CN115011674A - 一种孤独症肠菌移植有效性的体外评价方法 - Google Patents
一种孤独症肠菌移植有效性的体外评价方法 Download PDFInfo
- Publication number
- CN115011674A CN115011674A CN202210507756.XA CN202210507756A CN115011674A CN 115011674 A CN115011674 A CN 115011674A CN 202210507756 A CN202210507756 A CN 202210507756A CN 115011674 A CN115011674 A CN 115011674A
- Authority
- CN
- China
- Prior art keywords
- abundance
- transplantation
- autism
- enterobacteria
- donor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000002054 transplantation Methods 0.000 title claims abstract description 51
- 206010003805 Autism Diseases 0.000 title claims abstract description 48
- 208000020706 Autistic disease Diseases 0.000 title claims abstract description 48
- 238000000338 in vitro Methods 0.000 title claims abstract description 27
- 238000011156 evaluation Methods 0.000 title claims abstract description 24
- 241000305071 Enterobacterales Species 0.000 title claims abstract description 21
- 241000894006 Bacteria Species 0.000 claims abstract description 47
- 238000000034 method Methods 0.000 claims abstract description 22
- 238000012360 testing method Methods 0.000 claims abstract description 11
- 238000011002 quantification Methods 0.000 claims abstract description 5
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 21
- 241000605861 Prevotella Species 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 13
- 241000186000 Bifidobacterium Species 0.000 claims description 12
- 241000193403 Clostridium Species 0.000 claims description 11
- 230000000813 microbial effect Effects 0.000 claims description 11
- 244000005700 microbiome Species 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 10
- 238000012216 screening Methods 0.000 claims description 10
- 241000605716 Desulfovibrio Species 0.000 claims description 9
- 238000012163 sequencing technique Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 241000894007 species Species 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 108010059993 Vancomycin Proteins 0.000 claims description 4
- 239000003205 fragrance Substances 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 210000000936 intestine Anatomy 0.000 claims description 4
- 229960003165 vancomycin Drugs 0.000 claims description 4
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 claims description 4
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 claims description 4
- 241001202853 Blautia Species 0.000 claims description 3
- 229920001202 Inulin Polymers 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- YBHILYKTIRIUTE-UHFFFAOYSA-N berberine Chemical compound C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 YBHILYKTIRIUTE-UHFFFAOYSA-N 0.000 claims description 3
- 229940093265 berberine Drugs 0.000 claims description 3
