CN116808111B - 一种负载中药的水凝胶薄膜、其制备及抗骨肉瘤的应用 - Google Patents
一种负载中药的水凝胶薄膜、其制备及抗骨肉瘤的应用 Download PDFInfo
- Publication number
- CN116808111B CN116808111B CN202311048493.1A CN202311048493A CN116808111B CN 116808111 B CN116808111 B CN 116808111B CN 202311048493 A CN202311048493 A CN 202311048493A CN 116808111 B CN116808111 B CN 116808111B
- Authority
- CN
- China
- Prior art keywords
- weight
- parts
- pilose antler
- dried powder
- freeze
- 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.)
- Active
Links
- 201000008968 osteosarcoma Diseases 0.000 title claims abstract description 100
- 239000003814 drug Substances 0.000 title claims abstract description 58
- 239000000017 hydrogel Substances 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 210000003056 antler Anatomy 0.000 claims abstract description 118
- 239000000843 powder Substances 0.000 claims abstract description 60
- 229920001661 Chitosan Polymers 0.000 claims abstract description 41
- 230000006907 apoptotic process Effects 0.000 claims abstract description 41
- 108010022355 Fibroins Proteins 0.000 claims abstract description 31
- 230000001737 promoting effect Effects 0.000 claims abstract description 21
- 150000001875 compounds Chemical class 0.000 claims abstract description 19
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 19
- 230000001338 necrotic effect Effects 0.000 claims abstract description 17
- 230000035876 healing Effects 0.000 claims abstract description 16
- 241001116742 Drynaria Species 0.000 claims abstract description 12
- 241000893536 Epimedium Species 0.000 claims abstract description 12
- 241000736246 Pyrola Species 0.000 claims abstract description 12
- 241000405414 Rehmannia Species 0.000 claims abstract description 12
- 235000018905 epimedium Nutrition 0.000 claims abstract description 12
- 239000000203 mixture Substances 0.000 claims abstract description 12
- 241000220259 Raphanus Species 0.000 claims abstract description 11
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 claims abstract description 11
- 241000913745 Spatholobus Species 0.000 claims abstract description 11
- 241000336315 Cistanche salsa Species 0.000 claims abstract description 8
- 210000003734 kidney Anatomy 0.000 claims description 36
- 239000011259 mixed solution Substances 0.000 claims description 16
- 239000008176 lyophilized powder Substances 0.000 claims description 15
- 235000014347 soups Nutrition 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 13
- 229940079593 drug Drugs 0.000 claims description 12
- 230000012292 cell migration Effects 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 238000002791 soaking Methods 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- 239000000706 filtrate Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 230000004663 cell proliferation Effects 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 239000011521 glass Substances 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 3
- 230000007480 spreading Effects 0.000 claims description 3
- 238000003892 spreading Methods 0.000 claims description 3
