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CN106913566A - Applications of the silver wire grass alcohol M in antineoplastic sensitizer is prepared - Google Patents

Applications of the silver wire grass alcohol M in antineoplastic sensitizer is prepared Download PDF

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CN106913566A
CN106913566A CN201510990988.5A CN201510990988A CN106913566A CN 106913566 A CN106913566 A CN 106913566A CN 201510990988 A CN201510990988 A CN 201510990988A CN 106913566 A CN106913566 A CN 106913566A
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antineoplastic
tumor
cancer
silver wire
cells
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余龙
朱恒锐
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Fudan University
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
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    • AHUMAN NECESSITIES
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    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47Quinolines; Isoquinolines
    • A61K31/475Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
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    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • A61K31/7028Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

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Abstract

本发明属于细胞生物学和医药领域,涉及银线草醇M在制备抗肿瘤药物增敏剂中的应用。银线草醇M是天然产物,具有逆转肿瘤细胞多药耐药的作用,可以作为肿瘤多药耐药的逆转剂;银线草醇M还具有增加肿瘤多药耐药细胞对抗肿瘤药物敏感性的作用,可以作为化疗增敏剂使用。本发明还提供了抗肿瘤药物和银线草醇M联合使用的药物组合物抑制肿瘤多药耐药细胞增殖的方法。本发明中的小分子化合物银线草醇M作为新的抗肿瘤药物或者其辅助成分进行开发,抑瘤效果明显,绿色环保,将为治疗和治愈肿瘤提供了一种新的途径和手段。The invention belongs to the field of cell biology and medicine, and relates to the application of silvertendrol M in the preparation of antitumor drug sensitizers. Silverwort M is a natural product that can reverse the multidrug resistance of tumor cells and can be used as a reversal agent for tumor multidrug resistance; silvertendol M also has the ability to increase the sensitivity of tumor multidrug resistant cells to antitumor drugs It can be used as a chemosensitizer. The invention also provides a method for inhibiting tumor multidrug-resistant cell proliferation with the pharmaceutical composition combined with antineoplastic drugs and silver grass alcohol M. The small molecular compound silvertendrol M in the present invention is developed as a new anti-tumor drug or its auxiliary component, has obvious anti-tumor effect, is green and environment-friendly, and will provide a new way and means for treating and curing tumors.

Description

银线草醇M在制备抗肿瘤药物增敏剂中的应用Application of silvertendrol M in preparation of antitumor drug sensitizer

技术领域technical field

本发明属于细胞生物学和医药领域,具体而言,本发明涉及银线草醇M的新用途。本发明还涉及相应的药物组合物及其应用方法。The invention belongs to the field of cell biology and medicine, and in particular, the invention relates to a new application of silvertendrol M. The invention also relates to the corresponding pharmaceutical composition and its application method.

背景技术Background technique

肿瘤是机体在各种致癌因素作用下,局部组织的某一个细胞在基因水平上失去对其生长的正常调控,导致其克隆性异常增生而形成的异常病变。我国的肿瘤病例数相当庞大,有资料显示占全世界病例数的55%。Tumor is an abnormal lesion formed by the body under the action of various carcinogenic factors when a certain cell in a local tissue loses its normal regulation of its growth at the gene level, resulting in its clonal abnormal proliferation. The number of cancer cases in my country is quite large, accounting for 55% of the total number of cases in the world.

良性肿瘤对机体的影响较小,主要表现为局部压迫和阻塞症状。恶性肿瘤由于分化不成熟、生长较快,浸润破坏器官的结构和功能,并可发生转移,因而对机体影响严重。恶性肿瘤除可引起与上述良性肿瘤相似的局部压迫和阻塞症状外,还可有发热、顽固性疼痛,晚期可出现严重消瘦、乏力、贫血和全身衰竭的状态。Benign tumors have less impact on the body, mainly showing symptoms of local compression and obstruction. Malignant tumors have a serious impact on the body due to their immature differentiation, rapid growth, infiltration and damage to the structure and function of organs, and the possibility of metastasis. In addition to local compression and obstruction symptoms similar to those of benign tumors mentioned above, malignant tumors may also cause fever and intractable pain, and severe emaciation, fatigue, anemia, and systemic exhaustion may appear in the advanced stage.

