Mitochondrial transplantation therapy system and use thereof

Abstract

The present disclosure relates to the technical field of biomedical materials, and in particular, to a mitochondrial transplantation therapy system and use thereof. The mitochondrial transplantation therapy system provided by the present disclosure comprises: mitochondria obtained by isolation; and a transfer carrier. The mitochondrial transplantation therapy system is obtained by assembling the mitochondria and the transfer carrier. The transfer carrier comprises at least one of an artificial lipid vesicle, a vesicle derived from a cell membrane, and an exosome. The mitochondrial transplantation therapy system has a simple preparation process, and enables efficient and rapid delivery of healthy mitochondria into cells or tissues at the same time, thereby repairing the mitochondrial function of mitochondria-damaged cells. The mitochondrial transplantation therapy system has the potential to treat related diseases caused by mitochondrial dysfunction.

Description

Mitochondrial transplantation therapy system and its use Technical Field

[0001] The present disclosure relates to the technical field of biomedical materials, and in particular, to a mitochondrial transplantation therapy system and uses thereof. Background Art

[0002] Mitochondria are energy-supplying organelles in eukaryotic cells, playing a vital role in ATP synthesis, cellular metabolism, growth and development, and aging and death. Mitochondria are semi-autonomously replicating organelles within eukaryotic cells. Human mitochondria contain approximately 16.5 kb of circular double-stranded DNA (mtDNA), which encodes 13 proteins, 2 rRNAs, and 22 tRNAs. mtDNA encodes core components of mitochondrial respiratory complexes I-IV, which play a crucial role in maintaining normal cellular life. Mitochondrial oxidative phosphorylation exposes mtDNA to a highly reactive oxygen species environment. Lacking the protective protection of histones, mtDNA is particularly susceptible to oxidative stress damage compared to nuclear DNA, leading to mitochondrial dysfunction. Mitochondrial DNA can also undergo mutations during replication, which can be cleared through autophagy or mitochondrial fusion and fission. In dysfunctional or damaged mitochondria, mtDNA mutations accumulate within the cell, leading to an elevated mutation rate. Mitochondrial DNA exhibits a threshold effect. When the mtDNA mutation rate reaches this threshold, mitochondrial function is severely impaired, leading to cell death or tissue pathology, and ultimately, mitochondrial disease. Mitochondrial dysfunction, especially dysfunction of their metabolic activities, has been implicated in many diseases, including metabolic disorders, neurodegenerative diseases and cancer, as well as the aging process.

[0003] Mitochondrial diseases are hereditary disorders, largely transmitted through mtDNA mutations, with a prevalence of approximately 1 in 5,000 individuals. In the diseased state, mutant mtDNA often coexists with wild-type mtDNA. The severity of disease caused by mtDNA mutations correlates with the mutation rate, with a threshold effect. Mutations in more than 60% of the mtDNA must be present for some conditions to manifest, a defining characteristic of mtDNA diseases. Numerous researchers are currently attempting to reduce the mtDNA mutation rate below this threshold in order to treat these largely incurable diseases. Mitochondrial diseases are clinically heterogeneous and tissue-specific, with mutations within the same mitochondrial protein complex leading to diverse disease phenotypes. Several drugs have been used to alleviate the symptoms of mitochondrial diseases, such as idebenone for the treatment of Leber hereditary optic neuropathy (LHON) and sodium valproate for the treatment of myoclonic epilepsy in mitochondrial diseases. Mitochondrial replacement therapy (MRT) has made it possible for parents with mitochondrial diseases to have healthy offspring. Furthermore, several treatment options are available for mitochondrial diseases, including small molecule drugs and gene therapy targeting the mitochondrial genome. In addition, researchers have developed mitochondrial transplantation technology, which transplants healthy mitochondria into damaged cells or organs, restoring cellular energy metabolism and curing disease. Currently, mitochondrial transplantation has achieved positive results in treating or intervening in a variety of diseases, such as neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, sarcopenia, and heart transplantation.

[0004] However, current mitochondrial transplantation technology has many shortcomings, such as inappropriate vector selection resulting in a low number of healthy mitochondria, low efficiency of mitochondrial entry into the vector, or damage to mitochondrial structure or function. Therefore, further improvements in mitochondrial transplantation technology are urgently needed.

[0005] Public content

[0006] The present disclosure aims to address, at least to some extent, one of the technical problems in the related art. To this end, one object of the present disclosure is to provide a novel mitochondrial transplantation therapy system and its uses. The mitochondrial transplantation therapy system provided by the present disclosure has a simple preparation process and can efficiently and rapidly transfer healthy mitochondria simultaneously into cells or tissues, thereby repairing the mitochondrial function of cells with damaged mitochondria. The mitochondrial transplantation therapy system has the potential to treat diseases related to mitochondrial dysfunction.

[0007] The first aspect of the present disclosure provides a mitochondrial transplantation therapy system. According to an embodiment of the present disclosure, the mitochondrial transplantation therapy system comprises:

[0008] Isolated mitochondria; and

[0009] transfer vector,

[0010] Wherein, the mitochondrial transplantation therapeutic system is obtained by assembling the mitochondria and the transfer vector.

[0011] The transfer vector includes at least one of an artificial lipid vesicle, a cell-derived vesicle, and an exosome.

[0012] The cell membrane is a semipermeable membrane composed of bilayers of phospholipids, providing a relatively independent space for cellular life activities and ensuring their normal function. A variety of proteins, lipids, and carbohydrates are present on the cell membrane surface, exhibiting excellent fluidity and capable of forming vesicles for transport within and outside the cell. Currently, due to its excellent biocompatibility and biodegradability, the cell membrane has promising applications in drug development. Red blood cells are abundant in the blood, transporting oxygen and carbon dioxide, and playing a vital role in cellular respiration. During red blood cell maturation, the nucleus and organelles gradually disappear.

[0013] Parkinson's disease (PD) is a neurological disease in middle-aged and elderly people, which is associated with damage to dopaminergic neurons in the substantia nigra. The neurotoxic substance 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can be converted into MPP after entering the brain. + .MPP + It can selectively enter dopamine neurons in the substantia nigra of the midbrain, inhibit the activity of mitochondrial respiratory chain complex I, promote mitochondrial oxidative stress, lead to dopamine neuron death, and trigger Parkinson's symptoms. Therefore, mitochondrial damage may also be one of the causes of Parkinson's disease. The inventors used MPTP to construct a Parkinson's mouse model to explore whether the mitochondrial transplantation treatment system can work in vivo. The results showed that the mitochondrial transplantation treatment system provided by the present disclosure can restore the motor ability of Parkinson's mice and restore neurons.

