Mesenchymal stem cell immortalization method, related product and application

Abstract

The invention relates to a mesenchymal stem cell immortalization method, a related product and application, and belongs to the technical field of biotechnology and genetic engineering. The scheme of the invention provides a modification method capable of simply and quickly realizing immortalization of the mesenchymal stem cells through one-step double gene intervention, the immortalized mesenchymal stem cells are prepared by adopting a one-step method of combining gene knock-down and gene overexpression, and the gene modified plasmid constructed by the invention integrates gene knock-down and gene overexpression, so that the gene modified plasmid has a good application prospect. And the immortalized cell strain can be obtained by infecting the mesenchymal stem cells for one time.

Description

Technical Field

[0001] This application relates to a method for immortalizing mesenchymal stem cells and related products and applications, belonging to the fields of biotechnology and genetic engineering technology. Background Technology

[0002] Mesenchymal stem cell-derived exosomes have been widely used in biomedical research and development. As a fundamental raw material for exosome production, mesenchymal stem cells (MSCs) suffer from reduced cell proliferation and functional defects due to replicative senescence, severely impacting their application in industrial pharmaceutical manufacturing. Currently, the use of MSCs is typically limited to P5-P8 cells. Therefore, the biggest drawback of using MSC-derived exosomes in industrial pharmaceutical preparation lies in the limited cell productivity. Solving the problem of immortalizing MSCs is key to overcoming this limitation in industrial pharmaceutical production.

[0003] Currently, there are few publicly available studies on the immortalization of mesenchymal stem cells (MSCs), and there is no simple, rapid, one-step method for this purpose. For example, existing technologies for preparing immortalized cells other than MSCs include: Patent WO2014109696A1 discloses a method for immortalizing B cells and its uses; Patent CN103282498A discloses a method and carrier for cell immortalization, which does not involve MSCs; Patent EP1497413B1 discloses a method for preparing antibody-secreting cells that proliferate indefinitely; and Patent CN101189329A discloses a feeder cell that proliferates indefinitely, which is an embryonic fibroblast. There are many other studies on induced pluripotent stem cells in the existing technology, such as CN116829721A, US20210205369A1 and CN102286532B. Since induced pluripotent stem cells involve cell reprogramming, and their main purpose is to obtain the ability to redifferentiate, this may potentially affect the use of mesenchymal stem cells to secrete exosomes for production. As a completely different approach to modification, it is not of reference value.

[0004] Therefore, it is necessary to provide a method for the immortalization of mesenchymal stem cells, which can maintain the normal exosome secretion function of mesenchymal stem cells while minimizing changes to the mesenchymal stem cells to ensure their original state, and can also greatly improve the continuous proliferation capacity of mesenchymal stem cells, thereby solving the problem of limited production capacity of industrial pharmaceuticals using mesenchymal stem cells to produce exosomes in existing technologies. Summary of the Invention

[0005] To address the aforementioned issues, this application provides a method for immortalizing mesenchymal stem cells, along with related products and applications. The proposed method offers a simple and rapid one-step dual gene intervention approach to achieve mesenchymal stem cell immortalization. It employs a one-step gene knockdown combined with gene overexpression method to prepare immortalized mesenchymal stem cells. Furthermore, the gene-modified plasmid constructed in this invention integrates gene knockdown and gene overexpression, allowing for the acquisition of immortalized cell lines through a single infection of mesenchymal stem cells.

[0006] According to one aspect of this application, a method for preparing an immortalized mesenchymal stem cell line is provided, the method comprising the steps of p53 knockdown and C-MYC overexpression of mesenchymal stem cells. It should be noted that this method is not limited to a specific DNA sequence. For p53 knockdown, any other shRNA sequence targeting the p53 gene, as long as it can reduce p53 gene and protein expression, should be included within the scope of protection of this application. For C-MYC protein, any other mRNA sequence with C-MYC protein activity obtained through codon alteration should also be included within the scope of protection of this application. Any method that can increase C-MYC expression and improve C-MYC protein activity, such as by mutating C-MYC to increase its protein activity, will be understood by those skilled in the art to have the same effect as C-MYC overexpression and should also be within the scope of protection of this application.

[0007] This protocol does not limit the use of other aging-related genes, such as p16, p19, p27, and BMI1, or their combination with other proliferative genes such as N-MYC. Although this protocol only intervenes in the expression of p53 and C-MYC, those skilled in the art can combine it with other intervention methods, as long as the cellular characteristics of mesenchymal stem cells are preserved. For example, if the requirement is for exosome production of biopharmaceuticals, maintaining the normal exosome secretion characteristic is sufficient. Furthermore, this protocol does not limit other eukaryotic cell resistance screening methods, nor does it limit the method of plasmid introduction into cells, such as other viral introduction methods or non-viral transfection methods.

[0008] Optionally, the functional unit performing the p53 knockdown includes the shRNA nucleotide sequence shown in SEQ ID No. 1, and the functional unit performing the C-MYC overexpression expresses the C-MYC amino acid sequence shown in SEQ ID No. 2.

[0009] It should be noted that other shRNA nucleotide sequences that retain p53 knockdown function by simple substitutions or additions / deletions to the shRNA nucleotide sequence shown in SEQ ID No. 1 should also be included within the scope of protection of this application; similarly, other C-MYC amino acid sequences that retain C-MYC function by simple substitutions or additions / deletions to the C-MYC amino acid sequence shown in SEQ ID No. 2 should also be included within the scope of protection of this application. Furthermore, it will be understood by those skilled in the art that the aforementioned substitutions or additions / deletions do not constitute specific limitations on the changes but are merely a partial list of common modifications, and other technical solutions that perform the same function should be included within the scope of protection of this application.

