Methods for inducing colitis by cell-mediated transfer

Abstract

The present invention discloses a method for inducing colitis by cell-acquired transfer, which relates to the field of biomedicine technology. The method for inducing colitis by cell-acquired transfer comprises the following steps: S1, replicating a colitis model; S2, enriching mTh17 cells by flow cytometry; S3, inducing colitis by mTh17 cell-acquired transfer; S4, collecting colitis samples; S5, assessing the disease activity index; S6, processing pathological tissues: 1. paraffin sectioning; 2. hematoxylin-eosin staining. By using the method for inducing colitis by cell-acquired transfer provided in the present application, memory Th17 cells with high expression of IL-17A are obtained in vitro; mTh17 cells are acquired and transferred to SCID mice to successfully induce colitis. This helps to analyze the mechanism of action of mTh17 cells in the pathogenesis of UC, can be used to evaluate the efficacy of new anti-UC drugs, and promote the development of new anti-UC drugs.

Description

Technical Field

[0001] The present invention relates to biomedicine technology, and in particular to a method for inducing colitis by cell-mediated transfer. Background Art

[0002] Ulcerative colitis is a chronic immune-mediated inflammation of the colon, characterized by relapses and remissions. According to reports, the global incidence of UC has been on an upward trend, with incidences in North America and Europe at 8.8-23.1 / 100,000 people / year and 0.6-24.3 / 100,000 people / year, respectively, and slightly lower in Asia. Its chronicity and high recurrence rate have resulted in enormous healthcare and social costs. UC is more common in young and middle-aged people aged 15 to 30, with the main clinical symptoms being abdominal pain, diarrhea, and weight loss. Currently, no drugs or methods have been found to completely cure UC, and its specific cause and exact mechanism are unclear. Currently, the medical community generally recognizes that excessive Th17 cell response is a key factor in the initiation and remission of UC; however, it is difficult to directly obtain Th17 cells in vitro, and UC is difficult to be completely cured clinically and recurs repeatedly. This is closely related to the abnormal immune response of memory T cells to homologous antigens, and mTh17 is a typical representative of memory T cells; how to obtain memory Th17 cells with high expression of IL-17A in vitro and clarify the method of adaptive transfer of mTh17 cells to SCID mice to induce colitis can analyze the mechanism of action of mTh17 cells in the pathogenesis of UC, and can be used to evaluate the efficacy of new anti-UC drugs and promote the development of new anti-UC drugs. Summary of the Invention

[0003] In view of this, the present invention addresses the deficiencies in the prior art, and its main purpose is to provide a method for inducing colitis through cell-mediated transfer. By using the method provided in this application, memory Th17 cells that highly express IL-17A are obtained in vitro; mTh17 cells are adaptively transferred to SCID mice to successfully induce colitis; and the method is used to evaluate the efficacy of new anti-UC drugs and promote the development of new anti-UC drugs.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for inducing colitis by cell-mediated transfer, comprising the following steps:

[0006] S1. Colitis model replication: BALB / c mice were allowed to freely drink 2.5% DSS solution, water, and 2.5% DSS solution in turn;

[0007] S2. Flow cytometry enrichment of mTh17 cells: BALB / c mice were treated to obtain a single-cell suspension; cells were resuspended in staining buffer, centrifuged at 400 g / min for 4 min, and the supernatant was discarded. Repeat twice; cells were resuspended in staining buffer and blocked with receptor blockers; flow cytometry antibodies were added and incubated at room temperature in the dark for 30 min; cells were resuspended in staining buffer, centrifuged at 400 g / min for 4 min, and the supernatant was discarded twice; the volume was adjusted to the staining buffer, and after detection by flow cytometry, mTh17 cells from colitis mice were enriched in a collection tube;

[0008] S3. Induction of colitis by acquired transfer of mTh17 cells: After adaptive feeding, SCID mice were randomly divided into PBS and mTh17 groups. The mTh17 group was intraperitoneally injected with the cell mixture, while the PBS group was injected with an equal volume of PBS solution.

