CN117050934A - Preparation method of mouse prostate organoid and primary in situ prostate cancer animal model - Google Patents

Preparation method of mouse prostate organoid and primary in situ prostate cancer animal model Download PDF

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CN117050934A
CN117050934A CN202311310333.XA CN202311310333A CN117050934A CN 117050934 A CN117050934 A CN 117050934A CN 202311310333 A CN202311310333 A CN 202311310333A CN 117050934 A CN117050934 A CN 117050934A
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prostate
organoid
mouse
cells
organoids
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CN117050934B (en
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杨璐
李一璠
谭平
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West China Hospital of Sichuan University
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West China Hospital of Sichuan University
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Abstract

The application belongs to the field of biotechnology, and provides a preparation method of a mouse prostate organoid and a primary in-situ prostate cancer animal model, wherein the mouse prostate cell organoid is separated, digested and cultured in vitro, and simultaneously, the organoid is subjected to gene editing combination by taking different clinical hot spot mutation genes as backgrounds, and then is injected into the prostate in-situ tissue of a mouse, so that the prostate in-situ tissue of the mouse is developed into tumors; the mouse prostate tumor model constructed by the application can simulate the process of transforming normal cells into tumor cells in human body due to genetic change, can dynamically represent the process of generating and developing tumors, and is closer to the real situation in aspects of gene level, tumor microenvironment and the like. The method can efficiently prepare the prostate cancer model which is closer to the characteristics of the prostate cancer and meets the clinical research requirements, and the model can provide a favorable tool in the research fields of exploring the occurrence and development mechanism of the prostate cancer and the drug resistance characteristics, searching and optimizing the possible treatment modes of the new prostate cancer, and the like.

Description

Preparation method of mouse prostate organoid and primary in situ prostate cancer animal model
Technical Field
The application belongs to the technical field of biology, and particularly relates to a preparation method of a mouse prostate organoid and a primary in-situ prostate cancer animal model.
Background
Prostate cancer is one of 6 cancers with increased incidence rate of male cancers in China, and the incidence rate of the prostate cancer is 8% -10% per year, so that male cancers in China are seriously affected. The mouse prostate organoid culture and the establishment of a tumor model provide a possible technical platform for researching the occurrence and the development of the prostate cancer and exploring various treatment means.
The existing animal models of the prostate cancer mainly comprise a subcutaneous transplantation model, a patient-derived xenograft model (PDX), an in-situ transplantation model, a gene mouse model and a cancerogenic substance induction model. The subcutaneous transplantation model has the advantages of low cost, easy acquisition of tumor cell lines and the like, but a large number of tumor cell lines need to be transplanted subcutaneously in an immunodeficiency mouse, the cell lines are difficult to characterize tumor characteristics of patients, and subcutaneous tumors cannot reflect tissue ecology of interaction of prostate tumor cells and matrixes. The patient source xenogenesis planting model (PDX) has the advantages of approaching to the individual patient, reflecting individual disease characteristics, retaining tumor heterogeneity and the like, but the PDX model needs to be transplanted subcutaneously, and the success rate of the model is greatly dependent on the specimen itself due to the difference of the patient, the specimen material difference, and the raising cost of the severe immunodeficiency mice is too high and the difficulty is very high. Although the in-situ transplantation model can simulate the occurrence and development processes of human diseases, the operation difficulty is very high due to the small volume of the prostate tissue of the mice, and special equipment is required for monitoring the tumor formation. The background of the gene mouse model is very clear, the tumorigenic position can also occur in situ in the prostate tissue, but the model has higher cost and the preparation and propagation period of the gene mouse is overlong. The carcinogen induction model is largely dependent on the genetic background of mice, the individual tumors of the mouse prostate cancer model which induces formation are greatly different, the rate of tumor formation is low, and the model is difficult to simulate the tumor formation of patients.
