CX3CL1 overexpression microglial cell as well as preparation method and application of CX3CL1 overexpression microglial cell

Abstract

The invention discloses a microglial cell for overexpressing CX3CL1, which is characterized in that the microglial cell obtains the persistent CX3CL1 secretion capability similar to neurons through a lentivirus-mediated CX3CL1 overexpression system and a stem cell differentiation technology, so that a physiological microenvironment in which the neurons, chemotactic factors and the microglial cell interact is simulated. Pharmacological evaluation is carried out through the bionic model, the clinical transformation efficiency of central nervous system drug research and development can be remarkably improved, and a physiologically related verification platform is provided for analyzing a bidirectional regulation mechanism of neuroinflammation and tissue repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a microglia overexpressing CX3CL1, a preparation method thereof, and an application thereof. Background Art

[0002] The activation of microglia in the central nervous system is heterogeneous and can be divided into two opposite types: M1 phenotype (cytotoxic effect) and M2 phenotype (neuroprotective effect). In neurodegenerative diseases, there are dynamic changes in the M1 / M2 phenotype. Generally, M1 microglia dominate at the injury site at the end stage of the disease, and the tissue repair process of M2 microglia is inhibited at this time. Endogenous stimuli, including the presence of aggregated α-synuclein, mutant superoxide dismutase, Aβ, and tau oligomers, may continuously activate the M1 pro-inflammatory response and ultimately lead to irreversible damage to neurons. The change of microglial phenotype depends on the disease stage and severity; promoting the polarization of M1 phenotype to M2 within an appropriate time window may provide better therapeutic effects.

[0003] CX3CL1 has been shown to signal through its only receptor CX3CR1, which is located on microglia within the central nervous system (CNS). It is believed that the main function of CX3CL1 within the CNS is to reduce the pro-inflammatory response, and many studies have shown that it has a neuroprotective effect. Taking Alzheimer's disease (AD) as an example: AD is the most common dementia. Amyloid-β (Aβ) and protein Tau aggregates are disease markers and key participants in the pathogenesis of AD. CX3CL1 has an impact on Aβ clearance, tau phosphorylation, and microglial activation and initiation. The relationship between CX3CL1 / CX3CR1 signaling and the pathogenesis of AD has been widely studied, and its biochemical pathway may hide molecular targets for new treatment strategies for AD.

[0004] In the central nervous system, the chemokine CX3CL1 is continuously highly expressed in neurons and dynamically regulates the homeostatic interaction network between neurons and glial cells by forming a stable complex with the CX3CR1 receptor on the surface of microglia. In the traditional two-dimensional in vitro culture system, the isolated and cultured microglia cannot receive the continuous chemotactic stimulation of CX3CL1, and their phenotypic characteristics are significantly shifted: the expression of cell pro-inflammatory substances increases, and they are more polarized towards the M1 direction. This significant difference between the in vitro model and the in vivo environment suggests that there may be important limitations in the current microglial drug screening system - the pharmacodynamic data based on two-dimensional single-cell layer culture may not accurately reflect the true biological effects of drugs in the physiological CX3CL1 microenvironment. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art. To address the significant differences between traditional in vitro models and the in vivo microenvironment, the present invention uses a lentivirus-mediated CX3CL1 overexpression system and stem cell differentiation technology to endow microglia with the ability to continuously secrete CX3CL1 similar to neurons, thereby mimicking the physiological tripartite niche of neuron-chemokine-microglia interactions. Pharmacological evaluations carried out through this bionic model can significantly improve the clinical translation efficiency of central nervous system drug research and development, and provide a physiologically relevant verification platform for analyzing the bidirectional regulatory mechanism of neuroinflammation and tissue repair.

[0006] The present invention is achieved through the following technical solutions:

[0007] On the one hand, the present invention provides a method for preparing microglia overexpressing CX3CL1, comprising the following steps:

[0008] S1, construction of pluripotent stem cells: Add lentiviral particles overexpressing CX3CL1 to the pluripotent stem cells to be transfected, and after transfection, obtain pluripotent stem cells with low purity overexpressing CX3CL1;

[0009] S2, purification: Subject the pluripotent stem cells with low purity overexpressing CX3CL1 to Puro drug screening to obtain pluripotent stem cells with high purity overexpressing CX3CL1;

[0010] S3, differentiation: Differentiate the pluripotent stem cells with high purity overexpressing CX3CL1 to obtain microglia overexpressing CX3CL1.

[0011] In the above technical solution, the nucleotide sequence of CX3CL1 is as shown in SEQ ID NO.1.

[0012] In the above technical solution, the pluripotent stem cells to be transfected are H9.

[0013] On the other hand, the present invention provides a method for preparing microglia overexpressing CX3CL1, comprising the following steps:

[0014] Step 1, preparation of a lentiviral construct overexpressing CX3CL1:

[0015] Select the full-length cDNA sequence of CX3CL1 and clone it into a lentiviral expression vector carrying the EF-1α promoter; obtain a recombinant lentiviral expression vector;

[0016] For the above recombinant lentiviral expression vector with correct sequencing, use a plasmid large-scale extraction kit to extract and purify to obtain a high-quality recombinant plasmid, namely a lentiviral construct overexpressing CX3CL1, for lentiviral packaging and transfection of target cells;

[0017] Step 2, Preparation of Lentivirus Particles Overexpressing CX3CL1:

[0018] Transfect the cells to be transfected with the packaged lentiviral construct overexpressing CX3CL1, and collect the cell culture supernatant after culturing to obtain the lentivirus particles overexpressing CX3CL1;

[0019] Step 3, Preparation of Pluripotent Stem Cells with High Purity Overexpressing CX3CL1:

[0020] Infect the pluripotent stem cells to be transfected with the lentivirus particles overexpressing CX3CL1, and obtain pluripotent stem cells with high purity overexpressing CX3CL1 through Puro (Puromycin) screening;

[0021] Step 4, Differentiate into Microglia Overexpressing CX3CL1:

[0022] Digest the pluripotent stem cells with high purity overexpressing CX3CL1 into single cells, and further perform initial culture, amplification culture, and induced differentiation culture to obtain microglia overexpressing CX3CL1.

