CN116999451A - 柚皮苷在制备逆转卵巢癌细胞株的顺铂耐药性药物中的应用 - Google Patents
柚皮苷在制备逆转卵巢癌细胞株的顺铂耐药性药物中的应用 Download PDFInfo
- Publication number
- CN116999451A CN116999451A CN202310815433.1A CN202310815433A CN116999451A CN 116999451 A CN116999451 A CN 116999451A CN 202310815433 A CN202310815433 A CN 202310815433A CN 116999451 A CN116999451 A CN 116999451A
- Authority
- CN
- China
- Prior art keywords
- naringin
- ovarian cancer
- cisplatin
- ddp
- cells
- 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
- DFPMSGMNTNDNHN-ZPHOTFPESA-N naringin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC=2C=C3O[C@@H](CC(=O)C3=C(O)C=2)C=2C=CC(O)=CC=2)O[C@H](CO)[C@@H](O)[C@@H]1O DFPMSGMNTNDNHN-ZPHOTFPESA-N 0.000 title claims abstract description 45
- 239000001606 7-[(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxy-5-hydroxy-2-(4-hydroxyphenyl)chroman-4-one Substances 0.000 title claims abstract description 40
- 229930019673 naringin Natural products 0.000 title claims abstract description 40
- 229940052490 naringin Drugs 0.000 title claims abstract description 40
- 206010061535 Ovarian neoplasm Diseases 0.000 title claims abstract description 38
- 206010033128 Ovarian cancer Diseases 0.000 title claims abstract description 36
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 title claims abstract description 28
- 229960004316 cisplatin Drugs 0.000 title claims abstract description 28
- 239000003814 drug Substances 0.000 title claims abstract description 26
- 206010059866 Drug resistance Diseases 0.000 title claims description 9
- 229940079593 drug Drugs 0.000 title abstract description 13
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 230000000694 effects Effects 0.000 claims abstract description 10
- 230000019491 signal transduction Effects 0.000 claims abstract description 4
- 230000001105 regulatory effect Effects 0.000 claims abstract description 3
- 102100035186 DNA excision repair protein ERCC-1 Human genes 0.000 claims description 7
- 101000876529 Homo sapiens DNA excision repair protein ERCC-1 Proteins 0.000 claims description 7
- 210000002966 serum Anatomy 0.000 claims description 4
- 230000035755 proliferation Effects 0.000 claims description 2
- 238000011580 nude mouse model Methods 0.000 abstract description 5
- 241001465754 Metazoa Species 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 43
- 102100026808 Mitochondrial import inner membrane translocase subunit Tim8 A Human genes 0.000 description 31
