CN116121178B

CN116121178B - Intestinal stem cells and application thereof in improving intestinal microenvironment and resisting intestinal aging

Info

Publication number: CN116121178B
Application number: CN202211637693.6A
Authority: CN
Inventors: 史辛艺; 宫田秀明
Original assignee: Shenzhen Michael Biotech Co ltd
Current assignee: Shenzhen Taihua Sail Biotechnology Co.,Ltd.
Priority date: 2022-12-18
Filing date: 2022-12-18
Publication date: 2023-09-19
Anticipated expiration: 2042-12-18
Also published as: CN116121178A

Abstract

The application discloses an intestinal stem cell and application thereof in preparation of an intestinal canal microenvironment improving and intestinal canal aging resisting preparation. The intestinal stem cells comprise intestinal stem cells expressing Lgr5 and intestinal stem cells expressing B1, and the P2 generation cells of the intestinal stem cells and the intestinal stem cells are detected by WB detection and mRNA level detection, so that c-Myc-F, sox and MSI1 are respectively expressed. In addition, in vitro and in vivo experiments prove that the stem cells have the effects of maintaining the proliferation and normal steady state of small intestine crypts, improving the intestinal microenvironment to the probiotics direction and improving the intestinal differentiation function defect of mice.

Description

Intestinal stem cells and application thereof in improving intestinal microenvironment and resisting intestinal aging

Technical Field

The application relates to the technical field of intestinal microenvironments, in particular to an intestinal stem cell and application thereof in improving the intestinal microenvironment and resisting intestinal aging.

Background

During the aging process of the body, the self-renewal and multipotency of the stem cells of the intestinal tract are altered. It was found that the respiratory chain defect of the daughter cells of the old small intestine epithelial stem cells resulted in a decrease in proliferation and an increase in apoptosis 7. In addition, the number of epithelial cells of different types in the intestinal tract can be changed in the aging process of the organism, but the changes are different in different species and different intestinal tract parts. Sandstrom et al Dz-isl found that the number of endocrine cells in the duodenum of the elderly and in the large intestine of the aged mice was increased, but the number of endocrine cells in the rectum of the elderly was unchanged.

Current research on microenvironments during intestinal aging is mainly focused on smooth muscle cells, interstitial cells, enteric nerve cells and lymphocytes. Changes in digestive absorption function, immune barrier function, etc. of the intestinal tract during aging are closely related to changes in the micro-environmental cells of the intestinal tract, however, due to the complexity of the intestinal tract, the changes occurring during aging are still to be further studied.

Disclosure of Invention

In view of the above, it is an object of the present application to provide at least one formulation for alleviating or improving the intestinal microenvironment related factors during aging in order to improve a new solution in this field of application.

In a first aspect, the embodiment of the application discloses a subculture method of intestinal stem cells, which comprises the following steps:

separating to obtain an intestinal stem cell expressing the Lgr5 and an intestinal stem cell expressing the Bmi1, wherein the intestinal stem cell expressing the Lgr5 and the intestinal stem cell expressing the Bmi1 are both from twelve epithelial tissues;

and carrying out subculture on the intestinal stem cells expressing the Lgr5 and the intestinal stem cells expressing the Bmi1 in a primary culture solution and a subculture solution respectively, wherein the subculture time is not more than 4 generations, and the subculture time is not more than 48 hours each time.

In the embodiment of the application, the primary culture solution for culturing the intestinal stem cells expressing the Lgr5 is a DMEM/F12 culture medium containing 10-20 mM HEPES, 5-10 mM glutamine, 0.2-2 times N2 cell culture additive, 0.2-2 mg/mL CD Feed002, 80-100U/mL penicillin, 80-100 mg/mL streptomycin, 2-5 mug/mL transferrin, 10-20 mug/mL insulin, 0.10-0.25 mM nonessential amino acid and 0.2-1 mM sodium pyruvate; the subculture solution for subculturing the intestinal stem cells expressing the Lgr5 is a DMEM/F12 medium containing 8-15 mM HEPES, 3-8 mM glutamine, 0.2-1 mg/mL CD Feed002, 80-100U/mL penicillin, 80-100 mg/mL streptomycin, 2-5 mug/mL transferrin, 10-20 mug/mL insulin, 0.10-0.25 mM nonessential amino acid, and 0.2-1 sodium pyruvate.

In the embodiment of the application, the primary culture solution for culturing the intestinal stem cells expressing Bmi1 is a DMEM/F12 culture medium containing 10-20 mM HEPES, 5-10 mM glutamine, 0.5-2 mg/mL CD Feed002, 80-100U/mL penicillin, 80-100 mg/mL streptomycin, 2-5 μg/mL transferrin, 10-20 μg/mL insulin, 0.10-0.25 mM nonessential amino acid and 0.2-1 mM sodium pyruvate; the subculture solution for culturing the intestinal stem cells expressing Bmi1 is a DMEM/F12 culture medium containing 5-10 mM HEPES, 2-6 mM glutamine, 0.1-0.5 mg/mL CD Feed002, 50-80U/mL penicillin, 50-80 mg/mL streptomycin, 2-5 mug/mL transferrin, 10-20 mug/mL insulin, 0.10-0.25 mM nonessential amino acid and 0.2-1 mM sodium pyruvate.

In a second aspect, the embodiment of the application discloses a cell preparation, which comprises the intestinal stem cells expressing Lgr5 and the intestinal stem cells expressing Bmi1 prepared by the subculture method of the first aspect; the intestinal stem cells expressing Lgr5 and the intestinal stem cells expressing Bmi1 both express the c-Myc-F, sox and MSI1 genes at high levels.

In a third aspect, embodiments of the present application disclose a formulation for improving intestinal microenvironment and preventing intestinal aging, comprising an Lgr5 cell formulation and a Bmi1 cell formulation, each separately packaged, the Lgr5 cell formulation comprising not less than 106 stem cells expressing Lgr5 per mL and other pharmaceutically relevant protective agents maintaining cell viability, the Bmi1 cell formulation comprising not less than 106 stem cells expressing Bmi1 per mL and other pharmaceutically relevant protective agents maintaining cell viability;

wherein the Bmi 1-expressing intestinal stem cells and the Lgr 5-expressing intestinal stem cells are obtained by the subculture method of the first aspect; the intestinal stem cells expressing Lgr5 and the intestinal stem cells expressing Bmi1 both express the c-Myc-F, sox and MSI1 genes at high levels.

