Exosome overexpressing tm9sf1 gene and preparation method thereof and application thereof in preparation of tumor inhibiting drugs
By preparing exosomes overexpressing the TM9SF1 gene and targeting colorectal cancer cells, the problem of insensitivity to existing colorectal cancer treatments was solved, and the effect of effectively inhibiting tumor growth was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHAN UNIV OF SCI & TECH
- Filing Date
- 2026-03-12
- Publication Date
- 2026-06-09
Smart Images
Figure CN122168537A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, and in particular relates to exosomes overexpressing the TM9SF1 gene, their preparation methods, and their application in the preparation of tumor-inhibiting drugs. Background Technology
[0002] Colorectal cancer (CRC) is the second most common malignant tumor and the third leading cause of cancer death worldwide. Common symptoms of CRC include changes in stool shape, rectal bleeding, abdominal pain, weight loss, and anemia. In later stages, the tumor can cause intestinal obstruction due to blockage of the intestine or perforation of the intestinal wall, leading to peritonitis and endangering life. Currently, clinical treatment mainly involves surgery and adjuvant chemotherapy. However, a large proportion of patients are not responsive to conventional treatments, thus necessitating the development of new treatment strategies and drugs.
[0003] Exosomes are a class of membrane-bound vesicles secreted by cells, measuring approximately 30–150 nm in diameter, and widely distributed in body fluids. They are primarily formed via the endosomal pathway and carry various bioactive substances, including proteins, lipids, mRNA, miRNA, and lncRNA, serving as crucial mediators of intercellular communication. Exosomes possess inherent biocompatibility, stability, and the ability to carry multiple components, and in recent years have demonstrated significant potential in the treatment of diseases including oncology and cardiovascular diseases.
[0004] Transmembrane 9 superfamily proteins (TM9SF) are a class of proteins containing nine transmembrane regions and one non-cytoplasmic region. These proteins are highly conserved throughout biological evolution. TM9SF1, also known as MP70, is located on the long arm of human chromosome 14, with a full-length gene of 6594 bp encoding 606 amino acids. TM9SF1 is widely expressed in human tissues, but its biological function remains largely unknown. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention proposes exosomes overexpressing the TM9SF1 gene, their preparation method, and their application in the preparation of tumor-inhibiting drugs. The exosomes overexpressing the TM9SF1 gene provided by this invention target colorectal cancer cells, inhibiting their growth and thereby suppressing tumor growth.
[0006] To achieve the above objectives, the present invention provides an exosome overexpressing the TM9SF1 gene, wherein the NCBI accession number for the nucleotide sequence of the TM9SF1 gene is NM_001014842.3.
[0007] Preferably, the exosomes overexpressing the TM9SF1 gene are derived from HEK293T cells.
[0008] The present invention also provides a method for preparing exosomes overexpressing the TM9SF1 gene, comprising the following steps: 1) The TM9SF1 gene was ligated into the p3xFLAG vector to construct the p3xFLAG-TM9SF1 overexpression plasmid; 2) Transfect the p3xFLAG-TM9SF1 overexpression plasmid described in step 1) into HEK293T cells to obtain HEK293T cells overexpressing the TM9SF1 gene; 3) Expand the culture of HEK293T cells overexpressing the TM9SF1 gene as described in step 2), collect the supernatant, perform a first centrifugation to remove larger cell debris, perform a second centrifugation to remove smaller cell debris, collect the supernatant, perform a third centrifugation, collect the precipitate, filter it through a filter membrane, and obtain the exosomes.
[0009] Preferably, the culture time in step 3) is 36-60 hours.
[0010] Preferably, the conditions for the first centrifugation treatment in step 4) are 4°C, 800g, 5~15min.
[0011] Preferably, the conditions for the second centrifugation treatment in step 4) are 4°C, 10000g, 50~70min.
[0012] Preferably, the conditions for the third centrifugation treatment in step 4) are 4°C, 100,000g, 80~100min.