[0009] S4. Colitis sample collection: Mouse colons were collected and pretreated; colon tissue was blotted dry with filter paper and weighed; the proximal colon was fixed in 4% paraformaldehyde solution for 24 h, and the remaining tissue was snap-frozen in liquid nitrogen and transferred to a -80°C freezer for later use;

[0010] S5. Disease Activity Index Assessment: During the experiment, mice were weighed and their stool consistency and blood in stool were observed. The disease activity index was assessed based on weight loss, stool consistency, and blood in stool.

[0011] S6. Processing of pathological tissues: 1. Paraffin sections: a1. Fixation of colon tissue: Place the proximal colon in 4% paraformaldehyde solution and fix it for 24 hours; a2. Trimming: Trim the colon tissue to 0.5-1.0 cm thick, with the intestinal cavity parallel to the horizontal plane; a3. Dehydration; a4. Transparency; a5. Wax immersion: a6. Embed colon tissue; a7. Preparation of colon paraffin sections; 2. Hematoxylin-eosin staining: b1. Dewaxing: Dewaxing with xylene for 8 minutes; b2. Rehydration with gradient alcohol; b3. Hematoxylin staining; b4. Eosin staining: Place the colon tissue in eosin staining solution for 2 minutes; wash with tap water for 2 minutes; b5. Dehydration: Soak in 95% ethanol for 3 minutes; 100% ethanol for 3 minutes; 100% ethanol for 3 minutes; b6. Transparency: Soak in xylene for 5 minutes; b7. Sealing: Sealing with neutral gum and covering with a coverslip; b9. Collect colon pathology images of each group under an optical microscope.

[0012] As a preferred embodiment, the specific steps of treating BALB / c mice in S2 to obtain a single-cell suspension are as follows: isolating fresh BALB / c mouse spleen tissue, placing it in RMPI1640 culture medium, fully crushing the tissue, filtering it with a filter membrane, lysing the red blood cells with red blood cell lysis buffer, washing it twice with PBS solution, and preparing a single-cell suspension.

[0013] As a preferred embodiment: the flow cytometry antibodies in S2 are CD4 and CD73.

[0014] As a preferred solution: the cell mixture injected intraperitoneally into the mTh17 group in S3 is: 5.0×10 5 CD4 + CD73 + mTh17 cell mixture.

[0015] As a preferred embodiment, the steps of mouse colon pretreatment in S4 are as follows: pre-cool the mouse colon in 1×PBS solution at 4°C overnight, cut off the cecal end tissue, extract PBS solution with a 5.0 mL syringe, inject it into the colon tissue through a gavage needle, clean the intestinal contents, and repeat several times until the colon contents are cleaned.

[0016] As a preferred solution: in step a3 of dehydration in S6, the colon tissue is immersed in 60%, 70%, 80%, 90%, and 95% ethanol at room temperature for 1.0 h each; and anhydrous ethanol is used for immersion at room temperature for 0.5 h.

[0017] As a preferred solution: the a4 transparent film in S6 is soaked in anhydrous ethanol and xylene at a volume ratio of 2:1 at room temperature for 15 minutes, anhydrous ethanol and xylene at a volume ratio of 1:2 at room temperature for 7.5 minutes, and xylene at room temperature for 1.5 minutes.

[0018] As a preferred solution: the specific steps of preparing the paraffin sections of the a7 colon in S6 are: making 4 μm thick sections using a paraffin slicer; placing them in a slicing machine for flattening, adding ultrapure water in advance, and setting the water temperature at 45°C; removing the sections from the slides, and baking them in a slicing machine pre-set at 50°C until the water is evaporated.

[0019] As a preferred solution, b2 in S6 is rehydrated by washing with anhydrous ethanol for 4 minutes, 90% ethanol for 3 minutes, 80% ethanol for 3 minutes, 70% ethanol for 3 minutes, 60% ethanol for 3 minutes and tap water for 10 minutes.

