CN114478706A - 用于三维培养纤维网状结构多肽及其应用 - Google Patents
用于三维培养纤维网状结构多肽及其应用 Download PDFInfo
- Publication number
- CN114478706A CN114478706A CN202210182580.5A CN202210182580A CN114478706A CN 114478706 A CN114478706 A CN 114478706A CN 202210182580 A CN202210182580 A CN 202210182580A CN 114478706 A CN114478706 A CN 114478706A
- Authority
- CN
- China
- Prior art keywords
- lung cancer
- polypeptide
- culture
- culture medium
- organoids
- 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.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 37
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 34
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 34
- 239000000835 fiber Substances 0.000 title claims abstract description 10
- 210000002220 organoid Anatomy 0.000 claims abstract description 54
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims abstract description 47
- 201000005202 lung cancer Diseases 0.000 claims abstract description 47
- 208000020816 lung neoplasm Diseases 0.000 claims abstract description 47
- 238000000034 method Methods 0.000 claims abstract description 18
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 239000001963 growth medium Substances 0.000 claims description 24
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 22
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 22
- 210000001519 tissue Anatomy 0.000 claims description 18
- 239000003814 drug Substances 0.000 claims description 16
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 claims description 12
- 239000006285 cell suspension Substances 0.000 claims description 11
- 229960005309 estradiol Drugs 0.000 claims description 11
- 229960000890 hydrocortisone Drugs 0.000 claims description 11
- 229960005322 streptomycin Drugs 0.000 claims description 11
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims description 10
- 229960004308 acetylcysteine Drugs 0.000 claims description 10
- 229930182833 estradiol Natural products 0.000 claims description 10
- 102000045246 noggin Human genes 0.000 claims description 10
- 108700007229 noggin Proteins 0.000 claims description 10
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 9
- 101000825960 Homo sapiens R-spondin-3 Proteins 0.000 claims description 8
- 102100022766 R-spondin-3 Human genes 0.000 claims description 8
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 claims description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 7
- 229930182555 Penicillin Natural products 0.000 claims description 7
- 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 claims description 7
- 229940049954 penicillin Drugs 0.000 claims description 7
- 238000012216 screening Methods 0.000 claims description 6
- -1 B27 Chemical compound 0.000 claims description 5
- 206010028980 Neoplasm Diseases 0.000 claims description 5
