Primary porcine gastric mucosa epithelial cell, separation method, cell line, establishment method and application

Abstract

The invention discloses primary porcine gastric mucosa epithelial cells, a separation method, a cell line, an establishment method and application. The method comprises the following steps: 1) obtaining stomach body and stomach fundus tissues, and cleaning the obtained tissues in a cleaning solution; the cleaned tissue is placed in a cleaning culture medium to be soaked; 2) carrying out digestion treatment on the soaked tissue, and separating out an epithelial layer; performing digestion; 3) performing enzymolysis treatment on the epithelial layer; the enzymatic hydrolysate is prepared from the following components according to final concentration: 0.5 to 3 mg / mL of hyaluronidase, 1 to 5 mg / mL of collagenase I and water as a solvent; (4) terminating enzymolysis, filtering through a 100mu m cell filter membrane, then carrying out centrifugal treatment, and retaining a cell layer; 5) resuspending the cell layer in a proliferation culture medium, and performing cell culture under the conditions of 37 DEG C and 5% CO2, and 6) performing subculture when the cell growth density reaches 80 + / -5% confluence degree to obtain the primary porcine gastric mucosa epithelial cells, by adopting the method, the high-purity and high-activity primary porcine gastric mucosa epithelial cells can be efficiently separated and cultured.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to primary porcine gastric mucosal epithelial cells and isolation methods, cell lines and establishment methods and applications. Background Art

[0002] The porcine stomach is a key organ in the digestive process, and its digestive function directly affects the growth performance of pigs. Aflatoxin B1 (AFB1) is the most common and toxic mycotoxin in livestock feed, which has a serious impact on the growth and development of pigs. There have been progress in in vivo studies on the effects of AFB1 on animal gastric tissues. However, in vitro, the mechanism of action of AFB1 on porcine gastric epithelial cells is not fully understood. AFB1 may damage the gastric mucosa by inducing epithelial cell apoptosis, thereby leading to impaired digestion and absorption functions.

[0003] The isolation and culture of gastric mucosal epithelial cells have been well established in many species, including humans, mice, rabbits, and cattle. However, it still faces challenges to efficiently isolate and obtain highly pure porcine gastric epithelial cells.

[0004] Primary porcine gastric epithelial cells (PGECs) are important models for studying the molecular mechanisms and pathogenic processes of porcine gastric diseases, but their acquisition is difficult, the isolation process is costly, time-consuming, poor in stability and short in lifespan, which greatly limits their application prospects.

[0005] Immortalization refers to the process in which cells avoid senescence and death and have the ability of infinite division and proliferation during in vitro culture, either through genetic changes or external stimuli. In recent years, extending the lifespan of cells by viral gene transfection and telomerase activation has become a widely adopted method. There have been studies successfully establishing various immortalized epithelial cell lines from porcine oral cavity, ileum, colon, and small intestine for evaluating gene expression patterns and cell physiological states. However, at present, no porcine gastric epithelial cell line has been established, and an ideal cell model is still lacking. Currently, the enzyme digestion method is widely used in the isolation and culture of epithelial cells. However, when applying these techniques to isolate porcine gastric epithelial cells, there are some significant challenges, including low purity of epithelial cells and decreased viability. Summary of the Invention

[0006] The object of the present invention is to provide primary porcine gastric mucosal epithelial cells and isolation methods, cell lines and establishment methods and applications. By using the method of the present invention, highly pure and highly viable primary porcine gastric mucosal epithelial cells can be efficiently isolated and cultured, and the lifespan of porcine gastric epithelial cells cultured in vitro can be extended by the method of lentivirus infection of the HTERT gene and SV40T virus, and an immortalized cell line maintaining the physiological characteristics of primary cells can be established.

[0007] In order to solve the problems existing in the prior art, the technical solution adopted by the present invention is:

[0008] In the first aspect, the present invention provides a method for isolating primary porcine gastric mucosal epithelial cells, comprising the following steps:

[0009] 1) Obtain gastric body and fundus tissues, place the obtained tissues in a cleaning solution for cleaning; and soak the cleaned tissues in a cleaning medium;

[0010] 2) Transfer the soaked tissues to a dispase II-HBSS cell digestive solution for digestion treatment to isolate the epithelial layer; digest, and the dispase II-HBSS cell digestive solution is prepared by dissolving dispase II in HBSS;

[0011] 3) Perform enzymatic digestion on the epithelial layer; the enzymatic digestion solution has a final concentration composition of: hyaluronidase 0.5-3 mg / mL, collagenase I 1-5 mg / mL, and the solvent is water;

[0012] 4) Add RPMI1640 medium to terminate the enzymatic digestion, filter through a 100±1 μm cell filter membrane and then perform centrifugation treatment, and retain the cell layer;

[0013] 5) Resuspend the cell layer in a proliferation medium, and perform cell culture under the conditions of 37°C and 5% CO 2 ;

[0014] 6) When the cell growth density reaches 80±5% confluence, perform subculture at 37±0.5°C to obtain primary porcine gastric mucosal epithelial cells.

[0015] Further, in step 1), the cleaning solution is an HBSS solution containing 1±0.1% (m / m) penicillin / streptomycin and 100±1 μg / mL gentamicin / amphotericin B.

[0016] Further, in step 1), the cleaning medium is an HBSS solution containing 0.5%-1.5% (m / m) penicillin / streptomycin and 1%-2% (m / m) gentamicin / amphotericin B.

[0017] Further, in step 2), the concentration of dispase II in the dispase II-HBSS cell digestive solution is 1±0.1 mg / mL.

[0018] Further, in step 3), the composition of the enzymatic hydrolysate in terms of the final concentration is: hyaluronidase 1 - 2 mg / mL, collagenase I 2 - 3 mg / mL, and the solvent is water. Further, in step 4), the centrifugation process is carried out in two steps. After passing through a 100 ± 1 μm cell filter membrane for the first time, it is centrifuged at 380 - 410 xg for 4 - 6 minutes. For the second time, the obtained cells are added to HBSS containing 1 ± 0.1% (m / m) penicillin / streptomycin and centrifuged again at 380 - 410 xg for 4 - 6 minutes.