[0014] At present, researchers have developed a variety of methods for mitochondria to enter cells. The simplest is to directly incubate the extracted mitochondria with cells and use the endocytosis of the cells to enter the cells, but the efficiency of entering the cells is relatively low, and the mitochondria entering the cells are spherical, and the network structure of the mitochondria cannot be restored. At the same time, mitochondria can be transferred across cells. In addition, the extracted mitochondria can be directly injected into cells using microinjection, FluidFM technology, photothermal nanoblades and other methods, but they need to be operated at the single-cell level, and it takes a long time to obtain a large number of transplanted mitochondrial cells, and there must be equipment and operating experience. In the present disclosure, at least one of artificial lipid vesicles, cell-derived vesicles, and exosomes is used as a transfer vector. The developed mitochondrial transplantation treatment system is simple to operate and can efficiently and quickly transfer mitochondria into a large number of cells at the same time, thereby achieving the purpose of mitochondrial transfer.

[0015] According to an embodiment of the present disclosure, the transfer carrier is a cell membrane structure or a lipid vesicle structure derived from red blood cells.

[0016] According to the embodiments of the present disclosure, the mitochondrial transplantation therapy system provided by the present disclosure is obtained by assembling lipid vesicles prepared from red blood cell membranes and extracted mitochondria. In experiments, the inventors found that after incubating individual mitochondria with other cells, a small amount of mitochondria can enter the cells, but under time series observation, the mitochondria appear spherical in the cells and do not move, and the cell state does not improve. In particular, experimental exploration found that mitochondria encapsulated by the specific transfer vector of the present disclosure can efficiently enter cells and exert the functions and effects of mitochondria.

[0017] According to an embodiment of the present disclosure, the mitochondria are isolated from mammalian cells.

[0018] A second aspect of the present disclosure provides a drug. According to an embodiment of the present disclosure, the drug includes the mitochondrial transplantation treatment system described in the first aspect.

[0019] The third aspect of the present disclosure provides use of the mitochondrial transplantation treatment system described in the first aspect and the drug described in the second aspect in preparing a drug for repairing mitochondrial damage.

[0020] The inventors discovered that after incubating the mitochondrial transplantation treatment system disclosed herein with mitochondrial damaged cells, normal mitochondria can be efficiently and quickly transferred into a large number of recipient cells to achieve mitochondrial transplantation. The transplanted mitochondria restore the network structure within the cells and have mitochondrial function, thereby repairing the mitochondrial damage of the recipient cells.

[0021] According to an embodiment of the present disclosure, the mitochondrial damage includes mitochondrial function damage and mtDNA mutation.

[0022] According to an embodiment of the present disclosure, the mitochondrial function damage includes mitochondrial structure damage and mitochondrial metabolism damage.

[0023] According to an embodiment of the present disclosure, the mtDNA mutation includes mtDNA point mutation and mtDNA deletion mutation.

[0024] According to the embodiments of the present disclosure, the mitochondrial transplantation treatment system provided by the present disclosure can repair mtDNA damage mutations. Specifically, the inventors found through experiments that the function of transplanted mitochondria was detected in mtDNA-removed cells (ρ0 cells) and mtDNA large fragment-deficient cells GM04516. After mitochondrial transplantation, the mitochondrial morphology, mtDNA quantity and membrane potential of ρ0 cells were restored, mitochondrial ATP production was significantly restored, and the transcription level of mtDNA-encoded proteins was also restored. After mitochondrial transplantation, the mitochondrial morphology of GM04516 cells was restored, mitochondrial ATP production was significantly restored, and the transcription level of mtDNA-encoded proteins was also restored.

[0025] The fourth aspect of the present disclosure provides use of the mitochondrial transplantation treatment system described in the first aspect and the drug described in the second aspect in the preparation of drugs for repairing diseases related to mitochondrial dysfunction.

[0026] The inventors have discovered that the mitochondrial transplantation therapy system provided by the present disclosure can repair diseases associated with mitochondrial dysfunction. For example, it can alleviate Parkinson's disease caused by mitochondrial dysfunction. Specifically, through experiments, the inventors found that by using the small molecule drug MPTP to induce mitochondrial damage and constructing a Parkinson's mouse model, the inventors were able to repair mitochondrial damage in the Parkinson's mice using the mitochondrial transplantation therapy system disclosed herein. The results showed that dopaminergic neurons and the mice's motor skills were significantly restored, demonstrating that the developed mitochondrial transplantation therapy system can be used to repair mitochondrial damage.

[0027] According to an embodiment of the present disclosure, the mitochondrial dysfunction is caused by damage to the mitochondrial respiratory chain, abnormal proteins encoded by nuclear genes, and changes in physical and chemical properties.

[0028] According to an embodiment of the present disclosure, the mitochondrial dysfunction-related diseases include at least one of nervous system diseases caused by mitochondrial dysfunction, hearing impairment-related diseases, optic neuropathy, muscle lesions, cardiac function damage, liver function damage, kidney function damage, pancreatic function damage, gastrointestinal function damage, metabolic diseases, reproductive diseases, and bone lesions.

[0029] According to an embodiment of the present disclosure, the nervous system disease includes at least one of neuronal developmental delay, Parkinson's disease, Alzheimer's disease, stroke, epilepsy, migraine and amyotrophic lateral sclerosis.

[0030] According to an embodiment of the present disclosure, the hearing impairment-related disease includes at least one of deafness and sensorineural hearing loss.

[0031] According to an embodiment of the present disclosure, the optic neuropathy includes at least one of progressive external ophthalmoplegia, optic atrophy, and retinitis pigmentosa.

[0032] According to an embodiment of the present disclosure, the muscle pathology includes at least one of progressive muscle weakness and atrophy, sarcopenia, and exercise intolerance. Optionally, the cardiac function damage includes at least one of cardiomyopathy and myocardial conduction defect.

[0033] According to an embodiment of the present disclosure, the liver function damage includes at least one of liver failure, liver damage, fatty liver, and cirrhosis.

[0034] According to an embodiment of the present disclosure, the liver function impairment includes at least one of Fanconi syndrome, renal tubular acidosis, glomerulosclerosis, renal failure, and adrenal insufficiency.

[0035] According to an embodiment of the present disclosure, the pancreatic function impairment includes at least one of diabetes and pancreatitis.

[0036] According to an embodiment of the present disclosure, the gastrointestinal function impairment includes at least one of intestinal pseudo-obstruction and gastrointestinal motility disorder.

