CN116121184B

CN116121184B - Autologous stem cell preparation, preparation method thereof and application thereof in preparation of immunity-improving and organism-repairing composition

Info

Publication number: CN116121184B
Application number: CN202211628204.0A
Authority: CN
Inventors: 宫田秀明
Original assignee: Guangdong Cel Biotechnology Co ltd
Current assignee: Guangdong Cel Biotechnology Co ltd
Priority date: 2022-12-17
Filing date: 2022-12-17
Publication date: 2023-09-26
Anticipated expiration: 2042-12-17
Also published as: CN116121184A

Abstract

The application relates to the technical field of stem cells, in particular to an autologous stem cell preparation, a preparation method thereof and application thereof in preparing a composition for improving immunity and repairing organisms. The stem cell preparation comprises bone marrow stromal stem cells which highly express osteoprotegerin, runx2 and CCR7 genes and do not express CXCL13 genes. The bone marrow stromal cells can promote proliferation of B cells and secretion of IL-10 in vitro, down regulate CD19 after co-culture with the bone marrow stromal cells ⁺ B cell pair CD4 ⁺ The number of T cells and secretion of IFN-gamma have a certain immunosuppressive effect; and after autograft, can alleviate acute rejection after transplanting.

Description

Autologous stem cell preparation, preparation method thereof and application thereof in preparation of immunity-improving and organism-repairing composition

Technical Field

The application relates to the technical field of stem cells, in particular to an autologous stem cell preparation, a preparation method thereof and application thereof in preparing a composition for improving immunity and repairing organisms.

Background

The regenerated autologous stem cells have the functions of regeneration and hair phagocytosis. The autologous stem cells can also phagocytize tumors, proliferate bones and neoplasms by regenerating tissues, nerves, recovering functions of bones, muscle tissues, teeth and the like, and play a role in human body rehabilitation. The autologous bone marrow stromal cells and embryonic mesenchymal stem cells have strong proliferation capacity and multipotency, and the characteristic of massive expansion, and can differentiate into various tissues in a proper in vivo environment.

In addition to having lower immunogenicity (low expression of HLA-I molecules and no expression of HLA-II molecules), bone marrow stromal cells have the effect of regulating immune activity cell responses, and MSC can inhibit proliferation responses of T cells stimulated by mitogens, increase regulatory T cell ratios, promote proliferation and activation of B cells and antibody secretion thereof, and influence chemotactic functions of B cells.

Although there are a number of applications for bone marrow stromal stem cells, they have been less studied for body repair and immune enhancement applications and have not been effectively addressed for the inflammatory rejection of their repair after implantation into the body.

Disclosure of Invention

Accordingly, the present application is directed to a novel bone marrow stromal cells and a method for preparing the same, which solve the above-mentioned problems to a certain extent.

In a first aspect, the embodiment of the application discloses a preparation method of bone marrow stromal cells, comprising the following steps:

obtaining primary bone marrow stromal stem cells;

subculturing the primary bone marrow stromal cells to obtain the 3 rd generation bone marrow stromal cells;

culturing the 3 rd generation bone marrow stromal cells in two dimensions to obtain first cells;

and culturing the first cells in three dimensions to obtain second cells, wherein the second cells are final cells.

In the embodiment of the application, the step of subculturing specifically includes:

separating primary bone marrow stromal cells by digestion with 0.125% pancreatin, inoculating into serum-free culture solution to make initial concentration of BMSCs in culture system not less than 10 ⁵ Individual/mL, 5% CO at 37deg.C ₂ Shake culturing between conditions, changing liquid once every 2-4 days in the culturing process until the adherent cells are fused with each other, spreading the bottom of a culturing bottle, repeating the above operation, and transferring to the 3 rd generation.

In the embodiment of the application, the two-dimensional culture step specifically includes:

applying a two-dimensional film to the culture pore plates, pouring the serum-free liquid culture solution into each pore plate, and simultaneously inoculating the 3 rd generation bone marrow stromal stem cells, so that the cell concentration in each pore is not lower than 10 ⁵ Culture at a concentration of one mLAnd (5) culturing for 7-9 days.

In the embodiment of the application, the two-dimensional film is a regenerated silk fibroin film.

In the embodiment of the application, in the two-dimensional culture, every 2 days of culture, the liquid is completely changed, and simultaneously, after the liquid is changed, the 3 rd generation bone marrow stromal stem cells are inoculated freshly, and the cell concentration in each hole is not lower than 10 ⁵ And each mL.

In an embodiment of the present application, the three-dimensional culturing step includes:

taking the first cells, digesting and separating with 0.125% pancreatin, and regulating the concentration to be not lower than 10 by using the serum-free culture solution ⁵ Inoculating each of the above three-dimensional scaffolds at 37deg.C and 5% CO ₂ Culturing for 2 weeks outside the conditioned medium to obtain the second cells.

In the embodiment of the application, the three-dimensional scaffold is collagen hydrogel.

In a second aspect, the embodiment of the application discloses the bone marrow stromal cells prepared by the preparation method of the first aspect, which highly express the bone protecting protein, runx2 and CCR7 genes and do not express the CXCL13 gene.

