CN114908037A - Culture medium of human intestinal metaplasia gas-liquid interface model, construction method and application - Google Patents

Culture medium of human intestinal metaplasia gas-liquid interface model, construction method and application Download PDF

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CN114908037A
CN114908037A CN202210539797.7A CN202210539797A CN114908037A CN 114908037 A CN114908037 A CN 114908037A CN 202210539797 A CN202210539797 A CN 202210539797A CN 114908037 A CN114908037 A CN 114908037A
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liquid interface
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intestinal metaplasia
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CN114908037B (en
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刘思濛
温慧娟
孙向东
李富豪
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Fifth Affiliated Hospital of Zhengzhou University
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Abstract

The invention provides a culture medium for constructing a human intestinal metaplasia gas-liquid interface model, a construction method and application, wherein the proliferation culture medium comprises Wnt3a, R-spondin 1, human EGF, human FGF-10, human noggin, human gastrin, B27, N2, Y-27632, A83-01, Nicotinamide, HEPES, Glutamax and the like, and the differentiation culture medium comprises human EGF, human FGF-10, human noggin, human gastrin, B27, N2, Y-27632, A83-01, Nicotinamide, HEPES, Glutamax and the like. The culture medium and the cell culture chamber with the upper chamber and the lower chamber are adopted to successfully construct a human intestinal metaplasia gas-liquid interface model, the complementation of the existing animal model system is realized, the model can be used for drug effect evaluation, a bacterial infection or lentivirus infection model and an immune cell or interstitial cell co-culture model, can better research the mechanism of intestinal metaplasia generation, and can be used for preclinical experiments to determine possible treatment methods and perform personalized treatment on patients.

Description

Culture medium of human intestinal metaplasia gas-liquid interface model, construction method and application
Technical Field
The invention relates to the technical field of protein freeze-drying, in particular to a culture medium of a human intestinal metaplasia gas-liquid interface model, a construction method and application.
Background
Gastric Cancer (GC) is a leading cause of cancer death worldwide. The incidence of gastric cancer is 2 nd in China, and is second to lung cancer. Intestinal adenocarcinoma is the most common histological type of gastric cancer, and previous studies have shown that intestinal adenocarcinoma develops through a well-defined histological state, starting with chronic gastritis, to intestinal metaplasia, atypical hyperplasia and cancer. Intestinal Metaplasia (IM) refers to the replacement of gastric mucosal epithelium by intestinal glandular epithelium, with the appearance of absorptive, goblet and panne cells, sometimes with the formation of villous structures on the mucosal surface. Intestinal metaplasia is a recognized precancerous state and is currently the first key step in the development of intestinal-type gastric adenocarcinoma that is difficult to reverse. Research on the mechanism of intestinal metaplasia and possible treatment methods is restricted by the progress of models thereof, and no open report on intestinal metaplasia cell lines and in vitro experiments exists at present.
Since 2010, the technology of culturing organoids has rapidly developed, and is rated by "Science" as one of the largest scientific advances in 2013, and "Nature Methods" as the annual technology in the field of life Science in 2017. Organoids utilize stem cells to form "self-organization" in 3D culture, which can mimic the specific structure and function of their source tissues and organs in vitro, and have the ability to self-renew and differentiate. At present, human gastric epithelial organoids have been successfully constructed at home and abroad, and the advantages of the human gastric epithelial organoids include: the structure and the function are simulated, the long-term proliferation (semipermanent, the culture time is more than 1 year) can be realized, the material taking is small, the individual information is provided, and the method is suitable for individualized treatment and the like. However, organoids have certain disadvantages: the operation is complex (micro-injection in organoid is often needed), no mucus layer is provided, and the passage interval is short (generally 7-10 days, long-term drug treatment cannot be carried out). An air-liquid interface (ALI) culture model developed in recent years can make up for the deficiency of organoids and can realize complementation with organoid systems in primary cell culture, but successful establishment of the ALI model has certain difficulty and is a relatively popular and difficult research in the industry at present.