In the examples of the present application, the ratio of the administration of the Lgr5 cell preparation to the administration of the Bmi1 cell preparation was (2 to 5): 1.

In the embodiment of the application, the related protective agent for maintaining the cell viability in pharmacy comprises 50-80U/mL penicillin, 50-80 mg/mL streptomycin, 0.5-2 mg/mL gentamycin, 0.5-2 mg/mL amphotericin B, 0.1-0.5 mug/mL transferrin, 0.05-0.2 mug/mL insulin, 0.01-0.05 mM nonessential amino acid, 0.01-0.05 mM sodium pyruvate, 20-30 wt% matrigel and 50-60 wt% glycerol.

In an embodiment of the application, the formulation further comprises an independently packaged facilitation formulation comprising 5 to 20ng/mL of mouse SF20 factor, 1 to 5ng/mL of recombinant ErbB3 (ErbB 3-f), and 50 to 60wt% glycerol.

In the examples of the present application, the administration ratio of the Lgr5 cell preparation, the Bmi1 cell preparation and the accelerator preparation was (200 to 500): 100 (1 to 5).

In a fourth aspect, the embodiment of the application also discloses the application of the intestinal stem cells obtained by the subculture method of the first aspect or the cell preparation of the second aspect in preparing an intestinal microenvironment improving and intestinal aging resisting preparation.

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

the application extracts relevant cells from mouse duodenal epithelial tissue, and performs stem cell separation to obtain stem cells expressing Bmi1 and stem cells expressing Lgr5, and through WB detection and mRNA level detection, the P2 generation cells of the two cells are found to respectively express c-Myc-F, sox9 and MSI1, and the genes respectively play an important role in serving as transcription factors for intestinal tract proliferation differentiation and maintaining the steady state and repair capability of the small intestinal stem cells. Thus, it was demonstrated that the isolated Bmi 1-expressing stem cells and Lgr 5-expressing stem cells have the outstanding advantage of maintaining small intestine crypt proliferation and normal homeostasis, as well as their lesion repair ability.

In addition, the stem cells expressing Bmi1 and stem cells expressing Lgr5 obtained by separation are used for preparing a preparation for improving intestinal microenvironment and resisting intestinal aging, and in vivo experiments prove that the preparation has an effect of improving the intestinal microenvironment of old mice and a trend of resisting aging phase change gene expression, and the defect of intestinal differentiation function of the mice is improved, so that the intestinal microenvironment of the mice is improved in a probiotics direction.

Drawings

Fig. 1 shows a mouse duodenal primary stem cell provided by an embodiment of the application.

FIG. 2 is a graph showing the staining of primary Lgr5 cells provided in examples of the present application.

FIG. 3 is a staining chart of primary Bmi1 cells provided in the examples of the present application.

Fig. 4 is a WB detection diagram of primary Lgr5 cells and primary Bmi1 cells provided in the examples of the present application.

FIG. 5 is a WB detection map of P2-generation Lgr5 cells and P2-generation Bmi1 cells according to the present application.

Fig. 6 is a WB detection chart of P2-generation Lgr5 cells according to an embodiment of the present application.

FIG. 7 is a WB detection map of the P2 generation Bmi1 cells according to 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 identification of intestinal stem cells

1. Materials and methods

1. Material source

Balb/c mice, male, weight about 20g, feeding temperature 21+ -2deg.C, relative humidity 30-70%, illumination period 12/12h, purchased from Jiangsu Jiujiakang Biotech Co.

2. Acquisition of mouse duodenal epithelial cells

(1) Euthanizing the mice, taking the duodenal tissue of the mice under a dissecting microscope on a sterile operating table, removing redundant mesenteric tissue, placing the mice in a glass dish containing Hank's buffer solution, and flushing the mice with the buffer solution for a plurality of times until intestinal contents and mucus are removed;

(2) Cutting it into 1mm pieces with sterile scissors ³ Size, it was added to a kit containing 15U/mL type III collagenase (Sigma) and 0.3U/mL neutral protease (Shanghai Jizha Biotechnology Limited)Span), digesting at 80rpm for 1h in Hank's buffer, pipetting digested tissue up and down for 15min, transferring to a conical tube, and adding fetal bovine serum to a concentration of about 5wt% to inhibit collagenase/neutral protease activity;

(3) After the supernatant was removed, the culture flask was resuspended in DMEM/F12 complete medium and cultured to obtain the primary mouse duodenal epithelial cells, as shown in FIG. 1.

(4) After primary separation of cells, the cells were allowed to stand absolute for 2 days, then the cells were observed under an inverted microscope every 2 days, and the cells were changed in time, filtered through a sterile filter of 70. Mu.m, the filtrate was collected, resuspended in Hank's buffer to give a concentration of 10 ⁶ individual/mL of cell suspension as cell suspension to be further isolated.

(5) Fluorescent staining to identify intestinal stem cells:

inoculating the primary separated cells into a cell plate, immersing the primary separated cells in PBS for a plurality of times, fixing a cell slide for 15 to 30 minutes by using 4% paraformaldehyde, and cleaning a slide by using the PBS; discarding PBS, dripping goat serum on the cell slide, and sealing for 30min; discarding the sealing liquid, directly dripping the prepared primary antibody on the cell climbing sheet, and incubating overnight at 4 ℃ or incubating for 2 hours at 37 ℃; washing the cell climbing sheet for 3 times by PBS (phosphate buffer solution) for 3min each time, dripping Dapi, and incubating for 5-8 min in a dark place to dye the cell climbing sheet; washing the cell slide with PBS for 3 times, and washing off redundant Dapi 3min each time; the images were collected by liquid sealing with a sealing sheet containing an anti-fluorescence quencher and then observed under a fluorescence microscope.

3. Isolation and subculturing of intestinal stem cells

(1) Isolation of intestinal stem cells

In the isolation of intestinal stem cells of a specific example 1, specific steps include antibody preparation, incubation of cell antibodies, magnetic bead-antibody binding, and cell isolation and release steps.

