Coprophilous fungus transplantation combined preparation and method for combined treatment of intestinal diseases by combining coprophilous fungus transplantation with miR-146b

Abstract

The invention belongs to the technical field of coprophilous fungus transplantation, and particularly relates to a coprophilous fungus transplantation combined preparation and a method for combined treatment of intestinal diseases by combining coprophilous fungus transplantation with miR-146b. The coprophilous fungi transplantation combined preparation disclosed by the invention comprises coprophilous fungi and miR-146b. The invention creatively provides a coprophilous fungus transplantation and miR-146b combined therapy, and the coprophilous fungus transplantation and miR-146b combined therapy can achieve the following beneficial effects: the flora colonization rate is increased: the host intestinal microenvironment is adjusted through miR-146b, and the colonization capability of transplanted flora is enhanced; the miR-146b can inhibit the transcriptional activity of pathogenic bacteria (such as enterobacteriaceae) in a targeted manner, and can promote the functional expression of anti-inflammatory bacteria at the same time; and the safety risk is reduced: the proliferation of potential pathogenic bacteria in transplanted flora can be inhibited by miR-146b, and the adverse reaction is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of fecal microbiota transplantation, and particularly relates to a combined preparation for fecal microbiota transplantation and a method for combined treatment of intestinal diseases by fecal microbiota transplantation combined with miR-146b. Background Art

[0002] Fecal Microbiota Transplantation (FMT) is a treatment method that reconstructs the intestinal microecological balance by transplanting the intestinal flora of healthy donors into the patient's body. At present, FMT has been widely used in the treatment of recurrent Clostridium difficile infection (rCDI) and gradually extended to the clinical research of diseases such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). However, the existing FMT technology has the following significant defects:

[0003] (1) Unstable efficacy: Clinical data show that the remission rate of FMT for IBD is only 30-50%, and there are significant individual differences. The reason is that the colonization of transplanted flora is affected by the host intestinal microenvironment, and some beneficial bacteria are difficult to survive in the inflammatory environment;

[0004] (2) Insufficient regulation of flora function:

[0005] Traditional FMT only relies on the overall transplantation of flora and lacks precise regulation of the functions of specific flora. For example, the activity of anti-inflammatory flora (such as Prevotella) may be inhibited by the host inflammatory state;

[0006] (3) Safety risks:

[0007] Unmodified fecal bacteria may carry potential pathogenic bacteria or pro-inflammatory strains, resulting in post-transplant infection or aggravated inflammation. The existing technology reduces the risk through flora filtration, but cannot solve the functional defects.

[0008] MicroRNA (miRNA) is a class of endogenous small RNAs with a length of about 20-24 nucleotides, which have various important regulatory functions in cells. Each miRNA can have multiple target genes, and several miRNAs can also regulate the same gene. This complex regulatory network can either regulate the expression of multiple genes through one miRNA or finely regulate the expression of a certain gene through the combination of several miRNAs. It is speculated that miRNA regulates one-third of human genes. Recent studies have found that host-derived microRNA (such as miR-146b) can regulate the transcriptional activity of intestinal flora through extracellular vesicles (EVs). However, there are the following problems when using miRNA alone:

[0009] (1) Low delivery efficiency: Synthetic miRNA mimics are easily degraded in the intestine and difficult to target specific flora;

[0010] (2) Single function: Although iR-146b can inhibit the activity of pro-inflammatory bacteria (such as Escherichia coli), it cannot comprehensively reconstruct the diversity of the flora. Summary of the Invention

[0011] To solve the above technical problems existing in the prior art, the present invention proposes a combined therapy of fecal microbiota transplantation combined with miR-146b, aiming to achieve the following beneficial effects: improving the colonization rate of the flora: regulating the host intestinal microenvironment through miR-146b to enhance the colonization ability of the transplanted flora; precisely regulating the function of the flora: miR-146b targets and inhibits the transcriptional activity of pathogenic bacteria (such as Enterobacteriaceae), and at the same time promotes the functional expression of anti-inflammatory bacteria; reducing the safety risk: miR-146b can inhibit the proliferation of potential pathogenic bacteria in the transplanted flora and reduce adverse reactions.

[0012] The technical solution of the present invention is realized as follows:

[0013] One of the purposes of the present invention is to disclose a combined preparation for fecal microbiota transplantation, and the preparation includes fecal microbiota and miR-146b.

