Drug component for preventing and treating sequelae of cerebral thrombosis, preparation method and application thereof

Abstract

This application relates to the technical field of exosome preparation, specifically disclosing a pharmaceutical component for the prevention and treatment of sequelae of cerebral thrombosis, its preparation method, and its application. The pharmaceutical component comprises Exo-miR-17-92 and Exo-miR-30d-5p; it is obtained by secretion from mesenchymal stem cells capable of simultaneously overexpressing the genes miR-17-92 and miR-30d-5p; the sequence of the gene miR-17-92 is shown in SEQ ID NO:1, and the sequence of the gene miR-30d-5p is shown in SEQ ID NO:2. The pharmaceutical component of this application, comprising Exo-miR-17-92 and Exo-miR-30d-5p, exerts a significant and excellent therapeutic effect on sequelae of cerebral thrombosis through the synergistic effect of their expression products.

Description

Technical Field

[0001] This application relates to the technical field of drugs for the treatment of cerebral thrombosis, and more specifically, it relates to a drug component for the prevention and treatment of sequelae of cerebral thrombosis, a method for its preparation, and its application. Background Technology

[0002] Cerebral infarction, also known as ischemic stroke, refers to the ischemic necrosis or softening of localized brain tissue caused by ischemia and hypoxia due to impaired blood supply to the brain. Common clinical types of cerebral infarction include cerebral thrombosis, lacunar infarction, and cerebral embolism. The main clinical manifestations are sudden collapse, unconsciousness, hemiplegia, speech impairment, and intellectual disability.

[0003] Therefore, both prevention and treatment of cerebral thrombosis and its sequelae are essential. Current treatment methods for cerebral thrombosis include drug therapy and surgical treatment. Drug therapy includes the use of intravenous thrombolytic drugs, antiplatelet drugs, and antifibrinolytic drugs, but the efficacy of these drugs is generally limited. There are reports that exosomes are associated with thrombosis, infarction, and tumors. For example, exosome miR-21-3p is closely related to myocardial infarction and is associated with the occurrence and cellular activity of various tumors (gastric cancer, lung cancer, and kidney cancer, etc.); for example, in severe encephalitis, the upregulated expression of lncRNA RAB11B-AS1 targets and regulates microglial cell activation through miR-3d-5p, inducing an inflammatory response.

[0004] Therefore, it is necessary to provide a drug component based on microRNA exosomes for the treatment and prevention of cerebral thrombosis. Summary of the Invention

[0005] In order to improve the clinical treatment effect of cerebral thrombosis and its sequelae, this application provides a pharmaceutical component for the prevention and treatment of sequelae of cerebral thrombosis, its preparation method and application.

[0006] In a first aspect, this application provides a pharmaceutical component for the prevention and treatment of sequelae of cerebral thrombosis, employing the following technical solution:

[0007] A pharmaceutical component for the prevention and treatment of sequelae of cerebral thrombosis, the pharmaceutical component comprising exosome miR-17-92 and exosome miR-30d-5p.

[0008] Exo-miR-17-92 (exosomal miR-17-92) has the effect of repairing nerve cells. However, when Exo-miR-17-92 exosomes are used alone as a pharmaceutical component, their efficacy in treating cerebral thrombosis and its sequelae remains poor. Exo-miR-30d-5p (exosomal miR-30d-5p) can reverse M1 microglia that damage neurons into M2 microglia that have neuronal repair functions, but it does not have a therapeutic effect on the sequelae of cerebral thrombosis. By adopting the above technical solution, this application provides a drug component that can simultaneously produce Exo-miR-17-92 and Exo-miR-30d-5p. The two exosomes in this component work synergistically: Exo-miR-17-92 primarily functions to directly repair nerve cells and promote the cure of cerebral thrombosis and its sequelae, while Exo-miR-30d-5p plays an adjunctive therapeutic role, reversing neuronal-damaging factors (i.e., M1 type microglia that damage neuronal function) into neuronal-repairing factors (i.e., M2 type microglia with neuronal-repairing function). Through the collaboration of these two exosomes, the drug component ultimately exhibits significantly superior effects in preventing and treating the sequelae of cerebral thrombosis. Experimental results of this application show that: in animal experiments, it caused rat microglia to transform from the pro-inflammatory M1 type to the anti-inflammatory M2 type, significantly reducing the content of pro-inflammatory factors and significantly increasing the content of anti-inflammatory factors in rat plasma; simultaneously, it significantly reduced the size of cerebellar infarct lesions in rats. It is important to note that in this application, the choice of exosomes for overexpression will ultimately affect the therapeutic and preventative effects on sequelae of cerebral thrombosis. The applicant achieved significantly superior results by using the synergistic expression of these two types of exosomes.