[0037] According to an embodiment of the present disclosure, the metabolic disease includes at least one of aging, obesity, hyperglycemia, dyslipidemia, insulin resistance and cardiovascular disease.

[0038] According to an embodiment of the present disclosure, the reproductive disease includes at least one of premature ovarian failure and male infertility.

[0039] According to an embodiment of the present disclosure, the bone lesion includes at least one of kyphoscoliosis, dwarfism, and bone marrow failure.

[0040] A fourth aspect of the present disclosure provides use of the mitochondrial transplantation therapeutic system described in the first aspect and the drug described in the second aspect in the preparation of the following drugs:

[0041] (1) Drugs for skin repair;

[0042] (2) Drugs that improve ischemia-reperfusion injury during organ transplantation;

[0043] (3) drugs that prolong the storage time of transplanted organs;

[0044] (4) Drugs that treat or alleviate abnormal organ metabolic regulation.

[0045] According to an embodiment of the present disclosure, the skin repair includes at least one of wound healing and skin regeneration.

[0046] According to an embodiment of the present disclosure, the ischemia-reperfusion injury includes at least one of ischemia-reperfusion injury during heart, lung, and kidney transplantation, and ischemia-reperfusion injury in muscle or brain.

[0047] According to an embodiment of the present disclosure, the transplant organ includes at least one of a heart, a liver, a lung, and a kidney.

[0048] A fifth aspect of the present disclosure provides a method for obtaining heterologous mitochondrial hybrid cells. According to an embodiment of the present disclosure, the method comprises:

[0049] The mitochondrial transplantation therapeutic system described in the first aspect is co-incubated with recipient cells containing their own mitochondria, and the heterologous mitochondria contained in the mitochondrial transplantation therapeutic system enters the recipient cells, so as to obtain heterologous mitochondrial hybrid cells.

[0050] A sixth aspect of the present disclosure provides a heterologous mitochondrial hybrid cell. According to an embodiment of the present disclosure, the heterologous mitochondrial hybrid cell is obtained by the method described in the fifth aspect.

[0051] A seventh aspect of the present disclosure provides a method for repairing mitochondrial damaged cells in vitro. According to an embodiment of the present disclosure, the method comprises:

[0052] The mitochondrial transplantation therapeutic system described in the first aspect is mixed with mitochondrial damaged cells, and the normal mitochondria contained in the mitochondrial transplantation therapeutic system enters the mitochondrial damaged cells to repair the mitochondrial damaged cells.

[0053] In an eighth aspect, the present disclosure provides a method for treating and / or repairing diseases related to mitochondrial damage or mitochondrial dysfunction. According to an embodiment of the present disclosure, the method comprises administering the mitochondrial transplantation therapy system of the first aspect and / or the drug of the second aspect to a subject.

[0054] According to an embodiment of the present disclosure, the mitochondrial damage includes mitochondrial function damage and mtDNA mutation.

[0055] According to an embodiment of the present disclosure, the mitochondrial function damage includes mitochondrial structure damage and mitochondrial metabolism damage.

[0056] According to an embodiment of the present disclosure, the mtDNA mutation includes mtDNA point mutation and mtDNA deletion mutation.

[0057] According to an embodiment of the present disclosure, the mitochondrial dysfunction is caused by damage to the mitochondrial respiratory chain, abnormal proteins encoded by nuclear genes, and changes in physical and chemical properties.

[0058] According to an embodiment of the present disclosure, the mitochondrial dysfunction-related diseases include at least one of nervous system diseases caused by mitochondrial dysfunction, hearing impairment-related diseases, optic neuropathy, muscle lesions, cardiac function damage, liver function damage, kidney function damage, pancreatic function damage, gastrointestinal function damage, metabolic diseases, reproductive diseases, and bone lesions.

[0059] According to an embodiment of the present disclosure, the nervous system disease includes at least one of neuronal developmental delay, Parkinson's disease, Alzheimer's disease, stroke, epilepsy, migraine and amyotrophic lateral sclerosis.

[0060] According to an embodiment of the present disclosure, the hearing impairment-related disease includes at least one of deafness and sensorineural hearing loss.

[0061] According to an embodiment of the present disclosure, the optic neuropathy includes at least one of progressive external ophthalmoplegia, optic atrophy, and retinitis pigmentosa.

[0062] According to an embodiment of the present disclosure, the muscle pathology includes at least one of progressive muscle weakness and atrophy, sarcopenia, and exercise intolerance. Optionally, the cardiac function damage includes at least one of cardiomyopathy and myocardial conduction defect.

[0063] According to an embodiment of the present disclosure, the liver function damage includes at least one of liver failure, liver damage, fatty liver, and cirrhosis.

[0064] According to an embodiment of the present disclosure, the liver function impairment includes at least one of Fanconi syndrome, renal tubular acidosis, glomerulosclerosis, renal failure, and adrenal insufficiency.

[0065] According to an embodiment of the present disclosure, the pancreatic function impairment includes at least one of diabetes and pancreatitis.

[0066] According to an embodiment of the present disclosure, the gastrointestinal function impairment includes at least one of intestinal pseudo-obstruction and gastrointestinal motility disorder.

[0067] According to an embodiment of the present disclosure, the metabolic disease includes at least one of aging, obesity, hyperglycemia, dyslipidemia, insulin resistance and cardiovascular disease.

[0068] According to an embodiment of the present disclosure, the reproductive disease includes at least one of premature ovarian failure and male infertility.

[0069] According to an embodiment of the present disclosure, the bone lesion includes at least one of kyphoscoliosis, dwarfism, and bone marrow failure.

[0070] The ninth aspect of the present disclosure provides the use of the mitochondrial transplantation treatment system described in the first aspect and the drug described in the second aspect in treating and / or repairing diseases related to mitochondrial damage or mitochondrial dysfunction.

[0071] According to an embodiment of the present disclosure, the mitochondrial damage includes mitochondrial function damage and mtDNA mutation.

[0072] According to an embodiment of the present disclosure, the mitochondrial function damage includes mitochondrial structure damage and mitochondrial metabolism damage.

[0073] According to an embodiment of the present disclosure, the mtDNA mutation includes mtDNA point mutation and mtDNA deletion mutation.

[0074] According to an embodiment of the present disclosure, the mitochondrial dysfunction is caused by damage to the mitochondrial respiratory chain, abnormal proteins encoded by nuclear genes, and changes in physical and chemical properties.