(1) Preparation of antibodies:

a first tube: DSB-X reagent (Invitrogen) ^TM DSB-X ^TM Biotin protein marked kit product number D20655), adding 40 μl DMSO for sufficient dissolution;

a second tube: mu.L of Lgr5 antibody (mouse monoclonal antibody [ OTI2A2 ]]Ab273092, abcam China) and Bmi1 antibodies (mouse monoclonal antibody [ BMI1/2823 ]]Ab269678, abcam chinese) were added to different centrifuge tubes, respectively, and 0.2mL of PBS and 20 μl of heavy NaHCO were added to the centrifuge tubes, respectively, at the same time ₃ ；

Adding 2 mu L of DSB-X/DMSO mixed solution dissolved in the first tube into the second tube, and electromagnetically stirring for 1-1.5 hours at room temperature to obtain a sample loading solution;

the spin column is arranged on a glass tube, and 1mL of rosin suspension is added into the spin column for fixation; continuously adding rosin to increase the bed of the spin column to 1.5mL, allowing buffer solution to enter a collecting pipe under the action of gravity, retaining the column, and discarding the buffer solution; connecting the column with a collecting pipe, centrifuging the spin column at 1100G for 5min, discarding the buffer solution, and preparing the chromatographic column;

200 mu L of the sample solution is taken and added into the chromatographic column, spin strains are placed in a centrifugal force of 1100G for centrifugation for 5min, after centrifugation, the Lgr5 antibody marked by DSB-X and the Bmi antibody marked by DSB-X are respectively collected, and free DSB-X is absorbed by the spin column.

(2) Incubation of cell antibodies

Adding 10 mu L of each of the DSB-X labeled Lgr5 antibody and the DSB-X labeled Bmi1 antibody to 100 mu L of cell suspension to be separated; wherein, only the Lgr5 antibody marked by DSB-X is added into one tube, and only the Bmi1 antibody marked by DSB-X is added into one tube;

incubating the incubator at 37 ℃ for 30min, and shaking the re-suspension solution every 10 min;

after incubation, the supernatant was discarded by centrifugation at 150G and the cells were resuspended with separation buffer in the kit; the 3 steps were repeated once, at which time the cells had bound to the antibody.

(3) Magnetic bead-antibody binding

Preparation of magnetic beads: mixing magnetic beads with supernatant, adding 1 μl of magnetic beads and lmL into a centrifuge tube, standing on a magnetic rack for 3min, discarding supernatant, repeating for 3 times, and standing at 4deg.C;

adding a proper amount of magnetic beads into the cell suspension combined by the antibody;

incubate the incubator at 37℃for 30min, gently shake the solution every 10 min.

(4) Cell separation and release

Placing the solution after incubating the magnetic beads on a magnetic rack, and adsorbing for 5min;

resuspension the cells with separation buffer, 3 replicates;

placing the supernatant after resuspension on a magnetic rack to remove redundant magnetic beads as much as possible;

transferring the cells combined by the magnetic beads to a new centrifuge tube, and adding a proper amount of matrigel on ice for resuspension;

adding 24 pore plates in units of 50 mu L of each pore, and placing into a incubator;

after 30-45 min, the matrigel is solidified, and a small intestine organoid complete culture medium is added;

the growth of Lgr5 cells and Bmi1 cells was observed by regular liquid changes every 2 days.

In the isolation of intestinal stem cells provided in a specific comparative example 1, specific steps thereof including preparation of antibodies, incubation of cell antibodies, magnetic bead-antibody binding, and isolation and release of cells were basically the same as those of example 1, except that Lgr5 cells and Bmi1 cells, which only yielded Lgr5 cells, were not separately prepared.

(2) Subculture

In example 1 above, the subculture process includes:

(1) the above isolated Lgr5 cells were resuspended in DMEM/F12 medium containing 10mM HEPES, 10mM glutamine, 1 XN 2 cell culture supplement (invitrogen/Gibco, cat. No. 17502-048), 2mg/mL CD Feed002 (99014-302, jianshun), 100U/mL penicillin, 100mg/mL streptomycin, 5. Mu.g/mL transferrin, 10. Mu.g/mL insulin, 0.15mM non-essential amino acid, 1mM sodium pyruvate, designated P0 passage, placed at 37℃and 5% CO ₂ Culturing in an incubator.

(2) After the P0 generation grows to 80% of fusion rate, the culture solution is discarded, PBS containing 100U/mL penicillin and 100mg/mL streptomycin gentamicin is added for cleaning for 2 times, 1mL trypsin-EDTA digestive juice is added for digestion treatment for 1-2 min, and when the cells become round and float, 1-2 mL subculture solution is immediately added for stopping digestion. Wherein, the subculture solution is DMEM/F12 medium containing 10mM HEPES, 8mM glutamine, 1mg/mL CD Feed002, 100U/mL penicillin, 100mg/mL streptomycin, 5 μg/mL transferrin, 10 μg/mL insulin, 0.15mM nonessential amino acid, 1mM sodium pyruvate.

(3) After being blown uniformly by a pipetting gun, the mixture is transferred into a 15mL centrifuge tube after high-pressure sterilization, and the mixture is centrifuged for 5min at 75g at 4 ℃; the supernatant was discarded, and finally the pellet was resuspended in a pre-prepared subculture solution, cells were subcultured 1:2 or 1:3 depending on cell density, and transferred to 25cm previously coated with collagen I ² In a cell culture flask, labeled P1 generation, is placed at 37deg.C and 5% CO ₂ Culturing in an incubator. After 2d, the cell growth status and morphological characteristics were observed and passaged again.

Similarly, in the Bmi1 cell process of example 1, the primary culture broth contained 10mM HEPES, 10mM glutamine, 2mg/mL CD Feed002, 100U/mL penicillin, 100mg/mL streptomycin, 5. Mu.g/mL transferrin, 10. Mu.g/mL insulin, 0.15mM nonessential amino acid, 1mM sodium pyruvate in DMEM/F12 broth; the subculture medium was DMEM/F12 medium containing 10mM HEPES, 6mM glutamine, 0.5mg/mL CD Feed002, 80U/mL penicillin, 80mg/mL streptomycin, 5. Mu.g/mL transferrin, 10. Mu.g/mL insulin, 0.15mM nonessential amino acids, 1mM sodium pyruvate.

Thus, example 1 yielded Lgr5 cells and Bmi1 cells after subculturing, respectively, whereas comparative example 1 yielded only Lgr5 cells. Specifically, the number of passages is not more than 4, preferably only 2 passages, and the time for each passage is not more than 48 hours.