[0010] Optionally, the functional unit for C-MYC overexpression includes the C-MYC nucleotide sequence shown in SEQ ID No. 3.

[0011] It should be noted that, for those skilled in the art, the C-MYC amino acid sequence shown in SEQ ID No. 2 can be optimized to obtain a C-MYC nucleotide sequence other than that shown in SEQ ID No. 3, or other C-MYV nucleotide sequences obtained based on a C-MYC amino acid sequence other than that shown in SEQ ID No. 2 but with the same C-MYC activity. These sequences can also successfully express products with C-MYC activity, thereby performing the same function and achieving the same technical effect, and should therefore be included within the scope of protection of this application.

[0012] Optionally, the preparation method includes the step of transferring a single plasmid into mesenchymal stem cells, wherein the single plasmid includes a functional unit that simultaneously performs p53 knockdown and C-MYC overexpression.

[0013] It should be noted that this application does not limit the specific implementation method of p53 knockdown and C-MYC overexpression. As will be known to those skilled in the art from the content of this application, simultaneously reducing the expression level of p53 and increasing the expression level of C-MYC in mesenchymal stem cells can yield immortalized mesenchymal stem cells. The specific method used for p53 knockdown and C-MYC overexpression is a conventional skill possessed by those skilled in the art.

[0014] For example, in this application, lentiviruses are used to genetically modify mesenchymal stem cells, and p53 is knocked down by expressing shRNA that interferes with p53 expression. Those skilled in the art can also knock out and interfere with p53 expression-related genes to achieve the same p53 knockdown effect. In addition, this application uses a plasmid that contains functional units that achieve both p53 knockdown and C-MYC overexpression. Those skilled in the art can use multiple plasmids or non-plasmid methods for intervention. It is understood by those skilled in the art that other methods that can achieve the same effect of reducing p53 expression level and increasing C-MYC expression level should be included within the scope of protection of this application.

[0015] According to another aspect of this application, a mesenchymal stem cell immortalized cell line is provided, wherein the p53 expression level is reduced and the C-MYC expression level is increased compared with the unmodified mesenchymal stem cells.

[0016] Optionally, the cell line contains the shRNA nucleotide sequence shown in SEQ ID No. 1.

[0017] Optionally, the cells in the cell line express the C-MYC amino acid sequence shown in SEQ ID No. 2.

[0018] It should be noted that other shRNA nucleotide sequences that retain p53 knockdown function by simple substitutions or additions / deletions to the shRNA nucleotide sequence shown in SEQ ID No. 1 should also be included within the scope of protection of this application; similarly, other C-MYC amino acid sequences that retain C-MYC function by simple substitutions or additions / deletions to the C-MYC amino acid sequence shown in SEQ ID No. 2 should also be included within the scope of protection of this application. Furthermore, it will be understood by those skilled in the art that the aforementioned substitutions or additions / deletions do not constitute specific limitations on the changes but are merely a partial list of common modifications, and other technical solutions that perform the same function should be included within the scope of protection of this application.

[0019] Optionally, the cell line contains the C-MYC nucleotide sequence shown in SEQ ID No. 3.

[0020] It should be noted that, for those skilled in the art, the C-MYC amino acid sequence shown in SEQ ID No. 2 can be optimized to obtain a C-MYC nucleotide sequence other than that shown in SEQ ID No. 3, or other C-MYV nucleotide sequences obtained based on a C-MYC amino acid sequence other than that shown in SEQ ID No. 2 but with the same C-MYC activity. These sequences can also successfully express products with C-MYC activity, thereby performing the same function and achieving the same technical effect, and should therefore be included within the scope of protection of this application.

[0021] According to another aspect of this application, mesenchymal stem cell immortalized cell lines prepared by any of the above-described methods for preparing mesenchymal stem cell immortalized cell lines, or the application of any of the above-described mesenchymal stem cell immortalized cell lines in biomedical preparation, are provided.

[0022] Optionally, the application includes the step of using the immortalized mesenchymal stem cell line to express the target product via exosomes for biopharmaceutical preparation.

[0023] It should be noted that, as will be understood by those skilled in the art, due to limitations in current technological background and understanding, it may be impossible to list all potential application scenarios in detail. However, it is certain that the immortalized mesenchymal stem cells provided in this application have better passage and proliferation effects compared to unmodified or otherwise modified mesenchymal stem cells. The immortalized mesenchymal stem cells provided in this application can also play the same role in the fields where mesenchymal stem cells can be applied, and due to their excellent passage and proliferation effects, they are even more suitable for industrial production applications.

[0024] According to another aspect of this application, a biomaterial is provided, said biomaterial being one of the following:

[0025] A) An expression cassette or combination of expression cassettes comprising the shRNA nucleotide sequence shown in SEQ ID No. 1 and expressing the C-MYC amino acid sequence shown in SEQ ID No. 2;

[0026] B) An expression carrier comprising the expression box or combination of expression boxes described in A);

[0027] C) A viral vector containing the expression vector described in B);

[0028] D) Engineered cells containing the expression vector described in B) or the viral vector described in C).

[0029] The beneficial effects of this application include, but are not limited to:

[0030] This application provides a simple and rapid method for achieving mesenchymal stem cell immortalization through a one-step dual gene intervention. It uses a one-step method combining gene knockdown and gene overexpression to prepare immortalized mesenchymal stem cells. Furthermore, the gene-modified plasmid constructed in this invention integrates gene knockdown and gene overexpression, and an immortalized cell line can be obtained by a single infection of mesenchymal stem cells.

