Thymus tissue slice ectopic transplantation model and application thereof

Abstract

The invention belongs to the technical field of thymus tissue slice ectopic transplantation, and particularly relates to a thymus tissue slice ectopic transplantation model and application thereof, and a construction method of the model comprises the following steps: S1, collecting thymus tissue from a donor mouse to prepare slices; s2, transferring the slices to a sterile mixed cellulose ester film in a tissue culture dish for culture; s3, forming a capsular bag at the transplantation part of the receptor mouse; s4, placing the cultured allogeneic thymus tissue slice in a bag, and sewing the bag; s5, feeding the mouse after the mouse recovers autonomous activity, performing ectopic transplantation on the thymus tissue slice cultured in vitro to the inguinal lymph node of the nude mouse, analyzing the regeneration condition of T cells in peripheral blood cells through flow cytometry, and performing histological detection on ectopic thymus to evaluate the immune function reconstruction effect. The traditional Chinese medicine composition reconstructs the body central immunity, and the effect is better than that of being transplanted to the kidney capsule part.

Description

Technical Field

[0001] The invention belongs to the technical field of thymus tissue slice ectopic transplantation, and in particular relates to a thymus tissue slice ectopic transplantation model and application thereof. Background Art

[0002] The thymus is the place where T cells differentiate, develop, and mature. The thymus is composed of thymocytes and thymic stromal cells (TSC). Thymocytes are T cells at different stages of differentiation. TSC include thymic epithelial cells (TEC), macrophages, dendritic cells, and fibroblasts. Thymic epithelial cells are star-shaped, and their protrusions are interconnected to form a network. They are divided into two types: thymic cortical epithelial cells (cTECs) and thymic medullary epithelial cells (mTECs).

[0003] The thymus plays an important role in the establishment of the body's immune function and the reconstruction of lost immune function. It is also the place where T lymphocytes differentiate and mature. Some lymphocytes produced by the bone marrow continue to mature into T cells in the thymus, which can secrete thymosin and selectively play an immunoregulatory role in promoting T cell differentiation and maturation. T helper cells (TH) and T suppressor cells (TS) are both formed in the thymus. The thymus is also an organ for the body to maintain immune homeostasis.

[0004] Thymic deficiency is a disease in which T-cell function is defective due to congenital thymic maldevelopment or hypoplasia. The commonly used treatment method is thymus transplantation. At present, the main solid organs that are the subjects of transplantation surgery include kidneys, liver, heart and lungs. There have been varying degrees of success in transplanting these solid organs. The kidney is located in the abdominal cavity and is a solid organ, one on the left and one on the right, with a capsule covering the surface. Thymic tissue slices are transplanted into the renal capsule. During the operation, the abdominal wall muscles need to be cut to expose the kidneys. After the slices are implanted into the renal capsule, the kidneys are repositioned into the abdominal cavity, and the abdominal wall muscles and skin incisions are sutured in sequence. The whole process is complicated, with great damage and high risk. Summary of the invention

[0005] In order to solve the problems in the prior art, the present invention provides a thymus tissue slice ectopic transplantation model and its application, so as to achieve the purpose of ectopic transplantation of allogeneic thymus tissue slices cultured in vitro into the inguinal lymph nodes and rebuilding the central immunity of the body.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] The present invention aims to provide a thymus tissue slice ectopic transplantation model, and the method for constructing the model comprises the following steps:

[0008] S1: Thymus tissue was collected from donor mice to prepare sections;

[0009] S2: Transfer the slices to a sterile mixed cellulose ester membrane in a tissue culture dish for culture;

[0010] S3: pocket formation at the transplantation site in recipient mice;

[0011] S4: After placing the cultured allogeneic thymus tissue slices in the pouch, the pouch is sutured;

[0012] S5: After the mice recover their autonomous activities, feed them.

