Method for modifying umbilical cord mesenchymal stem cells by lipidosome loaded with total ginsenoside and application of umbilical cord mesenchymal stem cells

Abstract

The invention discloses an umbilical cord mesenchymal stem cell loaded with total ginsenoside liposome as well as a preparation method and application of the umbilical cord mesenchymal stem cell, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: dissolving total ginsenosides, lecithin and cholesterol in ethanol, adding into a phosphoric acid buffer solution for hydration, removing an ethanol solvent, and carrying out ultrasonic treatment and centrifugation to remove unencapsulated total ginsenosides and impurities so as to obtain total ginsenoside loaded lipidosome; resuspending the liposome loaded with the total ginsenoside in PBS (phosphate buffer solution), adding turbid liquid into a culture solution for culturing the umbilical cord mesenchymal stem cells for culturing, and separating to obtain the modified umbilical cord mesenchymal stem cells loaded with the liposome loaded with the total ginsenoside. The compound is applied to treatment of cisplatin-induced acute kidney injury, reaches the kidney injury part through blood circulation after intravenous injection, secretes cell factors to the outside and releases a part of total ginsenoside, so that accurate targeting of the kidney injury is realized, inflammatory response of the kidney injury in the acute stage is relieved, and later kidney repair of a patient with the acute kidney injury is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to umbilical cord mesenchymal stem cells loaded with ginsenoside total saponin liposomes, and a preparation method and application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) have great potential in treating degenerative diseases and tissue damage, but their clinical applications face challenges such as limited sources, complex extraction, and low survival efficiency. To overcome these limitations, existing methods include extracting exosomes, genetic engineering, stem cell membrane fusion liposomes, etc. However, these methods have disadvantages such as complex technology, high cost, and unknown safety. Therefore, it is particularly urgent to develop economical, efficient, and safe mesenchymal transformation methods.

[0003] Traditional methods of genetically modifying umbilical cord mesenchymal stem cells often involve complex gene editing techniques, which are difficult to operate and have high technical barriers. They may also cause gene mutations or unpredictable biosafety issues. As an advanced drug delivery carrier, liposomes have excellent biocompatibility, low toxicity, and efficient cell penetration, and can accurately deliver drugs or bioactive molecules into target cells. This property makes liposomes an ideal choice for modifying MSCs because it can ensure the effective loading and intracellular release of active ingredients. Ginseng total saponins, as the core active ingredient of traditional Chinese medicine ginseng, have multiple biological activities such as promoting cell proliferation, anti-oxidation, anti-inflammatory and immunomodulation. They can not only promote cell proliferation but also have significant effects on tissue repair and relief of inflammatory diseases.

[0004] Combining ginsenosides with MSCs may enhance the therapeutic effect by strengthening the proliferation ability of MSCs through its biological activity. The modified MSCs can also enhance the anti-inflammatory and repair effects when releasing ginsenosides, which opens up a new path for disease treatment. Summary of the invention

[0005] In view of the shortcomings and deficiencies in the prior art, the present invention provides an umbilical cord mesenchymal stem cell loaded with ginsenoside total saponin liposomes and a preparation method and application thereof. The preparation method is stable, efficient, universal and reproducible. The modified umbilical cord mesenchymal stem cells have stronger proliferation ability than conventional umbilical cord mesenchymal stem cells, longer storage time in the body, and better repair effect.

[0006] The present invention is achieved through the following technical solutions: A method for transforming umbilical cord mesenchymal stem cells by liposomes loaded with total ginsenosides, comprising the following steps: (1) dissolving total ginsenosides, lecithin and cholesterol in ethanol, adding them to phosphate buffer solution for hydration, removing the ethanol solvent, ultrasonicating and centrifuging to remove unencapsulated total ginsenosides and impurities, and obtaining ginsenoside-loaded liposomes; (2) Resuspending the liposomes loaded with total ginsenosides in PBS, adding the suspension to the culture medium containing umbilical cord mesenchymal stem cells for co-culture, and isolating the umbilical cord mesenchymal stem cells modified with the liposomes loaded with total ginsenosides.

[0007] Furthermore, in step (1), the mass ratio of ginsenosides, lecithin and cholesterol is 1.2: 0.8-1.2: 5-10.

[0008] Furthermore, the dissolution temperature of ginsenosides, lecithin and cholesterol is 50-55°C, the phosphate buffer solution is preheated to 50-55°C, and hydrated at 50-55°C for 25-30 minutes.

[0009] Furthermore, the concentration of the phosphate buffer in step (1) is 0.01-0.02 M and the pH is 7.0-7.4.

[0010] Furthermore, in step (2), the concentration of the ginsenoside liposomes loaded after resuspending in PBS is 1-2.5 mg / mL, and the concentration of the ginsenoside liposomes loaded after adding to the culture medium is 70-100 g / L, and the umbilical cord mesenchymal stem cells grow to occupy between 40-50% of the bottom of the culture bottle in the culture medium containing umbilical cord mesenchymal stem cells.

[0011] Furthermore, in step (2), the umbilical cord mesenchymal stem cell culture medium is a DMEM culture medium containing 1% penicillin-streptomycin mixed double antibody (penicillin 100 U / ml, streptomycin 0.1 mg / ml), 1% non-essential amino acids, and 10% fetal bovine serum.

[0012] Furthermore, the co-cultivation time is 24-48h and the temperature is 35~38°C.

[0013] In the present invention, the umbilical cord mesenchymal stem cells transformed by liposomes loaded with total ginsenosides are obtained by the method.

[0014] In the present invention, the umbilical cord mesenchymal stem cells loaded with total ginsenoside liposomes are used in the preparation of a drug for treating acute kidney injury.

[0015] Beneficial Effects (1) The present invention is mainly used for the treatment of cisplatin-induced acute kidney injury. The natural extract of total ginsenosides is loaded into umbilical cord mesenchymal stem cells via liposomes to achieve the transformation of umbilical cord mesenchymal stem cells. The transformed umbilical cord mesenchymal stem cells reach the site of kidney damage through blood circulation. The transformed umbilical cord mesenchymal stem cells secrete cytokines and release a portion of total ginsenosides, thereby achieving precise targeting of kidney damage, reducing the inflammatory response in the acute phase of kidney damage, and promoting kidney repair in the later stage of patients with acute kidney injury.

[0016] (2) The present invention has significant advantages over traditional methods for genetically modifying umbilical cord mesenchymal stem cells. Traditional methods often involve complex gene editing techniques, which are difficult to operate and have high technical barriers. They may also cause gene mutations or unpredictable biosafety issues. The present invention uses liposomes loaded with ginsenosides to co-culture with umbilical cord mesenchymal stem cells, achieving non-invasive drug loading and avoiding the potential risks of genetic modification. In addition, the method is simple to operate and easy to promote, greatly reducing the cost and time cost of technical implementation, and opening up a new path for the application of umbilical cord mesenchymal stem cells in the field of drug delivery.

[0017] (3) Compared with methods such as preparing umbilical cord mesenchymal stem cell membrane vesicles or collecting umbilical cord mesenchymal stem cell exosomes, the present invention has obvious advantages in resource utilization and efficiency. The preparation process of umbilical cord mesenchymal stem cell membrane vesicles and exosomes is cumbersome and the output is limited, which makes it difficult to meet the needs of large-scale treatment. The present invention, on the other hand, loads ginseng total saponins into liposomes and co-cultivates them with umbilical cord mesenchymal stem cells. The method is simple and can improve the proliferation efficiency of umbilical cord mesenchymal stem cells while achieving efficient drug loading and cell utilization, greatly improving the treatment efficiency. At the same time, the method retains the integrity and biological activity of umbilical cord mesenchymal stem cells, providing a strong guarantee for their long-term survival and functional performance in the body.

[0018] (4) Compared with direct intravenous injection of ginsenosides or liposomes loaded with ginsenosides and umbilical cord mesenchymal stem cells, the present invention achieves a significant improvement in drug delivery effect; through the co-culture strategy, the liposomes loaded with ginsenosides are effectively endocytosed by umbilical cord mesenchymal stem cells, allowing the drug to be more accurately targeted to the lesion site, thereby increasing the concentration and duration of the drug in the body. In addition, the modified umbilical cord mesenchymal stem cells survive longer in the body, further enhancing the therapeutic effect. This innovative method not only improves the efficiency and accuracy of drug delivery, but also provides new ideas and solutions for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a transmission electron micrograph of the ginseng total saponin loaded liposomes prepared in Example 2; Figure 2 Flow chart for modifying umbilical cord mesenchymal stem cells for loading ginsenoside liposomes; Figure 3 This is a fluorescence microscopic image of umbilical cord mesenchymal stem cells transformed with Dil-labeled liposomes loaded with total ginsenosides in Example 2. The liposomes are labeled with Dil (orange), and the cell nuclei are stained with Hoechst (blue). ; Figure 4 This is a graph showing the results of umbilical cord mesenchymal stem cell-related antibody detection of umbilical cord mesenchymal stem cells transformed with liposomes loaded with total ginsenosides in Example 2; Figure 5 Alizarin red staining (A) and oil red staining (B) of umbilical cord mesenchymal stem cells transformed with ginsenoside total saponin liposomes after differentiation into adipocytes and osteoblasts in Example 2; Figure 6 The Edu staining images of the umbilical cord mesenchymal stem cells transformed with liposomes loaded with total ginsenosides in Example 2 and the umbilical cord mesenchymal stem cells after culturing for 24 hours; Figure 7 The fluorescence imaging images of kidneys of mice model of acute kidney injury 2 weeks after DiD-labeled ginsenoside-loaded liposomes transformed with umbilical cord mesenchymal stem cells and umbilical cord mesenchymal stem cells were used; Figure 8 PAS staining of the kidneys of model mice injected with PBS, umbilical cord mesenchymal stem cells, and umbilical cord mesenchymal stem cells modified with ginsenoside total saponin liposomes 2 weeks after tail vein injection; Fig. 9 This is a graph of blood creatinine levels in model mice 2 weeks after tail vein injection of PBS, umbilical cord mesenchymal stem cells, and umbilical cord mesenchymal stem cells transformed with ginsenoside total saponin liposomes. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1 Extraction and culture of umbilical cord mesenchymal stem cells: (1) Place the umbilical cord in a 50 mL centrifuge tube filled with PBS and wash it three times by shaking. Move the washed umbilical cord to a new culture dish and use surgical scissors to accurately cut out tissue segments of appropriate length; (2) Use surgical scissors to finely chop the cut umbilical cord tissue until a uniform viscous tissue mince is formed; during this process, an appropriate amount of DMEM culture medium containing 20% ​​fetal bovine serum needs to be continuously added to ensure that the tissue remains moist and avoids drying; (3) Add sufficient cell culture medium to the culture dish containing the minced tissue, and gently stir to evenly distribute the tissue pieces in the culture medium. Use an 84-inch pipette to carefully and slowly transfer the mixed umbilical cord tissue suspension to the pre-prepared 35 mm culture dish, ensuring that it is evenly distributed and no large number of bubbles are generated. Gently cover the surface of the culture medium with a pre-dried coverslip, ensuring that the coverslip is close to the surface of the culture medium to maintain a sterile culture environment.

[0022] (4) Add an initial amount of 1.5 mL of cell culture medium to the culture dish to start the culture process. 8 hours after the start of culture, add 1 mL of culture medium to support cell growth. Thereafter, replace the culture medium every 3 days to maintain the nutrients and environmental conditions required for cell growth. Sufficient umbilical cord mesenchymal stem cells can be obtained after 3 weeks.

[0023] Example 2 The flow chart of preparing umbilical cord mesenchymal stem cells loaded with ginsenoside liposomes is as follows Figure 2 As shown: (1) Preparation of ginsenoside-loaded liposomes: Weigh 1.2 mg of ginsenosides, 1 mg of lecithin, and 5 mg of cholesterol, add them to 300 μL of anhydrous ethanol, and heat them in a 55°C water bath until they are completely dissolved to form a uniform solution; then, slowly inject the solution into a phosphate buffer solution (concentration 0.01 M and pH 7.4) preheated to 50°C while stirring, and continue stirring and hydrating at 50°C for 25 minutes; then, use a rotary evaporator to evaporate the ethanol; then, ultrasonicate the solution for 15 minutes; finally, centrifuge at 15,000 rpm for 30 minutes to remove unencapsulated ginsenosides and other impurities to obtain ginsenoside-loaded liposomes, the transmission electron microscopy image of which is shown in the figure below. Figure 1 As shown, the size of the liposomes loaded with total ginsenosides was about 100 nm.

[0024] (2) The ginsenoside-loaded liposomes obtained in step (1) are resuspended in a 0.01M PBS solution (pH 2.4). The ginsenoside-loaded liposomes in the suspension after resuspension have a concentration of 2.14 mg / ml. The suspension is added to an umbilical cord mesenchymal stem cell culture medium (DMEM culture medium containing 1% penicillin-streptomycin mixed double antibody (penicillin 100 U / ml, streptomycin 0.1 mg / ml), 1% non-essential amino acids, and 10% fetal bovine serum) to make the ginsenoside-loaded liposomes in the culture medium have a concentration of 80 mg / L. At this time, the umbilical cord mesenchymal stem cells grow to occupy 45% of the bottom of the culture bottle. The cells are cultured under this condition for 24 hours to obtain umbilical cord mesenchymal stem cells (transformed MSCs) transformed with ginsenoside-loaded liposomes.

[0025] The fluorescence microscopic image of the umbilical cord mesenchymal stem cells (transformed MSCs) transformed by the liposomes loaded with total ginsenosides in Example 2 is shown in FIG. Figure 3 As shown by Figure 3 It can be seen that most cells already have a large number of liposomes loaded with ginsenosides, which means that the cell transformation has been completed; The umbilical cord mesenchymal stem cell-related antibodies (CD29, CD73, CD90, CD11b, CD34, CD45) of the umbilical cord mesenchymal stem cells (transformed MSCs) transformed with ginsenoside total saponin liposomes obtained in Example 2 were tested by flow cytometry. Specifically, the transformed MSCs were resuspended in PBS to prepare a single cell suspension, and stained with CD29, CD73, CD90, CD11b, CD34, and CD45 flow cytometry-specific antibodies for 5 minutes, respectively. The proportion of cells positive for each antibody in the cells was detected by flow cytometry. The test results are as follows: Figure 4 As shown in the figure, it can be seen that the transformed MSCs are CD29, CD73, and CD90 positive, and CD11b, CD34, and CD45 negative, indicating that the stemness of the transformed MSCs has not changed at the cellular molecular level.

[0026] Alizarin red staining (A) and oil red staining (B) of the modified MSCs obtained in Example 2 after 2 weeks of induction into adipocytes and osteoblasts: The steps of osteogenic differentiation and alizarin red staining are as follows: cells in the logarithmic growth phase were taken at 2×10 4 cells / cm 2 The cells were inoculated into culture vessels at a density of 100 μg / mL and incubated at 37°C with 5% CO 2 Culture to 65% confluence under the culture environment, discard the supernatant, add osteogenic differentiation medium, replace osteogenic differentiation medium every 2-3 days, and incubate at 37°C, 5% CO 2 After culturing for 21 days in the culture environment, the culture medium was removed, and the cells were washed three times with an appropriate amount of PBS, and then dyed with an appropriate amount of alizarin red dye for 5 minutes. The alizarin red dye was removed, and the cells were washed twice with PBS, and an appropriate amount of 1×PBS was added to prevent the cells from drying out. The cells were observed under an inverted microscope. Adipocyte differentiation and identification method: Cells in the logarithmic growth phase were taken and 2×10 4 cells / cm 2 The cells were inoculated into the coated culture vessels at a density of 100 μg / mL and incubated at 37°C with 5% CO 2 Culture to 95% confluence under the culture environment, discard the supernatant, add adipogenic differentiation medium, and incubate at 37°C, 5% CO 2After culturing for 3 days in the culture environment, replace the adipogenic differentiation medium induction medium. After 1 day of culture, replace it with the adipogenic differentiation medium maintenance medium and continue to culture for 3 days. Induce for 21 days according to the above medium change frequency, aspirate the medium, wash once with an appropriate amount of PBS, fix with an appropriate amount of 4% neutral formaldehyde solution at room temperature for 50 minutes, discard the fixative and wash twice with PBS. Prepare Oil Red O working solution with physiological saline or 1×PBS and Oil Red O stock solution (Oil Red O stock solution: physiological saline = 3:2), prepare it before use; add an appropriate amount of Oil Red O working solution to the cleaned induction wells, let it stand for staining for 30 minutes; aspirate the Oil Red O working solution, wash twice with 1×PBS, and add an appropriate amount of 1×PBS to prevent the cells from drying out, and observe under an inverted microscope. The results are as follows Figure 5 As shown by Figure 5 It can be seen that the transformed MSCs still have differentiation ability, and their stemness is not changed after loading with ginsenoside liposomes.

[0027] The modified MSCs obtained in Example 2 were subjected to Edu fluorescence staining (Edu staining marks the proliferating cell nuclei in red) with the umbilical cord mesenchymal stem cells cultured for the same time but not treated. The results are as follows: Figure 6 As shown by Figure 6 It can be seen that the proliferation activity of transformed MSCs is stronger than that of untreated umbilical cord mesenchymal stem cells.

[0028] On the second day of the cisplatin-induced acute kidney injury experiment in mice, DiD-labeled modified umbilical cord mesenchymal stem cells and umbilical cord mesenchymal stem cells were injected into the mice through the tail vein of the mice, with a single injection concentration of 1×10 6 PBS cell suspension, the number of cells per mouse is 1×10 6 After 2 weeks, the mice were dissected and photographed using an animal imaging system. Figure 7 As shown, it can be seen that the signal intensity of the transformed MSCs is higher than that of the untransformed umbilical cord mesenchymal stem cells, proving that the retention of the transformed MSCs in the kidney is higher than that of the untransformed umbilical cord mesenchymal stem cells.

[0029] PAS staining was performed on the kidney tissues of mice two weeks after the experiment. The results are as follows: Figure 8 As shown, this indicates that modified MSCs are superior to unmodified umbilical cord mesenchymal stem cells in repairing renal injury.

[0030] The peripheral blood creatinine levels of the mice were tested two weeks after the experiment. Fig. 9 As shown, this indicates that modified MSCs are superior to unmodified umbilical cord mesenchymal stem cells in repairing renal injury.

[0031] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for transforming umbilical cord mesenchymal stem cells by liposomes loaded with total ginsenosides, characterized in that: The following steps are involved: (1) dissolving total ginsenosides, lecithin and cholesterol in ethanol, adding them to phosphate buffer solution for hydration, removing the ethanol solvent, ultrasonicating and centrifuging to remove unencapsulated total ginsenosides and impurities, and obtaining ginsenoside-loaded liposomes; (2) Resuspending the liposomes loaded with total ginsenosides in PBS, adding the suspension to the culture medium containing umbilical cord mesenchymal stem cells for co-culture, and isolating the umbilical cord mesenchymal stem cells modified with the liposomes loaded with total ginsenosides.

2. The method for transforming mesenchymal stem cells by liposomes loaded with total ginsenosides according to claim 1, characterized in that: In step (1), the mass ratio of ginsenosides, lecithin and cholesterol is 1.2:0.8-1.2:5-10.

3. The method for transforming umbilical cord mesenchymal stem cells by liposomes loaded with total ginsenosides according to claim 1, characterized in that: The dissolution temperature of ginsenosides, lecithin and cholesterol is 50-55°C. The phosphate buffer solution is preheated to 50-55°C and hydrated at 50-55°C for 25-30 minutes.

4. The method for transforming umbilical cord mesenchymal stem cells by liposomes loaded with total ginsenosides according to claim 1, characterized in that: The concentration of the phosphate buffer described in step (1) is 0.01-0.02 M and the pH is 7.0-7.

4.

5. The method for transforming umbilical cord mesenchymal stem cells by liposomes loaded with total ginsenosides according to claim 1, characterized in that: In step (2), the concentration of the ginsenoside liposomes loaded after resuspending in PBS is 1-2.5 mg / mL, and the concentration of the ginsenoside liposomes loaded after adding to the culture medium is 70-100 g / L. The umbilical cord mesenchymal stem cells grow to occupy between 40% and 50% of the bottom of the culture bottle in the culture medium containing umbilical cord mesenchymal stem cells.

6. The method for transforming umbilical cord mesenchymal stem cells by liposomes loaded with total ginsenosides according to claim 1, characterized in that: In step (2), the umbilical cord mesenchymal stem cell culture medium is a DMEM culture medium containing 1% penicillin-streptomycin mixed double antibody, 1% non-essential amino acids, and 10% fetal bovine serum.

7. The method for transforming umbilical cord mesenchymal stem cells by liposomes loaded with total ginsenosides according to claim 1, characterized in that: The co-cultivation time is 24-48h, and the temperature is 35~38℃.

8. Umbilical cord mesenchymal stem cells transformed with ginsenoside-loaded liposomes obtained by the method according to any one of claims 1 to 7.

9. Use of the umbilical cord mesenchymal stem cells loaded with total ginsenoside liposomes according to claim 8 in the preparation of a drug for treating acute kidney injury.

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