification is performed by viral transfection, lipofection, electrotransfer, gene editing, or mRNA transfection.
4. The FGF18 gene-modified mesenchymal stem cell of any one of claims 1-3, wherein the mesenchymal stem cell is a bone marrow tissue-derived mesenchymal stem cell, an adipose tissue-derived mesenchymal stem cell, an umbilical cord tissue-derived mesenchymal stem cell, or a placental tissue-derived mesenchymal stem cell.
5. The FGF18 gene-modified mesenchymal stem cell of any one of claims 1-4, wherein the mesenchymal stem cell is a human umbilical cord mesenchymal stem cell (hUC-MSCs).
6. The FGF18 gene-modified mesenchymal stem cell of any one of claims 1-5, wherein the transfection efficiency of FGF18 gene is tested by using GFP green fluorescence or Cherry red fluorescence as a reporter gene in the FGF18 gene-modified mesenchymal stem cell.
7. The method for preparing FGF18 gene-modified mesenchymal stem cells according to any one of claims 1-6, comprising the steps of:
step 1, obtaining primary human mesenchymal stem cells;
step 2, subculturing primary human mesenchymal stem cells;
step 3, constructing FGF18 overexpression lentiviruses;
and 4, transfecting the mesenchymal stem cells by the FGF18 overexpression lentiviruses, and screening a stable transfer cell line to obtain the FGF18 gene modified mesenchymal stem cells.
8. A composition comprising the FGF18 gene-modified mesenchymal stem cell of any one of claims 1-6 or the FGF18 gene-modified mesenchymal stem cell produced by the production method of claim 7.
9. Use of the FGF18 gene-modified mesenchymal stem cell of any one of claims 1-6, the FGF18 gene-modified mesenchymal stem cell prepared by the preparation method of claim 7, or the composition of claim 8 in the preparation of a medicament for the treatment of osteoarthritis.
10. The use of claim 10, wherein the medicament is in the form of an injection.
11. The use of claim 9 or 10, wherein the FGF18 genetically modified mesenchymal stem cell is administered by local joint cavity allo-injection.
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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 |
| WO2024051748A1 (en) * | 2022-09-06 | 2024-03-14 | 深圳瑞吉生物科技有限公司 | Mrna for treating osteoarthritis, preparation method therefor, and use thereof |
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