A photosynthetic cyanobacterium-mitochondrial metabolic coupling mitochondrial transplantation system, a preparation method and application thereof

Abstract

The application relates to a photosynthetic cyanobacterium-mitochondria metabolic coupling mitochondrial transplantation system, a preparation method and application thereof, and relates to the medical field.The application aims to solve the technical bottleneck that the existing mitochondrial transplantation technology has a short survival time of transplanted mitochondria, low functional activity and insufficient transplantation efficiency in the treatment of myocardial I / R injury, and simultaneously overcomes the inherent contradiction between'survival' and 'high-efficiency work'. Therefore, the application provides a photosynthetic cyanobacterium-mitochondria metabolic coupling mitochondrial transplantation system, a preparation method and application thereof, realizes accurate, efficient and long-acting treatment of myocardial I / R injury by constructing an in-situ bidirectional metabolic closed loop, and expands the application scene of the system in other mitochondrial dysfunction related diseases, thereby providing a new technical scheme for clinical treatment.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering, synthetic biology, organelle transplantation and regenerative medicine, and in particular to a photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system, and the application of this system in mitochondrial transplantation, especially in the treatment of myocardial ischemia-reperfusion (I / R) injury, and can also be extended to the treatment of other mitochondrial dysfunction-related diseases. Background Technology

[0002] Myocardial ischemia-reperfusion (I / R) injury is a core bottleneck hindering the prognosis of patients with acute ischemic heart disease (IHD) after reperfusion therapy. Taking myocardial ischemia-reperfusion injury as an example, its essence lies in energy metabolism disorders and oxidative stress damage caused by mitochondrial dysfunction. Currently, there are still no radical interventions to cure energy metabolism defects. Mitochondria are the cell's "energy factories," responsible for ATP production, and their dysfunction is the core pathological mechanism of myocardial I / R injury. Therefore, targeted repair of mitochondrial function and restoration of myocardial energy metabolism homeostasis are key breakthroughs in improving myocardial I / R injury.

[0003] Mitochondrial transplantation (MT), as an emerging targeted repair strategy, involves transplanting healthy mitochondria into damaged cardiomyocytes to replace dysfunctional mitochondria and restore energy metabolism. It has shown significant clinical potential in the treatment of myocardial I / R injury, and multiple clinical trials at home and abroad have verified its safety and efficacy.

[0004] However, current MT technology still faces two major challenges:

[0005] 1. Short survival time of transplanted mitochondria: As organelles without cell membrane protection, mitochondria have a short lifespan after being isolated in vitro. Their respiratory function is significantly lost after about 2 hours, and their survival time is even shorter in the pathological environment of high calcium and high reactive oxygen species (ROS) in the I / R transplantation area.

[0006] 2. Low functional activity and insufficient transplantation efficiency: Transplanted mitochondria have extremely low ATP synthesis efficiency, making it difficult to achieve long-term therapeutic benefits and often requiring multiple infusions.

[0007] Furthermore, current technology fails to resolve an inherent contradiction between "survival" and "efficient operation": for mitochondria to efficiently synthesize ATP, they must consume oxygen at a high rate, accompanied by the production of toxic ROS, which in turn damages their own structure and function, leading to a shortened lifespan. Moreover,

[0008] Therefore, developing a mitochondrial transplantation system that can maintain mitochondrial survival for a long time and drive its efficient operation in the I / R damage microenvironment is a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0009] This invention aims to address the technical bottlenecks of existing mitochondrial transplantation techniques in the treatment of myocardial ischemia / reperfusion injury (I / R) damage, namely, short survival time, low functional activity, and insufficient transplantation efficiency of transplanted mitochondria, while overcoming the inherent contradiction between "survival" and "efficient operation." To this end, this invention provides a photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system. By constructing an in-situ bidirectional metabolic closed loop, it achieves precise, efficient, and long-term treatment of myocardial I / R injury. Furthermore, it provides a method for preparing this system and its applications, expanding its application scenarios in other mitochondrial dysfunction-related diseases and providing a novel technical solution for clinical treatment.

[0010] This invention discloses a photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system, wherein the system comprises cyanobacteria and mitochondria.

[0011] And the carrier composition.