[0031] This application employs a single-stage modification method, simultaneously producing two different gene-modifying media for a single cell modification. This reduces workload and time, and avoids the problem of poor cell homogeneity caused by the randomness of the two gene-modifying media entering the cells. Compared to methods that first perform gene overexpression modification on cells and then gene knockdown modification on the overexpressing cells, or knockdown followed by overexpression, the cells modified in this application have high purity and strong uniformity.

[0032] This application only intervenes in the expression of two proteins, p53 and C-MYC, in mesenchymal stem cells. With minimal impact on mesenchymal stem cells, it preserves their cellular characteristics, such as the ability to secrete exosomes normally, achieves immortalization, and significantly improves their reproductive capacity. It is foreseeable that this application has a wide range of applications in industrial production and will bring huge economic value. Attached Figure Description

[0033] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0034] Figure 1 The plasmid maps constructed using the gene-modified plasmids involved in Example 1 of this application are as follows: (a: original lentiviral plasmid map, b: null interference and overexpression empty vector lentiviral plasmid map, c: p53 knockdown lentiviral plasmid map alone, d: C-MYC overexpression lentiviral plasmid map alone, e: p53 knockdown combined with C-MYC overexpression lentiviral plasmid map).

[0035] Figure 2 The results of virus infection and resistance screening of the target cells involved in Example 3 of this application are as follows (P9: normal passage culture without any intervention; CT: negative control of Puro screening without virus infection; S: control without knockdown and overexpression vector; 53: p53 knockdown alone; SM: MYC overexpression alone; 53M: p53 knockdown and MYC overexpression).

[0036] Figure 3The results of cell passage culture monitoring after resistance selection involved in Example 4 of this application are as follows (P9: normal passage culture without any intervention; S: control without knockdown or overexpression vector; 53: p53 knockdown alone; SM: MYC overexpression alone; 53M: p53 knockdown and MYC overexpression).

[0037] Figure 4 The results of the passage culture proliferation curves of cells after resistance screening involved in Example 4 of this application are shown in the figure above (the upper figure is the growth curve of cell passage, and the lower figure is the mean cell count of each group, unit: 100,000; P9: normal passage culture without any intervention; S: control without knockdown and overexpression vector; 53: p53 knockdown alone; SM: MYC overexpression alone; 53M: p53 knockdown and MYC overexpression).

[0038] Figure 5 The results of Western blot analysis of cell protein expression involved in Example 4 of this application are as follows (P13: normal passage culture to P13 without any intervention; S: control without knockdown or overexpression vector; 53: knockdown of p53 alone; SM: overexpression of MYC alone; 53M: knockdown of p53 and overexpression of MYC).

[0039] Figure 6 The results of SA-β-Gal staining for cell senescence involved in Example 4 of this application are as follows (S: no knockdown, no overexpression vector control; 53: p53 knockdown alone; SM: MYC overexpression alone; 53M: p53 knockdown, MYC overexpression).

[0040] Figure 7 The results of cell death PI staining detection involved in Example 5 of this application (SM: MYC overexpression alone; 53M: p53 knockdown and MYC overexpression);

[0041] Figure 8 This is a diagram showing the results of continuous passage culture of 53M cells involved in Example 5 of this application;

[0042] Figure 9 This is the cell growth curve of the 53M cells continuously passaged in Example 5 of this application;

[0043] Figure 10 This refers to the WB detection results of the 53M cell exosome protein involved in Example 6 of this application. Detailed Implementation

[0044] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present application are all purchased through commercial channels.

[0045] The main experimental equipment used in this application is shown in Table 1 below.

[0046] Table 1 Main Experimental Equipment

[0047]

[0048]

[0049] The main experimental reagents used in this application are shown in Table 2 below.

[0050] Table 2 Main Experimental Reagents

[0051]

[0052]

[0053]

[0054] The main experimental consumables used in this application are shown in Table 3 below.

[0055] Table 3 Main Experimental Consumables

[0056] name factory Item number 200ul PCR tube Sangon Biotech F611541 Bacterial culture dish Sangon Biotech F611003 0.45μm filter Sangon Biotech F512545 100mm cell culture plate Wuxi NEST 704001 6-well cell culture plate LABSELECT 11110 15ml centrifuge tube Wuxi NEST 601052 50ml centrifuge tube Wuxi NEST 601051 PVDF membrane Millipore IPVH00010 24-well cell culture plate LABSELECT 11310

[0057] The present application solution will be described below through specific embodiments.

[0058] Example 1: Construction of Gene-Modified Plasmids

[0059] like Figure 1 As shown, based on the lentiviral expression vector (PLVX-CMV-IRES-Puro) (a), lentiviral vectors for p53 knockdown and C-MYC overexpression (e), as well as control vectors for empty vector (b), p53 knockdown alone (c), and C-MYC overexpression alone (d) were constructed. The U6 promoter drove shRNA expression, and the CMV promoter drove C-MYC expression. Puromysin was ligated to C-MYC using the IRES element for eukaryotic resistance screening. Figure 1 In the lentivirus matrix, 3'LTR, HIV-1, RRE, WPRE, and cPPT / CTS are lentivirus-related elements; the U6 promoter drives shRNA expression, and the CMV enhancer and CMV promoter drive C-MYC expression; IRES is the ribosome entry site, used to drive Puro (puromycin resistance) expression; Puro is used for eukaryotic resistance selection; Ori is the plasmid replication origin in bacteria; the AmpR promoter drives AmpR (ampicillin resistance) expression in bacteria; AmpR is used for prokaryotic resistance selection. The main experimental steps are as follows.

[0060] S1. Annealing of double-stranded shRNA fragments: Transfer 10 μL each of the sense and antisense strands of the shRNA fragment to a 200 μL PCR tube, vortex to mix, and centrifuge briefly until the liquid reaches the bottom of the tube. Incubate the PCR tube in a PCR instrument: 95℃ for 30 s, 72℃ for 2 min, 37℃ for 2 min, and 25℃ for 2 min. After the reaction, place the PCR tube containing the annealing product in an ice bath for later use.

