Mitochondrial transplantation therapeutic systems and uses thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a mitochondrial transplantation treatment system and application thereof. The mitochondrial transplantation treatment system provided by the invention comprises mitochondria obtained by separation and a transfer vector. Wherein the mitochondria transplantation treatment system is obtained by assembling the mitochondria and the transfer vector, and the transfer vector comprises at least one of artificial lipid vesicles, vesicles derived from cell membranes and exosomes. The preparation process of the mitochondrial transplantation treatment system is simple, healthy mitochondria can be efficiently and rapidly transferred into cells or tissues at the same time, and the mitochondrial function of damaged mitochondrial cells is repaired. The mitochondrial transplantation treatment system has the potential of treating related diseases caused by mitochondrial dysfunction.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and specifically, to a mitochondrial transplantation treatment system and its uses. Background Art

[0002] Mitochondria are energy-supplying organelles in eukaryotic cells and play important roles in ATP synthesis, cell metabolism, growth and development, and the processes of aging and death. Mitochondria are semi-autonomous replicating organelles within eukaryotic cells. Human mitochondria contain approximately 16.5 kb of circular double-stranded DNA (mtDNA), which can encode 13 proteins, 2 kinds of rRNA, and 22 kinds of tRNA. The components encoded by mtDNA are the core components of mitochondrial respiratory complexes I-IV, and mitochondrial respiratory complexes play a key role in maintaining normal cell life activities. Mitochondrial oxidative phosphorylation places mtDNA in a highly reactive oxygen environment and, lacking histone protection, mtDNA is particularly vulnerable to oxidative stress damage compared to nuclear DNA, leading to abnormal mitochondrial function. Gene mutations also occur during the replication process of mitochondrial DNA and can be cleared through mitophagy or mitochondrial fusion and fission. In dysfunctional or damaged mitochondria, mtDNA mutations accumulate within cells, leading to an increased mutation rate. There is a threshold effect for mitochondrial DNA. When the mtDNA mutation rate reaches the threshold, mitochondrial function is severely damaged, leading to cell death or tissue lesions, and ultimately potentially resulting in mitochondrial diseases. Dysfunction of mitochondria, especially the dysfunction of their metabolic activities, is associated with many diseases, including metabolic diseases, neurodegenerative diseases, and cancer, as well as the aging process.

[0003] Mitochondrial diseases are genetic diseases, and a large part of them are transmitted through mtDNA mutations, with a prevalence rate of approximately 1 / 5,000. In the diseased state, mutant mtDNA usually coexists with wild-type mtDNA. The severity of the diseases caused by mtDNA mutations is related to the mutation rate, and there is a threshold effect. The occurrence of some diseases requires more than 60% of the mtDNA to mutate, which is a distinct feature of mtDNA diseases. Currently, many researchers have attempted to reduce the mtDNA mutation rate below the threshold to treat these basically incurable diseases. Mitochondrial diseases have clinical heterogeneity and tissue specificity. Mutations in the same mitochondrial protein complex can lead to different disease phenotypes. Some drugs have been used to relieve the symptoms of mitochondrial diseases. For example, idebenone is used to treat Leber hereditary optic neuropathy (LHON), and sodium valproate can be used to treat myoclonic epilepsy in mitochondrial diseases. Mitochondrial replacement (MRT) makes it possible for parents with mitochondrial diseases to have healthy offspring. At the same time, there are also some treatment methods for mitochondrial diseases, such as small molecule drugs, gene therapy targeting the mitochondrial genome, etc. In addition, some researchers have developed mitochondrial transplantation technology to transplant healthy mitochondria into damaged cells or organs to restore the energy metabolism of cells and cure diseases. Currently, mitochondrial transplantation has achieved many positive results in the treatment or intervention of various diseases, such as neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, sarcopenia, and heart transplantation.

[0004] However, there are many drawbacks to the current mitochondrial transplantation technology. For example, inappropriate vector selection can result in a small number of healthy mitochondria carried by it, low efficiency of mitochondria entering the vector, or damage to the structure or function of mitochondria. Therefore, there is an urgent need to further improve the mitochondrial transplantation technology. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems in the related art to some extent. For this reason, one object of the present invention is to provide a new mitochondrial transplantation treatment system and its uses. The preparation process of the mitochondrial transplantation treatment system provided by the present invention is simple, and it can efficiently and rapidly transfer healthy mitochondria into cells or tissues at the same time to repair the mitochondrial function of damaged cells. The mitochondrial transplantation treatment system has the potential to treat related diseases caused by mitochondrial dysfunction.

[0006] The first aspect of the present invention provides a mitochondrial transplantation treatment system. According to the embodiments of the present invention, the mitochondrial transplantation treatment system includes:

[0007] Isolated mitochondria; and

[0008] A transfer vector,

[0009] Among them, the mitochondrial transplantation treatment system is obtained by assembling the mitochondria with the transfer carrier.

[0010] The transfer carrier includes at least one of artificial lipid vesicles, cell-derived vesicles, and exosomes.

[0011] The cell membrane is a semi-permeable membrane composed of a phospholipid bilayer as a scaffold, providing a relatively independent space for the life activities of the cell and ensuring the normal life activities of the cell. There are various proteins, lipids, and carbohydrates on the cell membrane surface, which have good fluidity and can form vesicles for transportation inside and outside the cell. Currently, due to the good biocompatibility and degradability of the cell membrane, it has good applications in the field of drug development. Red blood cells are abundantly distributed in the blood, can transport O2 and CO2, and play an important role in the process of cell respiration. During the maturation of red blood cells, the nucleus and organelles gradually disappear.

[0012] Parkinson's disease (PD) is a neurological disease in middle-aged and elderly people, associated with the damage of substantia nigra dopaminergic neurons. The neurotoxic substance 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can be converted into MPP after entering the brain. + MPP + can selectively enter the midbrain substantia nigra dopaminergic neurons, inhibit the activity of mitochondrial respiratory chain complex I, trigger mitochondrial oxidative stress, cause the death of dopaminergic neurons, and trigger Parkinson's symptoms. Therefore, mitochondrial damage may also be one of the causes of Parkinson's disease. The inventor used MPTP to construct a Parkinson's mouse model to explore whether the mitochondrial transplantation treatment system can play a role in vivo, and found that the mitochondrial transplantation treatment system provided by the present invention can repair the motor ability of Parkinson's mice, and the neurons are also restored.

