Immunoregulatory stem cell, preparation method thereof and application of immunoregulatory stem cell in preparation of medicine for treating atherosclerosis

Abstract

The invention provides an immunoregulatory stem cell as well as a preparation method and application thereof in preparation of a medicine for treating atherosclerosis, and belongs to the technical field of stem cell application, the preparation method comprises the following steps: 1) culturing mesenchymal stem cells for 3-5 days; (2) then changing the liquid, adding an immune factor composition into the cell culture system, continuously culturing for 3-5 days, and carrying out passage for 1-2 times to obtain the modified mesenchymal stem cells; and 3) placing the modified mesenchymal stem cells in an environment with an oxygen concentration of 2-3.5% for hypoxia culture, and collecting the cells. The immune factor composition is added in the culture process to modify the cells, so that the anti-inflammatory factor secretion level of the cells is improved, the immune regulation function of the cells is enhanced, and the Treg cells can be induced and generated.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cell application, and in particular relates to an immunoregulatory stem cell, a preparation method thereof, and an application thereof in the preparation of a drug for treating atherosclerosis. Background Art

[0002] Atherosclerosis is a major chronic disease that threatens human health. Its pathological characteristics are vascular endothelial damage, lipid deposition, inflammatory cell infiltration, and arterial plaque formation, ultimately leading to serious complications such as myocardial infarction, stroke, and peripheral arterial disease. According to the World Health Organization, atherosclerosis-related diseases account for over 32% of annual deaths worldwide. Its core pathogenesis is closely related to an imbalance in immune inflammation: overactivation of pro-inflammatory M1 macrophages triggers chronic inflammation of the vascular wall, while deficient function of anti-inflammatory M2 macrophages and regulatory T cells (Tregs) leads to impaired lipid clearance and plaque instability.

[0003] Current clinical treatments are mainly lipid-lowering, antiplatelet and surgical interventions, but they have significant limitations: drug therapy (such as statins, PCSK9 inhibitors) can only delay plaque progression but cannot reverse lesions or repair damaged vascular endothelium; surgical treatment (such as stent implantation, bypass surgery) is aimed at advanced severe lesions and cannot solve the problems of underlying inflammation and plaque regeneration; although anti-inflammatory therapies (such as IL-1β inhibitors) can reduce the risk of cardiovascular events, systemic medications have significant side effects and fail to achieve precise regulation of the vascular microenvironment.

[0004] Mesenchymal stem cells (MSCs) have become an emerging direction for the treatment of atherosclerosis due to their immunoregulatory and tissue repair capabilities. MSCs can inhibit the activation of M1 macrophages by secreting cytokines such as IL-10 and TGF-β, promote M2 polarization and Treg differentiation, and repair damaged endothelium. However, existing technologies have the following key bottlenecks: Insufficient targeting: Traditional intravenous infusion of MSCs has a low retention rate in the body (only 1%-5% are enriched in the plaque site), and most are retained by organs such as the lungs and liver. They also lack the ability to respond to the plaque microenvironment (high reactive oxygen species ROS, low pH, high matrix metalloproteinases MMPs), resulting in insufficient local cell concentrations; Short functional persistence: MSCs are prone to apoptosis in oxidative stress and inflammatory microenvironments, and the secretion of natural immune regulatory factors is limited, making it difficult to maintain long-term anti-inflammatory effects.

[0005] Therefore, there is an urgent need to develop a stem cell therapy system that can accurately respond to the plaque microenvironment and maintain long-term immune regulation function, so as to achieve efficient targeted treatment of atherosclerosis. Summary of the Invention

[0006] In light of this, the present invention aims to provide immunomodulatory stem cells, a method for their preparation, and their use in the preparation of a drug for treating atherosclerosis. By constructing MSCs enhanced with a combination of immunomodulatory factors, the present invention addresses the shortcomings of the prior art and provides an innovative solution for cell therapy of this disease.

[0007] The present invention provides a method for preparing immunoregulatory stem cells, comprising the following steps:

[0008] 1) Cultivate mesenchymal stem cells for 3-5 days;

[0009] 2) Then, the medium is changed, and the immune factor composition is added to the cell culture system, and the culture is continued for 3 to 5 days, and passaged 1 to 2 times to obtain modified mesenchymal stem cells;

[0010] The immune factor composition includes recombinant human interferon IFN-α-2a, IL-10 and vitamin B6;

[0011] The final concentration of the recombinant human interferon IFN-α-2a in the cell culture system is 10-20 ng / ml;

[0012] The final concentration of IL-10 in the cell culture system is 5-15 ng / ml;

[0013] The final concentration of vitamin B6 in the cell culture system is 20-30 μg / ml;

[0014] 3) The modified mesenchymal stem cells were placed in a hypoxic culture environment with an oxygen concentration of 2% to 3.5%, and the cells were collected.

