CN111778325A - Method for using gene/protein as biliary tract atresia diagnosis mark and therapeutic target and application thereof - Google Patents
Method for using gene/protein as biliary tract atresia diagnosis mark and therapeutic target and application thereof Download PDFInfo
- Publication number
- CN111778325A CN111778325A CN202010657625.0A CN202010657625A CN111778325A CN 111778325 A CN111778325 A CN 111778325A CN 202010657625 A CN202010657625 A CN 202010657625A CN 111778325 A CN111778325 A CN 111778325A
- Authority
- CN
- China
- Prior art keywords
- notch3
- liver
- hepatic artery
- hepatic
- protein
- 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
- 238000000034 method Methods 0.000 title claims abstract description 72
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 61
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 35
- 210000003445 biliary tract Anatomy 0.000 title claims abstract description 24
- 238000003745 diagnosis Methods 0.000 title claims abstract description 17
- 230000001225 therapeutic effect Effects 0.000 title claims abstract description 16
- 210000004185 liver Anatomy 0.000 claims abstract description 74
- 210000002767 hepatic artery Anatomy 0.000 claims abstract description 68
- 102000001760 Notch3 Receptor Human genes 0.000 claims abstract description 55
- 108010029756 Notch3 Receptor Proteins 0.000 claims abstract description 55
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 21
- 239000003550 marker Substances 0.000 claims abstract description 18
- 201000010099 disease Diseases 0.000 claims abstract description 16
- 230000000694 effects Effects 0.000 claims abstract description 14
- 210000005228 liver tissue Anatomy 0.000 claims abstract description 14
- 238000007634 remodeling Methods 0.000 claims abstract description 14
- 238000004393 prognosis Methods 0.000 claims abstract description 13
- 239000003112 inhibitor Substances 0.000 claims abstract description 12
- 230000037361 pathway Effects 0.000 claims abstract description 12
- 238000011282 treatment Methods 0.000 claims abstract description 12
- 230000019491 signal transduction Effects 0.000 claims abstract description 7
- 238000011269 treatment regimen Methods 0.000 claims abstract description 4
- 206010010317 Congenital absence of bile ducts Diseases 0.000 claims description 125
- 201000005271 biliary atresia Diseases 0.000 claims description 125
- 230000002440 hepatic effect Effects 0.000 claims description 38
- 230000014509 gene expression Effects 0.000 claims description 31
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 30
- 241000699670 Mus sp. Species 0.000 claims description 23
- 210000002565 arteriole Anatomy 0.000 claims description 23
- 102000005650 Notch Receptors Human genes 0.000 claims description 22
- 108010070047 Notch Receptors Proteins 0.000 claims description 22
- 238000010186 staining Methods 0.000 claims description 17
- 238000001514 detection method Methods 0.000 claims description 15
- 210000001519 tissue Anatomy 0.000 claims description 15
- 210000004509 vascular smooth muscle cell Anatomy 0.000 claims description 15
- 210000001367 artery Anatomy 0.000 claims description 14
- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 claims description 14
- 229960004657 indocyanine green Drugs 0.000 claims description 14
- 238000002583 angiography Methods 0.000 claims description 13
- 230000003472 neutralizing effect Effects 0.000 claims description 13
- 241001465754 Metazoa Species 0.000 claims description 12
- 238000002474 experimental method Methods 0.000 claims description 11
- 239000007924 injection Substances 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 10
- 238000003757 reverse transcription PCR Methods 0.000 claims description 10
- 210000000329 smooth muscle myocyte Anatomy 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 210000004204 blood vessel Anatomy 0.000 claims description 9
- 238000005259 measurement Methods 0.000 claims description 9
- 239000011022 opal Substances 0.000 claims description 9
- 230000002018 overexpression Effects 0.000 claims description 9
- 230000008859 change Effects 0.000 claims description 8
- 210000003240 portal vein Anatomy 0.000 claims description 8
- 230000017531 blood circulation Effects 0.000 claims description 7
- 239000012188 paraffin wax Substances 0.000 claims description 7
- 230000000007 visual effect Effects 0.000 claims description 7
- 238000001262 western blot Methods 0.000 claims description 7
- 206010008635 Cholestasis Diseases 0.000 claims description 6
- 102000013127 Vimentin Human genes 0.000 claims description 6
- 108010065472 Vimentin Proteins 0.000 claims description 6
- 231100000359 cholestasis Toxicity 0.000 claims description 6
- 230000007870 cholestasis Effects 0.000 claims description 6
- 230000001575 pathological effect Effects 0.000 claims description 6
- 230000010412 perfusion Effects 0.000 claims description 6
- 238000002604 ultrasonography Methods 0.000 claims description 6
- 210000005048 vimentin Anatomy 0.000 claims description 6
- 108010081348 HRT1 protein Hairy Proteins 0.000 claims description 5
- 101150029234 Hes5 gene Proteins 0.000 claims description 5
- 102100032742 Histone-lysine N-methyltransferase SETD2 Human genes 0.000 claims description 5
- 101000654725 Homo sapiens Histone-lysine N-methyltransferase SETD2 Proteins 0.000 claims description 5
- 230000005764 inhibitory process Effects 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 5
- 101150092640 HES1 gene Proteins 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- 238000011327 histological measurement Methods 0.000 claims description 4
- 206010023126 Jaundice Diseases 0.000 claims description 3
- 241001529936 Murinae Species 0.000 claims description 3
- 101001046872 Mus musculus Hypoxia-inducible factor 1-alpha Proteins 0.000 claims description 3
- 101100348845 Mus musculus Notch3 gene Proteins 0.000 claims description 3
- 101000808007 Mus musculus Vascular endothelial growth factor A Proteins 0.000 claims description 3
- 102100036639 Myosin-11 Human genes 0.000 claims description 3
- 101710115164 Myosin-11 Proteins 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 230000001133 acceleration Effects 0.000 claims description 3
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 claims description 3
- 238000011532 immunohistochemical staining Methods 0.000 claims description 3
- 230000000877 morphologic effect Effects 0.000 claims description 3
- 210000000056 organ Anatomy 0.000 claims description 3
- 238000000131 plasma-assisted desorption ionisation Methods 0.000 claims description 3
- 230000001839 systemic circulation Effects 0.000 claims description 3
- 210000003462 vein Anatomy 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 2
- 230000011664 signaling Effects 0.000 claims description 2
- 238000007447 staining method Methods 0.000 claims description 2
- 238000012136 culture method Methods 0.000 claims 1
- 238000002601 radiography Methods 0.000 claims 1
- 210000000013 bile duct Anatomy 0.000 abstract description 12
- 230000004083 survival effect Effects 0.000 abstract description 11
- 206010021143 Hypoxia Diseases 0.000 abstract description 7
- 230000007954 hypoxia Effects 0.000 abstract description 6
- 230000007246 mechanism Effects 0.000 abstract description 5
- 235000017284 Pometia pinnata Nutrition 0.000 abstract description 4
- 240000007653 Pometia tomentosa Species 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 4
- 208000027418 Wounds and injury Diseases 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 230000006378 damage Effects 0.000 abstract description 3
- 230000002349 favourable effect Effects 0.000 abstract description 3
- 208000014674 injury Diseases 0.000 abstract description 3
- 230000001404 mediated effect Effects 0.000 abstract description 3
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 2
- 230000002300 anti-fibrosis Effects 0.000 abstract 1
- 230000002792 vascular Effects 0.000 description 18
- 239000012528 membrane Substances 0.000 description 16
- 238000010172 mouse model Methods 0.000 description 14
- DWJXYEABWRJFSP-XOBRGWDASA-N DAPT Chemical compound N([C@@H](C)C(=O)N[C@H](C(=O)OC(C)(C)C)C=1C=CC=CC=1)C(=O)CC1=CC(F)=CC(F)=C1 DWJXYEABWRJFSP-XOBRGWDASA-N 0.000 description 11
- 238000010171 animal model Methods 0.000 description 11
- 238000011977 dual antiplatelet therapy Methods 0.000 description 11
- 238000005406 washing Methods 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 239000000499 gel Substances 0.000 description 8
- 230000000903 blocking effect Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000007789 sealing Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 5
- 241000702670 Rotavirus Species 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 206010016654 Fibrosis Diseases 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- 102100031497 Heparan sulfate N-sulfotransferase 1 Human genes 0.000 description 4
- 101000588589 Homo sapiens Heparan sulfate N-sulfotransferase 1 Proteins 0.000 description 4
- 230000002159 abnormal effect Effects 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000004761 fibrosis Effects 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 239000002033 PVDF binder Substances 0.000 description 3
- BHTRKEVKTKCXOH-UHFFFAOYSA-N Taurochenodesoxycholsaeure Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCCS(O)(=O)=O)C)C1(C)CC2 BHTRKEVKTKCXOH-UHFFFAOYSA-N 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 210000003855 cell nucleus Anatomy 0.000 description 3
- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 description 3