[0012] Furthermore, the carrier is a hydrogel, and the cyanobacteria is a cyanobacterium that can perform photosynthesis under far-red light at 700-800 nm.

[0013] Furthermore, the concentration of mitochondria is 0.02-0.5 mg / mL, and the concentration of cyanobacteria is 10 mg / mL. 4 -10 7 per mL.

[0014] A method for preparing a photosynthetic cyanobacteria-mitochondrial metabolism-coupled mitochondrial transplantation system according to the present invention:

[0015] 1) Prepare suspensions of cyanobacteria and mitochondria separately;

[0016] 2) After mixing the cyanobacterial suspension and mitochondrial suspension with the carrier, the photosynthetic cyanobacterial-mitochondrial metabolic coupling mitochondrial transplantation system is obtained.

[0017] Furthermore, the cyanobacteria suspension is prepared as follows:

[0018] Cyanobacteria were placed in a culture medium and cultured until the logarithmic growth phase. The cyanobacterial cells were collected by centrifugation and washed to obtain the culture medium. The culture medium formula is as follows: NaNO3: 1.5 g / L, K2HPO4: 40 mg / L, MgSO4·7H2O: 75 mg / L, CaCl2·2H2O: 36 mg / L, citric acid: 6 mg / L, ferric ammonium citrate: 6 mg / L, Na2EDTA: 1 mg / L, Na2CO3: 20 mg / L, H3BO3: 2.86 mg / L, MnCl2·4H2O: 1.81 mg / L, ZnSO4·7H2O: 0.22 mg / L, Na2MoO4·2H2O: 0.39 mg / L, CuSO4·5H2O: 0.08 mg / L, and Co(NO3)2·6H2O: 0.05 mg / L. The pH of the culture medium is 7~7.5.

[0019] Furthermore, the mitochondrial suspension refers to the mitochondrial suspension obtained by resuspending mitochondria extracted from cells or tissues in a mitochondrial separation buffer; the mitochondrial separation buffer is composed of 250 mM sucrose, 15 mM KCl, 30 mM K2HPO2 and 5 mM MgCl2, and the pH of the mitochondrial separation buffer is 7~7.5.

[0020] Furthermore, the process of mixing the cyanobacterial suspension and mitochondrial suspension with the carrier to form the shape refers to:

[0021] After mixing the cyanobacterial suspension and mitochondrial suspension with hydrogel, a pregel mixture was obtained; it was then extruded into injectable microsphere gel, cured, and washed to obtain the photosynthetic cyanobacterial-mitochondrial metabolic coupling mitochondrial transplantation system.

[0022] Furthermore, the carrier is a hydrogel, which is prepared by mixing sodium alginate solution and gelatin solution in equal proportions.

[0023] The present invention relates to an application of a photosynthetic cyanobacteria-mitochondrial metabolism-coupled mitochondrial transplantation system, wherein the system is used to prepare drugs for treating mitochondrial dysfunction diseases.

[0024] Furthermore, the system is used to prepare drugs for treating myocardial ischemia-reperfusion injury.

[0025] This invention does not simply combine cyanobacteria with mitochondria, but rather, for the first time, constructs a bidirectional metabolic coupling closed-loop system. It overcomes the following technical challenges inherent in the combination of cyanobacteria and mitochondria:

[0026] Technical challenge 1: After mitochondria are isolated, they lack the protection of the cell membrane, resulting in a continuous decrease in membrane potential and reduced activity. Figure 3Technical challenge two: The normal in vitro culture conditions for cyanobacteria are 30℃ BG11 medium, and the mitochondrial survival environment is 4℃ (maintained).

[0027] Store at 37℃ (working temperature), using mitochondrial buffer. Maintaining homeostasis in the microenvironments of cyanobacteria and mitochondria is a challenge.

[0028] Technical challenge three: The hydrogenase (HydA) in cyanobacteria that catalyzes hydrogen production is extremely sensitive to oxygen. Under high oxygen partial pressure: hydrogenase...

[0029] Activity is reversibly inhibited, and electrons flow to the CO2 fixation pathway. Under low oxygen partial pressure, hydrogenase is activated, and electrons flow to the H2 production pathway. This is the first time a self-regulating positive feedback loop of "mitochondrial oxygen consumption → local hypoxia → hydrogenase activation → hydrogen production protection" has been constructed. This achieves the principle of "the more efficient the operation, the stronger the protection."

