WO2023082803A1 - 一种抑制肝癌细胞生长的活性多肽及其制备方法和应用 - Google Patents
一种抑制肝癌细胞生长的活性多肽及其制备方法和应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- 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
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates to the technical field of biomedicine, in particular to an active polypeptide for inhibiting the growth of liver cancer cells and its preparation method and application.
- hepatitis has been one of the major global public health problems. According to the latest data from WHO, there are 2 billion people infected with hepatitis virus in the world, of which about 500 million people are infected with chronic hepatitis, and about 1 million people suffer from it every year. die. Liver-related diseases, including various hepatitis, liver cirrhosis, and liver fibrosis, are major diseases that plague human health. Hepatitis, liver cirrhosis, and liver fibrosis will cause a large number of liver cell necrosis, leading to liver failure and even life-threatening.
- active peptides As a new type of protein drug, active peptides have aroused strong interest of scholars.
- the unique amino acid composition and amphiphilicity of the active polypeptide enable the polypeptide to combine with macromolecules such as nucleic acids, proteins, etc. Molecules that disrupt the normal function of cells and cause cell death.
- peptide drugs have made a lot of progress in the treatment of liver diseases, but so far, there is still a lack of effective therapeutic strategies and effective drugs for the treatment of liver diseases.
- the purpose of the present invention is to overcome the existing technical defects and provide an active polypeptide that inhibits the growth of liver cancer cells and its preparation method and application.
- the active polypeptide of the present invention can target liver organs, inhibit the proliferation and growth of liver cancer cells, and accelerate Tissue repair, restore liver function.
- the present invention provides the following technical solutions:
- an active polypeptide for inhibiting the growth of liver cancer cells is provided, the amino acid sequence of the active polypeptide is VNNSSLIGLGYTQTLKPGIK.
- the synthesis direction of the synthesis reaction is: sequential condensation from the C-terminal to the N-terminal according to the amino acid sequence of the active polypeptide.
- the cutting fluid includes the following components: TFA 95%, water 1%, EDT 2% and TIS 2%.
- the third aspect provides the use of the active polypeptide as described in the first aspect in the preparation of drugs for the prevention or treatment of hepatitis, liver cirrhosis and liver fibrosis.
- the medicine of the present invention can prevent or treat hepatitis, liver cirrhosis and liver fibrosis.
- the active polypeptide of the present invention can be formulated as an active ingredient in a non-toxic, inert and pharmaceutically acceptable carrier medium.
- the formulated drug can be administered by conventional routes, including but not limited to oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
- the dosage form of the drug is a drug for oral administration
- it contains a safe and effective amount of the active polypeptide of the present invention and a pharmaceutically acceptable carrier and/or adjuvant
- the drug for oral administration can be made into tablets, powders, granules
- the excipient used can be at least one of starch, lactose, sucrose, mannose, and hydroxymethylcellulose
- the disintegrant can be cross-linked polyvinylpyrrolidone, carboxylated At least one of sodium methyl starch
- binder can be at least one of gelatin, polyethylene glycol.
- Drugs for oral administration can also be formulated into emulsions, syrups, etc. in addition to the above-mentioned dosage forms.
- the medicine of the present invention can also be made into injections, which can be made into injections with water for injection, physiological saline, and glucose water, and the above-mentioned injections can be prepared by conventional methods.
- the active polypeptide of the present invention can also be used in combination with other drugs for treating hepatitis, liver cirrhosis and liver fibrosis.
- the present invention has the following beneficial effects:
- the active polypeptide provided by the invention can target the liver organ, significantly inhibit the DNA synthesis of tumor cells, inhibit the proliferation and growth of liver cancer cells, accelerate the repair of liver tissue, and restore liver function.
- Fig. 1 is the high performance liquid chromatogram of the active polypeptide provided by the present invention
- Fig. 2 is the mass spectrogram of the active polypeptide provided by the present invention.
- Figure 3 is a graph showing the inhibitory effect of active polypeptides provided by the present invention on the growth of SMMC-7721, LO-2, Chang liver, HepG-2 and Huh-7 cells.
- weight content herein can be represented by the symbol “%”.
- compositions and methods/processes of the present invention may comprise, consist of and consist essentially of the essential elements and limitations described herein and any additional or optional ingredients, components, steps or limitations described herein .
