CN117025504A

CN117025504A - Preparation method and application of gall bladder precursor-like cells

Info

Publication number: CN117025504A
Application number: CN202310519743.9A
Authority: CN
Inventors: 周伸奥; 周丽
Original assignee: Shanghai Celliver Biotechnology Co Ltd
Current assignee: Shanghai Celliver Biotechnology Co Ltd
Priority date: 2022-05-10
Filing date: 2023-05-09
Publication date: 2023-11-10
Also published as: CN117018033A; CN117025506A; WO2023217121A1; CN117018032A; WO2023217126A1; WO2023217134A1; CN117025503A; CN117025502A; WO2023217132A1; WO2023217130A1; WO2023217123A1; WO2023217129A1; WO2023217128A1; CN117025508A; CN117025505A; WO2023217125A1; CN117025507A

Abstract

The invention provides a preparation method and application of a gall bladder precursor-like cell, and the preparation method of the gall bladder precursor-like cell comprises the following steps: taking gallbladder tissue, and obtaining primary cholecystocytes after digestion and separation of the gallbladder tissue; culturing the primary cholecystocytes by using a reprogramming culture medium until the cell fusion degree is not lower than 80%, thereby obtaining the cholecyst precursor-like cells. The primary cholecyst cells are obtained after the cholecyst tissues are digested and separated, the primary cholecyst cells are cultured by using a reprogramming culture medium until the cell fusion degree is not lower than 80%, so that the cholecyst precursor-like cells are obtained, the primary cholecyst cells are dedifferentiated, the cholecyst precursor-like cells are rapidly and largely amplified in vitro, no exogenous genes exist, the operation is safe and reliable, and the batch yield is high; the gall bladder precursor-like cells can be applied to repair of damaged gall bladder tissues.

Description

Preparation method and application of gall bladder precursor-like cells

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method and application of gall bladder precursor-like cells.

Background

Along with the increasing standard of living of people, the diet structure is diversified, the incidence rate of gallbladder diseases is in a trend of rising year by year, and the laparoscopic cholecystectomy is a main mode for treating the gallbladder diseases, and is small in wound and few in complications. However, partial excision of the gallbladder or fistulization, etc. may cause localized damage to the gallbladder and thus affect related functions of the gallbladder, including affecting digestive function, increasing incidence of inflammation of the digestive tract, and incidence of colorectal cancer. With the development of science, cell therapy is increasingly being developed in the field of view of the public and is being applied to the treatment of various human diseases, such as gallbladder diseases. The gall bladder precursor-like cells can restore gall bladder function and reverse gall bladder injury, and are the most important cells for gall bladder tissue repair. By culturing and applying the gall bladder precursor-like cells, a new therapy of repairing the structure and the function of pathological gall bladder tissues and regenerating tissue cells by using cell preparations or cell secretion factors brings new hopes for gall bladder diseases. The method is characterized by constructing a gall bladder precursor-like cell culture system in vitro, researching the treatment effect of gall bladder precursor-like cells on a gall bladder disease model, and has important significance for researching postoperative recovery of gall bladder diseases and the like.

The sources of gallbladder precursor-like cells currently used for in vitro culture are mainly 3 kinds of cells, but all have defects of different degrees:

1. adult gall bladder and gall bladder of embryo: the precursor cells of gall bladder exist in different positions of the gall bladder of the adult, including mucous membrane cells, myometrium cells and the like, and a large number of precursor cells of gall bladder also exist in embryo gall bladder tissues. The gall bladder precursor-like cells can be obtained by separating gall bladder tissues and screening of adult gall bladder and embryo gall bladder. However, the separation of the embryo gall bladder from the adult gall bladder tissue requires sufficient initial gall bladder cells, and the cells have limited continuous passage capability and are difficult to apply to treatment.

2. ESCs/iPSCs differentiation: ESCs are pluripotent stem cells derived from mammalian fertilized eggs, whereas iPSCs are transduced from somatic cells by reprogramming, all of which can be induced to differentiate to give gall bladder precursor-like cells, using key transcription factors found in gall bladder development studies as markers of differentiation, such as CK-19, etc. However, few reports of ESCs/iPSCs for treating gallbladder diseases exist at present, mainly because the culture scheme of ESCs/iPSCs for in vitro induced differentiation into gallbladder cells is still immature, the standard of unified specification is lacking, the differentiation efficiency of ESCs/iPSCs is limited, and the residual ESCs have tumorigenicity, have the risk of developing teratomas and are still in ethical disputed.

3. Cholecystocyte reprogramming: mature cholecystocytes can be reprogrammed by activating a gene network consistent with cholecystokinin progenitor cells by expression of 6 transcription factors (SIX 1, SIX2, OSR1, EYA1, HOXA11 and SNAI 2) to obtain cholecyst precursor-like cells. However, mature cholecystocyte reprogramming requires enough initial cholecystocytes, and the continuous passage capability of the cholecyst precursor-like cells obtained by reprogramming is limited, so that the method is difficult to apply to treatment.

4. Other such as mesenchymal stem cells, hematopoietic stem cells, etc.: mesenchymal stem cells, hematopoietic stem cells and the like can be induced to differentiate to obtain gall bladder precursor-like cells, but have a problem of low differentiation efficiency and the like.

Therefore, it is necessary to provide a novel preparation method and application of the gall bladder precursor-like cells, so as to realize the large-scale expansion of the gall bladder precursor-like cells in vitro and apply the gall bladder precursor-like cells to the repair of damaged gall bladder tissues.

Disclosure of Invention

The invention aims to provide a preparation method and application of gall bladder precursor-like cells, which can realize the large-scale expansion of gall bladder precursor-like cells in vitro and are applied to the repair of damaged gall bladder tissues.

To achieve the above object, the method for preparing the gallbladder precursor-like cells of the present invention comprises the steps of:

S1: taking gallbladder tissue, and obtaining primary cholecystocytes after digestion and separation of the gallbladder tissue;

s2: culturing the primary cholecystocytes by using a reprogramming culture medium until the cell fusion degree is not lower than 80%, thereby obtaining the cholecyst precursor-like cells.

The preparation method of the gall bladder precursor-like cells has the beneficial effects that:

the primary cholecyst cells are obtained after the cholecyst tissues are digested and separated, the reprogramming culture medium is used for culturing the primary cholecyst cells until the cell fusion degree is not lower than 80%, so that the primary cholecyst precursor-like cells are obtained, the primary cholecyst cells are dedifferentiated, the primary cholecyst precursor-like cells are rapidly and largely amplified in vitro, no exogenous genes exist, the operation is safe and reliable, and the batch yield is high; the gall bladder precursor-like cells can be applied to repair of damaged gall bladder tissue.

Optionally, in the S1, the gallbladder tissue is derived from normal gallbladder tissue which cannot be used for transplantation.

