WO2023035949A1 - 一种建立眼部疾病模型的方法及其应用 - Google Patents

一种建立眼部疾病模型的方法及其应用 Download PDF

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WO2023035949A1
WO2023035949A1 PCT/CN2022/114537 CN2022114537W WO2023035949A1 WO 2023035949 A1 WO2023035949 A1 WO 2023035949A1 CN 2022114537 W CN2022114537 W CN 2022114537W WO 2023035949 A1 WO2023035949 A1 WO 2023035949A1
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model carrier
disease model
acinetobacter
eye
mutation
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PCT/CN2022/114537
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English (en)
French (fr)
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魏来
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珠海岐微生物科技有限公司
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Priority to EP22866419.9A priority Critical patent/EP4382112A1/en
Priority to CN202280061211.9A priority patent/CN117917962A/zh
Publication of WO2023035949A1 publication Critical patent/WO2023035949A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor

Definitions

  • the invention belongs to the technical field of diagnosis and treatment of eye diseases, and in particular relates to a method for establishing an eye disease model and the application of the eye disease model and model carrier obtained by the method in eye disease research and drug screening.
  • Inherited retinal degeneration a group of inherited disorders, is the most common cause of vision loss in the working population of industrialized countries.
  • Inherited retinal degeneration is a group of genetic disorders characterized by progressive loss of photoreceptor cells, including amaurosis congenita (LCA), retinitis pigmentosa (RP), early-onset rod-cone Malnutrition, rod-rod dystrophy, congenital stationary night and color blindness, and Stargardt disease (Broadgate et al., 2017). It is the most common cause of vision loss in working populations in industrialized countries, with an estimated incidence of 1:2000 (Kutluer et al., 2020).
  • CRB1 More than 200 genes are associated with IRD.
  • the CRB1 gene has long been recognized as an important gene whose mutations lead to various ophthalmic phenotypes, including LCA and RP (Ehrenberg et al., 2013).
  • LCA and RP As a key regulator of adhesion molecule junctions, CRB1 plays an important role in establishing cell polarity and maintaining ocular barrier integrity.
  • the frequency of IRD-causing CRB1 mutations varies widely around the world.
  • CRB1 mutant allele frequencies found in LCA cases were 6.7% in the United States, 1.7% in Canada, 0% in the Netherlands, 16.7% in Spain, and 11.5% in China (Li et al., 2011; Vallespin et al., 2007; Zernant et al., 2005), while CRB1 was the gene with the highest mutation frequency found in all Spanish patients with LCA, early-onset RP, and non-early-onset RP (Vallespin et al., 2007).
  • mutations in the CRB1 gene explain 4% of RP and 10-15% of LCA globally (Richard et al., 2006). Therefore, a therapeutic approach to rescue CRB1-associated vision loss is urgently needed.
  • An object of the present invention is to provide a method for establishing an eye disease model or an eye disease model carrier, especially a method for a retinal degeneration model or a model carrier, a model or a model carrier established by the method, and the method , disease models and/or applications of disease model carriers in eye disease research and related drug screening.
  • the invention provides a method for establishing an eye disease model, the method comprising infecting the eye disease model with microorganisms.
  • the infection includes direct contact with microorganisms or indirect contact with microorganisms.
  • the ocular disease model in which the eye is infected with microorganisms is obtained by raising the non-human animal in an SPF environment.
  • said microorganisms are from or identical to the intestinal bacteria of the same individual.
  • the eye disease includes retinal degeneration; more preferably, the retinal degeneration is progressive retinal degeneration.
  • the retinal degeneration is Inherited Retinal Degeneration (IRD).
  • IFD Inherited Retinal Degeneration
  • the eye diseases comprise LCA, RP, arRP, EORD, EORP, PPRPE, rettelangiectasia and/or choroideremia like fundus.
  • said eye disease comprises ocular inflammation, such as uveitis, glaucoma, and age-related macular degeneration (AMD), vitreitis, choroiditis, retinitis, retinal vasculitis, optic neuritis, and grapevine Meningitis, Behcet's disease, Vogt-Koyanagi-Harada syndrome, uveitis, retinopathy, sympathetic ophthalmia, cataract, conjunctivitis, glaucoma, etc.
  • ocular inflammation such as uveitis, glaucoma, and age-related macular degeneration (AMD)
  • vitreitis choroiditis
  • retinitis retinal vasculitis
  • optic neuritis and grapevine Meningitis
  • Behcet's disease Vogt-Koyanagi-Harada syndrome
  • uveitis retinopathy
  • sympathetic ophthalmia cataract
  • conjunctivitis glaucom
  • said model is a non-human animal, preferably monkey, dog, chimpanzee, rat, mouse.
  • the model carrier is a cell, tissue or organ, and the cell, tissue or organ is derived from a human or a non-human animal.
  • the cells are primary cells or cell lines.
  • said tissue is eye tissue
  • said organ is eye organ
  • said tissue or organ is a regenerative tissue or organ.
  • the said model has a pathogenic mutation of a gene.
  • the gene with pathogenic mutation is a gene related to maintaining the retinal barrier structure, and the retinal barrier is the outer blood-retinal barrier and/or the inner blood-retinal barrier.
  • the gene with the pathogenic eye gene mutation is selected from the following one or a combination of two or more genes: ABCA4 , ABCC6, ABCC9, ACBD5, ACO2, ACO2, ACTG1, ADGRV1, AHI1, AIPL1, ALMS1, AMY2B, APC, ARFGEF1, ARL13B, ARL13B, ARL6, ARMC9, ATOH7, B9D1, BAG3, BBS1, BBS1, BBS2, BBS5, BEST1 , C2CD3, CA4, CABP4, CACNA1F, CBS, CC2D2A, CDH23, CDH23, CDHR1, CEMIP2, CEP104, CEP250, CEP290, CEP290, CEP41, CEP78, CERKL, CFAP410, CFAP418, CHM, CLCC1, CLCN7, CLN3, CLN5, CLN8 , CLRN1, CLRN1, CNGA1, CNGA
  • the ocular disease-related genes in which pathogenic mutations have occurred in the model or model carrier include the CRB1 gene.
  • the mutation of the CRB1 gene of the model or model carrier includes one or more of the following mutations: c.107C>G, c.111delT, c.135C>G, c.257_258dupTG, c.258C >T, c.428_432delGATTC, c.430T>G, c.470G>C, c.481dupG, c.482C>T, c.584G>T, c.613_619del, c.717_718insG, c.750T>G, c .915T>A, c.929G>A, c.936T>G, c.998G>A, c.1084C>T, c.1125C>G, c.1148G>A, c.1208C>G, c.1269C >A, c.1298A>G, c.
  • the mutation of the CRB1 gene of the model or model carrier includes one or more of the following mutations: c.4006-1G>T, c.3686G>C, (p.Cys1229Ser), c. 2842+1delinsAA, c.4060G>A, (p.Ala1354Thr), c.3991C>T, (p.Arg1331Cys), c.3014A>T, (p.Asp1005Val), c.4005+1G>A, c.
  • 2680_2684del (p.Asn894fs), c.1733T>A, (p.Val578Glu), c.455G>A, (p.Cys152Tyr), c.3462_3463del, (p.Cys1154_Glu1155delinsTer), c.3037C>T, (p .Gln1013Ter), c.2673C>A, (p.Cys891Ter), c.2230C>T, (p.Arg744Ter), c.3676G>T, (p.Gly1226Ter), c.2842+5G>A, c.
  • the mutation of the CRB1 gene of the model or model carrier is Rd8 mutation.
  • the mutation is a homozygous mutation or a heterozygous mutation.
  • the above-mentioned gene mutations are congenitally present in the model or model carrier or the mutations are acquired acquired due to gene recombination operations.
  • the humanized CRB1 gene or human CRB1 gene exists in the model or model vector, and the endogenous CRB1 gene is deleted or not expressed.
  • said non-human animal has a defect in the colonic epithelial barrier and/or associated inflammation of the colonic wall.
  • the Occludin protein in the model body is significantly absent, and in a specific embodiment, the Occludin protein is significantly absent in the model body and the expression of Claudin1 is not obvious.
  • the microorganism is one or a combination of two or more of bacteria, archaea, protozoa, fungus or virus, preferably, the microorganism is a bacterium, and the bacterium is selected from: Anearostipes, Bifidobacterium (Bifidobacterium), Megamonas, Nitrosomonas, Oscillibacter, Tatumella, Thiobacillus sp., Clostridium, imine Acinetobacter, Streptococcus, Mannheimia, Fibrobacter, Prevotella, Campylobacter, Actinomyces , Hymenobacter, Escherichia, Tissierella, Klebsiella, Porphyromonas, Azospirillum (Azospira), Aquimarina, Achromobacter, Acidithiobacillus, Burkholderia, Marinobacter, Treponema ( Treponema), Actinosporangium, Vibrio
  • the bacteria are selected from: Anearostipes hadrus, Bifidobacterium pseudocatenulatum, Nitrosomonas sp.Is79A3, Oscillibacter valericigenes, Tatumella sp.TA1, Megamonas funiformis, Thiobacillus denitrificans, Clostridium tetani, Clostridium perfringens, Clostridium botulinum , Acinetobacter calcium acetate, Acinetobacter ruffii, Acinetobacter baumannii, Acinetobacter haemolytica, Acinetobacter johnsonii, Acinetobacter johnsonii, Streptococcus pyogenes, Streptococcus hemolyticus, succinic acid-producing filamentous Bacillus, Filamentobacter enterica, Porphyromonas nonsaccharolyticum, Porphyromonas pulpum, Porphyromonas gingivalis,
  • the infection method comprises direct contact or indirect contact between the microorganism and the site to be infected by the model carrier, the indirect contact means that there is a blood-retinal barrier between the microorganism and the site to be infected, preferably , for the outer blood-retinal barrier or the inner blood-retinal barrier.
  • the infection method comprises infecting the eye with intestinal bacteria through peripheral blood.
  • the intestinal epithelial barrier was significantly damaged in non-human animal models, allowing intestinal bacteria to enter the peripheral blood.
  • the retinal barrier was also significantly damaged, allowing intestinal bacteria entering the peripheral blood to infect the retina;
  • the non-human animal model has a mutation in the CRB1 gene; in a specific embodiment In the mode, the mutation of the CRB1 gene is Rd8 mutation.
  • the present invention provides a method for preparing an ocular inflammation model, wherein the method model is a non-human animal infection microorganism.
  • the ocular inflammation is caused by intestinal flora or the same flora as intestinal flora.
  • Said non-human animal is the animal as mentioned above.
  • microorganisms are as described above.
  • the present invention provides a method for preparing a retinal degenerative disease model, the method comprising infecting a non-human animal suffering from retinal degeneration with microorganisms.
  • the ocular inflammatory retinal degenerative disease is the aforementioned retinal disease.
  • Said non-human animal is the animal as mentioned above.
  • microorganisms are as described above.
  • the present invention provides a model carrier with ocular inflammation caused by microbial infection.
  • said microorganisms are from or identical to the intestinal bacteria of the same individual.
  • the eye disease includes retinal degeneration; more preferably, the retinal degeneration is progressive retinal degeneration.
  • the retinal degeneration is Inherited Retinal Degeneration (IRD).
  • IFD Inherited Retinal Degeneration
  • the retinal degeneration is a disease of the model animal itself or the retinal degeneration of the model animal is caused by genetic manipulation.
  • the eye diseases comprise LCA, RP, arRP, EORD, EORP, PPRPE, rettelangiectasia and/or choroideremia like fundus.
  • said eye disease comprises ocular inflammation, such as uveitis, glaucoma, and age-related macular degeneration (AMD), vitreitis, choroiditis, retinitis, retinal vasculitis, optic neuritis, and grapevine Meningitis, Behcet's disease, Vogt-Koyanagi-Harada syndrome, uveitis, retinopathy, sympathetic ophthalmia, cataract, conjunctivitis, glaucoma, etc.
  • ocular inflammation such as uveitis, glaucoma, and age-related macular degeneration (AMD)
  • vitreitis choroiditis
  • retinitis retinal vasculitis
  • optic neuritis and grapevine Meningitis
  • Behcet's disease Vogt-Koyanagi-Harada syndrome
  • uveitis retinopathy
  • sympathetic ophthalmia cataract
  • conjunctivitis glaucom
  • said model is a non-human animal, preferably monkey, dog, chimpanzee, rat, mouse.
  • the model carrier is a cell, tissue or organ, and the cell, tissue or organ is derived from a human or a non-human animal.
  • the cells are primary cells or cell lines.
  • said tissue is eye tissue
  • said organ is eye organ
  • said tissue or organ is a regenerative tissue or organ.
  • the said model has a pathogenic mutation of a gene.
  • the gene with pathogenic mutation is a gene related to maintaining the retinal barrier structure, and the retinal barrier is the outer blood-retinal barrier and/or the inner blood-retinal barrier.
  • one or more of the following genes are mutated in the model carrier: ABCA4, ABCC6, ABCC9, ACBD5, ACO2, ACO2, ACTG1, ADGRV1, AHI1, AIPL1, ALMS1, AMY2B, APC, ARFGEF1, ARL13B , ARL13B, ARL6, ARMC9, ATOH7, B9D1, BAG3, BBS1, BBS1, BBS2, BBS5, BEST1, C2CD3, CA4, CABP4, CACNA1F, CBS, CC2D2A, CDH23, CDH23, CDHR1, CEMIP2, CEP104, CEP250, CEP290, CEP290 , CEP41, CEP78, CERKL, CFAP410, CFAP418, CHM, CLCC1, CLCN7, CLN3, CLN5, CLN8, CLRN1, CLRN1, CNGA1, CNGA1, CNGA3, CNGB1, CNGB3, CNNM4, COL11A1, COL11A2, COL18A1, COL
  • the eye disease-related genes with pathogenic mutations in the model carrier include the CRB1 gene.
  • the mutation of the CRB1 gene of the model carrier includes one or more of the following mutations: c.107C>G, c.111delT, c.135C>G, c.257_258dupTG, c.258C>T , c.428_432delGATTC, c.430T>G, c.470G>C, c.481dupG, c.482C>T, c.584G>T, c.613_619del, c.717_718insG, c.750T>G, c.915T >A, c.929G>A, c.936T>G, c.998G>A, c.1084C>T, c.1125C>G, c.1148G>A, c.1208C>G, c.1269C>A , c.1298A>G, c.13
  • the mutation of the CRB1 gene of the model or model carrier includes one or more of the following mutations: c.4006-1G>T, c.3686G>C, (p.Cys1229Ser), c. 2842+1delinsAA, c.4060G>A, (p.Ala1354Thr), c.3991C>T, (p.Arg1331Cys), c.3014A>T, (p.Asp1005Val), c.4005+1G>A, c.
  • 2680_2684del (p.Asn894fs), c.1733T>A, (p.Val578Glu), c.455G>A, (p.Cys152Tyr), c.3462_3463del, (p.Cys1154_Glu1155delinsTer), c.3037C>T, (p .Gln1013Ter), c.2673C>A, (p.Cys891Ter), c.2230C>T, (p.Arg744Ter), c.3676G>T, (p.Gly1226Ter), c.2842+5G>A, c.
  • the mutation of the CRB1 gene of the model carrier is Rd8 mutation.
  • the mutation is a homozygous mutation or a heterozygous mutation.
  • the above-mentioned gene mutations are congenitally present in the model vector or the mutations are acquired acquired due to gene recombination operations.
  • the humanized CRB1 gene or human CRB1 gene exists in the model vector, and the endogenous CRB1 gene is deleted or not expressed.
  • said non-human animal has a defect in the colonic epithelial barrier and/or associated inflammation of the colonic wall.
  • the Occludin protein in the model body is significantly absent, and in a specific embodiment, the Occludin protein is significantly absent in the model body and the expression of Claudin1 is not obvious.
  • the microorganism is one or a combination of two or more of bacteria, archaea, protozoa, fungus or virus, preferably, the microorganism is a bacterium, and the bacterium is selected from: Anearostipes, Bifidobacterium (Bifidobacterium), Megamonas, Nitrosomonas, Oscillibacter, Tatumella, Thiobacillus sp., Clostridium, imine Acinetobacter, Streptococcus, Mannheimia, Fibrobacter, Prevotella, Campylobacter, Actinomyces , Hymenobacter, Escherichia, Tissierella, Klebsiella, Porphyromonas, Azospirillum (Azospira), Aquimarina, Achromobacter, Acidithiobacillus, Burkholderia, Marinobacter, Treponema ( Treponema), Actinosporangium, Vibrio
  • the bacteria are selected from: Anearostipes hadrus, Bifidobacterium pseudocatenulatum, Nitrosomonas sp.Is79A3, Oscillibacter valericigenes, Tatumella sp.TA1, Megamonas funiformis, Thiobacillus denitrificans, Clostridium tetani, Clostridium perfringens, Clostridium botulinum , Acinetobacter calcium acetate, Acinetobacter ruffii, Acinetobacter baumannii, Acinetobacter haemolytica, Acinetobacter johnsonii, Acinetobacter johnsonii, Streptococcus pyogenes, Streptococcus hemolyticus, succinic acid-producing filamentous Bacillus, Filamentobacter enterica, Porphyromonas nonsaccharolyticum, Porphyromonas pulpum, Porphyromonas gingivalis,
  • the infection method comprises direct contact or indirect contact between the microorganism and the site to be infected by the model carrier, the indirect contact means that there is a blood-retinal barrier between the microorganism and the site to be infected, preferably , for the outer blood-retinal barrier or the inner blood-retinal barrier.
  • the infection method comprises infecting the eye with intestinal bacteria through peripheral blood.
  • the intestinal epithelial barrier was significantly damaged in non-human animal models, allowing intestinal bacteria to enter the peripheral blood.
  • the retinal barrier was also significantly damaged, allowing intestinal bacteria entering the peripheral blood to infect the retina;
  • the non-human animal model has a mutation in the CRB1 gene; in a specific embodiment In the mode, the mutation of the CRB1 gene is Rd8 mutation.
  • the inflammatory model carrier is obtained from the disease model prepared by the method described above, or obtained from three-dimensional ocular cells, tissues or organs from non-human animals as described above with microorganism infection
  • the present invention provides an application of the above-mentioned method in evaluating the curative effect of targeted therapy for ocular diseases, and the ocular diseases include the above-mentioned ocular diseases.
  • the aforementioned model or model carrier is subjected to targeted therapy for the aforementioned gene mutation, and the model or model carrier that has not undergone targeted therapy is divided into two groups: Groups were established eye disease models according to the method described above, if in comparison, the group after targeted therapy can not be successfully modeled, indicating that targeted therapy has achieved beneficial effects.
  • the targeted therapy targets one of the following genes or a combination of two or more genes: ABCA4, ABCC6, ABCC9, ACBD5, ACO2, ACO2, ACTG1, ADGRV1, AHI1, AIPL1, ALMS1, AMY2B , APC, ARFGEF1, ARL13B, ARL13B, ARL6, ARMC9, ATOH7, B9D1, BAG3, BBS1, BBS1, BBS2, BBS5, BEST1, C2CD3, CA4, CABP4, CACNA1F, CBS, CC2D2A, CDH23, CDH23, CDHR1, CEMIP2, CEP104 , CEP250, CEP290, CEP290, CEP41, CEP78, CERKL, CFAP410, CFAP418, CHM, CLCC1, CLCN7, CLN3, CLN5, CLN8, CLRN1, CLRN1, CNGA1, CNGA1, CNGA3, CNGB1, CNGB3, CNNM4, COL11A1, COL11A2, COL
  • the targeted therapy targets one or more of the following mutations of the CRB1 gene: c.257_258dupTG, c.258C>T, c.428_432delGATTC, c.430T>G, c.
  • the targeted therapeutic drug comprises modified cells, modified proteins, RNA targeting the aforementioned gene or targeting the aforementioned mutation site, and/or DNA targeting a gene as previously described or targeting a mutation site as previously described.
  • the present invention provides an application of the disease model prepared by the aforementioned method in research related to eye diseases.
  • the eye diseases include the aforementioned eye diseases.
