CN116656733A

CN116656733A - Exosomes containing ATG7 siRNA and application thereof

Info

Publication number: CN116656733A
Application number: CN202310684401.2A
Authority: CN
Inventors: 李伟; 梁展文; 陈凯; 龚斐然; 何康; 徐梦丹; 吴梦瑶; 徐彩华
Original assignee: First Affiliated Hospital of Suzhou University
Current assignee: First Affiliated Hospital of Suzhou University
Priority date: 2023-06-12
Filing date: 2023-06-12
Publication date: 2023-08-29

Abstract

The invention belongs to the field of medicine, and in particular relates to application of an exosome of ATG7 siRNA in preparation of a medicine for treating biliary tract cancer. The method comprises the following steps: s1: extracting a targeting exosome expressing recombinant protein CD 63-APO-A1; s2: loading ATG7 siRNA into targeted exosomes in S1 results in engineered exosomes for biliary tract cancer treatment. The invention prepares an exosome of ATG7 siRNA; the engineered exosome realizes accurate drug delivery aiming at biliary tract cancer cells by targeting delivery of ATG7 siRNA to biliary tract cancer cells to kill tumor cells, and only aims at the therapeutic effect of biliary tract cancer cells to avoid toxic and side effects on normal cells.

Description

Exosomes containing ATG7 siRNA and application thereof

Technical Field

The invention belongs to the field of medicine, and particularly relates to an exosome containing ATG7 siRNA and application thereof.

Background

Bile duct cancer (CCA) is an invasive malignancy originating in biliary epithelial cells, and has high lethality, and can be classified into intrahepatic bile duct cancer, portal bile duct cancer, and distal bile duct cancer according to anatomical sites of its lesions. Although cholangiocarcinoma has a low overall incidence, its global incidence has gradually increased in recent years, accounting for about 15% of all primary liver tumors and 3% of gastrointestinal tumors. Bile duct cancer has extremely strong invasive transfer capability, surgical treatment is the main intervention means at present, and the postoperative recurrence transfer rate is as high as more than 80%. In addition, bile duct cancer is hidden from disease, and most patients find that they miss the opportunity to perform radical surgery due to late stage tumor metastasis. Despite advances in diagnosis and treatment of cholangiocarcinoma, patient prognosis has not been substantially improved in the last decade, and its 5-year survival rate (7% -20%) has not been satisfactory, and it is particularly important to explore molecular targeted therapies.

Autophagy is essentially a protein degradation system that can be used by tumor cells to cope with an "environmental stress" catabolic process that serves the purpose of recycling by degrading non-essential proteins, longevity proteins, damaged cell stages, etc. Autophagy is mainly of three types: giant autophagy (macroautophagy), microautophagy (microautophagy) and chaperone mediated autophagy (chaperone-mediated autophagy), which we commonly understand is giant autophagy. In the autophagy process, the degraded component is wrapped by a vesicle with a double-layer membrane structure, namely an autophagosome; the degradation process is completed after the autophagosome is fused with the lysosome to form an autophagic lysosome. Autophagy is involved in many pathological, physiological processes because of its unique recycling mechanism and the characteristic of coping with "environmental stress".

Autophagy in tumor cells is often associated with sensitivity to tumor chemotherapy. Salcher et al found that etoposide and doxorubicin induced autophagy in neuroblastoma, and that inhibition of autophagy significantly increased etoposide-and doxorubicin-induced apoptosis. In neuroblastomas, histone deacetylase 10 (HDAC 10) can promote autophagy-mediated cell survival. The combination of LY294002 (LY) and 5-Fu on a single carrier for Esophageal Squamous Cell Carcinoma (ESCC) has higher cytotoxicity than single drug. LY inhibits autophagy, thereby enhancing the sensitivity of cancer cells to 5-FU and resulting in more cell death.