[0030] Compared with existing mitochondrial transplantation technology, the present invention has the following significant advantages and beneficial effects:

[0031] 1. Significantly prolongs the active lifespan of mitochondria

[0032] The system of this invention protects mitochondrial functional activity through continuous hydrogen production by cyanobacteria. Experiments show that, under hydrogel encapsulation and cyanobacteria co-culture conditions, the duration of mitochondrial functional activity is extended from the conventional 2 hours to 72 hours.

[0033] 2. Significantly enhances ATP synthesis capacity

[0034] Cyanobacteria photosynthetic oxygen production provides mitochondria with sufficient electron acceptors, which can increase ATP production by about 8 times compared to naked mitochondria, significantly better than the existing Alda-1 drug treatment regimen (which only increases it by about 3 times).

[0035] 3. Reduce the need for multiple infusions

[0036] Because mitochondrial activity is significantly prolonged, a single administration of this system can achieve sustained release and continuous functional support for more than 72 hours, improving myocardial damage caused by myocardial ischemia-reperfusion for 72 hours in mice and miniature pigs, improving cardiac function, reducing infarct area, and avoiding the problem of multiple infusions within 24 hours due to rapid mitochondrial inactivation in existing technologies, thus improving treatment compliance and safety.

[0037] 4. Provides energy and provides antioxidant protection to the myocardium in a synergistic manner.

[0038] Transcriptomic analysis confirmed that this therapeutic system can effectively restore mitochondrial energy homeostasis, inhibit inflammation-related signaling pathways, and reduce cell apoptosis. Its cardioprotective mechanism can be attributed to the system providing oxygen through photosynthesis and synergistically exerting a cardioprotective effect through hydrogen-mediated antioxidant activity. Attached Figure Description

[0039] Figure 1 : A schematic diagram of the photosynthetic cyanobacteria-mitochondrial metabolic coupling system (Mito-9212-SA) of this invention;

[0040] Figure 2 : A graph showing the hydrogen and oxygen production of cyanobacteria under the action of this system;

[0041] Figure 3 Comparison of the effects of the system of this invention and free mitochondria;

[0042] Figure 4 : The therapeutic effect of the system of the present invention in a mouse I / R model;

[0043] Figure 5 : The therapeutic effect of the system of this invention in a miniature pig I / R model;

[0044] Figure 6 : A diagram illustrating the therapeutic mechanism of the system of the present invention in a mouse I / R model;

[0045] Figure 7 : Diagram illustrating the treatment mechanism of the system of the present invention in a miniature pig I / R model. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0047] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0048] 1. Extraction and functional modification of mitochondria

[0049] (1) Extraction of mitochondria: The collected cell suspension was centrifuged at 800 r / min for 3 min, resuspended in pre-cooled PBS, and a small sample was taken for counting. The remaining cell suspension was centrifuged and precipitated, and pre-cooled mitochondrial separation reagent was added. The cells were gently resuspended and placed on ice for 12 min. After incubation, the cell suspension was transferred to a glass homogenizer and homogenized at a constant speed for about 30 times to break the membrane and release the mitochondria from the cells to obtain a cell homogenate. The cell homogenate was centrifuged at 600 g and 4℃ for 10 min to remove unbroken cells and cell nuclei. The supernatant was centrifuged at 11000 g and 4℃ for 10 min. The precipitate at the bottom of the tube was the cell mitochondria. The mitochondria were resuspended in buffer (250 mM sucrose, 15 mM KCl, 30 mM K2HPO2, 5 mM MgCl2, pH 7.4), and then the protein concentration was determined.

[0050] (2) Extraction of mitochondria from tissue: A small piece of tissue was cut, washed once with PBS, and cut into very fine fragments with scissors. Trypsin was added, and the tissue was incubated on ice for 20 min. The trypsin was then discarded, and mitochondrial separation reagent was added. The tissue was homogenized on ice, and the homogenate was centrifuged at 600 g at 4°C for 5 min. The supernatant was centrifuged at 11000 g at 4°C for 10 min. The precipitate was the mitochondria. The mitochondria were resuspended in buffer, and then the protein concentration was determined.