  • the research described includes the interaction between diseases associated with inherited retinal degeneration and gut microbiota, among others.
  • the disease model is infected with an intestinal bacterium, and when the disease-causing mutation it carries is treated with cells, the curative effect of administration and non-administration is observed, and the drug is A small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic directed against the infected bacteria.
  • the disease model is infected with an intestinal bacterium, and when the pathogenic mutation it carries is treated with RNA, the curative effect of administration and non-administration is observed, and the drug is A small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic directed against the infected bacteria.
  • the disease model is infected with more than two kinds of intestinal bacteria, and when the pathogenic mutations carried by them are treated with cells, the curative effect of administration and non-administration is observed, and the The drug is a small molecule drug, preferably a broad spectrum antibiotic or an antibiotic directed against the infected bacteria.
  • the disease model is infected with two kinds of intestinal bacteria, and when the pathogenic mutations carried by them are treated with RNA, the curative effect of administration and non-administration is observed, and the drug is A small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic directed against the infected bacteria.
  • the pathogenic mutation occurs with the aforementioned gene, or, the pathogenic mutation is a mutation of the aforementioned CRB gene.
  • the present invention provides an application of the disease model carrier prepared by the above-mentioned method in research related to eye diseases.
  • the eye diseases include the aforementioned eye diseases.
  • the studies described include the synergistic effect of gut microbiota in diseases associated with hereditary retinal degeneration, among others.
  • the disease model carrier is infected with an intestinal bacterium, and when the disease-causing mutation it carries is treated by cells, the curative effect of administration and non-administration is observed, and the drug is a small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic directed against the infected bacteria.
  • the disease model carrier is infected with an intestinal bacterium, and when the pathogenic mutation carried by it is treated with RNA, the curative effect of administration and non-administration is observed, and the drug is a small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic directed against the infected bacteria.
  • the disease model carrier is infected with more than two kinds of intestinal bacteria, and when the pathogenic mutations carried by them are treated by cells, the curative effect of administration and non-administration is observed, and the The drug is a small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic directed against the infected bacteria.
  • the disease model carrier is infected with two kinds of intestinal bacteria, and when the pathogenic mutation it carries is treated with RNA, the curative effect of administration and non-administration is observed, and the drug is a small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic directed against the infected bacteria.
  • the pathogenic mutation occurs with the aforementioned gene, or, the pathogenic mutation is a mutation of the aforementioned CRB gene.
  • the present invention provides an application of the aforementioned disease model or disease model carrier in the screening of drugs related to eye diseases.
  • the medicines include one or a combination of small molecule medicines, chemical medicines, macromolecular medicines, biological medicines or natural medicines (such as traditional Chinese medicine or Chinese medicine extracts), cellular medicines, RNA medicines, and DNA medicines.
  • the eye diseases include the aforementioned eye diseases.
  • the small molecule compound is an antibiotic
  • the antibiotic is a broad-spectrum antibiotic drug generally known to those skilled in the art.
  • the small molecular compound is a non-broad-spectrum antibiotic targeted to specific bacteria.
  • the cells include modified immune cells, such as one or a combination of two or more groups of T cells, B cells or stem cells.
  • modified immune cells such as one or a combination of two or more groups of T cells, B cells or stem cells.
  • the RNA comprises mRNA, siRNA, sgRNA, miRNA, ASO and/or replicon RNA.
  • the disease model or disease model carrier is subjected to targeted therapy, and at the same time, the disease model or disease model carrier that has undergone targeted therapy and the disease model or disease model carrier that has not undergone targeted therapy are administered With or without administration of drugs as previously described, the progression of inflammation in several groups was observed, and the drug efficacy of targeted therapy was evaluated.
  • the disease model or disease model carrier is subjected to targeted therapy, and at the same time, the disease model or disease model carrier that has undergone targeted therapy and the disease model or disease model carrier that has not undergone targeted therapy are administered With or without drug administration as previously described, the progression of inflammation in several groups was observed to evaluate the efficacy of small molecule drugs.
  • the chemical drugs described in the present invention are selected from ⁇ -lactam antibiotics: including penicillins, cephalosporins, sulfurases, monolactams, ⁇ -lactamase inhibitors, methicillins, etc.; Aminoglycoside antibiotics: including streptomycin, gentamicin, kanamycin, tobramycin, amikacin, neomycin, ribomycin, micronomycin, asthromycin, etc.
  • Tetracycline antibiotics including tetracycline, oxytetracycline, chlortetracycline and doxycycline
  • chloramphenicol antibiotics including chloramphenicol, thiamphenicol, etc.
  • macrolide antibiotics including erythromycin, Leucomycin, tasteless erythromycin, acetylspiramycin, midecamycin, josamycin, azithromycin, etc.
  • glycopeptide antibiotics vancomycin, norvancomycin, teicoplanin, etc.
  • quinolone antibiotics including norfloxacin, ofloxacin, ciprofloxacin, pefloxacin, gatifloxacin
  • nitroimidazole antibiotics including metronidazole, tinidazole, ornidazole, etc.
  • rifamycin Steroid antibiotics including rifampicin; echinocandin antibiotics; polyene antibiotics; pyrimidine antibiotics; ally
  • the biomedicine described in the present invention is antimicrobial peptide, and described antimicrobial peptide is selected from insect antimicrobial peptide, for example, Lepidoptera antimicrobial peptide, Diptera antimicrobial peptide, Coleoptera antimicrobial peptide, Odonata antimicrobial peptide, Hymenoptera antimicrobial peptide Peptides, silkworm antimicrobial peptides, etc.; mammalian antimicrobial peptides, such as porcine antimicrobial peptides, sheep antimicrobial peptides, bovine antimicrobial peptides, human antimicrobial peptides, etc.; amphibian antimicrobial peptides: magainin, etc.; fish, mollusks, crustaceans Source of antimicrobial peptides: leopard sole antimicrobial peptides, catfish antimicrobial peptides, mussel antimicrobial peptides, shrimp antimicrobial peptides, etc.; plant antimicrobial peptides: Thi
  • the natural medicine described in the present invention is selected from astragalus, sealwort, angelica, notoginseng, rhubarb root, rhubarb charcoal, turmeric, zhebei, barley, pinellia, calcined ancient ink, salvia miltiorrhiza, comfrey, isatidis, houttuynia , honeysuckle, coptis, skullcap, dandelion, purslane, hawthorn, Folium Folium, forsythia, fungus Chen, andrographis paniculata, Bupleurum, Dijincao, hundred leaves, garlic, Phellodendron, Eucommia, Qinpi, Cnidium, Coptis, One or two or more of gallnut, viola, ebony, licorice, pomegranate peel, schisandra, saponins, myrobalan, flavescens, hibiscus bark, epimedium, artemis
  • the medicine described in the present invention may be oral medicine, injection medicine or external medicine, and the medicine for external use includes medicine for mucosal administration, preferably medicine for ophthalmic administration.
  • the dosage form of the medicine of the present invention can be solution, tablet, pill, capsule, injection, powder, powder for injection, patch, paint, mucosal administration preparation, and described mucosal administration preparation is preferably eye drops , eye ointments, eye spray preparations, etc.
  • Figure 1 Genotype and phenotype of Crb1 rd8/rd8 (rd8)-SPF mice.
  • Figure 1A is the genotype gel electrophoresis of Crb1 rd8/rd8 (rd8) and Crb1 wt/wt (C57BL/J, referred to as wt) mice
  • Figure 1B is the representative of Rd8-SPF and WT-SPF mice
  • Figure 1C shows the H&E staining of the pathological changes in both eyes of Rd8-SPF mice at E18.
  • Figure 2 Potential mechanism of retinal abnormalities and retinopathy in Rd8-SPF mice.
  • Figure 2A shows the pathological changes of the eyes of Rd8-SPF and WT-SPF mice at E18, P12 (before opening), P15 (after opening) and 8 weeks (8W) by H&E staining.
  • Rd8 mice showed typical retinal Abnormalities, including progressive retinal dysplasia (ruffles and false rosettes) and degeneration, whereas WT mice exhibited normal retinal architecture;
  • Figure 3 Comparison of gene expression profiles in the upper and lower retinal regions of Rd8-SPF mice by RNA-seq analysis.
  • Figure 3A is a diagram of the upper (no lesion) and lower (with lesion) regions of Rd8-SPF mice;
  • Figure 3B shows that RNA-seq analysis found that there is at least 2 times expression in the upper and lower retinal regions of Rd8-SPF mice Difference (P ⁇ 0.05);
  • Figure 3C shows the expression patterns of 179DEGs in the upper and lower retinal regions of Rd8- and WT-SPF mice.
  • Figure 4 Identification of bacteria within retinopathy of Rd8-SPF mice.
  • Figure 4A principal coordinates analysis (PCoA), showing that the bacterial composition in retinal tissues of Rd8 mice is significantly different from that of WT mice;
  • Figure 4C is the detection of retinal bacteria 16srDNA and vanco-bodipy in Rd8- and WT-SPF mice (4 weeks old) by fluorescence in situ hybridization;
  • Figure 4D is the observation of Rd8 by transmission electron microscope (TEM) The distribution of bacteria in the mouse retinopathy, the positive control is the coliform group;
  • TEM transmission electron microscope
  • Figure 5 Disruption of retinal adherens junctions in Rd8-SPF mice.
  • Figure 5A is the immunofluorescence staining of Rd8- and WT-SPF mouse retina CRB1 (red), BM is Bruch's membrane, CC is chorionic capillary, BL is basement membrane;
  • Figure 5B is Rd8- and WT-SPF small TEM observation of adherent junctions in the external membrane of the mouse retina, the red arrows are adherens junctions, AJ refers to adherens junctions, OLM refers to the external membrane, ONL refers to the outer nuclear layer, IS refers to the inner segment;
  • Figure 5C is the transmission electron microscope observation of Rd8-SPF Mouse retinal pigment epithelium (RPE) basal layer adhesion dissolution, chorionic capillary basal layer distortion, collagen layer disorder, CC is chorionic capillary, CH is choroid, BL is basement membrane;
  • Figure 5D shows Rd8
  • Fig. 6A Adhesive junction disruption (AJ) and outer nuclear layer (ONL) of Rd8-SPF mice outer membrane (OLM) shown for transmission electron microscopy (TEM).
  • AJ Adhesive junction disruption
  • ONL outer nuclear layer
  • TEM transmission electron microscopy
  • Fig. 6B Brunch membrane thickness of Rd8-SPF mice and WT-SPF mice shown by transmission electron microscope (TEM).
  • Figure 7 Defective colonic epithelial barrier and its associated inflammation in Rd8-SPF mice.
  • Figure 7B-D is the CRB1 protein of colonic enterocytes of Rd8- and WT-SPF mice (B) , Immunofluorescent staining of phalloidin (C) and occludin (D)
  • Figure 7E is the statistical analysis of the relative intensity of occludin in Rd8- and WT-SPF mice
  • Figure 7F is the westernblot detection of Rd8- and WT-SPF
  • Figure 7G is the statistical analysis of the relative intensity of Claudin1 in Rd8- and WT-SPF mice
  • Figure 7H is the transmission electron microscope observation of the adherent junction and Tight junctions
  • Figure 7I-L is the
  • Figure 8 Microbial species composition of the flora in different parts of the gastrointestinal tract of Rd8-SPF mice.
  • PCoA principal coordinate analysis
  • 8G is PCoA distinguishing the lower digestive tract (cecum, Microbial composition (H-J) of the colon and rectum) comparing the relative abundance of Akkermansia mucilage in the cecum (H), colon (I), and rectum (J) of Rd8 and WT mice. Data are expressed as mean ⁇ SEM *P ⁇ 0.05.
  • Figure 9 Tight junctions and adherens junctions of Rd8 mouse cecum.
  • Figure 9A is the immunofluorescence staining of Rd8- and WT-SPF mouse cecum enterocyte CRB1 protein (green);
  • Figure 9B is the immunofluorescence staining of Rd8- and WT-SPF mouse cecum enterocyte occlusivein (red);
  • Figure 9C is the statistical analysis of the relative intensity of occludin in Rd8- and WT-SPF mice *P ⁇ 0.05;
  • Figure 9D is the westernblot detection of the expression of Claudin1 in the cecum of Rd8- and WT-SPF mice;
  • Figure 9E is the expression of Rd8- and WT-SPF Statistical analysis of the relative intensity of mouse Claudin1, NS stands for not important;
  • Figure 9F shows transmission electron microscopy showing normal tight junctions and adherens junctions at the cecal epithelial barrier, MVs are microvesicles, and MCs are mitochondria
  • Figure 10 is the transmission electron microscope observation of the adherens junction (AJ) between the colonic epithelium and the tight junction (TJ) of Rd8- and WT-SPF mice, MV is the microvesicle, and MC is the mitochondria;
  • Figure 10B is the flow cytometry The frequency of fluorescence+ bacteria/cell in the peripheral blood of Rd8 and WT mice was detected.
  • Figure 11 Disruption of intestinal epithelial barrier function in Rd8-SPF mice.
  • Figure 11A shows the intestinal FICT-dextran permeability assay shows that the serum fluorescence intensity of Rd8 mice is significantly higher than that of WT mice***P ⁇ 0.001
  • Figure 11B shows the comparison of vanco-bodipy labeled fecal microbiota transplantation Percentage of fluorescence + bacteria/cell in peripheral blood of Rd8 and WT mice *P ⁇ 0.05
  • Figure 11C-D shows that both under the fluorescence microscope (C) and immunofluorescence staining (D) can be detected in the retinopathy of Rd8 mice vanco-bodipy+bacteria
  • Figure 11F-G compares the colon length, serum bacterial 16srrna (F) and bacterial LPS (G) levels of Rd8 and WT mice after DSS treatment for 13 days ****P ⁇ 0.0001
  • Figure 12 Retinal phenotypes of Rd8 GF and Rd8 GF SPF mice.
  • Figure 12A is the representative fundus images of Rd8-SPF and Rd8 GF mice at 4 weeks, 8 weeks, 12 weeks and 16 weeks;
  • Figure 12B is the retinal tissues of E18, P12, P15, and 8W eyes of Rd8-GF mice Medical observation;
  • Figure 12C is fluorescence in situ hybridization staining of bacterial 16srDNA and vanco-bodipy in Rd8-GF mouse retina;
  • Figure 12D is immunofluorescent staining of IBA1 (red) in retina of Rd8-SPF, Rd8 GF and WT-SPF mice ;
  • Figure 12E is the percentage of IBA1+ microglial cells in ONL of Rd8-SPF, Rd8 GF and WT-SPF mice ***P ⁇ 0.001;
  • NS represents not important;
  • Figure 12F is Rd8-SPF, Rd8-
  • C57BL/6N mice Crb1 rd8/Rd8 , named Rd8 mice
  • C57BL/6J mice Crb1 wt/wt , named wt mice carrying the Rd8 mutation
  • SPF pathogen-free
  • GF Germ-free RD8 mice were generated from embryos of female RD8 mice at the Animal Facility of the First affiliated Hospital of Sun Yat-sen University. GF mice were kept sterile, and facility staff performed weekly microbiological and parasite testing of fecal samples to ensure the sterility of the GF unit.
  • mice were sacrificed by cervical dislocation, eyeballs were enucleated, and fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4°C for 24 hours.
  • PFA paraformaldehyde
  • PBS phosphate buffered saline
  • Samples were washed 3 times with PBS, dehydrated in a series of alcohols and 2 times in xylene, then embedded in paraffin and serially sectioned at 10 ⁇ m with a microtome (RM 223; Leica, Wetzlar, Hesse-Darmstadt, Germany ). Sections were stained with hematoxylin and eosin (H&E). H&E images were acquired by Imager.Z2 (Zeiss).
  • Tissue sections were blocked with 10% donkey serum/PBST (0.1% tritonx-100/PBS) for 30 minutes and then incubated with primary antibodies overnight at 4°C. After washing with PBST, sections were incubated with fluorochrome-conjugated secondary antibodies and mounted with Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Phalloidin (A12379; Thermo-Fisher) staining was performed using the same immunohistochemical method, except that the secondary antibody was omitted.
  • a Zeiss confocal microscope Zeiss LSM880; Zeiss, Oberkochen, Germany
  • Imager.Z2 equipped with an ApoTome was used.
  • the primary antibodies used in this study were: anti-Crb1 (PA5-66373, ThermoFisher; 1:50), anti-Iba1 (ab178846, Abcam; 1:500), anti-ZO-1 (61-7300, ThermoFisher; 1:500 ), anti-Occludin (OC-3F10, Invitrogen; 1:200), AlexaFluor 488phalloidin (A12379, ThermoFisher; 1:500).
  • mice were anesthetized with dilated pupils. Regularly apply hypromellose eye drops to keep the cornea moist.
  • Mouse fundus photographs were obtained using a Micron IV mouse fundus camera (Phoenix Research Laboratories, Inc., Pleasanton, CA, USA).
  • Raw reads were first quality control assessed by FastQC (v0.11.8) and cutadapt (v1.15). Clean reads were aligned to the mouse genome (mm10) using HISAT2 (v2.1.0).
  • the gene expression data were imported into the DESeq2 package of R software (v3.6.1) for differential expression analysis. Differentially expressed genes (DEGs) were imported into intelligent pathway analysis (IPA) for functional enrichment analysis.
  • DEGs Differentially expressed genes
  • Retinal samples were collected and DNA was extracted using the MasterPure TM Intact DNA and RNA Purification Kit (epicentre). The contents of the stomach, jejunum, ileum, cecum, colon and rectum were collected and DNA was extracted using the QIAamp PowerFecal DNA kit (QIAGEN). After concentration measurement, DNA was subjected to sequencing library preparation using VAHTS TM MGI Universal DNA Library Prep Kit (Vazyme, China) according to the standard protocol provided by the manufacturer. Metagenome sequencing was performed with MGISEQ-2000RS. Raw reads were quality filtered by Trimmomatic (v0.36) and PRINSEQ (v0.20.4).
  • KneadData (v0.6.1) (https://bitbucket.org/biobakery/kneaddata).
  • Non-mouse-depleted reads were mapped to a pre-built MiniKraken database using Kraken 2 (v2.0.9). Classification results were screened with a confidence level of 0.20.
  • a negative blank control was processed with the samples. All species present in the negative blank control group were removed.
  • In vivo permeability assays were performed using the FITC-dextran method to evaluate barrier function. Food and water were removed overnight, and 8-week-old mice were orally administered 50 mg of FITC-labeled dextran (FD-70; Sigma-Aldrich) per 100 g (body weight). Serum was collected 5 hours after administration, and the fluorescence intensity (excitation, 492nm; emission, 525nm) of each sample was measured.
  • mice WT and Rd8 mice were fasted overnight and given 1 x 109 E by intragastric administration.
  • E. coli designed to consistently express RFP.
  • the mice were euthanized and 400 ⁇ L of peripheral blood was gently pipetted into a tube containing 4 mL of ACK lysis buffer (Gibco, USA) and incubated at RT for 3–5 min. After centrifugation at 300 x g for 5 min, cells were fixed and permeabilized (Cytofix/perm solution, BD Biosciences, USA) and then analyzed by flow cytometry (MACSQuant Analyzer 10, Miltenyi Biotec, Germany).
  • Colons and eyes were collected immediately after euthanasia and fixed in phosphate-buffered glutaraldehyde-paraformaldehyde solution for 1 h at room temperature. Colons were cut into 2 mm pieces. The anterior segment was resected, and the posterior segment was cut into 2mm ⁇ 2mm blocks. The dissected tissues were placed in fresh fixative for 12 hours, fixed with 1% osmium tetroxide, dehydrated and embedded in epon-resin. Regions of interest were pre-screened on micrometer thick sections stained with toluidine blue under a light microscope. Ultrasound images were then collected at 80 nm and counterstained with uranyl acetate and lead citrate. Ultrasonic sections were observed with a transmission electron microscope.
  • oligonucleotide probe was used in this study: EUB338, 5'-GCTGCCTCCGTAG-GAGT-3' (Amann et al., 1990). The 5' end of the probe bears a primary amino group to which tetramethylrhodamine isothiocyanate is covalently bonded.