[0013] Currently, several methods for mitochondria to enter cells have been developed by researchers. The simplest one is to directly co-incubate the extracted mitochondria with cells and enter the cells through the endocytosis of the cells, but the efficiency of entering the cells is low, and the mitochondria entering the cells are spherical and cannot restore the mitochondrial network structure. At the same time, mitochondria can perform transcellular transfer. In addition, methods such as microinjection, FluidFM technology, and photothermal nanoblades can directly inject the extracted mitochondria into cells, but it requires operation at the single-cell level, and it takes a long time to obtain a large number of mitochondria-transferred cells, and there are instrument equipment and operation experience requirements. In the present invention, at least one of artificial lipid vesicles, cell-derived vesicles, and exosomes is used as the transfer carrier, and the developed mitochondrial transplantation treatment system is simple to operate and can efficiently and rapidly transfer mitochondria into a large number of cells at the same time to achieve the purpose of mitochondrial transfer.

[0014] According to an embodiment of the present invention, the transfer vector is a cell membrane structure or lipid vesicle structure derived from red blood cells.

[0015] According to an embodiment of the present invention, the mitochondrial transplantation treatment system provided by the present invention is obtained by assembling lipid vesicles prepared from red blood cell membranes and extracted mitochondria. The inventors found in experiments that when isolated mitochondria were co-incubated with other cells, a small amount of mitochondria could enter the cells, but under time-series observation, the mitochondria were spherical in the cells, immobile, and the cell state did not improve. In particular, it was found in experimental exploration that mitochondria encapsulated by the specific transfer vector of the present invention could efficiently enter cells and exert the functions and roles of mitochondria.

[0016] According to an embodiment of the present invention, the mitochondria are isolated from mammalian cells.

[0017] The second aspect of the present invention provides a drug. According to an embodiment of the present invention, the drug comprises the mitochondrial transplantation treatment system described in the first aspect.

[0018] The third aspect of the present invention provides the use of the mitochondrial transplantation treatment system described in the first aspect and the drug described in the second aspect in the preparation of a drug for repairing mitochondrial damage.

[0019] The inventors found that after co-incubating the transplanted mitochondria of the present invention with mitochondria-damaged cells, normal mitochondria could be efficiently and rapidly transferred into a large number of recipient cells to achieve mitochondrial transfer. The transplanted mitochondria restored the network structure in the cells and had mitochondrial functions, thereby repairing the mitochondrial damage of the recipient cells.

[0020] According to an embodiment of the present invention, the mitochondrial damage includes mitochondrial function damage and mtDNA mutation.

[0021] According to an embodiment of the present invention, the mitochondrial function damage includes mitochondrial structure damage and mitochondrial metabolic damage.

[0022] According to an embodiment of the present invention, the mtDNA mutation includes mtDNA point mutation and mtDNA deletion mutation.

[0023] According to an embodiment of the present invention, the mitochondrial transplantation treatment system provided by the present invention can repair mtDNA damage. Specifically, the inventors found through experiments that the functions of transplanted mitochondria were detected in mtDNA-depleted cells (ρ0 cells) and cells with large mtDNA deletions GM04516. After transplantation of mitochondria, the mitochondrial morphology, the number of mtDNA and the membrane potential of ρ0 cells were restored, the mitochondrial ATP production was significantly restored, and the transcriptional level of mtDNA-encoded proteins was also restored. After transplantation of mitochondria, the mitochondrial morphology of GM04516 cells was restored, the mitochondrial ATP production was significantly restored, and the transcriptional level of mtDNA-encoded proteins was also restored.

[0024] The fourth aspect of the present invention provides the use of the mitochondrial transplantation treatment system described in the first aspect and the drug described in the second aspect in the preparation of a drug for repairing diseases related to mitochondrial dysfunction.

[0025] The inventors found that the mitochondrial transplantation treatment system provided by the present invention can repair diseases related to mitochondrial dysfunction. For example, it can alleviate Parkinson's disease caused by mitochondrial dysfunction. Specifically, the inventors found through experiments that a Parkinson's mouse model was constructed by inducing mitochondrial damage with the small molecule drug MPTP; then, through the mitochondrial transplantation treatment system of the present invention, the mitochondrial damage of Parkinson's mice could be repaired. As a result, it was found that dopaminergic neurons and the motor ability of mice were significantly restored, indicating that the developed mitochondrial transplantation treatment system can be used to repair mitochondrial damage.

[0026] According to an embodiment of the present invention, the mitochondrial dysfunction is caused by mitochondrial respiratory chain damage, abnormal proteins encoded by nuclear genes, and physicochemical property changes.

[0027] According to an embodiment of the present invention, the diseases related to mitochondrial dysfunction include at least one of neurological diseases, auditory impairment-related diseases, optic neuropathy, muscle diseases, heart function damage, liver function damage, kidney function damage, pancreatic function damage, gastrointestinal function damage, metabolic diseases, reproductive diseases, and bone lesions caused by mitochondrial dysfunction.

[0028] According to an embodiment of the present invention, the neurological diseases include at least one of neuronal developmental retardation, Parkinson's, Alzheimer's disease, stroke, epilepsy, migraine, and amyotrophic lateral sclerosis.

[0029] According to an embodiment of the present invention, the auditory impairment-related diseases include at least one of deafness and sensorineural hearing loss.

[0030] According to an embodiment of the present invention, the optic neuropathy includes at least one of progressive external ophthalmoplegia, optic atrophy, and retinitis pigmentosa.

[0031] According to an embodiment of the present invention, the muscle lesion includes at least one of progressive muscle weakness and atrophy, sarcopenia, and exercise intolerance. Optionally, the heart function impairment includes at least one of cardiomyopathy and myocardial conduction defect.

