Trem2 gene overexpressed macrophage for treating acute-to-chronic kidney disease and application of Trem2 gene overexpressed macrophage

Abstract

The invention relates to the technical field of cell carriers, in particular to Trem2 gene overexpressed macrophages for treating acute to chronic kidney diseases and application of the Trem2 gene overexpressed macrophages, and the macrophages comprise immortalized bone marrow-derived macrophages. The Trem2 gene overexpressed macrophage for treating the acute transfection chronic kidney disease, provided by the invention, is used for treating the acute transfection chronic kidney disease.

Description

Technical Field

[0001] The present invention relates to the technical field of cell carriers, and in particular to a Trem2 gene overexpressed macrophage for treating acute-to-chronic kidney disease and an application thereof. Background Art

[0002] Acute kidney injury (AKI) and chronic kidney disease (CKD) are related syndromes of contemporary concern due to their high morbidity and mortality. Recent studies have shown that AKI contributes to the development and progression of CKD. AKI in adults and children is strongly associated with an increased risk of developing CKD, which depends on the severity of AKI. It is estimated that approximately 30-70% of AKI cases progress to CKD or ESRD. The clinical features of acute kidney injury (AKI) are a sudden drop in glomerular filtration rate (GFR), increased serum creatinine (sCr), blood urea nitrogen (BUN), and electrolytes, leading to a temporary loss of renal function. If this condition persists for more than 90 days, it may lead to the development of CKD or ESRD.

[0003] The inflammatory nature of AKI involves resident macrophages and infiltrating immune cells of the renal mononuclear phagocytic system, suggesting that immune cells and their products may contribute to the progression of renal injury. During the acute phase of AKI, macrophages rapidly accumulate in the injured kidney and promote inflammation. They may play a key role in wound healing and repair as scavengers of proinflammatory cellular debris and promoters of regeneration. Early studies have shown that macrophages promote initial ischemia-reperfusion injury (IRI) in mice and promote fibrosis after IRI in rats. These observations are further supported by the fact that reducing monocytes / macrophages with clodronate liposomes before or 3 days after ischemic injury can reduce renal fibrosis.

[0004] Trem2 (triggering receptor expressed on myeloid cells 2) is a cell surface membrane protein that was originally discovered in monocyte-derived dendritic cells and mouse macrophages. Trem2 belongs to the immunoglobulin superfamily of single transmembrane receptors, and the transmembrane region is associated with the adaptor proteins DNAX activating protein 12 (DAP12) and DNAX activating protein 10 (DAP10). Trem2 regulates macrophage functions, such as phagocytosis and the release of inflammatory factors. At the same time, Trem2 is involved in the regulation of macrophage apoptosis, thereby affecting the degree and duration of inflammation and playing an immunomodulatory role. Trem2 macrophages may also play a role in the outcome of AKI-CKD. Studies have shown that Trem2 may be involved in regulating the inflammatory response and cellular stress response of kidney disease.

[0005] There are no reports in the prior art on the treatment of AKI to CKD using TREM2 macrophages. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a macrophage with overexpression of Trem2 gene for treating acute-to-chronic kidney disease and its application. The macrophage expressing Trem2 gene provided by the present invention can treat AKI-CKD.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a Trem2 gene overexpressing macrophage for treating acute-to-chronic kidney disease.

[0009] Preferably, the nucleotide sequence of the Trem2 gene is shown as SEQ ID No.1.

[0010] Preferably, the macrophages include immortalized bone marrow-derived macrophages.

[0011] Preferably, the method for preparing the Trem2 gene-overexpressing macrophages comprises the following steps:

[0012] 1) Clone the Trem2 gene into an expression vector to obtain a recombinant expression vector;

[0013] 2) Transfecting the recombinant expression vector obtained in step 1) into macrophages.

[0014] Preferably, the expression vector in step 1) includes GL122pSLenti-EF1-EGFP-F2A-BSR-CMV-MCS-WPRE.

[0015] Preferably, the step 2) transfection comprises the following steps:

[0016] Packaging the recombinant expression vector with packaging cells to obtain virus particles;

[0017] The virus particles were used to infect macrophages to establish stable Trem2 overexpressing cells.

[0018] Preferably, the packaging cells include 393T cells.

[0019] The present invention also provides a medicine for treating acute-to-chronic kidney disease, which comprises the Trem2 gene-overexpressing macrophages used in the above technical solution.

[0020] Preferably, the number of macrophages overexpressing the Trem2 gene in the drug is 1×10 6 indivual.

[0021] The mechanism of the Trem2 overexpressing gene macrophages provided by the present invention for treating acute-onset chronic kidney disease (AKI-CKD) is:

[0022] The Trem2 gene further activates the phagocytic function of macrophages by regulating the PI3K-AKT channel of macrophages, reduces the release of proinflammatory factors of macrophages, thereby reducing the damage and apoptosis of kidney cells, inhibiting renal fibrosis, and thus avoiding the progression of acute to chronic kidney disease.

[0023] Beneficial effects of the present invention:

[0024] 1. Trem2 may be involved in regulating inflammatory responses and cellular stress responses in kidney disease. It affects the development of kidney damage by regulating the phagocytic ability of macrophages and the release of inflammatory factors. After binding to DAP12, Trem2 can activate the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway, leading to an increase in intracellular calcium ions, which in turn affects the phagocytic function and inflammatory response of the cells.

[0025] 2. Currently, there is no better treatment for AKI, and there is no way to avoid or reduce kidney cell death. After large-scale death of kidney cells, kidney tissue may experience tubular atrophy, glomerular sclerosis, interstitial fibrosis, renal ischemia, capillary loss, etc., and its clinical manifestation is decreased renal function, i.e. CKD. Cell therapy is a cell-based treatment method that uses living cells as active drugs to treat diseases. It is currently widely regarded as the most clinically promising treatment method, and the construction of Trem2-overexpressing macrophages and the verification of in vivo effects provide a safer and more effective clinical treatment strategy for AKI-CKD. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0027] Figure 1 Figure 2 is a flow cytometry image of macrophages overexpressing Trem2. Generation and characterization of immortalized macrophages overexpressing TREM2 (TREM2OE-iMφ). Flow cytometry analysis of the expression level of TREM2 in uninfected (unt), empty lentivirus-infected (Ctrl), and TREM2 lentivirus-infected (TREM2OE-iMφ). N=3 per group. Mean ± SEM. ***p<0.001.

[0028] Figure 2Figure 2 is a graph showing the phagocytic function of Trem2-overexpressing macrophages. The phagocytic level of Ctrl- and Trem2OE-iMφ was assessed using the Zymosan phagocytosis kit. Left: Fluorescence images of phagocytosed Zymosan in Ctrl- and Trem2OE-iMφ. Right: Quantitative results of Zymosan-positive macrophages in two groups. N = 3 per group. Mean ± SEM. *p < 0.05.

[0029] Figure 3 To detect the expression level of Trem2 in mouse kidney macrophages after cell therapy, flow cytometry results of percentage of F4 / 80+TREM2+ macrophages in kidneys of three groups; N=3 per group; mean±SEM; ***p<0.001; and flow cytometry results of F4 / 80+CD11b+ macrophages in kidneys of three groups; N=3 per group; mean±SEM; *p<0.05;

[0030] Figure 4 The experimental effect diagram of detecting the renal morphology of mice after cell therapy, representative histological images and quantitative results of HE, PAS, Masson and Sirus red staining of renal tissues of Trem2KO mice intravenously transferred with PBS, Ctrl-iMφ and TREM2OE-iMφ; N = 3 per group; scale bar 50 μm; mean ± SEM; **p < 0.01;

[0031] Figure 5 To detect the protein expression level in the kidneys of mice after cell therapy, the Western blotting results of TNFα and αSMA protein levels in the three groups of mice on the 14th day; N = 3 per group; mean ± SEM; *p < 0.05, **p < 0.01;

[0032] Figure 6 This is the plasmid map of GL122 pSLenti-EF1-EGFP-F2A-BSR-CMV-MCS-WPRE. DETAILED DESCRIPTION

[0033] The present invention provides a Trem2 gene overexpressing macrophage for treating acute-to-chronic kidney disease.

[0034] In the present invention, the nucleotide sequence of the Trem2 gene is shown in SEQ ID No. 1, which is as follows:

[0035] .

[0036] In the present invention, the macrophages preferably include immortalized bone marrow-derived macrophages.

[0037] In the present invention, the method for preparing the Trem2 gene overexpressing macrophages comprises the following steps:

[0038] 1) Clone the Trem2 gene into an expression vector to obtain a recombinant expression vector;

[0039] 2) Transfecting the recombinant expression vector obtained in step 1) into macrophages to establish stable Trem2 overexpressing cells.

[0040] The present invention clones the Trem2 gene into an expression vector to obtain a recombinant expression vector. In the present invention, the expression vector preferably includes pSLenti-EF1-EGFP-F2A-BSR-CMV-MCS-WPRE. Item No.: GL122, Guangzhou Heyuan Biotechnology Co., Ltd., China. The present invention does not specifically limit the method for cloning the Trem2 gene into an expression vector, and those skilled in the art can use conventional methods.

[0041] The present invention transfects the obtained recombinant expression vector into macrophages. In the present invention, the transfection comprises the following steps: packaging the recombinant expression vector through packaging cells to obtain virus particles; infecting macrophages with the virus particles to establish stable Trem2 overexpressing cells. In the present invention, the packaging cells preferably include 393T cells. The present invention does not specifically limit the method for packaging the recombinant expression vector through packaging cells, and those skilled in the art can use conventional methods. The present invention does not specifically limit the method for infecting macrophages with virus particles, and those skilled in the art can use conventional methods.

[0042] The present invention also provides the use of the Trem2 gene overexpressing macrophages described in the above technical solution in the preparation of a drug for treating acute-chronic kidney disease.

[0043] The present invention also provides a drug for treating acute-to-chronic kidney disease, the drug comprising the Trem2 gene-overexpressing macrophages in the application of the above technical solution. In the present invention, the number of Trem2 gene-overexpressing macrophages in the drug is preferably 1×10 6 The present invention has no special limitation on the dosage form of the drug, and those skilled in the art can use conventional dosage forms.

[0044] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] A method for preparing macrophages based on overexpression of Trem2 gene and detecting the therapeutic effect thereof, comprising the steps of:

[0047] (1) Construction of cells overexpressing the Trem2 gene;

[0048] (2) Establish an AKI-CKD outcome model in Trem2KO mice.

[0049] (3) Cell therapy with overexpression of Trem2 gene.

[0050] The cells overexpressing the Trem2 gene in step (1) are prepared by the following steps: cloning the gene encoding Trem2 into an expression vector to obtain a recombinant expression vector; and transfecting the recombinant expression vector into macrophages to obtain macrophages overexpressing Trem2.

[0051] clone:

[0052] Obtain the target gene by PCR or enzyme digestion, construct it into the target vector by ligation or homologous recombination / assembly, and transform E.coli DH5α competent cells. Screen the transformants by colony PCR, and send the positive clones for sequencing. Extract the plasmid from the verified clones. Specific steps: Take out the competent cells at -80℃, place the Ep tube containing the competent cells on ice, and wait for the competent cells to melt naturally; use a pipette to take 1μL (concentration greater than 10ng / μL) of the plasmid to be transformed, slowly rotate and add it to the competent cells; ice bath for 15-30min; insert the Ep tube containing the competent cells into the float, put it in a 42℃ water bath for heat shock for 90s, and then immediately transfer it to ice for 2-3min. In the clean bench, add 500-700μL of antibiotic-free to the Ep tube containing the competent cells. The culture medium is placed in a constant temperature oscillator at 37°C and 220rpm for incubation for 40-60 minutes, and the plate is placed in a 37°C incubator for use; an alcohol lamp is lit in the clean bench, and the coating rod is disinfected (sterilized glass beads can also be used), and the plate is naturally cooled to room temperature; the plate is taken out of the 37°C incubator and placed in the clean bench for use; the transformation liquid Ep tube is taken out, 70-80 μL of the transformation liquid is taken out with a pipette, and the coating rod is evenly applied on the solid culture medium; the plate is inverted and incubated in a 37°C incubator overnight (14 hours).

[0053] The expression vector is a lentiviral expression vector:

[0054] GL122 pSLenti-EF1-EGFP-F2A-BSR-CMV-MCS-WPRE, plasmid map as shown Figure 6 shown.

[0055] Trem2×3flag sequence, the lowercase letters not in bold are the nucleotide sequence of the Trem2 gene:

[0056] GAATTCGCCACCatgggacctctccaccagtttctcctgctgctgatcacagccctgtcccaagccctca, the lowercase bold part is the 3-flag sequence.

[0057] The specific steps of transfection are preferably as follows: packaging the recombinant expression vector through 293T cells to obtain virus particles; and then infecting immortalized bone marrow-derived macrophages with the virus particles to obtain cells overexpressing Trem2.

[0058] Infection: The cells were digested, resuspended, counted, and plated in 24-well plates at 5×10 4The cells to be infected were added. The next day, after the cells adhered to the wall, the required volume of lentivirus was calculated according to the lentivirus titer and the optimal MOI of the lentivirus for the cells. (MOI = 20, the old culture medium was aspirated, and 500 μl of complete culture medium containing lentivirus and co-infection reagent was added. After 8-16 hours of virus infection, the medium was replaced with complete culture medium and culture was continued. After 72 hours of infection, the fluorescence intensity of the cells was observed under a fluorescence microscope. After the fluorescence was observed, the infection was considered successful, and the screening culture medium containing 10 μg / ml BSR was replaced. After one week of screening, subsequent detection experiments were carried out. During this period, the medium was replaced or subcultured according to the cell density.

[0059] The AKI-CKD outcome model in step (2) is prepared by the following steps:

[0060] A. Sterile surgical instruments (needle holder, surgical scissors, surgical forceps, chest expander, mouse artery clamp, ophthalmic scissors), 37℃ constant temperature pad, iodine tincture, 75% medical alcohol, medical cotton swabs, cotton balls, medical gauze, surgical lamp, shaver, suture needle and thread, saline, anesthetic, syringe, timer, medical tape, and weight measuring instrument.

[0061] B. Place the sterilized thermostatic pad on the operating table, cover the upper layer with a medical surgical pad, prepare two operating lamps for lighting, place the consumables and instruments required for the operation on the table, and arrange them in order for easy operation. Trim the medical gauze to 5 cm × 3 cm in size, soak it with physiological saline for use, prepare 1% pentobarbital anesthetic, and fill a 1 ml syringe. The experimental mice were divided into two groups, Group A Trem2KO mouse sham operation group, Group B Trem2KO mouse operation group, each group had 6 mice, each mouse was marked with an ear tag for easy identification, measure the weight of the mice, anesthetize the mice by intraperitoneal injection of 1% pentobarbital (anesthetic dosage: 0.15 ml / 20 g), shave the abdomen of the mice after anesthesia, fix the mouse limbs on the surgical pad with medical tape so that the abdomen is facing up, and disinfect the abdomen with a cotton swab dipped in iodine.

[0062] C. Cut the mouse skin and muscle layer along the midline of the abdomen, use a mouse chest expander to expand its abdomen to expose the abdominal contents, soak a cotton swab with saline, take the mouse intestines out of the abdomen, separate the mouse renal vein with forceps, expose the left kidney, and clamp the renal vein with an artery clamp. Observe the color change of the kidney. Gradually congested and dark purple-red indicates successful clamping. Time for 45 minutes, take back the mouse organs, pay attention to put them back in the original position, cover the mouse abdominal incision with a pre-prepared moist gauze, and keep the mouse at a constant body temperature of 37°C. , observe the status of the mice. After 45 minutes, use curved forceps to remove the mouse artery clamp and observe whether the color of the mouse kidney recovers from dark purple to bright red in a short time. If it can recover, the perfusion is considered successful. Finally, give about 200 μl of normal saline to the abdominal cavity, suture the muscle layer with continuous suture method, and suture the skin layer with interrupted suture method. Note that the sutures should be dense to facilitate wound healing. Wipe the blood on the mouse abdomen with normal saline, and then wipe the normal saline with dry medical gauze, observe the mouse status on a 37°C constant temperature pad, wait for the mouse to wake up, and put it in a clean cage after waking up.

[0063] D. On the thirteenth day of mouse feeding, the mice were placed in metabolic cages and fasted but not watered. The urine of the mice was collected for 24 hours. On the fourteenth day of mouse feeding, the weight of the mice was measured to see if there was a sharp decline or change. First, the mice were anesthetized, the eyeballs were removed, and blood was collected from the eyeballs to obtain about 500 μl of blood. The blood was placed at room temperature for 15 minutes, and then the serum and blood cells were separated at 4°C and 13,000 rpm. The serum was recovered and stored at -80°C for later use. Then, the mice were fixed on the operating board with medical tape, and the chest and abdomen of the mice were cut open along the midline with surgical scissors to expose the heart and kidneys of the mice. The left liver lobe of the mice was cut off, and the left ventricle was pierced from the apex of the heart with a scalp needle. 50 ml of normal saline was perfused. The color of the kidney of the mice was observed to recover from black purple to bright red in a short time. After suturing and modeling, the mice survived for 14 days, which was considered to have established the model. The proteinuria of the mice was collected, and the abnormal renal function of the mice was detected, which was considered to have successfully established the target model. The kidney of the mice was removed, the capsule of the kidney of the mice was removed, and the kidney was divided into four parts for freezing embedding, paraffin embedding, tissue protein and RNA extraction.

[0064] Treatment of macrophages with overexpression of Trem2 in step (3):

[0065] On the first day of establishing the mouse model, the mice in the surgical group were divided into two groups and injected with 1×10 6 The control group was injected with an equal volume of PBS and injected once on the second day of modeling.

[0066] Kidneys were collected after 14 days, and paraffin sections of mouse kidneys were used for morphological staining ( Figure 1-5), HE and PAS staining of renal sections of UIRI mice injected with PBS and GFP-iBMDM by tail vein showed several pathological features of CKD, including glomerular atrophy and sclerosis, while the glomerular morphology of UIRI mice injected with Trem2OE-iBMDM by tail vein was normal, and Masson and SirusRed staining showed that interstitial fibrosis appeared in the kidneys of UIRI mice injected with PBS and GFP-iBMDM by tail vein. Figure 4-5 Shown are representative histological images of renal tissue of Trem2KO mice stained with HE, PAS, Masson, and Sirus Red on day 14 after intravenous injection of PBS, GFP-iBMDMs, and Trem2OE-iBMDMs, as well as the detection results of the inflammatory factor TNF-a and the fibrosis indicator aSMA protein levels.

[0067] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A macrophage with overexpression of Trem2 gene for treating acute-to-chronic kidney disease.

2. The Trem2 gene-overexpressing macrophage according to claim 1, characterized in that: The nucleotide sequence of the Trem2 gene is shown in SEQ ID No.

1.

3. The Trem2 gene-overexpressing macrophage according to claim 1, characterized in that: The macrophages include immortalized bone marrow-derived macrophages.

4. The Trem2 gene-overexpressing macrophage according to claim 1, characterized in that: The method for preparing the Trem2 gene overexpressing macrophages comprises the following steps: 1) Clone the Trem2 gene into an expression vector to obtain a recombinant expression vector; 2) Transfecting the recombinant expression vector obtained in step 1) into macrophages to establish stable Trem2 overexpressing cells.

5. The Trem2 gene-overexpressing macrophage according to claim 4, characterized in that: The expression vector in step 1) includes GL122 pSLenti-EF1-EGFP-F2A-BSR-CMV-MCS-WPRE.

6. The Trem2 gene-overexpressing macrophage according to claim 4, characterized in that: The step 2) transfection comprises the following steps: Packaging the recombinant expression vector with packaging cells to obtain virus particles; The viral particles infect macrophages.

7. The Trem2 gene-overexpressing macrophage according to claim 6, characterized in that: The packaging cells include 393T cells.

8. Use of the Trem2 gene overexpressing macrophages according to any one of claims 1 to 7 in the preparation of a drug for treating acute-to-chronic kidney disease.

9. A drug for treating acute-onset chronic kidney disease, characterized in that: The drug comprises the macrophages overexpressing the Trem2 gene according to any one of claims 1 to 7.

10. The drug according to claim 8, characterized in that The number of macrophages overexpressing the Trem2 gene in the drug is 1×10 6 indivual.

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