Construction method and application of a mouse model of transplantation-related thrombotic microangiopathy

Abstract

The present invention discloses a method for constructing and applying a transplant-related thrombotic microangiopathy mouse model. The construction method comprises the following steps: first constructing a mouse allogeneic hematopoietic stem cell transplantation model, and then intraperitoneally injecting a small dose of LPS+H2O2 for induction on the 14th day after transplantation. The present invention verifies the feasibility of a TA-TMA model induced by a combination of a small dose of LPS and H2O2 through a variety of experimental means, thus filling a gap in the field of TA-TMA mouse model research and providing experimental tools and platforms for in-depth research on the pathogenesis and treatment strategies of TA-TMA. The present invention contributes to promoting the progress of TA-TMA-related drug research, provides new ideas and methods for the clinical treatment of TA-TMA, and has important practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal model construction, and particularly relates to a construction method and application of a transplantation-related thrombotic microangiopathy mouse model. Background Art

[0002] Transplant-associated thrombotic microangiopathy (TA-TMA) is a serious thrombotic complication after hematopoietic stem cell transplantation (HSCT). While its etiology remains unclear, it is currently believed to be related to complement system activation, formation of the membrane attack complex, and endothelial damage. TA-TMA shares clinical features with thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS), primarily manifesting as microangiopathic hemolytic anemia, peripheral blood thrombocytopenia, elevated lactate dehydrogenase, elevated serum creatinine, increased fragmented erythrocytes, microcirculatory fibrin deposition, and microvascular thrombosis, ultimately leading to multiorgan failure. A review of the literature indicates a 0.5% to 76% incidence of TA-TMA. A large review found a mortality rate of 75%, with most patients dying within 3 months of diagnosis. TA-TMA affects the kidneys, gastrointestinal tract, central nervous system, heart, lungs, and serosal surfaces. Risk factors for TA-TMA include calcineurin inhibitors, graft-versus-host disease (GVHD), infection, venous thromboembolic disease, ABO blood type incompatibility, and human leukocyte antigen mismatch.

[0003] Treatment of TA-TMA is based on an understanding of the underlying pathophysiology and is divided into preventive measures and supportive care. Preventive measures, such as avoiding endothelial toxins, avoiding infection, and optimizing transplant conditioning regimens, can minimize endothelial damage. Aggressive supportive care, including minimizing blood transfusions, controlling hypertension, and controlling any underlying infection, is the core of TA-TMA management. Other approaches include discontinuation of calcineurin inhibitors, therapeutic plasma exchange (TPE), rituximab, defibrotide, and eculizumab, with varying efficacy and survival benefits.

[0004] Currently, drugs for treating TA-TMA are only used in exploratory clinical cases, and there is still a lack of mouse models of TA-TMA. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides a method for constructing and applying a transplantation-associated thrombotic microangiopathy mouse model. By applying a small dose of inflammation plus oxidative stress to construct a TA-TMA mouse model, the present invention fills the gap in TA-TMA mouse models and provides reliable technical support for the exploration of drug research for the treatment of TA-TMA.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for constructing a transplantation-related thrombotic microangiopathy mouse model comprises the following steps:

[0008] Step 1) Establishing a mouse allogeneic hematopoietic stem cell transplantation model: Different strains of mice were selected as recipients and donors, respectively; starting on the seventh day before transplantation, the recipient mice were fed daily with acidified water containing gentamicin and levofloxacin. The acidified water containing antibiotics was continued until the seventh day after transplantation, after which the recipient mice were fed with standard acidified water. On the day of transplantation, the recipient mice were irradiated with 6.5 Gy lethal radiation. Within 4 to 6 hours after irradiation, bone marrow cells from the donor mice were transfused to complete the hematopoietic stem cell transplantation;

[0009] Step 2) Construction of a transplantation-related thrombotic microangiopathy mouse model: On the 14th day after transplantation, the mouse blood picture was basically restored. At this time, 3% H2O2 was first injected into the peritoneal cavity of the mouse, and then lipopolysaccharide (LPS) was injected into the peritoneal cavity 30 minutes later.