中医认为,癌症的起因首先是人体内阴阳平衡,组织细胞在不同的致癌因素长期作用下,细胞突变而引起的。其实癌组织也是人体的一部分,只有在人本阴阳平衡失调,五行生克乘侮发生变化的前提下,人体的免疫监控系统才会对其失去监控,任其发展。中草药能够以调理气血、调整阴阳平衡、维持正常生命体征而保命;以培补正气、产生抗体,清理"毒源"而治本。因而,中草药提取物成为治疗肿瘤一个重要方向。Traditional Chinese medicine believes that the cause of cancer is the balance of yin and yang in the human body, and the tissue cells are caused by cell mutations under the long-term action of different carcinogenic factors. In fact, cancer tissue is also a part of the human body. Only when the balance of yin and yang is out of balance and the five elements change, the immune monitoring system of the human body will lose its monitoring and let it develop. Chinese herbal medicine can preserve life by regulating qi and blood, adjusting the balance of yin and yang, and maintaining normal vital signs; Therefore, Chinese herbal medicine extracts have become an important direction for the treatment of tumors.

银线草醇M是中草药银线草的提取物,生物利用度高、性质比较稳定, 具有临床使用价值。随着人们对其的化学和生物学研究的深入, 其分子作用机制将逐步明确, 这将进一步推动此类化合物的化学结构修饰和构效关系研究, 并有助于提高银线草的药用价值。Silverwort M is the extract of Silverwort, a Chinese herbal medicine, with high bioavailability and relatively stable properties, and has clinical value. With the deepening of its chemical and biological research, its molecular mechanism of action will be gradually clarified, which will further promote the chemical structure modification and structure-activity relationship research of this kind of compound, and help to improve the medicinal use of Silvertracea value.

发明内容Contents of the invention

本发明的目的是提供银线草醇M的新的药物用途。The purpose of the present invention is to provide a new medicinal application of silvertradiol M.

本发明的目的是提供一种抗肿瘤药物增敏剂。The object of the present invention is to provide an antitumor drug sensitizer.

一方面,本发明提供了银线草醇M在制备抗肿瘤药物增敏剂中的应用。其中,银线草醇M的英文全称为Shizukaol M,其结构如图1所示。On the one hand, the present invention provides the application of silvertradiol M in the preparation of antitumor drug sensitizers. Among them, the full English name of silvertendrol M is Shizukaol M, and its structure is shown in FIG. 1 .

所述的肿瘤包括口腔癌、乳腺癌、肝癌、肺癌、宫颈癌、结肠癌或胰腺癌。Said tumors include oral cancer, breast cancer, liver cancer, lung cancer, cervical cancer, colon cancer or pancreatic cancer.

所述的抗肿瘤药物是长春新碱、柔红霉素、紫杉醇或者5-氟尿嘧啶。The antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil.

银线草醇M可以作为肿瘤多药耐药逆转剂。Silverwort M can be used as a reversal agent for tumor multidrug resistance.

另一方面,本发明提供了一种抗肿瘤药物组合物,所述的抗肿瘤药物组合物的有效成分是抗肿瘤药物和银线草醇M。On the other hand, the present invention provides an anti-tumor pharmaceutical composition, the active ingredients of which are anti-tumor drugs and silverwort M.

所述的抗肿瘤药物是细胞周期特异性药物或者细胞周期非特异性药物。The anti-tumor drugs are cell cycle specific drugs or cell cycle non-specific drugs.

所述的抗肿瘤药物是长春新碱、柔红霉素、紫杉醇或者5-氟尿嘧啶。The antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil.

本发明还提供了一种抑制体外肿瘤细胞生长增殖的方法,即在肿瘤细胞的培养基中加入银线草醇M和抗肿瘤药物。The present invention also provides a method for inhibiting the growth and proliferation of tumor cells in vitro, that is, adding silverwort M and antitumor drugs to the medium of tumor cells.

其中,加入银线草醇M的终浓度为1-50 μ mol/L。Wherein, the final concentration of adding silvergrass alcohol M is 1-50 μ mol/L.