Optionally, in the step S1, the step of obtaining primary cholecystocytes from the cholecystokinis tissue after digestion and separation includes:

s10: sequentially cleaning and sterilizing the gallbladder tissue with sterile PBS buffer solution, and then shearing the gallbladder tissue to obtain gallbladder fragments;

S11: adding cell digestive juice into the gallbladder fragments, and performing digestion treatment on the gallbladder fragments at 37 ℃ for 90 minutes to obtain primary gallbladder cell suspension;

s12: screening the primary cholecystocyte suspension by a screen, and collecting to obtain cholecystocyte filtrate;

s13: centrifuging the cholecystocyte filtrate, and discarding the supernatant to obtain cholecystocyte sediment;

s14: adding erythrocyte lysis balance liquid into the cholecyst cell sediment for re-suspension to obtain cholecyst cell mixed liquid, centrifuging the cholecyst cell mixed liquid, and discarding the supernatant to obtain cholecyst cell sediment;

s15: repeating said S14 until no erythrocytes are observed in said cholecystocyte pellet.

Optionally, in the processS10, cutting the gallbladder tissue into 1-2mm ³ 。

Optionally, in S11, the components of the cell digestive juice include, in percentage by volume of the cell digestive juice: 88% -96% of sterile PBS buffer solution, 3% -7% of pancreatin digestive solution and 1% -5% of type IV collagenase. The beneficial effects are that: the cell digestive juice enables the gall bladder tissue to be sufficiently digested.

Optionally, in S12, the primary cholecystocyte suspension is screened using a cell filter having a pore size of 60 to 80 microns.

Optionally, in S13, when the cholecystocyte filtrate is centrifuged, the centrifugation rate is 1000g and the centrifugation time is 3 minutes.

Optionally, in S14, when the cholecystocyte mixed solution is centrifuged, the rotational speed of the centrifugation is 1000g, and the centrifugation time is 3 minutes.

Optionally, in the S2, the reprogramming media comprises basal media, nutritional supplements, growth factors, TGF- β signaling inhibitors, wnt signaling pathway activators, and ROCK kinase inhibitors.

Optionally, the content of the growth factor is 15-30 nanograms per milliliter, the content of the ROCK kinase inhibitor is 5-20 micromoles, the content of the Wnt signal path activator is 1.2-6.7 micromoles, the content of the TGF-beta signal inhibitor is 0.4-3.3 micromoles, and the volume content of the nutritional supplement is not more than 10 percent.

Optionally, the basal medium is DMEM/F12 medium.

Optionally, the growth factor is at least one of an epidermal growth factor and a fibroblast growth factor.

Optionally, the nutritional supplement comprises at least one of N2, B27.

Optionally, the ROCK kinase inhibitor comprises Y-27632.

Optionally, the Wnt signaling pathway activator comprises CHIR-99021.

Alternatively, the TGF-beta signaling inhibitor comprises A-83-01.

Optionally, in the S2, the reprogramming media further comprises a GSK-3 inhibitor, the GSK-3 inhibitor comprising LY2090314. The beneficial effects are that: the LY2090314 is capable of promoting proliferation of the gall bladder precursor-like cells.

Optionally, the LY2090314 is present in an amount of 2 μg/ml.

Optionally, after the step S2, step S3 is further included: and after digestion treatment is carried out on the gall bladder precursor-like cells, carrying out subculture by using the reprogramming culture medium to obtain the subcultured gall bladder precursor-like cells, wherein the number of cell passages of the subculture is not less than 4. The beneficial effects are that: after the gall bladder precursor-like cells are digested, the reprogramming culture medium is used for subculturing to obtain the subcultured gall bladder precursor-like cells, and the gall bladder precursor-like cells can be stably subcultured under the condition of maintaining the epithelial precursor form, so that the large-scale expansion of the gall bladder precursor-like cells in vitro is realized.

Optionally, in the step S3, the step of subculturing using the reprogramming media includes:

s31: sucking and removing the supernatant of the reprogramming culture medium, washing the gall bladder precursor-like cells twice by using a sterile PBS buffer solution, and adding pancreatin digestive juice into the gall bladder precursor-like cells for digestion treatment for 1-5 minutes to obtain a mixed solution;

s32: centrifuging the mixed solution, and removing supernatant to obtain a gallbladder precursor-like cell precipitate;

s33: counting the cell of the gallbladder precursor-like cell pellet, and then inoculating the gallbladder precursor-like cell into the reprogramming culture medium to obtain a first-generation gallbladder precursor-like cell;

s34: subculturing the first generation gall bladder precursor-like cells using the reprogramming media.

Alternatively, in the step S32, when the mixed solution is subjected to centrifugation, the centrifugation rate is 200g and the centrifugation time is 5 minutes.

Optionally, the gallbladder-precursor-like cells positively express at least one characteristic marker.

Alternatively, the expression rate of the signature marker is not less than 50% and not more than 99%. The beneficial effects are that: the cells with the up-to-standard expression rate of the characteristic markers are the gall bladder precursor-like cells defined in the patent of the invention.

Further optionally, the expression rate of the signature marker is higher than 70%.

Optionally, the at least one of the signature markers comprises at least one of CD24, CD326, CK19 and Sox 9.

Alternatively, the gallbladder precursor-like cells negatively express at least one MHC class molecule, at least one of which comprises at least one of HLA-DR/DP/DQ. The beneficial effects are that: the gall bladder precursor-like cells do not express MHC-II antigens, and after the patient transplants the gall bladder precursor-like cells, the body of the patient cannot recognize the exogenous cells through MHC-II molecules and cannot generate immune attack on the exogenous cells, so that the possibility of rejection reaction is low.

Optionally, the gall bladder precursor-like cells are also negative for expression of at least one of CD34, CD 45. The beneficial effects are that: the gall bladder precursor-like cells do not express the hematopoietic stem cell antigen CD34 and the leukocyte common antigen CD45, and exhibit low immunogenicity.

The invention also provides application of the gall bladder precursor-like cells, and the gall bladder precursor-like cells prepared by the preparation method of the gall bladder precursor-like cells intervene in an in-vivo animal model. The beneficial effects are that: the gall bladder precursor-like cells can be directly returned to gall bladder tissue through gall bladder arteries and can be used for repairing damaged gall bladder tissue.

Optionally, the in vivo animal model comprises a mouse gall bladder injury model.

Drawings

FIG. 1 is a diagram of a cell culture on reprogramming media in example 2-1 provided by the present invention;

FIG. 2 is a graph of cell culture on control medium in example 2-2 provided by the present invention;

FIG. 3 is a graph of population doublings of P1-generation gall bladder precursor-like cells in reprogramming media and control media in examples 2-3 provided herein;

FIG. 4 is a graph showing the cell doubling time of the P1-generation gall bladder precursor-like cells of examples 2-3 according to the present invention subcultured in the reprogramming medium and the control medium;

FIG. 5 is an inverted microscopic image of the precursor-like cells of the gall bladder of the generation P1, P2, P3 in examples 2-3 provided by the present invention;

FIG. 6 is a graph showing the growth of cells subcultured with Pr-generation gall bladder precursor-like cells in reprogramming medium and DMEM+FBS medium in example 3-1 provided by the present invention;

FIG. 7 is a graph showing proliferation of Pr-substituted cholecystocytes from 4 different donor sources in reprogramming media according to example 3-2 provided herein;

FIG. 8 is a flow chart of the surface markers and the intracellular markers of the precursor cells of the gall bladder of generation P1 in example 4 provided by the present invention;

FIG. 9 is a graph showing survival of mice in healthy, modular and treated groups of example 5-1 provided herein;

FIG. 10 is a visual illustration of the anatomy of mice in the healthy, modular and treated groups of example 5-2 provided herein;

FIG. 11 is a graph showing total cholesterol levels in healthy, modular and treated mice of examples 5-3 provided herein;

FIG. 12 is a graph showing total bile acid content of mice in healthy, modular and treated groups of examples 5-3 provided herein;

FIG. 13 is a graph showing total bile acid content of mice in healthy, modular and treated groups of examples 5-3 provided herein;

FIG. 14 is a graph of direct bilirubin levels in healthy, modular and treated mice of examples 5-3 provided herein.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless otherwise defined, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. As used herein, the word "comprising" and the like means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof without precluding other elements or items.