[0013] Preferably, the filter membrane in step 4) is a 0.22 μm filter membrane.
[0014] The present invention also provides the application of the exosomes overexpressing the TM9SF1 gene in the preparation of tumor-inhibiting drugs.
[0015] Preferably, the exosomes overexpressing the TM9SF1 gene can target colorectal cancer cells, effectively inhibit the growth of colorectal cancer cells, and thus inhibit tumor growth.
[0016] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses exosomes overexpressing the TM9SF1 gene, their preparation method, and their application in the preparation of anti-tumor drugs. The method for preparing TM9SF1-overexpressing exosomes involves transfecting HEK293T cells with a plasmid overexpressing TM9SF1, followed by cell culture, exosome isolation, and purification. The prepared TM9SF1-overexpressing exosomes can target colorectal cancer cells, effectively inhibiting their growth and tumor progression, thus playing a positive role in the treatment of tumors and showing broad application prospects. Attached Figure Description
[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0018] Figure 1 This is a schematic diagram of the construction of the p3xFLAG-TM9SF1 plasmid in this invention; Figure 2 This is a graph validating the overexpression efficiency of HEK293T cells overexpressing the TM9SF1 gene in this invention. Figure 3 A schematic diagram of the extraction of exosomes from HEK293T cells overexpressing the TM9SF1 gene; Figure 4 Western blot validation image of exosomes overexpressing the TM9SF1 gene; Figure 5 This is a schematic diagram of nanoparticle tracking analysis technology for exosomes overexpressing the TM9SF1 gene, where A represents exosomes overexpressing empty vector plasmids and B represents exosomes overexpressing the TM9SF1 gene. Figure 6 A transmission electron microscope (TEM) schematic diagram of the morphology of exosomes overexpressing the TM9SF1 gene; scale bar is 20 nm. Figure 7 The images show plate colony detection results of exosomes overexpressing the TM9SF1 gene and colorectal cancer cells after co-incubation. In the images, A represents the plate colony of HCT116 cells co-incubated with exosomes overexpressing the TM9SF1 gene for 48 hours; B represents the plate colony of LoVo cells co-incubated with exosomes overexpressing the TM9SF1 gene for 48 hours; C represents the quantitative statistics of image A; and D represents the quantitative statistics of image B. In the figures, Con-Exos represents exosomes overexpressing the empty vector, and TM9SF1-Exos represents exosomes overexpressing TM9SF1. "and" "Representative difference significance analysis; Figure 8 The figures show the CCK8 assay results of cell proliferation after co-incubation of exosomes overexpressing the TM9SF1 gene with colorectal cancer cells. Figure A shows the CCK8 results of HCT116 cells co-incubated with exosomes overexpressing the TM9SF1 gene for 48 hours, and Figure B shows the CCK8 results of LoVo cells co-incubated with exosomes overexpressing the TM9SF1 gene for 48 hours. In the figures, Con-Exos represents exosomes overexpressing the empty vector, and TM9SF1-Exos represents exosomes overexpressing TM9SF1. "Representative difference significance analysis; Figure 9 The image shows the results of an animal model of colorectal cancer cells treated with exosomes overexpressing the TM9SF1 gene. In the image, A shows the tumor size in mice treated with exosomes overexpressing the TM9SF1 gene, and B is a statistical graph of tumor weight from image A. In the image, Con-Exos represents exosomes overexpressing the empty vector, and TM9SF1-Exos represents exosomes overexpressing TM9SF1. "Representative difference significance analysis; Figure 10 This is a growth curve of tumors after treatment with exosomes overexpressing the TM9SF1 gene. In the figure, Con-Exos represents exosomes overexpressing the empty vector, and TM9SF1-Exos represents exosomes overexpressing TM9SF1. "Representative difference significance analysis; Figure 11 Immunohistochemical images of tumor growth markers after treatment with exosomes overexpressing the TM9SF1 gene are shown. In the figure, Con-Exos represents exosomes overexpressing the empty vector, and TM9SF1-Exos represents exosomes overexpressing TM9SF1. The scale bar is 100 μm. Detailed Implementation
[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0024] The p3xFLAG vector used in this invention was purchased from Qingke Biotechnology; HEK293T cells were purchased from Wuhan Pronosai Co., Ltd.; serum-free DMEM complete medium and high glucose DMEM medium (containing 10% exosome-free fetal bovine serum) were purchased from Wuhan Jiang'an District Osto Biomaterials Business Department; colorectal cancer cells HCT116 and LoVo were purchased from Wuhan Pronosai Co., Ltd.