所述的肿瘤包括口腔癌、乳腺癌、肝癌、肺癌、宫颈癌或者胰腺癌。The tumor includes oral cancer, breast cancer, liver cancer, lung cancer, cervical cancer or pancreatic cancer.

实施上述方法时,先加入银线草醇M,然后加入抗肿瘤药物。也可以同时加入银线草醇M和抗肿瘤药物。When carrying out the above method, first add silvertrin M, and then add antineoplastic drugs. It is also possible to add silvertrin M and antineoplastic drugs at the same time.

本发明提供了银线草醇M的新用途,即其在制备抗肿瘤药物增敏剂中的应用。银线草醇M是天然产物,具有逆转肿瘤细胞多药耐药的作用,可以作为肿瘤多药耐药的逆转剂;银线草醇M还具有增加肿瘤多药耐药细胞对抗肿瘤药物敏感性的作用,可以作为化疗增敏剂使用。本发明中的小分子化合物银线草醇M作为新的抗肿瘤药物或者其辅助成分进行开发,抑瘤效果明显,绿色环保,将为治疗和治愈肿瘤提供了一种新的途径和手段。The invention provides a new application of silvertradiol M, that is, its application in the preparation of antitumor drug sensitizers. Silverwort M is a natural product that can reverse the multidrug resistance of tumor cells and can be used as a reversal agent for tumor multidrug resistance; silvertendol M also has the ability to increase the sensitivity of tumor multidrug resistant cells to antitumor drugs It can be used as a chemosensitizer. The small molecular compound silvertendrol M in the present invention is developed as a new anti-tumor drug or its auxiliary component, has obvious anti-tumor effect, is green and environment-friendly, and will provide a new way and means for treating and curing tumors.

附图说明Description of drawings

图1为银线草醇M的结构。Fig. 1 is the structure of silvergrass alcohol M.

具体实施方式detailed description

本发明提供了一种抗肿瘤药物,所述的抗肿瘤药物的活性成分是银线草醇M。所述的肿瘤可以是肝癌细胞、胃癌细胞、宫颈癌细胞或者血癌细胞。The invention provides an antineoplastic drug, and the active ingredient of the antineoplastic drug is silver grass alcohol M. The tumor can be liver cancer cells, gastric cancer cells, cervical cancer cells or blood cancer cells.

本发明的小分子化合物可以采用各种常规的制备方法制备。例如,采用人工化学合成的方法。The small molecule compounds of the present invention can be prepared by various conventional preparation methods. For example, the method of artificial chemical synthesis is adopted.

利用本发明小分子化合物,通过各种常规筛选方法,可筛选出与银线草醇M 发生相互作用的物质,如受体、抑制剂或拮抗剂等。Utilizing the small molecule compound of the present invention, substances that interact with Agnicola M, such as receptors, inhibitors or antagonists, can be screened out through various conventional screening methods.

本发明及其抑制剂、拮抗剂等,在治疗上进行施用(给药)时,可提供不同的效果。通常,可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中,其中pH通常约为5-8,较佳地pH约为6-8, pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药,其中包括(但并不限于):肌内、腹膜内、皮下、皮内、或局部给药。When the present invention and its inhibitors, antagonists, etc. are administered (administered) therapeutically, various effects can be provided. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, and the pH value can be formulated according to Depending on the nature of the substance and the condition being treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.

以本发明的银线草醇M为例,可以将其与合适的药学上可接受的载体联用。这类药物组合物含有治疗有效量的化合物和药学上可接受的载体或赋形剂。这类载体包括(但并不限于):盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的银线草醇M可以被制成针剂形式,例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。诸如片剂和胶囊之类的药物组合物,可通过常规方法进行制备。药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。活性成分的给药量是治疗有效量,例如每天约1微克/千克体重-约5毫克/千克体重。此外,本发明的银线草醇M 还可与其他治疗剂一起使用。当然,具体剂量还应考虑给药途径、病人健康状况等因素,这些都是熟练医师技能范围之内的。Taking silvertradiol M of the present invention as an example, it can be used in combination with a suitable pharmaceutically acceptable carrier. Such pharmaceutical compositions contain a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. Silverwort M of the present invention can be prepared in the form of injection, for example, by normal methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, silvergrassol M of the present invention can also be used together with other therapeutic agents. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dose, which are within the skill of skilled physicians.

