Construction and identification method of MRL / lpr lupus mouse kidney organoid and application of MRL / lpr lupus mouse kidney organoid

Abstract

A method for identifying MRL / lpr lupus kidney organoid specifically comprises the following steps: collecting an organoid culture medium to be detected, detecting the levels of ANA and anti-ds-DNA antibodies in the culture medium, and carrying out histomorphological identification on the organoid. According to the invention, the histomorphology of the MRL / lpr lupus kidney organ of a mouse is identified by detecting the levels of ANA and an anti-ds-DNA antibody as well as HE staining and immunofluorescence, so that whether the MRL / lpr lupus kidney organ is successfully constructed or not is detected.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to a construction and identification method of a MRL / lpr lupus mouse kidney organoid and an application thereof. Background Art

[0002] Systemic lupus erythematosus (SLE) is a chronic inflammatory disease that is more common in women than men in all age groups and populations; the male-female ratio is highest during the childbearing years. SLE can affect any organ, but it usually damages the kidneys. Lupus nephritis (LN) is a common complication of SLE, and 10% to 20% of LN patients will develop end-stage renal disease within 5 years of diagnosis, which is life-threatening.

[0003] The pathogenesis of LN is complex and is currently believed to be related to genetics, neuroendocrine, environment (ultraviolet light, drugs), viral infection, etc. These factors cause in situ immune complexes to deposit in renal tissue, leading to excessive proliferation of lymphoid tissue, formation of autoantibodies, activation of complement and multiple cytokines in the kidney, and abnormal proliferation and differentiation of parenchymal or interstitial cells such as glomerular mesangial cells, endothelial cells, and podocytes, leading to changes in renal function. The pathogenesis of LN involves multiple pathogenic mechanisms. The extrarenal causes of systemic lupus are based on a variety of combinations of genetic variations that impair the mechanisms that usually ensure immune tolerance to nuclear self-antigens.

[0004] The diagnosis and treatment of LN are challenging and mainly rely on renal biopsy, and there are no reliable non-invasive biomarkers for LN. So far, the complete remission rate of LN is only 20% to 30% after 6 months of standard treatment, which is far from satisfactory. In addition, the adverse reactions of immunosuppressants, especially glucocorticoids, further impair the prognosis of LN.

[0005] Biological agents targeting autoimmune reactions and inflammatory pathways have brought hope for the treatment of refractory lupus. Animal models are needed for the screening of preparations and the evaluation of drug efficacy. MRL / Lpr lupus mice are an animal model of autoimmune diseases similar to human systemic lupus erythematosus (SLE). However, MRL / lpr lupus mice are expensive and have a long cycle, which is not conducive to large-scale drug screening and evaluation.

[0006] Organoids are a newly developed in vitro three-dimensional culture technology. Compared with conventional cell line culture, the tissue structure and function formed by the organoid culture system are more complex, with advantages such as high fitting degree, stable passage, short culture cycle, and high success rate. It is currently an ideal in vitro culture model. Because it maintains the key characteristics of the parental tissue, organoids can be used for drug screening, disease modeling, etc., to contribute to personalized treatment. Therefore, the construction of MRL / lpr lupus mouse kidney organoids is of great significance for the study of systemic lupus erythematosus nephritis. However, there is currently no better technical solution for the construction of lupus nephritis organoids. There is no method to identify lupus nephritis organoids. Summary of the invention

[0007] The present invention aims to provide a method for identifying MRL / lpr lupus kidney organoids.

[0008] The invention provides a culture medium for constructing MRL / lpr lupus mouse kidney organoids. The culture medium of the invention optimizes the culture medium components and has a good supporting and promoting effect on the cultivation of MRL / lpr lupus mouse kidney organoids.

[0009] The present invention aims to provide a method for constructing kidney organoids of MRL / lpr lupus mice.

[0010] The object of the present invention is to provide the application of the above-mentioned MRL / lpr lupus mouse kidney organoids.

[0011] The purpose of the present invention is achieved through the following technical solutions: A method for identifying MRL / lpr lupus kidney organoids, characterized by collecting the culture medium of the organoid to be detected, detecting the levels of ANA and anti-ds-DNA antibodies in the culture medium, and performing tissue morphological identification of the organoids.

[0012] Furthermore, the levels of ANA and anti-ds-DNA antibodies in the renal organoid culture medium were detected by ELISA experimental scheme. When the expression of ANA and ds-DNA genes was detected in the culture medium, the organoids cultured in the culture medium were MRL / lpr lupus renal organoids.

[0013] Furthermore, the organoids to be tested were collected, fixed, embedded in paraffin, and sliced. Then, HE staining and immunofluorescence staining were used to detect the expression level of podocyte functional protein nephrin, and the tissue morphology of the organoids to be tested was identified.

[0014] A MRL / lpr lupus kidney organoid culture medium, characterized in that the MRL / lpr lupus kidney organoid culture medium is composed of a basal culture medium, a pH buffer, an antioxidant, a serum substitute, a TGF-β receptor inhibitor, a growth factor, a ROCK inhibitor, a cAMP pathway activator and a primary cell antibiotic.

[0015] Furthermore, the growth factors consist of Noggin, EGF, FGF 10 and Wnt-3a.

[0016] Furthermore, the basal culture medium is Advanced DMEM / F12 basal culture medium, the pH buffer is HEPES buffer; the antioxidant is N-acetylcysteine; the serum substitute is B-27supplement; the TGF-β receptor inhibitor is A83-01, the ROCK inhibitor is Y-27632; the cAMP pathway activator is Forskolin, and the primary cell antibiotic is primocin.

[0017] Furthermore, the MRL / lpr lupus kidney organoid culture medium also includes triple antibodies of penicillin / streptomycin / gentamicin.

[0018] Furthermore, in the MRL / lpr lupus kidney organoid culture medium, the concentration of B27 supplement is 0.8~1.2 x, the concentration of L-Glutamax is 0.8~1.2 x, the concentration of HEPES is 0.8~1.2 x, the concentration of A83-01 is 4~6 μM, the concentration of N-acetylcysteine ​​is 1.0~1.5 mM, the concentration of Noggin is 90~110 ng / mL, the concentration of EGF is 40~60 ng / mL, the concentration of Wnt-3a is 90~110 ng / mL, the concentration of Forskolin is 8~12 μM, the concentration of FGF 10 is 18~22 ng / mL, the concentration of Y-27632 is 8~12 μM, the concentration of primocin is 90~110 μg / mL, and the concentration of the triple antibody is 90~110 U / mL.

[0019] A method for constructing an MRL / lpr lupus kidney organoid, characterized in that: after obtaining a tissue sample, a cell solution is obtained by sequentially shearing the tissue, digesting, filtering, lysing red blood cells, washing, and counting; the cell solution is mixed with Matrigel matrix gel, and then inoculated into a well plate, and after the inoculation is completed, the plate is allowed to stand until the Matrigel matrix gel is solidified; after the Matrigel matrix gel is solidified, an MRL / lpr lupus kidney organoid culture medium is added for culturing to obtain an MRL / lpr lupus mouse kidney organoid.

[0020] Furthermore, the MRL / lpr lupus kidney organoid culture medium is composed of a basal culture medium, a pH buffer, an antioxidant, a serum replacement, a TGF-β receptor inhibitor, a growth factor, a ROCK inhibitor, a cAMP pathway activator and primary cell antibiotics.

[0021] Furthermore, the growth factors consist of Noggin, EGF, FGF 10 and Wnt-3a.

[0022] Furthermore, the basal culture medium is Advanced DMEM / F12 basal culture medium, the pH buffer is HEPES buffer; the antioxidant is N-acetylcysteine; the serum substitute is B-27supplement; the TGF-β receptor inhibitor is A83-01, the ROCK inhibitor is Y-27632; the cAMP pathway activator is Forskolin, and the primary cell antibiotic is primocin.

[0023] Furthermore, the MRL / lpr lupus kidney organoid culture medium also includes triple antibodies of penicillin / streptomycin / gentamicin.

[0024] Furthermore, in the MRL / lpr lupus kidney organoid culture medium, the concentration of B27 supplement is 0.8~1.2 x, the concentration of L-Glutamax is 0.8~1.2 x, the concentration of HEPES is 0.8~1.2 x, the concentration of A83-01 is 4~6 μM, the concentration of N-acetylcysteine ​​is 1.0~1.5 mM, the concentration of Noggin is 90~110 ng / mL, the concentration of EGF is 40~60 ng / mL, the concentration of Wnt-3a is 90~110 ng / mL, the concentration of Forskolin is 8~12 μM, the concentration of FGF 10 is 18~22 ng / mL, the concentration of Y-27632 is 8~12 μM, the concentration of primocin is 90~110 μg / mL, and the concentration of the triple antibody is 90~110 U / mL.

[0025] Most specifically, a method for constructing a MRL / lpr lupus kidney organoid is characterized by comprising the following steps: (1) Obtain MRL / lpr lupus kidney tissue and cut it into pieces of 1-2 mm 3Small pieces, then add digestion solution, digest in a 37℃ shaker until the cells are digested into 2-10 cell clusters; after digestion, add an appropriate amount of PBS to stop digestion, pass through a 100 μm sieve, and then wash the filter twice with 1 mL PBS, and centrifuge the filtrate; after centrifugation, discard the supernatant of the filtrate, lyse and resuspend with 2-3 mL red blood cell lysis buffer, let stand at 4℃ for 5 min, add 2-3 times the volume of red blood cell lysis buffer PBS to stop lysis, and centrifuge at 4℃, 1000 rpm for 5 min; discard the upper red blood cell lysis buffer, resuspend the cell pellet with PBS, and wash 1~2 times; count during the last wash, when used for 24-well plates, evenly divide 25w cells per well into 1.5 ml centrifuge tubes, and centrifuge; when used for 96-well plates, evenly divide 5w cells per well into 1.5 ml centrifuge tubes, and centrifuge; (2) After centrifugation, discard the supernatant and resuspend the cell pellet with 75%-80% Matrigel. After resuspension, add 50 μL of Matrigel to each well of a 24-well plate (use the middle 8 wells of each well); add 20 μL of Matrigel to each well of a 96-well plate (use the middle 60 wells of each well). Invert the 24-well plate and 96-well plate with Matrigel drops and place them in an incubator at 37 °C for 30 min to wait for the Matrigel to solidify. (3) After the matrix gel solidifies, remove the 24-well plate and the 96-well plate, add 500 μL of culture medium to each well of the 24-well plate, and add 200 μL of MRL / lpr lupus kidney organoid culture medium to each well of the 96-well plate. Seal the surrounding wells with PBS and replace the MRL / lpr lupus mouse kidney organoid culture medium every 3 to 5 days.

[0026] The MRL / lpr lupus kidney organoids constructed by the above method are used in screening LN-related drugs or disease modeling.

[0027] The present invention has the following technical effects: The MRL / lpr lupus mouse kidney organoid identification method of the present invention comprises the following steps: collecting culture medium, detecting the levels of ANA and anti-ds-DNA antibodies in the culture medium of the MRL / lpr lupus mouse kidney organoid by an ELISA experimental method; identifying the tissue morphology of the MRL / lpr lupus mouse kidney organoid by HE staining and immunofluorescence, thereby detecting whether the MRL / lpr lupus kidney organoid is successfully constructed.

[0028] The culture medium of the present invention optimizes the culture medium components, has a good supporting and promoting effect on the culture of MRL / lpr lupus mouse kidney organoids, observes the morphology of MRL / lpr lupus mouse kidney organoids under a bright field microscope, and has obvious typical vacuolar morphology of organoids on the 14th day, better retains the consistency of the tissue, optimizes the growth density, culture medium components, passage conditions and other aspects of lupus nephritis organoids, and successfully establishes lupus nephritis organoids, laying a foundation for further research and application of LN. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Routine testing of body weight, serum ANA and anti-ds-DNA antibody levels of MRL / lpr lupus female mice in Example 2.

[0030] Figure 2 : The morphological image of the MRL / lpr lupus kidney organoid stably cultured in the MRL / lpr lupus kidney organoid culture medium under bright field microscope on the 14th day in Example 2 ((a), magnification 10 times; (b), magnification 20 times) and the morphological image under bright field microscope on the 5th day after passaging ((c), magnification 40 times).

[0031] Figure 3 : HE staining and immunohistochemistry results of MRL / lpr lupus kidney organoids in Example 2.

[0032] Figure 4 : The levels of ANA and anti-ds-DNA antibodies in the culture medium of renal organoids of MRL / lpr lupus mice at different time periods in Example 2.

[0033] Figure 5 :Different growth factor combinations affect the growth status of lupus kidney organoids, (a): there is no Wnt-3a in the growth factor; (b): Wnt-3a is replaced by HGF in the growth factor; (c): HGF is added to the growth factor.

[0034] Figure 6 : Growth status of lupus kidney organoids when the concentration of ROCK inhibitor Y-27632 in MRL / lpr lupus kidney organoid culture medium is 10 mmol.

[0035] Figure 7 :The growth status of lupus kidney organoids after the cAMP pathway activator Forskolin was replaced with Sp-5,6-DCl-cBIMPS.

[0036] Figure 8 : Growth status of lupus kidney organoids after additional addition of R-spondins to the culture medium. DETAILED DESCRIPTION

[0037] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0038] Example 1 A MRL / lpr lupus kidney organoid culture medium, specifically composed of a basal culture medium, a pH buffer, L-glutamine, an antioxidant, a serum replacement, a TGF-β receptor inhibitor, a growth factor, a ROCK inhibitor, a cAMP pathway activator and a primary cell antibiotic; The basal medium is Advanced DMEM / F12 basal medium, the pH buffer is HEPES buffer; the antioxidant is N-acetylcysteine; the serum substitute is B-27supplement; the TGF-β receptor inhibitor is A83-01, the growth factor is composed of Noggin, EGF, FGF 10 and Wnt-3a; the ROCK inhibitor is Y-27632; the cAMP pathway activator is Forskolin, the primary cell antibiotic is primocin, and penicillin / streptomycin / gentamicin triple antibiotic. The components, brands and concentrations in the MRL / lpr lupus kidney organoid culture medium are shown in Table 1.

[0039] Table 1:

[0040] Example 2 A method for constructing MRL / lpr lupus kidney organoids, comprising the following steps: (1) Obtain kidney organ tissue from spontaneous MRL / lpr lupus female mouse model, about 30 weeks old, wash the tissue 1-2 times with PBS (containing 100 U / mL penicillin / streptomycin / gentamicin), and then cut into 1-2 mm pieces. 3Then add type IV collagenase digestion solution and digest in a 37°C shaker until the cells are digested into 2-10 cell clusters; after digestion, add an appropriate amount of PBS to stop digestion, pass through a 100 μm sieve, and then wash the filter twice with 1 mL PBS, and centrifuge the filtrate; after centrifugation, discard the supernatant of the filtrate, lyse and resuspend with 2-3 mL red blood cell lysis buffer, let stand at 4°C for 5 min, then add 2-3 times the volume of red blood cell lysis buffer PBS to stop lysis, and centrifuge at 4°C, 1000 rpm for 5 min; discard the upper layer of red blood cell lysis buffer, resuspend the cell pellet with PBS, and wash 1 to 2 times; count during the last wash, and when used for 24-well plates, evenly divide 25 w cells into 1.5 ml centrifuge tubes per well and centrifuge; when used for 96-well plates, evenly divide 5 w cells into 1.5 ml centrifuge tubes per well and centrifuge; (2) After centrifugation, discard the supernatant and resuspend the cell pellet with 75%-80% Matrigel. After resuspension, add 50 μL of Matrigel to each well of a 24-well plate (use the middle 8 wells of each well); add 20 μL of Matrigel to each well of a 96-well plate (use the middle 60 wells of each well). Invert the 24-well plate and 96-well plate with Matrigel drops and place them in an incubator at 37 °C for 30 min to wait for the Matrigel to solidify. (3) After the matrix gel solidifies, remove the 24-well plate and the 96-well plate, add 500 μL of culture medium to each well of the 24-well plate, and add 200 μL of the MRL / lpr lupus kidney organoid culture medium in Example 1 to each well of the 96-well plate. Seal the surrounding wells with PBS and replace the MRL / lpr lupus mouse kidney organoid culture medium every 3 to 5 days.

[0041] The daily index detection of the spontaneous MRL / lpr lupus female mouse model in Example 2 of the present invention is about 30 weeks old. The daily detection of the changes in the weight, anti-dsDNA, urine protein antibodies and ANA levels of the MRL / lpr lupus female mice is as follows: Figure 1 shown.

[0042] According to the construction method in Example 2, MRL / lpr lupus kidney organoids were obtained after 14 days of culture. The morphology of MRL / lpr lupus kidney organoids under a bright field microscope is as follows: Figure 2 As shown, on day 14, the typical vacuolar morphology of organoids is obvious.

[0043] Subculture after 14 to 21 days of culture: Aspirate and discard the old culture medium, add preheated Dispase Ⅱ working solution, 200 μL / well, and incubate in the incubator for 1 hour; after incubation, add 0.5 mL Advanced DMEM / F12 culture medium to the well plate, blow the matrix gel with a 1 mL pipette tip, blow the matrix gel off the well plate, collect it in a 15 mL centrifuge tube, and centrifuge it at 4°C, 1000 rpm for 5 min; discard the supernatant, add 1 mL TrypLE enzyme, blow it with a 1 mL pipette tip for 3-5 min, add 1 mL Advanced DMEM / F12 to terminate the digestion, centrifuge it at 4°C, 1000 rpm for 5 min, discard the supernatant, add 1 mL Advanced DMEM / F12 to resuspend and wash, transfer to a 1.5 mL EP tube, and centrifuge it at 4°C, 1000 rpm for 5 min.

[0044] Discard part of Advanced DMEM / F12, leaving the required volume of Advanced DMEM / F12, gently blow away the organoid tissue, add Matrigel and blow evenly, the final concentration of Matrigel is 75% (the Matrigel needs to be kept at 4°C throughout the process); Drop Matrigel containing organoids into 24-well plates and 96-well plates; place them in an incubator upside down for 30 min and then add complete culture medium.

[0045] After multiple passages, the cultured MRL / lpr lupus kidney organoids can still grow stably. The morphology of the MRL / lpr lupus kidney organoids under a bright field microscope after passage in the MRL / lpr lupus mouse kidney organoid culture medium in Example 1 is as follows: Figure 2 (c) shows that on day 5, there is obvious typical vacuolar morphology of organoids.

[0046] Example 3 The organoids constructed above were identified. The specific steps are as follows: (1) The MRL / lpr lupus mouse kidney organoids obtained in Example 2 were embedded and sectioned, and then conventional HE staining and immunohistochemical staining were performed to detect the expression of podocyte function protein nephrin.

[0047] like Figure 3 As shown, HE staining results showed that the organoids and the original MRL / lpr lupus kidney tissues had similar morphology. Immunofluorescence results showed that the MRL / lpr mouse kidney organoids expressed a high level of podocyte functional protein nephrin.

[0048] (2) The culture medium of the MRL / lpr lupus mouse kidney organoid cultured at different time stages obtained in Example 2 was collected, and the levels of ANA and anti-ds-DNA antibodies in the culture medium of the MRL / lpr lupus kidney organoid were detected by ELISA.

[0049] Antinuclear antibodies (ANA), also known as anti-nucleic acid antigen antibodies, are a group of autoantibodies produced against DNA, RNA, protein or molecular complexes of these substances in the cell nucleus; anti-ds-DNA antibodies are characteristic marker antibodies of systemic lupus erythematosus (SLE), and are present in the serum of more than 95% of active SLE patients. The titer of this antibody is closely related to the activity of SLE. The higher the titer, the greater the possibility that SLE is in the active stage. As the lupus kidney organoids grow, the concentration of the two antibodies detected in the secreted culture medium increases.

[0050] The results are as follows Figure 4 As shown in Figure 2, the renal organoids we constructed can secrete ANA and anti-ds-DNA antibodies. It is worth noting that as the culture time increases, the levels of ANA and anti-ds-DNA antibodies secreted by renal organoids gradually increase, suggesting that the efficacy of SLE drugs can be evaluated by detecting the secretion of autoantibodies in the culture medium.

[0051] Comparative Example 1: Regarding the effects of different growth factor combinations in the culture medium on the culture results of MRL / lpr lupus kidney organoids: When preparing the MRL / lpr lupus kidney organoid culture medium, the growth factors selected are as follows: (1) The growth factors are a combination of Noggin, EGF, and FGF 10; (2) HGF was used instead of Wnt-3a, and the growth factors were a combination of Noggin, EGF, FGF 10, and HGF; (3) The growth factors are a combination of Noggin, EGF, FGF 10, Wnt-3a and HGF.

[0052] After 14 days of stable culture in the culture medium, the results were as follows Figure 5As shown in the figure, it can be seen that when Wnt-3a is not added to the growth factor, the growth state of lupus kidney organoids is affected, and the growth state and speed are significantly inhibited; when Wnt-3a in the growth factor is replaced by the growth factor HGF, the growth state of lupus kidney organoids is also affected to a certain extent; adding a growth factor HGF, the growth state of lupus kidney organoids is not significantly affected. The above results show that the growth factor Wnt-3a is crucial for the culture of lupus kidney organoids, that is, the growth factor composed of Noggin, EGF, FGF 10 and Wnt-3a plays a synergistic role, and each component in this combination is indispensable.

[0053] Comparative Example 2: The MRL / lpr lupus kidney organoid culture medium includes basal culture medium, L-glutamine, pH buffer, antioxidants, serum replacement, TGF-β receptor inhibitor, growth factor, ROCK inhibitor, cAMP pathway activator, etc. Among them, when the cAMP pathway activator Forskolin is replaced with Sp-5,6-DCl-cBIMPS.

[0054] The results are as follows Figure 6 As shown, when Forskolin was replaced with Sp-5,6-DCl-cBIMPS, the density and growth state of lupus kidney organoids obtained after stable culture in the culture medium for 14 days were affected, and the growth state was relatively slow, that is, the cAMP pathway activator Forskolin was replaced with Sp-5,6-DCl-cBIMPS, which inhibited the formation of MRL / lpr lupus kidney organoids.

[0055] Comparative Example 3: The concentration of each component in the MRL / lpr lupus kidney organoid culture medium also has a significant effect on the culture results of the MRL / lpr lupus kidney organoids. When the concentration of the ROCK inhibitor Y-27632 was changed from 10 μM in Example 1 to 10 mM, we found that the growth state of the lupus kidney organoids was negatively affected by the increase in the concentration of the ROCK inhibitor Y-27632. When the concentration of the ROCK inhibitor Y-27632 was increased to 10 mM, the results of the organoids after stable culture in the culture medium for 14 days were as follows: Figure 7 As shown, it can be seen that increasing the concentration of ROCK inhibitor Y-27632 inhibited the formation of MRL / lpr lupus kidney organoids.

[0056] In addition, R-spondins (RSPOs) are enhancers of Wnt / β-catenin signaling and are generally added routinely. Therefore, during the study, we also tried to add R-spondins. The results after stable culture in the culture medium for 14 days are shown in Figure 8. We found that the growth state of lupus kidney organoids was relatively slow. That is, the addition of R-spondins inhibited the formation of MRL / lpr lupus kidney organoids.

Claims

1. A method for identifying MRL / lpr lupus kidney organoids, characterized in that The culture medium of the organoids to be tested was collected, the levels of ANA and anti-ds-DNA antibodies in the culture medium were detected, and the histomorphology of the organoids was identified.

2. A method for identifying MRL / lpr lupus kidney organoids as claimed in claim 1, characterized in that: The ELISA experimental scheme is used to detect the levels of ANA and anti-ds-DNA antibodies in the culture medium of renal organoids. When the expression of ANA and ds-DNA genes is detected in the culture medium, the organoids cultured in the culture medium are MRL / lpr lupus renal organoids.

3. A method for identifying MRL / lpr lupus kidney organoids as claimed in claim 2, characterized in that: The tissue morphology identification of the organoids is to collect the organoids to be tested, fix them, embed them in paraffin, slice them, and then use HE staining and immunofluorescence staining to detect the expression level of podocyte functional protein nephrin to identify the tissue morphology of the organoids to be tested.

4. A MRL / lpr lupus kidney organoid culture medium, characterized in that: The MRL / lpr lupus kidney organoid culture medium is composed of a basal culture medium, L-glutamine, a pH buffer, an antioxidant, a serum substitute, a TGF-β receptor inhibitor, a growth factor, a ROCK inhibitor, a cAMP pathway activator and primary cell antibiotics.

5. The MRL / lpr lupus kidney organoid culture medium according to claim 4, characterized in that: The growth factors consist of Noggin, EGF, FGF 10 and Wnt-3a.

6. A MRL / lpr lupus kidney organoid culture medium according to claim 4 or 5, characterized in that: The basal culture medium is Advanced DMEM / F12 basal culture medium, the pH buffer is HEPES buffer; the antioxidant is N-acetylcysteine; the serum substitute is B-27supplement; the TGF-β receptor inhibitor is A83-01, the ROCK inhibitor is Y-27632; the cAMP pathway activator is Forskolin, and the primary cell antibiotic is primocin.

7. The MRL / lpr lupus kidney organoid culture medium according to claim 6, characterized in that: In the MRL / lpr lupus kidney organoid culture medium, the concentration of B27 supplement is 0.8~1.2 x, the concentration of L-Glutamax is 0.8~1.2 x, the concentration of HEPES is 0.8~1.2 x, the concentration of A83-01 is 4~6 μM, the concentration of N-acetyl cysteine ​​is 1.0~1.5 mM, the concentration of Noggin is 90~110 ng / mL, the concentration of EGF is 40~60 ng / mL, the concentration of Wnt-3a is 90~110 ng / mL, the concentration of Forskolin is 8~12 μM, the concentration of FGF 10 is 18~22 ng / mL, the concentration of Y-27632 is 8~12 μM, and the concentration of primocin is 90~110 μg / mL.

8. A method for constructing MRL / lpr lupus kidney organoids based on the culture medium of claim 7, characterized in that: After obtaining the tissue sample, the cell solution is obtained by cutting the tissue, digesting, filtering, lysing red blood cells, washing and counting in sequence; the cell solution is mixed with Matrigel matrix gel, and then inoculated into the well plate. After the inoculation is completed, it is allowed to stand until the Matrigel matrix gel solidifies; after the Matrigel matrix gel solidifies, the MRL / lpr lupus kidney organoid culture medium is added for culture to obtain the MRL / lpr lupus mouse kidney organoid.

9. A method for constructing a MRL / lpr lupus kidney organoid as claimed in claim 8, characterized in that: The steps include: (1) Obtain MRL / lpr lupus kidney tissue and cut it into pieces of 1-2 mm 3 Small pieces, then add digestion solution, digest in a 37℃ shaker until the cells are digested into 2-10 cell clusters; after digestion, add an appropriate amount of PBS to stop digestion, pass through a 100 μm sieve, and then wash the filter twice with 1 mL PBS, and centrifuge the filtrate; after centrifugation, discard the supernatant of the filtrate, lyse and resuspend with 2-3 mL red blood cell lysis buffer, let stand at 4℃ for 5 min, add 2-3 times the volume of red blood cell lysis buffer PBS to stop lysis, and centrifuge at 4℃, 1000rpm for 5 min; discard the upper red blood cell lysis buffer, resuspend the cell pellet with PBS, and wash 1~2 times; count during the last wash, when used for 24-well plates, evenly divide 25w cells per well into 1.5 ml centrifuge tubes, and centrifuge; when used for 96-well plates, evenly divide 5w cells per well into 1.5 ml centrifuge tubes, and centrifuge; (2) After centrifugation, discard the supernatant and resuspend the cell pellet with 75%-80% Matrigel. After resuspension, add 50 μL of Matrigel to each well of a 24-well plate (use the middle 8 wells of each well); add 20 μL of Matrigel to each well of a 96-well plate (use the middle 60 wells of each well). Invert the 24-well plate and 96-well plate with Matrigel drops and place them in an incubator at 37 °C for 30 min to wait for the Matrigel to solidify. (3) After the matrix gel solidifies, remove the 24-well plate and the 96-well plate, add 500 μL of culture medium to each well of the 24-well plate, and add 200 μL of MRL / lpr lupus kidney organoid culture medium to each well of the 96-well plate. Seal the surrounding wells with PBS and replace the MRL / lpr lupus mouse kidney organoid culture medium every 3 to 5 days.

10. Use of the MRL / lpr lupus kidney organoid constructed by the method of claim 9 in screening LN-related drugs or disease modeling.

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