Example 2:

for Lgr5 cells: the P0 generation culture medium is DMEM/F12 medium containing 15mM HEPES, 8mM glutamine, 2 times N2 cell culture additive, 0.2mg/mL CD Feed002, 80U/mL penicillin, 80mg/mL streptomycin, 3 μg/mL transferrin, 16 μg/mL insulin, 0.25mM nonessential amino acid, 0.72mM sodium pyruvate; the subculture medium was DMEM/F12 medium containing 12mM HEPES, 5mM glutamine, 0.7mg/mL CD Feed002, 90U/mL penicillin, 90mg/mL streptomycin, 3. Mu.g/mL transferrin, 15. Mu.g/mL insulin, 0.25mM nonessential amino acids, 0.72 sodium pyruvate.

For Bmi1 cells: the P0-generation culture medium is DMEM/F12 culture medium containing 15mM HEPES, 8mM glutamine, 1.2mg/mL CD Feed002, 80U/mL penicillin, 80mg/mL streptomycin, 3 μg/mL transferrin, 14 μg/mL insulin, 0.25mM nonessential amino acid, 0.45mM sodium pyruvate; the subculture medium was DMEM/F12 medium containing 8mM HEPES, 4mM glutamine, 0.3mg/mL CD Feed002, 90U/mL penicillin, 90mg/mL streptomycin, 3. Mu.g/mL transferrin, 15. Mu.g/mL insulin, 0.25mM nonessential amino acids, 0.45mM sodium pyruvate.

Example 3:

for Lgr5 cells: the P0-generation culture medium is DMEM/F12 medium containing 20mM HEPES, 5mM glutamine, 0.2 times N2 cell culture additive, 1.4mg/mL CD Feed002, 90U/mL penicillin, 90mg/mL streptomycin, 2 μg/mL transferrin, 20 μg/mL insulin, 0.10mM nonessential amino acid, 0.2mM sodium pyruvate; the transfer medium was DMEM/F12 medium containing 20mM HEPES, 3mM glutamine, 1.0mg/mL CD Feed002, 100U/mL penicillin, 100mg/mL streptomycin, 2. Mu.g/mL transferrin, 20. Mu.g/mL insulin, 0.1mM nonessential amino acids, 0.2 sodium pyruvate.

For Bmi1 cells: the P0-generation culture medium is DMEM/F12 culture medium containing 20mM HEPES, 5mM glutamine, 0.5mg/mL CD Feed002, 90U/mL penicillin, 90mg/mL streptomycin, 2 μg/mL transferrin, 20 μg/mL insulin, 0.1mM nonessential amino acid, 0.2mM sodium pyruvate; the subculture medium was DMEM/F12 medium containing 5mM HEPES, 2mM glutamine, 0.1mg/mL CD Feed002, 100U/mL penicillin, 100mg/mL streptomycin, 2. Mu.g/mL transferrin, 20. Mu.g/mL insulin, 0.1mM nonessential amino acids, 0.2mM sodium pyruvate.

Comparative example 1: only Lgr5 cells were cultured in the same manner as in example 1.

Comparative example 2: the culture procedure for Lgr5 cells and Bmi1 cells was the same as in example 1, but the same culture solution was used for the culture solution:

DMEM medium containing 15wt% (weight percent) FBS, 100U/mL penicillin, 100mg/mL streptomycin, 2.5mg/mL gentamycin, 2.5mg/mL amphotericin B, 5 μg/mL transferrin, 10 μg/mL insulin, 0.15mM nonessential amino acid, imM sodium pyruvate, 1 μg/mL hydrocortisone.

Comparative example 3:

for Lgr5 cells: the primary culture medium is DMEM/F12 medium containing 10mM HEPES, 10mM glutamine, 100U/mL penicillin, 100mg/mL streptomycin, 5 μg/mL transferrin, 10 μg/mL insulin, 0.15mM nonessential amino acid, and 1mM sodium pyruvate; the transfer medium was 10mM HEPES, 8mM glutamine, 100U/mL penicillin, 100mg/mL streptomycin, 5. Mu.g/mL transferrin, 10. Mu.g/mL insulin, 0.15mM nonessential amino acids, 1mM sodium pyruvate in DMEM/F12 medium.

For Bmi cells: the primary culture medium contained 10mM HEPES, 10mM glutamine, 100U/mL penicillin, 100mg/mL streptomycin, 5. Mu.g/mL transferrin, 10. Mu.g/mL insulin, 0.15mM nonessential amino acids, 1mM sodium pyruvate in DMEM/F12 broth; the subculture medium was DMEM/F12 medium containing 10mM HEPES, 6mM glutamine, 80U/mL penicillin, 80mg/mL streptomycin, 5. Mu.g/mL transferrin, 10. Mu.g/mL insulin, 0.15mM nonessential amino acids, 1mM sodium pyruvate.

4. Identification of intestinal stem cells

(1) Immunohistochemical identification and detection of Lgr5 and Bmi expression in intestinal stem cells

Cells provided in each of the examples and comparative examples of subculture described above were fixed overnight with 10% normal buffered formalin and embedded in paraffin to prepare sections. After 3% hydrogen peroxide and goat serum treatment, rabbit mice were incubated with Bmi1 (1:800), lgr5 (1:6400) antibodies overnight (12 h) at 4 ℃, then with goat anti-rabbit antibodies and horseradish enzyme labeled streptavidin at 37 ℃, counterstained with DAPI, and desiccated patches. The negative control group used PBS instead of primary antibody. The image analysis software NIS-Elements analyzer 4.60.00 scans calculated the average absorbance value.

(2)RT-PCR

Detection of mRNA levels of intestinal stem cells Bmil, lgr5, and other related genes: extraction of intestinal tissue by Trizol methodThe total RNA is synthesized into cDNA through reverse transcription, and the target gene is subjected to RT-PCR; by relative quantification, with 2 ^-ΔΔCt And calculating the relative expression quantity of mRNA of the target gene/reference.

