CN116421718A - 人源抗proBDNF单克隆抗体的应用 - Google Patents
人源抗proBDNF单克隆抗体的应用 Download PDFInfo
- Publication number
- CN116421718A CN116421718A CN202310131390.5A CN202310131390A CN116421718A CN 116421718 A CN116421718 A CN 116421718A CN 202310131390 A CN202310131390 A CN 202310131390A CN 116421718 A CN116421718 A CN 116421718A
- Authority
- CN
- China
- Prior art keywords
- probdnf
- sepsis
- mice
- monoclonal antibody
- lung
- 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
- 206010040047 Sepsis Diseases 0.000 claims abstract description 61
- 208000004852 Lung Injury Diseases 0.000 claims abstract description 22
- 206010069363 Traumatic lung injury Diseases 0.000 claims abstract description 22
- 231100000515 lung injury Toxicity 0.000 claims abstract description 22
- 239000003814 drug Substances 0.000 claims abstract description 15
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 239000007928 intraperitoneal injection Substances 0.000 claims description 19
- 230000003472 neutralizing effect Effects 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- 229940079593 drug Drugs 0.000 claims description 5
- 238000009472 formulation Methods 0.000 claims description 3
- 238000001990 intravenous administration Methods 0.000 claims description 3
- 210000000440 neutrophil Anatomy 0.000 abstract description 28
- 230000000694 effects Effects 0.000 abstract description 22
- 230000004083 survival effect Effects 0.000 abstract description 13
- 230000001976 improved effect Effects 0.000 abstract description 10
- 230000004199 lung function Effects 0.000 abstract description 7
- 101800001848 BDNF precursor form Proteins 0.000 abstract description 6
- 102400001309 BDNF precursor form Human genes 0.000 abstract description 6
- 230000036737 immune function Effects 0.000 abstract description 6
- 230000035790 physiological processes and functions Effects 0.000 abstract description 6
- 230000005764 inhibitory process Effects 0.000 abstract description 3
- 210000004072 lung Anatomy 0.000 description 55
- 241000699670 Mus sp. Species 0.000 description 53
- 210000001519 tissue Anatomy 0.000 description 50
- 208000037487 Endotoxemia Diseases 0.000 description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 13
- 238000010172 mouse model Methods 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 230000002757 inflammatory effect Effects 0.000 description 12
- 238000001514 detection method Methods 0.000 description 11
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 10
- 201000000028 adult respiratory distress syndrome Diseases 0.000 description 10
- 239000002158 endotoxin Substances 0.000 description 10
- 229920006008 lipopolysaccharide Polymers 0.000 description 10
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 230000034994 death Effects 0.000 description 9
- 206010057249 Phagocytosis Diseases 0.000 description 8
- 230000008782 phagocytosis Effects 0.000 description 8
- 238000011282 treatment Methods 0.000 description 8
- 230000006870 function Effects 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 238000005520 cutting process Methods 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 230000008595 infiltration Effects 0.000 description 6
- 238000001764 infiltration Methods 0.000 description 6
- 230000000242 pagocytic effect Effects 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 210000000683 abdominal cavity Anatomy 0.000 description 5
- 206010069351 acute lung injury Diseases 0.000 description 5
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 5
- 230000028709 inflammatory response Effects 0.000 description 5
- 230000003902 lesion Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000012188 paraffin wax Substances 0.000 description 5
- 244000052769 pathogen Species 0.000 description 5
- 229960001412 pentobarbital Drugs 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 210000003437 trachea Anatomy 0.000 description 5
- 206010061218 Inflammation Diseases 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 210000002216 heart Anatomy 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 239000002504 physiological saline solution Substances 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 229920000742 Cotton Polymers 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 206010040070 Septic Shock Diseases 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 210000001015 abdomen Anatomy 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 210000004969 inflammatory cell Anatomy 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000008816 organ damage Effects 0.000 description 3
- 230000002685 pulmonary effect Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 230000036303 septic shock Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 210000000115 thoracic cavity Anatomy 0.000 description 3
- 210000000779 thoracic wall Anatomy 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 208000014644 Brain disease Diseases 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 208000032274 Encephalopathy Diseases 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 206010021143 Hypoxia Diseases 0.000 description 2
- 102000004889 Interleukin-6 Human genes 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 208000010718 Multiple Organ Failure Diseases 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 206010053159 Organ failure Diseases 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 2
- 208000004756 Respiratory Insufficiency Diseases 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000000038 chest Anatomy 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000006735 deficit Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- HJUFTIJOISQSKQ-UHFFFAOYSA-N fenoxycarb Chemical compound C1=CC(OCCNC(=O)OCC)=CC=C1OC1=CC=CC=C1 HJUFTIJOISQSKQ-UHFFFAOYSA-N 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 230000007954 hypoxia Effects 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 230000006996 mental state Effects 