[0020] As a preferred solution, the specific steps of b3 hematoxylin staining in S6 are: placing the colon tissue in hematoxylin staining solution for 5 minutes; washing with tap water for 10 minutes; separating with 1% hydrochloric acid and alcohol for 15 seconds; and returning to blue with tap water for 20 minutes.

[0021] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical scheme that through a method of cell-mediated transfer to induce colitis provided by the present application, memory Th17 cells with high expression of IL-17A are obtained in vitro; mTh17 cells are adaptively transferred to SCID mice to successfully induce colitis; this helps to analyze the mechanism of action of mTh17 cells in the pathogenesis of UC, can be used to evaluate the efficacy of new anti-UC drugs, and promote the development of new anti-UC drugs.

[0022] To more clearly illustrate the structural features and effects of the present invention, it is described in detail below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of flow cytometry enrichment of mTh17 cells in the present invention;

[0024] Figure 2 Schematic diagram of the mTh17 cells of the present invention highly expressing the pro-inflammatory cytokine IL-17A;

[0025] Figure 3 This is a schematic diagram of the weight change rate of colitis mice induced by mTh17 acquired transfer according to the present invention;

[0026] Figure 4 Schematic diagram of the disease activity index of colitis mice induced by mTh17 acquired transfer of the present invention;

[0027] Figure 5 Schematic diagram of the colon length of mice with colitis induced by mTh17 acquired transfer according to the present invention;

[0028] Figure 6 Schematic diagram of the colon weight of mice with colitis induced by mTh17 acquired transfer according to the present invention;

[0029] Figure 7 This is a schematic diagram of colon weight / colon length in colitis induced by mTh17 acquired transfer of the present invention;

[0030] Figure 8 Schematic diagram of the distribution of colon pathological tissues in each group of mice of the present invention;

[0031] Figure 9 Schematic diagram of the mouse pathological tissue damage scoring system of the present invention. DETAILED DESCRIPTION

[0032] The present invention Figures 1 to 9 As shown, a method for inducing colitis by cell-mediated transfer comprises the following steps:

[0033] S1. Colitis model replication: The mice were allowed to drink 2.5% (w / v) DSS solution freely from day 0 to day 7, water freely from day 7 to day 14, and 2.5% (w / v) DSS solution freely from day 14 to day 21.

[0034] S2. Flow cytometry enrichment of mTh17 cells: Colitis mice were euthanized with isoflurane, and the abdominal cavity was quickly opened. Fresh spleen tissue of BALB / c mice was isolated and placed in RMPI1640 medium. The tissue was thoroughly crushed, filtered through a filter membrane, and lysed with red blood cell lysis buffer. The red blood cells were washed twice with PBS solution to prepare a single-cell suspension; the cells were resuspended in staining buffer, centrifuged at 400g / min for 4 minutes, and the supernatant was discarded. This was repeated twice; the cells were resuspended in staining buffer and blocked with receptor blockers; flow cytometry antibodies CD4 and CD73 were added respectively, and the cells were incubated at room temperature in the dark for 30 minutes; the cells were resuspended in staining buffer, centrifuged at 400g / min for 4 minutes, and the supernatant was discarded twice; the volume of staining buffer was adjusted to the desired volume, and after detection by flow cytometry, the mTh17 cells of colitis mice were enriched in a collection tube;

[0035] S3. Induction of colitis by acquired transfer of mTh17 cells: Sixteen SCID mice were randomly divided into PBS and mTh17 groups after 3 days of adaptive feeding, n = 8 per group. On days 0 and 18 of the experiment, the mTh17 group was intraperitoneally injected with 5.0×10 5 CD4 + CD73 + mTh17 cell mixture (1.0 mL), while the PBS group was injected with an equal volume of PBS solution;

[0036] S4. Collection of colitis mouse samples: Mice were deeply anesthetized and euthanized by intraperitoneal injection of 1.0 mL of 2% sodium pentobarbital solution. The abdominal cavity was quickly opened, and the entire colon was separated from the cecum to the anus. The colon length of each mouse was measured with a ruler. The mouse colon was pre-cooled in 1× PBS solution at 4°C overnight. The cecum tissue was cut off. PBS solution was drawn with a 5.0 mL syringe and injected into the colon tissue through a gavage needle to clean the intestinal contents. This was repeated several times until the colon contents were completely cleaned. The colon tissue was blotted dry with filter paper and weighed. The formula: colon weight ÷ colon length = colon weight per unit colon length was used. The proximal colon was fixed in 4% paraformaldehyde solution for 24 h. The remaining tissue was quickly frozen in liquid nitrogen and transferred to a -80°C freezer for later use.