Aiming at the problems existing in the prior art, the embodiment of the invention provides a preparation method and application of gall bladder precursor-like cells, which can realize the large-scale expansion of gall bladder precursor-like cells in vitro and is applied to the repair of damaged gall bladder tissues.

The preparation method of the gall bladder precursor-like cell comprises the following steps:

The preparation method of the gall bladder precursor-like cells has the advantages that:

In some embodiments, in the S1, the gallbladder tissue is derived from normal gallbladder tissue that cannot be used for transplantation.

In some embodiments, in S1, the step of obtaining primary cholecystocytes from the cholecystokinis tissue after digestion and separation comprises:

s15: s14 is repeated until no erythrocytes are observed in the cholecystocyte pellet.

In some embodiments, in the step S10, the size of the gallbladder tissue after the shearing treatment is 1-2mm ³ 。

In some embodiments, in S11, the components of the cell digestive juice include, in volume percent of the cell digestive juice: 88% -96% of sterile PBS buffer solution, 3% -7% of pancreatin digestive solution and 1% -5% of type IV collagenase. The advantages are that: the cell digestive juice enables the gall bladder tissue to be sufficiently digested.

In some embodiments, in S12, the primary cholecystocyte suspension is screened using a cell filter having a pore size of 60 to 80 microns.

In some embodiments, in the step S13, the cholecystocyte filtrate is centrifuged at 1000g for 3 minutes.

In some embodiments, in the step S14, when the cholecystocyte mixed solution is centrifuged, the rotational speed of the centrifugation is 1000g, and the centrifugation time is 3 minutes.

In some embodiments, in the S2, the reprogramming media comprises basal media, nutritional supplements, growth factors, TGF- β signaling inhibitors, wnt signaling pathway activators, and ROCK kinase inhibitors.

In some embodiments, the growth factor is present in an amount of 15-30 nanograms per milliliter, the ROCK kinase inhibitor is present in an amount of 5-20 micromolar, the Wnt signaling pathway activator is present in an amount of 1.2-6.7 micromolar, the TGF- β signaling inhibitor is present in an amount of 0.4-3.3 micromolar, and the nutritional supplement is present in an amount of no more than 10% by volume.

In some embodiments, the basal medium is DMEM/F12 medium.

In some embodiments, the growth factor is at least one of an epidermal growth factor, a fibroblast growth factor.

In some embodiments, the nutritional supplement comprises at least one of N2, B27.

In some embodiments, the ROCK kinase inhibitor comprises Y-27632.

In some embodiments, the Wnt signaling pathway activator comprises CHIR-99021.

In some embodiments, the TGF-beta signaling inhibitor comprises A-83-01.

In some embodiments, in the S2, the reprogramming media further comprises a GSK-3 inhibitor, the GSK-3 inhibitor comprising LY2090314. The advantages are that: the LY2090314 is capable of promoting proliferation of the gall bladder precursor-like cells.

In some embodiments, the LY2090314 is present in an amount of 2 μg/ml.

In some embodiments, after the step S2, the method further includes step S3: and after digestion treatment is carried out on the gall bladder precursor-like cells, carrying out subculture by using the reprogramming culture medium to obtain the subcultured gall bladder precursor-like cells, wherein the number of cell passages of the subculture is not less than 4. The advantages are that: after the gall bladder precursor-like cells are digested, the reprogramming culture medium is used for subculturing to obtain the subcultured gall bladder precursor-like cells, and the gall bladder precursor-like cells can be stably subcultured under the condition of maintaining the epithelial precursor form, so that the large-scale expansion of the gall bladder precursor-like cells in vitro is realized.

In some embodiments, in S3, the step of subculturing using the reprogramming media comprises:

In some embodiments, in the step S32, the mixed solution is centrifuged at 200g for 5 minutes.

In some embodiments, the gall bladder precursor-like cells positively express at least one characteristic marker.

In some embodiments, the expression rate of the signature marker is not less than 50% and not more than 99%. The advantages are that: the cells with the up-to-standard expression rate of the characteristic markers are the gall bladder precursor-like cells defined in the patent of the invention.

In some embodiments, the expression rate of the signature marker is greater than 70%.

In some embodiments, at least one of the signature markers comprises at least one of CD24, CD326, CK19, and Sox 9.

In some embodiments, the gall bladder precursor-like cell negatively expresses at least one MHC class molecule, at least one of the MHC class molecules comprising at least one of HLA-DR/DP/DQ. The advantages are that: the gall bladder precursor-like cells do not express MHC-II antigens, and after the patient transplants the gall bladder precursor-like cells, the body of the patient cannot recognize the exogenous cells through MHC-II molecules and cannot generate immune attack on the exogenous cells, so that the possibility of rejection reaction is low.

In some embodiments, the gallbladder-precursor-like cells are also negative for expression of at least one of CD34, CD 45. The beneficial effects are that: the gall bladder precursor-like cells do not express the hematopoietic stem cell antigen CD34 and the leukocyte common antigen CD45, and exhibit low immunogenicity.

The invention also provides application of the gall bladder precursor-like cells, and the gall bladder precursor-like cells prepared by the preparation method of the gall bladder precursor-like cells intervene in an in-vivo animal model. The advantages are that: the gall bladder precursor-like cells can be directly returned to gall bladder tissue through gall bladder arteries and can be used for repairing damaged gall bladder tissue.

In some embodiments, the in vivo animal model comprises a mouse gall bladder injury model.

The following is a detailed description of specific examples:

example 1

This example provides for the acquisition of primary cholecystocytes

1. Initial organizational nature and source legitimacy declaration:

normal gallbladder tissue which cannot be used for transplantation is used as a starting material.

Specifically, the gallbladder tissue is shown to be normal gallbladder tissue by pathological examination.

Specifically, the normal gallbladder tissue which cannot be used for transplantation is a surgical sample derived from a patient aged no more than 70 years, the patient is not infected by an infectious virus through medical examination, and the patient does not use steroid hormone medicine within 6 months before surgery. The patient was fully informed of the purpose of the acquisition of the surgical sample prior to surgery and signed an informed consent form.