实验方法:experimental method:

1.细胞复苏1. Cell recovery

1) 从液氮罐中取出冻存管,直接投入37℃温水中,并不时摇动令其尽快融化。1) Take out the cryotube from the liquid nitrogen tank, put it directly into warm water at 37°C, and shake it from time to time to melt it as soon as possible.

2) 从37℃水浴中取出冻存管,用吸管吸出细胞悬液,注入离心管并加入10倍以上培养液,混合后低速离心,弃上清,再重复用培养液洗一次。2) Take out the cryotube from the 37°C water bath, suck out the cell suspension with a pipette, pour into the centrifuge tube and add more than 10 times of culture medium, mix and centrifuge at low speed, discard the supernatant, and wash again with culture medium again.

3) 用培养液适当稀释后,接种培养瓶,放在37℃培养箱静置培养,次日更换培养液,继续培养。培养至一定浓度时进行传代。PANC-1细胞培养在含10% Gibico胎牛血清的DMEM高糖培养基中;K562、HGC、QGY、MCF-7、PC-3等细胞培养在含10% 胎牛血清的1640培养基中,SK-hep1、HeLa、HepG2、等细胞培养在含10%胎牛血清的DMEM高糖培养基中,培养基中含100U/ml 青霉素和100μg/ml链霉素。3) After properly diluting with the culture medium, inoculate the culture bottle, place it in a 37°C incubator for static culture, replace the culture medium the next day, and continue the culture. Subculture when cultured to a certain concentration. PANC-1 cells were cultured in DMEM high-glucose medium containing 10% Gibico fetal bovine serum; K562, HGC, QGY, MCF-7, PC-3 and other cells were cultured in 1640 medium containing 10% fetal bovine serum. SK-hep1, HeLa, HepG2, and other cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum, and the medium contained 100U/ml penicillin and 100μg/ml streptomycin.

2.细胞传代培养2. Subculture of cells

每天观察细胞生长的情况,当细胞在培养瓶中长至约90%汇合时传代,约每隔2-4天传代一次。一瓶传代成三瓶,或一个25 cm2传代于一个75 cm2 的培养瓶中。方法:Observe the growth of the cells every day. When the cells grow to about 90% confluence in the culture flask, they should be subcultured every 2-4 days. Passage one bottle into three flasks, or one 25 cm 2 into one 75 cm 2 culture flask. method:

1) 用1×磷酸缓冲液洗涤细胞一次。1) Wash the cells once with 1X phosphate buffer.

2) 加入2-3ml胰酶消化液消化,置于37℃培养箱中数分钟。用手拍打细胞培养瓶,使细胞分离。2) Add 2-3ml trypsin digestion solution for digestion, and place in a 37°C incubator for several minutes. Tap the cell culture flask with your hands to detach the cells.

3) 用含10-15%的Gibico胎牛血清的合适培养基终止胰酶消化。将细胞分装于新的培养瓶中,继续培养。3) Terminate trypsinization with appropriate medium containing 10-15% Gibico fetal bovine serum. Aliquot the cells into new culture flasks and continue culturing.

悬浮细胞传代时,直接收集于离心管中离心,弃旧培养基。一般一瓶传代成三瓶的比例分装于新的培养瓶中,加入新鲜培养基继续培养。When the suspension cells are subcultured, they are directly collected in a centrifuge tube and centrifuged, and the old medium is discarded. Generally, the ratio of one bottle to three bottles is divided into new culture bottles, and fresh medium is added to continue the culture.

3.细胞冻存3. Cell cryopreservation

1) 取培养至对数生长期的细胞胰蛋白酶消化,收集于离心管中并计数,离心。1) Digest the cells cultured to the logarithmic growth phase with trypsin, collect them in a centrifuge tube, count them, and centrifuge.

2) 弃除胰蛋白酶及旧的培养液,加入配置好的冻存培养液(含10% DMSO,40%DMEM和50% Gibico胎牛血清),冻存液中细胞的最终浓度为0.5- 1×107/ml。用吸管轻轻吹打使细胞均匀,然后分装入无菌冻存管中,每管加1 -1.5ml。2) Discard the trypsin and the old culture medium, add the prepared frozen culture medium (containing 10% DMSO, 40% DMEM and 50% Gibico fetal bovine serum), the final concentration of cells in the frozen medium is 0.5-1 ×10 7 /ml. Gently pipette with a pipette to make the cells uniform, then divide into sterile cryopreservation tubes, add 1 -1.5ml to each tube.