[0014] Preferably, in the combined preparation for fecal microbiota transplantation, the concentration of miR-146b is: 0.1 μg / mL to 100 μg / mL; the concentration of fecal microbiota is 108 - 109 cfu / ml.

[0015] Another purpose of the present invention is to disclose a preparation method of the above combined preparation for fecal microbiota transplantation, including the following steps:

[0016] (1) Preparation of fecal microbiota suspension

[0017] Donor screening: Healthy donors need to meet the international FMT standards (such as no infectious diseases, metabolic diseases, history of antibiotic use, etc.); after the fecal samples are collected, they should be processed or cryopreserved (-80 °C) within 2 hours;

[0018] Preparation of fecal microbiota suspension: Take fecal samples (1 - 50 g) and mix them with sterile physiological saline or PBS buffer (50 - 500 mL), stir and homogenize; filter through 100 μm and 70 μm filters in sequence to remove large particle residues; centrifuge (3000 - 8000 rpm, 5 - 20 min) to collect the bacterial cell precipitate, and resuspend it in a sterile buffer (such as PBS containing 10% glycerol);

[0019] Optional step: Freeze-dry or microencapsulation treatment to improve stability;

[0020] (2) Addition and optimization of miR-146b

[0021] Source of miR-146b:

[0022] Synthesis of miR-146b mimic: Chemical modification to improve stability (such as 2'-O-methylation modification);

[0023] Expression of miR-146b in engineered bacteria: Engineered bacteria that stably secrete miR-146b;

[0024] Methods of adding miR-146b:

[0025] Direct mixing: Mix miR-146b mimic (0.1 - 100 μg / mL) with fecal bacteria suspension;

[0026] Co-transplantation of engineered bacteria: Mix engineered bacteria expressing miR-146b (10 6 -10 10 CFU / mL) with fecal bacteria suspension in a ratio of 1:1 to 1:10.

[0027] Preferably, the concentration of miR-146b is preferably 1 - 20 μg / mL, and the optimal range is 5 - 10 μg / mL.

[0028] Preferably, the ratio of the engineered bacteria to the fecal bacteria is preferably 1:5.

[0029] The third object of the present invention is to disclose a method for the combined treatment of intestinal diseases by fecal microbiota transplantation combined with miR-146b. This method uses the fecal microbiota transplantation combined preparation of the present invention for treatment. The administration method of the combined preparation of the present invention is as follows:

[0030] (1) Administration route

[0031] Enema: Suitable for colon inflammation, with a dose of 50 - 200 mL per time.

[0032] Oral enteric-coated capsules: Using pH-sensitive coating to ensure the release of the microbiota and miRNA in the intestine.

[0033] Colonoscopy-assisted administration: Precise positioning of the lesion site.

[0034] (2) Treatment cycle

[0035] Initial stage: 1 - 2 times per week for 2 - 4 weeks.

[0036] Maintenance stage: Once every 2 - 4 weeks, with a total treatment course of 4 - 12 weeks.

[0037] Beneficial effects

[0038] The beneficial effects of the present invention are summarized as follows:

[0039] The technical improvements and advantages of the technical solution of the present invention are as follows:

[0040] Precise regulation of the gut microbiota: miR-146b targets and inhibits the transcription of 16S rRNA of pro-inflammatory bacteria (such as Escherichia coli), while promoting the colonization of anti-inflammatory bacteria.

[0041] Enhanced stability: The engineered bacteria continuously secrete miR-146b to avoid the rapid degradation of synthetic miRNAs.

[0042] Safety optimization: miR-146b can inhibit the proliferation of potential pathogenic bacteria and reduce the infection risk of FMT.

[0043] The treatment method of fecal microbiota transplantation (FMT) combined with miR-146b provided by the present invention has significant advantages compared with traditional FMT or miRNA treatment alone. The synergistic effect of this method is reflected in that the fecal microbiota suspension provides microbiota diversity, while miR-146b selectively promotes the growth of anti-inflammatory bacteria and inhibits the activity of pathogenic bacteria, and enters bacteria through extracellular vesicles to down-regulate the expression of their pro-inflammatory genes. miR-146b can inhibit the expression of virulence factors of pathogenic bacteria, and the engineered bacteria delivery method can avoid the hepatotoxicity of synthetic miRNAs.