[0009] Optionally, the particle size of the mesenchymal stem cell exosomes is 50-140 nm.

[0010] Optionally, the median particle size of the mesenchymal stem cell exosomes is 62 nm, the D90 is 76 nm, and the D10 is 56 nm.

[0011] In this scheme, the median particle size refers to the particle size value at which the cumulative particle distribution is 50%, D90 refers to the particle size value at which the cumulative particle distribution is 90%, and D10 refers to the particle size value at which the cumulative particle distribution is 10%.

[0012] Secondly, this application provides a method for preparing the above-mentioned drug component for preventing and treating sequelae of cerebral thrombosis, using the following technical solution:

[0013] A method for preparing a pharmaceutical component for the prevention and treatment of sequelae of cerebral thrombosis includes the following steps:

[0014] S1. Obtain mesenchymal stem cells for later use; obtain recombinant lentivirus containing genes miR-17-92 and miR-30d-5p for later use;

[0015] The sequence of the gene miR-17-92 is shown in SEQ ID NO:1, and the sequence of the gene miR-30d-5p is shown in SEQ ID NO:2;

[0016] S2. The recombinant lentivirus and the mesenchymal stem cells are cultured in contact, centrifuged, and the supernatant is obtained to obtain the drug component.

[0017] Conventional methods involve introducing exogenous genes into mesenchymal stem cells via electroporation or liposomes, resulting in the presence of these exogenous genes within the cytoplasm of the mesenchymal stem cells. However, after repeated culture, the exogenous genes may be lost, leading to the absence of their expression products and severely impacting exosome secretion and therapeutic efficacy. This application addresses this issue by employing a genetically modified approach, using the aforementioned recombinant lentivirus transfection of mesenchymal stem cells to prepare genetically modified exosomes, ensuring the sustained presence of the expression products (i.e., the two types of exosomes). This method significantly enhances the expression intensity of miR-17-92 and miR-30d-5p genes at the mRNA level, thereby significantly improving the therapeutic effect of the drug component on sequelae of cerebral thrombosis. This method can simultaneously produce large quantities of both types of exosomes, significantly improving therapeutic efficacy.

[0018] Optionally, the recombinant lentivirus may also include a fluorescent selection marker gene and an resistance gene.

[0019] Optionally, the gene sequence of the lentivirus is shown in SEQ ID NO:3; the sequence of the fluorescent selection marker gene is shown in SEQ ID NO:4; the resistance gene is a puromycin resistance gene, and the sequence of the resistance gene is shown in SEQ ID NO:5.

[0020] Optionally, the method for contact culture of the recombinant lentivirus and the mesenchymal stem cells in S2 includes the following steps:

[0021] S21. The recombinant lentivirus and the mesenchymal stem cells are placed on mesenchymal stem cell basal culture medium and cultured at 35-38 ℃ for 20-30 h.

[0022] S22. Replace the mesenchymal stem cell basal culture medium with mesenchymal stem cell selection culture medium and then culture the subsequent sets until all mesenchymal stem cells express fluorescence;

[0023] S23. Then continue the amplification culture and collect the supernatant.

