Application of macrophages expressing TgMIF protein in the preparation of drugs for treating liver fibrosis

Abstract

The present invention discloses the use of macrophages expressing TgMIF protein in the preparation of a drug for treating liver fibrosis, and relates to the technical field of liver fibrosis. The amino acid sequence of the TgMIF protein is shown in SEQ ID NO. 1. The beneficial effect of the present invention is that the TgMIF protein in the present invention can continuously and directionally induce macrophages to shift to M1 for activation, and highly express the collagen-dissolving MMP9 protein, thereby reducing the volume of liver fibrosis and reducing the surface granularity, thereby providing an accurate and effective cell therapy method for treating liver fibrosis.

Description

Technical Field

[0001] The present invention relates to the technical field of liver fibrosis, and in particular to application of macrophages expressing TgMIF protein in preparing a medicine for treating liver fibrosis. Background Art

[0002] Liver fibrosis is a protective mechanism activated by liver cell damage and is resolved after the cause is eliminated. However, persistent damage can lead to the evolution of liver fibrosis into cirrhosis, which promotes the development of hepatocellular carcinoma. There is still no effective and specific anti-fibrosis therapy.

[0003] Macrophages play an important role in the pathological process of liver fibrosis. Cell therapy with macrophages as the main candidate cells is an emerging therapy for the treatment of liver fibrosis. In the CCl4-induced liver fibrosis mouse model, classically activated M1 macrophages induced by LPS and IFN-γ can improve liver fibrosis. For example, the patent application with publication number CN103977029A uses M1 macrophages to improve liver fibrosis, but how to stably and directionally regulate the phenotype of macrophages to achieve a more accurate and safer liver fibrosis treatment method is a difficulty in current research.

[0004] Toxoplasma gondii, also known as Toxoplasma gondii, is an opportunistic pathogen that is an obligate intracellular parasite. Toxoplasma gondii can infect almost all warm-blooded animals, including humans, causing the important zoonotic disease toxoplasmosis. It is estimated that about one-third of the world's population is chronically infected with Toxoplasma. During the innate immune response stage of Toxoplasma infection, type II strains can drive macrophages to polarize toward M1, allowing them to exert intracellular insecticidal effects to control the infection, and the host acquires strong immunity, thereby converting acute infection into chronic / latent infection.

[0005] Macrophage migration inhibitory factor (MIF) is composed of 115 amino acids, which is highly conserved among species and has tautomerase and oxidoreductase activities. MIF is a multifunctional protein with broad immunomodulatory properties that can be produced by a variety of cells, including monocytes, macrophages, dendritic cells, and lymphocytes. MIF can participate in innate and adaptive immunity by producing proinflammatory mediators such as TNF-α and IL-8. The homology between Toxoplasma gondii MIF-like protein (TgMIF) and human MIF protein is only 26%, which is a huge difference. There is currently no research on TgMIF in liver diseases. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an application of macrophages expressing TgMIF protein in the preparation of drugs for treating liver fibrosis.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] A use of macrophages expressing TgMIF protein in preparing a drug for treating liver fibrosis, wherein the amino acid sequence of the TgMIF protein is shown in SEQ ID NO.1.

[0009] Beneficial effects: The TgMIF protein in the present invention can induce macrophages to shift to M1 activation and highly express the collagen-dissolving MMP9 protein, thereby reducing the area of ​​liver fibrosis and the surface granularity, thereby providing a cell therapy method for treating liver fibrosis.

[0010] Preferably, the gene encoding the TgMIF protein has a nucleotide sequence as shown in SEQ ID NO.2.

[0011] Preferably, the primer sequences encoding the nucleotide sequence of the TgMIF protein are as follows: upstream primer: 5'-CCGGAATTCGCCACCATGCCCAAGTGCATGATCTTTTGCC-3', downstream primer: 5'-ATTTGCGGCCGCAGCCGAAAGTTCGGTCGCCCATGGCC-3'.

[0012] Preferably, the gene encoding the TgMIF protein is packaged with a lentivirus, stably infects macrophages, and then the macrophages are directed to be activated to obtain macrophages expressing the TgMIF protein.

[0013] Preferably, the lentivirus is a 293T cell containing a lentiviral plasmid.

[0014] Preferably, the lentiviral packaging comprises the following steps: inserting the TgMIF gene into the pLVX-C-FLAG-mCMV-ZsGreen-IRES-Puro vector.

[0015] The advantages of the present invention are that the TgMIF protein in the present invention can continuously and directionally induce macrophages to shift activation toward M1 and highly express the collagen-dissolving MMP9 protein, thereby reducing the volume of liver fibrosis and reducing surface granularity, thereby providing a cell therapy method for treating liver fibrosis.

