Preparation method and application of hypertonic stimulated bone marrow mesenchymal stem cells

Abstract

The invention discloses a preparation method of hypertonic stimulated bone marrow mesenchymal stem cells, and particularly relates to the technical field of stem cell culture. The method comprises the following steps: S1, adding a sodium chloride solution into a DMEM / f-12 complete medium to prepare a hypertonic medium system; s2, selecting third-generation bone marrow mesenchymal stem cells, and replacing the hypertonic culture medium when the cells are fused to 70-80%; s3, after the culture medium is replaced, culturing in a constant-temperature cell culture box to obtain the bone marrow mesenchymal stem cells subjected to hypertonic stimulation treatment. The method can effectively improve the cell activity and multiplication capacity of the bone marrow mesenchymal stem cells, delay cell senescence and enhance the anti-active oxygen and anti-hypertonic capacity, thereby improving the treatment effect on the radiation-induced lung injury.

Description

Technical Field

[0001] The present invention relates to the technical field of bone marrow mesenchymal stem cell culture, and in particular to a preparation method and application thereof for improving the biological efficacy of bone marrow mesenchymal stem cells under hypertonic stimulation, aiming to improve their survival rate after transplantation, thereby improving their therapeutic effect on radiation lung injury. Background Art

[0002] With the development of science and technology, nuclear energy and nuclear technology have been widely used in disease treatment. While bringing convenience to our lives, there are also many problems. In the radiotherapy of chest tumors, people will suffer from a variety of acute and chronic radiation sickness after being irradiated. Among them, radiation lung injury has attracted widespread attention due to its high incidence and high mortality rate.

[0003] At present, the clinical treatment for this type of injury is still mainly based on drugs, which achieve a certain effect by reducing inflammation and removing oxidative free radicals. However, because the mechanism of injury is relatively complex and easily affected by multiple factors, there are many problems such as drug-related pneumonia, allergies, and poor lung injury repair effects. Therefore, it is very urgent to find an effective treatment method.

[0004] To solve this problem, regenerative medicine provides a potential strategy, that is, based on the characteristics of mesenchymal stem cells (MSCs), such as multidirectional differentiation potential, low immunogenicity, strong regeneration ability, low tumorigenicity, no ethical controversy and easy gene transfection, it is applied to the treatment of radiation-induced lung injury, and relevant reports have confirmed that it has a certain therapeutic effect. However, due to the effect of ionizing radiation, radiation-induced lung injury will produce a large number of free radicals locally and cause ischemia and hypoxia in the microenvironment, causing local inflammation and leading to a high osmotic pressure environment, which seriously affects the homing ability of mesenchymal stem cells and their stay in damaged tissues. The therapeutic effect of radiation-induced lung injury is not obvious, which limits the function of BMSC. In addition, in clinical practice, multiple infusions of bone marrow mesenchymal stem cells are generally required to maintain treatment or enhance the efficacy. However, the in vitro expansion capacity of mesenchymal stem cells is limited. At the same time, the age and health of the donor also affect the proliferation capacity, immunosuppressive potential and survival rate of autologous stem cells, which in turn affects the action time and effect of stem cells. Therefore, finding a way to improve the biological efficacy of bone marrow mesenchymal stem cells has become an urgent problem to be solved.

[0005] Currently, there are many solutions to improve the biological efficacy of bone marrow mesenchymal stem cells, such as genetic modification, hypoxia pretreatment, improved culture medium, etc. However, most of them have problems such as complex operation and high raw material costs. It is very important to find a simple, feasible and low-cost method for optimizing the isolation and culture of mesenchymal stem cells. Summary of the invention

[0006] The purpose of the present invention is to provide a method for improving the biological efficacy of bone marrow mesenchymal stem cells under hypertonic stimulation, which solves the problem in the prior art that the survival rate of bone marrow mesenchymal stem cells in irradiated lung tissue after transplantation in vivo is low, thereby providing better therapeutic effects for applications such as cell transplantation and tissue engineering.

[0007] The first object of the present invention is to provide a method for preparing hypertonic stimulated bone marrow mesenchymal stem cells, comprising the following steps: S1, adding sodium chloride solution to DMEM / f-12 complete culture medium to prepare a hypertonic culture medium system; S2, selecting third-generation bone marrow mesenchymal stem cells, and replacing the hypertonic culture medium when the cell fusion reaches 70-80%; S3, after replacing the culture medium, culturing in a constant temperature cell culture incubator to obtain bone marrow mesenchymal stem cells treated with hypertonic stimulation.

[0008] As a preferred embodiment of the preparation of the hypertonic culture medium of the present invention, the culture medium is a complete culture medium to which a sterile hypertonic sodium chloride solution is added.

[0009] Furthermore, the culture medium is prepared by adding 30-275ul of sterile sodium chloride solution to a total volume of 15ml of complete culture medium, and the final concentration of sodium chloride is 0.16-0.26M.

[0010] Furthermore, the culture medium is prepared by adding 95 ul of sterile sodium chloride solution to a total volume of 15 ml of complete culture medium, and the final concentration of sodium chloride is 0.18M.

[0011] Furthermore, the complete culture medium is DMEM / f-12 culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0012] Furthermore, the sodium chloride concentration of the sterile sodium chloride solution is 5M.

[0013] Furthermore, the bone marrow mesenchymal stem cells are bone marrow mesenchymal stem cells from the femur of C57BL / 6 mice.

[0014] The second purpose of the present invention is to provide the use of the above-mentioned hypertonic stimulated bone marrow mesenchymal stem cells in the treatment of radiation-induced lung injury.

[0015] The radiation lung injury may be caused by an accident or may be caused by a patient receiving radiotherapy.

[0016] Generally speaking, the injury referred to in this article generally refers to a local chest radiation dose of less than 25Gy.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The present invention provides a method for preparing bone marrow mesenchymal stem cells stimulated by hypertonicity and application thereof. The proliferation ability, anti-aging ability and anti-active oxygen ability of stem cells are improved through hypertonic pretreatment, and the effect of repairing and treating radiation lung injury is better.

[0019] The present invention solves the problem that the current bone marrow mesenchymal stem cell pretreatment induction method is complicated and cumbersome. The exogenous substance sodium chloride solution used in the method of the present invention is non-toxic and harmless, easy to obtain, low-cost, does not require the application of special equipment or complex cell engineering technology, is simple to operate, can be promoted in clinical practice, and is conducive to the development of a standardized production platform. It provides a new way for the in vitro proliferation of bone marrow mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The cell morphology of BMSC under a microscope provided in Example 1 of the present invention;

[0021] Figure 2 A bar graph showing cell viability of cells provided in Example 2 of the present invention after being cultured in different osmotic pressure environments for 24-96 hours;

[0022] Figure 3 A shows the staining of cell senescence-specific β-galactosidase in the experimental group and the control group; Figure 3 B shows the schematic diagram of qPCR quantitative analysis of cell senescence-related genes in the experimental group and the control group;

[0023] Figure 4 Shown is the fluorescence flow cytometry detection of cellular ROS in the experimental group and the control group after H2O2 treatment;

[0024] Figure 5 Shown are the viability curves of cells in the experimental and control groups in different extremely hypertonic environments;

[0025] Figure 6 Shown is the H&E staining of mouse lung tissue after irradiation;

[0026] Figure 7 Shown is the Masson staining of mouse lung tissue after irradiation. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below with reference to the embodiments, but is not limited thereto.

[0028] Embodiment 1: Preparation of bone marrow mesenchymal stem cells (BMSCs)

[0029] 1.1 Preparation method: 4-5 week old C57BL / 6 mice were killed by dislocation, immersed in 75% ethanol solution for disinfection for 15 min, and then moved to a clean bench. Under sterile conditions, the bilateral femurs were completely removed and placed in PBS buffer with 10% double antibody. A 1 ml syringe was used to absorb DMEM / f-12 complete culture medium and rinsed repeatedly until the medullary cavity turned white. All the bone marrow was flushed into a culture dish and cultured in an incubator at 37°C and 5% CO2 saturated humidity; the whole solution was replaced once every 2 days until the cells were 80-90% confluent and passaged, and the 2nd-3rd generation cells were used for experiments.

[0030] 1.2 Experimental results: As the culture time increased, the number of floating cells in the culture dish gradually decreased, and the cells gradually changed from round to spindle-shaped. Figure 1 As shown, there were almost no suspended cells in the culture dish, and BMSCs were spindle-shaped, growing well with obvious colony formation.

[0031] Embodiment 2: Effects of different osmotic pressures on the activity of bone marrow mesenchymal stem cells

[0032] 2.1 Main experimental methods

[0033] (1) Prepare a series of hypertonic complete culture medium with a concentration gradient: Purchase sterile 5 mol / L sodium chloride solution as the hypertonic mother solution for this experiment. Dilute the hypertonic group with complete culture medium to make the concentration of sodium chloride in the culture medium 0.16M, 0.18M,

[0034] 0.2M, 0.22M, 0.24M and 0.26M.

[0035] (2) Pre-treat bone marrow mesenchymal stem cells with hypertonic complete medium: Inoculate the cell suspension in a 96-well plate and wait until the cells are fully attached to the wall.

[0036] The original culture medium was aspirated, and 200 μl of complete culture medium and a series of hypertonic complete culture medium with gradient concentrations were added to each well and cultured for 24 h, 48 h, 72 h and 96 h;

[0037] (3) Detection of cell viability using the CCK8 method: After hypertonic treatment for a corresponding period of time, the original culture medium was aspirated, and a mixture of 100 ul of complete culture medium and 10 ul of CCK-8 reaction solution was added to each well. After 2 h of reaction, the absorbance was detected at 450 nm using an ELISA reader.

[0038] 2.2 Main experimental results

[0039] (1) After 24 to 48 h of hypertonic treatment, the significance of cell viability inhibition gradually decreased, indicating that the cells gradually adapted to the hypertonic environment at this concentration.

[0040] (2) Figure 2As shown in the figure, after 72 hours of hypertonic treatment, cell viability gradually increased with the increase of concentration between 0.16-0.18 M; cell viability gradually decreased with the increase of concentration between 0.18-0.26 M; and cell viability increased significantly at 0.18 M (P<0.01). After 96 hours of hypertonic treatment, the significance of promoting cell viability decreased, and the inhibitory effect of higher concentrations became more obvious.

[0041] Embodiment 3: Effects of hypertonic preconditioning on the biological efficacy of bone marrow mesenchymal stem cells

[0042] 3.1 Main experimental methods

[0043] (1) Aging-specific β-galactosidase staining and qPCR were used to detect the expression of aging-related genes P16 and P21 to compare the cellular senescence of BMSCs under isotonic and hypertonic conditions.

[0044] (2) BMSCs pretreated with hypertonic medium (treated with 0.18 M hypertonic medium for 72 h) and BMSCs cultured under isotonic conditions were placed in three types of extremely hypertonic complete medium with sodium chloride concentrations of 0.3 M, 0.45 M, and 0.6 M, respectively, for different time periods.

[0045] (2h, 4h, 6h) After extremely hypertonic treatment, the original culture medium was removed and the activity was detected using the CCK8 method to determine whether the BMSCs after hypertonic treatment were resistant to the extremely hypertonic environment.

[0046] (3) H2O2 was used to simulate the high ROS environment in vitro. The third-generation BMSCs cultured under isotonic and hypertonic conditions were treated with 110uM H2O2 solution, respectively. The fluorescent probe DCFH-DA was used to detect reactive oxygen species, and its fluorescence intensity was detected by flow cytometry to compare the tolerance of BMSCs to high ROS under isotonic and hypertonic conditions.

[0047] 3.2 Experimental Results

[0048] (1) Figure 3 The β-galactosidase staining shown in A showed that the number of senescent cells in the hypertonic group was less than that in the isotonic group. At the same time, hypertonic pretreatment could downregulate the senescence-related genes P16 and P21 ( Figure 3 B), inhibited the senescence of BMSCs.

[0049] (2) Figure 4 As shown in the figure, after BMSCs were treated with hydrogen peroxide for 24 h, the fluorescence intensity of the hypertonic group was significantly lower than that of the control group, and the intracellular ROS level was significantly lower than that of the isotonic group.

[0050] (3) Figure 5As shown in the data, when cells were cultured in an extremely hypertonic environment, the viability of both isotonic and hypertonic BMSCs decreased; however, after appropriate hypertonic (0.18M) pretreatment, the adaptability of BMSCs to the extremely hypertonic environment was improved. In three different extremely hypertonic environments, the viability of the pretreated cells was significantly higher than that of the control group (P<0.01), indicating that hypertonic pretreatment can increase the sensitivity of cells to osmotic pressure, respond quickly to hypertonic stimulation, and reduce cell damage caused by sudden changes in osmotic pressure.

[0051] Embodiment 4: Therapeutic effect of hypertonic pretreatment of bone marrow mesenchymal stem cells on radiation-induced lung injury in mice

[0052] 4.1 Main experimental methods

[0053] (1) Place an anesthetized C57BL / 6 mouse in a mouse box for irradiation and fix its limbs. Only the mouse chest is exposed, and the rest of the mouse is shielded with a lead plate so that the chest is fully irradiated by the gamma-ray source. Perform local irradiation on the mouse chest (irradiation dose of 25 Gy, dose rate of 1 Gy / min).

[0054] (2) About 1 hour after irradiation, the mice were divided into a simple irradiation group, an isotonic BMSCs treatment group, and a hypertonic BMSCs treatment group. 5 Number / mouse injected into C57BL / 6 mice through tail vein. Correspondingly treated BMSCs, and the same number of mice were taken as blank control group.

[0055] (3) After local thoracic irradiation of mice, lung tissues were collected and subjected to H&E and Masson staining 1 week after irradiation to evaluate lung tissue damage and the degree of fibrosis.

[0056] 4.2 Experimental Results

[0057] (1) Figure 6 The H&E staining results shown in the figure show that the lung tissue of mice showed obvious congestion and hemorrhage after irradiation, a large number of red blood cells infiltrated in the lung interstitium, alveolar wall and vascular endothelium were damaged, and more inflammatory cells infiltrated in the alveolar cavity and bronchioles. The treatment of isotonic BMSCs can reduce the inflammatory exudation of damaged lung tissue, reduce the thickening of alveolar wall, and improve the damage of lung tissue. Compared with the isotonic BMSCs group, the hypertonic BMSCs have a more significant therapeutic effect.

[0058] (2) Figure 7 The Masson staining results shown in the figure show that blue collagen fibers began to appear in the lung tissue of mice 1 week after irradiation. Compared with the isotonic BMSCs treatment group, the expression of collagen fiber protein in the hypertonic BMSCs treatment group was significantly decreased.

[0059] It reduces the fibrinoid exudation and has a better therapeutic effect.

[0060] In summary, the hypertonic pretreated bone marrow mesenchymal stem cells of the present invention have better biological efficacy, can better achieve the treatment of radiation-induced lung injury, and can provide new ideas for other clinical applications of bone marrow mesenchymal stem cells.

[0061] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and cannot be understood as limiting the present invention. Any modifications, equivalent substitutions, improvements, etc. that are obvious to those skilled in the art within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing hypertonic stimulated bone marrow mesenchymal stem cells, characterized in that: The method comprises the following steps: S1, adding sodium chloride solution into DMEM / f-12 complete culture medium to prepare a hypertonic culture medium system; S2, selecting third-generation bone marrow mesenchymal stem cells, and replacing the hypertonic culture medium when the cell fusion reaches 70-80%; S3, after replacing the culture medium, culturing in a constant temperature cell culture box to obtain bone marrow mesenchymal stem cells treated with hypertonic stimulation.

2. The method according to claim 1, characterized in that The hypertonic medium is prepared by adding a sterile high-concentration sodium chloride solution into a complete medium to make it a hypertonic medium.

3. The method according to claim 1, characterized in that The complete culture medium is DMEM / f-12 culture solution containing 10% fetal bovine serum and 1% penicillin-streptomycin.

4. The method according to claim 2, characterized in that: The concentration of the sterile high concentration sodium chloride solution is 5M.

5. The method according to claim 1, characterized in that The bone marrow mesenchymal stem cells are bone marrow mesenchymal stem cells from the femur of C57BL / 6 mice.

6. The method according to claim 1, characterized in that The final concentration of sodium chloride in the hypertonic culture medium is 0.16-0.26M.

7. The method according to claim 1, characterized in that The final concentration of sodium chloride in the hypertonic medium is 0.18M.

8. The method according to claim 1, characterized in that The hypertonic stimulation time is 24-96h.

9. The method according to claim 1, characterized in that: The hypertonic stimulation time is 72h.

10. Use of bone marrow mesenchymal stem cells prepared according to the method according to any one of claims 1 to 9 in preparing a drug for treating radiation-induced lung injury.

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