- DCM dichloromethane
- HBTU benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
- DIEA is N,N-diisopropyl Ethylamine
- DMF is N,N-dimethylformamide
- TFA is trifluoroacetic acid
- EDT is 1,2-ethanedithiol
- Fmoc 9-fluorenylmethoxycarbonyl
- TIS is triisopropylsilane.
- This embodiment provides a method for preparing an active polypeptide, comprising the following steps:
- the cleaved polypeptide liquid is blown dry with nitrogen, washed 6-8 times with ether, dried at room temperature, and purified by preparative HPLC chromatography to obtain active polypeptide.
- the amino acid sequence of the active polypeptide is VNNSSLIGLGYTQTLKPGIK.
- the active polypeptide that embodiment 1 obtains is carried out liquid chromatography analysis, and its condition is:
- Mobile phase A 0.1% TFA aqueous solution
- Mobile phase B 0.1% TFA acetonitrile solution
- the present embodiment further verifies the inhibitory effect of the active polypeptide of embodiment 1 on the growth of SMMC-7721 cells, and the specific operation steps are as follows:
- Example 2 Take the active polypeptide prepared in Example 1, and use RPMI-1640 culture medium to make a solution containing 100 ⁇ g of active polypeptide per 1 mL.
- test product group For the test product group, add 100 ⁇ L of the test product solution to each well, and make 3 wells for each batch of the test product, and add 100 ⁇ L of RPMI-1640 culture solution to each well of the cell control group, and place it at 37 ° C, containing 5% carbon dioxide saturated water vapor Cultured in an incubator for 48 hours, took out the culture plate 4 hours before the end of the culture, sucked off the culture solution, added 0.01mol/L phosphate buffer (pH 7.3) to each well to wash once, and then added the above-mentioned phosphate buffer to each well solution (pH7.3) 100 ⁇ L and MTT solution 20 ⁇ L, continue to cultivate.
- phosphate buffer pH 7.7
- Results The value of the cell control group was 0.0631 ⁇ 0.0003, and that of the test product group was 0.0203 ⁇ 0.0009. There was a significant difference between the test product group and the cell control group, p ⁇ 0.01.
- the polypeptide of the present invention can obviously promote the growth of SMMC-7721 hepatocytes.
- Example 3 further verified the inhibitory effect of the active polypeptide of Example 1 on the growth of LO-2, Chang liver, HepG-2 and Huh-7 cells.
- LO-2, Chang liver, HepG-2 and Huh-7 cell replace SMMC-7721 cell with LO-2, Chang liver, HepG-2 and Huh-7 cell, and measure its absorbance; Wherein LO-2, Chang liver, HepG-2 and Huh- 7 are commercially purchased cells.
- Test result as shown in Figure 3 can be obtained by the test result of Figure 3, there is significant difference between test product group and cell control group, p ⁇ 0.01.
- Example 4 Combined with the test results of Example 3 and Example 4, it can be obtained that the active polypeptide of the present invention has obvious inhibitory effects on the growth of SMMC-7721, LO-2, Chang liver, HepG-2 and Huh-7 hepatocytes.
- the active polypeptide in the present invention can target the liver organ, obviously inhibit the DNA synthesis of tumor cells, inhibit the proliferation and growth of liver cancer cells, accelerate the repair of liver tissue, and restore liver function.
- the active polypeptide of the present invention can be used in the research and development of drugs for the prevention or treatment of hepatitis, liver cirrhosis and liver fibrosis.
- the drug described in the present invention uses the active polypeptide of Example 1 as the main active ingredient, and changes in the preparation system and administration methods, derivatives after simple chemical modification and adjustment of the active polypeptide are not excluded and the combination of multiple active substances.