- 238000013508 migration Methods 0.000 abstract description 21
- 230000000694 effects Effects 0.000 abstract description 20
- 230000005012 migration Effects 0.000 abstract description 18
- 230000035755 proliferation Effects 0.000 abstract description 16
- 238000009472 formulation Methods 0.000 abstract description 6
- 238000000338 in vitro Methods 0.000 abstract description 6
- 239000008187 granular material Substances 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 119
- 108090000765 processed proteins & peptides Proteins 0.000 description 23
- 238000002474 experimental method Methods 0.000 description 22
- 229920001184 polypeptide Polymers 0.000 description 22
- 102000004196 processed proteins & peptides Human genes 0.000 description 22
- 230000009545 invasion Effects 0.000 description 16
- 210000000988 bone and bone Anatomy 0.000 description 14
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 13
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 13
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 12
- 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 description 12
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 12
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 12
- 241000282994 Cervidae Species 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 150000004676 glycans Chemical class 0.000 description 8
- 229920001282 polysaccharide Polymers 0.000 description 8
- 239000005017 polysaccharide Substances 0.000 description 8
- 101001011663 Homo sapiens Mixed lineage kinase domain-like protein Proteins 0.000 description 7
- 102100030177 Mixed lineage kinase domain-like protein Human genes 0.000 description 7
- 239000004480 active ingredient Substances 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- DNXHEGUUPJUMQT-UHFFFAOYSA-N (+)-estrone Natural products OC1=CC=C2C3CCC(C)(C(CC4)=O)C4C3CCC2=C1 DNXHEGUUPJUMQT-UHFFFAOYSA-N 0.000 description 6
- DNXHEGUUPJUMQT-CBZIJGRNSA-N Estrone Chemical compound OC1=CC=C2[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 DNXHEGUUPJUMQT-CBZIJGRNSA-N 0.000 description 6
- 230000009471 action Effects 0.000 description 6
- 230000030833 cell death Effects 0.000 description 6
- 229960005309 estradiol Drugs 0.000 description 6
- 229960003399 estrone Drugs 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 208000005243 Chondrosarcoma Diseases 0.000 description 4
- 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 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000005728 strengthening Methods 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 101001089266 Homo sapiens Receptor-interacting serine/threonine-protein kinase 3 Proteins 0.000 description 3
- 102100033729 Receptor-interacting serine/threonine-protein kinase 3 Human genes 0.000 description 3
- 230000001174 ascending effect Effects 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 230000017074 necrotic cell death Effects 0.000 description 3
- 230000000149 penetrating effect Effects 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 2
- 206010005963 Bone formation increased Diseases 0.000 description 2