[0024] The mesenchymal stem cell selection culture medium contains antibiotics corresponding to the resistance gene as a screening criterion.

[0025] By employing the above-mentioned technical solution, mesenchymal stem cells are transfected with recombinant lentiviruses, enabling the high-intensity expression of genes miR-17-92 and miR-30d-5p, resulting in a mesenchymal stem cell exosome composition (i.e., the drug component) with excellent therapeutic effects.

[0026] Optionally, after S23 expansion culture, the supernatant is collected and concentrated to obtain a mesenchymal stem cell exosome culture medium in which the concentration of the mesenchymal stem cell exosomes is (1-5)×10⁻⁶. 11 per mL.

[0027] Optionally, the antibiotic is puromycin; in the mesenchymal stem cell selective culture medium, the amount of puromycin added is 1-3 μg / mL.

[0028] Optionally, the method for obtaining the recombinant lentivirus includes the following steps:

[0029] S121. The gene miR-17-92, the gene miR-30d-5p, the fluorescent selection marker gene, and the resistance gene are linked to a lentiviral gene to obtain a recombinant lentiviral vector.

[0030] S122. The recombinant lentiviral vector and host cells are mixed, the recombinant lentiviral is continuously packaged, and then the recombinant lentiviral is obtained from the host cell culture supernatant for later use.

[0031] Optionally, the host cell line is selected from either the 293T cell line or the 293FT cell line.

[0032] Optionally, methods for obtaining mesenchymal stem cells include the following steps:

[0033] S111. The obtained fat extract is rinsed and cut into blocks, then digested with type I collagenase, centrifuged, and the undigested tissue blocks and the lower cell clusters are resuspended to obtain a cell cluster resuspension.

[0034] S112. The cell cluster resuspension is cultured in a 34-38 ℃, 3-7 Vol.% CO2 incubator; after adherent culture for 2-4 days, mesenchymal stem cells are obtained, expanded and used for later use.

[0035] Thirdly, this application provides an application of the above-mentioned pharmaceutical component for the prevention and treatment of sequelae of cerebral thrombosis, employing the following technical solution:

[0036] Application of a pharmaceutical component for the prevention and treatment of sequelae of cerebral thrombosis, wherein the pharmaceutical component is used to prepare products for the prevention and treatment of sequelae of cerebral thrombosis.

[0037] In summary, this application has the following beneficial effects:

[0038] 1. This application selects two exosomes (Exo-miR-17-92 and Exo-miR-30d-5p) as drug components, in which Exo-miR-17-92 positively promotes neuronal repair, while Exo-miR-30d-5p mainly reverses neuronal damaging factors into neuronal repair factors; under the synergistic effect of the two, the efficacy of this drug component in preventing and treating sequelae of cerebral thrombosis is significantly improved.

[0039] 2. In this application, the drug component is preferably prepared using gene modification methods. Mesenchymal stem cells are transfected with lentiviruses containing the genes miR-17-92 and miR-30d-5p to obtain a recombinant lentivirus (i.e., a recombinant plasmid) capable of expressing the target gene stably. This method offers high preparation efficiency (i.e., obtaining a large amount of recombinant lentivirus with a high transfection rate) and high stability (the target gene is not lost even with continuous transfection). Attached Figure Description

[0040] Figure 1 It is a lentiviral vector map;

[0041] Figure 2 The expression intensity of the genes miR-17-92 and miR-30d-5p at the mRNA level in MSCs of the examples and ordinary MSCs;

[0042] Figure 3 The above are the concentration and particle size distribution diagrams of exosomes obtained in the examples;

[0043] Figure 4 The expression levels of pro-inflammatory and anti-inflammatory cytokines in MACO rats after injection of exosomes using different regimens;

[0044] Figure 5 The figure shows the intergroup comparison of mNSS scores and foot error test results in MACO rats after injection of exosomes with different regimens.

