Method for treating lupus nephritis by using exosome and related product thereof

Abstract

The invention discloses a method for treating lupus nephritis by using exosomes and related products thereof, new application of iPSC exosomes and iMac exosomes as drugs for treating lupus nephritis is discovered and confirmed for the first time, and the iPSC exosomes and iMac exosomes can significantly inhibit renal fibrosis, renal inflammatory response and renal pathological glycogen deposition, and can be used for treating lupus nephritis. The compound has an excellent treatment effect on the lupus nephritis, can be used for preparing the medicine for treating the lupus nephritis, provides a new direction for research and development of the medicine for treating the lupus nephritis, and has a wide application prospect in the field of treatment of the lupus nephritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a method for treating lupus nephritis using exosomes and related products. Background Art

[0002] Lupus nephritis (LN) is one of the most common and serious complications of systemic lupus erythematosus (SLE), mainly manifested as an immune inflammatory response in the kidney. The etiology of LN is complex, involving genetics, environmental factors and abnormal activation of the immune system, which leads to autoimmune reactions attacking kidney tissue and then causing nephritis. The clinical manifestations of LN include proteinuria, hematuria, hypertension and renal insufficiency, and in severe cases can lead to end-stage renal disease.

[0003] At present, the treatment of LN mainly relies on glucocorticoids and immunosuppressants, such as cyclophosphamide and mycophenolate mofetil. Although these treatments can control the condition of LN to a certain extent, there are many problems with long-term use, such as side effects of hormones, increased risk of infection due to immunosuppression, drug resistance and disease recurrence. In addition, the existing treatments are not effective for some patients, and a considerable number of LN patients still develop end-stage renal disease.

[0004] In recent years, with the development of cell biology and molecular biology, exosomes, as a new type of intercellular communication tool, have shown great potential in the field of disease treatment. Exosomes are tiny vesicles with a diameter of about 30-150 nanometers, which can be secreted by a variety of cells and carry a variety of biological molecules such as proteins, RNA, and DNA. Studies have found that exosomes play an important role in immune regulation, tissue repair, tumor treatment, and many other aspects.

[0005] Studies have shown that MSCs exosomes play a role by delivering anti-inflammatory factors, regulating immune cell function, and promoting tissue repair. However, the specific methods and clinical applications of exosomes in the treatment of LN are still unclear and require further research and development. Therefore, in view of the limitations of existing LN treatment methods, it is particularly important to find a safer, more effective treatment method with fewer side effects. Summary of the invention

[0006] In order to overcome the technical problem that the existing methods for treating lupus nephritis are limited, the present invention aims to provide a method for treating lupus nephritis using exosomes, in order to provide a new treatment option for patients with lupus nephritis.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] A first aspect of the present invention provides use of iPSC-derived macrophage exosomes and / or iPSC cell exosomes in the preparation of a medicament for treating and / or preventing lupus nephritis.

[0009] Furthermore, the iPSC-derived macrophage exosomes or iPSC cell exosomes are collected by any one or more of the following methods: hollow fiber column filtration, differential centrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, polymer co-precipitation, immunoaffinity technology, and microfluidic separation technology.

[0010] In the present invention, the iPSC or iPSC-derived macrophages can be prepared by conventional preparation methods known to those skilled in the art, or purchased through conventional purchasing channels. The present invention has no particular limitation on the specific source of the iPSC or iPSC-derived macrophages.

[0011] In some embodiments, the present invention has no particular restrictions on the method for collecting the iPSC-derived macrophage exosomes or iPSC cell exosomes, and any method for collecting the iPSC-derived macrophage exosomes or iPSC cell exosomes from the iPSC-derived macrophage supernatant or iPSC supernatant falls within the protection scope of the present invention.

[0012] In some embodiments, the hollow fiber column filtration method is a commonly used method for collecting exosomes, which specifically includes the following steps: hollow fiber column treatment, hollow fiber column balancing, exosome concentration, replacement, and harvesting. The hollow fiber column filtration method can effectively remove impurities and obtain relatively pure exosomes through multi-step filtration and chromatography. Compared with other methods, the hollow fiber column filtration method is relatively simple to operate and is suitable for routine laboratory operations.

[0013] In some embodiments, the differential centrifugation method is one of the most common exosome separation techniques. This method uses the different sedimentation coefficients of exosomes and other organelles to separate cells, cell fragments and other organelles at different centrifugal speeds to obtain pure exosomes. The differential centrifugation method has the advantages of simple extraction method, no other markers are introduced during the extraction process, and is suitable for large-dose sample processing.

[0014] In some embodiments, the density gradient centrifugation is an exosome extraction method that combines ultracentrifugation with sucrose density gradient, utilizing the density range of 1.13 g / mL-1.21 g / mL of exosomes to achieve separation of exosomes from non-vesicle particles (such as proteins, protein / RNA aggregates, etc.), thereby being able to extract low-content exosomes.