[0075] According to an embodiment of the present disclosure, the mitochondrial dysfunction-related diseases include at least one of nervous system diseases caused by mitochondrial dysfunction, hearing impairment-related diseases, optic neuropathy, muscle lesions, cardiac function damage, liver function damage, kidney function damage, pancreatic function damage, gastrointestinal function damage, metabolic diseases, reproductive diseases, and bone lesions.

[0076] According to an embodiment of the present disclosure, the nervous system disease includes at least one of neuronal developmental delay, Parkinson's disease, Alzheimer's disease, stroke, epilepsy, migraine and amyotrophic lateral sclerosis.

[0077] According to an embodiment of the present disclosure, the hearing impairment-related disease includes at least one of deafness and sensorineural hearing loss.

[0078] According to an embodiment of the present disclosure, the optic neuropathy includes at least one of progressive external ophthalmoplegia, optic atrophy, and retinitis pigmentosa.

[0079] According to an embodiment of the present disclosure, the muscle pathology includes at least one of progressive muscle weakness and atrophy, sarcopenia, and exercise intolerance. Optionally, the cardiac function damage includes at least one of cardiomyopathy and myocardial conduction defect.

[0080] According to an embodiment of the present disclosure, the liver function damage includes at least one of liver failure, liver damage, fatty liver, and cirrhosis.

[0081] According to an embodiment of the present disclosure, the liver function impairment includes at least one of Fanconi syndrome, renal tubular acidosis, glomerulosclerosis, renal failure, and adrenal insufficiency.

[0082] According to an embodiment of the present disclosure, the pancreatic function impairment includes at least one of diabetes and pancreatitis.

[0083] According to an embodiment of the present disclosure, the gastrointestinal function impairment includes at least one of intestinal pseudo-obstruction and gastrointestinal motility disorder.

[0084] According to an embodiment of the present disclosure, the metabolic disease includes at least one of aging, obesity, hyperglycemia, dyslipidemia, insulin resistance and cardiovascular disease.

[0085] According to an embodiment of the present disclosure, the reproductive disease includes at least one of premature ovarian failure and male infertility.

[0086] According to an embodiment of the present disclosure, the bone lesion includes at least one of kyphoscoliosis, dwarfism, and bone marrow failure.

[0087] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0089] Figure 1 shows a flow chart for the construction of a mitochondrial transplantation therapy system;

[0090] Figure 2 shows the results of transplanting mitochondria into cells and the functional results of the mitochondria in Example 2 of the present disclosure, wherein Figure a shows the mitochondrial morphology after the mitochondria are transplanted into cells according to the present disclosure, and the time series shows the dynamic changes of the transplanted mitochondria; (Host mito: cell's own mitochondria, Transfer mito: transplanted mitochondria, scale: 5 μm, enlarged scale: 2 μm); Figure b shows the transplantation efficiency of the mitochondrial transplantation treatment system of the present disclosure detected by flow cytometry; Figure c shows the changes in the mtDNA copy number in the cells 24 hours after the mitochondria transplantation according to the present disclosure; Figure d shows the changes in the mtDNA transcription level in the cells 24 hours after the mitochondria transplantation according to the present disclosure;

[0091] Figure 3 shows the results of repairing mitochondrial function of ρ0 cells by the mitochondrial transplantation treatment system of the present invention in Example 3, wherein Figure a shows the recovery effect of mtDNA levels in ρ0 cells after mitochondrial transplantation treatment; Figure b shows the recovery results of mitochondrial membrane potential in ρ0 cells after mitochondrial transplantation treatment; Figure c shows the changes in the copy number of mitochondria themselves in ρ0 cells after mitochondrial transplantation treatment; Figure d shows the changes in the content of mitochondrial-related proteins in ρ0 cells after mitochondrial transplantation treatment; Figure e shows the recovery of aerobic respiration oxidative phosphorylation levels and changes in ATP production in ρ0 cells after mitochondrial transplantation repair;

[0092] Figure 4 shows the effect of mitochondrial function recovery in GM04516 cells with large mitochondrial DNA fragment deletions after repair by the mitochondrial transplantation treatment system in Example 4. Figure a shows the mitochondrial DNA deletion fragments in GM04516 cells; Figure b shows the fluorescence image of exogenous mitochondria entering GM04516 cells after our transplantation treatment system; Figure c shows the mitochondrial morphological changes observed by transmission electron microscopy in GM04516 cells after treatment with the mitochondrial transplantation treatment system of the present disclosure; Figure d shows the recovery of cellular aerobic respiration metabolism and oxidative phosphorylation levels and changes in ATP production in GM04516 cells after treatment with the mitochondrial transplantation treatment system of the present disclosure; Figure e shows the changes in mtDNA copy number in GM04516 cells after treatment with the mitochondrial transplantation treatment system of the present disclosure; Figure f shows the changes in mtDNA transcription levels in GM04516 cells after treatment with the mitochondrial transplantation treatment system of the present disclosure;

[0093] 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 embodiment, wherein Figure a shows the construction of the Parkinson's mouse model and the process of transplanted mitochondria injection; Figure b shows the recovery of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra and striatum after mitochondrial transplantation treatment of the present invention, detected by immunohistochemistry; Figures c and d show the TH expression of substantia nigra neurons detected by immunoblotting after mitochondrial transplantation; Figures e and f show the trajectory diagrams and statistical results of the open field test on the movement ability of Parkinson's mice after 4 weeks of treatment with the mitochondrial transplantation treatment system of the present invention; Figure g shows the results of the pole climbing test on the movement coordination ability of Parkinson's mice after 4 weeks of treatment with the mitochondrial transplantation treatment system of the present invention.

[0094] Detailed Description of the Invention

[0095] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0096] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of the features. Furthermore, in the description of this disclosure, unless otherwise specified, "plurality" means two or more.

[0097] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0098] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, protein and nucleic acid chemistry, molecular biology, related terms, and laboratory procedures used herein are terms and routine procedures widely used in the corresponding fields. For example, standard recombinant DNA and molecular cloning techniques used in this disclosure are well known to those skilled in the art and are more fully described in Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook").

[0099] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0100] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0101] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0102] According to a specific embodiment of the present disclosure, the present disclosure provides a mitochondrial transplantation therapy system, comprising:

[0103] Isolated mitochondria; and

[0104] transfer vector,

[0105] Wherein, the mitochondrial transplantation therapeutic system is obtained by assembling the mitochondria and the transfer vector.

[0106] The transfer vector includes at least one of an artificial lipid vesicle, a cell-derived vesicle, and an exosome.

[0107] It should be noted that there are many ways to assemble mitochondria and transfer vectors, such as directly mixing and incubating the two, or adding other reagents that promote the entry of mitochondria into the transfer vector.