[0045] Figure 6 These are TTC staining images of sections of infarct lesions in MACO rats after injection of exosomes with different regimens.

[0046] Figure 7 This is an image showing the immunohistochemical results of MACO rats after injection of exosomes using different regimens. Implementation

[0047] The present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that: unless otherwise specified in the following embodiments, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from commercially available sources. Example

[0048] A method for preparing a drug component for the prevention and treatment of sequelae of cerebral thrombosis, comprising the following steps:

[0049] S1. Raw material preparation

[0050] S11. Obtain mesenchymal stem cells for later use; S12. Obtain lentivirus containing genes miR-17-92 and miR-30d-5p for later use.

[0051] Specifically, the method for obtaining mesenchymal stem cells is as follows:

[0052] S111. Obtain sterile adipose tissue from a cosmetic surgery clinic, and rinse the adipose tissue with 0.01 mol / L sterile PBS until it is bloodless; clean the adipose tissue with sterile ophthalmic scissors and forceps, and cut it into pieces of approximately 1 mm. 3 The tissue blocks were shaped into small pieces; then, an appropriate amount of 0.1 wt% type I collagenase was added, and the mixture was shaken and digested at 37°C for 30 min; the digestion was then terminated with Gibco cell culture medium containing 10 vol.% fetal bovine serum, and centrifuged at 1000 r / min for 15 min; the supernatant of the centrifuged suspension was discarded, and the incompletely digested tissue blocks and the lower cell clusters were resuspended in mesenchymal stem cell basal culture medium and mixed well to obtain the cell cluster resuspension.

[0053] S112. The cell clusters were resuspended and cultured in a 37 ℃, 5 Vol.% CO2 incubator. After 3 days of adherent culture, adipose-derived mesenchymal stem cells (MSCs) were obtained, which were then expanded in large quantities using a cell factory for later use.

[0054] Nanjing Zebrafish Biotechnology Co., Ltd. was commissioned to prepare a recombinant lentivirus containing the genes miR-17-92 and miR-30d-5p. The specific method is as follows:

[0055] S121. The genes miR-17-92, miR-30d-5p, green fluorescent protein selection marker gene, and puromycin resistance gene were linked to a lentiviral gene to obtain a recombinant lentivirus. The sequence of the miR-17-92 gene is shown in SEQ ID NO:1, the sequence of the miR-30d-5p gene is shown in SEQ ID NO:2, the lentiviral gene sequence is shown in SEQ ID NO:3, and the plasmid map is shown in... Figure 1As shown, the sequence of the green fluorescent selection marker gene is shown in SEQ ID NO:4, and the sequence of the puromycin resistance gene is shown in SEQ ID NO:5.

[0056] S122. After the 293T cells and recombinant lentivirus come into contact, cell culture is carried out. During this process, the recombinant lentivirus is continuously packaged in the 293T cells. Subsequently, the packaged recombinant lentivirus is obtained from the supernatant of the 293T cell culture medium and stored at -20 ℃ for later use.

[0057] S2. The recombinant lentivirus and adipose-derived mesenchymal stem cells are cultured in contact, centrifuged, and the supernatant is collected to obtain the drug component. The specific steps are as follows:

[0058] S21. Adipose-derived mesenchymal stem cells were cultured in T175 culture flasks with human mesenchymal stem cell serum-free basal medium (AM-V Serum Free Medium; catalog number SC-2013-GA, Tianjin Haoyang), and 1 wt% penicillin and an appropriate amount of recombinant lentivirus were added. The culture was carried out at 37 ℃ for 24 h.

[0059] S22. Subsequently, the serum-free basal medium for human mesenchymal stem cells in bottle T175 was replaced with serum-free selective medium for human mesenchymal stem cells: serum-free basal medium for human mesenchymal stem cells (AM-V Serum Free Medium; catalog number SC-2013-GA, Tianjin Haoyang) + 2 μg / mL puromycin. The culture was continuously screened at 37 ℃ until all MSCs expressed green fluorescence.