[0019] In a second aspect, the present invention provides a primary porcine gastric mucosal epithelial cell, which is obtained by the method for isolating and culturing primary porcine gastric mucosal epithelial cells described in the first aspect above.

[0020] In a third aspect, the present invention provides a porcine gastric mucosal epithelial cell line, which is deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: C202493, and the deposit date is November 22, 2024, named: porcine gastric mucosal epithelial cell line i - PGECs Sus scrofa.

[0021] In a fourth aspect, the present invention provides a method for establishing the porcine gastric mucosal epithelial cell line described in the third aspect above, including the following steps:

[0022] S1: Passage - culture the primary porcine gastric mucosal epithelial cells described in claim 7. When the confluence of the primary porcine gastric epithelial cells reaches 70 ± 5%, infect the primary porcine gastric epithelial cells overnight with two lentiviruses SV40T and hTERT at a concentration of MOI = 80 ± 1, and culture at 37°C.

[0023] S2: After culturing for 24 - 30 hours, add fresh complete medium, and then change the complete medium every other day.

[0024] S3: After 3.5 - 4.5 days, culture the primary porcine gastric epithelial cells in fresh complete medium containing 1 ± 0.1 μg / ml puromycin for 80 - 120 hours, and then perform amplification culture at 37°C.

[0025] S4: Detect the mycoplasma of the cells obtained by amplification culture. Culture the cell line with negative detection in DMEM / F12 (11320033 - Gibco, USA) medium containing 10% (m / m) FBS and 1% (m / m) penicillin / streptomycin at 37°C to obtain the porcine gastric mucosal epithelial cell line.

[0026] The present invention is the first to infect primary porcine gastric epithelial cells with SV40T and HTERT to construct an immortalized cell line, and this method provides a stable and reproducible model. This cell line provides a valuable tool for studying the molecular mechanisms and pathogenic processes affecting porcine gastric epithelium, especially processes related to immune responses and pathogen infections (such as exposure to aflatoxin B1). This model has great prospects in promoting livestock production and biomedical research, especially in gaining in-depth understanding of porcine gastrointestinal diseases and formulating effective treatment strategies.

[0027] In a fifth aspect, the present invention provides the use of the porcine gastric mucosal epithelial cell line described in the third aspect above in constructing an in vitro model for studying the molecular mechanisms and biological functions of the porcine stomach.

[0028] Using this cell line as a model for aflatoxin infection experiments, the results showed that genes related to immunity were upregulated.

[0029] The advantages and beneficial effects of the present invention are:

[0030] The present invention successfully used a combination of type I collagenase, hyaluronidase, and dispase II to isolate primary porcine gastric epithelial cells with high purity and high viability.

[0031] By using the method of the present invention, primary porcine gastric mucosal epithelial cells with high purity and high viability can be efficiently isolated and cultured. By lentiviral infection of the SV40T and HTERT genes, the in vitro lifespan of porcine gastric epithelial cells was extended, and an immortalized gastric epithelial cell line (i-PGECs) was successfully established, which retained the physiological characteristics of primary cells. This cell line provides an ideal experimental model for studying the digestion and metabolism of the porcine stomach, filling the gap in the existing porcine gastric epithelial cell line. Through techniques such as marker gene expression, proliferation detection, karyotype analysis, qRT-PCR, and RNA-seq, it was confirmed that i-PGECs retained the morphological and functional characteristics of primary PGECs.

[0032] We used transcriptome sequencing technology to identify key regulatory factors (PLAUR, NPC1) in the response of porcine gastric epithelial cells to aflatoxin B1 (AFB1), laying a foundation for using this cell line as an in vitro model for studying the molecular mechanisms and biological functions of the porcine stomach. i-PGECs can also serve as an important model for studying the pathogenesis of gastric infection-related diseases. It has important guiding significance and reference value for improving important growth traits of pigs in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Comparison of the primary epithelial cell isolation methods described in Example 1 and Comparative Example 1 and establishment of the immortalized cell line; in the figure:

[0034] A is a schematic diagram of the primary cell separation process described in Example 1 and Comparative Example 1;

[0035] B is a comparison of the total number of cells obtained in Example 1 and Comparative Example 1. B(1) is a comparison of cell numbers; B(2) is a comparison of the number of live cells; B(3) is a comparison of the percentage of live cells;

[0036] C is a microscopic image of the cells separated by the methods of Example 1 and Comparative Example 1 on the 1st day, 2nd day after culturing, and 4th day after passage;

[0037] D is a 10X and 20X microscopic image of PGECs and i-PGECs-P3;

[0038] E is the result of cytokeratin 18 immunostaining of PGECs, i-PGECs-P3, and GES-1;

[0039] F is a microscopic image of the immortalized cell line after establishment and its subsequent passage cells:

[0040] In the figure: iPGECs-P3 is the 3rd generation of immortalized porcine gastric epithelial cells; GES-1 is human gastric mucosal epithelial cells;

[0041] Figure 2 For the identification of PGECs and i-PGECs by RT-PCR and immunofluorescence; In the figure:

[0042] A is the analysis of target genes by QRT-PCR. (A-I) is the mRNA expression difference of SV40T in primary PGECs and i-PGECs (P<0.0001); (A-II) is the mRNA expression difference of HTERT in primary PGECs and i-PGECs ((P<0.0001).

[0043] B is the detection of the mRNA expression of KRT18, CDH1, and MUC1 in primary PGECs and i-PGECs by reverse transcription polymerase chain reaction (RT-PCR); LaneM: DNA MarkerⅡ; lane1, 3, 6: expression of KRT18, E-cadherin, and MUC1 in PGECs; lane2, 4, 7: expression of KRT18, E-cadherin, and MUC1 in i-PGECs; lane5: expression of GAPDH in PGECs;

[0044] C is the immunofluorescence identification of PGECs and i-PGECs.

[0045] In the figure: Immunostaining shows the expression of cytokeratin 18 (CK18), EPCAM, and E-Cadherin in PGECs and i-PGECs (20X); green fluorescence indicates CK18 and E-cadherin proteins, and red fluorescence indicates EPCAM protein; i-PGECs-P20 is the 20th passage of the immortalized porcine gastric epithelial cell line; i-PGECs-P42 is the 42nd passage of the immortalized porcine gastric epithelial cell line; i-PGECs-P50 is the 50th passage of the immortalized porcine gastric epithelial cell line;

[0046] Figure 3 For the apoptosis, cell cycle, and karyotype analysis of PGECs and i-PGECs; In the figure:

[0047] A is the transmission electron microscope image of i-PGECs; In A-I: De is the desmosome (arrow on the left side of Figure A-I), and TJ is the tight junction (arrow on the right side of Figure A-I); In A-II, MT is the mitochondrion (arrow in Figure A-II);

[0048] (B) is the karyotype analysis of i-PGECs, (B-I) is the karyotype analysis of i-PGECs-P20; (B-II) is the karyotype analysis of i-PGECs-P42;

[0049] (C) is the growth curve of primary PGECs and i-PGECs;

[0050] (D) is the apoptosis and cycle analysis of primary PGECs and i-PGECs,

[0051] (D-I) shows that the apoptosis percentages of primary PGECs and i-PGECs under normal growth conditions are 33.36% and 6.35% (Q2+Q4) respectively on the left and right;

[0052] (D-II) shows the cell cycle analysis of primary PGECs and i-PGECs on the left and right respectively; The percentages of primary PGECs and i-PGECs in the S phase are 10.96% and 24.63% respectively;

[0053] i-PGECs-P20 is the 20th passage of the immortalized porcine gastric epithelial cell line; i-PGECs-P42 is the 42nd passage of the immortalized porcine gastric epithelial cell line; i-PGECs-P50 is the 50th passage of the immortalized porcine gastric epithelial cell line.

[0054] Figure 4 For the transcriptome sequencing results of PGECs and i-PGECs; In the figure:

[0055] (A) is the principal component analysis (PCA) plot of RNA-Seq data of PGECs and i-PGECs, and different cell populations are represented by different colors;

[0056] (B) is the heatmap of differentially expressed gastric epithelial cell genes (DEGs) between PGECs and i-PGECs;

[0057] (C) is the functional enrichment analysis of PGECs and i-PGECs,

[0058] (C-I) are the top 5 KEGG pathways with the most upregulation;

[0059] (C-II) are the top 5 KEGG pathways with the most downregulation.

[0060] Figure 5 For the morphological observation and RNA-seq analysis of AFB1-infected i-PGECs, in the figure:

[0061] (A) are the microscopic images of i-PGECs 24 hours after infection with 2 μm AFB1. The upper figure is the control group, and the lower figure is the treatment group.

[0062] (B) is the principal component analysis (PCA) plot of RNA-Seq data for the control group and the treatment group. The control group is i-PGECs; the treatment group is AFB1-i-PGECs;

[0063] (C) is the volcano plot of differentially expressed genes (DEGs);

[0064] (D) is the KEGG pathway enrichment analysis for the control group and the treatment group;

[0065] (D-I) is the KEGG pathway enrichment analysis of upregulated genes in i-PGECs 24 hours after infection with 2 μm AFB1 compared with the uninfected control group;

[0066] (D-II) is the KEGG pathway enrichment analysis of downregulated genes in i-PGECs 24 hours after infection with AFB1 compared with the uninfected control group. Specific implementation manners

[0067] Next, the technical solutions in the embodiments of the present application will be clearly described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art belong to the scope protected by the present application.

[0068] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0069] The compositions of some of the culture media and reagents used in this application are shown in Table 1:

[0070] Table 1

[0071]

[0072] This application provides a porcine gastric mucosal epithelial cell line. The gastric body and fundus of pigs are selected as samples, and primary porcine gastric epithelial cells are isolated and identified using dispase II, type I collagenase, and hyaluronidase. Immortalized cell lines are constructed by infecting primary porcine gastric epithelial cells with lentiviral SV40T and hTERT. Currently, the immortalized porcine gastric primary cell line has been passaged more than 50 times and has been deposited at the China Center for Type Culture Collection for preservation certification. The deposit address is Wuhan University, Wuhan, China, the deposit date is November 22, 2024, the deposit number is CCTCC NO: C202493, and the name of the culture is: Porcine Gastric Mucosal Epithelial Cell Line i - PGECs Sus scrofa.

[0073] Example 1

[0074] This example provides a method for isolating porcine gastric mucosal epithelial cells, including the following steps:

[0075] 1) Select healthy pigs and anesthetize them under the premise of conforming to animal ethics; fix the pigs on the dissection table, carefully cut open the pig's abdomen with a sterilized scalpel to expose the stomach; after finding the stomach, cut the required tissue blocks from the middle part (gastric body) of the stomach and cut tissues from the upper part (fundus) of the stomach. Place the obtained gastric body and fundus tissues in a washing solution and wash them more than ten times. The washing solution is Hank's balanced salt solution (HBSS) containing 1% (m / m) penicillin / streptomycin and 100 μg / mL gentamicin / amphotericin B;

[0076] 2) Immerse the tissues in a culture dish containing washing medium for 20 minutes;

[0077] 3) Then transfer them to a dispase II - HBSS cell digestive solution and store them at 4°C for overnight digestion for 15 hours; the dispase II - HBSS cell digestive solution is prepared by dissolving dispase II (D4693 - Gibco, USA) in HBSS, and the concentration of dispase II in the dispase II - HBSS cell digestive solution is 1 mg / mL;

[0078] 4) Subsequently, use forceps to peel off the epithelial layer and wash it with HBSS;

[0079] 5) Next, use the blunt side of a scalpel to scrape the mucus layer on the epithelial surface and transfer it to a centrifuge tube containing a mixed enzymatic digestion solution; the mixed enzymatic digestion solution is composed of hyaluronidase (H3506-Merck, USA) and collagenase I (004196-Worthington, USA), and the concentration of hyaluronidase in the mixed enzymatic digestion solution is 1 mg / mL, and the concentration of collagenase I is 2 mg / mL;

