Preparation method of corydalis saxicola bunting external vesicles with hepatocellular carcinoma resisting function
By preparing high-purity extracellular vesicles of Coptis chinensis, the problem of insufficient preparation of extracellular vesicles of Coptis chinensis was solved, and an effective inhibitory effect on hepatocellular carcinoma was achieved.
Patent Information
- Application Number
- CN202510865449.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-26
- Publication Date
- 2025-10-28
AI Technical Summary
There is no existing research on the preparation of extracellular vesicles of Coptis chinensis, and its application in the treatment of hepatocellular carcinoma has not been fully developed.
High-purity extracellular vesicles of Coptis chinensis were prepared by drying fresh Coptis chinensis segments, soaking them in EDTA solution, treating them with a compound enzyme, and then combining differential centrifugation and filtration with polyethylene glycol solution.
It increases the content of dehydrocarvitine, stimulates cells to secrete extravesicles, decomposes the cell wall, and obtains extravesicles with higher purity, effectively inhibiting the proliferation, migration and clonal formation of hepatocellular carcinoma cells.
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Figure CN120843403A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of research technology of external vesicles of traditional Chinese medicine, and specifically relates to a method for preparing external vesicles of Coptis chinensis with anti-hepatocellular carcinoma function. Background Technology
[0002] Extracellular vesicles are natural nanomaterials containing small biological molecules such as lipids, proteins, and nucleic acids, produced by cellular metabolism. They play an important role in intercellular signaling, immune regulation, and the development of various diseases. Due to their natural targeting properties, good biocompatibility, and safety, extracellular vesicles can serve as ideal drug delivery carriers in the field of precision medicine. Compared with mammalian extracellular vesicles, plant-derived extracellular vesicles are abundant, low-cost, and safe and non-toxic.
[0003] In recent years, extracellular vesicles derived from Chinese medicinal materials have received increasing attention, but no research has yet been found on the preparation of extracellular vesicles from Coptis chinensis. Summary of the Invention
[0004] This invention provides a method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function, thus solving the above-mentioned technical problems.
[0005] To solve the above technical problems, the present invention adopts the following technical solution:
[0006] A method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function includes:
[0007] S1. Pretreatment: Wash and cut fresh Coptis chinensis into sections, dry at 25-40℃ for 10-24 hours, then soak in EDTA solution for 5-20 minutes, filter and wash to obtain pretreated Coptis chinensis.
[0008] S2. Add the pretreated Coptis chinensis from step S1 to the extract, then add the compound enzyme, stir evenly, soak for 8-20 hours, filter, and obtain the extract.
[0009] S3. Centrifuge the leachate from step S2 for 1-2 hours to obtain centrifuged material;
[0010] S4. The centrifuged material from step S3 was centrifuged again and enriched to obtain extracellular vesicles of Coptis chinensis.
[0011] in,
[0012] The extract consists of Na2HPO4, NaCl, and NaH2PO4;
[0013] The complex enzyme consists of cellulase, lignin peroxidase, and pectinase.
[0014] The concentration of the EDTA solution is 1-6 mM.
[0015] The concentrations of Na2HPO4 are 2-4 mM, NaCl is 50-80 mM, and NaH2PO4 is 8-20 mM.
[0016] Cellulase concentration: 80-150 U / mL; lignin peroxidase concentration: 50-100 U / mL; pectinase concentration: 30-60 U / mL.
[0017] The centrifugation treatment of the leachate in step S3 is as follows: first, centrifuge at 500-1000g for 10-25min, filter the supernatant, then centrifuge at 1500-2500g for 20-35min, filter the supernatant, add polyethylene glycol solution, centrifuge at 1500-4500g for 30-60min, filter the supernatant and treat it with an ultrafiltration membrane with a molecular weight cutoff of 30-100kDa to obtain the centrifuged product.
[0018] The concentration of the polyethylene glycol solution used was 1.5-4 g / mL.
[0019] The volume of the polyethylene glycol solution used should be 0.2-0.5 times the volume of the corresponding supernatant.