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- 239000007788 liquid Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 239000012980 RPMI-1640 medium Substances 0.000 description 6
- 230000004663 cell proliferation Effects 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 229910052697 platinum Inorganic materials 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 238000011577 humanized mouse model Methods 0.000 description 4
- 210000000987 immune system Anatomy 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 210000005104 human peripheral blood lymphocyte Anatomy 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000043136 MAP kinase family Human genes 0.000 description 2
- 108091054455 MAP kinase family Proteins 0.000 description 2
- 238000000134 MTT assay Methods 0.000 description 2
- 231100000002 MTT assay Toxicity 0.000 description 2
- ZONYXWQDUYMKFB-UHFFFAOYSA-N SJ000286395 Natural products O1C2=CC=CC=C2C(=O)CC1C1=CC=CC=C1 ZONYXWQDUYMKFB-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 235000020971 citrus fruits Nutrition 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229930003949 flavanone Natural products 0.000 description 2
- 235000011981 flavanones Nutrition 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000000306 recurrent effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 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 description 2
- 238000002054 transplantation Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- FTVWIRXFELQLPI-ZDUSSCGKSA-N (S)-naringenin Chemical compound C1=CC(O)=CC=C1[C@H]1OC2=CC(O)=CC(O)=C2C(=O)C1 FTVWIRXFELQLPI-ZDUSSCGKSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 206010061819 Disease recurrence Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 208000010201 Exanthema Diseases 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000634900 Homo sapiens Transcriptional-regulating factor 1 Proteins 0.000 description 1
- 239000002033 PVDF binder Substances 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
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940116193 Protein phosphatase inhibitor Drugs 0.000 description 1
- 241001506137 Rapa Species 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 102000000763 Survivin Human genes 0.000 description 1
- 108010002687 Survivin Proteins 0.000 description 1
- 102100029446 Transcriptional-regulating factor 1 Human genes 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 235000021028 berry Nutrition 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 230000003034 chemosensitisation Effects 0.000 description 1