- 206010006002 Bone pain Diseases 0.000 description 2
- 241000005787 Cistanche Species 0.000 description 2
- 241000336291 Cistanche deserticola Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 206010028851 Necrosis Diseases 0.000 description 2
- 102100023181 Neurogenic locus notch homolog protein 1 Human genes 0.000 description 2
- 102000014736 Notch Human genes 0.000 description 2
- 108010070047 Notch Receptors Proteins 0.000 description 2
- 101150079595 Notch1 gene Proteins 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 229940126680 traditional chinese medicines Drugs 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 1
- 102000051485 Bcl-2 family Human genes 0.000 description 1
- 108700038897 Bcl-2 family Proteins 0.000 description 1
- 241000721047 Danaus plexippus Species 0.000 description 1
- 206010061819 Disease recurrence Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101150092640 HES1 gene Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 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
- 238000000134 MTT assay Methods 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 101100284799 Mus musculus Hesx1 gene Proteins 0.000 description 1
- 230000005913 Notch signaling pathway Effects 0.000 description 1
- 206010057178 Osteoarthropathies Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 1
- 235000003704 aspartic acid Nutrition 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
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000006721 cell death pathway Effects 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 230000000482 effect on migration Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000004630 mental health Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000004634 pharmacological analysis method Methods 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/11—Pteridophyta or Filicophyta (ferns)
- A61K36/12—Filicopsida or Pteridopsida
- A61K36/126—Drynaria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
- A61K36/296—Epimedium
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/486—Millettia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/64—Orobanchaceae (Broom-rape family)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/80—Scrophulariaceae (Figwort family)
- A61K36/804—Rehmannia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
- A61K9/7007—Drug-containing films, membranes or sheets
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Alternative & Traditional Medicine (AREA)
- Mycology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Inorganic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Immunology (AREA)
- Rheumatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
一种负载中药的水凝胶薄膜、其制备及抗骨肉瘤的应用,它涉及生物医学工程领域,本发明要解决中药复合方剂在治疗骨肉瘤,尤其是抑制骨肉瘤细胞迁移,细胞增殖,骨肉瘤细胞的划痕愈合、促进骨肉瘤细胞的坏死性凋亡和促进骨肉瘤细胞的程序性凋亡效果不佳的问题。本发明中药复方冻干粉由鹿茸、熟地黄、淫羊藿、肉苁蓉、鸡血藤、鹿衔草、骨碎补和莱菔子组成。并加入壳聚糖及柞蚕丝素蛋白制成水凝胶薄膜。本发明改变了鹿茸补肾颗粒的传统剂型得到鹿茸补肾方,并将其制成冻干粉,已在体外试验中验证了鹿茸补肾方冻干粉具有抗骨肉瘤活性的能力,因此将三者结合制备补肾方冻干粉及负载鹿茸冻干粉水凝胶薄膜。本发明应用于骨肉瘤治疗领域。
Description
技术领域
本发明属于生物医学工程领域,具体涉及壳聚糖/丝素蛋白负载中药冻干粉的水凝胶薄膜在治疗骨肉瘤中的应用。
背景技术
