Application of induced pluripotent stem cell exosome in preparation of sciatic nerve injury medicine

Abstract

The invention provides application of an induced pluripotent stem cell exosome in preparation of sciatic nerve injury drugs, and relates to the technical field of exosome application. It is proved that the induced pluripotent stem cell exosome has a good treatment effect on sciatic nerve injury and can be widely applied to preparation of sciatic nerve injury drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome application, and in particular to the application of induced pluripotent stem cell exosomes in the preparation of a sciatic nerve injury drug. Background Art

[0002] Sciatic nerve injury is mainly caused by traumatic factors and disease factors, leading to sensory dysfunction, motor dysfunction and abnormal reflexes. Although existing treatments can alleviate the symptoms of sciatic nerve injury and promote nerve function recovery to a certain extent, there are still some problems. For example, the effect of conservative treatment is often limited, and it is difficult to achieve satisfactory results for severe nerve damage; although surgical treatment can directly relieve nerve compression and repair nerve damage, the surgical risk is high, and complications such as infection, bleeding, and nerve adhesion may occur after surgery. It takes a long time for nerve function to recover, and some patients may not be able to fully recover normal function.

[0003] Therefore, finding more effective treatment methods and improving the treatment effects of sciatic nerve injury are important issues facing the current medical field.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide an application of induced pluripotent stem cell exosomes in the preparation of a sciatic nerve injury drug, so as to improve the therapeutic effect of sciatic nerve injury and improve the problem that the existing conservative treatment of sciatic nerve injury has limited effect and the risk of surgical treatment is high.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] The present invention provides an application of induced pluripotent stem cell (iPSC) exosomes in the preparation of a sciatic nerve injury drug.

[0008] Furthermore, the induced pluripotent stem cell exosomes are derived from induced pluripotent stem cells generated by induction of peripheral blood mononuclear cells (PBMC).

[0009] The present invention provides a medicine for treating sciatic nerve injury, which comprises the above-mentioned induced pluripotent stem cell exosomes and a pharmaceutically acceptable excipient.

[0010] Furthermore, the dosage form of the drug includes any one of tablets, capsules, granules, pills, injections or sustained-release preparations.

[0011] Furthermore, the dosage form of the drug is an injection.

[0012] Furthermore, the induced pluripotent stem cell exosomes in the injectable drug are present in the form of an exosome suspension;

[0013] The exosome content in the exosome suspension is 2.00-5.00 mg / ml in terms of protein concentration.

[0014] Furthermore, the injectable drug also contains pharmaceutical excipients;

[0015] Preferably, the pharmaceutical excipient is matrix gel.

[0016] Furthermore, the volume ratio of matrix gel to exosome suspension in the injection drug is 2:1.

[0017] Furthermore, based on the exosome protein concentration, the application dosage of the induced pluripotent stem cell exosomes in the injection drug is 1 to 5 mg / kg body weight.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The induced pluripotent stem cell exosomes provided by the present invention can be widely used in the preparation of sciatic nerve injury drugs. The present application has confirmed that induced pluripotent stem cell exosomes have a good therapeutic effect on sciatic nerve injury and can be widely used in the preparation of sciatic nerve injury drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a morphological detection diagram of iPSC exosomes derived from peripheral blood PBMC provided in Example 1 of the present invention;

[0022] Figure 2 This is a diagram showing the HE staining test results of the sciatic nerve injury model of each experimental group provided in Example 3 of the present invention;

[0023] Figure 3 This is a graph showing the immunohistochemical detection results of the sciatic nerve injury model of each experimental group provided in Example 3 of the present invention;

[0024] Figure 4 This is a transmission electron microscopy test result diagram of the sciatic nerve injury model of each experimental group provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0027] According to one aspect of the present invention, an application of induced pluripotent stem cell exosomes in the preparation of a sciatic nerve injury drug.

[0028] The induced pluripotent stem cell exosomes provided by the present invention can be widely used in the preparation of sciatic nerve injury drugs. The present application has experimentally confirmed that induced pluripotent stem cell exosomes have a good therapeutic effect on sciatic nerve injury and can be widely used in the preparation of sciatic nerve injury drugs.