[0108] The mitochondria isolated and obtained in the mitochondrial transplantation therapy system described herein are healthy or normal mitochondria. "Healthy" and "normal" mitochondria herein refer to mitochondria with normal structure and function and without mtDNA damage. mtDNA damage includes mitochondrial structural damage, mitochondrial metabolic damage, mtDNA mutations, and mtDNA deletions.

[0109] There is no particular limitation on the source of the isolated mitochondria contained in the mitochondrial transplantation therapy system, and the mitochondria can be isolated from mammalian cells, for example, human cells.

[0110] It should be noted that when preparing the mitochondrial transplantation therapy system, there is no special restriction on the mass ratio of the transfer vector (such as artificial lipid vesicles) to the mitochondria. It is preferred to provide an excess of mitochondria to ensure that most of the artificial lipid vesicles can wrap the mitochondria to obtain the transfer vector. For example, the mass ratio of the transfer vector to the mitochondria can be 1:2. In addition, the transfer vector (such as artificial lipid vesicles) is a biodegradable component and does not affect the overall effect. The assembled transplanted mitochondrial particles 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. According to a specific embodiment of the present disclosure, the present disclosure provides a drug, which includes the mitochondrial transplantation therapy system described above. The drug can repair mitochondrial damage and can be used to repair diseases related to mitochondrial dysfunction.

[0111] According to a specific embodiment of the present disclosure, the damaged mitochondria are caused by mitochondrial DNA (mtDNA) damage.

[0112] Cells with mitochondrial dysfunction include cells with mtDNA ablation, cells with large mtDNA deletions, or cells with mtDNA point mutations. According to a specific embodiment of the present disclosure, cells with mitochondrial dysfunction can be cells with mitochondrial dysfunction caused by damage to the mitochondrial respiratory chain; cells with mitochondrial dysfunction caused by abnormalities in proteins encoded by nuclear genes; or cells with mitochondrial dysfunction caused by altered physicochemical properties.

[0113] According to a specific embodiment of the present disclosure, the mitochondrial transplantation therapy system provided by the present disclosure and the medicine containing the mitochondrial transplantation therapy system can be used to repair diseases related to mitochondrial dysfunction. The diseases related to mitochondrial dysfunction include at least one of nervous system diseases, hearing loss-related diseases, optic neuropathy, muscle lesions, cardiac 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, but the types of mitochondrial dysfunction-related diseases are not limited to the types 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 disclosure. According to a specific embodiment of the present disclosure, the mitochondrial transplantation therapy system is used for skin repair, including at least one form of wound healing and skin regeneration.

[0114] According to a specific embodiment of the present disclosure, the mitochondrial transplantation therapy 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, and muscle or brain ischemia-reperfusion injury.

[0115] According to a specific embodiment of the present disclosure, the mitochondrial transplantation therapy system is used to prolong the preservation time of transplanted organs, including at least one of heart, liver, lung and kidney transplants.

[0116] According to a specific embodiment of the present disclosure, a mitochondrial transplantation therapy system is used to slow down at least one form of aging, organ degeneration, and aging-related diseases, such as neurodegenerative diseases and cardiovascular diseases.

[0117] According to a specific embodiment of the present disclosure, the mitochondrial transplantation therapy system is used for at least one form of diseases such as weight loss and organ metabolism regulation.

[0118] According to an embodiment of the present disclosure, the present disclosure provides a method for obtaining heterologous mitochondrial hybrid cells, the method comprising:

[0119] The aforementioned mitochondrial transplantation therapeutic system is co-incubated with recipient cells containing autologous mitochondria, and the heterologous mitochondria contained in the mitochondrial transplantation therapeutic system enters the recipient cells, so as to obtain heterologous mitochondrial hybrid cells.

[0120] It should be noted that the healthy mitochondria contained in the mitochondrial transplantation therapy system provided herein can be derived from cells of the same or different species as the target cells to be repaired. Furthermore, the healthy mitochondrial-derived cells and recipient cells in the mitochondrial transplantation therapy system can originate from different organs, tissues, or species. The healthy mitochondrial-derived cells can be primary cells or cell lines.

[0121] It should be noted that although the inventors have mainly verified in the examples that the new mitochondrial transplantation treatment system provided by the present disclosure can repair mitochondria in cells with mtDNA removal; repair mitochondrial function in cells with mitochondrial DNA deletion; and repair Parkinson's disease symptoms in mice caused by mitochondrial dysfunction, the specific mitochondrial transplantation treatment system provided by the present disclosure can maintain the morphology and function of healthy mitochondria it encapsulates, and can efficiently enter recipient cells and exert the functions and effects of mitochondria. Therefore, in theory, the mitochondrial transplantation treatment system provided by the present disclosure can be used to repair all diseases related to mitochondrial dysfunction, such as neurological diseases, hearing loss-related diseases, optic neuropathy, muscle disease, cardiac function damage, liver function damage, kidney function damage, pancreatic function damage, gastrointestinal function damage, metabolic diseases, reproductive diseases, bone diseases, etc. caused by mitochondrial dysfunction. In addition, studies have reported that mitochondrial transplantation can also be used for skin repair, improving ischemia-reperfusion injury during organ transplantation, prolonging the preservation time of transplanted organs, treating or alleviating organ metabolic abnormalities, etc. Therefore, the mitochondrial transplantation treatment system disclosed in the present disclosure can also be used to treat these diseases.

[0122] According to preferred embodiments of the present disclosure, the inventors discovered that transplanting mitochondria from different species into different cell types—for example, transplanting mitochondria from HeLa and Cos7 cells into HeLa, Cos7, U20S, and 3T3 cells—resulted in the discovery that mitochondria from the same species can function within cells of the same species. Due to mitochondrial heterogeneity, it is preferred that the mitochondria used for transplantation originate from cells that are identical or similar to the cells being repaired. Cells from different locations vary in mitochondrial numbers and metabolic levels, so cells with high similarity should be selected as mitochondrial donors.