- 239000012894 fetal calf serum Substances 0.000 claims description 5
- 102400000058 Neuregulin-1 Human genes 0.000 claims description 4
- 108090000556 Neuregulin-1 Proteins 0.000 claims description 4
- 239000003560 cancer drug Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 241000124008 Mammalia Species 0.000 claims description 2
- 201000011510 cancer Diseases 0.000 claims description 2
- 230000000973 chemotherapeutic effect Effects 0.000 claims description 2
- 238000001959 radiotherapy Methods 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims description 2
- 239000012092 media component Substances 0.000 claims 1
- 150000001413 amino acids Chemical class 0.000 abstract description 8
- 238000012258 culturing Methods 0.000 abstract description 8
- 210000004072 lung Anatomy 0.000 abstract description 4
- 238000001338 self-assembly Methods 0.000 abstract description 3
- 230000035755 proliferation Effects 0.000 abstract description 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 44
- 229920005989 resin Polymers 0.000 description 35
- 239000011347 resin Substances 0.000 description 35
- 210000004027 cell Anatomy 0.000 description 31
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 22
- 239000000243 solution Substances 0.000 description 13
- 238000005406 washing Methods 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 12
- 229940079593 drug Drugs 0.000 description 11
- 108010087230 Sincalide Proteins 0.000 description 7
- 238000010609 cell counting kit-8 assay Methods 0.000 description 7
- 235000019441 ethanol Nutrition 0.000 description 7
- 239000012091 fetal bovine serum Substances 0.000 description 7
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000000306 component Substances 0.000 description 5
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 4
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 4
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 229960002949 fluorouracil Drugs 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 4
- 239000008096 xylene Substances 0.000 description 4
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 3
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 3
- 102000002322 Egg Proteins Human genes 0.000 description 3
- 108010000912 Egg Proteins Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 3
- 229960004316 cisplatin Drugs 0.000 description 3
- 239000002173 cutting fluid Substances 0.000 description 3
- 229960003668 docetaxel Drugs 0.000 description 3
- 235000014103 egg white Nutrition 0.000 description 3
- 210000000969 egg white Anatomy 0.000 description 3
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 3
- 229960005277 gemcitabine Drugs 0.000 description 3
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 3
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 229960003966 nicotinamide Drugs 0.000 description 3