[0023] In the above technical solution, the nucleotide sequence of CX3CL1 is as shown in SEQ ID NO.1.

[0024] In the above technical solution, in Step 2, the cells to be transfected are 293FT.

[0025] In the above technical solution, in Step 2, the culturing process is completed in a constant temperature environment of 37°C with 5% CO2.

[0026] In the above technical solution, in Step 2, the process of packaging the lentiviral construct overexpressing CX3CL1 is as follows:

[0027] Add the lentiviral construct overexpressing CX3CL1, the packaging helper plasmid PAX2, and the packaging helper plasmid VSVG to Opti-MEM; the mass ratio of the lentiviral construct overexpressing CX3CL1, the packaging helper plasmid PAX2, and the packaging helper plasmid VSVG is 2:1:1; add 16 micrograms of the lentiviral construct overexpressing CX3CL1 to every 0.92 milliliters of Opti-MEM; obtain the first mixture;

[0028] Add the PEI transfection reagent to the obtained first mixture and mix again, and incubate at room temperature for 15 minutes to obtain the second mixture, that is, obtain the packaged lentiviral construct overexpressing CX3CL1; among them, add 92 microliters of the PEI transfection reagent to every 0.92 milliliters of Opti-MEM.

[0029] In the above technical solution, in step 2, after culturing, the cell culture supernatant is collected, and the CX3CL1 overexpressing lentiviral particles are obtained through an ultracentrifugation concentration process.

[0030] In the above technical solution, in step 3, the pluripotent stem cells to be transfected are H9.

[0031] In the above technical solution, in step 3, the initial concentration of the Puro screening process is Puro at a concentration of 0.5 μg / ml, and the final concentration is Puro at 2.5 μg / ml.

[0032] In the above technical solution, in step 4, the medium used for the initial culture is EB formation medium, and its components are: bone morphogenetic protein 4: 15 - 25 μg / L, stem cell factor: 35 - 45 μg / L, vascular endothelial growth factor: 15 - 25 μg / L, ROCK inhibitor: 8 - 12 μmol / L, and the balance is the basic medium for stem cell differentiation;

[0033] The components of the EB amplification medium used for the amplification culture are: bone morphogenetic protein 4: 15 - 25 μg / L, stem cell factor: 35 - 45 μg / L, vascular endothelial growth factor: 15 - 25 μg / L, and the balance is the basic medium for stem cell differentiation;

[0034] The components of the induction differentiation medium used for the induction differentiation culture are: interleukin 34: 80 - 120 μg / L, FMS-like tyrosine kinase 3 ligand: 15 - 25 μg / L, and the balance is in the basic medium containing 15% fetal bovine serum.

[0035] On the other hand, the present invention provides a CX3CL1 gene overexpressing microglia, which is prepared by the method described in any one of the above.

[0036] The characteristic that the overexpressing CX3CL1 microglia provided by the present invention is more polarized towards the M2 direction is an ideal cell material for studying the specific function and regulatory mechanism of CX3CL1 in microglia and the like.

[0037] On the other hand, the present invention provides the application of the overexpressing CX3CL1 microglia as described above in studying the function of the CX3CL1 gene or its regulatory mechanism, or in screening drugs for treating neuroinflammation.

[0038] It should be noted that the application provided by the present invention is not limited to the above fields such as the study of the CX3CL1 gene function and its regulatory mechanism and drug screening. Using the CX3CL1 gene overexpressing microglia provided by the present invention for research or use in other fields also belongs to the protection scope of the present invention.

[0039] Optionally, in some embodiments of the present invention, the above research is for non-disease diagnosis or treatment purposes.

[0040] The advantages and beneficial effects of the present invention are as follows:

[0041] Through gene sequencing, it was found that the expression of related genes such as negative regulation of insulin-like growth factor receptor signaling pathway, regulation of neuroinflammatory response, and negative regulation of interleukin-13 production in microglia derived from pluripotent stem cells overexpressing CX3CL1 increased; at the same time, the expression of related genes such as vascular endothelial growth factor binding, platelet-derived growth factor binding, protein tyrosine kinase activity, glycosaminoglycan binding, and metallopeptidase activity increased. This indicates that overexpression of CX3CL1 in microglia will cause them to polarize more towards the M2 direction, promote cell proliferation, angiogenesis, wound healing, and enhance tissue repair.

[0042] It shows that overexpression of CX3CL1 will endow microglia cultured in vitro with certain anti-inflammatory effects and be closer to the functional state of microglia in the human brain, which is different from conventionally cultured microglia in vitro, which are in a pro-inflammatory state and polarize more towards the M1 direction. Overexpression of CX3CL1 enhances the anti-inflammatory and tissue repair effects of microglia, and constructs a new platform for basic medical research and clinical new drug development. Brief Description of the Drawings

[0043] Figure 1 is the qPCR verification of pluripotent stem cells overexpressing CX3CL1 of the present invention;

[0044] Figure 2 is the flow cytometry verification of pluripotent stem cells overexpressing CX3CL1 of the present invention;

[0045] Figure 3It is the characteristic marker staining of microglia from different stem cell sources in the present invention;

[0046] Figure 4 It is the qPCR verification of microglia with overexpressed CX3CL1 in the present invention;

[0047] Figure 5a It is the GO_f enrichment bubble chart of gene sequencing in the present invention;

[0048] Figure 5b It is the GO_p enrichment bubble chart of gene sequencing in the present invention.

