Chicken primary small intestine epithelial cell senescence model construction method, senescence model and application
Primary small intestinal epithelial cells of chickens were isolated by gradient centrifugation and collagenase digestion, and the gradient concentration D-galactose treatment combined with CCK8 detection was used to construct a stable and efficient aging model, solving the problem of model instability in the existing technology, and providing a reliable tool for poultry aging research.
Patent Information
- Application Number
- CN202510509708.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-22
- Publication Date
- 2025-07-25
AI Technical Summary
The existing technology has not yet established a stable and efficient model of primary small intestinal epithelial cell aging in chickens, which has restricted the research progress of poultry intestinal aging mechanism.
Gradient centrifugation combined with collagenase precision digestion method was used to separate primary small intestinal epithelial cells of chickens, and aging model was constructed by gradient concentration D-galactose treatment combined with CCK8 detection.
It significantly improved the purity and activity of cells, established a high repeatability aging model, solved the problems of instability in induction efficiency and large individual differences in traditional methods, and laid the foundation for high-throughput drug screening.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of cell technology, and particularly relates to a method for constructing an aging model of chicken primary intestinal epithelial cells, an aging model and applications thereof. Background Art
[0002] Intestinal epithelial cells (IECs) are a core component of the intestinal barrier and undertake key functions such as nutrient absorption, immune regulation, and mucosal repair. With aging, the aging of IECs leads to the degradation of intestinal function, the weakening of the barrier function, increases the risk of pathogen infection, and affects the growth performance of animals. In broiler chicken farming, intestinal health is directly related to feed conversion rate and disease resistance. Therefore, studying the aging mechanism of IECs is of great significance for improving the efficiency of poultry farming.
[0003] Existing research mainly focuses on the aging models of chicken embryonic stem cells or immortalized cell lines. However, primary IECs are closer to the physiological state, and the construction of their aging models is more crucial for studying the aging mechanism of the poultry intestine. However, due to the difficulty in isolating and culturing chicken primary intestinal epithelial cells (IECs) and the low efficiency of aging induction, a stable and efficient aging model of chicken intestinal epithelial cells (IECs) has not been established yet, which restricts the research progress in related fields. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for constructing an aging model of chicken primary intestinal epithelial cells with high efficiency and stability.
[0005] To achieve the above purpose, the present invention adopts the following technical solutions:
[0006] A method for constructing an aging model of chicken primary intestinal epithelial cells, comprising the following steps:
[0007] (1) After inoculating duodenal epithelial cells, culture them in DMEM / F12 complete medium containing 10% fetal bovine serum at 37 °C and 5% CO2 (volume ratio) until the cell confluence ≥ 80%; obtain sample duodenal epithelial cells;
[0008] (2) Treat the sample duodenal epithelial cells with D-galactose solutions at different concentrations for 24 h and 48 h respectively; then detect the survival rate of the duodenal epithelial cells to construct an aging model of chicken primary intestinal epithelial cells.
[0009] To obtain stable chicken primary intestinal epithelial cells, preferably, the method for obtaining the duodenal epithelial cells is:
[0010] S1. Separate the duodenum and duodenal mucosa of 18-day-old chicken embryos after removing the pancreas, put the separated duodenum into Hank's solution, centrifuge at 700 - 900 rcf for 8 - 10 min, discard the supernatant, and leave the remaining tissue;
[0011] S2, transferring the tissue to the mixed solution, stirring and digesting at 35-39° C. until digestion is complete, to obtain a digestion solution, wherein the mixed solution is composed of Hank's solution and collagenase I, the volume ratio of Hank's solution to collagenase I is (5-10):1; the concentration of collagenase I is 0.5 mg / mL;
[0012] S3, centrifuge the digestion solution at 700-900 rcf for 8-10 min and discard the supernatant to obtain the initial centrifuged tissue;
[0013] S4, washing the initial centrifuged tissue with Hank's solution, centrifuging at 700-900 rcf for 8-10 min, discarding the supernatant, then washing again with Hank's solution, centrifuging at 700-900 rcf for 8-10 min, discarding the supernatant, and obtaining initial cells;
[0014] S5. Dissolve the initial cells using DMEM / F12 containing 10% special grade fetal bovine serum, and then filter using a 200-mesh sieve to obtain duodenal epithelial cells.