[0051] (3) Mitochondrial protein concentration determination: The mitochondrial protein content was determined using the BCA protein quantification kit. BCA solution A and solution B (50:1) were mixed according to the kit ratio to prepare the BCA working solution. 20 μL of mitochondrial-containing buffer was added to each well of a 96-well plate, followed by 200 μL of BCA working solution, and the mixture was gently shaken to mix. The samples were incubated at 37°C in the dark for 30 min. The absorbance was then measured at 562 nm using a microplate reader, and the mitochondrial protein concentration was calculated using the standard curve method.

[0052] (4) Mitochondrial functionalization modification: Mitochondria were modified with the cell-penetrating peptide Pep-1 to promote the internalization of exogenous mitochondria. Pep-1 (KETWTWTWWTEWSQPKKKRKV cysteine) is an amphiphilic peptide with three domains: a hydrophobic tryptophan-rich motif, a hydrophilic lysine-rich domain (KKKRKV), and a spacer domain (SQP). Pep-1 can efficiently deliver mitochondria into cells through electrostatic and hydrophobic contact with the cell membrane. 105 μg of mitochondria was diluted to 200 μL in PBS, and an equal volume of Pep-1 diluted with water (0.06 mg) was added. The mixture was incubated at room temperature for 20 min to form a composite assembly.

[0053] 2. In vitro culture of cyanobacteria

[0054] Single colonies of cyanobacteria were inoculated into 50 mL of BG11 liquid medium for activation culture. The medium formulation was as follows: NaNO3: 1.5 g / L, K2HPO4: 40 mg / L, MgSO4·7H2O: 75 mg / L, CaCl2·2H2O: 36 mg / L, Citric acid: 6 mg / L, Ammonium ferric citrate: 6 mg / L, Na2EDTA: 1 mg / L, Na2CO3: 20 mg / L, trace elements: H3BO3 2.86 mg / L, MnCl2·4H2O 1.81 mg / L, ZnSO4·7H2O 0.22 mg / L, Na2MoO4·2H2O 0.39 mg / L, CuSO4·5H2O 0.08 mg / L, Co(NO3)2·6H2O 0.05 mg / L, pH... 7.1. The culture conditions were set as follows: a constant temperature shaker at 30℃, a shaking speed of 120 r / min, and full-spectrum illumination (light intensity 100 LUX).

[0055] 3. Preparation of hydrogel carriers

[0056] Weigh sodium alginate (SA) and gelatin (Gel) into 50 mL of deionized water. Stir magnetically in a 37°C water bath until SA and Gel are completely dissolved to prepare precursor solutions of different concentrations. Mix mitochondria (0.5 mg / mL) with cyanobacteria (10 mg / mL). 7 The precursor solution (number of spheres / mL) was resuspended in the precursor solution. Using a 1 mL sterile syringe, the precursor solution was added dropwise to a 0.1 mol / L CaCl2 crosslinking solution, controlling the droplet height to obtain uniform spherical shape. After crosslinking for 8-10 min, the hydrogel spheres were collected with sterile forceps and washed three times with buffer.

[0057] 4. System in vitro functional verification

[0058] (1) Mitochondrial morphology identification: Mitochondrial mitochondria were stained using a fluorescent probe with mitochondrial-specific staining, Mito-Tracker Red CMXROS. The principle is that Mito-Tracker Red CMXROS is a rhodamine derivative with a weak positive charge. It can passively diffuse into the mitochondrial matrix through the negative potential gradient (Δψm) of the inner mitochondrial membrane. When the mitochondrial membrane potential is high, the probe accumulates in the mitochondria and forms a stable fluorescent signal.

[0059] The mitochondria were resuspended in the diluted probe working solution and incubated at 37°C for 30 min. After incubation, the mitochondria were washed 2-3 times with PBS to remove unbound probes and observed under a fluorescence microscope.

[0060] (2) Mitochondrial ATP content determination: The ATP content determination kit was used to detect the mitochondrial ATP production capacity. The principle is that luciferase catalyzes the oxidation of luciferin in the presence of ATP, producing a fluorescent signal. The fluorescence intensity is linearly related to the ATP concentration. The ATP content can be calculated by referring to the standard curve. Prepare the working solution according to the kit ratio, and use it immediately. Mix the sample and working solution at a ratio of 1:10, incubate at room temperature in the dark for 15 min, and detect the fluorescence intensity using a fluorescence microplate reader at a wavelength of 582 nm. Calculate the ATP content.

