Kidney organoid and nephritis model rapid construction method and application and evaluation system thereof

Abstract

The invention relates to the technical field of biomedical research, in particular to a kidney organoid and nephritis model rapid construction method, application thereof and an evaluation system, and provides a brand-new, efficient and low-cost model system for nephritis research. The system not only can quickly simulate the occurrence and development process of nephritis, but also can ensure the effectiveness and reliability of the model through a comprehensive evaluation method; therefore, the method is expected to become an important tool for nephritis disease mechanism exploration, drug screening and personalized treatment in the future.

Description

Technical Field

[0001] The present invention relates to the field of biomedical research technology, and specifically provides a method for rapidly constructing a kidney organoid and a nephritis model, and an application and evaluation system thereof. Background Art

[0002] In the prior art, the construction of nephritis models mainly relies on animal experiments, such as complex surgical operations to remove most of the kidney tissue in stages to construct a nephritis model. Such models not only have a long modeling cycle, but are also accompanied by high mortality and a steep learning curve, which greatly limits their widespread application in nephritis research and affects the efficiency of clinical transformation of research results.

[0003] Organoids are similar to tissue organs; they are a model based on a 3D in vitro cell culture system that is highly similar to the source tissue or organ in the body. They can replicate the complex spatial morphology of differentiated tissues, and can show the interactions and spatial position morphology between cells and between cells and their surrounding matrix. They themselves can have similar physiological responses to differentiated tissues and organs in the body, and have extremely high similarity to the source tissues. Compared with traditional 2D cell culture models, 3D cultured organoids contain a variety of cell types, breaking through the simple physical contact between cells, forming a closer interaction between cells and between cells and the matrix, forming a functional "micro-organ", which can be better used to simulate the occurrence process and physiological and pathological state of organ tissues, and therefore has broad application prospects in basic research and clinical diagnosis and treatment.

[0004] However, there are few reports in the existing technology on how to construct kidney organoids that can be used for nephritis research, and how to quickly construct a nephritis model. Summary of the invention

[0005] The present invention simulates the occurrence and development of nephritis in vivo by culturing kidney organoids in vitro, and provides a rapid, economical and efficient method for constructing a nephritis model. A comprehensive evaluation system is constructed to evaluate the pathological characteristics, cell responses, gene expression changes and degree of functional damage of the nephritis model. The model overcomes the problems of complexity, high cost and high mortality in the construction process of traditional animal models, and provides strong support for the research on the disease mechanism of nephritis and the development of treatment strategies.

[0006] In order to achieve the above-mentioned object, the first aspect of the present invention discloses a method for rapid construction of kidney organoids, including the induction, culture and passage of kidney organoids. The specific steps are as follows: After enzymatic hydrolysis of kidney cells to obtain single cells, primary kidney cells were cultured in a cell culture incubator; After primary renal cells were passaged to P5, they were digested into single cells and plated at 1×10 5The concentration was inoculated into AIgiMatrix 3DCulture System, stimulated with STEMDIFF APEL2 plus CHIR99021 for one hour, and then induced with STEMDIFF APEL2+heparin+FGF9. The cells were passaged on the 7th day and cultured in Matrigel Matrix for 14 days to obtain kidney organoids.

[0007] The cell culture conditions were 5% CO2 and 37 degrees.

[0008] This culture method can enrich specific cell types in the kidney (such as podocytes, renal tubular epithelial cells, etc.), which is of great significance for studying specific kidney diseases or physiological functions. By standardizing the reagent combination and culture process, the consistency and stability between different batches of organoids can be ensured, and the reliability and repeatability of experimental results can be improved.

[0009] The second aspect of the present invention discloses a method for rapidly constructing a nephritis model based on the above-mentioned kidney organoids, wherein the above-prepared kidney organoids are exposed to an environment of doxorubicin and lipopolysaccharide to induce the formation of a nephritis model.

[0010] Preferably, the concentration of doxorubicin is 10 μg / ml, and the concentration of lipopolysaccharide is 1 μg / μl.

[0011] The third aspect of the present invention discloses the use of the above-mentioned kidney organoids or nephritis models in the development of drugs for treating kidney diseases.

[0012] The fourth aspect of the present invention discloses a method for constructing a nephritis model, and the nephritis model is evaluated by detecting the pathological characteristics, cell morphology changes, expression changes of key cell markers, gene expression spectrum changes and the degree of functional damage of the above-mentioned nephritis model.

[0013] Compared with the prior art, the present invention achieves the following beneficial effects: Compared with traditional animal models, the kidney organoid model used in the present invention has a short construction period and low cost, which can significantly reduce research investment. The organoid constructed by the present invention is close to the real kidney in structure and function, and can accurately simulate the occurrence and development of nephritis, providing strong support for disease mechanism research and drug screening. A method for constructing a kidney organoid nephritis model and its evaluation system are proposed, providing a new, efficient and low-cost model system for nephritis research. Currently, the commonly used model methods, doxorubicin injection and surgery, take about a month to complete the model, and it may take several months to achieve severe nephritis. The system designed by the present invention can not only quickly simulate the occurrence and development process of nephritis, but also ensure the effectiveness and reliability of the model through a comprehensive evaluation method. Therefore, the present invention is expected to become an important tool for future exploration of nephritis disease mechanisms, drug screening and personalized treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The morphology of the organoids in the control group and the nephritis group after 3D culture was observed under a microscope. It can be seen that the organoids in the control group have a compact structure and complete morphology, while the cell morphology in the nephritis group is obviously dispersed.

[0015] Figure 2 The survival rate of kidney cells in the renal organoid model under the induction of doxorubicin at concentrations of 1, 2, 5, and 10 (μg / ml) showed the process of pathological changes in the organoids after exposure to stimulating factors. The results showed that at a concentration of 10μg / ml, the survival rate of kidney cells was significantly reduced (p<0.0001) and the mean was less than 50%, which was consistent with the clinical situation of kidney cell damage in nephritis and could be used as an evaluation method for the degree of nephritis development in this model.

[0016] Figure 3 The immunofluorescence staining results of the nephritis model. The immunofluorescence results show that the morphology of kidney cells changes after nephritis, the intercellular space becomes larger, and the brightness of kidney cell markers Synaptopodin (SYN glomerular visceral epithelial cells), GATA3 (urothelial cells), Lotus tetragonolobus lectin (LTL renal proximal tubular cells) and Nephrin (NEP renal podocytes) decreases. This result proves that this culture method can produce normal renal cell structure, and multiple cells in the kidney of the nephritis group are damaged, which is consistent with the clinical manifestations of nephritis and can be used as an evaluation method for the development of nephritis in this model.

[0017] Figure 4 The results of the enzyme-linked immunosorbent assay to detect kidney damage marker proteins in the supernatant of nephritis organoids showed that after doxorubicin-induced nephritis, the kidney damage proteins KIM-1 (p<0.0001) and NALP3 (p<0.01) in the nephritis group increased significantly, which was consistent with the clinical manifestations of nephritis and could be used as an evaluation method for the degree of nephritis development in this model. DETAILED DESCRIPTION

[0018] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0019] Example 1 Induction and culture of kidney organoids 1. Preparation of starting materials: Mouse kidney cells were selected and kidney tissues were collected using sterile surgical techniques.

[0020] Perform delicate enzymatic digestion (e.g., using trypsin-EDTA solution) to obtain a single-cell suspension.

[0021] 2. Three-dimensional culture: The single cell suspension was inoculated into the AIgiMatrix 3D Culture System.

[0022] Initial stimulation was performed using STEMDIFF APEL2 medium combined with CHIR99021 for 1 hour.

[0023] The medium was replaced with freshly prepared STEMDIFF APEL2 medium supplemented with heparin and FGF9 as growth factors, and the culture was continued to promote cell growth, proliferation and differentiation in three-dimensional space.

[0024] 3. Microscopic observation on the third day showed that the cell colonies gradually aggregated into three-dimensional spherical structures.

[0025] 4. On the 7th day, the cells that have formed a stable three-dimensional structure are passaged again, and the spheres are dispersed into single cells or small cell clusters by enzymatic or mechanical dissociation, and transferred to a 6-well plate containing Matrigel Matrix for continued culture. The culture is continued for 14 days to obtain kidney group organoids. Figure 1 The right figure is of the control group (kidney organoids without lesions) in which the left figure is an image of the control group; it can be seen from the figure that the nephritis group organoids induced by the present invention have more specific cell types (such as podocytes, tubular epithelial cells, etc.), which is beneficial for studying specific kidney diseases or physiological functions; in addition, the nephritis group organoids of the present invention have a more dispersed structure, which provides a more favorable inflammation induction environment for the subsequent construction of a nephritis model.

[0026] Example 2: Construction of nephritis model 1. Treatment with stimulating factors: The mature kidney organoids cultured in Example 1 were exposed to 1, 2, 5, 10 (μg / mL) concentrations of doxorubicin and 1 μg / μL lipopolysaccharide to induce a simulated nephritis pathological environment for 24 hours, inducing mild fibrosis in the kidney organoids. Figure 2 The results showed that when the concentration of doxorubicin was 10μg / mL and the concentration of lipopolysaccharide was 1μg / μL, the survival rate of kidney cells was significantly reduced (p<0.0001) and the mean was less than 50%, and it would not lead to overall kidney organ failure and death; it was consistent with the kidney cell damage in the clinic of nephritis and could be used as an evaluation method for the degree of nephritis development in this model.

[0027] The mature kidney organoids cultured according to the method of Example 1 were induced with (1) 10 μg / mL doxorubicin (control group); (2) 10 μg / mL doxorubicin and 1 μg / μL lipopolysaccharide (nephritis group). 2. Model evaluation: In clinical practice, the severity of nephritis is usually assessed through: imaging examinations to observe changes in kidney morphology and structure; renal puncture biopsy, using immunofluorescence and other examination methods to observe the damage and cell viability of various kidney cells; biochemical indicators: observing whether kidney injury factors such as KIM-1 and NALP3 are elevated.

[0028] This experiment will also observe the morphological changes of organoids under a microscope, record the cell survival rate and morphological characteristics after treatment with different concentrations of doxorubicin, and verify the effectiveness of the nephritis model by combining molecular biology, immunology, histology and functional evaluation methods.

[0029] 3. Evaluation method (1) Immunofluorescence staining: Immunofluorescence staining of the nephritis model was performed using kidney cell specific antibodies such as Synaptopodin (SYN, visceral glomerular epithelial cells), GATA3 (urothelial cells), Lotus tetragonolobus lectin (LTL, proximal tubular cells) and Nephrin (NEP, renal podocytes).

[0030] After 24 hours of induction, the changes in renal cell morphology and marker expression after nephritis were immediately observed, and the changes in fluorescence and intercellular space were recorded. Figure 3 As shown; (2) Enzyme-linked immunosorbent assay: Detect changes in kidney injury marker proteins (such as specific cytokines, chemokines, etc.) in the supernatant of the nephritis model. The results are as follows Figure 4 shown.

[0031] Depend on Figure 3It can be seen that the nephritis group was observed immediately after 24 hours of induction, and kidney organoids such as SYN glomerular visceral epithelial cells, urothelial cells, LTL renal proximal tubule cells and NephrinNEP renal podocytes were significantly damaged (including decreased cell viability or death), which can be used as a nephritis model evaluation standard; in addition, Figure 4 The results showed that compared with the control group, the nephritis group significantly increased the expression of kidney injury marker proteins (KIM-1, NALP3 inflammasome) by related cells in the kidney organoids, so that the kidney organoids maintained the overall tissue vitality (no severe necrosis) and greatly accelerated the construction of the nephritis model (the nephritis model could be formed in 24 hours).

[0032] Comparative Example 1: Construction of conventional animal nephritis model Materials: 20 healthy male SD rats were randomly divided into an adriamycin nephropathy model group (10 rats) and a normal control group (10 rats). The rat adriamycin nephropathy model was established by injecting adriamycin (5 mg / kg) twice into the tail vein at an interval of 2 weeks. The changes of blood and urine biochemical indexes and renal tissue pathology of rats in each group were observed.

[0033] Pathological examination showed that 4 rats died in the adriamycin nephropathy model group, with a mortality rate of 40%. Statistics of surviving rats showed swelling of glomerular podocytes and mild proliferation of mesangial cells at the end of the 4th week; glomerular adhesion and atrophy of tubular epithelial cells at the end of the 8th week; and mild and moderate segmental sclerosis of some glomeruli at the end of the 12th week, with obvious focal lymphocyte infiltration and mild fibrosis in the interstitium. The results showed that the animal nephritis model constructed by high-dose injection of adriamycin not only had a high mortality rate, but also took a long time to construct the nephritis model, and it took at least one month to form a more severe nephritis.

[0034] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for rapid construction of kidney organoids, characterized in that: The specific steps for induction, culture and passage of kidney organoids are as follows: After enzymatic hydrolysis of kidney cells to obtain single cells, primary kidney cells were cultured in a cell culture incubator; After primary renal cells were passaged to P5, they were digested into single cells and plated at 1×10 5 The concentration was inoculated into AIgiMatrix 3DCulture System, stimulated with STEMDIFF APEL2 plus CHIR99021 for one hour, and then induced with STEMDIFF APEL2+heparin+FGF9. The cells were passaged on the 7th day and cultured in Matrigel Matrix for 14 days to obtain kidney organoids.

2. A method for rapid construction of kidney organoids according to claim 1, characterized in that: The cell culture conditions were 5% CO2 and 37 degrees.

3. A method for rapidly constructing a nephritis model, characterized in that: Take the kidney organoid described in any one of claims 1-2, expose it to an environment of doxorubicin and lipopolysaccharide, and induce a nephritis model.

4. The method for rapidly constructing a nephritis model according to claim 6, characterized in that: The concentration of doxorubicin is 10 μg / mL, and the concentration of lipopolysaccharide is 1 μg / μL.

5. Use of the kidney organoid as described in any one of claims 1-2, or the nephritis model as described in any one of claims 3-4 in the development of drugs for treating kidney diseases.

6. A method for constructing a nephritis model, characterized in that: The nephritis model is evaluated by detecting the pathological characteristics, cell morphology changes, expression changes of key cell markers, gene expression profile changes and degree of functional damage of the nephritis model described in any one of claims 3-4.

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