2. The specific operation steps are as follows:

preparing cell digestive juice: the volume ratio of the modified buffer solution PSB to the IV type collagenase is 100:1, dissolving collagenase powder of type IV by using an improved buffer solution PSB to obtain collagenase solution of type IV, adding 5mL of sterile PBS buffer solution and 3mL of pancreatin digestion solution into 2mL of collagenase solution of type IV to prepare cell digestion solution at room temperature, wherein the cell digestion solution contains 2% of collagenase of type IV and 3% of pancreatin digestion solution by volume percent of self-prepared collagenase.

Wherein the modified buffer is purchased from Thermo Fisher under the trade designation HBPP8621-100; collagenase type iv was purchased from YEASEN under the accession number 40510ES60; sterile PBS buffer was purchased from source culture, cat No. B310KJ; pancreatin digests were purchased from source cultures under the accession number S310JV.

Obtaining normal gallbladder tissue, cleaning the obtained gallbladder tissue by using 30mL of sterile PBS buffer (1X), sterilizing the cleaned gallbladder tissue, putting the cleaned gallbladder tissue into povidone iodine stock solution for sterilization and soaking for 30 seconds, and soaking the gallbladder tissue in 75% alcohol for 5 minutes; then cleaning three times of gallbladder tissues with sterile PBS buffer solution to sterilize, and shearing the sterilized gallbladder tissues to 1-2mm ³ The minced gallbladder tissue was transferred to a 15mL centrifuge tube, 3mL of cell digest was added to the tube, and the tube was placed at 37 ℃ for digestion for 90 minutes to obtain a cell suspension.

10mL of sterile PBS buffer was added to 3mL of the cell suspension for dilution, and then the cell suspension was screened using a 70 micron cell filter, and the cell filtrate was collected and the mucus and undigested tissue in the cell suspension were removed. Then, 13mL of the cell filtrate was put into a centrifuge, the cell filtrate was centrifuged at 1000rpm for 3 minutes, and the supernatant of the centrifuged cell filtrate was discarded to obtain a cell pellet.

Adding 2mL of erythrocyte lysis balance liquid into the obtained cell sediment to obtain a cell mixed liquid, re-suspending the cell mixed liquid, putting the re-suspended cell mixed liquid into a centrifugal machine again, centrifuging the cell mixed liquid for 3 minutes at the rotating speed of 1000rpm, discarding supernatant of the centrifuged cell mixed liquid to obtain the cell sediment after centrifuging, repeatedly adding erythrocyte lysis balance liquid into the cell sediment after centrifuging, re-suspending and centrifuging until no erythrocyte is observed in the cell sediment obtained by centrifuging again, completing erythrocyte lysis and removal, and obtaining primary cholecyst cells which are Pr generation cholecyst cells. Specifically, the rate of each centrifugation was 1000rpm, and the centrifugation time was 3 minutes.

Wherein the erythrocyte lysis balance liquid is purchased from HePengpeng organism under the product number HPBIO.

Example 2-1

Pr-substituted cholecystocytes obtained in example 1 were cultured at 1X 10 ⁴ Inoculation Density per square centimeter on 6-well plates, 2 ml of reprogramming media was added to each well of the plates and the plates were placed at 37℃and 5% CO ₂ The cell culture is carried out in the incubator of (2), and the new culture solution is replaced every three days until the cell fusion degree in each hole of the culture plate is not lower than 80% and the cell growth state is good, thus completing the amplification culture. The plates were purchased from NEST under the accession number 715001.

Absorbing and discarding the supernatant of the reprogramming culture medium on the culture plate, washing the culture plate twice by using a sterile PBS buffer solution, and dripping 1mL of pancreatin digestive juice into each hole of the culture plate for digestion treatment for 1-5 minutes to obtain a mixed solution; centrifuging the mixed solution at a centrifugation rate of 200g for 5 min to obtain cell precipitate, counting cells in the cell precipitate, and counting 1E+04 cells/cm ² Is inoculated in a reprogramming culture medium to obtain first-generation gall bladder precursor-like cells, which are marked as P1-generation gall bladder precursor-like cells. Wherein, pancreatin digest was purchased from source culture under the accession number S310JV.

The composition of the reprogramming media used was as follows: DMEM/F12 basal medium, N2 nutritional supplement in an amount of 1% and B27 nutritional supplement in an amount of 2% by volume of DMEM/F12 basal medium; an epithelial cell growth factor EGF with the content of 20ng/mL, a fibroblast growth factor bFGF with the content of 50ng/mL, a GSK-3 inhibitor LY2090314 with the content of 10uM, a ROCK kinase inhibitor Y-27632 with the content of 10uM, a Wnt signal path activator CHIR-99021 with the content of 3uM and a TGF-beta signal inhibitor A-83-01 with the content of 1 uM.

Wherein, DMEM/F12 basal medium is purchased from Procell under the trade designation PM150310-MR100; n2 nutritional supplements were purchased from Thermo Fisher under the accession number 175020481X; b27 nutritional supplement was purchased from Thermo Fisher under the accession number 125870101X; epithelial cell growth factor EGF was purchased from abcam under the accession number ab259398; fibroblast growth factor bFGF was purchased from biorbyt under accession number orb80024; ROCK kinase inhibitor Y-27632 was purchased from Yeasen under the trade designation 52604ES10; the Wnt signaling pathway agonist CHIR-99021 is purchased from MED bio under the designation MED18140; TGF-beta signaling inhibitor A-83-01 is available from Yeasen under the trade designation 53002ES08; GSK-3 inhibitor LY2090314 was purchased from Yeasen under the accession number 52991ES10.

Example 2-2

Pr-generation lung cells obtained in example 1 were inoculated onto a control medium for expansion culture to form a control group in which Pr-generation lung cells were expanded and cultured with a reprogramming medium in example 2-1, and the culture procedure in this example was identical to that in example 2-1.

The control medium lacked the GSK-3 inhibitor LY2090314 relative to the reprogramming medium, with the remaining components and amounts consistent with the reprogramming medium.

The plates of the experimental and control groups were removed from the incubator, placed under a microscope for inverted microscope imaging and photographed, and cell images were shown in figures 1 and 2. FIG. 1 is a diagram of a cell culture on reprogramming media in example 2-1 provided by the present invention; FIG. 2 is a graph of cell culture on control medium in example 2-2 provided by the present invention.

Referring to the controls of FIGS. 1 and 2, the cell density on the reprogramming media was greater than that on the control media, and the proliferation capacity and adherence of cells cultured in the reprogramming media were better than those of the control media, demonstrating that the GSK-3 inhibitor LY2090314 in the reprogramming media was able to promote proliferation of gall bladder precursor-like cells.

Examples 2 to 3

The operation steps of subculture are as follows: p1 generation cells were cultured according to a protocol of 2X 10 ⁴ Individual living cells/cm ² Is inoculated on a culture plate of a culture medium, and the culture plate is placed at 37 ℃ and contains 5 percent of CO ₂ The culture of cells was performed in the incubator of (a) until the cell fusion degree of the culture plate was not less than 80% and the cell growth state was good, the culture plate was taken out of the incubator, the supernatant of the reprogramming medium on the culture plate was sucked off, the cell layer on the culture plate was washed with 5mL of a sterile PBS buffer having a concentration of 10%, the washing liquid was discarded, and the cell layer was repeatedly washed again with 5mL of a sterile PBS buffer having a concentration of 10%.