- 239000006114 chemosensitizer Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 201000005884 exanthem Diseases 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 150000002207 flavanone derivatives Chemical class 0.000 description 1
- -1 flavanone glycoside Chemical class 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 235000006486 human diet Nutrition 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000006996 mental state Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- WGEYAGZBLYNDFV-UHFFFAOYSA-N naringenin Natural products C1(=O)C2=C(O)C=C(O)C=C2OC(C1)C1=CC=C(CC1)O WGEYAGZBLYNDFV-UHFFFAOYSA-N 0.000 description 1
- 235000007625 naringenin Nutrition 0.000 description 1
- 229940117954 naringenin Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000065 noncytotoxic Toxicity 0.000 description 1
- 230000002020 noncytotoxic effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000003934 phosphoprotein phosphatase inhibitor Substances 0.000 description 1
- 108091005981 phosphorylated proteins Proteins 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 206010037844 rash Diseases 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 208000016691 refractory malignant neoplasm Diseases 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/243—Platinum; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Inorganic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Endocrinology (AREA)
- Reproductive Health (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明属于药物技术领域,具体公开了柚皮苷在制备逆转卵巢癌细胞株的顺铂耐药性药物中的应用。本发明通过构建人源化卵巢癌裸鼠模型,研究了柚皮苷通过调节p38MAPK信号通路逆转A2780/DDP细胞耐顺铂的作用,并在动物体内进一步加以验证。
Description
技术领域
本发明属于生物医药技术领域,具体涉及柚皮苷在制备逆转卵巢癌细胞株的顺铂耐药性药物中的应用。
背景技术
卵巢癌(OC)作为一种女性常见的妇科恶性肿瘤,在世界范围内的死亡率较高,是常见的致死性肿瘤之一。手术及化疗作为目前最主要的治疗方式,铂类药物是一线化疗药物。在含铂治疗后6个月内疾病复发的患者被归类为铂类药物耐药,约25%的患者患有耐铂卵巢癌。因此,每年与OC相关的死亡病例在不断增加,虽然一些药物在复发情况下显示出益处,但关于其在铂耐药疾病复发情况下的疗效数据非常有限。由于该病的铂类耐药和高复发率给OC患者和社会带来了沉重负担,因此需要寻找一种药物能够逆转其耐药性来改善预后。
近来,有建议在人类饮食中添加更多的水果和浆果,尤其是柑橘类水果,能够帮助预防癌症并抑制癌症生长。柚皮苷(NG)作为一种衍生自黄烷酮柚皮素的黄烷酮糖苷,存在于许多植物物种中,尤其是柑橘类水果中,具有多种药理作用,包括抗氧化、抗炎和抗癌活性。在许多研究中,NG作为联合疗法的治疗效果比单一疗法要好得多,还可能能够作为有效的化学增敏剂,协同增强当前抗癌药物的细胞毒性作用,并克服耐药癌症细胞。它们已被证明在乳腺癌、膀胱癌和宫颈癌中可以抑制肿瘤细胞增殖并刺激细胞凋亡,但柚皮苷是否可以逆转卵巢癌耐顺铂(DDP)的作用仍然知之甚少。
发明内容
针对现有技术中的不足与难题,本发明旨在提供柚皮苷在制备逆转卵巢癌细胞株的顺铂耐药性药物中的应用。本发明拟验证柚皮苷可用于逆转卵巢癌细胞株的顺铂耐药,并在动物体内进一步加以验证。
本发明通过以下技术方案予以实现:
本发明提供柚皮苷在制备逆转卵巢癌细胞株的顺铂耐药性药物中的应用。
优选地,将柚皮苷与卵巢癌顺铂联合,制得所述药物。
优选地,所述药物中,卵巢癌顺铂的浓度为1.25~20μg/mL;所述柚皮苷的浓度为5~80μmol/L。
优选地,所述药物通过调节p38 MAPK信号通路逆转A2780/DDP细胞耐顺铂的作用。