- 235000005152 nicotinamide Nutrition 0.000 description 3
- 239000011570 nicotinamide Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- VDBKFYYIBLXEIF-GUBZILKMSA-N Arg-Gln-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O VDBKFYYIBLXEIF-GUBZILKMSA-N 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 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 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 229920003180 amino resin Polymers 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000010511 deprotection reaction Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- WVZWEMOFSIEEMU-UHFFFAOYSA-N indene-1,2,3-trione Chemical compound C1=CC=C2C(=O)C(=O)C(=O)C2=C1 WVZWEMOFSIEEMU-UHFFFAOYSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- DRDSQGHKTLSNEA-GLLZPBPUSA-N Gln-Glu-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DRDSQGHKTLSNEA-GLLZPBPUSA-N 0.000 description 1
- TWYSSILQABLLME-HJGDQZAQSA-N Glu-Thr-Arg Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O TWYSSILQABLLME-HJGDQZAQSA-N 0.000 description 1
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 208000019693 Lung disease Diseases 0.000 description 1
- 229940126560 MAPK inhibitor Drugs 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- LHUBVKCLOVALIA-HJGDQZAQSA-N Thr-Arg-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O LHUBVKCLOVALIA-HJGDQZAQSA-N 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- CUTSCJHLMGPBEJ-UHFFFAOYSA-N [N].CN(C)C=O Chemical compound [N].CN(C)C=O CUTSCJHLMGPBEJ-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000004630 atomic force microscopy Methods 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- UFMVNSFONAZFOT-UHFFFAOYSA-N dichloromethane;n-ethyl-n-propan-2-ylpropan-2-amine Chemical compound ClCCl.CCN(C(C)C)C(C)C UFMVNSFONAZFOT-UHFFFAOYSA-N 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 230000007613 environmental effect Effects 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
- 210000000981 epithelium Anatomy 0.000 description 1
- DZGCGKFAPXFTNM-UHFFFAOYSA-N ethanol;hydron;chloride Chemical compound Cl.CCO DZGCGKFAPXFTNM-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 230000037442 genomic alteration Effects 0.000 description 1
- 230000007773 growth pattern Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 208000037841 lung tumor Diseases 0.000 description 1
- 239000008204 material by function Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 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
- 238000004264 monolayer culture Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 102000002574 p38 Mitogen-Activated Protein Kinases Human genes 0.000 description 1