[0015] In order to ensure the effect of centrifugation, preferably, the relative centrifugal force of the centrifugation is 800 rcf and the centrifugation time is 8 min.
[0016] In order to ensure the acquisition of stable primary chicken intestinal epithelial cells, in step S2, the digestion temperature is 37°C, and the detection method for the completion of the digestion is: under a microscope, the digestion fluid can be observed, and 3 cells can be observed, and the 3 cells form a group or cluster; the volume ratio of Hank's solution to collagenase I is 10:1.
[0017] In order to ensure the effect of inoculation, preferably, the duodenal epithelial cells in step (1) are inoculated at 1×10 4 The cells were seeded in a 96-well plate at a density of 100 / mL.
[0018] In order to clarify the concentration of the D-galactose solution, preferably, the D-galactose solutions of different concentrations in step (2) are 0 mmol / L, 100 mmol / L, 200 mmol / L, 300 mmol / L, and 400 mmol / L, respectively.
[0019] In order to ensure the accuracy of the detection, the preferred method for detecting the survival rate of duodenal epithelial cells is as follows: add 10 μL of CCK8 solution to each cell culture well containing 100 μL of serum-free DMEM / F12 medium, add sample duodenal epithelial cells to the cell culture wells, and culture at 37°C for 2 hours. After the cells turn orange-yellow, take out the solution to another cell 96-well plate, and use an ELISA reader to detect the cell absorbance at a wavelength of 450 nm to obtain the cell survival rate.
[0020] The chicken primary intestinal epithelial cell senescence model constructed by the method for constructing a chicken primary intestinal epithelial cell senescence model described in the present invention.
[0021] Application of the chicken primary intestinal epithelial cell senescence model of the present invention in screening for drugs for alleviating, treating and / or preventing chicken primary intestinal epithelial cell senescence.
[0022] Compared with the prior art, the present invention has the following technical effects:
[0023] The present invention adopts a method of gradient centrifugation combined with precise digestion with collagenase. By optimizing the centrifugation parameters and digestion conditions, the purity and activity of duodenal epithelial cells are significantly improved, and the problems of cell membrane damage and cell debris contamination caused by the traditional mechanical scraping method are avoided, providing a high-quality cell basis for the subsequent construction of the senescence model; through treatment with gradient concentration of D-galactose combined with dynamic detection by CCK8, a senescence model is successfully established. This model has high repeatability and solves the technical bottleneck of unstable induction efficiency and large individual differences in traditional senescence models.
[0024] The present invention adopts a CCK8 detection system. By optimizing the detection conditions, the detection sensitivity is improved, the step of dissolving with organic solvents is avoided, and the detection efficiency and data accuracy are significantly improved, laying a technical foundation for high-throughput drug screening.
[0025] The method for constructing a chicken primary intestinal epithelial cell senescence model of the present invention provides a reliable tool for poultry senescence research and has significant scientific significance and industrial application prospects. Description of the Drawings
[0026] Figure 1 Schematic diagrams of the results of separating and culturing chicken primary intestinal epithelial cells for 0 h, 24 h, 48 h and 96 h in the present invention;
[0027] Figure 2 Cell state diagram of duodenal epithelial cells after treatment with D-galactose for 24 h in the present invention;
[0028] Figure 3 Cell state diagram of duodenal epithelial cells after treatment with D-galactose for 48 h in the present invention;
[0029] Figure 4 Cell viability diagrams of duodenal epithelial cells induced by D-galactose after culturing for 24 h and 48 h in the present invention;
[0030] Figure 5 Diagram showing the effect of chlorogenic acid on the cell viability of duodenal epithelial cells induced by D-galactose after 24 h and 48 h. Detailed Embodiments
[0031] The present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.