3) 将冻存管放入冻存盒置-80℃速冻,5小时后移入液氮罐中保存。3) Put the cryopreservation tube into the freezer box and place it at -80°C for quick freezing, and transfer it to a liquid nitrogen tank for storage after 5 hours.

4. 实验材料4. Experimental materials

药物准备:Drug preparation:

银线草醇M溶于DMSO(二甲基亚砜),配制成100mM或50mM的母液备用。Silverwort M was dissolved in DMSO (dimethyl sulfoxide), and prepared as 100mM or 50mM mother solution for later use.

银线草醇M可从上海远慕生物科技有限公司购买,其结构如图1所示。Silverwort M can be purchased from Shanghai Yuanmu Biotechnology Co., Ltd., and its structure is shown in Figure 1.

维拉帕米(VPL)、长春新碱(VCR)和阿霉素(ADR)购自罗氏化学公司,纯度都大于99%。Verapamil (VPL), vincristine (VCR) and doxorubicin (ADR) were purchased from Roche Chemical Company with a purity greater than 99%.

细胞来源:Cell source:

人口腔癌细胞株KB及其耐药细胞株KB/VCR由中科院药物所提供,人乳腺癌细胞株MCF-7及其耐药细胞株MCF-7/ADR,人结肠癌HCT-8及其耐药细胞株HCT-8/VCR均购自南京凯基生物公司。Human oral cancer cell line KB and its drug-resistant cell line KB/VCR were provided by the Chinese Academy of Sciences, human breast cancer cell line MCF-7 and its drug-resistant cell line MCF-7/ADR, human colon cancer HCT-8 and its drug-resistant cell line Drug cell line HCT-8/VCR were purchased from Nanjing Kaiji Biological Company.

试剂盒:Reagent test kit:

CCK-8试剂盒购自同仁公司。CCK-8 kit was purchased from Tongren Company.

CCK-8实验CCK-8 experiment

KB和KB/VCR细胞以及MCF-7和MCF-7/ADR细胞都按照3500/孔的密度接种到96孔板中,24 h后不同浓度的ADR,VCR,银线草醇M以及VCR和银线草醇M一起用含10%胎牛血清的α-MEM配好后被加到各个孔中。培养48 h后,弃去培养液,每孔加入90 μL 不含血清的培养基和10μL CCK-8试剂。37℃反应2 h后,酶标仪读取450 nm波长的吸光值(OD450)。通过计算得到用药组相对于对照组的细胞增殖比率。空白组为不加细胞只加培养基,对照组为加入与药物同体积的DMSO,细胞存活率=(实验组OD450-空白组OD450)/(对照组OD450-空白组OD450)。通过IC50值再计算耐药倍数(Resistance Fold,RF)。KB and KB/VCR cells as well as MCF-7 and MCF-7/ADR cells were inoculated into 96-well plates at a density of 3500/well, and after 24 h, different concentrations of ADR, VCR, Ag Linacidol M was prepared together with α-MEM containing 10% fetal bovine serum and added to each well. After culturing for 48 h, the culture medium was discarded, and 90 μL of serum-free medium and 10 μL of CCK-8 reagent were added to each well. After reacting at 37°C for 2 h, the absorbance value (OD450) at a wavelength of 450 nm was read with a microplate reader. The cell proliferation ratio of the medication group relative to the control group was obtained by calculation. In the blank group, only the culture medium was added without cells, and in the control group, DMSO with the same volume as the drug was added, and the cell survival rate = (OD450 of the experimental group-OD450 of the blank group)/(OD450 of the control group-OD450 of the blank group). The resistance fold (Resistance Fold, RF) was calculated based on the IC 50 value.

RF =耐药细胞株的 IC50 值/亲本细胞株的 IC50 值。 每个浓度设 3个重复孔,实验重复 3 次。RF = IC 50 value of drug-resistant cell line/IC 50 value of parental cell line. Three replicate wells were set up for each concentration, and the experiment was repeated 3 times.