Disclosure of Invention
In order to solve the problems, the application provides a preparation method of a mouse prostate organoid and a primary in situ prostate cancer animal model. The in-situ primary mouse prostate tumor model constructed by the application can simulate the process of converting normal cells into tumor cells in a human body due to genetic change, can dynamically represent the process of tumorigenesis and development, and is closer to the actual conditions of tumorigenesis and development in aspects of gene level, tumor microenvironment, tumor development, pathophysiology and the like.
The technical scheme of the application is as follows:
a method for culturing a prostate cell organoid of a mouse comprising the steps of:
(1) Taking prostate cells of mice for primary culture;
(2) Culturing the primary cells obtained by culture into organoids;
the operation steps for obtaining the prostate cells of the mice are specifically as follows:
(S1) euthanizing the mice, and repeatedly spraying the mouse carcasses with 75% ethanol;
(S2) fully exposing the viscera of the abdominal cavity of the mouse, and fully separating the bladder, testis and seminal vesicle of the mouse;
(S3) repeatedly rinsing the bladder, testis and seminal vesicle in pre-chilled PBS, followed by removal of the seminal vesicle, urethra and bladder, leaving only the anterior, middle, posterior and bilateral leaf tissue of the prostate;
(S4) shearing the prostate tissue obtained in the step (S3) into tissue blocks;
(S5) subjecting the obtained tissue mass to digestion treatment to obtain a tissue suspension;
and (S6) sequentially filtering and centrifuging the tissue suspension, and removing supernatant to obtain the primary cells of the prostate of the mice.
In the step (S5), adding a collagenase mixture for digestion treatment, wherein the collagenase mixture is a mixture formed by collagenase I and collagenase IV according to a volume ratio of 2:1;
the specific operation of the digestion treatment is as follows:
resuspension the tissue pieces with 15-20ml collagenase mixture in a 50ml centrifuge tube, digestion for 30-40min at 37 ℃ shaker at 220rpm, with upside down and/or blow with gun head 30-50 times every 10min;
the collagenase mixture was neutralized with an equal volume of DMEM and 10% fbs mixture and the digestion process was terminated.
The present inventors have found in long-term studies that by using collagenase i+iv in combination with Trypsin digestion, the following advantages are achieved: 1. collagenase (Collagenase) disperses cells by hydrolyzing the interstitial prolines of the cells. Collagenase has a strong digestion effect on collagen, and it has only a digestion effect on the cell matrix but has little damage to cells, so it is suitable for digestion and separation of fibrous tissues and harder cancer tissues. The calcium and magnesium ions and serum do not affect collagenase activity and digestion. The prostate tissue mainly comprises epithelial cells, stromal cells and the like, the epithelial tissue and the cell stroma can be effectively separated by using collagenase I+IV, the collagenase has small injury to the cells, and the aim of efficiently separating the epithelial tissue can be fulfilled under the help of mechanical force provided by vibration and blowing. 2. Trypsin (Trypsin) acts on peptide tendons linked to lysine or arginine to remove intercellular mucins and glycoproteins, and affects the cytoskeleton, thereby isolating cells. When the collagenase treated tissue mass has been substantially digested into an epithelial tissue mass, continued digestion with trypsin may loosen the cells within the tissue and more greatly digest them into individual cells, and the mechanical force provided by blowing during this process is equally important.
In step (2), the specific procedure for culturing the primary cells into organoids is as follows:
(a) Adding the obtained primary cells into matrigel, re-suspending on ice, and planting on a cell culture plate;
(b) Transferring the cell culture plate into a incubator for solidification;
(c) And adding an organoid culture medium into the holes of the cell culture plate, and culturing to obtain the organoid.
In the step (a), ACK is added to the obtained primary cells to be resuspended on ice, after standing, centrifugation is carried out, supernatant fluid is removed, and matrigel is added to be resuspended on ice; mixing the primary cells with matrigel with the protein concentration of 8-12 mg/ml;
in step (c), after the organoid medium is added, PBS is also added to adjacent wells to prevent evaporation of the medium.