[0032] According to an embodiment of the present invention, the liver function impairment includes at least one of liver failure, liver injury, fatty liver, and cirrhosis.

[0033] According to an embodiment of the present invention, the liver function impairment includes at least one of Fanconi syndrome, renal tubular acidosis, glomerulosclerosis, renal failure, and adrenocortical insufficiency.

[0034] According to an embodiment of the present invention, the pancreatic function impairment includes at least one of diabetes and pancreatitis.

[0035] According to an embodiment of the present invention, the gastrointestinal function impairment includes at least one of pseudo-obstruction and gastrointestinal motility disorder.

[0036] According to an embodiment of the present invention, the metabolic disease includes at least one of senescence, obesity, hyperglycemia, dyslipidemia, insulin resistance, and cardiovascular disease.

[0037] According to an embodiment of the present invention, the reproductive disease includes at least one of premature ovarian failure and male infertility.

[0038] According to an embodiment of the present invention, the bone lesion includes at least one of kyphoscoliosis, dwarfism, and bone marrow failure.

[0039] The fourth aspect of the present invention provides the use of the mitochondrial transplantation treatment system described in the first aspect and the drug described in the second aspect in the preparation of the following drugs:

[0040] (1) A drug for skin repair;

[0041] (2) A drug for improving ischemia-reperfusion injury during organ transplantation;

[0042] (3) A drug for prolonging the preservation time of transplanted organs;

[0043] (4) A drug for treating or alleviating abnormal organ metabolic regulation.

[0044] According to an embodiment of the present invention, the skin repair includes at least one of wound healing and skin regeneration.

[0045] According to an embodiment of the present invention, the ischemia-reperfusion injury includes at least one of ischemia-reperfusion injury during heart, lung, and kidney transplantation, and muscle or brain ischemia-reperfusion injury.

[0046] According to an embodiment of the present invention, the transplanted organ includes at least one of the heart, liver, lung, and kidney.

[0047] The fifth aspect of the present invention provides a method for obtaining heteroplasmic mitochondrial hybrid cells. According to an embodiment of the present invention, the method includes:

[0048] Co-incubating the transplanted mitochondria described in the first aspect with a recipient cell containing its own mitochondria, and the heteroplasmic mitochondria contained in the mitochondrial transplantation treatment system enter the recipient cell to obtain heteroplasmic mitochondrial hybrid cells.

[0049] The sixth aspect of the present invention provides a heteroplasmic mitochondrial hybrid cell. According to an embodiment of the present invention, the heteroplasmic mitochondrial hybrid cell is obtained by the method described in the fifth aspect.

[0050] The seventh aspect of the present invention provides a method for repairing mitochondria-damaged cells in vitro. According to an embodiment of the present invention, the method includes:

[0051] Co-incubating the mitochondrial transplantation treatment system described in the first aspect with mitochondria-damaged cells, and the normal mitochondria contained in the mitochondrial transplantation treatment system enter the mitochondria-damaged cells to repair the mitochondria-damaged cells.

[0052] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, where:

[0054] Figure 1 Shows the construction flow chart of the mitochondrial transplantation treatment system;

[0055] Figure 2 Shows the figure of transferring mitochondria into cells in Example 2 of the present invention and the functional results of the transferred mitochondria in the cells. Among them, Figure a shows the mitochondrial morphology after transferring mitochondria into cells by the present invention, and the time series shows the dynamic changes of the transplanted mitochondria; (Host mito: the cell's own mitochondria, Transfer mito: the transplanted mitochondria, scale bar: 5μm, enlarged scale bar: 2μm); Figure b shows the detection of the transfer efficiency of the mitochondrial transplantation treatment system of the present invention by flow cytometry; Figure c shows the change in the copy number of mtDNA in the cells 24 hours after transferring mitochondria by the present invention; Figure d shows the change in the transcriptional level of mtDNA in the cells 24 hours after transferring mitochondria by the present invention;

[0056] Figure 3 Shows the results of repairing the mitochondrial function of ρ0 cells by the mitochondrial transplantation treatment system of the present invention in Example 3. Among them, Figure a shows the recovery effect of the mtDNA level after the ρ0 cells are treated with mitochondrial transplantation; Figure b shows the recovery result of the mitochondrial membrane potential after the ρ0 cells are treated with mitochondrial transplantation; Figure c shows the change in the copy number of mitochondria themselves after the ρ0 cells are treated with mitochondrial transplantation; Figure d shows the change in the content of mitochondrial-related proteins after the ρ0 cells are treated with mitochondrial transplantation; Figure e shows the recovery of the aerobic respiration oxidative phosphorylation level and the change in ATP production of the cells after the ρ0 cells are repaired by mitochondrial transplantation;

[0057] Figure 4 Shows the recovery effect diagram of mitochondrial function after repair by the mitochondrial transplantation treatment system in the mitochondrial DNA large fragment deletion cell GM04516 in Example 4. Among them, Figure a shows the deleted fragment of mitochondrial DNA in GM04516 cells; Figure b shows the fluorescence image of exogenous mitochondria entering GM04516 cells through our transplantation system; Figure c shows the morphological change of mitochondria observed by transmission electron microscopy in GM04516 cells after treatment with the mitochondrial transplantation treatment of the present invention; Figure d shows the recovery of the aerobic respiration metabolic oxidative phosphorylation level and the change in ATP production of GM04516 cells after treatment with the mitochondrial transplantation treatment of the present invention; Figure e shows the change in the mtDNA copy number of GM04516 cells after treatment with the mitochondrial transplantation treatment of the present invention; Figure f shows the change in the mtDNA transcription level of GM04516 cells after treatment with the mitochondrial transplantation treatment of the present invention;

[0058] Figure 5 Shows the results of using the mitochondrial transplantation treatment system of the present invention to improve the symptoms of Parkinson's mice caused by mitochondrial dysfunction in the examples. Among them, Figure a shows the construction of the Parkinson's mouse model and the injection process of transplanted mitochondria; Figure b shows the recovery of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra and striatum detected by immunohistochemistry after treatment with the transplanted mitochondria of the present invention; Figures c and d show the TH expression in the substantia nigra neurons detected by immunoblotting after transplantation of mitochondria; Figures e and f show the trajectory diagram and statistical results of the motor ability of mice detected by the open field test 4 weeks after the Parkinson's mice are treated with the mitochondrial transplantation treatment of the present invention; Figure g shows the result diagram of the motor coordination ability of mice detected by the pole climbing test 4 weeks after the Parkinson's mice are treated with the mitochondrial transplantation treatment of the present invention. Detailed implementation manners

[0059] The embodiments of the present invention are described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention.