[0061] S2. PCR Amplification of the Target DNA Fragment: Prepare the PCR reaction solution in a 200 μL PCR tube: 20 ng template plasmid solution; 0.4 μL upstream primer (10 μM); 0.4 μL downstream primer; 5 μL 5X PrimeSTAR GXL buffer; 2 μL dNTP Mix; 1 μL PrimeSTAR GXL; Nuclease-Free Water to a final volume of 25 μL. Incubate the PCR tube in a PCR instrument: 98℃ for 2 min; 98℃ for 15 s, 60℃ for 15 s, 68℃ for 1 min, 38 cycles; 68℃ for 2 min. After the reaction, add 7.5 μL of the reaction solution to a 1.2% agarose gel and electrophoresis at 120V for 30 min. Store the remaining sample at 4℃ for later use. After electrophoresis, detect the amplification of the target fragment using a blue light gel cutter.

[0062] S3. Enzyme digestion of the cloning vector: Prepare the enzyme digestion reaction system (20 μL) in a 200 μL PCR tube: 5 μL cloning vector plasmid solution (200 ng / μL); 2 μL 10x Fast Digestion Buffer; 1 μL restriction endonuclease I; 1 μL restriction endonuclease II; 12 μL Nuclease-Free Water. Gently tap the bottom of the tube to mix the enzyme digestion reaction solution, briefly centrifuge, and incubate at 37°C for 60 min in a PCR instrument. After the reaction is complete, place the PCR tube containing the enzyme digestion product in an ice bath for later use.

[0063] S4. Ligation of double-stranded shRNA fragments with linearized vectors: Prepare the ligation reaction mixture (10 μL) in a 200 μL PCR tube: 2 μL linearized vector plasmid solution (50 ng / μL); 0.2 μL double-stranded shRNA annealing product (5 μM); 5 μL DNA ligase Solution I; 2.8 μL Nuclease-Free Water. Gently tap the bottom of the tube to mix the enzyme digestion solution, briefly centrifuge, and incubate at 16°C for 45 min in a PCR instrument. After the reaction, place the PCR tube containing the ligation product in an ice bath for later use.

[0064] S5. Recombination of the target DNA fragment with the linearized vector fragment: Prepare the ligation reaction mixture (10 μL) in a 200 μL PCR tube: 1 μL of linearized vector plasmid solution (50 ng / μL); 4 μL of the target DNA fragment PCR amplification product; and 5 μL of recombinase In-Fusion Premix. Gently tap the bottom of the tube to mix the enzyme digestion solution, briefly centrifuge, and incubate at 50°C for 25 min in a PCR instrument. After the reaction is complete, place the PCR tube containing the recombinant reaction product in an ice bath for later use.

[0065] S6. Transformation of DH5α competent cells with ligation and recombinant products: Remove DH5α competent cells from the cryogenic freezer and thaw on ice for 20 min. Add the ligation or recombinant product to the thawed competent cells, gently tap the bottom of the tube to mix thoroughly, and place on ice for 30 min. Incubate at 42°C for 75 s, then place on ice for 2 min. Add LB liquid medium to the competent cells and incubate at 37°C for 45 min in a shaking incubator. Transfer the cultured competent cells to LB solid medium plates, evenly cover the plates, and incubate statically at 37°C for 16 h.

[0066] S7. Transformant Single-Clon Amplification Culture: Using a 10µl sterile pipette tip, pick a single colony from an overnight solid plate and place it into a 15ml centrifuge tube containing 3ml LB liquid medium (containing 100µg / ml ampicillin). Incubate the centrifuge tube in a shaking incubator at 37°C for 16 hours.

[0067] S8. Plasmid Extraction: Transfer 2 ml of bacterial culture from each single colony to a separate 2 ml sterile EP tube. Extract plasmids using a rapid plasmid mini-prep kit. Elute each plasmid sample with 75 μL of Nuclease-Free Water. Transfer 5 μL from each sample to a new 1.5 ml EP tube for DNA sequencing. Store the remaining sample frozen at -20°C for later use.

[0068] Example 2 Lentiviral Production

[0069] HEK293T was transfected with lentiviral packaging plasmid and target plasmid. Cell culture supernatant containing virus particles was harvested. After concentrating the virus with PEG6000, the virus particles were resuspended to obtain four types of viral solutions. The main experimental steps are as follows.

[0070] S1 and 293T cell resuscitation and passage: Transfer 5 ml of 293T medium (DMEM medium containing 10% FBS and 1% P / S) to a 15 ml centrifuge tube. Remove the 293T cells frozen in liquid nitrogen and thaw them rapidly in a 37°C water bath. Transfer the thawed cell suspension to the aforementioned 15 ml centrifuge tube in a biosafety cabinet and centrifuge at 110g for 3 min. Discard the supernatant, add 1 ml of 293T medium, gently pipette to resuspend the cells, and add another 10 ml of medium. Seed the cells in 100 mm cell culture dishes, shake well back and forth and left and right, and then incubate statically in a cell culture incubator. Three days after cell resuscitation, examine the cells under a microscope; the cell coverage in the culture dish should be over 90%. For each 100 mm culture dish, discard the medium, add 3 ml of PBS to rinse the cell surface, discard the PBS, add 2 ml of 0.05% trypsin (0.25% trypsin diluted 1:5 in PBS) to evenly cover the cells, and incubate statically in an incubator for 3-5 min. Gently tap the culture dish to completely detach the cells. Add 3 ml of 293T medium and transfer the cell suspension to a 15 ml centrifuge tube. Centrifuge at 110 g for 3 min. Discard the supernatant, add 1 ml of 293T medium, and gently pipette to resuspend the cells. Transfer 150 μl of the cell suspension to three 100 mm cell culture dishes containing 10 ml of medium each. Shake well and incubate in a cell culture incubator.