骨肉瘤是青少年常见的恶性肿瘤,严重威胁青少年的生命安全和身心健康。骨肉瘤发病机制所涉及的分子过程尚不完全清楚。目前,常规的手术、化疗等治疗方法会存在复发和产生耐药性的问题。此外,手术后残留的肿瘤细胞会导致骨破坏和疾病复发。因此,有必要开发新药或增强当前使用的药物以及确定骨修复方法。药物靶向递送及手术植入骨修复材料以修复受损部位具有广泛的应用前景。壳聚糖具有优异的生物降解性和生物相容性,可用于生物工程各个领域。蚕丝蛋白是一种有机高分子材料,具备良好的韧性、成膜性及可生物降解特性,柞蚕丝素蛋白由于其分子链中具有由精氨酸、甘氨酸、天冬氨酸首尾相连而成的特殊的RGD三肽肽段,该肽段对哺乳动物细胞有良好的吸附性和促增殖性。鹿茸补肾颗粒是全国名中医赵文海教授,依据天池伤科流派的骨质增生丸(“骨质增生丸”治疗增生性骨关节病)。
公开号CN110368485A,发明名称《鹿茸多肽在制备用于治疗骨肉瘤的药物中的用途》公开了鹿茸多肽在制备用于治疗骨肉瘤的药物中的用途,该鹿茸多肽可显著抑制骨肉瘤细胞的增殖,并且诱导骨肉瘤细胞凋亡,尤其是人骨肉瘤细胞U2OS,在浓度为80μg/mL时效果最为显著。鹿茸多肽可以作为骨肉瘤治疗的潜在药物,减缓肿瘤的增长,促进治愈骨肉瘤。该专利虽然采用了鹿茸多肽治疗骨肉瘤,但是其并非中药制剂,而仅仅鹿茸众多活性成分中的一种多肽。
连湘倩,发表的《鹿茸活性成分调控骨肉瘤细胞死亡和迁移侵袭的机制研究》,公开了:通过网络药理学及体外细胞实验,探究鹿茸多肽及鹿茸其他活性成分调控骨肉瘤细胞死亡和迁移侵袭的作用机制。研究内容如下:(1)网络药理学分析获取鹿茸治疗骨肉瘤的物质基础和分子机制。结果发现TNF-α、Notch1等可作为鹿茸治疗骨肉瘤的关键靶点;17β-雌二醇和雌酮等可作为鹿茸治疗骨肉瘤的关键成分。TNF-α介导的坏死性凋亡可促进肿瘤细胞死亡,Notch1介导的Notch信号通路可调控肿瘤细胞的迁移和侵袭,推测鹿茸活性成分可通过这两种途径调控骨肉瘤细胞的死亡和迁移侵袭。(2)体外细胞实验探究17β-雌二醇、雌酮及鹿茸多肽调控骨肉瘤细胞死亡的机制。MTT实验结果发现,17β-雌二醇和雌酮均可有效抑制MG63细胞的增殖活性。酶联免疫吸附实验发现,17β-雌二醇和雌酮能够上调MG63细胞中TNF-α、RIPK3和MLKL蛋白的表达,TNF-α、RIPK3和MLKL介导骨肉瘤细胞坏死性凋亡。说明17β-雌二醇和雌酮可以促进骨肉瘤细胞MG63中坏死性凋亡的发生。同样通过酶联免疫吸附实验发现,鹿茸多肽也可上调骨肉瘤细胞MG63中TNF-α、RIPK3和MLKL的表达进而促进坏死性凋亡的发生。(3)体外细胞实验探究鹿茸多肽调控骨肉瘤细胞迁移侵袭的机制。细胞克隆形成实验结果显示,80μg/mL鹿茸多肽可以抑制骨肉瘤细胞MG63、U2OS的增殖能力;细胞划痕实验和Transwell实验结果显示,鹿茸多肽显著抑制骨肉瘤细胞MG63、U2OS的迁移和侵袭能力;Westernblot和qPCR实验结果显示,20μg/mL和80μg/mL鹿茸多肽处理细胞后,细胞内Notch1、Hes1蛋白及其mRNA表达量明显下降,说明鹿茸多肽可以抑制骨肉瘤细胞MG63、U2OS中Notch信号通路的表达。综上,鹿茸多肽、17β-雌二醇和雌酮可通过促进坏死性凋亡的发生进而促进骨肉瘤细胞死亡,鹿茸多肽下调Notch信号通路的表达从而抑制骨肉瘤细胞的迁移和侵袭。
目前骨肉瘤制备中的中药复方剂中,并没有涉及到含有鹿茸的中药复方剂,且在抑制骨肉瘤细胞迁移,细胞增殖,骨肉瘤细胞的划痕愈合、促进骨肉瘤细胞的坏死性凋亡和促进骨肉瘤细胞的程序性凋亡的效果上表现不佳。
发明内容
本发明的目的是为了解决中药复合方剂在治疗骨肉瘤,尤其是抑制骨肉瘤细胞迁移,细胞增殖,骨肉瘤细胞的划痕愈合、促进骨肉瘤细胞的坏死性凋亡和促进骨肉瘤细胞的程序性凋亡效果不佳的问题,而提供一种负载中药复方冻干粉的水凝胶薄膜、其制备方法及在抗骨肉瘤中的应用。
本发明的一种负载中药的水凝胶薄膜,是由5~15重量份的鹿茸、20~40重量份熟地黄、10~30重量份淫羊藿、10~30重量份肉苁蓉、10~30重量份鸡血藤、10~30重量份鹿衔草、10~30重量份骨碎补、10~30重量份莱菔子和壳聚糖及柞蚕丝素蛋白制成。
进一步地,所述的水凝胶薄膜是由10重量份的鹿茸、30重量份熟地黄、30重量份淫羊藿、20重量份肉苁蓉、20重量份鸡血藤、20重量份鹿衔草、20重量份骨碎补、20重量份莱菔子和壳聚糖及柞蚕丝素蛋白制成。
进一步地,所述的壳聚糖和柞蚕丝素蛋白的质量比为1:1,所述的壳聚糖和柞蚕丝素蛋白的混液溶液中壳聚糖的质量浓度为2%,采用盐酸溶解后与柞蚕丝素蛋白混合。
进一步地,所述的负载中药的水凝胶薄膜是将鹿茸、熟地黄、淫羊藿、肉苁蓉、鸡血藤、鹿衔草、骨碎补和莱菔子制成鹿茸补肾汤冻干粉,加入到壳聚糖及柞蚕丝素蛋白的混液溶液中,得到的负载鹿茸补肾汤冻干粉的丝素蛋白-壳聚糖共混溶液后,置于载玻片上,干燥后得到;其中,负载鹿茸补肾汤冻干粉的丝素蛋白-壳聚糖共混溶液中鹿茸补肾汤冻干粉的质量浓度为1.8mg/mL。
本发明的一种负载中药复方冻干粉的水凝胶薄膜的应用,所述的水凝胶薄膜用于制备抑制骨肉瘤细胞增殖的药物。
本发明的一种负载中药复方冻干粉的水凝胶薄膜的应用,所述的水凝胶薄膜用于制备抑制骨肉瘤细胞迁移的药物。
本发明的一种负载中药复方冻干粉的水凝胶薄膜的应用,所述的水凝胶薄膜用于制备抑制骨肉瘤细胞的划痕愈合、促进骨肉瘤细胞坏死性凋亡或促进骨肉瘤细胞程序性凋亡的药物。
本发明制备所述的负载中药的水凝胶薄膜的方法如下:
(1)、鹿茸煎煮液:
称取5~15重量份的鹿茸加20倍量的水浸泡,浸泡后进行3~4次煎煮,第一次煎煮与后续每次的煎煮时间均为20~30min,其中,第一次煎煮的前10min武火煎煮,后10~20min文火煎煮;后续每次煎煮的前10min均为武火煎煮,后10~20min均为文火煎煮;煎煮后用纱布过滤;
(2)、称取20~40重量份熟地黄、10~30重量份淫羊藿、10~30重量份肉苁蓉、10~30重量份鸡血藤、10~30重量份鹿衔草、10~30重量份骨碎补和10~30重量份莱菔子,加6倍量的水浸泡,浸泡后进行多次煎煮,每次煎煮20~30min,前10min武火,后10~20min文火;煎煮后用纱布过滤;
(3)、合并滤液:将步骤(1)煎煮后的鹿茸滤液与步骤(2)的滤液合并,将合并后的滤液制得鹿茸补肾汤冻干粉;
(4)、将步骤(3)得到的鹿茸补肾汤冻干粉加入到壳聚糖和柞蚕丝素蛋白的混液溶液,充分混匀,得到负载鹿茸补肾汤冻干粉的丝素蛋白-壳聚糖共混溶液;
(5)、将负载鹿茸补肾汤冻干粉的丝素蛋白-壳聚糖共混溶液铺在载玻片上,室温干燥后,得到所述的负载中药的水凝胶薄膜。
进一步地,所述的壳聚糖和柞蚕丝素蛋白的混液溶液中壳聚糖和柞蚕丝素蛋白的质量比为1:1,所述的壳聚糖和柞蚕丝素蛋白的混液溶液中壳聚糖的质量浓度为2%,采用盐酸溶解后与柞蚕丝素蛋白混合。
进一步地,步骤(4)中所述的负载鹿茸补肾汤冻干粉的丝素蛋白-壳聚糖共混溶液中鹿茸补肾汤冻干粉的质量浓度为1.8mg/mL。
进一步地,所制得的负载中药的水凝胶薄膜置于体积百分含量为80%的乙醇中进行不溶化处理。
进一步地,步骤(2)中所述的浸泡后进行多次煎煮的煎煮次数为3~4次。
本发明改变了鹿茸补肾颗粒的传统剂型得到鹿茸补肾方,并将其制成冻干粉,已在体外试验中验证了鹿茸补肾方冻干粉具有抗骨肉瘤活性的能力,因此将三者结合制备补肾方冻干粉及负载鹿茸冻干粉水凝胶薄膜。