[0049] Figure 6 It is the schematic diagram of the lentiviral vector structure in Example 1 of the present invention

[0050] For those of ordinary skill in the art, without creative efforts, other relevant drawings can be obtained based on the above drawings. Detailed implementation manners

[0051] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0052] Example 1

[0053] A method for preparing microglia with overexpressed CX3CL1, comprising the following steps:

[0054] S1, preparation of a lentiviral construct with overexpressed CX3CL1,

[0055] Select the full-length cDNA sequence of CX3CL1 and clone it into a lentiviral expression vector carrying the EF-1α promoter. At the same time, insert a reporter gene (such as green fluorescent protein) into the vector for screening and tracking expression; obtain a recombinant lentiviral expression vector;

[0056] For the above-mentioned recombinant lentiviral expression vector with correct sequencing, use PureLink TM HiPure Plasmid Maxiprep Kit to extract and purify to obtain a high-quality recombinant plasmid, that is, a lentiviral construct with overexpressed CX3CL1, for lentiviral packaging and transfection of target cells;

[0057] The viral plasmid was designed by Genewiz, as Figure 6 shown;

[0058] The full-length cDNA sequence of the said CX3CL1 is shown as SEQ ID NO.1:

[0059] SEQ ID NO.1

[0060]

[0061] The amino acid sequence encoded by the full-length cDNA sequence of CX3CL1 is shown in SEQ ID NO.2:

[0062] SEQ ID NO.2

[0063] MAPISLSWLLRLATFCHLTVLLAGQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGTFEKQIGEVKPRTTPAAGGMDESVVLEPEATGESSSLEPTPSSQEAQRALGTSPELPTGVTGSSGTRLPPTPKAQDGGPVGTELFRVPPVSTAATWQSSAPHQPGPSLWAEAKTSEAPSTQDPSTQASTASSPAPEENAPSEGQRVWGQGQSPRPENSLEREEMGPVPAHTDAFQDWGPGSMAHVSVVPVSSEGTPSREPVASGSWTPKAEEPIHATMDPQRLGVLITPVPDAQAATRRQAVGLLAFLGLLFCLGVAMFTYQSLQGCPRKMAGEMAEGLRYIPRSCGSNSYVLVPV*

[0064] S2. Preparation of lentiviral particles overexpressing CX3CL1

[0065] (1) Cell culture preparation:

[0066] Use 293FT cells, which is a fast-growing and highly transfectable cell line derived from human embryonic kidney cells and transformed with SV40 large T antigen, and is very suitable as a production host for lentivirus. When the cell density approaches 90%, digest the cells with trypsin preheated at 37°C to separate the cells.

[0067] After digestion, resuspend the cells evenly by centrifugation, and then seed them in a 10 cm culture dish.

[0068] Place the culture dish in an incubator at 37°C with 5% CO2 for 24 hours until the cells adhere and the coverage area reaches more than 50% of the total area of the culture dish. At this time, start the preparation for virus packaging.

[0069] (2) Virus packaging:

[0070] Prepare two sterile 50 ml centrifuge tubes. Add 920 μl of Opti-MEM to one centrifuge tube (Tube B), and add the overexpressed CX3CL1 lentiviral construct prepared in S1 at 16 μg, 8 μg of packaging plasmid PAX2 and 8 μg of packaging plasmid VSVG. The mass ratio here is overexpressed CX3CL1 lentiviral construct: PAX2: VSVG = 2:1:1.

[0071] Add 96 μl of PEI transfection reagent to another centrifuge tube (Tube A).

[0072] After leaving Tubes A and B standing for 5 minutes, slowly add the PEI mixture in Tube A to Tube B, mix well, and let stand for 15 minutes to obtain the packaged overexpressed CX3CL1 lentiviral construct, which is beneficial to improving the transfection efficiency.

[0073] (3) Transfect 293 cells with the packaging plasmid:

[0074] Dropwise and evenly add the packaged overexpressed CX3CL1 lentiviral construct to the 293FT cell culture dish prepared in step S2(1), and gently shake the culture dish to ensure that the mixture is evenly distributed.

[0075] Return the culture dish to the 37 °C incubator containing 5% CO2 and continue to incubate.

[0076] (4) Change the culture medium:

[0077] After 11 - 12 hours, aspirate the old culture medium, add fresh, pre-warmed to 37 °C complete culture medium, and continue to incubate the cells.

[0078] (5) Virus collection:

[0079] At about 48 hours after transfection, collect the cell culture supernatant containing lentiviral particles, and remove cells and cell debris by centrifugation to obtain a clear virus suspension.

[0080] (6) Ultracentrifuge to concentrate the virus

[0081] Ultracentrifuge to concentrate the clear virus suspension, carefully discard the supernatant, and gently resuspend the precipitate with sterile PBS. Aliquot the virus suspension into sterile cryotubes, 1 mL per tube, and avoid repeated freezing and thawing. Finally, measure the virus titer, and the qualified standard is not less than 1×10 5 TU / mL. After the above steps are completed, overexpressed CX3CL1 lentiviral particles are obtained.

[0082] The main operation in step (5) is to collect the virus. The centrifugation in step (5) is a routine low-speed centrifugation in the laboratory, aiming to remove impurities. The centrifugation in step (6) is ultracentrifugation, aiming to concentrate the virus so that it meets the qualified titer standard.

[0083] Preparation of pluripotent stem cells with high-purity overexpression of CX3CL1

[0084] Spread the pluripotent stem cells H9 to be transfected on a 24-well plate. After 12 - 24 hours, change the medium. The optimal density is 30% - 50%. Add the lentiviral particles with overexpression of CX3CL1 (obtained in step S2(6)) and culture. Re-infection with the virus solution can be carried out according to the situation. After the cells grow to confluence, detect the expression of the recombinant plasmid.

[0085] Observe the infected cells with fluorescence under a fluorescence microscope 48 - 72 hours after infection to preliminarily determine the infection effect, and then analyze the transfection rate by flow cytometry. The FITC channel is selected for the detection of EF-1α-HCX3CL1-Puro-EGFP.

[0086] The transfection rate is initially detected to be about 10%. At this time, it is pluripotent stem cells with low-purity overexpression of CX3CL1. Screen positive clones through Puro. The cells can tolerate 0.5 μg / ml concentration of Puro in about two weeks. Stain with CX3CL1 antibody and detect again by flow cytometry. It can be seen that the protein expression level of CX3CL1 increases significantly. Subsequently, gradually increase the concentration of the Puro drug until the cells can tolerate 2.5 μg / ml of Puro and the protein expression level of CX3CL1 no longer increases, obtaining pluripotent stem cells with high-purity overexpression of CX3CL1. Collect the cells into an ep tube for further verification experiments.

[0087] S4, Differentiate into microglia with stable enhanced expression of CX3CL1

[0088] The pluripotent stem cells with high-purity overexpression of CX3CL1 differentiate into microglia with overexpression of CX3CL1.

[0089] (1) EB (embryoid body) formation stage (Day 1)

[0090] a Prepare EB formation medium and EB amplification medium:

[0091] The EB formation medium contains: bone morphogenetic protein 4 at 20 μg / L, stem cell factor at 40 μg / L, vascular endothelial growth factor at 20 μg / L, ROCK inhibitor at 10 μmol / L, dissolved in the basic stem cell differentiation medium. The EB amplification medium contains: bone morphogenetic protein 4 at 20 μg / L, stem cell factor at 40 μg / L, vascular endothelial growth factor at 20 μg / L, dissolved in the basic stem cell differentiation medium;

[0092] b The highly purified CX3CL1-overexpressing pluripotent stem cells obtained in S3: First, use a pipette to aspirate the old medium, rinse the cells with 1 mL of 1×PBS solution, aspirate and discard the PBS, and then add 1 mL of Accutase enzyme. Incubate the cells in a 37°C incubator for about 3 minutes, tap the bottom of the flask to detach the cells, add 10 mL of DF12, transfer to a centrifuge tube, and centrifuge at 1000 g for 5 minutes.

[0093] c Resuspend the precipitate obtained by centrifugation with EB formation medium, add it to a V-bottom 96-well plate at 5000 cells per well with a volume of 100 μL, centrifuge at 500 g for 3 minutes, and culture the EB in an incubator.

[0094] (2) EB amplification stage (Day 4)

[0095] Observation with a microscope shows that the EB has significantly enlarged, with a diameter mostly between 200 - 400 μm. At this time, aspirate the original medium, add 100 μL of EB amplification medium, and continuously observe every day until the EB induction and differentiation stage. When the medium turns yellow, change the medium in a timely manner.

[0096] (3) EB induction and differentiation (Day 13)

[0097] a Coat a 24-well plate with 0.1% gelatin (2% gelatin diluted with PBS, 500 μL per well) one day in advance, place it in an incubator overnight for the next experiment

[0098] b Prepare the induction and differentiation culture medium for microglia:

[0099] The components of the induction and differentiation medium are: interleukin 34 at 100 μg / L, FMS-like tyrosine kinase 3 ligand at 20 μg / L, dissolved in a basal medium containing 15% fetal bovine serum;

[0100] c Transfer the EB in the 96-well plate to the coated 24-well plate, note that the EB should be evenly distributed and not too concentrated, place 5 - 6 EB in each well

[0101] d After the transfer is completed, add 500 μL of the microglia induction and differentiation medium (prepared in step b), and place it in an incubator for culture;

[0102] e According to this protocol, EB adherence can generally be observed on the 2nd - 3rd day. Observe the status of EB continuously every day thereafter. When the medium turns yellow, change the medium in a timely manner. Generally, change the medium once every 4 - 5 days.

[0103] (4) Microglia formation stage (Day 25)

[0104] Microscopic observation showed that colonies formed by microglia had appeared. At this time, microglia could be collected into an EP tube for further verification experiments.

[0105] Comparative Example 1

[0106] Preparation of microglia derived from pluripotent stem cells transfected with empty plasmid, including the following steps:

[0107] S1 EB (embryoid body) formation stage (Day 1)

[0108] a Prepare EB formation medium and EB amplification medium:

[0109] EB formation medium, the composition is: bone morphogenetic protein 4 20 μg / L, stem cell factor 40 μg / L, vascular endothelial growth factor 20 μg / L, ROCK inhibitor 10 μmol / L, dissolved in the basic medium for stem cell differentiation. The composition of EB amplification medium is: bone morphogenetic protein 4 20 μg / L, stem cell factor 40 μg / L, vascular endothelial growth factor 20 μg / L, dissolved in the basic medium for stem cell differentiation;

[0110] b Digest the pluripotent stem cells transfected with empty plasmid into single cells: First, use a pipette to aspirate the old medium, rinse the cells with 1 mL of 1×PBS solution, aspirate the PBS, and then add 1 mL of Accutase enzyme. Digest the cells in a 37°C incubator for about 3 minutes, tap the bottom of the flask to make the cells detach, add 10 ml of DF12, transfer to a centrifuge tube, and centrifuge at 1000 g for 5 min.

[0111] c Resuspend the precipitate obtained by centrifugation with EB formation medium, add 5000 cells per well with a volume of 100 μl to a V-bottom 96-well plate, centrifuge at 500 g for 3 min, and culture the EB in an incubator.

[0112] S2 EB amplification stage (Day 4)

[0113] Observation with a microscope showed that the EB had become significantly larger, with a diameter mostly between 200 - 400 μm. At this time, aspirate the original medium, add 100 μl of EB amplification medium, and continuously observe every day until the EB induction and differentiation stage. When the medium turns yellow, change the medium in a timely manner.

[0114] S3 EB induction and differentiation (Day 13)

[0115] a Coat a 24-well plate with 0.1% gelatin (2% gelatin diluted with PBS, 500 μl per well) one day in advance, place it in the incubator overnight for the next experiment

[0116] b Preparation of the induction and differentiation medium for microglia: interleukin-34 at 100 μg / L and FMS-like tyrosine kinase 3 ligand at 20 μg / L, dissolved in the basal medium containing 15% fetal bovine serum;

[0117] c Transfer the EBs in the 96-well plate to the pre-coated 24-well plate, ensuring that the EBs are evenly distributed and not overly concentrated. Place 5 - 6 EBs in each well.

[0118] d After the transfer, add 500 μl of the induction and differentiation medium (see step b) and place it in the incubator for culture;

[0119] e According to this protocol, EB adherence can generally be observed on the 2nd - 3rd day. Continuously observe the state of the EBs every day thereafter. When the medium turns yellow, change the medium in a timely manner. Generally, the medium needs to be changed once every 4 - 5 days.

[0120] S4 Microglia formation stage (Day 25)

[0121] Under microscopic observation, colonies of microglia have formed. At this time, the microglia can be collected into an EP tube for the next verification experiment.