[0037] S5. Disease Activity Index Assessment: During the experiment, mice were weighed and their stool consistency and blood in stool were observed. The disease activity index was assessed based on weight loss, stool consistency, and blood in stool.

[0038] Weight: no significant weight loss, 0 points; weight loss of 1-5%, 1 point; weight loss of 6-10%, 2 points; weight loss of 11-20%, 3 points; weight loss of more than 20%, 4 points;

[0039] Stool consistency: normal stool, 0 points; loose stool (dry), 2 points; loose stool or diarrhea (watery stool), 4 points;

[0040] Blood in stool: no bleeding, 0 points; positive fecal occult blood, 2 points; blood in stool, 3 points; anal bleeding, 4 points;

[0041] S6. Processing of pathological tissue: 1. Paraffin sectioning: a1. Fixation of colon tissue: Place the proximal colon in 4% paraformaldehyde solution for fixation for 24 hours; a2. Trimming: Trim the colon tissue to 0.5-1.0 cm thick, with the intestinal cavity parallel to the horizontal plane; a3. Dehydration: Soak the colon tissue in 60%, 70%, 80%, 90%, and 95% ethanol at room temperature for 1.0 hour each; Soak in anhydrous ethanol at room temperature for 0.5 hour; a4. Transparency: Use anhydrous ethanol and xylene in a volume ratio of 2:1, soak at room temperature for 15 minutes, anhydrous ethanol and xylene in a volume ratio of 1:2, soak at room temperature for 7.5 minutes, and xylene at room temperature for 1.5 minutes; a5. Waxing: a6. Embed colon tissue; a7. Preparation of colon paraffin sections: Use a paraffin slicer to make 4 μm thick sections; Place them flat on a slide spreader, pre-add ultrapure water, and set the water temperature at 45°C; Remove the sections from the slides and place them on a pre-set temperature of 5 Bake in a slide oven at 0℃ until the moisture is evaporated; 2. Hematoxylin-eosin staining: b1. Dewaxing: dewaxing with xylene for 8 minutes; b2. Gradient alcohol rehydration: rehydration with anhydrous ethanol for 4 minutes, 90% ethanol for 3 minutes, 80% ethanol for 3 minutes, 70% ethanol for 3 minutes, 60% ethanol for 3 minutes and washing with tap water for 10 minutes; b3. Hematoxylin staining: place the colon tissue in hematoxylin staining solution for 5 minutes; wash with tap water for 10 minutes; 1% hydrochloric acid alcohol for color separation for 15 seconds; tap water for blueing for 20 minutes; b4. Eosin staining: place the colon tissue in eosin staining solution for 2 minutes; wash with tap water for 2 minutes; b5. Dehydration: soak in 95% ethanol for 3 minutes, 100% ethanol for 3 minutes, and 100% ethanol for 3 minutes in sequence; b6. Transparentization: soak in xylene for 5 minutes; b7. Sealing: seal with neutral gum and cover with a coverslip; b9. Collect colon pathology pictures of each group under an optical microscope.

[0042] Statistical analysis:

[0043] SPSS 22.0 statistical software was used for data analysis. The measurement data were expressed as mean ± standard error (x ± s). One-way analysis of variance was used to analyze the differences between the groups. P < 0.05 was considered statistically significant.