[0020] In step S4, the centrifugation enrichment is carried out at 60,000-80,000g for 1-2 hours, and the precipitate is obtained by filtration. The precipitate is resuspended in PBS to obtain extracellular vesicles of Coptis chinensis.
[0021] The resuspension temperature should not exceed 4℃. After resuspension, centrifuge at 80,000-120,000g for 1-1.5h and filter to obtain the precipitate. The precipitate is resuspended again with PBS to obtain extracellular vesicles of Coptis chinensis.
[0022] The extracellular vesicles of Coptis chinensis prepared by the method of the present invention are used for the preparation of drugs for the prevention or treatment of hepatocellular carcinoma.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] This invention uses fresh Coptis chinensis cut into segments and dried, which not only increases the content of dehydrocarbeverine, but also stimulates its cells to secrete extravesicles; the added EDTA can chelate with calcium ions in the cells, which helps to carry out enzymatic hydrolysis of the cell wall in the subsequent process.
[0025] Soaking *Rhizophora stylosa* in the extract and then treating it with cellulase, lignin peroxidase, and pectinase breaks down the plant's cell walls, which helps release the exovesicles.
[0026] Differential centrifugation can remove large molecular components such as proteins from the leachate, while the added polyethylene glycol solution can react with the small particles such as lipoproteins remaining after centrifugation to precipitate them. After filtration, higher purity Coptis chinensis exovesicles can be obtained.
[0027] During centrifugation enrichment, two centrifugation processes help remove residual impurities, and resuspending in PBS at low temperature avoids the impact of high temperature during centrifugation on its activity. Attached Figure Description
[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.
[0029] Figure 1 This is an electron micrograph of the external vesicles of Coptis chinensis.
[0030] Figure 2 The particle size of the outer vesicles of *Coptis chinensis* is shown.
[0031] Figure 3 This image shows the phagocytosis of external vesicles of Coptis chinensis by cells.
[0032] Figure 4 The external vesicles of Coptis chinensis inhibited the proliferation of MHCC97H cells.
[0033] Figure 5 The external vesicles of Coptis chinensis inhibit the cell migration of MHCC97H.
[0034] Figure 6 To inhibit the formation of MHCC97H cell clones from the external vesicles of Coptis chinensis.
[0035] Figure 7 The external vesicles of Coptis chinensis inhibit the cell migration of MHCC97H. Detailed Implementation
[0036] To facilitate a better understanding of the present invention, the following examples are provided in conjunction with the accompanying drawings. These examples fall within the scope of protection of the present invention, but do not limit the scope of protection of the present invention.
[0037] Example 1
[0038] A method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function includes:
[0039] S1. Pretreatment: Wash and cut fresh Coptis chinensis into sections, dry at 25℃ for 24 hours, then soak in EDTA solution for 5 minutes, filter and wash to obtain pretreated Coptis chinensis.
[0040] S2. Add the pretreated Coptis chinensis from step S1 to the extract, then add the compound enzyme, stir evenly, soak for 8 hours, filter, and obtain the leachate.
[0041] S3. The leachate from step S2 is centrifuged for 1 hour to obtain centrifuged material;
[0042] S4. The centrifuged material from step S3 was centrifuged again and enriched to obtain extracellular vesicles of Coptis chinensis.
[0043] in,
[0044] The extract consists of Na2HPO4, NaCl, and NaH2PO4;
[0045] The complex enzyme consists of cellulase, lignin peroxidase, and pectinase.
[0046] The concentration of the EDTA solution is 1 mM.
[0047] The concentrations of Na2HPO4 were 2 mM, NaCl was 50 mM, and NaH2PO4 was 8 mM.
[0048] Cellulase concentration was 80 U / mL, lignin peroxidase concentration was 50 U / mL, and pectinase concentration was 30 U / mL.
[0049] The centrifugation treatment of the leachate in step S3 is as follows: first, centrifuge at 500g for 10min, filter the supernatant, then centrifuge at 1500g for 20min, filter the supernatant, add polyethylene glycol solution, centrifuge at 1500g for 30min, and filter the supernatant using an ultrafiltration membrane with a molecular weight cutoff of 30-100kDa to obtain the centrifuged product.
[0050] The concentration of the polyethylene glycol solution used was 1.5 g / mL.
[0051] The volume of the polyethylene glycol solution used was 0.2 times the volume of the corresponding supernatant.
[0052] In step S4, the centrifugation enrichment was carried out at 60,000g for 1 hour, and the precipitate was obtained by filtration. The precipitate was resuspended in PBS to obtain extracellular vesicles of Coptis chinensis.
[0053] The resuspension temperature should not exceed 4℃. After resuspension, centrifuge at 80000g for 1 hour and filter to obtain the precipitate. The precipitate is resuspended again with PBS to obtain extracellular vesicles of Coptis chinensis.
[0054] Example 2
[0055] A method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function includes:
[0056] S1. Pretreatment: Wash and cut fresh Coptis chinensis into sections, dry at 35℃ for 18 hours, then soak in EDTA solution for 12 minutes, filter and wash to obtain pretreated Coptis chinensis.
[0057] S2. Add the pretreated Coptis chinensis from step S1 to the extract, then add the compound enzyme, stir evenly, soak for 10 hours, filter, and obtain the leachate.
[0058] S3. The leachate from step S2 is centrifuged to obtain centrifuged material;
[0059] S4. The centrifuged material from step S3 was centrifuged again and enriched to obtain extracellular vesicles of Coptis chinensis.
[0060] in,
[0061] The extract consists of Na2HPO4, NaCl, and NaH2PO4;
[0062] The complex enzyme consists of cellulase, lignin peroxidase, and pectinase.
[0063] The concentration of the EDTA solution is 1-6 mM.
[0064] The concentrations of Na2HPO4 were 3 mM, NaCl was 65 mM, and NaH2PO4 was 12 mM.
[0065] Cellulase concentration was 90 U / mL, lignin peroxidase concentration was 60 U / mL, and pectinase concentration was 35 U / mL.
[0066] The centrifugation treatment of the leachate in step S3 is as follows: first, centrifuge at 600g for 15min, filter the supernatant, then centrifuge at 2000g for 25min, filter the supernatant, add polyethylene glycol solution, centrifuge at 2500g for 40min, filter the supernatant and treat it with an ultrafiltration membrane with a molecular weight cutoff of 30-100kDa to obtain the centrifuged product.
[0067] The concentration of the polyethylene glycol solution used was 2 g / mL.
[0068] The volume of the polyethylene glycol solution used was 0.35 times the volume of the corresponding supernatant.
[0069] In step S4, the centrifugation enrichment was carried out at 60,000g for 1 hour, and the precipitate was obtained by filtration. The precipitate was resuspended in PBS to obtain extracellular vesicles of Coptis chinensis.
[0070] The resuspension temperature should not exceed 4℃. After resuspension, centrifuge at 80000g for 1 hour and filter to obtain the precipitate. The precipitate is resuspended again with PBS to obtain extracellular vesicles of Coptis chinensis.
[0071] Example 3
[0072] A method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function includes:
[0073] S1. Pretreatment: Wash and cut fresh Coptis chinensis into sections, dry at 30℃ for 18 hours, then dry at 40℃ for 1 hour, then soak in EDTA solution for 15 minutes, filter and wash to obtain pretreated Coptis chinensis.
[0074] S2. Add the pretreated Coptis chinensis from step S1 to the extract, then add the compound enzyme, stir evenly, soak for 8-20 hours, filter, and obtain the extract.
[0075] S3. The leachate from step S2 is centrifuged to obtain centrifuged material;
[0076] S4. The centrifuged material from step S3 was centrifuged again and enriched to obtain extracellular vesicles of Coptis chinensis.
[0077] in,
[0078] The extract consists of Na2HPO4, NaCl, and NaH2PO4;
[0079] The complex enzyme consists of cellulase, lignin peroxidase, and pectinase.
[0080] The concentration of the EDTA solution is 3 mM.
[0081] The concentrations of Na2HPO4 were 3 mM, NaCl was 650 mM, and NaH2PO4 was 16 mM.
[0082] Cellulase concentration was 100 U / mL, lignin peroxidase concentration was 70 U / mL, and pectinase concentration was 40 U / mL.
[0083] The centrifugation treatment of the leachate in step S3 is as follows: first, centrifuge at 800g for 15min, filter the supernatant, then centrifuge at 2000g for 25min, filter the supernatant, add polyethylene glycol solution, centrifuge at 3500g for 50min, and filter the supernatant using an ultrafiltration membrane with a molecular weight cutoff of 30-100kDa to obtain the centrifuged product.
[0084] The concentration of the polyethylene glycol solution used was 3 g / mL.
[0085] The volume of the polyethylene glycol solution used was 0.3 times the volume of the corresponding supernatant.
[0086] In step S4, the centrifugation enrichment was carried out at 70,000g for 1.5 hours, and the precipitate was obtained by filtration. The precipitate was resuspended in PBS to obtain extracellular vesicles of Coptis chinensis.
[0087] The resuspension temperature should not exceed 4℃. After resuspension, centrifuge at 90000g for 1 hour and filter to obtain the precipitate. The precipitate is resuspended again with PBS to obtain extracellular vesicles of Coptis chinensis.
[0088] An application of extracellular vesicles of Coptis chinensis, wherein the extracellular vesicles of Coptis chinensis prepared by the method of the present invention are used for the preparation of drugs for the prevention or treatment of hepatocellular carcinoma.
[0089] Example 4
[0090] A method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function includes:
[0091] S1. Pretreatment: Wash and cut fresh Coptis chinensis into sections, dry at 38℃ for 13 hours, then soak in EDTA solution for 5-20 minutes, filter and wash to obtain pretreated Coptis chinensis.
[0092] S2. Add the pretreated Coptis chinensis from step S1 to the extract, then add the compound enzyme, stir evenly, soak for 16 hours, filter, and obtain the extract.
[0093] S3. The leachate from step S2 is centrifuged to obtain centrifuged material;
[0094] S4. The centrifuged material from step S3 was centrifuged again and enriched to obtain extracellular vesicles of Coptis chinensis.
[0095] in,
[0096] The extract consists of Na2HPO4, NaCl, and NaH2PO4;
[0097] The complex enzyme consists of cellulase, lignin peroxidase, and pectinase.
[0098] The concentration of the EDTA solution is 5 mM.
[0099] The concentrations of Na2HPO4 were 3 mM, NaCl was 70 mM, and NaH2PO4 was 18 mM.
[0100] Cellulase concentration was 120 U / mL, lignin peroxidase concentration was 90 U / mL, and pectinase concentration was 50 U / mL.
[0101] The centrifugation treatment of the leachate in step S3 is as follows: first, centrifuge at 900g for 22min, filter the supernatant, then centrifuge at 2200g for 30min, filter the supernatant, add polyethylene glycol solution, centrifuge at 4000g for 50min, filter the supernatant and treat it with an ultrafiltration membrane with a molecular weight cutoff of 30-100kDa to obtain the centrifuged product.
[0102] The concentration of the polyethylene glycol solution used was 3.5 g / mL.
[0103] The volume of the polyethylene glycol solution used was 0.4 times the volume of the corresponding supernatant.
[0104] In step S4, the centrifugation enrichment was carried out at 70,000g for 2 hours, and the precipitate was obtained by filtration. The precipitate was resuspended in PBS to obtain extracellular vesicles of Coptis chinensis.
[0105] The resuspension temperature should not exceed 4℃. After resuspension, centrifuge at 100,000g for 1.3h and filter to obtain the precipitate. The precipitate is resuspended again with PBS to obtain extracellular vesicles of Coptis chinensis.
[0106] Example 5
[0107] A method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function includes:
[0108] S1. Pretreatment: Wash and cut fresh Coptis chinensis into sections, dry at 40℃ for 10 hours, then soak in EDTA solution for 20 minutes, filter and wash to obtain pretreated Coptis chinensis.
[0109] S2. Add the pretreated Coptis chinensis from step S1 to the extract, then add the compound enzyme, stir evenly, soak for 8-20 hours, filter, and obtain the extract.
[0110] S3. The leachate from step S2 is centrifuged to obtain centrifuged material;
[0111] S4. The centrifuged material from step S3 was centrifuged again and enriched to obtain extracellular vesicles of Coptis chinensis.
[0112] in,
[0113] The extract consists of Na2HPO4, NaCl, and NaH2PO4;
[0114] The complex enzyme consists of cellulase, lignin peroxidase, and pectinase.
[0115] The concentration of the EDTA solution is 6 mM.
[0116] The concentrations of Na2HPO4 were 4 mM, NaCl was 80 mM, and NaH2PO4 was 20 mM.
[0117] Cellulase concentration was 150 U / mL, lignin peroxidase concentration was 100 U / mL, and pectinase concentration was 60 U / mL.
[0118] The centrifugation treatment of the leachate in step S3 is as follows: first, centrifuge at 1000g for 25min, filter the supernatant, then centrifuge at 2500g for 35min, filter the supernatant, add polyethylene glycol solution, centrifuge at 4500g for 60min, and filter the supernatant using an ultrafiltration membrane with a molecular weight cutoff of 30-100kDa to obtain the centrifuged product.
[0119] The concentration of the polyethylene glycol solution used was 4 g / mL.
[0120] The volume of the polyethylene glycol solution used was 0.5 times the volume of the corresponding supernatant.
[0121] In step S4, the centrifugation enrichment was carried out at 80,000g for 2 hours, and the precipitate was obtained by filtration. The precipitate was resuspended in PBS to obtain extracellular vesicles of Coptis chinensis.
[0122] The resuspension temperature should not exceed 4℃. After resuspension, centrifuge at 120000g for 1.5h and filter to obtain the precipitate. The precipitate is resuspended again with PBS to obtain extracellular vesicles of Coptis chinensis.
[0123] An application of extracellular vesicles of Coptis chinensis, wherein the extracellular vesicles of Coptis chinensis prepared by the method of the present invention are used for the preparation of drugs for the prevention or treatment of hepatocellular carcinoma.
[0124] Comparative Example 1
[0125] Same as Example 3, but without step S1.
[0126] Comparative Example 2
[0127] Same as Example 3, but without the polyethylene glycol solution.
[0128] Comparative Example 3
[0129] Same as Example 3, but without step S1 and the polyethylene glycol solution.
[0130] Experiment 1
[0131] The external vesicles of *Coptis chinensis* prepared in Examples 1-5 and Comparative Examples 1-3 were examined using electrophoresis, Brownian motion video analysis, and laser scattering microscopy at a laser wavelength of 520 nm. Peak analysis data are shown in Table 1.
[0132] Table 1. Analysis results of external vesicle peaks in Coptis chinensis.
[0133] Group diameter / nm Particle count / mL FWHM / nm percentage / % Example 1 125.7 <![CDATA[3.4×10 6 ]]> 108.2 86.0 Example 2 126.5 <![CDATA[3.7×10 7 ]]> 107.9 86.7 Example 3 121.3 <![CDATA[3.9×10 7 ]]> 106.5 88.9 Example 4 122.9 <![CDATA[3.2×10 7 ]]> 107.6 88.0 Example 5 124.6 <![CDATA[3.6×10 7 ]]> 108.2 87.3 Comparative Example 1 133.2 <![CDATA[2.9×10 7 ]]> 118.0 79.5 Comparative Example 2 129.4 <![CDATA[2.6×10 7 ]]> 113.2 80.2 Comparative Example 3 143.7 <![CDATA[1.3×10 7 ]]> 127.6 68.3
[0134] As shown in Table 1, Examples 1-5, the proportion of particles with diameters between 121.3-126.5 nm is relatively large, all exceeding 86.0%. Example 3 is the preferred embodiment, exhibiting smaller diameter, higher particle count, narrower peak width, and a larger percentage value compared to the other examples, indicating superior diameter, quantity, and purity of the exovesicles. Data from Example 3 and Comparative Examples 1-3 show that the particle count in Comparative Examples 1-3 is between 2.9 × 10⁻⁶. 7 -1.3×10 7 The larger peak width and smaller percentage indicate that the prepared exovesicles have uneven particle size distribution and small number, suggesting that step S1 and the polyethylene glycol solution used in the preparation process play key roles.
[0135] Experiment 2
[0136] Extracellular vesicles of Coptis chinensis: prepared using the method described in Example 3 of this invention.
[0137] MHCC97H cells: human highly metastatic liver cancer cells.
[0138] The p-values of the following experimental results were analyzed using Student's t-test in GraphPad software. *p<0.05 indicates that the difference is statistically significant.
[0139] Cell culture: Frozen MHCC97H cells were thawed at 37°C, transferred to centrifuge tubes, and RPMI-1640 medium was added. The mixture was then centrifuged at 1000 r / min for 5 min, the supernatant was discarded, and the cells were added to RPMI-1640 medium containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin. The cells were then cultured and passaged at 37°C in a 5% CO2 incubator.
[0140] Cell phagocytosis: MHCC97H cells in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension. The cells were then seeded into 6-well plates, with an appropriate amount of cell suspension added to each well to ensure a suitable cell density for subsequent observation. The plates were then incubated in a cell culture incubator. The suspension was diluted with an appropriate amount of PBS buffer to prepare a suitable concentration. A certain amount of the suspension was added to a new centrifuge tube, and an appropriate amount of PKH26 working solution was added according to the PKH26 dye instructions. The mixture was gently mixed and incubated at room temperature for a period of time (usually 15-30 min) to allow the *E. coli* bioparticles to be labeled with PKH26 red fluorescence. After incubation, an appropriate amount of serum-containing medium was added to terminate the reaction. The cells were then centrifuged at an appropriate speed (e.g., 1000-1500 rpm) for 5-10 min. The supernatant was discarded, and the labeled particles were washed 2-3 times with PBS buffer to remove unbound dye. Finally, the cells were resuspended in an appropriate amount of culture medium for later use.
[0141] Once the MHCC97H cells in the 6-well plate have grown to a suitable state, discard the original culture medium and gently wash the cells 2-3 times with PBS buffer. Add an appropriate amount of culture medium containing labeled pHrodo Red E. coli BioParticles to each well, ensuring sufficient contact between the particles and cells. Return the 6-well plate to the cell culture incubator and observe at different time points. At the predetermined observation time points, remove the 6-well plate, discard the culture medium, and gently wash the cells 3 times with PBS buffer to remove unphagocytosed particles. Add an appropriate amount of 4% paraformaldehyde to fix the cells and fix them at room temperature for 15-20 min. After fixation, discard the fixative and wash the cells 2-3 times with PBS buffer. Add an appropriate amount of DAPI staining solution and stain at room temperature in the dark for 5-10 min to stain the cell nuclei with blue fluorescence. After staining, wash the cells 3 times with PBS buffer to remove excess staining solution. Observe and photograph using a fluorescence microscope.
[0142] Depend on Figure 3 It can be seen that MHCC97H cells can phagocytose the outer vesicles derived from Coptis chinensis. No red fluorescent signal related to the outer vesicles was observed in the control group, highlighting the specificity of the phagocytosis phenomenon in the experimental group, indicating that the outer vesicles of Coptis chinensis can interact with MHCC97H cells.
[0143] Cell proliferation: HEPG2 cells passaged in an incubator were randomly divided into two groups: a control group (MHCC97H + PBS) and an experimental group (MHCC97H + Coptis chinensis Exo). Logarithmic growth phase cells were collected using 96-well plates, with a seeding density of 1 × 10⁶ cells / well. 4 Add 100 μL of cell resuspending solution to each well and incubate for 72 h; measure the absorbance of each well at 450 nm using a microplate reader.
[0144] Depend on Figure 4 It can be seen that the control group is above the experimental group, with a higher OD450 value, indicating that the treatment with extracellular vesicles of Coptis chinensis can effectively inhibit the proliferation of MHCC97H cells.
[0145] Cell migration: Draw horizontal lines on the back of a 6-well plate with a marker pen, every 0.5-1 cm, passing through the wells, with at least 5 lines passing through each well; take MHCC97H cells in logarithmic growth phase and divide them into two groups: control group (MHCC97H+PBS group) and experimental group (MHCC97H+Rhizoma Cynanchi Aquatica-Exo), digest them with trypsin to form single-cell suspensions, and seed them into 6-well culture plates; seed 6 wells per cell, with a total culture medium volume of 2 mL per well, and incubate at 37℃, 5% CO2 for 48 h; the next day, draw parallel to or perpendicular to the horizontal lines on the back of the 6-well plate lid; wash the cells 3 times with PBS to remove the drawn cells, and add serum-free culture medium; wipe off the marker horizontal lines on the back of the 6-well plate; take pictures under a 4x microscope at 0h, 24h, and 48h.
[0146] Depend on Figure 5 It can be seen that, compared with the control group, the wound width decreased more slowly in the experimental group, and the difference between the two groups was significant from 0-24h to 48h, indicating that the use of extracellular vesicles of Coptis chinensis has a better ability to inhibit the migration of MHCC97H cells.
[0147] Cell colony formation: MHCC97H cells in logarithmic growth phase were digested with trypsin to prepare a single-cell suspension, and then the cell concentration was adjusted to 100-200 cells / mL with culture medium. 2 mL of cell suspension was added to each well of a 6-well plate, resulting in approximately 200-400 cells per well. The plates were incubated at 37°C with 5% CO2. The culture medium was changed every 2-3 days. After 14 days of incubation, the culture medium in the 6-well plates was discarded, and the cell colonies were gently washed 2-3 times with PBS buffer. Cells were fixed with 4% paraformaldehyde for 15-20 minutes, then the fixative was discarded, and the plates were washed 2-3 times with PBS. Crystal violet staining solution was added, and the cells were stained at room temperature for 10-15 minutes. The stained cell colonies were slowly rinsed with water until the background was colorless, air-dried, and observed and counted under an inverted microscope. A colony with more than 50 cells was counted as one effective colony, and the colony formation rate was calculated.
[0148] Depend on Figure 6 It can be seen that the relative clone formation efficiency of the control group was 1±0.03656, while that of the experimental group was 0.4591±0.09910, with a P value of 0.0009, which showed a significant difference. This indicates that the extracellular vesicles of Coptis chinensis prepared by the present invention can inhibit the cloning of MHCC97H cells.
[0149] Cell migration:
[0150] Collect MHCC97H cells in the logarithmic growth phase, discard the old culture medium, wash twice with PBS; then add serum-free culture medium and starve them in an incubator for 12-24 hours; then discard the starved culture medium, add an appropriate amount of trypsin, digest at 37°C until the cells become round and detach, add serum-containing complete culture medium to stop digestion, gently pipette to collect the cells into centrifuge tubes; centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in PBS, and centrifuge again; resuspend the cells in serum-free culture medium and adjust the concentration to 1×10⁵-5×10⁵ cells / mL.
[0151] Add medium to the lower chamber: Add 600 μL of complete culture medium containing 10% PBS to the lower chamber of the 24-well plate; Add cell suspension to the upper chamber: Add 200 μL of cell suspension to the upper chamber of the Transwell plate.
[0152] Negative control (MHCC97H+PBS): serum-free culture medium was added to the upper chamber, and serum-containing culture medium was added to the lower chamber; Experimental group (MHCC97H+Rhizoma Coptidis Exo): Rhizoma Coptidis exovesicles prepared in this invention were added.
[0153] Carefully place the 24-well plate into a cell culture incubator and incubate at 37°C and 5% CO2 for 24 hours. Then remove the Transwell chamber and gently rinse the surface of the upper chamber with PBS. Place the chamber into the wells pre-filled with 4% paraformaldehyde and fix at room temperature for 20 minutes. Remove and rinse twice with PBS. Place the chamber into the wells containing 0.1% crystal violet staining solution and stain at room temperature for 15-20 minutes. Rinse three times with PBS to remove excess staining solution.
[0154] Gently wipe the upper surface of the chamber with a moistened cotton swab, place the chamber under an inverted microscope for observation, randomly select 5 fields of view, and count the number of cells on the lower surface of the filter membrane in each field of view.
[0155] Depend on Figure 7 It can be seen that the migration ability of MHCC97H cells in the experimental group was significantly weaker than that in the control group, indicating that the use of Coptis chinensis vesicles can significantly inhibit their migration ability.
[0156] In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, without necessarily requiring or implying any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, the phrase "comprising an element defined as..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.
[0157] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function, characterized in that, include: S1. Pretreatment: Wash and cut fresh Coptis chinensis into sections, dry at 25-40℃ for 10-24 hours, then soak in EDTA solution for 5-20 minutes, filter and wash to obtain pretreated Coptis chinensis. S2. Add the pretreated Coptis chinensis from step S1 to the extract, then add the compound enzyme, stir evenly, soak for 8-20 hours, filter, and obtain the extract. S3. Centrifuge the leachate from step S2 for 1-2 hours to obtain centrifuged material; S4. The centrifuged material from step S3 was centrifuged again and enriched to obtain extracellular vesicles of Coptis chinensis. in, The extract consists of Na2HPO4, NaCl, and NaH2PO4; The complex enzyme consists of cellulase, lignin peroxidase, and pectinase.
2. The method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function according to claim 1, characterized in that, The concentration of the EDTA solution is 1-6 mM.
3. The method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function according to claim 2, characterized in that, The concentrations of Na2HPO4 are 2-4 mM, NaCl is 50-80 mM, and NaH2PO4 is 8-20 mM.
4. The method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function according to claim 3, characterized in that, Cellulase concentration: 80-150 U / mL; lignin peroxidase concentration: 50-100 U / mL; pectinase concentration: 30-60 U / mL.
5. The method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function according to claim 4, characterized in that, Step S3, centrifugation treatment of the leachate, is as follows: First, centrifuge at 500-1000g for 10-25 min, filter the supernatant, then centrifuge at 1500-2500g for 20-35 min, filter the supernatant again, add polyethylene glycol solution, centrifuge at 1500-4500g for 30-60 min, filter the supernatant and treat it with an ultrafiltration membrane with a molecular weight cutoff of 30-100kDa to obtain the centrifuged product.
6. The method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function according to claim 5, characterized in that, The concentration of the polyethylene glycol solution used was 1.5-4 g / mL.
7. The method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function according to claim 6, characterized in that, The volume of the polyethylene glycol solution used should be 0.2-0.5 times the volume of the corresponding supernatant.
8. The method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function according to claim 7, characterized in that, In step S4, the centrifugation enrichment is carried out at 60,000-80,000 g for 1-2 hours, followed by filtration to obtain the precipitate. The precipitate was resuspended in PBS to obtain extracellular vesicles of Coptis chinensis.
9. The method for preparing Coptis chinensis exovesicles with anti-hepatocellular carcinoma function according to claim 8, characterized in that, The resuspension temperature should not exceed 4℃. After resuspension, centrifuge at 80,000-120,000g for 1-1.5 hours and filter to obtain the precipitate. The precipitate was resuspended in PBS to obtain extracellular vesicles of Coptis chinensis.