所有耐药细胞系在生长抑制实验之前在无药物培养基中生长3天。每个数值是3个独立实验结果,IC50以“均值±标准差”形式表示。VCR,长春新碱;ADR,柔红霉素;RF,耐药倍数。All drug-resistant cell lines were grown in drug-free medium for 3 days prior to growth inhibition experiments. Each value is the result of 3 independent experiments, and IC 50 is expressed in the form of "mean ± standard deviation". VCR, vincristine; ADR, daunorubicin; RF, resistance fold.

实施例1Example 1

实验结果:Experimental results:

KB/VCR和MCF-7/ADR是两种常用的多药耐药细胞株,在本实施例中,这两种细胞也表现出多药耐药的特性。如表1所示,KB/VCR细胞相对于KB细胞对VCR和ADR的耐药倍数分别为81.9倍和94.4倍,而MCF-7/ADR细胞跟MCF-7细胞相比对VCR和ADR的耐药倍数分别是38.1倍和20.9倍,显示实验所用耐药细胞具有多药耐药性,且MDR活性类似于文献报道的结果。KB/VCR and MCF-7/ADR are two commonly used multidrug-resistant cell lines, and in this example, these two kinds of cells also exhibit the characteristic of multidrug resistance. As shown in Table 1, compared with KB cells, the multiples of drug resistance of KB/VCR cells to VCR and ADR were 81.9 times and 94.4 times, respectively, while MCF-7/ADR cells were more resistant to VCR and ADR than MCF-7 cells. The drug multiples were 38.1 times and 20.9 times respectively, showing that the drug-resistant cells used in the experiment had multidrug resistance, and the MDR activity was similar to the results reported in the literature.

银线草醇M对亲本细胞株KB和MCF-7的半数抑制浓度IC50分别为71.3 μΜ和56.7μΜ,对耐药细胞株KB/VCR和MCF-7/ADR的IC50分别为170.8μΜ和141.2 μΜ,多药耐药细胞株KB/VCR和MCF-7/ADR没有表现出对化合物银线草醇M的交叉耐药,说明银线草醇M可以逃脱耐药细胞的多药耐药性。银线草醇M在10 μΜ 以下的浓度下,对细胞生长无明显的抑制,高于50 μΜ的浓度下会抑制细胞的增殖。The half-maximal inhibitory concentration IC 50 of silvertendol M to the parental cell lines KB and MCF-7 were 71.3 μM and 56.7 μM, respectively, and the IC 50 to the drug-resistant cell lines KB/VCR and MCF-7/ADR were 170.8 μM and 141.2 μM, the multidrug-resistant cell lines KB/VCR and MCF-7/ADR did not show cross-resistance to the compound silvertrin M, indicating that silvertrin M can escape the multidrug resistance of drug-resistant cells . Silverwort M did not significantly inhibit cell growth at a concentration below 10 μM, and inhibited cell proliferation at a concentration higher than 50 μM.

结论:银线草醇M能够克服耐药细胞株的耐药性,耐药细胞株对银线草醇M的敏感性基本相同。CONCLUSION: Silvertrin M can overcome the drug resistance of drug-resistant cell lines, and the sensitivity of drug-resistant cell lines to Silvertrin M is basically the same.

实施例2:CCK-8方法检测银线草醇M对肿瘤细胞多药耐药活性的逆转作用Example 2: The CCK-8 method detects the reversal effect of silvertendrol M on the multidrug resistance activity of tumor cells

KB/VCR, MCF-7/ADR和HCT-8/VCR细胞按照3500/孔的密度接种到96孔板中, 24 h后不同浓度的VCR,以及不同浓度的VCR和银线草醇M一起用含10%胎牛血清的α-MEM配好后被加到各个孔中。培养48 h后,弃去培养液,同实施实例1中方法,用CCK-8试剂盒测耐药细胞株及其亲本细胞对VCR和VCR+银线草醇M的活性,绘成曲线。每个浓度设 3个重复孔,实验重复3 次。KB/VCR, MCF-7/ADR and HCT-8/VCR cells were inoculated into 96-well plates at a density of 3500/well, and after 24 h, different concentrations of VCR, and different concentrations of VCR and silver grass alcohol M were used together α-MEM containing 10% fetal bovine serum was prepared and added to each well. After culturing for 48 h, the culture medium was discarded, and the CCK-8 kit was used to measure the activity of the drug-resistant cell line and its parental cells to VCR and VCR + silverwort M in the same manner as in Example 1, and a curve was drawn. Three replicate wells were set for each concentration, and the experiment was repeated 3 times.