- 108010068338 p38 Mitogen-Activated Protein Kinases Proteins 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- YYVGYULIMDRZMJ-UHFFFAOYSA-N propan-2-ylsilane Chemical compound CC(C)[SiH3] YYVGYULIMDRZMJ-UHFFFAOYSA-N 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 108091006082 receptor inhibitors Proteins 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 102000000568 rho-Associated Kinases Human genes 0.000 description 1
- 108010041788 rho-Associated Kinases Proteins 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002723 toxicity assay Methods 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 230000005760 tumorsuppression Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0688—Cells from the lungs or the respiratory tract
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
- C12N2501/392—Sexual steroids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/72—Transferases [EC 2.]
- C12N2501/727—Kinases (EC 2.7.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
-
- 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)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Pulmonology (AREA)
- Oncology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了用于三维培养纤维网状结构多肽及其应用，多肽的氨基酸序列如SEQ ID NO.1所示，该氨基酸呈纤维网状结构，能够用于细胞的三维培养构建类器官；本发明还公开了利用该多肽培养肺类器官或/和肺癌类器官的方法，本发明的方法可以加快肺癌细胞的增殖与自组装能力，加快类器官的建立以及提高类器官建立的成功率。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及用于三维培养纤维网状结构多肽，还涉及该肽在细胞三维培养中的应用。
背景技术
不适当的再生与包括肺癌在内的肺部疾病有关。肺癌是世界范围内的主要死亡原因之一，每年有近200万例新确诊病例。对于成功的治疗干预来说，诊断往往为时已晚。肺癌在个体之间表现出明显的表型和遗传异质性，这使得在动物身上建模很困难。类器官来源于肺上皮内的局部干/祖细胞，由于其在癌症治疗中的巨大潜力，在研究和临床中都引起了广泛的兴趣。各种肺癌类器官已被建立，以再现原发肺肿瘤的组织结构，并在体外长期扩张过程中维持原发肿瘤的基因组改变。
类器官是由多能干细胞或器官祖细胞分化，并自组装成有结构和功能的完整哺乳动物器官，类器官技术在研究广泛的对象方面潜力巨大，包括发育生物学、疾病病理学、细胞生物学、再生机制、精准医疗以及药物毒性和药效试验等。对于这些应用以及其他应用，类器官培养实现了对现有2D培养方法和动物模型系统的高信息量的互补，是二维培养和活体模型之间的重要桥梁，其较单层细胞培养模型更具生理学相关性，同时比活体模型更容易操纵监测信号通路和基因组编辑。类器官的价值在于它们能够自组织成最小的生物单位，表现出与原始组织相似的功能和复杂性。类器官的可操作性预示着类器官将为广泛的基础研究提供出色的模型系统，包括表达谱研究和体内难以获得的稀有细胞谱系的分析。
类器官在抗肿瘤药物筛选领域表现出了巨大的潜力，药物的筛选测试展示了药物对类器官最直观的表现，高度模拟了药物对器官的效果。因此，亟需一种可以加快肺癌细胞的增殖与自组装能力，加快类器官的建立以及提高类器官建立的成功率的培养方法，为肺癌类器官的培养提供了多样性。
多肽因其结构简单、可调、功能多样、成本较低等优点,在生物纳米技术领域备受关注。基于短肽自组装构建的纳米材料具有简单的制备工序、良好的生物相容性、较大的比表面积、易于修饰改造、较深的组织渗透性以及较高的生物活性等优势,已经成为生物催化、药物/基因控制释放、功能材料以及临床治疗等领域的热点研究对象。因此，合理设计短肽分子结构以及给予特定的环境刺激,能够多重非共价键相互作用自发的组织或聚集为具有特异形态和功能的纳米结构，可用于类器官三维培养对类器官培养具有重要意义。
发明内容
有鉴于此，本发明的目的之一在于提供一种用于三维培养纤维网状结构多肽；本发明的目的之二在于提供所述多肽在用于细胞三维培养中的应用；本发明的目的之三在于提供所述多肽建立肺癌类器官的方法；本发明的目的之四在于提供建立肺癌类器官的培养基；本发明的目的之五在于提供所述培养基在肺癌类器官筛选抗肺癌药物中的应用。
为达到上述目的，本发明提供如下技术方案：
1、用于三维培养纤维网状结构多肽，所述多肽的氨基酸序列如SEQ ID NO.1所示。
2、所述多肽在用于细胞三维培养中的应用。
优选的，所述细胞为正常组织细胞或癌细胞。
3、基于所述多肽建立肺癌类器官的方法，包括以下步骤：
(1)将获得的肺癌组织制备成单细胞悬液；
(2)将步骤(1)制备的单细胞悬液加入权利要求1所述多肽重悬，然后接种于培养装置中，加入所述的培养基，培养箱中扩大培养。
本发明中，步骤(1)中，所述肺癌组织来源于哺乳动物。
本发明中，步骤(1)中，所述乳腺癌组织制备成单细胞悬液具体过程是经过剪碎、研磨、过滤得到。
本发明中，所述培养基为含有如下组分的DMEM/F12完全培养基：雌二醇、氢化可的松、R-Spondin 3、Neuregulin 1、rh-EGF、Noggin、A83-01、Wnt3a、Y-27632、SB202190、B27、N-乙酰半胱氨酸、烟酰胺、GlutaMax、胎牛血清、青霉素/链霉素双抗。