本发明的中药复方冻干粉配伍:方中以鹿茸,生精补髓,养血益阴,强筋健骨为君,臣药熟地黄与淫羊藿调补阴阳、兴肾壮骨,肉苁蓉可达入肾充髓壮骨之效,取骨碎补、鹿衔草补骨治骨、止骨痛;佐以鸡血藤配合骨碎补等诸药,在补肾益精、填髓治骨基础上,通畅经络,行气活血,即可增强健骨作用,又能达“通则不痛”之效;使以莱菔子之健胃消食理气,以防补而滋腻之弊。诸药共用,以补肾壮骨,通络止痛。
本发明负载中药复方冻干粉的水凝胶薄膜与《鹿茸多肽在制备用于治疗骨肉瘤的药物中的用途》相比,其提取鹿茸的有效成分,直接使用鹿茸多肽治疗骨肉瘤,而本发明所选择的鹿茸并非蛋白单体鹿茸多肽,而是直接将鹿茸与熟地黄、淫羊藿、肉苁蓉、鸡血藤、鹿衔草、骨碎补、莱菔子制得中药复方冻干粉的水凝胶薄膜,而鹿茸由于未进行鹿茸多肽提取过程,因此,鹿茸多肽含量极少,并不能发挥出鹿茸多肽的功效。而本发明能够实现在抑制骨肉瘤细胞迁移,细胞增殖,改善骨肉瘤细胞的划痕愈合、促进骨肉瘤细胞的坏死性凋亡和促进骨肉瘤细胞的程序性凋亡的效果显著提升,是由于本发明采用的鹿茸与熟地黄、淫羊藿调补阴阳、兴肾壮骨,以及肉苁蓉入肾充髓壮骨,骨碎补、鹿衔草补骨治骨、止骨痛,从而达到改善或延缓骨肉瘤发展,可对抑制骨肉瘤细胞迁移,影响细胞增殖。
附图说明
图1为实施例1的中药冻干粉对骨肉瘤细胞增殖实验图;图A为鹿茸补肾方作用于U2OS细胞24h;图B为鹿茸补肾方作用于U2OS细胞48h;图C为鹿茸补肾方作用于SW1353细胞24h;图D为鹿茸补肾方作用于SW1353细胞48h;*代表与对照组比较,p<0.05;
图2为实施例1的中药冻干粉对骨肉瘤细胞划痕愈合能力结果图;图A、B表示U2OS细胞愈合能力及其定量分析;图C、D表示SW1353细胞愈合能力及其定量分析。Control表示对照组,Treatment表示加入1.8mg/mL的鹿茸补肾方冻干粉;*代表与对照组比较,p<0.05;
图3为实施例1的中药冻干粉对骨肉瘤细胞Transwell迁移、侵袭能力结果图;图A、C表示U2OS细胞愈合能力;图B、D表示SW1353细胞愈合能力。Control表示对照组,Treatment表示加入1.8mg/mL的鹿茸补肾方冻干粉;*代表与对照组比较,p<0.05;
图4为实施例1的中药冻干粉对骨肉瘤细胞坏死性凋亡结果图;图A表示SW1353细胞中TNF-α的表达量;图B、C表示U2OS细胞中TNF-α和MLKL的表达量。Control表示对照组,Treatment表示加入1.8mg/mL的鹿茸补肾方冻干粉;*代表与对照组比较,p<0.05;
图5为实施例1的中药冻干粉对骨肉瘤细胞凋亡结果图;图A和C分别为U2OS细胞中Bcl-2和Bax的表达情况及Bcl-2/Bax定量分析的比值,图B和D分别为SW1353细胞中Bcl-2和Bax的表达情况及Bcl-2/Bax定量分析的比值。Control组表示对照组,Treatment组表示加入1.8mg/mL的鹿茸补肾方冻干粉;*代表与对照组比较p<0.05;
图6为实施例1的中药冻干粉水凝胶薄膜照片;
图7为实施例1的中药冻干粉水凝胶薄膜对骨肉瘤细胞(U2OS)增殖能力结果图;
图8为实施例1的中药冻干粉水凝胶薄膜的多糖含量的测定图;其中,葡萄糖标准曲线,标准方程为y=0.6172x+0.1753,r2=0.9928;柱状图为不同煎煮次数鹿茸补肾方多糖含量。*代表p<0.05;
图9为不同煎煮次数鹿茸补肾方蛋白质含量检测结果图,其中,蛋白质标准曲线,标准方程为y=0.5231x+0.139,r2=0.9981;柱状图不同煎煮次数鹿茸补肾方蛋白质含量,*代表与煎煮一次相比p<0.05;
图10为不同煎煮次数鹿茸补肾方干膏含量检测结果,*代表p<0.05。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚明白,下面将详细叙述本发明所揭示内容的精神,任何所属技术领域技术人员在了解本发明内容的实施例后,当可由本发明内容所教示的技术,加以改变及修饰,其并不脱离本发明内容的精神与范围。
本发明的示意性实施例及其说明用于解释本发明,但并不作为对本发明的限定。
实施例1
本实施例的一种负载中药复方冻干粉的水凝胶薄膜制备方法如下:
第一步,按照鹿茸补肾方配方称取药材:鹿茸10g、熟地黄30g、淫羊藿20g、肉苁蓉20g、鸡血藤20g、鹿衔草20g、骨碎补20g、莱菔子20g。将鹿茸片和其他药材分别用蒸馏水浸泡一小时。
第二步,煎煮:鹿茸进行4次煎煮,每次加20倍水单煎,第一次煎煮30min,其中,前10min武火煎煮,后20min文火煎煮;后续煎煮时间均为20min,其中前10min武火煎煮,后10min文火煎煮,将煎煮的药液用纱布过滤;其他药材加6倍水每次煎煮30min,前10min武火,后10~20min文火;煎煮后用纱布过滤;将煎煮的药液用纱布过滤,分别将不同煎煮次数的鹿茸和其他药材的药液合并,浓缩至100mL后取出5mL再与下一次的滤液合并,重复浓缩及取样步骤。
第三步,多糖含量的测定:按照苯酚-硫酸法制备多糖标准曲线,将稀释100倍后的鹿茸补肾方溶液与5%苯酚、浓硫酸按照1:1:5的比例混匀静置10min,在40℃条件下水浴5min后冷却至室温后与490nm处测定OD值,根据标准曲线计算多糖含量,如图8所示,以OD值为纵坐标(y),葡萄糖浓度为横坐标(x)制作标准曲线,曲线方程为y=0.6172x+0.1753,r2=0.9928,说明葡萄糖含量在0~1mg/mL范围内时,酶标仪测定结果线性关系良好。根据标准曲线,按照鹿茸补肾方不同煎煮次数样品溶液测定的OD值计算多糖含量,测定结果显示从煎煮一次到煎煮四次多糖含量都是呈上升趋势,煎煮四次时多糖含量达到最大值,之后煎煮五次和六次与煎煮四次相比无明显差异。
第四步,蛋白质含量测定:根据BCA试剂盒使用方法制作蛋白质标准曲线,将稀释100倍后的鹿茸补肾方溶液与BCA试剂(BCA试剂A:BCA试剂B的体积比为50:1)按照1:10的比例混匀后于37℃烘箱中孵育30min,于562nm处测定OD值,根据标准曲线计算蛋白质含量,如图9所示,以OD值为纵坐标(y),葡萄糖浓度为横坐标(x)制作标准曲线,BCA法测定的曲线方程为y=0.5231x+0.139,r2=0.9981,由此可见,测定的OD值与蛋白质浓度之间的线性关系良好。根据标准曲线计算鹿茸补肾方不同煎煮次数样品溶液的蛋白质含量,测定结果显示与煎煮一次相比,其余煎煮次数蛋白质含量均呈上升趋势,但后续各煎煮次数之间无明显差异。
第五步,干膏含量的测定:将EP管干燥至恒重记录质量,取2mL上清液加入EP管中,55℃烘箱中干燥至恒重,称量,两次数量差值即为干膏含量,如图10所示,鹿茸补肾方经过5天的时间干燥至恒重,干膏含量随着煎煮次数增加呈上升趋势,在煎煮四次达到最大值,之后煎煮五次和煎煮六次与煎煮四次之间无明显差异,干膏含量表示所得中药汤剂的有效成分,煎煮过程中溶出的有效成分随煎煮次数的增加而增加。
第六步,将煎煮四次的鹿茸补肾方通过冻干干燥得到鹿茸补肾方冻干粉备用。
第七步,将0.1M盐酸溶液溶解的2%的壳聚糖溶液和柞蚕丝素蛋白按照1:1的质量比在磁力搅拌器上搅拌15min,充分混匀,得到壳聚糖/丝素蛋白共混溶液。将溶液分成三份备用。