[0060] S23. Continue expansion with serum-free selective medium for human mesenchymal stem cells and begin continuous collection of culture supernatant, storing it at -20 ℃ for later use. After thawing 10 L of culture supernatant, concentrate it to 500 mL using the 3D FloTrixvivaEXO exosome harvesting system manufactured by Beijing Huakan Company to obtain the drug component. This drug component can be used to prepare products for the prevention and treatment of sequelae of cerebral thrombosis.

[0061] The difference between this comparative example and the embodiment is that the recombinant lentivirus gene sequence does not contain the miR-17-92 gene, that is, the drug component does not contain Exo-miR-17-92. Otherwise, it is the same as the embodiment.

[0062] The difference between this comparative example and the embodiment is that the recombinant lentivirus gene sequence does not contain the gene miR-30d-5p, that is, the drug component does not contain Exo-miR-30d-5p. Otherwise, it is the same as the embodiment.

[0063] The difference between the drug component in this comparative example and the example is that the drug component contains Exo-miR-17-92 and Exo-miR-21-3p, and its preparation method is as follows: the Exo-miR-17-92 solution and the Exo-miR-21-3p solution are mixed at a volume ratio of 1:1.

[0064] The Exo-miR-17-92 solution was prepared using the method described in Comparative Example 1.

[0065] The preparation method of Exo-miR-21-3p solution is as follows: Fasting peripheral venous blood was collected from patients with myocardial infarction and added to an anticoagulant tube containing heparin sodium. After centrifugation at 2000 r / min and 4 ℃ for 10 min, the pale yellow plasma was transferred to an RNase-free centrifuge tube, and exosomes were extracted using the exoRNeasy Kit (Qiagen, Germany). The exosome concentration was adjusted to 3.3 × 10⁻⁶. 11 The concentration of cells / mL yields the Exo-miR-21-3p solution.

[0066] The difference between the drug component in this comparative example and the example is that the drug component contains Exo-miR-30d-5p and Exo-miR-21-3p, and its preparation method is as follows: the Exo-miR-30d-5p solution and the Exo-miR-21-3p solution are mixed at a volume ratio of 1:1.

[0067] The Exo-miR-30d-5p solution was prepared using the method described in Comparative Example 2; the Exo-miR-21-3p solution was prepared using the same method as Comparative Example 3.

[0068] 1. Real-time PCR detection

[0069] Real-time PCR was performed on adipose-derived mesenchymal stem cells overexpressing miR-17-92 and miR-30d-5p, as well as ordinary adipose-derived mesenchymal stem cells (i.e., those without modified miR-17-92 and miR-30d-5p). Specific results are shown in [link to relevant documentation]. Figure 2 .

[0070] from Figure 2 The results showed that after gene modification, the expression intensity of the genes miR-17-92 and miR-30d-5p in adipose-derived mesenchymal stem cells was significantly increased at the mRNA level. Specifically, the expression intensity of miR-17-92 increased 40-fold at the mRNA level, and the expression intensity of miR-30d-5p increased 36-fold. This result fully demonstrates the advantages of this method in preparing drug components: high efficiency and high yield.

[0071] 2. The concentration of exosomes in the concentrated exosomes obtained in the examples was determined by nanoparticle tracking analysis (NTA), specifically 3.3 × 10⁻⁶. 11 The exosomes were aliquoted into 1 mL vials and stored at -80°C. The particle size distribution of the exosomes was as follows: Figure 3 As shown, the particle size distribution of exosomes is 54-140 nm, the median particle size of exosomes is 62 nm, the D90 is 76 nm, and the D10 is 56 nm.