The organoid culture medium is prepared based on DMEM/F12 by adding the following cytokines:
leukocyte antigen HLA-B27 50-fold dilution, epidermal Growth Factor (EGF) 50-100ng/ml, human recombinant R-spondin-1 250-1000ng/ml, A83-01-200-500 nM, dihydrochloride (Y-27632) 10-50 mu M, L-alanyl-L-glutamine (Glutamax) 100-fold dilution, dihydrotestosterone (DHT) 1000-fold dilution, N-acetylcysteine (N-acetylcysteine) 1-2mM, GMP grade recombinant human Noggin 100-1000ng/ml, keratinocyte factor (FGF 10) 500ng/ml.
In the selection of the culture medium components, since the growth and development of the prostate is required to depend on androgens and metabolites thereof as hormone-dependent tissues, DHT (dihydrotestosterone) is added on the basis of a basic organoid culture medium, so that the growth of the in-vitro prostate organoid is supported, and the growth and differentiation processes of in-vivo prostate cells are better simulated. In addition, the culture medium for culturing the prostanoid in vitro is free from adding cytokines such as N2, wnt3a, and Gastin, so that the cost is reduced, and the preparation is simple.
The method for constructing the in-situ primary prostate cancer animal model based on the prostate cell organoid of the mouse comprises the following steps:
(A) Performing gene editing on the organoids;
(B) Injecting the organoid after gene editing into animal prostate tissue to obtain the in-situ primary prostate cancer animal model.
The animal prostate tissue may be human or mouse prostate tissue, or may be prostate tissue of other animals.
Before the organoids are subjected to gene editing, the organoids obtained by culture are also subjected to subculture, specifically:
(A1) Removing the culture medium in the organoids obtained by culture, and then adding recombinant digestive enzymes;
(B1) Transferring into a centrifuge tube, and incubating under the water bath condition of 37 ℃;
(C1) Repeatedly blowing and beating organoids, and centrifuging;
(D1) Removing the supernatant, adding matrigel into the obtained cells, re-suspending on ice, and then planting the cells on a cell culture plate;
(E1) Transferring the cell culture plate into an incubator for solidification;
(F1) Organoid medium was added to wells of the cell culture plates for subculturing organoids.
The gene editing operation for the organoids is specifically as follows:
(A2) Removing the culture medium in the organoids obtained by culture, and then adding recombinant digestive enzymes;
(B2) Transferring into a centrifuge tube, and incubating under the water bath condition of 37 ℃;
(C2) Repeatedly blowing and beating organoids, and centrifuging;
(D2) Removing supernatant, adding virus into the obtained cells to carry out resuspension, and then planting the cells in a cell culture plate;
(E2) After centrifugation at 2000rpm at 32℃for 1 hour, incubation at 37℃was carried out;
(F2) Transferring the organoids into a centrifuge tube, and centrifuging at 1500rpm for 10min;
(G2) Removing the supernatant, adding matrigel for resuspension, and then planting on a cell culture plate;
(H2) Transferring the cell culture plate into an incubator for solidification;
(F2) Organoid medium was added to wells of cell culture plates for organoid gene editing.
The gene editing is specifically mutation Trp53 and Pten genes, and the cMyc genes are expressed in a high degree. According to the application, by using the CRISPR-Cas9 technology, trp53 and Pten genes are knocked out through slow virus infection, and Myc genes are over-expressed through a retrovirus infection method, so that the gene background is clear and definite, the success rate of tumor formation is high, and the time period is short.
Preferably, the gene editing according to the present application further comprises the transfer of a fluorescence marker gene into the organoid.
The in-situ primary prostate animal model constructed by the method is applied to drug screening, drug toxicity test or animal model used as endocrine treatment test.