- QISXPYZVZJBNDM-UHFFFAOYSA-N berberine Natural products COc1ccc2C=C3N(Cc2c1OC)C=Cc4cc5OCOc5cc34 QISXPYZVZJBNDM-UHFFFAOYSA-N 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 210000003608 fece Anatomy 0.000 claims description 3
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 claims description 3
- 229940029339 inulin Drugs 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
- 238000003753 real-time PCR Methods 0.000 claims description 3
- ZLSOONVQLWLPMF-UHFFFAOYSA-M 3-(3,8-diamino-6-phenylphenanthridin-5-ium-5-yl)propyl-diethyl-methylazanium;dibromide Chemical compound [Br-].[Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 ZLSOONVQLWLPMF-UHFFFAOYSA-M 0.000 claims description 2
- TZRXHJWUDPFEEY-UHFFFAOYSA-N Pentaerythritol Tetranitrate Chemical compound [O-][N+](=O)OCC(CO[N+]([O-])=O)(CO[N+]([O-])=O)CO[N+]([O-])=O TZRXHJWUDPFEEY-UHFFFAOYSA-N 0.000 claims description 2
- 241000123710 Sutterella Species 0.000 claims description 2
- 238000010926 purge Methods 0.000 claims description 2
- 230000008901 benefit Effects 0.000 abstract description 5
- 238000013461 design Methods 0.000 abstract description 5
- 238000010171 animal model Methods 0.000 abstract description 4
- 230000010354 integration Effects 0.000 abstract description 2
- 238000012545 processing Methods 0.000 abstract description 2
- 230000000968 intestinal effect Effects 0.000 description 41
- 230000000694 effects Effects 0.000 description 14
- 208000029560 autism spectrum disease Diseases 0.000 description 11
- 241000736262 Microbiota Species 0.000 description 7
- 241000606125 Bacteroides Species 0.000 description 6
- 239000000370 acceptor Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 230000002550 fecal effect Effects 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 241000607598 Vibrio Species 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 241000233866 Fungi Species 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 244000005709 gut microbiome Species 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- 241000790511 Sauteria Species 0.000 description 3
- 208000013404 behavioral symptom Diseases 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000035479 physiological effects, processes and functions Effects 0.000 description 3
- 241000238658 Blattella Species 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241000792859 Enema Species 0.000 description 2
- 208000018522 Gastrointestinal disease Diseases 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 208000035781 Non-specific syndromic intellectual disability Diseases 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 241000192031 Ruminococcus Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- -1 azide propidium bromide Chemical class 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 208000013577 complex neurodevelopmental disease Diseases 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 230000007123 defense Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000007920 enema Substances 0.000 description 2