[0044] This protocol is simple to operate. It only needs to add a miR-146b mixing step to the existing FMT process. The lyophilized preparation can be stored for a long time (stability at -80°C > 6 months), effectively solving the two major technical problems of unstable efficacy of traditional FMT and difficult miRNA delivery. This combination therapy is significantly superior to the existing technologies in terms of microbiota colonization, anti-inflammatory effect and safety, providing a more reliable solution for intestinal diseases such as inflammatory bowel disease. Detailed implementation mode

[0045] To deepen the understanding of the present invention, the method of combined treatment of fecal microbiota transplantation combined with miR-146b for intestinal diseases of the present invention will be further described in detail below with reference to examples. These examples are only used to explain the present invention and do not limit the protection scope of the present invention.

[0046] Example 1

[0047] A combined preparation for fecal microbiota transplantation, which includes fecal microbiota and miR-146b.

[0048] In the combined preparation for fecal microbiota transplantation, the concentration of miR-146b is: 1 μg / mL; the concentration of fecal microbiota is 10^8 cfu / ml.

[0049] The preparation method of the above combined preparation for fecal microbiota transplantation includes the following steps:

[0050] (1) Preparation of fecal microbiota suspension

[0051] Donor screening: Healthy donors should meet the international FMT standards (such as no infectious diseases, metabolic diseases, history of antibiotic use, etc.); after the fecal samples are collected, they should be processed within 2 hours or stored frozen at -80°C.

[0052] Preparation of fecal microbiota suspension: Mix 1 g of fecal sample with 50 mL of sterile normal saline or PBS buffer, and stir to homogenize; filter successively through 100 μm and 70 μm filters to remove large particle residues; centrifuge (3000 rpm, 20 min) to collect the bacterial cell precipitate, and resuspend it in PBS solution containing 10% glycerol to obtain the fecal microbiota suspension.

[0053] (2) Addition and optimization of miR-146b

[0054] Source of miR-146b:

[0055] Synthesize miR-146b mimic: Modify with 2'-O-methylation to improve stability.

[0056] Addition method of miR-146b:

[0057] Direct mixing: Mix miR-146b mimic (1 μg / mL) with the fecal microbiota suspension to obtain the described fecal microbiota transplantation combined preparation.

[0058] Method for treating intestinal diseases by combining fecal microbiota transplantation with miR-146b. This method uses the fecal microbiota transplantation combined preparation obtained in this example for treatment. The administration method of the combined preparation of the present invention is:

[0059] (1) Administration route

[0060] Enema method: Suitable for colonic inflammation, with a dose of 50 - 200 mL / time.

[0061] Oral enteric-coated capsules: Use pH-sensitive coating to ensure the release of the microbiota and miRNA in the intestine.

[0062] Colonoscopy-assisted administration: Precisely locate the lesion site.

[0063] (2) Treatment cycle

[0064] Initial stage: 1 - 2 times per week, lasting for 2 - 4 weeks.

[0065] Maintenance stage: Once every 2 - 4 weeks, with a total treatment course of 4 - 12 weeks.

[0066] Example 2

[0067] A fecal microbiota transplantation combined preparation, which includes fecal microbiota and miR-146b in the preparation.

[0068] In the fecal microbiota transplantation combined preparation, the concentration of miR-146b is: 100 μg / mL; the fecal microbiota concentration is 109 cfu / ml.

[0069] The preparation method of the above fecal microbiota transplantation combined preparation includes the following steps:

[0070] (1) Preparation of fecal microbiota suspension

[0071] Donor screening: Healthy donors need to meet the international FMT standards (such as no infectious diseases, metabolic diseases, history of antibiotic use, etc.); after the fecal samples are collected, they should be processed or cryopreserved (-80 °C) within 2 hours.

[0072] Preparation of fecal microbiota suspension: Take 50 g of fecal samples and mix them with 500 mL of sterile physiological saline or PBS buffer, stir and homogenize; filter through 100 μm and 70 μm filters in sequence to remove large particle residues; centrifuge (8000 rpm, 5 min) to collect the bacterial cell precipitate and resuspend it in sterile buffer.

[0073] (2) Addition and optimization of miR-146b

[0074] Source of miR-146b:

[0075] Engineered bacteria express miR-146b;

[0076] Addition method of miR-146b:

[0077] Co-transplantation of engineered bacteria: Mix the engineered bacteria expressing miR-146b (10 6 -10 10 CFU / mL) with the fecal microbiota suspension in a ratio of 1:5 to obtain the fecal microbiota transplantation combined preparation.