[0071] S2. Plasmid Transfection of 293T Cells: After 3 days of passaged 293T cells, cells were collected for a second passage. On the second day after culture, under a microscope, the cell coverage reached more than 80%, and transfection was performed. Taking a 100mm culture dish as an example, take a 1.5ml sterile EP tube, add 0.5mL DMEM, 7.5μg pCMV-dR8.2 plasmid, 5μg pCMV-VSV-G plasmid, and 10μg expression plasmid, gently tap the bottom of the tube to mix thoroughly, and label it A. Take another 1.5ml sterile EP tube, add 0.5mL DMEM and 45μl Lipo293 transfection reagent, gently tap the bottom of the tube to mix thoroughly, and label it B. Transfer the plasmid mixture from tube A to tube B, invert to mix, and let stand at room temperature for 15min. After the mixture has been allowed to stand, it is evenly added dropwise to a 100mm cell culture dish containing 293T cells. After gently shaking it up and down and left and right to mix it, it is placed in a cell culture incubator for culture.

[0072] S3. Harvesting and Concentration of Lentiviral Cells: 48 hours post-transfection, collect cell supernatant from each 100mm culture dish into a 50ml centrifuge tube, and add 10ml of fresh 293T cell culture medium to the dish. Continue culturing the cells in an incubator, and refrigerate the collected cell supernatant. After culturing for another 24 hours, collect the cell supernatant again. Combine the two collections of cell supernatant and centrifuge at 200g for 10 minutes. Take the supernatant (approximately 20ml), filter it through a 0.45μm filter, and add 3.5ml of 3M NaCl solution and 7ml of 42.5% PEG6000 solution. Store at 2-8℃ overnight, inverting and mixing at least 4 times during this period, with each inversion at least 30 minutes apart. After centrifugation at 7500g for 1 hour at 10℃, discard the supernatant in a biosafety cabinet, removing as much residual liquid as possible from the tubes. Resuspend the precipitate in 1ml DMEM / F12 medium, pipetting several times before transferring to a 1.5mL sterile EP tube. Incubate overnight at 2-8℃, then centrifuge at 12000rpm for 3min. Transfer the supernatant to a new 1.5ml EP tube and store at 2-8℃ for later use. Note: All consumables that have come into contact with the virus solution and the virus supernatant after centrifugation must be soaked in chlorine-containing disinfectant overnight before disposal.

[0073] Example 3: Virus infection and resistance screening of target cells

[0074] The results are as follows Figure 2 As shown, adipose-derived mesenchymal stem cells (P9) were resuscitated and seeded into 6-well plates. On day 2 post-resuscitation (Day 1), they were infected with four different viruses. The groups were as follows: P9, normal passaged culture without any intervention; CT, negative control for Puro selection without virus infection; S, control without knockdown or overexpression vector; 53, p53 knockdown alone; SM, MYC overexpression alone; 53M, p53 knockdown and MYC overexpression. Three days post-virus infection (Day 4), Puromysin (Puro) was added for positive cell selection. Four days after Puro selection (Day 9), all uninfected cells (CT, Day 10) died under Puro treatment, and the surviving cells in the virus-infected group did not yield the initial genetically modified cells. The main experimental steps are as follows.

[0075] S1. Human adipose-derived mesenchymal stem cell (ADS) cell resuscitation: Transfer 5 ml of complete ADS culture medium (DMEM / F12 medium containing 1% PS, 10% FBS, and 10 μg / ml FGF8) to a 15 ml centrifuge tube. Remove the frozen ADS cells and thaw them rapidly in a 37°C water bath. Transfer the thawed cell suspension to the aforementioned 15 ml centrifuge tube in a biosafety cabinet and centrifuge at 110 g for 3 min. Discard the supernatant, add 1 ml of ADS culture medium, gently pipette to resuspend the cells, and add another 12 ml of medium, inverting to mix thoroughly. Seed the cells into 6-well cell culture plates, 2 ml per well, for a total of 6 wells, labeled as: P9, CT, S, SM, 53, and 53M. Shake well back and forth and left and right, then place in a cell culture incubator for static culture. Culture overnight, observe cell adhesion and growth under a microscope, and take photographs.

[0076] S2. Lentiviral infection of human adipose-derived mesenchymal stem cells: In a biosafety cabinet, aspirate 1 ml of culture medium from each well, add 1 ml of culture medium to each of P9 and CT wells, and add the corresponding viral solution to wells S, SM, 53, and 53M. Add Polybrene to each well to a final concentration of 5 μg / ml. After shaking well, incubate overnight. Observe cell adhesion and growth under a microscope and take photographs. In a biosafety cabinet, aspirate the culture medium from each well, add 2 ml of complete culture medium, and incubate statically in a CO2 cell culture incubator.

[0077] S3. Resistance Screening of Human Adipose-Derived Mesenchymal Stem Cells: Cells were collected 4 days after viral infection. Cell adhesion and growth were observed under a microscope, and photographs were taken. In a biosafety cabinet, the culture medium was aspirated from each well, and 2 ml of complete culture medium was added. Puromycin was added to each well to a final concentration of 0.5 μg / ml. After thorough mixing, the cells were placed in a cell culture incubator for static incubation. The culture medium was replaced with fresh puromycin every 3 days. Cell adhesion and growth were observed under a microscope, and photographs were taken.