[0032] Example 1
[0033] A method for constructing a chicken primary small intestinal epithelial cell aging model comprises the following steps:
[0034] (1) The duodenum and duodenal mucosa of an 18-day-old embryonic chicken with the pancreas removed were separated, and the separated duodenum was placed in Hank's solution, centrifuged at 800 rcf for 8 min, and the supernatant was discarded, leaving the remaining tissue;
[0035] (2) transferring the tissue to the mixed solution, stirring and digesting at 37° C. until digestion is complete, to obtain a digestion solution, wherein the mixed solution is composed of Hank's solution and collagenase I, the volume ratio of Hank's solution to collagenase I is 10:1, and the concentration of collagenase I is 0.5 mg / mL;
[0036] (3) Centrifuge the digestion solution at 800 rcf for 8 min and discard the supernatant to obtain the initial centrifuged tissue;
[0037] (4) Wash the initial centrifuged tissue with Hank's solution and centrifuge at 800 rcf for 8 min, discard the supernatant, then wash it again with Hank's solution and centrifuge at 800 rcf for 8 min, discard the supernatant, and obtain the initial cells;
[0038] (5) Use DMEM / F12 containing 10% special grade fetal bovine serum to dissolve the initial cells, and then filter them using a 200-mesh sieve to obtain duodenal epithelial cells.
[0039] (6) The successfully isolated duodenal epithelial cells (IEC) were cultured in DMEM / F12 containing 10% special grade fetal bovine serum at 25° C. and 5% CO2 by volume in a carbon dioxide incubator for later use;
[0040] (7) Duodenal epithelial cells were cultured at 1×10 4 The cells were inoculated at a density of 100 cells / mL in a 96-well plate and cultured in a DMEM / F12 complete medium containing 10% fetal bovine serum at 37°C and 5% CO2 by volume until the cell confluence was ≥80%; thus obtaining sample duodenal epithelial cells;
[0041] (8) Treat the sample duodenal epithelial cells with D-galactose solutions of concentrations of 0 mmol / L, 100 mmol / L, 200 mmol / L, 300 mmol / L, and 400 mmol / L for 24 h and 48 h, respectively; then detect the survival rate of the duodenal epithelial cells to construct a chicken primary small intestinal epithelial cell aging model; the survival rate detection method is as follows: add 10 μL of CCK8 solution to each cell culture well containing 100 μL of serum-free DMEM / F12 culture medium, add the sample duodenal epithelial cells to the cell culture wells, and culture at 37°C for 2 h. After the cells turn orange-yellow, take out the solution to another cell 96-well plate, and use an ELISA reader to detect the cell absorbance at a wavelength of 450 nm.
[0042] Example 2
[0043] A method for constructing a chicken primary small intestinal epithelial cell aging model comprises the following steps:
[0044] (1) The duodenum and duodenal mucosa of 18-day-old embryonic chickens with the pancreas removed were separated, and the separated duodenum was placed in Hank's solution, centrifuged at 700 rcf for 10 min, and the supernatant was discarded, leaving the remaining tissue;
[0045] (2) transferring the tissue to the mixed solution, stirring and digesting at 35° C. until digestion is complete, to obtain a digestion solution, wherein the mixed solution is composed of Hank's solution and collagenase I, the volume ratio of Hank's solution to collagenase I is 5:1, and the concentration of collagenase I is 0.5 mg / mL;
[0046] (3) Centrifuge the digestion solution at 700 rcf for 10 min and discard the supernatant to obtain the initial centrifuged tissue;
[0047] (4) Wash the initial centrifuged tissue with Hank's solution and centrifuge at 700 rcf for 10 min, discard the supernatant, then wash it again with Hank's solution and centrifuge at 700 rcf for 10 min, discard the supernatant, and obtain the initial cells;
[0048] (5) Use DMEM / F12 containing 10% special grade fetal bovine serum to dissolve the initial cells, and then filter them using a 200-mesh sieve to obtain duodenal epithelial cells.