本发明中，所述培养基组分的浓度如下：雌二醇10nM-1000nM、氢化可的松0.1-100μg/ml、R-Spondin 3 100-500ng/ml、Neuregulin 1 1-10nM、rh-EGF 1-10ng/ml、Noggin50-500ng/ml、A83-01 100-1000nM、Wnt3a 1-5nM、Y-27632 1-10μM、SB202190 100-1000ng、B27 10ng/ml、N-乙酰半胱氨酸1-5nM、烟酰胺1-10nM、GlutaMax 1-5nM、胎牛血清体积分数10％、青霉素/链霉素双抗体积分数1％。
4、一种建立肺癌类器官的培养基，所述培养基为含有如下组分的DMEM/F12完全培养基：雌二醇、氢化可的松、R-Spondin 3、Neuregulin 1、rh-EGF、Noggin、A83-01、Wnt3a、Y-27632、SB202190、B27、N-乙酰半胱氨酸、烟酰胺、GlutaMax、胎牛血清、青霉素/链霉素双抗。
5、所述培养基在肺癌类器官筛选抗肺癌药物中的应用，优选的，所述药物包含化疗药物、放疗药物或/和靶向药物。
本发明的有益效果在于：本发明公开了用于三维培养纤维网状结构多肽，该多肽成纤维网状结构，能够用于三维细胞培养，用于培养肺癌细胞配合本发明的培养基第2天可形成类器官，培养至14天最大能达到900μm，加快类器官的建立以及提高类器官建立的成功率，并且培养的类器官可以用于药物筛选，对肺癌的精准用药提供指导。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为多肽的投射显微镜图；
图2为配方1-3的培养基培养肺癌类器官14天生长形态图(标尺20μm)；
图3为配方1-3的培养基培养肺癌类器官培养14天的生长曲线图；
图4为配方1-3的培养基培养肺癌类器官和肺癌组织的第14天HE染色图(标尺50μm)；
图5为肺癌类器官筛选抗肿瘤药物的CCK-8肿瘤抑制率图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本发明实施例中涉及的培养基组分雌二醇(β-estradiol)，购自Sigma公司、氢化可的松(hydrocortisone)，购自Sigma公司、R-Spondin 3，购自R&D公司；Neuregulin 1，购自Peprotech公司；rh-EGF，购自Peprotech公司；Noggin，购自Peprotech公司；A83-01，购自Tocris公司；Wnt3a，购自Gibco公司；Y-27632，购自Abmole公司；SB202190，购自Sigma公司；B27，购自Gibco公司；N-Acetylcysteine，购自Gibco公司；Nicotinamide，购自Sigma公司；GlutaMax，购自Sigma公司；FBS，购自Gibco公司；Penicillin/Streptomycin，购自Gibco公司；DMEM/F12，购自Gibco公司。
实施例1、多肽的制备
多肽的制备方法，具体步骤如下：
1)称取氨基树脂3g(取代度0.3mmol/g)于150ml的反应器中，用50ml的二氯甲烷(DCM)浸泡1h溶胀树脂；
2)用3倍树脂体积的氮-二甲基甲酰胺(DMF)洗涤树脂，然后抽干液体，洗涤四次，将抽干树脂待用；
3)向反应器中加入50ml的20％哌啶(哌啶/DMF)，放在脱色摇床上摇晃20min，以此来脱去树脂上的Fmoc基团；脱完保护后用3倍树脂体积的DMF洗涤2次，3倍树脂体积的甲醇洗涤2次，3倍树脂体积的DMF洗涤2次，抽干树脂；
4)取少量树脂用茚三酮(九井水合茚三酮)法检测(检A、检B各两滴，100℃反应1min)，树脂有颜色，说明脱保护成功。
5)称取一定量的C端第一个天冬氨酸D和3倍摩尔量的1-羟基-苯骈三唑(HOBT)于50ml的离心管中，加入20ml的DMF将其溶解，然后加入3ml的N,N-二异丙基碳二亚胺(DIC)振荡摇匀1min，活化氨基酸，待溶液澄清后加入到反应器中，然后将反应器置于30℃的摇床中震荡反应。
6)2小时后，抽干反应溶液，DMF洗涤两遍，用一定量的醋酸酐封头(醋酸酐：DIEA:DCM＝1：1：2)半小时，然后用3倍树脂体积的DMF洗涤四次，抽干待用。
7)向反应器中加入一定量的20％哌啶(哌啶/DMF＝1:4)，放在脱色摇床上摇晃20min，以此来脱去氨基酸上的Fmoc基团。脱完保护后用3倍树脂体积的DMF洗涤2次，3倍树脂体积的甲醇洗涤2次，3倍树脂体积的DMF洗涤2次，抽干树脂。
8)取少量树脂用茚三酮(九井水合茚三酮)法检测(检A、检B各两滴，100℃反应1min)，树脂有颜色，说明脱保护成功。
9)称取后面一个谷氨酸和HOBT于50ml离心管,加入20ml的DMF将其溶解，然后加入3ml的N,N-二异丙基碳二亚胺(DIC)振荡摇匀1min，活化氨基酸，待溶液澄清后加入到反应器中，然后将反应器置于30℃的摇床中震荡反应1小时后，取少量树脂检测，用茚三酮法检测(检A、检B各两滴，100℃反应1min)，若树脂为无色，说明反应完全；若树脂有颜色，说明缩合不完全，需重投氨基酸再次反应。
10)待反应完全后，用DMF洗涤树脂四次，然后抽干，向反应器中加入一定量的20％哌啶(哌啶/DMF＝1:4)，放在脱色摇床上摇晃20min,以此来脱去树脂上的Fmoc基团。脱完保护后用3倍树脂体积的DMF洗涤2次，3倍树脂体积的甲醇洗涤2次，3倍树脂体积的DMF洗涤2次，抽干树脂，取少量树脂用茚三酮法检测是否脱去Fmoc基团。
11)按照步骤9-11依次接上后面的氨基酸；
12)待接完最后一个氨基酸后，脱去保护，用DMF洗涤四次，然后用甲醇洗涤4遍，然后将树脂抽干。然后用95切割液(三氟乙酸:1,2乙二硫醇:3，异丙基硅烷:水＝95:2:2:1)将多肽从树脂上切割下来(每克树脂加10ml切割液)，并用冰乙醚(切割液：乙醚＝1:9)离心沉降，并用乙醚洗涤离心3次，冷冻干燥粗品，最后用HPLC分离纯化，冻干纯品，得到一定纯度的多肽粉末，具体序列如下：Arg Gln Glu Thr Arg Gln Glu Thr Arg Gln Glu Thr ArgGln Glu Thr(SEQ ID NO.1)
13)最后用HPLC分离纯化，再冻干。使用时将多肽溶于PBS中形成多肽溶液，浓度大于10mg/ml。
经制得的多肽用原子力显微镜观察，结果如图1所示。结果显示，制得的多肽成纤维网状结构，表明具有三维细胞培养的潜力。
实施例2、建立肺类器官培养基
按下述浓度将各组分加入到DMEM/F12培养基中，得到用于培养肺癌类器官的培养基：