[0078] S4. Passaging of Human Adipose-Derived Mesenchymal Stem Cells: For cells cultured in 6-well cell culture plates, aspirate the culture medium from the wells, add 1 ml of PBS to cover the cell surface, rinse, aspirate the PBS, add 200 μL of 0.05% trypsin (0.25% trypsin diluted 1:5 in PBS), evenly cover the cells, and incubate for 2 min. Add 1 ml of complete mesenchymal stem cell culture medium (DMEM / F12 medium containing 1% PS, 10% FBS, 10 μg / ml FGF8), gently pipette several times with a 1 ml pipette, transfer the cell suspension to a 15 ml centrifuge tube, centrifuge at 110 g for 3 min, discard the supernatant, add 1 ml of mesenchymal stem cell culture medium, and gently resuspend. Take 20 μL of the cell suspension into a 1.5 ml centrifuge tube, add 20 μL of PBS, mix thoroughly, and count the cells using a cell counter. Take 0.3x... 10e5 cells were seeded into the original wells, and 2 ml of complete mesenchymal stem cell culture medium was added. After shaking well back and forth and left and right, the cells were placed in a cell culture incubator for static culture.

[0079] Example 4: Monitoring the Culture of Genetically Modified Mesenchymal Stem Cells

[0080] After resistance selection, cell cultures in each group were passaged and monitored. P9 group cells underwent normal passaged culture without any intervention; S group cells were controls without knockdown or overexpression vector introduction; 53 group cells had p53 knocked down alone; SM group cells had C-MYC overexpressed alone; and 53M group cells had p53 knocked down and C-MYC overexpressed. Cell growth, target protein expression, cell senescence, and cell death were monitored in different groups.

[0081] like Figure 3 As shown in the figure, the growth status monitoring of continuously cultured cells in each group shows that the 53M group of cells exhibits a higher growth density. Figure 4 As shown, cells in each group were passaged and counted. Cell growth curves showed that 53M cells had a significant growth advantage. Western blotting was performed on the p53 and c-MYC proteins in each group of cells, with ACTB protein used as an internal control. The results are as follows. Figure 5 As shown, Western blot analysis confirmed that p53 protein expression was decreased in cells of groups 53(a) and 53M(b), while C-MYC protein expression was increased in cells of groups SM(c) and 53M(d). Figure 6As shown, cell senescence (SA-β-Gal staining) assay revealed that after cell passage to P17, cells in group S showed severe senescence, cells in group 53 showed partial senescence, and cells in groups S and SM showed minimal senescence. Figure 7 As shown, cell death (PI staining) detection revealed that cell death was significantly increased in group S compared to group 53M.

[0082] The main experimental steps are as follows.

[0083] S1. Continuous passage culture of adipose-derived mesenchymal stem cells selected by Puromycin: For cells cultured in 6-well cell culture plates, aspirate the culture medium from the wells, add 1 ml of PBS to cover the cell surface, wash, aspirate the PBS, add 200 μl of 0.05% trypsin (0.25% trypsin diluted 1:5 in PBS), evenly cover the cells, and incubate for 2 min. Add 1 ml of complete mesenchymal stem cell culture medium (DMEM / F12 medium containing 1% PS, 10% FBS, 10 μg / ml FGF8), gently pipette several times, transfer the cell suspension to a 15 ml centrifuge tube, centrifuge at 110g for 3 min, discard the supernatant, add 1 ml of mesenchymal stem cell culture medium, and gently resuspend. Take 20 μl of the cell suspension into a 1.5 ml centrifuge tube, add 20 μl of PBS, mix thoroughly, and count the cells using a cell counter. Take 0.3x... 10e5 cells were seeded into the original wells, and 2 ml of complete mesenchymal stem cell culture medium was added. After shaking well back and forth and left and right, the cells were placed in a cell culture incubator for static culture.

[0084] S2. Western blot (WB) detection of proteins in adipose-derived mesenchymal stem cells: After cell passage and counting, 0.3 x 10e5 cells were placed in a 1.5 ml centrifuge tube, centrifuged at 600 g for 3 min, and the supernatant was discarded. The cells were resuspended in 0.5 ml of ice-cold PBS, and the mixture was inverted several times to mix. The cells were centrifuged at 600 g for 3 min, and the supernatant was discarded. 100 μl of Western blot and IP cell lysis buffer (containing 1 mM PMSF) was added to each tube, and the cells were thoroughly pipetted and placed on ice for 20 min. The cells were then centrifuged at 15000 g for 15 min at 4°C, and 80 μl of the supernatant was transferred to a new 1.5 ml tube. 20 μl of 5 x SDS protein loading buffer was added to each tube, and the mixture was mixed and then incubated in a boiling water bath for 10 min. 17.5 μl of 4–12% SimplePAGE precast gel was loaded into each well for gel electrophoresis at 60 V for 10 min and 90 V for 1 h. After electrophoresis, remove the protein gel, rinse it thoroughly, and place it in pre-cooled transfer buffer at 4°C. Following the electrode sequence (negative electrode - filter paper - gel - PVDF membrane - filter paper - positive electrode), transfer the membrane at 270 mA for 2-3 hours in an ice-water bath. After transfer, remove the PVDF membrane and rinse it once in TBST. Immerse the membrane in TBST, cut the target membrane according to the pre-stained protein markers, and incubate it in TBST containing 5% BSA at room temperature for 1 hour. Place the membrane in the corresponding primary antibody solution (MYC antibody, ACTB antibody 1:1000 diluted in TBST containing 5% BSA) and incubate overnight at 4°C. Rinse with TBST with shaking for 3 x 10 min. Incubate with secondary antibody (rabbit HRP-conjugated secondary antibody 1:5000 diluted in TBST containing 5% BSA) at room temperature for 1 hour, followed by TBST with shaking for 3 x 10 min. Detect the protein bands using an ECL chemiluminescence detection kit on a ChemiScope 6100 chemiluminescence imaging system.