[0015] In some embodiments, the size exclusion chromatography method uses porous gel balls to purify exosomes by molecular size. During the separation and extraction process, large molecules cannot enter the gel pores and are quickly eluted by the mobile phase along the gaps between the porous gels, while small molecules can enter the gel pores, have a longer retention time, and are eluted more slowly. The molecular size of exosomes is larger than that of general protein molecules, so exosomes will be eluted first, and proteins with small particle sizes will be eluted later, thereby separating high-purity exosomes.

[0016] In some embodiments, the ultrafiltration method is to filter the solvent and small molecules to the other side of the membrane, while retaining relatively large molecules on the ultrafiltration membrane to achieve the purpose of separation. The diameter of exosomes ranges from 30-150nm, and the exosomes in the sample can be separated by long-term low-speed centrifugation using an ultrafiltration membrane. The ultrafiltration method has the advantages of easy operation, relatively high enrichment efficiency, easy to match with other membranes or operations, and low cost.

[0017] In some embodiments, the polymer co-precipitation method uses a high molecular weight hydrophobic polymer (such as polyethylene glycol (PEG)) to change the solubility and dispersibility of exosomes, so that they can be precipitated under relatively low centrifugal force.

[0018] In some embodiments, the immunoaffinity technique is to enrich exosomes using antibodies that recognize exosome-specific markers.

[0019] In some embodiments, the microfluidic separation technology is an integrated technology combining exosome separation and analysis, and is suitable for rapid separation and characterization analysis of small amounts of body fluids.

[0020] In the present invention, the treatment and / or prevention refers to the medical management of a patient with the purpose of curing, improving, stabilizing or preventing a disease, pathological condition or condition. The term includes active therapy, i.e., treatment specifically for the purpose of improving a disease, pathological condition or condition, and also includes etiological treatment, i.e., treatment for the purpose of removing the cause of the relevant disease, pathological condition or condition.

[0021] In addition, the treatment and / or prevention also includes palliative treatment, i.e. treatment designed to relieve symptoms rather than cure a disease, pathological condition or condition; the term also includes preventive treatment, i.e. treatment aimed at minimizing or partially or completely inhibiting the development of the relevant disease, pathological condition or condition; and supportive treatment, i.e. treatment used to supplement another specific therapy aimed at improving the relevant disease, pathological condition or condition. In a specific embodiment of the present invention, the disease is particularly lupus nephritis.

[0022] Furthermore, the iPSC-derived macrophage exosomes or iPSC cell exosomes are collected by hollow fiber column filtration;

[0023] Optionally, the hollow fiber column filtration method comprises the following steps: hollow fiber column treatment, hollow fiber column balancing, exosome concentration, replacement, and harvesting.

[0024] Further, the hollow fiber column treatment includes flushing the hollow fiber column with sterile water;

[0025] Optionally, the balancing of the hollow fiber column comprises flushing the hollow fiber column with physiological saline;

[0026] Optionally, the exosome concentration comprises pressurizing the collected iPSC-derived macrophage supernatant or iPSC supernatant through a hollow fiber column to obtain a concentrated exosome solution;

[0027] Optionally, the replacement includes replacing the concentrated exosome solution with physiological saline to obtain replaced physiological saline;

[0028] Optionally, the harvesting includes circulating the replaced physiological saline in a hollow fiber column, and the obtained circulating fluid is the iPSC-derived macrophage exosomes or iPSC cell exosomes;

[0029] Optionally, the conditions for exosome concentration are inlet pressure 0.5 bar, outlet pressure ≤ 0.5 bar, and flow rate ≤ 100 mL / min;

[0030] Optionally, the replacement condition is 10 times the volume of normal saline, and the transmembrane pressure is ≤0.2 bar;

[0031] Optionally, the harvesting condition is that the cycle time is ≥5 min.

[0032] A second aspect of the present invention provides a pharmaceutical composition for treating and / or preventing lupus nephritis.

[0033] Furthermore, the pharmaceutical composition comprises the iPSC-derived macrophage exosomes and / or iPSC cell exosomes described in the first aspect of the present invention.

[0034] In some embodiments, the pharmaceutical composition may further comprise other second therapeutic drugs that can be used to treat and / or prevent lupus nephritis, and / or second therapeutic drugs that can be used to assist in the treatment and / or prevention of lupus nephritis.

[0035] In some embodiments, the other second therapeutic drugs that can be used to treat and / or prevent lupus nephritis, and / or second therapeutic drugs for adjuvant treatment and / or prevention of lupus nephritis include but are not limited to: hydroxychloroquine sulfate, prednisone, methylprednisolone, cyclophosphamide, tacrolimus, mycophenolate mofetil, azathioprine, leflunomide, tripterygium wilfordii, immunoglobulin, and rituximab.

[0036] In some embodiments, the second therapeutic drug is not particularly limited, and any drug that can be used to treat and / or prevent lupus nephritis, and / or any drug for auxiliary treatment and / or prevention of lupus nephritis falls within the scope of the second therapeutic drug of the present invention.