配方1：雌二醇100nM，氢化可的松50μg/ml、R-Spondin 3(250ng/ml)、Neuregulin1(5nM)、rh-EGF(5ng/ml)、Noggin(100ng/ml)、A83-01(TGF-βI型受体抑制剂，500nM)、Wnt3a(5nM)、Y-27632(蛋白激酶p160ROCK抑制剂，5μM)、SB202190(p38 MAPK抑制剂，500ng)、B27(10ng/ml)、N-乙酰半胱氨酸(N-Acetylcysteine，5nM)、烟酰胺(Nicotinamide，10nM)、GlutaMax添加剂(5nM)、胎牛血清(FBS，10％)、青链霉素(Penicillin/Streptomycin，1％)，。
配方2：雌二醇10nM，氢化可的松0.1μg/ml、R-Spondin 3(100ng/ml)、Neuregulin1(1nM)、rh-EGF(1ng/ml)、Noggin(50ng/ml)、A83-01(100nM)、Wnt3a(1nM)、Y-27632(1μM)、SB202190(100ng)、B27(10ng/ml)、N-Acetylcysteine(1nM)、Nicotinamide(1nM)、GlutaMax添加剂(1nM)、FBS(10％)、Penicillin/Streptomycin(1％)。
配方3：雌二醇1000nM，氢化可的松100μg/ml、R-Spondin 3(500ng/ml)、Neuregulin1(10nM)、rh-EGF(10ng/ml)、Noggin(500ng/ml)、A83-01(1000nM)、Wnt3a(5nM)、Y-27632(10μM)、SB202190(1000ng)、B27(10ng/ml)、N-Acetylcysteine(5nM)、Nicotinamide(10nM)、GlutaMax添加剂(5nM)、FBS(10％)、Penicillin/Streptomycin(1％)。
实施例3、建立肺癌类器官的方法
本实施例中肺癌样本均来自重庆市人民医院。
使用实施例1的培养基建立肺癌类器官的方法，具体操作步骤如下：
1)肺癌组织的获取
采集后将获得的肺癌组织/肺组织采用50ml的DMEM/F12+FBS(10％)+Penicillin/Streptomycin(1％)进行浸泡。
2)肺癌类器官的培养
(1)将得到的肺癌组织浸泡于1％的Penicillin/Streptomycin(青链霉素)中1h，将浸泡的肺癌样本取出，用一次性手术刀将细胞剪碎，剪碎的组织大约为1cm×1cm；用碾磨棒将1cm×1cm的肺癌手术样本碾碎，制备成为单细胞悬液；
(2)将单细胞悬液吸出，用200目的细胞滤网过滤2-3次，将多余的残留组织去除，加入PBS清洗3次，再加入多肽(10mg/ml)重悬细胞，接种于6孔板中；在六孔板中加入培养基A培养细胞，放置于37℃的培养箱中，每三天更换一次培养基，观察细胞的形态，光学显微镜下拍照记录，培养至14天。
配方1～3的培养基A培养1天，3天，5天，7天，10天和14天的结果如图2所示。类器官体积和细胞数量统计结果如图3所示，结果显示，培养过程中细胞逐渐增多，培养至第2天可形成类器官，培养至第4天类器官细胞数量快速增加，至7天呈对数增长；同时培养过程中类器官逐渐变大，培养至14天最大能达到900μm。
实施例5、肺癌类器官HE染色
将生长状态良好的类器官放入离心管内，200g转速离心10min，弃置部分上清液，在离心管内加入2ml蛋清，轻摇混匀，在蛋清液中悬浮细胞团，再加入5倍蛋清混合液体积的80％酒精，混匀，200g转速离心10min，去除上清液，加入体积分数10％甲醛固定3h。
使用体积分数4％多聚甲醛溶液浸泡组织样本，48h后采用石蜡包埋并切片。
HE染色步骤如下：二甲苯(Ⅰ)15min；二甲苯(Ⅱ)15min；无水乙醇(Ⅰ)5min；无水乙醇(Ⅱ)5min；95％乙醇3min；80％乙醇2min；70％乙醇2min；蒸馏水5min；苏木精染色液6min；自来水冲洗1min；1％盐酸乙醇1-2s；自来水冲洗10-30s；0.2％氨水返蓝6min；自来水冲洗10-30s；0.5％伊红染色液1-3min；蒸馏水冲洗1-2s；70％乙醇1min；80％乙醇1min；95％乙醇2min；无水乙醇(Ⅰ)3min；二甲苯(Ⅰ)10min；二甲苯(Ⅱ)10min。结果如图4所示。结果显示，肺癌类器官形成细胞团，且与肺癌细胞具有高度的形态一致性。
实施例6、肺癌类器官评估药物效果的CCK-8毒性实验
1)制作标准曲线(用于测定细胞具体数量)
(1)先用细胞计数板计数所制备的细胞悬液中的细胞数量，然后接种细胞到培养板内。
(2)按比例(例如：1/2比例)依次用培养基等比稀释成一个细胞浓度梯度，一般要做3-5个细胞浓度梯度，每个浓度建议3-6个复孔。
(3)接种后培养2-4小时使细胞贴壁，然后加CCK-8试剂培养一定时间后测定OD值，制作出一条以细胞数量为横坐标(X轴)，OD值为纵坐标(Y轴)的标准曲线。根据此标准曲线可以测定出未知样品的细胞数量，使用此标准曲线的前提是实验的条件要一致，便于确定细胞的接种数量以及加入CCK-8后的培养时间。
2)细胞活性检测
(1)在96孔板中接种细胞悬液100μL/孔，将培养板放在培养箱中37℃，5％CO2预培养一段时间。
(2)向每孔加入10μL CCK-8溶液，注意不要在孔中生成气泡，它们会影响OD值的读数。
(3)将培养板在培养箱内孵育1-4小时。
(4)用酶标仪测定在450nm处的吸光度。
(5)若暂时不测定OD值，可以向每孔中加入10μL 0.1M的HCL溶液或者1％w/v SDS溶液，并遮盖培养板避光保存在室温条件下。24小时内测定，吸光度不会发生变化。
3)细胞增殖-毒性检测
(1)在第七天，加入消化酶消化Matrigel，在96孔板中配制100μL的细胞悬液。将培养板放在培养箱37℃，5％CO2预培养24小时。
(2)向培养板加入10μL不同浓度的化疗药物和靶向药物，包括但不限于5-氟尿嘧啶、顺铂、多西他赛、吉西他滨。
(3)将培养板在培养箱孵育一段适当的时间(例如：6、12、24或48小时)。
(4)向每孔加入10μL CCK-8溶液，注意不要再孔中生成气泡，它们会影响OD值的读数。
(5)将培养板在培养箱内孵育1-4小时。
(6)用酶标仪测定在450nm处的吸光度。
(7)若暂时不测定OD值，可以向每孔中加入10μL 0.1M的HCL溶液或者1％w/v SDS溶液，并遮盖培养板避光保存在室温条件下。24小时内测定，吸光度不会发生变化。
(8)最终计算出5-氟尿嘧啶、顺铂、多西他赛、吉西他滨、生理盐水对肿瘤类器官的抑制率。
