CN113234681B - Method for establishing stomach precancerous state organoid model and special culture medium - Google Patents
Method for establishing stomach precancerous state organoid model and special culture medium Download PDFInfo
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Abstract
The invention discloses a method for establishing a stomach precancerous state organoid model and a special culture medium. The culture medium for constructing the organoid model in the precancerous state of the stomach comprises a complete culture medium for the organoids of the stomach and 1-methyl-3-nitro-1-nitrosoguanidine; the concentration of 1-methyl-3-nitro-1-nitrosoguanidine is 25 to 200. mu.M, preferably 25 to 50. mu.M, and more preferably 50. mu.M. The method for establishing the organoid model in the precancerous state of the stomach comprises the step of culturing the glandular organ of the gastric mucosa by using a culture medium for constructing the organoid model in the precancerous state of the stomach. The invention can effectively simulate and support continuous and dynamic observation of the change condition of morphological structure and gene characteristics in the process of malignant transformation of normal stomach organoid to precancerous state of stomach; compared with the animal model research of the previous stomach precancerous state, the method greatly reduces the research cost and the research period, and fills the blank of an in vitro organoid model; the gastric mucosa gland has high yield, good state, stable passage and long-term culture.
Description
Technical Field
The invention relates to a method for establishing a organoid model, in particular to a method for establishing a organoid model in a precancerous stomach state and a special culture medium, belonging to the technical field of biological medicines.
Background
Gastric cancer is the fifth most common cancer in the world and also the fourth leading cause of cancer-related death, and the five-year survival rate is less than 30 percent; 47.85 million new cases of gastric cancer (about 43.94 percent of the world) in 2020; the death cases are 37.38 million (about 48.62% of the world), and the disease burden is very serious. According to the corea cascade development pattern, over 80% of gastric cancers clinically develop through the multi-step evolution of "normal gastric mucosa → atrophic gastritis → intestinal metaplasia → intraepithelial neoplasia → gastric cancer", but an effective strategy for effectively blocking and/or reversing this cascade process is still lacking clinically. During gastrointestinal development, the stomach, small intestine and colon are differentiated from a primitive gut tube composed of endoderm cells, the foregut develops into the esophagus, stomach and proximal intestine, and the midand hindgut develops into the small intestine and colon. Intestinal metaplasia refers to the replacement of the normal epithelium of the gastric mucosa with intestinal epithelium, i.e., the presence of epithelial cells in the gastric mucosa resembling the mucosa of the small or large intestine, which is an important precancerous state of the stomach.
The etiology of the gastric cancer relates to complex factors of a host and the environment, wherein excessive intake of dietary carcinogens (mainly nitrate/nitrite) can increase cancer risks, and development of a near-physiological precancerous gastric state research model has important significance and value for screening effective early prevention and intervention strategies and reducing the incidence rate of gastric cancer. Although the research of successfully culturing the stomach organoids and the stomach cancer organoids by using the normal stomach mucosa tissue or the stomach cancer tumor tissue has been carried out before, the research on the culture method of the organoids in the precancerous state of the stomach is lacked at present.
Disclosure of Invention
The invention aims to provide a method for establishing a pre-gastric-cancer-state organoid model and a special culture medium thereof, which are used for inducing the malignant transformation of normal gastric mucosa in vivo by simulating the excessive intake of nitrates and/or nitrites in clinical diet.
The first purpose of the invention is to provide a culture medium for constructing an organoid model of the precancerous state of the stomach.
The culture medium for constructing the organoid model in the precancerous state (intestinal metaplasia) of the stomach comprises a complete stomach organoid culture medium and 1-Methyl-3-nitro-1-nitrosoguanidine (1-Methyl-3-nitro-1-nitrosoguanidine, MNNG);
The concentration of the 1-methyl-3-nitro-1-nitrosoguanidine in the culture medium for constructing the organoid model in the precancerous state of the stomach may be 25 to 200. mu.M, preferably 25 to 50. mu.M, and more preferably 50. mu.M.
In the above medium for constructing a pre-gastric carcinoma state organoid model, the gastric organoid complete medium may be a medium suitable for growth of gastric organoids comprising growth factors and extracellular matrix; specifically, the gastric organoid complete medium may consist of the following components in mass concentration:
epidermal Growth Factor (EGF) at 50 ng/mL;
100ng/mL human Noggin;
500ng/mL R-spondyloprotein (R-Spondin);
10nM Gastrin (Gastrin);
1 μ M N-acetyl-L-cysteine (nAcetylcysteine);
100ng/mL recombinant human fibroblast growth factor-10 (FGF 10);
100ng/mL wingless MMTV integration site family member 3A (Wnt 3A);
10 μ M4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES);
L-Glutamine (L-Glutamine) at 50. mu.g/mL;
100. mu.g/mL of a mixed solution of penicillin and Streptomycin (Penicilin-Streptomyces);
n2 supplement (N2 supplement) in a volume 100 times that of Advanced DMEM/F12 basal medium;
b27 supplement (B27 supplement) in a volume 50 times that of Advanced DMEM/F12 basal medium;
And the balance of Advanced DMEM/F12 basal medium.
In the above-mentioned medium for constructing a pre-gastric carcinoma state organoid model, the medium for constructing a pre-gastric carcinoma state organoid model further includes an organic solvent, such as dimethyl sulfoxide (DMSO), for dissolving the 1-methyl-3-nitro-1-nitrosoguanidine.
The second purpose of the invention is to provide the application of the culture medium in any one of the above items in constructing a pre-gastric cancer state (intestinal metaplasia) organoid model or preparing a product for constructing the pre-gastric cancer state (intestinal metaplasia) organoid model.
The third purpose of the invention is to provide a method for establishing an organoid model of the precancerous state of the stomach (intestinal metaplasia).
The method for establishing the organoid model in the precancerous state of the stomach (intestinal metaplasia) comprises the step of culturing the glands of the gastric mucosa by using any one of the culture mediums for establishing the organoid model in the precancerous state of the stomach.
In the above method, the culture conditions may be the following C1 or C2:
c1: at 37 ℃ and 5% CO2Culturing for 5-10 days under the concentration;
c2: at 37 ℃ with 5% CO2After culturing for 5 days at the concentration, dissociating and resuspending the stomach organoids into single cells, and adding the culture medium for constructing the organoid model in the precancerous state of the stomach based on 5% CO at 37 DEG C 2The culture was continued at concentration for 3 days.
In the above method, the method further comprises the step of taking the whole stomach and separating to obtain the gastric mucosal gland.
Further, the gastric mucosal gland is a mouse gastric mucosal gland; the whole stomach may be a mouse whole stomach; the whole stomach of the mouse can be specifically from 6-8 weeks old wild type C57/BL6 mice.
Further, the gastric mucosal glands are composed of a variety of cells; the steps for separating and obtaining the gastric mucosa gland can be as follows: and (3) after the whole stomach digestion incubation, placing the incubated whole stomach digestion in a shaking buffer (such as 5mL of the shaking buffer) for shaking, centrifuging, and collecting cell mass precipitates to obtain the gastric mucosal glands.
Furthermore, in the shaking step, 50-100 strip-shaped complete gastric mucosal glands are required to be obtained in each 20 mu L of suspension, and the number of the strip-shaped complete gastric mucosal glands can be specifically judged by taking 20 mu L of suspension and observing the number of the strip-shaped complete gastric mucosal glands under a microscope.
Further, the conditions of the digestion incubation may be as follows: incubation is carried out at 4 ℃ for 1-2 h, such as 1.5h, in a 5mM EDTA solution.
Still further, the digestion incubation further comprises the following steps: the whole stomach of the mouse is completely taken out and washed by DPBS, the content is extruded out, the mouse is washed again after being turned inside and outside, the upper part (cardia part) and the lower part (pylorus part) of the stomach are knotted by suture lines, and after the mouse is thoroughly washed, 1mL of DPBS buffer solution is injected into the stomach body to be full.
Still further, the shaking buffer may be prepared from 200 mL: 2 g: 3g of phosphate buffer solution, sorbitol and sucrose;
the conditions for the shaking may specifically be as follows: shaking for 1 time every 1 second at room temperature (such as 15-30 deg.C, and then 25 deg.C), and manually shaking for 30-60 times.
Further, the conditions for the centrifugation may be as follows: centrifuge at 300g for 5min at 4 ℃.
In the above method, the step of culturing the gastric mucosa gland comprises: and (3) solidifying the gastric mucosa gland in matrigel at 37 ℃ for 15min, and then adding the culture medium for constructing the organoid model in the precancerous state of the stomach for culture.
The invention has the following beneficial effects:
(1) the invention can effectively simulate and support continuous and dynamic observation of the change condition of morphological structure and gene characteristics in the process of malignant transformation of the stomach organoid from normal to precancerous state of stomach;
(2) compared with the previous animal model research of the stomach precancerous state, the method greatly reduces the research cost and the research period, and fills the blank of an in vitro organoid model.
(3) The gastric mucosa gland in the method has high yield and good state, and can be stably subcultured for a long time.
Drawings
FIG. 1 is a schematic diagram of the process for establishing the organoids in the pre-gastric cancer state induced by MNNG according to the present invention.
FIG. 2 is a photomicrograph of elongated intact gastric mucosal glands in 20 μ L of suspension after shaking in example 1.
FIG. 3 is a light microscope photograph of MNNG induced gut-like changes in gastric organoid morphology in example 1.
FIG. 4 shows MNNG-induced expression of CDX2, a transcription factor specific to intestinal epithelial cells in a pre-gastric carcinoma organoid in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the following describes technical solutions of the embodiments of the present invention clearly and completely with reference to the accompanying drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.
The culture medium for constructing the organoid model in the precancerous state (intestinal metaplasia) of the stomach comprises a complete culture medium of the stomach organoid and 1-methyl-3-nitro-1-nitrosoguanidine;
the concentration of 1-methyl-3-nitro-1-nitrosoguanidine in the culture medium for constructing the organoid model of the precancerous state of the stomach is 25 to 200. mu.M, preferably 25 to 50. mu.M, and more preferably 50. mu.M.
The method for establishing the organoid model in the precancerous state of the stomach (intestinal metaplasia) comprises the step of culturing the glands of the gastric mucosa by using the culture medium for establishing the organoid model in the precancerous state of the stomach.
The invention fills the blank of an in-vitro organoid model of a precancerous state of the stomach by simulating the process of inducing the malignant transformation of normal gastric mucosa in vivo by nitrate and/or nitrite carcinogens in clinical diet.
The method for establishing the organoid model in the precancerous state of the stomach of the present invention will be described in detail by way of specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.
The experimental materials and their sources referred to in the following examples are as follows:
6-8 week old C57/BL6 mice were purchased from Wintonlifa, Beijing.
PBS (phosphate buffered saline) was purchased from Cell signaling Inc. under the designation 9808S.
Ethylenediaminetetraacetic acid (EDTA) solution was purchased from Sigma under the designation V900106-500G.
Sorbitol (D-Sorbitol) was purchased from Sigma under the trade name S-1876.
Sucrose (Sucorese) was purchased from Sigma under the accession number S-9378.
Matrigel (Matrigel) was purchased from Cloning, Inc. under the designation 354277.
1-Methyl-3-nitro-1-nitrosoguanidine (1-Methyl-3-nitro-1-nitrosoguanidine, MNNG) was purchased from Tokyo chemical industry Co., Ltd., product number M0527.
Dimethyl sulfoxide (DMSO) was purchased from SIGMA corporation, USA under the product number D2650.
Basal medium (Advanced DMEM/F12) was purchased from Gibco, Inc. under the designation 12634-010.
Epidermal Growth Factor (EGF) was purchased from R & D Systems, Inc. under the accession number 2028-EG-200.
Human Noggin (Noggin) was purchased from R & D Systems under the trade designation 1967-NG-025/CF.
R-vertebrin (R-Spondin) was purchased from R & D Systems, Inc. under the reference 3474-RS-150.
Gastrin (Gastrin) is available from R & D Systems under the reference 3006/1.
N-acetyl-L-cysteine (nAcetYLCysteine) was purchased from Sigma under the designation 616-91-1.
Recombinant human fibroblast growth factor-10 (FGF10) available from R & D Systems, Inc. under the designation 345-FG-025.
Wingless MMTV integration site family member 3A (Wnt3A) was purchased from R & D Systems, Inc. under the accession number 5036-WN-010.
4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) was purchased from ThermoFisher, Inc. under the designation 15630-.
L-Glutamine (L-Glutamine) is available from Invitrogen as Cat 25030-.
The mixed solution of penicillin Streptomycin (Penicilin-Streptomyces) is purchased from Invitrogen company with the commodity number of 15070-.
N2 additive (N2 supplement) was purchased from ThermoFisher company under the cat number 17502048.
B27 additive (B27 supplement) was purchased from ThermoFisher corporation under the cat number 17504044.
The solutions and their formulations referred to in the following examples are as follows:
shaking buffer recipe: 200mL of DPBS; d-sorbitol 2 g; sucross 3 g.