第八步,将鹿茸补肾汤冻干粉按照1.8的量分别加入其中两份均为1mL的壳聚糖/丝素蛋白混合溶液,在磁力搅拌器上搅拌15min充分混匀即得两份浓度分为1.8mg/mL的负载鹿茸补肾汤冻干粉的丝素蛋白-壳聚糖共混溶液。
第九步,共混溶液铺在载玻片上,室温干燥两天,即得丝素蛋白-壳聚糖载药薄膜。
第十步,薄膜浸泡在80%的乙醇中进行不溶化处理。
第十一步,薄膜的灭菌处理:将制得的三种水凝胶薄膜(壳聚糖/丝素蛋白薄膜、负载1.8mg/mL鹿茸补肾方冻干粉的壳聚糖/丝素蛋白薄膜)先浸泡在75%的乙醇中30min,取出后浸泡PBS中1h(中间换液),之后再置于超净台中紫外照射30min,最后浸泡于1640培养基中24h。
第十二步,细胞实验;实验分为对照组(只在培养皿加入载玻片)、丝素蛋白-壳聚糖薄膜组及载药薄膜组,对处于对数生长期的骨肉瘤细胞U2OS进行培养,通过噻唑蓝试剂(MTT)对细胞增殖能力进行检测。
对本实施制备的水凝胶薄膜中的冻干粉进行骨肉瘤治疗考察如下:
1)鹿茸补肾方抑制骨肉瘤细胞的增殖
将实施例1步骤六煎煮四次的鹿茸补肾方冻干粉分别对骨肉瘤细胞U2OS和软骨肉瘤细胞SW1353增殖的半抑制浓度(IC50)进行研究,鹿茸补肾方冻干粉(0、1、2、3、4mg/mL,每mL培养基中所含冻干粉质量)作用于两种细胞24h、48h后,使用MTT法进行增殖实验的测定。实验结果如图1所示。随药物作用浓度和作用时间的增加对两种细胞的抑制效果更为显著,根据SPSS软件计算结果,鹿茸补肾方作用于U2OS细胞在24h和48h的IC50值分别为3.4mg/mL和1.833mg/mL;而鹿茸补肾方作用于SW1353细胞在24h和48h的IC50值分别为2.708mg/mL和1.899mg/mL。所以在后续实验中选用了1.8mg/mL作为实验浓度。
2)鹿茸补肾方抑制骨肉瘤细胞的划痕愈合能力
细胞划痕愈合能力是检测细胞迁移能力的经典实验,通过划痕前后迁移面积的比值来判定细胞迁移能力的强弱。实验发现经鹿茸补肾方冻干粉处理后的U2OS和SW1353细胞与对照组相比细胞的迁移能力明显降低,说明鹿茸补肾方冻干粉对骨肉瘤细胞的迁移能力有抑制作用,可以影响骨肉瘤细胞的划痕愈合能力,实验结果如图2所示。
3)鹿茸补肾方抑制骨肉瘤细胞的迁移侵袭
Transwell实验可以检测细胞的迁移和侵袭能力,如图3所示,通过穿出Transwell小室的细胞数量检测实施例1的鹿茸补肾方对骨肉瘤细胞迁移侵袭能力的影响,实验结果表明鹿茸补肾方冻干粉作用后U2OS细胞和SW1353细胞迁移和侵袭能力发生改变,加药处理组细胞穿出能力减弱,穿过小室的细胞数量减少。根据实验结果可以认为鹿茸补肾方冻干粉对骨肉瘤细胞的迁移侵袭能力有抑制作用,可以影响骨肉瘤细胞在体外的转移能力。
4)鹿茸补肾方促进骨肉瘤细胞的坏死性凋亡是受调节的坏死细胞死亡形式,坏死性凋亡是由肿瘤坏死因子α(TNF-α)下游的RIPK1-RIPK3-MLKL信号级联介导的细胞死亡途径。TNF-α可以诱导细胞坏死性凋亡,而MLKL是细胞坏死性凋亡的执行蛋白,因此,本发明首先检测了鹿茸补肾方冻干粉处理后的骨肉瘤细胞U2OS和软骨肉瘤细胞SW1353中TNF-α的表达情况,发现经药物处理后的U2OS细胞TNF-α的表达量升高,而SW1353细胞的TNF-α表达量无明显差异(图4的A、4的B)。猜测鹿茸补肾方对SW1353细胞的抑制可能不通过TNF-α的作用,因此只检测了TNF-α表达量有差异的U2OS细胞中MLKL的表达量,发现鹿茸补肾方处理后的U2OS细胞中MLKL的表达量呈升高趋势(图4的C),提示了鹿茸补肾方对骨肉瘤U2OS细胞的促进坏死性凋亡作用。
5)鹿茸补肾方促进骨肉瘤细胞的程序性凋亡
BCL-2和BAX是BCL-2家族内检测细胞程序性凋亡的明星分子, BCL-2与BAX的比值影响细胞的凋亡进程。当细胞内BAX高表达时,细胞对死亡信号敏感,促进了细胞发生程序性凋亡。当BCL-2高表达时,BCL-2可以和BAX形成异源二聚体,抑制细胞程序性凋亡。通过Western Blotting实验检测BCL-2和BAX在鹿茸补肾方冻干粉作用后的表达情况判断鹿茸补肾方对骨肉瘤细胞程序性凋亡的影响。检测了对照组和1.8mg/mL鹿茸补肾方冻干粉处理组的骨肉瘤细胞中BCL-2和BAX的表达量并进行了定量分析。鹿茸补肾方通过下调U2OS细胞中BCL-2和BAX的表达促进U2OS细胞的程序性凋亡,而通过上调SW1353细胞中BCL-2和BAX的表达促进SW1353细胞的程序性凋亡。如图5所示,实验结果表明,鹿茸补肾方冻干粉能够对骨肉瘤细胞的程序性凋亡产生促进作用。
本发明从骨肉瘤细胞增殖、迁移、侵袭、凋亡等方面实验进行了研究。细胞增殖实验主要研究鹿茸补肾方对骨肉瘤细胞的抑制作用,浓度为0-4mg/mL的鹿茸补肾方冻干粉分别作用于骨肉瘤细胞U2OS和软骨肉瘤细胞SW1353,作用时间为24h及48h,发现随鹿茸补肾方浓度升高及作用时间的延长抑制作用增加;细胞迁移侵袭的研究使用了细胞划痕愈合和Transwell实验,实验发现在1.8 mg/mL鹿茸补肾方冻干粉的作用下,细胞迁移率减弱,侵袭能力也下降,提示了鹿茸补肾方对骨肉瘤细胞迁移侵袭的抑制能力,可能在抑制骨肉瘤肺转移中发挥作用;细胞凋亡实验从TNF-α诱导的细胞坏死性凋亡和细胞程序性凋亡两方面进行了研究,分别采用了ELISA和Western Blotting实验,实验结果发现鹿茸补肾方对骨肉瘤细胞U2OS的坏死性凋亡及程序性凋亡均存在促进作用,对软骨肉瘤细胞SW1353的坏死性凋亡无明显影响,但促进其程序性凋亡,提示了鹿茸补肾方对骨肉瘤细胞发生凋亡的调控作用。
6)中药冻干粉水凝胶薄膜抑制骨肉瘤细胞的增殖
结果如图6和图7所示,图6 水凝胶薄膜,如图为水凝胶薄膜,从左到右分别为壳聚糖/丝素蛋白薄膜(CS/SF)、负载1.8mg/mL鹿茸补肾方冻干粉的壳聚糖/丝素蛋白薄膜(CS/SF-1.8mg/mL)。图7骨肉瘤细胞(U2OS)增殖能力的测定,如图检测了水凝胶薄膜作用骨肉瘤细胞U2OS之后的细胞增殖能力。
Claims (5)
1.一种负载中药复方冻干粉的水凝胶薄膜的应用,其特征在于所述的水凝胶薄膜在制备抑制骨肉瘤细胞增殖药物中的应用;
所述的水凝胶薄膜是由5~15重量份的鹿茸、20~40重量份熟地黄、10~30重量份淫羊藿、10~30重量份肉苁蓉、10~30重量份鸡血藤、10~30重量份鹿衔草、10~30重量份骨碎补、10~30重量份莱菔子和壳聚糖及柞蚕丝素蛋白制成;所述的壳聚糖和柞蚕丝素蛋白的混液溶液中壳聚糖和柞蚕丝素蛋白的质量比为1:1,所述的壳聚糖和柞蚕丝素蛋白的混液溶液中壳聚糖的质量浓度为2%,采用盐酸溶解后与柞蚕丝素蛋白混合;
所述的负载中药冻干粉的水凝胶薄膜制备方法如下:
一、鹿茸煎煮液:
称取5~15重量份的鹿茸加20倍量的水浸泡,浸泡后进行4次煎煮,第一次煎煮20~30min,其中,前10min武火煎煮,后10~20min文火煎煮;后续煎煮时间均为20~30min,其中前10min武火煎煮,后10~20min文火煎煮;煎煮后用纱布过滤;
二、称取20~40重量份熟地黄、10~30重量份淫羊藿、10~30重量份肉苁蓉、10~30重量份鸡血藤、10~30重量份鹿衔草、10~30重量份骨碎补和10~30重量份莱菔子,加6倍量的水浸泡,浸泡后进行多次煎煮,每次煎煮20~30min,前10min武火,后10~20min文火;煎煮后用纱布过滤;