Disclosure of Invention
Aiming at the problems and defects in the prior art, the invention provides a culture medium of a human intestinal metaplasia gas-liquid interface model, a construction method and application. The technical scheme of the invention is as follows:
in a first aspect, the invention provides a propagation medium for a human intestinal metaplasia gas-liquid interface model, comprising the following components in final concentrations: 30% -50% of Wnt3a 30; 115% -25% of R-spondin; 10-50ng/ml of recombinant human epidermal growth factor (human EGF); 50-500ng/ml of recombinant human fibroblast growth factor-10 (human FGF-10); 50-500ng/ml of recombinant human Noggin protein (human Noggin); human gastrin (human gastrin)5-50 nM; b272%; n21%; y-276321.5-7.5 mu M; a83-010.5-5 mu M; 5-50mM Nicotinamide (Nicotinamide); 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES)10 mM; glutamax 1%; advanced DMEM/F-1218.45% -48.45%.
In a second aspect, the invention provides a differentiation medium for a human intestinal metaplasia gas-liquid interface model, comprising the following components in final concentrations: 10-50ng/ml of recombinant human epidermal growth factor (human EGF); 50-500ng/ml of recombinant human fibroblast growth factor-10 (human FGF-10); 50-500ng/ml of recombinant human Noggin protein (human Noggin); human gastrin (human gastrin)5-50 nM; b272%; n21%; y-276321.5-7.5 mu M; a83-010.5-5 mu M; 5-50mM Nicotinamide (Nicotinamide); 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES)10 mM; glutamax 1%; advanced DMEM/F-1293.45%.
In a third aspect, the invention provides a method for constructing a human intestinal metaplasia gas-liquid interface model, which is to adopt a cell culture chamber with an upper chamber and a lower chamber for the obtained human intestinal metaplasia cells to be cultured, add the human intestinal metaplasia cells to be cultured in the upper chamber, add the proliferation culture medium in the lower chamber, and culture under the gas-liquid interface condition.
Further, the acquisition of the human intestinal metaplastic cells to be cultured comprises the following steps:
1) washing human intestinal metaplasia tissue with PBS solution for several times, removing obvious fat tissue, and cutting into 1 + -0.1 mm 2 Organizing the small blocks;
2) adding a digestive reagent into the cut tissue small blocks, performing shake digestion at 37 ℃ for 30 +/-10 min, standing to precipitate the tissue, and removing a supernatant;
3) the cells in the digested tissue were separated, the cells were resuspended in Advanced DMEM/F12 medium containing Fetal Bovine Serum (FBS), the cell suspension was centrifuged at 250g for 5min at 4 ℃ and the supernatant was discarded, and the cell pellet was collected and counted.
Further, the PBS solution contained 1% cyan/streptomycin, 50. mu.g/ml gentamicin, and 1.25. mu.l/ml amphotericin B.
Further, the digestion reagent is a chelating agent or collagenase.
Further, the cell culture chamber having upper and lower chambers was subjected to the following pretreatment before use: the cell culture chamber was placed in a 24-well cell culture plate at 10. mu.g/cm 2 Concentration of Collagen (Collagen) was diluted and added to the upper chamber, left at 37 ℃ for 30-60min, and the Collagen dilution was aspirated and washed 2 times with PBS.
Further, the upper chamber is added with human intestinal metaplasia cells to be cultured, the lower chamber is added with the culture medium, and the culture is carried out under the condition of a gas-liquid interface, which specifically comprises the following steps:
the human intestinal metaplasia cells to be cultured were resuspended in 200. mu.l of proliferation medium at 1000- 2 And removing the proliferation culture medium in the upper chamber after 3 days of culture, starting gas-liquid interface culture, removing mucus in the upper chamber every 3 days, replacing the proliferation culture medium in the lower chamber, and culturing for 10-14 days to obtain the model.
In a fourth aspect, the present invention provides a method for culturing and proliferating a human intestinal metaplasia gas-liquid interface model, which uses a cell culture chamber having an upper chamber and a lower chamber, comprising the steps of:
(1) digesting the obtained human intestinal epithelization gas-liquid interface model for 30min at 37 ℃ by using pancreatin;
(2) the cells were collected into FBS-containing Advanced DMEM/F12 medium to stop digestion and counted;
(3) the cell culture chamber was placed in a 24-well cell culture plate at 10. mu.g/cm 2 The concentration of Collagen (Collagen) is diluted and added to the upper chamber, and the mixture is placed at 37 ℃ for 30-60min,the Collagen dilutions were aspirated and washed 2 times with PBS;
(4) the cells obtained in step (2) were resuspended in 200. mu.l of the proliferation medium at 1000- 2 And (3) removing the proliferation culture medium in the upper chamber after culturing for 3 days, starting gas-liquid interface culture, removing mucus in the upper chamber every 3 days, replacing the proliferation culture medium in the lower chamber, and carrying out passage once every 2-6 weeks, namely repeating the steps (1) - (4), wherein the passage is carried out at least 6-10 times.
In a fifth aspect, the invention provides a differentiation culture method of a human intestinal metaplasia gas-liquid interface model, which adopts the human intestinal metaplasia gas-liquid interface model after proliferation culture, and the culture method comprises the following steps:
performing proliferation culture on human intestinal epithelialization gas-liquid interface model, removing mucus in upper small chamber, sucking proliferation culture medium, adding differentiation culture medium, culturing at 37 deg.C under 5% CO 2 Culturing in incubator, removing mucus in upper chamber every 3 days and replacing differentiation medium, and culturing for 6 days.
In a sixth aspect, the invention provides a human intestinal metaplasia gas-liquid interface model, which is obtained by the above construction method or proliferation culture method or differentiation culture method.
In a seventh aspect, the invention provides an application of the human intestinal metaplasia gas-liquid interface model in drug effect evaluation.
Further, the method of applying comprises: adopting a cell culture chamber with an upper chamber and a lower chamber, adding human intestinal epithelial metaplasia primary cells to be cultured into the upper chamber, adding the proliferation culture medium into the lower chamber, and performing proliferation culture under the condition of a gas-liquid interface; culturing for 7-14 days, adding proliferation culture medium containing certain drug concentration into the lower chamber, culturing for 2-4 weeks, removing mucus in the upper chamber every 3 days, and replacing proliferation culture medium containing drug in the lower chamber; and carrying out related cell detection according to the experimental requirements.
In an eighth aspect, the invention provides the use of the above human intestinal metaplasia fluid interface model as a model of bacterial infection or lentivirus infection.
Further, the bacteria include helicobacter pylori.
In a ninth aspect, the invention provides an application of the human intestinal metaplasia gas-liquid interface model as an immune cell or mesenchymal cell co-culture model.
The invention has the beneficial effects that:
the invention develops a proliferation culture medium and a differentiation culture medium constructed by a human intestinal metaplasia gas-liquid interface model, and successfully constructs the human intestinal metaplasia gas-liquid interface model by adopting a cell culture chamber with an upper chamber and a lower chamber, thereby realizing the complementation of the prior animal model system.
Drawings
FIG. 1 is a schematic diagram of a human intestinal epithelial gas-liquid interface culture model in example 1 of the present invention, in which 1-gas, 2-chamber, 3-mucus, 4-intestinal epithelial cells, 5-collagen pre-applied PC membrane, and 6-medium.
FIG. 2 is a diagram of a human intestinal epithelial cell gas-liquid interface model culture in example 1 of the present invention.
FIG. 3 is a schematic diagram of a metaplasia gas-liquid interface on human intestine obtained in example 1 of the present invention.
FIG. 4 stability passage results of the human intestinal metaplasia gas-liquid interface model of example 1 of the present invention.
FIG. 5 shows the expression of the intestinal markers ITLN1, TFF3 and LRIG1 after the intervention of rebamipide in the IM-ALI model in example 2 of the present invention.
FIG. 6 shows the enrichment of the domain of rebamipide interfering ALI-IM differentially expressed genes in example 2 of the present invention.
FIG. 7 shows the Rebamipide intervention ALI-IM differentially expressed gene enrichment in KEGG in example 2 of the present invention.
FIG. 8 shows the enrichment condition of rebamipide interfering ALI-IM differential expression gene on GO in example 2 of the invention.