Adding 2mL of TrypLE Express digestive juice into each well of the culture plate, shaking the culture plate to uniformly distribute the TrypLE Express digestive juice at the bottom of the culture plate, placing the culture plate at 37deg.C, and CO ₂ The content is 5 percent in an incubator; after 3 minutes, the gall bladder precursor-like cells fall off from the bottom of each hole of the culture plate, and the cells are observed to be in a suspension state under a microscope, and the cell morphology is round and transparent; adding the reprogramming culture medium into a culture plate to stop cell digestion, then washing the culture plate with 2mL of sterile PBS buffer solution with the concentration of 10%, transferring all the liquid on the culture plate into a centrifuge tube, centrifuging at the centrifugation rate of 200g for 5 minutes, taking out the centrifuge tube after centrifugation, spraying alcohol on the tube body of the centrifuge tube without shaking, wiping the tube, and putting the centrifuge tube into a biosafety cabinet; and opening a tube cover of the centrifuge tube, discarding supernatant in the centrifuge tube to obtain cell sediment, sucking a culture medium by using a dropper to blow the cell sediment in the centrifuge tube, blowing off and uniformly blowing the cell sediment, and collecting all cell suspension in the centrifuge tube to fix the volume to obtain P2 generation cells.

And repeating the steps to finish subculture of the P2 generation cells.

Wherein, the culture plate is purchased from NEST with the product number of 715001; trypLE Express digest was purchased from Gibco under the accession number 12604013; the incubator was purchased from ESCO under the trade designation CCL-170B-8; biosafety cabinet was purchased from Haier under the product number HR50-IIA2.

The P1-generation gall bladder precursor-like cells obtained in example 2-1 were subcultured in the reprogramming medium and the control medium, respectively, according to the procedure described above.

The population doubling level of the P1-generation gall bladder precursor-like cells in the reprogramming medium and the control medium is shown in fig. 3, and fig. 3 is a population doubling graph of the P1-generation gall bladder precursor-like cells in the reprogramming medium and the control medium subcultured in examples 2-3 provided by the invention. Referring to fig. 3, it can be seen that the P1-generation gall bladder precursor-like cells can be expanded to at least 9 th generation in the reprogramming medium, but the P1-generation gall bladder precursor-like cells can be expanded to at most 6 th generation in the control medium, which indicates that the proliferation capacity of the P1-generation gall bladder precursor-like cells in the reprogramming medium is greater than that of the P1-generation gall bladder precursor-like cells in the control medium, and that the GSK-3 inhibitor LY2090314 in the reprogramming medium can promote the proliferation of the gall bladder precursor-like cells.

The cell doubling time of the P1-generation gall bladder precursor-like cells in the reprogramming medium and the control medium is shown in fig. 4, and fig. 4 is a graph of the cell doubling time of the P1-generation gall bladder precursor-like cells in the reprogramming medium and the control medium in examples 2-3 provided by the invention. Referring to FIG. 4, it is apparent that the time taken for doubling the number of days for the P1-generation gall bladder precursor-like cells to multiply in the reprogramming medium is shortened, which indicates that the GSK-3 inhibitor LY2090314 in the reprogramming medium can promote the proliferation of gall bladder precursor-like cells and ensure the activity of gall bladder precursor-like cells.

When the reprogramming culture medium is used for subculturing the P1-generation gall bladder precursor-like cells, sampling the P2-generation gall bladder precursor-like cells and the P3-generation gall bladder precursor-like cells in the subculture, imaging the P1-generation gall bladder precursor-like cells, the P2-generation gall bladder precursor-like cells and the P3-generation gall bladder precursor-like cells by an inverted microscope, wherein the imaging results are shown in FIG. 5, and FIG. 5 is an inverted microscope imaging diagram of the P1-generation gall bladder precursor-like cells, the P2-generation gall bladder precursor-like cells and the P3-generation gall bladder precursor-like cells in examples 2-3 provided by the invention. Referring to FIG. 5, it can be seen that the cell morphology of the P2 and P3 gall bladder precursor-like cells was not significantly changed compared to the P1 gall bladder precursor-like cells. The method shows that when the reprogramming culture medium is used for subculturing the gall bladder precursor-like cells, the proliferation of the gall bladder precursor-like cells is stable, and the activity of the gall bladder precursor-like cells is good.

Example 3-1

Pr-generation cholecystocytes obtained in example 1 were subcultured in DMEM+FBS medium in accordance with the procedure in examples 2-3, and subcultured with Pr-generation lung cells subcultured using the reprogramming medium in examples 2-3 to form a control group, and the culture procedure in this example was identical to that in example 2-1.

The dmem+fbs medium used consisted of: comprises 90% DMEM medium and 10% FBS medium by volume percentage of DMEM+FBS medium.

Wherein, DMEM culture medium is purchased from source culture, and the commodity number is L110KJ; FBS medium was purchased from Corning under the trade designation 35081-CV.

The growth curves of the gall bladder precursor-like cells cultured by using the reprogramming media in examples 2-3 and the growth curves of the gall bladder precursor-like cells cultured by using the DMEM+FBS media in the present example are shown in FIG. 6, and FIG. 6 is a graph of the cell growth of the Pr-generation gall bladder precursor-like cells subcultured in the reprogramming media and the DMEM+FBS media in example 3-1 provided by the present invention; referring to fig. 6, it can be seen that the in vitro culture of the gallbladder precursor-like cells cultured using the reprogramming medium can be expanded at least to the 9 th generation, while the in vitro culture of the gallbladder precursor-like cells cultured using dmem+fbs medium can be expanded at most to the 4 th generation, indicating that the proliferation capacity of the gallbladder precursor-like cells cultured using the reprogramming medium is much greater than that of the gallbladder precursor-like cells using the primary core medium, and the cell culture capacity of the reprogramming medium is much greater than that of the primary core medium.

Example 3-2

4 cholecyst tissues from different donors were selected and subjected to tissue digestion using the experimental procedure of example 1, and Pr-substituted cholecyst cells from these 4 cholecyst tissues were subcultured in vitro using reprogramming media according to the experimental procedure of examples 2-3. The proliferation curve of 4 Pr-generation cholecyst cells from different donors in the reprogramming culture medium is shown in FIG. 7, and FIG. 7 is the proliferation curve of 4 Pr-generation cholecyst cells from different donors in the reprogramming culture medium in example 3-2 provided by the invention. Referring to fig. 7, it is apparent that the reprogramming medium has excellent cell culturing ability, and that the proliferation of gallbladder precursor-like cells is stable when the in vitro culture of gallbladder precursor-like cells is performed using the reprogramming medium.

Example 4

The gallbladder precursor-like cells obtained in example 2-1 were subjected to differential analysis using flow cytometry.