- 229940095399 enema Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 208000028774 intestinal disease Diseases 0.000 description 2
- 230000007413 intestinal health Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000006041 probiotic Substances 0.000 description 2
- 235000018291 probiotics Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- OTLLEIBWKHEHGU-UHFFFAOYSA-N 2-[5-[[5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy]-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-4-phosphonooxyhexanedioic acid Chemical compound C1=NC=2C(N)=NC=NC=2N1C(C(C1O)O)OC1COC1C(CO)OC(OC(C(O)C(OP(O)(O)=O)C(O)C(O)=O)C(O)=O)C(O)C1O OTLLEIBWKHEHGU-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000589013 Alcaligenaceae Species 0.000 description 1
- 241000588986 Alcaligenes Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- ZXFDSSZCWQFXAT-UHFFFAOYSA-N Br.[N-]=[N+]=[N-] Chemical compound Br.[N-]=[N+]=[N-] ZXFDSSZCWQFXAT-UHFFFAOYSA-N 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- 241000193163 Clostridioides difficile Species 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000223935 Cryptosporidium Species 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241001333951 Escherichia coli O157 Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 201000003741 Gastrointestinal carcinoma Diseases 0.000 description 1
- 241000224466 Giardia Species 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 241000567229 Isospora Species 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 208000024556 Mendelian disease Diseases 0.000 description 1
- 241000243190 Microsporidia Species 0.000 description 1
- 241001263478 Norovirus Species 0.000 description 1
- 229920002562 Polyethylene Glycol 3350 Polymers 0.000 description 1
- 241000192142 Proteobacteria Species 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 241000209051 Saccharum Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 108010079723 Shiga Toxin Proteins 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 241000607447 Yersinia enterocolitica Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 208000022379 autosomal dominant Opitz G/BBB syndrome Diseases 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007175 bidirectional communication Effects 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 108010068385 carbapenemase Proteins 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000006854 communication Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 229930182912 cyclosporin Natural products 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000000369 enteropathogenic effect Effects 0.000 description 1
