Preparation method of engineered multipotent macrophage for pulmonary fibrosis treatment

Abstract

The invention discloses a preparation method of engineered multipotent macrophages for pulmonary fibrosis treatment. The preparation method comprises the following steps: obtaining M1-l ike BMDM, preparing ABT (at) MP and constructing ABT (at) MP (at) M1, the method for obtaining the M1-l ike BMDM comprises the following steps: step 1, stripping bone substances of male rats, flushing out bone marrow cells, and putting the male rats into a DMEM (Dulbecco's Modified Eagle Medium) of FBS for culturing; 2, changing the liquid every 3 days and supplementing induction factors, and obtaining BMDM after 7 days; adding a stimulant into the BMDM, and stimulating for 24 hours, so as to finally obtain M1-l ike BMDM; the preparation method of the ABT (at) MP comprises the following steps: step one, dissolving poly-3-hydroxybutyrate-3-hydroxyhexanoate and ABT-263 in dichloromethane to serve as an oil phase; 2, filtering the sodium cholate aqueous solution through a filter to remove impurities to obtain a water phase; 3, dropwise adding the oil phase into the water phase under an ice-bath ultrasonic condition to form an O / W emulsion; according to the invention, the M1-l ike BMDM which is high in purity (greater than or equal to 97%) and expresses M1 type polarization markers such as iNOS, TNF-alpha, IL-6, IL-12 and the like is obtained; the polymer microparticles (ABT (at) MP) loaded with the aging ACE II scavenger ABT-263, which have the average particle size of about 4 microns and are in a uniform spherical shape, are prepared.

Description

Technical Field

[0001] The present invention relates to the technical field of pulmonary fibrosis treatment, and in particular to a method for preparing engineered multipotent macrophages for pulmonary fibrosis treatment. Background Art

[0002] Pulmonary fibrosis is an interstitial lung disease characterized by subpleural honeycomb scar areas, tractional bronchiectasis, and thickening of the surrounding alveolar septa. It can lead to decreased lung compliance, impaired gas exchange, and ultimately death from respiratory failure. Pulmonary fibrosis is common in men over 65 years old. The global prevalence in 2011 was as high as 400 cases per 100,000 people, and the median survival of patients is only 3-5 years. According to the clinical treatment guidelines for pulmonary fibrosis jointly issued by the American Thoracic Society, the European Respiratory Society, the Japanese Respiratory Society, and the Latin American Thoracic Association in 2015, pirfenidone and nintedanib are the only two effective drugs recommended for use as appropriate, but they can only delay the progression of the disease, but cannot ablate the existing fibrotic lesions to provide a healthy habitat for the regeneration of damaged lung tissue. Therefore, pulmonary fibrosis has become an increasingly serious public health problem, and it is imperative to explore new and efficient treatment strategies for it.

[0003] Pulmonary fibrosis can be regarded as a poor repair process of lung tissue injury due to disordered immune regulation. Macrophages play an indispensable role in every link of pulmonary fibrosis by changing their own activation mode: M1-like macrophages are dominant in the early stage of injury, playing a pro-inflammatory / anti-fibrotic role; M2-like macrophages increase disproportionately in the progressive stage of fibrosis, playing an anti-inflammatory / pro-fibrotic role. Therefore, manipulating macrophage subsets with different activation modes, that is, reducing the abnormally increased pro-fibrotic M2-like macrophages and / or supplementing the inferior anti-fibrotic M1-like macrophages, has become a new hope for the treatment of IPF. The applicant has previously attempted to adoptively transplant M1-like bone marrow derived macrophages (BMDM) to treat pulmonary fibrosis. The results showed that after non-exposed airway instillation adoptive transplantation, M1-like BMDM specifically colonized the lung tissue of mice with pulmonary fibrosis, alleviating fibrosis to a certain extent.