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Abstract
涉及一种抑制肝癌细胞生长的活性多肽及其制备方法和应用，所述活性多肽的氨基酸序列为VNNSSLIGLGYTQTLKPGIK。活性多肽可以靶向肿瘤细胞，抑制肝癌细胞的DNA合成，抑制肝癌细胞的增殖生长，加速肝脏组织的修复，恢复肝功能。合成的活性多肽，可用于制备预防或治疗肝炎、肝硬化和肝纤维化药物的研发。
Description
本发明涉及生物医药技术领域，具体涉及一种抑制肝癌细胞生长的活性多肽及其制备方法和应用。
近年来，肝炎一直是全球性重要公共卫生问题之一，世卫组织最新数据显示，全球共有20亿人感染肝炎病毒，其中约有5亿人感染了慢性肝炎，每年约有100万人因此而死亡。肝脏相关的疾病，包括各种肝炎以及肝硬化、肝纤维化，是困扰人类健康的重大疾病，肝炎、肝硬化及肝纤维化均会有大量的肝细胞坏死，致肝衰竭甚至危及生命。
活性多肽作为一种新型的蛋白药物引发了学者的强烈兴趣。活性多肽其独特的氨基酸组成及结构中的两亲性，使多肽可以和细胞核内大分子如核酸、蛋白质等，以及病毒或细菌表面带负电荷的成分相结合，进而破坏细胞膜结构或胞内大分子而扰乱细胞的正常功能并导致细胞死亡。近年来，多肽类药物治疗肝脏疾病已经取得了不少进步，但是，到目前为止，治疗肝脏疾病仍旧缺乏有效的治疗策略和有效药物。
因此，提供一种活性多肽，能抑制癌细胞的DNA合成，尤其是抑制肝癌细胞再生是本领域技术人员亟需解决的问题。
发明内容
本发明目的在于为克服现有的技术缺陷，提供了一种抑制肝癌细胞生长的活性多肽及其制备方法和应用，本发明的活性多肽可以靶向肝脏器官，抑制肝癌细的增殖生长，加速肝脏组织的修复，恢复肝功能。
为了解决上述技术问题，本发明提供了以下技术方案：
第一方面，提供了一种抑制肝癌细胞生长的活性多肽，所述活性多肽的氨基酸序列为VNNSSLIGLGYTQTLKPGIK。
第二方面，提供了一种如第一方面所述的活性多肽的制备方法，包括以下步骤：
(1)以氯三苯甲基氯树脂为起始原料，由9-芴甲氧羰基保护的氨基酸为单体进行合成反应，将氯三苯甲基氯树脂接上氨基酸以合成多肽溶液；
(2)合成反应完毕后，加入切割液，将多肽溶液从氯三苯甲基氯树脂上切割下来；
(3)使用乙醚沉淀多肽溶液和/或用氮气吹干多肽溶液，并采用制备型HPLC进行纯化，得到所述活性多肽。
进一步地，所述合成反应的合成方向为：从C端向N端按照所述活性多肽的氨基酸序列依次缩合。
进一步地，按质量百分比计，所述切割液包括以下组分：TFA 95％，水1％，EDT 2％和TIS 2％。
第三方面，提供了一种如第一方面所述的活性多肽在制备预防或治疗肝炎、肝硬化和肝纤维化药物中的应用。
如上所述，本发明的药物能预防或治疗肝炎、肝硬化和肝纤维化，通常，可将本发明的活性多肽作为活性成分配制于无毒的、惰性的和药学上可接受的载体介质。配制好的药物可以通过常规途径进行给药，包括但并不限口服、肌内、腹膜内、静脉内、皮下、皮内、或局部给药。
当药物的剂型为用于口服施用的药物时，其含有安全有效量的本发明的活性多肽以及药学上可接受的载体和/或辅剂，口服施用的药物可制成片剂、散剂、颗粒剂、胶囊剂等常用剂型，所用的赋型剂可以为淀粉、乳糖、蔗糖、甘露糖、羟甲基纤维素中的至少一种；崩解剂可以为交联聚乙烯比咯烷酮、羧甲基淀粉钠中的至少一种；粘合剂可以为明胶、聚乙二醇中的至少一种。用于口服施用的药物除上述剂型外，还可以制成乳剂、糖浆剂等。
本发明的药物还可以被制成注射剂，可以与注射用水、生理盐水、葡萄糖水制成注射剂，上述注射剂可通过常规方法进行制备。
此外，本发明的活性多肽还可以与其他治疗肝炎、肝硬化和肝纤维化的药物联用。
与现有技术相比，本发明具有以下有益效果：
本发明提供的的活性多肽，可以靶向肝脏器官，明显抑制肿瘤细胞的DNA合成，抑制肝癌细胞的增殖生长，加速肝脏组织的修复，恢复肝功能。
本发明附加的方面和优点将在下面的描述中部分给出，这些将从下面的描述中变得明显，或通过本发明的实践了解到。
此处所说明的附图用来提供对本发明的进一步理解，构成本申请的一部分，并不构成对本发明的不当限定，在附图中：
图1为本发明提供的活性多肽的高效液相色谱图；
图2为本发明提供的活性多肽的质谱图；
图3为本发明提供的活性多肽对SMMC-7721、LO-2、Chang liver、HepG-2和Huh-7细胞生长的抑制作用图。
为为了更充分的理解本发明的技术内容，下面将结合附图以及具体实施例对本发明作进一步介绍和说明；显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例；基于本发明中的实施例，本领域技术人员在没有作出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
对于本领域的技术人员来说，通过阅读本说明书公开的内容，本发明的特征、有益效果和优点将变得显而易见。
除非另外指明，所有百分比、分数和比率都是按本发明组合物的总重量计算的。本文术语“重量含量”可用符号“％”表示。
本文中“包括”、“包含”、“含”、“含有”、“具有”或其它变体意在涵盖非封闭式包括，这些术语之间不作区分。术语“包含”是指可加入不影响最终结果的其它步骤和成分。术语“包含”还包括术语“由...组成”和“基本上由...组成”。本发明的组合物和方法/工艺可包含、由其组成和基本上由本文描述的必要元素和限制项以及本文描述的任一的附加的或任选的成分、组分、步骤或限制项组成。
在本发明的方案中，DCM为二氯甲烷；HBTU为苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐；DIEA为N,N-二异丙基乙胺；DMF为N,N-二甲基甲酰胺；TFA为三氟乙酸；EDT为1,2-乙二硫醇；Fmoc为9-芴甲氧羰基；TIS为三异丙基硅烷。
实施例1
本实施例提供一种活性多肽的制备方法，包括以下步骤：
(1)将3g氯三苯甲基氯树脂(取代度为1.03mmol/g)放入反应管中，加入45mL DCM，震荡30min；
(2)接第一个氨基酸
通过砂芯抽滤掉溶剂，加入10mmol的Fmoc-Gly氨基酸，加入DMF溶解，再加入30mmol的DIEA，震荡60min；
(3)脱保护
去掉DMF，加20％哌啶DMF溶液45mL，反应5min；去掉再加20％哌啶DMF溶液，15min；
(4)洗
加入30mL的DMF清洗两次，再加入30mL的DCM清洗两次，最后加入30mL的DMF清洗两次；
(5)缩合
取保护氨基酸12mmol，HBTU 13mmol，使用15mL的DMF进行溶解，加入反应管，并立刻加入DIEA 13mmol，反应30min；
(6)洗
加入30mL的DMF清洗一次，加入30mL的DCM清洗两次，加入30mL的DMF清洗两次；
7)重复步骤3-6，从右到左依次接入序列中的氨基酸直至结束；
8)清洗树脂
待树脂接入序列中的全部氨基酸后，抽干，加入30mL的DMF清洗两次，加入30mL的甲醇清洗两次，加入30mL的DMF清洗两次；加入30mL的DCM清洗两次，抽干10min；
9)从树脂上切割多肽得到多肽液
配制切割液：TFA 95％，水1％，EDT 2％，TIS 2％；
用量：30mL；切割时间：120min；
10)纯化
将切割后的多肽液用氮气吹干，用乙醚洗涤6-8次，常温干燥，用制备型HPLC色谱将粗品提纯，得到活性多肽。
上述活性多肽的氨基酸序列为VNNSSLIGLGYTQTLKPGIK。
实施例2
将实施例1得到的活性多肽进行液相色谱分析，其条件为：
流动相A：0.1％的TFA水溶液；流动相B：0.1％的TFA乙腈溶液；
梯度：20％-35％流动相B，10min；
色谱柱：Kromasil 100-5 C18 4.6×300mm(5um)；
波长：220nm；
流速：1.0mL/min。
其高效液相色谱图如图1所示；测得样品纯度为98.42％。
质谱(ESI)：质谱图如图2所示；活性多肽的分子离子峰为2103.47。
实施例3
本实施例进一步验证实施例1的活性多肽对SMMC-7721细胞生长的抑制作用，具体操作步骤如下：
取实施例1制备的活性多肽，用RPMI-1640培养液制成每1mL中含活性多肽100μg的溶液。用10％的小牛血清培养液培养SMMC-7721细胞至对数增长期，其中SMMC-7721细胞为商品化购买的细胞；用0.25％胰蛋白酶-乙二胺四醋酸二钠消化液，消化对数增长期的SMMC-7721细胞，加10％小牛血清培养液，稀释至每1mL中含2.5×10
4～5×10
4个细胞，将上述细胞悬液在96孔细胞板上铺板，每孔100μL，。
供试品组，每孔加供试品溶液100μL，每批供试品均做3个孔，细胞对照组每孔分别加RPMI-1640培养液100μL，置于37℃，含5％二氧化碳饱和水汽的培养箱中培养48小时，结束培养前4小时取出培养板，吸去培养液，每孔加入0.01mol/L磷酸盐缓冲液(pH 7.3)洗一次，然后在每孔中加入上述磷酸盐缓冲液(pH7.3)100μL和MTT溶液20μL，继续培养。
培养结束后，吸出培养液，每孔加入100μL二甲基亚砜，摇匀，在酶标仪上以550nm的波长处分别测定其吸收度，测试结果如图3所示。
结果：细胞对照组为0.0631±0.0003，供试品组为0.0203±0.0009值，供试品组和细胞对照组之间有显著性差异，p≤0.01。
结论：本发明的多肽对SMMC-7721肝细胞的生长具有明显的促进作用。
实施例4
在实施例3的基础上，本实施例进一步验证了实施例1的活性多肽对LO-2、Chang liver、HepG-2和Huh-7细胞生长的抑制作用。
按照实施例3中的方法，以LO-2、Chang liver、HepG-2和Huh-7细胞替代SMMC-7721细胞，并测定其吸收度；其中LO-2、Chang liver、HepG-2和Huh-7均为商品化购买的细胞。
测试结果如图3所示，由图3的测试结果可得，供试品组和细胞对照组 之间有显著性差异，p≤0.01。
结合实施例3和实施例4的测试结果可得：本发明的活性多肽对SMMC-7721、LO-2、Chang liver、HepG-2和Huh-7肝细胞的生长均具有明显的抑制作用。
本发明中的活性多肽，可以靶向肝脏器官，明显抑制肿瘤细胞的DNA合成，抑制肝癌细胞的增殖生长，加速肝脏组织的修复，恢复肝功能。
此外，根据上述测试结果可以得出，本发明的活性多肽可用于预防或治疗肝炎、肝硬化和肝纤维化药物的研发。
值得注意的是，本发明所述的药物，该药物以实施例1的活性多肽为主要活性成分，不排除配制体系和给药方式的变动、对该活性多肽进行简单化学修饰调整后的衍生物以及多活性物质合用等情况。
以上对本发明实施例所提供的技术方案进行了详细介绍，本文中应用了具体个例对本发明实施例的原理以及实施方式进行了阐述，以上实施例的说明只适用于帮助理解本发明实施例的原理；同时，对于本领域的一般技术人员，依据本发明实施例，在具体实施方式以及应用范围上均会有改变之处，综上所述，本说明书内容不应理解为对本发明的限制。
Claims (8)
- 一种抑制肝癌细胞生长的活性多肽，其特征在于，所述活性多肽的氨基酸序列为VNNSSLIGLGYTQTLKPGIK。
- 一种如权利要求1所述的活性多肽的制备方法，其特征在于，包括以下步骤：(1)以氯三苯甲基氯树脂为起始原料，由9-芴甲氧羰基保护的氨基酸为单体进行合成反应，将氯三苯甲基氯树脂接上氨基酸以合成多肽溶液；(2)合成反应完毕后，加入切割液，将多肽溶液从氯三苯甲基氯树脂上切割下来；(3)使用乙醚沉淀多肽溶液和/或用氮气吹干多肽溶液，并采用制备型HPLC进行纯化，得到所述活性多肽。
- 根据权利要求2所述的制备方法，其特征在于，所述合成反应的合成方向为：从C端向N端按照所述活性多肽的氨基酸序列依次缩合。
- 根据权利要求2所述的制备方法，其特征在于，按质量百分比计，所述切割液包括以下组分：TFA 95％，水1％，EDT 2％和TIS 2％。
- 根据权利要求1所述的活性多肽在制备预防或治疗肝炎、肝硬化和肝纤维化药物中的应用。
- 根据权利要求5所述的应用，其特征在于，所述药物为可注射的溶液。
- 根据权利要求5所述的应用，其特征在于，所述药物为用于口服施用的药物。
- 根据权利要求7所述的应用，其特征在于，所述用于口服施用的药物还包括药学上可接受的载体和/或辅剂。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007134328A2 (en) * | 2006-05-15 | 2007-11-22 | Van Andel Research Institute | Geldanamycins and their quinone moieties inhibit cancer by acting on mitochondrial voltage-dependent anion channel (vdac) protein |
| US20120087862A1 (en) * | 2006-08-09 | 2012-04-12 | Homestead Clinical Corporation | Organ-specific proteins and methods of their use |
| US20180372754A1 (en) * | 2015-12-23 | 2018-12-27 | Polyquant Gmbh | Bladder cancer biomarker proteins |
| WO2019169697A1 (zh) * | 2018-03-09 | 2019-09-12 | 广东药科大学 | 一种肝癌细胞靶向抗菌肽嵌合体m27-39-htpp及其应用 |
| CN113845571A (zh) * | 2021-11-10 | 2021-12-28 | 华南理工大学 | 一种抑制肝癌细胞生长的活性多肽及其制备方法和应用 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2836750A1 (en) * | 2013-12-17 | 2015-06-17 | Agada Biosciences, LLC | Method and agents to quantify proteins from tissues |
| CN108676074B (zh) * | 2018-05-28 | 2021-05-04 | 广州博瑞佳生物科技有限公司 | 一种促肝细胞生长的活性多肽 |
| CN110669126B (zh) * | 2019-10-23 | 2022-08-16 | 广州领晟医疗科技有限公司 | 多肽gpr5及其促进肝细胞增殖和抑制肝细胞凋亡的用途 |
| CN110627866B (zh) * | 2019-10-23 | 2022-10-18 | 广州领晟医疗科技有限公司 | 多肽rly4及其促进肝再生和抑制肝细胞凋亡的用途 |
| CN110713521B (zh) * | 2019-10-23 | 2022-08-16 | 广州领晟医疗科技有限公司 | 多肽cak18n及其促进肝再生和抑制肝细胞凋亡的用途 |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007134328A2 (en) * | 2006-05-15 | 2007-11-22 | Van Andel Research Institute | Geldanamycins and their quinone moieties inhibit cancer by acting on mitochondrial voltage-dependent anion channel (vdac) protein |
| US20120087862A1 (en) * | 2006-08-09 | 2012-04-12 | Homestead Clinical Corporation | Organ-specific proteins and methods of their use |
| US20180372754A1 (en) * | 2015-12-23 | 2018-12-27 | Polyquant Gmbh | Bladder cancer biomarker proteins |
| WO2019169697A1 (zh) * | 2018-03-09 | 2019-09-12 | 广东药科大学 | 一种肝癌细胞靶向抗菌肽嵌合体m27-39-htpp及其应用 |
| CN113845571A (zh) * | 2021-11-10 | 2021-12-28 | 华南理工大学 | 一种抑制肝癌细胞生长的活性多肽及其制备方法和应用 |
Non-Patent Citations (2)
| Title |
|---|
| LEMASTERS, J.J. HOLMUHAMEDOV, E.: "Voltage-dependent anion channel (VDAC) as mitochondrial governator-Thinking outside the box", BIOCHIMICA ET BIOPHYSICA ACTA. MOLECULAR BASIS OF DISEASE., AMSTERDAM, NL, vol. 1762, no. 2, 1 February 2006 (2006-02-01), NL , pages 181 - 190, XP005230503, ISSN: 0925-4439, DOI: 10.1016/j.bbadis.2005.10.006 * |
| TAISLA SOPRANI;VINICIUS KUFFER ULIANA;ROGERIO FAUSTINO RIBEIRO;SERGIO LISBOA;GABRIELLA XAVIER MARETTO;ANDR? TEIXEIRA SILVA DA FERR: "Cardiac protein changes in rats after soybean oil treatment: a proteomic study", LIPIDS IN HEALTH AND DISEASE, BIOMED CENTRAL, LONDON, GB, vol. 14, no. 1, 14 April 2015 (2015-04-14), GB , pages 26, XP021223026, ISSN: 1476-511X, DOI: 10.1186/s12944-015-0024-3 * |
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