The activation enzyme encoded by the autophagy-related gene ATG7 is a key factor in autophagy membrane extension, and participates in two important ubiquitin binding systems in the autophagy process, namely, the conversion of water-soluble LC 3I into lipid-soluble LC3 II, and the binding of ATG12 to ATG5 and the binding of ATG16L1 molecule to form the pre-autophagy structure. ATG7 also has the feature of being independent of ubiquitin activating enzyme. ATG7 can bind to P53 and cooperate with P53 to participate in the regulation of cell cycle and apoptosis, activating autophagy in a stress state. We have found that the targeted inhibition of ATG7 can inhibit the growth and metastasis of bile duct cancer and has chemosensitization effect, which indicates that ATG7 can become a therapeutic target of bile duct cancer.

siRNA is a double stranded molecule, typically 19-21 base pairs, capable of modulating expression of a particular gene by cleaving homologous mRNA. Despite the high potential of siRNA technology in tumor treatment, clinical applications have been limited to date. On the one hand, siRNAs are large or negatively charged molecules that cannot penetrate the lipid bilayer of the cell membrane by passive diffusion. On the other hand, naked siRNA is vulnerable to enzymatic degradation and clearance in the in vivo environment, making it difficult for siRNA to reach tumor sites to function. Therefore, there is an urgent need for a vector capable of safely and efficiently delivering siRNA to cholangiocarcinoma to achieve its effect.

Exosomes (exosomes) are extracellular vesicles of about 40-160nm in diameter. The protein, lipid, nucleic acid and the like can be selectively packaged and released outside cells, and released to exosomes outside the cells, can be captured by cells residing in a microenvironment, and can also be carried to distant tissues and organs along with biological fluid to carry and transfer important signal molecules, so that a brand-new information transfer system between cells is formed.

In order to maximize the delivery efficiency and overcome the obstacle of the siRNA delivery process, thereby improving the autophagy level reduction of bile duct cancer caused by ATG7 siRNA, inhibiting the growth and having obvious advantages based on an exosome siRNA delivery system. The engineered design allows the surface to be modified by specific molecules to obtain specific targeting of bile duct cancer, which has the following advantages: (1) good biocompatibility and low toxicity; (2) The siRNA is prevented from being damaged by in vivo environment, and the circulation time of the siRNA in blood is prolonged; (3) siRNA can be specifically targeted to cholangiocarcinoma to reduce systemic side effects.

Although targeted inhibition of ATG7 can be achieved by exosome delivery of ATG7 siRNA, if the exosome is unable to specifically recognize cholangiocarcinoma cells, such exosome is also able to inhibit expression of ATG7 in normal cells, thereby potentially producing toxic side effects on normal cells, and thus exosomes delivering ATG7 siRNA are required to have cholangiocarcinoma cell specificity. Apolipoproteins are important constituent proteins of lipoproteins, which constitute biological macromolecules with nonpolar lipid cores and lipid monolayers of free cholesterol, involved in lipid transport in vivo. In addition, apolipoproteins act as ligands for lipoprotein receptors and are also involved in the specific binding of cell surface receptors to lipoproteins to ensure smooth downstream signaling. The current research shows that the apolipoproteins are roughly divided into five main classes A, B, C, E, wherein each class comprises different subclasses, such as class A is further divided into A-1, A-2 and A-4, more than 20 types of the apolipoproteins are found so far, and the receptor SR-B1 of the APO-A1 is found to be expressed on the surface of bile duct cancer cell membranes in a large quantity.

293T cells are a cell line with very low immunogenicity and paracrine properties. The secreted nano-sized exosomes can exert therapeutic effects by targeted delivery of ATG7 siRNA. The invention expresses CD63-APO-A1 recombinant protein in 293T cells to obtain biliary tract cancer targeted nanoscale exosomes, and the exosomes can perform therapeutic action only aiming at biliary tract cancer cells by targeting delivery of ATG7 siRNA to biliary tract cancer cells, so that toxic and side effects on normal cells are avoided.

However, in the prior art, there are few reports on ATG7 siRNA exosomes capable of specifically recognizing biliary tract cancer cells.

Disclosure of Invention

The invention expresses CD63-APO-A1 recombinant protein in 293T cells to obtain biliary tract cancer targeted nanoscale exosomes, and the exosomes can perform therapeutic action only aiming at biliary tract cancer cells by targeting delivery of ATG7 siRNA to biliary tract cancer cells, so that toxic and side effects on normal cells are avoided.

Specifically, the technical scheme of the invention is as follows:

the first aspect of the invention discloses a method for preparing an engineering exosome for treating biliary tract cancer, which comprises the following steps:

s1: extracting a targeting exosome expressing recombinant protein CD 63-APO-A1;

s2: loading ATG7 siRNA into targeted exosomes in S1 results in engineered exosomes for biliary tract cancer treatment.