[0004] However, the pathogenesis of pulmonary fibrosis is complex, and the current mainstream view in academia is that aging type II alveolar epithelial cells (ACEⅡ) are the initiating factor of pulmonary fibrosis. Lung RNAseq data of patients with pulmonary fibrosis showed that aging-related markers such as CDKN2A / p16INK4a and TP53 were positively correlated with the severity of the disease. These aged ACEⅡ produce apoptosis resistance and secrete a large amount of aging-related secretory phenotypes, lose stemness and continuously activate myofibroblasts, making it difficult for damaged lung tissue to repair normally and causing fibrosis progression. The applicant's preliminary experimental data showed that ACEⅡ in mice with bleomycin-induced pulmonary fibrosis highly expressed P16, Bcl-2 and Bcl-xL, and did not decrease after adoptive transplantation of M1-like BMDM. Therefore, engineering M1-like BMDM to give it the function of clearing aged ACEⅡ is a necessary measure to enhance its efficacy in treating pulmonary fibrosis. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing engineered multipotent macrophages for treating pulmonary fibrosis, aiming to improve the problem that the efficacy of treating pulmonary fibrosis needs to be improved.

[0006] The present invention is achieved as follows: a method for preparing engineered multipotent macrophages for the treatment of pulmonary fibrosis, comprising obtaining M1-like BMDM, preparing ABT@MP and constructing ABT@MP@M1;

[0007] M1-like BMDM acquisition includes

[0008] Step 1: strip the bone of the male mouse, flush out the bone marrow cells, and culture them in DMEM containing FBS;

[0009] Step 2: Change the medium and add induction factors every 3 days, and obtain BMDM after 7 days; add stimulatory factors to the above BMDM, stimulate for 24 hours, and finally obtain M1-like BMDM;

[0010] The preparation of ABT@MP includes

[0011] Step 1: poly 3-hydroxybutyrate-3-hydroxyhexanoate and ABT-263 are dissolved in dichloromethane as the oil phase;

[0012] Step 2: The sodium cholate aqueous solution is filtered through a filter to remove impurities and used as the aqueous phase;

[0013] Step 3: The oil phase was added dropwise to the water phase under ice bath ultrasonic conditions to form an O / W emulsion, and ultrasonication was continued for 15 min to reduce and stabilize the emulsion droplets;

[0014] Step 4: After the ultrasound is finished, the organic phase is removed by evaporation under reduced pressure, so that the poly 3-hydroxybutyrate-3-hydroxyhexanoate aggregates and solidifies into micron particles (MP);

[0015] Step 5: Place the system obtained in step 4 in an ultrafiltration tube, centrifuge at 3000 rpm for 15 min, discard the filtrate, and add fresh PBS to the retentate;

[0016] Step 6: Repeat step 5 to remove sodium cholate and collect ABT@MP microparticles;

[0017] The construction of ABT@MP@M1 includes

[0018] Step 1: EDC and NHS were added to the ABT@MP solution in sequence and shaken to activate the carboxyl groups; then, DSPE was added and stirred continuously at room temperature to achieve amide bond crosslinking;

[0019] Step 2: The system obtained in step 1 is placed in a dialysis bag to remove residual EDC and NHS to obtain DSPE@ABT@MP;

[0020] Step 3: Co-incubate DSPE@ABT@MP with M1-like BMDM, and use the lipid spear effect of DSPE to fix ABT@MP on the cell membrane surface of M1-like BMDM as a "cell backpack";

[0021] Step 4: Wash the cells with PBS to remove free DSPE@ABT@MP and assemble ABT@MP@M1.

[0022] Preferably, the male mouse bone is the femur and tibia of a 6-8 week old C57BL / 6 male mouse. After flushing out bone marrow cells with sterile PBS, erythrocytes are lysed and resuspended in DMEM containing 10% FBS and 25 ng / mL M-CSF for culture.

[0023] Preferably, the induction factor is 25 ng / mL of M-CSF, and the stimulatory hormone is 20 ng / mL of INF-γ.

[0024] Preferably, FACS detects CD11b and F4 / 80 to verify the BMDM identity of the obtained cells; qRT-PCR detects M1 polarization markers to verify the M1-like macrophage identity of the obtained cells, and the cell purity should be higher than 97%.

[0025] Preferably, poly 3-hydroxybutyric acid-3-hydroxyhexanoate is 50 mg, ABT-263 is 1 mg, and dichloromethane is 1 mL.

[0026] Preferably, the sodium cholate aqueous solution is 20 mL 0.1% (w / v), and is filtered through a 0.22 μm filter to remove impurities; the molecular weight cutoff of the ultrafiltration tube is 3.5 KD.