In a third aspect, embodiments of the present application disclose an autologous stem cell preparation comprising bone marrow stromal stem cells according to the second aspect.

In a fourth aspect, the embodiment of the application discloses an application of the autologous stem cell preparation in preparation of an immunity-improving and organism-repairing composition.

Compared with the prior art, the application has at least the following beneficial effects:

according to the embodiment of the application, the BMSCs primary cells from SPF grade New Zealand white rabbits are separated, and are subjected to passage, two-dimensional and three-dimensional culture, so that the bone marrow stromal stem cells capable of highly expressing bone protecting proteins, runx2 and CCR7 and not CXCL-13 are obtained. The bone marrow stromal cells can promote proliferation of B cells and secretion of IL-10 in vitro, down regulate CD19 after co-culture with the bone marrow stromal cells ⁺ B cell pair CD4 ⁺ T cell number and IFN-gamma secretion, has certain immunosuppressive effect.

According to the embodiment of the application, the bone marrow stromal stem cells obtained from the in vitro culture of SPF-grade New Zealand white rabbits are transplanted into the body of the bone marrow injury model white rabbits, so that the bone marrow stromal stem cells have a repairing effect on the exercise capacity of the injured white rabbits, and the acute rejection reaction after the transplantation can be relieved.

Drawings

Fig. 1 is a micrograph of primary bone marrow stromal cells provided in an embodiment of the present application.

Fig. 2 is a microscopic view of a 3 rd generation bone marrow stromal cell according to an embodiment of the present application.

Fig. 3 is a first cell micrograph of an embodiment of the present application.

Fig. 4 is a three-dimensional support electron microscope image provided by the embodiment of the application.

Fig. 5 is an electron microscope image of a three-dimensional scaffold and a second cell fusion provided in an embodiment of the present application.

FIG. 6 is a second cell micrograph of an embodiment of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.

Isolation and in vitro expansion of bone marrow stromal stem cells (BMSCs)

1. Experimental methods and materials

1. Cell origin

SPF grade New Zealand white rabbits, 8-9 weeks old, can reach about 1.8kg, purchased from Shanghai dried biological technology Co.

2. Primary rabbit bone marrow stroma stem cell material and isolated culture

Killing New Zealand white rabbits by air embolism, sterilizing, taking out femur on two sides, washing with heparin physiological saline, collecting washing water, centrifuging at 800rpm for 3min, removing supernatant, re-suspending precipitate with LG-DMEM culture solution, and concentrating at 37deg.C and 5% CO ₂ Culturing under the condition, changing liquid 1 time every 3 days, and carrying out passage when the cells grow to the fusion degree of more than 80%. Daily observations on an inverted phase contrast microscope are shown in fig. 1.

3. BMSCs passage and in vitro amplification

(1) Subculture

The primary cultured adherent cells can be digested and separated with 0.125% pancreatin, treated at 37deg.C for 2-5 min, and inoculated into serum-free McCoy's 5A liquid medium (product No. FK-GP00703, fan Ke company) containing 20 μg/mL of Luffa polypeptide (Luffa polypeptide for injection, specification 8mg, heilongjiang Jiang Shi pharmaceutical Co., ltd., national medicine standard H20050950), 50 μg/mL of chitooligosaccharide surfactant, so that the initial concentration of BMSCs in the culture system is not lower than 10 ⁵ Individual/mL, in T75 square flask at 37deg.C, 5% CO ₂ Shake culture between conditions. Wherein, the reference document of the chitooligosaccharide surfactant, the preparation of the chitooligosaccharide surfactant and the surface activity research thereof [ J ]]The functional polymer newspaper is prepared by a method disclosed in 12 months in 2003.

The culture process is divided into 2-4 days, liquid is changed once until the adherent cells are fused with each other, the bottom of the culture bottle is paved, the operation is repeated, 3 generations are transferred, and each generation of culture lasts for 7-9 days, and the following steps are continued. The 3 rd generation cells are shown in FIG. 2.

(2) Two-dimensional culture

Pasting a regenerated silk fibroin film (mask, product number MTY-038, meitian) into a 24-hole culture plate, pouring a serum-free McCoy's 5A liquid culture medium containing 20 mug/mL of deer and melon polypeptide and 50 mug/mL of chitooligosaccharide surfactant into each hole plate, inoculating 3 rd generation cells after passage in the steps, and enabling the cell concentration in each hole to be not lower than 10 ⁵ individual/mL;

every 2 days of culture, completely changing liquid, simultaneously inoculating fresh 3 rd generation cells after passage in the above steps after liquid change, and simultaneously making the cell concentration in each hole not lower than 10 ⁵ After culturing for 7-9 days, the following steps are carried out to obtain the first cells. As shown in fig. 3.