三、合并滤液:将步骤一煎煮后的鹿茸滤液与步骤二的滤液合并,将合并后的滤液制得鹿茸补肾汤冻干粉;
四、将步骤三得到的鹿茸补肾汤冻干粉加入到壳聚糖和柞蚕丝素蛋白的混液溶液,充分混匀,得到负载鹿茸补肾汤冻干粉的丝素蛋白-壳聚糖共混溶液;所述的负载鹿茸补肾汤冻干粉的丝素蛋白-壳聚糖共混溶液中鹿茸补肾汤冻干粉的质量浓度为1.8mg/mL或3.6mg/mL;
五、将负载鹿茸补肾汤冻干粉的丝素蛋白-壳聚糖共混溶液铺在载玻片上,室温干燥后,得到所述的负载中药冻干粉的水凝胶薄膜。
2.根据权利要求1所述的一种负载中药复方冻干粉的水凝胶薄膜的应用,其特征在于所述的水凝胶薄膜是由10重量份的鹿茸、30重量份熟地黄、30重量份淫羊藿、20重量份肉苁蓉、20重量份鸡血藤、20重量份鹿衔草、20重量份骨碎补、20重量份莱菔子和壳聚糖及柞蚕丝素蛋白制成。
3.根据权利要求1所述的一种负载中药复方冻干粉的水凝胶薄膜的应用,其特征在于所制得的负载中药冻干粉的水凝胶薄膜置于体积百分含量为80%的乙醇中进行不溶化处理。
4.根据权利要求1所述的一种负载中药复方冻干粉的水凝胶薄膜的应用,其特征在于所述的水凝胶薄膜在制备抑制骨肉瘤细胞迁移药物中的应用。
5.根据权利要求1所述的一种负载中药复方冻干粉的水凝胶薄膜的应用,其特征在于所述的水凝胶薄膜在制备抑制骨肉瘤细胞的划痕愈合、促进骨肉瘤细胞坏死性凋亡或促进骨肉瘤细胞程序性凋亡药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311048493.1A CN116808111B (zh) | 2023-08-21 | 2023-08-21 | 一种负载中药的水凝胶薄膜、其制备及抗骨肉瘤的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311048493.1A CN116808111B (zh) | 2023-08-21 | 2023-08-21 | 一种负载中药的水凝胶薄膜、其制备及抗骨肉瘤的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116808111A CN116808111A (zh) | 2023-09-29 |
CN116808111B true CN116808111B (zh) | 2023-12-26 |
Family
ID=88122392
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311048493.1A Active CN116808111B (zh) | 2023-08-21 | 2023-08-21 | 一种负载中药的水凝胶薄膜、其制备及抗骨肉瘤的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116808111B (zh) |
Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1742871A (zh) * | 2005-09-23 | 2006-03-08 | 北京阜康仁生物制药科技有限公司 | 藤黄健骨制剂及新的制备方法 |
CN101647897A (zh) * | 2009-07-16 | 2010-02-17 | 张艳 | 一种治疗骨质增生的中药丸剂 |
CN201558333U (zh) * | 2009-12-06 | 2010-08-25 | 湖北广仁药业有限公司 | 包薄膜衣的抗骨增生片 |
CN104327283A (zh) * | 2014-10-16 | 2015-02-04 | 苏州经贸职业技术学院 | 一种复合丝素蛋白水凝胶及其制备方法、应用 |
CN107468806A (zh) * | 2017-07-14 | 2017-12-15 | 大连理工大学 | 一种补肾中药组合物及其汤剂和冻干粉的制备方法 |
CN107551045A (zh) * | 2017-07-14 | 2018-01-09 | 大连理工大学 | 一种补肾中药汤剂冻干粉的制备方法及应用 |
CN110354254A (zh) * | 2019-07-09 | 2019-10-22 | 大连理工大学 | 鹿茸多肽在制备用于治疗骨肉瘤转移的药物中的用途 |
CN110368485A (zh) * | 2019-07-09 | 2019-10-25 | 大连理工大学 | 鹿茸多肽在制备用于治疗骨肉瘤的药物中的用途 |
CN113332232A (zh) * | 2021-06-15 | 2021-09-03 | 湖南绿蔓生物科技股份有限公司 | 一种含虎杖苷的组合物及其应用 |
CN115246938A (zh) * | 2020-12-18 | 2022-10-28 | 兰州理工大学 | 具有中药多糖活性的丝素蛋白水凝胶、及其制备方法和应用 |
CN116590231A (zh) * | 2023-04-13 | 2023-08-15 | 上海市第五人民医院 | 一种用于肠道肿瘤细胞培养的水凝胶支架及其制备方法 |
-
2023
- 2023-08-21 CN CN202311048493.1A patent/CN116808111B/zh active Active
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1742871A (zh) * | 2005-09-23 | 2006-03-08 | 北京阜康仁生物制药科技有限公司 | 藤黄健骨制剂及新的制备方法 |
CN101647897A (zh) * | 2009-07-16 | 2010-02-17 | 张艳 | 一种治疗骨质增生的中药丸剂 |
CN201558333U (zh) * | 2009-12-06 | 2010-08-25 | 湖北广仁药业有限公司 | 包薄膜衣的抗骨增生片 |
CN104327283A (zh) * | 2014-10-16 | 2015-02-04 | 苏州经贸职业技术学院 | 一种复合丝素蛋白水凝胶及其制备方法、应用 |
CN107468806A (zh) * | 2017-07-14 | 2017-12-15 | 大连理工大学 | 一种补肾中药组合物及其汤剂和冻干粉的制备方法 |
CN107551045A (zh) * | 2017-07-14 | 2018-01-09 | 大连理工大学 | 一种补肾中药汤剂冻干粉的制备方法及应用 |
CN110354254A (zh) * | 2019-07-09 | 2019-10-22 | 大连理工大学 | 鹿茸多肽在制备用于治疗骨肉瘤转移的药物中的用途 |
CN110368485A (zh) * | 2019-07-09 | 2019-10-25 | 大连理工大学 | 鹿茸多肽在制备用于治疗骨肉瘤的药物中的用途 |
CN115246938A (zh) * | 2020-12-18 | 2022-10-28 | 兰州理工大学 | 具有中药多糖活性的丝素蛋白水凝胶、及其制备方法和应用 |
CN113332232A (zh) * | 2021-06-15 | 2021-09-03 | 湖南绿蔓生物科技股份有限公司 | 一种含虎杖苷的组合物及其应用 |