[0025] Example 1 1) Homologous recombination of the TM9SF1 gene was ligated into the p3xFLAG vector, incubated at 50°C for 15 min, with EcoRI and BamHI restriction sites, to construct the p3xFLAG-TM9SF1 overexpression plasmid (e.g., ...). Figure 1 (As shown).
[0026] 2) Transfect the p3xFLAG-TM9SF1 overexpression plasmid transiently into HEK293T cells. First, mix 10 μg of p3xFLAG-TM9SF1 overexpression plasmid with 500 μL of serum-free DMEM complete medium. Then, prepare a new EP tube and mix 30 μL of PEI transfection reagent with 500 μL of serum-free DMEM complete medium. Let it stand for 5 min, then add the PEI-containing mixture to the plasmid mixture. Incubate at 37°C for 30 min, then add the mixture to HEK293T cells. After 4-6 h of transfection, replace with fresh serum-free DMEM complete medium. 48 h after transfection, a portion of cells can be harvested for Western blot verification of overexpression efficiency. The results are as follows: Figure 2As shown, HEK293T cells overexpressing the TM9SF1 gene were obtained.
[0027] 3) Subculture HEK293T cells overexpressing the TM9SF1 gene at a 1:3 ratio, at 37°C and 5% CO2, and then cultured statically in high-glucose DMEM medium (containing 10% exosome-free fetal bovine serum) for 48 hours. When the cells have reached the required volume, collect the culture supernatant into a 50mL sterile centrifuge tube. Centrifuge at 4°C and 800g for 10 minutes to remove larger cell debris. After the first centrifugation, transfer the supernatant to a new 50mL centrifuge tube and centrifuge at 4°C and 10000g for 60 minutes to remove smaller cell debris. Further transfer the supernatant to a dedicated exosome extraction centrifuge tube and centrifuge at 4°C and 100000g for 90 minutes. Collect the precipitate and resuspend it in a certain amount of pre-cooled sterile PBS in a laminar flow hood. Filter through a 0.22μm filter membrane to obtain exosomes overexpressing the TM9SF1 gene. Store at -80°C (e.g., ...). Figure 3 (As shown).
[0028] Western blot validation of exosomes overexpressing the TM9SF1 gene: Western blot detection of surface marker proteins in exosomes overexpressing the TM9SF1 gene: Prepare a 10% SDS-PAGE gel. After fully lysing the exosomes overexpressing the TM9SF1 gene and control exosomes (exosomes overexpressing the 3xFLAG empty vector plasmid), add 1 / 4 volume of 5×SDS loading buffer, boil for 10 min, load 60 μg of protein, and electrotransfer (200 mA, 90 min) to transfer the protein to a PVDF membrane. Block with 5% skim milk powder at room temperature for 2 h, react with the target primary antibody at 4℃ overnight. The next day, wash the membrane 3 times with 1×TBS, incubate with HRP-labeled goat anti-rabbit or anti-mouse IgG secondary antibody at room temperature for 1 h, wash the membrane 3 times with 1×TBS, add premixed HRP chemiluminescent substrate, and detect using a chemiluminescent gel imaging system. The exosome marker antibodies used in this experiment were TSG101, HSP70, ALIX, and Flotilin-1.
[0029] like Figure 4 The image shown is a Western blot result of exosomes overexpressing the TM9SF1 gene.
[0030] NTA validation of exosome size overexpressing the TM9SF1 gene: An appropriate amount of exosomes overexpressing the TM9SF1 gene were loaded onto the instrument. During loading, the exosomes were continuously diluted with sterile PBS to the optimal concentration, and the best field of view was selected for recording. Generally, 1×10⁻⁶ was used. 8 ~1×10 9particles / mL, save the data after the instrument is finished. You can create your own graphs or use the images exported from the instrument. Exosome size is 30~150nm (e.g., particles / mL). Figure 5 (As shown).
[0031] Transmission electron microscopy (TEM) images of exosomes overexpressing the TM9SF1 gene: First, the exosomes overexpressing the TM9SF1 gene were mixed with 4% PFA at a 1:1 ratio to obtain a sample. The mixture was incubated at room temperature for 10 min. A TEM copper grid was then placed on the grid with tweezers, and 10 μL of the sample was added. The grid was allowed to stand at room temperature for 10 min. Excess liquid was absorbed with filter paper. Then, 10 μL of PBS was added to the grid and allowed to stand for 1 min. Excess liquid was absorbed with filter paper again. Finally, 10 μL of 2% uranium oxalate was added to the grid and incubated at room temperature for 1 min. The staining solution was gently absorbed with filter paper. The grid was allowed to air dry for 20 min. The stained grid was then allowed to air dry naturally at room temperature for at least 10 min. The dried grid was then placed in the TEM sample holder. The accelerating voltage was set to 80-120 kV. The magnification was adjusted. Common observation conditions were: 20000× (overall morphology), 50000× (typical cup-shaped structure), and 100000× (membrane structure details). Images were recorded (e.g., ...). Figure 6 (As shown).
[0032] Example 2 We verified that exosomes overexpressing the TM9SF1 gene, when co-incubated with colorectal cancer cells, inhibited tumor cell growth.
[0033] The effect of exosomes overexpressing the TM9SF1 gene on in vitro colony formation of tumor cells was investigated as follows: ① HCT116 and LoVo colorectal cancer cells in good growth condition were selected for the experiment. HCT116 and LoVo cells were digested with trypsin. After digestion, complete culture medium was added to stop the digestion. The cells were centrifuged at 1600 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in fresh DMEM to prepare a cell suspension. ② Cell counting: 10 μL of cell suspension was mixed with 10 μL of trypan blue and pipetted into a hemocytometer. Cells were counted under an inverted microscope, generally recording the top and left cells. ③ The cell suspension was diluted to 500 cells per well and seeded into a six-well plate, with three replicates per group. The culture volume was 3 mL, and the cells were incubated at 37℃ in 5% CO2 for approximately 10 days. ④ After incubation, the culture medium in the wells was discarded, and 1 mL of DMEM was added to each well. Gently agitate the PBS, then aspirate completely; ⑤ Add 1.5 mL of tissue fixative and fix at room temperature for 30 min; ⑥ Discard the fixative, add 2 mL of 0.1% crystal violet solution for staining, and stain for 30 min; ⑦ After staining, wash the six-well plate, air dry, and photograph. Results are as follows: Figure 7 As shown, the results indicate that exosomes overexpressing the TM9SF1 gene significantly inhibited tumor cell growth after co-incubation with tumor cells.
[0034] Effects of exosomes overexpressing the TM9SF1 gene on in vitro cell proliferation and CCK8 formation in tumor cells: ① Colorectal cancer cells HCT116 and LoVo were seeded in 96-well plates, 2000 cells per well, and incubated at 37℃ in a 5% CO2 incubator for 24 h; ② After cell attachment, exosomes overexpressing the TM9SF1 gene were added to the cells; ③ After a certain treatment time, 10 μL of CCK8 solution was added to each well. For a culture volume of 100 μL, 10 μL was added; for a culture volume of 200 μL, 20 μL was added. Wells containing the corresponding amount of cell culture medium and CCK8 solution but without cells were used as blank controls; ④ The culture plates were incubated in an incubator for 1.5 h; ⑤ The absorbance at 450 nm was measured using a microplate reader. Finally, the data were processed. Results are as follows: Figure 8 As shown, the results indicate that after exosomes overexpressing the TM9SF1 gene were co-incubated with tumor cells, the proliferation capacity of tumor cells slowed down over time.
[0035] Effects of exosomes overexpressing the TM9SF1 gene on tumor cell proliferation in vivo: ① Colorectal cancer cells were subcutaneously injected into the axilla of nude mice, and observed for one week until tumor formation; ② Mice were intravenously injected with 100 μg of exosomes every two days for two weeks; ③ Changes in tumor volume were observed and recorded; ④ Immunohistochemical staining was performed on mouse tumors to detect the expression levels of Ki67 and Caspase-3. Results are as follows: Figure 9 As shown in the results, the tumors formed by the exosome group overexpressing the TM9SF1 gene were significantly smaller than those formed by the exosome group in the control group.
[0036] The effect of exosomes overexpressing the TM9SF1 gene on the proliferation and formation of tumor cells in vivo, and the results of tumor volume changes are as follows: Figure 10 As shown, the results indicate that tumor volume gradually decreases with increasing duration of exosome treatment overexpressing the TM9SF1 gene. Tumor immunohistochemical results are as follows: Figure 11 As shown, the results indicate that Ki67 expression in the exosome treatment group expressing the TM9SF1 gene was significantly lower than that in the control exosome treatment group.
[0037] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. An exosome overexpressing the TM9SF1 gene, characterized in that, The NCBI accession number for the nucleotide sequence of the TM9SF1 gene is NM_001014842.
3.
2. The exosomes overexpressing the TM9SF1 gene according to claim 1, characterized in that, The TM9SF1 gene is derived from HEK293T cells.
3. The method for preparing exosomes overexpressing the TM9SF1 gene as described in claim 1 or 2, characterized in that, Includes the following steps: 1) The TM9SF1 gene was ligated into the p3xFLAG vector to construct the p3xFLAG-TM9SF1 overexpression plasmid; 2) Transfect the p3xFLAG-TM9SF1 overexpression plasmid described in step 1) into HEK293T cells to obtain HEK293T cells overexpressing the TM9SF1 gene; 3) Expand the culture of HEK293T cells overexpressing the TM9SF1 gene as described in step 2), collect the supernatant, perform a first centrifugation to remove larger cell debris, perform a second centrifugation to remove smaller cell debris, collect the supernatant, perform a third centrifugation, collect the precipitate, filter it through a filter membrane, and obtain the exosomes.
4. The preparation method according to claim 3, characterized in that, The culture time described in step 3) is 36~60h.
5. The preparation method according to claim 3, characterized in that, In step 4), the conditions for the first centrifugation treatment are 4°C, 800g, and 5~15min.
6. The preparation method according to claim 3, characterized in that, In step 4), the conditions for the second centrifugation treatment are 4°C, 10000g, and 50~70min.
7. The preparation method according to claim 3, characterized in that, The conditions for the third centrifugation treatment in step 4) are 4°C, 100,000g, 80~100min.
8. The preparation method according to claim 3, characterized in that, The filter membrane mentioned in step 4) is a 0.22 μm filter membrane.
9. The use of exosomes overexpressing the TM9SF1 gene as described in claim 1 or 2 in the preparation of tumor-inhibiting drugs.
10. The application according to claim 9, characterized in that, The exosomes overexpressing the TM9SF1 gene can target colorectal cancer cells, effectively inhibiting their growth and thus suppressing tumor growth.