[0061] (3) Detection of exogenous mitochondrial internalization: Exogenous mitochondria were added to Mito Tracker Red CMXROS working solution and incubated at 37°C in the dark for 30 min. After incubation, the cells were centrifuged at 11000 g for 10 min at 4°C, and the supernatant was carefully aspirated. The precipitate was washed three times with fresh buffer to remove unbound dye. Native mitochondria in HL-1 cells were stained with Mito Tracker Green FM working solution and incubated at 37°C in the dark for 30 min. After incubation, the cells were washed three times with buffer to remove unbound dye. The exogenous mitochondria were resuspended in complete culture medium, and then the mitochondrial suspension and HL-1 cells were incubated together in a humidified incubator at 37°C and 5% CO2 for 2 h. The internalization of mitochondria was then observed using a fluorescence microscope.

[0062] (4) Measurement of mitochondrial hydrodynamic diameter and Zeta potential: The hydrodynamic diameter of mitochondria was measured by dynamic light scattering (DLS). The principle is to calculate the diffusion coefficient of the particles by detecting the scattered light fluctuations caused by the Brownian motion of the particles in the solution, and then converting the hydrodynamic diameter. Transfer the sample to a clean quartz cuvette, then place it in the particle size analyzer, turn on the instrument, and calibrate the instrument according to the instruction manual. Start the particle size analysis program, select the continuous particle size measurement mode, and obtain data such as the average particle size and particle size distribution of mitochondria. Transfer the sample to the sample cell with electrodes, start the particle size analysis program, select the continuous potential measurement mode, and obtain data such as the mitochondrial Zeta potential.

[0063] (5) Detection of oxygen and hydrogen production by cyanobacteria: Algal cells of a certain concentration were transferred to 100 mL conical flasks, and the changes in hydrogen and oxygen concentrations in the samples over time were monitored using a hydrogen detector (AP-B.H2-F; range 1000 ppm, resolution 1 ppm) and a dissolved oxygen meter (Mettler Toledo).

[0064] Experimental results

[0065] Observation of Mito-Tracker Red-labeled mitochondria under a fluorescence microscope, along with DLS and Zeta potentials, confirmed that Pep-1 successfully modified mitochondria and promoted internalization efficiency. Figure 3 The metabolic coupling system constructed in this invention has the ability to produce hydrogen and oxygen. Figure 2 PCC.9212 can sustain 8 times the ATP synthesis capacity for up to 48 hours (10). 7 The study demonstrated that cyanobacteria-mitochondrial metabolic coupling effects were extended to 72 hours, significantly outperforming previously reported Alda-1 drug treatment regimens (which only improved activity by approximately 3-fold). Furthermore, incorporating 0.5 μg / mL of UCNPs into the system under near-infrared light (980 nm) excitation achieved equivalent or even superior cyanobacterial-mitochondrial metabolic coupling effects, overcoming the limitation of insufficient light penetration depth in deep tissues.

[0066] 5. In vivo treatment of myocardial ischemia in animal models

[0067] (1) Experimental grouping: C57BL / 6J mice (male, 6-8 weeks old) and Bama miniature pigs (male, 4 months old) were randomly divided into normal sham operation group (Sham), myocardial ischemia-reperfusion group (I / R), mitochondrial transplantation group (Mito), mitochondrial hydrogel transplantation group (Mito-SA) and cyanobacteria-mitochondrial hydrogel transplantation group (Mito-9212-SA). They were fed in a normal environment for one week before the operation.