[0016] The present invention claims to protect the use of macrophages stably transfected with the Toxoplasma gondii parasite-derived molecule TgMIF in the preparation of drugs for treating liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a map of the vector pLVX-C-FLAG-mCMV-ZsGreen-IRES-Puro in the examples of the present invention;

[0018] Figure 2 In the embodiment of the present invention The fluorescence efficiency diagram, the left side of the figure is the fluorescence diagram, and the right side is the white light diagram;

[0019] Figure 3 This is a graph showing the expression results of TgMIF verified by qPCR in an embodiment of the present invention;

[0020] Figure 4 This is a diagram showing the expression results of TgMIF verified by WB in an embodiment of the present invention;

[0021] Figure 5 This is a diagram showing the result of TgMIF driving macrophages to polarize toward M1 in an embodiment of the present invention;

[0022] In the figure: Raw264.7; Raw264.7 of the stable LV vector; Raw264.7 stably expressing TgMIF

[0023] Figure 6 The results of TgMIF promoting macrophage secretion of MMP9 in the embodiment of the present invention are shown in Figure A, Figure B is a statistical analysis of MMP9 expression in cell supernatant, Figure C is a statistical analysis of iNOS, and Figure D is a statistical analysis of ARG1. Raw264.7 of the stable LV vector; Raw264.7 stably expressing TgMIF; Raw264.7 stably expressing TgMIF were stimulated with DMSO; Raw264.7 stably expressing TgMIF were stimulated with 20 μM U0126;

[0024] Figure 7 The frozen section of the present invention is shown in FIG. Survival time graph; DAPI represents cell nuclei; Merge is the merger of DAPI graph and TgMIF graph;

[0025] Figure 8 This is a graph of the serum levels of liver fibrosis mice in the examples of the present invention; NC in the figure: unmodeled group. The rest were modeled with CCL4 and divided into the following groups according to the different tail vein injections: PBS: PBS injection group; Raw264.7 injection group; Raw264.7 group injected with stable LV vector; The Raw264.7 group stably expressing TgMIF was injected.

[0026] Fig. 9 In the embodiment of the present invention Pathological damage image of liver fibrosis. A: Time of modeling and cell injection; B: Macroscopic view of liver; C: Pathological damage image of liver fibrosis. NC in the figure: No modeling group. The rest were modeled with CCL4 and divided into the following groups according to the different tail vein injections: PBS: PBS injection group; Raw264.7 injection group; Raw264.7 group injected with stable LV vector; The Raw264.7 group stably expressing TgMIF was injected. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0029] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0030] 1. Construction of pLVX-TgMIF-3flag-ZsGreen-Puro plasmid

[0031] 1. Extraction of total RNA of Toxoplasma gondii: Toxoplasma gondii, RH strain and ME49 strain were taken out of liquid nitrogen, thawed rapidly at 37°C and added to HFF cells for culture. After three consecutive passages, Toxoplasma gondii were collected and centrifuged at 4000 rpm for 10 min at room temperature. The precipitate was used for total RNA extraction, and Trizol was added to perform RNA extraction according to the conventional method.

[0032] 2. Specific amplification of TgMIF gene: Using total RNA of Toxoplasma gondii as template, refer to PrimeScript TM The instructions for use of the RT MasterMix (Perfect Real Time) kit (TaKaRa) were used to specifically amplify the TgMIF target gene. The reaction system was: 2 μl of 5× PrimeScript RT Master Mix (Perfect Real Time), 3 μl of total RNA template, and RNaseFree dH2O added to 10 μl. The reverse transcription reaction conditions were: 37°C for 15 min and inactivation at 85°C for 5 s.

[0033] The specific amplification reaction conditions of the TgMIF gene are as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 20 s, annealing at 60°C for 20 s, extension at 72°C for 30 s, for a total of 3 cycles, and finally extension at 72°C for 10 min. Upstream primer: 5'- CCGGAATTCGCCACC ATGCCCAAGTGCATGATCTTTTGCC-3' (underlined is the EcoRI restriction site), downstream primer: 5'- ATTTGCGGCCGCA GCCGAAAGTTCGGTCGCCCATGGCC-3' (the underline indicates the NotI restriction site).

[0034] 3. Connection, transformation and sequencing of target gene: After recovery and purification, the RT-PCR product was double-digested with EcoRI and NotI with the plasmid vector pLVX-C-FLAG-mCMV-ZsGreen-IRES-Puro (purchased from Wuhan Vinocell Biotechnology Co., Ltd.). Figure 1 As shown. Connect overnight at 16℃, take 10ul of the connection product and transform it into 100ul JM109 competent bacteria, and send the positive clone to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The sequencing primer is CMV-F, and its primer sequence is: 5'-CGCAAATGGGCGGTAGGCGTG-3'. The result is named pLVX-TgMIF-3flag-ZsGreen-Puro plasmid.

[0035] 2. rLV-TgMIF-3flag-ZsGreen-Puro lentiviral packaging and construction of stable Raw264.7 cell line

[0036] 1. Lentivirus packaging

[0037] The lentiviral plasmid pLVX-TgMIF-3flag-ZsGreen-Puro was introduced into 293T cells to produce a high titer lentivirus containing the target gene (referred to as rLV-TgMIF-3flag-ZsGreen-Puro). The specific steps are as follows:

[0038] (1) One day before transfection, prepare cells for subculturing: digest 293T cells with 0.25% trypsin, adjust the cell density with DMEM medium containing 10% serum, and inoculate 6 to 8 × 10 cells per 10 cm cell culture dish. 6 cells into a 10 cm cell culture dish and culture in a 37°C, 5% CO2 incubator. After 16 to 24 hours, when the cell density grows to 80% to 90%, it can be used for transfection.

[0039] (2) The next day, 2-4 hours before transfection, replace the medium with 5 ml of complete medium without P / S (DMEM + 10% FBS), then perform transfection and culture in a 37°C, 5% CO2 incubator.

[0040] (3) On the third day, i.e., after 12 to 16 hours of culture, the culture medium was discarded and replaced with 10 ml of fresh complete culture medium (DMEM + 10% FBS + P / S).

[0041] (4) On the fourth day, i.e. 24 h after the medium change, the supernatant was collected and stored in a 4°C refrigerator. Then, 10 ml of fresh complete medium (DMEM + 10% FBS + P / S) was added to a 10 cm cell culture dish and placed in a 37°C, 5% CO2 incubator for continued culture.

[0042] (5) Day 5: The supernatant was collected again, mixed with the supernatant collected for the first time, and centrifuged at 1000 rpm for 5 min. Cell debris was discarded and the supernatant was filtered through a 0.45 μm PVDF filter into a 50 ml round-bottom centrifuge tube.

[0043] (6) Centrifuge at 50,000 g for 2.5 h at 4°C. Mark the virus precipitate with a marker pen.

[0044] (7) Carefully discard the supernatant, dry it, and add DMEM (without serum or double antibody) or PBS to resuspend the virus precipitate at a volume of 100ul / 10cm culture dish. Let it stand at room temperature for 2 hours, then gently blow it evenly with a pipette (to avoid bubbles), and continue to stand at room temperature for 30 minutes. Dispense it into clean 1.5ml EP tubes according to the amount of virus used each time, and store it in a -80℃ refrigerator.

[0045] 2. Construction of stable cell lines

[0046] Lentivirus was used to infect Raw264.7 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) and screen mixed clone cell lines, hereinafter referred to as cells The specific steps are as follows:

[0047] (1) Resuscitate Raw264.7 cells and subculture them when the cells grow to 80-90% cell density for 2-3 times;

[0048] (2) Cultivate Raw264.7 cells, digest and count the cells when they grow to 80-90% cell density, and calculate 5×10 5 Cells were seeded into 6-well plates at a cell density of cells / well;

[0049] (3) On the second day, rLV-TgMIF-3flag-ZsGreen-Puro and control rLV-ZsGreen-Puro were added at an MOI of 50 and cultured overnight in a 37°C, 5% CO2 incubator;

[0050] (4) 24 h after adding the virus, the medium was replaced with DMEM+10% FBS+1% P / S complete medium;

[0051] (5) 48 hours after adding the virus, add Puromycin at a final concentration of 5 ug / ml for resistance screening, and change the medium (complete medium containing 5 ug / ml Puromycin) every 2-3 days;

[0052] (6) When the cells grow to 80-90% cell density, digest the cells and inoculate them into T-25cm 2 Continue to culture in cell culture flask;

[0053] (7) Continuously subculture for 2-3 generations to obtain a mixed clone stable cell line The control group was

[0054] The infected stable cell lines were observed using a fluorescence microscope. Figure 2 As shown, there are a large number of cells expressing green fluorescent protein in the field of view; Figure 3 and Figure 4 As shown, verified by qPCR and WB Expression of TgMIF in cells.

[0055] 3. Flow cytometry Phenotype

[0056] Flow cytometry was used to detect the expression of macrophage surface molecules F4-80, CD206, and CD86, and the macrophage marker F4-80, M2 marker CD206, and M1 marker CD86 were marked respectively. Figure 5 As shown, the results show Compared with the control group, the expression of M1 marker CD86 was higher, indicating that macrophages stably expressing TgMIF were polarized toward M1. Figure 6 As shown, WB detection of cell supernatant showed Compared with the control group, more collagen-dissolving MMP9 protein was secreted, proving Secretion of MMP9 protein.

[0057] 4. Has therapeutic effect on liver fibrosis

[0058] (1) CCl4 was used to establish a liver fibrosis model in mice (SPF grade C57 male mice, purchased from the Experimental Animal Center of Anhui Medical University) and infused into the tail vein. Cells were injected into the tail vein and observed under a fluorescence microscope Cells after mice, such as Figure 7 As shown, the frozen sections of the liver at 1, 3, 5, and 7 days all showed that the liver contained cells expressing green fluorescent protein, indicating that the cells entered the liver through the blood circulation after being injected into the mouse through the tail vein and survived in the liver for at least 7 days.

[0059] (2) The serum AST and ALT levels of mice with liver fibrosis were measured using a biochemical analyzer. Figure 8 As shown, It can significantly reduce the serum AST and ALT levels in mice with liver fibrosis, suggesting that it has a liver protective effect. Fig. 9 As shown, the gross anatomy shows It can reduce the volume of liver fibrosis and reduce the surface granularity. Liver HE staining, Sirius red staining and Masson staining all show that It can significantly reduce the area of ​​liver fibrosis and show good anti-liver fibrosis effect.

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the above embodiments, or to replace some of the technical features therein with equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. SEQUENCE LISTING <110> Anhui Medical University <120> Application of macrophages expressing TgMIF protein in the preparation of drugs for treating liver fibrosis <130> 2024.6.7 <160> 2 <170> PatentIn version 3.3 <210> 1 <211> 116 <212> PRT <213> Toxoplasma gondii <400> 1 Met Pro Lys Cys Met Ile Phe Cys Pro Val Ala Ala Thr Pro Ala Gln 1 5 10 15 Gln Asp Ala Leu Leu Lys Asp Ala Glu Lys Ala Val Ala Asp Ala Leu 20 25 30 Gly Lys Pro Leu Ser Tyr Val Met Val Gly Tyr Ser Gln Thr Gly Gln 35 40 45 Met Arg Phe Gly Gly Ser Ser Asp Pro Cys Ala Phe Ile Arg Val Ala 50 55 60 Ser Ile Gly Gly Ile Thr Ser Ser Thr Asn Cys Lys Ile Ala Ala Ala 65 70 75 80 Leu Ser Ala Ala Cys Glu Arg His Leu Gly Val Pro Lys Asn Arg Ile 85 90 95 Tyr Thr Thr Phe Thr Asn Lys Ser Pro Ser Glu Trp Ala Met Gly Asp 100 105 110 Arg Thr Phe Gly 115 <210> 2 <211> 351 <212> DNA <213> Toxoplasma gondii <400> 2 atgcccaagt gcatgatctt ttgccccgtc gcggcgacgc cggcgcagca ggacgccctc 60 ttgaaggacg ccgaaaaagc cgtcgcagac gctctgggga agcctctgag ctacgtcatg 120 gtgggatact cgcagaccgg gcagatgcgt ttcggcggga gcagcgaccc gtgtgcgttc 180 attcgcgttg cttccattgg aggcatcacc agttccacga actgcaaaat cgccgctgct 240 ctctccgctg catgcgaacg ccacctgggc gtccccaaga accgcatcta cacgacattc 300 acaaacaaga gcccctctga gtgggccatg ggcgaccgaa ctttcggctg a 351

Claims

1. Use of macrophages expressing TgMIF protein in the preparation of a drug for treating liver fibrosis, characterized in that: The TgMIF protein is encoded by a TgMIF protein encoding gene, and the TgMIF protein encoding gene is amplified using cDNA of Toxoplasma gondii RH strain and ME49 strain as templates and a primer pair consisting of an upstream primer and a downstream primer; The upstream primer is: 5'-CCGGAATTCGCCACCATGCCCAAGTGCATGATCTTTTGCC-3', and the downstream primer is: 5'-ATTTGCGGCCGCAGCCGAAAGTTCGGTCGCCCATGGCC-3'.

2. The use of macrophages expressing TgMIF protein according to claim 1 in the preparation of a drug for treating liver fibrosis, characterized in that: The coding gene of TgMIF protein is packaged with lentivirus, stably infects macrophages, and then the macrophages are activated in a targeted manner to obtain macrophages expressing TgMIF protein.

3. The use of macrophages expressing TgMIF protein according to claim 2 in the preparation of a drug for treating liver fibrosis, characterized in that: The lentivirus is a 293T cell containing a lentiviral plasmid.

4. The use of macrophages expressing TgMIF protein according to claim 2 in the preparation of a drug for treating liver fibrosis, characterized in that: The lentiviral packaging comprises the following steps: inserting the TgMIF encoding gene into the pLVX-C-FLAG-mCMV-ZsGreen-IRES-Puro vector.

Images