[0072] 3. Animal experiments

[0073] Using a rat MACO model, the treatment group was divided into three groups (n=10 per group): Exo-miR-17-92 (Comparative Example 1), Exo-miR-30d-5p (Comparative Example 2), and Exo-miR-17-92 / 30d-5p (Example). Another 10 MACO rats served as the control group. After recanalization of the blocked cerebral blood vessels, the treatment group received a single injection of 100 μL of exosomes into the internal carotid artery, while the control group received 100 μL of saline.

[0074] 3.1 Expression level of anti-inflammatory factors

[0075] One week after treatment, blood samples were drawn from each group of animals to detect the expression levels of pro-inflammatory and anti-inflammatory cytokines in their plasma. Specific results are shown below. Figure 4 The expression trends of each group showed that the expression levels of pro-inflammatory cytokines in the control group were significantly higher than those in other groups, while the expression levels of anti-inflammatory cytokines in the Exo-miR-17-92 / 30d-5p group were significantly higher than those in other groups. This fully demonstrates that the co-existence of the miR-17-92 and miR-30d-5p genes in Exo-miR-17-92 / 30d-5p exhibits a synergistic effect, significantly increasing the levels of anti-inflammatory factors and decreasing the levels of pro-inflammatory factors in the blood.

[0076] 3.2. Within 28 days of treatment, intergroup comparisons were conducted on mNSS scores and foot fault test results. See below for details. Figure 5 Among them, the Exo-miR-17-92 / 30d-5p group had the lowest mNSS score and the fewest foot errors, while the control group showed no significant changes in either indicator within 28 days. These results indicate that using exosomes from the example for related treatments has a better therapeutic effect.

[0077] Meanwhile, the trial was also conducted on the drug components of Comparative Examples 3 and 4, with the following results: In Comparative Example 3, after 28 days of treatment, the mNSS score was 6.5 and the number of foot errors was 12; while in Comparative Example 4, after 28 days of treatment, the mNSS score was 5.0 and the number of foot errors was 8. These results clearly demonstrate that, for this drug component, the synergistic therapeutic effect of Exo-miR-17-92 and Exo-miR-30d-5p is significantly higher than that of the other groups.

[0078] 3.3. Animals were euthanized 28 days later, and sections of the infarct lesion were stained with TTC to determine the size of the infarct lesion. See attached results. Figure 6 In the image, dark red represents the infarct lesion, and light pink represents normal brain tissue; from Figure 6 It is evident that the control group had the largest infarct lesion, while the Exo-miR-17-92 / 30d-5p group showed no obvious macroscopically visible infarct lesions. This result fully demonstrates that when the drug component containing both Exo-miR-17-92 and Exo-miR-30d-5p from this application is used to treat cerebral infarction, the synergistic effect of the two components significantly reduces the area of ​​the infarct lesion. In other words, the synergistic effect of these two components in preventing and treating sequelae of cerebral thrombosis is excellent.

[0079] 3.4. Animals were sacrificed after 28 days, and M1 type microglia (CD32) were detected by immunohistochemistry. + M2 type microglia (CD206) + The degree of expression of ) is shown in the following figures. Figure 7 . Figure 7 The results showed that the proportion of M1 microglia in the control group was significantly higher than that in other groups, and the proportion of M2 microglia in the Exo-miR-17-92 / 30d-5p group was significantly higher than that in other groups.

[0080] The results of this experiment on the drug components of Comparative Examples 3 and 4 were as follows: In Comparative Example 3, the proportion of M1-type microglia was 35% and 65% respectively; while in Comparative Example 4, the proportion of M1-type microglia was 30% and 70% respectively. These results clearly demonstrate that for this drug component, the synergistic treatment with Exo-miR-17-92 and Exo-miR-30d-5p has a superior effect on promoting microglia transformation, thus contributing to improving the therapeutic efficacy of the drug component to some extent.