FIG. 9 shows the enrichment of rebamipide in ALI-IM differentially expressed genes in protein localization in example 2 of the invention.
FIG. 10 shows the difference in cell number between the human intestinal metaplasia and gas-liquid interface models obtained in example 1, comparative example 2 and comparative example 3 of the present invention.
Figure 11 provides the cell status on day 7 of lentivirus infection of the human intestinal metaplasia ALI model for example 3 and comparative example 4.
Detailed Description
The percentages in the present invention, unless otherwise specified, are expressed in terms of volume percent concentration.
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
Example 1
The embodiment provides a method for constructing a human intestinal metaplasia gas-liquid interface model, and the obtained human intestinal metaplasia gas-liquid interface model is subjected to proliferation culture and differentiation culture, wherein the model construction method is to adopt a cell culture chamber with an upper chamber and a lower chamber for the obtained human intestinal metaplasia cells to be cultured, the human intestinal metaplasia cells to be cultured are added into the upper chamber, the proliferation culture medium is added into the lower chamber, the culture is carried out under the gas-liquid interface condition, the culture principle is shown in figure 1, and the culture substance is shown in figure 2. The whole process specifically comprises the following steps:
1) sample cleaning: multiple washes of human intestinal metaplastic tissue were performed with PBS containing 1% penicillin/streptomycin, 50. mu.g/ml gentamicin, and 1.25. mu.l/ml amphotericin B.
2) Sample shearing: removing significant adipose tissue, and cutting the tissue to about 1mm 2 Small blocks with the same size as much as possible.
3) Tissue digestion: transferring the sheared tissue into a 15ml centrifuge tube, adding 8-10ml of chelating solution or collagenase, shaking and digesting for 30min at 37 ℃, standing to precipitate the tissue, and removing supernatant.
4) Separation and filtration: gently squeezing the digested tissue in the step 3) by using a syringe piston, and fully separating glands or cells. ② suspending glands or cells in a centrifuge tube by using FBS-containing Advanced DMEM/F12 culture medium, separating the cells by using an injector, centrifuging for 5min at 4 ℃ and 250g, removing supernatant, collecting cell precipitates and counting.
5) Pretreatment of the chamber: taking out the Cell culture insert, placing the Cell culture insert in a 24-hole Cell culture plate, and culturing the Cell culture insert at the concentration of 10 mu g/cm 2 The Collagen was diluted and added to the upper layer of the cell. The cells were placed in a cell incubator at 37 ℃ for 30-60min, and before use the Collagen diluent was aspirated and washed 2 times with PBS.
6) Primary culture: cells were resuspended at 1000/μ L in 200 μ L of medium and added to the upper layer of the chamber. The lower chamber was filled with 500. mu.l of human intestinal metaplasia ALI proliferation medium (Wnt3a 30%; R-spondin 115%; human EGF 20 ng/ml; human FGF-10150 ng/ml; human noggin150 ng/ml; human gastrin10 nM; B272%; N21%; Y-276327.5. mu.M (reduced from day 3 to 1.5. mu.M); A83-011. mu.M; Nicotinamide10 mM; HEPES10 mM; Glutamax 1%; Advanced DMEM/F-1248.45%) at 37 ℃ in 5% CO 2 Culturing in an incubator. And on the third day, removing the upper culture medium and starting gas-liquid interface culture. During the period, mucus in the upper chamber is removed and the human intestinal metaplasia ALI medium is replaced every 3 days, and a human intestinal metaplasia gas-liquid interface model is successfully obtained after 10 days of culture, as shown in fig. 3, and the model is shown to have polarity, and the top of the model expresses an intestinal marker MUC 2.