[0049] (6) The successfully isolated duodenal epithelial cells (IEC) were cultured in DMEM / F12 containing 10% special grade fetal bovine serum at 25° C. and 5% CO2 by volume in a carbon dioxide incubator for later use;
[0050] (7) Duodenal epithelial cells were cultured at 1×10 4The cells were inoculated at a density of 100 cells / mL in a 96-well plate and cultured in a DMEM / F12 complete medium containing 10% fetal bovine serum at 37°C and 5% CO2 by volume until the cell confluence was ≥80%; thus obtaining sample duodenal epithelial cells;
[0051] (8) Treat the sample duodenal epithelial cells with D-galactose solutions of concentrations of 0 mmol / L, 100 mmol / L, 200 mmol / L, 300 mmol / L, and 400 mmol / L for 24 h and 48 h, respectively; then detect the survival rate of the duodenal epithelial cells to construct a chicken primary small intestinal epithelial cell aging model; the survival rate detection method is as follows: add 10 μL of CCK8 solution to each cell culture well containing 100 μL of serum-free DMEM / F12 culture medium, add the sample duodenal epithelial cells to the cell culture wells, and culture at 37°C for 2 h. After the cells turn orange-yellow, take out the solution to another cell 96-well plate, and use an ELISA reader to detect the cell absorbance at a wavelength of 450 nm.
[0052] Example 3
[0053] A method for constructing a chicken primary small intestinal epithelial cell aging model comprises the following steps:
[0054] (1) The duodenum and duodenal mucosa of an 18-day-old embryonic chicken with the pancreas removed were separated, and the separated duodenum was placed in Hank's solution, centrifuged at 900 rcf for 9 min, and the supernatant was discarded, leaving the remaining tissue;
[0055] (2) transferring the tissue to the mixed solution, stirring and digesting at 39° C. until digestion is complete, to obtain a digestion solution, wherein the mixed solution is composed of Hank's solution and collagenase I, the volume ratio of Hank's solution to collagenase I is 7:1, and the concentration of collagenase I is 0.5 mg / mL;
[0056] (3) Centrifuge the digestion solution at 900 rcf for 9 min and discard the supernatant to obtain the initial centrifuged tissue;
[0057] (4) Wash the initial centrifuged tissue with Hank's solution, centrifuge at 900 rcf for 9 min, discard the supernatant, then wash it again with Hank's solution, centrifuge at 900 rcf for 9 min, discard the supernatant, and obtain the initial cells;
[0058] (5) Use DMEM / F12 containing 10% special grade fetal bovine serum to dissolve the initial cells, and then filter them using a 200-mesh sieve to obtain duodenal epithelial cells.
[0059] (6) The successfully isolated duodenal epithelial cells (IEC) were cultured in DMEM / F12 containing 10% special grade fetal bovine serum at 25° C. and 5% CO2 by volume in a carbon dioxide incubator for later use;
[0060] (7) Duodenal epithelial cells were cultured at 1×10 4 The cells were inoculated at a density of 100 cells / mL in a 96-well plate and cultured in a DMEM / F12 complete medium containing 10% fetal bovine serum at 37°C and 5% CO2 by volume until the cell confluence was ≥80%; thus obtaining sample duodenal epithelial cells;
[0061] (8) Treat the sample duodenal epithelial cells with D-galactose solutions of concentrations of 0 mmol / L, 100 mmol / L, 200 mmol / L, 300 mmol / L, and 400 mmol / L for 24 h and 48 h, respectively; then detect the survival rate of the duodenal epithelial cells to construct a chicken primary small intestinal epithelial cell aging model; the survival rate detection method is as follows: add 10 μL of CCK8 solution to each cell culture well containing 100 μL of serum-free DMEM / F12 culture medium, add the sample duodenal epithelial cells to the cell culture wells, and culture at 37°C for 2 h. After the cells turn orange-yellow, take out the solution to another cell 96-well plate, and use an ELISA reader to detect the cell absorbance at a wavelength of 450 nm.
[0062] Example 4
[0063] A method for constructing a chicken primary small intestinal epithelial cell aging model comprises the following steps:
[0064] (1) The duodenum and duodenal mucosa of 18-day-old embryonic chickens with the pancreas removed were separated, and the separated duodenum was placed in Hank's solution, centrifuged at 800 rcf for 9 min, and the supernatant was discarded, leaving the remaining tissue;
[0065] (2) transferring the tissue to the mixed solution, stirring and digesting at 37° C. until digestion is complete, to obtain a digestion solution, wherein the mixed solution is composed of Hank's solution and collagenase I, the volume ratio of Hank's solution to collagenase I is 9:1, and the concentration of collagenase I is 0.5 mg / mL;
[0066] (3) Centrifuge the digestion solution at 800 rcf for 9 min and discard the supernatant to obtain the initial centrifuged tissue;
[0067] (4) Wash the initial centrifuged tissue with Hank's solution and centrifuge at 800 rcf for 9 min, discard the supernatant, then wash it again with Hank's solution and centrifuge at 800 rcf for 9 min, discard the supernatant, and obtain the initial cells;
[0068] (5) Use DMEM / F12 containing 10% special grade fetal bovine serum to dissolve the initial cells, and then filter them using a 200-mesh sieve to obtain duodenal epithelial cells.
[0069] (6) The successfully isolated duodenal epithelial cells (IEC) were cultured in DMEM / F12 containing 10% special grade fetal bovine serum at 25° C. and 5% CO2 by volume in a carbon dioxide incubator for later use;
[0070] (7) Duodenal epithelial cells were cultured at 1×10 4 The cells were inoculated at a density of 100 cells / mL in a 96-well plate and cultured in a DMEM / F12 complete medium containing 10% fetal bovine serum at 37°C and 5% CO2 by volume until the cell confluence was ≥80%; thus obtaining sample duodenal epithelial cells;
[0071] (8) Treat the sample duodenal epithelial cells with D-galactose solutions of concentrations of 0 mmol / L, 100 mmol / L, 200 mmol / L, 300 mmol / L, and 400 mmol / L for 24 h and 48 h, respectively; then detect the survival rate of the duodenal epithelial cells to construct a chicken primary small intestinal epithelial cell aging model; the survival rate detection method is as follows: add 10 μL of CCK8 solution to each cell culture well containing 100 μL of serum-free DMEM / F12 culture medium, add the sample duodenal epithelial cells to the cell culture wells, and culture at 37°C for 2 h. After the cells turn orange-yellow, take out the solution to another cell 96-well plate, and use an ELISA reader to detect the cell absorbance at a wavelength of 450 nm.
[0072] The cell status of the duodenal epithelial cells cultured in step (6) of Example 1 was observed at 0 h, 24 h, 48 h and 96 h, as shown in FIG. Figure 1 As shown in the figure, it can be seen that the duodenal epithelial cells separated in the method of the present invention are intact, and the cell proliferation state and activity are good after culture.
[0073] like Figure 2 and 3 The cell states of duodenal epithelial cells induced by D-galactose solution in Example 1 after 24h and 48h of culture are shown. The results show that D-galactose solution has a certain effect on aging of duodenal epithelial cells; Figure 2 The unit mM is mmol / L.
[0074] like Figure 4The cell viability after culturing duodenal epithelial cells with D-galactose solution for 24 h and 48 h is shown. In the figure, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001; after treating duodenal epithelial cells (IEC) with D-galactose for 24 h, the cell viability decreased significantly, and the four concentrations of 100 mmol / L, 200 mmol / L, 300 mmol / L, and 400 mmol / L all decreased significantly (P < 0.01); when the D-galactose concentration increased to 200 mmol / L, the cell viability decreased extremely significantly (P < 0.001). After treating duodenal epithelial cells (IEC) with D-galactose for 48 h, compared with the control group, the cell viability of the D-galactose group decreased extremely significantly (P < 0.001).
[0075] According to the CCK-8 cell viability detection results, the senescence model of duodenal epithelial cells (IEC) induced by D-galactose was successfully constructed, and the cell senescence state was relatively stable when treated with a D-gal concentration of 200 mmol / L for 24 h, which was beneficial for the next experiment.
[0076] To further verify the application of the chicken primary small intestinal epithelial cell senescence model constructed by the method of the present invention in the screening of alleviating and / or preventing and treating drugs, the following experiments were carried out in the present invention:
[0077] Chlorogenic acid was added to duodenal epithelial cells (IEC) at concentrations of 5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, and 200 μg / ml respectively. After 12 h, a D-galactose solution of 200 mmol / L was added to induce cell senescence. After 12 h and 36 h, the CCK8 method was used to detect the cell viability of each group. As Figure 5 shown, in the figure, * indicates P < 0.05, and **** indicates P < 0.0001; with the increase of the CGA addition level, compared with the model group of the present invention, the cell viability of the cells treated with 100 μg / ml of chlorogenic acid for 24 h increased extremely significantly (P < 0.01), and the cell viability of the 200 μg / ml chlorogenic acid group increased compared with the control model group of the present invention, but not significantly (P > 0.05). The test results show that chlorogenic acid can improve cell viability and resist cell senescence, and the best effect is achieved when 100 μg / mL is added to duodenal epithelial cells.
