Anti-aging mesenchymal stromal cell as well as construction method and application thereof

Abstract

The invention belongs to the technical field of cell biology, and particularly relates to an anti-aging mesenchymal stromal cell as well as a construction method and application thereof. The construction method of the anti-aging mesenchymal stromal cells comprises the following steps: S1, culturing in vitro and carrying out passage on the mesenchymal stromal cells to P5 to P10 generations; s2, placing the mesenchymal stromal cells in a microgravity environment for rotary culture; and S3, digesting the cultured cells to obtain the anti-aging mesenchymal stromal cells. Compared with the prior art of simulating microgravity to explore phenotypes such as morphology, proliferation, immunity and the like of the mesenchymal stromal cells, a new breakthrough is made, and a new strategy and thought are provided for delaying stromal cell senescence, researching mechanisms of space weightlessness related diseases and increasing clinical application of stromal cells.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to an anti-aging mesenchymal matrix cell, its construction method, and its application. Background Technology

[0002] Human mesenchymal stromal cells (hMSCs) possess the capacity for self-renewal and differentiation, playing a crucial role in maintaining tissue and organ integrity and promoting damage repair. However, stromal cells inevitably enter a senescent state during long-term in vitro culture and expansion, and in vivo stromal cell senescence is considered an important marker and driving force of aging. Therefore, effectively delaying or inhibiting the senescence process of hMSCs and maintaining their vitality and function has become a key challenge in improving their clinical therapeutic effects and the industrial application value of stromal cell-based technologies.

[0003] There are few existing methods to combat cellular senescence. Adding chemical molecules may have other side effects; while physical methods offer advantages such as greater safety, reliability, and stability. Existing research shows that MSCs maintain their phenotype and proliferative capacity, and exhibit enhanced immunosuppressive function under conditions such as the International Space Station or simulated microgravity. In disease models such as cerebral infarction and spinal cord injury, MSCs cultured under microgravity conditions can improve their function and significantly enhance therapeutic effects, but research on the effects of simulated microgravity on MSC senescence is lacking.

[0004] Therefore, we look forward to a scheme that applies the microgravity environment to cell therapy to combat cell aging, improve cell vitality, and provide a research tool for the mechanism of aging. Summary of the Invention

[0005] The purpose of this invention is to provide an anti-aging mesenchymal matrix cell, its construction method, and its application, so as to realize the construction of anti-aging cells and improve cell vitality.

[0006] Therefore, the present invention provides the following technical solution.

[0007] The first aspect of this invention provides a method for constructing anti-aging mesenchymal stromal cells, comprising the following steps: S1: Mesenchymal stromal cells were cultured in vitro and passaged to P5 to P10 generations; S2: The above-mentioned mesenchymal matrix cells were placed in a microgravity environment and rotated for culture. S3: Digest the cultured cells to obtain anti-aging mesenchymal matrix cells.

[0008] In a preferred embodiment of the present invention, in step S1, the mesenchymal stromal cells include umbilical cord mesenchymal stromal cells, bone marrow mesenchymal stromal cells, adipose mesenchymal stromal cells, dental pulp mesenchymal stromal cells, placental mesenchymal stromal cells, amniotic membrane mesenchymal stromal cells, synovial mesenchymal stromal cells, and thymic mesenchymal stromal cells.

[0009] In a preferred embodiment of the present invention, the mesenchymal stromal cells are human umbilical cord mesenchymal stromal cells.

[0010] In a preferred embodiment of the present invention, step S2 includes: (i) The obtained P5 to P10 generation mesenchymal stromal cells were seeded into culture flasks containing complete culture medium; (ii) Place the above culture flasks in a three-dimensional rotary instrument for incubation.

[0011] In a preferred embodiment of the present invention, in step (i), the complete culture medium consists of 10-15% fetal bovine serum, 80-100 U / mL penicillin and 80-100 g / mL streptomycin added to each 1L of low-glucose MEM culture medium.

[0012] In a preferred embodiment of the present invention, in step (i), the inoculum size for the 2D plane adherent culture of mesenchymal matrix cells is 10,000 cells / mL; The inoculation amount of the mesenchymal matrix cell 3D culture pellets is 8000-10000 pellets / pill.

[0013] In a preferred embodiment of the present invention, in step (ii), the parameters of the three-dimensional gyroscope are set as follows: the forward rotation speed is 60-80 rpm, the reverse rotation speed is 60-80 rpm, and the frequency is 2800-3000Hz.

[0014] In a preferred embodiment of the present invention, in step (ii), the culture conditions are: a temperature of 37°C, a time of 70-72 h, and 5% CO2.

[0015] A second aspect of the present invention provides anti-aging mesenchymal matrix cells constructed according to the construction method described above.

[0016] A third aspect of the present invention provides the use of anti-aging mesenchymal stromal cells constructed according to the construction method described above in the preparation of a medicament for treating osteoporosis.

[0017] By employing the above technical solution, the present invention has at least the following advantages: This invention provides a method for delaying the senescence of human mesenchymal stromal cells (MSCs) through physical means. This method involves culturing human MSCs in a simulated microgravity environment within a three-dimensional rotary apparatus to slow their senescence. Results show that, compared to human MSCs cultured using traditional 2D and 3D methods under the influence of Earth's natural gravity, the microgravity environment of this invention significantly downregulates the gene and protein expression levels of classic cellular senescence markers p16, p21, and p53. This invention represents a significant breakthrough compared to previous methods for simulating microgravity to explore the morphology, proliferation, and immune phenotypes of MSCs, providing new strategies and ideas for delaying MSC senescence, studying the mechanisms of space weightlessness-related diseases, and expanding the clinical applications of MSCs.

[0018] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the structure of the three-dimensional rotary instrument of the present invention.

[0020] Figure 2 Flow cytometry analysis of human umbilical cord mesenchymal matrix cells.

[0021] Figure 3 This image shows the ability of human umbilical cord mesenchymal stromal cells to differentiate into adipocytes, osteoblasts, and chondrocytes.

[0022] Figure 4 The results of detecting aging indicators of 2D planar adherent hMSCs after static culture under natural gravity and culture in a simulated microgravity environment for 72 h are shown. Among them, (A) is the Western Blot result; (B) is the semi-quantitative Western Blot result.

[0023] Figure 5 The results of qPCR detection of 2D planar adherent hMSCs after static culture under natural gravity and culture in a simulated microgravity environment for 72 h are shown. Among them, (A) is the relative expression level of p16 mRNA; (B) is the relative expression level of p21 mRNA; and (C) is the relative expression level of p53 mRNA.

[0024] Figure 6 The results show the detection of senescence indicators of 3D hMSCs cell spheres after static culture under natural gravity and culture in a simulated microgravity environment for 72 hours; (A) is the Western Blot result; (B) is the semi-quantitative Western Blot result.

[0025] Figure 7The results of qPCR detection of 3D hMSCs cell spheres after static culture under natural gravity and culture under simulated microgravity environment for 72 h are shown. Among them, (A) is the relative expression level of p16 mRNA; (B) is the relative expression level of p21 mRNA; and (C) is the relative expression level of p53 mRNA. Detailed Implementation

[0026] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] The following embodiments involve and mention: 1. Western blot analysis of protein expression levels of cellular senescence markers p16, p21, and p53. Proteins in the test cells were lysed using RIPA lysis buffer (catalog number P0013B, Beyotime) containing a protease inhibitor, and protein concentrations were determined using a BCA protein assay kit (catalog number P0009, Beyotime). Proteins were separated by 12.5% ​​sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The resulting membrane was blocked in 5% skim milk for 1.5 hours, then incubated with primary antibody overnight at 4°C, followed by elution. After incubation with secondary antibody for 1 hour, the protein bands were finally visualized using an enhanced chemiluminescence assay kit (catalog number E423-01, Vazyme).

[0028] 2. qPCR detection of gene expression levels of cellular senescence markers p16, p21, and p53. DNA from the p16, p21, and p53 genes in cells was extracted using a DNA extraction kit (catalog number DM101-01, Vazyme). Then, qPCR primers were designed with p16, p21, p5, and GAPDH (internal reference) as target genes (as shown in Table 1) and qPCR amplification reactions were performed.

[0029] The qPCR amplification reaction system is 20 μL, including: 2 μL cDNA template, 10 μL SYBR GreenMaster fluorescent dye, 1 μL each of upstream and downstream primers (10 μM) for each gene, and the remainder ddH2O.

[0030] The amplification reaction conditions were: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 15 s, 60℃ annealing for 1 min, 40 cycles, and melting curve analysis at 60~95℃.

[0031] The nucleotide sequences of the amplification primers used are shown in Table 1 below: Table 1 Primer Sequences

[0032] 3. Three-dimensional rotary instrument The three-dimensional gyroscope used in this embodiment of the invention is a self-assembly gyroscope, and its structural diagram is shown below. Figure 1 .like Figure 1 As shown, the three-dimensional gyroscope consists of a gyroscope base 2, a gyroscope culture chamber 1, a gyroscope culture chamber outer frame 3, a gyroscope motor 4, and gyroscope bearings and a circuit box 5, all mounted on the gyroscope base 2. The gyroscope culture chamber 1 is surrounded by the gyroscope culture chamber outer frame 3.

[0033] When the three-dimensional gyroscope of this invention is in operation, the sample is first placed in the culture chamber 1 of the three-dimensional gyroscope, fixed and balanced, and then the forward and reverse rotation parameters are set, for example, to 60 rpm / min respectively. After that, the device is started, thereby realizing the rotational culture of the sample. The three-dimensional gyroscope of this invention changes the orientation of the cell culture container in the gravitational field by continuously and randomly rotating at multiple angles, thereby making the direction of gravity felt by the cells change rapidly and uniformly, averaging close to "isotropy", that is, simulating the state of "microgravity" or "weightlessness".

[0034] Example 1: Isolation and expansion of human umbilical cord mesenchymal stromal cells Human umbilical cords were collected under aseptic conditions. Blood was cleaned from the cord, and the umbilical cord veins and vessels were removed. The cords were then cut into small pieces, adhered to a culture vessel, digested, centrifuged, resuspended, and inoculated until 80-90% confluence. The cells were then digested, passaged, and expanded to the 5th generation. The specific steps are as follows: 1) Select pregnant women who meet the health requirements for biological sample donors to collect samples, ensuring the safety of biological samples and excluding HIV, HBV, HCV, CMV, HIV-1 & 2, EBV and other potentially detectable viruses to avoid the risk of spreading infectious diseases.

[0035] 2) Umbilical cord source: With informed consent from the mother, the experimental protocol was approved by the Ethics Committee for Stromal Cell Research at Gulou Hospital. The collection requirements met the following two points: ① Healthy vaginally delivered infants or infants delivered by cesarean section with an Apgar score of 8-10; ② Under aseptic conditions, the umbilical cord, approximately 10-20 cm in length, was collected from the proximal end near the fetus. 2 cm was cut off from each end, and the umbilical cord was immersed in DPBS containing 2% penicillin and streptomycin, transported in a medical incubator, and processed within 4 hours.

[0036] 3) Staff members enter the facility and operate within the biosafety cabinet throughout the entire process.

[0037] 4) Cut the umbilical cord into small segments of about 2 cm and wash with DPBS containing 2% penicillin and streptomycin until no blood is present.

[0038] 5) Remove the three blood vessels from the umbilical cord: two arteries and one vein. After removing the blood vessels, cut the umbilical cord into pieces approximately 1 mm in size using ophthalmic scissors. 3 Small tissue blocks.

[0039] 6) After the tissue is minced, it is directly applied to a T 75 culture flask, inverted for 4 h, then the culture flask is upright, 10 mL of human mesenchymal stem cell complete culture medium is added, and the flask is placed in an incubator for culture. The composition of the human mesenchymal stem cell complete culture medium is: 1 L of low glucose MEM medium (product number 10567014, Gibco) with 10% fetal bovine serum (product number 10099141C, Gibco), 100 U / mL penicillin and 100 g / mL streptomycin (product number 15140122, penicillin and streptomycin, Gibco).

[0040] 7) After about 14 days, the cells will crawl out and form colonies (CFU-F). Gently tap the culture flask to detach the tissue blocks and discard them.

[0041] 8) Gently wash the cell surface with PBS, replace with 10 mL of fresh human mesenchymal matrix complete culture medium, place in an incubator and continue culturing. Passage the cells when they reach 40%-50% confluence.

[0042] 9) Discard the old culture medium, wash once with room temperature DPBS, and remove the DPBS. Add an appropriate amount of Tryple and incubate at 37°C for 3 min for digestion.

[0043] 10) Gently pipette the cells to collect the cell suspension into a centrifuge tube. Centrifuge at 1200 r / min for 5 min at room temperature and discard the supernatant. Resuspend the cells in human mesenchymal stem cell culture medium, gently pipette to mix evenly, and seed at a density of approximately 1.5 × 10⁻⁶ cells / mL. 4 / cm 2 .

[0044] 11) Place in a 37 ℃, 5% CO2 incubator and incubate statically until 80-90% confluence.

[0045] 12) Then, following steps 9)-11) above, the cells are digested and passaged again to obtain human umbilical cord mesenchymal stromal cells.

[0046] The purity and homogeneity of the obtained human umbilical cord mesenchymal matrix cells were detected by flow cytometry. The results are shown in [Figure number missing]. Figure 2 .like Figure 2 As shown, the purity and uniformity of the human umbilical cord mesenchymal matrix cells in this embodiment passed the test (referencing GMP cell bank construction standards); among them, the proportion of CD90, CD73, and CD105 positive cells was >95%, and the proportion of CD45, CD34, CD19, CD14, and HLA-DR positive cells was <2%, indicating that the prepared human umbilical cord mesenchymal matrix cells had good purity and uniformity.

[0047] Differentiation induction assays were performed to assess the adipogenic, osteogenic, and chondrogenic differentiation capabilities of cells, specifically including the following steps: (1) Adipogenic differentiation induction: The prepared P5 generation human umbilical cord mesenchymal stromal cells were seeded into 6-well plates, 100,000 cells per well, and 2 mL of complete human mesenchymal stromal cell culture medium was added to each well. The plates were then incubated statically at 37 ℃ and 5% CO2 until 80-90% confluence. After that, the complete culture medium was replaced with OriCell. ® The adipogenic differentiation medium in the human-related stem cell adipogenic differentiation kit was continuously replaced every 3 days until 21 days of culture. Finally, the cells were washed, fixed with 4% paraformaldehyde, and identified by Oil Red O staining.

[0048] (2) Osteogenic differentiation induction: The prepared P5 generation human umbilical cord mesenchymal stromal cells were seeded into 6-well plates, 100,000 cells per well, and 2 mL of complete human mesenchymal stromal cell culture medium was added to each well. The plates were then incubated statically at 37 ℃ in a 5% CO2 incubator until 80-90% confluence. The culture medium was then replaced with OriCell culture medium. ® The osteogenic induction medium in the human-related stem cell osteogenic induction differentiation kit was continuously replaced every 3 days until 21 days of culture. Finally, the cells were washed, fixed with 4% paraformaldehyde, and identified by alizarin red staining.

[0049] (3) Chondrogenic differentiation induction: The prepared P5 generation human umbilical cord mesenchymal stromal cells were seeded into 1.5 mL spiral-topped Eppendorf tubes, 200,000 cells per tube. The tubes were centrifuged at 3000 rpm for 5 min, and 500 μL of complete human mesenchymal stromal cell culture medium was added to each tube. The tubes were then incubated at 37 ℃ in a 5% CO2 incubator for 24 h until the cells formed small spherical shapes. Afterwards, the complete culture medium was replaced with OriCell medium. ® The chondrogenic differentiation induction medium in the human-related stem cell chondrogenic differentiation kit was continuously replaced every 3 days, with the bottom of the tube gently agitated daily to suspend the cell pellets in the culture medium until 21 days of culture. Finally, the cells were washed, fixed in 4% paraformaldehyde, embedded using OCT, frozen sectioned, and identified by alcine blue staining.

[0050] The results of the cell differentiation capacity assays for adipogenesis, osteoogenesis, and chondrogenesis are shown in the figure. Figure 3 .like Figure 3 The results showed that the human umbilical cord mesenchymal stromal cells prepared in this embodiment had trilineal differentiation ability and met the standard for human umbilical cord mesenchymal stromal cells.

[0051] Example 2: A comparative study on the aging of 2D planar adherent human mesenchymal stem cells cultured under natural gravity and simulated microgravity environments. This embodiment investigated the senescence of 2D planar adherent human mesenchymal stem cells after static culture under natural gravity and culture in a simulated microgravity environment. The specific experimental steps are as follows: Eight flasks of T25 adherent human umbilical cord mesenchymal stromal cells (P5 generation human umbilical cord mesenchymal stromal cells prepared in Example 1) were collected, with an initial seeding density of 500,000 cells per flask. The cells were divided into two groups: a 2D microgravity group and a 2D control group, with four flasks in each group. The cells in each group were then treated as follows: 2D Microgravity Group: Four bottles of T25 adherent human umbilical cord mesenchymal stem cells were placed in a three-dimensional rotary apparatus to simulate a microgravity environment for rotational culture. Before culture, the four bottles of T25 adherent human mesenchymal stem cells were filled with complete human mesenchymal stem cell culture medium and balanced. During culture, the rotation speed of the three-dimensional rotary apparatus was set to 80 rpm for both forward and reverse rotation, with a frequency of 3000 Hz. The three-dimensional rotary apparatus was then placed in a cell culture incubator at 37℃ and 5% CO2 for rotational culture. The complete human mesenchymal stem cell culture medium consisted of 1L of low-glucose MEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 g / mL streptomycin. 2D control group: Four bottles of T25 adherent human mesenchymal stem cells were placed in a cell culture incubator at 37℃ and 5% CO2 for static culture. Before culture, the four bottles of T25 adherent human mesenchymal stem cells were filled with complete human mesenchymal stem cell culture medium. The composition of the complete human mesenchymal stem cell culture medium was: 1L of low glucose MEM medium with 10% fetal bovine serum, 100 U / mL penicillin and 100 g / mL streptomycin added. After culturing the cells for 72 hours as described above, the cells from each group were digested, and the cell pellets were harvested. Two vials from each group were used for Western blotting to detect the protein expression levels of cell senescence markers. The results are shown below. Figure 4 The remaining two vials from each group were used for qPCR detection of gene expression levels of cell senescence markers; results are shown below. Figure 5 .

[0052] like Figure 4As shown, compared with the 2D control group, the expression level of p16 (a classic marker of cell senescence) in the 2D planar adherent human mesenchymal matrix cells of the 2D microgravity group was significantly reduced under simulated microgravity environment. The average expression level of the loss-of-cell group was 0.5859 times that of the control group. p =0.0023), p21 (the average expression level of the loss group was 0.5780 times that of the control group), p =0.0063) and p53 (the average expression level of the loss group was 0.4391 times that of the control group, p Protein expression level (=0.0020).

[0053] like Figure 5 As shown, compared with the 2D control group, the expression level of p16 (a classic marker of cell senescence) in the 2D planar adherent human mesenchymal matrix cells of the 2D microgravity group was significantly reduced under simulated microgravity environment. The average expression level of the loss-of-cell group was 0.0172 times that of the control group. p <0.0001), p21 (the average expression level of the loss group was 0.4407 times that of the control group), p =0.0069) and p53 (the mean expression level of the loss group was 0.3154 times that of the control group, p mRNA expression levels <0.0001).

[0054] The results were consistent when the experiment was repeated three times.

[0055] Example 3: Comparative Study of Senescence in 3D hMSCs Cell Spheroids Cultured under Static Gravity and Simulated Microgravity Environment This embodiment investigated the aging of 3D hMSCs cell spheroids after static culture under natural gravity and culture in a simulated microgravity environment. The specific experimental steps are as follows: In a 96-well U-shaped plate with ultra-low adhesion, 10,000 human mesenchymal stem cell suspensions of generation P5 prepared in Example 1 were seeded into each well. Then, 200 μL of complete human mesenchymal stem cell culture medium was added to each well. After 48 hours of culture, the cells fused and shrank into small spheres. The resulting cell spheres were collected into two T25 cell culture flasks, with 1 million cell spheres seeded into each flask. The experiment was divided into two groups: a 3D microgravity group and a 3D control group, with one flask in each group. The cells in each group were then treated as follows: 3D Microgravity Group: One flask of cells was placed in a three-dimensional rotary apparatus to simulate a microgravity environment for rotational culture. Before culture, the T25 cell culture flask was filled with complete human mesenchymal stem cell culture medium and balanced. During culture, the rotation speed of the three-dimensional rotary apparatus was set to 80 rpm for both forward and reverse rotation, with a frequency of 3000 Hz. The three-dimensional rotary apparatus was then placed in a cell culture incubator at 37℃ and 5% CO2 for rotational culture. The complete human mesenchymal stem cell culture medium used consisted of 1L of low-glucose MEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 g / mL streptomycin. 3D control group: One T25 cell culture flask was placed in a cell culture incubator at 37℃ and 5% CO2 for static culture. Before culture, the first four flasks of T25 adherent human mesenchymal stem cells were filled with complete human mesenchymal stem cell culture medium. The complete human mesenchymal stem cell culture medium consisted of 1L of low-glucose MEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 g / mL streptomycin. After culturing for 72 hours using the above method, cell pellets were harvested. Total protein was extracted from the ground cell pellets and analyzed by Western blotting. Results are shown below. Figure 6 Total RNA was extracted and used for qPCR detection; results are shown below. Figure 7 .

[0056] like Figure 6 As shown, compared with the 3D control group, the expression level of p16 (a classic marker of cell senescence) in the 3D microgravity group was significantly reduced in simulated microgravity environment cultured in the 3D microgravity group. The average expression level of the loss-of-cell group was 0.6867 times that of the control group. p =0.0004), p21 (the average expression level of the loss group was 0.6166 times that of the control group, p =0.0003) and p53 (the average expression level of the loss group was 0.6151 times that of the control group, p The protein expression level was 0.0003.

[0057] like Figure 7 As shown, compared with the 3D control group, the expression level of p16 (a classic marker of cell senescence) in the 3D microgravity group was significantly reduced in simulated microgravity environment cultured in the 3D microgravity group, with the average expression level of the loss group being 0.3284 times that of the control group. p <0.0001), p21 (the average expression level of the loss group was 0.3714 times that of the control group), p <0.0001) and p53 (the average expression level of the loss group was 0.0446 times that of the control group, p Gene expression levels <0.0001).

[0058] The results were consistent when the experiment was repeated three times.

[0059] The results of Examples 2 and 3 above show that, compared with human mesenchymal matrix cells cultured under natural gravity on Earth, both 2D planar adherent and 3D cell spheroid-like human mesenchymal matrix cells exhibited a decrease in aging indicators at the gene and protein levels under simulated microgravity conditions, demonstrating significant anti-aging effects; and the results obtained from the three repeated experiments were consistent, indicating that the culture method of the present invention is reproducible.

[0060] Example 4: Application of anti-aging cells in the treatment of osteoporosis In this embodiment, an osteoporosis model animal was constructed, and different cells were used for treatment to evaluate the therapeutic effect of each cell on osteoporosis mice. The specific steps included: 1. Experimental animals ICR mice, 3 months old, male, weighing 25±2g, clean grade.

[0061] 2. Grouping and Treatment 2.1 Grouping Groups A, B, C, and D, with 8 mice in each group.

[0062] 2.2 Processing Group A: Normal group, intraperitoneal injection of an equal volume of normal saline (300μL), once every 2 weeks, for a total of 2 injections; Group B: Osteoporosis group. First, D-galactose solution (150 mg / kg) was injected intraperitoneally three times a week for 12 weeks; then, normal saline (300 μL) was injected via the tail vein once every two weeks for a total of two injections. Group C: Umbilical cord MSC group, firstly, D-galactose solution (150mg / kg) was injected intraperitoneally 3 times a week for 12 weeks; then normal umbilical cord MSCs (cells obtained from the 2D control group in Example 2, 1 million / time, 300μL) were injected into the tail vein once every 2 weeks for a total of 2 injections; Group D: Umbilical cord MSC anti-aging group. First, D-galactose solution (150mg / kg) was injected intraperitoneally three times a week for 12 weeks. Then, microgravity-treated umbilical cord MSCs (cells obtained from the 2D microgravity group culture in the example, 1 million / time, 300μL) were injected into the tail vein once every two weeks for a total of 2 injections.

[0063] The changes in bone mineral density (BMD), trabecular number (Tb.N), and trabecular separation (Tb.Sp) of each group of mice were then statistically analyzed, and the results are shown in Table 2.

[0064] Table 2. Changes in BMD, Tb.N, and Tb.Sp levels in mice.

[0065] As shown in Table 2, compared with group A, mice in group B showed significantly lower BMD and Tb.N levels and significantly higher Tb.Sp levels, indicating successful establishment of the senile osteoporosis mouse model and severe bone loss. Compared with group B, mice in group C showed significantly higher BMD and Tb.N levels and significantly lower Tb.Sp levels, indicating that umbilical cord MSCs have a significant therapeutic effect on senile osteoporosis. Group D: The umbilical cord MSCs treated with microgravity showed better performance, with significantly improved BMD and Tb.N levels, indicating that microgravity rotation promotes the treatment of osteoporosis with MSCs. These results indicate that MSCs treated with microgravity rotation according to this invention (group D) have anti-MSC aging effects and better osteoporosis improvement effects compared with the normal MSC group (group C).

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for constructing anti-aging mesenchymal stromal cells, characterized in that, Includes the following steps: S1: Mesenchymal stromal cells were cultured in vitro and passaged to P5 to P10 generations; S2: The above-mentioned mesenchymal matrix cells were placed in a microgravity environment and rotated for culture. S3: Digest the cultured cells to obtain anti-aging mesenchymal matrix cells.

2. The construction method according to claim 1, characterized in that, In step S1, the mesenchymal stromal cells include umbilical cord mesenchymal stromal cells, bone marrow mesenchymal stromal cells, adipose mesenchymal stromal cells, dental pulp mesenchymal stromal cells, placental mesenchymal stromal cells, amniotic membrane mesenchymal stromal cells, synovial mesenchymal stromal cells, and thymic mesenchymal stromal cells.

3. The construction method according to claim 2, characterized in that, The mesenchymal stromal cells are human umbilical cord mesenchymal stromal cells.

4. The construction method according to claim 1, characterized in that, Step S2 includes: (i) The obtained P5 to P10 generation mesenchymal stromal cells were seeded into culture flasks containing complete culture medium; (ii) Place the above culture flasks in a three-dimensional rotary instrument for incubation.

5. The construction method according to claim 4, characterized in that, In step (i), the complete culture medium consists of 10-15% fetal bovine serum, 80-100 U / mL penicillin, and 80-100 g / mL streptomycin added to each 1L of low-glucose MEM culture medium.

6. The construction method according to claim 4, characterized in that, In step (i), the inoculum size for the 2D plane adherent culture of mesenchymal matrix cells is 10,000 cells / mL; The inoculation amount of the mesenchymal matrix cell 3D culture pellets is 8000-10000 pellets / pill.

7. The construction method according to claim 4, characterized in that, In step (ii), the parameters of the three-dimensional gyroscope are set as follows: the forward rotation speed is 60-80 rpm, the reverse rotation speed is 60-80 rpm, and the frequency is 2800-3000Hz.

8. The construction method according to claim 4, characterized in that, In step (ii), the culture conditions are: temperature of 37°C, time of 70-72h, and 5% CO2.

9. Anti-aging mesenchymal matrix cells constructed according to any one of claims 1-8.

10. The use of the anti-aging mesenchymal stromal cells constructed according to any one of claims 1-8 in the preparation of a drug for treating osteoporosis.

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