[0122] Comparative Example 2

[0123] Preparation of microglia from ordinary pluripotent stem cells, including the following steps:

[0124] S1 EB (embryoid body) formation stage (Day 1)

[0125] a Preparation of EB formation medium and EB amplification medium:

[0126] The EB formation medium contains: bone morphogenetic protein 4 at 20 μg / L, stem cell factor at 40 μg / L, vascular endothelial growth factor at 20 μg / L, and ROCK inhibitor at 10 μmol / L, dissolved in the stem cell differentiation basal medium. The EB amplification medium contains: bone morphogenetic protein 4 at 20 μg / L, stem cell factor at 40 μg / L, and vascular endothelial growth factor at 20 μg / L, dissolved in the stem cell differentiation basal medium;

[0127] b Digest the ordinary pluripotent stem cells into single cells: First, use a pipette to aspirate the old medium, rinse the cells with 1 mL of 1×PBS solution, aspirate the PBS, and then add 1 mL of Accutase enzyme. Incubate the cells in a 37°C incubator for about 3 minutes, tap the bottom of the flask to detach the cells, add 10 ml of DF12, transfer to a centrifuge tube, and centrifuge at 1000 g for 5 min.

[0128] The precipitate obtained by centrifugation was resuspended in EB formation medium, and 5000 cells per well with a volume of 100 μl were added to a V-bottom 96-well plate, followed by centrifugation at 500 g for 3 min to obtain EB, which was then cultured in an incubator.

[0129] S2 EB amplification stage (Day 4)

[0130] Observation under a microscope showed that the EB had significantly enlarged, with diameters mostly ranging between 200 - 400 μm. At this time, the original medium was aspirated, and 100 μl of EB amplification medium was added. Subsequently, continuous observation was required every day until the EB induction and differentiation stage. When the medium turned yellow, it needed to be replaced in a timely manner.

[0131] S3 EB induction and differentiation (Day 13)

[0132] a Coat a 24-well plate with 0.1% gelatin (2% gelatin diluted with PBS, 500 μl per well) one day in advance and place it in an incubator overnight for the next experiment.

[0133] b Prepare the induction and differentiation medium for microglia: The components of the induction and differentiation medium are: interleukin 34 at 100 μg / L and FMS-like tyrosine kinase 3 ligand at 20 μg / L, dissolved in a basal medium containing 15% fetal bovine serum.

[0134] c Transfer the EB in the 96-well plate to the coated 24-well plate, ensuring that the EB is evenly distributed and not overly concentrated. Place 5 - 6 EB in each well.

[0135] d After the transfer, add 500 μl of the induction and differentiation medium (see step b) and place it in an incubator for culture.

[0136] e According to this protocol, EB attachment can generally be observed on the 2nd - 3rd day. Subsequently, continuous observation of the EB status is required every day. When the medium turns yellow, it needs to be replaced in a timely manner, and generally, the medium needs to be replaced every 4 - 5 days.

[0137] S4 Microglia formation stage (Day 25)

[0138] Observation under a microscope showed that colonies of microglia had formed. At this time, the microglia could be collected into an EP tube for the next verification experiment.

[0139] Example 2

[0140] Verification in the preparation process of microglia overexpressing CX3CL1, including:

[0141] Cell culture preparation: Prepare ordinary pluripotent stem cells H9 and pluripotent stem cells H9-GFP transfected with empty plasmid, and place the culture dishes in a 37°C incubator containing 5% CO2 for daily culture for subsequent verification experiments.

[0142] 1. Verification of the expression level of the target gene of pluripotent stem cells with high-purity overexpression of CX3CL1

[0143] After extracting RNA from the pluripotent stem cells with high-purity overexpression of CX3CL1 (labeled as H9-CX3CL1-Stem cell) obtained in step S3 of Experimental Example 1, the pluripotent stem cells transfected with empty plasmid (labeled as H9-GFP-Stem cell), and ordinary pluripotent stem cells (labeled as H9-Stem cell), 4 replicate wells were set up for each group of cells, and qPCR was performed after reverse transcription. (See Appendix Figure 1 )

[0144] Appendix Figure 1 is the qPCR result graph. The vertical axis is the relative RNA expression level of CX3CL1, and the horizontal axis is the group. It can be seen that compared with the control groups H9-Stem cell and H9-GFP-Stem cell, the RNA expression level of CX3CL1 in the experimental group H9-CX3CL1-Stem cell was significantly increased.

[0145] After staining the pluripotent stem cells with high-purity overexpression of CX3CL1 (labeled as H9-CX3CL1-Stem cell) obtained in step S3 of Experimental Example 1, the pluripotent stem cells transfected with empty plasmid (labeled as H9-GFP-Stem cell), and ordinary pluripotent stem cells (labeled as H9-Stem cell) with the antibody CX3CL1, flow cytometry was performed. (See Appendix Figure 2 )

[0146] Appendix Figure 2 is the flow cytometry histogram. The vertical axis is the cell count, and the horizontal axis is the fluorescence signal intensity of CX3CL1. The fluorescence channel is APC. Different colors represent different groups. Black represents the control group H9-Stem cell, green represents the control group H9-GFP-Stem cell, and red represents the experimental group H9-CX3CL1-Stem cell. It can be seen that compared with the control groups H9-Stem cell and H9-GFP-Stem cell, the protein expression level of CX3CL1 in the experimental group H9-CX3CL1-Stem cell was significantly increased.

[0147] 2. Verification of microglial cell differentiation

[0148] The microglia overexpressing CX3CL1 obtained in S4 of Experimental Example 1 (labeled as H9-CX3CL1-microglia), the microglia derived from pluripotent stem cells transfected with empty plasmid obtained in Comparative Example 1 (labeled as H9-GFP-microglia), and the microglia derived from ordinary pluripotent stem cells obtained in Comparative Example 2 (labeled as H9-microglia) were stained with antibodies IBA1 and CD11b and then detected by flow cytometry. (IBA1 is a calcium-binding protein specifically expressed in microglia. IBA1 antibody has currently been widely used as a marker for microglia. CD11b mainly labels monocytes and macrophages, and microglia are macrophages located in the brain and spinal cord, responsible for the immune defense of the central nervous system (CNS)).