[0029] Exosomes derived from induced pluripotent stem cells (iPSCs) are rich in a variety of bioactive molecules related to the maintenance and induction of stemness. These molecules include specific proteins, nucleic acids (such as miRNAs, lncRNAs, etc.), and lipids. For example, certain miRNAs (such as miR-200c, miR-302, etc.) can regulate the expression of target genes, thereby affecting the stemness characteristics of cells. By delivering these miRNAs to recipient cells, iPSC-derived exosomes can potentially confer or enhance the stemness of recipient cells, giving them higher self-renewal ability and multidirectional differentiation potential. Proteins carried in exosomes, such as transcription factors and signaling pathway-related proteins, can also participate in regulating stemness-related gene expression and signaling pathways, such as the Wnt / β-catenin pathway and the Notch pathway, thereby maintaining or inducing the stemness state of cells.

[0030] In a preferred embodiment of the present invention, the induced pluripotent stem cell exosomes are derived from induced pluripotent stem cells generated by inducing human peripheral blood mononuclear cells (PBMC).

[0031] As a preferred embodiment, the present application confirms through mouse experiments that induced pluripotent stem cells induced by peripheral blood mononuclear cells have a better repair effect on sciatic nerve injury than other stem cell exosomes (such as mesenchymal cell exosomes).

[0032] This may be because the iPSCs induced by peripheral blood PBMC (mononuclear cells) have high consistency and stability in genetic background and cell characteristics, which helps to ensure the stability of the quality and function of the produced exosomes; in addition, the iPSC exosomes derived from peripheral blood PBMC have low immunogenicity, which reduces the risk of causing immune rejection when used in vivo, making them more suitable for allogeneic therapeutic applications. This low immunogenicity allows exosomes to be used multiple times and repeatedly, improving the feasibility and effectiveness of treatment.

[0033] According to one aspect of the present invention, a drug for treating sciatic nerve injury comprises the above-mentioned induced pluripotent stem cell exosomes and a pharmaceutically acceptable excipient.

[0034] The present invention provides a drug for treating sciatic nerve injury, which comprises the above-mentioned induced pluripotent stem cell exosomes and a pharmaceutically acceptable excipient. In view of the mouse experiment provided in the present application, the injection with induced pluripotent stem cell exosomes as the active ingredient can effectively improve the reflex abnormalities caused by sciatic nerve injury and restore a certain degree of motor and sensory function.

[0035] Active ingredient means any ingredient that provides pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or affects the structure or any function of the human or other animal body.

[0036] Pharmaceutically acceptable excipients refer to pharmaceutically acceptable auxiliary materials or carriers that are compatible with other ingredients of the drug preparation and suitable for contact with tissues or organs of the recipient (such as humans or animals). When used, they have no or very little toxicity, irritation, allergic reaction, immunogenicity or other complications.

[0037] In a preferred embodiment of the present invention, the dosage form of the drug includes any one of tablets, capsules, granules, pills, injections or sustained-release preparations.

[0038] In a preferred embodiment of the present invention, the dosage form of the drug is an injection drug.

[0039] In the above preferred embodiment, the induced pluripotent stem cell exosomes in the injection drug are present in the form of an exosome suspension, and the exosome content in the exosome suspension is 2.00 to 5.00 mg / ml in terms of protein concentration.

[0040] As a preferred embodiment, the preparation method of induced pluripotent stem cell exosome suspension is as follows:

[0041] 1. Use density gradient centrifugation to collect peripheral blood samples. After centrifugation, PBMCs are between the layered liquid and the plasma layer and should be carefully collected.

[0042] 2. The isolated PBMCs were mixed with a vector kit carrying reprogramming factors (CytoTune TM -iPS 2.0 Sendai virus reprogramming kit Invitrogen TM ) Extracellular matrix such as Matrigel and Shouning biological culture medium were mixed and inoculated into a cell culture plate without a feeder layer for co-culture.