[0085] Dilute 1:5000 in TBST containing 5% BSA and incubate at room temperature for 1 h, followed by TBST oscillation and washing 3 x 10 min. Detect protein bands using the ECL chemiluminescence detection kit on a ChemiScope 6100 chemiluminescence imaging system.

[0086] (Diluted 5000 μL in TBST containing 5% BSA) and incubated at room temperature for 1 h, followed by 3 x 10 min oscillation washes with TBST. Protein bands were detected using the ECL chemiluminescence detection kit on a ChemiScope 6100 chemiluminescence imaging system.

[0087] S3. SA-β-gal assay for senescence in adipose-derived mesenchymal stem cells: Cells were collected 4 days after viral infection. Cell adhesion and growth were observed under a microscope, and photographs were taken. In a biosafety cabinet, the culture medium was discarded from each well, and 2 ml of complete culture medium was added. Puromycin was added to each well to a final concentration of 0.5 ug / ml. After thorough mixing, the cells were placed in a cell culture incubator for static incubation. The culture medium was replaced with fresh puromycin every 3 days. Cell adhesion and growth were observed under a microscope, and photographs were taken.

[0088] S4. Detection of cell death in adipose-derived mesenchymal stem cells: Cells were collected 41 days after viral infection, with 0.1 x 10^6 cells each of SM and 53M cells added to 24-well cell culture plates. After overnight culture, 10 μL of PI staining solution was added to each well, shaken to mix, and then incubated statically. Microscopic examination and photographs were taken at 4 h, 10 h, and 24 h after staining.

[0089] Example 5: Monitoring of Continuous Passage Culture of Immortalized Cells

[0090] The aforementioned 53M cells exhibited better proliferative capacity and lower levels of cell senescence and death compared to other cell groups. Further continuous passage culture monitoring of the 53M cells was conducted to determine whether they possess immortalization characteristics. Figure 7 As shown, 53M cells were continuously passaged to 48 generations (P48), maintaining stable morphology and state. The cell status images of 53M cells from day 23 to day 128 post-viral infection are shown below. Figure 8 As shown, 53M cells were continuously passaged to P48, maintaining a stable cell proliferation rate. The number of 53M cells increased from the initial 0.3 x 10e6 to 4.8 x 10e17, an increase of 1.6 x 10e18 times. The main experimental steps are as follows.

[0091] Continuous passage culture of 53M cells: For cells cultured in 6-well cell culture plates, aspirate the culture medium from the wells, add 1 ml of PBS to cover the cell surface, rinse, aspirate the PBS, add 200 μl of 0.05% trypsin (0.25% trypsin diluted 1:5 in PBS), evenly cover the cells, and incubate for 2 min in an incubator. Add 1 ml of complete mesenchymal stem cell culture medium (DMEM / F12 medium containing 1% PS, 10% FBS, 10 μg / ml FGF8), gently pipette several times with a 1 ml pipette, transfer the cell suspension to a 15 ml centrifuge tube, centrifuge at 110 g for 3 min, discard the supernatant, add 1 ml of mesenchymal stem cell culture medium, and gently pipette to resuspend the cells. Take 20 μl of the cell suspension into a 1.5 ml centrifuge tube, add 20 μl of... After thoroughly mixing with PBS, cell counting was performed using a cell counter. 0.3 x 10⁶ cells were seeded into the original wells, and 2 ml of complete mesenchymal stem cell culture medium was added. The cells were shaken well and then placed in a cell culture incubator for static culture. Cells were passaged and counted every 3 days. Cell growth curves were plotted based on the cell count results. The results are shown below. Figure 9 As shown, cells were passaged and counted every 3 days, and cell growth curves were plotted based on the cell count results. The number of 53M cells increased from the initial 0.3 x 10e6 to 4.8 x 10e17, an increase of about 1.6 x 10e18 times.

[0092] Example 6: Detection of exosome proteins in immortalized 53M cells

[0093] 53M cells were cultured, and total cellular protein samples were prepared. Exosomes were collected and exosomal protein samples were prepared. Western blotting was used to detect the expression of the target protein in cells and exosomes. 53M cells indicates 53M total cellular protein samples, PEG indicates 53M exosomal protein samples obtained using the PEG6000 precipitation method, and Thermo indicates 53M exosomal protein samples obtained using Thermo exosome separation reagent. Western blotting results are shown below. Figure 10 As shown, the results indicate that, using the total protein sample from 53M cells as a positive control, exosomes derived from 53M cells contain exosome marker proteins but not C-MYC protein. The main experimental steps are as follows.

[0094] S1. Preparation of Total Cell Protein Samples: 0.5 x 10e6 cells were seeded into a 6-well cell culture plate containing 2 ml of mesenchymal stem cell culture medium. After thorough shaking, the plate was placed in a cell culture incubator for static culture. After 48 h of cell culture, the cell supernatant was collected into a 15 ml centrifuge tube and stored in a refrigerator for later use. Cells in the wells were washed with PBS, digested with trypsin, and digestion was terminated. Cells were then collected and counted. 1 x 10e6 cells were placed into a 1.5 ml centrifuge tube, centrifuged at 600 g for 3 min, and the supernatant was discarded. The cells were resuspended in 0.5 ml of ice-cold PBS, inverted several times to mix, and centrifuged at 600 g for 3 min, discarding the supernatant. 200 μl of Western blot and IP cell lysis buffer (containing 1 mM PMSF) was added, and the mixture was thoroughly pipetted. The plate was placed on ice for 20 min, centrifuged at 15000 g for 15 min at 4°C, and 160 μl of the supernatant was transferred to a new 1.5 ml tube. Add 40 μL of 5x SDS protein loading buffer, mix well, and then boil in a water bath for 10 minutes. Store frozen for later use.

