GB2618634A - 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 PDFInfo
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- GB2618634A GB2618634A GB2216760.5A GB202216760A GB2618634A GB 2618634 A GB2618634 A GB 2618634A GB 202216760 A GB202216760 A GB 202216760A GB 2618634 A GB2618634 A GB 2618634A
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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
RECOMBINANT PANCREATIC CARCINOMA CELL FOR EXOSOME TRACKING
AND USE THEREOF
TECHNICAL FIELD
100011 The present disclosure belongs to the technical field of biomedicine, and specifically relates to a recombinant pancreatic carcinoma cell for exosome tracking and use thereof
BACKGROUND
[0002] Exosomes are a group of saucer-shaped extracellular vesicles of 30-150 nm in diameter, enveloped in a lipid bilaycr membrane; exosomes are widely found in cell culture supernatants and various body fluids, including blood, lymph, saliva, urine, follicular fluid, semen, pericardial fluid, and milk. Exosomes originate from endosomes. Continuous cytoplasmic membrane invagination finally leads to the formation of the multi-vesicular body (MVB). Interaction of MVBs with other vesicles and organelles in the cell leads to the final formation of exosomes and promotes the diversity of exosome components. Cellular components undergo intercellular communication and transport via exosomes. Exosomes secreted by tumor cells can be migrated by creating tumor microenvironments; the number of secreted exosomes has a direct effect on neoplasm metastasis and plays a very important role in promoting the processes of tumorigenesis and proliferation. Reportedly, exosomes are present in all biological fluids, and the composition of the exosome complex can be readily obtained by sampling biological fluids; biological liquid biopsy can show that exosomes have an important potential for diagnosing tumors and other diseases and revealing prognoses thereof Research on the specific pathogenic behaviour mechanism of exosomes is difficult to break through and is mostly limited to the visual presentation of the behaviour process thereof [0003] Pancreatic carcinoma is one of the most common digestive tract malignancies, known as the king of cancer in the field of oncology. Pancreatic carcinoma is characterized by significant enhancement of exosome secretion. A difficult problem in investigating the underlying mechanism of the pancreatic carcinoma is the observation, measurement and visualization of the behaviour of exosomes.
[0004] Tracking exosomes in vivo can provide important knowledge regarding their biodistribution, migration abilities, toxicity, biological role, communication capabilities, and mechanism of action. In the prior art, researchers use a molecular biology method for fusion expression of a fluorescent protein (for example, green fluorescent protein (GFP); red fluorescent protein (RFP)) with a marker protein on the cxosomal membrane, for example, CD63, CD9, and CD81, so as to track the movement of exosomes from a donor to a recipient cell. Monitoring the fluorescence in the membrane protein can realize the direct visualization of the neutralization of in vivo exosome transfer by in vivo culture. For example, the specific protein CD63 of the exosome and the expression element of the GFP are constructed into a plasmid and packaged into a lentivirus; subsequently, cells arc infected with the lentivirus to allow exosomes secreted by the cells to carry green fluorescence. However, in the conventional GFP-CD63-based exosome tracking method, the fluorescence is unstable and not bright enough, and difficult to implement the visualization of the release and uptake of dynamic exosomes. Exosomes are a group of saucer-shaped extracellular vesicles of 30-150 nm in diameter, enveloped in a lipid bilayer membrane; exosomes are widely found in cell culture supernatants and various body fluids, including blood, lymph, saliva, urine, follicular fluid, semen, pericardial fluid, and milk. Exosomes originate from endosomes. Continuous cytoplasmic membrane invagination finally leads to the formation of the multi-vesicular body (MVB). Interaction of MVBs with other vesicles and organelles in the cell leads to the final formation of exosomes and promotes the diversity of exosome components. Cellular components undergo intercellular communication and transport via exosomes. Exosomes secreted by tumor cells can be migrated by creating tumor microcnvironments; the number of secreted exosomes has a direct effect on neoplasm metastasis and plays a very important role in promoting the processes of tumorigenesis and proliferation. Reportedly, exosomes are present in all biological fluids, and the composition of the exosome complex can be readily obtained by sampling biological fluids; biological liquid biopsy can show that exosomes have an important potential for diagnosing tumors and other diseases and revealing prognoses thereof Research on the specific pathogenic behaviour mechanism of exosomes is difficult to break through and is mostly limited to the visual presentation of the behaviour process thereof [0005] Pancreatic carcinoma is one of the most common digestive tract malignancies, known as the king of cancer in the field of oncology. Pancreatic carcinoma is characterized by significant enhancement of exosome secretion. A difficult problem in investigating the underlying mechanism of the pancreatic carcinoma is the observation, measurement and visualization of the behaviour of exosomes.
[0006] Tracking exosomes in vivo can provide important knowledge regarding their biodistribution, migration abilities, toxicity, biological role, communication capabilities, and mechanism of action. In the prior art, researchers use a molecular biology method for fusion expression of a fluorescent protein (for example, g-een fluorescent protein (GFP); red fluorescent protein (RFP)) with a marker protein on the exosomal membrane, for example, CD63, CD9, and CD81, so as to track the movement of exosomes from a donor to a recipient cell. Monitoring the fluorescence in the membrane protein can realize the direct visualization of the neutralization of in vivo exosome transfer by in vivo culture. For example, the specific protein CD63 of the exosome and the expression element of the GFP are constructed into a plasmid and packaged into a lentivirus; subsequently, cells are infected with the lentivirus to allow exosomes secreted by the cells to carry green fluorescence. However, in the conventional GFP-CD63-based exosome tracking method, the fluorescence is unstable and not bright enough, and difficult to implement the visualization of the release and uptake of dynamic exosomes.
SUMMARY
[0007] In view of this, an objective of the present disclosure is to provide a recombinant pancreatic carcinoma cell for exosome tracking and use thereof. In a method for exosome tracking based on the recombinant pancreatic carcinoma cell provided by the present disclosure, fluorescence is stable and bright and can implement the visualization of the release and uptake of dynamic exosomes.
[0008] The present disclosure provides a recombinant pancreatic carcinoma cell for exosome tracking, where the recombinant pancreatic carcinoma cell includes a recombinant vector; a target gene pl linorin_M I 53R-CD63 is inserted into the recombinant vector; the target gene pi Ihiorin_114153R-CD63 is an encoding gene of a protein having an amino acid sequence shown in SEQ ID NO:!.
[0009] Preferably, an original vector of the recombinant vector includes pCDH-CMV-MCS-EF1-PURO.
[0010] Preferably, restriction sites at 5'-end and 3'-end of an insertion site of the pHIuorinMJ53R-CD63 in the pCDH-CMV-MCS-EFI-PURO are EcoRI and Nod, respectively. [0011] The present disclosure further provides use of the recombinant pancreatic carcinoma cell according to foregoing solution in preparation of a reagent or kit for exosome tracking in a pancreatic carcinoma cell.
[0012] The present disclosure further provides a method for exosome tracking in a pancreatic carcinoma cell, including the following steps: [0013] conducting microscopic observation on the recombinant pancreatic carcinoma cell according to the foregoing solution under a fluorescence microscope.
[0014] Preferably, the microscopic observation includes living cell observation and/or cell immunotluorescence observation.
[0015] Preferably, the observation includes one or more of the following items: [0016] (1) observing positive fluorescent labels in extracellular exosomes; [0017] (2) observing a pathfinding trajectory of the extracellular exosomes; and [0018] (3) observing a fusion of a multi-vesicular body (MVB) with a cytoplasmic membrane.
[0019] The present disclosure provides a recombinant pancreatic carcinoma cell for exosome tracking, where the recombinant pancreatic carcinoma cell includes a recombinant vector; a target gene pl Iluorin_M I 53R-CD63 is inserted into the recombinant vector; the target gene pHluorin A1153R-CD63 is an encoding gene of a protein having an amino acid sequence shown in SEQ ID NO: 1. In the present disclosure, excessive brightness of CD63-GFP fusion protein in an endosome is reduced by modifying an encoding gene of the CD63-GFP fusion protein and adding a pH-sensitive GFP derivative pHluorin, so as to better observe dynamic fusion events between MVBs and plasma membranes; moreover, a single amino acid variant Ml 53R is added to prevent pHluorin-CD63 from easy photobleaching and quenching under 2D and 3D cell culture conditions. In the present disclosure, a pancreatic carcinoma stable cell line is conducted based on a recombinant vector inserted with the target gene pIlluorin_A4153R-CD63, a recombinant pancreatic carcinoma stable cell line transfected with a recombinant plasmid is subjected to microscopic living cell observation or cell immunolluorescence observation, and extracellular exosomes and tail spots thereof released into an extracellular matrix are effectively labelled under a confocal scanning microscope, so that a pathfinding trajectory thereof is observed more readily and thus pathogenic behaviours of migrating living cells are revealed. The present disclosure solves a visualization problem in research on the mechanism of the pathogenic behaviour of the exosome, constructs and obtains a bright and stable fluorescent tracer substance suitable for a plurality of cell culture environments, monitors the release and uptake of exosomes during the migration of living cells, and observes the pathfinding trajectory. The present disclosure is used in the research on exosomes in the pathogenesis of a disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 illustrates a fluorescence imaging result of Example I; [0021] FIG. 2 illustrates a result of verification of efficiency based on Western blot; [0022] FIG. 3 illustrates a fluorescence imaging result of Comparative Example.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0023] The present disclosure provides a recombinant pancreatic carcinoma cell for exosome tracking, where the recombinant pancreatic carcinoma cell includes a recombinant vector; a target gene pHluorin A4153R-CD63 is inserted into the recombinant vector; the target gene pHluorin A4153R-CD63 is an encoding gene of a protein having an amino acid sequence shown in SEQ ID NO: I. [0024] In the present disclosure, excessive brightness of CD63-GFP fusion protein in an endosome is reduced by modifying an encoding gene of the CD63-GFP fusion protein and adding a pH-sensitive GFP derivative pHluorin, so as to better observe dynamic fusion events between MVBs and plasma membranes; moreover, a single amino acid variant M153R is added to enable stable expression of pHluorin-CD63 under 2D and 3D cell culture conditions and prevent fluorescence from early photobleaching and quenching.
100251 In the present disclosure, the target gene pHluorin 1V1153R-CD63 is an encoding gene of a protein having an amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence of the protein shown in SEQ ID NO: 1 is specifically as follows: 100261 SKGEELFTGVVPILVELDGDVNGHICSVSGEGEGDATYGICTLKFICTTGKLPVP WPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKF EGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIRADKQKNGIKANFKIRHNIEDG GVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEFVTAAGITHG MDELYK.
[0027] In the present disclosure, a nucleotide sequence of the target gene pHluorin M153R-CD63 is found in the literature (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.).
[0028] In the present disclosure, an original vector of the recombinant vector may preferably include pCDH-CMV-MCS-EF1-PURO. In the present disclosure, restriction sites at 5'-end and 3'-end of an insertion site of the pl lluorin_11/1153R-CD63 in the pCDH-CMV-MCS-EF1-PURO are EcoRI and Not!, respectively.
100291 The present disclosure has no special limitation on a construction method of the recombinant vector, as long as a conventional construction method in the art may be used. [0030] In the present disclosure, the target gene pHluorin M153R-CD63 and the vector pCDH-CMV-MCS-EF1-PURO are recombined to synthesize a recombinant vector pCDH-CMV-pHluorin M153R-CD63-EF1-Puro having an advantage of high transfection efficiency.
[0031] In the present disclosure, the pancreatic carcinoma cell may preferably include a pancreatic carcinoma cell line PANC-1.
[0032] The present disclosure further provides use of the recombinant pancreatic carcinoma cell according to foregoing solution in preparation of a reagent or kit for exosome tracking in a pancreatic carcinoma cell.
[0033] The present disclosure further provides a method for exosome tracking in a pancreatic carcinoma cell, including the following steps: [0034] conducting microscopic observation on the recombinant pancreatic carcinoma cell according to the foregoing solution under a fluorescence microscope.
[0035] In the present disclosure, the observation may preferably include one or more of the following items: [0036] (1) observing positive fluorescent labels in extracellular exosomes; [0037] (2) observing a pathfinding trajectory of the cxtracellular exosomes; and [0038] (3) observing a fusion of a multi-vesicular body (MVB) with a cytoplasmic membrane. [0039] The technical solution of the present disclosure will be described below clearly and completely in conjunction with the examples of the present disclosure.
[0040] Example 1
[0041] 1) A target gene pHluorin)11153R-CD63 was obtained with EcoRI as a 5'-end restriction endonuclease and Noll as a 3'-end restriction endonuclease, and recombined with a vector pCDH-CMV-MCS-EF1-PURO to synthesize a plasmid pCDH-CMV-pHluorin M153R-CD63-EF1-Puro (see [Sung B 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 plasmid was synthesized by Shanghai Transheep Biotechnology Co., Ltd. [0042] 2) The plasmid pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro was verified by sequencing, and closed-loop quality identification of the plasmid was conducted by agarose gel electrophoresis of DNA.
[0043] 3) DH-5a competent Escherichia colt cells were taken out from a -80°C freezer and left to stand on ice fors min. 10 IAL of plasmid pCDH-CMV-pHluorin_M153R-CD63-EF1-Puro and 30 [IL of DH-5a competent E. coil cells were pipetted gently to mix and left to stand on ice for 2 min. [0044] 4) The above mixture was accurately incubated on a 42°C metal bath for 45 s, taken out, and left to stand on ice for 2 min. [0045] 5) The mixture was completely added to a culture tube, and 4 mL of LB broth supplemented with AMP was added; the culture tube was placed on a shaker (in a slantwise manner) and shaken at 37°C and 250 rpm for 12-16 h; 4 mL of LB broth supplemented with AMP was added to every 100 RL of bacterial suspension.
[0046] 6) After shaking, each tube of bacterial suspension was divided into two tubes; each tube was made up with 4 mL of LB broth supplemented with AMP and shaken for additional 14-16 h. [0047] 7) A plasmid pCDH-CMV-pHluorin M153R-CD63-EF1-Puro of a higher concentration was extracted by using a kit.
[0048] 8) PANC-1 cells in a 100 mm-size culture dish were digested and re-suspended trypsin, spread evenly on a 6-well plate (approximately 0.5 x 106 cells per well), and transfected after 6 h. [0049] 9) 80 pL of Basal Serum-free Medium was pipetted, supplemented with 4-5 pL of the plasmid pCDH-CM V-pHluorin_M153R-CD63-EF I -Puro (approximately 3 jig) extracted in step 7), and left to stand at room temperature.
[0050] 10) After 5 min, 3 pL of lipo2000 was added to increase the transfection efficiency of the plasmid, the tube wall was flicked (violent pipetting was not allowed), and the tube was left to stand at room temperature for 20 min. [0051] 11) After incubating in an incubator for 48 h, successfully infected cell lines were obtained, and a stably expressed PANC-1 cell line was obtained by screening with puromycin. [0052] 12) Microscopic observation was conducted under a fluorescence microscope; positive fluorescent labels of extracellular exosomes and pathfinding trajectories thereof were observed, and fusion events between MVBs and cytoplasmic membranes were observed. The result is shown in FIG. 1. FIG. 1 illustrates a positive tailing of an extracellular exosome released into the extracellular matrix.
[0053] 13) After stably transfected cells were cultured and stimulated with serum-free Dulbecco's Modified Eagle Medium (DMEM) for 48 h, a supernatant was collected and partly subjected to nanoparticle tracking analysis (NTA). The stimulation herein was starvation, and the conventional cell culture was a complete medium supplemented with 5% fetal bovine serum (FBS). The exosome supernatant was collected and starved with Basal Serum-free Medium. [0054] 14) The collected supernatant was separated using a common high speed centrifuge; [0055] i) At 3,000 rpm and 4°C for 15 min (300 G); [0056] The tube was changed, the supernatant was collected, and pellets were discarded.
[0057] ii) At 5,000 rpm and 4°C for 20 min (2,000 G); [0058] The tube was changed, the supernatant was collected, and pellets were discarded.
[0059] iii) At 12,000 rpm and 4°C for 30 min (16,500 G); [0060] The tube was changed, and particles with a diameter of greater than 0.22 were filtered through a filter tip.
[0061] 15) Using an ultracentrifuge, ultracentrifugation was conducted at 54,000 rpm and 4°C; after 2 h, the supernatant was discarded; pellets were re-suspended with 100 pL of phosphate buffered saline (PBS) or a strong lysis buffer and transferred into a new EP tube.
[0062] 16) The content of CD63 protein in exosomes of stably transfected PANC-1 cells and untreated PANC-1 cells was compared by Western blot and fluorescence microscopy. The results are shown in FIG. 2. As can be seen from FIG. 2, the expression level of CD63 in transfected cell protein is significantly upregulated.
[0063] Comparative Example [0064] Transthction was conducted using GFP-CD63 without modified label pHluorin. The rest of treatments were the same as those in Example 1, and the fluorescence was observed under a fluorescence microscope.
[0065] The result is shown in FIG. 3. As can bc seen from FIG. 3, in the ordinary GFP-CD63-based cxosome tracking method, in the absence of the pHluorin Ml 53R label, thc fluorescence is unstable and not bright enough, and difficult to implement the visualization of the release and uptake of dynamic exosomes.
[0066] In the present disclosure, the pancreatic carcinoma cell line PANC1 was transfected using a recombinant plasmid inserted with a bright tracer target gene pHluorin A/1153R-CD63, and a stable cell line is constructed. Moreover, cells were treated by starvation and using an exosome inhibitor, leading to increased or decreased content of exosomes released by cells; further, observation of different expressions of GFP in the living cell state can show changes in the shape and position of green fluorophore, as well as the presentation of the tailings of positive spots of the tails of the exosomes.
[0067] The method provided by the present disclosure can become a powerful tool in the research on the mechanism of pancreatic carcinoma and has very important innovation significance.
[0068] Although the above example has described the present disclosure in detail, it is only a part of, not all of, the examples of the present disclosure. Other examples may also be obtained by persons based on the example without creative efforts, and all of these examples shall fall within the protection scope of the present disclosure.
Claims (7)
- WHAT IS CLAIMED IS: 1. A recombinant pancreatic carcinoma cell for exosome tracking, wherein the recombinant pancreatic carcinoma cell comprises a recombinant vector; a target gene pH1uorin M153R-CD63 is inserted into the recombinant vector; the target gene pHluorin MI 53R-CD63 is an encoding gene of a protein having an amino acid sequence shown in SEQ ID NO: 1.
- 2. The recombinant pancreatic carcinoma cell according to claim 1, wherein an original vector of the recombinant vector comprises pCDH-CMV-MCS-EF1-PURO.
- 3. The recombinant pancreatic carcinoma cell according to claim 2, wherein restriction sites at 5'-end and 3'-end of an insertion site of the target gene pHluorin M153R-CD63 in the pCDH-CMV-MCS-EFI-PURO are &URI and Non, respectively.
- 4. Use of the recombinant pancreatic carcinoma cell according to any one of claims 1 to 3 in preparation of a reagent or kit for exosome tracking in a pancreatic carcinoma cell.
- 5. A method for exosome tracking in a pancreatic carcinoma cell, comprising the following step: conducting microscopic observation on the recombinant pancreatic carcinoma cell according to any one of claims 1 to 3 under a fluorescence microscope.
- 6. The method according to claim 5, wherein the microscopic observation comprises living cell observation and/or cell immunofluoresccnce observation.
- 7. The method according to claim 5 or 6, wherein the observation comprises one or more of the following items: (1) observing positive fluorescent labels in extracellular exosomes; (2) observing a pathfinding trajectory of the extracellular exosomes; and (3) observing a fusion of a multi-vesicular body (MVB) with a cytoplasmic membrane.
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Non-Patent Citations (4)
| Title |
|---|
| Messenger, S. W. et al, "A Ca2+-stimulated exosome release pathway in cancer cells is regulated by Munc13-4", J Cell Biol, Vol. 217, No. 8, (20180621), see abstract, and p. 2881, right-hand col, para 1, and fig 6, p. 2883, right-hand col, para 1, p. 2886, right-hand col, par 2, and p.2888 * |
| NG, M. et al, "AAS6682.1", Genbank, (20040321), see sequence information * |
| Sung, B. H. et al, "A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells", Nature Communications, vol. 11, no. 1, (20200429), see abstract, and p. 2, left-hand col, par 1 and left-right col. connecting para, p. 6, left-hand col, par 3, and fig 4E-F * |
| Verweij, F.J. et al, "Live Tracking of Inter-organ Communicaiton by Endogenous Exosomes In Vivo", Dev Cell., Vol. 48, No. 4, (20190225), see abstract * |
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