[0123] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0124] Example 1 Construction of a new mitochondrial transplantation therapy system

[0125] The mitochondrial transplantation therapy system was constructed according to the following methods:

[0126] 1. Preparation of Artificial Lipid Vesicles

[0127] Twenty C57BL / 6J mice were obtained from the experimental animal center and anesthetized with an intraperitoneal injection of 1.25% avertin (0.2 mL / 10 g). The mice were observed for anesthesia and blood was collected from the eye socket using a glass capillary tube. Approximately 100-200 μL of blood was collected from each mouse. To prevent coagulation, an appropriate amount of anticoagulant can be added to the collection tube. The collected blood was centrifuged at 800 g for 10 minutes, and the supernatant was gently aspirated to obtain a dark red cell pellet. An appropriate amount of pre-chilled PBS solution was added to the blood cell pellet, and after pipetting and resuspension, the cells were filtered through a 0.45 μm filter to remove the agglomerated cells in the pellet. The cell suspension was centrifuged at 800 g for 10 minutes, the supernatant was removed, and an appropriate amount of pre-chilled PBS solution was added and centrifuged. This operation was repeated 2-3 times. An appropriate amount of pre-chilled 25% PBS solution was added to the cell pellet, and the mixture was rotated on a rotary mixer for 15-30 minutes. The cell pellet was centrifuged at 800 g for 10 minutes, and the supernatant was removed. Add an appropriate amount of pre-chilled PBS solution and centrifuge, repeating the operation 2-3 times. Then, aliquot the artificial lipid vesicles and store them at -80°C.

[0128] 2. Mitochondrial Extraction

[0129] For the target, select any cells of the corresponding species, including primary cells or cell lines, and culture and expand them in vitro. 7When the cells are collected, use 0.25% trypsin to digest and collect them, centrifuge at 300g for 3 minutes, remove the upper culture medium, add DPBS solution to resuspend the cells, centrifuge at 300g for 3 minutes, and aspirate the supernatant. Add an appropriate amount of mitochondrial extraction solution, place on ice for 10-20 minutes, use a 5mL syringe to blow the cells on ice 25-35 times, and centrifuge at 800g for 5 minutes. Transfer the supernatant to a new centrifuge tube, being careful not to aspirate the lower sediment, and centrifuge at 800g for 5 minutes. Subsequently, aspirate the supernatant into a centrifuge tube, centrifuge at 12000g for 5 minutes, pour out the supernatant, and obtain a preliminary mitochondrial pellet. The mitochondrial pellet needs to be stored on ice for the construction of the transplantation system and is now used.

[0130] The extracted mitochondria were mixed evenly with the artificial lipid vesicles and stirred using a magnetic stirrer for 1 hour. The mixed solution was centrifuged at 800 g for 5 minutes, and the supernatant was removed to obtain the encapsulated mitochondrial transplantation therapy system shown in Figure 1.

[0131] Example 2 Mitochondrial transplantation therapy system enters cells and has function

[0132] 1. Mitochondrial transplantation into cells

[0133] The counted HeLa cells were seeded into 6 cm cell culture dishes, with 3-5×10 cells per well. 5 Cells were cultured for 12-24 hours to allow the cells to fully adhere to the wall. The mitochondria encapsulated in Example 1 were diluted with DMEM medium (10% fetal bovine serum, 1% MEM NEAA and GlutaMax) containing penicillin and streptomycin and added to the culture plate. After culturing for 6-12 hours, the cells were washed three times with DPBS and cultured with medium containing penicillin and streptomycin. Cell samples were collected, and the genome and RNA were extracted according to the kit instructions. The expression levels of mitochondrial-related genes in the cells were detected after the mitochondria were transplanted into the cells.

[0134] 2. Mitochondrial Transplantation Efficiency and Mitochondrial Morphology

[0135] After being encapsulated by the cell membrane, mitochondria enter the recipient cell through endocytosis. After membrane fusion, the mitochondria are released into the cell. Mitochondria are labeled with the fluorescent proteins EGFP and DsRed, respectively. After the cells are expanded, fluorescent mitochondria are extracted. After the fluorescent mitochondria are transplanted into the cells, live cell imaging is used to determine whether the exogenous mitochondria can survive within the cells. HeLa cells were used for the transfection experiment. As shown in Figure 2a, DsRed-labeled mitochondria were extracted from HeLa mtDsRed cells and transplanted into HeLa mtGFP cells. Most of the transplanted mitochondria were observed to be spherical, while some returned to a linear shape. Time-lapse images show that tubular and spherical mitochondria can transform into each other, indicating that the transplanted mitochondria can undergo dynamic changes in vivo, preliminarily indicating that the transplanted mitochondria can survive within the cell. To test the efficiency of the mitochondrial transplantation therapy system, flow cytometry was used to measure the efficiency of the exogenous mitochondria. As shown in Figure 2b, 24 hours after transplantation, the proportion of cells containing DsRed-labeled mitochondria exceeded 90%, demonstrating that the mitochondrial transplantation therapy system can transfer mitochondria into a large number of cells in a short period of time.

[0136] The number of mitochondria in different cells varies greatly, and is abundant in cells and tissues with active metabolism. Mitochondria contain mtDNA. The quantity and quality of mtDNA are crucial for maintaining mitochondrial function and mitochondrial homeostasis. Changes in the number of mitochondria can be preliminarily assessed by detecting the amount of mtDNA. The inventors collected cell samples 24 hours after mitochondrial transplantation, extracted the genome, and used qPCR to detect changes in mtDNA copy number. As shown in Figure 2c, the number of mtDNA copies in the cells increased significantly after mitochondrial transplantation. In order to explore the differences in cellular mtDNA transcription levels after mitochondrial transplantation, qPCR was used to detect the transcription levels of 13 proteins and 2 rRNAs after mitochondrial transplantation. RNA samples were extracted 24 hours after mitochondrial transplantation, and the relative expression of mtDNA transcription was detected. As shown in Figure 2d, the transcription level of mitochondrial-encoded proteins was significantly increased 24 hours after mitochondrial transplantation. In summary, the mitochondrial transplantation therapy system can effectively transplant mitochondria into cells and have function.

[0137] Example 3: Mitochondrial transplantation therapy system repairs mitochondrial function in mtDNA-depleted cells

[0138] 1. Construction and Identification of Mitochondrial DNA-Depleted Cells

[0139] To investigate whether the mitochondrial transplantation therapy system could repair mitochondrial damage, we constructed a mtDNA-depleted cell line (ρ0 cells). HeLa cells were treated with EB, and passaged cells were collected. Genome and protein extraction was performed, and mitochondrial DNA copy number and mitochondrial protein expression were measured until mtDNA was nearly completely eliminated. ρ0 cells were then obtained to investigate whether the mitochondrial transplantation therapy system could repair mitochondrial function.