实验结果:Experimental results:

KB/VCR细胞对VCR的耐药倍数为81.9倍,MCF-7/ADR对ADR的耐药倍数为20.9倍。当加入10 μM的银线草醇M后,使KB/VCR细胞对VCR的敏感性增加了4.57倍,而ADR对耐药细胞MCF-7/ADR的敏感性增加了5.02倍。银线草醇M能够逆转肿瘤多药耐药细胞对化疗药物的耐药性。而这种效应在亲本细胞中表现并不明显。The multiplicity of drug resistance of KB/VCR cells to VCR was 81.9 times, and that of MCF-7/ADR cells to ADR was 20.9 times. After adding 10 μM silvertrin M, the sensitivity of KB/VCR cells to VCR was increased by 4.57 times, while the sensitivity of ADR to drug-resistant cells MCF-7/ADR was increased by 5.02 times. Silverwort M can reverse the drug resistance of tumor multidrug-resistant cells to chemotherapy drugs. However, this effect was not obvious in the parental cells.

结论:in conclusion:

银线草醇M可以逆转肿瘤多药耐药细胞KB/VCR和MCF-7/ADR的耐药性,可以作为抗肿瘤药物的增敏剂,或者与抗肿瘤药物制成药物组合物。Silvertrin M can reverse the drug resistance of tumor multi-drug resistant cells KB/VCR and MCF-7/ADR, and can be used as a sensitizer for anti-tumor drugs, or made into a pharmaceutical composition with anti-tumor drugs.

在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. applications of the silver wire grass alcohol M in antineoplastic is prepared, described application is that silver wire grass alcohol M is preparing antineoplastic Application in sensitizer.
2. application as claimed in claim 1, it is characterised in that silver wire grass alcohol M is tumor multi-drug resistance reversal agents.
3. application as claimed in claim 1, it is characterised in that described tumour includes carcinoma of mouth, nasopharyngeal carcinoma, breast cancer, liver Cancer, lung cancer, cervical carcinoma, colon cancer or cancer of pancreas.
4. application as claimed in claim 1, it is characterised in that described antineoplastic is vincristine, daunorubicin, purple China fir alcohol or 5 FU 5 fluorouracil.
5. a kind of antineoplastic pharmaceutical compositions, it is characterised in that the active ingredient of described antineoplastic pharmaceutical compositions is anti-swollen Tumor medicine and silver wire grass alcohol M.
6. antineoplastic pharmaceutical compositions as claimed in claim 5, it is characterised in that described antineoplastic is the cell cycle Specific drug or cell cycle nonspecific agent (CCNSA).
7. antineoplastic pharmaceutical compositions as claimed in claim 5, it is characterised in that described antineoplastic is new Changchun Alkali, daunorubicin, taxol or 5 FU 5 fluorouracil.
8. it is a kind of suppress tumor cell in vitro growing multiplication method, it is characterised in that in the culture medium of tumour cell add Silver wire grass alcohol M and antineoplastic.
9. method as claimed in claim 8, it is characterised in that described tumour includes carcinoma of mouth, breast cancer, nasopharyngeal carcinoma, liver Cancer, lung cancer, cervical carcinoma or cancer of pancreas.
10. method as claimed in claim 8, it is characterised in that first add silver wire grass alcohol M, be subsequently adding antineoplastic.
CN201510990988.5A 2015-12-28 2015-12-28 Applications of the silver wire grass alcohol M in antineoplastic sensitizer is prepared Pending CN106913566A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113101337A (en) * 2021-04-22 2021-07-13 张媛 Medicine for slowing breast cancer cell division and preparation method thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113101337A (en) * 2021-04-22 2021-07-13 张媛 Medicine for slowing breast cancer cell division and preparation method thereof

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Application publication date: 20170704