Surface marker staining of the P1-generation gallbladder precursor-like cells obtained in example 2-1:

sampling the P1-generation gall bladder precursor-like cells in the example 2-1, sucking and removing the reprogramming culture medium, using 5mL of sterile PBS buffer solution for rinsing, then dripping 2mL of pancreatin digestion solution into a culture dish for digestion treatment to obtain a cell mixture, placing the cell mixture into a 15mL centrifuge tube, placing the centrifuge tube into a centrifuge, carrying out centrifugation treatment at the speed of 200g for 5 minutes, and discarding supernatant of the cell mixture after the centrifugation treatment to obtain a cell precipitate.

To the cell pellet was added 100. Mu.L of staining buffer to resuspend the cell pellet, and the resuspended cell pellet was transferred to 6 1.5mL centrifuge tubes, 100. Mu.L/tube. Respectively adding 5 mu L of the antibody to be tested into the 6 1.5mL centrifuge tubes, and blowing and uniformly mixing; after the centrifuge tubes were placed in a refrigerator at 2-8℃for 30 minutes, sterile PBS buffer was added to each centrifuge tube at a dose of 800. Mu.L/tube, and the centrifuge tubes were placed in a centrifuge and subjected to centrifugation at 300g for 5 minutes. After centrifugation, the supernatant was discarded, the centrifuge tubes were placed on a tube rack and incubated at 37℃for 20 minutes, 400. Mu.L of staining buffer was added to each centrifuge tube after incubation to resuspend the cell pellet, and then the cells were transferred to a flow tube, and the surface markers were detected by flow from the resuspended cell mixture.

The antibody names used for the surface marker flow detection are as follows: CD24, CD326, CD34, CD45 and HLA-DR/DP/DQ.

Wherein CD34 is purchased from abcam, cat No. ab81289; CD45 is purchased from abcam, cat No. ab40763; HLV-DR/DP/DQ is purchased from abcam under the trade designation ab7856; CD326 was purchased from BD Biosciences under the trade designation 565399; CD24 was purchased from abcam under the accession number ab290730; staining buffer was purchased from Biosharp under the accession number BL1136A; pancreatin digests were purchased from source cultures under the accession number S310JV.

Intracellular marker staining was performed on the P1-generation gallbladder precursor-like cells obtained in example 2-1:

sampling the P1 generation lung precursor-like cells in example 2-1, sucking and removing the reprogramming media, rinsing with 5mL of sterile PBS buffer solution, then dripping 2mL of pancreatin digestion solution into a culture dish for digestion treatment to obtain a cell mixture, placing the cell mixture into a 15mL centrifuge tube, placing the centrifuge tube into a centrifuge, carrying out centrifugation treatment at a speed of 200g for 5 minutes, and discarding supernatant of the cell mixture after the centrifugation treatment to obtain a cell precipitate.

1mL of the fixed membrane penetrating fluid is added into the cell sediment, the centrifuge tube is placed in a refrigerator at the temperature of 2-8 ℃ for standing for 50 minutes, 2mL of sterile PBS buffer solution is added into the centrifuge tube, the centrifuge tube is subjected to centrifugation at the rotating speed of 300g for 5 minutes, and after centrifugation, 500 microliters of staining buffer solution is added into the centrifuge tube for resuspension. The resuspended cell pellet was transferred to 4 1.5mL centrifuge tubes, 100. Mu.L/tube format. Adding 5 μl of the antibody to be tested into the 4 1.5mL centrifuge tubes, blowing, mixing, and placing the centrifuge tubes into a centrifuge tube containing 5% CO at 37deg.C ₂ Is allowed to stand for 30 minutes in the incubator of (C). After incubation, sterile PBS buffer was added to each centrifuge tube at a dose of 800. Mu.L/tube, and the tubes were placed in a centrifuge and centrifuged at 300g for 5 minutes. After centrifugation, the supernatant was discarded, the tubes were placed on a tube rack and incubated at room temperature for 30 minutes, and each tube after incubation was added with 400. Mu.L of staining buffer to resuspend the cell pellet, and then transferred to a flow tube, and the cell mixture after resuspension was subjected to flow detection of intracellular markers.

Wherein Sox9 is purchased from abcam under the trade designation ab208427; fixed transmembrane fluids were purchased from BD Biosciences under the accession number 554714; CK19 is purchased from abcam under the trade designation ab205445; pancreatin digest was purchased from source culture under accession number S310JV; staining buffer was purchased from Biosharp under the accession number BL1136A.

The results of the flow detection of the surface markers and intracellular markers of the P1-generation gall bladder precursor-like cells are shown as 8. FIG. 8 is a flow chart of the surface markers and the intracellular markers of the precursor cells of the gall bladder of generation P1 in example 4 provided by the present invention.

Referring to FIG. 8, example 2-1 positive expression of surface markers CD24 and CD326 and negative expression of surface markers CD34, CD45, HLA-DR/DP/DQ using P1 generation gall bladder precursor-like cells cultured in reprogramming medium; the intracellular markers SOX9 and CK19 were expressed positively in the P1-generation gall bladder precursor-like cells cultured using the reprogramming media in example 2-1. The P1-generation gallbladder precursor-like cells cultured using the reprogramming media in example 2-1 positively expressed the gallbladder precursor-related markers CD24, CD326, SOX9, CK19, and the expression rate of CD24/CD326/SOX9/CK19 was higher than 70%, and the P1-generation gallbladder precursor-like cells exhibited gallbladder precursor-like cell characteristics; in example 2-1, the P3-generation gall bladder precursor-like cells cultured by the reprogramming media negatively express CD34, CD45 and HLA-DR/DP/DQ, which shows that the cells do not express MHC-II antigens, the patient cannot recognize the cells through the MHC-II antigens of the immune system after transplanting the cells, the immune system of the patient cannot generate immune attack, and thus the possibility of rejection reaction of the patient is low; meanwhile, the cell does not express hematopoietic stem cell antigen CD34 and leukocyte common antigen CD45, and shows low immunogenicity.

Example 5

The present example provides a method for modeling a model of a mouse gall bladder injury, and uses the gall bladder precursor-like cells of example 2 to intervene in the model of a mouse gall bladder injury, and examines the effect of the gall bladder precursor-like cells of example 2 on gall bladder injury.

This example was modeled using 34 male C57BL/6 mice of 5 week old purchased from Peking Vitrehua laboratory animal technologies Co., ltd, the weight of which averaged 45g.

Before the model of the damage of the mouse gall bladder is molded, a mouse gall bladder damage pre-molding experiment is carried out, and the molding effect of the molding inducer is verified.

The operation steps of the mouse gall bladder injury pre-molding experiment are as follows:

(1) Four mice were randomly selected, of which 3 were randomly selected for modeling, and the remaining 1 were used as normal controls.

(2) Pouring 0.6 g/bottle of lincomycin hydrochloride into a beaker, adding 1mL of physiological saline into the beaker, vortex-dissolving until the lincomycin hydrochloride is completely dissolved in the physiological saline to obtain a lincomycin hydrochloride solution, subpackaging the lincomycin hydrochloride solution with the specification of 0.1 mL/tube, and storing the subpackaged lincomycin hydrochloride solution in a refrigerator at-80 ℃.