[0013] Further, in S2, the thymus tissue slices are cultured in a carbon dioxide incubator for 5 to 7 days, the sterile mixed cellulose ester membrane in the tissue culture dish is immersed in the thymus tissue culture medium, and the thymus tissue culture medium is replaced daily.

[0014] Furthermore, the transplantation site of the recipient mice was the inguinal lymph nodes.

[0015] Furthermore, the dosage of the implanted thymus tissue slice is 1.0 to 7.0 mg.

[0016] Furthermore, the recipient mice are athymic nude mice, aged mice or severe immunodeficient mice.

[0017] Furthermore, in S4, the in vitro cultured allogeneic thymus tissue slices were rinsed with sterile saline, the slices were placed at the opening of the pouch, and the slices were delivered along the edge of the pouch into the pouch of fat and lymph nodes using an intravenous indwelling needle hose, and the thymus tissue slices covered the surface of the lymph nodes.

[0018] Furthermore, in S5, the mice were fed sterile pure water with antibiotics added for one week after transplantation to prevent infection, and were fed mouse feed. The surgical incision of the mice was observed every day. After one week of continuous observation of the mice, the water was changed to sterile pure water and fed with mouse feed.

[0019] Furthermore, 4 weeks after the thymus tissue slices were transplanted, the content of CD3+TCR-β+T cells in the peripheral blood mononuclear cells of mice was detected by flow cytometry every two or three weeks.

[0020] A thymic tissue slice heterotopic transplantation model for research / application in the treatment of thymic defects.

[0021] Research / application of a thymus tissue slice heterotopic transplantation model in the reconstruction of the body's central immunity.

[0022] Compared with the prior art, the beneficial technical effects of the present invention are:

[0023] 1. The present invention ectopically transplants in vitro cultured allogeneic thymus tissue slices into the inguinal lymph nodes of nude mice, and evaluates the effect of immune function reconstruction by analyzing the regeneration of T cells in peripheral blood cells by flow cytometry and histological detection of the ectopic thymus. The present invention reconstructs the central immunity of the body, and the effect is better than transplantation into the renal capsule.

[0024] 2. Compared with transplantation to the renal capsule, the transplantation to the inguinal lymph nodes of the present invention is simpler and less damaging to the recipient. At the same time, transplantation to the inguinal lymph nodes does not require incision of the abdominal wall muscles, and the risk of infection during the whole process is lower.

[0025] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the dynamic culture process of the thymus tissue slices cultured in vitro in the present invention.

[0027] Figure 2 The schematic diagram is of the heterotopic transplantation of allogeneic thymus tissue slices cultured in vitro into the inguinal lymph nodes of the present invention.

[0028] Figure 3 This is a postoperative picture of the allogeneic thymus tissue slice cultured in vitro according to the present invention being ectopically transplanted into the right inguinal lymph node of nude mice.

[0029] Figure 4 It is a scatter plot of the percentage of CD3+TCR-β+ cells in transplanted lymph nodes and non-transplanted peripheral blood in the present invention.

[0030] Figure 5 This is a logical relationship diagram of the streaming analysis in the present invention.

[0031] Figure 6 The percentages of various cells are shown in Figure 10 weeks after the allogeneic thymus tissue slices cultured in vitro were ectopically transplanted into the right inguinal lymph nodes of nude mice.

[0032] Figure 7 This is a bar graph showing the percentage of CD3+TCR-β+ cells in peripheral blood 10 weeks after inguinal lymph node implantation according to the present invention.

[0033] Figure 8 The scatter plots are of the percentage of CD3+TCR-β+ cells in peripheral blood detected by flow cytometry 4, 7 and 10 weeks after surgery for the thymus tissue sections not transplanted, transplanted to the inguinal lymph nodes and transplanted to the renal capsule of the present invention.

[0034] Fig. 9 The figure is a histological comparison of the heterotopic transplantation of the allogeneic thymus tissue slices cultured in vitro into the inguinal lymph nodes and the renal capsule of the present invention. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0036] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0037] The implanted object is BALB / c Nude mouse. Athymic nude mice are mutant mice with congenital thymus defects (referred to as nude mice). Due to the lack of mature T lymphocytes, cellular immune function is low. The lack of T cell function makes athymic mice not reject allogeneic and xenogeneic transplants, making them the most suitable experimental animals for studying thymus function. In addition, elderly mice or mice with severe immunodeficiency can also be selected.

[0038] 1. Preparation and culture of thymus tissue slices from newborn mice or adult mice

[0039] The thymus of newborn mice or adult mice was collected aseptically, and the capsule and connective tissue on the surface of the thymus tissue were removed with small animal dissecting forceps and small animal dissecting scissors. The thymus tissue slicer was used to cut the thymus tissue slices into 0.5-1 mm thick slices along the natural seams of the longitudinal line of the thymus tissue, and transferred to the sterile mixed cellulose ester membrane immersed in the thymus tissue culture medium in the tissue culture dish. In a carbon dioxide incubator (5% CO 2 , 37°C) for 5 to 7 days, and the thymus tissue culture medium was replaced daily.

[0040] In vitro culture can eliminate T lymphocytes from the donor thymus tissue, effectively reducing the production of GVDH (graft-versus-host disease) after thymus tissue slice transplantation, while retaining the tissue structure and stromal cells of the thymus. The tissue skeleton and stromal cells of the implanted thymus tissue can provide an immune microenvironment for the development of lymphocytes and promote the recipient's hematopoietic stem cells to develop into mature T lymphocytes in the thymus tissue slices.

[0041] Figure 1The dynamic culture process of thymic tissue slices cultured in vitro is shown. Day 0 indicates the day when the thymic tissue slices were prepared, Day 1 indicates that the thymic tissue slices have been cultured for 1 day, and so on. We observed that the appearance of the thymic tissue slices changed as follows: on day 0, the thymic tissue slices were light pink, and the slices with more blood were bright red, plump and shiny; as the culture time increased, a large number of lymphocytes were washed out of the thymic tissue slices, the thymic tissue culture medium became turbid, and the slice thickness gradually decreased. On day 3 of culture, it was clearly visible that the thymic tissue slices gradually changed from light pink to light yellow, and the slice thickness became slightly thinner; on day 5 of culture, the slices became significantly thinner due to the large removal of lymphocytes.

[0042] 2. Heterotopic transplantation of in vitro cultured thymus tissue slices into the inguinal lymph nodes of nude mice

[0043] 1. The experimental animals were randomly divided into an experimental group (transplantation group) and a control group (non-transplantation group).

[0044] 2. Intraperitoneally inject Avertin anesthetic (according to the recommended dose in the instructions) into 6- to 8-week-old BALB / c nude mice.

[0045] 3. Transfer the fully anesthetized nude mouse to the clean bench in the sterile operating room. Place the nude mouse in supine position, and secure its limbs with tape on the warm animal operating table.

[0046] 4. After routine iodine disinfection of the surgical site, use small animal dissecting scissors to make a longitudinal incision about 1.0 cm long along the midline of the lower abdomen of the nude mouse at 0.5 to 0.7 cm.

[0047] 5. Use small animal tissue forceps to bluntly separate the subcutaneous tissue to expose the fat-wrapped inguinal lymph nodes.

[0048] 6. Use spring scissors to cut a small hole in the fat. Use ophthalmic forceps in your left hand to clamp the fat tissue. Use fine forceps in your right hand to bluntly separate the fat tissue wrapped around the lymph nodes to form a pouch (about 0.3 cm in length and 0.3 cm in depth).

[0049] 7. Rinse the in vitro cultured thymus tissue slices with sterile saline, place the slices at the opening of the pouch, and use a 24G intravenous catheter hose to deliver the slices along the edge of the pouch into the pouch of fat and lymph nodes. The thymus tissue slices cover the surface of the lymph nodes.

[0050] 8. Use absorbable sutures (type 5-0) to close the pocket of the implanted thymus tissue slice. Suture the skin incision with a single layer of interrupted sutures, keep the incision in a natural state, and avoid suturing too tightly. Figure 2 , Schematic diagram of ectopic transplantation of in vitro cultured allogeneic thymic tissue slices into inguinal lymph nodes.

[0051] 9. Disinfect the surgical incision with iodine and place the mouse on a heating pad to wake up. Figure 3 , The allogeneic thymus tissue slices cultured in vitro were ectopically transplanted into the right inguinal lymph nodes of nude mice after surgery. After the mice recovered their independent activities, they were placed in cages and raised in separate cages.

[0052] 10. The control group did not implant allogeneic thymus tissue slices cultured in vitro.

[0053] 11. After the operation, feed the mice with sterile pure water with antibiotics added for one week to prevent infection, and feed them with normal mouse feed. Observe the surgical incision of the mice every day (if the incision heals well and there is no infection, it is normal for the suture to fall off; if the incision is split, it needs to be sutured after re-anesthesia; if there is a small amount of exudation from the incision, it needs to be disinfected with iodine). After observing the mice for one week, replace them with sterile pure water and feed them with normal mouse feed.

[0054] 12. Four weeks after the thymus tissue slice transplantation surgery, the content of CD3+TCR-β+T cells in the peripheral blood mononuclear cells (PBMC) of the mice was detected by flow cytometry every two or three weeks (such as 4 weeks, 6 weeks, 8 weeks, 10 weeks, etc., or 4 weeks, 7 weeks, 10 weeks, 13 weeks, etc.).

[0055] 13. If the content of CD3+TCR-β+T cells in mouse PBMCs does not increase significantly after 4 to 6 weeks, new thymus tissue slices can be implanted into the inguinal lymph node on the other side of the mouse according to the above transplantation scheme.

[0056] 3. Dosage of implanted thymus tissue slices

[0057] The dose range of thymus tissue slices implanted in each nude mouse in the experiment was 1.0 to 7.0 mg. One, two or more thymus tissue slices may be implanted.

[0058] 4. Selection of transplantation site

[0059] Left inguinal lymph nodes, right inguinal lymph nodes, or bilateral inguinal lymph nodes.

[0060] V. Experimental Example: Heterotopic transplantation of in vitro cultured allogeneic thymus tissue slices into the right inguinal lymph nodes of nude mice

[0061] Experimental subjects: 11 BALB / c athymic nude mice, 6 weeks old, ♂, weighing 18-26g, specific pathogen free (SPF) grade, purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. (Experimental mouse quality certificate number: B202402210289). They were randomly divided into an experimental group (transplanted lymph nodes) and a control group (untransplanted group). The doses of thymus tissue slices implanted in the 7 nude mice in the experimental group were divided into two types (1.0 < transplanted dose ≤ 3.0 mg, 3.0 < transplanted dose ≤ 7.0 mg), and the remaining 4 nude mice were the control group. The donors were thymus tissue slices from newborn 8-day-old BALB / c wild-type mice, which were transplanted into the right inguinal lymph nodes of nude mice after 5 days of in vitro culture (referred to as the transplanted lymph node group). The doses of thymus tissue slices implanted in the 7 nude mice were 1.7 mg, 2.1 mg, 2.3 mg, 4.1 mg, 5.6 mg, 5.9 mg and 6.6 mg. Flow cytometry was used to detect the content of T cells in the peripheral blood of the experimental group (transplanted lymph nodes) and the control group (non-transplanted group) at 4, 7 and 10 weeks after surgery.

[0062] Figure 4 The scatter plot shows the percentage of CD3+TCR-β+ cells in the peripheral blood of the experimental group and the control group 4, 7 and 10 weeks after surgery. The results showed that the content of CD3+TCR-β+ cells in the peripheral blood of mice in the experimental group was significantly higher than that in the control group 4, 7 and 10 weeks after surgery (* indicates P value < 0.05, ** indicates P value < 0.01, **** indicates P value < 0.0001), indicating that transplantation of allogeneic thymus tissue slices cultured in vitro into the inguinal lymph nodes of BALB / c nude mice can reconstruct the immune function of nude mice.

[0063] Figure 5 This is a logical relationship diagram for streaming analysis. Figure 5 Shown are the changes in CD3+TCR-β+ cells, CD4+ cells, and CD8+ cells in the peripheral blood 10 weeks after in vitro cultured thymus tissue sections were implanted into the inguinal lymph nodes of BALB / c nude mice.

[0064] We further compared the content of CD3+TCR-β+ cells in peripheral blood 10 weeks after thymic tissue sections were implanted into inguinal lymph nodes at two transplantation doses: 1.0 < transplantation dose ≤ 3.0 mg and 3.0 < transplantation dose ≤ 7.0 mg. Figure 7 The bar graph shows the percentage of CD3+TCR-β+ cells. The results showed that 10 weeks after thymus tissue sections were transplanted into the right inguinal lymph nodes of nude mice in the dose range of 1.0<transplantation dose ≤3.0 mg and 3.0<transplantation dose ≤7.0 mg, there was no significant difference in the content of CD3+TCR-β+ cells in the peripheral blood of nude mice (P value 0.80, ns means not significant, i.e. P value>0.05).

[0065] Effect of different transplantation sites on immune reconstitution: Allogeneic thymus tissue slices with a transplantation dose of 1.0 < transplantation dose ≤ 3.0 mg prepared by the same method as that for implantation into the inguinal lymph nodes were transplanted into the renal capsule. Flow cytometry was used to detect the levels of T cells in the peripheral blood of the experimental group (renal capsule transplantation) and the control group (non-transplantation group) at 4, 7, and 10 weeks after surgery.

[0066] Figure 8 The scatter plots show the percentage of CD3+TCR-β+ cells in the peripheral blood of the experimental group and the control group detected by flow cytometry 4, 7 and 10 weeks after surgery. The results show that the content of CD3+TCR-β+ cells in the peripheral blood of mice in the experimental group was significantly higher than that in the control group at 4, 7 and 10 weeks after surgery (** indicates P value < 0.01, **** indicates P value < 0.0001), indicating that ectopic transplantation of allogeneic thymus tissue slices cultured in vitro into the renal capsule can rebuild the immunity of nude mice. At the same time, Figure 8 Comparison of the changes in the content of CD3+TCR-β+ cells in peripheral blood 4, 7 and 10 weeks after transplantation of allogeneic thymic tissue slices into inguinal lymph nodes and renal capsule: The results showed that there was no significant difference in the percentage of CD3+TCR-β+ cells in the peripheral blood of mice with thymic tissue slices transplanted into inguinal lymph nodes and renal capsule 4 weeks after transplantation (ns means not significant, i.e. P value>0.05), but at the seventh and tenth weeks after transplantation, the percentage of CD3+TCR-β+ cells in the peripheral blood of mice in the inguinal lymph node transplantation group was significantly higher than that in the renal capsule transplantation group (* means P value <0.05, *** means P value <0.001). In summary, the immune function reconstruction effect of allogeneic thymic tissue slices cultured in vitro transplanted into the inguinal lymph nodes is better than that transplanted into the renal capsule.

[0067] We further collected tissues from the transplanted sites 12 weeks after mouse transplantation and evaluated the developmental effects of in vitro cultured thymus tissue sections in the inguinal lymph nodes and renal capsule by HE and immunohistochemistry. Fig. 9 HE staining results showed that the allogeneic thymus tissue sections cultured in vitro and implanted in the inguinal lymph nodes and the thymus tissue sections implanted in the renal capsule were clearly demarcated from the surrounding tissues, and obvious grafts were visible. No grafts were found in the control group, indicating that the grafts could survive normally. A large number of lymphocytes gathered in the grafts, among which the deep HE staining was in the cortical area of ​​the thymus, with a high cell density, indicating that immature T lymphocytes were in a state of rapid proliferation. The lighter staining was in the medullary area of ​​the thymus, where the immature T cells were undergoing negative selection.

[0068] Immunohistochemistry was used to analyze the content of T cells and epithelial cells in the grafts. Anti-CD3 marked the CD3+T cells of mice, Anti-cytokeratin 5 (CK5) marked the mTECs of mice, and Anti-cytokeratin 8 (CK8) simultaneously marked the cTECs of mice. The results showed that the lymphocytes gathered in the grafts were CD3+T cells. Anti-CK5 / CK8 can mark the "network" of thymic epithelial cells, including CK5+ mTECs, CK8+ mTECs and cTECs. These results indicate that allogeneic thymic tissue slices cultured in vitro can develop normally into thymic tissue when transplanted into inguinal lymph nodes, and epithelial cells in thymic tissue slices can provide an immune microenvironment that supports the development of thymic T cells, and induce the development and maturation of T cells by chemotaxis of mouse hematogenous stem cells.

[0069] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A thymus tissue slice heterotopic transplantation model, characterized in that: The model construction method includes the following steps: S1: Thymus tissue was collected from donor mice to prepare sections; S2: Transfer the slices to a sterile mixed cellulose ester membrane in a tissue culture dish and culture; S3: pockets formed at the transplantation site in recipient mice; S4: After placing the cultured allogeneic thymus tissue slices in the pouch, the pouch is sutured; S5: After the mice recover their autonomous activities, feed them.

2. A thymus tissue slice heterotopic transplantation model as claimed in claim 1, characterized in that: In S2, the thymus tissue slices were cultured in a carbon dioxide incubator for 5 to 7 days, and the sterile mixed cellulose ester membrane in the tissue culture dish was immersed in thymus tissue culture medium, and the thymus tissue culture medium was replaced daily.

3. A thymus tissue slice heterotopic transplantation model as claimed in claim 1, characterized in that: The transplantation site of the recipient mice was the inguinal lymph nodes.

4. A thymus tissue slice heterotopic transplantation model as claimed in claim 3, characterized in that: The dose of implanted thymus tissue slices ranges from 1.0 to 7.0 mg.

5. A thymus tissue slice heterotopic transplantation model as claimed in claim 1, characterized in that: The recipient mice were athymic nude mice, aged mice or severe immunodeficient mice.

6. A thymus tissue slice heterotopic transplantation model as claimed in claim 1, characterized in that: In S4, the ex vivo cultured allogeneic thymic tissue slices were rinsed with sterile saline, placed at the opening of the pouch, and delivered along the edge of the pouch into the pouch of fat and lymph nodes using an intravenous indwelling needle hose, with the thymic tissue slices covering the surface of the lymph nodes.

7. A thymus tissue slice heterotopic transplantation model as claimed in claim 1, characterized in that: In S5, the mice were fed sterile pure water with antibiotics added for one week after transplantation to prevent infection, and were fed mouse feed. The surgical incision of the mice was observed every day. After one week of continuous observation of the mice, the water was changed to sterile pure water and fed with mouse feed.

8. A thymus tissue slice heterotopic transplantation model as claimed in claim 1, characterized in that: Four weeks after thymus tissue slice transplantation, the content of CD3+TCR-β+T cells in mouse peripheral blood mononuclear cells was detected by flow cytometry every two or three weeks.

9. Research / application of a thymus tissue slice heterotopic transplantation model as described in any one of claims 1 to 8 in the treatment of thymus deficiency.

10. Research / application of a thymus tissue slice heterotopic transplantation model as claimed in any one of claims 1 to 8 in reconstructing the central immunity of the body.

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