Preferably, in S2, the nucleotide sequence of the ATG7 siRNA is SEQ ID NO: 5-9.

Preferably, the S1 includes:

s11: preparing a CD63-APO-A1 fusion protein;

s12: constructing an expression vector pLentai-PuroR-CMV-CD63-APO-A1-EGFR;

s13: extracting recombinant plasmid pLentai-PuroR-CMV-CD63-APO-A1-EGFR;

s14: transfecting recombinant plasmid pLentai-Puror-CMV-CD63-APO-A1-EGFR into cells, and extracting the targeted exosomes expressing recombinant protein CD63-CXCL 17.

More preferably, in S12, pLenta-Puror-CMV-EGFR vector is ligated with the CD63-APO-A1 recovery product, and the ligation product is transfected into E.coli.

More preferably, the nucleotide sequence of the CD63-APO-A1 fusion gene is shown in SEQ ID NO:14, said CD63-APO-A1 fusion gene is translated into said CD63-APO-A1 fusion protein.

In a second aspect, the invention discloses an engineered exosome for use in the treatment of biliary tract cancer, the engineered exosome comprising an ATG7 siRNA.

Preferably, the engineered exosome envelope expresses a recombinant CD63-APO-A1 fusion protein.

The invention also discloses the engineering exosome prepared by the method or the application of the engineering exosome in preparing medicaments for treating biliary tract cancer.

The fourth aspect of the invention discloses a medicament for treating biliary tract cancer, which comprises the engineering exosome or the engineering exosome prepared by the method.

In a fifth aspect, the invention discloses the use of the above-described treatment of biliary tract cancer in the field of cancer treatment.

Compared with the prior art, the invention has the following advantages:

according to the invention, the autophagy related oncogene ATG7 is taken as a treatment target, and the engineering exosome is adopted, so that tumor tissue specific drug delivery aiming at bile duct cancer cells is realized, and the ATG7 is only inhibited in the bile duct cancer cells, thereby avoiding toxic and side effects on normal cells.

Drawings

FIG. 1 shows the result of enzyme digestion verification. * Nc=no cleavage control, 1 kb=1 kb dnamarker, e=xmai-BamHI cleavage.

Fig. 2 is a schematic diagram of flow cytometry detection of fluorescence intensity statistical analysis of empty vector and recombinant plasmid EGFP, P <0.01.

FIG. 3 is a graph demonstrating the expression of CD63 and APO-A1 in 293T cells.

Fig. 4 is a statistical analysis of red fluorescence intensity of normal cells and bile duct cancer cells incubated with engineered exosomes, P <0.01.

Fig. 5 is a schematic diagram of down-regulation of LRPPRC expression by engineered exosomes-mediated biliary cancer cells.

Detailed Description

The technical solution of the present invention will be described in detail below with reference to the drawings and examples, but the present invention is not limited to the scope of the examples.

The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. The reagents and materials used in the present invention are commercially available.

Example 1

1. The method comprises the following steps:

construction of 1.1CD63-APO-A1 fusion Gene

1.1.1 design and Synthesis of primers

Based on the gene sequence of fusion protein CD63-APO-A1, the PCR primer was designed by itself as follows

P1:5'-CCACCGCCATGGTGGCGCCCGGGA-3' (CCCGGG XmaI recognition site) (SEQ ID NO: 1)

P2：5’-AGTGGCTACGAGGTGATGT-3’(SEQ ID NO：2)

P3：5’-ATGTAGATGAAAGCTGCGG-3’(SEQ ID NO：3)

P4:5'-TCACTGGGTGTTGAGGGATCCGC-3' (GGATCC is a BamHI recognition site) (SEQ ID NO: 4)

1.1.2CD63 amplification of APO-A1 Gene fragments

The 293T cell cDNA is used as a template, the P1 and the P2 of the CD63 gene are used as primers, and the CD63 gene is amplified, and the reaction system is as follows:

the PCR reaction conditions were: pre-denaturation at 98℃for 5min, denaturation at 98℃for 30s, annealing at 61℃for 1min, extension at 72℃for 2min,35cycle, extension at 72℃for 10min, and preservation at 4 ℃.

The 293T cDNA is used as a template, the P3 and the P4 of the APO-A1 sequence are used as primers, and the APO-A1 sequence is amplified, and the reaction system is as follows:

Purification of 1.1.3PCR product

And (3) after the amplified PCR product is subjected to 1% agarose gel electrophoresis analysis, cutting off a target band, and purifying by a DNA gel recovery kit to obtain a purified PCR product.

Amplification of 1.1.4CD63-APO-A1 fusion Gene

The fusion gene CD63-APO-A1 is amplified by using a method of overlapping, extending and splicing PCR (SOE-PCR), taking PCR recovery products of CD63 and APO-A1 as templates and P1 and P4 as primers, and the reaction system is as follows:

the PCR reaction conditions were: pre-denaturation at 98℃for 8min, denaturation at 98℃for 30s, annealing at 64℃for 1min, extension at 72℃for 2min,42cycle, extension at 72℃for 10min, and preservation at 4 ℃.

After the amplified PCR product is analyzed by 1% agarose gel electrophoresis, a target band is cut off, and the PCR product is purified by a DNA gel recovery and purification kit.

1.2 construction of the expression vector pLentai-Puror-CMV-CD63-APO-A1-EGFR

1.2.1pLentai-Puror-CMV-EGFR vector cleavage step

The vector pLentai-PuroR-CMV-EGFR was digested with BamHI and XmaI to prepare a reaction system:

after being evenly mixed, the mixture is incubated in a water bath kettle at 37 ℃ for 2 hours; 1.5% agarose gel electrophoresis, it can be seen that 7700bp and 848bp bands are obtained, and 7700bp vector bands are recovered. (10XNEBufferr 3.1 from New Englandbiolabs cat# B6003S) 1.2.2T4 ligation step

Establishing T4 connection system

After mixing, the mixture was left to join overnight at 16 ℃. (10XT4 connection Buffer from Thermo Scientific cargo number B69)

1.2.3 conversion of ligation products

(1) Taking out competent TOP10 E.coli cells in a refrigerator at-80deg.C, placing on an ice box of an ultra-clean workbench, adding 5 μl of the connection product when the cells are melted into ice-water mixture, gently blowing to mix the connection product, and ice-bathing for 30min;

(2) Water bath at 42 ℃ for 90s and ice bath for 4min;

(3) Adding 800 μl of culture medium, and shake culturing at 37deg.C for 4 hr;

(4) Mu.l of the culture product was pipetted onto a solid culture plate with Amp resistance (10. Mu.g/ml) and incubated overnight at 37 ℃.

1.3 extraction of recombinant plasmid pLentai-Puror-CMV-CD63-APO-A1-EGFR

Plasmid extraction was performed using a plasmid miniprep kit (Labselect, inc.).

(1) Taking 1 to 5ml of bacterial liquid, centrifuging at 12000rpm for 1 minute, absorbing and discarding the supernatant, then adding 200 mu l of solution P1, adding 200 mu l of solution P2 after fully mixing, gently turning up and down for 6 to 8 times until the solution becomes clear and viscous, immediately adding 250 mu l of solution P3, and gently turning up and down for 6 to 8 times. Followed by centrifugation at 12000rpm for 10 minutes.

(2) Transferring the supernatant to an adsorption column at 12000rpm, and centrifuging for 1 minute; the waste liquid was discarded, 700. Mu.l of Washing buffer was added thereto, and the mixture was centrifuged at 12000rpm for 1 minute, and the mixture was repeated twice. Placing the adsorption column into a recovery tube, centrifuging at 12000rpm for 2 minutes, discarding the waste liquid, standing at room temperature until the residual liquid in the adsorption column is dried. The column was transferred to a fresh EP tube, 20. Mu.l of eluent was added dropwise, and the mixture was allowed to stand at room temperature for 5 minutes at 12000rpm and centrifuged for 2 minutes. The solution was collected and the Thermo NanoDrop-2000 was used to measure plasmid concentration and stored at-20 ℃.

1.4293T cell transfection recombinant plasmid pLentai-Puror-CMV-CD63-APO-A1-EGFR

Cell transfection was performed using the Thermo Fisher Scientific company lipofectamine kit, as follows:

(1) Experiments were performed using 293T cells in the logarithmic growth phase. After cell digestion and counting, the cells were used in a 2X 10 per well ⁵ Is plated in 6-well plates, cultured overnight, and transfected after cells adhere.