[0037] In the case where the present invention has clearly confirmed through experiments that the iPSC-derived macrophage exosomes and / or iPSC cell exosomes described in the first aspect of the present invention have a therapeutic effect on lupus nephritis, those skilled in the art may combine the iPSC-derived macrophage exosomes and / or iPSC cell exosomes with other drugs for the treatment of lupus nephritis according to actual needs. Therefore, using only iPSC-derived macrophage exosomes and / or iPSC cell exosomes as active ingredients or using iPSC-derived macrophage exosomes and / or iPSC cell exosomes in combination with the other drugs as active ingredients in the preparation of drugs for the treatment of lupus nephritis will fall within the scope of protection of the present invention.

[0038] In some embodiments, the pharmaceutical composition can be prepared according to any preparation method known in the art. For this purpose, if necessary, the iPSC-derived macrophage exosomes and / or iPSC cell exosomes can be combined with one or more solid or liquid excipients to prepare a suitable administration form or dosage form for human use.

[0039] A third aspect of the present invention provides a pharmaceutical preparation for treating and / or preventing lupus nephritis.

[0040] Furthermore, the pharmaceutical preparation comprises the iPSC-derived macrophage exosomes and / or iPSC cell exosomes described in the first aspect of the present invention.

[0041] Furthermore, the dosage forms of the pharmaceutical preparation include in situ injection preparations, intravenous reinfusion preparations, intramuscular injection preparations, capsule preparations, oral preparations, sustained-release preparations, granule preparations, lyophilized powder injections, soluble powder injections, tablets or pills.

[0042] In some embodiments, the pharmaceutical composition or pharmaceutical formulation may further comprise a pharmaceutically acceptable carrier and / or excipient.

[0043] In some embodiments, in order to prepare the unit dosage form containing the iPSC-derived macrophage exosomes and / or iPSC cell exosomes into an injectable preparation, such as a solution, emulsion, lyophilized powder injection and suspension, all diluents commonly used in the art can be used, including but not limited to: water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injectable preparation. In addition, conventional cosolvents, buffers, pH regulators, etc. can also be added.

[0044] In some embodiments, colorants, preservatives, spices, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations if necessary.

[0045] In some embodiments, the dosage of the pharmaceutical composition or pharmaceutical preparation of the present invention depends on many factors, such as the nature and severity of the disease to be treated and / or prevented (in the present invention, particularly lupus nephritis), the sex, age, weight and individual response of the patient or animal, the route of administration and the number of administrations, etc. The above dosage can be administered in a single dosage form or divided into several, such as two, three or four dosage forms.

[0046] In some embodiments, a variety of prescriptions may be made depending on factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, diet, administration time, administration route, excretion rate and sensitivity, and a skilled physician can usually easily determine the prescription and the dosage that is effective for the desired treatment and / or prevention.

[0047] The fourth aspect of the present invention provides a method for preparing the iPSC-derived macrophage exosomes and / or iPSC cell exosomes described in the first aspect of the present invention.

[0048] Further, the method comprises the following steps: hollow fiber column treatment, hollow fiber column balancing, exosome concentration, replacement, and harvesting;

[0049] Optionally, the hollow fiber column treatment includes flushing the hollow fiber column with sterile water;

[0050] Optionally, the balancing of the hollow fiber column comprises flushing the hollow fiber column with physiological saline;

[0051] Optionally, the exosome concentration comprises pressurizing the collected iPSC-derived macrophage supernatant or iPSC supernatant through a hollow fiber column to obtain a concentrated exosome solution;

[0052] Optionally, the replacement includes replacing the concentrated exosome solution with physiological saline to obtain replaced physiological saline;

[0053] Optionally, the harvesting includes circulating the replaced physiological saline in a hollow fiber column, and the obtained circulating fluid is the iPSC-derived macrophage exosomes or iPSC cell exosomes;

[0054] Optionally, the conditions for exosome concentration are inlet pressure 0.5 bar, outlet pressure ≤ 0.5 bar, and flow rate ≤ 100 mL / min;

[0055] Optionally, the replacement condition is 10 times the volume of normal saline, and the transmembrane pressure is ≤0.2 bar;

[0056] Optionally, the harvesting condition is that the cycle time is ≥5 min.

[0057] In addition, the present invention also provides a method for treating and / or preventing lupus nephritis in a subject in need thereof, the method comprising: administering to a subject in need thereof a therapeutically and / or preventively effective amount of the iPSC-derived macrophage exosomes and / or iPSC cell exosomes described in the first aspect of the present invention, the pharmaceutical composition described in the second aspect of the present invention, or the pharmaceutical preparation described in the third aspect of the present invention.

[0058] In some embodiments, the route of administration includes, but is not limited to, topical, intravenous, intramuscular, subcutaneous, oral, intraperitoneal, intradermal, intranasal, intrapulmonary, intrarectal, etc. In a specific embodiment, the dosage that is beneficial to the subject can be determined by a clinician based on factors such as the type, age, weight, general disease condition, and administration method of the subject for administration.