[0140] 2. Mitochondrial transplantation therapy system repairs mitochondrial function of ρ0 cells

[0141] Mitochondria labeled with EGFP or DsRed were extracted and assembled with artificial lipid vesicles derived from red blood cells according to the method of Example 1. After the fluorescent mitochondria were transplanted into the cells using the assembled mitochondrial transplantation treatment system, the exogenous mitochondria were detected by taking photos of living cells to see if they could survive in the cells. After 24 hours of mitochondria being transferred to ρ0 cells using the transplantation treatment system disclosed herein, Picogreen staining and photo detection revealed that the mitochondria of ρ0 cells basically did not contain mtDNA, and after the mitochondrial transplantation treatment disclosed herein, most of the mitochondria of ρ0 cells obtained mtDNA; and after the mitochondrial transplantation treatment disclosed herein, the morphology of the mitochondria of ρ0 cells was restored from an abnormal fragmented state to a linear shape of normal mitochondria (as shown in a in Figure 3). At the same time, TMRM staining was performed using the mitochondrial membrane potential fluorescent reagent, as shown in b in Figure 3. After 1-7 days of transplantation, the morphology and membrane potential of the mitochondria were restored, proving that the mitochondrial metabolic function was restored. In addition, qPCR was used to detect the relative copy number of mtDNA. It was found that the mtDNA copy number was well restored after 24 hours of mitochondrial transplantation treatment disclosed herein (c in Figure 3). Subsequently, mitochondrial-related proteins TFAM and Tom20 were used to assess the number and transcription level of mitochondria. Seven days after transplantation, the expression levels of TFAM and Tom20 proteins increased, preliminarily indicating that the number and transcription level of mitochondria in ρ0 cells were restored (Figure 3d). At the same time, in order to detect whether mitochondrial respiration has recovered, Seahorse was used to detect mitochondrial respiration. The results showed that after 24 hours of mitochondrial transplantation treatment of the present invention, the mitochondrial oxidative phosphorylation metabolism level of ρ0 cells was well restored, and the production of ATP was significantly improved (Figure 3e). In summary, the mitochondrial transplantation treatment system disclosed in the present invention can effectively repair the mitochondrial function of mitochondrial-deficient cells.

[0142] Example 4: Mitochondrial transplantation therapy system repairs mitochondrial function in cells lacking mitochondrial DNA

[0143] During mitochondrial DNA (mtDNA) replication, mutations and deletions can occur, impairing mitochondrial function and ultimately leading to mitochondrial diseases. In this disclosure, the inventors used GM04516 cells (purchased from the Coriell Institute) harboring a 7031bp mtDNA deletion, as shown in Figure 4(a), to investigate whether a mitochondrial transplantation therapy system could repair these mutant cell lines.

[0144] From primary human skin fibroblasts GZF2 (derived from Guangzhou Institute of Biomedicine and Health), mitochondria labeled with DsRed were extracted from the cells, and the mitochondria were transferred into GM04516 cells using the transplantation treatment system disclosed herein (the transplantation treatment system assembled in Example 3). After 24 hours of transplantation treatment disclosed herein, the mitochondria were labeled using the mitochondrial fluorescent dye MitoTracker deep red, and it was observed that most of the mitochondria transplanted into GM04516 were spherical, and a small number of mitochondria returned to linear shape (Fig. 4b). GM04516 cells were collected 7 days after transplantation, and after electron microscope samples were prepared, the morphology of the mitochondria after transplantation was observed using a transmission electron microscope. It can be observed from Fig. 4c that the GM04516 cells with large mtDNA fragment deletions had abnormal ultrastructures such as the inner cristae of the mitochondria, and after 7 days of treatment with the mitochondrial transplantation treatment system disclosed herein, the clear ultrastructure of the inner cristae of the mitochondria could be clearly observed, and the mitochondrial morphology returned to normal. The transmission electron microscopy results showed that normal mitochondria were transplanted into mitochondrially damaged cells through the mitochondrial transplantation treatment system, and the repair of damaged mitochondria could be accelerated.

[0145] After a large fragment of mitochondrial DNA is deleted, the synthesis of respiratory chain complexes is blocked, resulting in a decrease in the oxidative phosphorylation level of the cells. After 24 hours of culture, the mitochondria were transplanted into GM04516 cells with a large fragment deletion, and the oxygen consumption rate of the cells was detected. As shown in Figure 4d, using Seahorse to detect cell metabolism, it was found that after the mitochondrial transplantation treatment disclosed in the present invention, the basal respiration level of GM04516 cells was significantly improved. The ability of mitochondria to synthesize ATP is improved, and the maximum oxygen consumption of the cells also increases significantly, which can better resist the influence of external factors on mitochondria. The results of cell metabolism detection show that treatment with the mitochondrial transplantation treatment system disclosed in the present invention can effectively improve the mitochondrial function of cells with large mitochondrial DNA fragments, which helps cells have a stable energy supply.

[0146] After treatment with the mitochondrial transplantation therapy system disclosed herein, the inventors detected an increase in the number of mtDNA copies in GM04516 cells (e in FIG. 4 ). To verify whether the increased mtDNA would affect mtDNA transcription, qPCR was used to detect mtDNA transcription. As shown in FIG. 4 f, 24 hours after mitochondrial transplantation, the transcription levels of mitochondrial DNA-encoded proteins and rRNA were significantly increased, and the transcription levels of the proteins ATP6, ND3, DN4L, ND4, ND5, ND6, and CYTB encoded by the deletion fragments were significantly increased, indicating that the mitochondrial transplantation therapy system disclosed herein can effectively improve mitochondrial function in cells with large mitochondrial DNA fragment deletions.

[0147] Example 5: Mitochondrial transplantation therapy repairs Parkinson's disease symptoms in mice caused by mitochondrial dysfunction

[0148] 1. Construction of Parkinson's disease mouse model

[0149] C57BL / 6J mice (6-8 weeks) were purchased from Guangdong Yaokang Biotechnology Co., Ltd. After quarantine, they were divided into cages with 5-6 mice per cage and housed in an SPF-grade animal room. The temperature was maintained at 22°C and the relative humidity was about 30%. There was sufficient food and water supply, a clean living environment, and a light and dark cycle every 12 hours. The mice were marked with ear tags, weighed, and their weight was recorded. The experiment was started when the mice grew to about 20g, which lasted about 6-8 weeks. 1.25% Avertin (0.2mL / 10g) mouse anesthetic was used for intraperitoneal injection. The mouse toes were pinched to confirm whether the anesthesia was complete. Then, 2.5mg / kg of MPTP was injected intraperitoneally for 5 consecutive times per day. The survival status of the mice was observed on the day of injection. If the condition was not good, they were euthanized in time. After about 14 days, the mice were killed and brain tissue was taken to identify whether the Parkinson's mouse model was successfully constructed.