[0095] S2. Preparation of exosome protein samples: Seed 0.5 x 10e6 cells into a 6-well cell culture plate containing 2 ml of mesenchymal stem cell culture medium. Shake well and incubate statically in a cell culture incubator. After 48 h of cell culture, collect the cell supernatant into a 15 ml centrifuge tube. Filter the cell supernatant through a 0.45 μm filter, and transfer 1.2 ml of the filtered supernatant to a 2 ml centrifuge tube. Add 0.2 ml of 3M NaCl solution and 0.4 ml of 42.5% PEG6000 solution, labeling this tube as a PEG tube. Take another 1.2 ml of the filtered supernatant and transfer it to a 2 ml centrifuge tube. Add 0.6 ml of Thermo exosome extraction reagent, labeling this tube as a Thermo tube. Mix thoroughly by inverting, and store overnight at 2-8 °C, inverting at least 4 times during storage, with an interval of at least 30 min between each inversion. Centrifuge at 10000g for 1 hour at 4°C, discard the supernatant, and remove as much residual liquid as possible from the tubes. Add 100ul of Western blot and IP cell lysis buffer (containing 1mM PMSF) to each tube, thoroughly pipette, add 25ul of 5x SDS protein loading buffer, mix well, and incubate in a boiling water bath for 10 minutes. Store frozen for later use.

[0096] S3. Western blot analysis of cell and exosome protein samples: Using a 4-12% SimplePAGE pre-prepared gel, load 17.5 μL per well for gel electrophoresis at 60V for 10 min, then 90V for 1 h. After electrophoresis, remove the protein gel, rinse thoroughly, and place in pre-cooled transfer buffer at 4°C. Following the electrode setup (negative electrode - filter paper - gel - PVDF membrane - filter paper - positive electrode), transfer the membrane in an ice-water bath at 270 mA for 2-3 h. After transfer, rinse the PVDF membrane once in TBST, immerse the membrane in TBST, cut the target membrane according to the pre-stained protein markers, and incubate in TBST containing 5% BSA at room temperature for 1 h. The membrane was placed in the corresponding primary antibody solution (antibody 1:1000 diluted in TBST containing 5% BSA) and incubated overnight at 4°C. After TBST oscillation and washing, the membrane was incubated for 1 hour at room temperature with secondary antibody (rabbit HRP-conjugated secondary antibody 1:5000 diluted in TBST containing 5% BSA). After TBST oscillation and washing, the membrane was incubated for 1 hour. Protein bands were detected using the ECL chemiluminescence detection kit on a ChemiScope 6100 chemiluminescence imaging system.

[0097] It should be noted that other methods that can reduce intracellular p53 activity, such as replacing the p53-targeting shRNA with other methods that can reduce p53 expression, can also achieve the same technical effect as the proposed solution. Other methods that can increase intracellular C-MYC activity, such as overexpressing C-MYC proteins with different amino acid sequences but the same C-MYC activity, and overexpressing C-MYC using the same amino acid sequence but different nucleotide sequences, can also achieve the same technical effect as the proposed solution and should be within the scope of protection of the proposed solution.

[0098] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for preparing an immortalized mesenchymal stem cell line, characterized in that, The preparation method includes the steps of p53 knockdown and C-MYC overexpression of mesenchymal stem cells.

2. The preparation method according to claim 1, characterized in that, The functional unit for p53 knockdown includes the shRNA nucleotide sequence shown in SEQ ID No. 1, and the functional unit for C-MYC overexpression expresses the C-MYC amino acid sequence shown in SEQ ID No.

2.

3. The preparation method according to claim 2, characterized in that, The functional unit for C-MYC overexpression includes the C-MYC nucleotide sequence shown in SEQ ID No.

3.

4. The preparation method according to claim 3, characterized in that, The preparation method includes the step of transferring a single plasmid into mesenchymal stem cells, wherein the single plasmid includes a functional unit that simultaneously performs p53 knockdown and C-MYC overexpression.

5. A mesenchymal stem cell immortalization cell line, characterized in that, Compared to unmodified mesenchymal stem cells, the immortalized mesenchymal stem cell line exhibited decreased p53 expression and increased C-MYC expression.

6. The mesenchymal stem cell immortalization cell line according to claim 5, characterized in that, The cell line contains the shRNA nucleotide sequence shown in SEQ ID No.

1.

7. The mesenchymal stem cell immortalization cell line according to claim 5, characterized in that, The cells in the cell line expressed the C-MYC amino acid sequence shown in SEQ ID No.

2.

8. The mesenchymal stem cell immortalization cell line according to claim 7, characterized in that, The cell line contains the C-MYC nucleotide sequence shown in SEQ ID No.

3.

9. The application of the mesenchymal stem cell immortalized cell line prepared by the method of any one of claims 1 to 4, or the mesenchymal stem cell immortalized cell line of any one of claims 5 to 8, in biomedical preparation.

10. A biomaterial, characterized in that, The biomaterial is one of the following: A) An expression cassette or combination of expression cassettes comprising the shRNA nucleotide sequence shown in SEQ ID No. 1 and expressing the C-MYC amino acid sequence shown in SEQ ID No. 2; B) An expression carrier comprising the expression box or combination of expression boxes described in A); C) A viral vector containing the expression vector described in B); D) Engineered cells containing the expression vector described in B) or the viral vector described in C).

Images