[0078] The method for the combined treatment of intestinal diseases by fecal microbiota transplantation combined with miR-146b uses the fecal microbiota transplantation combined preparation obtained in this example for treatment. The administration method of the combined preparation of the present invention is:

[0079] (1) Administration route

[0080] Enema method: Suitable for colonic inflammation, the dose is 50 - 200 mL / time.

[0081] Oral enteric-coated capsules: Use pH-sensitive coating to ensure the release of the microbiota and miRNA in the intestine.

[0082] Colonoscopy-assisted administration: Precise positioning of the lesion site.

[0083] (2) Treatment cycle

[0084] Initial stage: 1 - 2 times a week, lasting for 2 - 4 weeks.

[0085] Maintenance phase: once every 2 - 4 weeks, with a total treatment course of 4 - 12 weeks.

[0086] Example 3

[0087] A fecal microbiota transplantation combined preparation, which includes fecal microbiota and miR - 146b.

[0088] In the fecal microbiota transplantation combined preparation, the concentration of miR - 146b is: 10 μg / mL; the concentration of fecal microbiota is 10^8 cfu / ml.

[0089] The preparation method of the above - mentioned fecal microbiota transplantation combined preparation includes the following steps:

[0090] (1) Preparation of fecal microbiota suspension

[0091] Donor screening: Healthy donors need to meet the international FMT standards (such as no infectious diseases, metabolic diseases, history of antibiotic use, etc.); after the fecal sample is collected, it should be processed or cryopreserved (-80 °C) within 2 hours.

[0092] Preparation of fecal microbiota suspension: Take 30 g of fecal sample and mix it with 300 mL of sterile physiological saline or PBS buffer, stir and homogenize; filter successively through 100 μm and 70 μm filters to remove large - particle residues; centrifuge (5000 rpm, 10 min) to collect the bacterial cell precipitate, and resuspend it in PBS sterile buffer containing 10% glycerol.

[0093] (2) Addition and optimization of miR - 146b

[0094] Source of miR - 146b:

[0095] Engineered bacteria express miR - 146b;

[0096] Addition method of miR - 146b:

[0097] Co - transplantation of engineered bacteria: Mix the engineered bacteria expressing miR - 146b (10^ 6 -10^ 10 CFU / mL) with the fecal microbiota suspension in a ratio of 1:10 to obtain the fecal microbiota transplantation combined preparation.

[0098] A method for the combined treatment of intestinal diseases by fecal microbiota transplantation combined with miR - 146b, which uses the fecal microbiota transplantation combined preparation obtained in this example for treatment. The administration method of the combined preparation of the present invention is:

[0099] (1) Administration route

[0100] Enema method: applicable to colonic inflammation, with a dose of 50 - 200 mL per time.

[0101] Oral enteric-coated capsules: Using pH-sensitive coatings to ensure the release of the microbiota and miRNAs in the intestine.

[0102] Colonoscopy-assisted drug delivery: Precise localization of the lesion site.

[0103] (2) Treatment cycle

[0104] Initial stage: 1 - 2 times per week for 2 - 4 weeks.

[0105] Maintenance stage: Once every 2 - 4 weeks, with a total treatment course of 4 - 12 weeks.

[0106] Example 4

[0107] A fecal microbiota transplantation combined preparation, the preparation comprising fecal microbiota and miR-146b.

[0108] In the fecal microbiota transplantation combined preparation, the concentration of miR-146b is: 100 μg / mL; the concentration of the fecal microbiota is 109 cfu / ml.

[0109] The preparation method of the above fecal microbiota transplantation combined preparation comprises the following steps:

[0110] (1) Preparation of the fecal microbiota suspension

[0111] Donor screening: Healthy donors need to meet the international FMT standards (such as no infectious diseases, metabolic diseases, history of antibiotic use, etc.); after the fecal sample is collected, it should be processed within 2 hours or cryopreserved (-80 °C);

[0112] Preparation of the fecal microbiota suspension: Take 50 g of the fecal sample and mix it with 500 mL of sterile physiological saline or PBS buffer, stir and homogenize; filter successively through 100 μm and 70 μm filters to remove large particle residues; centrifuge (8000 rpm, 5 min) to collect the bacterial cell precipitate, and resuspend it in a sterile buffer to obtain the fecal microbiota suspension;

[0113] The obtained fecal microbiota suspension is freeze-dried or microencapsulated to improve stability.