优选地,所述药物对A2780和A2780/DDP人卵巢癌细胞增殖进行抑制。
优选地,所述药物抑制ERCC1蛋白含量。
优选地,所述药物抑制血清中的CA125和HE4蛋白。
将柚皮苷、或柚皮苷与卵巢癌顺铂联合,再与药学上可接受的辅料联合,制得所述的逆转卵巢癌细胞株的顺铂耐药性的药物。
所述的逆转卵巢癌细胞株的顺铂耐药性的药物制备成药学上可接受的任意治疗卵巢癌的药物剂型。
与现有技术相比,本发明考虑到卵巢癌的高复发率、铂类化疗的耐药性及治疗后的胃肠道毒副作用,我们通过构建人源化卵巢癌裸鼠模型,研究了柚皮苷通过调节p38MAPK信号通路逆转A2780/DDP细胞耐顺铂的作用,并在动物体内进一步加以验证。
附图说明
图1为MTT法检测细胞增殖。
图2为Western blot检测A2780/DDP蛋白表达。
图3为人源化免疫小鼠卵巢癌移植模型构建。
具体实施方式
下面结合附图,对本发明作进一步地说明。
实施例1
柚皮苷逆转卵巢癌细胞株的顺铂耐药性的药效实验
1.试剂配制
将5.8mg柚皮苷(中国北京索莱宝科技有限公司)溶解于1ml二甲基亚砜(DMSO)中制备10mmol/L柚皮苷储备溶液,使用0.22μm膜过滤,并于-20℃储存。然后,通过在RPMI-1640培养基中稀释制备5μmol/l、10μmol/l、20μmol/l、40μmol/l和80μmol/l的工作溶液。顺铂(DDP,齐鲁制药有限公司,中国山东)在RPMI-1640培养液中稀释制成浓度为200μg/ml母液,避光保存。
2.细胞培养
人卵巢癌细胞系A2780和顺铂耐药卵巢癌细胞系A2780/DDP由南昌大学医学院提供,在RPMI-1640培养基中培养,其中含10%胎牛血清、100U/mL青霉素和100U/mL链霉素,置于37℃、5% CO2培养箱中培养48小时,2~3天进行一次传代,对呈指数生长的细胞进行实验,剩余的细胞保存于液氮中。
3.MTT试验
采用MTT法检测单用柚皮苷、单用顺铂及柚皮苷+顺铂对A2780和A2780/DDP人卵巢癌细胞的抑制作用。将细胞(2*104cells/well)接种于96孔板中,培养箱中孵育过夜使细胞贴壁生长,然后用不同浓度的顺铂(1.25、2.5、5、10、20μg/ml)分别处理A2780和A2780/DDP细胞,用不同浓度的柚皮苷(5、10、20、40和80μmol/l)处理A2780/DDP细胞,对照组加入不含药物的RPMI-1640培养基,每组设5个复孔,共孵育48小时后,每孔加入20μl MTT(5mg/ml)避光培养4小时,再弃去培养液加入150μl DMSO使紫色结晶完全溶解,使用酶标仪检测波长为490nm时的OD值,计算细胞增殖抑制率,以细胞生长率>90%的柚皮苷浓度(20μmol/L)用作柚皮苷的非细胞毒性计量。再用20μmol/l的柚皮苷联合不同浓度的顺铂(1.25、2.5、5、10、20μg/ml)处理A2780/DDP人卵巢癌细胞,通过测量OD值,计算柚皮苷治疗后顺铂耐药性的逆转倍数,每个实验重复3次并计算平均值。
图1为MTT法检测细胞增殖,其中A为不同浓度柚皮苷对细胞增殖的抑制率;B为不同浓度DDP对细胞增殖的抑制率。进而验证了不同浓度DDP作用的A2780和A2780/DDP细胞的耐药值、不同浓度柚皮苷作用A2780/DDP的细胞增殖性以及柚皮苷联合DDP对A2780/DDP细胞耐药性的逆转。
4.提取A2780/DDP细胞胞浆蛋白
(1)在对数生长期间,在沉淀的细胞A2780/DDP里加双抗培养基,然后放在37℃、5%CO2浓度的环境条件下,通过细胞孵育箱进行培养,待细胞90%以上贴壁后,分为如下各组:C-对照组;D-DDP组;N-柚皮苷组;ND-DDP+柚皮苷组。每组加入p38MAPK抑制剂SB203580,放回孵育箱中继续培养24h。
(2)24h后将旧培养基弃净,用PBS缓冲液冲洗细胞两次后,连同培养皿一起放在冰上。每5-10×106个细胞中加入500μl RAPA裂解液,每组细胞分别加入1μl蛋白磷酸酶抑制剂以防止细胞裂解时释放的磷酸酶将胞内磷酸化蛋白降解,用刮勺刮擦培养皿底部以确保细胞能够的到完全裂解。裂解后将细胞放入到1.5ml的离心管,并轻轻震荡重悬,放入4℃冰箱静置2min。
(3)细胞裂解液:按照蛋白抽提试剂=1:2的比例进行混合、摇匀,并将摇匀后的液体置于4℃冰箱中进行进行静置,静置的时间为10min。
(4)将EP管放入低温(4℃)高速(12000rpm)离心机中离心15min。离心后,离心管内的液体呈现两相分化情况。然后去除上下两相的液体,对两相间的絮状蛋白进行保存。
(5)在离心管里放无水乙醇1ml,轻轻震荡使其与蛋白沉淀混匀,把离心管放入低温(4℃)高速(12000rpm)离心机中离心15min。离心后蛋白沉淀于管底。
(6)吸除EP管中所有液体,敞开管口,待无水乙醇完全蒸发后,加入适量3% SDS溶液,置于沸水中煮沸至蛋白絮状物完全溶解消失。
5.BCA法测定蛋白含量
(1)标准曲线的绘制:取一块酶标板,依次加入试剂。
(2)根据样品数量,BCA试剂A与BCA试剂B按50:1配制适量BCA工作液,充分混匀。
(3)各孔加入200μL BCA工作液。
(4)把酶标板放在振荡器上振荡30s,37℃放置30分钟,然后在562nm下比色测定。以蛋白含量(μg)为横坐标,吸光值为纵坐标绘出标准曲线。
(5)稀释待测样品至合适浓度,使样品稀释液总体积为20μL,加入BCA工作液200μL,充分混匀,37℃放置30分钟后,以标准曲线号管做参比,在562nm波长下比色,记录吸光值。
(6)根据所测样品的吸光值,在标准曲线上即可查得相应的蛋白量(μg),除以样品稀释液总体积(20μL),乘以样品稀释倍数即为样品实际浓度(单位:μg/μL)。
6.Western blot实验