(3) Taking 3 lincomycin hydrochloride solutions packaged in the above way, respectively adding 2.9mL of physiological saline into each lincomycin hydrochloride solution, mixing uniformly by vortex to obtain 3 lincomycin hydrochloride solutions with the concentration of 0.033mg/mL as a molding reagent, and temporarily storing the molding reagent at the temperature of 2-8 ℃.

(4) 3 molding reagents were injected into 3 mice for molding through tail veins of the mice within 2 hours of completion of the molding reagent preparation, respectively. The injection frequency is 1 day and 1 time, 7 consecutive days, and the mice are put back into the cage for feeding after each injection.

(5) After 7 days, 4 mice were sacrificed and gallbladder was removed by laparotomy, and the gallbladder morphology of the 4 mice was observed under a microscope.

Compared with normal control mice, 3 mice used for molding have serious gallbladder wall fibrosis, obvious gallbladder atrophy and good molding effect of molding inducer, and can be used for molding of a mouse gall bladder injury model.

Wherein, the batch number of the lincomycin hydrochloride is H52020346.

Intervention experiments of gallbladder precursor-like cells on mouse gallbladder injury model:

cell preparation of gallbladder precursor-like cells:

taking out the mixture of a plurality of matrigel and gall bladder precursor-like cells from a liquid nitrogen tank, then placing the mixture on dry ice for transportation, immediately immersing the mixture in a water bath at 37 ℃ until the mixture is completely melted to liquid, obtaining a mixed solution, and shaking the water bath to uniformly distribute the mixed solution in the water bath. Taking out the whole mixed solution by using a pipette, adding the mixed solution into a 50mL sterile centrifuge tube, and placing the centrifuge tube in a separation mode In the heart machine, the centrifugal treatment was carried out at a rotational speed of 200g for 5 minutes. After the centrifugation, the supernatant in the centrifuge tube was discarded to obtain a mixed solution precipitate, and the mixed solution precipitate was diluted with physiological saline to a concentration of 5X 10 ⁶ And (3) carrying out filtration treatment on the gall bladder precursor-like cell solution by using a 70 mu m cell filter screen to obtain a cell preparation of gall bladder precursor-like cells.

The preparation method comprises the following steps of preparing lincomycin hydrochloride solution with concentration of 0.55mg/kg according to the preparation steps of dissolving lincomycin hydrochloride in a mouse gall bladder injury pre-molding experiment.

The mice were grouped as follows: randomly selecting 20 mice to perform modeling of a mouse gall bladder injury model, injecting lincomycin hydrochloride according to the concentration of 0.55mg/kg into the abdominal cavity of the mice for a single time to finish modeling, and randomly dividing the mice in a model group into two groups, namely a modeling group and a treatment group, wherein 10 mice in each group are respectively treated after 4 hours of injection of lincomycin hydrochloride solution into the mice in the model group; the remaining 10 mice were treated without any treatment and served as healthy groups.

The mice in the model and treatment groups were injected with cell preparations of gall bladder precursor-like cells according to the data in Table 1.

TABLE 1

Mice in the model and treatment groups were dosed via the gallbladder vein, the procedure for dosing was as follows:

a. Filling the mice into an induction box of a gas anesthesia machine for anesthesia by using isoflurane;

b. taking out the mice after losing consciousness, and shaving the abdomen of the mice;

c. fixing the mice on an operation plate, accessing a mask, and using isoflurane to maintain anesthesia;

d. coating iodic voltage on the abdomen of the mouse for disinfection;

e. cutting the abdominal skin and muscle layer of the mice, and freeing the spleens of the mice;

f. blowing the test reagent uniformly, and sucking 0.2mL of liquid medicine by using a syringe;

h. the needle of the injector is penetrated into the gall bladder of the mouse, the liquid medicine is injected, and the injection time is about 1 minute;

i. after the completion, the injector is withdrawn, and a puncture point of the gall bladder of the mouse is pressed by a dry cotton swab for stopping bleeding and preventing leakage of liquid;

j. closing the abdominal incision of the mouse by stitching layer by layer, and coating an iodophor on the operation part of the mouse for sterilization;

k. and (5) placing the mice on a heating pad for rewarming, and placing the mice back into a cage box for feeding after waking up.

Example 5-1

And (3) feeding mice of the model building group and the treatment group back to a cage box for 7 days after injection, observing the behavior, spirit and survival condition of the mice, recording the death time of the mice, and drawing the survival curve of the mice.

The survival curves of mice are shown in FIG. 9, and FIG. 9 is a graph showing the survival of mice in healthy, modular and treated groups in example 5-1 provided by the present invention. Referring to fig. 9, it can be seen that the survival rate of mice in the model group was reduced to 60% and the survival rate of mice in the treatment group was reduced to 75% on day 2 of rearing in the cage; on day 2 of cage rearing, the survival rate of mice in the model group was reduced to 35%, while the survival rate of mice in the treatment group was stabilized at 75%; on day 6 of cage rearing, the survival rate of mice in the model group was stabilized at 35%, while the survival rate of mice in the treatment group was reduced to 55%; on day 6 of cage rearing, the survival rate of mice in the model group was reduced to 5%, while the survival rate of mice in the treatment group was stabilized at 55%.

In general, the survival rate of mice in the model building group and the treatment group is reduced relative to that of mice in the normal group, and the survival rate of the mice in the model building group and the treatment group is lower than that of the mice in the normal group within 7 days of the feeding in a back cage, which means that the biliary sac tissues of the mice in the model building group and the treatment group are damaged, thereby influencing the survival rate of the mice; however, the survival rate of mice in the treatment group is lower slowly and higher than that of mice in the model group, which means that the damage of the gall bladder of the mice in the treatment group can be inhibited to a certain extent after the gall bladder precursor-like cells are used, and the damaged gall bladder tissues are repaired, so that the survival rate of the mice is improved.

Example 5-2

The mice of the model building group and the treatment group after 7 days of rearing the returned cage box are uniformly sacrificed, and the specific operation steps comprise: the mouse was injected with sultai 50 from its tail vein, and after deep anesthesia, the mouse was exsanguinated from the abdominal aorta, and finally the cervical dislocation was sacrificed. Among them, sultai 50 was purchased from vitamin, cat No. 785T.

The gall bladder of the mice in the model building group, the treatment group and the healthy group is extracted by laparotomy, the gall bladder of the mice in the model building group, the treatment group and the healthy group are observed visually and microscopically, and the photographing result is shown in fig. 10, and fig. 10 is an external view of the anatomical tissues of the mice in the healthy group, the model building group and the treatment group in the embodiment 5-2 provided by the invention. Referring to fig. 10, it is clear that the mouse gall bladder mucosa gland of the model group has hypersecretion, the gall bladder mucosa submucosa and the lamina propria edema have a large amount of exudation fibers, the gall bladder mucosa beryllium wall and the hidden which is sunk in the lamina propria are increased, the gall bladder mucosa sink deep to Luoa (Roki-tan sky-ascoff) formed by the muscular layer, the epithelial cell hyperplasia is obvious, the fibrosis of the mouse gall bladder wall of the model group is serious, and the gall bladder atrophy is obvious; however, the mice in the treatment group have lighter gallbladder fibrosis, relatively smaller gallbladder injury and no obvious hyperplasia of epithelium, which proves that the precursor-like cells of the gallbladder have the function of inhibiting gallbladder wall fibrosis and epithelial hyperplasia and can inhibit gallbladder injury.