- 230000000688 enterotoxigenic effect Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000002095 exotoxin Substances 0.000 description 1
- 231100000776 exotoxin Toxicity 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019138 food restriction Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000007236 host immunity Effects 0.000 description 1
- 230000007412 host metabolism Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- 208000037817 intestinal injury Diseases 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- ZDWVWKDAWBGPDN-UHFFFAOYSA-O propidium Chemical compound C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 ZDWVWKDAWBGPDN-UHFFFAOYSA-O 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 210000004767 rumen Anatomy 0.000 description 1
- 230000001523 saccharolytic effect Effects 0.000 description 1
- 150000004666 short chain fatty acids Chemical class 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000003997 social interaction Effects 0.000 description 1
- CJUDSKIRZCSXJA-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(OC)=C1 CJUDSKIRZCSXJA-UHFFFAOYSA-M 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 229940098232 yersinia enterocolitica Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明设计了一种孤独症肠菌移植的体外评价方法，采用模拟临床试验的整合方案，以移植前后香浓指数对比和孤独症特征菌相对定量指标为主要评价指标，解决了孤独症肠菌移植在临床应用细节中的主要不确定性问题。相比于动物模型和行为评价量表方法，具有试验周期短和可定量等优点，适于大批样本量的处理，提高了孤独症肠菌移植的效率。
Description
技术领域
本发明涉及肠菌移植的体外评价方法，更具体地涉及孤独症肠菌移植有效性的体外评价方法，属于生物医药新技术领域。
背景技术
孤独症谱系障碍(Autism Spectrum Disorder，ASD)是复杂的神经发育障碍，其核心症状是行为障碍和社交互动、沟通缺陷。患有ASD的人还经常出现包括肠道问题在内的共患疾病，这可能反映了大脑和肠道之间双向交流的异常。基于微生物群的孤独症疾病诊断模型揭示了ASD儿童存在明显偏离正轨的肠道菌群发育特征，这些特征与ASD儿童核心症状的严重程度显著正相关；大样本量临床研究发现是否患有 ASD对肠道菌群的影响不随年龄的变化而改变，提示组成异常的肠道菌群是ASD患儿的一个相对恒定的特征(Deviated andearly unsustainable stunted development of gut microbiota in children withautism spectrum disorder，Gut，2021；0：1-12)，称之为孤独症特征菌群。Cell刊登题为“Human gut microbiota from Autism Spectrum Disorder promote behavioralsymptoms in mice”的论文通过小鼠模型进一步证实了孤独症特征菌群和孤独症核心症状的关联性，提示肠道菌群改变是孤独症发病与治疗的关键环节。肠菌移植(FecalMicrobiota Transplantation，FMT)是将健康人粪便微生物通过一定方式移植到患者肠内，以调节肠道菌群失衡，重建具有正常功能的肠道微生态***以治疗肠道疾病的一种新医疗技术。
目前肠菌移植已应用于改善孤独症行为症状和胃肠道症状。但肠菌移植对孤独症等肠外疾病的治愈率并不理想，治愈率在20-23.5％。在孤独症治疗的临床应用中还存在以下问题：首先，由于ASD不是单一的病因，临床应用中缺乏有效鉴定用于诊断的生物标志物；再者，ASD是复杂的神经发育障碍，缺乏理想的动物模型来概括ASD的疾病状态，基于孤独症行为的评价量表(ABC、ADOS、DSM-5、ADI-R或者CARS)，不能有效与小鼠的行为症状相对应。由于临床试验受到伦理审查、动物模型试验质量等因素限制，肠菌移植干预措施对ASD患者胃肠道症状和行为症状的疗效还只能获得小样本量的提示性证据。而在肠菌移植临床试验手术过程中，不仅鼻肠管、鼻胃管、肠镜、结肠镜、灌肠手术环节给孤独症儿童带来难以接受的痛苦；而且这些临床方法还会发生误吸或者肠损伤等副作用(Future directions inreducing gastrointestinal disorders in children with ASD using FecalMicrobiota Transplantation，Frontiers in Cellular and Infection Microbiology，2021，11，1-10)。因此，需要建立基于肠道微生物大样本量的体外试验来预评估肠菌移植的效果，以改善粪菌移植的依从性，提高孤独症肠菌移植临床应用的治愈率。
发明内容