[0010] Preferably, in step 1), the recipient mice are 6-8 week old wild-type Balb / C mice, and the donor mice are 6-8 week old wild-type C57BL / 6 mice.

[0011] Preferably, in step 1), the dosage of gentamicin is 400,000 U / L, and the concentration of levofloxacin is 50 mg / L.

[0012] Preferably, the bone marrow cells in step 1) are erythrocyte-free, and the number of nucleated cells is 5×10 6 of bone marrow cells.

[0013] Preferably, in step 2), the injection volume of the 3% H2O2 is 5 mL / kg; and the injection volume of the lipopolysaccharide LPS is 2.5 mg / kg.

[0014] Preferably, the lipopolysaccharide LPS in step 2) is derived from Escherichia coli 0111:B4.

[0015] The mouse model constructed by the above construction method is used in the preparation of drugs for treating transplantation-related thrombotic microangiopathy.

[0016] The mouse model constructed by the above construction method is used to screen drugs for treating transplantation-related thrombotic microangiopathy.

[0017] The mouse model constructed by the above construction method is used in the study of drug efficacy and side effects related to transplantation-related thrombotic microangiopathy.

[0018] The mouse model constructed by the above construction method is used in the study of the causes and pathological mechanisms of the occurrence and development of transplantation-related thrombotic microangiopathy.

[0019] The beneficial effects of the present invention are as follows:

[0020] The present invention comprehensively verifies the feasibility of a TA-TMA model induced by a small dose of LPS and H2O2 by comprehensively utilizing a variety of experimental methods such as observation of mouse survival, platelet count analysis, biochemical index detection, peripheral blood smear search for broken red blood cells, and kidney section HE staining, thereby filling the gap in the research field of TA-TMA mouse models and providing experimental tools and platforms for in-depth study of the pathogenesis and treatment strategies of TA-TMA. The TA-TMA mouse model constructed by the method of the present invention allows researchers to more accurately simulate the pathological process of human TA-TMA disease, thereby helping to reveal its pathogenesis and pathophysiological changes. At the same time, the model can also be used to evaluate the therapeutic effects of different drugs on TA-TMA, providing a reliable basis for drug screening and optimization. In addition, the TA-TMA mouse model established by the present invention has the advantages of simple operation, good repeatability, and high stability, and is suitable for large-scale drug screening and in-depth research. Therefore, the present invention helps to promote the progress of TA-TMA-related drug research, provides new ideas and methods for the clinical treatment of TA-TMA, and has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Flowchart for the construction of TA-TMA mouse model and control group;

[0022] Figure 2 For mouse survival prognosis analysis;

[0023] Figure 3 The platelet count in the peripheral blood of mice was 4 hours after intraperitoneal injection of drugs;

[0024] Figure 4 These are the peripheral blood biochemical indicators of mice 4 hours after intraperitoneal injection of drugs: a is the statistical graph of mouse serum creatinine; b is the statistical graph of mouse serum lactate dehydrogenase;

[0025] Figure 5 This is a mouse peripheral blood smear stained with Liu's stain observed under a 100× microscope: the red arrows point to fragmented red blood cells and atypical red blood cells;

[0026] Figure 6 HE staining observation images of mouse tissues and organs: a is a 100× microscopic observation image of the glomerulus, the red arrow points to the fibrous material deposition, and the red triangle points to the platelet thrombus; b is a 20× microscopic observation image of the lung, the red arrow points to the inflammatory cell infiltration; c is a 100× microscopic observation image of the lung, the red arrow points to the inflammatory cell infiltration, and the red triangle points to the platelet aggregation. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] The sources of the experimental mice involved in the following examples are as follows:

[0029] Wild-type Balb / C mice and wild-type C57BL / 6 mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0030] Example 1

[0031] TA-TMA is a syndrome of multi-organ damage caused by microvascular thrombosis. Clinically, its main manifestations are microangiolytic anemia (with red blood cell fragments) and peripheral thrombocytopenia, and it is often accompanied by acute renal damage and central nervous system abnormalities. It is a serious complication after transplantation and is closely related to the prognosis of transplant patients. Professor Jodele of Cincinnati Children's Hospital performed differential analysis of RNA-seq data of TA-TMA patients and showed that TA-TMA patients had changes in inflammation and oxidative stress. The median onset time of TA-TMA patients is 40 days after hematopoietic stem cell transplantation, and hematopoiesis has been basically reconstructed. Therefore, based on the mouse allogeneic hematopoietic stem cell transplantation model, we gave them a small dose of inflammation and oxidative stress when their hematopoiesis was basically reconstructed, detected the relevant indicators of TA-TMA, and determined whether the mice were ill. The specific methods are as follows:

[0032] 1. Construction of TA-TMA mouse model

[0033] (1) Construction of a mouse allogeneic hematopoietic stem cell transplantation model

[0034] 6-8 week old SPF Balb / C wild-type female mice were selected as recipients, and 6-8 week old SPF C57BL / 6 wild-type female mice were selected as donors. Starting from the 7th day before transplantation, the recipient mice were fed with acidified water containing gentamicin (400,000 U / L) and levofloxacin (50 mg / L) daily. The acidified water containing antibiotics was continued until the 7th day after transplantation, after which it was changed to ordinary acidified water for continued feeding. On the day of transplantation, the recipient mice received 6.5 Gy lethal (myeloablative) irradiation. Within 4-6 hours after irradiation, the donor mouse-derived bone marrow cells (red blood cells removed, nucleated cells count 5×10 6 ).

[0035] (2) Assessment of hematopoietic reconstitution after transplantation

[0036] On the 14th day after transplantation, peripheral blood was collected from the mice for three-category testing. The results showed that the white blood cells, hemoglobin, and platelets of the mice were basically restored, which can be used as the baseline values ​​of the TA-TMA mouse model.

[0037] (3) Construction of TA-TMA mouse model

[0038] like Figure 1 As shown, on the 14th day after transplantation, the blood picture was basically restored, and the experimental groups were divided into induced TA-TMA group (LPS+H2O2), H2O2 group, LPS group, and control group (PBS), as follows:

[0039] ① TA-TMA induction group: intraperitoneal injection of 3% H2O2 5mL / kg, 30 minutes later, intraperitoneal injection of a small dose of lipopolysaccharide (LPS) (derived from Escherichia coli 0111:B4, the same below) 2.5mg / kg;

[0040] ②H2O2 group: intraperitoneal injection of 3% H2O2 5mL / kg;

[0041] ③LPS group: intraperitoneal injection of a small dose of lipopolysaccharide (LPS) 2.5 mg / kg;

[0042] ④Control group: intraperitoneal injection of PBS.

[0043] (4) The survival of mice was observed after administration. In addition, blood was collected from the orbital vein of mice 4 hours after intraperitoneal administration. The mouse TA-TMA model was verified by platelet count, biochemical indicators, fragmented red blood cells in peripheral blood smears, and HE staining of kidney sections.

[0044] 2. Validation and Analysis of the TA-TMA Mouse Model

[0045] (1) Observation of mouse survival status

[0046] like Figure 2 As shown, no mice in the PBS control group and the H2O2 group died, and all mice survived until the final observation time, 14 days after intraperitoneal injection; the mice in the low-dose LPS+H2O2-induced TA-TMA model group died as early as 7 hours after administration, and all mice died within 24 hours, with a median survival time of only 14 hours; the mice in the low-dose LPS group began to die 2 days after administration and did not reach the median survival time within the 14-day observation period. Therefore, compared with the PBS control group, the survival time of the mice in the low-dose LPS+H2O2-induced TA-TMA model group was significantly shortened ( **** P<0.0001).

[0047] (2) Platelet count in mouse peripheral blood

[0048] Because no mice in the H2O2 group or the PBS group died, the experiment was subsequently divided into three groups: the TA-TMA group (LPS + H2O2), the LPS group, and the control group (PBS). Four hours after intraperitoneal injection, blood was collected from the medial canthus of the orbital vein of the mice, and routine blood tests were performed to record the platelet count.

[0049] like Figure 3As shown in the figure, compared with the PBS control group, the platelet count of mice in the TA-TMA model group induced by low-dose LPS+H2O2 was significantly decreased (162±76 vs.456±91×10 9 / L, *** P<0.001). This indicates that mice in the low-dose LPS+H2O2 group developed thrombocytopenia.

[0050] (3) Biochemical indicators of mouse peripheral blood

[0051] The clinical manifestations of microcirculatory thrombosis in the TA-TMA model are similar to those of thrombotic thrombocytopenic purpura (TTP) pentad, including fever and platelet count, as well as microangiopathic hemolytic anemia, renal function impairment, and neuropsychiatric abnormalities. Among them, renal function impairment includes abnormal kidney-related indicators in peripheral blood biochemical indicators and the discovery of microcirculatory thrombosis in renal pathological biopsy. Elevated lactate dehydrogenase is included in the diagnostic criteria of each version of TA-TMA. Therefore, we detected two indicators, creatinine (CREA) and lactate dehydrogenase (LDH), in the peripheral blood serum of mice, such as Figure 4 shown.

[0052] Serum creatinine levels such as Figure 4 As shown in middle a, compared with the PBS control group injected intraperitoneally, the creatinine level of mice in the TA-TMA model group induced by low-dose LPS+H2O2 was significantly increased (50.39±5.39 vs. 31.29±0.92 μmol / L, ** LPS alone did not increase serum creatinine levels (32.73±5.06 vs. 31.29±0.92 μmol / L, P>0.05).

[0053] Serum lactate dehydrogenase levels such as Figure 4 As shown in middle b, compared with the PBS control group injected intraperitoneally, the lactate dehydrogenase level of mice in the TA-TMA model group induced by low-dose LPS+H2O2 was significantly increased (5554±1727 vs. 626±91 U / L, ** There was no statistically significant difference in the changes of serum lactate dehydrogenase levels after LPS alone (1171±653 vs. 626±91 U / L, P>0.05).

[0054] The above experimental results suggest that the serum creatinine and lactate dehydrogenase of mice in the TA-TMA model group induced by low-dose LPS+H2O2 increased, which is consistent with the biochemical indicators of TA-TMA.

[0055] (4) Liu staining of mouse peripheral blood smear

[0056] We then performed peripheral blood smears on the mice, stained them with Liu's stain, and looked for broken red blood cells in the cell monolayer under a microscope.

[0057] like Figure 5 As shown, erythrocytes (RBCs) are the most abundant cells in the peripheral blood smear, appearing orange-red with a pale central stain, consistent with a biconcave disc-shaped morphology. In the low-dose LPS+H₂O₂-induced TA-TMA model group, fragmented and atypical RBCs (indicated by red arrows) were observed in the peripheral blood smear. In the other two groups, the percentage of fragmented and atypical RBCs was less than 1%. This experiment further demonstrates the feasibility of inducing the TA-TMA model using a low-dose LPS+H₂O₂-induced model.

[0058] (5) HE staining of mouse tissues and organs

[0059] The mice were then killed by cervical dislocation, and their kidney and lung tissues were removed. After being fixed with paraformaldehyde, they were embedded in paraffin and sectioned, and HE staining was performed. The glomeruli and lung tissues were observed under a microscope. Figure 6 shown.

[0060] Most of the glomeruli are located in the renal cortex and near the medulla. The glomeruli are composed of the renal capsule and the capillary glomeruli inside the capsule. The glomeruli are observed under a 100× microscope (oil lens). Figure 6 As shown in (a), platelet microthrombi (indicated by the red triangle) and local fibrinoid deposition (indicated by the red arrow) were observed in the kidneys of mice in the TA-TMA model induced by low-dose LPS+H2O2; no platelet thrombi were observed in the other two groups.

[0061] Lung tissue was observed under a 20× microscope. Figure 6 As shown in middle b, compared with the mice in the intraperitoneal injection of PBS group, the lung tissue structure of the mice in the low-dose LPS+H2O2-induced TA-TMA model group was significantly damaged, the alveolar structure was disordered, and a large number of inflammatory cells infiltrated (indicated by the red arrows).

[0062] Lung tissue was observed under a 100× microscope. Figure 6 As shown in middle c, compared with the mice in the intraperitoneal injection of PBS group, platelet-aggregated microthrombi (indicated by red triangles) and inflammatory cell infiltration (indicated by red arrows) were observed in the lungs of mice in the low-dose LPS+H2O2-induced TA-TMA model group.

[0063] The above tissue section staining showed that the evidence of low-dose LPS+H2O2-induced TA-TMA model was complete, and microthrombi were visible in the pathology.

[0064] The experimental data above validate the feasibility of the low-dose LPS+H₂O₂-induced TA-TMA model, including mouse survival, platelet count, biochemical markers, fragmented red blood cells on peripheral blood smears, and H&E staining of kidney sections. This model helps to elucidate the pathogenesis and pathophysiological changes of TA-TMA. It can also be used to evaluate the therapeutic effects of different drugs against TA-TMA, providing a reliable basis for drug screening and optimization, and offering new ideas and approaches for the clinical treatment of TA-TMA.

[0065] The embodiments described above are only some of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be based on the scope required by the claims. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making creative efforts shall fall within the scope of protection of the present invention.

Claims

1. A method for constructing a transplantation-related thrombotic microangiopathy mouse model, characterized in that: The following steps are involved: Step 1) Establishing a mouse allogeneic hematopoietic stem cell transplantation model: Different strains of mice were selected as recipients and donors, respectively; starting on the seventh day before transplantation, the recipient mice were fed daily with acidified water containing gentamicin and levofloxacin. The acidified water containing antibiotics was continued until the seventh day after transplantation, after which the recipient mice were fed with standard acidified water. On the day of transplantation, the recipient mice were irradiated with 6.5 Gy lethal radiation. Within 4 to 6 hours after irradiation, bone marrow cells from the donor mice were transfused to complete the hematopoietic stem cell transplantation; Step 2) Construction of a transplantation-related thrombotic microangiopathy mouse model: On the 14th day after transplantation, the mouse blood picture was basically restored. At this time, 3% H2O2 was first injected into the peritoneal cavity of the mouse, and then lipopolysaccharide LPS was injected into the peritoneal cavity 30 minutes later.

2. The method for constructing a transplantation-related thrombotic microangiopathy mouse model according to claim 1, characterized in that: In the step 1), the recipient mice are 6-8 week old wild-type Balb / C mice, and the donor mice are 6-8 week old wild-type C57BL / 6 mice.

3. The method for constructing a transplantation-related thrombotic microangiopathy mouse model according to claim 1, characterized in that: In step 1), the dosage of gentamicin is 400,000 U / L, and the concentration of levofloxacin is 50 mg / L.

4. The method for constructing a transplantation-related thrombotic microangiopathy mouse model according to claim 1, characterized in that: Step 1) The bone marrow cells are erythrocyte-free, and the number of nucleated cells is 5×10 6 of bone marrow cells.

5. The method for constructing a transplantation-related thrombotic microangiopathy mouse model according to claim 1, characterized in that: In step 2), the injection volume of the 3% H2O2 is 5 mL / kg; the injection volume of the lipopolysaccharide LPS is 2.5 mg / kg.

6. The method for constructing a transplantation-related thrombotic microangiopathy mouse model according to claim 1, characterized in that: In step 2), the lipopolysaccharide LPS is derived from Escherichia coli 0111:B4.

7. Use of the mouse model constructed by the method for constructing a transplantation-related thrombotic microangiopathy mouse model according to any one of claims 1 to 6 in the preparation of a drug for treating transplantation-related thrombotic microangiopathy.

8. Use of the mouse model constructed by the method for constructing a transplantation-related thrombotic microangiopathy mouse model according to any one of claims 1 to 6 in screening drugs for treating transplantation-related thrombotic microangiopathy.

9. Use of the mouse model constructed by the method for constructing a transplantation-related thrombotic microangiopathy mouse model according to any one of claims 1 to 6 in the study of the efficacy and side effects of drugs related to transplantation-related thrombotic microangiopathy.

10. Use of the mouse model constructed by the method for constructing a transplantation-related thrombotic microangiopathy mouse model according to any one of claims 1 to 6 in the study of the causes and pathological mechanisms of the occurrence and development of transplantation-related thrombotic microangiopathy.

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