TABLE 1 primers for detection

Primer name	Sequence of steps
		Bmi1-F	acgtcatgtatgaagaggaacct as shown in SEQ ID NO.1
Bmi1-R	tggccgaactctgtatttcaaag as shown in SEQ ID NO.2
		Lgr5-F	acattcccaagggagcgttc as shown in SEQ ID NO.3
Lgr5-R	atgtggttggcatctaggcg as shown in SEQ ID NO.4
		β-actin-F	tgttaccaactgggacgaca as shown in SEQ ID NO.5
β-actin-R	ctgggtcatcttttcacggt as shown in SEQ ID NO.6
		c-Myc-F	aagaggctaaagttggacagtgg as shown in SEQ ID NO.7
c-Myc-R	cgcagggcaaagaaactca as shown in SEQ ID NO.8
		Sox9-F	gtgggagcgacaactttacc as shown in SEQ ID NO.9
Sox9-R	gcgagcacttagcagaaac as shown in SEQ ID NO.10
		MSI1-F	agagtgaggacatcgtggaga as shown in SEQ ID NO.11
MSI1-R	ggcgtaggttgtggcttg as shown in SEQ ID NO.12

(3) Western blot detection

The intestinal stem cells provided in each example and comparative example were subjected to lysis, protein denaturation, electrophoresis, and primary dilution concentrations Bmil (1:2000) and Lgr5 (1:1000) were added for color development and luminescence, and the average absorbance value was measured by using Gel-Pro analyzer for image analysis.

5. Statistical analysis

All test data are expressed as mean and standard deviation, data were processed using SPSS13.0 software and multiple comparisons and significance signatures were performed on each column of data.

2. Results

As shown in fig. 2 and 3, the staining patterns of the primary Lgr5 and Bmi1 cells obtained by separation are respectively shown, the nuclei of the DAP1 staining result are blue, and the other parts of the cells are dark red; and the surface of the Bmi1 cell of the DAPI staining result turns yellow green, and the stem cell specific markers Lgr5 and Bmi1 are respectively expressed positively, which indicates that the cell separation is successful.

TABLE 2 mRNA levels relative to beta-actin

As can be seen from table 2, the level of Lgr5 mRNA of the P2 generation Lgr5 cells provided in examples 1 to 3 is significantly higher than that in comparative examples 1 to 3, and the level of Bmi1 mRNA of the P2 generation Bmi1 cells provided in examples 1 to 3 is significantly higher than that in comparative examples 2 to 3, thereby demonstrating that the subculture method provided in the examples of the present application has significant promotion advantages for promoting the expression of Lgr5 and Bmi1, respectively.

Further, the embodiment of the application also adopts WB detection on the expression of Lgr5 and Bmi1 proteins, the expression gray level diagrams of which are shown in FIGS. 4 and 5, and the expression results of which are counted in Table 3. Therefore, the subculture method provided by the embodiment of the application has obvious promotion advantages for promoting the expression of Lgr5 and Bmi1 respectively.

TABLE 3 protein expression levels relative to beta-actin

TABLE 4 mRNA levels relative to beta-actin (Lgr 5 cells of the P2 generation)

Description of the embodiments	c-Myc-F	Sox9	MSI1
				Example 1	1.71±0.05a	1.62±0.07a	1.46±0.04a
Example 2	1.69±0.08a	1.61±0.06a	1.52±0.06a
				Example 3	1.68±0.06a	1.63±0.04a	1.51±0.04a
Comparative example 1	1.68±0.05a	1.61±0.08a	1.43±0.06a
				Comparative example 2	1.26±0.12c	1.25±0.03b	－
Comparative example 3	1.34±0.08b	1.26±0.05b	－

TABLE 5 mRNA levels relative to beta-actin (Bmi 1 cells of P2 generation)

Tables 4 and 5 also show the measurement of mRNA levels of other genes related to the Lgr5 cells and Bmi1 cells provided in examples 1 to 3 and comparative examples 1 to 3, respectively, as shown in FIGS. 6 and 7, which are WB measurement charts of c-Myc-F, sox9 and MSI1 proteins. In tables 4 and 5, "-" indicates no detection or absence. c-Myc is an important transcription factor for regulating stem cell proliferation, differentiation and other life functions, and is also one important protein for maintaining the normal function of crypt cell. SOX9, a member of the family of high mobility group box transcription factors, is predominantly distributed in the crypt part of the small intestine and regulated by Wnt signaling pathways, and is also positively correlated with small intestine crypt proliferation. MSI1 belongs to the family of RNA binding proteins, and has the common gene driving mode and target effect in intestinal cells, and MSI1 has obvious influence on the steady state and repair capability of small intestinal stem cells. As can be seen, the expression levels of c-Myc-F, sox and MSI1 in the P2 generation Lgr5 cells provided in examples 1-3 are significantly higher than those in comparative examples 1-3, and the expression levels of c-Myc-F, sox and MSI1 in the P2 generation Bmi1 cells provided in examples 1-3 are significantly higher than those in comparative examples 1-3. Therefore, the Lgr5 cells and Bmi1 cells provided by the embodiments of the present application may have outstanding advantages in maintaining the proliferation and normal homeostasis of the crypt of the small intestine and its ability to repair lesions.

In vivo experiments

1. Materials and methods

1. Experimental animals: balb/c mice, male, 36 month old mice and 5 month old young mice, the raising temperature is 21+/-2 ℃, the relative humidity is 30-70%, the illumination period is 12/12h, and the feed is purchased from Jiangsu Jiuzhikang biotechnology Co.

2. Test article

In order to further verify the application of the Lgr5 cells and Bmi1 cells provided in the examples and the comparative examples to aspects of microenvironment of intestinal tracts, anti-intestinal aging and the like, the application further carries out in vivo experiments by using the cells as test samples.

Therefore, the embodiment of the application discloses a method for improving intestinal microenvironment and resisting intestinal agingComprises an Lgr5 cell preparation and a Bmi1 cell preparation which are individually packaged, wherein the Lgr5 cell preparation comprises not less than 10 ⁶ individual/mL Lgr5 cells and other pharmaceutically relevant protective agents that maintain cell viability, bmi1 cell preparations comprising not less than 10 ⁶ individual/mL Bmi1 cells and other related protective agents that pharmaceutically maintain cell viability. When the preparation is applied, the application ratio of the Lgr5 cell preparation to the Bmi1 cell preparation is (2-5): 1.

Wherein, the related protective agent for maintaining the cell viability in pharmacy comprises 50-80U/mL penicillin, 50-80 mg/mL streptomycin, 0.5-2 mg/mL gentamycin, 0.5-2 mg/mL amphotericin B, 0.1-0.5 mug/mL transferrin, 0.05-0.2 mug/mL insulin, 0.01-0.05 mM nonessential amino acid, 0.01-0.05 mM sodium pyruvate, 20-30 wt% matrigel and 50-60 wt% glycerol.