[0149] Then, the highly purified pluripotent stem cells overexpressing CX3CL1 obtained in S3 of Experimental Example 1 (labeled as H9-CX3CL1-Stem cell), the pluripotent stem cells transfected with empty plasmid (labeled as H9-GFP-Stem cell), and the ordinary pluripotent stem cells (labeled as H9-Stem cell) were stained with IBA1 and CD11b and then detected by flow cytometry. (See attached Figure 3 )

[0150] Attached Figure 3 is a flow scatter plot. The vertical axis is the fluorescence signal intensity of CD11b, and the fluorescence channel is PC7A. The horizontal axis is the fluorescence signal intensity of IBA1, and the fluorescence channel is APC. Different colors represent different cells. Black is pluripotent stem cells, and red is microglia derived from the corresponding pluripotent stem cells. The groups from left to right are Comparative Example H9-Stem cell and H9-microglia, Comparative Example H9-GFP-Stem cell and H9-GFP-microglia, and Example H9-CX3CL1-Stem cell and H9-CX3CL1-microglia). It can be seen that compared with pluripotent stem cells, the proportion of IBA1 and CD11 double-positive cells in microglia derived from the corresponding pluripotent stem cells is about 95%, indicating that they have been successfully differentiated into microglia.

[0151] 3. Verification of the expression level of the target gene in microglia overexpressing CX3CL1

[0152] After extracting RNA from the microglia overexpressing CX3CL1 obtained in S4 of Experimental Example 1 (labeled as H9-CX3CL1-microglia) and the microglia derived from ordinary pluripotent stem cells obtained in Comparative Example 2 (labeled as H9-microglia), reverse transcription was performed for qPCR. (See attached Figure 4 )

[0153] Attached Figure 4This is the qPCR result graph. The vertical axis represents the relative RNA expression level of CX3CL1, and the horizontal axis represents the groups. It can be seen that compared with the comparative example H9-Stem cell, the RNA expression level of CX3CL1 in the example H9-CX3CL1-Stem cell is significantly increased.

[0154] Functional verification of microglia overexpressing CX3CL1 in Example 3

[0155] The microglia overexpressing CX3CL1 obtained in S4 of Experimental Example 1 (labeled as H9-CX3CL1-microglia), the microglia derived from pluripotent stem cells transfected with empty plasmid obtained in Comparative Example 1 (labeled as H9-GFP-microglia), and the microglia derived from ordinary pluripotent stem cells obtained in Comparative Example 2 (labeled as H9-microglia) were subjected to RNA sequencing. GO functional enrichment analysis was performed on the intersection gene set of "differentially expressed genes between H9-CX3CL1-microglia and H9-GFP-microglia" and "differentially expressed genes between H9-CX3CL1-microglia and H9-microglia". The enrichment results of gene molecular function (go_f) and biological process (go_p) were focused on, and the association between the enrichment degree and gene count was presented using a bubble plot during visualization. The results showed that in the intersection gene set, the expressions of genes related to negative regulation of insulin-like growth factor receptor signaling pathway, regulation of neuroinflammatory response, negative regulation of interleukin-13 production, etc. in H9-CX3CL1-microglia increased; meanwhile, the expressions of genes related to vascular endothelial growth factor binding, platelet-derived growth factor binding, protein tyrosine kinase activity, glycosaminoglycan binding, metallopeptidase activity, etc. in H9-CX3CL1-microglia increased. This indicated that overexpression of CX3CL1 in microglia would cause them to polarize more towards the M2 direction, promote cell proliferation, angiogenesis, wound healing, and enhance tissue repair function. (See Figure 5)

[0156] It is indicated that overexpression of CX3CL1 can endow microglia cultured in vitro with certain anti-inflammatory effects, making them closer to the functional state of microglia in the human brain, which is different from microglia cultured conventionally in vitro, the latter being in a pro-inflammatory state and more polarized towards the M1 direction. Overexpression of CX3CL1 enhances the anti-inflammatory and tissue repair effects of microglia, constructing a new platform for basic medical research and clinical new drug development.

[0157] The specific implementation steps not described in the above embodiments are generally common steps. The following are some examples:

[0158] Steps for large-scale plasmid extraction

[0159] Before starting:

[0160] RNase A needs to be added to the resuspension buffer (R3), mixed well, and marked as added. Store at 4°C.

[0161] If the lysis buffer (L7) contains salt precipitation, heat it in a 37°C water bath until clear. Do not shake L7.

[0162] (1) Obtain the enriched Escherichia coli precipitate. Centrifuge at 4000g for 10 minutes. Divide into 6 tubes, 50 ml for each enrichment.

[0163] (2) Add 10 ml of RNase A buffer (R3) to the precipitate and resuspend by pipetting up and down.

[0164] (3) Add 10 ml of lysis buffer (L7) and invert the tube 5 times up and down. Let it stand at room temperature for 5 minutes.

[0165] During the waiting period, place the column on a conical flask, add 30 ml of equilibration buffer (EQ1) to the column, and let it flow out naturally by gravity.

[0166] (4) Add 10 ml of precipitation buffer (N3). Immediately invert the tube to mix well. Centrifuge at 7000g for 10 minutes at 4°C in a 4°C centrifuge.

[0167] (5) Transfer the supernatant obtained by centrifugation to the column. (Try not to aspirate the white material)

[0168] (6) Add 60 ml of W8 until it has all flowed out.

[0169] (7) Transfer the column to a new 50 ml centrifuge tube, add 15 ml of elution buffer (E4) to the column, and let it flow out naturally. (At this time, the centrifuge tube contains the purified plasmid)

[0170] (8) Add 10.5 ml of isopropanol to the centrifuge tube and mix well. Centrifuge at 7000g for 2 h at 4°C in a 4°C centrifuge. At this time, white precipitate can be seen on the side wall of the centrifuge tube.

[0171] (9) After aspirating the supernatant with a pipette, enter the cell culture room, add 1 mL of 70% ethanol, dissolve and mix well, transfer to a centrifuge tube with a screw thread, and centrifuge at 12,000 rpm for 1 minute.

[0172] (10) After centrifugation, aspirate the supernatant (try to aspirate it as clean as possible) and air dry it naturally (ethanol is volatile).

[0173] (11) Add 300 - 600 μL of sterile ultrapure water and mix well.

[0174] (12) Use a nanodrop to detect the plasmid concentration.

[0175] Daily culture of pluripotent stem cells:

[0176] (1) Cell medium change:

[0177] Pluripotent stem cells are cultured daily in a T25 flask. During the culture process, pay attention to ensuring that the cells form colonies and do not be dispersed into single cells. The cell medium is changed every 24 hours.