[0043] 3. After multiple changes of fluid, the cell clusters that form clones, i.e., PBMC-derived iPSCs, are screened out, and their supernatants are collected. The exosomes in the supernatant are obtained by ultracentrifugation and suspended in PBS to obtain the iPSC exosome suspension derived from peripheral blood PBMCs.

[0044] In the above preferred embodiment, the injection medicine further comprises pharmaceutical excipients;

[0045] Preferably, the pharmaceutical excipient is matrix gel.

[0046] In a preferred embodiment of the present invention, the volume ratio of matrix gel to exosome suspension in the injection drug is 2:1.

[0047] In a preferred embodiment of the present invention, the dosage of induced pluripotent stem cell exosomes in the injection drug is 1-5 mg / kg body weight based on the exosome protein concentration.

[0048] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0049] Example 1 Preparation of iPSC exosome preparation derived from peripheral blood PBMC

[0050] (1) Induction of iPSCs from human peripheral blood PBMCs:

[0051] (1.1) 30 ml of human peripheral blood was centrifuged at 800 g for 10 minutes at room temperature. The plasma was taken as the supernatant and the red blood cells in the lower layer were diluted to the original volume with physiological saline. The cells were slowly spread into a centrifuge tube containing 15 ml of human lymphocyte separation solution. Be careful not to break the liquid surface of the separation solution. The cells were centrifuged at 700 g for 20 minutes at room temperature. The middle white film layer (mononuclear cell layer) was carefully aspirated into another centrifuge tube. An appropriate amount of physiological saline was added to mix and wash. The cells were centrifuged at 500 g for 10 minutes, washed twice, and the supernatant was discarded to obtain the cell pellet, i.e., the PBMC of peripheral blood.

[0052] (1.2) The obtained precipitated cells and the Sendai virus vector carrying reprogramming factors (Oct4, Sox2, Klf4, c-Myc) are suspended and mixed with an extracellular matrix such as Matrigel and a specific culture medium and then inoculated;

[0053] Day -4 (i.e. 4 days before day 0): Use CytoTune TM -iPS 2.0 complete PBMC medium Peripheral blood mononuclear cells (PBMCs) were plated at a density of 5 × 105 in the center of a 24-well plate;

[0054] Day -3 to -1 (i.e., 3 to 1 day before day 0): replace half of the culture medium with 0.5 ml of complete PBMC culture medium;

[0055] Day 0: Using CytoTune TM - Transduce cells with iPS 2.0 Sendai reprogramming vector at an appropriate MOI. Incubate cells overnight;

[0056] Day 1: Replace the culture medium with complete PBMC medium and remove the CytoTune 2.0 Sendai reprogramming vector;

[0057] Day 3: Place the transduced cells on coated culture dishes and culture in complete StemPro-34 medium without cytokines;

[0058] Days 4-6: Replace complete StemPro-34 medium without cytokines every other day;

[0059] Day 7: Begin transition to Essential 8 Medium by replacing half of the cytokine-free StemPro-34 medium with Essential 8 Medium.

[0060] Day 8: Replace the entire culture medium with Essential 8 Medium to complete the transition and continue culturing cells on the in vitro coated dish;

[0061] Days 9-28: Replace medium with fresh Essential 8 Medium daily and monitor the incubator for the emergence of iPSC colonies. When iPSC colonies are ready for transfer, perform vital staining, pick and transfer undifferentiated iPSCs to fresh in vitro coated dishes for expansion.

[0062] (2) Preparation of iPSC exosomes from human peripheral blood PBMCs:

[0063] iPSCs derived from peripheral blood PBMC obtained in step (1) were digested and collected and inoculated into serum-free medium ( hiPSC / hESC culture medium-ncTarget), when the cells grew to about 90% confluence, the culture supernatant was collected and filtered by ultracentrifugation, i.e., centrifugation at 4°C, 2000g for 10 min, and the supernatant was taken; after filtering with a 0.22μm filter, the supernatant was concentrated to 10ml using a 100kDa MW ultrafiltration tube; centrifugation at 4°C, 100,000g for 1h, the supernatant was discarded, and 0.5ml PBS was added for resuspending.

[0064] Repeat this step, and finally use PBS to adjust the concentration of the preparation to 2.00-5.00 mg / ml (total protein concentration). The obtained preparation is the peripheral blood PBMC-derived iPSC exosome preparation.

[0065] Figure 1 This is a morphological detection diagram of peripheral blood PBMC-derived iPSC exosomes provided in this example.

[0066] Depend on Figure 1 It can be seen that the exosomes prepared in this example are observed to be cup-shaped or disc-shaped bodies under a transmission electron microscope.

[0067] Example 2

[0068] (I) Preparation of sciatic nerve injury mouse model:

[0069] Eight-week-old ICR male mice were anesthetized by injection of 50 mg / kg 1% sodium pentobarbital (prepared in PBS). The tip of the tail and the paw were clamped with forceps to check the reflexes and ensure that the animal had no reaction. Using a shaver, the surgical field was shaved from below the knee to the buttocks, and the left leg was used uniformly for modeling. The animal was placed on its right side and the left hind limb was placed on a small platform to keep it elevated, and the leg was fixed with tape. Find the knee, make a 1 cm longitudinal incision proximal to the knee joint, bluntly separate the skin, and separate the sciatic nerve just below the muscle near the blood vessels close to the thigh bone (femur). Part of the sciatic nerve was cut with a blade 1 cm below the femoral tuberosity to construct a mouse model of sciatic nerve injury.

[0070] (II) Model grouping:

[0071] The mouse sciatic nerve injury model established by the method described in step (i) was used for the experiment. The experiment was divided into 3 groups (10 mice in each group):

[0072] Experimental group 1 was the sciatic nerve injury model group;

[0073] Experimental group 2 was the sciatic nerve injury model peripheral blood PBMC-derived iPSC exosome preparation injection group;

[0074] Experimental group 3 was the sciatic nerve injury model umbilical cord-derived mesenchymal stem cell exosome (UCMSC-EXO) preparation injection group.

[0075] After the above experimental groups were modeled, experimental group 2 was injected with 20-30 μl of injection gel A containing induced pluripotent stem cell exosomes around the incision, and the injection gel was allowed to gel for 20 seconds before suturing layer by layer. Experimental group 3 was injected with 20-30 μl of injection gel B containing UCMSC-EXO exosomes around the incision, and the injection gel was allowed to gel for 20 seconds before suturing layer by layer. Experimental group 1 was injected with an equal volume of matrigel mixed with PBS as experimental groups 2 and 3.

[0076] Wherein: the injection gel A is prepared by mixing the peripheral blood PBMC-derived iPSC exosome preparation prepared in Example 1 with matrigel, the volume ratio of the matrigel to the iPSC exosome preparation is 2:1, and the exosome content in the injection gel A is 100 μg (total protein amount, equivalent to 1-5 mg / kg body weight based on the experimental mouse weighing 18-25 g),

[0077] Injection gel B is prepared by mixing UCMSC-EXO exosomes with matrigel. The volume ratio of the matrigel to the UCMSC-EXO exosome preparation is 2:1. The exosome content in injection gel B is 100 μg (total protein amount, equivalent to 1-5 mg / kg body weight based on the experimental mouse weighing 18-25 g).

[0078] Example 3

[0079] Two weeks after the injection of experimental groups 1 to 3 in Example 2, samples were taken for HE staining, immunohistochemistry and transmission electron microscopy to analyze the effects of exosomes from different sources on sciatic nerve injury.

[0080] Note: During the two-week culture process after injection of experimental groups 1 to 3, mice in each group were cultured using conventional methods and were allowed to freely access food and water.

[0081] (I) HE staining detection:

[0082] After 2 weeks of culture, the mice were killed by cervical dislocation, the skin of the left hind limb was bluntly separated, the sciatic nerve just below the muscle was separated, and 1 cm was cut off. HE staining was used to observe the morphological changes of the damaged sciatic nerve.

[0083] Figure 2 This is a diagram showing the HE staining test results of the sciatic nerve injury model in each experimental group provided in this example.