[0114] (2) Addition and optimization of miR-146b

[0115] Source of miR-146b:

[0116] Engineered bacteria express miR-146b;

[0117] Addition method of miR-146b:

[0118] Co-transplantation of engineered bacteria: The engineered bacteria expressing miR-146b (10 6 -10 10Mix the CFU / mL) with the fecal bacteria suspension in a ratio of 1:5 to obtain the described fecal microbiota transplantation combined preparation.

[0119] A method for the combined treatment of intestinal diseases by fecal microbiota transplantation combined with miR-146b, which uses the fecal microbiota transplantation combined preparation obtained in this example for treatment. The administration method of the combined preparation of the present invention is as follows:

[0120] (1) Administration route

[0121] Enema method: Suitable for colonic inflammation, with a dose of 50 - 200 mL / time.

[0122] Oral enteric-coated capsules: Use pH-sensitive coating to ensure the release of the flora and miRNA in the intestine.

[0123] Colonoscopy-assisted administration: Precise localization of the lesion site.

[0124] (2) Treatment cycle

[0125] Initial stage: 1 - 2 times a week for 2 - 4 weeks.

[0126] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A fecal microbiota transplantation combined preparation, characterized in that: The preparation contains fecal microbiota and miR-146b.

2. The fecal microbiota transplantation combined preparation according to claim 1, wherein: In the combined preparation of fecal microbiota transplantation, the concentration of miR-146b is 0.1 μg / mL to 100 μg / mL; the concentration of fecal microbiota is 10^8 - 10^9 cfu / ml.

3. The preparation method of the fecal microbiota transplantation combined preparation according to any one of claims 1-2, characterized in that, It includes the following steps: (1) Preparation of fecal microbiota suspension Donor screening; Preparation of fecal microbiota suspension: Take a fecal sample and mix it with a solution, stir and homogenize; filter through a filter screen to remove large particle residues; centrifuge to collect the bacterial cell precipitate and resuspend it in a sterile buffer to obtain a fecal microbiota suspension; (2) Addition and optimization of miR-146b Source of miR-146b: Synthesize miR-146b mimic or express miR-146b with engineered bacteria; Addition method of miR-146b: Direct mixing or co-transplantation with engineered bacteria.

4. The preparation method of the fecal microbiota transplantation combined preparation according to claim 3, wherein: In the preparation step of the fecal microbiota suspension in step (1), take 1 - 50 g of fecal sample and mix it with 50 - 500 mL of sterile physiological saline or PBS buffer, stir and homogenize; filter successively through 100 μm and 70 μm filter screens to remove large particle residues; centrifuge at 3000 - 8000 rpm for 5 - 20 min to collect the bacterial cell precipitate and resuspend it in a sterile buffer; the sterile buffer is preferably PBS containing 10% glycerol.

5. The preparation method of the fecal microbiota transplantation combined preparation according to claim 3, characterized in that: To improve stability, the fecal microbiota suspension can be optionally freeze-dried or microencapsulated.

6. The preparation method of the fecal microbiota transplantation combined preparation according to claim 3, wherein: The source of miR-146b in step (2) specifically includes: Synthesize miR-146b mimic: Chemical modification to improve stability; Express miR-146b with engineered bacteria: Engineered bacteria stably secrete miR-146b.

7. The preparation method of the fecal microbiota transplantation combined preparation according to claim 3, wherein: The addition method of miR-146b in step (2) specifically includes: Direct mixing: Mix 0.1 - 100 μg / mL of miR-146b mimic with the fecal microbiota suspension; Engineering bacteria co-transplantation: Mix the engineered bacteria expressing miR-146b with the fecal bacteria suspension at a ratio of 1:1 to 1:

10. The concentration of the engineered bacteria is 10 6 -10 10 CFU / mL.

8. A method for the combined treatment of intestinal diseases by fecal microbiota transplantation and miR-146b, characterized in that: This method uses the combined preparation of fecal microbiota transplantation according to any one of claims 1 - 2 for treatment.

9. The method for treating intestinal diseases by fecal microbiota transplantation combined with miR-146b according to claim 8, characterized in that The administration method of the combined preparation of fecal microbiota transplantation is: (1) Administration routes include: Enema: Suitable for colon inflammation, with a dose of 50 - 200 mL / time; Oral enteric-coated capsules: Use pH-sensitive coating to ensure the release of the microbiota and miRNA in the intestine; Colonoscopy-assisted administration: Accurately locate the lesion site; (2) Treatment cycle Initial stage: 1 - 2 times per week for 2 - 4 weeks; Maintenance stage: Once every 2 - 4 weeks, with a total treatment course of 4 - 12 weeks.