[0178] (2) Cell passage:

[0179] Pre - coat a new T25 flask with Matrix gel (2.5 mL of basal medium + 30 μL of Matrix concentrated gel) overnight for the next cell passage.

[0180] First, use a pipette to aspirate the old medium, rinse the cells with 1 mL of 1×PBS buffer, aspirate the PBS, then add 1 mL of ReLeSR to rinse the cells, and aspirate the ReLeSR. Digest the cells in a 37°C incubator for about 3 minutes, tap the bottom of the flask to make the cells detach, and add 1 mL of basal medium to terminate the digestion. Aspirate the 1 mL of cell suspension and drop 8 drops into the pre - coated T25 flask, and check under the microscope whether the cells in the T25 flask are evenly distributed.

[0181] (3) Cell cryopreservation:

[0182] Pre - warm the cell cryopreservation box to room temperature in advance. Prepare the cryopreservation solution according to the ratio of serum - free medium: DMSO = 9:1. After obtaining the cell suspension according to the method of cell passage, centrifuge at 1000 rpm for 5 minutes, resuspend the cell pellet with the cryopreservation solution, add 1 - 1.5 mL of the mixture to each cryopreservation tube, place it in the cryopreservation box, store at - 80°C, and transfer to a liquid nitrogen tank for storage after the cells are completely frozen.

[0183] (4) Cell thawing:

[0184] Pre - coat a new T25 flask with Matrix gel (2.5 mL of basal medium + 30 μL of Matrix concentrated gel) overnight for the next cell thawing.

[0185] Adjust the temperature of the water bath to 37°C. Take out the cryotubes stored in the liquid nitrogen tank or -80°C refrigerator according to the record. Use forceps to carefully hold the cryotube body and place it in the water bath, gently shaking until the solid inside the tube completely melts. Add about 10 mL of stem cell medium to a 15 mL centrifuge tube to dilute the concentration of DMSO in the cryopreservation solution. Add the cell suspension to the 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. During this period, prepare the cell recovery medium (4 mL stem cell medium + 4 μL YZ7632). After centrifugation, discard the supernatant. Carefully pipette the cell pellet with the cell recovery medium until it forms a lump (be careful not to disperse the cells in this step), transfer it to a coated T25 culture flask, and culture it in a 37°C incubator.

[0186] qPCR step (1) RNA extraction

[0187] Use a column-type total RNA extraction kit to extract RNA. The specific steps are as follows:

[0188] Collect the cells and wash them once with PBS.

[0189] Lysis: After obtaining the cell pellet, gently flick the bottom of the ep tube, add an appropriate amount of lysis buffer RL, and vortex.

[0190] Filtration: Transfer the obtained solution to the filter column CS (the filter column CS is placed in the collection tube), and centrifuge at 12000 rpm for 2 minutes.

[0191] Add 1 volume of 70% ethanol to the filtrate and mix well (there may be precipitation at this time). Transfer the obtained solution to the adsorption column CR3 (the adsorption column CR3 is placed in the collection tube), centrifuge at 12000 rpm for 1 minute, pour out the filtrate, and put it back into the collection tube.

[0192] Add 350 μL of protein removal solution RW1 to CR3, centrifuge at 12000 rpm for 1 minute, pour out the filtrate, and put it back into the collection tube.

[0193] Add 500 μL of washing solution RW (please check if ethanol is added) to CR3, let it stand at room temperature for 2 minutes, centrifuge at 12000 rpm for 1 minute, pour out the filtrate, and put the adsorption column CR3 back into the collection tube.

[0194] Repeat the previous step, centrifuge at 12000 rpm for 2 minutes, pour out the waste liquid, place CR3 at room temperature for several minutes to dry the washing solution.

[0195] Transfer CR3 to a new RNase-Free centrifuge tube, add 30 - 100 μL of RNase-Free ddH2O, let it stand at room temperature for 2 minutes, centrifuge at 12000 rpm for 2 minutes to obtain the RNA solution (store it at -80°C).

[0196] (2) Reverse transcription

[0197] Use All in one First-Strand Synthesis to reverse transcribe RNA into cDNA. The sample addition for each PCR tube is as follows:

[0198] RNA 2ul MasterMix 4ul dsDNase 1ul ddH20 13ul

[0199] The PCR program is as follows:

[0200] 37℃ 2 min 55℃ 15 min 85℃ 5 min 12℃ ∞

[0201] (3) qPCR

[0202] The sample addition for each PCR tube is as follows:

[0203] cDNA 17 / 9ul ddH20 64 / 9ul Taq SYBR Green 10ul CX3CL1 primer 1ul

[0204] Attachment: Steps for flow cytometry staining of CX3CL1

[0205] CX3CL1 mainly exists in the cytoplasm. Therefore, before staining with the CX3CL1 antibody, it is necessary to use BD

[0206] Cytofix / Cytoperm Plus to permeabilize the membrane, and then stain with the CX3CL1 antibody. The specific steps are as follows:

[0207] (1) After washing the digested cells twice with PBS, resuspend them with 200 μl of Fixation / Permeabilization. Fix at 4°C for 20 min. (During this period, prepare 1×Perm / Wash washing solution, that is, 4.5 ml of ultrapure water + 0.5 ml of 10×Perm / wash washing solution)

[0208] (2) Wash the fixed cells twice with 250 μl of 1×Perm / Wash washing solution

[0209] (3) Resuspend with 50 μl of 1×Pem / Wash washing solution, add 1 μl of CX3CL1 antibody, and incubate at room temperature in the dark for 20 min

[0210] (4) After the staining is completed, wash once with 250 μl of 1×Perm / Wash washing solution, then wash once with PBS, and then resuspend with PBS to 200 μl, filter with gauze, and detect on the machine. (The detection channel for this CX3CL1 antibody is APC)

[0211] List of main reagents

[0212] Reagent name Catalog number Company <![CDATA[PureLink TM HiPure Plasmid Maxiprep Kit]]> K210006 Thermo Fisher Opti-MEM 31985062 Gibco PEI 40816ES02 Yeasen Accutase enzyme 07922 07920 Stem Cell mTeSR Plus 100-0276 100-1130 Stem Cell DMEM-F12 medium KGM12500NH-500 Kaiji Biotechnology ReLeSR 100-0483 Stem Cell DMSO D806645 MACKLIN Total RNA extraction kit DP430 TIANGEN All in one First-Strand Synthesis EG15133S Yugong Biotechnology Taq SYBR Green EG20117M Yugong Biotechnology BD Cytofix / Cytoperm Plus 554715 BD CX3CL1-APC 130-108-114 Miltenyi Biotec

[0213] Main self-prepared reagents

[0214]

[0215] The above has made an exemplary description of the present invention. It should be noted that without departing from the core of the present invention, any simple deformation, modification or equivalent replacement that can be made by those skilled in the art without creative efforts shall fall within the protection scope of the present invention.