The beneficial effects of the application are as follows:
the application provides a preparation method of a mouse prostate organoid and a primary in-situ prostate cancer animal model, which are characterized in that the mouse prostate organoid is isolated, digested and cultured in vitro, and simultaneously, the organoids are subjected to gene editing combination by taking different clinical hot spot mutation genes as backgrounds and then injected into the prostate in-situ tissue of the mouse, so that the prostate in-situ tissue of the mouse is developed into tumors; the in-situ primary mouse prostate tumor model constructed by the application can simulate the process of converting normal cells into tumor cells in a human body due to genetic change, can dynamically represent the process of tumorigenesis and development, and is closer to the actual conditions of tumorigenesis and development in aspects of gene level, tumor microenvironment, tumor development, pathophysiology and the like. The method can efficiently prepare the prostate cancer model which is closer to the characteristics of the prostate cancer and meets the clinical research requirements, and the model can provide a favorable tool in the research fields of exploring the occurrence and development mechanism of the prostate cancer and the drug resistance characteristics, searching and optimizing the possible treatment modes of the new prostate cancer, and the like. Compared with a genetic engineering tumor animal model, the method provided by the application has the advantages that the time consumption is short, the death of the animal before the tumor formation is not caused, and the tumor formation rate is high; compared with an animal model for transplanting tumor, the method has the characteristics of clear driving factors of tumor generation, more true organ microenvironment and the like.
Drawings
In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic representation of a three-dimensional cultured prostate organoid;
FIG. 2 shows a light-microscopic view of a prostate organoid; p3 in the figure represents the organoid obtained after the third passage after tissue ablation to the primary organoid (P0),
FIG. 3 shows a light-passaging map of a prostate organoid; p1 in the figure represents the organoid obtained after the first passage after tissue ablation to the primary organoid (P0), and P2 represents the organoid obtained after the second passage after tissue ablation to the primary organoid (P0);
FIG. 4 shows the fluorescence detection for the prostate organoid gene editing;
FIG. 5 shows the editing of T7 endonuclease I assays for mouse organogene;
FIG. 6 shows tumor growth observed over time after prostate surgery in mice;
FIG. 7A shows a photograph of a mouse prostate tumor tissue formed as an organoid transplant;
FIG. 7B is a graph of HE staining of prostate tumor tissue of the mice shown in FIG. 7A.
Description of the embodiments
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, based on the examples herein, which are within the scope of the application as defined by the claims, will be within the scope of the application as defined by the claims.
The following describes the above technical scheme in detail with reference to specific embodiments.
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Some of the english abbreviations in the present application are explained as follows:
DMEM: is a medium with very wide application, can be used for culturing a plurality of mammalian cells and is purchased from GIBCO company.
DMEM/F12: is F12 medium and DMEM medium combined according to a ratio of 1:1, and is called DMEM/F12 medium. Combines the advantages of the F12 containing rich components and the DMEM containing high-concentration nutrients. Purchased from GIBCO.
Matrigel: the main components of the composition are laminin, type IV collagen, nidogen, heparan sulfate glycoprotein and the like, and the composition also comprises growth factors, matrix metalloproteinase and the like. Purchased from BD company.
B27, a B27 supplement, a commercially available product, can be used to formulate the culture medium. B27 supplement was provided as a 50-fold liquid concentrate comprising, among other ingredients, biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol acetate, sodium selenite, triiodothyronine (T3), DL-alpha-tocopherol (vitamin E), albumin, insulin, and transferrin. Purchased from Life Technologies company.
N-acetylcysteine: n-acetylcysteine, purchased from Sigma company.
EGF: epidermal growth factor, commercially available from R & D company.
Noggin: cell growth protein fraction, commercially available from Peprotech company.
R-spondin 1: human cell growth encodes a protein, commercially available from Peprotech.
A83-01: TGF-beta inhibitors were purchased from Tocris Bioscience company.
FGF10: fibroblast growth factor, purchased from Peprotech corporation.
Nicotinamide: nicotinamide, purchased from Sigma.
Y-27632: ROCK-specific on-way blockers were purchased from Abmole Bioscience company.
Glutamax: commercial cell culture additives, purchased from GIBCO.
TrypLE: recombinant digestive enzymes for dissociation of adherent mammalian cells were purchased from GIBCO.
Trypsin: trypsin is widely used for cell digestion, conventional cell culture passaging and primary tissue digestion, purchased from GIBCO.