[0080] 6) Place the centrifuge tube in a reciprocating shaking water bath at 37 °C for 1 hour for digestion;

[0081] 7) Add 2 volumes of RPMI1640 medium for terminating enzymatic digestion to terminate the enzymatic digestion. The RPMI1640 medium for terminating enzymatic digestion is added with 10% (m / m) fetal bovine serum (FBS; 16140071-Gibco, USA), 1% (m / m) penicillin / streptomycin, and 1 ml of Prophylactic (ant-mpp-InvivoGen, USA) with a concentration of 2.5 mg / ml in RPMI1640 medium (11875093-Gibco, USA); Prophylactic (ant-mpp-InvivoGen, USA);

[0082] 8) Mix the suspension well, filter it through a 100-μm cell filter membrane, and then centrifuge at 400 xg for 5 minutes;

[0083] 9) Then add HBSS containing 1% (m / m) penicillin / streptomycin to the centrifuge tube and centrifuge again at 400 xg for 5 minutes;

[0084] 10) Discard the supernatant, resuspend the precipitate in the proliferation medium, and perform cell culture in an incubator at 37 °C and 5% CO 2 2;

[0085] The proliferation medium is added with 20% (m / m) fetal bovine serum (FBS; 16140071-Gibco, USA), 1% (m / m) penicillin-streptomycin, 2.5 mg / mL Prophylactic (2.5 mg / mL, ant-mpp-Invivogen, USA), insulin (4 μg / mL, 491077C-Sigma, USA), Epidermal Growth Factor (EGF) (25 ng / mL, C029-Novoprotin, China), HEPES (25 mM, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) (15630080-Gibco, USA), gentamicin / amphotericin B (100 μg / mL, R01510-Gibco, USA). 11) On the next day, the culture dish was washed twice with pre-warmed HBSS to remove non-adherent cells. The cells were observed daily to ensure no contamination;

[0086] 12) When the cell growth density reached 80% confluence, subculture was carried out at 37 °C for 24 h to obtain primary porcine gastric mucosal epithelial cells, denoted as primary PGECs.

[0087] The separation method of this example is denoted as (Method 2, DCH).

[0088] Comparative Example 1

[0089] This comparative example provides a method for separating porcine gastric mucosal epithelial cells. The difference between this comparative example and Example 1 is only that step 3) is omitted, and the rest are the same as in Example 1.

[0090] The separation method of this comparative example is denoted as (Method 1, CH).

[0091] Example 2

[0092] This example provides a porcine gastric mucosal epithelial cell line, and its construction method includes the following steps:

[0093] S1: When the confluence of the primary porcine gastric epithelial cells obtained in Example 1 reached 70%, the primary porcine gastric epithelial cells were infected overnight with two lentiviruses, SV40T (Vector Builder, China) and hTERT (Vector Builder, China), at a concentration of MOI = 80, and cultured at 37 °C;

[0094] S2: After culturing for 1 day, fresh complete medium was added, and during the subsequent culture process, the complete medium was changed every other day; the complete medium was DMEM / F12 supplemented with 10% (m / m) FBS and 1% (m / m) penicillin / streptomycin (P1400-Solarbio, China);

[0095] S3: Four days later, the primary porcine gastric epithelial cells were cultured in fresh complete medium containing 1 μg / ml puromycin (Sigma-Aldrich, St. Louis, MO) for 96 hours, and then cultured and amplified at 37 °C for 48 h;

[0096] S4: After mycoplasma detection was negative, the newly established cell line was cultured at 37 °C in DMEM / F12 (11320033 - Gibco, USA) medium containing 10% (m / m) FBS and 1% (m / m) penicillin / streptomycin to obtain an immortalized porcine gastric mucosal epithelial cell line, denoted as (i-PGECs).

[0097] Experimental Example 1

[0098] I. Methods

[0099] 1. Experimental animals

[0100] Female two-way crossbred pigs at 4 to 5 weeks of age were purchased from Wangjiang Farm, Sichuan Province, China. During the experiment, the pigs were all humanely treated. After being anesthetized until euthanasia, the gastric body and fundus tissues were collected for subsequent experiments. All animal experiments were approved by the Laboratory Animal Ethics Committee of Sichuan Agricultural University and were strictly conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals.

[0101] 2. Chemical reagents

[0102] Aflatoxin B1 (AFB1) (A6636, Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) (CAS: 67 - 68 - 5, MP-Biomedicals) to prepare a stock solution with a concentration of 1000 μg / mL. After aliquoting, the stock solution was stored at -20 °C for later use.

[0103] 3. The methods described in Example 1 and Comparative Example 1 were respectively used to isolate porcine gastric mucosal epithelial cells, and the method described in Example 2 was used to establish a porcine gastric mucosal epithelial cell line.

[0104] 4. Cell line culture and AFB1 treatment

[0105] Human gastric epithelial cells (GES-1) were purchased from Meisen Cell Bank (CCTCC-00200010, Zhejiang, China). GES-1 cells were cultured in RPMI 1640 medium containing 10% (m / m) fetal bovine serum (FBS, Gibco) and 1% (m / m) penicillin-streptomycin, and placed at 37 °C, 5% CO 2Cultured under the conditions stated. Unless otherwise specified, the primary porcine gastric epithelial cells (PGECs) and immortalized porcine gastric epithelial cells (i-PGECs) used in all subsequent experiments were the 2nd passage and the 42nd passage respectively. The i-PGECs were seeded in 6-well culture plates and cultured until the cell confluence exceeded 85%. Twenty-four hours before treatment, the complete medium was replaced with serum-free medium (DMEM / F12 medium). After pretreatment of the i-PGECs, 2 μmol / L of aflatoxin B1 (AFB1) was added and the cells were treated for 24 hours. Subsequently, the cells in the control group and the treatment group were both collected for RNA sequencing (RNA-seq) analysis.