本发明设计了一种孤独症肠菌移植有效性的体外评价方法：其特征在于包括以下步骤和方法：
(1)健康供体初筛：采集健康供体和孤独症患者粪便样品通过宏基因组或16SrRNA测序检测供体(D) 和受体(R)样品多样性香浓指数Sd、Sr和其特征菌原始丰度值D、R；其特征在于健康供体初筛指标应满足(a)多样性香浓指数Sd大于3；(b)普雷沃氏菌属(Prevotella)、双歧杆菌属(Bifidobacter)、布劳特菌属(Blautia)原始丰度D/R＞1和脱硫弧菌属(Desulfovibrio)、梭菌属(Clostridium)、萨特菌属(Sutterella)原始丰度D/R＜1；
(2)受体肠菌体外准备：将新鲜受体粪便样品添加万古霉素、聚乙二醇、菊粉、黄连素中至少一种，在氧浓度为0-5％条件下处理4-24h，获得清肠预处理受体肠菌；
(3)供体、受体体外预配型：将步骤(1)检测获得的合格供体粪便样品按照重量百分比10％至40％移植接种到步骤(2)清肠预处理的受体肠菌中，并加3倍重量比的MG液体培养基混匀；供、受体混合菌液在氧浓度为0-5％条件下培养48h，获得供、受体肠菌重组培养液；通过宏基因组或16SrRNA测序检测，确定重组培养液的多样性香浓指数St和孤独症特征菌群培养丰度终值T；
(4)移植方案有效性评价：通过正交试验筛选有效移植的供体和受体组合及受体体外准备方案，有效移植判断指标为重组培养液的微生物组成满足：(a)香浓多样性指数St/Sr＞1；(c)普雷沃氏菌属丰度T/R＞1、双歧杆菌属丰度T/R＞1、布劳特菌属丰度T/R＞1和脱硫弧菌属丰度T/R＜1、梭菌属丰度T/R＜1、萨特菌属丰度T/R＜1中的至少三项。
所述的孤独症肠菌移植有效性的体外评价方法，其特征在于步骤(4)所述有效移植判断指标还包括重组培养液微生物组成满足普雷沃氏菌属/脱硫弧菌属丰度T的比值显著高于受体样品原始丰度R的比值。
所述的孤独症肠菌移植有效性的体外评价方法，所述孤独症特征菌丰度检测还可以采用叠氮溴化丙锭交联结合种属特异性16SrDNA荧光定量PCR方法进行活菌数绝对定量。
本发明的创新点和有益效果：
粪便微生物群移植(FMT)的潜在机制包括供体和受体肠道微生物群之间的直接相互作用或竞争以实现稳态、供体微生物群对宿主免疫的影响以及微生物群调节宿主代谢和生理的影响。基于供体微生物组成指标“和/或”微生物群的功能选择供体，可能对提高FMT的疗效很重要。针对孤独症FMT需要合理设计并严格控制，同时考虑到微生物多样性、组成功能和供体-受体因素。
本发明所述肠菌移植的体外评价方法，采用模拟临床试验的整合方案，进行合理的孤独症肠菌移植流程设计，解决了肠菌移植在临床应用细节中的主要不确定性问题，：(1)首先基于本实验室供体/受体肠道检测的数据库以及国内外孤独症肠道菌群生物信息学分析成果(The human gut microbiome as a potential factor in Autism SpectrumDisorder.Inernational Journal of Molecular Sciences，2022，23，1363：1-22；)，确定影响孤独症的关键微生物物种和FMT预测因素；相比于动物模型和行为评价量表，具有试验周期短和可定量等优点，适于大批样本量的处理，实质性改进孤独症肠菌移植的效率，解决肠菌移植临床应用的瓶颈问题；(2)本发明采用了移植前后孤独症特征菌群比值变化和菌群多样性指数作为评价指标；既解决了孤独症个体异质性难于普遍适用的问题，又在健康供体筛选的关键环节给出相对量化指标，相较于目前行业内肠菌简单的的配型(普雷沃氏型、拟杆菌型和瘤胃球菌型)分析，使移植关键步骤供体筛选更精准； (3)患者的肠道菌群具有获得性细菌防御***，供体菌群怎样能克服患者自身菌群防御从而有效的定植也是临床试验前需要考察的关键问题；本发明的方法设计了抗生素、医用灌肠试剂以及益生元和中药等肠菌移植前干预方案，从而促进异种微生物群定植并增强肠道微生物群的重编程，为探索孤独症肠道菌群机制，提高肠菌移植的治愈率奠定基础。
本发明评估微生物群组成变化的定量分析中孤独症特征菌属采用了叠氮溴化丙锭(PMA)交联死亡细胞体DNA，再进行荧光定量PCR方法；相比宏基因组/16SrRNA测序分析的样本微生物群变化评价方法，不仅经济便捷，而且能够区别活菌和死菌，对特征菌属的定量和移植效果关系评价更准确。
本发明的设计基于有效的肠道微生物的体外培养方法，即对于粪便样本采用本实验室开发的高度还原肠道微生物结构和多样性的体外培养方法(专利申请号202210371491)，建立了模拟肠道的体外移植***和检测方法，具有操作方便、重复性好、可调控的优势。
具体实施方式
步骤1：健康供体初筛
本发明肠菌健康供体样品采自年龄5～35岁，通过生理、心理、个人史、稳定性、持续性、限食耐受性6个维度筛选标准，排除了因提供者个体的传染性疾病、遗传性疾病以及服用药物、不健康饮食等使受体产生不良反应的风险，并且在过去的一年里未食用抗生素类药物。所述供体样本经过致病菌艰难梭菌、弯曲杆菌、大肠杆菌O157、沙门氏菌、贺氏菌、弧菌、志贺菌毒素产生大肠埃希氏菌、肠致病性大肠埃希氏菌、肠集聚性大肠埃希氏菌、肠产毒性大肠埃希氏菌、肠侵袭性大肠埃希氏菌、小肠耶尔森菌、志贺单胞菌、万古霉素耐药菌、超广谱内酰胺酶产生菌、碳青霉烯酶产生革兰氏阴性菌的筛查，寄生虫贾第鞭毛虫、隐孢子虫、环孢菌、溶组织内阿米、等孢属的筛查、微孢子虫，以及轮状病毒、诺如病毒、腺病毒、新冠病毒的筛查。
针对孤独症的健康供体初筛不仅在肠道菌群多样性的香浓指数指标，而且增加了孤独症特征菌的相对量化指标：普雷沃氏菌属、双歧杆菌属、布劳特菌属丰度大于其受体丰度；脱硫弧菌属、梭菌属、萨特菌属的供体丰度小于其受体丰度。
香浓指数表征微生态***的稳定性，以及面对外界致病菌等入侵的抵御能力。其计算公式H＝-∑(Pi) (log2Pi)其中Pi为每个菌的占比例，值越大代表物种种类越多，均匀性也更好，相应的多样性也越高。正常人群中香浓指数在2～9之间，一般大于3以上表明具有一定多样性。
孤独症特征菌群的确定参考孤独症肠菌生物信息学荟萃分析Alteredcomposition and tunction of intestinal microbiota in Autism SpectrumDisorders：a systematic review. Translational Psychiatry，2019，9(43)：1-13及本实验室的肠道菌群宏基因组检测数据库。具体的特征菌群与孤独症的相关性如下：