EXAMPLE 1 organoid culture
The embodiment provides a method for culturing a kind of organ, which comprises the steps of obtaining fresh prostate tissue cells, carrying out the combination of collagenase I and IV and pancreatin to be single cells, and culturing the prostate tissue organoid under in vitro 3D culture conditions, wherein a process flow chart is shown in figure 1.
1, culture medium for prostate organoids of mice (composition see Table 1)
Collagenase I+IV (GIBCO)
3. 0.25%Trypsin-EDTA（GIBCO）
Matrigel (corDing)
5.DPBS basic（gobo）
6.1× ACK
7.75% ethanol solution
8.DMEM（GIBCO）+10%FBS。
TABLE 1 composition of the mouse prostate organoid medium (prepared with DMEM/F12)
Cytokines and methods of use Optional concentration ranges Cytokines and methods of use Optional concentration ranges
B27 50 times dilution EGF 50~100ng/ml
R-spondin 1 250~1000ng/ml A83-01 200~500nM
Y-27632 10~50uM GlutaMAX 100-fold dilution
DHT Dilution by 1000 times N-acetylcysteine 1~2mM
Noggin 100~1000ng/ml FGF10 500ng/ml
The required apparatus is as follows:
1. scissor and flat forceps
2 six hole plates
3.50 ml BD tube
4.48 orifice plate
5 ice chest
6, absorbing water paper
7.70 um cell sieve
The operation method comprises the following steps:
euthanized mice, repeatedly spraying the mouse carcasses with 75% ethanol;
placing the mice on absorbent paper, placing the absorbent paper in an ultra-clean cell workbench, fully exposing organs of the abdominal cavity by scissors, finding organs of the reproductive system of the male mice such as bladder, testis, seminal vesicle gland and the like below the abdominal cavity, fully separating by flat forceps, and cutting ureters and urethra by scissors;
repeatedly rinsing the male mouse reproductive system organs in precooled PBS, and removing the other tissues such as seminal vesicle glands, urethra, bladder and the like by forceps under a stereoscopic dissecting microscope, and only leaving the front, middle, rear and two side lobes of the prostate; the forceps should be carefully used to separate the prostate under a stereoscopic dissecting microscope to prevent the mixing of residual tissues;
shearing the prostate tissue with scissors to a size of 1-3mm 3 Organizing the blocks;
5. resuspending the sheared prostate tissue with 15-20ml collagenase (collagenase i2±1 mg/ml+collagenase in1±0.5 mg/ml) in a 50ml BD tube, shaking at 37 ℃ and speed 220rpm, digesting for 30-40min, taking out upside down every ten minutes and/or blowing with 1ml gun head 30-50 times;
6. after observing that the tissue mass is obviously reduced or the cell suspension is obviously turbid, sucking 0.1-0.2ml of the tissue suspension into a cell culture dish, and observing the separation condition of the digestive cells under an optical microscope after standing for 1-2 min;
if it is observed that the digested cells are still in the form of a cell mass, the following procedure can be continued: 1) Centrifugation at 1500rpm for 10 mins; 2) Removing supernatant, re-suspending digested prostate tissue with 10-15ml trypsin in 50ml BD tube, digesting for 10-15min in 37 degree water bath, taking out every five minutes, inverting and/or blowing with 1.5ml gun head for 20-40 times; 3) The digestion process was terminated by neutralizing trypsin with an equal volume of dmem+10% fbs; 4) Passing the tissue suspension obtained above through a 70um sieve; 5) Centrifugation at 1500rpm for 5mins; 6) Removing supernatant, adding 120ul matrigel on ice to re-suspend cells, seeding in 48 well plate, 25-30 ul/well; 7) Solidifying for 15mins in a 37 DEG incubator; 8) 150-170ul of culture medium is added to each well, and 0.5-1ml of PBS is added to the adjacent wells to prevent the culture medium from evaporating; 9) Daily observations indicate that the medium turns dark or yellow, either by changing to new medium or by passage in combination with the cell growth density and status.