[0044] Result analysis:

[0045] Th17 cells are recognized as pro-inflammatory cells that highly express the pro-inflammatory cytokine IL-17A and are key factors in the initiation and recurrence of ulcerative colitis; CD4 + CD73 + (mTh17) cells accounted for 76.1%, which were CD45RA + CD62L + CCR7 + CD4 + (CD4 + Naive) cells (6 times Figure 2 This indicates that mTh17 cells highly express the pro-inflammatory cytokine IL-17A and have an inflammatory phenotype.

[0046] To investigate whether the acquired transfer of mTh17 cells induces colitis in SCID mice, CD4 + CD73 + mTh17 cells were injected intraperitoneally with 5.0×10^5 CD4 + CD73 + mTh17 cells induced colitis; compared with the normal SCID mice in the PBS group, the body weight change rate of the colitis SCID mice in the mTh17 group ( Figure 3 ) decreased significantly from the 28th to the 34th day of the experiment (p < 0.05 or p < 0.01), and the disease activity index ( Figure 4 ) began to increase significantly from the 8th day of the experiment (p<0.05 or p<0.01); on the 30th day of the experiment, the feces of mice were negative in the PBS group but positive or strongly positive in the mTh17 group, and some mice in the mTh17 group were obviously observed to have typical colitis symptoms such as blood in the stool, diarrhea, and depression; the colon length of SCID mice with colitis in the mTh17 group ( Figure 5 ) were significantly lower than those of normal SCID mice in the PBS group (p<0.05), while the colon weight ( Figure 6 ), colon weight / colon length ( Figure 7 ) increased significantly; this indicates that SCID mice induced by the acquired transfer of mTh17 cells showed typical clinical symptoms of ulcerative colitis.

[0047] Pathological tissue analysis further revealed that the colon mucosal lamina propria of SCID mice induced by acquired transfer of mTh17 cells had a large number of inflammatory cells infiltrating and obvious ulcer formation in the mucosa. The pathological tissue injury score was also significantly higher than that of the PBS group (p < 0.05) ( Figure 9); This indicates that colitis induced by the acquired transfer of mTh17 cells successfully induced an inflammatory response in colonic tissue; colitis induced by the acquired transfer of mTh17 cells is an effective strategy for replicating the colitis model; memory Th17 cells with high expression of IL-17A were obtained in vitro; and colitis was successfully induced by the acquired transfer of mTh17 cells into SICD mice; this helps to understand the mechanism of action of mTh17 cells in the pathogenesis of UC, can be used to evaluate the efficacy of new anti-UC drugs, and promote the development of new anti-UC drugs.