[0068] (2) Construction of myocardial ischemia-reperfusion model: Mouse model construction: The weight of mice was accurately measured by electronic balance. The mice were anesthetized by intraperitoneal injection of tribromoethanol working solution (250 mg / kg). After the righting reflex disappeared, the hair in the sternal area was treated with depilatory cream and the skin of the surgical area was disinfected with 75% ethanol solution. The mice were fixed in a supine position on a constant temperature operating table. The endotracheal tube was inserted through the mouth and the small animal ventilator was connected to establish mechanical ventilation. The effectiveness of ventilation was evaluated by observing the rhythmic rise and fall of the chest before entering the surgical stage. The 3rd to 4th intercostal space on the left chest was disinfected again. The skin was cut open and the pectoral muscles and intercostal muscles were bluntly separated layer by layer. A mini thoracotomy device was inserted to expose the pericardium. The pericardium was lifted with micro forceps and the pericardium was cut open with ophthalmic scissors. The left anterior descending coronary artery (LAD) was carefully separated. The LAD was ligated by 8-0 needle suture for 30 min to induce acute myocardial ischemia. The model was confirmed to be successful by observing whether the area below the ligation line turned white.

[0069] Pig model establishment: Male Bama miniature pigs (25–30 kg, 4 months old) were selected. After weighing, anesthesia was induced by intramuscular injection. The pigs were transported to the operating table and fixed in a supine position with limbs immobilized and both sides of the chest fully exposed. A cannula was inserted through the marginal ear vein, and compound sodium chloride injection (10 ml / kg / L) was injected intravenously. Tracheal intubation was performed, and positive pressure mechanical ventilation was connected. The respiratory rate was 25 breaths / min, the inspiratory-to-expiratory ratio was 1:2, and the tidal volume was 8–10 mL / kg. Anesthesia was maintained by inhalation of 2% isoflurane. The thoracotomy was performed through the 4th intercostal incision on the left chest wall or through a median sternal incision (1 cm above the sternal angle and 1 cm below the xiphoid process, with an incision length of 8–10 cm). cm, cut the sternum to the second intercostal space with an electric saw (keeping the manubrium of the sternum continuous), retract the sternum with a thoracotomy instrument, open the pericardium and suspend it, expose the heart and locate the LAD, bluntly dissect the artery and vein, pass through the LAD with a 5-0 suture below the origin of the first oblique branch, with a needle depth of 1–1.5 mm, ligate the LAD for 30 minutes, observe the color of the myocardium below the ligation line to confirm successful ischemia; loosen the ligation line during reperfusion, close the chest layer by layer, continuously observe vital signs such as spontaneous breathing and pharyngeal reflex after surgery, remove the endotracheal tube after suctioning, and administer intramuscular penicillin within 3 days after surgery to prevent infection.

[0070] (3) Experimental interventions: The sham-operated group underwent open-chest surgery without ligation of the LAD; the I / R group received an injection of PBS solution before reperfusion; the Mito group received an injection of exogenous mitochondria; the Mito-SA group received an injection of a mixture of exogenous mitochondria and hydrogel; and the Mito-9212-SA group received an injection of a mixture of exogenous mitochondria, PCC 9212, and hydrogel. All were injected at multiple points at the edge of the ischemic area of ​​the myocardium using a 30 G 1 mL insulin needle. The injection volume for mice was 45 μL, and the injection volume for Bama miniature pigs was 200 μL.

[0071] Functional testing:

[0072] (1) Echocardiography: Cardiac function was assessed using an M-mode Doppler ultrasound machine before surgery, 24 h after reperfusion, and 3 d after reperfusion. The experimental animals were anesthetized and fixed in a supine position. After skin preparation of the chest, an appropriate amount of coupling gel was applied, and the probe was placed at the left sternal border to observe cardiac structure and myocardial motion. Cardiac function was then measured using M-mode Doppler ultrasound in the long-axis view of the left ventricle, and the images were recorded. Ejection fraction (EF) and systolic fraction (FS) were analyzed.

[0073] (2) Measurement of myocardial infarction area: To assess the degree of myocardial injury, the infarction area was measured using TTC staining. The heart was quickly removed, rinsed with physiological saline, and immediately frozen at -80°C for 30 min. The frozen heart was cut from the apex of the left ventricle to the base, approximately 4-5 short-axis sections were prepared, and incubated at 37°C in 1% TTC solution in the dark for 30 min, ensuring that each myocardial section was in full contact with the TTC staining solution. After removal, the sections were fixed in 4% paraformaldehyde solution. The staining was observed; the infarcted area was white, and the non-infarcted area was red. Images were captured using a digital camera.

[0074] (3) Myocardial transcriptome sequencing: Myocardial tissue was placed in pre-cooled RNase-free cryovials, flash-frozen in liquid nitrogen, and sent to Lianchuan Biotechnology Co., Ltd. for transcriptome sequencing analysis. Samples that passed quality inspection were tested on the Illumina Novaseq 6000 sequencing platform. Differentially regulated genes between groups were screened using DESeq2 software, and GO and KEGG functional enrichment analyses were performed on upregulated and downregulated differentially regulated genes, respectively.

[0075] Experimental results

[0076] In mice ( Figure 4 ) and miniature pigs ( Figure 5 In the model, the system of this invention significantly improves cardiac function, reduces the infarct area after I / R (infarction / reperfusion injury), and effectively alleviates abnormal biochemical changes in serum caused by myocardial I / R injury. By restoring mitochondrial energy metabolism, blocking inflammatory and apoptotic signals, correcting abnormal cell cycle activity, and promoting tissue repair, it effectively achieves myocardial protection. Figure 6 , 7 ).

Claims

1. A photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system, characterized in that, The system consists of cyanobacteria, mitochondria, and a carrier.

2. The photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system according to claim 1, characterized in that, The carrier is a hydrogel, and the cyanobacteria are cyanobacteria that can perform photosynthesis under far-red light at 700-800 nm.

3. The photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system according to claim 1, characterized in that, The concentration of mitochondria was 0.02-0.5 mg / mL, and the concentration of cyanobacteria was 10 mg / mL. 4 -10 7 per mL.

4. A method for preparing a photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system according to any one of claims 1 to 3, characterized in that, The system is prepared according to the following method: 1) Prepare suspensions of cyanobacteria and mitochondria separately; 2) After mixing the cyanobacterial suspension and mitochondrial suspension with the carrier, the photosynthetic cyanobacterial-mitochondrial metabolic coupling mitochondrial transplantation system is obtained.

5. The method for preparing a photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system according to claim 4, characterized in that, The cyanobacteria suspension was prepared as follows: Cyanobacteria were placed in a culture medium and cultured until the logarithmic growth phase. The cyanobacterial cells were collected by centrifugation and washed to obtain the culture medium. The culture medium formula is as follows: NaNO3: 1.5 g / L, K2HPO4: 40 mg / L, MgSO4·7H2O: 75 mg / L, CaCl2·2H2O: 36 mg / L, citric acid: 6 mg / L, ferric ammonium citrate: 6 mg / L, Na2EDTA: 1 mg / L, Na2CO3: 20 mg / L, H3BO3: 2.86 mg / L, MnCl2·4H2O: 1.81 mg / L, ZnSO4·7H2O: 0.22 mg / L, Na2MoO4·2H2O: 0.39 mg / L, CuSO4·5H2O: 0.08 mg / L, and Co(NO3)2·6H2O: 0.05 mg / L. The pH of the culture medium is 7~7.

5.

6. The method for preparing a photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system according to claim 4, characterized in that, The mitochondrial suspension refers to the mitochondria extracted from cells or tissues and resuspended in a mitochondrial separation buffer. The mitochondrial separation buffer is composed of 250 mM sucrose, 15 mM KCl, 30 mM K2HPO2 and 5 mM MgCl2, and the pH of the mitochondrial separation buffer is 7~7.

5.

7. The method for preparing a photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system according to claim 4, characterized in that, The process of mixing cyanobacterial suspension, mitochondrial suspension, and carrier to form a shape refers to: After mixing the cyanobacterial suspension and mitochondrial suspension with hydrogel, a pregel mixture was obtained; it was then extruded into injectable microsphere gel, cured, and washed to obtain the photosynthetic cyanobacterial-mitochondrial metabolic coupling mitochondrial transplantation system.

8. A method for preparing a photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system according to claim 4 or 7, characterized in that, The carrier is a hydrogel, which is prepared by mixing sodium alginate solution and gelatin solution in equal proportions.

9. The application of the photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system as described in any one of claims 1 to 3, characterized in that, The system is used to prepare drugs for treating mitochondrial dysfunction.

10. The application of the photosynthetic cyanobacteria-mitochondrial metabolic coupling mitochondrial transplantation system according to claim 9, characterized in that, The system is used to prepare drugs for the treatment of myocardial ischemia-reperfusion injury.

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