Still further, the formulation for improving intestinal microenvironment and preventing intestinal aging disclosed in the examples of the present application further comprises an independently packaged enhancer formulation comprising 5-20 ng/mL mouse SF20 factor (product MEOPP-19011, guangzhou Sitting Biotechnology Co., ltd.), 1-5 ng/mL recombinant ErbB3 (ErbB 3-f) (product HEOPP-05081, guangzhou Sitting Biotechnology Co., ltd.) and 50-60 wt% glycerol. When the preparation is applied, the application ratio of the Lgr5 cell preparation, the Bmi1 cell preparation and the accelerating preparation is (200-500): 100 (1-5).

Furthermore, the preparation for improving intestinal micro-ring and resisting intestinal aging disclosed by the embodiment of the application can be independently packaged with an Lgr5 cell preparation, a Bmi1 cell preparation and a promoting preparation respectively, is frozen overnight at 4 ℃, or can be directly put into a cell gradient cooling box and finally can be transferred into liquid nitrogen for freezing storage so as to be stored for a long time.

For in vivo experiments, the test samples co-provided by the present application are shown in table 6. In Table 6, the facilitation formulation comprises 5ng/mLSF20, 1ng/mLErbB3-f and 50wt% glycerol, where "wt%" represents weight percent.

TABLE 6

4. Model preparation, grouping and administration

10 young mice are randomly selected as young groups, 36 month old mice are selected as old groups, any intervention measures are supplemented, and the young mice are used for establishing an intestinal microenvironment disturbance model group.

Establishing an intestinal microenvironment disturbance model: the tail of each mouse is clamped by a plastic clamp, and the tail is treated for 1 time per day for 30min, and after continuous stimulation for 15 days, the tail is used as a sign of successful model replication, wherein the mice are in emotion irritability, low falling, tiredness, low lying, lusterless hair, stool pool, and gradually reduced sound.

The mice in the model group and the aged group were used as administration groups, and the mice in the administration groups were respectively injected with the above test substances 1 to 8 at a weight of 70. Mu.L/kg, 2 times per week, and 2 weeks continuously. The model group and the administration group continued to perform intervention measures inducing spleen deficiency except for the young and the old.

5. Selective culture of mice primary intestinal flora

And respectively taking fecal samples at the 0 th week, the 2 nd week, the 4 th week and the 8 th week after the administration, adding 10 times of diluent, swirling for 30min, filtering by double-layer gauze, centrifuging the filtrate at-4 ℃ for 10min by l500g, and taking supernatant to obtain fecal suspension. 1mL of fecal suspension is absorbed and inoculated on a selective culture medium plate respectively, evenly coated and anaerobically cultured at 37 ℃. Selective culture of enterobacteria, clostridium, bifidobacterium and lactobacillus were performed, respectively. The calculation formula of the probiotic index is as follows: intestinal Probiotics Index (PI) = [1 gcpu ^-1 (Bif)+ZgCFU ^-1 (Lac)－1gCFU ^-1 (Clos)－1gCFU ^-1 (Bac)]/1gCFU ^-1 (Total) where Bif is the number of bifidobacteria in the sample, lac is the number of lactobacilli in the sample, clos is the number of clostridia in the sample, bac is the number of enterobacteria in the sample, and Total is the number of Total enterobacteria in the sample. The B/E value refers to the ratio of bifidobacteria to enterobacteriaceae, and is obtained by dividing the number of bifidobacteria by the value of the corresponding enterobacteriaceae in the same system.

6. Intestinal mucosa barrier function index detection

Mouse serum was collected and assayed according to the instructions of mouse D-Lac (Kameshu/Kmaels), DAO (Shanghai Reed Biotechnology Co., ltd.) and ZO-1ELISA assay kit (product model: IHC0100061, gekko organism).

7、qRT-PCR

(1) With reference to the above method, the intestinal epithelial tissue of the mouse is taken on an operating table, digested with collagenase and neutral protease, and then the cells are completely suspended in DMEM/F12, filtered with a sterile filter of 70 μm, and the filtrate is collected and resuspended with Hank's buffer to give a concentration of 10 ⁶ Cell suspensions of individual/mL;

(2) Extraction of RNA and Synthesis of cDNA

The supernatant was discarded after centrifugation at 1200rpm for 5 minutes to obtain a cell pellet, 1ml of Trizol reagent was added, and after repeated pipetting, the cell pellet was allowed to stand at room temperature for 5 minutes to completely separate the protein nucleic acid complex. After that, 0.2ml of chloroform was added thereto, the tube was covered with a cap, and then vigorously shaken for 15 seconds, and left at room temperature for 2 minutes. After centrifugation at 12000rpm for 10 minutes at 4℃the upper colorless aqueous phase in the centrifuge tube was removed and transferred to a new RNase-Free centrifuge tube. Then, an equal volume of absolute ethyl alcohol (RNase-Free) was added to the aqueous phase solution, and the mixture was mixed upside down. The whole solution was slowly added to a collection tube equipped with an adsorption column, and then centrifuged at 12000rpm for 20 seconds, after which the waste liquid in the collection tube was poured out, and the adsorption column was again placed in the collection tube. Then, 700. Mu.L of Buffer RW 1,12000rpm was added to the adsorption column, centrifuged for 20 seconds, and the waste liquid in the collection tube was discarded, and the adsorption column was again placed in the collection tube. After that, an equal volume of Buffer RW2 was added to the adsorption column, and the mixture was centrifuged at 12000rpm for 20 seconds, and the waste liquid in the collection tube was discarded, and the adsorption column was again put back into the collection tube. Then adding Buffer RW2 with equal volume into the adsorption column again, centrifuging at 12000rpm for 20 seconds, pouring out the waste liquid in the collecting pipe, and putting the adsorption column into the collecting pipe again. Centrifuge at 12000rpm for 2 minutes at 4 ℃, pour the waste liquid in the collection tube. The adsorption column was left to dry thoroughly at room temperature. Placing the adsorption column in a new RNase-Free centrifuge tube, adding 20 μl of RNase-Free Water into the adsorption column, standing at room temperature for 1 min, centrifuging at 12000rpm at 4deg.C for 10min, collecting RNA solution, and storing the obtained RNA at-70deg.C to prevent degradation.

The obtained RNA template was reverse transcribed into cDNA using the TransScript One-Step gDNARemoval and cDNASynthesis SuperMix kit (Beijing full gold Biotechnology Co., ltd.). qRT-PCR was performed using a TransStart Tip Green qPCR SuperMix kit (Beijing full gold Biotechnology Co., ltd.) two-step method.