[0106] 5. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis

[0107] The primary porcine gastric epithelial cells isolated in Example 1 and Comparative Example 1 and the immortalized porcine gastric epithelial cell line in Example 2 were seeded in 6-well plates at a density of 2×10 5 cells / mL, and 2 mL of cell suspension was added to each well. Total RNA was extracted using TRIzol reagent (Invitrogen) according to the requirements of the reagent instructions. The quality and purity of RNA were determined by measuring the optical density (OD) at wavelengths of 260 nm and 280 nm using a spectrophotometer. 1 μg of RNA was reverse transcribed into cDNA using HiScript III RT SuperMix (Vazyme, China). The diluted cDNA was used as a template for quantitative real-time PCR (qRT-PCR). On a CFX Connect TM Real-Time Fluorescent Quantitative System (Bio-Rad, USA), qRT-PCR amplification was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). The reaction conditions were pre-denaturation at 95°C for 30 seconds, followed by 40 cycles with the cycle conditions of 95°C for 10 seconds and 60°C for 30 seconds. After PCR amplification, the primer specificity was verified by melting curve analysis, and the melting curve conditions were: 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds. GAPDH was used as the internal reference gene in the experiment. The PCR primer sequences and the target gene fragment sizes are listed in Table 2. The Ct values were calculated in Excel according to the standard procedure, and the relative expression level of mRNA was calculated using the 2-ΔΔCt method (Zhang et al., 2022), where △△Ct = (Ct 实验组目标基因 - Ct 实验组GAPDH ) - (Ct 对照组目标基因 - Ct 对照组GAPDH ).

[0108] Table 2 Primer sequences

[0109]

[0110] 6. Immunofluorescence Experiment

[0111] The primary cells obtained in Example 1 and Comparative Example 1 and the immortalized cells established in Example 2 cultured in 12-well plates were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 minutes. Subsequently, the cells were rinsed three times with PBS1X and infiltrated in a 0.02% Triton X-100 solution at room temperature for 20 minutes. After infiltration, a PBS solution containing 5% bovine serum albumin (BSA) and 2% goat serum was added and blocked at room temperature for 1 hour to block nonspecific binding sites. After blocking, the cells were incubated with a primary antibody at room temperature for 1 hour. The primary antibodies included: Cytokeratin 18 (Cytokeratin 18, Catalog No. A19778, ABclonal, China), E-cadherin (Catalog No. 20874-1-AP, Proteintech, China), and EPCAM (Catalog No. ER1802-80, HUABIO, China), all diluted with PBS containing 5% BSA at a volume ratio of 1:100. After the primary antibody incubation was completed, the cells were washed three times with PBS solution. Subsequently, the cells were placed in a PBS solution containing 5% BSA, and a 1:500 dilution of goat anti-rabbit IgG (H+L) secondary antibody (Cat. No. AS053, ABclonal, China) was added and incubated at room temperature for 1 hour. After the secondary antibody incubation was completed, the cells were washed again three times with 1X PBS. Then, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, China) for 5 minutes. Finally, the experimental results were observed and recorded by fluorescence microscopy (Nikon, Japan).

[0112] 7. Growth Curve Determination

[0113] The primary porcine stomach cells obtained in Example 1 and Comparative Example 1 and the immortalized cell line established in Example 2 were inoculated into a 96-well plate, and 100 μL of 1×10 3 Each treatment group was set up with 6 replicates per day, and the cell activity was tested every day according to the instructions of the Cell Counting Kit-8 (CCK-8, Solarbio).

[0114] After adding 10 μL of CCK-8 reagent to each well, the plate was incubated at 37°C for 2 hours, and then the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Science Inc.). The cell growth curve was plotted with time as the x-axis and the average absorbance (A450) as the y-axis.

[0115] 8. Karyotyping steps

[0116] Detect the chromosome number of immortalized cells according to the standard method. When the immortalized cells reach 80% confluence, treat the cells with colchicine (Solarbio, Beijing, China) at a final concentration of 0.025 μg / mL at 37 °C for 5 hours. Subsequently, the cells are collected after trypsin digestion and the cells are precipitated by centrifugation (200 g, 5 minutes).

[0117] After preheating 0.075 M KCl, resuspend the cells in 5 mL of potassium chloride solution and incubate at 37 °C for 20 minutes. Then, fix the cells with freshly prepared ice-cold glacial acetic acid-methanol (volume ratio of glacial acetic acid to methanol is 1:3) fixative and gently mix with a pipette. Repeat this step twice after collecting the cells by centrifugation (1000 rpm, 10 minutes), discard the supernatant, and add 5 mL of fixative to resuspend the cells.

[0118] Subsequently, drop 100 μL of the cell suspension onto a pre-cooled glass slide, quickly dry it with a flame, and then stain the chromosomes with Giemsa stain (Giemsa stock, 9:1) diluted in phosphate buffer at pH: 7.4 for 15 - 20 minutes. After staining, rinse with water and air dry. Select cells in metaphase with well-dispersed chromosomes under a low-power microscope and observe with an oil immersion lens. The samples are then dehydrated, covered with a coverslip, and sealed. Observe and digitally record the chromosome number of metaphase cells (n = 100 cells) through a microscope.

[0119] 9. TEM imaging

[0120] After digesting the cultured cells with 0.25% trypsin, collect the cells by centrifugation (1000 rpm, 5 minutes). Discard the supernatant, fix the cells with 3% glutaraldehyde (diluted 1:5 with PBS) at 4 °C for 5 minutes. Subsequently, transfer the cell suspension to a 1.5 mL EP tube by centrifugation (12000 rpm, 10 minutes). The fixed samples are dehydrated step by step with gradient acetone, and the concentration gradient of the dehydrating agent is 30% → 50% → 70% → 80% → 90% → 95% → 100%. Subsequently, embed them in epoxy resin. Section the embedded samples and stain them with uranium salts. Finally, observe the cells under a transmission electron microscope (Hitachi, Tokyo, Japan) and collect images.

[0121] 10. Apoptosis detection

[0122] The primary porcine gastric cells obtained in Example 1 and Comparative Example 1 and the immortalized cell line established in Example 2 were seeded at 1×10 5After collecting the number of cells with PBS, centrifuge at 250 g for 5 minutes, repeat twice, and discard the supernatant. Add 500 μL of Annexin V-FITC binding buffer, 5 μL of Annexin V-FITC, and 10 μL of propidium iodide (PI) staining solution (from an apoptosis detection kit, Beyotime, Shanghai, China) to the cells, and incubate for 10 minutes at room temperature in the dark. Subsequently, detect the apoptosis ratio of the cells by flow cytometry, and perform data analysis using CytExpert software.

[0123] 11. Cell cycle detection

[0124] The primary porcine gastric cells obtained in Example 1 and Comparative Example 1 and the immortalized cell line cells established in Example 2 (1×10 6 cells) were fixed overnight with pre-cooled 75% ethanol, then washed twice with 1X PBS and the supernatant was discarded. Add 500 μL of PI / RNase A staining solution and incubate for 30 minutes in the dark. Subsequently, detect the cell cycle distribution by flow cytometry, and perform data analysis using CytExpert software.

[0125] II. Results

[0126] 1. The present invention successfully and efficiently isolated primary porcine gastric epithelial cells (PGECs), thereby establishing an immortalized porcine gastric epithelial cell line (i-PGECs).

[0127] In Comparative Example 1, type I collagenase and hyaluronidase (Method 1, CH) were used to digest and isolate epithelial cells. In Example 1 of the present invention, dispase II was added on the basis of type I collagenase and hyaluronidase (Method 2, DCH), which can significantly improve the efficiency of primary cell isolation. In this study, we isolated primary gastric epithelial cells (PGECs) from the porcine gastric epithelium by two different digestion methods (CH and DCH, see the method section and Figure 1 A). The results of trypan blue staining showed that at the same digestion time, the DCH method significantly increased the total cell yield and the number of live cells ( Figure 1 B). Comparison of the total number of cells: The number of cells obtained by the (CH) method was the least, while the number of cells obtained by the DCH method was the most, (P<0.01); Comparison of the number of live cells: The number of live cells obtained by the (DCH) method was significantly higher than that of other methods, (P<0.001); Comparison of the percentage of live cells: The percentage of live cells in the (DCH) method was significantly higher than that in the CH method, (P<0.001).

[0128] Figure 1(C) are the microscopic images of cells separated by two methods on the 1st day, 2nd day after culturing, and 4th day after passage. It was found by optical microscopy that the cells separated by the DCH method showed better morphological characteristics on the first day of culture, including that the average diameter of epithelial cells remained stable, and the cell contour still presented a slate-like structure ( Figure 1 C). After 48 hours, the cells in the DCH group migrated out of the aggregated state, began to divide and spread, showed clonal growth characteristics and had a higher refractive index. In addition, compared with the CH method, the purity of epithelial cells separated by the DCH method was higher ( Figure 1 C). After 96 hours of culture and passage, both groups of cells showed a certain degree of cell death and slow proliferation, and the cell death in the CH group was more significant. To establish stably passaged porcine gastric epithelial cells, the primary PGECs in the DCH group with higher viability and purity were selected, and the expression of SV40T and HTERT was enhanced using a lentiviral vector. After infecting with SV40T and HTERT, immortalized cells (i-PGECs) were obtained by puromycin screening. Figure 1 (D) are the microscopic images of PGECs and i-PGECs-P3. It was found by optical microscopy that there were no obvious morphological changes between i-PGECs-P3 and primary PGECs ( Figure 1 D). Immunofluorescence results showed that CK18 was expressed in primary PGECs, i-PGECs-P3 and GES-1 cells ( Figure 1 D). In addition, i-PGECs cells at different passages ( Figure 1 F) all showed typical cobblestone-like characteristics, and there were no obvious differences compared with primary cells.

[0129] Figure 1 (E) are the immunostaining results of cytokeratin 18 of PGECs, i-PGECs-P3 and GES-1. Immunofluorescence results showed that cytokeratin 18 was expressed in PGEC, i-PGECs-P3 and GES-1, proving that both i-PGECs-P3 and PGECs are epithelial cells.

[0130] 2. Biological characteristics of PGECs and i-PGECs

[0131] 2.1 i-PGECs maintained their biological characteristics

[0132] QRT-PCR analysis showed that SV40T ( Figure 2 A-I) and HTERT ( Figure 2 A-II) were successfully infected in i-PGECs ( Figure 2A). To compare the biological characteristics of primary PGECs and i-PGECs, the results of RT-PCR experiments confirmed that the three markers, epithelial markers KRT18, CDH1, and MUC1, were positively expressed in both primary PGECs and i-PGECs ( Figure 2 B). In addition, immunofluorescence experiments showed that the epithelial marker proteins CK18, E-cadherin, and EPCAM were all positively expressed in primary PGECs, i-PGECs-P42, and i-PGECs-P5 ( Figure 2 C).

[0133] Transmission electron microscopy (TEM) images showed that i-PGECs had intact structural integrity, and tight junctions and desmosomes were visible between adjacent cells ( Figure 3 A-I), and mitochondria were also clearly distributed in the cytoplasm ( Figure 3 A-II), and there were significant differences in the mRNA expression of SV40T and HTERT between primary PGECs and i-PGECs (P < 0.0001);

[0134] 2.2 Karyotype analysis results of i-PGECs-P20 and i-PGECs-P42 showed that the cells contained 38 chromosomes, consistent with a diploid karyotype ( Figure 3 B). These results confirmed the genetic stability of i-PGECs.

[0135] Growth curve, apoptosis, and cell cycle analysis of PGECs and i-PGECs:

[0136] Cell proliferation was evaluated by the CCK-8 method, and the growth curve is shown in Figure 3 C. The growth of primary PGECs was relatively gentle and stable, while i-PGECs entered the logarithmic growth phase on the 2nd day, reached the maximum proliferation rate on the 5th day, and then entered the plateau phase. Compared with primary PGECs, the proliferation ability of i-PGECs was significantly enhanced. The apoptosis experiment showed that the proportion of apoptotic cells in primary PGECs was 33.36%, while in i-PGECs it was only 6.35% (Q2+Q4) ( Figure 3 D-I). In addition, the results of cell cycle analysis showed that the proportions of cells in the S phase of primary PGECs and i-PGECs were 10.96% and 24.63% respectively ( Figure 3 D-II). In summary, these results indicate that i-PGECs have stronger proliferation activity and a significantly lower apoptosis rate compared to primary PGECs.

[0137] Overall, the epithelial marker genes KRT18, MUC1, CDH1, and EPCAM were expressed in both primary PGECs and i-PGECs. Both showed stable cell structure and karyotype. However, the proliferation rate of primary PGECs was slower than that of i-PGECs.

[0138] 3. Transcriptomic characteristics of PGECs and i-PGECs

[0139] 4. We analyzed the RNA sequencing data to comprehensively evaluate the characteristics of PGECs and i-PGECs. The quality control results confirmed the accuracy of the RNA sequencing data, and in the principal component analysis (PCA), a clear separation was shown between PGECs and i-PGECs. The triplicate samples of each group showed high consistency, ensuring the reliability of the data ( Figure 4 A). To verify whether i-PGECs maintained the epithelial characteristics of PGECs, we collected 50 key marker genes reported in gastric epithelial cells, namely: TUBA1B, NRG1, PSAT1, CPT1A, RNF157, NUPR1, RPS12, MAN1A1, ASF1A, ANXA4, ID2, PROX1, HES6, HEPACAM2, STMN1, ALDH1A1, ADI1, AVIL, MARCKSL1, SOX4, TUBB2B, HMGN1, TUBA1A, MDK, SLIT2, GRK5, CACNA2D1, KIF13A, CKB, PTPN13, GATA6, ATP1B1, MUC1, NBR1, TP53INP1, CAPN8, FUT1, RIMS2, ZNRF3, CENPF, TOP2A, KIF11, COL17A1, ALDOA, PGK1, GDF15, CA9, NDUFA4L2, KIT, ZFHX3. The results showed that these marker genes were expressed in both PGECs and i-PGECs, indicating that both retained the epithelial characteristics of gastric epithelial cells ( Figure 4 B). KEGG database analysis showed that the pathways upregulated in i-PGECs were mainly related to cell proliferation, including cell population proliferation (FGF2), positive regulation of cell motility (BCL2), and embryonic organ development (HOXA10). The downregulated pathways were mainly related to cell differentiation and secretion regulation, including epithelial cell differentiation (SOX9), cell secretion regulation (GCK), and regulation of MAPK cascade (BMP2) ( Figure 4 C). Overall, i-PGECs upregulated the pathways related to proliferation and maintained the characteristics of PGECs. Genes and pathways in PGECs found by transcriptome sequencing analysis after AFB1 treatment.

[0140] After treating i-PGECs with 2 μM AFB1 for 24 hours, obvious morphological changes occurred in the i-PGECs of the experimental group compared with the control group, and some cells detached ( Figure 5 A), indicating that AFB1 inhibited the normal proliferation of i-PGECs. To study the response of PGECs to AFB1 infection, we used RNA-seq to detect the effect of 24-hour AFB1 infection on host gene expression. Principal component analysis (PCA, Figure 5 B) results showed that the samples of the control group and the treatment group were clearly separated in the figure; the three replicates within each group showed high consistency. The results of the volcano plot ( Figure 5 C) showed that there were 871 differentially expressed genes (DEGs) in the i-PGECs of the treatment group compared with the control group, among which 572 genes were up-regulated and 299 genes were down-regulated.

[0141] Among the differentially expressed genes, the most significantly up-regulated genes PLAUR, NPC1, and MAFF play important roles in the immune response. The most significantly down-regulated gene NRG1, as a member of the epidermal growth factor family, regulates the growth and differentiation of epithelial tissues.

[0142] The homologous genes were classified using the KEGG database to gain an in-depth understanding of the biological processes related to the DEGs. The top 10 up-regulated pathways were mainly related to the immune response. In addition to the positive regulation of leukocyte and monocyte migration (CXCL8), these pathways also involved leukocyte activation (ADAM17), inflammatory response (IL6R, IL17RA), T cell activation, and regulation of kinase activity (IRS2) ( Figure 5 D-I). The top 10 down-regulated pathways included the enzyme-linked receptor protein signaling pathway (IRS1), organ growth (FGF9), and BMP signaling pathway (SMAD6) ( Figure 5 D-II). These results indicate that AFB1 activates the immune response of i-PGECs, but at the same time AFB1 inhibits their proliferation ( Figure 5 D).

[0143] In summary, the present invention adopts a two-step enzymatic digestion method: first, dispase II is used to separate the epithelial layer from the lamina propria, and then a combination of type I collagenase and hyaluronidase is used to digest the scraped mucosal layer. This method not only effectively separates epithelial cells without fibroblast contamination but also maintains the integrity of epithelial cells. Type I collagenase is a relatively mild enzyme and causes less damage to primary cells during digestion; however, it requires a longer digestion time to obtain a sufficient number of cells, thereby reducing the efficiency. The combination of type I collagenase and hyaluronidase significantly shortens the separation time and increases the yield of non-mesenchymal epithelial cells.

[0144] Our results showed that during the passage of cell lines, the expression level of hTERT gradually decreased, while the expression of SV40T increased. This highlights the importance of infecting PGECs with both genes, as the presence of hTERT and SV40T can ensure stable immortalization by maintaining the expression of at least one gene, thus stabilizing the immortalized characteristics of the cells.

[0145] The gene clusters with high expression in i-PGECs were mainly related to cell proliferation. In primary cultures extracted from the developing cortex from E14 to E16, studies have shown that FGF2 regulates cell proliferation by shortening the G1 phase and promoting proliferative division, thus playing its role in regulating the duration of the G1 phase. BCL2 promotes cell survival by reducing the cell proliferation rate. HOXA10 is significantly upregulated in gastric cancer and promotes the proliferation of gastric cancer cells. Most of the downregulated genes are related to cell differentiation. Sox9 is a key regulator of the differentiation of mucous neck cells during gastric development. BMP2 is a morphogenetic factor in the TGF-β superfamily and plays a key role in regulating the differentiation of uterine stromal cells. Glucokinase (GCK) is a key enzyme in glucose metabolism and plays an important role in regulating cell secretion.