(2) Preparing a solution 1: MEM medium (250. Mu.l) +lipo3000 (3.75. Mu.l). Preparing a solution 2: MEM medium (250. Mu.l) +plasmid (5. Mu.g) +P3000. Solutions 1 and 2 were left at room temperature for 15 minutes, respectively, followed by mixing and incubation for 5 minutes.

(3) After the cells were rinsed with PBS, the mixture of (2) was added, and the cells were cultured for 6 hours and changed to DMEM complete medium.

1.5 identification of CD63-APO-A1 fusion protein expression in 293T cells by Western blot.

(1) Protein cleavage: firstly, preparing a lysate: 200. Mu.l of RIPA lysate was mixed with 4. Mu.l of protease inhibitor, 4. Mu.l of phosphatase inhibitor and 2. Mu.l of PMSF on ice and prepared for use. To the cell pellet or exosome pellet, 200. Mu.l of lysate was added, followed by incubation on ice for 20 minutes, and after 12000 Xg, centrifugation was performed at 4℃for 5 minutes, and the supernatant was collected. Protein concentration was determined using BCA assay.

(2) Protein denaturation: SDS-PAGE protein loading buffer (5X) was added to the supernatant followed by a water bath at 100deg.C for 10 minutes to allow complete denaturation of the protein.

(3) Electrophoresis: an 8% SDS-PAGE gel was prepared, run at 80V constant pressure for 30 minutes after loading, then run at 120V constant pressure until bromophenol blue was 0.5cm from the lower edge of the gel.

(4) Transferring to membrane, taking out SDS-PAGE gel, fixing in the order of power cathode, sponge, filter paper, SDS-PAGE gel, PVDF membrane, filter paper, sponge and power anode, placing in a transferring tank, transferring to membrane for 90 min under constant pressure of 100V.

(5) The PVDF membrane was removed and placed in a blocking solution containing 5% nonfat dry milk formulated with TBST and blocked for 1 hour at room temperature.

(6) Incubating primary antibodies: the PVDF membrane was removed and blocked with a blocking solution according to 1: the primary antibody was diluted in a ratio of 1000, the membrane was placed in the primary antibody solution, gently shaken on a shaker, and incubated overnight at 4 ℃.

(7) Incubating a secondary antibody: the PVDF membrane was removed, washed 3 times with TBST, and then the secondary antibody was incubated at room temperature, and after 1 hour, washed 3 times with TBST.

(8) Color development: preparing ECL luminous liquid: reinforcing liquid: stabilizing solution = 1:1. ECL luminescent droplets were applied to the protein-binding side of PVDF membranes. After the excessive luminous liquid is sucked by filter paper, the film is pressed into a sheet by an X-ray film, and then the film is sequentially put into a developing solution and a fixing solution, washed by water, photographed and counted.

1.6 extraction of Targeted exosomes expressing recombinant protein CD63-APO-A1

The old 293T cell culture broth was aspirated, rinsed 3 times with PBS, and in order to ensure that the collected culture supernatant was free from the influence of foreign body confounding factors, the culture medium was replaced with DMEM medium containing 10% of FBS from which the foreign body was removed (ultracentrifuge removal of the foreign body), and the culture broth was collected after a conventional culture for 36 hours. 50ml of the culture was centrifuged at 300 Xg at 4℃for 10 minutes, and the pellet was discarded. Then, 2000 Xg was centrifuged at 4℃for 10 minutes, and the precipitate was discarded. Then 10000 Xg, centrifuged at 4℃for 30 minutes, and the precipitate was discarded. Followed by centrifugation at 100000 Xg at 4℃for 70 minutes, at which time the pellet was the exosome. The supernatant was pipetted off, followed by a 200. Mu.l PBS resuspension, followed by 100000 Xg, centrifugation at 4℃for 70 minutes, the supernatant was pipetted off, and 50. Mu.l PBS resuspension was added. Placing in a refrigerator at-80deg.C for preservation.

1.7ATG7 siRNA sequence and target site

The siRNA targeting the ATG7 site was designed and synthesized as shown in the following table:

1.8 construction of engineered exosomes by electroporation of ATG7 siRNA into the targeted exosomes

Purified targeted exosomes and ATG7 were mixed in electroporation buffer, electroporated in electroporation cuvette using Bio-Rad gene pulserXcell electroporation system at 350V, followed by incubation of the mixture at 37 ℃ for 30min to restore exosome envelope.

1.9 observation of exosomes by transmission electron microscopy

Mu.l of the exosome suspension was added dropwise to the Formvar-carbon film coated copper-loaded mesh, allowed to stand for 1 minute and then air-dried. The Formvar membrane side was rinsed 3 times with PBS and the copper mesh side was kept dry. Fixation was performed for 20 minutes at room temperature using 1% glutaraldehyde. Using ddH ₂ After O rinse twice, stain with 2% uranyl acetate for 15 minutes. Followed by rinsing with methylcellulose-UA for 10 minutes and air-drying. Observed under a transmission electron microscope and photographed.

1.10 identification of particle size of exosomes

With ddH ₂ O dilutes exosome suspension, and the exosome particle concentration is controlled to be 1x10 ⁷ Ml to 1x10 ⁹ Between/ml, the number and size of examples in the samples were determined using a ZetaView PMX110 instrument at 405nm laser and the exosome size was analyzed using nanoparticle tracer analysis (Nanoparticle Tracking Analysis, NTA) software.

1.11 exosome tracing and targeting identification

(1) Exosome staining

The exosome content was determined by BCA assay, 500. Mu.g exosome was mixed with 4. Mu.g/ml of Dil red dye in equal volume and incubated at 4℃for 2 hours in the absence of light. After subsequently re-suspending the exosomes with 1ml of PBS, 100000×g, centrifuged at 4 ℃ for 70 min, the supernatant was pipetted off and re-suspended with 50 μl of PBS, which is red fluorescent-labeled exosomes (Dil-exosomes). Placing in a refrigerator at-80deg.C, and storing in dark place.

(2) Exosome and cell incubation

And (3) incubating the exosomes with the Dil fluorescent markers by adopting different cell lines, and observing the uptake condition of bile duct cancer compared with the engineered exosomes by other cell lines in the same time. The following operations were all performed in the dark. Will be 5x10 ⁵ Individual cells were seeded in 6-well plates with cell slide placed thereon, 50. Mu.g of Dil-Exosome was added to the culture solution after 12 hours, the culture solution was pipetted off after 24 hours, followed by rinsing 3 times with PBS and 1ml of 4% paraformaldehyde was added thereto for fixation for 15 minutes at room temperature. After aspiration of the fixative, 1ml of 0.1% Triton X-100 was added, the cells were incubated for 5 minutes, followed by 3 washes with PBS. Finally, use premixingDAPI anti-fluorescence quencher, and protected from light for 15 minutes. Exosome phagocytosis was observed under a fluorescence microscope. And detecting exosome phagocytosis by using a flow cytometer.

1.12 detection of bile duct cancer cell ATG7 expression level by real-time fluorescent quantitative PCR experiment

The primers required for the experiment were synthesized by Shanghai Biotechnology Co. The primer sequences involved in this section are shown in the following table:

experiments were performed using SYBR Green PCR Master Mix from Roche, the system is as follows:

the reaction system is placed in a LightCycler 96 real-time fluorescence quantitative PCR instrument. PCR amplification of cDNA was performed in a two-step method.

The conditions are as follows: step 1, pre-denaturation: setting 1 cycle at 95 ℃ for 30 seconds; step 2, PCR reaction: at 95 ℃,5 seconds, 60 ℃,30 seconds, 40 cycles are set. 3 duplicate wells were set for each sample. Using GAPDH as an internal reference, the Ct value (the number of cycles required for the fluorescence intensity to reach a set threshold) of each sample was then measured, and the relative expression level of the target gene was 2 ^-△△CT And (3) calculating:

Δct (experimental group) =ct (gene of interest, experimental group) -CT (reference gene, experimental group)

Δct (control) =ct (target gene, control) -CT (reference gene, control)

ΔΔct= Δct (experimental group) - Δct (control group)

2. Results

2.1 identification of recombinant plasmid pLentai-Puror-CMV-CD63-APO-A1-EGFR

The recombinant plasmid pLentai-PuroR-CMV-CD63-APO-A1-EGFR was digested with XmaI and BamHI, and as a result, two approximately 9000 and 3500bp size bands, namely a pLentai-PuroR-CMV-EGFR linear fragment and a CD63-APO-A1 fusion gene fragment, were seen in FIG. 1, and the recombinant expression plasmid pLentai-PuroR-CMV-CD63-APO-A1-EGFR was successfully constructed.

2. Transfection of recombinant plasmid pLentai-PuroR-CMV-CD63-APO-A1-EGFR into 293T cells

The recombinant plasmid was transfected into 293T cells, and the 293T cells expressed EGFP to emit green fluorescence, and the fluorescence intensity was detected using a flow cytometer. Indicating that the engineering plasmid can express the integrated recombinant CD63-APO-A1 gene in cells, as shown in FIG. 2.

2.3 verification of fusion Gene CD63-APO-A1

The expression of CD63 and APO-A1 in 293T cells transfected with empty vector was low, while the expression of CD63 and APO-A1 in 293T cells transfected with engineering plasmid was high, indicating that the recombinant plasmid was able to successfully express integrated CD63 and APO-A1 in 293T cells, as shown in FIG. 3.

2.4 extraction, preparation and identification of engineered exosomes

The 293T cell culture solution transfected with the engineering plasmid is collected, exosomes in the culture solution are extracted by an ultracentrifugation method, ATG7 siRNA is delivered into the exosomes by electroporation, the exosomes are constructed as engineering exosomes, the structure of the exosomes is observed under an electron microscope, and the structure is not obviously different from that of a general exosomes, so that the modified engineering exosomes do not change the structural properties of the exosomes. Particle size of the engineered exosomes was detected by NTA. The particle sizes of the engineering exosomes are not obviously different, which indicates that the structural properties of the modified engineering exosomes are not changed.

2.5 determination of engineered exosome targeting

To verify the targeting of the engineered exosomes, the engineered exosomes were labeled with red dye Dil and used to incubate human normal bile duct cells (HIBEpiC) and bile duct cancer cells (RBE), red fluorescence was measured by flow cytometry, and more red fluorescence was found in bile duct cancer cells, indicating that the engineered exosomes tended to be enriched in tumor cells rather than normal tissue cells, indicating that the engineered exosomes had targeting to bile duct cancer, as shown in fig. 4.

2.6 engineering exosomes to down-regulate ATG7 expression in cholangiocarcinoma cells

Expression of ATG7 in bile duct cancer cells transfected with common exosomes (control) and engineered exosomes (CD 63-APO-A1) was detected by a real-time fluorescent quantitative PCR method, as shown in FIG. 5.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A method of preparing an engineered exosome for use in the treatment of biliary tract cancer, comprising:

s1: extracting a targeting exosome expressing recombinant protein CD 63-APO-A1;

2. The method of claim 1, wherein in S2, the nucleotide sequence of the ATG7 siRNA is SEQ ID NO: 5-9.

3. The method according to claim 1, wherein S1 comprises:

s11: preparing a CD63-APO-A1 fusion protein;

s12: constructing an expression vector pLentai-PuroR-CMV-CD63-APO-A1-EGFR;

s13: extracting recombinant plasmid pLentai-PuroR-CMV-CD63-APO-A1-EGFR;

4. A method according to claim 3, wherein in S12, the pLentai-PuroR-CMV-EGFR Vector is ligated with the CD63-APO-A1 recovery product, followed by transfection of the ligation product into e.

5. A method according to claim 3, wherein the nucleotide sequence of the CD63-APO-A1 fusion gene is set forth in SEQ ID NO:14, said CD63-APO-A1 fusion gene is translated into said CD63-APO-A1 fusion protein.

6. An engineered exosome for use in the treatment of biliary tract cancer, wherein the engineered exosome comprises an ATG7 siRNA.

7. The engineered exosome of claim 6, wherein the engineered exosome has expressed on its envelope a recombinant CD63-APO-A1 fusion protein.

8. Use of an engineered exosome prepared according to the method of claims 1-5 or an engineered exosome of claims 6-7 in the manufacture of a medicament for the treatment of biliary tract cancer.

9. A medicament for treating biliary tract cancer, comprising the engineered exosome of claims 6-7 or the engineered exosome produced by the method of claims 1-5.

10. Use of the composition according to claim 9 for the treatment of biliary tract cancer in the field of cancer treatment.