7. Neutralizing collagenase i+iv with an equal volume of dmem+10% fbs, and terminating the digestion process;
8. passing the tissue suspension obtained above through a 70um sieve;
centrifugation at 9.1500 rpm for 5mins;
10. the supernatant was removed, the cells were resuspended in ice with 5ml of ACK and left to stand for 3mins; (this step can be omitted if no obvious red blood cells are visible)
Centrifugation at 11.1500 rpm for 5mins;
12. removing supernatant, adding 120ul matrigel on ice to re-suspend cells, seeding in 48 well plate, 25-30 ul/well; melting matrigel in a 4-degree refrigerator 20min in advance, and performing the whole operation on ice as much as possible;
13. solidifying the cell 48-hole culture plate in a 37-degree incubator for 15mins;
14. 150-170ul of culture medium is added to each well, and 0.5-1ml of PBS is added to the adjacent wells to prevent the culture medium from evaporating;
15. the decision to change the medium or to carry out the passaging process is based on the color of the medium and the cell status.
The resulting organoid effect is shown in fig. 2, from which it can be seen that: the normal prostate organoid "vacuole" morphology and its HE-stained appearance were similar.
EXAMPLE 2 organoid passaging
The required reagents:
1. mouse prostate organoid medium (same table 1)
2.TryplE(GIBCO)
3. Matrigel (burning)
4.DPBS basic(GIBCO)
The required apparatus is as follows:
1.50 ml BD tube
2.15 ml BD tube
3.48 orifice plate
4. Ice chest.
The specific operation method comprises the following steps:
1. sucking off the culture medium on the organoids, adding 1ml of Tryple to each well; the passages are distinguished according to different purposes, if the passages are used for amplifying the organoids, the organoids are required to be as dense as possible, the organoids are beneficial to growth, and if the passages are used for photographing and staining the organoids, the organoids are required to be as sparse as possible, and the organoids are beneficial to differentiation;
2. mashing matrigel with 1ml gun tip, and sucking all into 15ml BD tube; the choice of 15ml bd tube and 50ml bd tube should depend on the number of organoids, if the number is large, 50ml bd tubes should be used, and the number is small with 15ml bd tubes;
incubating for 15mins in a water bath kettle at the temperature of 3.37 ℃;
4. repeatedly blowing the organoids with a 10ml pipette;
centrifuging at 5.1500 rpm for 10 mins;
6. removing supernatant, adding 120ul matrigel on ice to re-suspend cells, seeding in 48 well plate, 30 ul/well;
7. solidifying for 15mins in a 37 DEG incubator;
8. 150-170ul of culture medium is added to each hole, PBS is added around the culture medium, and evaporation to dryness is prevented;
9. daily observations were made that the medium turned yellow and replaced with new medium or passaged.
The passaging effect is shown in fig. 3, from which it can be seen that: the cell density after passage treatment is moderate and the cell state is good.
Example 3 organoid Gene editing
The required reagents:
1. mouse prostate organoid medium (same table 1)
2.TryplE（GIBCO）
3. Matrigel (burning)
plenti-Virus (Cas 9 gene-carrying vector, and sgRNA-expressing vector, which targets Trp53 and Pten genes, causes frame shift mutation of Trp53 gene, causes frame shift mutation of Pten gene);
retro-Virus (carrying cMyc protooncogene, and D-luciferase reporter gene, such that cMyc protooncogene is highly expressed);
6. polycuramide (4 mg/ml)
7.DPBS basic(GIBCO)
The required apparatus is as follows:
1.50 ml BD tube
2.15 ml BD tube
3.48 orifice plate
4.24 orifice plate
5. Ice box
The specific operation method comprises the following steps:
1. sucking off the culture medium on the organoids, adding 1ml of Tryple to each well;
2. mashing matrigel with 1ml gun tip, and sucking all into 15ml BD tube;
incubating for 15mins in a water bath kettle at the temperature of 3.37 ℃;
4. repeatedly blowing the organoids with a 10ml pipette;
centrifugation at 5.1500 rpm for 10 mins.