[0060] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0061] The endpoints and any values disclosed in this text for a range are not limited to the exact range or value. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and a single point value, and between single point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this text.

[0062] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the protein and nucleic acid chemistry, molecular biology, related terms, and laboratory operation procedures used herein are all terms and conventional procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well-known to those skilled in the art and are more comprehensively described in the following literature: Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook").

[0063] In this text, the term "comprising" or "including" is an open expression, that is, it includes the content specified in the present invention, but does not exclude other aspects.

[0064] In this text, the terms "optionally", "optional", or "option" generally mean that the subsequent event or condition may or may not occur, and this description includes the cases where the event or condition occurs and the cases where the event or condition does not occur.

[0065] As used herein, the term "and / or" covers all combinations of the items connected by this term, and should be regarded as each combination having been separately listed in this text. For example, "A and / or B" covers "A", "A and B", and "B". For example, "A, B, and / or C" covers "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0066] According to a specific embodiment of the present invention, the present invention provides a mitochondrial transplantation treatment system, comprising:

[0067] isolated mitochondria; and

[0068] a transfer carrier,

[0069] wherein, the mitochondrial transplantation treatment system is obtained by assembling the mitochondria with the transfer carrier,

[0070] the transfer carrier includes at least one of artificial lipid vesicles, cell-derived vesicles, and exosomes.

[0071] It should be noted that there can be various ways of assembling the mitochondria and the transfer carrier. For example, the two can be directly mixed and incubated, or some other reagents that promote the entry of mitochondria into the transfer carrier can be added.

[0072] The isolated mitochondria in the mitochondrial transplantation treatment system mentioned in the present invention are healthy or normal mitochondria. Here, the "healthy" and "normal" mitochondria refer to mitochondria with normal structural functions and without mtDNA mutations. mtDNA mutations include mtDNA point mutations and mtDNA deletion mutations, etc.

[0073] Regarding the isolated mitochondria contained in the mitochondrial transplantation treatment system, their sources are not particularly limited. For example, they can be isolated from mammalian cells. For another example, the mammalian cells can be human cells.

[0074] It should be noted that when preparing the mitochondrial transplantation treatment system, the mass ratio of the transfer carrier (artificial lipid vesicles) to the mitochondria is not particularly limited. It is preferred to provide an excess of mitochondria to ensure that most of the artificial lipid vesicles can encapsulate the mitochondria to obtain the transfer carrier. For example, the mass ratio of the transfer carrier (artificial lipid vesicles) to the mitochondria can be 1:2. In addition, the transfer carrier (artificial lipid vesicles) is a biodegradable component and does not affect the overall effect. After assembly, the transplanted mitochondria are relatively large and can be precipitated to the bottom of the centrifuge tube by centrifugation, and the excess mitochondria are discarded in the supernatant.

[0075] According to a specific embodiment of the present invention, the present invention provides a drug, which comprises the aforementioned mitochondrial transplantation treatment system. Using this drug can repair mitochondrial damage and can be used to repair mitochondrial dysfunction-related diseases.

[0076] According to a specific embodiment of the present invention, the damaged mitochondria are caused by mitochondrial DNA (mtDNA) mutations.

[0077] Cells with mitochondrial dysfunction include cells with mtDNA depletion, cells with large mtDNA deletions, or cells with mtDNA mutations. According to a specific embodiment of the present invention, the cells with mitochondrial dysfunction can be cells with mitochondrial dysfunction caused by mitochondrial respiratory chain damage; or cells with mitochondrial dysfunction caused by abnormal proteins encoded by nuclear genes; or cells with mitochondrial dysfunction caused by physicochemical property changes.

[0078] According to a specific embodiment of the present invention, the mitochondrial transplantation treatment system provided by the present invention and the drug containing the mitochondrial transplantation treatment system can be used to repair diseases related to mitochondrial dysfunction. The diseases related to mitochondrial dysfunction include at least one of neurological diseases, hearing impairment-related diseases, optic neuropathy, muscle diseases, heart function damage, liver function damage, kidney function damage, pancreatic function damage, gastrointestinal function damage, metabolic diseases, reproductive diseases, and bone diseases caused by mitochondrial dysfunction. However, the types of diseases related to mitochondrial dysfunction are not limited to those listed here, and can also be any other diseases caused by mitochondrial dysfunction known in the art, all of which are included in the scope of the present invention. According to a specific embodiment of the present invention, the mitochondrial transplantation treatment system is used for skin repair, including at least one form of wound healing and skin regeneration.

[0079] According to a specific embodiment of the present invention, the mitochondrial transplantation treatment system is used to improve ischemia-reperfusion injury during organ transplantation, including at least one form of ischemia-reperfusion injury during heart, lung, and kidney transplantation, as well as muscle or brain ischemia-reperfusion injury.

[0080] According to a specific embodiment of the present invention, the mitochondrial transplantation treatment system is used to extend the preservation time of transplanted organs, including at least one form of heart, liver, lung, and kidney transplantation.

[0081] According to a specific embodiment of the present invention, the mitochondrial transplantation treatment system is used to slow down aging, organ degeneration, and diseases related to aging, such as at least one form of neurodegenerative diseases and cardiovascular diseases.

[0082] According to a specific embodiment of the present invention, the mitochondrial transplantation treatment system is used for at least one form of diseases such as weight loss and organ metabolism regulation.