Claims

1. A method for preparing microglia overexpressing CX3CL1, characterized in that, It includes the following steps: S1. Pluripotent stem cell construction: Add lentiviral particles overexpressing CX3CL1 to the pluripotent stem cells to be transfected. After transfection, pluripotent stem cells with low purity overexpressing CX3CL1 are obtained. S2. Purification: Subject the pluripotent stem cells with low purity overexpressing CX3CL1 to Puro drug screening to obtain pluripotent stem cells with high purity overexpressing CX3CL1. S3. Differentiation: Differentiate the pluripotent stem cells with high purity overexpressing CX3CL1 to obtain microglia overexpressing CX3CL1.

2. The method for preparing microglia overexpressing CX3CL1 according to claim 1, characterized in that, The nucleotide sequence of CX3CL1 is as shown in SEQ ID NO.1; preferably, the pluripotent stem cells to be transfected are H9.

3. A method for preparing microglia overexpressing CX3CL1, characterized in that, It includes the following steps: Step 1. Preparation of lentiviral construct overexpressing CX3CL1: Select the full-length cDNA sequence of CX3CL1 and clone it into a lentiviral expression vector carrying the EF-1α promoter; a recombinant lentiviral expression vector is obtained. For the above recombinant lentiviral expression vector with correct sequencing, use a plasmid large-scale extraction kit to extract and purify to obtain a high-quality recombinant plasmid, that is, the lentiviral construct overexpressing CX3CL1, for lentiviral packaging and transfection of target cells. Step 2. Preparation of lentiviral particles overexpressing CX3CL1: Use the packaged lentiviral construct overexpressing CX3CL1 to transfect the cells to be transfected. After culturing, collect the cell culture supernatant to obtain the lentiviral particles overexpressing CX3CL1. Step 3. Preparation of pluripotent stem cells with high purity overexpressing CX3CL1: Infect the pluripotent stem cells to be transfected with the lentiviral particles overexpressing CX3CL1 and obtain pluripotent stem cells with high purity overexpressing CX3CL1 through Puro screening. Step 4. Differentiate to generate microglia overexpressing CX3CL1: Digest the pluripotent stem cells with high purity overexpressing CX3CL1 into single cells, and further perform initial culture, amplification culture, and induced differentiation culture to obtain microglia overexpressing CX3CL1.

4. The method for preparing microglia overexpressing CX3CL1 according to claim 1, wherein The nucleotide sequence of CX3CL1 is as shown in SEQ ID NO.

1.

5. The method for preparing microglia overexpressing CX3CL1 according to claim 1, wherein, In step 2, the cells to be transfected are 293FT; Preferably, the culturing process is completed in a constant temperature environment of 37°C containing 5% CO2; Preferably, the process of packaging the lentiviral construct overexpressing CX3CL1 is: Add the lentiviral construct overexpressing CX3CL1, packaging helper plasmid PAX2, and packaging helper plasmid VSVG to Opti-MEM; the mass ratio of the lentiviral construct overexpressing CX3CL1, packaging helper plasmid PAX2, and packaging helper plasmid VSVG is 2:1:1; add 16 micrograms of the lentiviral construct overexpressing CX3CL1 to every 0.92 milliliters of Opti-MEM; a first mixture is obtained. Add PEI transfection reagent to the obtained first mixture and mix again, and incubate at room temperature for 15 minutes to obtain a second mixture, that is, the packaged lentiviral construct overexpressing CX3CL1; among them, add 92 microliters of PEI transfection reagent to every 0.92 milliliters of Opti-MEM. Preferably, in step 2, after culturing, the cell culture supernatant is collected, and the CX3CL1-overexpressing lentiviral particles are obtained through an ultracentrifugation concentration process.

6. The method for preparing microglia overexpressing CX3CL1 according to claim 1, wherein In step 3, the pluripotent stem cells to be transfected are H9; Preferably, the initial concentration of the Puro screening process is Puro at a concentration of 0.5 μg / ml, and the final concentration is Puro at 2.5 μg / ml.

7. The method for preparing microglia overexpressing CX3CL1 according to claim 1, characterized in that, In step 4, the medium used for the initial culture is an EB formation medium, and the components are: bone morphogenetic protein 4: 15 - 25 μg / L, stem cell factor: 35 - 45 μg / L, vascular endothelial growth factor: 15 - 25 μg / L, ROCK inhibitor: 8 - 12 μmol / L, and the balance is a basic medium for stem cell differentiation; The components of the EB amplification medium used for the amplification culture are: bone morphogenetic protein 4: 15 - 25 μg / L, stem cell factor: 35 - 45 μg / L, vascular endothelial growth factor: 15 - 25 μg / L, and the balance is a basic medium for stem cell differentiation; The components of the induction differentiation medium used for the induction differentiation culture are: interleukin 34: 80 - 120 μg / L, FMS-like tyrosine kinase 3 ligand: 15 - 25 μg / L, and the balance is a basic medium containing 15% fetal bovine serum.

8. A microglia overexpressing CX3CL1, characterized in that, It is prepared by the method according to any one of claims 1 to 7.

9. A cell material, characterized in that, It comprises the microglia overexpressing CX3CL1 as claimed in claim 8.

10. Use of the microglia overexpressing CX3CL1 as claimed in claim 8 in studying the function of the CX3CL1 gene or its regulatory mechanism, or in screening drugs for treating neuroinflammation.

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