CN117442585A - Tripterine delivery body for targeting pancreatic cancer and preparation method thereof - Google Patents
Tripterine delivery body for targeting pancreatic cancer and preparation method thereof Download PDFInfo
- Publication number
- CN117442585A CN117442585A CN202311785807.6A CN202311785807A CN117442585A CN 117442585 A CN117442585 A CN 117442585A CN 202311785807 A CN202311785807 A CN 202311785807A CN 117442585 A CN117442585 A CN 117442585A
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- tripterine
- pancreatic cancer
- membrane protein
- exosome
- targeting
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Abstract
The invention relates to the technical field of biological medicine, in particular to a tripterine delivery body targeting pancreatic cancer and a preparation method thereof. The delivery body is obtained by loading tripterine on membrane protein engineering exosomes obtained by separating after steady cell transfer of exosome membrane protein lentiviral expression vectors; the functional sequence structure of the exosome membrane protein lentiviral expression vector comprises a kozak sequence, an initiation codon, an exogenous targeting gene PTP, an exosome membrane protein LAMP2B, P A cleavage peptide, an initiation codon, an EGFP fluorescent sequence and a stop codon from the N end. The engineering exosome for targeting pancreatic cancer is prepared by a genetic engineering method, so that the targeting capability of the exosome is greatly enhanced, the engineering exosome is used for loading tripterine, the tripterine can better exert the drug effect, the drug absorption efficiency is improved, the drug toxicity is reduced, the apoptosis of pancreatic cancer cells is promoted, and the control of pancreatic cancer is facilitated.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a tripterine delivery body targeting pancreatic cancer and a preparation method thereof.
Background
Pancreatic cancer is a digestive tract tumor with extremely poor prognosis, and has the characteristics of difficult early diagnosis, low surgical excision rate, easy recurrence and metastasis after surgery and the like. Advanced pancreatic cancer systemic treatment, again based on chemotherapy. In recent years, rapid progress of immunotherapy has not been approved for pancreatic cancer. The targeted therapeutic drugs have remarkable treatment effects of PARP inhibitors and KRAS-G12C inhibitors, but are limited to pancreatic cancer patients with lower mutation frequency for adapting to the inhibitors, and less benefited patients. Although the molecular mechanism and drug studies of pancreatic cancer have been in progress, prognosis of pancreatic cancer patients has not been significantly improved. Because of the uniqueness of pancreatic cancer tumor microenvironment components, the pancreatic cancer tumor microenvironment components have great influence on various aspects such as proliferation, metastasis, immune escape, drug resistance and the like of tumors, and deep research on pathogenesis of pancreatic cancer TME components, novel targeted drugs and combined administration schemes is urgent and necessary.
Celastrol (Celastrol, cel for short), also called celastrine, a natural friedelane type pentacyclic triterpene, is one of the effective active ingredients of the tripterygium wilfordii, and has remarkable inhibition effect on various tumor types. Tripterine can induce apoptosis of pancreatic cancer cells by targeted inhibition of PAK1 kinase signaling pathway, and has potential value for treating pancreatic cancer (tripterine has effects of inhibiting PAK1 against pancreatic cancer and mechanism research thereof, zhu Lingxia, etc.). The tripterine-chitosan oligosaccharide conjugate (Cel-CSO) remarkably inhibits the tumor growth of human pancreatic cancer cells (BxPC-3), induces apoptosis and effectively inhibits tumor metastasis (Celastrol-conjugated chitosan oligosaccharide for the treatment of pancreatic cancer, xiaohu Zeng, ect), but has poor water solubility, low bioavailability, easy occurrence of adverse reaction when taken orally and other problems, thus affecting the wide clinical application.
Exosomes refer to small vesicles (30-150 nm) containing complex RNAs and proteins, which nowadays are specifically disc-shaped vesicles with diameters of 40-100 nm. All cultured cell types secrete exosomes, and exosomes naturally occur in body fluids, including blood, saliva, urine, cerebrospinal fluid and milk. Mainly derived from the multivesicular body formed by the invagination of intracellular lysosome particles, and released into extracellular matrix after fusion of the outer membrane of the multivesicular body with the cell membrane. When exosomes were first discovered in 1983, they were considered a way for cells to excrete waste, and today a wide variety of functions are found with extensive research into their biological sources, their material constitution and transport, intercellular signaling, and distribution in body fluids. The function of exosomes depends on the cell type from which they are derived, and they can be involved in a variety of aspects such as immune responses in the body, antigen presentation, cell migration, cell differentiation, tumor invasion, etc.
However, fusion of exosomes with recipient cells is not random, and natural exosomes have the problem of poor targeting.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a tripterine delivery body for targeting pancreatic cancer and a preparation method thereof, wherein the exosome can effectively inhibit proliferation of pancreatic cancer and promote apoptosis of pancreatic cancer cells.
In a first aspect of the invention, there is provided a delivery body of tripterine for targeting pancreatic cancer, which is obtained by loading tripterine on a membrane protein engineering exosome obtained by separating a cell stably transformed by an exosome membrane protein lentiviral expression vector; the functional sequence structure of the exosome membrane protein lentiviral expression vector comprises a kozak sequence, an initiation codon, an exogenous targeting gene PTP, an exosome membrane protein LAMP2B, P A cleavage peptide, an initiation codon, an EGFP fluorescent sequence and a stop codon from the N end.
PTP (KTLLPTP) specifically binds to pancreatic cancer cells. Through proteomics analysis, the targeting peptide PTP realizes the targeting effect through specific binding with pleectin-1 which is mispositioned on the surface of a cell membrane when pancreatic cancer occurs. Plectin1 is a traditional scaffold cytoskeletal protein that is abundant in almost all mammalian tissues and cell types, especially in tissues that are resistant to high mechanical stress. In normal physiological conditions Plectin1 is usually expressed only in the cytoplasm and not on the cell membrane. However, if the tissue is cancerous, the cellular localization of Plectin1 may change and may exist on both the cytoplasm and the cell membrane. The presence of Plectin1 was found in murine and human PDAC cells. Plectin1 is of considerable interest to researchers because of its specific abnormal localization to the surface of pancreatic cancer epithelial cells. In 2008, a targeting peptide PTP with 7 amino acids (KTLLPTP) in a functional short peptide is screened out by utilizing a gene mouse PDAC model and a phage display library technology, so that pancreatic cancer can be specifically targeted. The targeting peptide can form liposome distearoyl phosphatidylethanolamine-polyethylene glycol-pancreatic cancer targeting peptide PTP (DSPE-PEG-PTP), and the formed liposome can directly act on a tumor target to form an active targeting effect, but the combination is not expressed in a genetic layer, so that the liposome is unstable.
Lysosomal associated membrane protein 2B (LAMP 2B), a member of the Lysosomal Associated Membrane Protein (LAMP) family, is localized mainly in lysosomes and endosomes within cells, is distributed on cell surfaces in small amounts, and is also expressed in large amounts on exosomes. LAMP protein provides carbohydrate ligand for selectin, plays a role in tumor cell metastasis, and also plays a role in protecting, maintaining and adhering lysosomes. Alternative splicing of the gene results in multiple transcript variants encoding different proteins. Human LAMP2B has a molecular weight of 39 kDa and has 363 amino acids, comprising a 29 amino acid signal peptide, an N-terminal membrane ectodomain and a C-terminal transmembrane region, comprising a very short cytoplasmic tail. In the invention, targeting peptide PTP and LAMP2B are adopted to fuse and display a targeting motif, so as to realize targeting effect.
Preferably, the sequence of the exogenous targeting gene PTP is shown as SEQ ID NO:1, wherein the LAMP2B sequence of the exosome membrane protein is shown in SEQ ID NO: 2.
Preferably, the kozak sequence is GCCACC, and the P2A cleavage peptide sequence is shown in SEQ ID NO:3, the EGFP fluorescent sequence is shown as SEQ ID NO: 4.
Preferably, the functional sequence structure of the exosome membrane protein lentiviral expression vector is shown in SEQ ID NO: shown at 5.
Preferably, the exosome membrane protein lentiviral expression vector is constructed from psPAX2, pCMV-VSV-G and pLVX-PTP-LAMP2B-EGFP-puro plasmids.
Preferably, the pLVX-PTP-LAMP2B-EGFP-Puro plasmid is obtained by constructing pLVX-Puro after recombination of an A fragment (SEQ ID NO: 6) and a B fragment (SEQ ID NO: 7) into XhoI-EcoRI double cleavage.
In a second aspect of the present invention, there is provided a method for preparing the pancreatic cancer-targeted tripterine delivery body, comprising the steps of:
s1, constructing an exosome membrane protein lentiviral expression vector containing an exogenous targeting gene PTP and exosome membrane protein LAMP 2B;
s2, transfecting the exosome membrane protein lentiviral expression vector constructed in the step S1 into HEK293T cells, and screening a stable transgenic cell line;
s3, the stable transgenic HEK293T cells screened in the step S2 secrete membrane protein engineering exosomes;
s4, loading the membrane protein engineering exosomes secreted in the step S3 and the tripterine to construct the tripterine delivery body targeting pancreatic cancer.
Preferably, puromycin is used in step S2 to screen for stable transgenic cell lines.
Preferably, the step S3 specifically includes: centrifuging to remove HEK293T cells from the cell culture liquid, applying a larger centrifugal force to the supernatant to remove large cell fragments, and performing high-speed centrifugation again to collect membrane protein engineering exosomes from the supernatant.
Preferably, step S4 specifically includes: dissolving tripterine in dimethyl sulfoxide, diluting with PBS, mixing membrane protein engineering exosome with diluted tripterine solution, incubating at room temperature, and filtering with ultrafilter tube to remove free tripterine.
The beneficial effects are that:
according to the technical scheme, the engineering exosomes for targeting pancreatic cancer are prepared by a genetic engineering method, so that the targeting ability of the exosomes is greatly enhanced, and tripterine is loaded for pancreatic cancer targeting treatment. The preparation method provided by the invention is simple and easy to implement, the obtained delivery body is stable, an effective amplification process is realized, the effect of the tripterine can be better exerted, the drug absorption efficiency is improved, the drug toxicity is reduced, the drug is conveyed more safely, the pancreatic cancer control is facilitated, and a new way is opened up for the tripterine transportation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows a plasmid map of pLVX-PTP-Lamp2B-EGFP-puro provided by the present invention.
FIG. 2 shows Exo, exo provided by the present invention PTP 、Exo PTP Three exosome western blotting identification result diagrams of @ Cel.
Fig. 3 is a transmission electron microscope image of Exo exosomes provided by the present invention.
FIG. 4 shows Exo provided by the present invention PTP Transmission electron microscopy of exosomes.
FIG. 5 is a graph showing the particle size distribution of Exo exosomes provided by the present invention.
FIG. 6 is a graph showing the particle count of Exo exosomes provided by the present invention.
FIG. 7 shows Exo provided by the present invention PTP Particle size distribution profile of exosomes.
FIG. 8 shows Exo provided by the present invention PTP Particle count diagram of exosomes.
Fig. 9 is a graph of detection results of Exo visible light spectrum provided by the present invention.
FIG. 10 shows Exo according to the present invention PTP And (5) a visible spectrum detection result diagram.
FIG. 11 shows Exo provided by the present invention PTP And (5) a visible spectrum detection result diagram of the @ Cel.
FIG. 12 shows Exo provided by the present invention PTP Results of pro-apoptotic effects of Control on PANC-1 cells.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms also include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Examples
The embodiment provides a tripterine delivery body for targeting pancreatic cancer, which is obtained by loading tripterine on a membrane protein engineering exosome obtained by separating after steady cell transfer of an exosome membrane protein lentiviral expression vector; the functional sequence structure of the exosome membrane protein lentiviral expression vector comprises a kozak sequence, an initiation codon, an exogenous targeting gene PTP, an exosome membrane protein LAMP2B, P A cleavage peptide, an initiation codon, an EGFP fluorescent sequence and a termination codon from the N end.
In this embodiment, the sequence of the exogenous targeting gene PTP is shown in SEQ ID NO:1, the LAMP2B sequence of the exosome membrane protein is shown as SEQ ID NO: 2.
In this example, the kozak sequence GCCACC, P2A cleavage peptide sequence is set forth in SEQ ID NO:3, EGFP fluorescent sequence is shown as SEQ ID NO: 4.
In this example, the functional sequence structure of the exosome membrane protein lentiviral expression vector is shown in SEQ ID NO: shown at 5.
In this example, the exosome membrane protein lentiviral expression vector was constructed from psPAX2, pCMV-VSV-G and pLVX-PTP-LAMP2B-EGFP-puro plasmids.
In this example, the pLVX-PTP-LAMP2B-EGFP-Puro plasmid was obtained by recombinant of fragment A (SEQ ID NO: 6) and fragment B (SEQ ID NO: 7) into the double XhoI-EcoRI digested pLVX-Puro construct.
The embodiment also provides a preparation method of the tripterine delivery body for targeting pancreatic cancer, which comprises the following steps:
s1, constructing an exosome membrane protein lentiviral expression vector containing an exogenous targeting gene PTP and exosome membrane protein LAMP 2B;
s2, transfecting the exosome membrane protein lentiviral expression vector constructed in the step S1 into HEK293T cells, and screening a stable transgenic cell line;
s3, the stable transgenic HEK293T cells screened in the step S2 secrete membrane protein engineering exosomes;
s4, loading the membrane protein engineering exosomes secreted in the step S3 and the tripterine to construct the tripterine delivery body targeting pancreatic cancer.
In this example, puromycin is used in step S2 to screen for stably transfected cell lines.
In this embodiment, step S3 specifically includes: centrifuging to remove HEK293T cells from the cell culture liquid, applying a larger centrifugal force to the supernatant to remove large cell fragments, and performing high-speed centrifugation again to collect membrane protein engineering exosomes from the supernatant.
In this embodiment, step S4 specifically includes: dissolving tripterine in dimethyl sulfoxide, diluting with PBS, mixing membrane protein engineering exosome with diluted tripterine solution, incubating at room temperature, and filtering with ultrafilter tube to remove free tripterine.
In one embodiment, the method of preparing a pancreatic cancer targeted tripterine delivery body is as follows:
1. synthesis and cloning of plasmid (pLVX-PTP-Lamp 2B-EGFP-puro)
The primer of the target plasmid is amplified by a PCR method to obtain a fragment PCR product (the primer information is shown in table 1), and the full-length construction of the target plasmid is obtained by recombining the A fragment (SEQ ID NO: 6) and the B fragment (SEQ ID NO: 7) into a pLVX-Puro linearization vector after XhoI-EcoRI double digestion.
TABLE 1 primer information
The connection method is as follows:
the connection reaction system of the treated target fragment and the carrier is shown in Table 2.
TABLE 2
The method of transformation is as follows:
(1) Plasmid was aspirated at a plasmid concentration of about 100 ng/. Mu.L by adding 1-3. Mu.L to about 100. Mu.L of competent cells (E.coli Top 10), and the mixture was gently swirled to mix and left on ice for 3 minutes. The plasmid concentration is higher and the plasmid is properly added less, and the plasmid concentration is lower and some plasmids can be added more.
(2) The 42℃water bath 90 s is not shaken.
(3) The solution was left in the ice bath for about 3 minutes.
(4) 500-800. Mu.L of pre-warmed LB medium at 37℃was added to each tube, and shaking table 200 rpm at 37℃was gently performed for 40 minutes.
Verification of recombinants:
(1) Puromycin-containing agar plates were prepared.
(2) mu.L of the bacterial liquid was taken, then spread on an agar plate containing the corresponding resistance, gently spread on the surface of the plate with a sterile glass applicator, and the plate was incubated at 37℃for 15 minutes.
(3) Colonies can appear by incubating the inverted plate at 37℃for 12-16 hours.
(4) The plate is picked up, the bacteria are shaken for 14 hours at 37 ℃ and 250 revolutions per minute, PCR identification is carried out by using bacterial liquid, and the positive clone bacterial liquid is sent to sequencing.
2. Method for identifying cloning plasmid
(1) The target plasmid-A fragment was PCR amplified and the identified primers are shown in Table 1.
The expected amplified fragment length was 1118, and the PCR reaction used a 20. Mu.L system: primer 0.5. Mu.L, template bacteria 2. Mu.L, polymerase buffer 0.5. Mu.L, buffer 3. Mu.L, ddH2O 14. Mu.L. Cycle parameters: pre-denaturation at 96℃for 3 min;95℃15S, 58℃15S, 72℃20S, 23 cycles, 72℃final extension 1 min.
(2) Screening positive clones by a bacterial liquid PCR method, shaking bacteria and extracting plasmids at 37 ℃ for the obtained positive bacterial liquid, and sequencing.
(3) The correct plasmid was sequenced (primers shown in Table 3) and digested with XhoI to obtain the 2097/8119 fragments.
TABLE 3 sequencing primers
The pLVX-PTP-Lamp2B-EGFP-puro plasmid map is shown in figure 1, and PTP is used as a targeting peptide for targeting pancreatic cancer cells at the N end of an exosome surface protein Lamp 2B; EGFP is at the C-terminus, indicating transfection effect.
3. Transfection
(1) Packaging cells (HEK 293T cells) were plated in 6 well plates. Plates were plated with 6E+5 cells/6 well plates using 3 mL complete medium (DMEM+10% FBS) at 37℃with 5% CO 2 Incubate overnight.
(2) Opti-MEM diluted lip 3000 (μl) was well mixed in tube A, opti-MEM diluted psPAX2 (lentiviral packaged helper plasmid), pCMV-VSV-G (lentiviral packaged helper plasmid), pLVX-PTP 2B-EGFP-puro were well stirred in tube B, then P3000 ™ reagent was added to tube B and well mixed, diluted Lipofectamine ™ 3000 reagent was added from tube A to tube B (1:1 ratio) of diluted DNA, well mixed and incubated at room temperature for 10-15 min. Gently adding the mixture to the cells, gently shaking the 6-well plate to mix, and mixing at 37deg.C, 5%, CO 2 Incubate for 16 hours.
(3) The medium was changed and at 37℃5% CO 2 Cells were cultured under for 48 hours.
(4) Inoculating the target cells. Plates were plated with 2 mL complete medium at 3E+5 cells/6 well plate.
(5) The virus supernatant was harvested.
a. The virus supernatant was collected from 293T flasks, centrifuged at 200g for 5min and then filtered through a 0.45. Mu.M filter to remove cellular particles and debris.
If not used immediately, the virus supernatant should be stored in a refrigerator at-80 ℃. However, during the freeze and thaw process, virus titer can decrease.
b. Polybrene (stock 10 mg/mL) was added to the virus supernatant to a final concentration of 8 ug/mL and mixed thoroughly prior to use.
(6) Transfecting the target cells.
a. The 0.5mL medium was removed from HEK293T cell flasks and discarded.
b. 1.5mL Polybrene-enhanced viral supernatant was added to HEK293T cells, mixed well and incubated at 37℃with 5% CO 2 Incubate for 72 hours.
(7) Puromycin selection of stably transformed cell lines.
(8) And (5) monoclonal culture.
(9) Extracting exosomes. Centrifugation was first performed at 300g for 10min to remove cells from the cell culture broth; then, applying a larger centrifugal force of 10000-20000g for 30min to the supernatant to remove large cell fragments and broken organelles; finally, the exosomes were collected from the supernatant by centrifugation again at a high speed of 100000-150000g for 70min, all at 4 ℃.
(10) Tripterine loading. For loading Cel into exosomes, a room temperature incubation method was used. Cel was dissolved in dimethyl sulfoxide, followed by Exo PTP The suspension was mixed with Cel solution diluted with PBS at 25℃for 2h with a rotary mixer. Exo was filtered through a 0.5mL30kDa ultrafiltration tube PTP Ultrafiltration of 2 times to wash free Cel, the upper layer of the ultrafiltration tube was resuspended in PBS to trap particles.
The exosomes have a lipid bilayer membrane structure, and the membrane surface is rich in sphingomyelin, cholesterol and ceramide lipid raft lipids; abundant proteins are included, including membrane transport factors, signal transduction factors (e.g., β -catenin, wnt), cytoskeletal proteins (e.g., β -actin, myosin, and tubulin), transmembrane proteins that play an important role in cell adhesion (e.g., CD9, CD63, CD81, and CD 82), and heat shock proteins involved in the protein folding process. The proteins such as TSG101, CD63, LAMP2, HSP70 and the like can be used as biomarkers of exosomes and used for identifying the exosomes. Therefore, the HEK293T cell exosome (Exo) extracted in the above step and the exosome (Exo) of the HEK293T cell transfected by the lentivirus were subjected to PTP ) Cel-loaded lentivirus transfected exosomes of HEK293T cells (Exo PTP Western blot identification of three exosomes @ Cel) is performed, the results are shown in fig. 2, from which it can be seen: exosome protein markers CD63, LAMP2, HSP70 and TSG101 in Exo, exo PTP Middle, exo PTP The endoplasmic reticulum protein Calnexin is an exosome negative protein which can be obviously detected.
Exo、Exo PTP The results of the transmission electron microscopy of the exosomes are shown in figures 3-4, from which it can be seen that: exo, exo PTP The exosomes are basically consistent in shape and size, all show obvious phospholipid bilayer spherical shape, have obvious membrane boundaries under an electron microscope, and are in a tea tray or cup-shaped structure with different sizes of 30-200nm, which shows that the transfected pLVX-PTP-Lamp2B-EGFP-puro plasmid basically does not change the shape and size of exosomes.
The particle size distribution and particle number of Exo exosomes are shown in fig. 5-6, from which it can be seen that: the particle size distribution is between 40 and 120nm, the average particle size is 68.8nm, and the particle number is 1.7E+12 Particles/mL; exo (Exo) PTP The particle size distribution and particle number of the exosomes are shown in fig. 7-8, as can be seen from fig. 7: the particle size distribution is between 40 and 120nm, the average particle size is 70.8nm, the particle number is 4.2E+11particles/mL, and the particle size distribution is basically consistent with Exo exosomes; furthermore, it can be seen from fig. 8 that: transfected exosomes are clustered, precisely because EGFP fluorescence is expressed in transfected exosomes, further demonstrating successful plasmid transfection.
For Exo, exo PTP 、Exo PTP Visible spectrum detection is carried out on three exosomes @ Cel, and the results are shown in figures 9-11, from which it can be seen that: exo exosomes show characteristic absorption peaks for nucleic acids and proteins (fig. 9); exo (Exo) PTP Exosomes showed absorption peaks substantially identical to Exo exosomes (fig. 10); exo (Exo) PTP The @ Cel exosomes showed characteristic peaks of absorption of tripterine in addition to nucleic acid and protein, indicating successful loading of tripterine (FIG. 11).
(11) Apoptosis test. 200 w/well of PANC-1 cells (human pancreatic cancer cells) were plated with 6 well plates, 37℃and 5% CO 2 The incubator was incubated for 24 hours. Each well was filled with 4. Mu.M Cel, exo@Cel, exo PTP Incubate @ Cel for 8h. The cell culture broth was carefully collected into a centrifuge tube for use. Cells were digested with pancreatin without EDTA until they could be gently blown down with a pipette or gun head, the cell culture broth collected previously was added, all adherent cells were blown down, and the cells were gently blown away. The tube was collected again. The cells were pelleted by centrifugation at about 1000rpm for 5 min. Approximately 1mL of 4℃pre-chilled PBS was added, the cells were resuspended, the pelleted cells were centrifuged again, and the supernatant carefully removed. Resuspension of fine particles with 1x binding bufferCells, the concentration of which is regulated to 1-5×10 6 And (3) per mL, taking 100 mu L of cell suspension in a 5mL flow tube, adding 5 mu L of Lannexin V/FITC, uniformly mixing, and then incubating for 5 minutes at room temperature in a dark place. Add 5 mu L's propidium iodide solution (PI) to add 400 mu L LPBS, carry out the flow immediately and detect. Analysis was performed using flowjo software, a two-color scattergram was drawn, FITC was the abscissa, PI was the ordinate, and the results are shown in fig. 12, from which the pro-apoptotic effect of different drug systems on PANC-1 cells can be seen: exo (Exo) PTP @Cel>Cel>Exo@Cel>Control shows that the tripterine has a pro-apoptosis effect on PANC-1 cells, and the targeting peptide PTP specifically targets pancreatic cancer cell surface receptor Plectin-1, so that the anti-tumor effect of the medicine is effectively improved.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A tripterine delivery body for targeting pancreatic cancer is characterized in that the tripterine delivery body is obtained by carrying out stable cell transfer on an exosome membrane protein lentiviral expression vector and then carrying out membrane protein engineering exosome loading tripterine obtained by separation; the functional sequence structure of the exosome membrane protein lentiviral expression vector comprises a kozak sequence, an initiation codon, an exogenous targeting gene PTP, an exosome membrane protein LAMP2B, P A cleavage peptide, an initiation codon, an EGFP fluorescent sequence and a stop codon from the N end.
2. The pancreatic cancer-targeted tripterine delivery body according to claim 1, wherein the exogenous targeting gene PTP sequence is set forth in SEQ ID NO:1, wherein the LAMP2B sequence of the exosome membrane protein is shown in SEQ ID NO: 2.
3. The pancreatic cancer-targeting tripterine delivery body according to claim 1, wherein the kozak sequence is GCCACC and the P2A cleavage peptide sequence is set forth in SEQ ID NO:3, the EGFP fluorescent sequence is shown as SEQ ID NO: 4.
4. The pancreatic cancer-targeted tripterine delivery vehicle of claim 1, wherein the functional sequence structure of the exosome membrane protein lentiviral expression vector is as set forth in SEQ ID NO: shown at 5.
5. The pancreatic cancer-targeting tripterine delivery vehicle of claim 1, wherein the exosome membrane protein lentiviral expression vector is constructed from psPAX2, pCMV-VSV-G, and pLVX-PTP-LAMP2B-EGFP-puro plasmids.
6. The pancreatic cancer-targeting tripterine delivery vehicle of claim 5, wherein the pLVX-PTP-LAMP2B-EGFP-puro plasmid is a polypeptide that encodes SEQ ID NO:6 and SEQ ID NO:7, recombinant to XhoI-EcoRI double enzyme cutting pLVX-Puro construction.
7. A method of preparing a pancreatic cancer-targeted tripterine delivery body according to any one of claims 1-6, comprising the steps of:
s1, constructing an exosome membrane protein lentiviral expression vector containing an exogenous targeting gene PTP and exosome membrane protein LAMP 2B;
s2, transfecting the exosome membrane protein lentiviral expression vector constructed in the step S1 into HEK293T cells, and screening a stable transgenic cell line;
s3, the stable transgenic HEK293T cells screened in the step S2 secrete membrane protein engineering exosomes;
s4, loading the membrane protein engineering exosomes secreted in the step S3 and the tripterine to construct the tripterine delivery body targeting pancreatic cancer.
8. The method for preparing a pancreatic cancer-targeted tripterine delivery body according to claim 7, wherein puromycin is used for screening of stably transformed cell lines in step S2.
9. The method for preparing a pancreatic cancer-targeted tripterine delivery body according to claim 7, wherein step S3 specifically comprises: centrifuging to remove HEK293T cells from the cell culture liquid, applying a centrifugal force to the supernatant to remove cell debris, and centrifuging again to collect membrane protein engineering exosomes from the supernatant.
10. The method for preparing a pancreatic cancer-targeted tripterine delivery body according to claim 7, wherein step S4 specifically comprises: dissolving tripterine in dimethyl sulfoxide, diluting with PBS, mixing membrane protein engineering exosome with diluted tripterine solution, incubating at room temperature, and filtering with ultrafilter tube to remove free tripterine.
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