[0084] Depend on Figure 2The HE staining results showed that after two weeks of treatment in experimental group 2, the sciatic nerves of mice had relatively complete and neatly arranged nerve bundles, while the nerve bundles of experimental groups 1 and 3 were arranged in a disorderly and incomplete manner, indicating that experimental group 2 of the present application using iPSC exosomes derived from peripheral blood PBMC had a certain protective and repair effect on sciatic nerve injury in mice and could significantly improve the integrity of the sciatic nerve bundles.

[0085] (II) Immunohistochemistry detection:

[0086] The sciatic nerves of mice in each experimental group were obtained, and the expression of microtubule-associated protein MAP-2 in the damaged nerve segments of each experimental group was detected by immunohistochemistry.

[0087] Figure 3 This is a graph showing the immunohistochemical detection results of the sciatic nerve injury model in each experimental group provided in this example.

[0088] Figure 3 The arrow in the figure indicates MAP-2 protein. Figure 3 The immunohistochemistry results showed that after two weeks of treatment, there was a large amount of MAP-2 expression in experimental group 2, indicating that there were more nerve cells, while the nerve cells in experimental groups 1 and 3 were less than those in the stem cell group, suggesting that experimental group 2 could promote the expression of microtubule-associated protein MAP-2 and have a protective effect on nerve cells.

[0089] (III) Transmission electron microscopy detection:

[0090] The sciatic nerves of mice in each experimental group were obtained, and the integrity of the nerve myelin sheath structure was detected by transmission electron microscopy.

[0091] Figure 4 1 is a transmission electron microscopy test result diagram of the sciatic nerve injury model of each experimental group provided in this embodiment;

[0092] Depend on Figure 4 It can be seen that transmission electron microscopy showed that the myelin sheath structure of experimental group 1 was damaged, while the nerve bundles of experimental group 2 were neatly arranged and the myelin sheath structure was intact, indicating that the complex can promote the regeneration and repair of the damaged sciatic nerve myelin sheath. While experimental group 3 used the implementation method of mesenchymal stem cell exosomes, although it had a certain repair effect on the myelin sheath structure of experimental group 1, the neatness of the arrangement of the nerve bundles and the integrity of the myelin sheath structure were significantly worse than the implementation method of experimental group 2 of the present application using induced pluripotent stem cell exosomes.

[0093] In summary, induced pluripotent stem cell exosomes have a good therapeutic effect on sciatic nerve injury, can significantly improve nerve compression in sciatic nerve injury and repair nerve damage, and have a better modification effect on sciatic nerve injury than stem cell exosomes from other sources, and can be widely used in the preparation of sciatic nerve injury drugs.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of induced pluripotent stem cell exosomes in the preparation of sciatic nerve injury drugs.

2. The use according to claim 1, characterized in that: The induced pluripotent stem cell exosomes are derived from induced pluripotent stem cells generated by induction of peripheral blood mononuclear cells.

3. A drug for treating sciatic nerve injury, characterized in that: The drug comprises the induced pluripotent stem cell exosomes according to claim 1 or 2 and a pharmaceutically acceptable excipient.

4. The drug for treating sciatic nerve injury according to claim 3, characterized in that: The dosage form of the drug includes any one of tablets, capsules, granules, pills, injections or sustained-release preparations.

5. The drug for treating sciatic nerve injury according to claim 4, characterized in that: The dosage form of the drug is injection.

6. The drug for treating sciatic nerve injury according to claim 5, characterized in that: The induced pluripotent stem cell exosomes in the injectable drug are in the form of an exosome suspension; The exosome content in the exosome suspension is 2.00-5.00 mg / ml in terms of protein concentration.

7. The drug for treating sciatic nerve injury according to claim 5, characterized in that: Injectable drugs also contain pharmaceutical excipients; Preferably, the pharmaceutical excipient is matrix gel.

8. The drug for treating sciatic nerve injury according to claim 7, characterized in that: The volume ratio of matrix gel to exosome suspension in the injection drug is 2:

1.

9. The drug for treating sciatic nerve injury according to claim 7, characterized in that: Based on the exosome protein concentration, the application dosage of the induced pluripotent stem cell exosomes in the injection drug is 1 to 5 mg / kg body weight.

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