[0081] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A pharmaceutical component for the prevention and treatment of sequelae of cerebral thrombosis, characterized in that, The drug components include exosomes miR-17-92 and miR-30d-5p, the sequence of miR-17-92 is shown in SEQ ID NO:1, the sequence of miR-30d-5p is shown in SEQ ID NO:2, and the particle size of the mesenchymal stem cell exosomes is 50-140 nm.

2. A pharmaceutical component for the prevention and treatment of sequelae of cerebral thrombosis according to claim 1, characterized in that, The median particle size of the mesenchymal stem cell exosomes was 62 nm, the D90 was 76 nm, and the D10 was 56 nm.

3. A method for preparing a pharmaceutical component for the prevention and treatment of sequelae of cerebral thrombosis according to any one of claims 1-2, characterized in that, Includes the following steps: S1. Obtain mesenchymal stem cells for later use; obtain recombinant lentivirus containing genes miR-17-92 and miR-30d-5p for later use; The sequence of the gene miR-17-92 is shown in SEQ ID NO:1, and the sequence of the gene miR-30d-5p is shown in SEQ ID NO:2; S2. The recombinant lentivirus and the mesenchymal stem cells are cultured in contact, centrifuged, and the supernatant is obtained to obtain the drug component.

4. A method for preparing a pharmaceutical component for the prevention and treatment of sequelae of cerebral thrombosis according to claim 3, characterized in that, The recombinant lentivirus also includes a fluorescent selection marker gene and an resistance gene.

5. The method for preparing the pharmaceutical component for preventing and treating sequelae of cerebral thrombosis according to claim 4, characterized in that, The gene sequence of the lentivirus is shown in SEQ ID NO:3; the sequence of the fluorescent selection marker gene is shown in SEQ ID NO:4; the resistance gene is a puromycin resistance gene, and the sequence of the resistance gene is shown in SEQ ID NO:

5.

6. The method for preparing the pharmaceutical component for preventing and treating sequelae of cerebral thrombosis according to claim 4, characterized in that, The method for contact culture of the recombinant lentivirus and the mesenchymal stem cells described in S2 includes the following steps: S21. The recombinant lentivirus and the mesenchymal stem cells are placed on mesenchymal stem cell basal culture medium and cultured at 35-38°C for 20-30 h. S22. Replace the mesenchymal stem cell basal culture medium with mesenchymal stem cell selection culture medium and then culture the subsequent sets until all mesenchymal stem cells express fluorescence; S23. Then continue the amplification culture and collect the supernatant. The mesenchymal stem cell selection culture medium contains antibiotics corresponding to the resistance gene as a screening criterion.

7. The method for preparing the pharmaceutical component for preventing and treating sequelae of cerebral thrombosis according to claim 4, characterized in that, The method for obtaining the recombinant lentivirus includes the following steps: S121. The gene miR-17-92, the gene miR-30d-5p, the fluorescent selection marker gene, and the resistance gene are linked to a lentiviral gene to obtain a recombinant lentiviral vector. S122. The recombinant lentiviral vector and host cells are mixed, the recombinant lentiviral is continuously packaged, and then the recombinant lentiviral is obtained from the host cell culture supernatant for later use.

8. The method for preparing the pharmaceutical component for preventing and treating sequelae of cerebral thrombosis according to claim 3, characterized in that, The method for obtaining mesenchymal stem cells includes the following steps: S111. The obtained fat extract is rinsed and cut into blocks, then digested with type I collagenase, centrifuged, and the undigested tissue blocks and the lower cell clusters are resuspended to obtain a cell cluster resuspension. S112. The cell cluster resuspension is cultured in a 34-38 ℃, 3-7 Vol.% CO2 incubator; after adherent culture for 2-4 days, mesenchymal stem cells are obtained, expanded and used for later use.

9. The application of the pharmaceutical component according to any one of claims 1-2 for the prevention and treatment of sequelae of cerebral thrombosis, characterized in that, The drug components are used to prepare products for the prevention and treatment of sequelae of cerebral thrombosis.

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