[0083] According to an embodiment of the present invention, the present invention provides a method for obtaining heterologous mitochondrial hybrid cells, the method comprising:

[0084] Co - incubate the mitochondrial transplantation treatment system described above with recipient cells containing autologous mitochondria, such that the heterologous mitochondria contained in the mitochondrial transplantation treatment system enter the recipient cells to obtain heterologous mitochondrial hybrid cells.

[0085] It should be noted that the healthy mitochondria contained in the mitochondrial transplantation treatment system provided by the present invention can be derived from cells of the same or different types as the target cells to be repaired. Even the cells from which the healthy mitochondria in the mitochondrial transplantation treatment system are derived and the recipient cells can be from different organs, different tissues, and different species. The cells from which the healthy mitochondria are derived can be primary cells or cell lines.

[0086] It should be noted that although the inventors mainly verified in the examples that the new mitochondrial transplantation treatment system provided by the present invention can repair the mitochondria of mtDNA - depleted cells, repair the mitochondrial function of cells with mitochondrial DNA deletions, and repair the symptoms of Parkinson's mice caused by mitochondrial dysfunction. However, because the specific mitochondrial transplantation treatment system provided by the present invention can maintain the morphology and function of the healthy mitochondria it encloses, can efficiently enter recipient cells, and can exert the functions and roles of mitochondria, therefore, theoretically, the mitochondrial transplantation treatment system provided by the present invention can be used to repair all diseases related to mitochondrial dysfunction, such as neurological diseases, hearing - impaired related diseases, optic neuropathy, muscle diseases, heart function damage, liver function damage, kidney function damage, pancreatic function damage, gastrointestinal function damage, metabolic diseases, reproductive diseases, bone lesions, etc. caused by mitochondrial dysfunction. In addition, there have been research reports that mitochondrial transplantation can also be used for skin repair, improving ischemia - reperfusion injury during organ transplantation, extending the preservation time of transplanted organs, treating or alleviating abnormal organ metabolic regulation, etc. Therefore, these diseases can also be treated using the mitochondrial transplantation treatment system of the present invention.

[0087] According to a preferred embodiment of the present invention, the inventors found that when different types of mitochondria are transplanted into different types of cells, for example, when HeLa and Cos7 cell mitochondria are transplanted into HeLa, Cos7, U20S, and 3T3 cells, it was found that mitochondria between the same species can play a role in cells of the same species. Due to the heterogeneity of mitochondria, preferably, the cells from which the mitochondria for transplantation are derived are the same or similar cells as the cells to be repaired. There are differences in the number of mitochondria and metabolic levels in cells from different parts, and cells with a high degree of similarity are preferably selected as mitochondrial donors.

[0088] The solution of the present disclosure will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. For those without specific techniques or conditions noted in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0089] Example 1 Construction of a new mitochondrial transplantation treatment system

[0090] Construct a mitochondrial transplantation treatment system according to the following method:

[0091] 1. Preparation of artificial lipid vesicles

[0092] Take out 20 C57BL / 6J mice from the experimental animal center and anesthetize them by intraperitoneal injection with 1.25% avertin (0.2 mL / 10 g). Observe the anesthetic state of the mice, and use a glass capillary blood collection tube to collect blood from the orbital cavity. Approximately 100 - 200 μL of blood can be collected from each mouse. To prevent blood clotting, an appropriate amount of anticoagulant can be added to the collection tube. Centrifuge the collected blood at 800 g for 10 min, gently aspirate the supernatant to obtain a dark red cell pellet. Add an appropriate amount of pre-cooled PBS solution to the cell pellet, resuspend by pipetting, and filter the cells through a 0.45 μm filter to remove the agglutinated cells in the pellet. Centrifuge the cell suspension at 800 g for 10 min, discard the supernatant, add an appropriate amount of pre-cooled PBS solution and then centrifuge, and repeat the operation 2 - 3 times. Add an appropriate amount of pre-cooled 25% PBS solution to the cell pellet, rotate on a rotary mixer for 15 - 30 min, centrifuge at 800 g for 10 min, and discard the supernatant. Add an appropriate amount of pre-cooled PBS solution and then centrifuge, and repeat the operation 2 - 3 times. Subsequently, aliquot the artificial lipid vesicles and store them at -80 °C.

[0093] 2. Mitochondrial extraction

[0094] For the targeted target, select any cells of the corresponding species, including primary cells or cell lines, for in vitro culture and amplification. Wait until the cells grow to 5×10 7When the number reaches a certain level, collect the cells by digesting them with 0.25% trypsin, centrifuge at 300 g for 3 min, discard the upper layer of the culture medium, resuspend the cells with DPBS solution, centrifuge at 300 g for 3 min, and aspirate the supernatant. Add an appropriate amount of mitochondrial extraction solution, place it on ice for 10 - 20 min, pipette the cells on ice 25 - 35 times using a 5 mL syringe, and centrifuge at 800 g for 5 min in a freezer. Transfer the supernatant to a new centrifuge tube, being careful not to aspirate the lower precipitate, and centrifuge at 800 g for 5 min in a freezer. Subsequently, aspirate the supernatant into a centrifuge tube, centrifuge at 12,000 g for 5 min in a freezer, pour out the supernatant, and initially obtain a mitochondrial precipitate. The mitochondrial precipitate for the construction of the transplantation system needs to be stored on ice and used immediately upon extraction.

[0095] Mix the extracted mitochondria evenly with artificial lipid vesicles and stir for 1 h using a magnetic stirrer. Centrifuge the mixed solution at 800 g for 5 min, discard the supernatant, and obtain the encapsulated mitochondrial transplantation treatment system as Figure 1 shown.

[0096] Example 2 Transfer of mitochondria into cells and their functionality

[0097] 1. Process of mitochondrial transplantation into cells

[0098] Seed the counted HeLa cells into 6 cm cell culture dishes, inoculating 3 - 5×10 5 cells per well, and culture for 12 - 24 h to allow the cells to adhere well. Dilute the encapsulated mitochondria in Example 1 with DMEM medium containing penicillin and streptomycin (10% fetal bovine serum, 1% MEM NEAA, and GlutaMax), and add it to the culture plate. After culturing for 6 - 12 h, wash 3 times with DPBS and continue culturing with the medium containing penicillin and streptomycin. Collect cell samples, extract genomic DNA and RNA respectively according to the kit instructions, and detect the expression levels of cell mitochondrial-related genes after mitochondrial transplantation into the cells.

[0099] 2. Mitochondrial transplantation efficiency and mitochondrial morphology

[0100] After being encapsulated by the cell membrane, mitochondria enter the recipient cells through endocytosis. After membrane fusion, the mitochondria are released into the cells. Label the mitochondria with fluorescent proteins EGFP and DsRed respectively. After expanding the culture of the cells, extract the fluorescent mitochondria. After transferring the fluorescent mitochondria into the cells, detect whether the exogenous mitochondria can survive in the cells by live cell photography. Select HeLa cells for the import experiment, as Figure 2As shown in a, DsRed-labeled mitochondria were extracted from HeLa mtDsRed cells and transplanted into HeLa mtGFP cells. Most of the transplanted mitochondria were spherical, and a part of the mitochondria restored to linear. It was observed in the time-lapse images that tubular mitochondria and spherical mitochondria could transform into each other, and the imported mitochondria could undergo dynamic changes in vivo, preliminarily indicating that the transplanted mitochondria could survive in cells. To detect the transplantation efficiency of the mitochondrial transplantation treatment system, flow cytometry was further used to detect the transplantation efficiency of exogenous mitochondria. As Figure 2 shown in b, after 24 h of transplantation, the proportion of cells containing DsRed-labeled mitochondria accounted for more than 90% of the total cells, indicating that the mitochondrial transplantation treatment system could transplant mitochondria into a large number of cells within a short time.

[0101] The number of mitochondria varies greatly in different cells and is abundant in metabolically active cells and tissues. Mitochondria contain mtDNA, and the quantity and quality of mtDNA are crucial for maintaining mitochondrial function and mitochondrial homeostasis. The change in the number of mitochondria can be preliminarily evaluated by detecting the quantity of mtDNA. The inventor collected cell samples 24 h after transplanting mitochondria, extracted the genome, and used qPCR to detect the change in the copy number of mtDNA. As Figure 2 shown in c, it indicated that the copy number of mtDNA in cells increased significantly after transplanting mitochondria. To explore the difference in the transcriptional level of cell mtDNA after transplanting mitochondria, qPCR was used to detect the transcriptional levels of 13 proteins and 2 rRNAs after transplanting mitochondria. The RNA samples were extracted 24 h after transplanting mitochondria, and the relative expression levels of mtDNA transcription were detected. As Figure 2 shown in d, the transcriptional levels of mitochondrially encoded proteins increased significantly 24 h after transplanting mitochondria. In summary, the mitochondrial transplantation treatment system can effectively transplant mitochondria into cells and has functions.

[0102] Example 3 Repair of mitochondrial function in mtDNA-depleted cells by the mitochondrial transplantation treatment system

[0103] 1. Construction and identification of mtDNA-depleted cells

[0104] To explore whether the mitochondrial transplantation treatment system has the effect of repairing mitochondrial damage, mtDNA-depleted cells (ρ0 cells) were constructed. HeLa cells were treated with EB, the passaged cells were collected, the genome and proteins were extracted, and the mitochondrial DNA copy number and mitochondrial protein expression were detected until the mtDNA was almost completely cleared to obtain ρ0 cells, which were used to explore whether the mitochondrial transplantation treatment system has the function of repairing cell mitochondrial function.

[0105] 2. Repair of mitochondrial function in ρ0 cells by the transplanted mitochondrial transplantation treatment system

[0106] Extract mitochondria labeled with EGFP or DsRed, and assemble them with erythrocyte-derived artificial lipid vesicles according to the method of Example 1. After transplanting the fluorescent mitochondria into cells using the assembled mitochondrial transplantation treatment system, detect whether the exogenous mitochondria can survive in the cells by live cell photography. 24 hours after transplanting mitochondria into ρ0 cells using the transplantation system of the present invention, detect by Picogreen staining and photography, and it is found that the mitochondria of ρ0 cells basically do not contain mtDNA. After mitochondrial transplantation treatment of the present invention, most of the mitochondria of ρ0 cells have obtained mtDNA; and after mitochondrial transplantation treatment of the present invention, the morphology of the mitochondria of ρ0 cells has changed from an abnormal fragmented state to a linear shape of normal mitochondria (as shown in Figure 3 a in). At the same time, use the mitochondrial membrane potential fluorescent reagent TMRM for staining, as shown in Figure 3 b in. 1-7 days after transplantation, both the morphology and membrane potential of the mitochondria have recovered, indicating that the mitochondrial metabolic function has recovered. In addition, qPCR is used to detect the relative copy number of mtDNA. The results show that the copy number of mtDNA has been well recovered 24 hours after mitochondrial transplantation treatment of the present invention ( Figure 3 c in). Subsequently, the mitochondrial-related proteins TFAM and Tom20 are used to evaluate the number and transcription level of mitochondria. 7 days after transplantation, the protein expression levels of TFAM and Tom20 increase, preliminarily indicating that the number and transcription level of mitochondria in ρ0 cells have recovered ( Figure 3 d in). At the same time, in order to detect whether mitochondrial respiration has recovered, Seahorse is used to detect mitochondrial respiration. The results show that 24 hours after treatment with the mitochondrial transplantation treatment system of the present invention, the mitochondrial oxidative phosphorylation metabolism level of ρ0 cells has been well recovered, and the production of ATP has been significantly increased ( Figure 3 e in). In summary, the mitochondrial transplantation treatment system of the present invention can effectively repair the mitochondrial function of mitochondrial-deficient cells.