The formula of the complete culture medium for the mouse stomach organoid comprises the following components: advanced DMEM/F12; EGF (50 ng/mL); noggin (100 ng/mL); R-Spondin (500 ng/mL); gastrin (10 nM); nacytelcystein (1 μ M); FGF10(100 ng/ml); wnt3A (100 ng/mL); HEPES (10. mu.M); L-Glutamine (50. mu.g/mL); Pen-Strep (100. mu.g/mL); n2 supplement (100X, 1:100, 100 fold volume of Advanced DMEM/F12 basal medium); b27 supplement (50X, 1:50, expressed in volume 50-fold of Advanced DMEM/F12 basal medium).
Example 1 establishment of a model of the Pre-gastric carcinoma State (intestinal metaplasia) organoids
Establishing a stomach precancerous state (intestinal metaplasia) organoid model according to a flow diagram shown in figure 1, and specifically comprising the following steps:
1) Taking 6-8 weeks old wild type C57/BL6 mice, after abdominal dissection, knotting the upper part of the stomach (cardia) by using a suture needle with a thread, respectively cutting off the connection between the stomach and the esophagus and the small intestine, taking out the whole stomach, putting the whole stomach into a 10cm cell culture dish containing precooled DPBS (dendritic cell-mediated coagulation) for rinsing, and removing impurities on the surface.
2) A small opening is cut at the pylorus of the stomach by scissors, the stomach is gently squeezed by forceps to extrude the content in the stomach, then the forceps are used to prop against one end of the knotted cardia, the whole stomach is turned over outwards, and the stomach is put in pre-cooled DPBS to be cleaned again, and then the pylorus is knotted and sealed.
3) About 1mL of DPBS was aspirated with a 1mL syringe, injected into the stomach, allowed to swell, and placed in a 15mL centrifuge tube containing 5mM EDTA solution and incubated for 1.5h at 4 ℃ in a shaker.
4) After incubation was complete, the 15mL centrifuge tubes were transferred to the cells on ice for subsequent manipulations. Absorbing the EDTA solution, adding 5mL of shaking buffer, and manually shaking at room temperature (25 ℃), wherein the EDTA solution is shaken about once per second for 30-60 times, and 20 mu L of suspension is taken out during the shaking and is observed under a microscope until 50-100 strip-shaped complete gastric mucosa glands reach the required density and number, and in the case of shaking for 30 times, the 20 mu L of suspension reaches 30-50 strip-shaped complete gastric mucosa glands (see figure 2). Centrifuging the liquid in a 15mL tube for 5min at 4 ℃ by using an Eppendorf Centrifuge 5810R type Centrifuge;
5) After the centrifugation is finished, sucking the supernatant, adding pre-melted matrigel into the precipitate for resuspension, dividing the resuspended system into four dishes preheated at 37 ℃, adding 25uL matrigel into each hole, and placing the dishes in an incubator at 37 ℃ for 15min to fully solidify the sediment.
6) Preparing MNNG solution, firstly dissolving MNNG by DMSO to prepare mother liquor with the concentration of 100mM, taking 2mL of mouse stomach organoid complete culture medium, adding 1 mu L of MNNG mother liquor to prepare the final concentration of 50 mu M, adding 380 mu L of culture solution containing MNNG into each hole, and replacing the culture solution once every 2-3 days. An equivalent amount of DMSO solution was added as a control.
7) By the 5 th day of culture, the stem cells in the stomach organoids are differentiated, and the expression of the intestinal epithelial cell specific transcription factor CDX2 in part of the cell nuclei can be observed under a confocal microscope, as shown in FIG. 3.
8) After continuous culture to 8 days, part of organoids are seen to present similar intestinal type changes; or after dissociating and resuspending the gastronomic organ into single cells on the 5 th day, the gastronomic organ is redistributed into culture dishes, and MNNG with the concentration of 50 mu M is added continuously for 3 days to culture, so that most organs show similar intestinal type change. As shown in fig. 4.
Example 2 establishment of a Pre-gastric carcinoma State (intestinal metaplasia) organoid model
The organoid model for the precancerous state of the stomach was established according to the procedure of example 1, except that the final concentration of MNNG in the medium containing MNNG was replaced with 1 μ M.
No obvious change of organoid morphological structure is observed after continuous culture to 8 days, and the sixth day, cells in very individual organoids are positive to CDX2, mainly the severely aged organoids are observed, and organoid proliferation is not obviously inhibited.
Example 3 establishment of a Pre-gastric carcinoma State (intestinal metaplasia) organoid model
The pre-gastric carcinoma organoid model was constructed according to the procedure of example 1, except that the final concentration of MNNG in the medium containing MNNG was replaced with 25 μ M.
After the continuous culture is carried out until the day 8, the obvious changes of the form and the structure of part of organoids can be seen, and the cells in about 1/3 organoids are positive in CDX2, the positive proportion of CDX2 in severely aged organoids is larger, and the organoid proliferation is not obviously inhibited by the day 6.
Example 4 establishment of a Pre-gastric carcinoma State (intestinal metaplasia) organoid model
The gastric precancerous condition organoid model was constructed according to the procedure of example 1, except that the final concentration of MNNG in the medium containing MNNG was replaced with 100 μ M.
After continuous culture to the 6 th day, obvious changes of the shape and structure of a large proportion of organoids can be seen; approximately 2/3 organoids were CDX2 positive for a large number of cells and organoid proliferation was significantly inhibited.
Example 5 establishment of a Pre-gastric carcinoma (intestinal metaplasia) organoid model
The gastric precancerous condition organoid model was constructed according to the procedure of example 1, except that the final concentration of MNNG in the medium containing MNNG was replaced with 200 μ M.
After continuous culture to the 6 th day, obvious changes of the shape and structure of a large proportion of organoids can be seen; about 2/3 organoids were CDX2 positive in a large number of cells, and even some organoids had a percentage of CDX2 positive cells of over 85%, and the organoid proliferation rate was slower than that of the 100 μ M group.
Claims (5)
1. The method for establishing the stomach precancerous state organoid model comprises the step of culturing the stomach mucosa glands by using a culture medium for constructing the stomach precancerous state organoid model;
the culture medium for constructing the organ-like model in the precancerous state of the stomach comprises a complete culture medium of the organ-like model of the stomach and 1-methyl-3-nitro-1-nitrosoguanidine;
the concentration of the 1-methyl-3-nitro-1-nitrosoguanidine in the culture medium for constructing the organ-like model in the precancerous state of the stomach is 25-200 mu M;
the stomach organoid complete medium consists of the following components in mass concentration:
50 ng/ml epidermal growth factor;
100 ng/ml human noggin;
500 ng/ml R-spondyloprotein;
10 nM gastrin;
1 μ M N-acetyl-L-cysteine;
100 ng/ml recombinant human fibroblast growth factor-10;
100 ng/ml wingless MMTV integration site family member 3A;
10 μ M4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid;
l-glutamine at 50. mu.g/ml;
100 mu g/ml streptomycin mixed solution;
n2 additive in a volume 100 times that of Advanced DMEM/F12 basal medium;
b27 supplement in a volume 50 times that of Advanced DMEM/F12 basal medium;
and the balance of Advanced DMEM/F12 basal medium;
the culture medium for constructing the organoid model of the precancerous state of the stomach further comprises an organic solvent for dissolving the 1-methyl-3-nitro-1-nitrosoguanidine; the organic solvent is dimethyl sulfoxide;
the culture conditions are C1 or C2:
c1: at 37 ℃ with 5% CO2Culturing for 5-10 days under the concentration;
c2: at 37 ℃ with 5% CO2After 5 days of culture at concentration, the gastric organoids were dissociated, resuspended into single cells, and the culture medium was added at 37 ℃ with 5% CO2The culture was continued at concentration for 3 days.
2. The method of claim 1, wherein: the method also comprises the steps of taking the whole stomach, separating and obtaining the gastric mucosal gland.
3. The method of claim 2, wherein: the separation steps are as follows: and after the whole stomach digestion incubation, placing the incubated whole stomach digestion solution in a shaking buffer solution for shaking, centrifuging, and collecting cell mass precipitates to obtain the gastric mucosal gland.
4. The method of claim 3, wherein: the digestion incubation conditions were as follows: incubating in 5 mM EDTA solution for 1-2 h at 4 ℃;
in the shaking step, 50-100 strip-shaped complete gastric mucosal glands are required to be contained in each 20 mu L of suspension;
the shaking buffer consisted of 200 ml: 2 g: 3 g of phosphate buffer solution, sorbitol and sucrose;
the conditions of the centrifugation were as follows: centrifuge at 300 g for 5 min at 4 ℃.
5. The method of claim 1, wherein: the steps for culturing the gastric mucosa gland are as follows: and (3) curing the gastric mucosa gland in matrigel at 37 ℃ for 15 min, and then adding the culture medium for constructing the organoid model in the precancerous state of the stomach for culture.
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