[0059] In the present invention, the effective amount refers to an amount having a therapeutic effect or an amount required to produce a therapeutic effect in a treatment subject. For example, a drug therapeutically or pharmaceutically effective amount refers to the amount of a drug required to produce a desired therapeutic effect, and the therapeutic effect can be reflected by the results of clinical trials, model animal studies and / or in vitro studies. The pharmaceutically effective amount depends on several factors, including but not limited to: characteristic factors of the treatment subject (such as height, weight, gender, age and medication history), and the severity of the disease.

[0060] In the present invention, the subject refers to any animal, including humans and non-human animals. The non-human animals include all vertebrates, for example, mammals, such as non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a specific embodiment of the present invention, the subject is preferably a human.

[0061] A fifth aspect of the present invention provides an intelligent diagnosis and treatment system for lupus nephritis based on Internet medical services, the system comprising:

[0062] A diagnostic element information collector, used to collect diagnostic element information of lupus nephritis related to the patient and send the collected data results to the disease diagnosis matcher;

[0063] A disease diagnosis matcher, used to match the diagnosis element information of the diagnosis element information collector with the disease type, and send the matched data results to the treatment plan matcher;

[0064] A treatment plan matcher, used to determine the matching result of the disease diagnosis matcher and call the treatment plan according to the matching result, and then send the treatment plan data result to the disease diagnosis and treatment information outputter;

[0065] A disease diagnosis and treatment information output device, used to output the data results received from the disease diagnosis matcher and the treatment plan matcher to a receiving unit;

[0066] The lupus nephritis diagnostic elements include urine routine examination data, blood routine examination data, renal function examination data, and renal puncture examination data;

[0067] In the disease diagnosis matcher, if the urine routine test data shows positive urine protein, the blood routine test data shows leukopenia and thrombocytopenia, the renal function test data shows renal changes, and the renal puncture test data shows renal tubular deformation, necrosis, vascular wall thickening, renal interstitial fibrosis, and active inflammation, the patient is matched as a lupus nephritis patient;

[0068] In the disease diagnosis matcher, if the urine routine test data shows that urine protein is negative, the blood routine test data shows that there is no abnormality, the renal function test data shows that there is no abnormality, and the renal puncture test data shows that there is no abnormality, the patient to be diagnosed is matched as a patient with non-lupus nephritis;

[0069] In the treatment plan matcher, if the disease diagnosis matcher matches the patient to be diagnosed as a patient with lupus nephritis, the following treatment plan is matched and called: administering a therapeutically effective amount of the iPSC-derived macrophage exosomes and / or iPSC cell exosomes described in the first aspect of the present invention to the subject;

[0070] In the treatment regimen matcher, if the disease diagnosis matcher matches the patient as a non-lupus nephritis patient, there is no need to adopt the following treatment regimen: administering a therapeutically effective amount of the iPSC-derived macrophage exosomes and / or iPSC cell exosomes described in the first aspect of the present invention to the subject.

[0071] Furthermore, the receiving unit is a display screen, a computer client, a mobile phone client or a tablet.

[0072] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0073] The present invention discovers and confirms for the first time the new use of iPSC exosomes and iMac exosomes as drugs for treating lupus nephritis. iPSC exosomes and iMac exosomes can significantly inhibit renal fibrosis, inhibit renal inflammatory response, and inhibit renal pathological glycogen deposition. They have excellent therapeutic effects on lupus nephritis and can be used in the preparation of drugs for treating lupus nephritis. The present invention provides a new direction for the research and development of drugs for treating lupus nephritis and has broad application prospects in the field of lupus nephritis treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 This is the result of the detection of albumin and creatinine in mouse urine;

[0075] Figure 2 This is a graph showing the results of the IL-4 and IL-2 content test in mouse blood;

[0076] Figure 3 This is the result of PAS staining of mouse kidney;

[0077] Figure 4 This is the Masson staining result of mouse kidney;

[0078] Figure 5 This is the result of HE staining of mouse kidney;

[0079] Figure 6 This is the result graph corresponding to Austin score of mouse kidney;

[0080] Figure 7 A schematic diagram of an intelligent diagnosis and treatment system for lupus nephritis based on Internet medical services provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0081] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0082] The reagents and raw materials used in the present invention are easily obtained by ordinary technicians in the field and can be obtained from commercial channels unless otherwise specified. The experimental methods in the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way.

[0083] Example 1 Preparation of iPSC-Exosomes and iMac-Exosomes

[0084] 1. Experimental Materials

[0085] Hollow fiber column (manufacturer: Ripleyne, catalog number S02-E300-05-N).

[0086] 2. Experimental methods

[0087] (1) iPSC-differentiated macrophages (iMac)

[0088] Macrophages were prepared from iPSCs using the preparation method in patent CN115433715B previously applied for by the applicant of the present application.

[0089] (2) Preparation of iPSC-Exosomes and iMac-Exosomes (iPSC-Exosomes are used as an example here)

[0090] 1) Hollow fiber column treatment

[0091] Rinse the hollow fiber column with 1 L of sterile water, and then rinse with 1 L of 0.5 M NaOH for storage.