注意：如果待测物质有氧化性或还原性的话，可在加CCK-8之前更换新鲜培养基(除去培养基，并用培养基洗涤细胞两次，然后加入新的培养基)，去掉药物影响。当然药物影响比较小的情况下，可以不更换培养基，直接扣除培养基中加入药物后的空白吸收即可，结果如图5所示。相比较于生理盐水，5-氟尿嘧啶、顺铂、多西他赛、吉西他滨对肺癌类器官都有一定的抑制率，且5-氟尿嘧啶对肿瘤类器官的抑制率最高，高达78％。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (10)
1.用于三维培养纤维网状结构多肽，其特征在于：所述多肽的氨基酸序列如SEQ IDNO.1所示。
2.权利要求1所述多肽在用于细胞三维培养中的应用。
3.根据权利要求2所述的应用，其特征在于：所述细胞为癌细胞。
4.基于权利要求1所述多肽建立肺癌类器官的方法，其特征在于，包括以下步骤：
(1)将获得的肺癌组织制备成单细胞悬液；
(2)将步骤(1)制备的单细胞悬液加入权利要求1所述多肽重悬，然后接种于培养装置中，加入所述的培养基，培养箱中扩大培养。
5.根据权利要求4所述的方法，其特征在于，步骤(1)中，所述肺癌组织来源于哺乳动物。
6.根据权利要求4所述的方法，其特征在于，步骤(1)中，所述肺癌组织制备成单细胞悬液具体过程是经过剪碎、研磨、过滤得到。
7.根据权利要求4所述的方法，其特征在于：所述培养基为含有如下组分的DMEM/F12完全培养基：雌二醇、氢化可的松、R-Spondin 3、Neuregulin 1、rh-EGF、Noggin、A83-01、Wnt3a、Y-27632、SB202190、B27、N-乙酰半胱氨酸、烟酰胺、GlutaMax、胎牛血清、青霉素/链霉素双抗。
8.根据权利要求4所述的方法，其特征在于：所述培养基组分的浓度如下：雌二醇10nM-1000nM、氢化可的松0.1-100μg/ml、R-Spondin 3 100-500ng/ml、Neuregulin 1 1-10nM、rh-EGF 1-10ng/ml、Noggin 50-500ng/ml、A83-01 100-1000nM、Wnt3a 1-5nM、Y-27632 1-10μM、SB202190 100-1000ng、B27 10ng/ml、N-乙酰半胱氨酸1-5nM、烟酰胺1-10nM、GlutaMax1-5nM、胎牛血清体积分数10％、青霉素/链霉素双抗体积分数1％。
9.一种建立肺癌类器官的培养基，其特征在于：所述培养基为含有如下组分的DMEM/F12完全培养基：雌二醇、氢化可的松、R-Spondin 3、Neuregulin 1、rh-EGF、Noggin、A83-01、Wnt3a、Y-27632、SB202190、B27、N-乙酰半胱氨酸、烟酰胺、GlutaMax、胎牛血清、青霉素/链霉素双抗。
10.权利要求9所述培养基在肺癌类器官筛选抗肺癌药物中的应用，其特征在于：所述药物包含化疗药物、放疗药物或/和靶向药物。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210182580.5A CN114478706B (zh) | 2022-02-25 | 2022-02-25 | 用于三维培养纤维网状结构多肽及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210182580.5A CN114478706B (zh) | 2022-02-25 | 2022-02-25 | 用于三维培养纤维网状结构多肽及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114478706A true CN114478706A (zh) | 2022-05-13 |
| CN114478706B CN114478706B (zh) | 2023-08-01 |
Family
ID=81484130
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210182580.5A Active CN114478706B (zh) | 2022-02-25 | 2022-02-25 | 用于三维培养纤维网状结构多肽及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114478706B (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114958754A (zh) * | 2022-06-06 | 2022-08-30 | 重庆嘉士腾生物科技有限公司 | 用于三维培养类器官纳米纤维gc水凝胶及其应用和培养直肠癌类器官的方法 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104334718A (zh) * | 2012-03-13 | 2015-02-04 | 中国科学院遗传与发育生物学研究所 | 通过三维培养重新编程细胞 |
| CN106967672A (zh) * | 2017-03-24 | 2017-07-21 | 四川大学华西医院 | 一种肺及肺癌组织培养方法以及用其构建肺癌小鼠动物模型方法 |
| CN109554346A (zh) * | 2018-12-05 | 2019-04-02 | 首都医科大学附属北京胸科医院 | 一种肺癌类器官模型及其在肿瘤研究中的应用 |
| CN110592022A (zh) * | 2019-09-17 | 2019-12-20 | 罗国安 | 一种肺肿瘤类器官专用培养基及无支架3d培养方法 |
| CN112080472A (zh) * | 2020-09-01 | 2020-12-15 | 南通大学 | 一种培养专用于生物医药功能研究的人肺癌类器官3d模型的方法 |
| CN112501110A (zh) * | 2020-11-26 | 2021-03-16 | 海西纺织新材料工业技术晋江研究院 | 三维培养肺及肺癌组织类器官的标准化培养基及培养方法 |
-
2022
- 2022-02-25 CN CN202210182580.5A patent/CN114478706B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104334718A (zh) * | 2012-03-13 | 2015-02-04 | 中国科学院遗传与发育生物学研究所 | 通过三维培养重新编程细胞 |
| CN106967672A (zh) * | 2017-03-24 | 2017-07-21 | 四川大学华西医院 | 一种肺及肺癌组织培养方法以及用其构建肺癌小鼠动物模型方法 |