[0150] 2. Mitochondrial transplantation therapy repairs Parkinson's disease symptoms in mice caused by mitochondrial dysfunction

[0151] Parkinson's disease is a neurological disease. MPTP is decomposed in the body to produce MPP +It can damage the mitochondria of dopamine neurons and cause Parkinson's symptoms in mice. In the experiment, after 2 weeks of treatment with MPTP in mice (a in Figure 5), the mice moved slowly and trembled. After 4 weeks of treatment with the mitochondrial transplantation treatment system of the present invention (obtained by assembling mitochondria derived from mouse fibroblasts with mouse red blood cells according to the method of Example 1), the mice were euthanized, and the brain tissue was fixed and sliced. The expression of tyrosine hydroxylase (TH) in the substantia nigra region and the striatum region of the midbrain was detected to characterize the repair of TH-positive neurons and evaluate the therapeutic effect of the mitochondrial transplantation treatment system of the present invention. As shown in b in Figure 5, after injection of MPTP, the number of TH-containing neuronal cells in the substantia nigra region and the striatum region decreased significantly. After treatment with the mitochondrial transplantation treatment system of the present invention, the number of dopaminergic neuronal cells containing TH in the substantia nigra region and the striatum region was restored. Brain tissue was taken from the substantia nigra, and TH expression was detected after protein extraction. It can be clearly seen that TH expression decreased significantly after the addition of MPTP. After treatment with the mitochondrial transplantation therapy system, TH expression was significantly upregulated (Figure 5c, d), and the secretion of tyrosine hydroxylase by dopaminergic neurons was restored.

[0152] In order to evaluate whether the motor ability of Parkinson's mice has been restored, the open field and climbing pole experiments were used to analyze the activities of the mice. The results showed that the activity distance and the number of times the Parkinson's mice entered the central area were significantly reduced, and the mice had a certain degree of depression; after treatment with the mitochondrial transplantation treatment system disclosed in the present invention, the motor ability of the mice improved (e, f in Figure 5). As shown in Figure 5 g, after MPTP treatment, the time taken by the mice to climb from the top of the climbing pole to the bottom was significantly increased. After treatment with the mitochondrial transplantation treatment system disclosed in the present invention, the time taken by the mice was significantly shortened, and the motor ability was relatively well restored. In summary, the mitochondrial transplantation treatment system can alleviate the symptoms of Parkinson's mice and has a certain therapeutic effect.

[0153] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic expressions 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 any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0154] Although embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations of the present disclosure. A person skilled in the art may change, modify, replace, and vary the above embodiments within the scope of the present disclosure.

Claims

1. A mitochondrial transplantation treatment system, wherein, Comprising: Isolated mitochondria; 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, wherein, The mitochondria are isolated from mammalian cells.

4. A drug, wherein, 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 metabolic 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, 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 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 auditory 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 kidney function damage includes at least one of Fanconi syndrome, renal tubular acidosis, glomerulosclerosis, renal failure, and adrenocortical 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 senility, 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 organs include at least one of the heart, liver, lung, and kidney.

10. A method for obtaining heteroplasmic mitochondrial hybrid cells, wherein, 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 the heterologous mitochondria contained in the mitochondrial transplantation treatment system enter the recipient cells to obtain heterologous mitochondrial hybrid cells.

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

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

13. A method for treating and / or repairing diseases associated with mitochondrial damage or mitochondrial dysfunction, wherein, The method includes: Administering to a subject the mitochondrial transplantation treatment system according to any one of claims 1-3 and / or the drug according to claim 4 of the drug.

14. The method according to claim 13, wherein The mitochondrial damage includes mitochondrial function damage and mtDNA mutation; Optionally, the mitochondrial function damage includes mitochondrial structure damage and mitochondrial metabolic damage; optionally, the mtDNA damage includes mtDNA point mutation and mtDNA deletion mutation; Optionally, the mitochondrial dysfunction is caused by mitochondrial respiratory chain damage, abnormal proteins encoded by nuclear genes, and physicochemical property changes; Optionally, the mitochondrial dysfunction-related diseases 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 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 injury includes at least one of Fanconi syndrome, renal tubular acidosis, glomerulosclerosis, renal failure, and adrenal cortical insufficiency; Optionally, the pancreatic function injury includes at least one of diabetes and pancreatitis; Optionally, the gastrointestinal function injury includes at least one of pseudo-obstruction and gastrointestinal motility disorder; Optionally, the metabolic disease includes at least one of senescence, obesity, hyperglycemia, dyslipidemia, insulin resistance, and cardiovascular disease; Optionally, the reproductive disease includes at least one of premature ovarian failure and male infertility; Optionally, the skeletal lesion includes at least one of kyphoscoliosis, dwarfism, and bone marrow failure.

15. Use of the mitochondrial transplantation treatment system according to any one of claims 1-3 and the drug according to claim 4 in the treatment and / or repair of diseases related to mitochondrial injury or mitochondrial dysfunction.

16. The use according to claim 15, wherein, The mitochondrial injury includes mitochondrial function injury and mtDNA mutation; Optionally, the mitochondrial function injury includes mitochondrial structural injury and mitochondrial metabolic injury; Optionally, the mtDNA injury includes mtDNA point mutation and mtDNA deletion mutation; Optionally, the mitochondrial dysfunction is caused by mitochondrial respiratory chain injury, 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 nerve diseases, muscle diseases, heart function injury, liver function injury, kidney function injury, pancreatic function injury, gastrointestinal function injury, metabolic diseases, reproductive diseases, and skeletal lesions caused by mitochondrial dysfunction; Optionally, the neurological diseases include at least one of neuronal developmental delay, 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 nerve diseases include 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 injury includes at least one of cardiomyopathy and myocardial conduction defect; Optionally, the liver function injury includes at least one of liver failure, liver injury, fatty liver, and cirrhosis; Optionally, the liver function injury includes at least one of Fanconi syndrome, renal tubular acidosis, glomerulosclerosis, renal failure, and adrenal cortical insufficiency; Optionally, the pancreatic function injury includes at least one of diabetes and pancreatitis; Optionally, the gastrointestinal function injury includes at least one of pseudo-obstruction and gastrointestinal motility disorder; Optionally, the metabolic disease includes at least one of senescence, obesity, hyperglycemia, dyslipidemia, insulin resistance, and cardiovascular disease; Optionally, the reproductive disease includes at least one of premature ovarian failure and male infertility; Optionally, the bone lesion includes at least one of kyphoscoliosis, dwarfism, and bone marrow failure.

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