[0092] 2) Hollow fiber column balance

[0093] Rinse the hollow fiber column with 1 L of normal saline.

[0094] 3) Exosome Concentration

[0095] The supernatant of the collected iPSCs (prepared by the method of the prior patent CN113462638B of the present applicant) was all pressurized to pass through the hollow fiber column, the inlet pressure was controlled at 0.5 bar, the outlet pressure was controlled below 0.5 bar, and the flow rate did not exceed 100 mL / min.

[0096] 4) Replacement

[0097] After deconcentration, the concentrated exosome solution was replaced with 10 times the volume of normal saline, and the transmembrane pressure was controlled below 0.2 bar.

[0098] 5) Harvest

[0099] The replaced saline solution was circulated in the hollow fiber column for more than 5 minutes, and then the circulating fluid was collected in a collection bottle for later use.

[0100] The preparation method of iMac exosomes is the same as the preparation method of iPSC exosomes mentioned above.

[0101] Example 2 In vivo treatment of lupus nephritis with the exosomes prepared in Example 1

[0102] 1. Experimental Materials

[0103] 10-week-old MRL / Lpr female mice (purchased from Yusibeifu (Beijing) Biotechnology Co., Ltd.), mouse urine albumin ELISA detection kit (manufacturer: BioAssay, catalog number DICT-500), mouse urine creatinine ELISA detection kit (manufacturer: Zhongbei Green, catalog number E-90A), mouse blood IL-4 ELISA detection kit (manufacturer: Qisong Biological, catalog number 96T-QS42901), mouse blood IL-2 ELISA detection kit (manufacturer: Qisong Biological, catalog number 96T-QS42903), PAS staining kit (Manufacturer: Biyuntian, catalog number C0142S), MASSON staining kit (Manufacturer: Solebow, catalog number G1346), HE staining kit (Manufacturer: Solebow, catalog number G1121), Telitacicept (Manufacturer: MCE, catalog number HY-P99568), iPSC-derived macrophage exosomes (iMac-Exosome, prepared in Example 1), iPSC cell exosomes (iMSC-Exosome, prepared in Example 1), PBS (Manufacturer: Hyclone, catalog number SH30256.01).

[0104] 2. Experimental methods

[0105] 1) Grouped medication

[0106] Twenty-four mice were randomly divided into control group, positive drug group, iMac-Exosome group and iPSC-Exosome group, with 6 mice in each group. The control group was injected with 200μL of PBS through the tail vein; the positive drug group was injected with 200μL of Telitacicept, 15mg / kg through the tail vein; the iMac-Exosome group was injected with 200μL of iPSC-derived macrophage exosomes, containing 40 billion particles; the iPSC-Exosome group was injected with 200μL of iPSC exosomes, containing 40 billion particles. Each group was given the drug once a week for a total of 10 weeks.

[0107] 2) Urine index test

[0108] Urine was collected from each mouse at the 5th and 10th weeks, and the albumin and creatinine content in the urine of each mouse was measured according to the instructions of the mouse urine albumin ELISA detection kit and the mouse urine creatinine ELISA detection kit.

[0109] 3) Blood index testing

[0110] In the 10th week, blood was collected from the eye sockets of each mouse, and the levels of IL-4 and IL-2 in the blood of each mouse were measured according to the instructions of the mouse blood IL-4 ELISA detection kit and the mouse blood IL-2 ELISA detection kit.

[0111] 4) Mouse kidney histochemical staining

[0112] At the 10th week, the mice were euthanized, and the kidneys of each mouse were embedded and fixed. The largest cross-sectional slices of the mouse kidneys were cut, and the slice thickness was 4 μm. The kidney paraffin sections of each mouse were then stained and analyzed using the PAS staining kit, MASSON staining kit, and HE staining kit.

[0113] 3. Experimental results

[0114] 1) Urine index detection

[0115] The albumin and creatinine in the urine of each group of mice were detected, and it was found that after five weeks of medication, there was no significant difference in the albumin and creatinine content in the urine of each group of mice compared with the control group. However, after 10 weeks of medication, the albumin and creatinine content in the urine of mice in the positive drug group and the two exosome groups decreased significantly compared with the control group, indicating that the two exosomes prepared in Example 1 of the present invention have significant therapeutic effects on lupus nephritis ( Figure 1 ).

[0116] 2) Blood index test

[0117] The IL-4 and IL-2 in the blood of mice in each group were tested respectively, and it was found that after 10 weeks of medication, the IL-4 content in the blood of mice in each group decreased significantly compared with the control group, and the IL-2 content in the blood of mice in each group increased significantly. In addition, compared with the positive drug group, the decrease in IL-4 and the increase in IL-2 in the blood of mice in the two exosome groups were more significant ( Figure 2 ). Because IL-4 is proportional to the severity of lupus nephritis, IL-2 has an inhibitory effect on lupus nephritis. Therefore, the experimental results show that the two exosomes have a significantly stronger therapeutic effect on lupus nephritis than the positive drug. This technical effect of the two exosomes prepared in Example 1 of the present invention is a technical effect that is unexpected by those skilled in the art based on the prior art.