| CN109554346A (zh) * | 2018-12-05 | 2019-04-02 | 首都医科大学附属北京胸科医院 | 一种肺癌类器官模型及其在肿瘤研究中的应用 |
| CN110592022A (zh) * | 2019-09-17 | 2019-12-20 | 罗国安 | 一种肺肿瘤类器官专用培养基及无支架3d培养方法 |
| CN112080472A (zh) * | 2020-09-01 | 2020-12-15 | 南通大学 | 一种培养专用于生物医药功能研究的人肺癌类器官3d模型的方法 |
| CN112501110A (zh) * | 2020-11-26 | 2021-03-16 | 海西纺织新材料工业技术晋江研究院 | 三维培养肺及肺癌组织类器官的标准化培养基及培养方法 |
Non-Patent Citations (1)
| Title |
|---|
| MARK H.PAUSCH ET AL: "Multiple Ca2 + /calmodulin-dependent protein kinase genes in a unicellular eukaryote", THE EMBO JOURNAL, vol. 10, no. 6, pages 1511 - 1522, XP002146843 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114958754A (zh) * | 2022-06-06 | 2022-08-30 | 重庆嘉士腾生物科技有限公司 | 用于三维培养类器官纳米纤维gc水凝胶及其应用和培养直肠癌类器官的方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114478706B (zh) | 2023-08-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101195838B1 (ko) | 분리된 전분화능 성체줄기세포 및 그의 분리 및 배양 방법 | |
| US20050014255A1 (en) | Stem cells for clinical and commercial uses | |
| CN105960411A (zh) | 自组装肽、肽模拟物和肽缀合物作为构件用于生物制造和打印 | |
| CN104212760B (zh) | 肌肉干细胞体外培养方法及其应用 | |
| WO2019035618A1 (ko) | 3차원 폐암 오가노이드 배양 방법 및 이를 이용한 환자 유래 이종이식 동물모델의 제조 방법 | |
| CN111057680A (zh) | 用于肺部肿瘤细胞的培养基和三维培养方法 | |
| Daniels et al. | Micromass cultures of limb and other mesenchyme | |
| US10928383B2 (en) | Method for predicting effect of drug | |
| CN113774027A (zh) | 一种蜂巢状GelMA微球在构建肿瘤模型中的应用 | |
| CN115011560A (zh) | 一种脑胶质瘤类器官、培养基及培养方法 | |
| CN114478706A (zh) | 用于三维培养纤维网状结构多肽及其应用 | |
| CN102282250A (zh) | 使哺乳动物祖细胞分化为产生胰岛素的胰岛细胞的方法 | |
| JP2007512857A (ja) | 三次元の哺乳動物卵巣上皮性卵細胞の調製方法及び生物適合性マトリックスにおける卵胞培養システム | |
| JPH10118103A (ja) | 皮膚付属器官様構造体を含む人工皮膚及びその製造方法 | |
| BRPI0710522A2 (pt) | cultura de células tridimensional | |
| CN114317442A (zh) | 一种建立乳腺类器官或/和乳腺癌类器官的培养基及方法和应用 | |
| Grover | The enzymatic dissociation and reproducible reaggregation in vitro of 11-day embryonic chick lung | |
| CN114958756B (zh) | 一种用于前列腺癌类器官培养的培养液及其制备方法 | |
| CN116590232A (zh) | 一种甲状腺癌类器官、培养基及培养方法 | |
| CN112608899B (zh) | 一种无血清培养基在培养癌组织起源球状体中的应用 | |
| JP2004500855A (ja) | 感染モデル | |
| CN114181284B (zh) | 纳米短肽drf3在药物、nk细胞载体和生物医学的应用 | |
| Wang et al. | Metastatic tumor cell invasion of brain organ tissue cultured on cellulose polyacetate strips | |
| JP5720980B2 (ja) | クッパー細胞の効率的増殖方法およびその利用 | |
| US20150337260A1 (en) | Hybrid Matrix |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240809 Address after: Room 2, Floor 15, No. 8, West Section of Mount Taishan Avenue, Liangjiang New District, Yubei District, Chongqing 401121 Patentee after: Chongqing Jiashiteng Pharmaceutical Co.,Ltd. Country or region after: China Address before: 401120 No. 21-03-001343, 3 / F, No. 210, west section of Jinkai Avenue, Dazhulin street, Liangjiang New Area, Chongqing Patentee before: Chongqing jiashiteng Biotechnology Co.,Ltd. Country or region before: China |