TABLE 7 primers for detection

Primer name	Sequence(s)
		Bmi1-F	acgtcatgtatgaagaggaacct as shown in SEQ ID NO.1
Bmi1-R	tggccgaactctgtatttcaaag as shown in SEQ ID NO.2
		Lgr5-F	acattcccaagggagcgttc as shown in SEQ ID NO.3
Lgr5-R	atgtggttggcatctaggcg as shown in SEQ ID NO.4
		β-actin-F	tgttaccaactgggacgaca as shown in SEQ ID NO.5
β-actin-R	ctgggtcatcttttcacggt as shown in SEQ ID NO.6
		Axin2-F	gagagtgagcggcagagc as shown in SEQ ID NO.13
Axin2-R	cggctgactcgttctcct as shown in SEQ ID NO.14
		Ascl2-F	gagagctaagcccgatgga as shown in SEQ ID NO.15
Ascl2-R	tcagtagccccctaaccaac as shown in SEQ ID NO.16
		Alpi-F	gctcaaagaggcccatga as shown in SEQ ID NO.17
Alpi-R	atgatcagaacctggtgcaa as shown in SEQ ID NO.18
		Atoh-F	tccctgaaaactgagacaacc as shown in SEQ ID NO.19
Atoh-R	gctaacaacgatcaccacaga as shown in SEQ ID NO.20
		Defa24-F	aggaccaggctgtgtctgtc as shown in SEQ ID NO.21
Defa24-R	tcttcctttgcagcctcttg as shown in SEQ ID NO.22
		Chga-F	cgatccagaaagatgatggtc as shown in SEQ ID NO.23
Chga-R	cggaagcctctgtctttcc as shown in SEQ ID NO.24

8. Statistical analysis

2. Results

TABLE 8PI values

Table 8 lists the intestinal PI values of young, aged, model and dosed mice at weeks 2, 4 and 8 post-dosing. Compared with young mice, the PI value of the mice in the model group is obviously reduced, and the PI value of the mice in the aging group is also reduced; compared with the model group, the intestinal PI value of the mice in the administration group is obviously increased. Specifically, in the administration group, after the mice are administered with the test substances 1 to 4, the intestinal PI values of the test substances are significantly higher than those of the test substances 5 to 8 in both weeks 4 and 8, so that the test substances 1 to 4 can significantly improve the intestinal flora structure of the mice based on the examples 1 to 3 and the protective preparation, and the intestinal microenvironment of the mice is improved in the direction of probiotics.

TABLE 9B/E values

Table 9 lists the intestinal B/E values at weeks 2, 4 and 8 post-dose for young, aged, model and dosed mice. Compared with young animals, the B/E value of the mice in the model group is obviously reduced, and the B/E value of the mice in the aging group also has a reduced trend, which represents that the intestinal microenvironment of the mice in the aging group has a disturbance trend. Compared with the model group, the intestinal B/E value of the mice in the administration group is obviously increased. Specifically, in the administration group, after the mice are administrated with the test products 1-4, the intestinal B/E values of the test products are obviously higher than those of the test products 5-8 in the 4 th week and the 8 th week, so that the test products 1-4 can obviously improve the intestinal flora structure of the mice based on the test products 1-3 and the protective preparation, and the bifidobacterium proportion of the test products is increased, so that the intestinal microenvironment of the test products is improved towards the probiotics.

TABLE 10 intestinal mucosa barrier function

Table 10 lists the intestinal mucosa barrier function index for the serum of each group of mice. Compared with young mice, the serum D-Lac concentration and DAO activity content of the mice in the model group are obviously increased, which indicates that SS leads to obvious increase of intestinal barrier permeability of the mice, and ZO-1 expression quantity is obviously reduced, which indicates that intestinal barrier function of the mice is damaged; the content of D-Lac, DAO and ZO-1 in the serum of the aged mice also has the same trend relative to the young mice, which indicates that the intestinal mucosa barrier function of the serum of the aged mice is atrophic. In the administration group, the serum D-Lac content and DAO activity of the mice administered with the test substances 1 to 4 were significantly reduced and ZO-1 was significantly increased, compared with the model group, thereby indicating that the test substances 1 to 4, based on examples 1 to 3 and the protective agent, were able to improve the intestinal barrier permeability of the model mice and repair the intestinal lesions thereof.

TABLE 11 mRNA levels relative to beta-actin

As can be seen from table 11, the expression levels of Bmi1 and Lgr5 were significantly reduced and expression levels of Axin and Ascl2 were significantly increased in the aged and model mice compared to the young mice, thus indicating that the levels of Bmi1 and Lgr5 stem cells with differentiation ability were reduced in the aged and model mice, the intestinal tissues tended to be aged, and Axin and Ascl2 served as Wnt signaling pathway target genes, wherein overexpression of Axin could activate negative feedback regulation of Wnt signaling pathway, and overexpression of Ascl2 could lead to tumorigenesis. Compared with the aging group and the model group, in the administration group, after the mice are administrated with the test products 1-4, the expression level of Bmi1 and Lgr5 is obviously increased, and the expression level of Axin and Ascl2 is obviously reduced, thereby indicating that the test products 1-4 based on the examples 1-3 and the protective preparation can provide the mice with the effects of activating stem cells, improving intestinal differentiation capacity, reducing negative feedback regulation of Wnt, reducing the possibility of tumorigenesis and resisting the aging trend of the intestinal tract of the mice.

TABLE 12 mRNA levels relative to beta-actin

To further explore the effect of the test sample provided by the application on the aging of the intestinal tissues of mice, the application further explores the expression levels of the intestinal tissues Alpi, atoh, defa and Chga of each group of mice. As shown in table 12, the expression levels of Alpi, atoh, defa and Chga were significantly reduced in the intestinal tissues of the mice in the aged and model groups compared to the young groups, indicating that the differentiation marker genes, ALPI (intestinal cells), ato 1 (secretory lineage), DEF24 (paneth cells) and Chga (enteroendocrine cells) were significantly reduced in the intestinal tissues of the mice in the aged and model groups, and that the differentiation function defects were present in the intestinal tissues of the mice in the aged and model groups. In the administration group, after the test samples 1 to 4 are administered to the mice, the expression levels of the intestinal tissues Alpi, atoh, defa and the Chga of the mice are obviously higher than those of the aged group and the model group, and the test samples 5 to 8 do not have obvious rising trend after being administered. From this, it was demonstrated that the test pieces 1 to 4, based on examples 1 to 3 and the protective agent, were able to improve the defect of intestinal differentiation function in mice and to combat the trend of aging of intestinal function in mice.