Claims
1. A method for constructing a senescence model of primary chicken intestinal epithelial cells, characterized in that, The following steps are involved: (1) After inoculating duodenal epithelial cells, culture them in DMEM / F12 complete medium containing 10% fetal bovine serum at 37° C. and 5% CO2 by volume until the cell confluence is ≥80% to obtain sample duodenal epithelial cells; (2) The sample duodenal epithelial cells were treated with D-galactose solution of different concentrations for 24 h and 48 h respectively; then the survival rate of the duodenal epithelial cells was detected to construct an aging model of primary chicken small intestinal epithelial cells.
2. The method for constructing a primary chicken small intestinal epithelial cell senescence model according to claim 1, characterized in that The method for obtaining duodenal epithelial cells is as follows: S1. Separate the duodenum and duodenal mucosa of an 18-day-old embryonic chicken with the pancreas removed, and place the separated duodenum in Hank's solution. Centrifuge at 700-900 rcf for 8-10 min, discard the supernatant, and leave the remaining tissue. S2, transferring the tissue to the mixed solution, stirring and digesting at 35-39° C. until digestion is complete, to obtain a digestion solution, wherein the mixed solution is composed of Hank's solution and collagenase I, the volume ratio of Hank's solution to collagenase I is (5-10):1; the concentration of collagenase I is 0.5 mg / mL; S3, centrifuge the digestion solution at 700-900 rcf for 8-10 min and discard the supernatant to obtain the initial centrifuged tissue; S4, washing the initial centrifuged tissue with Hank's solution, centrifuging at 700-900 rcf for 8-10 min, discarding the supernatant, then washing again with Hank's solution, centrifuging at 700-900 rcf for 8-10 min, discarding the supernatant, and obtaining initial cells; S5. Dissolve the initial cells using DMEM / F12 containing 10% special grade fetal bovine serum, and then filter using a 200-mesh sieve to obtain duodenal epithelial cells.
3. The method for constructing a primary chicken intestinal epithelial cell senescence model according to claim 2, characterized in that, The relative centrifugal force of the centrifugation is 800 rcf, and the centrifugation time is 8 min.
4. The method for constructing a primary chicken small intestinal epithelial cell senescence model according to claim 2, wherein In step S2, the digestion temperature is 37°C, and the digestion completion detection method is: under a microscope, the digestion solution can be observed, and 3 cells can be observed, and the 3 cells form a group or cluster; the volume ratio of Hank's solution to collagenase I is 10:
1.
5. The method for constructing a primary chicken intestinal epithelial cell senescence model according to claim 1, wherein, In step (1), when inoculating duodenal epithelial cells, inoculate them in a 96-well plate at a density of 1×10 4 cells / mL.
6. The method for constructing a primary chicken small intestinal epithelial cell senescence model according to claim 1, wherein The D-galactose solutions of different concentrations in step (2) are 0 mmol / L, 100 mmol / L, 200 mmol / L, 300 mmol / L, and 400 mmol / L, respectively.
7. A method for constructing a primary chicken small intestinal epithelial cell senescence model according to claim 5, characterized in that The method for detecting the survival rate of duodenal epithelial cells is as follows: add 10 μL of CCK8 solution to each cell culture well containing 100 μL of serum-free DMEM / F12 medium, add sample duodenal epithelial cells to the cell culture wells, and culture at 37°C for 2 hours. After the cells turn orange-yellow, take out the solution to another cell 96-well plate, and use an enzyme reader to detect the cell absorbance at a wavelength of 450 nm to obtain the cell survival rate.
8. A chicken primary small intestinal epithelial cell aging model constructed using the construction method described in claims 1-7.
9. Use of the chicken primary small intestinal epithelial cell aging model according to claim 8 in screening drugs for alleviating and / or preventing aging of chicken primary small intestinal epithelial cells.