CN115305253A - Recombinant pancreatic cancer cell for exosome tracing and application thereof - Google Patents

Recombinant pancreatic cancer cell for exosome tracing and application thereof Download PDF

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CN115305253A
CN115305253A CN202210040013.6A CN202210040013A CN115305253A CN 115305253 A CN115305253 A CN 115305253A CN 202210040013 A CN202210040013 A CN 202210040013A CN 115305253 A CN115305253 A CN 115305253A
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pancreatic cancer
exosome
recombinant
cancer cell
phluorin
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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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Affiliated Hospital of Nantong University
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Abstract

The invention provides a recombinant pancreatic cancer cell for exosome tracing and application thereof, belonging to the technical field of biomedicine; the recombinant pancreatic cancer cell comprises a recombinant vector; the destination gene pHluorin _ M153R-CD63 is inserted into the recombinant vector. The invention utilizes the recombinant vector inserted with the target gene pHluorin _ M153R-CD63 to construct a pancreatic cancer stable cell line, and can more easily observe the path-seeking track after effectively marking extracellular exosomes and tail spots released in extracellular matrix, thereby revealing the pathogenic behavior of migrating living cells. The invention solves the visualization problem in the research of the exosomal pathogenic behavior mechanism, constructs the fluorescent tracer substance which is bright and stable and is suitable for various cell culture environments, and can monitor the release and uptake of the exosome in the migration process of living cells and observe the path-finding track.

Description

Recombinant pancreatic cancer cell for exosome tracing and application thereof
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a recombinant pancreatic cancer cell for exosome tracing and application thereof.
Background
Exosomes are a kind of extracellular vesicles which are 30-150 nm in size and are in a saucer shape and are wrapped by lipid bilayer membranes, and are widely present in cell culture supernatants and various body fluids, including blood, lymph fluid, saliva, urine, follicular fluid, semen, pericardial fluid, milk and the like. Exosomes originate in the endosome, and the continuous invagination of the cytoplasmic 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 contributing to the diversity of exosome components. Cell components are communicated and transported among cells through exosomes, the exosomes secreted by tumor cells can help the tumor cells to transfer by shaping a tumor microenvironment, the secretion quantity of the exosomes can directly influence the tumor transfer, and the exosomes play an important role in the process of promoting the formation and proliferation of tumors. Exosomes are reported to be present in all biological fluids, the composition of the complex of exosomes can be readily obtained by sampling the biological fluids, and exosomes can be shown to have significant potential for diagnosing tumors and other diseases and revealing their prognosis based on biological fluid biopsies. However, research on the specific pathogenic behavior mechanism of exosome is difficult to break through, and is mostly limited by visual presentation of the behavior process.
Pancreatic cancer is one of the common malignant tumors in the digestive tract, and is called the king of cancer in the field of tumors. Pancreatic cancer has the characteristic of obviously enhancing secretion of exosome, and a great problem in researching a deep mechanism of pancreatic cancer disease is observation, measurement and visualization of the behavior of exosome.
In vivo tracking of exosomes may provide important knowledge roles, communication abilities and action mechanisms regarding the biodistribution, migration ability, toxicity, biology of exosomes. In the prior art, researchers have used molecular biology methods to express fluorescent proteins (e.g., green fluorescent protein GFP; red fluorescent protein RFP) fused to marker proteins on exosome membranes, such as CD63, CD9, CD81, etc., to track the movement of exosomes from donor to recipient cells. By monitoring the fluorescence of the membrane proteins, direct visualization of exosome transfer in vivo and in vivo cultures can be achieved. For example, the expression elements of the exosome specific proteins CD63 and green fluorescent protein GFP are constructed as plasmids and packaged into lentiviruses, which are then used to infect cells to make the exosomes secreted by the cells green fluorescent. However, in the conventional GFP-CD63 exosome tracing method, the fluorescence is unstable and is not bright enough, and it is difficult to visualize the dynamic exosome release and uptake.
Disclosure of Invention
In view of the above, the present invention provides a recombinant pancreatic cancer cell for exosome tracing and an application thereof, and an exosome tracing 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 invention provides a recombinant pancreatic cancer cell for exosome tracing, which comprises a recombinant vector; the target gene pHluorin _ M153R-CD63 is inserted into the recombinant vector; the target gene pHluorin _ M153R-CD63 is a coding gene of a protein with an amino acid sequence shown in SEQ ID No. 1.
Preferably, the original vector of the recombinant vector comprises pCDH-CMV-MCS-EF1-PURO.
Preferably, the restriction site 5 'to the insertion site of pHluorin _ M153R-CD63 on pCDH-CMV-MCS-EF1-PURO is EcoR I, and the restriction site 3' to the insertion site is NotI.
The invention also provides application of the recombinant pancreatic cancer cell in the scheme in preparation of a reagent or a kit for tracing pancreatic cancer cell exosomes.
The invention also provides a pancreatic cancer cell exosome tracing method, which comprises the following steps:
the recombinant pancreatic cancer cells described in the above protocol were observed under a mirror using a fluorescence microscope.
Preferably, the under-mirror observation comprises live cell observation and/or cellular immunofluorescence observation.
Preferably, the observed content includes one or more of the following items:
(1) Observing the positive fluorescent marker of the extracellular exosomes;
(2) Observing the path-seeking track of the extracellular exosome;
(3) The fusion of the multivesicular bodies with the cytoplasmic membrane was observed.
The invention provides a recombinant pancreatic cancer cell for exosome tracing, which comprises a recombinant vector; the target gene pHluorin _ M153R-CD63 is inserted into the recombinant vector; the target gene pHluorin _ M153R-CD63 is a coding gene of a protein with an amino acid sequence shown as SEQ ID No. 1. According to the invention, the coding gene of the CD63-GFP fusion protein is modified, the pH-sensitive GFP derivative pHluorin is added to reduce the too high brightness of the CD63-GFP fusion protein in the endosome of the cell, so that the dynamic fusion event of a multivesicular body (MVB) and a plasma membrane can be better observed, and the single amino acid mutation M153R is added to ensure that the pHluorin-CD63 is not easy to bleach and quench under the culture conditions of 2D and 3D cells. The invention constructs a pancreatic cancer stable cell line based on a recombinant vector inserted with a target gene pHluorin _ M153R-CD63, performs living cell observation or cell immunofluorescence observation under a microscope on the recombinant pancreatic cancer stable cell line transfected with a recombinant plasmid, effectively marks extracellular exosomes and tail spots released in extracellular matrix under a confocal microscope, and can more easily observe a path-finding track, thereby revealing the pathogenic behavior of migrating living cells. The invention solves the visualization problem in the research of the pathogenic behavior mechanism of the exosome, constructs the fluorescent tracer substance which is bright and stable and is suitable for various cell culture environments, can monitor the release and uptake of the exosome in the migration process of living cells and observe the path-finding track, and is used for the research of the pathogenic mechanism of the exosome in diseases.
Drawings
FIG. 1 shows the fluorescence imaging results of example 1;
FIG. 2 shows the results of WesternBlot efficiency validation;
FIG. 3 shows the fluorescence imaging results of the comparative example.
Detailed Description
The invention provides a recombinant pancreatic cancer cell for exosome tracing, which comprises a recombinant vector; the target gene pHluorin _ M153R-CD63 is inserted into the recombinant vector; the target gene pHluorin _ M153R-CD63 is a coding gene of a protein with an amino acid sequence shown as SEQ ID No. 1.
The invention improves the coding gene of the CD63-GFP fusion protein, adds a pH-sensitive GFP derivative pHluorin to reduce the overhigh brightness of the CD63-GFP fusion protein in a cell endosome so as to better observe the dynamic fusion event of a multivesicular body (MVB) and a plasma membrane, and adds a single amino acid mutation M153R so that the pHluorin-CD63 is stably expressed under the culture conditions of cells 2D and 3D, thereby avoiding the premature bleaching and quenching of fluorescence.
In the invention, the target gene pHluorin _ M153R-CD63 is a coding gene of a protein with an amino acid sequence shown as SEQ ID NO. 1; the amino acid sequence of the protein shown in SEQ ID NO.1 is specifically as follows:
SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIRADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK。
in the present invention, the nucleotide sequence of the gene of interest pHluorin _ M153R-CD63 is described in (Sun B H, lersner A V, guerrero J, et al. A. Live cell reporter of exosome precipitation and uptake of nucleic acids purification primers [ J ]. Nature communications.).
In the present invention, the original vector of the recombinant vector preferably comprises pCDH-CMV-MCS-EF1-PURO. In the present invention, the restriction site 5 'to the insertion site of pHluorin _ M153R-CD63 into pCDH-CMV-MCS-EF1-PURO is EcoR I, and the restriction site 3' to the insertion site is NotI.
The method for constructing the recombinant vector is not particularly limited, and a conventional construction method in the art can be used.
The recombinant vector pCDH-CMV-pHluorin _ M153R-CD 63-PERO is formed by recombining the target gene pHluorin _ M153R-CD63 and the vector pCDH-CMV-MCS-EF1-PURO, and has the advantage of high transfection efficiency.
In the present invention, the pancreatic cancer cell preferably comprises a pancreatic cancer cell line PANC-1.
The invention also provides application of the recombinant pancreatic cancer cell in the scheme in preparation of a reagent or a kit for tracing pancreatic cancer cell exosomes.
The invention also provides a pancreatic cancer cell exosome tracing method, which comprises the following steps:
the recombinant pancreatic cancer cells described in the above protocol were observed under a mirror using a fluorescence microscope.
In the present invention, the observed content preferably includes one or more of the following items:
(1) Observing the positive fluorescent marker of the extracellular exosomes;
(2) Observing the path-seeking track of the extracellular exosome;
(3) The fusion of the multivesicular bodies with the cytoplasmic membrane was observed.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1
1) EcoRI is used as 5 'end restriction enzyme, notI is used as 3' end restriction enzyme, a target gene pHluorin _ M153R-CD63 is obtained, and the target gene pHluorin _ M153R-CD63-EF1-PURO is recombined with a vector pCDH-CMV-MCS-EF1-PURO into a plasmid pCDH-CMV-pHluorin _ M153R-CD63-EF1-PURO (see [ Sung B H, lersner A V, guerrero J, et al. A. Live cell reporter of exosome differentiation and uptake cells Pathofinning behavior of hybridization cells [ J ]. Nature communications.), which is synthesized by Shanghai rights Yang Biotech Limited.
2) The plasmid pCDH-CMV-pHluorin _ M153R-CD63-EF1-Puro is subjected to sequencing verification, and the closed-loop quality of the plasmid is identified by DNA gel electrophoresis.
3) The DH-5. Alpha. Competence was removed from the freezer at-80 ℃ and allowed to stand on ice for 5min. 10 μ l of plasmid pCDH-CMV-pHluorin _ M153R-CD63-EF1-Puro and 30 μ lDH-5 α were aspirated, mixed by gentle blowing, and allowed to stand on ice for 2min.
4) After incubation in a metal bath at 42 ℃ for 45s, the cells were removed and placed on ice for 2min.
5) Adding all the components into a shake tube, adding 4ml of LB liquid containing AMP to cultivate, putting the mixture into a shaking table (inclined placement), shaking at 37 ℃ and 250 rpm for 12-16 h, and adding 4ml of LB liquid containing AMP to each 100 mu l of bacterial liquid to cultivate.
6) After shaking, each tube of the bacterial liquid is divided into two tubes averagely, 4ml of liquid containing AMP is supplemented for cultivation, and the bacteria are shaken again for 14-16 h.
7) Extracting by using a kit to obtain a plasmid pCDH-CMV-pHluorin _ M153R-CD63-EF1-Puro with higher concentration.
8) Pancreatin was used to digest and resuspend PANC-1 cells in the Petri dish, and then plated evenly into six-well plates (approximately 0.5 × 10 cells per well) 6 One), transfection was performed after 6h.
9) Sucking 80. Mu.l of a serum-free basal medium, adding 4 to 5. Mu.l of the plasmid pCDH-CMV-pHluorin _ M153R-CD63-EF1-Puro (about 3. Mu.g) extracted in step 7), and standing at room temperature.
10 5min later 3 μ l lipo2000 was added to increase plasmid transfection efficiency and gently flicked (without forceful flicking) tube walls were left at room temperature for 20min.
11 Culturing in an incubator for 48h to obtain a cell strain successfully infected, and adding puromycin for screening to obtain a PANC-1 cell strain stably expressed.
12 Using a fluorescence microscope for under-lens observation, observing the positive fluorescence label of the extracellular exosome and the path-finding track thereof, and observing the fusion event of the MVB and the cytoplasmic membrane, and the result is shown in figure 1. As can be seen from fig. 1, the extracellular exosomes are released to the positive tail of the extracellular matrix.
13 Culture and stimulation of the stably transformed cell lines in serum-free DMEM medium for 48 hours, and then the supernatant was collected and partially subjected to NTA analysis. The stimulation is starvation stimulation, the conventional cell culture is complete culture medium containing 5% fetal calf serum, and the supernatant of the exosome is collected and subjected to starvation stimulation by using a serum-free basal medium.
14 Separating the collected supernatant with a common high-speed centrifuge;
①3000rpm 15min4℃(300G)；
and (4) replacing the tube, collecting the supernatant, and discarding the precipitate.
②5000rpm 20min 4℃(2000G)；
And (4) replacing the tube, collecting the supernatant, and discarding the precipitate.
③12000rpm 30min4℃(16500G)；
The pipe is changed, and the filter tip filters out particles with the diameter of more than 0.22.
15 Ultracentrifuge, 4 ℃, 54000rpm ultracentrifuge, 2h after discarding the supernatant, 100 u L PBS or strong lysate heavy suspension precipitation, transfer to new EP tube.
16 Westernblotting and fluorescence microscopy compared the amount of CD63 protein in stable PANC-1 extracellular exosomes to untreated PANC-1 cells, and the results are shown in FIG. 2. As can be seen in FIG. 2, the expression level of CD63 in the cell protein after transfection was significantly increased.
Comparative example
The transfection was carried out using GFP-CD63 without the modified tag pHluorin, and the rest of the treatment was the same as in example 1, and the fluorescence state was observed under a fluorescence microscope.
The results are shown in FIG. 3. As can be seen from fig. 3, in the conventional GFP-CD63 exosome tracing method, the pHluorin _ M153R tag is absent, the fluorescence is unstable and not bright enough, and it is difficult to visualize the dynamic exosome release and uptake.
The invention uses a recombinant plasmid inserted with a bright tracer target gene pHluorin _ M153R-CD63 to transfect a pancreatic cancer cell line PANC1 and construct a stable cell line. And the hunger stimulation and the treatment of the cells by using an exosome inhibitor lead to the increase or decrease of the content of exosomes released by the cells, and the different expressions of green fluorescence are observed under the state of living cells, so that the change of the shape and the position of a green light mass can be seen, and the trailing presentation of a positive spot at the tail part of the exosome can be seen.
The method can become a powerful tool in the pancreatic cancer mechanism research, and has very important innovative significance.
Although the present invention has been described in detail with reference to the above embodiments, it is to be understood that the present invention is not limited to the details of the embodiments, and that other embodiments may be devised without departing from the spirit and scope of the present invention.
Sequence listing
<110> affiliated hospital of Nantong university
<120> recombinant pancreatic cancer cell for exosome tracing and application thereof
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Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly
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Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr
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Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr
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Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr
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Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys
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Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp
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Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr
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Asn Asp His Gln Val Tyr Ile Arg Ala Asp Lys Gln Lys Asn Gly Ile
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Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln
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Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val
180 185 190
Leu Leu Pro Asp Asn His Tyr Leu Phe Thr Thr Ser Thr Leu Ser Lys
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Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr
210 215 220
Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys
225 230 235

Claims (7)

1. A recombinant pancreatic cancer cell useful for exosome tracing, comprising a recombinant vector; the target gene pHluorin _ M153R-CD63 is inserted into the recombinant vector; the target gene pHluorin _ M153R-CD63 is a coding gene of a protein with an amino acid sequence shown in SEQ ID No. 1.
2. The recombinant pancreatic cancer cell of claim 1, wherein the original vector of said recombinant vector comprises pCDH-CMV-MCS-EF1-PURO.
3. The recombinant pancreatic cancer cell according to claim 2, wherein the restriction enzyme site 5 'to the insertion site of the gene of interest pHluorin _ M153R-CD63 into pCDH-CMV-MCS-EF1-PURO is EcoR I, and the restriction enzyme site 3' to the insertion site is Not I.
4. Use of the recombinant pancreatic cancer cell of any one of claims 1 to 3 for the preparation of a reagent or kit for exosome-tracking of pancreatic cancer cells.
5. A method of pancreatic cancer cell exosome tracking comprising the steps of:
the recombinant pancreatic cancer cell of any one of claims 1 to 3, which is observed under a microscope using a fluorescence microscope.
6. The method of claim 5, wherein the under-the-mirror observation comprises live cell observation and/or cellular immunofluorescence observation.
7. The method of claim 5 or 6, wherein the observed content comprises one or more of the following items:
(1) Observing the positive fluorescent marker of the extracellular exosomes;
(2) Observing the path-finding track of the extracellular exosome;
(3) The fusion of the multivesicular bodies with the cytoplasmic membrane was observed.
CN202210040013.6A 2022-01-14 2022-01-14 Recombinant pancreatic cancer cell for exosome tracing and application thereof Pending CN115305253A (en)

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Application publication date: 20221108