Examples 5 to 3

In example 5-2, after gallbladder was removed by laparotomy in mice of the model, treatment group and healthy group, the model, treatment group and healthy group were extracted and biochemically analyzed by a biochemical analyzer (manufacturer: roche medical treatment, model: cobas 8000) to obtain experimental results as shown in FIGS. 11 to 14.

FIG. 11 is a graph showing total cholesterol levels in healthy, modular and treated mice of examples 5-3 provided by the present invention. The ordinate of fig. 11 shows the total cholesterol value in the bile, and referring to fig. 11, it is understood that the total cholesterol content in the bile of mice in the model and treatment group is higher than that in the healthy group, which indicates abnormal lipid metabolism in the mice in the model and treatment group, and indicates damage to the tissues of the cholesterol in the mice in the model and treatment group; the total cholesterol content in the bile of the mice in the treatment group is lower than that in the modeling group, the digestion capacity of the mice to high-fat substances is improved, and the method shows that the gall bladder precursor-like cells can inhibit the gall bladder injury of the mice and repair the damaged gall bladder tissues of the mice.

FIG. 12 is a graph showing total bile acid content of mice in healthy, modular and treated groups of examples 5-3 provided by the present invention. Referring to fig. 12, it can be seen that the total bile acid in the bile of the mice in the model and the treatment group has a higher value than that in the healthy group, which indicates that the bile of the mice in the model and the treatment group cannot be concentrated and cholestasis occurs, indicating that the biliary sac tissue of the mice in the model and the treatment group is damaged; the total bile acid value in the bile of the mice in the treatment group is lower than that in the modeling group, so that the accumulation condition of the bile juice of the mice is slowed down, which indicates that the use of the gall bladder precursor-like cells can inhibit the damage of gall bladder of the mice and repair the damaged gall bladder tissues of the mice.

FIG. 13 is a graph showing total bile acid content of mice in healthy, modular and treated groups of examples 5-3 provided by the present invention. The ordinate of fig. 13 shows the total bilirubin values in bile, and referring to fig. 13, it is clear that the total bilirubin values in bile of mice in the model group and the treatment group are higher than those in the healthy group, which indicates that there is gall bladder stones in the mice in the model group and the treatment group, which indicates that the tissues of the gall bladder of the mice in the model group and the treatment group are damaged; the total bilirubin in the bile of mice in the treatment group has a lower value than that of the modeling group, and the occurrence probability of the gall bladder stones of the mice is reduced, which indicates that the gall bladder precursor-like cells can be used for inhibiting the gall bladder injuries of the mice and repairing the damaged gall bladder tissues of the mice so as to avoid the gall bladder injuries of the mice caused by the gall stones. Cholestasis promotes lipid metabolism of the liver.

FIG. 14 is a graph of direct bilirubin levels in healthy, modular and treated mice of examples 5-3 provided herein. The ordinate of fig. 14 shows the values of direct bilirubin in bile, and referring to fig. 14, it is clear that the values of direct bilirubin in bile of mice in the model group and the treatment group are higher than those in the healthy group, which indicates that cholestasis occurs in the mice in the model group and the treatment group, and indicates that the tissues of the biliary sac of the mice in the model group and the treatment group are damaged; the total bile acid value in the bile of the mice in the treatment group is lower than that of the modeling group, and the accumulation condition of the bile juice of the mice is slowed down, which indicates that the use of the gall bladder precursor-like cells can inhibit the damage of gall bladder of the mice, repair the damaged gall bladder tissues of the mice and improve the lipid metabolism of liver of the mice.

In combination with examples 5-1 to 5-3, it was concluded that the gallbladder precursor-like cells cultured in the reprogramming media of examples 2-3 can inhibit mouse gall bladder injury and repair damaged gall bladder tissue in mice when treating acute injury of the mouse gall bladder.

While embodiments of the present invention have been described in detail hereinabove, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. It is to be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the following claims. Moreover, the invention described herein is capable of other embodiments and of being practiced or of being carried out in various ways.

Claims

1. A method for preparing a gall bladder precursor-like cell, comprising the steps of:

2. A method of preparing gallbladder precursor-like cells according to claim 1, wherein in S1, the step of obtaining primary gallbladder cells after digestion and separation of the gallbladder tissue comprises:

3. A method of preparing gallbladder precursor-like cells according to claim 2, wherein in said 11, the components of said cell digestive juice comprise, in volume percent of said cell digestive juice: 88% -96% of sterile PBS buffer solution, 3% -7% of pancreatin digestive solution and 1% -5% of type IV collagenase.

4. A method of preparing a gall bladder precursor-like cell according to claim 1, wherein in S2, the reprogramming media comprises basal media, nutritional supplements, growth factors, TGF- β signaling inhibitors, wnt signaling pathway activators, and ROCK kinase inhibitors.

5. A method of preparing gall bladder precursor-like cells according to claim 4, wherein the growth factor is present in an amount of 15-30 nanograms per milliliter, the ROCK kinase inhibitor is present in an amount of 5-20 micromolar, the Wnt signaling pathway activator is present in an amount of 1.2-6.7 micromolar, the TGF- β signaling inhibitor is present in an amount of 0.4-3.3 micromolar, and the nutritional supplement is present in an amount of no more than 10% by volume based on the volume of basal medium.

6. A method of preparing gall bladder precursor-like cells according to claim 1, wherein in said S2, said reprogramming media further comprises a GSK-3 inhibitor comprising LY2090314.

7. A method of preparing a gallbladder-precursor-like cell according to claim 1, wherein the gallbladder-precursor-like cell positively expresses at least one signature marker.

8. A method of preparing a gall bladder precursor-like cell according to claim 7, wherein the expression rate of the signature marker is not less than 50% and not more than 99%.

9. A method of preparing a gall bladder precursor-like cell according to claim 7, wherein at least one of said signature markers comprises at least one of CD24, CD326, CK19 and Sox 9.

10. A method of preparing a gall bladder precursor-like cell according to claim 1, wherein said gall bladder precursor-like cell negatively expresses at least one MHC class molecule, at least one of said MHC class molecules comprising at least one of HLA-DR/DP/DQ.

11. A method of preparing a gallbladder-precursor-like cell according to claim 10, wherein the gallbladder-precursor-like cell is also negative for the expression of at least one of CD34, CD 45.

12. Use of a gallbladder precursor-like cell according to claim 1, wherein said gallbladder precursor-like cell is prepared by a method of preparing a gallbladder precursor-like cell according to claim 1, for intervention in an in vivo animal model.

13. The use of gallbladder-precursor-like cells according to claim 12, wherein said in vivo animal model comprises a mouse biliary injury model.