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 208000029744 multiple organ dysfunction syndrome Diseases 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000004768 organ dysfunction Effects 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 210000005084 renal tissue Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 201000004193 respiratory failure Diseases 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000008719 thickening Effects 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 239000008096 xylene Substances 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- JRBJSXQPQWSCCF-UHFFFAOYSA-N 3,3'-Dimethoxybenzidine Chemical compound C1=C(N)C(OC)=CC(C=2C=C(OC)C(N)=CC=2)=C1 JRBJSXQPQWSCCF-UHFFFAOYSA-N 0.000 description 1
- 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
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 241000004176 Alphacoronavirus Species 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 1
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 1
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 1
- 244000306301 Caesalpinia sappan Species 0.000 description 1
- 235000015162 Caesalpinia sappan Nutrition 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000628997 Flos Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 206010024642 Listless Diseases 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 208000034486 Multi-organ failure Diseases 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007339 Nerve Growth Factor Receptors Human genes 0.000 description 1
- 108010032605 Nerve Growth Factor Receptors Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 208000001388 Opportunistic Infections Diseases 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 101800004937 Protein C Proteins 0.000 description 1
- 102000017975 Protein C Human genes 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 240000006413 Prunus persica var. persica Species 0.000 description 1
- 206010037423 Pulmonary oedema Diseases 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 206010061481 Renal injury Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 101800001700 Saposin-D Proteins 0.000 description 1
- 206010051379 Systemic Inflammatory Response Syndrome Diseases 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 210000002821 alveolar epithelial cell Anatomy 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 1
- 210000001043 capillary endothelial cell Anatomy 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 230000001447 compensatory effect Effects 0.000 description 1
- 230000004154 complement system Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000003831 deregulation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 210000005069 ears Anatomy 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
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000000744 eyelid Anatomy 0.000 description 1
- 238000000556 factor analysis Methods 0.000 description 1
- 210000003195 fascia Anatomy 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 210000005161 hepatic lobe Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000003960 inflammatory cascade Effects 0.000 description 1
- 208000037806 kidney injury Diseases 0.000 description 1
- 238000004898 kneading Methods 0.000 description 1
- 210000005240 left ventricle Anatomy 0.000 description 1
- 208000017971 listlessness Diseases 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000008383 multiple organ dysfunction Effects 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 208000037891 myocardial injury Diseases 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000031990 negative regulation of inflammatory response Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000019581 neuron apoptotic process Effects 0.000 description 1
- 230000000508 neurotrophic effect Effects 0.000 description 1
- 239000003900 neurotrophic factor Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 229960000856 protein c Drugs 0.000 description 1
- 208000005333 pulmonary edema Diseases 0.000 description 1
- 230000009325 pulmonary function Effects 0.000 description 1
- 210000004879 pulmonary tissue Anatomy 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 231100000272 reduced body weight Toxicity 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 210000001562 sternum Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000000475 sunscreen effect Effects 0.000 description 1
- 239000000516 sunscreening agent Substances 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 238000011870 unpaired t-test Methods 0.000 description 1
- 230000008728 vascular permeability Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pulmonology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明公开了一种人源抗proBDNF单克隆抗体的应用,用于制备治疗脓毒症以及脓毒症所致的肺损伤的药物或制剂。本发明中抗proBDNF单克隆抗体主要发挥维持中性粒细胞的免疫功能稳态的作用,通过抗proBDNF单克隆抗体中和proBDNF,可以改善脓毒症以及脓毒症所致的肺损伤模型小鼠的肺功能、提高脓毒症模型小鼠的生存率。ProBDNF几乎无生理功能,且生理状态下含量极低,proBDNF单克隆抗体的副作用很小,即生物安全性高,同时半衰期长,从而达到免疫功能亢进和免疫功能抑制的阴阳平衡。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种人源抗proBDNF单克隆抗体用于制备治疗对脓毒症及脓毒症所致的肺损伤的药物或制剂的应用。
背景技术
冠状病毒病(COVID-19),重症患者会出现呼吸衰竭、急性呼吸窘迫综合征(acuterespiratory distress syndrome,ARDS)、脓毒症休克、脓毒症合并其他器官功能衰竭,死亡病例高达670万人。脓毒症可导致肺损伤、肝损伤、肾脏损伤、心肌损伤和脓毒症脑病等多器官功能障碍,是重症监护病房患者死亡的首位原因。脓毒症累及肺脏会发展为急性肺损伤(Acutelung injury,ALI)或ARDS。约40%脓毒症患者会发生ALI,是ICU常见危重症之一,常表现为顽固性低氧血症、弥漫性双肺炎症浸润、肺水肿等。如果ALI得不到有效控制而发展为ARDS,进而出现呼吸功能衰竭和死亡。脓毒症肺损伤病理特征表现为肺泡上皮细胞和毛细血管内皮细胞受损,炎性细胞浸润,肺间质充血水肿,弥漫性肺部炎症,肺血管通透性增加、参与通气的肺组织减少。其临床特征为低氧血症,双肺透光度降低,肺内分流和生理无效腔增加,肺顺应性降低。早在2006年JAMA综述指出,机体经历重大创伤后,早期促炎介质大量释放、微循环紊乱等,而后期则伴随发生代偿性的抗炎反应综合征,导致机会感染风险增加、脓毒症肺损伤、器官功能障碍甚至死亡。
目前关于治疗脓毒症及脓毒症所致肺损伤的研究方兴未艾,针对病原相关分子模式(PAMPs)、组织损伤相关分子模式(DAMPs)、补体系统或者单核细胞、中性粒细胞激活和趋化的动物研究或者观察性研究广泛开展,并证实可以一定程度上改善患者炎症反应水平/肺损伤。研究表明,抗生素治疗、复苏策略、血糖水平管理和呼吸机使用仍然是对抗该疾病的唯一经过验证的行动,然而,人口老龄化和抗生素耐药细菌的出现带来了新的挑战。初始高免疫反应与ARDS患者的免疫抑制和死亡率之间的联系尚不清楚。既往专注于高炎症状态阶段的治疗包括抗TLR4、重组活化蛋白C和抗TNF在临床试验中的失败证明以上说法,因为炎症是对抗感染的必要过程。此外,大量研究充分证明,脓毒症所致肺损伤病程的主要驱动因素是宿主对感染的反应而不是入侵生物体本身。因而需要重新思考我们目前对脓毒症所致肺损伤病理生理学的理解。不仅专注于脓毒症所致肺损伤早期高炎症状态时病原体的清除,而且注重患者进入免疫抑制状态的契机及期间各免疫细胞状态的改变。
ALI/ARDS是脓毒症所致全身炎症反应综合症在肺部的表现,病理过程是大量中性粒细胞浸润和炎症介质释放,通过激活巨噬细胞/单核细胞分泌炎症细胞因子来扩增炎症级联。其中,肺部中性粒细胞异常活化和募集是其脓毒症肺损伤病理机制的核心环节。炎症组织中过度浸润和活化的中性粒细胞不仅清除病原物还导致周围组织损伤和不可控的炎症,最终导致器官衰竭和死亡。因此,肺组织炎症损伤部位中性粒细胞及时凋亡并被清除在调控炎症消退过程中发挥关键性作用。此外,中性粒细胞抗原递呈的变化不仅可以作为脓毒症肺损伤发生的生物标记物,同时还可用于预判脓毒症肺损伤患者的转归。以上研究提示,精准靶向调节中性粒细胞功能稳态,是治疗脓毒症以及脓毒症所致肺损伤的关键。
研究表明脑源性神经营养因子前体(pro brain-derived neurotrophicfactor,proBDNF)通过结合其高亲和力受体总神经营养因子受体p75(neurotrophic pan receptorp75,p75NTR)激活下游信号通路,促进神经元的凋亡以及负性调控神经突触可塑性等生物学效应。我们团队一系列研究揭示了病理状态下,多种免疫细胞高表达proBDNF,并通过自分泌或者旁分泌的形式分泌proBDNF扰乱免疫炎症微环境,加重器官损伤甚至衰竭。前期研究已证实,在腹腔注射脂多糖(LPS)诱导的脓毒症模型小鼠,肠系膜淋巴结髓质T细胞高表达proBDNF。此外,外周血免疫细胞分泌proBDNF并下调脑膜CD4+T细胞的抗炎功能,从而促进脓毒症脑病进程。
基于此,本发明提供一种人源抗proBDNF单克隆抗体的应用。
发明内容
为解决现有技术存在的缺陷,本发明提供一种人源抗proBDNF单克隆抗体的应用。
为了解决上述技术问题,本发明提供了如下的技术方案:
本发明提供一种人源抗proBDNF单克隆抗体的应用,用于制备治疗脓毒症以及脓毒症所致肺损伤的药物或制剂。
优选的,所述人源抗proBDNF单克隆抗体用于中和proBDNF。
优选的,所述药物或制剂通过静脉或腹腔注射方式给药。
本发明相较于现有技术,具有以下有益效果:
本发明中抗proBDNF单克隆抗体主要发挥维持中性粒细胞的免疫功能稳态的作用,通过人源抗proBDNF单克隆抗体中和proBDNF,可以改善脓毒症所致模型小鼠肺功能,提高脓毒症模型小鼠的生存率。由于proBDNF几乎无生理功能,且生理状态下含量极低,proBDNF单克隆抗体的副作用很小,即生物安全性高,同时半衰期长,从而达到免疫功能亢进和免疫功能抑制的阴阳平衡,从而实现精准治疗脓毒症肺损伤。
附图说明
图1是本发明ProBDNF在肺组织中性粒细胞的表达示意图;
图2是本发明腹腔注射人源抗proBDNF单克隆抗体改善内毒素血症模型小鼠生存率的示意图;
图3是本发明中和proBDNF对内毒素血症模型小鼠肺脏病变的影响的示意图;
图4是本发明ProBDNF对内毒素血症模型小鼠肺脏中性粒细胞吞噬功能的影响的示意图。
具体实施方式
以下对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
本实施例提供一种人源抗proBDNF单克隆抗体的应用,用于制备治疗脓毒症肺损伤的药物或制剂。所述人源抗proBDNF单克隆抗体用于中和proBDNF。优选的,所述药物或制剂通过静脉或腹腔注射方式给药。
一、试验对象
野生型C57BL/6J小鼠(wild type,WT),8周,体重20-25g,雄性,由中南大学实验动物学部提供。小鼠均饲养在中南大学实验动物学部,通风良好,室温26℃,12:12光照:黑暗循环,相对湿度55%。动物实验均由中南大学湘雅二医院实验动物伦理委员会批准(批准文号:2018334),按照国家卫生研究院《实验动物护理和使用指南》(国家卫生研究院出版物第8023号,1978年校改)进行,并遵循美国国家卫生研究院实验动物的护理和使用指南。尽一切努力减少使用的小鼠数量。实验动物许可证号:SYXK(湘)2012-003。
二、内毒素血症小鼠模型构建
(1)模型构建:8周大的C57雄性小鼠随机分为两组,采用腹腔注射(20mg/kg,intraperitoneal injection,i.p.)LPS(from E.coli 055:B5,L2880,Sigma,St.Louis,MO,USA)建立脓毒症小鼠模型,对照组小鼠腹腔注射同体积的生理盐水。各组小鼠在无病原体条件下自由进食和饮水。注射药物溶液不能有溢出,且注射速度要快,腹腔注射时要回抽,以免打入血管或者肠道内。
(2)生存率评估:从模型构建后开始,每日双盲法观察并记录小鼠的生命体征及生存情况,包括活动、进食、体重和精神状态变化,第1-3天内每3-4小时观察一次,之后每12小时观察一次连续观察7天,至小鼠状态好转不出现死亡为止。
三、内毒素血症模型小鼠肺组织proBDNF的检测
组织的获取:腹腔注射1%戊巴比妥麻醉小鼠,将小鼠置于冰上的解剖盘中,用手术剪迅速剪开处于深度麻醉状态的小鼠的腹腔打开胸腔,沿腹白线将小鼠腹部和胸部剪开,剪至胸腔完全暴露,将两侧隔膜剪开,用止血钳拉开左侧胸骨,暴露心脏部位针从心尖插入到左心室,剪开右心耳,打开蠕动泵,用40r/min的速度对小鼠灌注预冷的生理盐水约50mL,用手术镊托住心脏,将灌注针插入小鼠心尖,剪开小鼠肝叶,使血液可以从肝脏流出,待小鼠肝脏颜色由深红色变为桃黄色,提示小鼠全身灌注完成。然后灌注提前预冷的4%多聚甲醛直到小鼠抽搐消失,身体的四肢僵硬。分离获取心脏、肝脏、肺脏和肾脏组织在4%多聚甲醛中固定,浸泡过夜,用于后期组织学染色。
染色前准备:送病理科进行石蜡包埋后按最大面进行石蜡切片,厚度为5微米。石蜡切片在65℃的烤箱中烘烤两小时,待其冷却后放入二甲苯溶液中10分钟进行脱蜡后,分别放入无水乙醇、95%乙醇和75%乙醇溶液各5分钟。切片取出后将其放置到柠檬酸抗原修复液高压上汽修复5分钟,自然冷却到室温后加入PBS清洗切片3次,每次5分钟。
免疫荧光染色:将切片擦干,注意避开组织,再用阻水笔围绕组织画圈,圈可稍微画大。滴加封闭液(10%驴血清)到切片上的组织中,再将切片放入密闭反应盒内置室温下封闭1小时;封闭结束后将配置好的一抗(1:200,anti-Ly6G;anti-proBDNF antibody,1:200)加入到切片上的组织中,置于4℃冰箱过夜。次日取出反应盒待恢复至室温后,用PBS清洗切片,10分钟×3次。次日取出反应盒待恢复至室温后,用PBS清洗切片,10分钟×3次,PBS稀释荧光二抗(Goat Anti-Rabbit(488),1:500for proBDNF;Goat Anti-Rat(594),1:500for Ly6G)并滴加到切片的组织上,再将切片放入密闭反应盒内置室温下反应1小时,PBS清洗切片10分钟×3次,滴加DAPI(H-1200,Vector lab)封片后通过荧光显微镜(NikonECLIPSE 80i,Nikon Corporation,Tokyo,Japan)观察。
四、人源抗proBDNF单克隆抗体干预对内毒素血症模型小鼠生存率的影响
(1)模型构建:8周大的C57雄性小鼠随机分为两组,采用腹腔注射(20mg/kg,i.p.)LPS建立脓毒症小鼠模型,对照组小鼠腹腔注射同体积的生理盐水。各组小鼠在无病原体条件下自由进食和饮水。注射药物溶液不能有溢出,且注射速度要快,腹腔注射时要回抽,以免打入血管或者肠道内。
(2)proBDNF中和抗体干预:在给予LPS前30分钟,腹腔注射100μg人源抗proBDNF单克隆抗体(McAb-proB)或同型对照IgG(catalog no.AT1596,CMCTAG)进行干预治疗。每日双盲法观察并记录小鼠的生命体征及生存情况,包括活动、进食、体重和精神状态变化,第1-3天内每3-4小时观察一次,之后每12小时观察一次连续观察7天,至小鼠状态好转不出现死亡为止。
四、McAb-proB干预对内毒素血症模型小鼠肺组织炎症反应的影响
染色前准备:按最大面进行石蜡切片,厚度为5微米。石蜡切片在65℃的烤箱中烘烤两小时,待其冷却后放入二甲苯溶液中10分钟进行脱蜡后,分别放入无水乙醇、95%乙醇和75%乙醇溶液各5分钟。切片取出后将其放置到柠檬酸抗原修复液高压上汽修复5分钟,自然冷却到室温后加入PBS清洗切片3次,每次5分钟。
HE染色:采用HE染色试剂盒(Vector Laboratories,catalog:H-3502)进行染色。步骤为先把切片取出后滴加苏木苏染色液,显微镜下观察到蓝色细胞核后,水洗返蓝,若细胞核染色太深可用1%盐酸酒精反色。滴加伊红染色液染细胞质。待切片完全干透后,进行脱水、透明、封片和镜检(Nikon ECLIPSE 80i,Nikon Corporation,Japan)拍照。
五、McAb-proB干预对内毒素血症模型小鼠肺组织炎症因子的影响
生存率实验观察完毕后,将各组小鼠同时间取材。小鼠腹腔注射10%水合氯醛深度麻醉,脱颈处死小鼠,立即取出心脏、肝脏、肺脏和肾脏组织,放入标记好的EP管中,迅速放入液氮中暂存,随后置于-80℃冰箱中。
具体实验步骤如下:
1)离心机开机,4℃预冷。
2)组织称重后,每个组织按100mg的组织/1mL的比例加入Trizol提取液,匀浆机匀浆1分钟,冰上静置10分钟。
3)EP管中加入200μl氯仿,充分混匀15s,室温静置10分钟待其分层。
4)4℃条件下12000rpm,离心10分钟。
5)从EP管中吸取上层水相至新的EP管中,每管加入等体积的异丙醇,上下颠倒混匀15s,-20℃冰箱中沉淀1h。
6)4℃条件下12000rpm,离心10分钟。
7)弃上清液得到RNA沉淀,加入1ml预冷的75%乙醇清洗沉淀,4℃条件下8000rpm,离心5分钟,弃上清。将离心管倒扣置晾干获得RNA沉淀。
8)离心管中加入20μl的DEPC水溶解RNA沉淀,放入65℃的反应5分钟。
9)用DEPC水作为本底,吸取1μl的RNA在酶标仪(BioTek Instruments,Inc,USA)中检测RNA浓度。
10)EP管中加入1μl的Oligod(T)、2μg total RNA、补足RNA酶free水至12μl。
11)每个样品中加入8μl反应体系(试剂盒反应体系见下表),震荡混匀离心后在37℃水浴箱中反应30分钟,85℃水浴箱中反应5分钟使逆转录酶失活后得到cDNA溶液。
12)引物设计,在pubmed数据库上设计相关指标的引物序列。
13)使用BioRad公司mix染液进行实时定量PCR反应。反应体系见下表:
试剂 | 体积/μl |
Supermix(2×) | 5 |
反式引物(10μM) | 0.2 |
顺式引物(10μM) | 0.2 |
去RNA酶水 | 3.6 |
cDNA | 1 |
总体积 | 10 |
14)每个样品同时做检测指标IL-1β(其上游引物序列如SEQ ID NO:3所示,其下游引物序列如SEQ ID NO:4所示)、IL-6(其上游引物序列如SEQ ID NO:5所示,其下游引物序列如SEQ ID NO:6所示)、TNF-α(其上游引物序列如SEQ ID NO:7所示,其下游引物序列如SEQ ID NO:8所示)与GAPDH(其上游引物序列如SEQ ID NO:1所示,其下游引物序列如SEQID NO:2所示),做2个复孔。按照mix说明书设定PCR仪(CFX96,Eppendorf)的反应步骤:95℃/5分钟,95℃/10秒,60℃/30秒(40个循环)。反应终止后导出Ct值的excel表,目的基因表达用GAPDH校正,利用2-△△CT计算目的基因的mRNA相对表达水平。
六、McAb-proB干预对内毒素血症模型小鼠肺功能的影响
(1)肺组织干湿比重:小鼠腹腔注射戊巴比妥(70mg/kg)深度麻醉,将小鼠置于冰盘上的解剖盘中,用手术剪迅速剪沿腋前线剪断肋骨并将胸前壁向上翻折暴露胸腔,沿气管走行分离肺组织。立即在微量电子秤上称量并记录肺组织重量(湿重,Wet),接着转入60摄氏度烤箱内放置72h。72h后重新称量肺组织的体重(干重,Dry),计算湿重/干重(Wet/dry)并采用prism 8.0统计分析。
(2)肺组织MPO活性检测:小鼠腹腔注射戊巴比妥(70mg/kg)深度麻醉,将小鼠置于冰盘上的解剖盘中,用手术剪迅速沿腋前线剪断肋骨并将胸前壁向上翻折暴露胸腔,沿气管走行分离肺组织。称量10mg肺组织并加入1mL pH 6.0预冷的十六烷基三甲基溴化铵缓冲液(50mM KPO4,0.5%hexadecyltrimethylammonium bromide),匀浆机4℃匀浆10秒。随后4℃条件下14000g离心30分钟,取上清液(20μL)转移到96孔板中。然后立即向96孔板中加入200μL 37℃盐酸邻联茴香胺溶液(16.7mg O-联茴香胺,100ml:90%水和10%50mM KPO4+0.0005%H2O2),最后在450nm处读取光密度OD值(BioTek Instruments,Inc,美国)。
(3)肺泡灌洗液(bronchoalveolar lavage fluid,BALF)蛋白浓度检测:腹腔注射1%戊巴比妥麻醉小鼠,将小鼠置于冰盒上的解剖盘中,固定四肢,将头部用牙线或者组织钳固定在正位,用酒精擦拭颈部和腹部皮肤,轻轻提起两侧耳下缘连线处皮肤,沿腹白线剪开,逐层分离皮肤、筋膜和肌层,显露气管后,将棉线穿过气管下方,将注射器针头穿透气管并沿气管方向向内推进,系上棉线以固定针尖和减少向上反流,然后向气管内慢慢注入0.5mL生理盐水,轻柔揉捏小鼠肺部片刻后,抽回液体,放松棉线并拔出针尖。以上步骤重复3次,将3次收集的液体注入2mL EP管中待用。将收集获得的BALF进行离心,5000rpm,10min。采用蛋白检测试剂盒(Invent Biotechnologies Inc.,catalog:SD-001)并参照试剂盒说明书对脓毒症小鼠BALF上清液的总蛋白水平进行检测。
七、ProBDNF干预对内毒素血症模型小鼠肺组织中性粒细胞吞噬功能的影响
(1)小鼠肺组织单个细胞悬液制备:小鼠腹腔注射戊巴比妥(70mg/kg)深度麻醉,将小鼠置于冰盘上的解剖盘中,用手术剪迅速剪沿腋前线剪断肋骨并将胸前壁向上翻折暴露胸腔,沿气管走行分离肺组织。剪刀剪碎肺组织,越小越好,在加入500μL预冷的PBS缓冲溶液,轻揉研磨剪碎的肺组织。在加入3mL PBS,充分混匀细胞悬液,并用40μm晒目过滤获取肺组织单细胞悬液。体外加入proBDNF重组蛋白或中和抗体处理6h。6h后离心弃上清待吞噬染色。
(2)吞噬能力检测:采用Invitrogen pHrodoTM Green ZymosanBioparticlesTMConjugate for Phagocytosis Assay Kit,Catalog Number:P35365商业化试剂盒进行检测。首先将1mg的干粉用PBS溶解成0.5mg/mL。再将100μL的培养基换成吞噬试剂,添加到阳性对照,实验组,和阴性(无细胞)对照组。将培养板盖好,移入37℃细胞培养箱,反应2小时。最后,待反应时间终止后,即刻用流式细胞仪进行检测。
八、数据收集与统计分析
组间两两比较采用未配对t检验,多组间比较采用单因素方差分析。所有数据均有平均值±标准误(Mean±SEM)表示。采用Graphpad Prism 8.3.5软件(SanFrancisco,CA)和23.0版本的IBM SPSS统计软件(IBM,San Francisco,CA)对实验数据进行统计学分析,P值小于0.05代表差异具有统计学意义。
九、实验结果
1.内毒素血症模型小鼠肺组织中性粒细胞上proBDNF表达增加。
腹腔注射LPS诱导脓毒症模型,24h和48h后收集对照组小鼠(Con)和LPS组小鼠肺组织,采用组织学染色首先明确肺组织proBDNF的表达变化及来源。结果显示,肺组织proBDNF的表达明显增加并与中性粒细胞共定位(图1)。图1表示ProBDNF在肺组织中性粒细胞的表达。与Con相比,免疫荧光染色检测发现内毒素血症模型小鼠肺组织Ly6G+中性粒细胞与proBDNF共定位。Scale bar=20μm。
2.人源抗proBDNF单克隆抗体干预改善内毒素血症模型小鼠生存率。
采用课题组自主研发的人源化mAb-proB干预LPS诱导的脓毒症模型小鼠。双盲法观察并统计mAb-proB干预组和Con组小鼠的生存率。在腹腔注射LPS提前30分钟(-30min),分别给予脓毒症模型小鼠腹腔注射100mg mAb-proB或同型对照IgG(图2)。结果发现,腹腔注射LPS后两组小鼠均出现精神萎靡,活动减少,行动迟缓,饮食饮水减少,体重下降,出现寒颤,眼睑部有黏稠分泌物,肛门处有拉稀样粪便。腹腔注射Ab-proB干预组脓毒症模型小鼠均早于IgG组开始好转且生存率均高于IgG组,具有统计学意义(P<0.01)(图2)。图2表示腹腔注射人源抗proBDNF单克隆抗体改善内毒素血症模型小鼠生存率。n=10/组,**P<0.01。
3、McAb-proB干预对内毒素血症模型小鼠肺组织的影响。
接着,我们观察了mAb-proB抗体对脓毒症模型小鼠肺脏的影响,我们收集了对照组和干预组小鼠肺脏和BALF探究proBDNF对于脓毒症所致肺功能损伤的作用。通过HE染色、RT-qPCR检测肺脏组织的炎性病变,测量肺组织的湿/干重比和MPO活性检测,以及BALF总蛋白定量检测,评估mAb-proB干预对脓毒症肺损伤模型小鼠肺功能损伤的影响。HE染色显示腹腔注射mAb-proB干预显著改善模型小鼠肺脏组织病变(图3A),IgG组表现出大量炎性细胞浸润,肺泡壁明显增厚,可见肺泡腔内红细胞,而mAb-proB干预组炎性细胞浸润及肺泡壁增厚情况均相对较轻,且肺脏损伤评分显著低于IgG组(图3A),伴随肺组织中性粒细胞数减少(图3B)。炎症因子IL-1β、TNF-α和IL-6的转录水平显著降低,均具有统计学差异(图3C-E)。与IgG组相比,mAb-proB干预组模型小鼠肺组织的湿/干重比(图3F)、MPO活性(图3G)和BALF中的总蛋白浓度(图3H)均显著下调。提示,腹腔注射mAb-proB抗体改善脓毒症模型小鼠肺功能损伤以及肺组织炎症反应。
图3表示中和proBDNF对内毒素血症模型小鼠肺脏病变的影响。模型小鼠取BALF和肺组织,评估肺组织的炎症水平和功能改变。模型小鼠肺组织炎症水平检测:肺组织的HE染色(A)和中性粒细胞免疫组织化学染色(B)。炎症因子转录水平检测(C-E)。模型小鼠肺功能检测:肺组织的湿/干重比(F)、肺组织MPO活性(G)以及BALF总蛋白量(H)。Scale Bar=100μm(A图),ScaleBar=20μm(B图),n=5/组。
4、ProBDNF干预对内毒素血症模型小鼠肺组织中性粒细胞吞噬功能的影响。
最后,我们观察了proBDNF干预对脓毒症模型小鼠肺脏中性粒细胞吞噬功能的影响,我们收集了内毒素血症模型组小鼠肺脏中性粒细胞,体外加入proBDNF重组蛋白和mAb-proB进行干预。结果发现,proBDNF重组蛋白处理抑制中性粒细胞吞噬功能,而拮抗proBDNF后可以增强中性粒细胞吞噬功能。
图4表示ProBDNF对内毒素血症模型小鼠肺脏中性粒细胞吞噬功能的影响。ProBDNF重组蛋白处理抑制中性粒细胞吞噬功能,而拮抗proBDNF后可以逆转吞噬功能的下降,n=5/组,*P<0.05,***P<0.001。
在全球,COVID-19患者合并ARDS发生的患者的总体死亡率高达39%。COVID-19相关性ARDS患者预后极差(即呼吸机脱机天数、重症监护病房住院时长或医院住院时长,以及死亡),其主要死亡原因包括脓毒症或脓毒症休克、脓毒症相关性多器官衰竭。脓毒症是由宿主对感染的反应失调引起的危及生命的器官功能障碍,是创伤、烧伤和感染等临床急危重患者的严重并发症之一,也是诱发脓毒症休克、多器官功能障碍综合征的重要原因。肺是脓毒症患者受影响最严重的器官之一,脓毒症合并ARDS发生率和死亡率高达40%以上。到目前为止,尚缺乏有效的脓毒症以及脓毒症所致靶器官损伤的治疗方法。因此,评估与脓毒症以及脓毒症所致的靶器官损伤相关的生物标志物并探究脓毒症的治疗策略具有重要意义。我们的实验结果发现脓毒症模型小鼠中性粒细胞来源的proBDNF显著增加,拮抗proBDNF可改善模型小鼠肺组织炎症反应、中性粒细胞吞噬功能和肺功能,进而提高脓毒症模型小鼠的生存率。
明确自主研发的人源抗proBDNF单抗作为防治脓毒症以及脓毒症所致肺损伤新型药物的临床转化价值。由于proBDNF几乎无生理功能,且生理状态下含量极低,因此我们可以预见抗proBDNF单抗的副作用很小,即生物安全性高,同时半衰期长;另一方面,脓毒症适当的炎症反应有利于坏死组织的吸收、防治感染,故维持脓毒症感染患者炎症稳态是关键。我们的实验结果发现,抗proBDNF单抗主要发挥维持中性粒细胞的免疫功能稳态的作用。从这点提示,阻断proBDNF类似于免疫修饰疗法,达到“免疫功能亢进和免疫功能抑制”的阴阳平衡,从而达到精准治疗脓毒症以及脓毒症所致的靶器官损伤。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (3)
1.人源抗proBDNF单克隆抗体的应用,其特征在于,用于制备治疗脓毒症以及脓毒症所致肺损伤的药物或制剂。
2.根据权利要求1所述的人源抗proBDNF单克隆抗体的应用,其特征在于,所述人源抗proBDNF单克隆抗体用于中和proBDNF。
3.根据权利要求1所述的人源抗proBDNF单克隆抗体的应用,其特征在于,所述药物或制剂通过静脉或腹腔注射方式给药。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310131390.5A CN116421718A (zh) | 2023-02-17 | 2023-02-17 | 人源抗proBDNF单克隆抗体的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310131390.5A CN116421718A (zh) | 2023-02-17 | 2023-02-17 | 人源抗proBDNF单克隆抗体的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116421718A true CN116421718A (zh) | 2023-07-14 |
Family
ID=87091453
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310131390.5A Pending CN116421718A (zh) | 2023-02-17 | 2023-02-17 | 人源抗proBDNF单克隆抗体的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116421718A (zh) |
-
2023
- 2023-02-17 CN CN202310131390.5A patent/CN116421718A/zh active Pending
Non-Patent Citations (3)
Title |
---|
RU-YI LUO 等: "ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells", JOURNAL OF NEUROINFLAMMATION, vol. 17, pages 6 - 8 * |
WEI-YUN SHEN 等: "Up-regulation of proBDNF/p75NTR signaling in antibody-secreting cells drives systemic lupus erythematosus", SCI. ADV., vol. 8, pages 1 - 14 * |
ZHE WANG等: "Upregulation of proBDNF in the Mesenteric Lymph Nodes in Septic Mice", NEUROTOXICITY RESEARCH, pages 7 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Dai et al. | Negative regulation of PI3K/AKT/mTOR axis regulates fibroblast proliferation, apoptosis and autophagy play a vital role in triptolide-induced epidural fibrosis reduction | |
Yu et al. | The modulation of endometriosis by lncRNA MALAT1 via NF-κB/iNOS. | |
WO2016184427A1 (zh) | 低氧处理的间充质干细胞及其应用 | |
WO2020030097A1 (zh) | 促进细胞生长和组织修复的方法及组合物 | |
CN112121042B (zh) | Pso用于制备抗败血症及其诱发的心肌损伤药物的应用 | |
CN108864311A (zh) | 一种抑制md2与cirp蛋白结合的短肽及其应用 | |
Zhu et al. | Autologous blood transfusion stimulates wound healing in diabetic mice through activation of the HIF‐1α pathway by improving the blood preservation solution | |
CN102552935B (zh) | 肝细胞核因子1α治疗慢性肝病的用途 | |
CN110755450B (zh) | 间充质干细胞来源的细胞外囊泡在治疗蛛网膜下腔出血中的应用 | |
WO2020030091A1 (zh) | 用于治疗组织坏死或改善心脏功能的药物 | |
CN116421718A (zh) | 人源抗proBDNF单克隆抗体的应用 | |
RU2309744C1 (ru) | Способ лечения послеродового эндометрита | |
CN111481535B (zh) | Idhp用于制备抗败血症及其诱发的心肌损伤药物的应用 | |
CN108685906A (zh) | 小分子化合物p7c3的新应用 | |
CN107625781B (zh) | miRNA抑制子在制备防治心肌梗死药物中的应用 | |
CN114470196B (zh) | CCL3/CCR4中和抗体、NF-κB抑制剂在制备NEC治疗药物中的用途 | |
CN109730994A (zh) | 组蛋白甲基转移酶ezh2的抑制剂在制备防治腹膜透析后腹膜纤维化的药物中的用途 | |
CN110200976A (zh) | 隐丹参酮在制备促进糖尿病患者创面愈合的药物中的用途 | |
CN113181163B (zh) | 千层纸素a在制备治疗肺纤维化药物中的应用 | |
WO2024045071A1 (zh) | 表皮生长因子在制备治疗间质性膀胱炎的药物的应用 | |
Angelova et al. | Effects of partial liquid ventilation on lipopolysaccharide-induced inflammatory responses in rats | |
CN114560817B (zh) | 一种用于抑制纤维化的小分子药物及其应用 | |
CN115252631B (zh) | 三七提取物在制备治疗糖尿病肾病药物中的应用 | |
CN114796265B (zh) | S-nanoFe用于制备抗败血症及其诱发的心肌损伤药物的应用 | |
CN114533726B (zh) | 一种用于抑制纤维化的小分子药物及其应用 |
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 |