- 229960001091 chenodeoxycholic acid Drugs 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- BHTRKEVKTKCXOH-AYSJQVDDSA-N taurochenodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)C1C2C2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)CC1 BHTRKEVKTKCXOH-AYSJQVDDSA-N 0.000 description 3
- 230000008719 thickening Effects 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 239000008096 xylene Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 101000817629 Homo sapiens Dymeclin Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 108700011259 MicroRNAs Proteins 0.000 description 2
- 208000031481 Pathologic Constriction Diseases 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 210000003017 ductus arteriosus Anatomy 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 210000002603 extrahepatic bile duct Anatomy 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 210000000232 gallbladder Anatomy 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000000366 juvenile effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000002679 microRNA Substances 0.000 description 2
- 230000004089 microcirculation Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000008722 morphological abnormality Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 238000000751 protein extraction Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000036262 stenosis Effects 0.000 description 2
- 208000037804 stenosis Diseases 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 208000004476 Acute Coronary Syndrome Diseases 0.000 description 1
- 241000239290 Araneae Species 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010008609 Cholangitis sclerosing Diseases 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 1
- 206010023129 Jaundice cholestatic Diseases 0.000 description 1
- 108010029755 Notch1 Receptor Proteins 0.000 description 1
- 201000005267 Obstructive Jaundice Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- DZGWFCGJZKJUFP-UHFFFAOYSA-N Tyramine Natural products NCCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-N 0.000 description 1
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003510 anti-fibrotic effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 210000001043 capillary endothelial cell Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000013189 cholangiography Methods 0.000 description 1
- 230000001587 cholestatic effect Effects 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 231100000317 environmental toxin Toxicity 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000000521 hyperimmunizing effect Effects 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000001969 hypertrophic effect Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000003886 intestinal anastomosis Effects 0.000 description 1
- 210000003228 intrahepatic bile duct Anatomy 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000003836 peripheral circulation Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 208000002815 pulmonary hypertension Diseases 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 208000010157 sclerosing cholangitis Diseases 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910001285 shape-memory alloy Inorganic materials 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 229960003732 tyramine Drugs 0.000 description 1
- DZGWFCGJZKJUFP-UHFFFAOYSA-O tyraminium Chemical group [NH3+]CCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-O 0.000 description 1
- 108010080213 vascular factor Proteins 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000009278 visceral effect Effects 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
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
- C12Q1/6876—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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
Abstract
The invention discloses a method for using gene/protein as biliary tract atresia diagnosis mark and therapeutic target and its application. The Notch3 signal pathway and the hepatic artery system remodeling phenomenon mediated by the Notch3 signal pathway are proposed for the first time in the BA background, and are perfected on the existing theoretical mechanism. The Notch3-Hey1 gene/protein highly expressed in liver tissues can be converted into a BA diagnostic marker, and the method is favorable for assisting in predicting the disease prognosis of children and selecting an operation mode. The current anti-inflammatory and anti-fibrosis treatment in clinic does not achieve an ideal curative effect, the Kasai operation cannot completely prevent the continuous development of BA, and the Notch3 pathway specific inhibitor provides a new auxiliary treatment strategy, which is beneficial to improving the hypoxia and bile duct injury of the junction area, slowing down the progress of the disease course and improving the liver survival rate of BA.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for using genes/proteins as biliary atresia diagnosis markers and therapeutic targets and application thereof.
Background
Biliary Atresia (BA) is a severe obstructive jaundice disease especially in newborns, and is characterized by progressive atresia of the extrahepatic bile duct and sclerosing cholangitis of the extensive intrahepatic bile duct system, with rapid disease progression, with a natural survival period of only two years, and with an incidence rate of 1/5000-1/12000 in continental regions. Although the hepatoportal intestinal anastomosis (Kasai) can restore biliary flow through surgical reconstruction and prolong the survival time of the liver, the disease cannot be cured, at least 75 percent of the autologous livers of children patients still continue fibrosis to progress, and the liver transplantation operation becomes the only choice. At present, the etiology of biliary atresia is not studied, and the mechanism hypothesis mainly includes: gene susceptibility, bile duct dysplasia, environmental toxin and virus infection, immunoregulation disorder and the like. The strong bile duct reaction triggered by cholangiophilis to cause hyperimmune inflammatory lesions is considered to be the mainstream theory, but the anti-inflammatory and anti-fibrotic treatments under the guidance of this theory do not reach the expected efficacy. Since any single theory hypothesis can not be fully applied to elucidating the clinical subtypes with distinct features of biliary atresia, it is also suggested that biliary atresia is not caused by a single cause but by a superposition of multiple factors.
The hepatic artery system is the only source of blood supply for the biliary tract system, and both strictly accompany the Glisson system and maintain close anatomical and functional relationships. Bile duct epithelial cells are a cell group with high oxygen consumption and low oxygen deficiency tolerance and are easily damaged by free radicals, so that the vascular plexus around the bile duct formed by the branch at the tail end of the hepatic artery is particularly important. The vascular morphological abnormality in biliary atresia is published as early as the end of the 20 th century, and a plurality of liver histological research evidences mention that the hepatic arteriole hyperplasia and the thickened structure change in biliary atresia, and the middle layer of the hepatic artery tube wall is remarkably thickened. Doppler ultrasound measurements suggest an increase in the hepatic artery resistance to blood flow index in biliary atresia, a change that correlates with poor prognosis of the disease. In 2004, ufacker described abnormal vascular phenomena of "truncation" at the end of hepatic artery and "clustering" of peripheral small vessels in infants with BA when performing hepatic artery angiography. Subsequently, the results of the vascular-associated cytokine study suggested that the hepatic artery system was deficient in oxygen supply, and HIF-1 α and VEGFA were significantly elevated in the BA hepatic vascular zone compared to non-BA cholestasis cases. These evidences have not been able to define that hepatic artery morphological abnormalities correlate closely with BA, nor are they suitable as specific diagnostic markers.
Until two breakthrough studies, in 2009 Lee observed abnormal blood flow under the liver capsule in color doppler ultrasound, which was considered as an early diagnosis index; the source of the abnormal blood flow signal is observed under direct vision of a laparoscope in 2017, and is named as spider vessel sign under the hepatic capsule (HSST sign), histologically confirmed as hepatic arteriole, the sensitivity is 100% when the hepatic arteriole is used for diagnosing biliary atresia, and the specificity is 97.8%. In the later stage, by means of an upright microscope, liver envelope vascular plexus similar to HSST is also observed in a rotavirus-induced biliary atresia mouse model, and because the survival period of the biliary atresia mouse model is not enough to form obvious hepatic fibrosis, the liver vascular factor is presumed to participate in the BA pathological mechanism in the early stage. A microscopic study in 2019 recently reported that capillary endothelial cells around a bile duct appear shriveling and broken in an early stage in a biliary atresia mouse model, no virus particles are seen in the cells, and meanwhile, a capillary bed around the bile duct is gradually sparse and reduced, so that early-stage microcirculation disturbance exists. These findings all suggest that structural changes in the hepatic artery and its branches may be a contributor to BA. Notch is involved in the regulation of a variety of physiological and disease processes as an important developmental signal, critical to cell fate decisions in embryogenesis and early postnatal stages, with Notch3 signaling being particularly critical for angiogenesis.
The patent of granted publication No. CN102818866B discloses the application of a diagnostic marker for neonatal biliary atresia, in which the diagnostic marker of the medical device is the content ratio of taurochenodeoxycholic acid and chenodeoxycholic acid in serum. The invention has the advantages that: the application of taurochenodeoxycholic acid and chenodeoxycholic acid as biliary atresia diagnostic markers is firstly proposed, and the taurochenodeoxycholic acid and chenodeoxycholic acid can be used for distinguishing biliary atresia and other infant biliary juice stasis diseases, striving for the optimal operation time for the infant patients and improving the operation curative effect. Although the diagnostic marker and the application thereof can be used as a diagnostic reference for judging whether the child is blocked by the biliary tract, the technical reference cannot be provided for the treatment of the blocked by the biliary tract on the basis of the technology.
The patent of the publication No. CN104774914B is used for diagnosing biliary atresia by comparing the difference of serum microRNA expression profiles of infants suffering from biliary atresia and cholestatic infant hepatitis syndrome by utilizing a microRNA chip, but on the basis of the technology, a technical scheme for treating biliary atresia is not provided.
Disclosure of Invention
Technical problem to be solved
The invention aims to provide a method for taking genes/proteins as biliary atresia diagnosis markers and treatment targets and application thereof, and aims to solve the problems that no method for diagnosing and treating biliary atresia is provided in the prior art from vascular morphology, and the existing biliary atresia diagnosis method cannot be used for treatment of biliary atresia on the basis of the technology of the biliary atresia diagnosis method.
(II) technical scheme
In order to realize the method for diagnosing and treating biliary atresia based on gene/protein and the application thereof, and solve the problems that the prior art does not provide a method for diagnosing and treating biliary atresia from vascular morphology, and the existing biliary atresia diagnostic method can not be used for treating biliary atresia on the basis of the technology thereof, the invention provides the following technical scheme:
a method for using gene/protein as biliary tract atresia diagnosis mark and therapeutic target point includes the following steps:
summarizing the structural characteristics of a human biliary atresia hepatic artery system, and selecting hepatic artery main and hepatic ductus arteriosus for observation, wherein the hepatic artery main structure and functional indexes are measured by Doppler ultrasound and indocyanine green angiography methods, and the hepatic ductus arteriosus structure is measured by histology;
studying the expression of Notch in the liver of a human biliary atresia model and an animal biliary atresia model, wherein the detection method adopts RT-PCR, Western-blots, OPAL multispectral staining and primary culture and staining of mouse hepatic portal vascular smooth muscle cells, and Notch3-Hey1 gene/protein in liver tissues is used as a biliary atresia diagnostic marker;
diversification intervention is carried out on Notch3 in an animal biliary tract occlusion model, and overexpression and inhibition experiments of Notch3 are carried out in an animal biliary tract occlusion model for verification.
Preferably, the method for detecting the main structure and the functional indexes of the hepatic artery by the Doppler ultrasonic measurement and the indocyanine green angiography method specifically comprises the following steps:
clinical Doppler ultrasonic detection hepatic artery main structure and functional indexes: dividing the infant with pathological jaundice into a BA group and a non-BA cholestasis group, recording case basic data, ultrasonically measuring the diameter of a hepatic artery, the diameter of a portal vein, a hepatic artery resistance index and the acceleration time of the hepatic artery, taking the section of the proximal end of the hepatic right artery parallel to the portal vein as an anatomical mark for ultrasonic measurement of the diameter of the hepatic artery and the diameter of the portal vein, and reading blood flow parameters after confirming that a stable hepatic artery pulse signal is captured;
indocyanine green angiography detects hepatic artery trunk structure and functional index: the method comprises the steps of selecting a patient with highly suspected BA and needing laparoscopic biliary tract angiography examination, setting the patient to be a BA group and an age-matched Non-BA group, injecting ICG (acute coronary syndrome) from veins, entering the liver from the hepatic artery through systemic circulation for developing, reflecting the perfusion condition of the hepatic artery to organs, preparing a solution by using 0.9% NaCl injection, adjusting a laparoscopic image acquisition system to a fluorescence mode, focusing a visual field center on the liver by using an adjusting lens, starting timing immediately after the rapid injection from a venous channel is finished, maintaining the lens to stably observe the fluorescence intensity change and acquire dynamic images.
Preferably, the histological measurement of the arteriole structure in the hepatic region is specifically as follows: carrying out arteriole structure measurement after HE (high intensity intrinsic contrast) staining and alpha-SMA (shape memory alloy) immunohistochemical staining of paraffin tissue slices, randomly selecting a arteriole with 200 or 400 times of visual field and formed by 2-5 layers of smooth muscle cells on the tube wall, measuring the Inner Diameter (ID), selecting the maximum transverse diameter and the minimum transverse diameter to take the average value, measuring the wall thickness (AW), selecting 3 uniformly-spaced tube walls, measuring the thickness by taking the intersection point of the connecting lines of the maximum transverse diameter and the minimum transverse diameter as the center to take the average value, and recording the AW/ID ratio.
Preferably, the expression of Notch in the liver of a human biliary tract occlusion model and an animal biliary tract occlusion model is researched, and RT-PCR is adopted to detect the Notch1-4 in the liver tissue of a human body or a mouse and downstream target genes of the Notch1, Hes5, Hey1, Hey2 and HeyL; western-blot detection is adopted to compare the expression of human liver Notch3-Hey1 pathway protein and the dynamic expression trend of mouse Notch3, HIF-1 alpha and VEGFA protein.
Preferably, the RT-PCR is adopted to detect Notch1-4 in human liver or mouse liver tissue and downstream target genes of Hes1, Hes5, Hey1, Hey2 and HeyL thereof, wherein the humanized primer sequence is as follows:
the murine primer sequences were as follows:
preferably, in the study of the expression of Notch in the liver of the human biliary atresia model and the animal biliary atresia model, the antibody/fluorescence color matching scheme adopting OPAL multispectral staining is as follows: notch 3/blue-green, Hey 1/gray, HIF-1 α/yellow, Vimentin/magenta, SMMHC/red, CK 19/green, PADI/blue.
Preferably, the primary culture and staining of mouse hepatic portal vascular smooth muscle cells are specifically performed by selecting mice born from Day 10-Day 14 to dissect hepatic portal blood vessels to separate VSMCs, and observing and comparing the morphological and phenotypic characteristics of the mouse hepatic portal VSMCs of BA and normal mice.
The application of gene/protein as biliary tract locking diagnosis mark and treating target includes: notch3-Hey1 gene/protein in liver tissue as biliary tract atresia diagnostic marker; the activation degree of the Notch3 signal pathway is used for assisting in predicting the disease prognosis of the infant with BA and optimizing the operation decision; the anti-hepatic artery remodeling effect produced by specific inhibitors of Notch3, including anti-human Notch3 neutralizing antibody, synthetic Notch3 receptor recognition blocker, or directed administration method against hepatic artery system Notch3, is used as a clinical treatment strategy.
Preferably, the upstream or downstream key signal molecule of the Notch3 signal path is used as a biliary tract occlusion diagnostic marker, or assists in predicting the disease prognosis of the infant with BA and optimizing the operation decision.
Preferably, the specific inhibitor acts on key signal molecules upstream or downstream of the Notch3 pathway and is capable of producing an anti-hepatic arterial remodeling effect.
(III) advantageous effects
Compared with the prior art, the invention provides a method for taking a gene/protein as a biliary atresia diagnosis mark and a treatment target and application thereof, and the method has the following beneficial effects:
1. the method and the application thereof, namely the Notch3 signal channel and the hepatic artery system remodeling phenomenon mediated by the Notch3 signal channel are firstly applied to the technology of biliary tract occlusion diagnosis and treatment, and are the improvement of the existing clinical theoretical mechanism.
2. The method and the application thereof have the advantages that the Notch3-Hey1 gene/protein highly expressed in liver tissues is used as a BA diagnosis marker, the activation degree of the Notch3 signal channel can be used as a prediction index for judging disease prognosis and guiding operation decision, and the method is favorable for assisting in predicting disease prognosis of children and selecting operation modes.
3. According to the method and the application of the invention, the Notch3 signal path can become an effective intervention target point for clinical treatment, and the hepatic artery remodeling resisting effect generated by the Notch3 specific inhibitor can be used as a clinical treatment strategy, so that the method is beneficial to improving the hypoxia and bile duct injury of the junction area, slowing down the progress of the disease course and improving the liver survival rate of BA itself.
Drawings
FIG. 1 is a diagram of the hepatic artery trunk structure and function index for clinical ultrasonic detection;
FIG. 2 is a graph comparing results of angiography of indocyanine green;
FIG. 3 is a representation of the arteriole structure of the human hepatic region of confluence;
FIG. 4 is a structural comparison of small hepatic artery in the region of human hepatic junction under different prognosis conditions;
FIG. 5 is a structural comparison of the small hepatic artery in the hepatic region of human BA at different stages of the disease process;
FIG. 6 is a graph showing a comparison of liver histopathology of BA patients less than 1 month old showing no significant fibrosis, but showing liver envelope surface spider-like vascular features typical of BA patients 2 months old;
FIG. 7 shows HSST-like vascular characterization of the surface of the liver envelope under an upright microscope in a rotavirus-induced mouse model;
FIG. 8 is a graph showing abnormally high expression of Notch3-Hey1 in human BA liver;
FIG. 9 is a comparison of multispectral staining results for human BA and non-BA liver OPAL;
FIG. 10 is a graph showing the results of Notch expression detection in the BA mouse model;
FIG. 11 is a graph showing mouse hepatic portal vascular smooth muscle cell culture and phenotype identification;
FIG. 12 is a mechanistic map of Notch3 pathway facilitating BA processivity by mediating hepatic arterial system structural remodeling;
FIG. 13 is a graph of the therapeutic effect of Notch3 neutralizing antibody on a BA mouse model;
FIG. 14 is a graph showing that Notch3 neutralizing antibody intervention can improve hypoxia in the zona of the mouse sink in BA;
FIG. 15 is a graph showing that Notch3 neutralizing antibody intervention can significantly reduce liver envelope surface vascular plexus growth in BA mice;
FIG. 16 is a graph of the therapeutic effect of the Notch broad-spectrum inhibitor DAPT on a BA mouse model.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The invention provides a method for taking genes/proteins as biliary atresia diagnosis markers and therapeutic targets, which comprises the following steps:
summarizing the structural characteristics of a human BA hepatic artery system, wherein in order to solve the structural characteristics of the human hepatic artery system, the level of a resistance arteriole in a hepatic artery main region and a hepatic region is selected for observation, the hepatic artery main region is measured by Doppler ultrasound and an indocyanine green angiography method, and the hepatic artery main region and the hepatic region are measured by histology;
secondly, the expression of the Notch in human BA and animal model liver is researched, in order to research the expression of the Notch from multiple layers, the detection method adopts RT-PCR, Western-blots, OPAL multispectral staining, primary culture and staining of smooth muscle cells and the like;
③ carrying out diversified intervention aiming at Notch3 in animal models. Specifically, an overexpression and inhibition experiment of Notch3 is carried out in an animal model, the overexpression experiment is carried out by means of an NICD3 overexpression adenovirus tool, a blocking strategy adopts a Notch3 neutralizing antibody and a Notch receptor broad-spectrum inhibitor DAPT, and the phenotype change and the survival curve of a mouse are observed respectively to judge how the corresponding intervention effect is.
The method comprises the following steps:
1. clinical ultrasonic detection hepatic artery trunk structure and functional index: infants with pathological jaundice are classified into BA group and Non-BA cholestasis group (Non-BA cholestasis, Non-BA group). Recording basic data (age, sex and weight) of a case, ultrasonically measuring Hepatic Artery Diameter (HAD), Portal Vein Diameter (PVD), Hepatic Artery Resistance Index (HARI) and Hepatic artery acceleration time (HAT), selecting a section of the proximal end of the right Hepatic artery parallel to the Portal artery as an anatomical marker for uniform positioning standard, and reading blood flow kinetic parameters after confirming that a stable Hepatic artery pulse signal is captured. Fig. 1 shows the hepatic artery main structure and function index of clinical ultrasonic detection, which indicates that hepatic artery diameter, portal vein diameter and hepatic artery resistance index of BA patients are all higher than those of non-BA cholestasis group patients (P < 0.05);
2. indocyanine green (ICG) angiography method: case selection was performed on children who had high suspicion of BA requirement for laparoscopic cholangiography, and the groups were set to BA group and age-matched Non-BA group. ICG enters the liver from the hepatic artery through the systemic circulation after being injected from the vein, the perfusion condition of the hepatic artery to organs can be reflected, the recommended dosage is 0.2-0.5 mg/kg, in the embodiment, 0.9% NaCl injection is used for preparing solution, the laparoscopic image acquisition system is adjusted to a fluorescence mode, the lens is adjusted to concentrate the center of the visual field to the liver, timing is started immediately after the rapid injection from the venous channel is finished, the lens is maintained to stably observe the fluorescence intensity change and acquire dynamic images. Fig. 2 is a comparison of indocyanine green angiography results showing that the liver of infant patients without BA was rapidly illuminated after several seconds when subjected to indocyanine green angiography, whereas about 2/3 in the liver of infant patients with BA showed delayed hepatic arterial perfusion.
3. Histological measurement of arteriole structure in the region of human liver sinks: carrying out arteriole structure measurement after HE staining and alpha-SMA immunohistochemical staining of paraffin tissue sections, randomly selecting arterioles with 200 or 400 times of visual field and pipe walls composed of 2-5 layers of smooth muscle cells, measuring the Inner Diameter (ID), selecting the length of the maximum transverse diameter and the minimum transverse diameter to take the average value, measuring the thickness of the pipe walls (AW), selecting 3 uniformly spaced pipe walls, measuring the thickness by taking the intersection point of the connecting line of the maximum transverse diameter and the minimum transverse diameter as the center to take the average value, and recording the AW/ID ratio.
The conventional HE staining procedure included:
1) paraffin section xylene dewaxing and gradient alcohol step-by-step hydration;
2) adding hematoxylin dropwise for dyeing for 5min, and washing with tap water for 1 time;
3) dropwise adding eosin for dyeing for 2min, and washing with tap water for 1 time;
4) gradient alcohol dehydration, xylene transparent treatment and neutral resin sealing.
The immunohistochemical method adopts a DAB color development method, and comprises the following specific steps:
5) paraffin sections are dewaxed by dimethylbenzene and hydrated by gradient alcohol;
6) selecting EDTA or sodium citrate solution for antigen retrieval according to the optimal pH value of the antibody, boiling in a boiling water bath for 20min, and gradually cooling to room temperature;
7) treating with 3% hydrogen peroxide to block peroxidase, standing at 37 deg.C for 20min, and washing with PBS for 2 times and 5 min/time;
8) blocking with 5% BSA, standing at 37 deg.C for 20 min;
9) adding primary antibody dropwise, and incubating overnight at 4 ℃;
10) washing with PBS for 2 times, adding biotin-labeled secondary antibody dropwise, and incubating at 37 deg.C for 30 min;
11) washing with PBS for 2 times, dripping the ready-prepared DAB color developing solution for 3-10 min, and washing with PBS again;
12) counterstaining with hematoxylin for 2-4 min, differentiating with 1% hydrochloric acid alcohol for 10s, and turning the tap water to blue;
13) gradient alcohol dehydration, xylene transparent treatment, drying and then neutral gum sealing.
Fig. 3 shows the structural expression of the small artery in the human liver manifold area, and the measurement and analysis of the ratio of the wall thickening of the resistance small artery in the manifold area of the BA group, the phenomenon of the stenosis of the lumen and the wall thickness (AW)/the Inner Diameter (ID) of the tube are shown, and the BA group is obviously higher than the non-BA and non-hepatitis disease group (P is less than 0.001).
FIG. 4 is a structural comparison diagram of hepatic arterioles in human liver manifold area under different prognosis conditions, which shows that the hepatic arteriole tube wall in liver manifold area of infant with bad prognosis after Kasai operation among the infant patients with BA is obviously thickened compared with the hepatic arteriole tube wall in liver manifold area of infant with better prognosis; FIG. 5 is a graph comparing the structure of the small hepatic artery in the area of human BA liver junction at different stages of the disease process, showing that the liver of a child with BA shows a marked sign of stenosis at the time of late liver transplantation compared to the Kasai procedure; the tissue staining method used in FIGS. 4 and 5 is as described in item 3 above.
Example 2
The method for observing spider-like blood vessel characteristics (HSST characteristics) on the surface of the liver envelope comprises the following steps: the observation of HSST sign and the collection of photos can be finished under the direct vision of a laparoscope system. In this example, photographs of the visceral surface and the diaphragm surface of the liver are collected before the operation of the free gallbladder is started after the laparoscope is connected. Fig. 6 shows that BA patients less than 1 month old did not exhibit significant fibrosis in liver tissue pathology, but exhibited spider-like vascular characteristics of the liver envelope surface as typical of BA patients 2 months old.
A rotavirus (RRV) induced mouse model method and a mouse liver envelope surface blood vessel characteristic observation method are as follows: adult wild type SPF-grade BALB/c mice (7w) were bred at a 2:1 or 3:1 ratio of males to females, pregnant mice body weight changes were recorded daily, and production was daily checked during the expected period of time to delivery for timely handling. Newborn mice were randomly divided into NC group and RRV group. RRV group mice were intraperitoneally injected with 30. mu.l of virus solution via a micro-syringe within 12h after birth, Day1 is 1d after RRV injection, and NC group mice were intraperitoneally injected with the same amount of MEM medium at the same time. Observing the blood vessel plexus on the surface of the mouse liver under a microscope in a normal open field, quickly dissecting and exposing the liver after the neck of the mouse is cut off, placing the mouse under a 40-time microscope in the open field, finely adjusting the focal length and the visual field, and obtaining a liver envelope surface picture. FIG. 7 shows HSST-like vascular features on the surface of the liver envelope under an upright microscope in a rotavirus-induced mouse model.
The embodiment is a vascular phenomenon observation method for a biliary atresia disease model, and the liver envelope vascular characteristic is the externalization expression of remodeling change of biliary atresia hepatic artery and can be used as one of effective indexes for intervention of a posterior blocking strategy.
Example 3
In the step of the second step of the example 1, the method for detecting the expression of the Notch3-Hey1 pathway in the liver of human BA in the research of the expression of the Notch in the liver of human BA and animal model comprises the following steps: the quantitative detection method uses RT-PCR and Western-blot. Wherein, RT-PCR detects Notch1-4 in human liver tissues and main downstream target genes of Hes1, Hes5, Hey1, Hey2 and HeyL thereof. FIG. 8 shows an abnormally high expression pattern of Notch3-Hey1 in human BA liver. The primer sequences are as follows:
firstly, extracting total RNA from tissues, and preparing cDNA by conventional reverse transcription, wherein the specific steps are as follows:
1) collecting liver tissue processed by RNAlater and frozen in-80 refrigerator, grinding 0.05g in homogenizer, adding 1ml Trizol, homogenizing, transferring homogenate to 1.5ml EP tube;
2) adding 0.2ml of chloroform into each tube, rapidly shaking for 15s, standing for 2min, observing the initial stratification of homogenate, and then centrifuging at 12000rpm for 15 min;
3) sucking the supernatant into a sterile enzyme-free 1.5ml EP tube, adding isopropanol with the same volume, mixing uniformly, centrifuging at 12000rpm at 4 ℃ for 15min, and removing the supernatant to obtain RNA precipitate;
4) treating the precipitate with 1ml of 75% ethanol, repeating the above operations for purification, discarding the supernatant, and drying the precipitate at room temperature;
5) adding 50 mul DEPC water to dissolve the RNA precipitate, detecting the concentration and the purity, and preparing reverse transcription;
6) the following ingredients were added and incubated at 37 ℃ for 30min → 65 ℃ for 10min to inactivate DNaseI:
7) the reverse transcription reaction system is as follows:
8) the cDNA can be added with a proper amount of deionized water, diluted to a proper concentration, and stored in a refrigerator at-20 ℃, and an RT-PCR reaction system is configured as follows (3 auxiliary holes are arranged, the annealing temperature is 60 ℃):
the Ct value (the number of amplification cycles that have passed when the fluorescence signal intensity in the reaction tube reaches a set threshold) derived from the PCR instrument is determined by taking the expression level of the internal reference as a reference standard and adopting 2 as the original data-ΔΔCTThe method is used for analysis.
Example 4
In the step of the second step of the example 1, in the study of the expression of human BA and Notch in animal liver, Western-blots detection method was used to compare the expression of human liver Notch3-Hey1 pathway protein and the dynamic expression trend of mouse Notch3, HIF-1 alpha and VEGFA protein. The whole process comprises protein extraction and quantification, SDS-PAGE electrophoresis, membrane transfer and color development analysis. The specific steps of protein extraction and concentration determination are as follows:
1) taking 0.05g of liver tissue, placing the liver tissue in a homogenizer, adding 0.5ml of lysate, repeatedly placing the homogenate on ice, and uniformly grinding the lysate until the lysate is completely lysed;
2) transferring the mixed solution to a centrifuge tube, centrifuging at 4 ℃ and 12000rpm for 15min, taking the supernatant and subpackaging;
3) preparing a BSA standard according to a reference table provided by the BCA protein concentration determination kit;
4) measuring the concentration by a microplate method, and respectively adding 25 mul of standard substance and a sample to be measured into an ELISA plate;
5) adding 200 mu l of BCA working solution into each hole, oscillating for 30s, and then placing in a 37 ℃ incubator for incubation for 30min at constant temperature;
6) and (5) detecting the absorbance by using a microplate reader, and calculating the protein concentration (mu g/mu l) of the sample to be detected according to the absorbance curve of the BSA standard.
SDS-PAGE electrophoresis, membrane transfer and color development experiments can be prepared, and the steps are as follows:
1) gels of different concentrations were prepared according to the size of the protein molecule of interest, and the reference provided in the kit instructions was as follows:
2) the separation gel is configured according to the required volume and gel concentration, taking 12% gel as an example:
3) the concentrated gum was prepared as follows:
4) preparing glue according to the formula, pouring the glue between glass plates of the electrophoresis tank, inserting a comb, and standing by after solidification;
5) the extracted protein samples were mixed according to 5: adding SDS (sodium dodecyl sulfate) loading buffer solution in a proportion of 1, and continuously boiling in a boiling water bath for 3-5 min until the protein is completely denatured;
6) adding 10 μ g of sample into each well with a sample adding gun, connecting with an electrophoresis apparatus, concentrating gel for 80V 20min, separating gel for 120V60min, and cutting off power supply when bromophenol blue reaches the edge of the gel;
7) opening the glass clamping plate, taking out the colloid, covering the PVDF membrane, carefully removing bubbles, performing membrane rotation in a sandwich arrangement mode (filter paper/glue/membrane/filter paper), placing the colloid in a clamping groove, placing the membrane in a cathode, placing the membrane in an anode, and performing electrophoresis for 120min under a current of 200 mA;
8) after the membrane is transferred, taking out the PVDF membrane, putting the PVDF membrane into 5% skimmed milk, and sealing for 2 hours at room temperature;
9) diluting primary antibody to appropriate proportion, incubating with membrane, and refrigerating at 4 deg.C overnight;
10) washing the membrane for 3 times by TBST, diluting the secondary antibody, incubating with the membrane, and incubating at 37 ℃ for 1 h;
11) washing the membrane for 3 times by TBST, preparing ECL luminescent liquid (uniformly mixing the solution A and the solution B in equal volume), dripping a liquid shifter on the front surface of the membrane, reacting in a dark room in a dark place for 2min, completely sucking out excessive liquid, putting the membrane into a gel imager, generally setting the exposure time to be 1-5 min, and observing and recording protein bands;
12) ImageJ software reads WB stripe grayscale values.
Example 5
In the step of the second step of the example 1, the method for researching the expression of Notch in human BA and animal liver by using OPAL multispectral staining and flow-like analysis specifically comprises the following steps: tissue staining specificity evaluation is carried out on the primary antibody and paraffin liver sections through a conventional immunohistochemical pre-experiment, and multispectral staining can be started after verification is qualified. OPAL technology utilizes TSA labeling to achieve a multi-label counterstaining protocol, allowing multiple applications of primary antibodies to different target proteins (up to 7 color fluorescent labels) on the same paraffin section. After the previous round of antibody is removed by microwave heating, tyramine signals with different fluorescent labels are left to be stably and covalently bound on a plurality of corresponding target proteins, so that the cross reaction of the previous antibody when the antibody is repeatedly labeled is effectively avoided. The method can obtain images with high signal-to-noise ratio by matching with a Vectra full-spectrum image acquisition system, split and correct multicolor spectra by matching with InForm image analysis software, intelligently identify specific tissue types by adopting a self-learning algorithm for segmentation, can perform immunofluorescence staining at the single cell level, and perform quantitative analysis on expressions in different regions. The antibody/fluorescent color scheme in this example was: notch 3/blue-green, Hey 1/gray, HIF-1 α/yellow, Vimentin/magenta, SMMHC/red, CK 19/green, PADI/blue.
FIG. 9 is a comparison of human BA and non-BA liver OPAL results of multispectral staining, which shows that the proliferation of small artery and small bile duct in BA sink area is obvious, high-expression Notch3 is located in hepatic artery system, the hypoxia condition of BA sink area is heavier, bile duct cells in anoxic state are more, and synthetic smooth muscle cells marked by vimentin are more.
Example 6
In the step of 'study of expression of Notch in liver of human BA and animal model' in example 1, the method for detecting expression of Notch in mouse model of BA is the same as that described in example 3, wherein the murine PCR primers used are as follows:
as shown in FIG. 10, the results of the detection of Notch expression in the BA mouse model showed that the liver of the BA mouse also had early overactivation of Notch3 pathway, and the expression peak was around day 7.
Example 7
In the step of the second step of the example 1, in the step of studying the expression of Notch in human BA and animal model liver, the method for culturing and identifying phenotype by using mouse hepatic portal vascular smooth muscle cells specifically comprises the following steps: and selecting mice born with Day 10-Day 14, dissecting hepatic portal blood vessels, separating VSMCs, and observing and comparing the morphological and phenotypic characteristics of the hepatic portal VSMCs of the BA mice and normal mice. The specific steps of extracting the primary SMCs comprise:
1) killing the mouse by breaking the neck, disinfecting abdominal skin by 75% alcohol, quickly separating and exposing a Glisson system connecting a hepatic portal part and duodenum, and putting the mouse into a 100mm sterile plate filled with PBS buffer solution after completely cutting as much as possible;
2) carefully separating the tissues of the blood vessel by a dissecting microscope, separating the tissues of the blood vessel, putting the tissues into another sterile plate filled with PBS buffer solution, and transferring the tissues to a clean bench for operation;
3) carefully cutting the tissue blocks into tissue blocks with the size of 1-2 mm by using an ophthalmic scissors, transferring a pipette to a 15ml centrifuge tube, adding type 2 collagenase, and slightly oscillating and digesting the tissue blocks in a water area at 37 ℃ until the tissue blocks are digested into emulsion;
4) gently blowing and beating, adding a DMEM medium to stop digestion, filtering a screen to obtain a single cell suspension, and centrifuging at 1200rpm for 5 min;
5) discarding the supernatant to obtain cell precipitate, adding DMEM/F12+ 5% FBS + 5% PS culture medium for resuspension, transferring to a culture dish, observing the cell state under a mirror, and placing in a 37 ℃ incubator with 5% CO2 and a humid environment;
the growth state was observed every day, and the culture medium was changed 2 to 3 days. When adherent cells grew to about 70% confluency, the cells were digested with 0.25% trypsin and subcultured or plated at 1:2 for subsequent experiments. The double-labeled fluorescent staining of alpha-SMA and vimentin is used for evaluating VSMCs cell subtypes, and the steps are as follows:
1) inoculating SMCs on a slide in a 12-hole plate until the cells are fused to more than 80%;
2) fixing the slide with 4% paraformaldehyde for 15min, and washing the slide with PBS for 3 times, each for 3 min;
3) removing the washing liquid, and dripping 0.5% Triton X-100 for 20min at room temperature;
4) dropping normal goat serum onto the slide, sealing at room temperature for 30min, and completely sucking the sealing liquid;
5) dropwise adding the diluted primary antibody onto the glass slide, and placing the glass slide in a moisture preservation box at 4 ℃ for incubation overnight;
6) soaking and washing for 3 times by using PBST buffer solution, dripping fluorescent secondary antibody after completely sucking liquid, and incubating for 1h at 37 ℃ in a dark place;
7) staining with another fluorescent antibody of a different species origin repeating the steps 3) to 5);
8) and (3) dropping DAPI to counter stain the cell nucleus, incubating for 5min in a dark place, sucking out liquid, sealing the cell nucleus by using an anti-fluorescence quencher, and observing the cell nucleus under a fluorescence microscope.
As shown in FIG. 11, the conditions of mouse hepatic portal vascular smooth muscle cell culture and phenotype identification show that the phenotype of hepatic portal vascular smooth muscle cells of BA mice is more juvenile than the form of the control group, and the synthetic type/contractile type ratio imbalance of vimentin/alpha-SMA marker is biased to the more juvenile synthetic type.
Example 8
Fig. 12 is a mechanical diagram of promoting BA persistent progression by mediating hepatic artery system structure remodeling in the Notch3 pathway, and aims at the step of three, "performing diversified intervention to Notch3 in an animal model, performing overexpression and inhibition experiment of Notch3 in the animal model, verifying the overexpression experiment (i.e., performing intraperitoneal injection of NICD3 overexpression adenovirus in a neonatal mouse) by means of an NICD3 overexpression adenovirus tool, and observing phenotype change and survival curve of the mouse respectively by using a Notch3 neutralizing antibody and a Notch receptor broad-spectrum inhibitor DAPT in the blocking strategy to judge how the corresponding intervention effect is, specifically adopting the following method:
an application method and an evaluation method of a Notch3 neutralizing antibody in a BA animal model are as follows: the Notch3 neutralizing antibody experiment designs action time gradient, after 12h of postnatal intra-abdominal RRV injection of intervention group mice, the first group starts to inject Notch3 antibody from the birth 2d of the mice, and the operation is continued for 2 d; the second group started injection from the 5 th postnatal day of mice, with unchanged mode; third group mice started to dry after birth at 7 d. Each group had 10 mice, and each antibody injection was administered at a dose of 10. mu.g. RRV + PBS/MEM + PBS was used as a control. As shown in FIG. 13, the therapeutic effect of Notch3 neutralizing antibody on BA mouse model is shown, wherein (c) the group starts intervention respectively on days 2, 5 and 7, the earlier the intervention is, the better the protection effect is, the survival rate of mouse is obviously improved compared with BA control group, the phenotype is improved, and no fibrosis of gallbladder and extrahepatic bile duct appears on day 14.
II, evaluating the hypoxia condition of the mouse sink area: the specific procedure for selecting HIF-1 α immunofluorescence histochemical staining was as described in example 1 above for "histological measurement of arteriole structures in the region of human liver sinks". As shown in fig. 14, it was shown that Notch3 neutralizing antibody intervention could improve hypoxia in the zone of the mouse sink in BA.
The method for observing the vascular plexus on the surface of the mouse liver envelope is as described in the concrete contents of the rotavirus (RRV) induced mouse model method and the observation method of the vascular plexus on the surface of the mouse liver envelope in example 2. As shown in fig. 15, it was shown that Notch3 neutralizing antibody intervention could significantly reduce hepatic envelope surface vascular plexus growth in BA mice.
Fourthly, the blocking strategy adopts a Notch3 neutralizing antibody and a Notch receptor broad-spectrum inhibitor DAPT, and the application method and the evaluation method of the DAPT in the BA animal model are specifically as follows: in order to study the effective dosage, drug dosage comparison is designed, after mice are intraperitoneally injected with RRV/DMSO within 12h after birth, 5 mug of DAPT (dissolved in DMSO), 10 mug of DAPT and 15 mug of DAPT are respectively intraperitoneally injected the next day, and each group comprises 12 mice. As shown in figure 16, is the therapeutic effect of the Notch broad-spectrum inhibitor DAPT on a BA mouse model, where BA mouse survival was improved upon application of a 10 μ g dose of DAPT.
Through the various experimental detection methods of the above embodiments, the experimental results are as follows:
notch3-Hey1 gene protein and a liver artery remodeling phenomenon mediated by the same can be used as a histological auxiliary diagnosis index of biliary atresia liver diseases, and the remodeling phenomenon related to the whole liver artery system is found in human BA, and specifically comprises the steps of widening the diameter of a liver artery, thickening the middle layer of an artery wall, increasing the resistance index of the liver artery, delaying the perfusion of the liver artery, increasing the ratio of the thickness of a wide arteriole wall to the inner diameter of the duct wall and spider-shaped blood vessel characteristics on the surface of a liver envelope. The degree of hepatic artery structural pathology may be related to the prognosis and course of BA. Meanwhile, the Notch3-Hey1 pathway which is expressed and positioned in the hepatic artery system has early abnormal activation in the BA liver, unbalanced smooth muscle cell phenotype and enhanced proliferative activity. The Notch3 pathway is known to promote arterial hypertrophic remodeling by maintaining a dedifferentiated state of the smooth muscle cell phenotype, which is also an initial pathological feature of pulmonary hypertension disease.
Therefore, the involvement of the Notch3-Hey1 signaling pathway in the pathological mechanism of BA is summarized as: the Notch3 passage of the hepatic artery system is abnormally activated, so that the smooth muscle cells are excessively proliferated to cause the thickening of the wall of an arteriole and a arteriole, the narrowing and even the blocking of a lumen, the peripheral circulation resistance is obviously increased due to continuous remodeling, the main trunk of the upstream hepatic artery is passively widened, the blood flow resistance is increased, and the effective perfusion volume of the hepatic artery is reduced; meanwhile, the open area of the downstream capillary network is correspondingly reduced, microcirculation around the bile duct is disturbed, the anoxic injury of the epithelial cells of the bile duct is aggravated, and the double attack is formed with other exogenous factors. Therefore, the abnormally high-expression Notch3-Hey1 protein/mRNA can be used as a BA diagnostic marker, and is favorable for assisting in predicting the disease prognosis of children and selecting a surgical mode.
Meanwhile, the Notch3 inhibition experiment performed in the BA animal model shows ideal protection effect. It is observed that the Notch3 blocker (antibody/DAPT) can generate certain protection effect on RRV infected mice, and better protection effect is obtained when specificity blocking is carried out in the early stage, the mouse BA phenotype is improved, and the survival rate is obviously improved. Therefore, the Notch3 signaling pathway may become an effective intervention target for clinical treatment, and the inhibitor thereof can be applied to BA adjuvant therapy and prolong the survival time of the liver.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A method for using gene/protein as biliary tract atresia diagnosis mark and therapeutic target point is characterized by comprising the following steps:
selecting hepatic artery main and hepatic region arterioles for observation, wherein the hepatic artery main structure and functional indexes are measured by Doppler ultrasound and indocyanine green angiography methods, and the hepatic region arteriole structure is measured by means of histology;
studying the expression of Notch in the liver of a human biliary tract occlusion model and an animal biliary tract occlusion model, wherein the detection method adopts RT-PCR, Western-blots, OPAL multispectral staining and mouse hepatic portal vascular smooth muscle cell primary culture and staining;
diversified intervention is carried out on Notch3 in an animal biliary tract occlusion model, and overexpression and inhibition experiments of Notch3 are carried out in the animal biliary tract occlusion model for verification.
2. The method for using gene/protein as biliary atresia diagnostic marker and therapeutic target according to claim 1, wherein the structural and functional indexes of hepatic artery trunk are detected by doppler ultrasound measurement and indocyanine green angiography methods, specifically:
clinical Doppler ultrasonic detection hepatic artery main structure and functional indexes: dividing the infant with pathological jaundice into a BA group and a non-BA cholestasis group, recording case basic data, ultrasonically measuring the diameter of a hepatic artery, the diameter of a portal vein, a hepatic artery resistance index and hepatic artery acceleration time, measuring the diameter of the hepatic artery and the diameter of the portal vein by ultrasonic measurement, selecting a section parallel to the portal vein at the proximal end of the right hepatic artery as an anatomical mark, and reading blood flow parameters after confirming that a stable hepatic artery pulse signal is captured;
indocyanine green angiography detects hepatic artery trunk structure and functional index: the method comprises the steps of selecting a patient with high suspicion that BA needs laparoscopic biliary tract radiography examination, setting the patient to be a BA group and an age-matched Non-BA group, injecting ICG from veins, entering liver from hepatic artery through systemic circulation for developing, reflecting perfusion condition of the hepatic artery to organs, preparing a solution by using 0.9% NaCl injection, adjusting a laparoscopic image acquisition system to a fluorescence mode, adjusting a lens to concentrate the center of a visual field on the liver, immediately starting timing when the rapid injection of a venous channel is finished, maintaining the lens to stably observe fluorescence intensity change and acquire dynamic images.
3. The method as claimed in claim 1, wherein the histological measurement of arteriole structure in hepatic region is specifically as follows: carrying out arteriole structure measurement after HE staining and alpha-SMA immunohistochemical staining of paraffin tissue sections, randomly selecting arterioles with 200 or 400 times of visual field and pipe walls composed of 2-5 layers of smooth muscle cells, measuring the Inner Diameter (ID), selecting the length of the maximum transverse diameter and the minimum transverse diameter to take the average value, measuring the thickness of the pipe walls (AW), selecting 3 uniformly spaced pipe walls, measuring the thickness by taking the intersection point of the connecting line of the maximum transverse diameter and the minimum transverse diameter as the center to take the average value, and recording the AW/ID ratio.
4. The method for studying the expression of Notch in liver of human and animal biliary atresia models according to claim 1, wherein RT-PCR is used to detect Notch1-4 in human liver or mouse liver tissue and its downstream target genes Hes1, Hes5, Hey1, Hey2, HeyL;
western-blot detection is adopted to compare the expression of human liver Notch3-Hey1 pathway protein and the dynamic expression trend of mouse Notch3, HIF-1 alpha and VEGFA protein.
5. The method of claim 4, wherein the gene/protein is used as diagnostic marker and therapeutic target for biliary atresia, and the method comprises the following steps: the method adopts RT-PCR to detect Notch1-4 in human liver or mouse liver tissue and downstream target genes Hes1, Hes5, Hey1, Hey2 and HeyL thereof, wherein the human primer sequence is as follows:
the murine primer sequences were as follows:
6. the method for using gene/protein as diagnostic marker and therapeutic target of biliary atresia according to claim 1, wherein the antibody/fluorescence color scheme using OPAL multispectral staining in the study of Notch expression in liver of human biliary atresia model and animal biliary atresia model is: notch 3/blue-green, Hey 1/gray, HIF-1 α/yellow, Vimentin/magenta, SMMHC/red, CK 19/green, PADI/blue.
7. The method of claim 1, wherein the gene/protein-based diagnostic marker and therapeutic target for biliary atresia are as follows: the primary culture and staining method of mouse hepatic portal vascular smooth muscle cells comprises the specific steps of selecting mice born with Day 10-Day 14 to dissect hepatic portal blood vessels to separate VSMCs, and observing and comparing the morphological and phenotypic characteristics of the hepatic portal VSMCs of BA mice and normal mice.
8. The application of the gene/protein as biliary tract atresia diagnosis mark and treatment target point includes: notch3-Hey1 gene/protein in liver tissue as biliary atresia diagnostic marker; the activation degree of the Notch3 signal pathway is used for assisting in predicting the disease prognosis of the infant with BA and optimizing the operation decision; the anti-hepatic artery remodeling effect produced by specific inhibitors of Notch3, including anti-human Notch3 neutralizing antibodies, synthetic Notch3 receptor recognition blockers, or directed administration methods against hepatic artery system Notch3, is a clinical treatment strategy.
9. The application of claim 8, wherein the upstream or downstream key signaling molecule of Notch3 signaling pathway is used as biliary atresia diagnostic marker or auxiliary for predicting the disease prognosis of infant with BA and optimizing the operation decision.
10. The application of the gene/protein as a biliary tract occlusion diagnosis marker and a therapeutic target according to claim 8 or 9, wherein the specific inhibitor acts on upstream or downstream key signal molecules of Notch3 pathway to generate an anti-hepatic artery remodeling effect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010657625.0A CN111778325A (en) | 2020-07-09 | 2020-07-09 | Method for using gene/protein as biliary tract atresia diagnosis mark and therapeutic target and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010657625.0A CN111778325A (en) | 2020-07-09 | 2020-07-09 | Method for using gene/protein as biliary tract atresia diagnosis mark and therapeutic target and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111778325A true CN111778325A (en) | 2020-10-16 |
Family
ID=72758706
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010657625.0A Pending CN111778325A (en) | 2020-07-09 | 2020-07-09 | Method for using gene/protein as biliary tract atresia diagnosis mark and therapeutic target and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111778325A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060140943A1 (en) * | 2002-09-05 | 2006-06-29 | Champion Brian R | Immunotherapy using modulators of notch signalling |
| CN102818866A (en) * | 2012-08-03 | 2012-12-12 | 上海交通大学医学院附属新华医院 | Applications of diagnostic markers in biliary atresia of newborns |
| CN104774914A (en) * | 2014-01-09 | 2015-07-15 | 复旦大学附属儿科医院 | Detection and applications of microRNA serum marker of biliary atresia and cholestatic infant hepatitis syndrome |
-
2020
- 2020-07-09 CN CN202010657625.0A patent/CN111778325A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060140943A1 (en) * | 2002-09-05 | 2006-06-29 | Champion Brian R | Immunotherapy using modulators of notch signalling |
| CN102818866A (en) * | 2012-08-03 | 2012-12-12 | 上海交通大学医学院附属新华医院 | Applications of diagnostic markers in biliary atresia of newborns |
| CN104774914A (en) * | 2014-01-09 | 2015-07-15 | 复旦大学附属儿科医院 | Detection and applications of microRNA serum marker of biliary atresia and cholestatic infant hepatitis syndrome |
Non-Patent Citations (5)
| Title |
|---|
| 冯定浩等: "Notch 1、Notch 2在肾脏发育及肾纤维化中的作用研究进展", 《广西医学》 * |
| 姜??等: "基于超声、肝胆核素显像和磁共振胆胰管成像影像学检查诊断胆道闭锁准确性研究的系统评价和Meta分析", 《中国循证儿科杂志》 * |
| 毛永忠等: "Notch配体、受体在小儿胆道畸形肝组织中的表达及意义", 《中华小儿外科杂志》 * |
| 蔡雄等: "Notch信号转导通路与原发性肝癌", 《中国肿瘤生物治疗杂志》 * |
| 饶建华等: "活体肝移植在中国的发展与前景", 《中国普外基础与临床杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JPWO2014061419A1 (en) | Novel cancer markers and their use | |
| CN107362366A (en) | Application of the ALOX12 inhibitor in ischemical reperfusion injury medicine is prepared | |
| CN107441100A (en) | Treat the compound of ischemical reperfusion injury | |
| CN110575540B (en) | Use of PDGF inhibitors for the production of a medicament for the treatment of inflammatory intestinal diseases | |
| Cole et al. | Congenital heart disease linked to maternal autoimmunity against cardiac myosin | |
| Sato et al. | Detection of TUNEL-positive cardiomyocytes and c-kit-positive progenitor cells in children with congenital heart disease | |
| CN105560255B (en) | Application and its medicine of the obakulactone in treatment pulmonary fibrosis, chronic pneumonia, chronic respiratory disease medicine is prepared | |
| CN111778325A (en) | Method for using gene/protein as biliary tract atresia diagnosis mark and therapeutic target and application thereof | |
| CN108619149A (en) | Applications of the SIS3 in that modulates fibrosis relevant disease | |
| CN115245575B (en) | Application of vascular smooth muscle cell specific DDX24 and downstream molecules thereof in vascular development | |
| CN110967490A (en) | Application of ABIN3 in preparation of medicine for preventing, relieving and/or treating liver ischemia-reperfusion injury | |
| Eastman Jr et al. | Fibroblast growth factor-10 signals development of von Brunn's nests in the exstrophic bladder | |
| CN108535480A (en) | Application of the EphA8 genes in preparing anti-breast cancer medicines and its diagnostic kit | |
| CN105396136B (en) | CCN1（Cyr61）Application in treatment skin injury and atrophoderma relevant disease | |
| Jeong et al. | An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver | |
| CN107648257A (en) | Hydrogen is for preparing the purposes in treating kidney fibrosis preparation | |
| Wang et al. | Correlations between claudin-1 and PIGF expressions in retinoblastoma. | |
| CN111228504A (en) | Application of ribosome S6 kinase RSK in preparing medicine for treating or repairing peripheral nerve injury | |
| CN116148471B (en) | Biomarker for pulmonary arterial hypertension and application thereof | |
| Wang et al. | Preliminary research of relationship between acute peritonitis and celiac primo vessels | |
| CN115337260B (en) | Composite acellular scaffold/hyaluronic acid temperature-sensitive hydrogel and application thereof | |
| CN107741497A (en) | For assessing the immunofluorescence detection agent box of liver cancer patient therapeutic effect | |
| CN107625777A (en) | Purposes of the rhodioside in complement inhibitor medicine is prepared | |
| CN115804773A (en) | Application of pazopanib in preparing neuroinflammation inhibitor | |
| Seval-Celik et al. | Expression of vascular endothelial growth factor and transforming growth factor alpha in rat testis during chronic renal failure |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201016 |
|
| RJ01 | Rejection of invention patent application after publication |