6. The cell organoids were resuspended with lentiviruses or retroviruses according to 1: the addition of the polybrene (1 ul of polybrene added to 1ml of virus liquid) was made in a 1000 ratio and placed in a 24-well plate; before infection, the number of cells should be ensured as much as possible, because a considerable number of cells die due to infection; the virus is preferably filtered by 0.22um filter membrane during infection to avoid pollution. Or centrifuging at 800rpm to remove 293T cells when virus is packaged; the organoids should be planted as densely as possible after infection is completed so that organoids grow;
centrifugation at 32℃for 1 hour at 7.2000 rpm;
incubating in an 8.37 degree incubator for 4 hours;
9. organoids were harvested into 15ml small BD tubes;
10.1500rpm 10mins centrifuging;
11. removing supernatant, adding 120ul matrigel on ice to re-suspend cells, seeding in 48 well plate, 30 ul/well;
12. solidifying for 15mins in a 37 DEG incubator;
13. 150-170ul of culture medium is added to each hole, PBS is added around the culture medium, and evaporation to dryness is prevented;
fluorescence was observed for 14.48 hours.
Identification result:
the fluorescence identification of the organoid gene editing is shown in fig. 4, the left image is a light-microscopic image, the organoid morphology of the prostate after editing under the common light-microscopic image is changed compared with that of the normal prostate, and the streak part shown by the arrow in the right image is a "successfully edited prostanoid", so that the Pten gene is successfully edited into the prostanoid; the enzyme digestion identification of the gene-edited T7 endonuclease I (namely T7E 1) is shown in FIG. 5, the molecular weight of the marker is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, NC is a negative control group, pten in the PCR product is not mutated, so that the T7 endonuclease cannot identify mutation sites to work; sgPten is an experimental group, and shows that Pten genes in PCR products are successfully edited and mutated, so that T7 endonuclease successfully recognizes mutation sites to work, and Pten genes are successfully edited.
EXAMPLE 4 animal model construction
Performing organoid culture, passage, gene editing sequentially by the method of examples 1-3, transplanting the obtained organoid with gene editing into mice prostate tissue in situ, monitoring tumor size and position (as shown in FIG. 6) by in vivo imaging technique, and waiting for tumor signal value to reach 10 7 And above or when the obvious hard lump tissue is touched from the implantation position, the mice are sacrificed, and the prostate tissue is directly observed and HE stained and observed, as shown in fig. 7A and 7B, and the pathological results show that the prostate cancer and the tumor cell nucleus have high heterogeneity.
The foregoing is merely illustrative of the present application, and the present application is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims (10)

1. A method for preparing a prostate cell organoid of a mouse, comprising the steps of:
(1) Taking prostate cells of mice for primary culture;
(2) Culturing the primary cells obtained by culture into organoids;
the operation steps for obtaining the prostate cells of the mice are specifically as follows:
(S1) euthanizing the mice, and repeatedly spraying the mouse carcasses with 75% ethanol;
(S2) fully exposing the viscera of the abdominal cavity of the mouse, and fully separating the bladder, testis and seminal vesicle of the mouse;
(S3) repeatedly rinsing the bladder, testis and seminal vesicle in pre-chilled PBS, followed by removal of the seminal vesicle, urethra and bladder, leaving only the anterior, middle, posterior and bilateral leaf tissue of the prostate;
(S4) shearing the prostate tissue obtained in the step (S3) into tissue blocks;
(S5) subjecting the obtained tissue mass to digestion treatment to obtain a tissue suspension;
and (S6) sequentially filtering and centrifuging the tissue suspension, and removing supernatant to obtain the primary cells of the prostate of the mice.
2. The method for preparing a prostate cell organoid of a mouse according to claim 1, wherein in the step (S5), a collagenase mixture is added for digestion treatment, and the collagenase mixture is a mixture of collagenase i and collagenase iv in a volume ratio of 2:1;
the specific operation of the digestion treatment is as follows:
resuspension the tissue pieces with 15-20ml collagenase mixture in a 50ml centrifuge tube, digestion for 30-40min at 37 ℃ shaker at 220rpm, with upside down and/or blow with gun head 30-50 times every 10min;
the collagenase mixture was neutralized with an equal volume of DMEM and 10% fbs mixture and the digestion process was terminated.