普雷沃氏菌属是革兰氏阴性菌、非运动性、杆状、单细胞，可在厌氧条件下生长。普雷沃氏菌属是一个大属，包括50多个不同的物种。大多数可以从口腔和肠道中分离出来。被诊断患有孤独症的儿童的普雷沃氏菌属丰度较低；
双歧杆菌属细菌为典型的益生菌，在肠道菌群评价中作为肠道平衡指数B/E的正向指标；早期研究表明孤独症儿童肠道中双歧杆菌较正常儿童显著降低，而乳杆菌属则相反。(Gastrointestinal flora and gastrointestinal status in children withautism--comparisons to typical children and correlation with autismseverity.BMC Gastroenterol.2011 Mar 16；11：22)；
布劳特菌属在结直肠癌患者中显著减少，具有防止炎症发生，促进短链脂肪酸产生等维持肠道稳态活性的作用，具有潜在益生菌特性；
脱硫弧菌属在孤独症受试者中比在对照组中更常见，并发现孤独症儿童的兄弟姐妹有中等数量的脱硫弧菌，表明该生物体可能在家庭环境中传播。脱硫弧菌是一种厌氧杆菌，不产生孢子，但通过其他机制对有氧条件和其他不利条件具有抗性，脱硫弧菌属还产生重要的毒力因子及脂多糖，它的生理和代谢使其独特地解释了孤独症中的大部分病理生理；
梭菌属已经有多个研究证实，这种菌在孤独症患者的体内更多；其中，梭状芽孢杆菌XVIII 群是可以产生外毒素并促进炎症发生的菌，它们可能促进了炎症和孤独症的发生；
萨特菌属是变形菌门最丰富的菌之一，是一种厌氧或微需氧的、耐胆汁、解糖、非运动性且不形成孢子的革兰氏阴性球杆菌。对伴有胃肠功能肠癌的孤独症儿童肠道中检测到了产碱菌科Alcaligenaceae的某些成员；而这些产碱菌属水平的升高是由于萨特菌的水平较高。诊断患有孤独症和胃肠道紊乱的儿童中，超过一半的肠道活检组织中存在萨特菌，而正常发育的胃肠道紊乱儿童的活检组织中没有萨特菌。
步骤2：受体肠菌体外准备
将步骤1中宏基因组或16SrRNA测序分析的孤独症患者样品记录编号，称取1g加入厌氧管中，参考《菌群移植标准化方法学的建立与临床应用中国专家共识2020年版》及清肠剂和药剂服用说明，进行受体肠道体外准备。具体处理方案如下：(1)按照重量百分比0.2％万古霉素作为主要的FMT前的抗生素预处理4h：先配成0.4％溶液等体积添加混匀，厌氧条件下处理4h后，无菌生理盐水离心洗涤备用；(2) 按照1.5倍重量给予肠道清洁剂聚乙二醇PEG-3350混匀，厌氧条件下处理24小时后，无菌生理盐水离心洗涤备用；(3)菊粉按照重量百分比1％添加量直接添加到受体样品中，厌氧条件下处理24h备用；(4) 黄连素按照重量百分比0.6％直接添加到受体样品中，厌氧条件下处理24h直接备用或者离心洗涤备用。
步骤3：供体、受体体外预配型
将步骤1检测获得的合格供体粪便样品按照重量百分含量10％至40％移植接种到步骤(2)预处理的受体肠菌中，并加3倍重量比的MG液体培养基混匀；记录供体和受体组合的肠菌编号，预处理方式及其他相关信息，包括供受体肠菌类型；供、受体混合菌液在氧浓度为0-5％条件下培养48h，获得供、受体肠菌重组培养液；通过宏基因组或16SrRNA测序检测，确定重组培养液的多样性香浓指数St和孤独症特征菌群培养丰度终值T。
健康供体和孤独症患者受体肠道菌群根据微生物优势物种分为：
肠型1，又可以表示为ET_B，拟杆菌(Bacteroides)是其最好的指示类群，该菌属是人体肠道广泛分布的一类细菌，该菌擅长分解碳水化合物，能够将肠道中复杂有机物分解成小分子，有效保护肠粘膜，促进肠道健康；
肠型2，又可以表示为ET_P，是由普氏菌(Prevotella)驱动的，它的丰度通常与拟杆菌的丰度成反比，该菌属是肠道的常见菌群，能够分解食物中的蛋白质和碳水化合物，参与合成多种维生素；
肠型3，又可以表示为ET_F，是通过厚壁菌(Firmicutes)占比高低来区分的，其中最主要的类群为瘤胃球菌(Ruminococcus)，该菌属能发酵纤维素、纤维二糖、产生甲酸、乙酸、乳酸、B族维生素等物质，促进肠道健康。
步骤4移植方案有效性评价
通过供体、受体及清肠预处理3因素正交试验筛选组合，有效移植判断指标为重组培养液的微生物组成满足：(a)香浓多样性指数St/Sr＞1；(c)普雷沃氏菌属丰度T/R＞1、双歧杆菌属丰度T/R＞1、布劳特菌属丰度T/R＞1和脱硫弧菌属丰度T/R＜1、梭菌属丰度T/R＜1、萨特菌属丰度T/R＜1中的至少三项；
孤独症患者肠道菌群的多样性香浓指数在门水平显著低于正常人群，而在属水平上相较于健康儿童也呈现不随发育而增长的停滞趋势(Altered gut microbial profileis associated with abnormal metabolism activity ofAutism SpectrumDisorder.GUT MICROBES，2020，11(5)：1246-1267)。因此香浓指数也是评价孤独症肠道菌群重建的基础指标(Scientific frontiers in faecal microbiota transplantation：joint document of Asia-Pacific Association of Gastroenterology(APAGE)andAsiaPacific Society for digestive endoscopy(APSDE). Gut，2020，69：83-91.)。
孤独症患者有益菌属普雷沃氏菌属、双歧杆菌属、布劳特菌属增加，或者孤独症患者有害菌属脱硫弧菌属、梭菌属、萨特菌属减少，可能预示孤独症患者重建的肠道菌群向健康人群转变。研究表明肠道菌群存在平衡和互相作用，多数菌属功能是复杂的，对于孤独症的治疗效果同时存在正副作用，既与菌量有关也与菌群效应有关；而多种临床试验的生信分析出现了互相矛盾的统计结果(Alteration of gut microbiota： new strategy fortreating Autism Spectrum Disorder.Frontiers in Cell and DevelopmentalBiology，2021，11，1-10)。因此，本发明在有效数据统计分析的基础上，不以单一菌属的增加和减少作为菌群重建和移植疗效的判断标准，而是将微生物组成阈值设置为满足普雷沃氏菌属丰度T/R＞1、双歧杆菌属丰度T/R＞1、布劳特菌属丰度T/R＞1和脱硫弧菌属丰度T/R＜1、梭菌属丰度T/R＜1、萨特菌属丰度 T/R＜1中的至少3项指标，获得更高的移植成功率。