In summary, the application extracts relevant cells from mouse duodenal epithelial tissue, separates stem cells to obtain stem cells expressing Bmi1 and stem cells expressing Lgr5, and detects WB and mRNA levels to find that P2 generation cells of the two cells express c-Myc-F, sox9 and MSI1 respectively, and the genes play important roles as transcription factors for intestinal tract proliferation differentiation and maintenance of small intestinal stem cell homeostasis and repair capability respectively. Thus, it was demonstrated that the isolated Bmi 1-expressing stem cells and Lgr 5-expressing stem cells have the outstanding advantage of maintaining small intestine crypt proliferation and normal homeostasis, as well as their lesion repair ability.

In addition, the stem cells expressing Bmi1 and stem cells expressing Lgr5 obtained by separation are used for preparing a preparation for improving intestinal microenvironment and resisting intestinal aging, and in vivo experiments prove that the preparation has an effect of improving the intestinal microenvironment of old mice and the trend of resisting aging phase change gene expression, and can improve the intestinal differentiation function defect of the mice, so that the intestinal microenvironment of the mice is improved in a probiotics direction.

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 subculturing intestinal stem cells, comprising the following steps:

separating to obtain an intestinal stem cell expressing the Lgr5 and an intestinal stem cell expressing the Bmi1, wherein the intestinal stem cell expressing the Lgr5 and the intestinal stem cell expressing the Bmi1 are both from duodenal epithelial tissues; and

performing subculture on the intestinal stem cells expressing the Lgr5 and the intestinal stem cells expressing the Bmi1 in a primary culture solution and a subculture solution respectively, wherein the subculture time is not more than 4 generations, and the subculture time is not more than 48 hours each time;

wherein the primary culture solution for culturing the intestinal stem cells expressing the Lgr5 is a DMEM/F12 culture medium which comprises 10-20 mM HEPES, 5-10 mM glutamine, 0.2-2 times N2 cell culture additive, 0.2-2 mg/mL CD Feed002, 80-100U/mL penicillin, 80-100 mg/mL streptomycin, 2-5 mug/mL transferrin, 10-20 mug/mL insulin, 0.10-0.25 mM nonessential amino acid and 0.2-1 mM sodium pyruvate;

wherein the subculture solution for subculturing the intestinal stem cells expressing the Lgr5 is a DMEM/F12 culture medium of 8-15 mM HEPES, 3-8 mM glutamine, 0.2-1 mg/mL CD Feed002, 80-100U/mL penicillin, 80-100 mg/mL streptomycin, 2-5 mug/mL transferrin, 10-20 mug/mL insulin, 0.10-0.25 mM nonessential amino acid, and 0.2-1 mM sodium pyruvate;

wherein the primary culture solution for culturing the intestinal stem cells expressing Bmi1 is a DMEM/F12 culture medium of 10-20 mM HEPES, 5-10 mM glutamine, 0.5-2 mg/mL CD Feed002, 80-100U/mL penicillin, 80-100 mg/mL streptomycin, 2-5 mug/mL transferrin, 10-20 mug/mL insulin, 0.10-0.25 mM nonessential amino acid and 0.2-1 mM sodium pyruvate;

wherein the subculture solution for culturing the intestinal stem cells expressing Bmi1 is a DMEM/F12 culture medium of 5-10 mM HEPES, 2-6 mM glutamine, 0.1-0.5 mg/mL CD Feed002, 50-80U/mL penicillin, 50-80 mg/mL streptomycin, 2-5 μg/mL transferrin, 10-20 μg/mL insulin, 0.10-0.25 mM nonessential amino acid and 0.2-1 mM sodium pyruvate.

2. A cell preparation comprising an Lgr 5-expressing intestinal stem cell produced by the subculture method of claim 1 and a Bmi 1-expressing intestinal stem cell; the intestinal stem cells expressing Lgr5 and the intestinal stem cells expressing Bmi1 both express the c-Myc-F, sox and MSI1 genes at high levels.

3. An agent for improving intestinal microenvironment and resisting intestinal aging comprises an Lgr5 cell preparation and a Bmi1 cell preparation which are packaged independently, wherein the Lgr5 cell preparation contains not less than 10 ⁶ individual/mL of Lgr 5-expressing intestinal stem cells and other pharmaceutically relevant protective agents that maintain cell viability, bmi1 cell preparations comprising not less than 10 ⁶ individual/mL of Bmi1 expressing intestinal stem cells and other related protective agents that pharmaceutically maintain cell viability;

wherein the Bmi 1-expressing intestinal stem cells and the Lgr 5-expressing intestinal stem cells are obtained by the subculture method of claim 1; the intestinal stem cells expressing Lgr5 and the intestinal stem cells expressing Bmi1 both express the c-Myc-F, sox and MSI1 genes at high levels.

4. The preparation according to claim 3, wherein the ratio of the Lgr5 cell preparation to the Bmi1 cell preparation is (2 to 5): 1 when the preparation is administered.

5. The formulation of claim 4, wherein the pharmaceutically relevant protective agent for maintaining cell viability comprises 50-80U/mL penicillin, 50-80 mg/mL streptomycin, 0.5-2 mg/mL gentamycin, 0.5-2 mg/mL amphotericin B, 0.1-0.5 μg/mL transferrin, 0.05-0.2 μg/mL insulin, 0.01-0.05 mM nonessential amino acid, 0.01-0.05 mM sodium pyruvate, 20-30 wt% matrigel, and 50-60 wt% glycerol.

6. The formulation of claim 5, further comprising an independently packaged booster formulation comprising 5-20 ng/mL mouse SF20 factor, 1-5 ng/mL recombinant ErbB3 (ErbB 3-f), and 50-60 wt% glycerol.

7. The preparation according to claim 6, wherein the administration ratio of the Lgr5 cell preparation, the Bmi1 cell preparation and the accelerator preparation is (200-500): 100 (1-5).

8. Use of the intestinal stem cells obtained by the subculture method of claim 1 or the cell preparation of claim 2 for preparing an agent for improving intestinal microenvironment and resisting intestinal aging.