[0048] The above description is merely a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for inducing colitis by cell-mediated transfer, characterized in that: The steps include: S1. Reproduction of colitis model: BALB / c mice were allowed to freely drink 2.5% DSS solution, water, and 2.5% DSS solution in turn; S2. Flow cytometry enrichment of mTh17 cells: Treat BALB / c mice with colitis in step S1 to obtain a single-cell suspension; resuspend the cells in staining buffer, centrifuge at 400g / min for 4 minutes, and discard the supernatant, repeat twice; resuspend the cells in staining buffer and block with receptor blockers; add flow cytometry antibodies CD4 and CD73, respectively, and incubate at room temperature in the dark for 30 minutes; resuspend the cells in staining buffer, centrifuge at 400g / min for 4 minutes, and discard the supernatant twice; adjust the volume with staining buffer, and after detection by flow cytometry, collect the enriched mTh17 cells from the colitis mice in a collection tube; S3. Induction of colitis by acquired transfer of mTh17 cells: After adaptive feeding, SCID mice were randomly divided into PBS and mTh17 groups. The mTh17 group was intraperitoneally injected with a mixture of mTh17 cells, while the PBS group was injected with an equal volume of PBS solution. S4. Colitis sample collection: Mouse colons were collected and pretreated; colon tissue was blotted dry with filter paper and weighed; the proximal colon was fixed in 4% paraformaldehyde solution for 24 h, and the remaining tissue was snap-frozen in liquid nitrogen and transferred to a -80°C freezer for later use; S5. Disease Activity Index Assessment: During the experiment, mice were weighed and their stool consistency and blood in stool were observed. The disease activity index was assessed based on weight loss, stool consistency, and blood in stool. S6. Processing of pathological tissues: I. Paraffin sections: a1. Fixation of colon tissue: Fix the proximal colon in 4% paraformaldehyde solution for 24 h; a2. Trimming: Trim the colon tissue to 0.5-1.0 cm thick, with the intestinal lumen parallel to the horizontal plane; a3. Dehydration; a4. Transparency; a5. Wax immersion: a6. Embed colon tissue; a7. Preparation of colon paraffin sections; II. Hematoxylin-eosin staining: b1. Dewaxing: Dewaxing in xylene for 8 min; b2. Rehydration in graded alcohols; b3. Hematoxylin staining; b4. Eosin staining: Place the colon tissue in eosin solution for 2 min; then wash with tap water for 2 min; b5. Dehydration: Soak in 95% ethanol for 3 min, 100% ethanol for 3 min, and 100% ethanol for 3 min, respectively; b6. Transparency: Soak in xylene for 5 min; b7. Mounting: Mount the slides with neutral gum and cover with a coverslip; b9. Collect colon pathology images of each group under an optical microscope; The flow cytometry antibodies in S2 are CD4 and CD73; The cell mixture injected intraperitoneally into the mTh17 group in S3 was: 5.0×10 5 CD4 + CD73 + mTh17 cell mixture; The a4 transparent film in the S6 is soaked at room temperature for 15 minutes in a volume ratio of anhydrous ethanol to xylene of 2:1, soaked at room temperature for 7.5 minutes in a volume ratio of anhydrous ethanol to xylene of 1:2, soaked at room temperature for 3.5 minutes in xylene I, and soaked at room temperature for 1.5 minutes in xylene II.

2. The method for inducing colitis by cell-mediated transfer according to claim 1, characterized in that: The specific steps of processing the colitis BALB / c mice in step S1 to obtain a single cell suspension in S2 are: isolating fresh spleen tissue of the colitis BALB / c mouse, placing it in RMPI1640 culture medium, fully crushing the tissue, filtering it with a filter membrane, lysing the red blood cells with red blood cell lysis solution, washing it with PBS solution twice, and preparing a single cell suspension.

3. The method for inducing colitis by cell-mediated transfer according to claim 1, wherein: The steps of mouse colon pretreatment in S4 are as follows: pre-cool the mouse colon in 1×PBS solution at 4°C overnight, cut off the cecal end tissue, extract PBS solution with a 5.0 mL syringe, inject it into the colon tissue through a gavage needle, and wash the intestinal contents. Repeat several times until the colon contents are cleaned.

4. The method for inducing colitis by cell-mediated transfer according to claim 1, wherein: In the dehydration step a3 of S6, the colon tissue was immersed in 60%, 70%, 80%, 90%, and 95% ethanol at room temperature for 1.0 h each; and in anhydrous ethanol at room temperature for 0.5 h.

5. The method for inducing colitis by cell-mediated transfer according to claim 1, characterized in that: The specific steps of preparing the paraffin sections of the a7 colon in S6 are as follows: using a paraffin slicer to make 4 μm thick sections; placing them in a slicing machine to flatten them, adding ultrapure water in advance, and setting the water temperature at 45°C; removing the sections from the slides, and baking them in a slicing machine pre-set at 50°C until the water is evaporated.

6. The method for inducing colitis by cell-mediated transfer according to claim 1, characterized in that: The rehydration of b2 in S6 was performed by washing with anhydrous ethanol for 4 minutes, 90% ethanol for 3 minutes, 80% ethanol for 3 minutes, 70% ethanol for 3 minutes, 60% ethanol for 3 minutes and tap water for 10 minutes.

7. The method for inducing colitis by cell-mediated transfer according to claim 1, characterized in that: The specific steps of b3 hematoxylin staining in S6 are: placing the colon tissue in hematoxylin staining solution for 5 minutes; washing with tap water for 10 minutes; separating with 1% hydrochloric acid and alcohol for 15 seconds; and returning to blue with tap water for 20 minutes.

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