7) And (3) proliferation culture: cell digestion: mucus and culture medium were removed from the obtained human intestinal metaplasia gas-liquid interface model, and the upper and lower chambers were washed with PBS 3 times, respectively. 200 mul and 400 mul of pancreatin are respectively added into the upper chamber and the lower chamber to be digested at 37 ℃ for 30min, and the mixture is blown and beaten by a gun head every 10min and is mixed evenly. Collecting cells: the cells were collected into FBS-containing Advanced DMEM/F12 medium to terminate digestion and counted. Pretreatment of the small chamber: same as step 5). Multiplication culture: resuspending the cells obtained in step (ii) in a proliferation medium at 1000 cells/. mu.l, adding the cell to the upper chamber, adding the proliferation medium to the lower chamber, and culturing at 37 ℃ with 5% CO 2 And (3) removing the proliferation culture medium in the upper chamber after culturing for 3 days, starting gas-liquid interface culture, removing mucus in the upper chamber every 3 days, replacing the proliferation culture medium in the lower chamber, and carrying out passage once every 2-6 weeks, namely repeating the steps (1) - (4), wherein the passage is carried out at least 6-10 times.
8) Differentiation culture: after the proliferation culture was completed, mucus in the upper chamber was removed, the proliferation medium was aspirated, and a differentiation medium (human EGF 20ng/ml, human FGF-10150 ng/ml, human noggin150ng/ml, human gastrin10nM, B272%, N21%, Y-276327.5. mu.M, A83-011. mu.M, Nicotinamide10mM, HEPES10mM, Glutamax 1%, Advanced DMEM/F-1293.45%) was added thereto at 37 ℃ with 5% CO 2 The culture was carried out in an incubator for 6 days, during which mucus in the upper chamber was removed and the differentiation medium was changed every 3 days.
Identification of the human intestinal metaplasia fluid interface model obtained in this example.
Fig. 4 shows the results of stable passage of the human intestinal metaplasia gas-liquid interface model, each passage being 28 days of proliferation culture, showing that the intestinal marker (a), the epithelial stem cell marker (B), the epithelial cell marker (C) and the gland marker (D) all have stability with passage of the human intestinal metaplasia gas-liquid interface model.
Example 2
The embodiment is used for detecting the action effect of rebamipide on the human intestinal metaplasia gas-liquid interface model, and the specific process is as follows:
1) sample cleaning: multiple washes of human intestinal metaplastic tissue were performed with PBS containing 1% penicillin/streptomycin, 50. mu.g/ml gentamicin, and 1.25. mu.l/ml amphotericin B.
2) Sample shearing: removing significant adipose tissue, and cutting the tissue to about 1mm 2 Small blocks with the same size as much as possible.
3) Tissue digestion: firstly, transferring the sheared tissue into a 15ml centrifuge tube, adding 8-10ml of chelating solution or collagenase, carrying out shake digestion at 37 ℃ for 30min, standing to precipitate the tissue, and removing supernatant.
4) Separation and filtration: gently squeezing the digested tissue in the step 3) by using a syringe piston, and fully separating glands or cells. ② suspending glands or cells in a centrifuge tube by using FBS-containing Advanced DMEM/F12 culture medium, separating the cells by using an injector, centrifuging for 5min at 4 ℃ and 250g, removing supernatant, collecting cell precipitates and counting.
5) Pretreatment of the small chamber: taking out the Cell culture insert, placing the Cell culture insert in a 24-hole Cell culture plate, and culturing the Cell culture insert at the concentration of 10 mu g/cm 2 The Collagen was diluted and added to the upper layer of the cell. The cells were placed in a cell incubator at 37 ℃ for 30-60min, and before use the Collagen dilutions were aspirated and washed 2 times with PBS.
6) Primary culture: cells were resuspended in 200. mu.l of medium at 200000 and added to the upper layer of the chamber. The lower chamber was filled with 500. mu.l of human intestinal metaplasia ALI proliferation medium (Wnt3a 30%; R-spondin 115%; human EGF 20 ng/ml; human FGF-10150 ng/ml; human noggin150 ng/ml; human gastrin10 nM; B272%; N21%; Y-276327.5. mu.M (reduced to 1.5. mu.M from day 3), A83-011. mu.M; Nicotinamide10 mM; HEPES10 mM; Glutamax 1%; Advanced DMEM/F-1248.45%, proliferation medium of the same example 1) at 37 ℃ with 5% CO 2 Culturing in an incubator. And on the third day, removing the upper culture medium and starting gas-liquid interface culture. During this period mucus was removed from the upper chamber and human intestinal metaplasia ALI medium was replaced every 3 days. On the 7 th day of human intestinal metaplasia gas-liquid interface culture, ALI proliferation medium containing 100. mu.M rebamipide was added to the lower chamber for 4 weeks, during which time mucus in the upper chamber was collected every 3 days and the ALI proliferation medium containing the drug was replaced. The upper chamber mucus was collected for proteomics (MS) detection at the time of fluid change. Collecting cell extracted RNA at the termination of the experiment, carrying out RNA-seq portioning, and detecting the expression condition of the intestinal marker by qPCR (quantitative polymerase chain reaction) so as to evaluate the effect of the rebamipide on a human intestinal epithelialization in vitro model.