[0107] Example 4 Repair of Mitochondrial Function in Mitochondrial DNA-Deficient Cells by Mitochondrial Transplantation Treatment System

[0108] During the replication process of mitochondrial DNA (mtDNA), mutations and deletions of mtDNA will occur, which will affect mitochondrial function and ultimately lead to mitochondrial diseases. In the present invention, the inventor selects GM04516 cells with a 7031bp mtDNA deletion (purchased from Coriell Institute), and the specific deletion fragment is as shown in Figure 4 a in, to explore whether the mitochondrial transplantation treatment system has a repair effect in mutant cell lines.

[0109] Extract mitochondria labeled with DsRed from primary human skin fibroblasts GZF2 (from Guangzhou Institute of Biomedicine and Health). Use the transplantation system of the present invention (the assembled transplantation system in Example 3) to transplant mitochondria into GM04516 cells. After 24 hours of transplantation treatment according to the present invention, use the mitochondrial fluorescent dye MitoTracker deepred to label the mitochondria. It can be observed that most of the mitochondria transplanted into GM04516 are spherical, and a small part of the mitochondria return to linearity ( Figure 4 in b). Collect GM04516 cells 7 days after transplantation, prepare them into electron microscopy samples, and use a transmission electron microscope to observe the morphology of the mitochondria after transplantation. From Figure 4 it can be observed in c that in GM04516 cells with large fragment deletions of mtDNA, the ultrastructures such as the inner cristae of the mitochondria are abnormal. However, 7 days after mitochondrial transplantation treatment according to the present invention, clear ultrastructures of the mitochondrial inner cristae can be clearly observed, and the mitochondrial morphology returns to normal. The transmission electron microscope results show that the mitochondrial transplantation treatment system transfers normal mitochondria into mitochondria-damaged cells and can accelerate the repair of damaged mitochondria.

[0110] After large fragment deletions occur in mitochondrial DNA, the synthesis of respiratory chain complexes is blocked, resulting in a decrease in the oxidative phosphorylation level of cells. After transferring mitochondria into GM04516 cells with large fragment deletions and culturing them for 24 hours, the oxygen consumption rate of the cells was detected. As Figure 4 shown in d, using Seahorse to detect cell metabolism, it is found that after mitochondrial transplantation treatment according to the present invention, the basal respiration level of GM04516 cells is significantly improved. The ability of mitochondria to synthesize ATP is increased, and the maximum oxygen consumption of the cells also increases significantly, enabling better resistance to the influence of external factors on mitochondria. The cell metabolism detection results show that after mitochondrial transplantation treatment according to the present invention, the mitochondrial function of cells with large fragment deletions of mitochondrial DNA can be effectively improved, which helps the cells to have a stable energy supply.

[0111] After treatment with the mitochondrial transplantation treatment system of the present invention, the inventors detected an increase in the mtDNA copy number in GM04516 cells ( Figure 4 in e). In order to verify whether the increased mtDNA would affect mtDNA transcription, qPCR was used to detect mtDNA transcription. As Figure 4 shown in f, 24 hours after transplanting mitochondria, the transcription levels of proteins and rRNAs encoded by mitochondrial DNA are significantly increased. The transcription levels of proteins ATP6, ND3, DN4L, ND4, ND5, ND6, and CYTB encoded by the deleted fragments are all significantly increased, indicating that the mitochondrial transplantation treatment system of the present invention can effectively improve the mitochondrial function in cells with large fragment deletions of mitochondrial DNA.

[0112] Example 5 Treatment System of Mitochondrial Transplantation for Repairing Symptoms of Parkinson's Mice Caused by Mitochondrial Dysfunction

[0113] 1. Construction of Parkinson's Mouse Model

[0114] C57BL / 6J mice (6 - 8 weeks old) were purchased from Guangdong Medicilon Biotech Co., Ltd. After quarantine, they were caged separately, with 5 - 6 mice in each cage. They were raised in an SPF - level animal house, maintained at a constant temperature (22°C), with a relative humidity of about 30%, sufficient food and water supply, a clean living environment, and a 12 - h cycle of light and darkness. The mice were marked with ear tags, weighed and their weights were recorded. The experiment started when the mice grew to about 20 g, which took about 6 - 8 weeks. The mice were anesthetized by intraperitoneal injection with 1.25% avertin (0.2 mL / 10 g). After pinching the mouse's toes to confirm complete anesthesia, 2.5 mg / kg of MPTP was continuously injected intraperitoneally 5 times a day. Note that the survival status of the mice was observed on the injection day. If the status was poor, the mice were euthanized in time. After about 14 days, the mice were sacrificed, and their brain tissues were taken to identify whether the Parkinson's mouse model was successfully constructed.

[0115] 2. Treatment System of Mitochondrial Transplantation for Repairing Symptoms of Parkinson's Mice Caused by Mitochondrial Dysfunction

[0116] Parkinson's disease is a neurological injury disease. MPP+ produced by the decomposition of MPTP in the body + can damage the mitochondria of dopaminergic neurons, causing Parkinson's symptoms in mice. In the experiment, after treating the mice with MPTP for 2 weeks( Figure 5 as shown in a) below), the mice showed slow movement and tremors. After treating with the mitochondrial transplantation treatment system of the present invention (obtained by assembling mitochondria derived from mouse fibroblasts and mouse red blood cells according to the method of Example 1) for 4 weeks, the mice were euthanized. After taking out the brain tissues and fixing and sectioning them, the expression of tyrosine hydroxylase (TH) in the substantia nigra region and striatum region of the brain was detected to characterize the repair of TH - positive neurons and evaluate the treatment effect of the mitochondrial transplantation treatment system of the present invention. As Figure 5 shown in b) below, after injecting MPTP, the number of TH - containing neurons in the substantia nigra region and striatum region decreased significantly. After treatment with the mitochondrial transplantation treatment system of the present invention, the number of dopaminergic neurons containing TH in the substantia nigra region and striatum region was restored. Taking the brain tissues in the substantia nigra region, extracting proteins and detecting TH expression, it can be clearly seen that after adding MPTP, the TH expression decreased significantly, and after treatment with the mitochondrial transplantation treatment system, the TH expression was significantly up - regulated( Figure 5 as shown in c, d) below), and the secretion of tyrosine hydroxylase by dopaminergic neurons was restored.