(3) Three-dimensional culture

1) Construction of a three-dimensional matrix

Regulating pH of collagen solution (MDL number: MFCD00130825, sigma) to neutral, adding into low viscosity orifice plate (Corning), placing in 37 deg.C incubator for 2 hr to crosslink collagen hydrogel, and lyophilizing to obtain collagen scaffold. The dissociation method of the collagen scaffold comprises the following steps: the digestion solution was collected by allowing 3% collagenase B (EC number: 3.4.24.3 (BRENDA, IUBMB), roche) to act at 37℃for 5 to 8 minutes, and centrifuged at 400g for 5 minutes, and the supernatant was discarded. As shown in fig. 4.

2) Inoculating culture

Taking the cells (first cells) in the two-dimensional culture, separating cells adhered to silk membrane by digestion with 0.125% pancreatin, and regulating concentration to not lower than 10 with serum-free McCoy's 5A liquid medium containing 20 μg/mL Luffa polypeptide and 50 μg/mL chitooligosaccharide surfactant ⁵ Inoculating each of the above three-dimensional scaffolds at 37deg.C and 5% CO ₂ Culturing for 2 weeks outside the condition culture to obtain the second cell. The materials are obtained in the culture process respectively to determine that the cells are fused with the three-dimensional scaffold. As shown in fig. 5. The second cells obtained from the three-dimensional collagen scaffold using the method described above are shown in FIG. 6.

The present application uses the above-described passaging and in vitro amplification procedures for BMSCs as example 1.

Example 2 was also disclosed, which subjected to the same subculture, two-dimensional culture and three-dimensional culture procedures as in example 1 for BMSCs, wherein the medium used was serum-free McCoy's 5A liquid medium containing 10. Mu.g/mL of deer-melon polypeptide, 10. Mu.g/mL of rapeseed polypeptide and 50. Mu.g/mL of chitooligosaccharide surfactant.

Example 3 was also disclosed, which subjected to the same subculture, two-dimensional culture and three-dimensional culture procedures as in example 1 on BMSCs using a serum-free McCoy's 5A liquid medium containing 20. Mu.g/mL of deer-melon polypeptide, 15. Mu.g/mL of rapeseed polypeptide and 50. Mu.g/mL of chitooligosaccharide surfactant.

Comparative example 1 was also disclosed in which BMSCs were subjected to the same subculture and two-dimensional culture as in example 1, and were not subjected to three-dimensional culture, using the same medium as in example 1.

Comparative example 2, in which BMSCs were subcultured as in example 1, was not subjected to two-dimensional culture and three-dimensional culture, and the culture medium was the same as in example 1.

Comparative example 3, in which BMSCs were subjected to subculture as in example 1 and then directly subjected to three-dimensional culture (two-dimensional culture was not performed), was also disclosed, and the culture medium was the same as in example 1.

Comparative example 4 was also disclosed in which BMSCs were subjected to the same subculture, two-dimensional culture and three-dimensional culture procedures as in example 1 using a serum-free McCoy's 5A liquid medium.

Comparative example 5 was also disclosed in which BMSCs were subjected to the same subculture, two-dimensional culture and three-dimensional culture procedures as in example 1 using a serum-free McCoy's 5A liquid medium containing 50. Mu.g/mL of chitooligosaccharide surfactant.

Comparative example 6 was also disclosed, which performed the same subculture, two-dimensional culture and three-dimensional culture procedures as in example 1 on BMSCs using a serum-free McCoy's 5A liquid medium containing 20. Mu.g/mL of deer-melon polypeptide.

4. Identification of BMSCs

Taking cell suspension 10 ⁶ 1mL PBS was added for resuspension, 10 flow tubes were equally divided, 1 tube was left as a blank, and the remaining 9 tubes were respectively added with 7 surface marker antibodies (anti-CD 34, CD73, CD105, CD45, CD90, CDIIa and HLA-DR and peer controls (IgG 1 and IgG2a, 5. Mu.L), incubated at 4℃for 20min in the absence of light, washed 2 times with PBS, and examined by flow cytometry.

5. RT-PCR detection of mRNA expression

Test article: isolating a suspension of cultured BMSCs cells at a cell concentration of 10 ⁶ About one/mL; the BMSCs cells subjected to passage and in vitro expansion provided in each of examples 1 to 3 and comparative examples 1 to 6 were adjusted to a concentration of 10 ⁶ About one/mL.

Extracting total RNA from BMSCs cells of a test sample according to the specification of TIANCEN company total RNA extraction kit,analyzing the purity and the integrity of the extracted total RNA, and synthesizing cDNA from the total RNA with high purity and good integrity according to a Fermentas one-step method; using the synthesized cDNA as template according to SYBR Premix Ex Taq ^TM II (Tli RNaseH Plus) kit instructions for preparing the liquid and reacting: SYBR PremixEx TaqTaq in 20. Mu.L of reaction System ^TM II 10. Mu.L, upstream and downstream of the primer 0.8. Mu.L, ROX Refer-energy Dye (50X) 0.4. Mu.L, cDNA template 2. Mu.L, ddH, respectively ₂ O6. Mu.L; fluorescent PCR reaction parameters: 95 ℃ for 30min;95 ℃ for 5s; 30s at 60 ℃;40 cycle periods, fluorescence collection and detection are carried out after the 3 rd step of each cycle is finished, and data collection and data conversion are carried out according to the ratio of 2 by using ABI StepOne software ^-ΔΔT The relative quantitative analysis of the osteogenic related gene, CCR7 and CXCL13 mRNA expression was performed.

Wherein the primers were provided by Shanghai, and the sequences are shown in Table 1.

TABLE 1

6. Data statistics

The experimental data are subjected to data analysis by Excel 2013 and SPSS 22.0 statistical software for statistical arrangement, each data is measured for a plurality of times and represented by an average value and a standard deviation thereof, and single-factor analysis of variance (One-way ANOVA) and DunCan's multiple comparison are respectively carried out by SPSS 22.0, and a significant difference mark is carried out.

2. Results

TABLE 2

TABLE 3 Table 3

Tables 2 and 3 show the results of positive expression of the cell surface markers of the primary BMSCs of the examples of the present application and after in vitro passaging and expansion culture, wherein "a" indicates that no positive cells were detected. As can be seen from tables 2 and 3, BMSCs provided in each of examples and comparative examples highly expressed CD73, CD90 and CD105, and were identified as BMSCs cells with or without expressing CD34, CDlla and HLA-DR. However, the positive rates of CD73, CD90 and CD105 for BMSCs provided in examples 1-3 were significantly higher than those of the comparative examples, while CD34, CDIIa and HLA-DR were significantly lower than those of the comparative examples, indicating that the BMSCs provided in the examples of the present application were of higher cell purity.

TABLE 4 relative mRNA expression

Table 4 shows the relative expression levels of the osteogenic related genes, CCR7 and CXCL13 mRNA of primary BMSCs cells after in vitro passaging and expansion culture, where "a" indicates no detection. The results revealed that the expanded BMSCs cells of comparative examples 1 to 6 showed no significant difference in the expression of the osteoblast differentiation-related genes relative to the isolated BMSCs; whereas the mRNA expression of the bone formation related genes of BMSCs provided in examples 1-3 was significantly higher than that of the comparative examples, it was demonstrated that the examples of the present application were able to significantly promote the expression of bone formation related genes of BMSCs by subculture, two-dimensional culture and three-dimensional culture processes.

In addition, CCR7 mRNA was expressed in the BMSCs provided in examples 1-3 and comparative examples 1-3, 6, whereas CCR7 mRNA expression was not detected in the primary BMSCs cells and the cells provided in comparative examples 4, 5. However, the BMSCs cells provided in examples 1-3 did not detect CXCL13 expression, and both the primary BMSCs cells and the cells provided in comparative examples 1-6 were detected. According to comparative analysis, the subculture, the two-dimensional culture and the three-dimensional culture provided by the embodiment of the application can promote the expression of CCR7 of primary BMSCs cells, but not CXCL13. Also, comparative example 1 and comparative examples 1 to 6 found that CCR7 expression occurred after subculturing primary BMSCs cells, while CXCL13 had a decreased expression tendency, thus demonstrating that CCR7 expression could be up-regulated and CXCL13 expression could be down-regulated by subculture, two-dimensional culture and three-dimensional culture of the present application. In addition, the BMSCs provided in comparative example 6 can also promote the expression of BMSCs CCR7 by culturing the serum-free McCoy's 5A liquid medium containing only the deer melon polypeptide, and the culture medium not used in comparative examples 4 and 5 contains no deer melon polypeptide, and the expression of CCR7 is not detected, which indicates that the addition of the deer melon polypeptide to the culture medium provided in the embodiment of the application can promote the expression of BMSCs CCR 7.

Cell experiment

1. Materials and methods

1. Co-culture of BMSCs with B lymphocytes

B lymphocytes were purchased from Feng Hui organism, cat: CL0711. The test cells were BMSCs cells obtained by passaging and expanding only the primary BMSCs described above and examples 1 to 3 and comparative examples 1 to 6. CD19 ⁺ B lymphocytes, which are sorted from the above B lymphocytes. T lymphocytes were purchased from Feng Hui organism, cat: CL0078.CD4 ⁺ T lymphocytes, which are sorted from the above T lymphocytes.

(1) Inoculating into a Transwell 24-well plate containing RPMI-1640 according to the ratio of BMSCs to B=1:5, inoculating BMSCs into an upper chamber, and inoculating B cells into a lower chamber to obtain BMSCs with the number of 1×10 in each well plate ⁵ The number of B lymphocytes per mL was 5X 10 ⁵ The total volume of the culture system per well was 500. Mu.L per mL, and co-cultured for 3d. Negative controls were set without addition of BMSCs cells.

(2) BMSCs: CD19 were prepared in the same manner ⁺ B=1:5 for 3d co-cultivation. Negative controls were set without addition of BMSCs cells.

(3) Cells were harvested for staining and analyzed for CD19 using flow cytometry ⁺ B cell percentage change and its biological function.

(4) B lymphocyte proliferation assay

Co-culturing with MSC at ratio of MSC to B=1:5, adding anti-human IgM 10mg/L and CD40L 1mg/L (stimulation time 96 h) of white rabbit, and setting 3 multiple holes for all cell groups. Collecting B cells after 96h, and respectively detecting proliferation conditions of B cells cultured independently and B cells co-cultured with MSC by flow cytometry

(5) B lymphocyte secretion cytokine detection

Co-culturing with MSC at a ratio of MSC: B=1:5, culturing for 3d by the above co-culturing method, adding PMA (Phorbol 12-Myristate 13-Acetate, BD company) at a final concentration of 50 μg/L, ionomycin (iono-mycine, BD company) at 500 μg/L, and BFA (Bfeeldin A, BD company) at 10mg/L at the last 6h of the culture;

after a further incubation time of 72h, the cells were harvested and incubated with 0.1% BSA+0.05% NaN ₃ Washed once with PBS pH7.4, the supernatant was discarded, and the cell concentration was adjusted to 10 ⁶ Taking 100 mu L of cell suspension in a flow tube, adding a phenotype flow antibody CD 19-FITC, fully mixing, and incubating for 15min at room temperature in a dark place; adding 100 μl of cell fixative (solution A), mixing thoroughly, incubating at room temperature in the dark for 15min, washing with PBS (pH 7.4) containing 0.1% BSA+0.05% NaN3, and discarding the supernatant; adding 100 mu L of cell rupture liquid (solution B) to resuspend cells, adding intracellular cytokine flow antibody IL-10, fully shaking and uniformly mixing, incubating for 30min at 4 ℃ in dark, and then adding 2mL of pH7.4 PBS containing 0.1% BSA+0.05% NaN3 to wash twice to remove redundant antibodies; the supernatant was discarded, and the cells were resuspended with 200. Mu.L of 10g/L paraformaldehyde and flow-detected.

2. Co-cultured CD19 ⁺ B cell pair CD4 ⁺ Effect of T cell proliferation

Taking CFSE tagged CD4 ⁺ T cell (2X 10) ⁵ Cell/well) and CD19 co-cultured with BMSCs cells as provided in the examples or comparative examples of the present application described above ⁺ B, washing with PBS one time, discarding the supernatant, and pressing to CD19 ⁺ B∶CD4 ⁺ T=1:1 in quantity ratio to 96-well plates containing 200. Mu.g/L of anti-CD3mAb, 1mg/L of anti-CD28mAb in RPMI-1640 medium, total volume of culture system 200. Mu.L, after co-culture for 96h, flow-detected CD4 ⁺ T cell proliferation.

3. Co-cultured CD19 ⁺ B cell pair CD4 ⁺ T cell secretion IFN-Influence of gamma

Taking CD4 ⁺ T cells and CD19 co-cultured with BMSCs cells provided in the examples or comparative examples of the present application as described above ⁺ B, washing with PBS one time, discarding the supernatant, and pressing to CD19 ⁺ B∶CD4 ⁺ T=1:1 ratio was co-cultured with MSC for 3d, total volume of culture system was 500 μl, and leukocyte activation cocktail (BD company) was added for the last 6 hours.

The cells were collected using a solution containing 0.1% BSA+0.05% NaN ₃ The cells were washed once with PBS at pH7.4, the supernatant was discarded, and the cell concentration was adjusted to 10 ⁶ 100. Mu.L of the cell suspension was taken in a flow tube and the phenotypic flow antibody CD4 was added ^- FITC is fully and uniformly mixed, and incubated for 15min at room temperature in dark; adding 1 OO. Mu.L of solution A cell fixative, mixing thoroughly, incubating at room temperature in the dark for 15min, and adding a solution containing 0.1% BSA+0.05% NaN ₃ Washed once with PBS pH7.4, and the supernatant was discarded; adding 100 μLs solution of B cell membrane rupture), adding intracellular cytokine flow antibody IFN-gamma-APC, shaking, mixing, incubating at 4deg.C for 30min, and adding 0.1%BSA+0.05%Na N ₃ Washing twice with PBS at pH7.4 to remove excessive antibody; the supernatant was discarded and the cells were resuspended with 200 μl 1% pfa and flow-tested.

4. Data statistics

2. Results

TABLE 5

According to the application, the primary BMSCs, the BMSCs provided by the examples and the comparative examples are respectively co-cultured with B cells, the proliferation results of the B cells are evaluated by a CFSE fluorescent labeling system, the CFSE positive rate in a negative control is only about 26.25%, the BMSCs provided by the examples 1-3 have obvious effect of promoting proliferation of the B cells, and the BMSCs provided by the comparative examples 1-6 have no obvious proliferation promoting effect on the B cells.

The results of flow detection of B cells secreting IL-10 by co-culturing the primary BMSCs, the BMSCs provided by the examples and the comparative examples with the B cells are shown in Table 5, the positive rate of the B cells secreting IL-10 in the negative control is only about 0.62%, the primary BMSCs and the BMSCs provided by the comparative examples 1-6 have a remarkable reducing effect on the IL-10 secretion of the B cells, and the BMSCs provided by the examples 1-3 obviously promote the secretion of the B cells after co-culturing with the B cells. As can be seen from the results of Table 4, both the primary BMSCs and the BMSCs provided in comparative examples 1-6 expressed CXCL13 in small amounts, whereas the BMSCs provided in examples 1-3 did not express CXCL13, thereby possibly causing down-regulation of IL-10 secretion by B cells due to CXCL13.

The application discloses CD19 after co-culture with BMSCs in one step ⁺ Immunosuppression modulating effect of B cells. As can be seen from Table 5, CD4 was detected by flow ⁺ The number of T cells and the positive rate of IFN-gamma secretion indicate that the BMSCs cells and CD19 provided in examples 1-3 of the present application ⁺ After co-culture of B cells, CD19 ⁺ B cells re-associated with CD4 ⁺ T cell co-culture significantly reduced both in number and positive rate of IFN-gamma secretion, whereas primary BMSCs and BMSCs provided in comparative examples 1-6 were compared to CD19, respectively ⁺ After B cell co-culture, there is no obvious inhibition of CD4 ⁺ T cell number and IFN-gamma secretion.

As can be seen from the results of Table 4, the BMSCs provided in the examples of the present application were subjected to respective CCR7 chemokines, and the migration of the chemokines was utilized to migrate from the upper and lower Transwell chambers to effect proliferation of B cells, up-regulate IL-10 secretion of B cells, and down-regulate CD19 co-cultured therewith ⁺ B cell pair CD4 ⁺ T cell number and IFN-gamma secretion, has certain immunosuppressive effect.

Animal experiment

1. Materials and methods

1. Experimental animals: the SPF grade New Zealand white rabbits.

2. Test article

The BMSCs suspensions provided in the primary BMSCs, examples 1 to 3 and comparative examples 1 to 6 were adjusted to have a final cell concentration of 10 by using the culture solution used in the culture of each of the above examples or comparative examples ⁶ Each mL of primary BMSCs was conditioned with LG-DMEM medium and used as a test sample. Wherein, examples 1-3 and comparative examples 1-6 were collected after three-dimensional culture, cells were dissociated from the three-dimensional scaffold using the method provided above.

3. Modeling and grouping experiments

The white rabbit is anesthetized by intraperitoneal injection of 3% sodium pentobarbital, and covered with gauze soaked by physiological saline to make T-shaped white rabbit ₉ ～T ₁₀ Laminectomy was followed by impingement of the exposed spinal cord at a height of 25mm using an Impactor M-III spinal cord Impactor. After the operation, in order to prevent postoperative infection, gentamicin (8 mg/kg) is injected into the muscle of the white rabbit for 1 time every day, and the white rabbit can stop after being observed for 3 days without symptoms of infection such as hematuria and the like. The operation is carried out for the white rabbits for 2 times every day until the spontaneous urination is recovered. This was used as a model white rabbit.

And selecting part of the model white rabbits as a transplanting group. The micro-injector is fixed at the center depth of 1.5mm of the damaged part of the white rabbit by a brain stereotactic instrument at the 0 th day after the operation, and the 10 mu L of the test sample is respectively injected by a micro-injection pump at the speed of 1 mu L/min. After each injection, the needle was left for 2min to prevent extravasation of fluid.

The white rabbits subjected to the above operation without BMSCs were used as a model group.

4. Spinal cord function scoring

And (3) evaluating the functional recovery condition after the spinal cord injury by using a spinal cord injury functional evaluation method, and respectively scoring the white rabbits in a double-blind mode at the 0 th, 5 th and 10 th days of the operation to evaluate the exercise capacity of the hind limbs of the white rabbits.

5、Western Blot

On the 7 th day after three groups of white rabbits are subjected to operation, part of the white rabbits are selected to be euthanized, spinal cord sections of damaged parts of 3 groups of white rabbits are respectively extracted on ice, total protein is extracted by RIPA lysate and protein concentration is measured by BCA method, protein content in each sample is calculated by using a standard curve, the loading amount is adjusted to 40 mug, the protein is transferred to a PVDF membrane after being separated by polyacrylamide gel electrophoresis, the protein is subjected to primary incubation at 4 ℃ for overnight (NGF antibody dilution ratio is 1:750 and BDNF antibody dilution ratio is 1:400), the membrane is washed by TEST for 3 times for 10min, secondary antibodies are incubated for 2h at room temperature (HRP marked goat anti-rabbit IgG and dilution ratio is 1:10000), the membrane is washed by TEST for 3 times, each time for 10min, ECL luminescent agent is finally added for imaging and storing, the grey value of a strip is analyzed by using Lab software of the ECL luminescent agent, the grey value of the strip is indicated by grey value, and the grey value of NGF or BDNF expression is indicated by the grey value of each group grey value as the grey value of the corresponding group of the white rabbit, and the grey value of the group is transplanted as the relative grey value of the group.

6. Flow cytometry detection of T lymphocyte differentiation

(1) Lymph node and spleen were collected

Injecting anesthetic into the abdominal cavity of a white rabbit 3 days after BMSCs cells are implanted for anesthesia, removing abdominal hair, cutting off skin along the midline of the abdomen, passively separating skin and abdominal muscle along the subcutaneous fascia layer to two sides, exposing lymph nodes by using forceps to carry out the fascia layer separation, and taking out the lymph nodes to be placed in RPMI 1640 culture medium; the left upper abdominal muscle was cut, and the spleen was removed and placed in RPMI 1640 medium.

(2) Preparation of cell suspension

Rolling the lymph combined with spleen, filtering with screen, centrifuging filtrate at 1500rpm for 3min, removing supernatant, and preparing into suspension with RPMI 1640 solution, which is lymphocyte suspension. After the spleen is crushed and tissue fluid is centrifuged, supernatant is removed, and then 2mL of erythrocyte lysate diluted 10 times by deionized water is added for 5min, PBS is used for washing for 2 times, centrifugation is carried out at 100rpm for 5min, supernatant is discarded, and RPMI 1640 solution is used for preparing spleen cell suspension.

(3) Treg detection

Mixing 100 μL of lymph node cell suspension with 1.5 μLCD4 antibody, mixing 50 μL of spleen cell suspension with 2 μL of CD25 antibody, incubating at 4deg.C in dark for 15min, adding 700 μL of membrane breaker (eBioscience) respectively, treating at normal temperature for 30min, adding 1mL of washing liquid (eBioscience) respectively, washing for 5min, centrifuging at 1500rpm for 3min, discarding supernatant, and washing repeatedly for 1 time; add 2. Mu.L Foxp3 antibody (Abcam China) respectively, incubate at 4deg.C for 30min, wash 2 times with PBS, discard supernatant, and perform flow cytometry.

(4) Detection of ThI/Th2/Th17

1) Stimulation of T lymphocytes to secrete cytokines: 200. Mu.L of lymph node cell suspension, 500. Mu.L of stimulator (BD Pharmingen, 500. Mu.L/EP tube) and 260. Mu.L of LFBS were mixed, 100. Mu.L of spleen cell suspension, 500. Mu.L of stimulator (BD Pharmingen, 500. Mu.L/EP tube) and 260. Mu.L of LFBS were mixed, the liquid was supplemented to 1300. Mu.L with PEMI 1640 medium and incubated for 5h at 37 ℃;

2) Transferring the sample after stimulation into a flow tube, washing twice with PBS, adding 3 mu LCD3 and 3 mu LCD4 antibody into each tube, mixing, and incubating at 4deg.C in dark place for 15min; standing at normal temperature for 17min; adding 1ml of cleaning solution into each tube, flicking, standing for cleaning for 2 times, centrifuging, discarding the supernatant, adding 700 mu L of membrane breaker into each tube, centrifuging at 1500rpm for 3min after 5min, discarding the supernatant, and repeating centrifuging for 1 time; samples in the flow tubes were equally divided into two tubes, one tube was added with 2.5. Mu. LIFN-y and IL-4 antibody, the other tube was added with 2.5. Mu. LIL-17 antibody, incubated at 4℃for 30min in the absence of light, washed 2 times with PBS, the supernatant was discarded, and the pellet was homogenized for detection by flow cytometry.

7. Data statistics

2. Results

TABLE 6

Table 6 shows the motor scores of the rabbits on the left 3 th row, at the 0 th, 5 th and 10 th days after the transplantation, and at the 0 th day after the operation, the BMS scores of all the rabbits are 0 point, thus proving that the molding is successful. The scores of the white rabbits in each group are increased with the increase of time, but the increase amplitude is different; at 5, examples 1-3, comparative examples 3 and comparative example 6 in the transplanted group had a significant promotion of the white rabbit exercise score relative to the primary BMSCs group, while the other comparative examples did not significantly promote, and at 10d, examples 1-3, comparative examples 1-3 and comparative example 6 in the transplanted group had a significant promotion of the white rabbit exercise score relative to the primary BMSCs group. Further, in the transplanted group, examples 1 to 3 have a remarkable promoting effect on the motor scores of the white rabbits compared with each comparative example, thus demonstrating that the repairing effect on the motor ability of the white rabbits after the BMSCs cells provided in example 3 of the application are transplanted to the spinal cord injured rabbits is most remarkable.

The relative amounts of NGF and BDNF protein expression on day 10 after the transplantation of the white rabbits of each group are shown in the right two columns of table 6. As is clear from Table 6, examples 1 to 3 and comparative examples 1 to 3 have an effect of promoting NGF and BDNF expression in the spinal cord of white rabbits, and examples 1 to 3 have a remarkable effect of promoting. NGF and BDNF are markers of spinal cord nerve function and repair regeneration, thus demonstrating that BMSCs cells provided in examples 1-3 of the present application have an obvious repair function for nerve function of spinal cord injured white rabbits.

TABLE 7

Table 7 shows the ratio of ThI/Th2 and Treg/Th17 in spleen and lymphoid tissues at day 9 after transplantation in each group of white rabbits. As can be seen from table 7, in the transplanted group, the ratio of ThI/Th2 was significantly reduced with respect to the model group, whereas the ratio of Treg/Th17 was significantly increased with respect to the model group, whereas the ratio of ThI/Th2, the ratio of Treg/Th17 of comparative examples 1 to 6 did not completely exhibit such a trend, or partially exhibited such a trend, in both of the lymphoid tissue and spleen tissue. Therefore, after the BMSCs cells provided by the embodiment of the application are implanted into bone marrow damaged white rabbits, T cells in spleen tissues and lymphoid tissues are not differentiated towards Th, but are differentiated towards tregs more, and the T cell balance of the white rabbits implanted with the BMSCs cells is developed towards an anti-inflammatory direction, so that the tendency of acute rejection after transplantation is reduced as the balance of Th1/Th2 becomes a key factor for starting immune rejection, and tregs are cell groups with immunoregulation/immunosuppression functions in CD4+ T cells.

To sum up:

Therefore, the stem cell preparation prepared from the bone marrow stromal cells disclosed by the embodiment of the application has application prospects in the fields of improving immunity, repairing organisms and the like.

The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.

Claims

1. A method for preparing bone marrow stromal cells, comprising the steps of:

obtaining primary bone marrow stromal stem cells;

subculturing the primary bone marrow stromal cells to obtain the 3 rd generation bone marrow stromal cells; the subculture step specifically comprises the following steps: separating primary bone marrow stromal cells by digestion with 0.125% pancreatin, inoculating into serum-free culture solution to make initial concentration of BMSCs in culture system not less than 10 ⁵ Individual/mL, 5% CO at 37deg.C ₂ Shake culturing between conditions, changing liquid once every 2-4 days in the culturing process until adherent cells are fused with each other, spreading the bottom of a culturing bottle, repeating the above operation, and transferring to the 3 rd generation;

culturing the 3 rd generation bone marrow stromal cells in two dimensions, wherein the two-dimensional culturing step specifically comprises the following steps: pasting a regenerated silk fibroin film into culture pore plates, pouring the serum-free liquid culture solution into each pore plate, and simultaneously inoculating the 3 rd generation bone marrow stromal stem cells, so that the cell concentration in each pore is not lower than 10 ⁵ Culturing for 7-9 days at a volume of one mL to obtain a first cell; and

three-dimensionally culturing the first cell, the three-dimensionally culturing comprising: taking the first cells, digesting and separating with 0.125% pancreatin, and regulating the concentration to be not lower than 10 by using the serum-free culture solution ⁵ Inoculating into collagen hydrogel at 37deg.C and 5% CO ₂ Culturing for 2 weeks outside the conditioned medium to obtain the second cells;

wherein, the culture medium of subculture is: serum-free McCoy's 5A liquid medium containing 20 μg/mL deer-melon polypeptide, 50 μg/mL chitooligosaccharide surfactant; or serum-free McCoy's 5A liquid medium containing 10 μg/mL deer-melon polypeptide, 10 μg/mL rapeseed polypeptide and 50 μg/mL chitooligosaccharide surfactant; or serum-free McCoy's 5A liquid medium containing 20 μg/mL deer-melon polypeptide, 15 μg/mL rapeseed polypeptide and 50 μg/mL chitooligosaccharide surfactant;

the culture medium for the two-dimensional culture is as follows: serum-free McCoy's 5A liquid medium containing 20 μg/mL deer-melon polypeptide, 50 μg/mL chitooligosaccharide surfactant; or serum-free McCoy's 5A liquid medium containing 10 μg/mL deer-melon polypeptide, 10 μg/mL rapeseed polypeptide and 50 μg/mL chitooligosaccharide surfactant; or serum-free McCoy's 5A liquid medium containing 20 μg/mL deer-melon polypeptide, 15 μg/mL rapeseed polypeptide and 50 μg/mL chitooligosaccharide surfactant;

the culture medium for three-dimensional culture is as follows: serum-free McCoy's 5A liquid medium containing 20 μg/mL deer-melon polypeptide, 50 μg/mL chitooligosaccharide surfactant; or serum-free McCoy's 5A liquid medium containing 10 μg/mL deer-melon polypeptide, 10 μg/mL rapeseed polypeptide and 50 μg/mL chitooligosaccharide surfactant; or serum-free McCoy's 5A liquid medium containing 20 μg/mL deer-melon polypeptide, 15 μg/mL rapeseed polypeptide and 50 μg/mL chitooligosaccharide surfactant.

2. The method according to claim 1, wherein the two-dimensional culture is performed with a total amount of liquid change every 2 days, and fresh 3 rd generation bone marrow stromal cells are inoculated after liquid change, and the cell concentration in each well is made to be not less than 10 ⁵ And each mL.

3. The bone marrow stromal cells prepared by the method of claim 1, which highly express osteoprotegerin, runx2 and CCR7 genes, and do not express CXCL13 genes.

4. An autologous stem cell preparation comprising the bone marrow stromal stem cells of claim 3.

5. The use of bone marrow stromal cells obtained by said method of claim 1 for the preparation of an immunity enhancing and repair composition.