Fig. 5 shows the expression of the intestinal markers ITLN1, TFF3 and LRIG1 after rebamipide intervention in the IM-ALI model, indicating that the expression of the intestinal markers ITLN1, TFF3 and LRIG1 is decreased after rebamipide intervention in the IM-ALI model, suggesting that rebamipide can improve intestinal metaplasia.
FIGS. 6-9 show that the MS differential expression gene after the rebamipide IM-ALI model is mainly enriched in inflammatory and cancer related pathways such as NF-kappa B, TGF-beta and JAK-STAT. And (3) interfering the mass spectrum differential protein enrichment result after ALI-IM with rebamipide. FIGS. 6-9 show the enrichment of rebamipide in the domains, KEGG, GO and protein localization of the ALI-IM differentially expressed genes.
Example 3
The embodiment provides an operation method for human intestinal metaplasia gas-liquid interface model lentivirus infection, which comprises the following specific processes:
1) culturing according to the method of the steps 1) to 6) of the embodiment 1 to obtain a human intestinal metaplasia and gas-liquid interface model;
2) cell digestion: mucus and medium were removed and the upper and lower chambers were washed 3 times with PBS. Adding 200 μ L and 400 μ L pancreatin into the upper chamber and the lower chamber respectively, digesting at 37 deg.C for 30min, and blowing and mixing uniformly every 10min with a gun head.
3) Cell collection: the cells were collected into FBS-containing Advanced DMEM/F12 medium to terminate digestion and counted.
4) Pretreatment of the chamber: taking out the Cell culture insert, placing the Cell culture insert in a 24-hole Cell culture plate, and culturing the Cell culture insert at the concentration of 10 mu g/cm 2 The Collagen was diluted and added to the upper layer of the cell. The cells were placed in a cell incubator at 37 ℃ for 30min, and before use the Collagen dilutions were aspirated and washed 2 times with PBS.
5) Lentivirus infection and propagation culture: 200000 cells, 8. mu.M Polybrane and HBLV-ZsGreen-Puro lentivirus at MOI 25 were mixed well in 200. mu.l of medium per well and added to the upper chamber layer. Mu.l of human intestinal metaplasia ALI growth medium (the same growth medium as in example 1) was added to the lower chamber, and the mixture was incubated at 37 ℃ and 5% CO 2 Culturing in an incubator. After 24h of culture, the upper medium was replaced. And removing the upper culture medium on the third day of culture to start gas-liquid interface culture. During this period mucus in the upper chamber was removed and human intestinal metaplasia ALI medium was changed every 3 days, with passages at a ratio of 1:4 every 4 weeks. Passage 3 times, cell infection efficiency was measured by flow cytometry. The infection efficiency of human intestinal metaplasia ALI cells is 92.7 percent through detection.
Comparative example 1
The comparative example provides a method for constructing a human intestinal metaplasia gas-liquid interface model, which is different from the method in example 1 in that: the proliferation medium consists of, at final concentration: human EGF 20 ng/ml; human FGF-10150 ng/ml; human noggin150 ng/ml; human gastrin10 nM; b272%; n21%; y-276327.5. mu.M (reduced from day 3 to 1.5. mu.M); a83-011 mu M; nicotinamide10 mM; HEPES10 mM; glutamax 1%; advanced DMEM/F-1293.45%; the culture was carried out for 14 days.
Comparative example 2
The comparative example provides a method for constructing a human intestinal metaplasia gas-liquid interface model, which is different from the method in example 1 in that: the proliferation medium consists of, at final concentration: wnt3a 30%; r-spondin 115%; human EGF 20 ng/ml; human FGF-10150 ng/ml; human noggin150 ng/ml; human gastrin10 nM; b272%; n21%; a83-011 mu M; nicotinamide10 mM; HEPES10 mM; glutamax 1%; advanced DMEM/F-1248.45%; the culture was carried out for 14 days.
Comparative example 3
The comparative example provides a method for constructing a human intestinal metaplasia gas-liquid interface model, which is different from the method in example 1 in that: the proliferation medium consists of the following components according to final concentration: wnt3a 30%; r-spondin 115%; b272%; n21%; y-276327.5. mu.M (decreasing from day 3 to 1.5. mu.M); HEPES10 mM; glutamax 1%; advanced DMEM/F-1248.45%); the culture was carried out for 14 days.
FIG. 10 provides the cell numbers of the human intestinal metaplasia gas-liquid interface models obtained in example 1, comparative example 2, and comparative example 3, wherein the cell number of the model of about 978000 can be obtained by culturing in example 1 for 14 days; secondly, comparative example 2, after removing Y-27632 from the proliferation medium, the number of cells in the model obtained by 14 days of culture was reduced to about 796000; the proliferation medium composition of comparative example 1, from which 4 components of Wnt3a and R-spondin 1 were removed, and the proliferation medium composition of comparative example 3 from which 4 components of recombinant human epidermal growth factor, recombinant human fibroblast growth factor, recombinant human Noggin protein and human gastrin were removed, had a great effect on the successful construction of the model.
Comparative example 4
The comparative example provides an operation method for lentivirus infection of a human intestinal metaplasia gas-liquid interface model, which is different from the operation method in example 3 in that: this comparative example was cultured in a gas-liquid interface model on day 7, and 8. mu.M Polybrane and HBLV-ZsGreen-Puro lentivirus with an MOI of 25 were thoroughly mixed in 200. mu.l of the medium and added to the upper layer of the chamber. After 24h, the upper medium was aspirated. Cell fluorescence was observed with a fluorescence microscope on days 1, 3 and 7 of infection, respectively, and figure 11 provides the cells on day 7 of the lentivirus-infected human intestinal metaplasia ALI model for example 3 and comparative example 4. FIG. 11 shows that the infection of comparative example 4 was unsuccessful, suggesting that the time point for lentiviral infection on the gas-liquid interface model is critical.
In summary, the invention has the advantages that:
1) the invention develops a proliferation culture medium and a differentiation culture medium constructed by a human intestinal epithelization gas-liquid interface model, and adopts a cell culture chamber with an upper chamber and a lower chamber to successfully construct the human intestinal epithelization gas-liquid interface model, thereby realizing the complementation of the prior animal model system.
2) The human intestinal metaplasia gas-liquid interface model constructed by the invention can proliferate for a long time, the culture time is more than 6 months and passage is stable, the passage time is 2 weeks to 6 weeks, the model can be treated by adding medicines for a long time, and the model can be used as an in vitro medicine screening tool for IM treatment and can also be used as a model for long-term infection of helicobacter pylori and the like.
3) The human intestinal metaplasia gas-liquid interface model constructed by the invention has high simulation performance, and can highly simulate the in-vivo real gastric epithelial enteroplasia characteristics, 1) the model has cell polarity, can distinguish a basal surface and a free surface, and can secrete mucus; 2) various cell types with gastric gland lineages; 3) has the phenotype of intestinal metaplasia of gastric mucosa of organism.
4) The model can be used for evaluating the effect of medicines, as a bacterial infection or lentivirus infection model and as an immune cell or interstitial cell co-culture model, can better research the mechanism of enterogenesis, and can be used for preclinical experiments to determine possible treatment methods and perform personalized treatment on patients.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. A proliferation culture medium of a human intestinal metaplasia gas-liquid interface model is characterized in that: the following components were included at the final concentrations: 30% -50% of Wnt3a 30; 115% -25% of R-spondin; human EGF10-50 ng/ml; 500ng/ml of human FGF-1050; human noggin50-500 ng/ml; human gastrin5-50 nM; b272%; n21%; y-276321.5-7.5 mu M; a83-010.5-5 mu M; nicotinamide5-50 mM; HEPES10 mM; glutamax 1%; advanced DMEM/F-1218.45% -48.45%.