[0015] Preferably, during the hypoxic culture process in step 2), IL-6 and bFGF are added to the culture system.

[0016] Preferably, the final concentration of IL-6 in the cell culture system is 5 to 15 ng / ml; the final concentration of bFGF in the cell culture system is 20 to 40 ng / ml.

[0017] Preferably, the culture medium for culturing the mesenchymal stem cells is a serum-free complete culture medium.

[0018] The present invention provides immunoregulatory stem cells prepared by the preparation method.

[0019] The present invention also provides the use of the immunoregulatory stem cells in preparing medicine for treating atherosclerosis.

[0020] Compared with the existing technology, the present invention has the following beneficial effects: the preparation method of immunoregulatory stem cells provided by the present invention modifies the cells by adding an immune factor composition during the culture process, thereby enhancing the immune regulatory function of the cells, and further acclimating and culturing them in a hypoxic environment. On the one hand, it enhances the adaptability of the cells to the hypoxic environment, providing a basis for their subsequent survival in the plaque microenvironment in the body; on the other hand, it enhances the immune regulatory ability of the cells and improves the elimination effect of atherosclerotic plaques.

[0021] Furthermore, the present invention adds IL-6 and bFGF during the hypoxic culture process; IL-6 can regulate the secretion of multiple immunoregulatory factors by stem cells, induce the production of regulatory T cells (Tregs), and inhibit the secretion of proinflammatory cytokines, thereby exerting immunosuppressive and anti-inflammatory effects, and helping to maintain immune homeostasis. In a hypoxic environment, IL-6 can reduce the apoptosis of immunoregulatory stem cells and increase cell survival rate by activating anti-apoptotic signaling pathways in cells. bFGF can enhance the vitality and metabolic function of stem cells, allowing them to better adapt to hypoxic environments. The immunoregulatory stem cells prepared by the present invention have significant immunoregulatory effects, strong adaptability to the lesion microenvironment, and significant therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are the test results of the secretion levels of the anti-inflammatory factors obtained in the cells prepared in Examples 1 to 3 and Comparative Examples 1 to 3;

[0023] Figure 2 The cell proliferation in step 6 of Examples 1 to 3 and Comparative Examples 1 to 3 is shown. DETAILED DESCRIPTION

[0024] The present invention provides a method for preparing immunoregulatory stem cells, comprising the following steps: 1) culturing mesenchymal stem cells for 3 to 5 days; 2) then changing the medium, adding an immune factor composition to the cell culture system, continuing to culture for 3 to 5 days, and passaged 1 to 2 times to obtain modified mesenchymal stem cells; 3) placing the modified mesenchymal stem cells in a hypoxic culture environment at an oxygen concentration of 2% to 3.5%, and collecting the cells.

[0025] In the present invention, mesenchymal stem cells are first cultured for 3 to 5 days. The mesenchymal stem cells are preferably human umbilical cord mesenchymal stem cells. The present invention does not specifically limit the source of the human umbilical cord mesenchymal stem cells. They can be prepared by themselves or commercially available products, which can be purchased from Wuhan Punosai Life Science Technology Co., Ltd. In the present invention, the culture medium for the mesenchymal stem cells is preferably a serum-free complete medium.

[0026] In the present invention, the immune factor composition includes recombinant human interferon IFN-α-2a, IL-10 and vitamin B6; the final concentration of the recombinant human interferon IFN-α-2a in the cell culture system is preferably 10-20 ng / ml, more preferably 12-18 ng / ml, and more preferably 16 ng / ml; the final concentration of the IL-10 in the cell culture system is preferably 5-15 ng / ml, more preferably 7-13 ng / ml, and more preferably 10 ng / ml; the final concentration of the vitamin B6 in the cell culture system is preferably 20-30 μg / ml, and more preferably 24-26 μg / ml. In the present invention, the role of recombinant human interferon IFN-α-2a is to promote the secretion of cytokines by mesenchymal stem cells, enhance their immune regulatory function, and on the other hand promote the proliferation of mesenchymal stem cells. In the present invention, IL-10 can activate the PI3K-AKT pathway, among other things, thereby promoting cell cycle progression, enabling stem cells to enter the division phase more rapidly, increasing their number and facilitating large-scale stem cell expansion and culture. Furthermore, IL-10 helps maintain the stemness of stem cells, preventing the loss of stemness during subculture of mesenchymal stem cells. In the present invention, vitamin B6 plays an important role in maintaining a stable intracellular environment. By adding the aforementioned immune factor composition during culture, the present invention significantly enhances the immune regulatory function of cells.

[0027] In the present invention, preferably, the cells are passaged when the fusion is 80% to 90%; the number of passages is 1 or 2; during the passage culture process, the immune factor composition is added; and finally, modified mesenchymal stem cells are obtained.

[0028] To enhance the adaptability of mesenchymal stem cells to hypoxic environments and further enhance their immunomodulatory function, the modified mesenchymal stem cells are cultured in an oxygen concentration environment of 2% to 3.5%. In the present invention, the oxygen concentration is preferably 2.5% to 3%. The present invention preferably performs hypoxic culture in a hypoxic incubator. During the hypoxic culture process, IL-6 and bFGF are preferably added to the culture medium; the final concentration of IL-6 in the cell culture system is preferably 5 to 15 ng / ml, more preferably 8 to 12 ng / ml, and even more preferably 10 ng / ml; the final concentration of bFGF in the cell culture system is preferably 20 to 40 ng / ml, more preferably 25 to 35 ng / ml, and even more preferably 30 ng / ml. In the present invention, the hypoxic culture time is preferably 14 to 20 hours, and even more preferably 16 to 18 hours. After the hypoxic culture is completed, the cells are harvested; the harvested cells are used directly or stored in liquid nitrogen for later use.

[0029] The present invention also provides immunoregulatory stem cells prepared by the preparation method. Compared with ordinary mesenchymal stem cells, the immunoregulatory stem cells prepared by the present invention have strong immunoregulatory ability, can promote M2 macrophage polarization, and can increase Treg cell differentiation.

[0030] The present invention also provides the use of the immunoregulatory stem cells in the preparation of a medicament for treating atherosclerosis. In the present invention, the use of the immunoregulatory stem cells can reduce inflammatory responses and promote plaque stabilization and reduction. In the present invention, the immunoregulatory stem cells are preferably administered by injection; in the present invention, the immunoregulatory stem cells are preferably coupled to a targeting element during administration to accurately localize the immunoregulatory stem cells to the lesion site. The present invention does not specifically limit the targeting element; any targeting element known in the art, such as a conventional delivery system in the art, may be employed.

[0031] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Human umbilical cord mesenchymal stem cells (Cat. No. CP-CL11), human mesenchymal stem cell serum-free culture medium (Cat. No. CM-SC01), and human peripheral blood mononuclear cells (CP-H182) were purchased from Wuhan Punosai Life Science Technology Co., Ltd.

[0033] IFN-α-2a, IL-10, vitamin B6, IL-6 and bFGF were all commercially available products.

[0034] Example 1

[0035] Step 1. Take out the T25 cell culture flask, disinfect the flask with 75% alcohol, remove the sealing film, and place it in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 3 hours to stabilize the cells.

[0036] Step 2: Aspirate the culture medium from the T25 cell culture flask and wash the cells once with PBS;

[0037] Step 3. Add 1 mL of 0.25% trypsin digestion solution to the T25 culture flask. Gently rotate the culture flask until the digestion solution covers the entire bottom of the culture flask. Aspirate the excess trypsin digestion solution and incubate at 37°C for 2 minutes. Observe under an inverted microscope. After the cells shrink and become round, add 5 mL of complete culture medium to terminate the digestion.

[0038] Step 4: Gently pipette to mix, inoculate the cells into T25 culture flasks at a ratio of 1:2, and then add fresh serum-free complete medium to 5 mL. Place the cells in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 3 days before changing the medium.

[0039] Step 5. Add IFN-α-2a (final concentration 16 ng / ml), IL-10 (final concentration 10 ng / ml) and vitamin B6 (final concentration 25 μg / ml) to the cell culture system after the medium is changed. After continuing to culture for 3 days (change the medium after 2 days of culture), the cells reach 80% to 90% confluence and are passaged. After passage, add IFN-α-2a (final concentration 16 ng / ml), IL-10 (final concentration 10 ng / ml) and vitamin B6 (final concentration 25 μg / ml) to the cell culture system and continue to culture for 2 days.

[0040] Step 6: The cells were then placed in a hypoxic incubator for 16 hours, maintaining an oxygen concentration of 2.5% to obtain immunoregulatory stem cells.

[0041] Example 2

[0042] Step 1. Take out the T25 cell culture flask, disinfect the flask with 75% alcohol, remove the sealing film, and place it in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 4 hours to stabilize the cells.

[0043] Step 2: Aspirate the culture medium from the T25 cell culture flask and wash the cells once with PBS;

[0044] Step 3. Add 1 mL of 0.25% trypsin digestion solution to the T25 culture flask. Gently rotate the culture flask until the digestion solution covers the entire bottom of the culture flask. Aspirate the excess trypsin digestion solution and incubate at 37°C for 3 minutes. Observe under an inverted microscope. After the cells shrink and become round, add 5 mL of complete culture medium to terminate the digestion.

[0045] Step 4: Gently pipette to mix, inoculate T25 culture flasks at a ratio of 1:2, and then add fresh serum-free complete medium to 5 mL. Place in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 4 days before changing the medium;

[0046] Step 5. Add IFN-α-2a (final concentration 15 ng / ml), IL-10 (final concentration 20 ng / ml) and vitamin B6 (final concentration 30 μg / ml) to the cell culture system after the medium is changed. After continuing to culture for 4 days (the medium is changed every two days), the cells reach 80% to 90% confluence and are passaged. After passage, add IFN-α-2a (final concentration 16 ng / ml), IL-10 (final concentration 10 ng / ml) and vitamin B6 (final concentration 25 μg / ml) to the cell culture system and continue to culture for 2 days.

[0047] Step 6: The cells were then placed in a hypoxic incubator for 20 hours, maintaining an oxygen concentration of 3.0% to obtain immunoregulatory stem cells.

[0048] Example 3

[0049] Step 1. Take out the T25 cell culture flask, disinfect the flask with 75% alcohol, remove the sealing film, and place it in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 3 hours to stabilize the cells.

[0050] Step 2: Aspirate the culture medium from the T25 cell culture flask and wash the cells once with PBS;

[0051] Step 3. Add 1 mL of 0.25% trypsin digestion solution to the T25 culture flask. Gently rotate the culture flask until the digestion solution covers the entire bottom of the culture flask. Aspirate the excess trypsin digestion solution and incubate at 37°C for 2 minutes. Observe under an inverted microscope. After the cells shrink and become round, add 5 mL of complete culture medium to terminate the digestion.

[0052] Step 4: Gently pipette to mix, inoculate the cells into T25 culture flasks at a ratio of 1:2, and then add fresh serum-free complete medium to 5 mL. Place the cells in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 3 days before changing the medium.

[0053] Step 5. Add IFN-α-2a (final concentration 16 ng / ml), IL-10 (final concentration 10 ng / ml) and vitamin B6 (final concentration 25 μg / ml) to the cell culture system after the medium is changed. After continuing to culture for 3 days (change the medium after 2 days of culture), the cells reach 80% to 90% confluence and are passaged. After passage, add IFN-α-2a (final concentration 16 ng / ml), IL-10 (final concentration 10 ng / ml) and vitamin B6 (final concentration 25 μg / ml) to the cell culture system and continue to culture for 2 days.

[0054] Step 6: The cells were then cultured in a hypoxic incubator, and IL-6 (final concentration 10 ng / ml) and bFGF (final concentration 30 ng / ml) were added to the culture system. ) The oxygen concentration was maintained at 2.5% and the cells were cultured for 16 hours to obtain immune regulatory stem cells.

[0055] Comparative Example 1

[0056] The operation is as in Example 3, except that:

[0057] Step 5: IFN-α-2a, IL-10, and vitamin B6 were not added;

[0058] Step 6: The oxygen concentration was maintained at a normal 20% without adding IL-6 and bFGF.

[0059] Comparative Example 2

[0060] The operation is as in Example 3, except that:

[0061] Step 5: IFN-α-2a, IL-10, and vitamin B6 were not added;

[0062] Comparative Example 3

[0063] The operation is as in Example 3, except that:

[0064] Step 6: The oxygen concentration was maintained at a normal 20% without adding IL-6 and bFGF.

[0065] Experimental Example 1

[0066] The secretion levels of the cellular anti-inflammatory factors prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were detected. The anti-inflammatory factors detected included IL-10, IDO, and TGF-β.

[0067] The cell culture supernatant was collected for ELISA detection. The cell culture supernatant was detected using ELISA kits for detecting IL-10, IDO, and TGF-β. The results were as follows: Figure 1 As shown, the secretion levels of anti-inflammatory factors of cells prepared in Examples 1 to 3 are significantly higher than those in Comparative Examples 1 to 3; according to the comparison of Comparative Examples 1 to 3, it can be seen that both the addition of the immune factor composition and the hypoxic culture steps have an impact on the secretion of anti-inflammatory factors, and the addition of the immune composition has a greater impact.

[0068] Cell proliferation detection

[0069] Before the start of step 6 of the cultivation in Examples 1 to 3 and Comparative Examples 1 to 3, the same number (1×10 4 / mL) were added to a 96-well plate, 200 μl of cells were added to each well, and 6 replicates were performed for each treatment; the culture conditions of Examples 1 to 3 and Comparative Examples 1 to 3, step 6, were respectively cultured, and samples were taken at 12h, 24h, and 48h of culture to detect cell proliferation; the specific detection method was as follows: 20 μl of MTT (5g / L) was added to each well, placed at 37°C for 4h, and then 150 μl of DMSO was added to each well, shaken for 10min, and the absorbance value (OD value, 490nm) of each well was measured using a microplate reader. The OD value represented the relative number of cells. The results are shown in FIG. Figure 2 As shown, hypoxic culture can significantly promote cell proliferation.

[0070] Experimental Example 2

[0071] Detection of the ability of the immunoregulatory stem cells prepared in Example 3 to induce the generation of regulatory T cells

[0072] The culture medium of the immunomodulatory stem cells prepared in Example 3 was aspirated and washed once with PBS. 1 mL of 0.25% trypsin digestion solution was added to a T25 culture flask. The culture flask was gently rotated until the digestion solution covered the entire bottom of the culture flask. Excess trypsin digestion solution was aspirated and the cells were incubated at 37°C for 2 minutes. Observation was performed under an inverted microscope. After the cells shrank and rounded, 5 mL of complete culture medium was added to terminate digestion and obtain a single-cell suspension.

[0073] The single cell suspension was inoculated into the culture plate, with 10 cells per well. 5 Cells were cultured overnight; then pre-cultured peripheral blood mononuclear cells were added for co-culture, with the ratio of immune regulatory stem cells to peripheral blood mononuclear cells being 1:3. After culturing for 8 days, the cells were harvested for flow cytometry detection of CD4+CD25+FoxP3+ regulatory T cells (Treg). A control group was set up to culture peripheral blood mononuclear cells alone. The flow cytometry results showed that after co-culturing of the immune regulatory stem cells in Example 3 with peripheral blood mononuclear cells, Treg cells were induced to form, with a ratio of 14.55% for Treg cells, while the ratio of Treg cells in peripheral blood mononuclear cells that were not co-cultured was 6.18%.

[0074] As can be seen from the above examples, the preparation method of immunoregulatory stem cells provided by the present invention can modify the cells by adding an immune factor composition during the culture process, thereby increasing the level of cell anti-inflammatory factor secretion, enhancing the cell's immunoregulatory function, and inducing the generation of Treg cells.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing immunoregulatory stem cells, characterized in that: The following steps are involved: 1) Cultivate mesenchymal stem cells for 3-5 days; 2) Then, the medium is changed, and the immune factor composition is added to the cell culture system, and the culture is continued for 3 to 5 days, and passaged 1 to 2 times to obtain modified mesenchymal stem cells; The immune factor composition includes recombinant human interferon IFN-α-2a, IL-10 and vitamin B6; The final concentration of the recombinant human interferon IFN-α-2a in the cell culture system is 10-20 ng / ml; The final concentration of IL-10 in the cell culture system is 5-15 ng / ml; The final concentration of vitamin B6 in the cell culture system is 20-30 μg / ml; 3) The modified mesenchymal stem cells were placed in a hypoxic culture environment with an oxygen concentration of 2% to 3.5%, and the cells were collected.

2. The preparation method according to claim 1, characterized in that Step 3) During the hypoxic culture process, IL-6 and bFGF are added to the culture system.

3. The preparation method according to claim 2, characterized in that The final concentration of the IL-6 in the cell culture system is 5-15 ng / ml; the final concentration of the bFGF in the cell culture system is 20-40 ng / ml.

4. The preparation method according to claim 2, characterized in that The hypoxic culture time is 14 to 20 hours.

5. The preparation method according to any one of claims 1 to 4, characterized in that The culture medium for culturing the mesenchymal stem cells is a serum-free complete culture medium.

6. Immunomodulatory stem cells obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the immunoregulatory stem cells according to claim 6 in the preparation of a medicament for treating atherosclerosis.

Images