[0117] To evaluate whether the motor ability of Parkinson's mice has been restored, the open field and pole climbing experiments were used to analyze the activities of the mice. It was found that the activity distance and the number of times of entering the central area of Parkinson's mice were significantly reduced, and the mice had a certain degree of depression; after treatment with the mitochondrial transplantation treatment system of the present invention, the motor ability of the mice was improved ( Figure 5 in e,f). As Figure 5 shown in g, after MPTP treatment, the time for the mice to climb from the top to the bottom of the pole increased significantly. After treatment with the mitochondrial transplantation treatment system of the present invention, the time used by the mice was significantly shortened, and the motor ability had a relatively good recovery. In summary, the mitochondrial transplantation treatment system can alleviate the symptoms of Parkinson's mice and has a certain therapeutic effect.

[0118] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", "some implementation manners" or "some examples", etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0119] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A mitochondrial transplantation treatment system, characterized in that, Comprising: Mitochondria obtained by separation; And A transfer vector, wherein, the mitochondrial transplantation treatment system is obtained by assembling the mitochondria and the transfer vector, The transfer vector includes at least one of artificial lipid vesicles, cell-derived vesicles, and exosomes.

2. The mitochondrial transplantation treatment system according to claim 1, wherein The transfer vector is a cell membrane structure or lipid vesicle structure derived from red blood cells.

3. The mitochondrial transplantation treatment system according to claim 1, characterized in that, The mitochondria are obtained by separating from mammalian cells.

4. A drug, characterized in that, Comprising the mitochondrial transplantation treatment system according to any one of claims 1-3.

5. Use of the mitochondrial transplantation treatment system according to any one of claims 1-3 and the drug according to claim 4 in the preparation of a drug for repairing mitochondrial damage.

6. The use according to claim 5, wherein The mitochondrial damage includes mitochondrial function damage and mtDNA mutation; Optionally, the mitochondrial function damage includes mitochondrial structure damage and mitochondrial metabolism damage; Optionally, the mtDNA mutation includes mtDNA point mutation and mtDNA deletion mutation.

7. Use of the mitochondrial transplantation treatment system according to any one of claims 1-3 and the drug according to claim 4 in the preparation of a drug for repairing diseases related to mitochondrial dysfunction.

8. The use according to claim 7, wherein, The mitochondrial dysfunction is caused by mitochondrial respiratory chain damage, abnormal proteins encoded by nuclear genes, and physicochemical property changes; Optionally, the diseases related to mitochondrial dysfunction include at least one of neurological diseases, hearing impairment-related diseases, optic neuropathy, muscle diseases, heart function damage, liver function damage, kidney function damage, pancreatic function damage, gastrointestinal function damage, metabolic diseases, reproductive diseases, and skeletal lesions caused by mitochondrial dysfunction; Optionally, the neurological diseases include at least one of neuronal developmental retardation, Parkinson's disease, Alzheimer's disease, stroke, epilepsy, migraine, and amyotrophic lateral sclerosis; Optionally, the hearing impairment-related diseases include at least one of deafness and sensorineural hearing loss; Optionally, the optic neuropathy includes at least one of progressive external ophthalmoplegia, optic atrophy, and retinitis pigmentosa; Optionally, the muscle diseases include at least one of progressive muscle weakness and atrophy, sarcopenia, and exercise intolerance; Optionally, the heart function damage includes at least one of cardiomyopathy and myocardial conduction defect; Optionally, the liver function damage includes at least one of liver failure, liver injury, fatty liver, and cirrhosis; Optionally, the liver function damage includes at least one of Fanconi syndrome, renal tubular acidosis, glomerulosclerosis, renal failure, and adrenal cortical insufficiency; Optionally, the pancreatic function damage includes at least one of diabetes and pancreatitis; Optionally, the gastrointestinal function damage includes at least one of pseudo-obstruction and gastrointestinal motility disorder; Optionally, the metabolic diseases include at least one of senescence, obesity, hyperglycemia, dyslipidemia, insulin resistance, and cardiovascular diseases; Optionally, the reproductive diseases include at least one of premature ovarian failure and male infertility; Optionally, the skeletal lesions include at least one of kyphoscoliosis, dwarfism, and bone marrow failure.

9. Use of the mitochondrial transplantation treatment system according to any one of claims 1-3 and the drug according to claim 4 in the preparation of the following drugs: (1) A drug for skin repair; (2) A drug for improving ischemia-reperfusion injury during organ transplantation; (3) A drug for prolonging the preservation time of transplanted organs; (4) A drug for treating or alleviating abnormal organ metabolic regulation, Optionally, the skin repair includes at least one of wound healing and skin regeneration; Optionally, the ischemia-reperfusion injury includes at least one of ischemia-reperfusion injury during heart, lung, and kidney transplantation, and muscle or brain ischemia-reperfusion injury; Optionally, the transplanted organ includes at least one of the heart, liver, lung, and kidney.

10. A method for obtaining a heteroplasmic mitochondrial hybrid cell, characterized in that, The method includes: Co-incubating the mitochondrial transplantation treatment system according to any one of claims 1-3 with recipient cells containing autologous mitochondria, and allowing the heterologous mitochondria contained in the mitochondrial transplantation treatment system to enter the recipient cells to obtain heterologous mitochondrial hybrid cells.

11. A heteroplasmic mitochondrial hybrid cell, characterized in that, The heterologous mitochondrial hybrid cells are obtained by the method according to claim 10.

12. A method for repairing mitochondria-damaged cells in vitro, characterized in that, The method includes: Co-incubating the mitochondrial transplantation treatment system according to any one of claims 1-3 with mitochondrial damaged cells, and allowing the normal mitochondria contained in the mitochondrial transplantation treatment system to enter the mitochondrial damaged cells to repair the mitochondrial damaged cells.

Images