2. A differentiation medium of a human intestinal metaplasia gas-liquid interface model is characterized in that: the following components were included at the final concentrations: human EGF10-50 ng/ml; 500ng/ml of human FGF-1050; human noggin50-500 ng/ml; human gastrin5-50 nM; b272%; n21%; y-276321.5-7.5 mu M; a83-010.5-5 mu M; nicotinamide5-50 mM; HEPES10 mM; glutamax 1%; advanced DMEM/F-1293.45%.
3. A method for constructing a human intestinal metaplasia gas-liquid interface model is characterized by comprising the following steps: the method comprises culturing human intestinal metaplasia cells to be cultured in a cell culture chamber having an upper chamber and a lower chamber, wherein the human intestinal metaplasia cells to be cultured are added to the upper chamber, and the proliferation medium according to claim 1 is added to the lower chamber, and culturing is performed under the condition of gas-liquid interface.
4. The method for constructing the human intestinal metaplasia gas-liquid interface model according to claim 3, wherein the method comprises the following steps: the human intestinal metaplasia cells to be cultured are added into the upper small chamber, the proliferation culture medium is added into the lower small chamber, and the culture is carried out under the condition of a gas-liquid interface, and the method specifically comprises the following steps:
resuspending the human intestinal metaplasia cells to be cultured in the proliferation medium at 1000- 2 And removing the proliferation culture medium in the upper chamber after 3 days of culture, starting gas-liquid interface culture, removing mucus in the upper chamber every 3 days, replacing the proliferation culture medium in the lower chamber, and culturing for 1-2 weeks to obtain the model.
5. A proliferation culture method of a human intestinal metaplasia gas-liquid interface model is characterized by comprising the following steps: a cell culture chamber having an upper chamber and a lower chamber is used, comprising the steps of:
(1) digesting the obtained human intestinal metaplasia gas-liquid interface model for 30min at 37 ℃ by adopting pancreatin;
(2) the cells were collected into FBS-containing Advanced DMEM/F12 medium to stop digestion and counted;
(3) the cell culture chamber was placed in a 24-well cell culture plate at 10. mu.g/cm 2 The concentration of Collagen (Collagen) was diluted and added to the upper chamber, left at 37 ℃ for 30-60min, and the Collagen dilution was aspirated and washed 2 times with PBS;
(4) resuspending the cells obtained in step (2) in the proliferation medium described in claim 1 at 1000- 2 And (3) removing the proliferation culture medium in the upper chamber after culturing for 3 days, starting gas-liquid interface culture, removing mucus in the upper chamber every 3 days, replacing the proliferation culture medium in the lower chamber, and carrying out passage once every 2-6 weeks, namely repeating the steps (1) - (4), wherein the passage is carried out at least 6-10 times.
6. A differentiation culture method of a human intestinal metaplasia gas-liquid interface model is characterized by comprising the following steps: is a human intestinal metaplasia gas-liquid interface model obtained by the proliferation culture method according to claim 5, which comprises the steps of:
carrying out proliferation culture on human intestinal metaplasia gas-liquid interface model, removing mucus in upper layer small chamber, and culturingRemoving the proliferation medium by aspiration, adding the differentiation medium of claim 2, and culturing at 37 deg.C with 5% CO 2 Culturing in an incubator, wherein mucus in the upper chamber is removed and the differentiation medium is replaced every 3 days, and culturing is carried out for 6 days.
7. A human intestinal metaplasia gas-liquid interface model is characterized in that: is obtained by the method of constructing according to claim 3 or 4, the method of growing culture according to claim 5, or the method of differentiating culture according to claim 6.
8. Use of the human intestinal metaplasia gas-liquid interface model of claim 7 for the assessment of drug efficacy.
9. Use of the human intestinal metaplasia fluid interface model of claim 7 as a model for bacterial or lentiviral infection.
10. Use of the human intestinal metaplasia gas-liquid interface model of claim 7 as a co-culture model of immune cells or stromal cells.
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