WO2020211599A1 - 一种依帕司他在制备胰腺癌药物中的应用及对胰腺癌细胞分泌外泌体的抑制作用的验证方法 - Google Patents

一种依帕司他在制备胰腺癌药物中的应用及对胰腺癌细胞分泌外泌体的抑制作用的验证方法 Download PDF

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WO2020211599A1
WO2020211599A1 PCT/CN2020/080793 CN2020080793W WO2020211599A1 WO 2020211599 A1 WO2020211599 A1 WO 2020211599A1 CN 2020080793 W CN2020080793 W CN 2020080793W WO 2020211599 A1 WO2020211599 A1 WO 2020211599A1
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pancreatic cancer
exosomes
epalrestat
protein
secretion
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PCT/CN2020/080793
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English (en)
French (fr)
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肖明兵
纪易斐
顾志峰
施炜
季洁
范义辉
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南通大学附属医院
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Priority to US17/435,832 priority Critical patent/US20220151997A1/en
Priority to ZA2020/06359A priority patent/ZA202006359B/en
Publication of WO2020211599A1 publication Critical patent/WO2020211599A1/zh

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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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Definitions

  • the invention belongs to the technical field of application of epalrestat, and specifically relates to an application of epalrestat in preparing pancreatic cancer drugs and a verification method for its inhibitory effect on pancreatic cancer cells secreting exosomes.
  • pancreatic cancer plays a key role in the occurrence, development, metastasis and drug resistance of pancreatic cancer.
  • Pancreatic cancer helps its progression by secreting exosomes to shape the microenvironment of itself and other organs. Compared with normal cells and other tumors, pancreatic cancer cells secrete more exosomes, and highly malignant pancreatic cancer cells can transfer their cancerous characteristics to low-grade cancer cells through exosomes to promote their proliferation, Migration and invasion to accelerate disease progression.
  • Exosomes derived from pancreatic cancer can also be taken up by Kupffer cells in the liver, causing Kupffer cells to secrete TGF ⁇ , which in turn promotes the production of fibronectin by hepatic stellate cells, and creates a microenvironment suitable for tumor growth to promote liver metastasis. It can be seen that finding effective methods to inhibit exosomes secretion is of great significance for the clinical treatment of pancreatic cancer.
  • GW4869 a non-competitive inhibitor of neutral Smase (sphingomyelinase), is basically a recognized exosome inhibitor and can inhibit the secretion of exosomes.
  • farnesyl transferase inhibitor Manumycin A
  • Ras signaling pathway and hnRNP H1 Ras signaling pathway and hnRNP H1.
  • these drugs can inhibit the generation and release of exosomes, they have not yet started clinical trials, and the development of a new drug requires high investment and unknown risks. Therefore, how to find a new use of an old medicine has become a problem that needs to be solved at present.
  • the technical problem to be solved by the present invention is to provide a method for verifying the application of epalrestat in the preparation of pancreatic cancer drugs and its inhibitory effect on pancreatic cancer cells secreting exosomes, which has solved the problems raised in the background art.
  • the embodiment of the present invention provides an application of epalrestat in the preparation of pancreatic cancer drugs.
  • pancreatic cancer drug is used to inhibit the secretion of pancreatic cancer cell exosomes.
  • the embodiment of the present invention provides an anti-pancreatic cancer pharmaceutical composition, characterized in that the pharmaceutical composition contains epalrestat.
  • the pharmaceutical composition is used to inhibit the secretion of pancreatic cancer cell exosomes.
  • the embodiment of the present invention also provides a method for verifying the inhibitory effect of epalrestat on the secretion of exosomes of pancreatic cancer cells, which is characterized by including the following processes: (1) establishing a pancreatic cancer cell group treated with epalrestat And the control pancreatic cancer cell group, the cell supernatant exosomes were extracted by low-temperature ultracentrifugation; (2) After the collected exosomes were lysed, the BCA kit was used to quantify the protein, and the measured protein amount was used to reflect the exosomes (3) Transmission electron microscopy to verify the cup-shaped structure of exosomes wrapped in a double lipid membrane; (4) Protein polyacrylamide gel electrophoresis, Coomassie brilliant blue, to detect the concentration of exosomal proteins.
  • the step (1) specifically includes the following process: the pancreatic cancer cell group and the control pancreatic cancer cell group treated with epalrestat are treated with 80 ml of cell supernatant under the standard of the same cell number, and treated at 4°C. Centrifuge at 300 ⁇ g for 15 min, 2,000 ⁇ g for 30 min, 16,500 ⁇ g for 30 min, and then remove the precipitation. The supernatant is filtered through a 0.22 ⁇ m filter and then 150,000 ⁇ g low-temperature ultracentrifugation for 120 min. The precipitate is collected and resuspended in 100ul PBS. Store in aliquots at 80°C.
  • the step (2) specifically includes the following process: take 10 ⁇ L of the collected exosomes and fix them with 2.5% glutaraldehyde for 2 hours, wash the exosomes with PBS and resuspend them in 100 ⁇ L PBS, and drop 20 ⁇ L on a small copper sheet , 3% phosphotungstic acid water solution negative staining for 1 min, observation by transmission electron microscope.
  • the step (3) specifically includes the following process: dilute the protein standard solution to 0.5mg/mL with PBS; prepare the BCA working solution according to the number of samples, the BCA working solution is now used, and the 0.5mg/ml
  • the protein standard diluent is added to the 96-well plate in the order of 0, 1, 2, 4, 8, 12, 16, and 20L, and it is made up to 20L with PBS.
  • the concentration of the diluted standard is 0, 0.025, 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5mg/mL, add 1 ⁇ L exosomal protein sample per well, add PBS to make up to 20 ⁇ L.
  • the step (3) specifically includes the following process: preparing a 12% SDS-PAGE gel. Add 5 ⁇ loading buffer to the exosomes, boil for 20 minutes and load the sample. After the electrophoresis, wash the gel with distilled water for 10 minutes, add an appropriate amount of Coomassie Brilliant Blue Quick Staining Solution, shake on the shaker for 1 hour, and stain until a clear target is seen For protein bands, discard the staining solution, add an appropriate amount of distilled water, and take photos to observe the results.
  • the beneficial effects of the above-mentioned technical scheme of the present invention are as follows:
  • the present invention provides a new use of epalrestat, which is to inhibit exosome secretion.
  • epalrestat is There is huge application potential in clinical tumor treatment.
  • Figure 1A is a transmission electron microscope observation of the morphology and number of exosomes in the control pancreatic cancer cell group
  • Figure 1B shows the morphology and quantity of exosomes in the pancreatic cancer cell group treated with epalrestat
  • Figure 2 is a graph showing the concentration of exosomal protein detected by the BCA method in the control pancreatic cancer cell group and the pancreatic cancer cell group treated with epalrestat;
  • Figure 3 is a band diagram of protein gel electrophoresis with Coomassie brilliant blue staining to detect the concentration of exosomal protein
  • Figure 4 is a grayscale statistical diagram of protein gel electrophoresis with Coomassie brilliant blue staining to detect exosomal protein concentration bands
  • Figure 5A is a diagram of subcutaneous tumor formation in nude mice in the control group
  • Figure 5B is a diagram of subcutaneous tumor formation in nude mice in the experimental group
  • Figure 6 is a diagram showing the detection of CD63 protein expression in peripheral serum exosomes of nude mice by dot immunoblotting.
  • the terms “installed”, “connected”, and “connected” should be understood in a broad sense unless otherwise clearly specified and limited.
  • it can be a fixed connection or a detachable connection.
  • Connected or integrally connected it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the internal communication between two components.
  • the specific meaning of the above-mentioned terms in the present invention can be understood in specific situations.
  • pancreatic cancer drug is used to inhibit the secretion of pancreatic cancer cell exosomes.
  • the embodiment of the present invention provides an anti-pancreatic cancer pharmaceutical composition, characterized in that the pharmaceutical composition contains epalrestat.
  • the pharmaceutical composition is applied to inhibit exosomes secreted by pancreatic cancer cells.
  • the embodiment of the present invention also provides a method for verifying the inhibitory effect of epalrestat on the secretion of exosomes of pancreatic cancer cells, which is characterized by including the following processes: (1) establishing a pancreatic cancer cell group treated with epalrestat And the control pancreatic cancer cell group, the cell supernatant exosomes were extracted by low-temperature ultracentrifugation; (2) After the collected exosomes were lysed, the BCA kit was used to quantify the protein, and the measured protein amount was used to reflect the exosomes (3) Transmission electron microscopy to verify the cup-shaped structure of exosomes wrapped in a double lipid membrane; (4) Protein polyacrylamide gel electrophoresis, Coomassie brilliant blue, to detect the concentration of exosomal proteins.
  • a method for verifying the inhibitory effect of epalrestat on the secretion of pancreatic cancer cells includes the following steps: extracting cell supernatant exosomes using a low-temperature ultracentrifugation method.
  • the detailed method is: After epalrestat treatment, the pancreatic cancer cell group and the control pancreatic cancer cell group collect 80ml of cell supernatant under the same standard of cell number, and centrifuge at 300 ⁇ g for 15min at 4°C, and centrifuge at 2,000 ⁇ g. Centrifuge at 16,500 ⁇ g for 30 min and remove the precipitation. The supernatant is filtered through a 0.22 ⁇ m filter and then ultracentrifuged at 150,000 ⁇ g for 120 min at low temperature. The precipitate is collected and resuspended in 100ul PBS and stored in aliquots at -80°C.
  • Transmission electron microscopy verifies the cup-shaped structure of exosomes wrapped in a double lipid membrane. Take 10 ⁇ L of the collected exosomes and fix them with 2.5% glutaraldehyde for 2 hours. Wash the exosomes with PBS and resuspend them in 100 ⁇ L PBS. Take 20 ⁇ l and drop them on a small copper plate, and stain with 3% phosphotungstic acid in water for 1 min. Observe by transmission electron microscope . Among them, the morphology and number of exosomes were observed by transmission electron microscope, as shown in Figure 1A and Figure 1B.
  • Figure 1A is the morphology and number of exosomes observed by transmission electron microscope in the control pancreatic cancer cell group;
  • Figure 1B is the treatment of epalrestat The morphology and quantity of exosomes were observed by transmission electron microscope in the posterior pancreatic cancer cell group.
  • the BCA kit was used to quantify the protein, and the measured amount of protein was used to reflect the amount of exosomes.
  • Figure 2 is a statistical diagram of the concentration of exosomes detected by the BCA method in the control pancreatic cancer cell group, where contorl is the control pancreatic cancer cell group
  • Protein polyacrylamide gel electrophoresis Coomassie brilliant blue
  • Detailed method prepare 12% SDS-PAGE gel. Add 5 ⁇ loading buffer to the exosomes, boil for 20 minutes and load the sample. After the electrophoresis, wash the gel with distilled water for 10 minutes, add an appropriate amount of Coomassie Brilliant Blue Quick Staining Solution, and shake for 1 hour on a shaker. Stain until you see a clear target protein band, discard the staining solution, add an appropriate amount of distilled water, and take a photo to observe the result.
  • Use protein gel electrophoresis Coomassie brilliant blue staining to detect exosomal protein concentration as shown in Figure 3 and Figure 4.
  • Figure 3 is a band diagram of protein gel electrophoresis Coomassie brilliant blue staining to detect exosomal protein concentration
  • Figure 4 shows The gray-scale statistics of the bands of protein gel electrophoresis with Coomassie brilliant blue staining to detect the concentration of exosomal protein. The measured amount of protein was used to reflect the amount of exosomes. The above test results showed that the amount of exosomes and protein secreted by pancreatic cancer cells decreased significantly after inhibiting aldone reductase compared with the control group.
  • MARKE is a pre-stained protein Marker, which is used as a standard for determining molecular weight in SDS-PAGE (protein electrophoresis); Contorl is a control pancreatic cancer cell group; Epalrestat is a pancreatic cancer cell group treated with epalrestat .
  • nude mouse experiment was carried out in the present invention, which specifically includes the following steps: S1, nude mouse model preparation: construction of an untreated Capan2 cell line Nude mouse subcutaneous tumor model, the model forming time is 20 days; S2, the nude mouse model is divided into experimental group nude mice and control group nude mice, each group has 10 nude mice, and the experimental group nude mice are injected intraperitoneally with 50mg/day.
  • FIG. 5A is a diagram of subcutaneous tumor formation in nude mice in the control group
  • Figure 5B is a diagram of subcutaneous tumor formation in nude mice in the experimental group. Comparing 5A with Fig.
  • Figure 6 is a dot immunoblot method to detect the expression of CD63 protein in peripheral serum exosomes of nude mice.
  • the expression of CD63 is used to reflect the number of exosomes.
  • Contorl is the CD63 in peripheral serum exosomes of nude mice in the control group.
  • Protein expression; Epalrestat is the expression of CD63 protein in peripheral serum exosomes of nude mice in the experimental group; first repetition, second repetition, and third repetition are three repeated experiments.
  • the test results showed that compared with the control group of nude mice, the number of exosomes secreted by pancreatic cancer cells in the experimental group of nude mice, namely the group injected with epalrestat, was significantly reduced.
  • the specific process of the dot immunoblotting method includes 1 preparation: connect the negative pressure device, cut the NC membrane to the size of the 70-well plate, place it on the 70-well plate, turn on the negative pressure device, and set the negative pressure value to 0.06 MPa.
  • 2Sampling Set up control wells and sample wells, each well is set with 3 multiple holes, add 1 ⁇ l of control serum (several normal human mixed serum) to the control wells, add 1 ⁇ l of each sample to be tested into the sample wells, wait for the completion of the sample loading After that, the nitrocellulose membrane (NC membrane) was allowed to dry naturally for 30 minutes.
  • the reagents in the embodiment of the present invention are prepared as follows:
  • 1 ⁇ PBST buffer Dissolve 0.24g KH2PO4, 0.2g KCl, 1.44g Na2HPO4 ⁇ 12H2O and 1mLTween-20 in deionized water, dilute to 1000mL, and store at room temperature.
  • Epalrestat-an aldosterone reductase inhibitor used to prevent, improve and treat peripheral nerve disorders (numbness, pain) complicated by diabetes has a low incidence of adverse reactions, is safe and effective, and has been clinically widely used.
  • the present invention provides a new use of epalrestat that can significantly inhibit the secretion of pancreatic cancer cell exosomes after blocking the polyol pathway. This feature makes epalrestat possible to have important value in the treatment of clinical tumors, and is useful for clinical patients. Looking for potential drug targets provides a new theoretical basis.
  • Epalrestat (Epalrestat) is a clinically used aldosterone reductase inhibitor for the treatment of diabetic complications. Our results show that Epalrestat has a new purpose, which is to inhibit the secretion of exosomes. Exosomal secretion plays a key role in tumors, and epalrestat has great potential in clinical tumor treatment.

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Abstract

本发明提供一种依帕司他在制备胰腺癌药物中的应用，胰腺癌药物应用于抑制胰腺癌细胞的外泌体分泌。一种抗胰腺癌的药品组合物，药品组合物含有依帕司他。所述药品组合物应用于抑制胰腺癌细胞的外泌体分泌。本发明的实施例还提供一种依帕司他对胰腺癌细胞分泌外泌体的抑制作用的验证方法，包括以下过程：利用低温超速离心法提取细胞上清外泌体；将收集的外泌体裂解后用BCA试剂盒定量蛋白，用测得的蛋白量来反映外泌体的量；透射电镜验证外泌体的双层脂质膜包裹的杯状结构；蛋白质聚丙烯酰胺凝胶电泳考马斯亮蓝检测外泌体蛋白浓度。本发明中提供了依帕司他有一个新的用途，即抑制外泌体分泌，依帕司他在临床肿瘤治疗中有巨大的应用潜力。

Description

一种依帕司他在制备胰腺癌药物中的应用及对胰腺癌细胞分泌外泌体的抑制作用的验证方法 技术领域
本发明属于依帕司他的应用技术领域，具体涉及一种依帕司他在制备胰腺癌药物中的应用及对胰腺癌细胞分泌外泌体的抑制作用的验证方法。
背景技术
特殊的肿瘤微环境在胰腺癌的发生、发展、转移及耐药中发挥了关键作用。胰腺癌通过分泌外泌体来塑造自身及其它器官的微环境来帮助其进展。与正常细胞和其他肿瘤相比，胰腺癌细胞分泌的外泌体更多，且高度恶性的胰腺癌细胞可通过外泌体将其癌性特征转移至低度恶性的癌细胞，促进其增殖、迁移和侵袭以加速疾病进展。胰腺癌来源的外泌体也能够被肝脏中的Kupffer细胞摄取，引起Kupffer细胞分泌TGFβ，进而促进了肝星状细胞产生纤连蛋白，营造一个适宜肿瘤生长的微环境从而促进肝转移。由此可见，寻找有效的抑制外泌体分泌手段对于胰腺癌的临床治疗具有重要意义。
目前，中性Smase(鞘磷脂酶)的非竞争性抑制剂GW4869基本上是公认的外泌体抑制剂，可以抑制外泌体的分泌。并且有研究发现在前列腺癌细胞中，法尼基转移酶抑制剂(Manumycin A)通过抑制Ras信号通路和hnRNP H1的表达来抑制外泌体的生成和分泌。虽然这些药物都可以抑制外泌体生成释放，但都尚未开展临床试验，且一个新药的开发需要承担高额的投入和未知的风险。因此如何寻找一种老药的新用途已成为目前需要解决的问题。
发明内容
本发明要解决的技术问题是提供一种依帕司他在制备胰腺癌药物中的应用及对胰腺癌细胞分泌外泌体的抑制作用的验证方法，已解决背景技术中所提出的问题。
为解决上述技术问题，本发明的实施例提供一种依帕司他在制备胰腺癌药物中的应用。
进一步的，所述胰腺癌药物应用于抑制胰腺癌细胞外泌体的分泌。
本发明的实施例提供一种抗胰腺癌的药品组合物，其特征在于，所述药品组合物含有依帕司他。
进一步的，所述药品组合物应用于抑制胰腺癌细胞外泌体的分泌。
本发明的实施例还提供一种依帕司他对胰腺癌细胞分泌外泌体的抑制作用的验证方法，其特征在于，包括以下过程：(1)建立依帕司他处理后胰腺癌细胞组和对照胰腺癌细胞组，利用低温超速离心法提取细胞上清外泌体；(2)将收集的外泌体裂解后用BCA试剂盒定量蛋白，用测得的蛋白量来反映外泌体的量；(3)透射电镜验证外泌体的双层脂质膜包裹的杯状结构；(4)蛋白质聚丙烯酰胺凝胶电泳考马斯亮蓝检测外泌体蛋白浓度。
进一步的，所述步骤(1)具体包括以下过程：将依帕司他处理后胰腺癌细胞组和对照胰腺癌细胞组在细胞数量相同的标准下留取细胞上清液80ml，在4℃经300×g离心15min，2,000×g离心30min，16,500×g离心30min后去沉淀，所得上清液经过0.22μm滤嘴过滤再150,000×g低温超速离心120min，收集沉淀重悬于100ul PBS中，-80℃分装保存。
进一步的，所述步骤(2)具体包括以下过程：取10μL收集的外泌体用2.5％的戊二醛固定2h，PBS洗涤外泌体并重悬于100μL PBS中，取20ul滴于小铜片上，3％磷钨酸水溶负染1min，透射电镜观察。
进一步的，所述步骤(3)具体包括以下过程：用PBS将蛋白标准溶液稀释到0.5mg/mL；根据样品数量，配制BCA工作液，BCA工作液现配现用，将0.5mg/ml的蛋白标准稀释液按0、1、2、4、8、12、16、20L的顺序加到96孔板中，用PBS补足到20L，稀释后的标准品浓度分别为0、0.025、0.05、0.1、0.2、0.3、0.4、0.5mg/mL，每孔加1μL外泌体蛋白样品，加PBS补足到20μL。每孔加200μL BCA工作液，37℃孵育20-30min，酶标仪测定562nm的吸光度，根据标准曲线和使用的样品体积计算出样品的蛋白浓度。
进一步的，所述步骤(3)具体包括以下过程：制备12％SDS-PAGE凝胶。外泌体中加入5×loading buffer，煮沸20min后上样，电泳结束后，用蒸馏水洗涤凝胶10min，加入适量的考马斯亮蓝快速染色液，在摇床上摇动1h，染色至看到清晰的目标蛋白条带，弃染色液，加适量蒸馏水，拍照观察结果。
本发明的上述技术方案的有益效果如下：本发明中提供了依帕司他有一个 新的用途，即抑制外泌体分泌，鉴于外泌体分泌在肿瘤中的关键作用，依帕司他在临床肿瘤治疗中有巨大的应用潜力。
附图说明
图1A为对照胰腺癌细胞组透射电镜观察外泌体形态和数量图；
图1B为依帕司他处理后胰腺癌细胞组透射电镜观察外泌体形态和数量图；
图2为对照胰腺癌细胞组和依帕司他处理后胰腺癌细胞组用BCA法检测外泌体蛋白浓度图；
图3为蛋白凝胶电泳考马斯亮蓝染色检测外泌体蛋白浓度的条带图；
图4为蛋白凝胶电泳考马斯亮蓝染色检测外泌体蛋白浓度条带的灰度统计图；
图5A为对照组裸鼠皮下成瘤情况图；
图5B为实验组裸鼠皮下成瘤情况图；
图6为点免疫印迹法检测裸鼠外周血清外泌体中CD63蛋白表达量图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合附图及具体实施例进行详细描述。
在本发明的描述中，需要说明的是，术语“中心”、“上”“下”、“左”、“右”、“竖直”、“水平”、“内”、“外”、“前”、“后”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。此外，术语“第一”、“第二”、“第三”仅用于描述目的，而不能理解为指示或暗示相对重要性。
在本发明的描述中，需要说明的是，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”应作为广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通。对于本领域的普通技术人员而言，可以具体情况理解上述术语在本发明中的具体含义。
一种依帕司他在制备胰腺癌药物中的应用。
进一步的，所述胰腺癌药物应用于抑制胰腺癌细胞外泌体的分泌。
本发明的实施例提供一种抗胰腺癌的药品组合物，其特征在于，所述药品组合物含有依帕司他。
进一步的，所述药品组合物应用于抑制胰腺癌细胞分泌的外泌体。
本发明的实施例还提供一种依帕司他对胰腺癌细胞分泌外泌体的抑制作用的验证方法，其特征在于，包括以下过程：(1)建立依帕司他处理后胰腺癌细胞组和对照胰腺癌细胞组，利用低温超速离心法提取细胞上清外泌体；(2)将收集的外泌体裂解后用BCA试剂盒定量蛋白，用测得的蛋白量来反映外泌体的量；(3)透射电镜验证外泌体的双层脂质膜包裹的杯状结构；(4)蛋白质聚丙烯酰胺凝胶电泳考马斯亮蓝检测外泌体蛋白浓度。
在本发明的实施例中，一种依帕司他对胰腺癌细胞分泌外泌的抑制作用的验证方法，具体包括以下步骤：利用低温超速离心法提取细胞上清外泌体。详细方法为：将依帕司他处理后胰腺癌细胞组和对照胰腺癌细胞组在细胞数量相同的标准下留取细胞上清液80ml，在4℃经300×g离心15min，2,000×g离心30min，16,500×g离心30min后去沉淀，所得上清液经过0.22μm滤嘴过滤再150,000×g低温超速离心120min，收集沉淀重悬于100ul PBS中，-80℃分装保存。
透射电镜验证外泌体的双层脂质膜包裹的杯状结构。取10μL收集的外泌体用2.5％的戊二醛固定2h，PBS洗涤外泌体并重悬于100μL PBS中，取20ul滴于小铜片上，3％磷钨酸水溶负染1min，透射电镜观察。其中，透射电镜观察外泌体形态和数量，如图1A和图1B所示，其中，图1A为对照胰腺癌细胞组透射电镜观察外泌体形态和数量图；图1B为依帕司他处理后胰腺癌细胞组透射电镜观察外泌体形态和数量图。
将收集的外泌体裂解后用BCA试剂盒定量蛋白，用测得的蛋白量来反映外泌体的量。详细方法为：用PBS将蛋白标准溶液稀释到0.5mg/mL；根据样品数量，配制BCA工作液(试剂A:试剂B＝50:1)，BCA工作液现配现用。将0.5mg/ml的蛋白标准稀释液按0、1、2、4、8、12、16、20L的顺序加到96孔板中，用PBS补足到20L，稀释后的标准品浓度分别为0、0.025、0.05、0.1、0.2、0.3、0.4、0.5mg/mL。每孔加1μL外泌体蛋白样品，加PBS补足到20μL。每孔加200μL BCA工作液，37℃孵育20-30min。酶标仪测定562nm的 吸光度。根据标准曲线和使用的样品体积计算出样品的蛋白浓度。通过BCA试剂盒对分离得到的外泌体进行蛋白定量，如图2所示，图2为对照胰腺癌细胞组用BCA法检测外泌体蛋白浓度统计图，其中，contorl为对照胰腺癌细胞组的外泌体蛋白浓度数据；Epalrestat为依帕司他处理后胰腺癌细胞组的外泌体蛋白浓度数据。
蛋白质聚丙烯酰胺凝胶电泳考马斯亮蓝检测外泌体蛋白浓度。详细方法：制备12％SDS-PAGE凝胶。外泌体中加入5×loading buffer，煮沸20min后上样。电泳结束后，用蒸馏水洗涤凝胶10min，加入适量的考马斯亮蓝快速染色液，在摇床上摇动1h。染色至看到清晰的目标蛋白条带，弃染色液，加适量蒸馏水，拍照观察结果。用蛋白凝胶电泳考马斯亮蓝染色检测外泌体蛋白浓度，如图3和图4所示，图3为蛋白凝胶电泳考马斯亮蓝染色检测外泌体蛋白浓度的条带图，图4为蛋白凝胶电泳考马斯亮蓝染色检测外泌体蛋白浓度的条带灰度统计图。用测得的蛋白量来反映外泌体的量，通过以上的检测结果显示：与对照组相比，抑制醛酮还原酶后，胰腺癌细胞分泌的外泌体数量及蛋白量均明显下降。在图3中，MARKE为预染蛋白Marker，在SDS-PAGE(蛋白电泳)中作为测定分子量大小的标准品使用；Contorl为对照胰腺癌细胞组；Epalrestat为依帕司他处理后胰腺癌细胞组。
为验证依帕司他能抑制胰腺癌生长并且抑制外泌体分泌，在本发明中进行了裸鼠实验验证，具体包括以下步骤：S1、裸鼠模型制备：用未处理过的Capan2细胞系构建裸鼠皮下成瘤模型，模型成型时间为20天；S2、将裸鼠模型分为实验组裸鼠和对照组裸鼠，每组裸鼠为10只，对实验组裸鼠每天腹腔注射50mg/kg/d依帕司他，连续注射三天；S3、7天后，观察比较实验组裸鼠和对照组裸鼠的肿瘤体积大小；S4、提取裸鼠外周血清中的外泌体，用点免疫印迹法检测外泌体表面标志物CD63的表达量来反映外泌体的数量。其中，裸鼠皮下成瘤情况对比图，如图5A和如5B所示，如图5A为，对照组裸鼠皮下成瘤情况图；图5B为实验组裸鼠皮下成瘤情况图，由图5A和图5B进行比较可见，实验组即腹腔注射依帕司他组裸鼠肿瘤体积变小。图6为点免疫印迹法检测裸鼠外周血清外泌体中CD63蛋白表达量图，用CD63的表达量来反映外泌体的数量，其中，Contorl为对照组裸鼠外周血清外泌体中CD63蛋白表达情况；Epalrestat为 实验组裸鼠外周血清外泌体中CD63蛋白表达情况；first repetition、second repetition、third repetition为三次重复实验。检测结果显示：与裸鼠对照组相比，裸鼠实验组即腹腔注射依帕司他组胰腺癌细胞分泌的外泌体数量明显下降。
在本发明中，点免疫印迹法的具体过程包括①准备：连接负压装置，将NC膜裁剪至70孔板相当大小，放置在70孔板上，打开负压装置，设置负压值为0.06MPa。②加样：设置对照品孔和样本孔，每孔设置3个复孔，对照品孔加入1μl对照血清(数例正常人混合血清)，样本孔分别加入各待测样本1μl，待加样完毕后，将硝酸纤维素膜(NC膜)自然晾干30min。③封闭：将NC膜置于5％的脱脂奶粉(TBS-T配制)中，摇床上缓慢摇动，室温封闭1.5h。弃去封闭液，用TBS-T漂洗2～3次。④孵育一抗：一抗用5％BSA按1:500比例稀释，将NC膜浸入其中，恒温摇床摇动，37℃孵育4h。将NC膜放入TBS-T中洗涤3次，每次5min。⑤孵育二抗：二抗用1％脱脂奶粉按1:5000比例稀释，将NC膜浸入其中，室温孵育1.5h。将NC放入TBS-T中洗涤3次，每次15min。⑥显影：将洗涤完毕的NC膜平铺于显影仪的适当位置，将显影剂均匀滴加于膜上，凝胶成像系统拍照、保存。
其中，本发明的实施例中试剂配制如下：
完全培养基：将50mL胎牛血清(美国Gibco公司)，5mL Penicillin Streptomycin溶液(美国Gibco公司)加入至450mL的DMEM高糖培养基(Hyclone公司)，混匀后4℃保存。
细胞PBS(Sangon公司)。
1×PBST缓冲液：将0.24g KH2PO4，0.2g KCl，1.44g Na2HPO4·12H2O和1mLTween-20溶于去离子水中，定容至1000mL，室温保存。
一抗：兔抗小鼠CD63单克隆抗体(英国Abcam公司)。
二抗：HRP标记的山羊抗兔IgG(Jackson ImmunoResearch公司)。
依帕司他(epalrestat)——一种用于预防、改善和治疗糖尿病并发的末梢神经障碍(麻木感、疼痛)的醛酮还原酶抑制药，不良反应发生率较低，安全有效，已在临床广泛应用。本发明中提供了依帕司他阻断多元醇通路后能显著抑制胰腺癌细胞外泌体分泌的新用途，这特性使得依帕司他在临床肿瘤治疗中可 能具有重要的价值，为临床患者寻找潜在的药物靶点提供新的理论依据。
依帕司他(Epalrestat)是一种临床上用于治疗糖尿病并发症的醛酮还原酶抑制药物，我们的结果表明，依帕司他有一个新的用途，即抑制外泌体分泌，鉴于外泌体分泌在肿瘤中的关键作用，依帕司他在临床肿瘤治疗中有巨大的应用潜力。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (9)

  1. 一种依帕司他在制备胰腺癌药物中的应用。
  2. 根据权利要求1所述的一种依帕司他在制备胰腺癌的药物中的应用，其特征在于，所述胰腺癌药物应用于抑制胰腺癌细胞外泌体的分泌。
  3. 一种抗胰腺癌的药品组合物，其特征在于，所述药品组合物含有依帕司他。
  4. 根据权利要求1所述的一种抗胰腺癌的药品组合物，其特征在于，所述药品组合物应用于抑制胰腺癌细胞外泌体的分泌。
  5. 一种依帕司他对胰腺癌细胞分泌外泌体的抑制作用的验证方法，其特征在于，包括以下过程：(1)建立依帕司他处理后胰腺癌细胞组和对照胰腺癌细胞组，利用低温超速离心法提取细胞上清外泌体；(2)将收集的外泌体裂解后用BCA试剂盒定量蛋白，用测得的蛋白量来反映外泌体的量；(3)透射电镜验证外泌体的双层脂质膜包裹的杯状结构；(4)蛋白质聚丙烯酰胺凝胶电泳考马斯亮蓝检测外泌体蛋白浓度。
  6. 根据权利要求5所述的一种依帕司他对胰腺癌细胞分泌外泌体的抑制作用的验证方法，其特征在于，所述步骤(1)具体包括以下过程：将依帕司他处理后胰腺癌细胞组和对照胰腺癌细胞组在细胞数量相同的标准下留取细胞上清液80ml，在4℃经300×g离心15min，2,000×g离心30min，16,500×g离心30min后去沉淀，所得上清液经过0.22μm滤嘴过滤再150,000×g低温超速离心120min，收集沉淀重悬于100ul PBS中，-80℃分装保存。
  7. 根据权利要求5所述的一种依帕司他对胰腺癌细胞分泌外泌体的抑制作用的验证方法，其特征在于，所述步骤(2)具体包括以下过程：取10μL收集的外泌体用2.5％的戊二醛固定2h，PBS洗涤外泌体并重悬于100μL PBS中，取20ul滴于小铜片上，3％磷钨酸水溶负染1min，透射电镜观察。
  8. 根据权利要求5所述的一种依帕司他对胰腺癌细胞分泌外泌体的抑制作用的验证方法，其特征在于，所述步骤(3)具体包括以下过程：用PBS将蛋白标准溶液稀释到0.5mg/mL；根据样品数量，配制BCA工作液，BCA工作液现配现用，将0.5mg/ml的蛋白标准稀释液按0、1、2、4、8、12、16、20L的顺序加到96孔板中，用PBS补足到20L，稀释后的标准品浓度分别为0、0.025、0.05、0.1、0.2、0.3、0.4、0.5mg/mL。每孔加1μL外泌体蛋白样品，加PBS 补足到20μL。每孔加200μL BCA工作液，37℃孵育20-30min，酶标仪测定562nm的吸光度，根据标准曲线和使用的样品体积计算出样品的蛋白浓度。
  9. 根据权利要求5所述的一种依帕司他对胰腺癌细胞分泌外泌体的抑制作用的验证方法，其特征在于，所述步骤(3)具体包括以下过程：制备12％SDS-PAGE凝胶，外泌体中加入5×loading buffer，煮沸20min后上样，电泳结束后，用蒸馏水洗涤凝胶10min，加入适量的考马斯亮蓝快速染色液，在摇床上摇动1h，染色至看到清晰的目标蛋白条带，弃染色液，加适量蒸馏水，拍照观察结果。
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