[0118] 3) Histochemical staining

[0119] PAS staining results of the paraffin sections of the kidneys of mice in each group showed that a large amount of glycogen accumulated in the kidneys of mice in the control group ( Figure 3 , red arrows), but glycogen deposition was significantly reduced in the positive drug and two exosome groups ( Figure 3 ).

[0120] Lupus nephritis can also cause renal fibrosis, so Masson staining was performed on the kidney sections. The results showed that the kidneys of the control group mice showed obvious and more blue staining (black arrows), indicating more severe fibrosis. In comparison, the blue staining of the kidneys of the positive drug group and the two exosome-treated mice was significantly reduced ( Figure 4 ). This indicates that the two exosomes prepared in Example 1 of the present invention can effectively inhibit renal fibrosis.

[0121] To further evaluate the severity of nephritis in mice, we performed HE staining on the sections of mice. The results showed that the kidneys of mice in the control group showed obvious inflammatory reactions, including lymphocyte infiltration (purple arrows), connective tissue hyperplasia (orange arrows), epithelial cell water lesions (green arrows), glomerular mesangial matrix hyperplasia (dark green), protein casts (light blue), increased cells in capillaries (yellow), cell fragmentation (dark red), crescents (brown), uniform acidic substances (dark blue), and renal tubular atrophy (white); while the exosome treatment group only showed partial and small amounts of inflammatory reactions ( Figure 5 ). This further illustrates that the two exosomes prepared in Example 1 of the present invention can effectively treat lupus nephritis.

[0122] 4) Austin Rating

[0123] Scoring was performed based on PAS, HE, and Masson results. The results showed that the Austin score of the kidneys of mice in the exosome group was significantly lower than that of the control group. In addition, the exosome group also showed a trend of lower scores compared with the positive drug ( Figure 6 ), the above results once again proved the effectiveness of the two exosomes prepared in Example 1 of the present invention in the treatment of lupus nephritis, and their therapeutic effect on lupus nephritis is better than that of the positive drug Telitacicept.

[0124] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0125] In some of the processes described in the specification and claims of the present invention and the above-mentioned drawings, multiple operations appearing in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this document or may be executed in parallel. The serial numbers of the operations, such as 101, 102, 103, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel.

[0126] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0127] Figure 7 Schematic diagram of an intelligent diagnosis and treatment system for lupus nephritis based on Internet medical services provided by an embodiment of the present invention. Specifically, the system includes:

[0128] 101: a diagnostic element information collector, used to collect diagnostic element information of lupus nephritis related to the patient and send the collected data results to a disease diagnosis matcher;

[0129] 102: a disease diagnosis matcher, used to match the diagnosis element information of the diagnosis element information collector with the disease type, and send the matched data results to a treatment plan matcher;

[0130] 103: a treatment plan matcher, used to determine the matching result of the disease diagnosis matcher and call the treatment plan according to the matching result, and then send the treatment plan data result to the disease diagnosis and treatment information output device;

[0131] 104: a disease diagnosis and treatment information output device, used to output the data results received from the disease diagnosis matcher and the treatment plan matcher to a receiving unit;

[0132] The lupus nephritis diagnostic elements include urine routine examination data, blood routine examination data, renal function examination data, and renal puncture examination data;

[0133] In the disease diagnosis matcher, if the urine routine test data shows positive urine protein, the blood routine test data shows leukopenia and thrombocytopenia, the renal function test data shows renal changes, and the renal puncture test data shows renal tubular deformation, necrosis, vascular wall thickening, renal interstitial fibrosis, and active inflammation, the patient is matched as a lupus nephritis patient;

[0134] In the disease diagnosis matcher, if the urine routine test data shows that urine protein is negative, the blood routine test data shows that there is no abnormality, the renal function test data shows that there is no abnormality, and the renal puncture test data shows that there is no abnormality, the patient to be diagnosed is matched as a patient with non-lupus nephritis;

[0135] In the treatment plan matcher, if the disease diagnosis matcher matches the patient to be diagnosed as a patient with lupus nephritis, the following treatment plan is matched and called: administering a therapeutically effective amount of the iPSC-derived macrophage exosomes and / or iPSC cell exosomes of the present invention as described above to the subject;

[0136] In the treatment plan matcher, if the disease diagnosis matcher matches the patient as a non-lupus nephritis patient, there is no need to adopt the following treatment plan: administering a therapeutically effective amount of the iPSC-derived macrophage exosomes and / or iPSC cell exosomes of the present invention as described above to the subject.

[0137] In one embodiment, the receiving unit is a display screen, a computer client, a mobile phone client or a tablet.

[0138] In one embodiment, the disease categories include lupus nephritis and non-lupus nephritis.

[0139] In one embodiment, the present invention detects the albumin and creatinine in the urine of mice in the control group, positive drug group, iMac-Exosome group and iPSC-Exosome group as described above. The results show that after five weeks of medication, there is no significant difference in the albumin and creatinine content in the urine of mice in each group compared with the control. However, after 10 weeks of medication, the content of albumin and creatinine in the urine of mice in the positive drug group and the two exosome groups decreased significantly compared with the control group, indicating that the two exosomes prepared in Example 1 of the present invention have significant therapeutic effects on lupus nephritis ( Figure 1 ).

[0140] In one embodiment, the present invention detects IL-4 and IL-2 in the blood of mice in the control group, positive drug group, iMac-Exosome group and iPSC-Exosome group as described above. The results show that after 10 weeks of medication, the IL-4 content in the blood of mice in each group decreased significantly compared with the control, and the IL-2 content in the blood of mice increased significantly. In addition, compared with the positive drug group, the decrease in IL-4 and the increase in IL-2 in the blood of mice in the two exosome groups were more significant ( Figure 2 ).

[0141] In one embodiment, the present invention respectively performed PAS staining of the kidney paraffin sections of mice in the control group, positive drug group, iMac-Exosome group and iPSC-Exosome group as described above, and the results showed that a large amount of glycogen accumulation appeared in the kidneys of the mice in the control group ( Figure 3 , red arrows), but glycogen deposition was significantly reduced in the positive drug and two exosome groups ( Figure 3 ).

[0142] In one embodiment, the present invention performed Masson staining on kidney sections. The results showed that the kidneys of the mice in the control group showed obvious and more blue staining (black arrows), that is, more severe fibrosis occurred. In comparison, the blue staining of the kidneys of the mice in the positive drug group and the two exosomes-treated mice was significantly reduced ( Figure 4 ). This indicates that the two exosomes prepared in Example 1 of the present invention can effectively inhibit renal fibrosis.

[0143] In one embodiment, the present invention further performed HE staining on the sections of mice. The results showed that the kidneys of mice in the control group showed obvious inflammatory reactions, including lymphocyte infiltration (purple arrows), connective tissue hyperplasia (orange arrows), epithelial cell water lesions (green arrows), glomerular mesangial matrix hyperplasia (dark green), protein casts (light blue), increased cells in capillaries (yellow), cell fragmentation (dark red), crescents (brown), uniform acidic substances (dark blue), and renal tubular atrophy (white); while the exosome treatment group only showed partial and small amounts of inflammatory reactions ( Figure 5 ). This further illustrates that the two exosomes prepared in Example 1 of the present invention can effectively treat lupus nephritis.

[0144] In one embodiment, the present invention is scored according to the results of PAS, HE, and Masson. The results show that the Austin score of the kidney of mice in the exosome group is significantly lower than that of the control group; in addition, the exosome group also shows a trend of lower scores compared with the positive drug ( Figure 6 ).

[0145] The above results demonstrate the effectiveness of the two types of exosomes, iPSC-derived macrophage exosomes and / or iPSC cell exosomes, in the treatment of lupus nephritis as described in the aforementioned system, and that their therapeutic effect on lupus nephritis is better than that of the positive drug Telitacicept, which proves the clinical feasibility and effectiveness of the above-mentioned intelligent diagnosis and treatment system for lupus nephritis based on Internet medical services.

[0146] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0147] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0148] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0149] In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units.

[0150] A person of ordinary skill in the art can understand that all or part of the steps in the various methods of the above embodiments can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium, and the storage medium may include: a read-only memory (ROM), a random access memory (RAM), a disk or an optical disk, etc.

[0151] A person skilled in the art can understand that all or part of the steps in the above-mentioned embodiment method can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium. The above-mentioned storage medium can be a read-only memory, a disk or an optical disk, etc.

[0152] The above is a detailed introduction to a computer device provided by the present invention. For a person skilled in the art, according to the concept of the embodiments of the present invention, there may be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. Use of iPSC-derived macrophage exosomes and / or iPSC cell exosomes in the preparation of a drug for treating and / or preventing lupus nephritis.

2. The use according to claim 1, characterized in that: The iPSC-derived macrophage exosomes or iPSC cell exosomes are collected by any one or more of the following methods: hollow fiber column filtration, differential centrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, polymer co-precipitation, immunoaffinity technology, and microfluidic separation technology.

3. The use according to claim 1, characterized in that: The iPSC-derived macrophage exosomes or iPSC cell exosomes are collected by hollow fiber column filtration method; Optionally, the hollow fiber column filtration method comprises the following steps: hollow fiber column treatment, hollow fiber column balancing, exosome concentration, replacement, and harvesting.