3. The method of claim 1, wherein in step (2), the primary cells are cultured as organoids by the following steps:
(a) Adding the obtained primary cells into matrigel, re-suspending on ice, and planting on a cell culture plate;
(b) Transferring the cell culture plate into a incubator for solidification;
(c) And adding an organoid culture medium into the holes of the cell culture plate, and culturing to obtain the organoid.
4. The method of claim 3, wherein in the step (a), ACK is added to the obtained primary cells to resuspend them on ice, and after the primary cells are left to stand, the primary cells are centrifuged to remove the supernatant, and matrigel is added to the primary cells to resuspend them on ice; mixing the primary cells with matrigel with the protein concentration of 8-12 mg/ml;
in step (c), after the organoid medium is added, PBS is also added to adjacent wells to prevent evaporation of the medium.
5. The method for preparing a prostate cell organoid of a mouse according to claim 3 or 4, wherein the organoid medium is DMEM/F12 based, and is formulated by adding the following cytokines:
leukocyte antigen HLA-B27-fold dilution, epidermal cell growth factor 50-100ng/ml, human recombinant R-spondin-1 250-1000ng/ml, A83-01-200-500 nM, dihydrochloride 10-50 mu M, L-alanyl-L-glutamine 100-fold dilution, dihydrotestosterone 1000-fold dilution, N-acetylcysteine 1-2mM, GMP-grade recombinant human Noggin 100-1000ng/ml, keratinocyte factor 500ng/ml.
6. A method for constructing an in situ primary prostate cancer animal model from a mouse prostate organoid prepared by the method of any one of claims 1-5, comprising the steps of:
(A) Performing gene editing on the organoids;
(B) Injecting the organoid after gene editing into animal prostate tissue to obtain the in-situ primary prostate cancer animal model.
7. The method for constructing an in situ primary prostate cancer animal model according to claim 6, wherein the organoids obtained by the culture are further subcultured before the organoids are subjected to gene editing, specifically:
(A1) Removing the culture medium in the organoids obtained by culture, and then adding recombinant digestive enzymes;
(B1) Transferring into a centrifuge tube, and incubating under the water bath condition of 37 ℃;
(C1) Repeatedly blowing and beating organoids, and centrifuging;
(D1) Removing the supernatant, adding matrigel into the obtained cells, re-suspending on ice, and then planting the cells on a cell culture plate;
(E1) Transferring the cell culture plate into an incubator for solidification;
(F1) Organoid medium was added to wells of the cell culture plates for subculturing organoids.
8. The method of constructing an in situ primary prostate cancer animal model according to claim 6, wherein the operation of gene editing the organoids is specifically:
(A2) Removing the culture medium in the organoids obtained by culture, and then adding recombinant digestive enzymes;
(B2) Transferring into a centrifuge tube, and incubating under the water bath condition of 37 ℃;
(C2) Repeatedly blowing and beating organoids, and centrifuging;
(D2) Removing the supernatant, adding lentivirus or retrovirus to the obtained cells for resuspension, and then planting the cells on a cell culture plate;
(E2) After centrifugation at 2000rpm at 32℃for 1 hour, incubation at 37℃was carried out;
(F2) Transferring the organoids into a centrifuge tube, and centrifuging at 1500rpm for 10min;
(G2) Removing the supernatant, adding matrigel for resuspension, and then planting on a cell culture plate;
(H2) Transferring the cell culture plate into an incubator for solidification;
(F2) Organoid medium was added to wells of cell culture plates for organoid gene editing.
9. The method of constructing an in situ primary prostate cancer animal model according to claim 8, wherein said gene editing is specifically the mutant Trp53 and Pten genes, and cMyc genes are highly expressed.
10. Use of an in situ primary prostate cancer animal model constructed according to the method of any one of claims 6-9 in drug screening, drug toxicity testing or as an endocrine treatment test animal model.
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