取对数生长期的A2780/DDP细胞,并分别作用于DDP组(10μg/ml)、Nar组(20μmol/l)和联合组(DDP 10μg/ml,Nar 20μmol/l)以及SB203580组(10、20、40μmol/l)24小时后,在冰上用蛋白酶RIPA裂解液裂解30min,将裂解后的细胞置于离心管中,在4℃离心机中12000rpm离心15min后收集上清液制备蛋白质样品。用10%的SDS-PAGF凝胶进行聚丙烯酰胺凝胶电泳分离蛋白质,并转移至PVDF膜上,在室温下将其封闭于5%脱脂乳中90min后,将膜在4℃下与特异性一级抗体进行孵育过夜,包括兔抗β-actin(1:1000,CST、4970)、兔抗P38 MAPK(1:2000,Abmart、T55600F)、兔抗磷酸化P38 MAPK(p-P38 MAPK,1:2000,Abmart、TP56391F)、兔抗ERCC1(1:2500,Proteintech、14586-1-AP)、兔抗P-gp(1:500,Proteintech、22336-1-AP),室温孵育二抗1h,最后加入ECL发光试剂显影,以β-actin为内参,采用Image J统计蛋白条带灰度值。
图2为Western blot检测A2780/DDP蛋白表达,其中A为各组p38MAPK和ERCC1相对含量的比较;B为使用SB203580后各组Survivin和ERCC1相对含量的比较。图中C组表示对照组,N组表示柚皮苷组,D组表示DDP组,ND组表示柚皮苷+DDP联合组。
由图2可以看出,柚皮苷、柚皮苷联合DDP均能抑制人卵巢癌胞p38MAPK和ERCC1蛋白水平的表达,且柚皮苷联合DDP效果更优。
7.免疫系统人源化小鼠模型的构建、饲养及鉴定
7.1小鼠饲养
购买40只SPF级雌性裸鼠,体重22±2g,鼠龄5-6周龄,SPF级屏障设施中饲养。饲养环境的温度维持在(23±2)℃,相对湿度维持在50%~60%,光照12h/12h正常明暗交替,小鼠笼盒、垫料、饲料及饮用水经过高温高压蒸汽灭菌处理,小鼠自由饮水、摄食。
7.2人外周血淋巴细胞FBL的分离
采用Ficoll密度梯度离心法将人新鲜外周血中的淋巴细胞提取出来。
具体步骤如下:原料:全血、无菌PBS溶液、人外周血淋巴细胞分离液、RPMI 1640培养基、胎牛血清(FBS)、双抗P/S
(1)配制所需的溶液:细胞培养基:RPMI 1640+10% FBS+1% P/S
(2)将获得的抗凝新鲜外周全血标本10ml转入50ml离心管中,加入10ml PBS溶液稀释,轻轻混匀;
(3)取四支15ml离心管,先加入5ml人外周血淋巴细胞分离液,然后将稀释的血液轻轻加到四支离心管的分离液上层,一定要轻柔,避免两种溶液混合在一起,每只离心管各5ml稀释血液;离心,2000rpm,20min;
(4)离心结束后,可以观察到从离心管底部到管口分为四层。红细胞比重最大,主要分布在离心管底部为第一层;接着是人淋巴细胞分离液层;第三层为淋巴细胞层,最上面一层为血浆层;用吸管将第三层白膜层即淋巴细胞层吸取在另一干净的15ml离心管中;
(5)向白膜层细胞管中加入10ml PBS洗涤白膜层细胞,离心,1500rpm,10min,20℃;弃掉上清液,再加入5mL的PBS重悬细胞,离心,1500rpm,10min,20℃,重复该步骤一次;
(6)弃上清后加入培养基重悬目的细胞,并计数及台盼蓝染色细胞活力检测;
(7)0.4%台盼蓝染色:用100μL移液器吸取90μL分离的目的细胞悬液,转移至1.5ml的无菌离心管中,再取10μL台盼蓝溶液加入管中,吹打混匀,使其终浓度为0.04%;室温静置染色3-5min;
(8)利用细胞计数仪计数并显微镜下观察细胞存活情况:死细胞被台盼蓝染成蓝色,而活细胞不着色具有较强的折光性。
7.3免疫系统人源化小鼠模型的构建
将分离的PBL以3×107/200μL,通过腹腔注射入裸鼠体内。具体操作步骤如下:
(1)选取5-6周龄雌性裸鼠,适应性饲养1周;
(2)用培养基将新鲜分离的PBL细胞重悬至浓度为3×107/ml,并在注射前短暂放置于冰上保存;
(3)使用1ml无菌注射器吸取200μL PBL悬液,腹腔注射入小鼠体内;
(4)监测小鼠各项指标的变化,如小鼠精神状态、饮食、活动力,有无皮疹等,并在整个研究过程中评估GVHD的产生。
7.4流式细胞术检测外周血中人免疫细胞的比例
人免疫系统重建2天后,通过剪鼠尾采集100-200μL,流式细胞仪检测小鼠外周血中的人源性CD3+T细胞,以证实人源化T细胞免疫重建。
图3为人源化免疫小鼠卵巢癌移植模型构建,其中,A为流式细胞术检测空白小鼠和人源化小鼠的T细胞。B为ELISA法检测各组血清CA125和HE4。C为各组卵巢肿瘤大小。D为各组正常组织和移植瘤的苏木精-伊红(HE)和免疫组化(IHC)检测;E为Western blot检测各组p38MAPK、p-p38MAPK、ERCC1、p-gp蛋白表达的比较。
图3中B组表示空白组,C组表示对照组,N组表示柚皮苷组,D组表示DDP组,ND组表示柚皮苷+DDP联合组。
通过构建免疫系统人源化小鼠模型,可以看出,柚皮苷组、柚皮苷联合DDP均能降低血清CA125和HE4,经柚皮苷组、柚皮苷联合DDP治疗后,卵巢肿瘤大小明显减低,且均能抑制人卵巢癌胞p38MAPK、p-p38MAPK、ERCC1、p-gp等蛋白表达;柚皮苷联合DDP的效果更好。
以上所述仅表达了本发明的优选实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形、改进及替代,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (7)
1.柚皮苷在制备逆转卵巢癌细胞株的顺铂耐药性药物中的应用。
2.根据权利要求1所述的应用,其特征在于:将柚皮苷与卵巢癌顺铂联合制得所述药物。
3.根据权利要求2所述的应用,其特征在于:所述药物中,卵巢癌顺铂的浓度为1.25~20μg/mL;所述柚皮苷的浓度为5~80μmol/L。