  • Dye-oligonucleotide conjugates (100 ⁇ ) were stored at -20°C.
  • Prefixed retinal sections were rinsed 3 times with DEPC-treated PBS. After treatment with 0.2% Triton X-100/DEPC-treated PBS, sections were hybridized with probe (500 nM) overnight at 37°C and mounted with Fluoromount-G.
  • Plasma LPS concentration was measured with an enzyme-linked immunosorbent assay kit (SEB526Ge; Cloud-Clone Corp., USA).
  • Fresh stool samples were collected in 50 ml conical tubes with sterile 1xPBS and spun down until homogeneous. The contents were filtered with a 0.22 ⁇ m filter (Millipore) to remove fecal residues and centrifuged to obtain gut microbiota before incubation with vanco-bodipy for 30 min at RT. Gut bacteria labeled with vanco-bodipy were gavaged at 1x108 cfu/mouse in PBS. After gavage for 24 hours, the mouse retinal slices were taken for observation.
  • 2.5% DSS MW 36000–50000d, Yeasen, China
  • FIG. 1A The characteristics of the retinal microenvironment of Crb1rd8/rd8 (rd8) and Crb1wt/wt (C57BL/J, named wt) mice were observed (Fig. 1A). Phenotyped in Rd8 mice raised in a specific pathogen-free (Rd8-SPF) environment, they exhibited all the typical retinal findings, including leukoplakia in the inferior nasal quadrant of the eye on fundoscopy (Fig. 1B), and progressive retinal Dysplasia (ruffles and false rosettes) and retinal histological degeneration (H&E staining) ( Figure 2A). The results showed that 7 out of 32 Rd8 mice developed mild retinal dysplasia (E18) before birth (Fig.
  • mice revealed that seven bacterial species were significantly enriched in the subretinal region, including Taenia hadrus, Pseudomonas Streptococcus Bifidobacterium pseudocatenulatum, Megamonas funiformis, Nitrosomonas Is79A3, valerian-producing Oscillibacter, Tatumella sp.TA1, and Thiobacillus denitrificans (Figure 4B).
  • Example 5 demonstrates that the major defect in the Rd8-SPF mouse retina is the outer blood-retinal barrier
  • Immunofluorescence staining data confirmed reduced or absent expression of CRB1 protein in the outer retinal membrane of Rd8 (Fig. 5A). The data confirmed that weak expression of CRB1 protein could be found on Bruch's membrane (Fig. 5A). Using transmission electron microscopy, it was found that in the retinopathy site of Rd8-SPF mice, the adherens junctions on the external membrane were disrupted, which was related to the outward migration of the outer nuclear layer (Fig. 5B and Fig. 6A).
  • CRB1 protein in cecum enterocytes of wild-type mice was identified by immunofluorescence staining, while its expression was significantly attenuated in Rd8 mice (Fig. 9A).
  • Fig. 9B-C a significant loss of Occludin
  • Fig. 9D-E Claudin1
  • CRB1 protein was found to be significantly expressed on the apical and basal surfaces of colonic enterocytes (Fig. 7B). Loss of CRB1 protein was associated with significantly reduced expression of Phalloidin (Fig. 7C) and Occludin (Fig. 7D and 4E), but not Claudin1 (Fig. 7F and 4G) protein.
  • Fig. 7C Phalloidin
  • Fig. 7D and 4E Occludin
  • Fig. 7F and 4G Claudin1
  • Example 8 Examining whether intestinal epithelial barrier defects lead to altered intestinal permeability and microbiota like peripheral blood flow and retinal tissue migration in Rd8 mice
  • Intestinal FICT-dextran permeability assays were performed on WT and Rd8 mice. As shown in FIG. 11A , compared with the fluorescence intensity in the blood of WT mice, the fluorescence in the peripheral blood of Rd8 mice increased significantly 5 hours after administration of FICT dextran. Furthermore, using Vancompy-labeled fecal microbiota transplantation assays, a significant increase in fluorescence+ bacteria/cell was found in the peripheral blood of Rd8 mice compared with WT mice at 24 h after fecal transplantation (Fig. 10B and Fig. 11B). Importantly, the presence of Vancomopy+ bacteria was detectable in Rd8 mouse retinopathy (Fig.
  • Rd8 mice were re-isolated under sterile (GF) conditions and tested for changes in their retinal degeneration phenotype.
  • GF sterile
  • FIG. 12A retinopathy (white spots) found in Rd8-SPF mice was hardly observed in germ-free Rd8(Rd8 GF) mice.
  • Retinal histology of Rd8-GF mice showed normally developing retinal tissue (Fig. 12B) without the typical lesions found in Rd8-SPF mice (Fig. 2A and Fig.

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Abstract

提供一种建立眼部疾病模型的方法，包括将微生物感染到模型载体上。以及提供了用这种方法制备的感染了微生物的眼部模型载体。所述眼部模型载体可用于眼部疾病研究、眼部疾病药物的筛选。其中，所述眼部疾病为视网膜变性，所述微生物为肠道细菌或与肠道细菌相同的细菌。

Description

一种建立眼部疾病模型的方法及其应用 技术领域
本发明属于眼科疾病的诊断与治疗技术领域，具体涉及一种建立眼部疾病模型的方法和通过该方法得到的眼部疾病模型和模型载体在眼部疾病研究、药物筛选中的应用。
背景技术
遗传性视网膜变性(IRD)是一组遗传性疾病，是工业化国家劳动人口视力下降的最常见原因。遗传性视网膜变性(IRD)是一组以光感受器细胞进行性丢失为特征的遗传性疾病，包括先天性黑蒙(LCA)、色素性视网膜炎(RP)、早发性视杆-视锥细胞营养不良、视杆-视杆营养不良、先天性静止性夜盲和色盲，和Stargardt病(Broadgate等人，2017年)。它是工业化国家工作人口视力丧失的最常见原因，估计发病率为1:2000(Kutluer等人，2020年)。由于遗传和临床的异质性，IRDs的治疗需要高度个性化的治疗策略。虽然神经保护、基因治疗和细胞替代治疗是治疗不同阶段IRD的拟议方法，但到目前为止，只有一种纠正RPE65基因突变的基因治疗(Luxturna)被FDA批准用于治疗LCA(Botto等人，2021；Ikelle等人，2020年；Kutluer等人，2020年)
超过200个基因与IRD有关。其中，CRB1基因长期以来被认为是一个重要的基因，其突变导致多种眼科表型，包括LCA和RP(Ehrenberg et al.，2013)。CRB1作为粘附分子连接的关键调节因子，在建立细胞极性和维持眼屏障完整性方面起着重要作用。IRD引起CRB1突变的频率在世界范围内有很大差异。例如，在LCA病例中发现的CRB1突变等位基因频率在美国为6.7％，在加拿大为1.7％，在荷兰为0％，在西班牙为16.7％，在中国为11.5％(Li等人，2011；Vallespin等人，2007年；Zernant等人，2005)，而CRB1是西班牙所有LCA、早发性RP和非早发性RP患者中发现的突变频率最高的基因(Vallespin等人，2007)。总的来说，CRB1基因突变解释了全球4％的RP和10-15％的LCA(Richard et al.，2006)。因此，迫切需要一种挽救CRB1相关视力丧失的治疗方法。
发明内容
本发明的一个目的是提供一种建立眼部疾病模型或眼部疾病模型载体的方法，尤其是视网膜变性模型或模型载体的方法及通过所述方法建立的模型或模型载体，以及所述的方法、疾病模型和/或疾病模型载体在眼部疾病研究、相关药物筛选方面的应用。
本发明的目的是通过以下技术方案实现的：
本发明提供一种建立眼部疾病模型的方法，所述方法包括让眼部疾病模型感染微生物。
优选的，所述的感染包括直接与微生物接触或者间接与微生物接触。在一个具体实施方式中，是通过将非人动物饲养于SPF环境中获得眼部感染了微生物的眼部疾病模型。
优选的，所述的微生物来自同一个体的肠道细菌或者与该个体的肠道细菌相同。
优选的，所述的眼部疾病包含视网膜变性；更优选的，所述的视网膜变性为进行性视网膜变性。
优选的，所述的视网膜变性为遗传性视网膜变性(IRD)。
优选的，所述的眼部疾病包含LCA、RP、arRP、EORD、EORP、PPRPE、rettelangiectasia和/或choroideremia like fundus。
优选的，所述的眼部疾病包含眼部炎症，例如葡萄膜炎、青光眼、和年龄相关的黄斑变性(AMD)、玻璃体炎，脉络膜炎、视网膜炎，视网膜血管炎，视神经炎，还有葡萄膜炎、白塞病、伏格特-小柳-原田综合征、葡萄膜炎、视网膜病变、交感性眼炎、白内障、结膜炎、青光眼等。
优选的，所述的模型是非人动物，优选猴、犬、黑猩猩、大鼠、小鼠。
优选的，所述的模型载体是细胞、组织或器官，所述的细胞、组织或器官来源于人或非人动物。
优选的，所述的细胞为原代细胞或细胞系。
优选的，所述的组织为眼部组织，所述的器官为眼部器官。
优选的，所述的组织或器官为再生组织或器官。
优选的，所述的模型存在基因的致病突变。
优选的，所述的发生了致病突变的基因为与维持视网膜屏障结构有关的基因，所述的视网膜屏障为外血-视网膜屏障和/或内血-视网膜屏障。
优选的，所述的模型或模型载体中存在致病的眼部疾病相关基因的突变，所述的发生了致病的眼部基因突变的基因选自以下一个或两个以上基因的组合：ABCA4、ABCC6、ABCC9、ACBD5、ACO2、ACO2、ACTG1、ADGRV1、AHI1、AIPL1、ALMS1、AMY2B、APC、ARFGEF1、ARL13B、ARL13B、ARL6、ARMC9、ATOH7、B9D1、BAG3、BBS1、BBS1、BBS2、BBS5、BEST1、C2CD3、CA4、CABP4、CACNA1F、CBS、CC2D2A、CDH23、CDH23、CDHR1、CEMIP2、CEP104、CEP250、CEP290、CEP290、CEP41、CEP78、CERKL、CFAP410、CFAP418、CHM、CLCC1、CLCN7、CLN3、CLN5、CLN8、CLRN1、CLRN1、CNGA1、CNGA1、CNGA3、CNGB1、CNGB3、CNNM4、COL11A1、COL11A2、COL18A1、COL2A1、COL4A1、COL9A1、COL9A2、CP、CP、CPLANE1、CRB1、ERCC4、CSPP1、CTNNA1、CYP4V2、DHDDS、DYNC2H1、DYNC2I1、DYNC2I2、ENPP1、ERCC4、EVC2、EYS、EYS、F5、FAM161A、FBN1、FKRP、FKTN、FLG、FLVCR1、FOXE3、FUZ、GLB1、GMPPB、GNAT1、GRK1、GRM6、GUCA1A、GUCA1B、GUCY2D、HADHA、HGSNAT、HPS3、HPS5、IDH3B、IFT122、IFT140、IFT140、IFT43、IFT52、IFT74、IFT80、IFT80、IFT81、IFT88、IKBKG、IMPDH1、IMPG2、INPP5E、INTU、IQCB1、IQCE、IREB2、KCNJ13、KCNQ1、KCNV2、KIAA0586、KIAA0753、KIF7、KIZ、KIZ-AS1、KLHL7、KRIT1、LBR、LCA5、LOC101927157、 LOC111365204、LRP2、LRP5、MAK、MAPKAPK3、MATK、MCOLN1、MERTK、MKS1、MPDZ、MT-ATP6、MT-CO3、MT-TE、MT-TL1、MTHFR、MUTYH、MYO7A、MYO7A、NMNAT1、NPHP1、NR2E3、OCA2、OTX2、PANK2、PAX6、PCARE、PCDH15、PDE6A、PDE6B、PDE6B、PDE6D、PEX1、PEX1、PEX12、PEX26、PEX6、PHF3、PITPNM3、PKD2、PLA2G5、POC5、POMT1、PRCD、PRDM13、PROM1、PRPF3、PRPF31、PRPF8、PRPH2、RAD51C、RBP3、RBP4、RD3、RDH12、RDH5、RGR、RGR、RHO、RIMS1、RLBP1、ROM1、RP1、RP1L1、RP2、RPE65、RPE65、RPGR、RPGRIP1、RPGRIP1L、RS1、SACS、SAG、SCAPER、SDCCAG8、SIX6、SLC19A1、SLC22A5、SLC26A4、SLC2A9、SLTM、SNRNP200、SPAG17、SPATA7、SPG11、TFAP2A、TGFB2、TGFBR2、TMEM107、TMEM237、TMEM67、TOGARAM1、TOPORS、TPP1、TRAF3IP1、TREX1、TRIM59-IFT80、TSPAN12、TTC21B、TTC21B、TTC8、TULP1、USH1C、USH2A、USH2A、USH2A、USH2A、USH2A-AS1、VAC14、VCAN、VCAN、VCAN-AS1、VHL、VPS13B、WDR19、WDR19、WDR35、WDR73、YARS1、ZFYVE26、ZFYVE26、ZNF408。
在一个具体实施方式中，所述的模型或模型载体发生了致病突变的眼部疾病相关的基因中包含CRB1基因。
优选的，所述的模型或模型载体的CRB1基因的突变包含如下的一种或两种以上的突变：c.107C>G、c.111delT、c.135C>G、c.257_258dupTG、c.258C>T、c.428_432delGATTC、c.430T>G、c.470G>C、c.481dupG、c.482C>T、c.584G>T、c.613_619del、c.717_718insG、c.750T>G、c.915T>A、c.929G>A、c.936T>G、c.998G>A、c.1084C>T、c.1125C>G、c.1148G>A、c.1208C>G、c.1269C>A、c.1298A>G、c.1313G>A、c.1438T>C、c.1438T>G、c.1576C>T、c.1604T>C、c.1690G>T、c.1733T>A、c.1750G>T、c.1760G>A、c.1834T>C、c.1963delC、c.2025G>T、c.2042G>A、c.2128G>C、c.2129C>T、c.2185_2186insAlu、c.2219C>T、c.2222T>C、c.2234C>T、c.2245_2247del 3bp(TCA)、c.2258T>C、c.2290C>T、c.2365_2367del AAT,in frame deletion、c.2401A>T、c.2438_2439ins>100A、c.2441_2442del、c.2465G>A、c.2479G>T、c.2506C>A、c.2509G>C、c.2536G>A、c.2548_2551delGGCT、c.2548G>A、c.2555T>C、c.2611_2613insT、c.2671T>G、c.2676delG、c.2681A>G、c.2688T>A、c.2816G>A、c.2843G>A、c.2853dupT、c.2884_2886delTTA、c.2957A>T、c.2966T>C、c.2983G>T、c.3002A>T、c.3008T>C、c.3035T>C、c.3037C>T、c.3074G>A、c.3074G>T、c.3122T>C、c.3212T>C、c.3296C>A、c.3299T>C、c.3299T>G、c.3307G>A/C、c.3320T>C、c.3320T>G、c.3331G>T、c.3343_3352del、c.3347delT、c.3343_3352del、c.3347delT、c.3427delT、c.3482A>G、c.3493T>C、c.3655T>G、c.3541T>C、c.3542dupG、c.3593A>G、c.3613G>A、c.3653G>T、c.3659_3660delinsA、c.3664C>T、c.3668G>C、c.3676G>T、c.3713_3716dup、c.3879G>A、c.3914C>T、c.3949A>C、c.3961T>A、c.3988delG、c.3988G>T、c.3995G>T、c.3996C>A、c.3997G>T、c.4094C>A、c.4121_4130del、c.4142C>T、c.4148G>A、c.2128+2T>G、c.2842+5G>A、c.3878+1G>T、c.4005+1G>A、c.4005+2T>G、c.4006-2A>G、c.4006-1G>T、c.619G>A、 c.614T>C、c.1472A>T、c.1903T>C、c.2809G>A、c.3103C>T、c.4082G>A、c.4060G>A、c.866C>T、c.1463T>C、c.2035C>G、c.2306_2307GC>AG、c.2306G>A、c.2714G>A、c.2875G>A、c.3992G>A。
进一步优选的，所述的模型或模型载体的CRB1基因的突变包含如下的一种或两种以上的突变：c.4006-1G>T、c.3686G>C、(p.Cys1229Ser)、c.2842+1delinsAA、c.4060G>A、(p.Ala1354Thr)、c.3991C>T、(p.Arg1331Cys)、c.3014A>T、(p.Asp1005Val)、c.4005+1G>A、c.2680_2684del、(p.Asn894fs)、c.1733T>A、(p.Val578Glu)、c.455G>A、(p.Cys152Tyr)、c.3462_3463del、(p.Cys1154_Glu1155delinsTer)、c.3037C>T、(p.Gln1013Ter)、c.2673C>A、(p.Cys891Ter)、c.2230C>T、(p.Arg744Ter)、c.3676G>T、(p.Gly1226Ter)、c.2842+5G>A、c.2842T>C、(p.Cys948Arg)、c.3988del、(p.Glu1330fs)、c.2506C>A、(p.Pro836Thr)、c.2291G>A、(p.Arg764His)、c.1576C>T、(p.Arg526Ter)、c.613_619del、(p.Ile205fs)、c.3320T>C、(p.Leu1107Pro)、c.2688T>A、(p.Cys896Ter)、c.2555T>C、(p.Ile852Thr)、c.2222T>C、(p.Met741Thr)、c.1148G>A、(p.Cys383Tyr)、c.2843G>A、(p.Cys948Tyr)、c.4121_4130del、(p.Ala1374fs)、c.3307G>A、(p.Gly1103Arg)、c.484G>A、(p.Val162Met)、c.2401A>T、(p.Lys801Ter)、c.2234C>T、(p.Thr745Met)、c.2290C>T、(p.Arg764Cys)、c.3122T>C、(p.Met1041Thr)。
优选的，所述的模型或模型载体的CRB1基因的突变为Rd8突变。
优选的，所述的突变为纯和突变或杂合突变。
优选的，所述的模型或模型载体体内先天存在如前所述的基因的突变或者所述的突变是由于基因重组操作而后天携带的。
优选的，所述的模型或模型载体体内存在人源化CRB1基因或人CRB1基因，其内源CRB1基因缺失或不表达。
优选的，所述的非人动物具有结肠上皮屏障缺陷和/或相关的结肠壁炎症。
在一个具体实施例中，所述的模型体内Occludin蛋白的显著缺失，在一个具体实施例中，所述的模型体内Occludin蛋白显著缺失和Claudin1表达不明显。
所述微生物为细菌、古细菌、原生生物、真菌或病毒中的一种或两种以上的组合，优选的，所述微生物为细菌，所述细菌选自：Anearostipes、双歧杆菌(Bifidobacterium)、巨单胞菌(Megamonas)、亚硝基单胞菌(Nitrosomonas)，颤螺旋菌(Oscillibacter)、塔特姆菌(Tatumella)、硫杆菌(Thiobacillus sp.)、梭菌属(Clostridium)、不动杆菌属(Acinetobacter)、链球菌属(Streptococcus)、曼氏杆菌属(Mannheimia)、纤维杆菌属(Fibrobacter)、普氏菌属(Prevotella)、弯曲杆菌属(Campylobacter)、放线菌属(Actinomyces)、薄层菌属(Hymenobacter)、埃希氏杆菌属(Escherichia)、泰氏菌属(Tissierella)、克雷白氏杆菌属(Klebsiella)、卟啉单胞菌属(Porphyromonas)、固氮螺菌属(Azospira)、海水菌属(Aquimarina)、无色菌属(Achromobacter)、嗜酸硫杆菌属(Acidithiobacillus)、伯克霍尔德菌属(Burkholderia)、海杆菌属(Marinobacter)、密螺旋体属(Treponema)、孢裹放线菌属(Actinosporangium)、 弧菌属(Vibrio)、瘤胃球菌属(Ruminococcus)、甲烷短杆菌(Methanobrevibacter)、志贺氏杆菌(Shigella)、弗兰克氏菌属(Frankia)、链霉菌属厌氧原体属(Anaeroplasma)、粪球菌属(Coprococcus)中的一种或两种以上。
具体的，所述细菌选自：Anearostipes hadrus、Bifidobacterium pseudocatenulatum、Nitrosomonas sp.Is79A3、Oscillibacter valericigenes、Tatumella sp.TA1、Megamonas funiformis、Thiobacillus denitrificans、破伤风梭菌、产气荚膜梭菌、肉毒梭菌、醋酸钙不动杆菌、鲁菲不动杆菌、鲍曼不动杆菌、溶血不动杆菌、琼氏不动杆菌、约翰逊不动杆菌、化脓性链球菌、溶血性链球菌、产琥珀酸丝状杆菌、肠道丝状杆菌、非解糖卟啉单胞菌、牙髓卟啉单胞菌、牙龈卟啉单胞菌、空肠弯曲菌、结肠弯曲菌、海鸟弯曲菌、乌普萨拉弯曲菌、简明弯曲菌、胎儿弯曲菌、衣氏放线菌、奈氏放线菌、溶齿放线菌、粘稠放线菌、纽氏放线菌、大肠埃希菌、蟑螂埃希菌、弗格森埃希菌、赫尔曼埃希菌、伤口埃希菌、极尖泰氏菌、肺炎克雷伯氏菌、臭鼻克雷伯氏菌、巴西固氮螺菌、无色杆菌、脱氮硫杆菌、氧化亚铁硫杆菌、氧化硫硫杆菌、那不勒斯硫杆菌、伯克霍尔德菌、海洋分枝杆菌、苍白密螺旋体、猪痢疾密螺旋体、梅氏弧菌、白色瘤胃球菌、生黄瘤胃球菌、瘤胃甲烷短杆菌、痢疾志贺菌、福氏志贺菌、鲍氏志贺菌、宋内志贺菌、弗兰克氏菌、规则粪球菌、白色链霉菌、门多萨假单胞菌，栖息微球菌，脂环素反硝化菌，氧化木糖无色杆菌，鞘脂单胞杆菌，脓肿分枝杆菌，金黄节杆菌，普氏菌，草木樨中华根瘤菌，酸性酵母，表皮葡萄球菌，铜绿假单胞菌，金黄色葡萄球菌，溶血性葡萄球菌，恶臭假单胞菌，嗜麦芽寡养单胞菌，蜡状芽孢杆菌，巨大芽孢杆菌，罗氏乳酸杆菌，阴道嗜血杆菌，蜜蜂屎肠球菌，正哈氏嗜纤维菌，地衣芽孢杆菌，白叶枯菌，鲍曼不动杆菌，醋酸钙不动杆菌，睾丸酮丛毛单胞菌，堪萨斯分枝杆菌，斯润金芽孢杆菌，柯氏柠檬酸杆菌，发酵成对杆菌，粘质沙雷氏菌，维氏鞘氨醇单胞菌，肺炎克雷伯菌，荧光假单胞菌，皮氏罗尔斯顿菌，卷曲乳杆菌，伯克霍尔德菌，德氏乳杆菌，Meiothermus silvanus(D)，大肠杆菌，藤黄微球菌，枯草芽孢杆菌，粘金色棒状杆菌，大芬戈尔德菌中的一种或两种以上。
优选的，所述的感染方法包含是让微生物与模型载体待感染的部位直接接触或间接接触，所述的间接接触是指所述的微生物和待感染的部位之间存在血视网膜屏障，优选的，为外血-视网膜屏障或内血-视网膜屏障。
在一个具体实施方式中，所述的感染方法包含通过外周血使肠道细菌感染眼部。具体的，非人动物模型的肠上皮屏障收到显著损伤，致使肠道细菌进入外周血。在一个模型体内，视网膜屏障也收到了显著受损，使得进入外周血的肠道细菌感染视网膜；在一个具体实施方式中，所述的非人动物模型发生了CRB1基因的突变；在一个具体实施方式中，所述的CRB1基因的突变是Rd8突变。
进一步，本发明提供一种制备眼部炎症模型的方法，所述的方法模型为非人动物感染微生 物。
优选的，所述的眼部炎症是由肠道菌群或与肠道菌群相同的菌群引起的。
所述的非人动物为如前所述的动物。
所述的微生物为如前所述的微生物。
进一步，本发明提供一种制备视网膜变性疾病模型的方法，所述的方法包括使患有视网膜变性的非人动物感染微生物。
优选的，所述的眼部炎症视网膜变性疾病为如前所述的视网膜疾病。
所述的非人动物为如前所述的动物。
所述的微生物为如前所述的微生物。
进一步，本发明提供一种有眼部炎症的模型载体，所述的眼部炎症是由微生物感染引起的。
优选的，所述的微生物来自同一个体的肠道细菌或者与该个体的肠道细菌相同。
优选的，所述的眼部疾病包含视网膜变性；更优选的，所述的视网膜变性为进行性视网膜变性。
优选的，所述的视网膜变性为遗传性视网膜变性(IRD)。
优选的，所述的视网膜变性是模型动物自身的疾病或者是由于基因操作使模型动物患视网膜变性。
优选的，所述的眼部疾病包含LCA、RP、arRP、EORD、EORP、PPRPE、rettelangiectasia和/或choroideremia like fundus。
优选的，所述的眼部疾病包含眼部炎症，例如葡萄膜炎、青光眼、和年龄相关的黄斑变性(AMD)、玻璃体炎，脉络膜炎、视网膜炎，视网膜血管炎，视神经炎，还有葡萄膜炎、白塞病、伏格特-小柳-原田综合征、葡萄膜炎、视网膜病变、交感性眼炎、白内障、结膜炎、青光眼等。
优选的，所述的模型是非人动物，优选猴、犬、黑猩猩、大鼠、小鼠。
优选的，所述的模型载体是细胞、组织或器官，所述的细胞、组织或器官来源于人或非人动物。
优选的，所述的细胞为原代细胞或细胞系。
优选的，所述的组织为眼部组织，所述的器官为眼部器官。
优选的，所述的组织或器官为再生组织或器官。
优选的，所述的模型存在基因的致病突变。
优选的，所述的发生了致病突变的基因为与维持视网膜屏障结构有关的基因，所述的视网膜屏障为外血-视网膜屏障和/或内血-视网膜屏障。
优选的，所述的模型载体中有以下一个或两个以上基因发生了突变：ABCA4、ABCC6、 ABCC9、ACBD5、ACO2、ACO2、ACTG1、ADGRV1、AHI1、AIPL1、ALMS1、AMY2B、APC、ARFGEF1、ARL13B、ARL13B、ARL6、ARMC9、ATOH7、B9D1、BAG3、BBS1、BBS1、BBS2、BBS5、BEST1、C2CD3、CA4、CABP4、CACNA1F、CBS、CC2D2A、CDH23、CDH23、CDHR1、CEMIP2、CEP104、CEP250、CEP290、CEP290、CEP41、CEP78、CERKL、CFAP410、CFAP418、CHM、CLCC1、CLCN7、CLN3、CLN5、CLN8、CLRN1、CLRN1、CNGA1、CNGA1、CNGA3、CNGB1、CNGB3、CNNM4、COL11A1、COL11A2、COL18A1、COL2A1、COL4A1、COL9A1、COL9A2、CP、CP、CPLANE1、CRB1、ERCC4、CSPP1、CTNNA1、CYP4V2、DHDDS、DYNC2H1、DYNC2I1、DYNC2I2、ENPP1、ERCC4、EVC2、EYS、EYS、F5、FAM161A、FBN1、FKRP、FKTN、FLG、FLVCR1、FOXE3、FUZ、GLB1、GMPPB、GNAT1、GRK1、GRM6、GUCA1A、GUCA1B、GUCY2D、HADHA、HGSNAT、HPS3、HPS5、IDH3B、IFT122、IFT140、IFT140、IFT43、IFT52、IFT74、IFT80、IFT80、IFT81、IFT88、IKBKG、IMPDH1、IMPG2、INPP5E、INTU、IQCB1、IQCE、IREB2、KCNJ13、KCNQ1、KCNV2、KIAA0586、KIAA0753、KIF7、KIZ、KIZ-AS1、KLHL7、KRIT1、LBR、LCA5、LOC101927157、LOC111365204、LRP2、LRP5、MAK、MAPKAPK3、MATK、MCOLN1、MERTK、MKS1、MPDZ、MT-ATP6、MT-CO3、MT-TE、MT-TL1、MTHFR、MUTYH、MYO7A、MYO7A、NMNAT1、NPHP1、NR2E3、OCA2、OTX2、PANK2、PAX6、PCARE、PCDH15、PDE6A、PDE6B、PDE6B、PDE6D、PEX1、PEX1、PEX12、PEX26、PEX6、PHF3、PITPNM3、PKD2、PLA2G5、POC5、POMT1、PRCD、PRDM13、PROM1、PRPF3、PRPF31、PRPF8、PRPH2、RAD51C、RBP3、RBP4、RD3、RDH12、RDH5、RGR、RGR、RHO、RIMS1、RLBP1、ROM1、RP1、RP1L1、RP2、RPE65、RPE65、RPGR、RPGRIP1、RPGRIP1L、RS1、SACS、SAG、SCAPER、SDCCAG8、SIX6、SLC19A1、SLC22A5、SLC26A4、SLC2A9、SLTM、SNRNP200、SPAG17、SPATA7、SPG11、TFAP2A、TGFB2、TGFBR2、TMEM107、TMEM237、TMEM67、TOGARAM1、TOPORS、TPP1、TRAF3IP1、TREX1、TRIM59-IFT80、TSPAN12、TTC21B、TTC21B、TTC8、TULP1、USH1C、USH2A、USH2A、USH2A、USH2A、USH2A-AS1、VAC14、VCAN、VCAN、VCAN-AS1、VHL、VPS13B、WDR19、WDR19、WDR35、WDR73、YARS1、ZFYVE26、ZFYVE26、ZNF408的一种或两种以上的组合。
在一个具体实施方式中，所述的模型载体发生了致病突变的眼部疾病相关的基因中包含CRB1基因。
优选的，所述的模型载体的CRB1基因的突变包含如下的一种或两种以上的突变：c.107C>G、c.111delT、c.135C>G、c.257_258dupTG、c.258C>T、c.428_432delGATTC、c.430T>G、c.470G>C、c.481dupG、c.482C>T、c.584G>T、c.613_619del、c.717_718insG、c.750T>G、c.915T>A、c.929G>A、c.936T>G、c.998G>A、c.1084C>T、c.1125C>G、c.1148G>A、c.1208C>G、c.1269C>A、c.1298A>G、c.1313G>A、c.1438T>C、c.1438T>G、c.1576C>T、c.1604T>C、c.1690G>T、c.1733T>A、c.1750G>T、c.1760G>A、c.1834T>C、c.1963delC、c.2025G>T、c.2042G>A、c.2128G>C、c.2129C>T、 c.2185_2186insAlu、c.2219C>T、c.2222T>C、c.2234C>T、c.2245_2247del 3bp(TCA)、c.2258T>C、c.2290C>T、c.2365_2367del AAT,in frame deletion、c.2401A>T、c.2438_2439ins>100A、c.2441_2442del、c.2465G>A、c.2479G>T、c.2506C>A、c.2509G>C、c.2536G>A、c.2548_2551delGGCT、c.2548G>A、c.2555T>C、c.2611_2613insT、c.2671T>G、c.2676delG、c.2681A>G、c.2688T>A、c.2816G>A、c.2843G>A、c.2853dupT、c.2884_2886delTTA、c.2957A>T、c.2966T>C、c.2983G>T、c.3002A>T、c.3008T>C、c.3035T>C、c.3037C>T、c.3074G>A、c.3074G>T、c.3122T>C、c.3212T>C、c.3296C>A、c.3299T>C、c.3299T>G、c.3307G>A/C、c.3320T>C、c.3320T>G、c.3331G>T、c.3343_3352del、c.3347delT、c.3343_3352del、c.3347delT、c.3427delT、c.3482A>G、c.3493T>C、c.3655T>G、c.3541T>C、c.3542dupG、c.3593A>G、c.3613G>A、c.3653G>T、c.3659_3660delinsA、c.3664C>T、c.3668G>C、c.3676G>T、c.3713_3716dup、c.3879G>A、c.3914C>T、c.3949A>C、c.3961T>A、c.3988delG、c.3988G>T、c.3995G>T、c.3996C>A、c.3997G>T、c.4094C>A、c.4121_4130del、c.4142C>T、c.4148G>A、c.2128+2T>G、c.2842+5G>A、c.3878+1G>T、c.4005+1G>A、c.4005+2T>G、c.4006-2A>G、c.4006-1G>T、c.619G>A、c.614T>C、c.1472A>T、c.1903T>C、c.2809G>A、c.3103C>T、c.4082G>A、c.4060G>A、c.866C>T、c.1463T>C、c.2035C>G、c.2306_2307GC>AG、c.2306G>A、c.2714G>A、c.2875G>A、c.3992G>A。
进一步优选的，所述的模型或模型载体的CRB1基因的突变包含如下的一种或两种以上的突变：c.4006-1G>T、c.3686G>C、(p.Cys1229Ser)、c.2842+1delinsAA、c.4060G>A、(p.Ala1354Thr)、c.3991C>T、(p.Arg1331Cys)、c.3014A>T、(p.Asp1005Val)、c.4005+1G>A、c.2680_2684del、(p.Asn894fs)、c.1733T>A、(p.Val578Glu)、c.455G>A、(p.Cys152Tyr)、c.3462_3463del、(p.Cys1154_Glu1155delinsTer)、c.3037C>T、(p.Gln1013Ter)、c.2673C>A、(p.Cys891Ter)、c.2230C>T、(p.Arg744Ter)、c.3676G>T、(p.Gly1226Ter)、c.2842+5G>A、c.2842T>C、(p.Cys948Arg)、c.3988del、(p.Glu1330fs)、c.2506C>A、(p.Pro836Thr)、c.2291G>A、(p.Arg764His)、c.1576C>T、(p.Arg526Ter)、c.613_619del、(p.Ile205fs)、c.3320T>C、(p.Leu1107Pro)、c.2688T>A、(p.Cys896Ter)、c.2555T>C、(p.Ile852Thr)、c.2222T>C、(p.Met741Thr)、c.1148G>A、(p.Cys383Tyr)、c.2843G>A、(p.Cys948Tyr)、c.4121_4130del、(p.Ala1374fs)、c.3307G>A、(p.Gly1103Arg)、c.484G>A、(p.Val162Met)、c.2401A>T、(p.Lys801Ter)、c.2234C>T、(p.Thr745Met)、c.2290C>T、(p.Arg764Cys)、c.3122T>C、(p.Met1041Thr)。
优选的，所述的模型载体的CRB1基因的突变为Rd8突变。
优选的，所述的突变为纯和突变或杂合突变。
优选的，所述的模型载体体内先天存在如前所述的基因的突变或者所述的突变是由于基因重组操作而后天携带的。
优选的，所述的模型载体体内存在人源化CRB1基因或人CRB1基因，其内源CRB1基因缺失或不表达。
优选的，所述的非人动物具有结肠上皮屏障缺陷和/或相关的结肠壁炎症。
在一个具体实施例中，所述的模型体内Occludin蛋白的显著缺失，在一个具体实施例中，所述的模型体内Occludin蛋白显著缺失和Claudin1表达不明显。
所述微生物为细菌、古细菌、原生生物、真菌或病毒中的一种或两种以上的组合，优选的，所述微生物为细菌，所述细菌选自：Anearostipes、双歧杆菌(Bifidobacterium)、巨单胞菌(Megamonas)、亚硝基单胞菌(Nitrosomonas)，颤螺旋菌(Oscillibacter)、塔特姆菌(Tatumella)、硫杆菌(Thiobacillus sp.)、梭菌属(Clostridium)、不动杆菌属(Acinetobacter)、链球菌属(Streptococcus)、曼氏杆菌属(Mannheimia)、纤维杆菌属(Fibrobacter)、普氏菌属(Prevotella)、弯曲杆菌属(Campylobacter)、放线菌属(Actinomyces)、薄层菌属(Hymenobacter)、埃希氏杆菌属(Escherichia)、泰氏菌属(Tissierella)、克雷白氏杆菌属(Klebsiella)、卟啉单胞菌属(Porphyromonas)、固氮螺菌属(Azospira)、海水菌属(Aquimarina)、无色菌属(Achromobacter)、嗜酸硫杆菌属(Acidithiobacillus)、伯克霍尔德菌属(Burkholderia)、海杆菌属(Marinobacter)、密螺旋体属(Treponema)、孢裹放线菌属(Actinosporangium)、弧菌属(Vibrio)、瘤胃球菌属(Ruminococcus)、甲烷短杆菌(Methanobrevibacter)、志贺氏杆菌(Shigella)、弗兰克氏菌属(Frankia)、链霉菌属厌氧原体属(Anaeroplasma)、粪球菌属(Coprococcus)中的一种或两种以上。
具体的，所述细菌选自：Anearostipes hadrus、Bifidobacterium pseudocatenulatum、Nitrosomonas sp.Is79A3、Oscillibacter valericigenes、Tatumella sp.TA1、Megamonas funiformis、Thiobacillus denitrificans、破伤风梭菌、产气荚膜梭菌、肉毒梭菌、醋酸钙不动杆菌、鲁菲不动杆菌、鲍曼不动杆菌、溶血不动杆菌、琼氏不动杆菌、约翰逊不动杆菌、化脓性链球菌、溶血性链球菌、产琥珀酸丝状杆菌、肠道丝状杆菌、非解糖卟啉单胞菌、牙髓卟啉单胞菌、牙龈卟啉单胞菌、空肠弯曲菌、结肠弯曲菌、海鸟弯曲菌、乌普萨拉弯曲菌、简明弯曲菌、胎儿弯曲菌、衣氏放线菌、奈氏放线菌、溶齿放线菌、粘稠放线菌、纽氏放线菌、大肠埃希菌、蟑螂埃希菌、弗格森埃希菌、赫尔曼埃希菌、伤口埃希菌、极尖泰氏菌、肺炎克雷伯氏菌、臭鼻克雷伯氏菌、巴西固氮螺菌、无色杆菌、脱氮硫杆菌、氧化亚铁硫杆菌、氧化硫硫杆菌、那不勒斯硫杆菌、伯克霍尔德菌、海洋分枝杆菌、苍白密螺旋体、猪痢疾密螺旋体、梅氏弧菌、白色瘤胃球菌、生黄瘤胃球菌、瘤胃甲烷短杆菌、痢疾志贺菌、福氏志贺菌、鲍氏志贺菌、宋内志贺菌、弗兰克氏菌、规则粪球菌、白色链霉菌、门多萨假单胞菌，栖息微球菌，脂环素反硝化菌，氧化木糖无色杆菌，鞘脂单胞杆菌，脓肿分枝杆菌，金黄节杆菌，普氏菌，草木樨中华根瘤菌，酸性酵母，表皮葡萄球菌，铜绿假单胞菌，金黄色葡萄球菌，溶血性葡萄球菌，恶臭假单胞菌，嗜麦芽寡养单胞菌，蜡状芽孢杆菌，巨大芽孢杆菌，罗氏乳酸杆菌，阴道嗜血杆菌，蜜蜂屎肠球菌，正哈氏嗜纤维菌，地衣芽孢杆菌，白叶枯菌，鲍曼不动杆菌，醋酸钙不动杆菌，睾丸酮丛毛单胞菌，堪萨斯分枝杆菌，斯润金芽孢杆菌，柯氏柠檬酸杆菌，发酵成对杆菌，粘 质沙雷氏菌，维氏鞘氨醇单胞菌，肺炎克雷伯菌，荧光假单胞菌，皮氏罗尔斯顿菌，卷曲乳杆菌，伯克霍尔德菌，德氏乳杆菌，Meiothermus silvanus(D)，大肠杆菌，藤黄微球菌，枯草芽孢杆菌，粘金色棒状杆菌，大芬戈尔德菌中的一种或两种以上。
优选的，所述的感染方法包含是让微生物与模型载体待感染的部位直接接触或间接接触，所述的间接接触是指所述的微生物和待感染的部位之间存在血视网膜屏障，优选的，为外血-视网膜屏障或内血-视网膜屏障。
在一个具体实施方式中，所述的感染方法包含通过外周血使肠道细菌感染眼部。具体的，非人动物模型的肠上皮屏障收到显著损伤，致使肠道细菌进入外周血。在一个模型体内，视网膜屏障也收到了显著受损，使得进入外周血的肠道细菌感染视网膜；在一个具体实施方式中，所述的非人动物模型发生了CRB1基因的突变；在一个具体实施方式中，所述的CRB1基因的突变是Rd8突变。
所述有炎症的模型载体来自如前所述的方法制备得到的疾病模型，或，用微生物感染来自立体的来自如前所述的非人动物的眼部细胞、组织或器官得到的
本发明提供一种如前所述的方法在评估眼部疾病靶向治疗疗效中的应用，所述的眼部疾病包含如前所述的眼部疾病。
在一个具体实施方式中，对如前所述的模型或模型载体进行针对如前所述的基因突变的靶向治疗，将经过靶向治疗和未经过靶向治疗的模型或模型载体分为两组，分别按照如前所述的方法建立眼部疾病模型，如果相比之下，经过靶向治疗的组不能成功建模，说明靶向治疗取得了有益的效果。
在一个具体实施方式中，所述靶向治疗靶向如下一种基因或两种以上的基因的组合：ABCA4、ABCC6、ABCC9、ACBD5、ACO2、ACO2、ACTG1、ADGRV1、AHI1、AIPL1、ALMS1、AMY2B、APC、ARFGEF1、ARL13B、ARL13B、ARL6、ARMC9、ATOH7、B9D1、BAG3、BBS1、BBS1、BBS2、BBS5、BEST1、C2CD3、CA4、CABP4、CACNA1F、CBS、CC2D2A、CDH23、CDH23、CDHR1、CEMIP2、CEP104、CEP250、CEP290、CEP290、CEP41、CEP78、CERKL、CFAP410、CFAP418、CHM、CLCC1、CLCN7、CLN3、CLN5、CLN8、CLRN1、CLRN1、CNGA1、CNGA1、CNGA3、CNGB1、CNGB3、CNNM4、COL11A1、COL11A2、COL18A1、COL2A1、COL4A1、COL9A1、COL9A2、CP、CP、CPLANE1、CRB1、ERCC4、CSPP1、CTNNA1、CYP4V2、DHDDS、DYNC2H1、DYNC2I1、DYNC2I2、ENPP1、ERCC4、EVC2、EYS、EYS、F5、FAM161A、FBN1、FKRP、FKTN、FLG、FLVCR1、FOXE3、FUZ、GLB1、GMPPB、GNAT1、GRK1、GRM6、GUCA1A、GUCA1B、GUCY2D、HADHA、HGSNAT、HPS3、HPS5、IDH3B、IFT122、IFT140、IFT140、IFT43、IFT52、IFT74、IFT80、IFT80、IFT81、IFT88、IKBKG、IMPDH1、IMPG2、INPP5E、INTU、IQCB1、IQCE、IREB2、KCNJ13、KCNQ1、KCNV2、KIAA0586、KIAA0753、KIF7、KIZ、KIZ-AS1、KLHL7、KRIT1、LBR、LCA5、LOC101927157、 LOC111365204、LRP2、LRP5、MAK、MAPKAPK3、MATK、MCOLN1、MERTK、MKS1、MPDZ、MT-ATP6、MT-CO3、MT-TE、MT-TL1、MTHFR、MUTYH、MYO7A、MYO7A、NMNAT1、NPHP1、NR2E3、OCA2、OTX2、PANK2、PAX6、PCARE、PCDH15、PDE6A、PDE6B、PDE6B、PDE6D、PEX1、PEX1、PEX12、PEX26、PEX6、PHF3、PITPNM3、PKD2、PLA2G5、POC5、POMT1、PRCD、PRDM13、PROM1、PRPF3、PRPF31、PRPF8、PRPH2、RAD51C、RBP3、RBP4、RD3、RDH12、RDH5、RGR、RGR、RHO、RIMS1、RLBP1、ROM1、RP1、RP1L1、RP2、RPE65、RPE65、RPGR、RPGRIP1、RPGRIP1L、RS1、SACS、SAG、SCAPER、SDCCAG8、SIX6、SLC19A1、SLC22A5、SLC26A4、SLC2A9、SLTM、SNRNP200、SPAG17、SPATA7、SPG11、TFAP2A、TGFB2、TGFBR2、TMEM107、TMEM237、TMEM67、TOGARAM1、TOPORS、TPP1、TRAF3IP1、TREX1、TRIM59-IFT80、TSPAN12、TTC21B、TTC21B、TTC8、TULP1、USH1C、USH2A、USH2A、USH2A、USH2A、USH2A-AS1、VAC14、VCAN、VCAN、VCAN-AS1、VHL、VPS13B、WDR19、WDR19、WDR35、WDR73、YARS1、ZFYVE26、ZFYVE26、ZNF408。
在一个具体实施方式中，所述的靶向治疗靶向CRB1基因的如下突变的一种或两种以上：c.257_258dupTG、c.258C>T、c.428_432delGATTC、c.430T>G、c.470G>C、c.481dupG、c.482C>T、c.584G>T、c.613_619del、c.717_718insG、c.750T>G、c.915T>A、c.929G>A、c.936T>G、c.998G>A、c.1084C>T、c.1125C>G、c.1148G>A、c.1208C>G、c.1269C>A、c.1298A>G、c.1313G>A、c.1438T>C、c.1438T>G、c.1576C>T、c.1604T>C、c.1690G>T、c.1733T>A、c.1750G>T、c.1760G>A、c.1834T>C、c.1963delC、c.2025G>T、c.2042G>A、c.2128G>C、c.2129C>T、c.2185_2186insAlu、c.2219C>T、c.2222T>C、c.2234C>T、c.2245_2247del 3bp(TCA)、c.2258T>C、c.2290C>T、c.2365_2367del AAT,in frame deletion、c.2401A>T、c.2438_2439ins>100A、c.2441_2442del、c.2465G>A、c.2479G>T、c.2506C>A、c.2509G>C、c.2536G>A、c.2548_2551delGGCT、c.2548G>A、c.2555T>C、c.2611_2613insT、c.2671T>G、c.2676delG、c.2681A>G、c.2688T>A、c.2816G>A、c.2843G>A、c.2853dupT、c.2884_2886delTTA、c.2957A>T、c.2966T>C、c.2983G>T、c.3002A>T、c.3008T>C、c.3035T>C、c.3037C>T、c.3074G>A、c.3074G>T、c.3122T>C、c.3212T>C、c.3296C>A、c.3299T>C、c.3299T>G、c.3307G>A/C、c.3320T>C、c.3320T>G、c.3331G>T、c.3343_3352del、c.3347delT、c.3343_3352del、c.3347delT、c.3427delT、c.3482A>G、c.3493T>C、c.3655T>G、c.3541T>C、c.3542dupG、c.3593A>G、c.3613G>A、c.3653G>T、c.3659_3660delinsA、c.3664C>T、c.3668G>C、c.3676G>T、c.3713_3716dup、c.3879G>A、c.3914C>T、c.3949A>C、c.3961T>A、c.3988delG、c.3988G>T、c.3995G>T、c.3996C>A、c.3997G>T、c.4094C>A、c.4121_4130del、c.4142C>T、c.4148G>A、c.2128+2T>G、c.2842+5G>A、c.3878+1G>T、c.4005+1G>A、c.4005+2T>G、c.4006-2A>G、c.4006-1G>T、c.619G>A、c.614T>C、c.1472A>T、c.1903T>C、c.2809G>A、c.3103C>T、c.4082G>A、c.4060G>A、c.866C>T、c.1463T>C、c.2035C>G、c.2306_2307GC>AG、c.2306G>A、c.2714G>A、c.2875G>A、c.3992G>A。
在一个具体实施方式中，所述的靶向治疗药物包含经过修饰的细胞、经过修饰的蛋白、靶向如前所述的基因或靶向如前所述的突变位点的RNA，和/或靶向如前所述的基因或靶向如前所述的突变位点的DNA。
本发明提供一种如前所述的方法制备得到的疾病模型在眼部疾病相关的研究中的应用。所述的眼部疾病包含如前所述的眼部疾病。所述的研究包括与遗传性视网膜变性相关的疾病与肠道菌群的相互响应等。
在一个具体实施方式中，所述的疾病模型感染了一种肠道细菌，当其所携带的致病突变被细胞治疗的情况下，观察给药和不给药的疗效，所述的药物为小分子药物，优选为广谱抗生素或针对所感染的细菌的抗生素。在一个具体实施方式中，所述的疾病模型感染了一种肠道细菌，当其所携带的致病突变被RNA治疗的情况下，观察给药和不给药的疗效，所述的药物为小分子药物，优选为广谱抗生素或针对所感染的细菌的抗生素。在一个具体实施方式中，所述的疾病模型感染了两种以上的肠道细菌，当其所携带的致病突变被细胞治疗的情况下，观察给药和不给药的疗效，所述的药物为小分子药物，优选为广谱抗生素或针对所感染的细菌的抗生素。在一个具体实施方式中，所述的疾病模型感染了两种肠道细菌，当其所携带的致病突变被RNA治疗的情况下，观察给药和不给药的疗效，所述的药物为小分子药物，优选为广谱抗生素或针对所感染的细菌的抗生素。
优选的，所述的致病突变发生与如前所述的基因，或，所述的致病突变为如前所述的CRB基因的突变。
本发明提供一种如前所述的方法制备得到的疾病模型载体在眼部疾病相关的研究中的应用。所述的眼部疾病包含如前所述的眼部疾病。所述的研究包括与遗传性视网膜变性相关的疾病与肠道菌群的协同作用等。
在一个具体实施方式中，所述的疾病模型载体感染了一种肠道细菌，当其所携带的致病突变被细胞治疗的情况下，观察给药和不给药的疗效，所述的药物为小分子药物，优选为广谱抗生素或针对所感染的细菌的抗生素。在一个具体实施方式中，所述的疾病模型载体感染了一种肠道细菌，当其所携带的致病突变被RNA治疗的情况下，观察给药和不给药的疗效，所述的药物为小分子药物，优选为广谱抗生素或针对所感染的细菌的抗生素。在一个具体实施方式中，所述的疾病模型载体感染了两种以上的肠道细菌，当其所携带的致病突变被细胞治疗的情况下，观察给药和不给药的疗效，所述的药物为小分子药物，优选为广谱抗生素或针对所感染的细菌的抗生素。在一个具体实施方式中，所述的疾病模型载体感染了两种肠道细菌，当其所携带的致病突变被RNA治疗的情况下，观察给药和不给药的疗效，所述的药物为小分子药物，优选为广谱抗生素或针对所感染的细菌的抗生素。
优选的，所述的致病突变发生与如前所述的基因，或，所述的致病突变为如前所述的CRB 基因的突变。
本发明提供一种如前所述的疾病模型或疾病模型载体在眼部疾病相关药物筛选中的应用。所述的药物包含小分子药物，化学药物，高分子药物，生物药物或天然药物(例如中药或中药提取物)、细胞药物、RNA药物、DNA药物的一种或两种以上的组合。
所述的眼部疾病包含如前所述的眼部疾病。
优选的，小分子化合物为抗生素，所述的抗生素为一般为本领域技术人员所熟知的广谱抗生素药物。
优选的，所述的小分子化合物为对特定的细菌具有针对性的非广谱性的抗生素。
优选的，所述的细胞包含经过修饰的免疫细胞，例如T细胞、B细胞或者干细胞的一种或两组以上的组合。
优选的，所述的RNA包含mRNA、siRNA、sgRNA、miRNA、ASO和/或复制子RNA。
在一个具体实施方式中，对所述的疾病模型或疾病模型载体进行靶向治疗，同时对经过靶向治疗的疾病模型或疾病模型载体和未经过靶向治疗的疾病模型或疾病模型载体施与如前所述的药物或不给药，观察几组的炎症进展，评估靶向治疗的药物疗效。
在一个具体实施方式中，对所述的疾病模型或疾病模型载体进行靶向治疗，同时对经过靶向治疗的疾病模型或疾病模型载体和未经过靶向治疗的疾病模型或疾病模型载体施与如前所述的药物或不给药，观察几组的炎症进展，评估小分子药物的疗效。
本发明所述的化学药物选自β-内酰胺类抗生素：包括青霉素类、头孢菌素类、硫酶素类、单内酰环类，β-内酰酶抑制剂、甲氧青霉素类等；氨基糖苷类抗生素：包括链霉素、庆大霉素、卡那霉素、妥布霉素、丁胺卡那霉素、新霉素、核糖霉素、小诺霉素、阿斯霉素等；四环素类抗生素：包括四环素、土霉素、金霉素及强力霉素等；氯霉素类抗生素：包括氯霉素、甲砜霉素等；大环内脂类抗生素：包括红霉素、白霉素、无味红霉素、乙酰螺旋霉素、麦迪霉素、交沙霉素、阿奇霉素等；糖肽类抗生素：万古霉素、去甲万古霉素、替考拉宁等；喹诺酮类抗生素：包括诺氟沙星、氧氟沙星、环丙沙星、培氟沙星、加替沙星；硝基咪唑类抗生素：包括甲硝唑、替硝唑、奥硝唑等；利福霉素类抗生素：包括利福平等；棘白菌素类抗生素；多烯类抗生素；嘧啶类抗生素；烯丙胺类抗生素；氮唑类抗生素；其它抗生素：磷霉素、卷霉素、环丝氨酸、林可霉素、氯林可霉素、丝裂霉素、放线菌素D、博莱霉素、阿霉素、异烟肼、吡嗪酰胺、环孢霉素等中的一种或者两种以上。
本发明所述的生物药物为抗菌肽，所述的抗菌肽选自昆虫抗菌肽，例如，鳞翅目抗菌肽、双翅目抗菌肽，鞘翅目抗菌肽，蜻蜓目抗菌肽，膜翅目抗菌肽，家蚕抗菌肽等；哺乳动物抗菌肽，例如，猪抗菌肽，绵羊抗菌肽，牛抗菌肽，人抗菌肽等；两栖动物抗菌肽：爪蟾素等；鱼类、软体动物、甲壳类动物来源的抗菌肽：豹鳎抗菌肽，鲶抗菌肽，贻贝抗菌肽，虾抗菌肽等； 植物抗菌肽：Thi-onins等，细菌抗菌肽：杆菌肽、短杆菌肽、多粘菌素和乳链菌肽等中的一种或者两种以上。
本发明所述的天然药物选自黄芪、黄精、当归、三七、白茅根、大黄炭、郁金、浙贝、薏仁、法半夏、煅古墨、丹参、紫草、板蓝根、鱼腥草、金银花、黄连、黄芩、蒲公英、马齿苋、山楂、大青叶、连翘、菌陈、穿心莲、柴胡、地锦草、百部、大蒜、黄柏、杜仲、秦皮、蛇床子、黄连、五倍子、紫花地丁、乌梅、甘草、石榴皮、五味子、皂刺、诃子、苦参、土槿皮，淫羊藿，青蒿或其提取物中的一种或者两种以上。
本发明所述的药物可以是口服药物、注射药物或者外用药物，所述的外用药物包括粘膜给药药物，优选为眼部给药药物。
本发明所述的药物的剂型可以是溶液、片剂、丸剂、胶囊、注射液、粉末、注射用粉末、贴剂、涂剂、粘膜给药制剂，所述的粘膜给药制剂优选为眼药水、眼药膏、眼喷雾制剂等。
附图说明
图1：Crb1 rd8/rd8(rd8)-SPF小鼠的基因型和表型。其中，图1A为Crb1 rd8/rd8(rd8)和Crb1 wt/wt(C57BL/J，简称wt)小鼠的基因型凝胶电泳图，图1B为Rd8-SPF和WT-SPF小鼠的代表性眼底图像，Rd8-SPF小鼠视网膜下鼻象限出现典型的白色斑点，图1C为检测Rd8-SPF小鼠E18时双眼病理变化的H&E染色。
图2：Rd8-SPF小鼠发生视网膜异常及视网膜病变的潜在机制。其中图2A为H&E染色检测Rd8-SPF和WT-SPF小鼠在E18、P12(睁眼前)、P15(睁眼后)和8周(8W)的双眼病理变化，Rd8小鼠表现出典型的视网膜异常，包括进行性视网膜发育不良(皱褶和假花环)和变性，而WT小鼠表现出正常的视网膜结构；图2B为Rd8-和WT-SPF小鼠在E18、P12、P15和8W时眼睛有或无损伤的统计数据，从P12、P15和8W的Rd8小鼠的所有视网膜中都可以看到典型的病变，但WT-SPF小鼠中没有任何一个，WT-SPF：在E18、P12、P15和8W时，每组n＝6；Rd8-SPF:E18处n＝32，P12处n＝6，P15处n＝8；图2C为Rd8小鼠视网膜上(无病变)区和下(有病变)区DEGs火山图分析。Slamf1(信号淋巴细胞活化分子家族成员1)和Ncf4(中性粒细胞胞浆因子)是Rd8-SPF小鼠视网膜病变中增殖倍数最高的两个基因；图2D为DEGs的IPA功能分析；图2E为免疫荧光染色显示Rd8小鼠视网膜病变区有丰富的IBA1+小胶质细胞(红色)。
图3：RNA序列分析比较Rd8-SPF小鼠视网膜上、下区基因表达谱。其中，图3A为Rd8-SPF小鼠上(无病变)和下(有病变)区域的图示；图3B为RNA-seq分析发现Rd8-SPF小鼠视网膜上、下区存在至少2倍的表达差异(P<0.05)；图3C为Rd8-和WT-SPF小鼠视网膜上下区179DEGs的表达模式。
图4：Rd8-SPF小鼠视网膜病变内细菌的鉴定。其中，图4A为主坐标分析(PCoA)，显 示Rd8小鼠视网膜组织中细菌组成与WT小鼠有显著差异；图4B通过宏基因组测序鉴定Rd8小鼠视网膜上(S)和下(I)组织的微生物种类组成(n＝4)；图4C为荧光原位杂交法检测Rd8-和WT-SPF小鼠(4周龄)视网膜细菌16srDNA和vanco-bodipy；图4D用透射电镜(TEM)观察Rd8小鼠视网膜病变内细菌的分布，阳性对照为大肠菌群；
图5：Rd8-SPF小鼠视网膜粘附连接的破坏。其中，图5A为Rd8-和WT-SPF小鼠视网膜CRB1(红色)的免疫荧光染色，BM为Bruch膜，CC为绒毛膜毛细血管，BL为基膜；图5B为Rd8-和WT-SPF小鼠视网膜外界膜粘附连接的透射电镜观察，红色箭头为粘附连接，AJ指粘附连接，OLM为外界膜，ONL为外核层，IS指内段；图5C为透射电镜观察Rd8-SPF小鼠视网膜色素上皮(RPE)基底层粘连溶解、绒毛膜毛细血管基底层扭曲、胶原层紊乱，CC为绒毛膜毛细血管，CH为脉络膜，BL为基膜；图5D为Rd8-和WT-SPF小鼠Bruch膜厚度的统计分析****P<0.0001。
图6A：为透射电镜(TEM)显示Rd8-SPF小鼠外界膜(OLM)粘附连接断裂(AJ)和外核层(ONL)。
图6B：透射电镜(TEM)显示的Rd8-SPF小鼠和WT-SPF小鼠Brunch膜的厚度。
图7：Rd8-SPF小鼠结肠上皮屏障缺陷及其相关炎症。其中，图7A为Rd8和WT小鼠胃肠道不同部位Rd8视网膜中细菌的相对丰度(n＝5)；图7B-D为Rd8-和WT-SPF小鼠结肠肠细胞CRB1蛋白(B)、鬼笔环化蛋白(C)和闭塞蛋白(D)的免疫荧光染色；图7E为Rd8-和WT-SPF小鼠咬合蛋白相对强度的统计分析；图7F为westernblot检测Rd8-和WT-SPF小鼠结肠肠上皮细胞Claudin1的表达；图7G为Rd8-和WT-SPF小鼠Claudin1相对强度的统计分析；图7H为透射电镜观察Rd8-和WT-SPF小鼠结肠上皮间的贴壁连接和紧密连接；图7I-L为Rd8-和WT-SPF小鼠上皮微绒毛数(I)、微绒毛长度(J)、微绒毛宽度(K)和完整粘附连接总数(L)的统计分析；图7M-N为比较Rd8-和WT-SPF小鼠Tnfa、Il1b、Il12a、(M)和Mucin2(N)的mRNA表达水平。
图8：Rd8-SPF小鼠胃肠道不同部位菌群的微生物种类组成。其中，图8A-F为基于Bray-Curtis距离的WT(n＝5)和Rd8(n＝5)小鼠胃肠道样品微生物种类组成的主坐标分析(PCoA)，包括胃(图8A)、空肠(图8B)、回肠(图8C)、盲肠(图8D)、结肠(图8E)和直肠(图8F)；图8G为PCoA区分Rd8小鼠和WT-SPF小鼠下消化道(盲肠、结肠和直肠)的微生物组成(H-J)比较Rd8和WT小鼠盲肠(H)、结肠(I)和直肠(J)中粘液阿克曼菌的相对丰度。数据表示为平均值±SEM*P<0.05。
图9：Rd8小鼠盲肠的紧密连接和粘附连接。其中，图9A为Rd8-和WT-SPF小鼠盲肠肠细胞CRB1蛋白(绿色)的免疫荧光染色；图9B为Rd8-和WT-SPF小鼠盲肠肠细胞闭塞素(红色)的免疫荧光染色；图9C为Rd8-和WT-SPF小鼠咬合蛋白相对强度的统计分析*P<0.05； 图9D为westernblot检测Rd8-和WT-SPF小鼠盲肠Claudin1的表达；图9E为Rd8-和WT-SPF小鼠Claudin1相对强度的统计分析，NS代表不重要；图9F为透射电镜检查显示盲肠上皮屏障处有正常的紧密连接和粘着连接，MV为微泡，MC为线粒体。
图10：图10A为透射电镜观察Rd8-和WT-SPF小鼠结肠上皮与紧密连接(TJ)间的粘附连接(AJ)，MV为微泡，MC为线粒体；图10B为流式细胞仪检测Rd8和WT小鼠外周血中荧光+细菌/细胞的频率。
图11：Rd8-SPF小鼠肠上皮屏障功能破坏。其中，图11A为肠道FICT-葡聚糖通透性测定显示Rd8小鼠血清荧光强度明显高于WT小鼠***P<0.001；图11B为用vanco-bodipy标记粪便微生物群移植法比较Rd8和WT小鼠外周血中荧光+细菌/细胞的百分比*P<0.05；图11C-D为在荧光显微镜下(C)和免疫荧光染色(D)均能在Rd8小鼠视网膜病变中检测到vanco-bodipy+细菌；图11F-G为比较DSS治疗13d后Rd8和WT小鼠的结肠长度、血清细菌16srrna(F)和细菌LPS(G)水平****P<0.0001；图11H为每天监测用2.5％DSS处理的Rd8(n＝20)和WT(n＝19)小鼠的体重；图11I为Rd8(n＝20)和WT(n＝19)小鼠经2.5％DSS处理后的Kaplan-Meier生存曲线。
图12：Rd8 GF和Rd8 GF SPF小鼠的视网膜表型。其中，图12A为Rd8-SPF和Rd8 GF小鼠在4周、8周、12周和16周时的代表性眼底图像；图12B为Rd8-GF小鼠E18、P12、P15、8W双眼视网膜组织学观察；图12C为Rd8-GF小鼠视网膜细菌16srDNA和vanco-bodipy的荧光原位杂交染色；图12D为Rd8-SPF、Rd8 GF和WT-SPF小鼠视网膜中IBA1(红色)的免疫荧光染色；图12E为Rd8-SPF、Rd8 GF和WT-SPF小鼠ONL中IBA1+小胶质细胞的百分比***P<0.001；NS代表不重要；图12F为Rd8-SPF、Rd8-GF和WT-SPF小鼠视网膜ZO-1(红色)和Phalloidin(绿色)蛋白的免疫荧光染色；图12G为H&E染色检测出生后SPF环境中饲养的Rd8-GF小鼠(Rd8-GF-SPF小鼠)P15和8W视网膜病变。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述。在如下提供的实施例中，仅采用了将Crb1基因突变的小鼠饲养于SPF环境下的建模方法，通过验证其视网膜局部炎症反应的存在和细菌在病变部位的存在，确定所述的模型视网膜感染了细菌。进一步通过接下来的实验，验证所述的细菌来自肠道。这个具体实施例并不排除其他的建模方法，比如饲养于微生物条件更复杂的环境里，或者向眼部组织施与来自肠道的微生物或与肠道微生物相同的微生物，让眼部组织直接或间接与如前所述的微生物接触等。
显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
1.老鼠
携带Rd8突变的C57BL/6N小鼠(Crb1 rd8/Rd8，命名为Rd8小鼠)和C57BL/6J小鼠(Crb1 wt/wt，命名为wt小鼠)购自北京维通利华实验动物技术有限公司，并在中山眼科中心动物设施的无特定病原体(SPF)条件下进行维护。孙中山大学附属第一附属医院的动物设施，用雌性RD8小鼠的胚胎产生无菌(GF)RD8小鼠。GF小鼠保持无菌状态，设施工作人员每周对粪便样本进行微生物和寄生虫检测，以确保GF单元的无菌性。如前所述进行小鼠基因分型(Mattapallil等人，2012)。两个小鼠品系的Crb1基因型得到证实(图4A)。所有动物实验均经中山眼科中心机构动物护理和使用委员会批准，并遵守《ARVO眼科和视力研究动物使用声明》。
2.方法
2.1组化
小鼠颈椎脱位处死，眼球摘除，用4％多聚甲醛(PFA)在4℃磷酸盐缓冲液(PBS)中固定24h。样品用PBS洗涤3次，在一系列醇中脱水，在二甲苯中脱水2次，然后包埋在石蜡中，并用切片机在10μm处连续切片(RM 223；Leica,Wetzlar,Hesse-Darmstadt,Germany)。切片用苏木精和伊红(H&E)染色。H&E图像由Imager.Z2(蔡司)获得。
取眼，室温下放置于4％PFA中5min，然后解剖并固定眼罩45min，取结肠组织，室温下固定于4％PFA中4h，PBS冲洗后，用30％蔗糖渗透分离的眼罩和结肠过夜进行冷冻保护，嵌入OCT化合物(Cat.4583；SAKURA,USA)，并储存在-80℃切片前。在12μm处切割切片用于所有免疫染色目的。
用10％驴血清/PBST(0.1％tritonx-100/PBS)阻断组织切片30分钟，然后在4℃下与一级抗体孵育过夜。在用PBST洗涤后，用荧光染料结合的二级抗体孵育切片，并用Fluoromount-G(Southern Biotech，Birmingham，AL，USA)固定。采用相同的免疫组织化学方法进行Phalloidin(A12379；Thermo-Fisher)染色，除了遗漏二级抗体。用蔡司共聚焦显微镜(蔡司LSM880；蔡司，Oberkochen，德国)和Imager.Z2配备ApoTome(蔡司)。本研究中使用的主要抗体是：抗Crb1(PA5-66373，ThermoFisher；1:50)，抗Iba1(ab178846，Abcam；1:500)，抗-ZO-1(61-7300，ThermoFisher；1:500)，抗Occludin(OC-3F10，Invitrogen；1:200)，AlexaFluor 488phalloidin(A12379，ThermoFisher；1:500).
2.2眼底摄影
小鼠被麻醉，瞳孔扩大。定期应用羟丙甲纤维素滴眼液保持角膜湿润。使用Micron IV小鼠眼底照相机(Phoenix Research Laboratories,Inc.,Pleasanton,CA,USA)获得小鼠眼底照片。
2.3视网膜上下部RNA-seq分析
用MasterPure TM全DNA和RNA纯化试剂盒(epicentre)从视网膜上下提取总RNA。 用Qbit-RNA-HS分析试剂盒测定RNA浓度。测序文库是按照制造商提供的标准协议，使用VAHTS TM Total RNA seq(H/M/R)库准备试剂盒(Vazyme，中国)制备，并在MGISEQ 2000RS平台上测序。
原始读数首先由FastQC(v0.11.8)和cutadapt(v1.15)进行质量控制评估。使用HISAT2(v2.1.0)将干净的读数与小鼠基因组(mm10)对齐。将基因表达数据导入R软件(v3.6.1)的DESeq2包进行差异表达分析。将差异表达基因(DEGs)导入智能通路分析(IPA)进行功能富集分析。
2.4宏基因组测序
收集视网膜样本，并使用MasterPure TM完整DNA和RNA纯化试剂盒(epicentre)提取DNA。收集胃、空肠、回肠、盲肠、结肠和直肠的内容物，并使用QIAamp PowerFecal DNA试剂盒(QIAGEN)提取DNA。浓度测量后，按照制造商提供的标准方案，使用VAHTS TM MGI通用DNA文库制备试剂盒(Vazyme，中国)对DNA进行测序文库制备。用MGISEQ-2000RS进行宏基因组测序。原始读数通过Trimmomatic(v0.36)和PRINSEQ(v0.20.4)进行质量过滤。使用KneadData删除鼠标读取(v0.6.1)(https://bitbucket.org/biobakery/kneaddata).使用Kraken 2(v2.0.9)将非鼠标清除读取映射到预构建的MiniKraken数据库。分类结果用0.20的置信度进行筛选。阴性空白对照与样品一起处理。阴性空白对照组中存在的所有物种均被去除。
2.5 FITC-葡聚糖体内肠道通透性测定
用FITC标记的葡聚糖法进行体内通透性测定以评价屏障功能。隔夜取出食物和水，8周大的小鼠每100克(体重)口服50毫克FITC标记的葡聚糖(FD-70；Sigma-Aldrich)。给药后5h采集血清，测定每个样品的荧光强度(激发，492nm；发射，525nm)。
2.6流式细胞术
WT和Rd8小鼠禁食过夜，并灌胃给予1 x 109 E。大肠杆菌(设计为一致表达RFP)。灌胃给药6小时后，对小鼠实施安乐死，将400μL外周血轻轻移液到含有4mL ACK溶解缓冲液(Gibco，美国)的试管中，并在RT下培养3-5分钟。在300x g离心5分钟后，将细胞固定并渗透(Cytofix/perm溶液，BD Biosciences，USA)，然后通过流式细胞仪(MACSQuant Analyzer 10，Miltenyi Biotec，Germany)分析。
2.7电子显微镜组织制备
安乐死后立即收集结肠和眼睛，并在室温下在磷酸盐缓冲的戊二醛-多聚甲醛溶液中固定1h。结肠被切成2毫米的块。眼前段切除，后段切割成2mm×2mm块。解剖后的组织在新鲜固定液中放置12h，用1％四氧化锇固定，脱水后包埋于epon-resin中。在光学显微镜下用甲苯胺蓝染色，在微米厚的切片上预先筛选感兴趣的区域。然后收集80nm的超声图像，用醋酸铀和柠檬酸铅复染。用透射电子显微镜观察超声切片。
2.8荧光原位杂交(FISH)
本研究使用了以下寡核苷酸探针：EUB338，5'-GCTGCCTCCGTAG-GAGT-3'(Amann等人，1990)。探针的5'端带有一个伯氨基，四甲基罗丹明异硫氰酸酯与该氨基共价结合。将染料寡核苷酸结合物(100μM)储存在-20℃下。
预固定的视网膜切片用DEPC处理的PBS冲洗3次。在用0.2％Triton X-100/DEPC处理的PBS处理后，在37℃下将切片与探针(500nM)杂交过夜，并用Fluoromount-G安装。
2.9实时定量PCR检测结肠组织中基因表达水平
新鲜的小鼠结肠组织(～1cm)在液氮中快速冷冻，研磨并用RNA提取裂解缓冲液裂解。然后使用Qiagen RNeasy Plus试剂盒纯化总RNA，并使用Takara PrimeScript RT试剂盒和gDNA擦除器将其反向转录为cDNA。qPCR检测相应基因的表达水平。数据标准化为β-肌动蛋白。
2.10 QPCR定量测定血浆中16srrna基因水平
经2.5％DSS处理13天后，从WT和Rd8小鼠全血中分离血浆。大约50μL血浆用于使用MasterPure分离总核酸TM完整的DNA和RNA纯化试剂盒(epicentre，美国)。将沉淀的核酸溶解在20μL无核酸酶的水中。qPCR分析(ChamQ-SYBR-Color-qPCR-Master-Mix，Vazyme，中国)使用LightCycler 96系统(美国罗氏)进行。由于所有样品中的DNA浓度都极低，因此使用等体积的每个样品作为模板(20μL中的4μL)。使用以下通用16S rRNA基因引物测量细菌总负荷：27F 5’-AGAGTTTGATCCTGGCTCAG-3’,534R 5’-GCATTACCGCGGCTGCTGG-3’.
2.11酶联免疫吸附试验(ELISA)
每一个样品直接用100μL血浆进行测量。用酶联免疫吸附试剂盒(SEB526Ge；Cloud-Clone Corp.,USA)测定血浆LPS浓度。
2.12荧光团vanco-bodipy标记肠道细菌在Rd8小鼠体内的定殖
新鲜粪便样本收集在带有无菌1xPBS的50ml锥形管中，并旋转直至均质。内容物用0.22μm过滤器(Millipore)过滤以去除粪便残留物，并离心以获得肠道微生物群，然后在RT下与vanco-bodipy孵育30分钟。用vanco-bodipy标记的肠道细菌在PBS中以1x10 8cfu/小鼠灌胃。灌胃24小时后，取小鼠视网膜切片观察。
2.13右旋糖酐硫酸钠(DSS)诱导结肠炎的实验研究
通过饮用水长期口服2.5％DSS(MW 36000–50000d，Yeasen，China)诱导小鼠肠道炎症。第0天至第13天每天监测体重，第13天处死小鼠，测量结肠长度。为了生存分析，小鼠被允许在其饮用水中随意饮用2.5％DSS 43天，并且在43天期间每24小时监测小鼠的死亡率状态。
2.14肠道共生体耗竭
氨苄西林的广谱抗生素混合物(A；1克/升；Sigma)，甲硝唑(M；1克/升；新霉素(N；1克/升；Sigma)和万古霉素(V；500毫克/升；在饮用水中给怀孕的母鼠喂食Sigma(AMNV)，断奶后继续给幼崽喂食。对照组小鼠被安置在同一机架上的常规装置中。
3.实施例
实施例1建模
观察Crb1rd8/rd8(rd8)和Crb1wt/wt(C57BL/J，命名为wt)小鼠的视网膜微环境特征(图1A)。在无特定病原体(Rd8-SPF)环境中饲养的Rd8小鼠，观察其表型，发现其呈现所有典型的视网膜表现，包括眼底镜检查下眼下鼻象限的白斑(图1B)，以及进行性视网膜发育不良(皱褶和假花环)和视网膜组织学变性(H&E染色)(图2A)。结果显示32只Rd8小鼠中有7只在出生前出现轻微的视网膜发育不良(E18)(图2A和图2B)。此外，E18处的所有异常都是单侧发生的(图2A和图1C)，而在P12(睁眼前)、P15(睁眼后)和8周龄(8W)Rd8小鼠的所有视网膜中都可以看到视网膜皱褶和假花环等典型病变，但在WT-SPF小鼠中没有任何一只(图2A和图2B)。数据表明Rd8小鼠的视网膜异常早在胚胎E18时就发生了，眼内环境在Crb1突变的遗传影响下能够驱动视网膜表型。
实施例2检测建模后的小鼠(Rd8-SPF)视网膜是否存在病变内病原体及其相关的局部免疫反应
使用RNA-seq技术进行转录组学分析，比较Rd8-SPF小鼠上(无病变)和下(有病变)区域的基因表达谱(图3A)。在去除WT-SPF小鼠视网膜上下区域之间具有差异表达模式(2倍变化和P<0.05)的基因后，显示在Rd8-SPF小鼠中发现179个基因(DEG)在视网膜上下区域之间具有至少2倍的表达差异(P<0.05)(图3B和图2C)。在Rd8-SPF小鼠中，这些179个DEG中的大多数在损伤的下半区高度表达(图3C)。使用Ingenuity Pathway analysis(IPA)工具对这些DEG进行的功能分析显示，参与细菌和病毒识别的基因在Rd8-SPF小鼠视网膜病变中高度表达的所有DEG中显著富集(图2D)。重要的是，病变增加倍数最多的前两个基因Ncf4和Slamf1都是吞噬细胞抗菌反应的调节因子。数据表明，Rd8视网膜中存在病变内病原体及其相关的局部免疫反应，这些结果与具有促炎功能的基因的丰富表达(图2D)和IBA1+小胶质细胞向视网膜病变区域的浸润(图2E)相结合。
实施例3检测建模后的小鼠视网膜病变部位是否存在细菌
对WT-SPF(n＝5，年龄＝4周)和Rd8-SPF(n＝4，年龄＝4周)小鼠的视网膜组织进行宏基因组分析。分析发现，WT和Rd8小鼠的视网膜中细菌DNA含量极低，在所有质量控制和去污步骤后，均未检测到病毒或真菌DNA。然而，如图4A所示，主坐标分析(PCoA)表明WT-SPF和Rd8-SPF视网膜的细菌组成存在明显差异。重要的是，对Rd8-SPF(n＝4，年龄＝4周)小鼠视网膜上下区域进行的进一步宏基因组分析显示，在视网膜下区域有7种细菌显著富 集，包括无角绦虫hadrus、假链球菌Bifidobacterium pseudocatenulatum、漏斗状巨单胞菌Megamonas funiformis、亚硝基单胞菌Is79A3，产缬草Oscillibacter、Tatumella sp.TA1和脱氮硫杆菌(图4B)。为了直接观察视网膜病变中是否存在细菌，对Rd8-SPF小鼠(4周龄)进行荧光原位杂交(FISH)和vanco-bodipy染色分析，特别是对革兰氏阳性细菌的细胞壁进行染色。如图4C所示，细菌只能在病变部位发现，而不能在正常视网膜区域发现。使用透射电子显微镜(TEM)进一步证实了病变内细菌的存在(图4D)。数据表明，细菌可以在Rd8-SPF小鼠的视网膜病变中发现。
实施例5证明Rd8-SPF小鼠视网膜的主要缺陷是外血-视网膜屏障
免疫荧光染色数据证实了Rd8视网膜外界膜CRB1蛋白表达减少或缺失(图5A)。数据证实在Bruch膜上可以发现CRB1蛋白的弱表达(图5A)。利用透射电镜，发现在Rd8-SPF小鼠的视网膜病变部位，外界膜上的粘附连接被破坏，这与外核层的向外迁移有关(图5B和图6A)。透射电镜检查还显示Rd8-SPF视网膜中视网膜色素上皮(RPE)基膜处的溶解粘附连接和扭曲的绒毛膜毛细血管基膜，以及它们之间的胶原层的紊乱，这两种情况在任何WT-SPF小鼠中均未发现(图5C和图6B)。这与Rd8-SPF小鼠的Bruch膜破裂和Bruch膜厚度显著减少有关(图5C、图6B和图5D)。另一方面，透射电镜检查没有发现视网膜毛细血管内皮细胞和视网膜色素上皮细胞之间紧密连接的任何变化。因此，结果提示Rd8-SPF小鼠视网膜的主要缺陷是外血-视网膜屏障，而不是内血-视网膜屏障。
实施例6对肠道不通部位的微生物群进行宏基因组分析
在Rd8视网膜中发现的所有七种细菌(图4B)以前都是胃肠道(GI)细菌。因此，对WT-SPF(n＝5)和Rd8-SPF(n＝5)小鼠胃肠道不同部位(包括胃、空肠、回肠、盲肠、结肠和直肠)的微生物群进行宏基因组分析(图8A-F)。Rd8-SPF小鼠下消化道中的微生物成分与WT-SPF小鼠中的微生物成分明显不同(图8G)。在盲肠(图8H)、结肠(图8I)和直肠(图8J)的WT和Rd8肠道菌群中，Akkermansia mucinphila被确定为最不同的细菌种类，尽管在Rd8视网膜中没有追踪到(图4B)。实际上，Rd8视网膜中的7种细菌都在WT和Rd8小鼠的下消化道中发现，盲肠中的细菌最多(图7A)。然而，在Rd8小鼠的上消化道中几乎检测不到它们。这些数据表明，这些视网膜细菌存在于Rd8-SPF而不是在WT-SPF小鼠的胃肠道菌群。
通过免疫荧光染色鉴定了野生型小鼠盲肠肠细胞中CRB1蛋白的表达，而在Rd8小鼠中其表达明显减弱(图9A)。此外，观察到Rd8小鼠盲肠中Occludin(图9B-C)表达显著缺失，而Claudin1(图9D-E)表达不明显。进一步的透射电镜检查显示盲肠上皮屏障处有正常的紧密连接和粘附连接(图9F)。
实施例7检查Rd8小鼠结肠是否存在屏障缺陷
与盲肠的结果相似，发现CRB1蛋白在结肠肠上皮细胞的顶面和底面有明显的表达(图7B)。CRB1蛋白的丢失与Phalloidin(图7C)和Occludin(图7D和图4E)的表达显著减少相关，但与Claudin1(图7F和图4G)蛋白的表达无关。重要的是，利用透射电镜，观察到大多数细胞中结肠上皮间的粘附连接无法定位，而Rd8小鼠中的紧密连接似乎正常(图7H和图S6A)。在Rd8小鼠结肠上皮中，除连接改变外，还观察到线粒体空泡化。尽管上皮微绒毛的数量没有变化(图7I)，但Rd8小鼠的微绒毛变得更长(图7J)和更细(图7K)，与WT小鼠相比，完整粘附连接的总数显著减少(图7L)。连接和微绒毛的超微结构改变也伴随着结肠壁中Il12a表达的显著升高(图7M)，而Tnfa、Il1b和黏蛋白2的表达没有任何变化(图7N)。综上所述，数据表明Rd8小鼠具有结肠上皮屏障缺陷及其相关的结肠壁炎症。
实施例8检查肠上皮屏障缺陷是否会导致Rd8小鼠肠道通透性改变和微生物群像周围血流和视网膜组织迁移
对WT和Rd8小鼠进行肠FICT-dextran通透性测定。如图11A所示，与WT小鼠血液中荧光强度相比，给予FICT右旋糖酐后5小时，Rd8小鼠外周血中荧光显著增加。此外，使用Vancompy标记的粪便微生物群移植试验，发现Rd8小鼠外周血中荧光+细菌/细胞的显著升高，与WT小鼠在粪便移植后24小时相比(图10B和图11B)。重要的是，Rd8小鼠视网膜病变中可检测到Vancomopy+细菌的出现(图11C)，但WT视网膜病变中没有(未显示数据)，Rd8小鼠视网膜组织的免疫荧光染色进一步证实了这一点(图11D)。这些数据表明，肠道对多糖分子和细菌细胞的通透性增加，导致细菌从肠腔转移到血流和视网膜病变。
实施例9 Rd8在增加肠道通透性的情况下对肠道应激的反应实验
将WT和Rd8小鼠暴露于含1.5％右旋糖酐硫酸钠(DSS)的饮用水中，引起WT小鼠的轻微结肠炎。DSS治疗13天后，发现Rd8小鼠的结肠长度明显短于野生型小鼠(图11E)，Rd8小鼠血液中的细菌比野生型小鼠血液中的细菌多，细菌16S rRNA(图11F)和细菌LPS(图11G)的血清水平表明了这一点。
实施例10细菌对Rd8小鼠视网膜表型的逆转作用
虽然在Rd8小鼠视网膜中发现了由外血-视网膜屏障和肠上皮屏障破坏引起的病变内细菌，但这些细菌是Rd8小鼠视网膜变性的原因还是后果尚不清楚。因此，在无菌(GF)条件下重新分离Rd8小鼠，并检测其视网膜变性表型是否发生改变。如图12A所示，在Rd8-SPF小鼠中发现的视网膜病变(白斑)在无菌Rd8(Rd8 GF)小鼠中几乎没有观察到。Rd8-GF小鼠的视网膜组织学显示正常发育的视网膜组织(图12B)没有Rd8-SPF小鼠中发现的典型病变(图2A和图1C)，这证实了Rd8-GF小鼠中缺乏视网膜变性。Rd8 GF小鼠中无法检测到视网膜细菌的事实(图12C)与Rd8 GF小鼠中视网膜外核层(ONL)(图12D和图12E)中小胶质细胞的急剧减少有关，尽管ZO-1和Phalloidin蛋白染色显示外界膜的破坏，在Rd8-SPF 和Rd8 GF小鼠中均发现(图12F)。此外，当出生后在SPF环境中饲养Rd8 GF小鼠(Rd8 GF SPF小鼠)时，视网膜病变再次出现(图12G)。这些数据支持Rd8小鼠视网膜变性是细菌依赖性的事实。
以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (36)

  1. 一种建立眼部疾病模型或模型载体的方法，所述方法包括将眼部疾病模型或模型载体感染微生物。
  2. 根据权利要求1所述的方法，其特征在于，所述的感染包括直接与微生物接触或者间接与微生物接触，所述的间接与微生物接触为在眼部和微生物之间存在视网膜屏障。
  3. 根据权利要求1-2任一所述的方法，其特征在于，所述的微生物来自同一个体的肠道细菌或者与该个体的肠道细菌相同。
  4. 根据权利要求1-3任一所述的方法，其特征在于，所述的眼部疾病包含视网膜变性；优选的，所述的视网膜变性为进行性视网膜变性；进一步优选的，所述的视网膜变性为遗传性视网膜变性。
  5. 根据权利要求1-4任一所述的方法，其特征在于，所述的眼部疾病包含眼部炎症，优选的，所述的眼部炎症包含视网膜炎。
  6. 根据权利要求1-5任一所述的方法，其特征在于，所述的模型是非人动物，优选猴、犬、黑猩猩、大鼠、小鼠。
  7. 根据权利要求1-6任一所述的方法，其特征在于，所述的模型载体选自细胞、组织或器官，所述的组织或器官来源于非人动物，所述的细胞来源于人或非人动物的原代细胞或细胞系，所述的组织或器官来源于非人动物或来源于人干细胞发育得到的眼部组织或器官。
  8. 根据权利要求1-7任一所述的方法，其特征在于，所述的模型或模型载体中眼部基因存在致病突变，优选的，所述的发生了致病突变的眼部基因包括以下一个或两个以上基因发生了突变：ABCA4、ABCC6、ABCC9、ACBD5、ACO2、ACO2、ACTG1、ADGRV1、AHI1、AIPL1、ALMS1、AMY2B、APC、ARFGEF1、ARL13B、ARL13B、ARL6、ARMC9、ATOH7、B9D1、BAG3、BBS1、BBS1、BBS2、BBS5、BEST1、C2CD3、CA4、CABP4、CACNA1F、CBS、CC2D2A、CDH23、CDH23、CDHR1、CEMIP2、CEP104、CEP250、CEP290、CEP290、CEP41、CEP78、CERKL、CFAP410、CFAP418、CHM、CLCC1、CLCN7、CLN3、CLN5、CLN8、CLRN1、CLRN1、CNGA1、CNGA1、CNGA3、CNGB1、CNGB3、CNNM4、COL11A1、COL11A2、COL18A1、COL2A1、COL4A1、COL9A1、COL9A2、CP、CP、CPLANE1、CRB1、ERCC4、CSPP1、CTNNA1、CYP4V2、DHDDS、DYNC2H1、DYNC2I1、DYNC2I2、ENPP1、ERCC4、EVC2、EYS、EYS、F5、FAM161A、FBN1、FKRP、FKTN、FLG、FLVCR1、FOXE3、FUZ、GLB1、GMPPB、GNAT1、GRK1、GRM6、GUCA1A、GUCA1B、GUCY2D、HADHA、HGSNAT、HPS3、HPS5、IDH3B、IFT122、IFT140、IFT140、IFT43、IFT52、IFT74、IFT80、IFT80、IFT81、IFT88、IKBKG、IMPDH1、IMPG2、INPP5E、INTU、IQCB1、IQCE、IREB2、KCNJ13、KCNQ1、KCNV2、KIAA0586、KIAA0753、KIF7、KIZ、KIZ-AS1、KLHL7、KRIT1、LBR、LCA5、LOC101927157、LOC111365204、LRP2、 LRP5、MAK、MAPKAPK3、MATK、MCOLN1、MERTK、MKS1、MPDZ、MT-ATP6、MT-CO3、MT-TE、MT-TL1、MTHFR、MUTYH、MYO7A、MYO7A、NMNAT1、NPHP1、NR2E3、OCA2、OTX2、PANK2、PAX6、PCARE、PCDH15、PDE6A、PDE6B、PDE6B、PDE6D、PEX1、PEX1、PEX12、PEX26、PEX6、PHF3、PITPNM3、PKD2、PLA2G5、POC5、POMT1、PRCD、PRDM13、PROM1、PRPF3、PRPF31、PRPF8、PRPH2、RAD51C、RBP3、RBP4、RD3、RDH12、RDH5、RGR、RGR、RHO、RIMS1、RLBP1、ROM1、RP1、RP1L1、RP2、RPE65、RPE65、RPGR、RPGRIP1、RPGRIP1L、RS1、SACS、SAG、SCAPER、SDCCAG8、SIX6、SLC19A1、SLC22A5、SLC26A4、SLC2A9、SLTM、SNRNP200、SPAG17、SPATA7、SPG11、TFAP2A、TGFB2、TGFBR2、TMEM107、TMEM237、TMEM67、TOGARAM1、TOPORS、TPP1、TRAF3IP1、TREX1、TRIM59-IFT80、TSPAN12、TTC21B、TTC21B、TTC8、TULP1、USH1C、USH2A、USH2A、USH2A、USH2A、USH2A-AS1、VAC14、VCAN、VCAN、VCAN-AS1、VHL、VPS13B、WDR19、WDR19、WDR35、WDR73、YARS1、ZFYVE26、ZFYVE26、ZNF408的一种或两种以上的组合。
  9. 根据权利要求1-8任一所述的方法，其特征在于，所述的模型或模型载体的CRB1基因包含如下的一种或两种以上的突变：c.107C>G、c.111delT、c.135C>G、c.257_258dupTG、c.258C>T、c.428_432delGATTC、c.430T>G、c.470G>C、c.481dupG、c.482C>T、c.584G>T、c.613_619del、c.717_718insG、c.750T>G、c.915T>A、c.929G>A、c.936T>G、c.998G>A、c.1084C>T、c.1125C>G、c.1148G>A、c.1208C>G、c.1269C>A、c.1298A>G、c.1313G>A、c.1438T>C、c.1438T>G、c.1576C>T、c.1604T>C、c.1690G>T、c.1733T>A、c.1750G>T、c.1760G>A、c.1834T>C、c.1963delC、c.2025G>T、c.2042G>A、c.2128G>C、c.2129C>T、c.2185_2186insAlu、c.2219C>T、c.2222T>C、c.2234C>T、c.2245_2247del 3bp(TCA)、c.2258T>C、c.2290C>T、c.2365_2367del AAT,in frame deletion、c.2401A>T、c.2438_2439ins>100A、c.2441_2442del、c.2465G>A、c.2479G>T、c.2506C>A、c.2509G>C、c.2536G>A、c.2548_2551delGGCT、c.2548G>A、c.2555T>C、c.2611_2613insT、c.2671T>G、c.2676delG、c.2681A>G、c.2688T>A、c.2816G>A、c.2843G>A、c.2853dupT、c.2884_2886delTTA、c.2957A>T、c.2966T>C、c.2983G>T、c.3002A>T、c.3008T>C、c.3035T>C、c.3037C>T、c.3074G>A、c.3074G>T、c.3122T>C、c.3212T>C、c.3296C>A、c.3299T>C、c.3299T>G、c.3307G>A/C、c.3320T>C、c.3320T>G、c.3331G>T、c.3343_3352del、c.3347delT、c.3343_3352del、c.3347delT、c.3427delT、c.3482A>G、c.3493T>C、c.3655T>G、c.3541T>C、c.3542dupG、c.3593A>G、c.3613G>A、c.3653G>T、c.3659_3660delinsA、c.3664C>T、c.3668G>C、c.3676G>T、c.3713_3716dup、c.3879G>A、c.3914C>T、c.3949A>C、c.3961T>A、c.3988delG、c.3988G>T、c.3995G>T、c.3996C>A、c.3997G>T、c.4094C>A、c.4121_4130del、c.4142C>T、c.4148G>A、c.2128+2T>G、c.2842+5G>A、c.3878+1G>T、c.4005+1G>A、c.4005+2T>G、c.4006-2A>G、c.4006-1G>T、c.619G>A、c.614T>C、c.1472A>T、c.1903T>C、c.2809G>A、c.3103C>T、c.4082G>A、c.4060G>A、c.866C>T、c.1463T>C、 c.2035C>G、c.2306_2307GC>AG、c.2306G>A、c.2714G>A、c.2875G>A、c.3992G>A。
  10. 根据权利要求1-9任一所述的方法，其特征在于，所述的模型或模型载体的CRB1基因包含Rd8突变。
  11. 根据权利要求9-10任一所述的方法，其特征在于，所述的突变为纯和突变或杂合突变。
  12. 根据权利要求1-11任一所述的方法，其特征在于，所述的模型或模型载体存在基因突变是先天的或通过基因重组操作后天携带的。
  13. 根据权利要求1-12任一所述的方法，其特征在于，所述的疾病模型具有结肠上皮屏障缺陷和/或相关的结肠壁炎症。
  14. 根据权利要求1-13任一所述的方法，其特征在于，所述微生物为细菌、古细菌、原生生物、真菌或病毒中的一种或两种以上的组合，优选的，所述微生物为细菌，所述细菌选自：Anearostipes、双歧杆菌(Bifidobacterium)、巨单胞菌(Megamonas)、亚硝基单胞菌(Nitrosomonas)，颤螺旋菌(Oscillibacter)、塔特姆菌(Tatumella)、硫杆菌(Thiobacillus sp.)、梭菌属(Clostridium)、不动杆菌属(Acinetobacter)、链球菌属(Streptococcus)、曼氏杆菌属(Mannheimia)、纤维杆菌属(Fibrobacter)、普氏菌属(Prevotella)、弯曲杆菌属(Campylobacter)、放线菌属(Actinomyces)、薄层菌属(Hymenobacter)、埃希氏杆菌属(Escherichia)、泰氏菌属(Tissierella)、克雷白氏杆菌属(Klebsiella)、卟啉单胞菌属(Porphyromonas)、固氮螺菌属(Azospira)、海水菌属(Aquimarina)、无色菌属(Achromobacter)、嗜酸硫杆菌属(Acidithiobacillus)、伯克霍尔德菌属(Burkholderia)、海杆菌属(Marinobacter)、密螺旋体属(Treponema)、孢裹放线菌属(Actinosporangium)、弧菌属(Vibrio)、瘤胃球菌属(Ruminococcus)、甲烷短杆菌(Methanobrevibacter)、志贺氏杆菌(Shigella)、弗兰克氏菌属(Frankia)、链霉菌属厌氧原体属(Anaeroplasma)、粪球菌属(Coprococcus)中的一种或两种以上。
  15. 根据权利要求1-14任一所述的方法，其特征在于，所述细菌选自：Anearostipes hadrus、Bifidobacterium pseudocatenulatum、Nitrosomonas sp.Is79A3、Oscillibacter valericigenes、Tatumella sp.TA1、Megamonas funiformis、Thiobacillus denitrificans、破伤风梭菌、产气荚膜梭菌、肉毒梭菌、醋酸钙不动杆菌、鲁菲不动杆菌、鲍曼不动杆菌、溶血不动杆菌、琼氏不动杆菌、约翰逊不动杆菌、化脓性链球菌、溶血性链球菌、产琥珀酸丝状杆菌、肠道丝状杆菌、非解糖卟啉单胞菌、牙髓卟啉单胞菌、牙龈卟啉单胞菌、空肠弯曲菌、结肠弯曲菌、海鸟弯曲菌、乌普萨拉弯曲菌、简明弯曲菌、胎儿弯曲菌、衣氏放线菌、奈氏放线菌、溶齿放线菌、粘稠放线菌、纽氏放线菌、大肠埃希菌、蟑螂埃希菌、弗格森埃希菌、赫尔曼埃希菌、伤口埃希菌、极尖泰氏菌、肺炎克雷伯氏菌、臭鼻克雷伯氏 菌、巴西固氮螺菌、无色杆菌、脱氮硫杆菌、氧化亚铁硫杆菌、氧化硫硫杆菌、那不勒斯硫杆菌、伯克霍尔德菌、海洋分枝杆菌、苍白密螺旋体、猪痢疾密螺旋体、梅氏弧菌、白色瘤胃球菌、生黄瘤胃球菌、瘤胃甲烷短杆菌、痢疾志贺菌、福氏志贺菌、鲍氏志贺菌、宋内志贺菌、弗兰克氏菌、规则粪球菌、白色链霉菌、门多萨假单胞菌，栖息微球菌，脂环素反硝化菌，氧化木糖无色杆菌，鞘脂单胞杆菌，脓肿分枝杆菌，金黄节杆菌，普氏菌，草木樨中华根瘤菌，酸性酵母，表皮葡萄球菌，铜绿假单胞菌，金黄色葡萄球菌，溶血性葡萄球菌，恶臭假单胞菌，嗜麦芽寡养单胞菌，蜡状芽孢杆菌，巨大芽孢杆菌，罗氏乳酸杆菌，阴道嗜血杆菌，蜜蜂屎肠球菌，正哈氏嗜纤维菌，地衣芽孢杆菌，白叶枯菌，鲍曼不动杆菌，醋酸钙不动杆菌，睾丸酮丛毛单胞菌，堪萨斯分枝杆菌，斯润金芽孢杆菌，柯氏柠檬酸杆菌，发酵成对杆菌，粘质沙雷氏菌，维氏鞘氨醇单胞菌，肺炎克雷伯菌，荧光假单胞菌，皮氏罗尔斯顿菌，卷曲乳杆菌，伯克霍尔德菌，德氏乳杆菌，Meiothermus silvanus(D)，大肠杆菌，藤黄微球菌，枯草芽孢杆菌，粘金色棒状杆菌，大芬戈尔德菌中的一种或两种以上。
  16. 一种眼部疾病模型载体，所述眼部疾病是由于微生物感染了所述的模型载体引起的，优选的，所述的模型载体选自细胞、组织或器官，进一步优选的，所述的细胞来源于人或非人动物的原代细胞或细胞系，所述的组织或器官来源于非人动物或来源于人干细胞发育得到的眼部组织或器官。
  17. 根据权利要求16所述的眼部疾病模型载体，其特征在于，所述的眼部疾病包含视网膜变性；优选的，所述的视网膜变性为进行性视网膜变性；进一步优选的，所述的视网膜变性为遗传性视网膜变性。
  18. 根据权利要求16-17任一所述的眼部疾病模型载体，其特征在于，所述的眼部疾病包含眼部炎症，优选的，所述的眼部炎症包含视网膜炎。
  19. 根据权利要求16-18任一所述的眼部疾病模型载体，其特征在于，所述的微生物来自同一个体的肠道细菌或者与该个体的肠道细菌相同。
  20. 根据权利要求16-19任一所述的眼部疾病模型载体，其特征在于，所述的模型载体中以下一个或两个以上基因发生了突变：ABCA4、ABCC6、ABCC9、ACBD5、ACO2、ACO2、ACTG1、ADGRV1、AHI1、AIPL1、ALMS1、AMY2B、APC、ARFGEF1、ARL13B、ARL13B、ARL6、ARMC9、ATOH7、B9D1、BAG3、BBS1、BBS1、BBS2、BBS5、BEST1、C2CD3、CA4、CABP4、CACNA1F、CBS、CC2D2A、CDH23、CDH23、CDHR1、CEMIP2、CEP104、CEP250、CEP290、CEP290、CEP41、CEP78、CERKL、CFAP410、CFAP418、CHM、CLCC1、CLCN7、CLN3、CLN5、CLN8、CLRN1、CLRN1、CNGA1、CNGA1、CNGA3、CNGB1、CNGB3、CNNM4、COL11A1、COL11A2、COL18A1、COL2A1、COL4A1、COL9A1、COL9A2、CP、CP、CPLANE1、 CRB1、ERCC4、CSPP1、CTNNA1、CYP4V2、DHDDS、DYNC2H1、DYNC2I1、DYNC2I2、ENPP1、ERCC4、EVC2、EYS、EYS、F5、FAM161A、FBN1、FKRP、FKTN、FLG、FLVCR1、FOXE3、FUZ、GLB1、GMPPB、GNAT1、GRK1、GRM6、GUCA1A、GUCA1B、GUCY2D、HADHA、HGSNAT、HPS3、HPS5、IDH3B、IFT122、IFT140、IFT140、IFT43、IFT52、IFT74、IFT80、IFT80、IFT81、IFT88、IKBKG、IMPDH1、IMPG2、INPP5E、INTU、IQCB1、IQCE、IREB2、KCNJ13、KCNQ1、KCNV2、KIAA0586、KIAA0753、KIF7、KIZ、KIZ-AS1、KLHL7、KRIT1、LBR、LCA5、LOC101927157、LOC111365204、LRP2、LRP5、MAK、MAPKAPK3、MATK、MCOLN1、MERTK、MKS1、MPDZ、MT-ATP6、MT-CO3、MT-TE、MT-TL1、MTHFR、MUTYH、MYO7A、MYO7A、NMNAT1、NPHP1、NR2E3、OCA2、OTX2、PANK2、PAX6、PCARE、PCDH15、PDE6A、PDE6B、PDE6B、PDE6D、PEX1、PEX1、PEX12、PEX26、PEX6、PHF3、PITPNM3、PKD2、PLA2G5、POC5、POMT1、PRCD、PRDM13、PROM1、PRPF3、PRPF31、PRPF8、PRPH2、RAD51C、RBP3、RBP4、RD3、RDH12、RDH5、RGR、RGR、RHO、RIMS1、RLBP1、ROM1、RP1、RP1L1、RP2、RPE65、RPE65、RPGR、RPGRIP1、RPGRIP1L、RS1、SACS、SAG、SCAPER、SDCCAG8、SIX6、SLC19A1、SLC22A5、SLC26A4、SLC2A9、SLTM、SNRNP200、SPAG17、SPATA7、SPG11、TFAP2A、TGFB2、TGFBR2、TMEM107、TMEM237、TMEM67、TOGARAM1、TOPORS、TPP1、TRAF3IP1、TREX1、TRIM59-IFT80、TSPAN12、TTC21B、TTC21B、TTC8、TULP1、USH1C、USH2A、USH2A、USH2A、USH2A、USH2A-AS1、VAC14、VCAN、VCAN、VCAN-AS1、VHL、VPS13B、WDR19、WDR19、WDR35、WDR73、YARS1、ZFYVE26、ZFYVE26、ZNF408的一种或两种以上的组合。
  21. 根据权利要求16-20任一所述的眼部疾病模型载体，其特征在于，所述的模型载体的CRB1基因突变包含如下的一种或两种以上的突变：c.107C>G、c.111delT、c.135C>G、c.257_258dupTG、c.258C>T、c.428_432delGATTC、c.430T>G、c.470G>C、c.481dupG、c.482C>T、c.584G>T、c.613_619del、c.717_718insG、c.750T>G、c.915T>A、c.929G>A、c.936T>G、c.998G>A、c.1084C>T、c.1125C>G、c.1148G>A、c.1208C>G、c.1269C>A、c.1298A>G、c.1313G>A、c.1438T>C、c.1438T>G、c.1576C>T、c.1604T>C、c.1690G>T、c.1733T>A、c.1750G>T、c.1760G>A、c.1834T>C、c.1963delC、c.2025G>T、c.2042G>A、c.2128G>C、c.2129C>T、c.2185_2186insAlu、c.2219C>T、c.2222T>C、c.2234C>T、c.2245_2247del 3bp(TCA)、c.2258T>C、c.2290C>T、c.2365_2367del AAT,in frame deletion、c.2401A>T、c.2438_2439ins>100A、c.2441_2442del、c.2465G>A、c.2479G>T、c.2506C>A、c.2509G>C、c.2536G>A、c.2548_2551delGGCT、c.2548G>A、c.2555T>C、c.2611_2613insT、c.2671T>G、c.2676delG、c.2681A>G、c.2688T>A、c.2816G>A、c.2843G>A、c.2853dupT、c.2884_2886delTTA、c.2957A>T、c.2966T>C、c.2983G>T、c.3002A>T、c.3008T>C、c.3035T>C、c.3037C>T、c.3074G>A、c.3074G>T、c.3122T>C、c.3212T>C、c.3296C>A、c.3299T>C、 c.3299T>G、c.3307G>A/C、c.3320T>C、c.3320T>G、c.3331G>T、c.3343_3352del、c.3347delT、c.3343_3352del、c.3347delT、c.3427delT、c.3482A>G、c.3493T>C、c.3655T>G、c.3541T>C、c.3542dupG、c.3593A>G、c.3613G>A、c.3653G>T、c.3659_3660delinsA、c.3664C>T、c.3668G>C、c.3676G>T、c.3713_3716dup、c.3879G>A、c.3914C>T、c.3949A>C、c.3961T>A、c.3988delG、c.3988G>T、c.3995G>T、c.3996C>A、c.3997G>T、c.4094C>A、c.4121_4130del、c.4142C>T、c.4148G>A、c.2128+2T>G、c.2842+5G>A、c.3878+1G>T、c.4005+1G>A、c.4005+2T>G、c.4006-2A>G、c.4006-1G>T、c.619G>A、c.614T>C、c.1472A>T、c.1903T>C、c.2809G>A、c.3103C>T、c.4082G>A、c.4060G>A、c.866C>T、c.1463T>C、c.2035C>G、c.2306_2307GC>AG、c.2306G>A、c.2714G>A、c.2875G>A、c.3992G>A。
  22. 根据权利要求16-21任一所述的眼部疾病模型载体，其特征在于，所述的模型载体的CRB1基因的突变为Rd8突变。
  23. 根据权利要求16-22任一所述的眼部疾病模型载体，其特征在于，所述的突变为纯和突变或杂合突变。
  24. 根据权利要求16-23任一所述的眼部疾病模型载体，其特征在于，所述的突变是所述的模型载体先天携带的或经过基因重组操作后天携带的。
  25. 根据权利要求16-24任一所述的眼部疾病模型载体，其特征在于，所述的非人动物具有结肠上皮屏障缺陷和/或相关的结肠壁炎症。
  26. 根据权利要求16-25任一所述的眼部疾病模型载体，其特征在于，所述微生物为细菌、古细菌、原生生物、真菌或病毒中的一种或两种以上的组合，优选的，所述微生物为细菌，所述细菌选自：Anearostipes、双歧杆菌(Bifidobacterium)、巨单胞菌(Megamonas)、亚硝基单胞菌(Nitrosomonas)，颤螺旋菌(Oscillibacter)、塔特姆菌(Tatumella)、硫杆菌(Thiobacillus sp.)、梭菌属(Clostridium)、不动杆菌属(Acinetobacter)、链球菌属(Streptococcus)、曼氏杆菌属(Mannheimia)、纤维杆菌属(Fibrobacter)、普氏菌属(Prevotella)、弯曲杆菌属(Campylobacter)、放线菌属(Actinomyces)、薄层菌属(Hymenobacter)、埃希氏杆菌属(Escherichia)、泰氏菌属(Tissierella)、克雷白氏杆菌属(Klebsiella)、卟啉单胞菌属(Porphyromonas)、固氮螺菌属(Azospira)、海水菌属(Aquimarina)、无色菌属(Achromobacter)、嗜酸硫杆菌属(Acidithiobacillus)、伯克霍尔德菌属(Burkholderia)、海杆菌属(Marinobacter)、密螺旋体属(Treponema)、孢裹放线菌属(Actinosporangium)、弧菌属(Vibrio)、瘤胃球菌属(Ruminococcus)、甲烷短杆菌(Methanobrevibacter)、志贺氏杆菌(Shigella)、弗兰克氏菌属(Frankia)、链霉菌属厌氧原体属(Anaeroplasma)、粪球菌属(Coprococcus)中的一种或两种以上。
  27. 根据权利要求16-26任一所述的眼部疾病模型载体，其特征在于，所述细菌选自： Anearostipes hadrus、Bifidobacterium pseudocatenulatum、Nitrosomonas sp.Is79A3、Oscillibacter valericigenes、Tatumella sp.TA1、Megamonas funiformis、Thiobacillus denitrificans、破伤风梭菌、产气荚膜梭菌、肉毒梭菌、醋酸钙不动杆菌、鲁菲不动杆菌、鲍曼不动杆菌、溶血不动杆菌、琼氏不动杆菌、约翰逊不动杆菌、化脓性链球菌、溶血性链球菌、产琥珀酸丝状杆菌、肠道丝状杆菌、非解糖卟啉单胞菌、牙髓卟啉单胞菌、牙龈卟啉单胞菌、空肠弯曲菌、结肠弯曲菌、海鸟弯曲菌、乌普萨拉弯曲菌、简明弯曲菌、胎儿弯曲菌、衣氏放线菌、奈氏放线菌、溶齿放线菌、粘稠放线菌、纽氏放线菌、大肠埃希菌、蟑螂埃希菌、弗格森埃希菌、赫尔曼埃希菌、伤口埃希菌、极尖泰氏菌、肺炎克雷伯氏菌、臭鼻克雷伯氏菌、巴西固氮螺菌、无色杆菌、脱氮硫杆菌、氧化亚铁硫杆菌、氧化硫硫杆菌、那不勒斯硫杆菌、伯克霍尔德菌、海洋分枝杆菌、苍白密螺旋体、猪痢疾密螺旋体、梅氏弧菌、白色瘤胃球菌、生黄瘤胃球菌、瘤胃甲烷短杆菌、痢疾志贺菌、福氏志贺菌、鲍氏志贺菌、宋内志贺菌、弗兰克氏菌、规则粪球菌、白色链霉菌、门多萨假单胞菌，栖息微球菌，脂环素反硝化菌，氧化木糖无色杆菌，鞘脂单胞杆菌，脓肿分枝杆菌，金黄节杆菌，普氏菌，草木樨中华根瘤菌，酸性酵母，表皮葡萄球菌，铜绿假单胞菌，金黄色葡萄球菌，溶血性葡萄球菌，恶臭假单胞菌，嗜麦芽寡养单胞菌，蜡状芽孢杆菌，巨大芽孢杆菌，罗氏乳酸杆菌，阴道嗜血杆菌，蜜蜂屎肠球菌，正哈氏嗜纤维菌，地衣芽孢杆菌，白叶枯菌，鲍曼不动杆菌，醋酸钙不动杆菌，睾丸酮丛毛单胞菌，堪萨斯分枝杆菌，斯润金芽孢杆菌，柯氏柠檬酸杆菌，发酵成对杆菌，粘质沙雷氏菌，维氏鞘氨醇单胞菌，肺炎克雷伯菌，荧光假单胞菌，皮氏罗尔斯顿菌，卷曲乳杆菌，伯克霍尔德菌，德氏乳杆菌，Meiothermus silvanus(D)，大肠杆菌，藤黄微球菌，枯草芽孢杆菌，粘金色棒状杆菌，大芬戈尔德菌中的一种或两种以上。
  28. 根据权利要求16-27任一所述的眼部疾病模型载体，其特征在于，所述的眼部疾病模型载体是按照权利要求1-15的方法得到的。
  29. 根据权利要求16-28所述的眼部疾病模型载体，其特征在于，所述的眼部疾病模型载体来自权利要求1-15的方法构建的眼部疾病模型。
  30. 一种权利要求1-15任一所述的方法、权利要求1-15任一所述的方法制备得到的眼部疾病模型、权利要求16-29任一所述的眼部疾病模型载体在筛选眼部疾病靶向治疗药物中的应用。
  31. 根据权利要求30所述的应用，其特征在于，所述的靶向治疗靶向与眼部疾病相关的基因。
  32. 根据权利要求30-31任一所述的应用，其特征在于，所述的与眼部疾病相关的基因为以下一种基因或两种以上的基因的组合：ABCA4、ABCC6、ABCC9、ACBD5、ACO2、 ACO2、ACTG1、ADGRV1、AHI1、AIPL1、ALMS1、AMY2B、APC、ARFGEF1、ARL13B、ARL13B、ARL6、ARMC9、ATOH7、B9D1、BAG3、BBS1、BBS1、BBS2、BBS5、BEST1、C2CD3、CA4、CABP4、CACNA1F、CBS、CC2D2A、CDH23、CDH23、CDHR1、CEMIP2、CEP104、CEP250、CEP290、CEP290、CEP41、CEP78、CERKL、CFAP410、CFAP418、CHM、CLCC1、CLCN7、CLN3、CLN5、CLN8、CLRN1、CLRN1、CNGA1、CNGA1、CNGA3、CNGB1、CNGB3、CNNM4、COL11A1、COL11A2、COL18A1、COL2A1、COL4A1、COL9A1、COL9A2、CP、CP、CPLANE1、CRB1、ERCC4、CSPP1、CTNNA1、CYP4V2、DHDDS、DYNC2H1、DYNC2I1、DYNC2I2、ENPP1、ERCC4、EVC2、EYS、EYS、F5、FAM161A、FBN1、FKRP、FKTN、FLG、FLVCR1、FOXE3、FUZ、GLB1、GMPPB、GNAT1、GRK1、GRM6、GUCA1A、GUCA1B、GUCY2D、HADHA、HGSNAT、HPS3、HPS5、IDH3B、IFT122、IFT140、IFT140、IFT43、IFT52、IFT74、IFT80、IFT80、IFT81、IFT88、IKBKG、IMPDH1、IMPG2、INPP5E、INTU、IQCB1、IQCE、IREB2、KCNJ13、KCNQ1、KCNV2、KIAA0586、KIAA0753、KIF7、KIZ、KIZ-AS1、KLHL7、KRIT1、LBR、LCA5、LOC101927157、LOC111365204、LRP2、LRP5、MAK、MAPKAPK3、MATK、MCOLN1、MERTK、MKS1、MPDZ、MT-ATP6、MT-CO3、MT-TE、MT-TL1、MTHFR、MUTYH、MYO7A、MYO7A、NMNAT1、NPHP1、NR2E3、OCA2、OTX2、PANK2、PAX6、PCARE、PCDH15、PDE6A、PDE6B、PDE6B、PDE6D、PEX1、PEX1、PEX12、PEX26、PEX6、PHF3、PITPNM3、PKD2、PLA2G5、POC5、POMT1、PRCD、PRDM13、PROM1、PRPF3、PRPF31、PRPF8、PRPH2、RAD51C、RBP3、RBP4、RD3、RDH12、RDH5、RGR、RGR、RHO、RIMS1、RLBP1、ROM1、RP1、RP1L1、RP2、RPE65、RPE65、RPGR、RPGRIP1、RPGRIP1L、RS1、SACS、SAG、SCAPER、SDCCAG8、SIX6、SLC19A1、SLC22A5、SLC26A4、SLC2A9、SLTM、SNRNP200、SPAG17、SPATA7、SPG11、TFAP2A、TGFB2、TGFBR2、TMEM107、TMEM237、TMEM67、TOGARAM1、TOPORS、TPP1、TRAF3IP1、TREX1、TRIM59-IFT80、TSPAN12、TTC21B、TTC21B、TTC8、TULP1、USH1C、USH2A、USH2A、USH2A、USH2A、USH2A-AS1、VAC14、VCAN、VCAN、VCAN-AS1、VHL、VPS13B、WDR19、WDR19、WDR35、WDR73、YARS1、ZFYVE26、ZFYVE26、ZNF408。
  33. 根据权利要求30-32任一所述的应用，其特征在于，所述的靶向治疗靶向CRB1基因的如下一种或两种以上的突变：c.257_258dupTG、c.258C>T、c.428_432delGATTC、c.430T>G、c.470G>C、c.481dupG、c.482C>T、c.584G>T、c.613_619del、c.717_718insG、c.750T>G、c.915T>A、c.929G>A、c.936T>G、c.998G>A、c.1084C>T、c.1125C>G、c.1148G>A、c.1208C>G、c.1269C>A、c.1298A>G、c.1313G>A、c.1438T>C、c.1438T>G、c.1576C>T、c.1604T>C、c.1690G>T、c.1733T>A、c.1750G>T、c.1760G>A、c.1834T>C、c.1963delC、c.2025G>T、c.2042G>A、c.2128G>C、c.2129C>T、c.2185_2186insAlu、c.2219C>T、c.2222T>C、c.2234C>T、c.2245_2247del 3bp(TCA)、c.2258T>C、c.2290C>T、c.2365_2367del AAT,in frame deletion、c.2401A>T、c.2438_2439ins>100A、 c.2441_2442del、c.2465G>A、c.2479G>T、c.2506C>A、c.2509G>C、c.2536G>A、c.2548_2551delGGCT、c.2548G>A、c.2555T>C、c.2611_2613insT、c.2671T>G、c.2676delG、c.2681A>G、c.2688T>A、c.2816G>A、c.2843G>A、c.2853dupT、c.2884_2886delTTA、c.2957A>T、c.2966T>C、c.2983G>T、c.3002A>T、c.3008T>C、c.3035T>C、c.3037C>T、c.3074G>A、c.3074G>T、c.3122T>C、c.3212T>C、c.3296C>A、c.3299T>C、c.3299T>G、c.3307G>A/C、c.3320T>C、c.3320T>G、c.3331G>T、c.3343_3352del、c.3347delT、c.3343_3352del、c.3347delT、c.3427delT、c.3482A>G、c.3493T>C、c.3655T>G、c.3541T>C、c.3542dupG、c.3593A>G、c.3613G>A、c.3653G>T、c.3659_3660delinsA、c.3664C>T、c.3668G>C、c.3676G>T、c.3713_3716dup、c.3879G>A、c.3914C>T、c.3949A>C、c.3961T>A、c.3988delG、c.3988G>T、c.3995G>T、c.3996C>A、c.3997G>T、c.4094C>A、c.4121_4130del、c.4142C>T、c.4148G>A、c.2128+2T>G、c.2842+5G>A、c.3878+1G>T、c.4005+1G>A、c.4005+2T>G、c.4006-2A>G、c.4006-1G>T、c.619G>A、c.614T>C、c.1472A>T、c.1903T>C、c.2809G>A、c.3103C>T、c.4082G>A、c.4060G>A、c.866C>T、c.1463T>C、c.2035C>G、c.2306_2307GC>AG、c.2306G>A、c.2714G>A、c.2875G>A、c.3992G>A。
  34. 根据权利要求30-32任一所述的应用，其特征在于，所述的靶向治疗药物包含经过修饰的细胞、蛋白、靶向权利要求32所述基因或靶向权利要求33所述的突变位点的RNA和/或靶向权利要求32所述基因或靶向权利要求33所述的突变位点的RNA DNA。
  35. 一种权利要求1-15任一所述的方法、权利要求1-15任一所述的方法制备得到的眼部疾病模型、权利要求16-29任一所述的眼部疾病模型载体在眼部疾病相关的研究中的应用。
  36. 一种权利要求1-15任一所述的方法、权利要求1-15任一所述的方法制备得到的眼部疾病模型、权利要求16-29任一所述的眼部疾病模型载体在眼部疾病相关药物筛选中的应用，所述的药物包含小分子药物，化学药物，高分子药物，生物药物或天然药物(例如中药或中药提取物)、细胞药物、RNA药物、DNA药物的一种或两种以上的组合。
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