CN116590231A (zh) * | 2023-04-13 | 2023-08-15 | 上海市第五人民医院 | 一种用于肠道肿瘤细胞培养的水凝胶支架及其制备方法 |
Also Published As
Publication number | Publication date |
---|---|
CN116808111A (zh) | 2023-09-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Xie et al. | Ganoderma lucidum extract inhibits proliferation of SW 480 human colorectal cancer cells | |
Fernandez et al. | Efficacy of Rhizophora mangle aqueous bark extract in the healing of open surgical wounds | |
Liu et al. | Camptothecine encapsulated composite drug delivery system for colorectal peritoneal carcinomatosis therapy: biodegradable microsphere in thermosensitive hydrogel | |
Xu et al. | Cinnamon cassia oil chitosan nanoparticles: Physicochemical properties and anti-breast cancer activity | |
Davarmanesh et al. | Protective effect of bilberry extract as a pretreatment on induced oral mucositis in hamsters | |
Wang et al. | ASP2-1, a polysaccharide from Acorus tatarinowii Schott, inhibits osteoclastogenesis via modulation of NFATc1 and attenuates LPS-induced bone loss in mice | |
Foote et al. | Medicinal herb use and the renal patient | |
CN116808111B (zh) | 一种负载中药的水凝胶薄膜、其制备及抗骨肉瘤的应用 | |
Ren et al. | Optimization of Hericium erinaceus polysaccharide-loaded Poly (lactic-co-glycolicacid) nanoparticles by RSM and its absorption in Caco-2 cell monolayers | |
Luo et al. | MPEG-PCL nanomicelles platform for synergistic metformin and chrysin delivery to breast cancer in mice | |
Hassani et al. | Evaluation of collagen type I and III, TGF-β1, and VEGF gene expression in rat skin wound healing treated by alginate/chitosan hydrogel containing crocetin | |
CN111494318B (zh) | 一种肿瘤靶向和还原敏感型复合胶束及其制备方法和应用 | |
CN112891559B (zh) | 促进牙髓干细胞增殖和成骨分化的多肽共修饰柚皮苷纳米脂质体及其制备方法和用途 | |
CN116942847B (zh) | 一种靶向毛囊的载药植物外泌体及其制备方法和应用 | |
CN103520439B (zh) | 一种治疗热毒蕴结型慢性骨髓炎的中药及其制备方法 | |
Jia et al. | Targeting tumor-derived exosome-mediated premetastatic niche formation: The metastasis-preventive value of traditional Chinese medicine | |
CN109771497A (zh) | 一种降压胶囊及制备方法 | |
Hou et al. | Preparation and characterization of vaccarin, hypaphorine and chitosan nanoparticles and their promoting effects on chronic wounds healing | |
CN106309758B (zh) | 一种抗胃肠癌的药物组合物 | |
Rosyid et al. | Improving Diabetic Foot Ulcer Healing with Adjuvant Bitter Melon Leaf Extract (Momordica charantia L.) | |
Manmuan et al. | Evaluation of standardized extract of Centella Asiatica on cell viability and repressive cancer migration in metastatic colorectal cancer cells in vitro | |
Kasiram et al. | Combination therapy of cisplatin and other agents for osteosarcoma: a review | |
Mahmoodi Khatonabadi et al. | Umbelliprenin Inhibited Angiogenesis and Metastasis of MDA-MB-231 Cell Line through Downregulation of CoCl2/EGFMediated PI3K/AKT/ERK Signaling | |
Gong et al. | Danggui Buxue Tang improves therapeutic efficacy of doxorubicin in triple negative breast cancer via ferroptosis | |
Xu et al. | The hematopoietic function of medicinal wine Maoji Jiu revealed in blood deficiency model rats |
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 |