[0146] The gastric epithelial marker genes we collected, such as GATA6 (expressed in parietal cells and involved in the development and functional regulation of gastric epithelial cells as a transcription factor) and FUT1 (expressed in mucous cells and involved in regulating the function and structure of mucus), were expressed in both PGECs and i-PGECs.

[0147] AFB1 is absorbed by the gastrointestinal epithelium through the ingestion of contaminated food and feed. We revealed the potential effects of AFB1 on porcine gastric epithelial cells. This study found that AFB1 activated the genes ADAM17 and IL6R related to the inflammatory response and caused abnormal changes in the cell morphology of i-PGECs. AFB1 exposure induced autophagy in i-PGECs and upregulated autophagy-related genes including CTSB and CTSD. In addition, IRS2, which promotes cell proliferation, also plays a role in immune response and monocyte proliferation. The downregulation of genes related to organ development such as IRS1, FGF9, and SMAD6 indicates that the proliferation of the cell line is inhibited.

[0148] Through morphological, transcriptomic, and protein expression analyses, we confirmed that immortalized porcine gastric epithelial cells (i-PGECs) maintained the key physiological characteristics of gastric epithelial cells. In addition, i-PGECs showed a positive immune response to aflatoxin B1 exposure. This newly developed cell line provides a valuable tool for studying the mechanisms of porcine gastric diseases and host-pathogen interactions, and is expected to promote future research progress in animal growth and human gastric health.

[0149] The embodiments of the present application have been described above in conjunction with the accompanying drawings. However, the present application is not limited to the above specific embodiments. The above specific embodiments are merely illustrative rather than restrictive. Under the inspiration of the present application, those of ordinary skill in the art can also make many forms without departing from the purpose of the present application and the scope protected by the claims, and all of them fall within the protection scope of the present application.

Claims

1. A method for isolating primary porcine gastric mucosal epithelial cells, characterized in that: The following steps are involved: 1) Obtaining gastric body and gastric fundus tissues, washing the obtained tissues in a washing solution; and soaking the washed tissues in a washing culture medium; 2) transferring the soaked tissue to a dispase II-HBSS cell digestion solution for digestion to separate the epithelial layer; digestion, wherein the dispase II-HBSS cell digestion solution is prepared by dissolving dispase II in HBSS; 3) performing enzymatic hydrolysis on the epithelial layer; the enzymatic hydrolysis solution comprises the following final concentrations: hyaluronidase 0.5-3 mg / mL, collagenase I 1-5 mg / mL, and the solvent is water; 4) Add RPMI1640 medium to terminate enzymatic hydrolysis, filter through 100±1μm cell filter and centrifuge to retain the cell layer; 5) Resuspend the cell layer in proliferation medium and culture the cells at 37°C and 5% CO2. 6) When the cell growth density reaches 80±5% confluence, culture the cells at 37±0.5°C to obtain primary porcine gastric mucosal epithelial cells.

2. A method for isolating primary porcine gastric mucosal epithelial cells according to claim 1, characterized in that: In step 1), the cleaning solution is a HBSS solution containing 1±0.1% (m / m) penicillin / streptomycin and 100±1 μg / mL gentamicin / amphotericin B.

3. A method for isolating primary porcine gastric mucosal epithelial cells according to claim 1, characterized in that: In step 1), the washing medium is a HBSS solution containing 0.5%-1.5% (m / m) penicillin / streptomycin and 1%-2% (m / m) gentamicin / amphotericin B.

4. The method for isolating primary porcine gastric mucosal epithelial cells according to claim 1, characterized in that: In the step 2), the concentration of the dispase II in the dispase II-HBSS cell digestion solution is 1±0.1 mg / mL.

5. The method for isolating primary porcine gastric mucosal epithelial cells according to claim 1, characterized in that: In step 3), the enzymatic solution is composed of the following final concentrations: hyaluronidase 1-2 mg / mL, collagenase I 2-3 mg / mL, and the solvent is water.

6. The method for isolating primary porcine gastric mucosal epithelial cells according to claim 1, characterized in that: In step 4), the centrifugation is performed twice. The first time, the cells are filtered through a 100±1 μm cell filter and then centrifuged at 380-410×g for 4-6 minutes. The second time, the cells are added with HBSS containing 1±0.1% (m / m) penicillin / streptomycin and centrifuged again at 380-410×g for 4-6 minutes.

7. A primary porcine gastric mucosal epithelial cell, characterized in that: The primary porcine gastric mucosal epithelial cells are obtained by the isolation and culture method of any one of claims 1 to 6.

8. A porcine gastric mucosal epithelial cell line, characterized in that: The porcine gastric mucosal epithelial cell line is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C202493 and a deposit date of November 22, 2024, and is named: porcine gastric mucosal epithelial cell line i-PGECs Sus scrofa.

9. The method for establishing a porcine gastric mucosal epithelial cell line according to claim 8, characterized in that: The following steps are involved: S1: Subculturing the primary porcine gastric mucosal epithelial cells described in claim 7, and when the confluence of the primary porcine gastric epithelial cells reaches 70±5%, infecting the primary porcine gastric epithelial cells with two lentiviruses SV40T and hTERT at a concentration of MOI=80±1 overnight, and culturing at 37°C; S2: Add complete medium after 24-30 hours of culture, and then replace the complete medium every other day; S3: After 3.5-4.5 days, the porcine gastric primary epithelial cells were cultured in fresh complete medium containing 1±0.1 μg / ml puromycin for 80-120 hours, and then expanded at 37°C; S4: The cells obtained by amplification culture were tested for mycoplasma, and the cell lines that tested negative were cultured in DMEM / F12 medium containing 10% (m / m) FBS and 1% (m / m) penicillin / streptomycin at 37°C to obtain porcine gastric mucosal epithelial cell lines.

10. Use of the porcine gastric mucosal epithelial cell line according to claim 8 in constructing an in vitro model for studying the molecular mechanism and biological function of porcine stomach.

Images