4.根据权利要求1或2所述的应用,其特征在于:所述药物通过调节p38 MAPK信号通路逆转A2780/DDP细胞耐顺铂的作用。
5.根据权利要求1或2所述的应用,其特征在于:所述药物对A2780和A2780/DDP人卵巢癌细胞增殖进行抑制。
6.根据权利要求1或2所述的应用,其特征在于:所述药物抑制ERCC1蛋白含量。
7.根据权利要求1或2所述的应用,其特征在于:所述药物抑制血清中的CA125和HE4蛋白。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310815433.1A CN116999451A (zh) | 2023-07-05 | 2023-07-05 | 柚皮苷在制备逆转卵巢癌细胞株的顺铂耐药性药物中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310815433.1A CN116999451A (zh) | 2023-07-05 | 2023-07-05 | 柚皮苷在制备逆转卵巢癌细胞株的顺铂耐药性药物中的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116999451A true CN116999451A (zh) | 2023-11-07 |
Family
ID=88573670
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310815433.1A Pending CN116999451A (zh) | 2023-07-05 | 2023-07-05 | 柚皮苷在制备逆转卵巢癌细胞株的顺铂耐药性药物中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116999451A (zh) |
-
2023
- 2023-07-05 CN CN202310815433.1A patent/CN116999451A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108402472B (zh) | 一种女用蚕蛹提取生物蛋白 | |
CN111346105A (zh) | Cx43介导的青蒿素B联合顺铂抗肺癌作用及相关机制 | |
CN113230259A (zh) | 逆转紫杉醇耐药性的化合物伊曲茶碱的药物用途 | |
CN116999451A (zh) | 柚皮苷在制备逆转卵巢癌细胞株的顺铂耐药性药物中的应用 | |
Li et al. | Effect of procyanidins from Pinus koraiensis bark on growth inhibition and expression of PCNA and TNF-[alpha][alpha] in mice with U14 cervical cancer | |
CN102145030B (zh) | 具有调节免疫抑制肿瘤作用的奇异果软胶囊的制备方法 | |
CN111000851B (zh) | 丹参酮i在制备肿瘤细胞自噬诱导剂药物中的应用 | |
CN108721313A (zh) | 一种维生素d受体激动剂 | |
CN114699370A (zh) | 一种共载多粘菌素e和姜黄素的脂质体的制备方法及其应用 | |
CN113633639A (zh) | 一种德法替尼在治疗血小板减少症药物中的应用 | |
CN113577086A (zh) | 异戊酰螺旋霉素类化合物或其组合物在制备治疗免疫失调的药物中的应用 | |
CN109260197A (zh) | 靛玉红类化合物和硼替佐米在制备治疗多发性骨髓瘤的药物中的用途 | |
CN111643488A (zh) | 补骨脂酚在提高化疗药抗肿瘤作用中的应用 | |
Lu et al. | Effect of Acanthopanax giraldii Harms Var. Hispidus Hoo polysaccharides on the human gastric cancer cell line SGC-7901 and its possible mechanism | |
CN110215523B (zh) | 甲基hispolon在制备抗宫颈癌的药物中的应用 | |
CN115192570B (zh) | 仙鹤草内酯在制备预防和/或治疗肺癌的药物中的用途 | |
CN104825507A (zh) | 一种藤梨根提取物及其在制备治疗胆管癌药物中的应用 | |
CN108434133A (zh) | 一种小白菊内酯用作骨形成蛋白9抑制剂的生物医药用途 | |
CN117045639B (zh) | 一种用于治疗胃癌的药物组合物及其应用 | |
CN111729074B (zh) | 多肽rl25在制备抗肿瘤药物中的应用 | |
TW201304789A (zh) | 台灣綠蜂膠萃取物用於減緩患者病情發展之用途 | |
CN117122605A (zh) | 杨梅苷在治疗血小板减少症药物制备中的应用 | |
SN et al. | Therapeutic Comparison Between Alcoholic and Aqueous Plant Extract of Tannins with Metronidazole in Experimentally Infected Laboratory Mice Cryptosporidium parvum Oocysts | |
CN107174595B (zh) | 桐花树叶正丁醇提取物及其制备和治疗前列腺癌的应用 | |
CN117159525A (zh) | Cert抑制剂在制备治疗急性髓系白血病的药物中的应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |