WO2023133778A1 - Recombinant pancreatic cancer cell for tracking of exosomes, and use thereof - Google Patents

Recombinant pancreatic cancer cell for tracking of exosomes, and use thereof Download PDF

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WO2023133778A1
WO2023133778A1 PCT/CN2022/071906 CN2022071906W WO2023133778A1 WO 2023133778 A1 WO2023133778 A1 WO 2023133778A1 CN 2022071906 W CN2022071906 W CN 2022071906W WO 2023133778 A1 WO2023133778 A1 WO 2023133778A1
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pancreatic cancer
exosomes
cancer cell
recombinant
phluorin
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PCT/CN2022/071906
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French (fr)
Chinese (zh)
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肖明兵
吴桐
陈飞
林仁杰
季洁
马彭
崔小鹏
钱坤艳
徐伟松
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南通大学附属医院
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Priority to PCT/CN2022/071906 priority Critical patent/WO2023133778A1/en
Priority to GB2216760.5A priority patent/GB2618634A/en
Publication of WO2023133778A1 publication Critical patent/WO2023133778A1/en

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Definitions

  • the invention belongs to the technical field of biomedicine, and specifically relates to a recombinant pancreatic cancer cell that can be used for exosome tracing and its application.
  • Exosomes are a type of saucer-shaped extracellular vesicles wrapped by a lipid bilayer membrane with a size of 30-150 nm, which are widely present in cell culture supernatants and various body fluids, including blood, lymph, saliva, Urine, follicular fluid, semen, pericardial fluid, milk, etc. Exosomes originate from endosomes, and the continuous invagination of the plasma membrane eventually leads to the formation of multivesicular bodies, which interact with other vesicles and organelles in the cell, leading to the eventual formation of exosomes and promoting Diversity of exosome components. Cell components communicate and transport between cells through exosomes.
  • Exosomes secreted by tumor cells can help them metastasize by shaping the tumor microenvironment.
  • the amount of exosomes secreted can directly affect tumor metastasis and also promote tumor formation and proliferation. play a very important role in the process. It has been reported that exosomes exist in all biological fluids, and the composition of exosome complexes can be easily obtained by sampling biological fluids. Based on biological liquid biopsies, exosomes can be shown to be useful in diagnosing tumors and other diseases and revealing their prognosis. has great potential. However, the research on the specific pathogenic behavior mechanism of exosomes is difficult to break through, and it is mostly limited by the visualization of its behavior process.
  • Pancreatic cancer is one of the common malignant tumors of the digestive tract and is known as the "King of Cancer" in the field of tumors. Pancreatic cancer is characterized by increased secretion of exosomes. A major challenge in studying the underlying mechanism of pancreatic cancer is the observation, measurement and visualization of exosome behavior.
  • exosome tracking can provide important knowledge about exosome biodistribution, migration capacity, toxicity, biological roles, communication capabilities and mechanisms of action.
  • researchers use molecular biology methods to fuse fluorescent proteins (such as green fluorescent protein GFP; red fluorescent protein RFP) with marker proteins on the exosome membrane, such as CD63, CD9, CD81, etc., to track exosomes Movement from donor to recipient cells.
  • fluorescent proteins such as green fluorescent protein GFP; red fluorescent protein RFP
  • marker proteins on the exosome membrane such as CD63, CD9, CD81, etc.
  • the expression elements of the specific protein CD63 and the green fluorescent protein GFP of exosomes are constructed into plasmids and packaged into lentiviruses, and then the cells are infected with the lentiviruses, so that the exosomes secreted by the cells have green fluorescence.
  • the fluorescence is unstable and not bright enough, making it difficult to visualize the dynamic exosome release and uptake.
  • Exosomes are a type of saucer-shaped extracellular vesicles wrapped by a lipid bilayer membrane with a size of 30-150 nm, which are widely present in cell culture supernatants and various body fluids, including blood, lymph, saliva, Urine, follicular fluid, semen, pericardial fluid, milk, etc. Exosomes originate from endosomes, and the continuous invagination of the plasma membrane eventually leads to the formation of multivesicular bodies, which interact with other vesicles and organelles in the cell, leading to the eventual formation of exosomes and promoting Diversity of exosome components. Cell components communicate and transport between cells through exosomes.
  • Exosomes secreted by tumor cells can help them metastasize by shaping the tumor microenvironment.
  • the amount of exosomes secreted can directly affect tumor metastasis and also promote tumor formation and proliferation. play a very important role in the process. It has been reported that exosomes exist in all biological fluids, and the composition of exosome complexes can be easily obtained by sampling biological fluids. Based on biological liquid biopsies, exosomes can be shown to be useful in diagnosing tumors and other diseases and revealing their prognosis. has great potential. However, the research on the specific pathogenic behavior mechanism of exosomes is difficult to break through, and it is mostly limited by the visualization of its behavior process.
  • Pancreatic cancer is one of the common malignant tumors of the digestive tract and is known as the "King of Cancer" in the field of tumors. Pancreatic cancer is characterized by increased secretion of exosomes. A major challenge in studying the underlying mechanism of pancreatic cancer is the observation, measurement and visualization of exosome behavior.
  • exosome tracking can provide important knowledge about exosome biodistribution, migration capacity, toxicity, biological roles, communication capabilities and mechanisms of action.
  • researchers use molecular biology methods to fuse fluorescent proteins (such as green fluorescent protein GFP; red fluorescent protein RFP) with marker proteins on the exosome membrane, such as CD63, CD9, CD81, etc., to track exosomes Movement from donor to recipient cells.
  • fluorescent proteins such as green fluorescent protein GFP; red fluorescent protein RFP
  • marker proteins on the exosome membrane such as CD63, CD9, CD81, etc.
  • the expression elements of the specific protein CD63 and the green fluorescent protein GFP of exosomes are constructed into plasmids and packaged into lentiviruses, and then the cells are infected with the lentiviruses, so that the exosomes secreted by the cells have green fluorescence.
  • the fluorescence is unstable and not bright enough, making it difficult to visualize the dynamic exosome release and uptake.
  • the purpose of the present invention is to provide a recombinant pancreatic cancer cell that can be used for exosome tracking and its application.
  • the exosome tracking method based on the recombinant pancreatic cancer cell of the present invention has stable and bright fluorescence, and can Visualize dynamic exosome release and uptake.
  • the present invention provides a recombinant pancreatic cancer cell that can be used for exosome tracing
  • the recombinant pancreatic cancer cell includes a recombinant vector; the recombinant vector inserts the target gene pHluorin_M153R-CD63; the target gene pHluorin_M153R-CD63 is The coding gene of the protein whose amino acid sequence is shown in SEQ ID NO.1.
  • the original vector of the recombinant vector includes pCDH-CMV-MCS-EF1-PURO.
  • the restriction enzyme cutting site at the 5' end of the insertion site of the pHluorin_M153R-CD63 on pCDH-CMV-MCS-EF1-PURO is EcoR I, and the restriction enzyme cutting site at the 3' end is Not I.
  • the present invention also provides the application of the recombinant pancreatic cancer cells described in the above scheme in the preparation of reagents or kits for tracing pancreatic cancer cell exosomes.
  • the present invention also provides a method for tracing pancreatic cancer cell exosomes, comprising the following steps:
  • the microscopic observation includes live cell observation and/or cell immunofluorescence observation.
  • the content of the observation includes one or more of the following items:
  • the present invention provides a recombinant pancreatic cancer cell that can be used for exosome tracing
  • the recombinant pancreatic cancer cell includes a recombinant vector; the recombinant vector inserts the target gene pHluorin_M153R-CD63; the target gene pHluorin_M153R-CD63 is The coding gene of the protein whose amino acid sequence is shown in SEQ ID NO.1.
  • the present invention is based on the construction of a stable pancreatic cancer cell line based on the recombinant vector inserted with the target gene pHluorin_M153R-CD63, and the recombinant pancreatic cancer stable cell line after the transfection of the recombinant plasmid is subjected to living cell observation under a microscope or cell immunofluorescence observation, confocal microscope Effectively labeling extracellular exosomes and their tail spots released in the extracellular matrix can make it easier to observe their path-finding trajectories, thereby revealing the pathogenic behavior of migrating living cells.
  • the invention solves the visualization problem in the study of exosome pathogenic behavior mechanism, constructs a bright, stable fluorescent tracer substance suitable for various cell culture environments, and can monitor the release of exosomes during the migration process of living cells And uptake, as well as the observation of path-finding trajectory, are used for the study of the pathogenic mechanism of exosomes in diseases.
  • Fig. 1 is the fluorescence imaging result of embodiment 1;
  • FIG. 1 is the WesternBlot efficiency verification results
  • Fig. 3 is the fluorescence imaging result of the comparative example.
  • the present invention provides a recombinant pancreatic cancer cell that can be used for exosome tracing
  • the recombinant pancreatic cancer cell includes a recombinant vector; the recombinant vector inserts the target gene pHluorin_M153R-CD63; the target gene pHluorin_M153R-CD63 is The coding gene of the protein whose amino acid sequence is shown in SEQ ID NO.1.
  • the present invention by modifying the gene encoding the CD63-GFP fusion protein, adding pHluorin, a pH-sensitive GFP derivative, to reduce the excessive brightness of the CD63-GFP fusion protein in the endosome, so as to better observe the multivesicular body ( MVB) and the plasma membrane dynamic fusion event, and adding a single amino acid mutation M153R enables stable expression of pHluorin-CD63 in 2D and 3D cell culture conditions, avoiding premature bleaching and quenching of fluorescence.
  • the target gene pHluorin_M153R-CD63 is the coding gene of the protein shown in the amino acid sequence as SEQ ID NO.1; the amino acid sequence of the protein shown in the SEQ ID NO.1 is specifically:
  • nucleotide sequence of the target gene pHluorin_M153R-CD63 is referred to (Sung B H, Lersner A V, Guerrero J, et al. A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells[J ]. Nature Communications.).
  • the original vector of the recombinant vector preferably includes pCDH-CMV-MCS-EF1-PURO.
  • the restriction enzyme cutting site at the 5' end of the insertion site of pHluorin_M153R-CD63 on pCDH-CMV-MCS-EF1-PURO is EcoR I, and the restriction enzyme cutting site at the 3' end is Not I.
  • the present invention has no special limitation on the construction method of the recombinant vector, and conventional construction methods in the art can be used.
  • the present invention recombines the target gene pHluorin_M153R-CD63 with the carrier pCDH-CMV-MCS-EF1-PURO to synthesize the recombinant vector pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro, which has the advantage of high transfection efficiency.
  • the pancreatic cancer cells preferably include pancreatic cancer cell line PANC-1.
  • the present invention also provides the application of the recombinant pancreatic cancer cells described in the above scheme in the preparation of reagents or kits for tracing pancreatic cancer cell exosomes.
  • the present invention also provides a method for tracing pancreatic cancer cell exosomes, comprising the following steps:
  • the content of the observation preferably includes one or more of the following items:
  • the target gene pHluorin_M153R-CD63 was obtained and recombined with the vector pCDH-CMV-MCS-EF1-PURO to synthesize the plasmid pCDH-CMV- pHluorin_M153R-CD63-EF1-Puro (see ⁇ Sung B H, LersnerAV, Guerrero J, et al. A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells[J]. Nature Communications. ⁇ ), provided by Shanghai Quan Synthesized by Yang Biotechnology Co., Ltd.
  • GFP-CD63 without the improved tag pHluorin was used for transfection, and the rest of the treatment was the same as in Example 1, and the fluorescence state was observed under a fluorescence microscope.
  • the present invention uses the recombinant plasmid inserted with the bright tracer target gene pHluorin_M153R-CD63 to transfect the pancreatic cancer cell line PANC1, and constructs a stable cell line. And through starvation stimulation and the use of exosome inhibitors to treat the cells, resulting in the increase or decrease of the exosome content released by the cells, and the different expressions of green fluorescence were observed in the state of living cells, and the shape and position of the green light group can be seen. Changes, and the appearance of positive spot tailing of exosome tails can be seen.
  • the method of the invention can become a powerful tool in the study of the mechanism of pancreatic cancer, and has very important innovative significance.

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Abstract

Provided are a recombinant pancreatic cancer cell for tracking of exosomes, and the use thereof, which belong to the technical field of biomedicine. The recombined pancreatic cancer cell contains a recombination vector, wherein the pHluorin_M153R-CD63 target gene is inserted into the recombination vector. A stable pancreatic cancer cell line is constructed using the recombinant vector in which the pHluorin_M153R-CD63 target gene is inserted; and extracellular exosomes and tail spots released thereby in an extracellular matrix are effectively labeled, and then pathfinding trajectories thereof can be more easily observed, thereby showing the pathogenic behavior of migrated living cells. The visualization problem in the study of the pathogenic behavior mechanism of exosomes is solved; a bright and stable fluorescent tracer substance suitable for a variety of cell culture environments is constructed; the release and intake amounts of exosomes in the migration process of living cells can be monitored; and observation of pathfinding trajectories is achieved.

Description

一种可用于外泌体示踪的重组胰腺癌细胞及其应用A recombinant pancreatic cancer cell that can be used for exosome tracking and its application 技术领域technical field
本发明属于生物医学技术领域，具体涉及一种可用于外泌体示踪的重组胰腺癌细胞及其应用。The invention belongs to the technical field of biomedicine, and specifically relates to a recombinant pancreatic cancer cell that can be used for exosome tracing and its application.
背景技术Background technique
外泌体是一类大小为30～150nm、呈茶托状的由脂质双层膜包裹的细胞外囊泡，广泛存在于细胞培养上清及各种体液中，包括血液、淋巴液、唾液、尿液、卵泡液、精液、心包液、乳汁等。外泌体起源于细胞内体，细胞质膜的连续内陷最终导致多囊泡体的形成，多囊泡体与细胞内的其他囊泡和细胞器相互作用，导致外泌体的最终形成，并促进外泌体成分的多样性。细胞成分通过外泌体进行细胞间的交流与运输，肿瘤细胞分泌的外泌体可以通过塑造肿瘤微环境来帮助其转移，其分泌数量可直接影响到肿瘤的转移，也在促进肿瘤形成和增殖的过程中发挥十分重要的作用。据报道，外泌体存在于所有生物体液中，通过生物体液取样可以很容易地获得外泌体复合物的组成，基于生物液体活检可显示出外泌体在诊断肿瘤和其他疾病以及揭示其预后的方面有着重大潜力能。而外泌体具体致病行为机制的研究难以突破，多受限于其行为过程的可视化呈现。Exosomes are a type of saucer-shaped extracellular vesicles wrapped by a lipid bilayer membrane with a size of 30-150 nm, which are widely present in cell culture supernatants and various body fluids, including blood, lymph, saliva, Urine, follicular fluid, semen, pericardial fluid, milk, etc. Exosomes originate from endosomes, and the continuous invagination of the plasma membrane eventually leads to the formation of multivesicular bodies, which interact with other vesicles and organelles in the cell, leading to the eventual formation of exosomes and promoting Diversity of exosome components. Cell components communicate and transport between cells through exosomes. Exosomes secreted by tumor cells can help them metastasize by shaping the tumor microenvironment. The amount of exosomes secreted can directly affect tumor metastasis and also promote tumor formation and proliferation. play a very important role in the process. It has been reported that exosomes exist in all biological fluids, and the composition of exosome complexes can be easily obtained by sampling biological fluids. Based on biological liquid biopsies, exosomes can be shown to be useful in diagnosing tumors and other diseases and revealing their prognosis. has great potential. However, the research on the specific pathogenic behavior mechanism of exosomes is difficult to break through, and it is mostly limited by the visualization of its behavior process.
胰腺癌是消化道常见恶性肿瘤之一，在肿瘤领域素有“癌症之王”的称号。胰腺癌具有外泌体明显分泌增强的特点，研究胰腺癌疾病深层机制的一大难题就是对外泌体行为的观察、测量及可视化。Pancreatic cancer is one of the common malignant tumors of the digestive tract and is known as the "King of Cancer" in the field of tumors. Pancreatic cancer is characterized by increased secretion of exosomes. A major challenge in studying the underlying mechanism of pancreatic cancer is the observation, measurement and visualization of exosome behavior.
体内追踪外泌体可以提供有关外泌体的生物分布、迁移能力、毒性、生物学的重要知识角色，沟通能力和行动机制。现有技术中，研究人员使用分子生物学方法将荧光蛋白(例如绿色荧光蛋白GFP；红色荧光蛋白RFP)与外泌体膜上的标记蛋白如CD63、CD9、CD81等融合表达可以跟踪外泌体从供体到受体细胞的运动。通过监测膜蛋白的荧光，可以实现活体培养物中和体内外泌体转移的直接可视化。例如，将外泌体的特定蛋白CD63和绿色荧光蛋白GFP的表达元件构建成质粒再包装到慢病毒中，随后用此慢病毒感染细胞，使细胞分泌的外泌体带有绿色荧光。但是，常规的GFP-CD63的外泌体示踪方法中，荧光不稳定且不够明亮，难以对动 态的外泌体释放及摄取实行可视化。外泌体是一类大小为30～150nm、呈茶托状的由脂质双层膜包裹的细胞外囊泡，广泛存在于细胞培养上清及各种体液中，包括血液、淋巴液、唾液、尿液、卵泡液、精液、心包液、乳汁等。外泌体起源于细胞内体，细胞质膜的连续内陷最终导致多囊泡体的形成，多囊泡体与细胞内的其他囊泡和细胞器相互作用，导致外泌体的最终形成，并促进外泌体成分的多样性。细胞成分通过外泌体进行细胞间的交流与运输，肿瘤细胞分泌的外泌体可以通过塑造肿瘤微环境来帮助其转移，其分泌数量可直接影响到肿瘤的转移，也在促进肿瘤形成和增殖的过程中发挥十分重要的作用。据报道，外泌体存在于所有生物体液中，通过生物体液取样可以很容易地获得外泌体复合物的组成，基于生物液体活检可显示出外泌体在诊断肿瘤和其他疾病以及揭示其预后的方面有着重大潜力能。而外泌体具体致病行为机制的研究难以突破，多受限于其行为过程的可视化呈现。In vivo tracking of exosomes can provide important knowledge about exosome biodistribution, migration capacity, toxicity, biological roles, communication capabilities and mechanisms of action. In the prior art, researchers use molecular biology methods to fuse fluorescent proteins (such as green fluorescent protein GFP; red fluorescent protein RFP) with marker proteins on the exosome membrane, such as CD63, CD9, CD81, etc., to track exosomes Movement from donor to recipient cells. Direct visualization of exosome transfer in live culture and in vivo can be achieved by monitoring the fluorescence of membrane proteins. For example, the expression elements of the specific protein CD63 and the green fluorescent protein GFP of exosomes are constructed into plasmids and packaged into lentiviruses, and then the cells are infected with the lentiviruses, so that the exosomes secreted by the cells have green fluorescence. However, in the conventional GFP-CD63 exosome tracking method, the fluorescence is unstable and not bright enough, making it difficult to visualize the dynamic exosome release and uptake. Exosomes are a type of saucer-shaped extracellular vesicles wrapped by a lipid bilayer membrane with a size of 30-150 nm, which are widely present in cell culture supernatants and various body fluids, including blood, lymph, saliva, Urine, follicular fluid, semen, pericardial fluid, milk, etc. Exosomes originate from endosomes, and the continuous invagination of the plasma membrane eventually leads to the formation of multivesicular bodies, which interact with other vesicles and organelles in the cell, leading to the eventual formation of exosomes and promoting Diversity of exosome components. Cell components communicate and transport between cells through exosomes. Exosomes secreted by tumor cells can help them metastasize by shaping the tumor microenvironment. The amount of exosomes secreted can directly affect tumor metastasis and also promote tumor formation and proliferation. play a very important role in the process. It has been reported that exosomes exist in all biological fluids, and the composition of exosome complexes can be easily obtained by sampling biological fluids. Based on biological liquid biopsies, exosomes can be shown to be useful in diagnosing tumors and other diseases and revealing their prognosis. has great potential. However, the research on the specific pathogenic behavior mechanism of exosomes is difficult to break through, and it is mostly limited by the visualization of its behavior process.
胰腺癌是消化道常见恶性肿瘤之一，在肿瘤领域素有“癌症之王”的称号。胰腺癌具有外泌体明显分泌增强的特点，研究胰腺癌疾病深层机制的一大难题就是对外泌体行为的观察、测量及可视化。Pancreatic cancer is one of the common malignant tumors of the digestive tract and is known as the "King of Cancer" in the field of tumors. Pancreatic cancer is characterized by increased secretion of exosomes. A major challenge in studying the underlying mechanism of pancreatic cancer is the observation, measurement and visualization of exosome behavior.
体内追踪外泌体可以提供有关外泌体的生物分布、迁移能力、毒性、生物学的重要知识角色，沟通能力和行动机制。现有技术中，研究人员使用分子生物学方法将荧光蛋白(例如绿色荧光蛋白GFP；红色荧光蛋白RFP)与外泌体膜上的标记蛋白如CD63、CD9、CD81等融合表达可以跟踪外泌体从供体到受体细胞的运动。通过监测膜蛋白的荧光，可以实现活体培养物中和体内外泌体转移的直接可视化。例如，将外泌体的特定蛋白CD63和绿色荧光蛋白GFP的表达元件构建成质粒再包装到慢病毒中，随后用此慢病毒感染细胞，使细胞分泌的外泌体带有绿色荧光。但是，常规的GFP-CD63的外泌体示踪方法中，荧光不稳定且不够明亮，难以对动态的外泌体释放及摄取实行可视化。In vivo tracking of exosomes can provide important knowledge about exosome biodistribution, migration capacity, toxicity, biological roles, communication capabilities and mechanisms of action. In the prior art, researchers use molecular biology methods to fuse fluorescent proteins (such as green fluorescent protein GFP; red fluorescent protein RFP) with marker proteins on the exosome membrane, such as CD63, CD9, CD81, etc., to track exosomes Movement from donor to recipient cells. Direct visualization of exosome transfer in live culture and in vivo can be achieved by monitoring the fluorescence of membrane proteins. For example, the expression elements of the specific protein CD63 and the green fluorescent protein GFP of exosomes are constructed into plasmids and packaged into lentiviruses, and then the cells are infected with the lentiviruses, so that the exosomes secreted by the cells have green fluorescence. However, in the conventional GFP-CD63 exosome tracking method, the fluorescence is unstable and not bright enough, making it difficult to visualize the dynamic exosome release and uptake.
发明内容Contents of the invention
有鉴于此，本发明的目的在于提供一种可用于外泌体示踪的重组胰腺癌细胞及其应用，基于本发明重组胰腺癌细胞进行的外泌体示踪方法，荧光稳定且明亮，能够对动态的外泌体释放及摄取实行可视化。In view of this, the purpose of the present invention is to provide a recombinant pancreatic cancer cell that can be used for exosome tracking and its application. The exosome tracking method based on the recombinant pancreatic cancer cell of the present invention has stable and bright fluorescence, and can Visualize dynamic exosome release and uptake.
本发明提供了一种可用于外泌体示踪的重组胰腺癌细胞，所述重组胰腺癌细胞包含重组载体；所述重组载体上插入有目的基因pHluorin_M153R-CD63；所述目的基因pHluorin_M153R-CD63为氨基酸序列如SEQ ID NO.1所示的蛋白的编码基因。The present invention provides a recombinant pancreatic cancer cell that can be used for exosome tracing, the recombinant pancreatic cancer cell includes a recombinant vector; the recombinant vector inserts the target gene pHluorin_M153R-CD63; the target gene pHluorin_M153R-CD63 is The coding gene of the protein whose amino acid sequence is shown in SEQ ID NO.1.
优选的，所述重组载体的原始载体包括pCDH-CMV-MCS-EF1-PURO。Preferably, the original vector of the recombinant vector includes pCDH-CMV-MCS-EF1-PURO.
优选的，所述pHluorin_M153R-CD63在pCDH-CMV-MCS-EF1-PURO上的插入位点的5’端的限制性酶切位点为EcoR I，3’端的限制性酶切位点为Not I。Preferably, the restriction enzyme cutting site at the 5' end of the insertion site of the pHluorin_M153R-CD63 on pCDH-CMV-MCS-EF1-PURO is EcoR I, and the restriction enzyme cutting site at the 3' end is Not I.
本发明还提供了上述方案所述的重组胰腺癌细胞在制备用于胰腺癌细胞外泌体示踪的试剂或试剂盒中的应用。The present invention also provides the application of the recombinant pancreatic cancer cells described in the above scheme in the preparation of reagents or kits for tracing pancreatic cancer cell exosomes.
本发明还提供了一种胰腺癌细胞外泌体示踪的方法，包括以下步骤：The present invention also provides a method for tracing pancreatic cancer cell exosomes, comprising the following steps:
使用荧光显微镜对上述方案所述的重组胰腺癌细胞进行镜下观察。Microscopically observe the recombinant pancreatic cancer cells described in the protocol above using a fluorescence microscope.
优选的，所述镜下观察包括活细胞观察和/或细胞免疫荧光观察。Preferably, the microscopic observation includes live cell observation and/or cell immunofluorescence observation.
优选的，所述观察的内容包括如下项目中的一种或几种：Preferably, the content of the observation includes one or more of the following items:
(1)观察细胞外外泌体的阳性荧光标记；(1) Observe the positive fluorescent labeling of extracellular exosomes;
(2)观察细胞外外泌体的寻径轨迹；(2) Observing the path-finding trajectory of extracellular exosomes;
(3)观察多囊泡体与细胞质膜的融合情况。(3) Observe the fusion of multivesicular body and plasma membrane.
本发明提供了一种可用于外泌体示踪的重组胰腺癌细胞，所述重组胰腺癌细胞包含重组载体；所述重组载体上插入有目的基因pHluorin_M153R-CD63；所述目的基因pHluorin_M153R-CD63为氨基酸序列如SEQ ID NO.1所示的蛋白的编码基因。本发明通过对CD63-GFP融合蛋白的编码基因进行改造，添加pH敏感的GFP衍生物pHluorin来降低CD63-GFP融合蛋白在细胞内体中的过高亮度，以更好地观察多囊泡体(MVB)与质膜的动态融合事件，并加入单一氨基酸突变M153R使得pHluorin-CD63在细胞2D及3D培养条件下不易漂白和淬灭。本发明基于插入有目的基因pHluorin_M153R-CD63的重组载体进行胰腺癌稳定细胞系的构建，转染重组质粒后的重组胰腺癌稳定细胞系进行显微镜下的活细胞观察或细胞免疫荧光观察，共聚焦显微镜下对细胞外的外泌体及其释放于细胞外基质中的尾部斑点进行有效标记，可更容易地观察其寻径轨迹，从而揭示迁移活细胞的致病行为。本发明解决了外泌体致病行为机制 研究中的可视化难题，构建得到了明亮、稳定、且适用于多种细胞培养环境的荧光示踪物质，可监测活细胞在迁移过程中外泌体的释放和摄取量，以及寻径轨迹的观察，用于外泌体在疾病的致病机制的研究。The present invention provides a recombinant pancreatic cancer cell that can be used for exosome tracing, the recombinant pancreatic cancer cell includes a recombinant vector; the recombinant vector inserts the target gene pHluorin_M153R-CD63; the target gene pHluorin_M153R-CD63 is The coding gene of the protein whose amino acid sequence is shown in SEQ ID NO.1. In the present invention, by modifying the gene encoding the CD63-GFP fusion protein, adding pHluorin, a pH-sensitive GFP derivative, to reduce the excessive brightness of the CD63-GFP fusion protein in the endosome, so as to better observe the multivesicular body ( MVB) and the plasma membrane dynamic fusion event, and adding a single amino acid mutation M153R makes pHluorin-CD63 difficult to bleach and quench under cell 2D and 3D culture conditions. The present invention is based on the construction of a stable pancreatic cancer cell line based on the recombinant vector inserted with the target gene pHluorin_M153R-CD63, and the recombinant pancreatic cancer stable cell line after the transfection of the recombinant plasmid is subjected to living cell observation under a microscope or cell immunofluorescence observation, confocal microscope Effectively labeling extracellular exosomes and their tail spots released in the extracellular matrix can make it easier to observe their path-finding trajectories, thereby revealing the pathogenic behavior of migrating living cells. The invention solves the visualization problem in the study of exosome pathogenic behavior mechanism, constructs a bright, stable fluorescent tracer substance suitable for various cell culture environments, and can monitor the release of exosomes during the migration process of living cells And uptake, as well as the observation of path-finding trajectory, are used for the study of the pathogenic mechanism of exosomes in diseases.
附图说明Description of drawings
图1为实施例1的荧光成像结果；Fig. 1 is the fluorescence imaging result of embodiment 1;
图2为WesternBlot效率验证结果；Figure 2 is the WesternBlot efficiency verification results;
图3为比较例的荧光成像结果。Fig. 3 is the fluorescence imaging result of the comparative example.
具体实施方式Detailed ways
本发明提供了一种可用于外泌体示踪的重组胰腺癌细胞，所述重组胰腺癌细胞包含重组载体；所述重组载体上插入有目的基因pHluorin_M153R-CD63；所述目的基因pHluorin_M153R-CD63为氨基酸序列如SEQ ID NO.1所示的蛋白的编码基因。The present invention provides a recombinant pancreatic cancer cell that can be used for exosome tracing, the recombinant pancreatic cancer cell includes a recombinant vector; the recombinant vector inserts the target gene pHluorin_M153R-CD63; the target gene pHluorin_M153R-CD63 is The coding gene of the protein whose amino acid sequence is shown in SEQ ID NO.1.
本发明通过对CD63-GFP融合蛋白的编码基因进行改造，添加pH敏感的GFP衍生物pHluorin来降低CD63-GFP融合蛋白在细胞内体中的过高亮度，以更好地观察多囊泡体(MVB)与质膜的动态融合事件，并加入单一氨基酸突变M153R使得pHluorin-CD63在细胞2D及3D培养条件下稳定表达，避免荧光的过早漂白和淬灭。In the present invention, by modifying the gene encoding the CD63-GFP fusion protein, adding pHluorin, a pH-sensitive GFP derivative, to reduce the excessive brightness of the CD63-GFP fusion protein in the endosome, so as to better observe the multivesicular body ( MVB) and the plasma membrane dynamic fusion event, and adding a single amino acid mutation M153R enables stable expression of pHluorin-CD63 in 2D and 3D cell culture conditions, avoiding premature bleaching and quenching of fluorescence.
在本发明中，所述目的基因pHluorin_M153R-CD63为氨基酸序列如SEQ ID NO.1所示的蛋白的编码基因；SEQ ID NO.1所示的蛋白的氨基酸序列具体为：In the present invention, the target gene pHluorin_M153R-CD63 is the coding gene of the protein shown in the amino acid sequence as SEQ ID NO.1; the amino acid sequence of the protein shown in the SEQ ID NO.1 is specifically:
Figure PCTCN2022071906-appb-000001
Figure PCTCN2022071906-appb-000001
在本发明中，所述目的基因pHluorin_M153R-CD63的核苷酸序列参见(Sung B H,Lersner A V,Guerrero J,et al.A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells[J].Nature Communications.)。In the present invention, the nucleotide sequence of the target gene pHluorin_M153R-CD63 is referred to (Sung B H, Lersner A V, Guerrero J, et al. A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells[J ]. Nature Communications.).
在本发明中，所述重组载体的原始载体优选的包括pCDH-CMV-MCS-EF1-PURO。在本发明中，所述pHluorin_M153R-CD63在pCDH-CMV-MCS-EF1-PURO上的插入位点的5’端的限制性酶切位点为EcoR I，3’端的限制性酶切位点为Not I。In the present invention, the original vector of the recombinant vector preferably includes pCDH-CMV-MCS-EF1-PURO. In the present invention, the restriction enzyme cutting site at the 5' end of the insertion site of pHluorin_M153R-CD63 on pCDH-CMV-MCS-EF1-PURO is EcoR I, and the restriction enzyme cutting site at the 3' end is Not I.
本发明对所述重组载体的构建方法没有特殊限制，采用本领域的常规构建方法即可。The present invention has no special limitation on the construction method of the recombinant vector, and conventional construction methods in the art can be used.
本发明将目的基因pHluorin_M153R-CD63与载体pCDH-CMV-MCS-EF1-PURO重组合成重组载体pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro，具有转染效率高的优点。The present invention recombines the target gene pHluorin_M153R-CD63 with the carrier pCDH-CMV-MCS-EF1-PURO to synthesize the recombinant vector pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro, which has the advantage of high transfection efficiency.
在本发明中，所述胰腺癌细胞优选的包括胰腺癌细胞株PANC-1。In the present invention, the pancreatic cancer cells preferably include pancreatic cancer cell line PANC-1.
本发明还提供了上述方案所述的重组胰腺癌细胞在制备用于胰腺癌细胞外泌体示踪的试剂或试剂盒中的应用。The present invention also provides the application of the recombinant pancreatic cancer cells described in the above scheme in the preparation of reagents or kits for tracing pancreatic cancer cell exosomes.
本发明还提供了一种胰腺癌细胞外泌体示踪的方法，包括以下步骤：The present invention also provides a method for tracing pancreatic cancer cell exosomes, comprising the following steps:
使用荧光显微镜对上述方案所述的重组胰腺癌细胞进行镜下观察。Microscopically observe the recombinant pancreatic cancer cells described in the protocol above using a fluorescence microscope.
在本发明中，所述观察的内容优选的包括如下项目中的一种或几种：In the present invention, the content of the observation preferably includes one or more of the following items:
(1)观察细胞外外泌体的阳性荧光标记；(1) Observe the positive fluorescent labeling of extracellular exosomes;
(2)观察细胞外外泌体的寻径轨迹；(2) Observing the path-finding trajectory of extracellular exosomes;
(3)观察多囊泡体与细胞质膜的融合情况。(3) Observe the fusion of multivesicular body and plasma membrane.
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.
实施例1Example 1
1)以EcoRI为5’端限制性内切酶，NotI为3’端限制性内切酶，获得目的基因pHluorin_M153R-CD63，与载体pCDH-CMV-MCS-EF1-PURO重组合成质粒pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro(参见【Sung B H,LersnerAV,Guerrero J,et al.A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells[J].Nature Communications.】)，由上海权阳生物科技有限公司合成。1) Using EcoRI as the 5' restriction endonuclease and NotI as the 3' restriction endonuclease, the target gene pHluorin_M153R-CD63 was obtained and recombined with the vector pCDH-CMV-MCS-EF1-PURO to synthesize the plasmid pCDH-CMV- pHluorin_M153R-CD63-EF1-Puro (see 【Sung B H, LersnerAV, Guerrero J, et al. A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells[J]. Nature Communications.】), provided by Shanghai Quan Synthesized by Yang Biotechnology Co., Ltd.
2)对质粒pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro进行测序验证，并用DNA凝胶电泳进行质粒闭环质量鉴定。2) The plasmid pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro was sequenced and verified, and the quality of the closed circle of the plasmid was identified by DNA gel electrophoresis.
3)将DH-5α感受态从-80℃冰箱取出，冰上静置5min。吸取10μl质 粒pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro和30μlDH-5α感受态，轻吹混合，冰上静置2min。3) Take the competent DH-5α out of the -80°C refrigerator and let it stand on ice for 5 minutes. Pipette 10 μl plasmid pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro and 30 μl DH-5α competent, blow gently to mix, and let stand on ice for 2 minutes.
4)42℃金属浴精确孵育45s后取出，冰上放置2min。4) Take it out after precise incubation in a metal bath at 42°C for 45 seconds, and place it on ice for 2 minutes.
5)全部加入摇菌管，加入4ml含AMP的LB液培，入摇床(倾斜放置)，37℃，250转速，摇12～16h，每100μl菌液中加入4ml含AMP的LB液培。5) Add all the bacteria into the shaking tube, add 4ml of LB liquid culture containing AMP, place on a shaking table (placed on a tilt), 37°C, 250 rpm, shake for 12-16 hours, add 4ml of LB liquid culture containing AMP to every 100 μl of bacterial liquid.
6)摇完后，将每管菌液平均分成两管，再补足4ml含AMP的液培，再次摇菌14～16h。6) After shaking, divide each tube of bacteria liquid into two tubes equally, and make up 4ml of liquid culture containing AMP, and shake the bacteria again for 14-16 hours.
7)使用试剂盒抽提，得到浓度较高的质粒pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro。7) Use the extraction kit to obtain the plasmid pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro with a higher concentration.
8)使用胰蛋白酶将中皿中的PANC-1细胞消化重悬，再均匀铺至六孔板中(每孔细胞数约0.5×10 6个)，6h后进行转染。 8) Digest and resuspend the PANC-1 cells in the medium dish with trypsin, spread them evenly in a six-well plate (about 0.5×10 6 cells per well), and perform transfection 6 hours later.
9)吸取80μl无血清基础培养基，加4～5μl步骤7)提取得到的质粒pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro(约3μg)，室温静置。9) Take 80 μl of serum-free basal medium, add 4-5 μl of the plasmid pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro (about 3 μg) extracted in step 7), and let stand at room temperature.
10)5min后加3μl lipo2000以增加质粒转染效率，轻弹管壁(不可用力吹打)室温静置20min。10) After 5 minutes, add 3 μl lipo2000 to increase the efficiency of plasmid transfection, lightly flick the tube wall (do not forcefully pipette) and let stand at room temperature for 20 minutes.
11)培养箱培养48h后，得到感染成功的细胞株，再加入嘌呤霉素进行筛选，得到稳定表达的PANC-1细胞株。11) After culturing in an incubator for 48 hours, a successfully infected cell line was obtained, and then puromycin was added for screening to obtain a stably expressing PANC-1 cell line.
12)使用荧光显微镜进行镜下观察，观察细胞外外泌体的阳性荧光标记及其寻径轨迹，观察MVB与细胞质膜的融合事件，结果参见图1。由图1可以看出，细胞外泌体释放到细胞外基质的阳性拖尾。12) Use a fluorescence microscope to observe under the microscope, observe the positive fluorescent markers of extracellular exosomes and their path-finding tracks, and observe the fusion event of MVB and the plasma membrane of the cell. See Figure 1 for the results. It can be seen from Figure 1 that the positive tailing of exosomes released into the extracellular matrix.
13)用无血清DMEM培养基对稳转细胞株进行培养和刺激48小时后收集上清，部分进行NTA分析。这里的刺激为饥饿刺激，常规细胞培养为含5％胎牛血清的完全培养基，收取外泌体上清则使用无血清基础培养基进行饥饿刺激。13) After culturing and stimulating the stably transfected cell line with serum-free DMEM medium for 48 hours, the supernatant was collected, and some of them were analyzed by NTA. The stimulation here is starvation stimulation, conventional cell culture is a complete medium containing 5% fetal bovine serum, and the supernatant of exosomes is collected using a serum-free basal medium for starvation stimulation.
14)用普通高速离心机将收集的上清进行分离；14) Separating the collected supernatant with an ordinary high-speed centrifuge;
①3000rpm 15min 4℃(300G)；①3000rpm 15min 4℃(300G);
换管，收上清，弃沉淀。Change the tube, collect the supernatant, and discard the precipitate.
②5000rpm 20min 4℃(2000G)；②5000rpm 20min 4℃(2000G);
换管，收上清，弃沉淀。Change the tube, collect the supernatant, and discard the precipitate.
③12000rpm 30min 4℃(16500G)；③12000rpm 30min 4℃(16500G);
换管，滤嘴过滤掉直径0.22以上的颗粒。Change the tube, and filter out particles with a diameter of 0.22 or more.
15)用超速离心机，4℃下，以54000rpm进行超离，2h后弃上清，100μlPBS或强效裂解液重悬沉淀，转移至新的EP管中。15) Use an ultracentrifuge at 54000 rpm at 4°C for ultracentrifugation, discard the supernatant after 2 hours, resuspend the pellet in 100 μl of PBS or powerful lysate, and transfer to a new EP tube.
16)Western blotting及荧光显微镜下对比稳转PANC-1细胞外泌体与未处理的PANC-1细胞中CD63蛋白的含量，结果参见图2。由图2可以看出，转染后细胞蛋白中的CD63表达含量明显增加。16) Western blotting and fluorescence microscope were used to compare the CD63 protein content in the exosomes of stably transfected PANC-1 cells and untreated PANC-1 cells. See Figure 2 for the results. It can be seen from Figure 2 that the expression level of CD63 in the cell protein was significantly increased after transfection.
比较例comparative example
使用无改进标签pHluorin的GFP-CD63进行转染，其余处理同实施例1，并在荧光显微镜下观察荧光状态。GFP-CD63 without the improved tag pHluorin was used for transfection, and the rest of the treatment was the same as in Example 1, and the fluorescence state was observed under a fluorescence microscope.
结果参见图3。由图3可以看出，普通GFP-CD63的外泌体示踪方法中，无pHluorin_M153R标签，荧光不稳定且不够明亮，难以对动态的外泌体释放及摄取实行可视化。See Figure 3 for the results. It can be seen from Figure 3 that in the common GFP-CD63 exosome tracking method, there is no pHluorin_M153R label, the fluorescence is unstable and not bright enough, and it is difficult to visualize the dynamic exosome release and uptake.
本发明使用插入有明亮示踪物目的基因pHluorin_M153R-CD63的重组质粒对胰腺癌细胞系PANC1进行转染，并构建稳定细胞系。并通过饥饿刺激以及使用外泌体抑制剂对细胞进行处理，导致细胞释放的外泌体含量增高或减低，并在活细胞状态下观察绿色荧光的不同表达，可见绿色光团的形态及位置的变化，并可见外泌体尾部阳性斑点拖尾的呈现。The present invention uses the recombinant plasmid inserted with the bright tracer target gene pHluorin_M153R-CD63 to transfect the pancreatic cancer cell line PANC1, and constructs a stable cell line. And through starvation stimulation and the use of exosome inhibitors to treat the cells, resulting in the increase or decrease of the exosome content released by the cells, and the different expressions of green fluorescence were observed in the state of living cells, and the shape and position of the green light group can be seen. Changes, and the appearance of positive spot tailing of exosome tails can be seen.
本发明的方法能够成为胰腺癌机制研究中一大有力工具，具有十分重要的创新意义。The method of the invention can become a powerful tool in the study of the mechanism of pancreatic cancer, and has very important innovative significance.
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。Although the foregoing embodiment has described the present invention in detail, it is only a part of the embodiments of the present invention rather than all embodiments, and people can also obtain other embodiments according to the present embodiment without inventive step, and these embodiments are all Belong to the protection scope of the present invention.

Claims (7)

  1. 一种可用于外泌体示踪的重组胰腺癌细胞，所述重组胰腺癌细胞包含重组载体；所述重组载体上插入有目的基因pHluorin_M153R-CD63；所述目的基因pHluorin_M153R-CD63为氨基酸序列如SEQ ID NO.1所示的蛋白的编码基因。A recombinant pancreatic cancer cell that can be used for exosome tracking, the recombinant pancreatic cancer cell contains a recombinant vector; the recombinant vector inserts the target gene pHluorin_M153R-CD63; the target gene pHluorin_M153R-CD63 has an amino acid sequence such as SEQ The coding gene of the protein indicated by ID NO.1.
  2. 根据权利要求1所述的重组胰腺癌细胞，其特征在于，所述重组载体的原始载体包括pCDH-CMV-MCS-EF1-PURO。The recombinant pancreatic cancer cell according to claim 1, wherein the original vector of the recombinant vector comprises pCDH-CMV-MCS-EF1-PURO.
  3. 根据权利要求2所述的重组胰腺癌细胞，其特征在于，所述目的基因pHluorin_M153R-CD63在pCDH-CMV-MCS-EF1-PURO上的插入位点的5’端的限制性酶切位点为EcoR I，3’端的限制性酶切位点为NotI。The recombinant pancreatic cancer cell according to claim 2, wherein the restriction enzyme cutting site at the 5' end of the insertion site of the target gene pHluorin_M153R-CD63 on pCDH-CMV-MCS-EF1-PURO is EcoR I, the restriction enzyme site at the 3' end is NotI.
  4. 权利要求1～3任意一项所述的重组胰腺癌细胞在制备用于胰腺癌细胞外泌体示踪的试剂或试剂盒中的应用。Use of the recombinant pancreatic cancer cell according to any one of claims 1 to 3 in the preparation of reagents or kits for tracing pancreatic cancer cell exosomes.
  5. 一种胰腺癌细胞外泌体示踪的方法，包括以下步骤：A method for tracing pancreatic cancer cell exosomes, comprising the following steps:
    使用荧光显微镜对权利要求1～3任意一项所述的重组胰腺癌细胞进行镜下观察。Using a fluorescence microscope to observe the recombinant pancreatic cancer cells described in any one of claims 1-3.
  6. 根据权利要求5所述的方法，其特征在于，所述镜下观察包括活细胞观察和/或细胞免疫荧光观察。The method according to claim 5, characterized in that the microscopic observation comprises live cell observation and/or cell immunofluorescence observation.
  7. 根据权利要求5或6所述的方法，其特征在于，所述观察的内容包括如下项目中的一种或几种：The method according to claim 5 or 6, wherein the observed content includes one or more of the following items:
    (1)观察细胞外外泌体的阳性荧光标记；(1) Observe the positive fluorescent labeling of extracellular exosomes;
    (2)观察细胞外外泌体的寻径轨迹；(2) Observing the path-finding trajectory of extracellular exosomes;
    (3)观察多囊泡体与细胞质膜的融合情况。(3) Observe the fusion of multivesicular body and plasma membrane.
PCT/CN2022/071906 2022-01-14 2022-01-14 Recombinant pancreatic cancer cell for tracking of exosomes, and use thereof WO2023133778A1 (en)

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WO2018160699A1 (en) * 2017-03-01 2018-09-07 University Of Notre Dame Du Lac Biomarkers for diagnosis, prediction and/or prognosis of pancreatic cancer and uses thereof

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WO2018160699A1 (en) * 2017-03-01 2018-09-07 University Of Notre Dame Du Lac Biomarkers for diagnosis, prediction and/or prognosis of pancreatic cancer and uses thereof

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