重组培养液中普雷沃氏菌属丰度T较受体菌原始丰度R增加，脱硫弧菌丰度T较受体菌原始丰度R 减少都预示孤独症患者重建的肠道菌群向健康人群转变。因此二者的比值升高，即重组培养液微生物组成满足普雷沃氏菌属/脱硫弧菌属丰度T的比值显著高于受体样品原始丰度R的比值，表明孤独症患者肠道菌群重建效果越好，即移植方案有效。
评价肠菌移植微生物重建效率的关键技术方法是特征菌的定量。相比与16SrRNA测序和宏基因组分析，采用肠菌活菌定量对于微生物重建效率评价更为精准。叠氮溴化丙锭(propidium monoazide，PMA)为核酸结合染料，可以穿过受损的细胞膜进入细胞内同DNA发生共价交联，阻断死亡菌体DNA在后续PCR 中的扩增。本发明将核酸结合染料叠氮溴化丙锭预处理后，通过种属特异性16SrDNA qPCR对孤独症特征菌属的活菌进行绝对定量。本发明所用引物见表1。
表1菌属特异性16SrDNA qPCR引物
按照本发明所述孤独症肠菌移植有效性的体外评价的正交实验实施例如表2所示；各实施例的检测结果如表3所示。按照实施例I～XII的移植方案治疗的患者随访记录，表明其ABC (Autism Behavior Checklist，ABC)量表评分降至至53分以下，获得平均73％以上的改善率；按照实施例II、IV、V、X、XI、XII的移植方案，患者ABC量表评分降至31分以下，达到50％的治愈率，显著高于平均水平20-23.5％。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
表2肠菌移植有效性的体外评价的正交实验
表3肠菌移植有效性体外评价实施例检测结果
a孤独症患者按照实施例方案进行肠菌移植治疗后ABC量表评分与确诊时ABC量表评分差值。
Claims (3)
1.一种孤独症肠菌移植有效性的体外评价方法；其特征在于包括以下步骤：
(1)健康供体初筛：采集健康供体和孤独症患者粪便样品通过宏基因组或16SrRNA测序检测供体(D)和受体(R)样品多样性香浓指数Sd、Sr和其特征菌原始丰度值D、R；其特征在于健康供体初筛指标应满足(a)多样性香浓指数Sd大于3；(b)普雷沃氏菌属(Prevotella)、双歧杆菌属(Bifidobacter)、布劳特菌属(Blautia)原始丰度D/R＞1和脱硫弧菌属(Desulfovibrio)、梭菌属(Clostridium)、萨特菌属(Sutterella)原始丰度D/R＜1；
(2)受体肠菌体外准备：将新鲜受体粪便样品添加万古霉素、聚乙二醇、菊粉、黄连素中至少一种，在氧浓度为0-5％条件下处理4-24h，获得清肠预处理受体肠菌；
(3)供体、受体体外预配型：将步骤(1)检测获得的合格供体粪便样品按照重量百分比10％至40％移植接种到步骤(2)清肠预处理的受体肠菌中，并加3倍重量比的MG液体培养基混匀；供、受体混合菌液在氧浓度为0-5％条件下培养48h，获得供、受体肠菌重组培养液；通过宏基因组或16SrRNA测序检测，确定重组培养液的多样性香浓指数St和孤独症特征菌群培养丰度终值T；
(4)移植方案有效性评价：通过正交试验筛选有效移植的供体和受体组合及受体体外准备方案，有效移植判断指标为重组培养液的微生物组成满足：(a)香浓多样性指数St/Sr＞1；(c)普雷沃氏菌属丰度T/R＞1、双歧杆菌属丰度T/R＞1、布劳特菌属丰度T/R＞1和脱硫弧菌属丰度T/R＜1、梭菌属丰度T/R＜1、萨特菌属丰度T/R＜1中的至少三项。
2.根据权利要求1所述的孤独症肠菌移植有效性的体外评价方法，其特征在于步骤(4)所述有效移植判断指标还包括重组培养液微生物组成满足普雷沃氏菌属/脱硫弧菌属丰度T的比值显著高于受体样品原始丰度R的比值。
3.根据权利要求1所述的孤独症肠菌移植有效性的体外评价方法，所述孤独症特征菌丰度检测还可以采用叠氮溴化丙锭交联结合种属特异性16SrDNA荧光定量PCR方法进行活菌数绝对定量。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210507756.XA CN115011674A (zh) | 2022-05-11 | 2022-05-11 | 一种孤独症肠菌移植有效性的体外评价方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210507756.XA CN115011674A (zh) | 2022-05-11 | 2022-05-11 | 一种孤独症肠菌移植有效性的体外评价方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115011674A true CN115011674A (zh) | 2022-09-06 |
Family
ID=83068200
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210507756.XA Pending CN115011674A (zh) | 2022-05-11 | 2022-05-11 | 一种孤独症肠菌移植有效性的体外评价方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115011674A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117037914A (zh) * | 2023-07-25 | 2023-11-10 | 中科国康(浙江)生命科学有限公司 | 一种基于个体特征定向制备的肠道菌群的*** |
-
2022
- 2022-05-11 CN CN202210507756.XA patent/CN115011674A/zh active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117037914A (zh) * | 2023-07-25 | 2023-11-10 | 中科国康(浙江)生命科学有限公司 | 一种基于个体特征定向制备的肠道菌群的*** |
| CN117037914B (zh) * | 2023-07-25 | 2024-04-02 | 中科国康(浙江)生命科学有限公司 | 一种基于个体特征定向制备的肠道菌群的*** |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cox et al. | Gut microbiome in progressive multiple sclerosis | |
| Wang et al. | Dynamic distribution of gut microbiota in goats at different ages and health states | |
| Aragon et al. | The urinary tract microbiome in health and disease | |
| Johnson et al. | Identification of urovirulence traits in Escherichia coli by comparison of urinary and rectal E. coli isolates from dogs with urinary tract infection | |
| Quigley et al. | Small intestinal bacterial overgrowth | |
| Snel et al. | Dietary strategies to influence the gastro-intestinal microflora of young animals, and its potential to improve intestinal health | |
| Wang et al. | Gastrointestinal microbiota and metabolite biomarkers in children with autism spectrum disorders | |
| CN110592246A (zh) | 一种人肠道菌群定量检测方法及其应用 | |
| US20220119864A1 (en) | Canid microbiome monitoring tools and diagnostic methods | |
| GAŁĘCKA et al. | Faecalibacterium prausnitzii and Crohn’s Disease–is There any Connection? | |
| Tannock | The search for disease-associated compositional shifts in bowel bacterial communities of humans | |
| Costa et al. | Evaluation of changes in microbiota after fecal microbiota transplantation in 6 diarrheic horses | |
| Fleury et al. | Development and validation of a new dynamic in vitro model of the piglet colon (PigutIVM): application to the study of probiotics | |
| CN115011674A (zh) | 一种孤独症肠菌移植有效性的体外评价方法 | |
| Al Humam | Special biochemical profiles of Escherichia coli strains isolated from humans and camels by the VITEK 2 automated system in Al-Ahsa, Saudi Arabia | |
| SULEIMAN et al. | Bacterial diarrhea among infants in developing countries: An overview of diarrheagenic Escherichia coli (DEC) | |
| Phyo et al. | Fecal microbiome alterations in pediatric patients with short bowel syndrome receiving a rotating cycle of gastrointestinal prophylactic antibiotics | |
| Zhou et al. | The relationship between urinary stones and gut microbiomeby 16S sequencing | |
| Lindroth et al. | Faecal bacterial composition in horses with and without free faecal liquid: a case control study | |
| RU2616280C1 (ru) | Способ диагностики состояния микробиоты кишечника на фоне эрадикационной терапии helicobacter pylori и его применение | |
| CN110613057A (zh) | 双乙酸钠在提高动物肠道菌群多样性中的应用 | |
| Chen et al. | Bacitracin methylene disalicylate improves intestinal health by modulating its development and microbiota in weaned rabbits | |
| US9962416B2 (en) | Probiotics for treating neuropathic bladder associated urinary tract infection | |
| Smirnova et al. | Study of microbiome aberrations in patients with irritable bowel syndrome with diarrhea by next-generation sequencing | |
| Kiebooms et al. | Thiouracil-forming bacteria identified and characterized upon porcine in vitro digestion of Brassicaceae feed |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |