CN117092348A - 多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用 - Google Patents
多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用 Download PDFInfo
- Publication number
- CN117092348A CN117092348A CN202311035071.0A CN202311035071A CN117092348A CN 117092348 A CN117092348 A CN 117092348A CN 202311035071 A CN202311035071 A CN 202311035071A CN 117092348 A CN117092348 A CN 117092348A
- Authority
- CN
- China
- Prior art keywords
- kidney
- transplanted kidney
- transplanted
- acox1
- polyunsaturated fatty
- 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
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 title claims abstract description 62
- 208000001647 Renal Insufficiency Diseases 0.000 title claims abstract description 30
- 201000006370 kidney failure Diseases 0.000 title claims abstract description 30
- 239000003814 drug Substances 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 210000003734 kidney Anatomy 0.000 claims abstract description 110
- 102100026798 Peroxisomal acyl-coenzyme A oxidase 1 Human genes 0.000 claims abstract description 85
- 101000833892 Homo sapiens Peroxisomal acyl-coenzyme A oxidase 1 Proteins 0.000 claims abstract description 83
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims abstract description 41
- 206010016654 Fibrosis Diseases 0.000 claims abstract description 41
- 230000004761 fibrosis Effects 0.000 claims abstract description 41
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims abstract description 40
- 210000002744 extracellular matrix Anatomy 0.000 claims abstract description 40
- 238000007634 remodeling Methods 0.000 claims abstract description 32
- 206010023421 Kidney fibrosis Diseases 0.000 claims abstract description 26
- 101100108090 Caenorhabditis elegans acox-1.1 gene Proteins 0.000 claims abstract description 8
- 230000002265 prevention Effects 0.000 claims abstract description 4
- 230000014509 gene expression Effects 0.000 claims description 72
- 108090000623 proteins and genes Proteins 0.000 claims description 58
- MBMBGCFOFBJSGT-KUBAVDMBSA-N all-cis-docosa-4,7,10,13,16,19-hexaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 claims description 42
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 claims description 39
- 235000020669 docosahexaenoic acid Nutrition 0.000 claims description 23
- 229940090949 docosahexaenoic acid Drugs 0.000 claims description 21
- 235000020661 alpha-linolenic acid Nutrition 0.000 claims description 20
- 229960004488 linolenic acid Drugs 0.000 claims description 20
- YMTINGFKWWXKFG-UHFFFAOYSA-N fenofibrate Chemical compound C1=CC(OC(C)(C)C(=O)OC(C)C)=CC=C1C(=O)C1=CC=C(Cl)C=C1 YMTINGFKWWXKFG-UHFFFAOYSA-N 0.000 claims description 14
- 229960002297 fenofibrate Drugs 0.000 claims description 13
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 238000012216 screening Methods 0.000 claims description 10
- 101150077457 ACOX1 gene Proteins 0.000 claims description 8
- 230000002829 reductive effect Effects 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 230000001737 promoting effect Effects 0.000 claims description 4
- 238000002054 transplantation Methods 0.000 abstract description 21
- 229940079593 drug Drugs 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 7
- 230000007812 deficiency Effects 0.000 abstract description 5
- 101000833887 Yarrowia lipolytica (strain CLIB 122 / E 150) Acyl-coenzyme A oxidase 1 Proteins 0.000 abstract description 2
- 230000008685 targeting Effects 0.000 abstract description 2
- 210000002919 epithelial cell Anatomy 0.000 abstract 1
- 210000000738 kidney tubule Anatomy 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 126
- 210000004027 cell Anatomy 0.000 description 51
- 241000700159 Rattus Species 0.000 description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 22
- 238000000034 method Methods 0.000 description 21
- 210000001519 tissue Anatomy 0.000 description 21
- 239000007788 liquid Substances 0.000 description 20
- 239000003112 inhibitor Substances 0.000 description 19
- 239000002207 metabolite Substances 0.000 description 19
- 238000010186 staining Methods 0.000 description 16
- 239000003153 chemical reaction reagent Substances 0.000 description 15
- 230000003827 upregulation Effects 0.000 description 15
- 238000005406 washing Methods 0.000 description 15
- 230000030279 gene silencing Effects 0.000 description 14
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 12
- 235000020778 linoleic acid Nutrition 0.000 description 12
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 12
- 230000009469 supplementation Effects 0.000 description 12
- 238000003197 gene knockdown Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 10
- 210000004369 blood Anatomy 0.000 description 10
- 239000008280 blood Substances 0.000 description 10
- 238000001514 detection method Methods 0.000 description 10
- 238000011534 incubation Methods 0.000 description 10
- 201000002793 renal fibrosis Diseases 0.000 description 10
- 210000004926 tubular epithelial cell Anatomy 0.000 description 10
- DIEDVCMBPCRJFQ-UHFFFAOYSA-N 10,12-tricosadiynoic acid Chemical compound CCCCCCCCCCC#CC#CCCCCCCCCC(O)=O DIEDVCMBPCRJFQ-UHFFFAOYSA-N 0.000 description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 102000008186 Collagen Human genes 0.000 description 9
- 108010035532 Collagen Proteins 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 102100033010 Integrin beta-5 Human genes 0.000 description 9
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 9
- 229920001436 collagen Polymers 0.000 description 9
- 238000012163 sequencing technique Methods 0.000 description 9
- 230000001502 supplementing effect Effects 0.000 description 9
- 239000008096 xylene Substances 0.000 description 9
- 238000004043 dyeing Methods 0.000 description 8
- 150000002500 ions Chemical class 0.000 description 8
- 108020004999 messenger RNA Proteins 0.000 description 8
- 239000012071 phase Substances 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 210000002254 renal artery Anatomy 0.000 description 8
- 238000012546 transfer Methods 0.000 description 8
- 210000000626 ureter Anatomy 0.000 description 8
- 239000012224 working solution Substances 0.000 description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 7
- 102000004318 Matrilysin Human genes 0.000 description 7
- 108090000855 Matrilysin Proteins 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 7
- 230000003828 downregulation Effects 0.000 description 7
- 239000000975 dye Substances 0.000 description 7
- 239000012188 paraffin wax Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 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 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 230000003176 fibrotic effect Effects 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 150000002632 lipids Chemical class 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 230000037361 pathway Effects 0.000 description 6
- 210000002796 renal vein Anatomy 0.000 description 6
- 101001015059 Homo sapiens Integrin beta-5 Proteins 0.000 description 5
- 101000990912 Homo sapiens Matrilysin Proteins 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 5
- 239000012154 double-distilled water Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 230000002496 gastric effect Effects 0.000 description 5
- 238000003119 immunoblot Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 210000005084 renal tissue Anatomy 0.000 description 5
- 101100328892 Arabidopsis thaliana COL4 gene Proteins 0.000 description 4
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 4
- 102100030417 Matrilysin Human genes 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 101100237842 Xenopus laevis mmp18 gene Proteins 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 230000018044 dehydration Effects 0.000 description 4
- 238000006297 dehydration reaction Methods 0.000 description 4
- 238000010199 gene set enrichment analysis Methods 0.000 description 4
- 239000003292 glue Substances 0.000 description 4
- 229960002897 heparin Drugs 0.000 description 4
- 229920000669 heparin Polymers 0.000 description 4
- 238000003125 immunofluorescent labeling Methods 0.000 description 4
- 238000011065 in-situ storage Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 108010021518 integrin beta5 Proteins 0.000 description 4
- 239000007928 intraperitoneal injection Substances 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 235000020660 omega-3 fatty acid Nutrition 0.000 description 4
- 201000008611 peroxisomal acyl-CoA oxidase deficiency Diseases 0.000 description 4
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- 238000011552 rat model Methods 0.000 description 4
- 238000010839 reverse transcription Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000008399 tap water Substances 0.000 description 4
- 235000020679 tap water Nutrition 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- 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 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000002033 PVDF binder Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 210000001367 artery Anatomy 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 235000014113 dietary fatty acids Nutrition 0.000 description 3
- 238000010201 enrichment analysis Methods 0.000 description 3
- 229930195729 fatty acid Natural products 0.000 description 3
- 239000000194 fatty acid Substances 0.000 description 3
- 150000004665 fatty acids Chemical class 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 210000002216 heart Anatomy 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 230000002055 immunohistochemical effect Effects 0.000 description 3
- 238000011532 immunohistochemical staining Methods 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 235000020665 omega-6 fatty acid Nutrition 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 238000004451 qualitative analysis Methods 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 239000012192 staining solution Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 150000003626 triacylglycerols Chemical class 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
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 101100348341 Caenorhabditis elegans gas-1 gene Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 2
- 101001015064 Homo sapiens Integrin beta-6 Proteins 0.000 description 2
- 101001090047 Homo sapiens Peroxiredoxin-4 Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102100033011 Integrin beta-6 Human genes 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 101100161764 Mus musculus Acoxl gene Proteins 0.000 description 2
- 101100447658 Mus musculus Gas1 gene Proteins 0.000 description 2
- 102100034768 Peroxiredoxin-4 Human genes 0.000 description 2
- 229940122907 Phosphatase inhibitor Drugs 0.000 description 2
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 230000003872 anastomosis Effects 0.000 description 2
- 210000000702 aorta abdominal Anatomy 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 230000001054 cortical effect Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000000151 deposition Methods 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 229960003444 immunosuppressant agent Drugs 0.000 description 2
- 230000001861 immunosuppressant effect Effects 0.000 description 2
- 239000003018 immunosuppressive agent Substances 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 210000000231 kidney cortex Anatomy 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 229960004232 linoleic acid Drugs 0.000 description 2
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 2
- 230000008327 renal blood flow Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- DVSZKTAMJJTWFG-SKCDLICFSA-N (2e,4e,6e,8e,10e,12e)-docosa-2,4,6,8,10,12-hexaenoic acid Chemical compound CCCCCCCCC\C=C\C=C\C=C\C=C\C=C\C=C\C(O)=O DVSZKTAMJJTWFG-SKCDLICFSA-N 0.000 description 1
- GHVNFZFCNZKVNT-KIJKOTCYSA-N 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-nonadecadeuteriodecanoic acid Chemical compound [2H]C([2H])([2H])C([2H])([2H])C([2H])([2H])C([2H])([2H])C([2H])([2H])C([2H])([2H])C([2H])([2H])C([2H])([2H])C([2H])([2H])C(O)=O GHVNFZFCNZKVNT-KIJKOTCYSA-N 0.000 description 1
- TUNFSRHWOTWDNC-KLTYLHELSA-N 2,2-dideuteriotetradecanoic acid Chemical compound OC(=O)C([2H])([2H])CCCCCCCCCCCC TUNFSRHWOTWDNC-KLTYLHELSA-N 0.000 description 1
- 101150033839 4 gene Proteins 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
- GZJLLYHBALOKEX-UHFFFAOYSA-N 6-Ketone, O18-Me-Ussuriedine Natural products CC=CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O GZJLLYHBALOKEX-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 102000005867 Fibrillin-1 Human genes 0.000 description 1
- 108010030229 Fibrillin-1 Proteins 0.000 description 1
- 240000008168 Ficus benjamina Species 0.000 description 1
- 108700042658 GAP-43 Proteins 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
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 241000555745 Sciuridae Species 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000008901 benefit 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
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 238000009835 boiling Methods 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
- 239000007853 buffer solution Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 150000001721 carbon Chemical group 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- DVSZKTAMJJTWFG-UHFFFAOYSA-N docosa-2,4,6,8,10,12-hexaenoic acid Chemical class CCCCCCCCCC=CC=CC=CC=CC=CC=CC(O)=O DVSZKTAMJJTWFG-UHFFFAOYSA-N 0.000 description 1
- KAUVQQXNCKESLC-UHFFFAOYSA-N docosahexaenoic acid (DHA) Natural products COC(=O)C(C)NOCC1=CC=CC=C1 KAUVQQXNCKESLC-UHFFFAOYSA-N 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 238000000132 electrospray ionisation Methods 0.000 description 1
- 208000028208 end stage renal disease Diseases 0.000 description 1
- 201000000523 end stage renal failure Diseases 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
- 235000004626 essential fatty acids Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 235000021323 fish oil Nutrition 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 1
- 235000021388 linseed oil Nutrition 0.000 description 1
- 239000000944 linseed oil Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000002552 multiple reaction monitoring Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000010349 pulsation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005173 quadrupole mass spectroscopy Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- MHQHHBYRYFICDV-UHFFFAOYSA-M sodium;pyrimidin-3-ide-2,4,6-trione Chemical compound [Na+].O=C1CC(=O)[N-]C(=O)N1 MHQHHBYRYFICDV-UHFFFAOYSA-M 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000009966 trimming Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000002618 waking effect Effects 0.000 description 1
- XOSXWYQMOYSSKB-LDKJGXKFSA-L water blue Chemical compound CC1=CC(/C(\C(C=C2)=CC=C2NC(C=C2)=CC=C2S([O-])(=O)=O)=C(\C=C2)/C=C/C\2=N\C(C=C2)=CC=C2S([O-])(=O)=O)=CC(S(O)(=O)=O)=C1N.[Na+].[Na+] XOSXWYQMOYSSKB-LDKJGXKFSA-L 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
-
- 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
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Pathology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- General Physics & Mathematics (AREA)
- Emergency Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供了多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用,属于生物药物技术领域。本发明针对目前未有较好疗效的靶向药物可延缓移植肾纤维化(IF)的进展,提供了多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用,具体通过改善移植肾纤维化水平防治移植肾功能衰竭。以肾移植模型大鼠为研究对象,多不饱和脂肪酸可缓解移植肾肾小管上皮细胞因酰基辅酶A氧化酶1(ACOX1)缺失所致的细胞外基质重塑,来有效改善移植肾的纤维化,从而达到预防和/或治疗移植肾功能衰竭的目的。
Description
技术领域
本发明属于生物药物技术领域,具体涉及多不饱和脂肪酸(PUFA)在制备预防和/或治疗移植肾功能衰竭的药物中的应用。
背景技术
肾移植是终末期肾病的最佳治疗手段。近年来随着医疗水平的提高,包括移植手术技术的发展和新型免疫抑制剂的联合应用,移植肾的短期存活率已得到提高,但长期存活率仍未有明显改善。间质纤维化和小管萎缩(IF/TA)是导致远期移植肾功能衰竭的最主要原因。有研究显示,肾移植3~6月后,约有40%的患者存在移植肾IF,2年以后约有65%的患者存在移植肾IF。目前,仍未有较好疗效的靶向药物可延缓移植肾IF进展,临床上仅能通过合理搭配免疫抑制剂的使用以最大程度上避免药物毒性诱导的损伤,以延长移植肾的存活时间。针对移植肾IF靶向药物的缺乏意味着部分病人在首次移植后的移植肾不可避免的再次功能衰竭,面临再次肾移植,这无疑加重了移植肾患者的经济、心理和生理负担。此外,由于肾源的极度短缺,抑制移植肾IF进展,延长移植肾的工作年限是肾移植领域亟需解决的重要问题。
发明内容
有鉴于此,本发明的目的在于提供ACOX1作为分子靶点在制备、筛选移植肾功能衰竭的药物中的应用。
本发明的目的还在于提供一种多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用,多不饱和脂肪酸能有效阻止细胞外基质重塑,从而降低移植肾纤维化水平。
本发明提供了ACOX1作为分子靶点在制备、筛选移植肾功能衰竭的药物中的应用。
优选的,一种促进ACOX1基因或蛋白表达的试剂在制备、筛选移植肾功能衰竭的药物中的应用。
优选的,所述试剂包括非诺贝特。
本发明提供了多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用。
优选的,所述多不饱和脂肪酸包括亚油酸、α-亚麻酸和二十二碳六烯酸。
优选的,所述亚油酸、α-亚麻酸和二十二碳六烯酸的质量比为(1~2)∶(1~2)∶(1~2)。
优选的,所述亚油酸、α-亚麻酸和二十二碳六烯酸的质量比为1∶1∶1。
优选的,所述移植肾功能衰竭为移植肾间质纤维化。
优选的,所述多不饱和脂肪酸通过阻止细胞外基质重塑来改善移植肾纤维化。
优选的,所述多不饱和脂肪酸抑制ACOX1表达降低导致的移植肾的纤维化。
本发明提供了ACOX1作为分子靶点在制备、筛选移植肾功能衰竭的药物中的应用。实验证明,ACOX1缺失导致多不饱和脂肪酸水平下调和细胞外基质重塑基因表达上调,而多不饱和脂肪酸的回补能够逆转因ACOX1缺失导致的肾纤维化进程和下调细胞外基质重塑基因表达量。而使用非诺贝特能够上调ACOX1表达,从而抑制移植肾纤维化;通过抑制剂使这正常肾脏中ACOX1活性受损,导致细胞外基质重塑基因表达上调,促进肾纤维化。可见ACOX1的抑制促进肾纤维化。因此,ACOX1基因或蛋白可作为治疗靶点,在开发、筛选或制备移植肾功能衰竭的药物中的应用。
本发明提供了多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用。本发明以构建的大鼠肾移植模型为研究对象,给予大鼠模型亚油酸、亚麻酸和二十二碳六烯酸混合液灌胃处理,同时设置生理盐水灌胃的对照组。结果表明,PUFA灌胃的肾移植模型大鼠可有效改善移植肾纤维化,且未发现显著副作用。可见,本发明通过改善移植肾纤维化水平来达到防治移植肾功能衰竭的目的,具有一定的转化应用价值,填补了市场上尚未有延缓移植肾IF的药物或制剂的空白。
同时本发明提供的应用还具有以下优势:
1)PUFA可从日常饮食中获取,如鱼油、亚麻籽油都富含大量ω-3PUFA,大豆油中富含ω-6PUFA,有针对性的日常饮食即可达到改善移植肾的纤维化水平;
2)PUFA作为必需脂肪酸,安全可靠,容易获得和被接受;
3)PUFA无毒副作用,在移植肾纤维化的治疗中可避免进一步损伤肾脏;
4)PUFA制备方法成熟,成本低廉。
5)补充PUFA可缓解移植肾肾小管上皮细胞因酰基辅酶A氧化酶1(ACOX1)缺失所致的细胞外基质重塑,抑制移植肾纤维化进展,防治移植肾功能衰竭的目的。
附图说明
图1为移植肾中ACOX1表达缺失促进移植肾纤维化,其中A为人移植肾穿刺样本中同一视野下纤维化水平(Masson染色,蓝染为胶原沉积)和ACOX1表达水平(免疫组织化学染色,红染为ACOX1表达)对比结果;ci评分代表纤维化分级;B为不同纤维化水平的人移植肾穿刺样本中ACOX1表达水平统计图;C为移植年限超10年的组织样本中ACOX1表达水平热图;D为肾小管上皮细胞系HK-2中敲低ACOX1的免疫印记结果;E为敲低ACOX1后差异基因火山图(红点为上调基因,绿点为下调基因);F为差异基因富集结果;G为与肾纤维化相关的基因表达热图;H为基因富集分析(Gene Set EnrichmentAnalysis,GSEA)结果;
图2非诺贝特处理抑制移植肾纤维化,其中A为大鼠肾移植模型构建后给药模式图;B为移植肾组织的He染色和Masson染色结果;C为基于Masson染色的纤维化水平统计图;D为免疫荧光染色显示ACOX1的表达情况(蓝色荧光为细胞核,绿色荧光标记肾小管上皮细胞,紫色荧光为ACOX1,红色荧光为平滑肌肌动蛋白);
图3为ACOX1缺失促进细胞外基质重塑结果,其中A为移植肾线上数据库中ACOX1表达水平与细胞外基质重塑基因相关性分析结果;B为人移植肾样本中同一视野下ACOX1表达正常和缺失的肾小管上皮细胞中细胞外基质重塑基因表达情况(蓝色荧光为细胞核,黄色荧光为ACOX1,绿色荧光为四型胶原,红色荧光为基质金属蛋白酶7,紫色荧光为整合素β5);C为免疫印记检测HK-2细胞中敲低ACOX1后细胞外基质重新基因表达情况;D为免疫荧光实验在敲低ACOX1细胞和对照组细胞中检测基质金属蛋白酶7的表达水平(红色荧光为基质金属蛋白酶7);E为免疫荧光实验在敲低ACOX1细胞和对照组细胞中检测整合素β5的表达水平(红色荧光为整合素β5);F为使用ACOX1抑制剂10,12-Tricosadiynoic acid处理HK-2细胞后细胞外基质重塑基因的表达水平;G为使用ACOXl抑制剂10,12-Tricosadiynoic acid处理小鼠后肾脏的纤维化水平;H为使用ACOX1抑制剂10,12-Tricosadiynoic acid处理小鼠后肾脏中四型胶原的表达水平(蓝色荧光为细胞核,绿色荧光标记肾小管上皮细胞,红色荧光为四型胶原);I为四型胶原表达的统计图;J为使用ACOX1抑制剂10,12-Tricosadiynoicacid处理小鼠后肾脏中基质金属蛋白酶7的表达水平(蓝色荧光为细胞核,绿色荧光标记肾小管上皮细胞,红色荧光为基质金属蛋白酶7);K为基质金属蛋白酶7表达的统计图;
图4为敲低ACOX1的HK-2细胞非靶脂质组的检测结果,其中A为脂质代谢物热图(橘色为增加,绿色为减少);B为代谢物改变的火山图(红点为增加的代谢物,绿点为减少的代谢物);C为上调前10的代谢物;D为下调前10的代谢物;
图5为补充多不饱和脂肪酸抑制ACOX1缺失诱导的细胞外基质基因表达结果,其中A为在敲低ACOX1的HK-2细胞中单独添加亚油酸后细胞外基质重塑基因的mRNA水平;B为在敲低ACOX1的HK-2细胞中单独添加α-亚麻酸后细胞外基质重塑基因的mRNA水平;C为在敲低ACOX1的HK-2细胞中单独添加二十二碳六烯酸后细胞外基质重塑基因的mRNA水平;D为在敲低ACOX1的HK-2细胞中共同添加亚油酸、α-亚麻酸和二十二碳六烯酸后细胞外基质重塑基因的mRNA水平;E为在敲低ACOX1的HK-2细胞中单独或共同添加亚油酸、α-亚麻酸和/或二十二碳六烯酸后细胞外基质重塑基因的蛋白表达水平;F为使用ACOX1抑制剂10,12-Tricosadiynoic acid处理和补充多不饱和脂肪酸(亚油酸、α-亚麻酸和或二十二碳六烯酸混合物,每种脂肪酸计量为100mg/kg)后小鼠肾脏纤维化水平结果;G为使用ACOX1抑制剂10,12-Tricosadiynoic acid处理和补充多不饱和脂肪酸(亚油酸、α-亚麻酸和或二十二碳六烯酸混合物)后小鼠肾脏中细胞重塑基因表达情况;H为使用ACOX1抑制剂10,12-Tricosadiynoic acid处理和补充多不饱和脂肪酸(亚油酸、α-亚麻酸和或二十二碳六烯酸混合物)后小鼠肾脏四型胶原表达情况(蓝色荧光为细胞核,绿色荧光标记肾小管上皮细胞,红色荧光为四型胶原);
图6为肾移植患者血清中多不饱和脂肪酸水平,其中A为血清中三酰甘油水平;B为ω-3多不饱和脂肪酸水平;C为ω-6多不饱和脂肪酸水平;
图7为补充多不饱和脂肪酸抑制大鼠模型中移植肾纤维化的结果,A为大鼠肾移植模型灌胃多不饱和脂肪酸后纤维化水平的结果;B为处理组和对照组移植肾纤维化水平的统计图;C为大鼠肾移植模型灌胃多不饱和脂肪酸后移植肾中细胞外基质重塑基因的表达水平(黄色为四型胶原,绿色荧光为基质金属蛋白酶7,浅红色为原纤维蛋白1,红色荧光为整合素β5);D为处理组和对照组移植肾中细胞外基质重塑基因表达水平的统计图;E为大鼠肾移植模型灌胃多不饱和脂肪酸后心、肝、脾、肺、原位肾的苏木素-伊红(HE)染色结果。
具体实施方式
本发明提供了ACOX1作为分子靶点在制备、筛选移植肾功能衰竭的药物中的应用。
在本发明中,所述药物优选为促进ACOX1基因(SEQ ID NO:1)或ACOX1蛋白(SEQ IDNO:2)表达的药物。在本发明实施例中,结果证明人移植肾样本中,ACOX1低表达患者表现更高的纤维化水平;选取移植时间大于10年的患者,高表达ACOX1的患者不进展为较严重的纤维化;沉默ACOX1导致的上调基因富集在纤维化通路;沉默ACOX1导致的与肾纤维化通路相关基因的上调;沉默ACOX1导致的上调基因富集在肾衰竭和肾纤维化途径。可见移植肾中ACOX1表达缺失参与移植肾纤维化进展。使用非诺贝特能够上调ACOX1表达,抑制移植肾纤维化;通过抑制剂使这正常肾脏中ACOX1活性受损,导致细胞外基质重塑基因表达上调,促进肾纤维化。可见ACOX1的抑制促进肾纤维化。实验证明,ACOX1的抑制导致多不饱和脂肪酸水平下调和细胞外基质重塑基因表达上调,而多不饱和脂肪酸的回补能够逆转因ACOX1缺失导致的肾纤维化进程和下调细胞外基质重塑基因表达量。因此,ACOX1基因或蛋白可作为治疗靶点,在开发、筛选或制备移植肾功能衰竭的药物中的应用。
非诺贝特结构式如式I所示:
本发明提供了多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用。
在本发明中,在化学结构上,PUFA是一条由碳、氢原子相互连结而成的长链(18个碳原子以上),其间带有3-6个不饱和键(即双键)。因其第一个不饱和键位于甲基一端的第3或6个碳原子上,故名ω-3/6多不饱和脂肪酸。所述多不饱和脂肪酸优选包括亚油酸(LA)、α-亚麻酸(α-LA)和二十二碳六烯酸(DHA),结构式具体见式II~式IV。
规格均为纯度大于95%。在本发明实施例中,所述多不饱和脂肪酸购自麦克林公司。
在本发明中,所述预防和/或治疗移植肾功能衰竭优选包括延缓移植肾间质纤维化实现。在本发明实施例中,首先在人移植肾样本中确定ACOX1表达减少,并与移植肾纤维化进展密切相关。ACOX1的缺少导致多种参与移植肾纤维化的细胞外基质重塑基因表达上调,此结果被认为是ACOX1缺少导致移植肾纤维化的重要因素。为明确ACOX1缺少如何导致细胞外基质重塑,对敲低HK-2的细胞进行非靶脂质组检测,确定ACOX1缺陷导致多不饱和脂肪酸水平下降。对移植肾患者血液的检测结果显示,与肾脏未发生纤维化的患者相比,纤维化患者血液中的多不饱和脂肪酸水平减少。为进一步探究补充多不饱和脂肪酸是否有助于抑制细胞外基质重塑、延缓纤维化。首先在体外细胞实验中验证了补充多不饱和脂肪酸抑制细胞外基质重塑基因的表达;进一步在ACOX1抑制剂处理的小鼠肾脏中验证了上述结果;最后构建大鼠肾移植模型,通过灌胃的方法为肾移植肾大鼠补充多不饱和脂肪酸,结果显示,补充多不饱和脂肪酸能够显著抑制移植肾纤维化水平,改善肾功能,抑制细胞外基质重塑基因的表达。
下面结合实施例对本发明提供的多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用。进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
确定ACOX1表达水平与移植肾纤维化的关系
1.人移植肾样本选取
随机选取吉林大学第一医院组织库中的移植肾穿刺样本,对每一样本根据Banff评分标准确定纤维化水平,标准如下:
ci0:肾间质纤维化区域为肾皮质实质<5%;
ci1:肾间质纤维化区域占肾皮质实质6%~25%(间质轻度纤维化);
ci2:肾间质纤维化区域占肾皮质实质26%~50%(间质中度纤维化);
ci3:肾间质纤维化区域占肾皮质实质>50%(间质重度纤维化);
2.Masson染色
染色用试剂盒购买于索莱宝公司,货号G1346;具体步骤如下:
(1)组织切片常规脱蜡至水洗:石蜡切片二甲苯脱蜡2次×30min,脱蜡后的切片依次浸入100%乙醇I、100%乙醇II、95%乙醇I、95%乙醇II、80%乙醇和70%乙醇中5min,自来水洗;
(2)配置铁苏木素染色液(A1染液∶A2染液=1∶1),并染色5min;
(3)使用酸性乙醇(试剂B)分化5-10s,自来水洗;
(4)Masson蓝化液(试剂C)染色3-5min,自来水洗后蒸馏水洗1min;
(5)丽春红品红染色液(试剂D)染色10min;
(6)弱酸工作液(蒸馏水∶弱酸溶液(试剂E)=2∶1)洗1min;
(7)磷钼酸溶液(试剂F)洗1min,弱酸工作液洗1min;
(8)苯胺蓝染色液(试剂G)中染色1-2min,弱酸工作液洗1min;
(9)95%乙醇快速脱水2-3s,100%无水乙醇脱水3次×30s;
(10)100%二甲苯透明3次×5min,中性树胶封片。
3.免疫组织化学(IHC)染色
IHC染色用试剂盒购买于迈新生物技术有限公司,货号9720;
(1)组织切片常规脱蜡至水洗:石蜡切片二甲苯脱蜡2次×30min,脱蜡后的切片依次浸入100%乙醇I、100%乙醇II、95%乙醇I、95%乙醇II、80%乙醇和70%乙醇中5min,自来水洗;
(2)切片浸入柠檬酸盐修复液中,95℃热修复5min,室温冷却;
(3)PBS洗5min×3次;
(4)滴加阻断剂(试剂1)至切片上,完全覆盖组织,以去除内源性过氧化酶,室温孵育15min;
(5)PBS洗5min×3次;
(6)滴加血清(试剂2)至切片上,完全覆盖组织,以封闭非特异性蛋,室温孵育15min;
(7)擦去试剂2,滴加适当比例经稀释的一抗,4℃过夜;
(8)次日PBS洗5min×3次;
(9)滴加生物素标记的抗鼠/兔IgG的第二抗体(试剂3)至切片上,完全覆盖组织,室温孵育15min;
(10)PBS洗5min×3次;
(11)滴加辣根过氧化酶标记的链霉菌抗生物素蛋白(试剂4)室温孵育15min;
(12)PBS洗5min×3次;
(13)滴加配置好的DAB工作液,光镜下观察,观察到胞浆或胞核呈棕黄色颗粒后,根据需要在适当显色时间后用水终止显色;
(14)使用Mayer苏木素复染细胞核5min,自来水洗;
(15)弱氨水返蓝,自来水洗
(16)梯度酒精脱水、二甲苯透明、中性树胶封片。
4.免疫组化评分(H-score)
(1)染色强度分数:阴性0;弱1;中等2;强3。
(2)阳性细胞频率:少于5%,0分;5%~25%,1分;26%~50%,2分;51%~75%,3分;大于75%,4分。
(3)评分标准:每个评分独立,结果综合。
(4)例:一个标本包含75%的肿瘤细胞中等强度染色(3×2=6分),另外25%的肿瘤细胞是弱强度的(1×1=1),最后结果6+1=7。
(5)数据分析时,0-7被认为是低表达,而8-12被认为是高表达。
5.慢病毒转染沉默ACOX1表达
通过慢病毒转染方式,利用沉默载体构建shCtrl-HK2/shACOX1-HK-2细胞系;
(1)慢病毒载体由上海吉凯基因设计提供,均携带嘌呤霉素抗性基因和绿色荧光蛋白基因;
(2)细胞转染
①将对数期生长状态良好的HK-2细胞接种于6孔板中;
②待细胞生长融合至60%时,PBS清洗3次,将病毒液、助转染试剂和1mL培养基混合后加入6孔板中;
③37℃、5%CO2孵箱中培养72h,荧光显微镜下观察绿色荧光;
④使用1μg/mL浓度的嘌呤霉素筛选2周,去除未被转染的HK-2细胞,获得具有稳定抗性的克隆,传代、扩增、冻存备用。
6.免疫印记
(1)蛋白提取
取状态良好且汇合度达80%-90%的细胞,弃去培养基,PBS清洗两次后将培养皿置于-80℃冰箱中2h,取出细胞后在培养皿中加入适量的RIPA裂解液、蛋白酶抑制剂和磷酸酶抑制剂,三者比例为100∶1∶1,冰上裂解30min,期间每隔5min晃动培养皿,使裂解液与细胞充分接触。裂解完成后使用细胞刮刮下细胞,将含有细胞碎片的混合液体置于1.5ml EP管中,12000rpm 4℃离心10min,吸取上层清液,置于新的EP管中,-80℃保存。
(2)BCA法蛋白浓度测定
①配置BCA工作液:根据美伦BCA检测试剂盒说明书配置工作液,A液∶B液=50∶1,每孔200μL,根据所测样本数量确定需配置的工作液体积。
②加样:将0.5μg/μLBSA标准品按0、1、2、4、8、12、16、20μL体积一次加入96孔板中,每孔用PBS补足,使总体积均为20μL。待测样本孔中加入19μL PBS和1μL待测样本。然后每孔加入200μL BCA工作液。
③孵育:样品添加完成后将孔板置于37℃烘箱中孵育30min。待完全变色后检查吸光度。
④吸光度(OD值)测定:将孔板放入酶标仪中,检测562nm波长下各孔的OD值。
⑤样品浓度换算:使用Excel表格根据标准品浓度0、0.025、0.05、0.1、0.2、0.3、0.4、0.5和对应的OD值计算出公式,将待测样品OD值带入公式得到数值乘以20则为待测样品的蛋白浓度。
(3)蛋白溶液处理
根据各样品浓度按相同体积将所有样品浓度调一致,加入选取体积1/4的SDS蛋白上样缓冲液,充分混匀后,沸水浴加热5min使蛋白变性,水浴后直接置于冰上迅速降温,处理后的样品-20℃保存。
(4)SDS-PAGE凝胶配置
参照表1配置10%的分离胶和5%的浓缩胶。将各试剂按所需体积一次加入至50mL小烧杯中,充分混匀后使用1mL移液器将液体逐次加入至胶板中。待加入适量液体后,摇晃胶板,使气泡上浮、溢出。将胶板放置水平位置,使用1mL移液器沿胶板上沿从左至右缓慢滴加双蒸水,直至双蒸水溢出。常温下静置20分钟直至分离胶凝固。倒出双蒸水,按相同的方式加入配置好的浓缩胶,将梳子插入胶板内,静置直至凝固。
表1SDS-PAGE凝胶配方
(5)加样
将电泳装置安装好后,加入1×电泳缓冲液,拔去梳子,每孔上样体积10~20μL,可根据已测得的样品浓度和实验需求调整上样体积,使样品质量在30~60μg之间为宜。上样完成后,在旁边的空孔中加入3~4μL蛋白marker,使用相同体积的1×Loading Buffer加入到最边缘的两孔中。最后,补足电泳液。
(6)电泳
连接电源,80V电泳30min,120V电泳约1.5h,待marker条带完全分离,溴酚蓝条带跑至接近底部,终止电泳。
(7)转膜
根据蛋白marker条带的分子量位置切取目的蛋白和内参蛋白所在位置的凝胶,测量凝胶长度和宽度,剪取略大于凝胶的滤纸板和略大于滤纸板的PVDF膜,按照从下至上为滤纸一膜一胶一滤纸的顺序在半干转膜仪上摆放好,且每层间隙均需滴加半干转膜液,最后在整个三明治结构上滴加足量的半干转膜液。盖上上层转膜仪和外盖,使用20V电压,根据膜大小转膜25-40min。
(8)封闭
转膜结束后直接将膜置于5%脱脂奶粉溶液中,摇床上封闭1h。
(9)抗体孵育
使用5%脱脂奶粉按适当比例配置ACOX1一抗(抗体购自Affinity公司,货号DF12046)和GAPDH一抗(抗体购自Proteintech公司,货号10494-1-AP)孵育液,将膜浸入一抗孵育液中,4℃过夜。次日,取出PVDF膜,TBST清洗5min×3次。使用TBST按适当比例配置二抗(抗体购自Proteintech公司,货号SA00001-2)孵育液,将膜浸入二抗孵育液中,摇床上常温孵育1h。孵育完成后,使用TBST清洗5min×3次。
(10)显色
按照ECL试剂盒(购自大连美仑公司,货号MA0186)说明书将A液、B液按1∶1配置适当体积的工作液,将工作液滴加至PVDF膜上,在显色仪内曝光后得到带有条带的Westernblot图片。
7.RNA-sequence检测
本检测委托北京诺禾致源公司完成。
(1)RNA提取与检测
Agilent 2100bioanalyzer:精确检测RNA完整性和总量。
(2)文库构建与质检
建库起始RNA为总RNA。使用总量>=1μg的RNA通过Oligo(dT)磁珠富集带有polyA尾的mRNA,随后在Fragmentation Buffer中随机打断得到的mRNA。以片段化mRNA作为模版,引物为随机寡核苷酸,逆转录合成cDNA第一条链,随后用RNase H降解RNA链,并在DNApolymerase I体系下,以dNTPs为原料合成cDNA第二条链。双链cDNA纯化后通过末端修复、加A尾并连接测序接头,AMPure XP beads筛选370~420bp的cDNA,PCR扩增后再次使用AMPure XP beads纯化PCR产物,最终获得文库。文库构建完成后,使用Qubit2.0Fluorometer初步定量,并将文库稀释至1.5ng/μL,随后使用Agilent2100bioanalyzer对文库的insert size进行检测,如符合预期,通过qRT-PCR方法准确定量文库有效浓度,以保证文库质量。
(3)上机测序
把不同文库按照有效浓度及目标下机数据量的需求pooling后进行Illumina测序,并产生150bp配对末端读数。测序的基本原理是边合成边测序。在测序的flow cell中加入四种荧光标记的dNTP、DNA聚合酶以及接头引物进行扩增,测序簇延伸互补链时,每加入一个带有荧光的dNTP就能够释放出相应荧光,测序仪通过对荧光信号的捕获,使用计算机软件可以将光信号转化为测序峰,获得待测片段的序列信息。
(4)数据分析
①数据质控
测序片段被高通量测序仪测得的图像数据经CASAVA碱基识别转化为序列数据,其中主要包含测序片段的序列信息以及其对应的测序质量信息。测序获得的原始数据中包含少量带有测序接头或测序质量较低的reads。为了保证数据质量,需通过去除带接头的reads、去除含N(无法确定的碱基信息)的reads、去除低质量reads等方法对原始数据进行过滤。
②序列比对到参考基因组
在基因组网站下载参考基因组和基因模型注释文件。使用HISAT2 v2.0.5构建参考基因组的索引,并将配对末端的clean reads与参照基因组比对。
③基因表达水平定量
通过Feature Counts(1.5.0-p3)计算映射到每个基因的读数。然后根据基因的长度计算FPKM,并计算映射到该基因的读数。
④差异表达分析
使用DESeq2软件比较两个组合之间的差异表达分析。Benjamini和Hochberg两种方法用于调整所得P值以控制错误发现率。Adjust P值<0.05的基因为差异表达基因。
⑤差异基因富集分析
Cluster Profiler(3.4.4)软件用于差异表达基因的DisGeNET富集分析。DisGeNET数据库整合了人类疾病相关基因。DisGeNET富集以小于0.05的adjust P值作为显著性富集的阈值。
⑥基因集富集分析
基因集富集分析(Gene Set EnrichmentAnalysis,GSEA)使用预先定义的基因集,不需要指定明确的差异基因阈值,将基因按照在两类样本中的差异表达程度排序,然后检验预先设定的基因集合是否在这个排序表的顶端或者底端富集。使用GSEA分析工具http://www.broadinstitute.org/gsea/index.jsp,对该物种的DisGeNET数据集进行GSEA分析。
8.试验结果
8.1人移植肾样本中,ACOX1低表达患者表现更高的纤维化水平(图1中A和B);选取移植时间大于10年的患者,高表达ACOX1的患者不进展为较严重的纤维化(图1中C);
8.2人肾小管上皮细胞系中沉默ACOX1(图1中D)导致387种基因上调表达和380种基因下调表达(图1中E);
8.3沉默ACOX1导致的上调基因富集在纤维化通路;
8.4基因表达热图显示,沉默ACOX1导致的与肾纤维化通路相关的上调基因;
8.5GSEA分析显示沉默ACOX1导致的上调基因富集在肾衰竭和肾纤维化途径。
由上述结果可知,移植肾中ACOX1表达缺失参与移植肾纤维化进展。
实施例2
非诺贝特通过上调ACOX1抑制移植肾纤维化
1.构建大鼠肾移植模型
健康成年的SD大鼠购自吉林大学实验动物中心。体重180~220g,术前禁食12h,自由饮水。手术在室温25℃,相对湿度60%的清洁非无菌环境中进行。使用巴比妥钠腹腔注射麻醉。
供体手术:取SD大鼠左肾作为供肾。术中从腰静脉给予含肝素125U/ml的生理盐水0.5ml肝素化;自左肾动脉水平下方1.5cm腹主动脉用静脉留置针穿刺进针至左肾动脉水平,然后于左肾动脉水平上下方分别套丝线结扎腹主动脉阻断肾脏血运,同时用0~4℃乳酸林格氏液挂高60.0cm重力原位连续灌注左肾;于左肾静脉起始处切断左肾静脉,观察左肾颜色变至淡黄色,左肾静脉流出清亮的灌注液后,于左肾动脉起始处切断左肾动脉,游离输尿管至肾下极处离断,将左肾连同输尿管取出置于含肝素的冰盐水中略加修剪后保存、备用。
受体手术:受体SD大鼠麻醉后,上腹部横行切口,于左肾动、静脉起始处上血管夹,靠近肾门处剪断左肾动、静脉,分别用肝素盐水冲洗动静脉;于肾下极水平游离剪断输尿管后切除左肾。将供肾放于左肾窝,在10倍外科显微镜下用9-0的无损伤缝线先后端端吻合肾动脉和静脉;吻合过程中用冰屑防止供肾温度升高。吻合完毕并检查无出漏血,恢复肾血流,数秒内可见移植肾实质变鲜红、均匀、明亮,肾静脉充盈,吻合口饱满,肾动脉搏动明显,3~5min内可见输尿管有清亮尿液流出。调整好输尿管位置,10-0的无损伤缝合丝线间断吻合供受体输尿管。
术后处理:关腹后在大鼠腹腔内注入温生理盐水5ml,腹腔注射青霉素10万U,将大鼠置于加热垫上复温,苏醒后喂5%葡萄糖生理盐水,恢复行走后放入鼠笼正常喂养。
药物处理:模型构建成功后通过腹腔注射的方式给予大鼠非诺贝特,计量为100mg/kg/天,给药7周。非诺贝特购买于MedChemExpress(MCE)公司,货号#HY-17356。
2.肾组织苏木精-伊红染色
使用组织为肾移植大鼠的移植肾脏(左肾)。进行石蜡包埋后切片,石蜡切片二甲苯脱蜡2次×30min,脱蜡后的切片依次浸入100%乙醇I、100%乙醇II、95%乙醇I、95%乙醇II、80%乙醇和70%乙醇中5min,自来水洗后使用苏木精染核5min,盐酸酒精分化,弱氨水反蓝。水洗后伊红染色10min,然后依次在70%乙醇、80%乙醇、95%乙醇I、95%乙醇II、95%乙醇III、100%乙醇I和100%乙醇II中涮洗和浸泡脱水,脱水后的切片浸入100%二甲苯I和100%二甲苯II中脱去乙醇,最后使用中性树脂封片。
3.Masson染色
方法同上,使用组织为大鼠移植肾组织。
4.组织免疫荧光染色
(1)组织切片常规脱蜡至水洗:石蜡切片二甲苯脱蜡2次×30min,脱蜡后的切片依次浸入100%乙醇I、100%乙醇II、95%乙醇I、95%乙醇II、80%乙醇和70%乙醇中5min,自来水洗;
(2)切片浸入柠檬酸盐修复液中,95℃热修复5min,室温冷却;
(3)PBS洗5min×3次;
(4)5%胎牛血清(BSA)封闭40分钟;
(5)使用0.1%BSA按适当比例配置ACOX1一抗(抗体购自Affinity公司,货号DF12046)、α-SMA一抗(抗体购自Abcam公司,货号ab7817)孵育液,将一抗孵育液覆盖组织,4℃过夜。
(6)去除一抗,PBST清洗后,避光条件下加入荧光二抗,室温孵育1小时;
(7)去除二抗,PBS清洗后,使用PBS按适当比例配置LTL荧光染料(染料购自Vector公司,货号L-1320-5),避光条件下加入LTL染料,室温孵育半小时;
(8)去除LTL染料,PBS清洗后,使用PBS按适当比例配置DAPI染料,避光条件下加入DAPI染料,室温孵育10分钟;
(9)去除DAPI染料,PBS清洗后,荧光显微镜下观察细胞荧光。
5.试验结果
非诺贝特处理能有效抑制大鼠移植肾纤维化(图2中B和C);同时非诺贝特处理上调大鼠移植肾样本中ACOX1表达水平,抑制纤维化标志分子α-平滑肌肌动蛋白(α-SMA)表达水平(图2中D)。
由上述结果可知,大鼠移植肾中非诺贝特可上调ACOXl表达,抑制移植肾纤维化进展。
实施例3
ACOX1通过上调细胞外基质重塑相关分子表达促进移植肾纤维化
1.使用线上数据库获取移植肾样本中ACOX1及细胞外基质重塑相关分子的mRNA表达水平,并分析相关性;
2.组织/细胞免疫荧光染色
组织:
(1)组织切片常规脱蜡至水洗:石蜡切片二甲苯脱蜡2次×30min,脱蜡后的切片依次浸入100%乙醇I、100%乙醇II、95%乙醇I、95%乙醇II、80%乙醇和70%乙醇中5min,自来水洗;
(2)切片浸入柠檬酸盐修复液中,95℃热修复5min,室温冷却;
(3)PBS洗5min×3次;
(4)5%胎牛血清(BSA)封闭40分钟;
(5)使用0.1%BSA按适当比例配置ACOX1一抗(抗体购自Affinity公司,货号DF12046)、MMP7一抗(抗体购自Affinity公司,货号AF0218)ITGB5一抗(抗体购自Affinity公司,货号AF0185)和COL4一抗(抗体购自Affinity公司,货号AF0510)孵育液,将一抗孵育液覆盖组织,4℃过夜。’
(6)去除一抗,PBST清洗后,避光条件下加入荧光二抗,室温孵育1小时;
(7)去除二抗,PBS清洗后,共聚焦显微镜下观察细胞荧光。
细胞:
(1)取对数生长期细胞胰蛋白酶消化、离心、重悬后取适当密度铺于玻璃底共聚焦专用培养皿中;
(2)次日,待细胞铺展后,PBS清洗3次,4%多聚甲醛固定细胞10分钟;
(3)弃去固定液,PBS清洗3次,双蒸水清洗,室温下干燥;
(4)PBST清洗3次,使用0.1%Triton-X100打孔,静置20分钟;
(5)吸去Triton-X100溶液,使用2%BSA封闭非特异性抗体,室温放置1小时;
(6)PBST清洗后,加入适当配比浓度的一抗,4℃孵育过夜;
(7)去除一抗,PBST清洗后,避光条件下加入荧光二抗,室温孵育1小时;
(8)去除二抗,PBS清洗后,共聚焦显微镜下观察细胞荧光。
3.ACOX1抑制剂处理细胞及小鼠
3.1细胞系:人胚肾细胞系HEK-293、人肾小管上皮细胞系HK-2、大鼠肾小管上皮细胞系NRK
3.2细胞实验分组:对照组,给予溶剂(DMSO)处理;实验组:分别按0.25μM、0.5μM和1μM处理细胞24小时
3.3动物来源:BALB/c小鼠
3.4动物实验分组:对照组,给予溶剂(DMSO)处理;实验组:给予ACOX1抑制剂(10,12-Tricosadiynoic acid)处理,计量为100μg/kg/天,给药方式为腹腔注射,给药时间为一个月;
4.免疫印记
方法同上。
5.组织/细胞免疫荧光化学
方法同上。
6.试验结果
6.1线上数据库中,人移植肾样本的ACOX1表达与多种细胞外基质重塑基因表达呈显著负相关(图3中A);
6.2人移植肾肾穿刺样本中ACOX1表达与MMP7、ITGB5和COL4表达呈负相关(图3中B);
6.3沉默ACOX1导致细胞外基质重塑基因,包括COL4、MMP7、ITGB5、ITGB6和PRDX4,表达增加(图3中C);
6.4沉默ACOX1导致,细胞外基质重塑基因MMP7和ITGB5表达增加(图3中D和E);
6.5 ACOX1抑制剂处理导致细胞外基质重塑基因,包括COL4、MMP7、ITGB5、ITGB6和PRDX4,表达增加(图3中F);
6.6ACOX1抑制剂处理导致小鼠肾脏发生纤维化(图3中G);
6.7ACOX1抑制剂处理导致小鼠肾脏中细胞外基质重塑基因表达增加(图3中H-K);
由上述结果可知,移植肾中ACOX1表达缺失通过诱导细胞外基质重塑基因表达上调促进移植肾纤维化。
实施例3
ACOX1缺失导致多不饱和脂肪酸水平下调
1.非靶脂质组学检测
本检测委托北京诺禾致源公司完成。
(1)样本提取
①将装有实施例2处理的样本的EP管放入液氮中2min,取出后置于冰上解冻5min,涡旋混匀;重复3次后,5000rpm/min、4℃条件下离心1min;
②加入1mL脂质提取液后,涡旋15min,加入200μL双蒸水;
③涡旋1min,12000rpm/min、4℃条件下离心10min;
④离心后吸取500μL上清液至新的EP管中,浓缩后,使用200μL流动相B复溶,用于液相色谱串联质谱(LC-MS/MS)分析。
(2)色谱质谱采集条件
①液相条件:色谱柱-ThermoAccucore TM C30柱,i.d.2.1×100mm,2.6μm;流动相-A相,乙晴/水(60/40,V/V)(含0.1%甲酸,10mmol/L甲酸铵);B相,乙晴/异丙醇(10/90,V/V)(含0.1%甲酸,10mmol/L甲酸铵);流动相梯度,0min为A/B(80/20,V/V),2min为(70/30,V/V),4min为(40/60,V/V),9min为(15/85,V/V),14min为(10/90,V/V),15min为(5/95,V/V),17.3min为(5/95,V/V),17.5min为(80/20,V/V),20min为(80/20,V/V);流速—0.35mL/min;柱温45℃;进样量2μL。
②质谱条件:电喷雾离子源稳定500℃,正离子模式下质谱电压5550V,负离子模式下质谱电压-4500V,离子源gas1(GS1)15psi,gas1(GS1)55psi,帘气35psi,碰撞诱导电离参数Medium;三重四级杆中,每个离于对根据优化的去蔟电压和碰撞能进行扫描检测。
(3)代谢物定性与定量
基于标准品数据孔MWDB,根据检测物质的保留时间RT(Retention time)、子母离子对信息及二级谱数据进行定性分析;利用三重四级杆质谱的多反应监测模式进行代谢物定量分析;使用软件Analyst 1.6.3处理质谱数据。根据代谢物RT与峰型信息,对每个代谢物在不同样本中检测到的色谱峰进行定量,以确保定性定量的准确性。
(4)聚类分析
按照样本的特征进行分类,使同一类别内的个体具有尽可能高的同质性,将代谢物含量数据采用unit variance sealing归一化处理,通过R软件,对代谢物在不同样本间的积累进行聚类。
(5)差异代谢物统计
对所检测到的代谢物进行定性定量分析后,结合分组情况,对各分组中代谢物定量信息的差异倍数变化进行比较。
2.实验分组
对照组:对照载体转染的shCtrl-HK-2细胞作为对照组;实验组:靶向沉默ACOX1得得到的shACOX1-HK-2细胞作为实验组组;每组包含3个样本;
3.试验结果
3.1 HK-2细胞中沉默ACOX1导致35种脂质代谢物上调和105种脂质代谢物下调(图4中A和B);
3.2上调最显著的10种脂质代谢物以富含饱和脂肪酸的三酰甘油为主(图4中C);
3.3下调最显著的10种脂质代谢物以富含多不饱和脂肪酸的三酰甘油为主(图4中D);
实施例4
补充PUFA缓解ACOX1缺少诱导的细胞外基质重塑基因表达上调
1.实时荧光定量PCR
(1)TrizoL法提总RNA
①取细胞汇合率达80-90%的细胞,PBS清洗2次,在培养皿中加入1mL TrizoL,裂解细胞,使用移液器反复吹打细胞,使细胞脱落;
②将含有细胞的裂解液转移至1.5mL EP管中,移液器反复吹打至裂解野种无明显沉淀,室温静置5-10min;
③12000rpm 4℃离心5min,吸取上清液置于新的EP管中,弃沉淀;
④在EP管中加入200μL氯仿,振荡混匀15s,静置15min;
⑤12000rpm 4℃离心15min;
⑥吸取上层水相至新的EP管中;
⑦加入0.5mL异丙醇,振荡混匀后静置10min;
⑧12000rpm 4℃离心10min,弃去上层液体,RNA沉于EP管底部;
⑨EP管中加入75%乙醇,温和振荡EP管,悬浮沉淀,洗去有机溶剂;
⑩8000rpm 4℃离心5min,弃上清,室温干燥后,使用10-30μL DEPC水溶解DNA,-80℃冰箱保存。
(2)逆转录合成cDNA
①取提取好的总RNA,使用微量紫外分光光度计检测RNA浓度,根据测定的浓度吸取1μg RNA置于PCR管中;
②按表2中的反应体系加入相应试剂(试剂盒购自翌圣生物公司货号11119 ES)
表2逆转录反应体系
③PCR仪设置逆转录程序:25℃5min-42℃30min-85℃5min-4℃,将添加好的PCR管放入PCR仪中,运行程序。
(3)qPCR检测
①按表3中的反应体系加入cDNA和相应试剂(试剂盒购自翌圣生物公司货号11203ES)
表3aPCR反应体系
表4待测基因的扩增引物序列
/>
②qPCR仪设置程序:95℃5min;95℃10s,60℃34s,40个循环,溶解曲线,将添加好的PCR板放入qPCR仪中,运行程序。
2.免疫印记
方法同上。
3.Masson染色
方法同上。
4.组织免疫荧光染色
方法同上。
5.实验分组
细胞:以沉默ACOX1的细胞为对照组,实验组分别或共同添加多不饱和脂肪酸,包括LA、α-LA和DHA;
动物:采用BALB/c小鼠;对照组,给予溶剂(DMSO)处理;实验组1,给予ACOX1抑制剂(10,12-Tricosadiynoic acid)处理,计量为100μg/kg/天,给药方式为腹腔注射,给药时间为一个月;实验组2,再ACOX1抑制剂处理的基础上,给予多不饱和脂肪酸混合液灌胃,混合液包括LA、α-LA和DHA(质量比为1∶1∶1),每种计量为100 mg/kg/天,处理时间为一个月;LA、α-LA和DHA均购自上海麦克林生化科技股份有限公司。
6.试验结果
6.1单独添加LA、α-LA或DHA不可恢复由于沉默ACOX1导致的细胞外基质重塑基因表达上调(图5中A-C);
6.2补充LA、α-LA和DHA混合液可恢复由于沉默ACOX1导致的细胞外基质重塑基因表达上调(图5中D和E);
6.3补充LA、α-LA和DHA混合液可抑制ACOX1抑制剂导致的小鼠肾脏纤维化;
6.4补充LA、α-LA和DHA混合液可抑制ACOX1抑制剂导致的小鼠肾脏中四型胶原表达上调;
实施例5
纤维化肾移植患者血液中存在多不饱和脂肪酸水平下降
1.肾移植患者血液选取
收集肾移植患者血液,根据患者移植肾纤维化水平分组,分组如下:无纤维化组(Group A)共7例,纤维化水平根据Banff评分为ci 0;纤维化组(Group B)共7例,纤维化水平根据Banff评分为6例ci1和1例ci2;
2.脂肪酸靶向脂质组检测
测试服务委托北京诺禾致源公司完成。
(1)样品前处理
准确称取脂肪酸标准品,制备为浓度2000μg/mL的混标线性母液,使用甲醇将母液稀释为40000、20000、10000、4000、2000、1000、400、200、100、40、20、10ng/mL的工作液。配置一定浓度的Decanoic acid-d19、Myristic acid-d2、Octadecanoic、acid-d35、Eicosanoicacid-d39和Lignoceric acid-d4溶液,混匀后得到内标溶液。
(2)色谱和质谱方法
①色谱条件:色谱柱,Waters ACQUITY UPLC BEH C18(2.1×100mm,1.7μm);流动相,A相-0.05%甲酸水溶液;B相-50%异丙醇/乙晴溶液;柱温,50℃;进样量2μL;流速0.3mL/min;
②质谱条件:电喷雾电离源,负离子电离模式;离子源温度550℃,离子源电压-4500V,气帘气35psi,雾化气和辅助气均为60psi;多反应监测进行扫描。
(3)标准曲线和定量限
对标准也的浓度系列进行LC-MS检测,浓度为横坐标,标准品比内标峰面积的比值为纵坐标,得到各脂肪酸的线性方程;采用信噪比法确定定量限。
(4)样本定量
根据建立的线性方程,将样本的检测结果带入方程中,对待测样品进行定量分析。
3.试验结果
3.1两组患者血液中的三酰甘油(TG)水平无明显差异(图6中A);
3.2两组患者血液中ω-3(n-3)多不饱和脂肪酸水平差异显著,表现为纤维化组患者血液中多种ω-3多不饱和脂肪酸显著下调,包括C18:3、C20:3、C22:5(n-3)和C22:6(图6中B);
3.3两组患者血液中ω-6(n-6)多不饱和脂肪酸水平差异显著,表现为纤维化组患者血液中多种ω-6多不饱和脂肪酸显著下调,包括C18:3(n-6)、C22:2、C20:4、C22:4和C22:5(n-6)(图6中C);
实施例6
补充PUFA抑制肾移植模型大鼠移植肾的纤维化进展
1.构建大鼠肾移植模型
使用同品系大鼠构建单侧肾移植模型,即先切取供者大鼠和受者大鼠左肾,将供者大鼠左肾移植给受者大鼠,缝合肾动脉、静脉和输尿管。
2.实验分组
模型构建后分为2组,每组5只大鼠,对照组不做出来,实验组给予给予多不饱和脂肪酸混合液灌胃,混合液包括LA、α-LA和DHA,每种计量为100mg/kg/天,处理时间为一个月;LA、a-LA和DHA均购自上海麦克林生化科技股份有限公司;
3.Masson染色
方法同上,使用的组织为移植肾组织
4.免疫组织化学染色及免疫荧光化学染色
方法同上,使用的组织为移植肾组织
5.苏木精-伊红染色
方法同上,使用组织为肾移植大鼠的心、肝、脾、肺、原位肾脏(右肾)。
6.试验结果
6.1补充多不饱和脂肪酸能有效抑制大鼠模型中移植肾纤维化(图7中A和B);
6.2补充多不饱和脂肪酸能有效抑制大鼠模型中移植肾中细胞外基质重塑基因的表达(图7中C和D);
6.3补充多不饱和脂肪酸对大鼠对心、肝、脾、肺和原位肾脏(右肾)均无显著影响,具有良好的安全性(图7中E)。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.ACOX1作为分子靶点在制备、筛选移植肾功能衰竭的药物中的应用。
2.根据权利要求1所述应用,其特征在于,一种促进ACOX1基因或蛋白表达的试剂在制备、筛选移植肾功能衰竭的药物中的应用。
3.根据权利要求2所述应用,其特征在于,所述试剂包括非诺贝特。
4.多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用。
5.根据权利要求1所述应用,其特征在于,所述多不饱和脂肪酸包括亚油酸、α-亚麻酸和二十二碳六烯酸。
6.根据权利要求5所述应用,其特征在于,所述亚油酸、α-亚麻酸和二十二碳六烯酸的质量比为(1~2):(1~2):(1~2)。
7.根据权利要求6所述应用,其特征在于,所述亚油酸、α-亚麻酸和二十二碳六烯酸的质量比为1:1:1。
8.根据权利要求1~7中任意一项所述应用,其特征在于,所述移植肾功能衰竭为移植肾间质纤维化。
9.根据权利要求4所述应用,其特征在于,所述多不饱和脂肪酸通过阻止细胞外基质重塑来改善移植肾纤维化。
10.根据权利要求8所述应用,其特征在于,所述多不饱和脂肪酸抑制ACOX1表达降低导致的移植肾的纤维化。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311035071.0A CN117092348A (zh) | 2023-08-17 | 2023-08-17 | 多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311035071.0A CN117092348A (zh) | 2023-08-17 | 2023-08-17 | 多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117092348A true CN117092348A (zh) | 2023-11-21 |
Family
ID=88769233
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311035071.0A Pending CN117092348A (zh) | 2023-08-17 | 2023-08-17 | 多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117092348A (zh) |
-
2023
- 2023-08-17 CN CN202311035071.0A patent/CN117092348A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Komada et al. | ASC in renal collecting duct epithelial cells contributes to inflammation and injury after unilateral ureteral obstruction | |
Wang et al. | High urea induces depression and LTP impairment through mTOR signalling suppression caused by carbamylation | |
Zhu et al. | The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress‐Mediated Cell Injury | |
CN106620694B (zh) | Cornulin作为靶点在制备防治银屑病的药物中的应用 | |
Daniel et al. | Transgelin is a marker of repopulating mesangial cells after injury and promotes their proliferation and migration | |
CN109568583B (zh) | Ripk3作为靶点在制备防治银屑病的药物中的应用 | |
Zou et al. | Learning and memory impairment and transcriptomic profile in hippocampus of offspring after maternal fructose exposure during gestation and lactation | |
Cheng et al. | Dysregulated expression of mRNA and SNP in pulmonary artery remodeling in ascites syndrome in broilers | |
CN113908283A (zh) | Prmt5抑制剂及其与pd-l1抗体阻断剂联合在治疗肺癌上的应用 | |
US20130203048A1 (en) | Wound Healing Metakaryotic Stem Cells and Methods of Use Thereof | |
Yang et al. | Cardioprotective effect of NRG-4 gene expression on spontaneous hypertension rats and its mechanism through mediating the activation of ErbB signaling pathway | |
CN117092348A (zh) | 多不饱和脂肪酸在制备预防和/或治疗移植肾功能衰竭的药物中的应用 | |
CN112569354B (zh) | tau蛋白及其基因作为药物靶点在制备治疗糖尿病药物中的应用 | |
CN116953247A (zh) | 一种用于早期诊断阿尔茨海默症的生物标志物及其用途 | |
CN111826442B (zh) | 预防肺癌靶标plekhn1及其应用 | |
Kasinath et al. | Urine podoplanin heralds the onset of ischemia-reperfusion injury of the kidney | |
CN112716940A (zh) | 卡格列净在制备治疗与stat6蛋白相关疾病的药物中的应用 | |
CN110694067A (zh) | 一种抑制血管生成素样蛋白8的物质的应用 | |
Zhang et al. | HOXC8/TGF-β1 positive feedback loop promotes liver fibrosis and hepatic stellate cell activation via activating Smad2/Smad3 signaling | |
Wang et al. | Retracted: DLX5 gene regulates the Notch signaling pathway to promote glomerulosclerosis and interstitial fibrosis in uremic rats | |
Okamoto et al. | Altered clinicopathology and renal pathology in dogs treated with a clinical dose of cisplatin | |
Cui et al. | Dendritic cells originating exosomal miR-193b-3p induces regulatory T cells to alleviate liver transplant rejection | |
Wang et al. | Renal lipid accumulation and aging linked to tubular cells injury via ANGPTL4 | |
Li et al. | The long noncoding RNA uc003pxg. 1 interacts with miR-339-5p to promote TGF-β1 expression and vascular endothelial cell proliferation, migration and angiogenesis in coronary heart disease | |
김정현 | The Role of Blood-derived Exosomal hsa-miR-130a-5p from Abdominal Aortic Aneurysm patients in Human Aortic Smooth Muscle Cells |
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 |