[0027] Preferably, the particle size of ABT@MP observed under optical microscopy and transmission electron microscopy should be about 4 μm.

[0028] Preferably, EDC is 20 mg, NHS is 20 mg, the pH of the ABT@MP solution is 6.0, the concentration of DSPE is 1 mg / mL, and the dosage is 1 mL.

[0029] Preferably, the modification efficiency of ABT@MP fixed on the surface of M1-like BMDM is detected by imaging flow cytometry and the like, and if it is higher than 80%, it can be used for the treatment of pulmonary fibrosis.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention obtains high-purity (≥97%) M1-like BMDM expressing M1-type polarization markers such as iNOS, TNF-α, IL-6, and IL-12; prepares polymer microparticles (ABT@MP) with an average particle size of about 4 μm and a uniform spherical shape loaded with the aging ACEⅡ scavenger ABT-263; and utilizes non-genetic cell surface engineering technology to fix ABT@MP as a "cell backpack" on the cell membrane surface of M1-like BMDM to assemble engineered multipotent macrophages ABT@MP@M1 for the treatment of pulmonary fibrosis. The modification efficiency of ABT@MP fixed on the cell membrane surface of M1-like BMDM in this group of engineered multipotent macrophages should be above 80%. Adoptive therapy based on this group of engineered multipotent macrophages showed significant advantages in the treatment of pulmonary fibrosis. Compared with the non-adoptive transplantation group and the simple M1-like BMDM adoptive transplantation group: ①Pro-SPC and p16INK4a immunofluorescence staining showed that ABT@MP@M1 could efficiently clear aged ACEⅡ; ②Masson and Sirius red staining of lung tissue confirmed that ABT@MP@M1 could significantly reduce excessive deposition of extracellular matrix; ③Lung tissue reconstruction after small animal CT suggested that ABT@MP@M1 could significantly increase the volume of non-fibrotic lung; ④Small animal respiratory function testing showed that ABT@MP@M1 could effectively improve key lung function indicators such as tidal volume (TV), minute ventilation (MV), and peak inspiratory flow (RIF) in mice with pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of engineered multipotent macrophages (ABT@MP@M1) for the treatment of pulmonary fibrosis;

[0032] Figure 2 It is the purity and phenotype detection of M1-like BMDM;

[0033] Figure 3 It is the particle size and morphology analysis of ABT@MP;

[0034] Figure 4 It is the modification efficiency test of ABT@MP fixed on the cell membrane surface of M1-like BMDM;

[0035] Figure 5 The establishment and validation of a radiation-induced pulmonary fibrosis mouse model;

[0036] Figure 6 Pro-SPC and p16INK4a immunofluorescence staining showed that ABT@MP@M1 can effectively remove aging ACEⅡ;

[0037] Figure 7 Masson and Sirius red staining of lung tissue confirmed that ABT@MP@M1 could significantly reduce the excessive deposition of extracellular matrix;

[0038] Figure 8 The lung tissue reconstruction after small animal CT showed that ABT@MP@M1 could significantly increase the non-fibrotic lung volume;

[0039] Fig. 9 Small animal respiratory function tests showed that ABT@MP@M1 can effectively improve key lung function indicators such as tidal volume (TV), minute ventilation (MV), and peak inspiratory flow (RIF) in mice with pulmonary fibrosis.

[0040] Figure 2 A in the middle is the detection of CD11c and F4 / 80 expression in M1-like BMDM by FACS; B is the detection of M1-type polarization marker expression in BMDM by qRT-PCR;

[0041] Figure 3 A is a typical image of ABT@MP under 40x bright field microscope, with a scale of 50μm; B is the statistical particle size distribution of Image pro plus; C is the scanning electron microscope with a magnification of 10000x, with a scale of 5μm; D is the scanning electron microscope with a magnification of 40000x, with a scale of 1μm;

[0042] Figure 4 A is an imaging flow cytometry diagram; B is a typical imaging flow cytometry image; Ch01 is bright field, Ch02 is the green channel indicating M1-ike BMDM, Ch09 is the red channel indicating ABT@MP, and the scale bar is 10μm; C is a typical image of ABT@MP@M1 under bright field at 10x, and the scale bar is 200μm; D is a typical image of ABT@MP@M1 under confocal microscopy at 40x, and the scale bar is 50μm;

[0043] Figure 5 A is the timeline; B is Masson staining of lung tissue, with a scale of 200 μm; C is the statistics of Masson staining results; D is the hydroxyproline HYP content in lung tissue 26 weeks after irradiation. DETAILED DESCRIPTION

[0044] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0045] The following is a further description with reference to the accompanying drawings and specific embodiments:

[0046] In summary, the present invention uses non-genetic cell surface engineering technology to fix polymer microparticles (ABT@MP) loaded with the senescent ACEⅡ scavenger ABT-263 on the surface of the M1-like BMDM cell membrane to construct engineered multipotent macrophages (ABT@MP@M1, Figure 1 ). After ABT@MP@M1 is infused back into the airway through non-invasive instillation, M1-like BMDM can carry ABT@MP to the fibrotic lesions and continuously release ABT-263, inducing apoptosis of aging ACEⅡ for a long time, thereby assisting M1-like BMDM and improving its therapeutic efficacy. This engineered multipotent macrophage adoptive therapy is a new and efficient treatment strategy for pulmonary fibrosis, and has important clinical significance for promoting the recovery of lung function in patients. In addition, the macrophage engineering technology established by the present invention will provide new ideas and methods for the modification of other types of cells (such as T cells and NK cells), and has a broader application prospect.

[0047] The details of the technical solution are as follows:

[0048] ① Obtaining M1-like BMDM: The femur and tibia of 6-8 week old C57BL / 6 male mice were peeled off, and the bone marrow cells were carefully flushed out with sterile PBS; after lysing the red blood cells, the cells were resuspended in DMEM containing 10% FBS and 25 ng / mL M-CSF and cultured; the medium was changed and induction factors were added every 3 days, and BMDM were obtained after 7 days; INF-γ (20 ng / mL) was added to the above BMDM, and stimulated for 24 hours to finally obtain M1-like BMDM; CD11b and F4 / 80 were detected by FACS to verify the BMDM identity of the obtained cells;

[0049] qRT-PCR was used to detect M1-like polarization markers such as iNOS, TNF-α, IL-6, and IL-12 to verify the M1-like macrophage identity of the obtained cells. The cell purity should be higher than 97% ( Figure 2 ).

[0050] ② Preparation of ABT@MP: 50 mg poly 3-hydroxybutyric acid-3-hydroxyhexanoate (PHBHHx-PEG) and 1 mg ABT-263 were dissolved in 1 mL dichloromethane as the oil phase; 20 mL 0.1% (w / v) sodium cholate aqueous solution was filtered through a 0.22 μm filter to remove impurities as the water phase; the oil phase was added dropwise to the water phase (about 1 min) under ice bath ultrasound (300 W, ultrasound 1 s, pause 1 s) to form an O / W emulsion, and ultrasound was continued for 15 min to reduce and stabilize the emulsion droplets; after the ultrasound was completed, the organic phase was removed by evaporation under reduced pressure to allow PHBHHx-PEG to aggregate and solidify into micron particles (MP); then, the system was placed in an ultrafiltration tube (molecular weight cutoff 3.5 KD), centrifuged at 3000 rpm for 15 min, the filtrate was discarded, and fresh PBS was added to the retentate. The above process was repeated 3 times to remove sodium cholate and free ABT-263 as much as possible and collect ABT@MP micron particles. The particle size of ABT@MP was examined under light microscope, and its morphology was observed under scanning electron microscope ( Figure 3 ).

[0051] ③ Construction of ABT@MP@M1: 20 mg EDC and 10 mg NHS were added to the ABT@MP solution (pH = 6.0) in sequence, and shaken for 30 minutes to activate the carboxyl group; then, 1 mL DSPE (1 mg / mL) was added thereto, and stirred continuously at room temperature for 24 hours to achieve amide bond crosslinking; after the reaction, the system was placed in a dialysis bag (molecular weight cutoff 3.5KD) and dialyzed for 48 hours to remove residual EDC and NHS to obtain DSPE@ABT@MP. DSPE@ABT@MP was co-incubated with M1-like BMDM, and the lipid spear effect of DSPE was used to fix ABT@MP as a "cell backpack" on the cell membrane surface of M1-like BMDM. The cells were washed 3 times with PBS to remove free DSPE@ABT@MP, and ABT@MP@M1 was assembled. The modification efficiency of ABT@MP fixed on the cell membrane surface of M1-like BMDM was detected by imaging flow cytometry and other means. If it is higher than 80%, it can be used for the treatment of pulmonary fibrosis ( Figure 4 ).

[0052] ④ Establishment of pulmonary fibrosis mouse model: 6-8 week old C57BL / 6 male mice were taken and placed in lead suits after anesthesia to protect the other organs and only expose the chest cavity. The chest cavity of the mice was irradiated with X-rays at 160 kV, 10 mA, and 1.785 Gy / min*11 min. After 26 weeks, lung tissue pathology analysis was performed to evaluate whether the model was successfully established ( Figure 5 ).

[0053] Evaluation of the efficacy of adoptive therapy of ABT@MP@M1 in the treatment of pulmonary fibrosis: Using healthy mice and non-transplanted model mice as controls, 2×106ABT@MP@M1 were adoptively transplanted into mice with pulmonary fibrosis at the 22nd week of X-ray irradiation modeling by non-exposure airway instillation. At the 4th week after transplantation, i.e. the 26th week of X-ray irradiation modeling, the pulmonary ventilation function of the mice was tested, and lung CT was performed in parallel. The mice were then killed, and lung tissue collagen staining was performed and the clearance of senescent ACEⅡ was detected to comprehensively evaluate the efficacy of adoptive therapy of ABT@MP@M1 in the treatment of pulmonary fibrosis ( Figure 6-9 ).

[0054] The above contents are further described below in conjunction with specific embodiments.

[0055] Example 1 Acquisition of M1-like BMDM

[0056] 1. Experimental Materials

[0057] 1. Experimental Animals

[0058] Healthy 6-8 week old male C57BL / 6 mice, weighing 18-20 g, were purchased from the Experimental Animal Center of Air Force Medical University and maintained in an SPF animal room.

[0059] 2. Experimental Equipment

[0060] Cell culture incubator, flow cytometer, fluorescence quantitative PCR instrument

[0061] 3. Experimental Reagents

[0062] Fetal bovine serum, DMEM medium, penicillin-streptomycin solution, PBS, cytokine M-CSF, reverse transcription and fluorescence quantitative PCR kit

[0063] 2. Experimental Methods

[0064] 1. The femur and tibia of 6-week-old C57BL / 6 mice were peeled off, and the bone marrow cells were carefully flushed out with sterile PBS; after lysing the red blood cells, they were resuspended in DMEM containing 10% FBS with 25 ng / mL M-CSF and cultured; the medium was changed and the induction factor was added every 3 days, and BMDM was obtained after 7 days; INF-γ (20 ng / mL) was added to the above BMDM, and stimulated for 24 hours to finally obtain M1-like BMDM;

[0065] 2. FACS detection of CD11b and F4 / 80 to verify the BMDM identity of the obtained cells;

[0066] 3.qRT-PCR was used to detect M1 polarization markers such as iNOS, TNF-α, IL-6, and IL-12 to verify the M1-like macrophage identity of the obtained cells.

[0067] 3. Experimental Results

[0068] Bone marrow cells were extracted from C57BL / 6 mice, and the cells were continuously cultured in vitro. After induction with a certain concentration of M-CSF for 7 days and stimulation with INF-γ for 1 day, M1-like BMDM was obtained. FACS results showed that more than 99% of the cells in this group showed a BMDM phenotype with double positive CD11c and F4 / 80. In addition, the cells highly expressed TNF-α, IL-1β, IL-6 and other macrophage M1-like polarization markers ( Figure 2 ), indicating that the M1-like BMDM was successfully obtained.