4. The use according to claim 3, characterized in that: The hollow fiber column treatment comprises flushing the hollow fiber column with sterile water; Optionally, the balancing of the hollow fiber column comprises flushing the hollow fiber column with physiological saline; Optionally, the exosome concentration comprises pressurizing the collected iPSC-derived macrophage supernatant or iPSC supernatant through a hollow fiber column to obtain a concentrated exosome solution; Optionally, the replacement includes replacing the concentrated exosome solution with physiological saline to obtain replaced physiological saline; Optionally, the harvesting includes circulating the replaced physiological saline in a hollow fiber column, and the obtained circulating fluid is the iPSC-derived macrophage exosomes or iPSC cell exosomes; Optionally, the conditions for exosome concentration are inlet pressure 0.5 bar, outlet pressure ≤ 0.5 bar, and flow rate ≤ 100 mL / min; Optionally, the replacement condition is 10 times the volume of normal saline, and the transmembrane pressure is ≤0.2 bar; Optionally, the harvesting condition is that the cycle time is ≥5 min.

5. A pharmaceutical composition for treating and / or preventing lupus nephritis, characterized in that: The pharmaceutical composition comprises the iPSC-derived macrophage exosomes and / or iPSC cell exosomes as described in any one of claims 1 to 4.

6. A pharmaceutical preparation for treating and / or preventing lupus nephritis, characterized in that: The pharmaceutical preparation comprises the iPSC-derived macrophage exosomes and / or iPSC cell exosomes as described in any one of claims 1 to 4.

7. The pharmaceutical preparation according to claim 6, characterized in that The dosage forms of the pharmaceutical preparation include in situ injection preparations, intravenous reinfusion preparations, intramuscular injection preparations, capsule preparations, oral preparations, sustained-release preparations, granule preparations, lyophilized powder injections, soluble powder injections, tablets or pills.

8. A method for preparing the iPSC-derived macrophage exosomes and / or iPSC cell exosomes as claimed in any one of claims 1 to 4, characterized in that: The method comprises the following steps: hollow fiber column treatment, hollow fiber column balancing, exosome concentration, replacement, and harvesting; Optionally, the hollow fiber column treatment includes flushing the hollow fiber column with sterile water; Optionally, the balancing of the hollow fiber column comprises flushing the hollow fiber column with physiological saline; Optionally, the exosome concentration comprises pressurizing the collected iPSC-derived macrophage supernatant or iPSC supernatant through a hollow fiber column to obtain a concentrated exosome solution; Optionally, the replacement includes replacing the concentrated exosome solution with physiological saline to obtain replaced physiological saline; Optionally, the harvesting includes circulating the replaced physiological saline in a hollow fiber column, and the obtained circulating fluid is the iPSC-derived macrophage exosomes or iPSC cell exosomes; Optionally, the conditions for exosome concentration are inlet pressure 0.5 bar, outlet pressure ≤ 0.5 bar, and flow rate ≤ 100 mL / min; Optionally, the replacement condition is 10 times the volume of normal saline, and the transmembrane pressure is ≤0.2 bar; Optionally, the harvesting condition is that the cycle time is ≥5 min.

9. An intelligent diagnosis and treatment system for lupus nephritis based on Internet medical services, characterized in that: The system comprises: A diagnostic element information collector, used to collect diagnostic element information of lupus nephritis related to the patient and send the collected data results to the disease diagnosis matcher; A disease diagnosis matcher, used to match the diagnosis element information of the diagnosis element information collector with the disease type, and send the matched data results to the treatment plan matcher; A treatment plan matcher, used to determine the matching result of the disease diagnosis matcher and call the treatment plan according to the matching result, and then send the treatment plan data result to the disease diagnosis and treatment information outputter; A disease diagnosis and treatment information output device, used to output the data results received from the disease diagnosis matcher and the treatment plan matcher to a receiving unit; The lupus nephritis diagnostic elements include urine routine examination data, blood routine examination data, renal function examination data, and renal puncture examination data; In the disease diagnosis matcher, if the urine routine test data shows positive urine protein, the blood routine test data shows leukopenia and thrombocytopenia, the renal function test data shows renal changes, and the renal puncture test data shows renal tubular deformation, necrosis, vascular wall thickening, renal interstitial fibrosis, and active inflammation, the patient is matched as a lupus nephritis patient; In the disease diagnosis matcher, if the urine routine test data shows that urine protein is negative, the blood routine test data shows that there is no abnormality, the renal function test data shows that there is no abnormality, and the renal puncture test data shows that there is no abnormality, the patient to be diagnosed is matched as a patient with non-lupus nephritis; In the treatment plan matcher, if the disease diagnosis matcher matches the patient to be diagnosed as a patient with lupus nephritis, the following treatment plan is matched and called: administering a therapeutically effective amount of the iPSC-derived macrophage exosomes and / or iPSC cell exosomes described in any one of claims 1 to 4 to the subject; In the treatment regimen matcher, if the disease diagnosis matcher matches the patient as a non-lupus nephritis patient, there is no need to adopt the following treatment regimen: administering a therapeutically effective amount of the iPSC-derived macrophage exosomes and / or iPSC cell exosomes described in any one of claims 1 to 4 to the subject.

10. The intelligent diagnosis and treatment system for lupus nephritis based on Internet medical services according to claim 9 is characterized in that: The receiving unit is a display screen, a computer client, a mobile phone client or a tablet.

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