[0069] Example 2 Preparation of ABT@MP

[0070] 1. Experimental Materials

[0071] 1. Experimental Equipment

[0072] Ultrasonic crusher, fluorescence microscope, scanning electron microscope, electronic balance

[0073] 2. Experimental Reagents

[0074] Poly 3-hydroxybutyrate-3-hydroxyhexanoate, ABT-263, dichloromethane

[0075] 2. Experimental Methods

[0076] 1.50 mg poly 3-hydroxybutyric acid-3-hydroxyhexanoate (PHBHHx-PEG) and 1 mg ABT-263 were dissolved in 1 mL dichloromethane as the oil phase; 20 mL 0.1% (w / v) sodium cholate aqueous solution was filtered through a 0.22 μm filter to remove impurities and used as the water phase; the oil phase was added dropwise to the water phase (taking about 1 min) under ice bath ultrasound (300 W, ultrasound 1 s, pause 1 s) to form an O / W emulsion, and ultrasound was continued for 15 min to reduce and stabilize the emulsion droplets; after the ultrasound was completed, the organic phase was removed by reduced pressure evaporation to allow the PHBHHx-PEG to aggregate and solidify into micron particles (MP);

[0077] 2. Place the system in an ultrafiltration tube (molecular weight cutoff 3.5KD), centrifuge at 3000rpm for 15min, discard the filtrate, and add fresh PBS to the retentate. Repeat the above process 3 times to remove sodium cholate and free ABT-263 as much as possible, and collect ABT@MP microparticles;

[0078] 3. The particle size of ABT@MP was examined under light microscope and its morphology was observed under scanning electron microscope.

[0079] 3. Experimental Results

[0080] ABT@MP was prepared by a modified emulsification / solvent evaporation method. A certain concentration of ABT@MP was dropped on a glass slide, and after it was slightly dried, its particle size was analyzed under a light microscope. The particle size of ABT@MP showed a normal distribution in the range of 2-6μm, and its average particle size was about 4μm ( Figure 3 A, B). Scanning electron microscopy images show that ABT@MP is a uniform spherical shape with a basically smooth surface ( Figure 3 C,D).

[0081] Example 3 Construction of ABT@MP@M1

[0082] 1. Experimental Materials

[0083] 1. Experimental Equipment

[0084] Cell culture incubator, low-temperature high-speed centrifuge, imaging flow cytometer, fluorescence microscope

[0085] 2. Experimental Reagents

[0086] Fetal bovine serum, DMEM medium, penicillin-streptomycin solution, PBS, 0.25% trypsin II. Experimental methods

[0087] 1. 20 mg EDC and 10 mg NHS were added to the ABT@MP solution (pH = 6.0) in sequence and shaken for 30 min to activate the carboxyl group; then, 1 mL DSPE (1 mg / mL) was added thereto and stirred at room temperature for 24 h to achieve amide bond crosslinking; after the reaction, the system was placed in a dialysis bag (molecular weight cutoff 3.5 KD) and dialyzed for 48 h to remove residual EDC and NHS to obtain DSPE@ABT@MP;

[0088] 2. Incubate it with M1-like BMDM for 6 h, wash the cells three times with PBS to remove free DSPE@ABT@MP, and assemble ABT@MP@M1;

[0089] 3. The modification efficiency of ABT@MP fixed on the cell membrane surface of M1-like BMDM was detected by imaging flow cytometry, laser confocal microscopy and other means.

[0090] 3. Experimental Results

[0091] 2×10 6 M1-like BMDM with 6×10 6 After ABT@MP was incubated for 6 hours, free microparticles were removed by repeated gradient dilution, and the adherent cells were digested to obtain ABT@MP@M1. An appropriate amount of ABT@MP@M1 was resuspended in 300 μL flow cytometry solution and analyzed by imaging flow cytometry. Figure 4 As shown in A, the efficiency of ABT@MP modification on the cell membrane surface of M1-like BMDM is over 80%. Figure 4 B is a typical image of ABT@MP@M1 imaging flow cytometry. As expected, it can be seen that different numbers of red ABT@MPs are mounted on the surface of green M1-like BMDM cell membranes, rather than being engulfed by macrophages. When ABT@MP@M1 was examined under a light microscope, more than 80% of the long spindle-shaped "antennae" M1-like BMDM cell membranes in the field of view had darker, regular spherical ABT@MPs ( Figure 4 C) Figure 4 As shown in Figure D, red ABT@MP and green M1-like BMDM can be seen attached under laser confocal microscopy, and there is no overlap between the two, that is, ABT@MP exists on the surface of the cell membrane of M1-like BMDM but is not engulfed by it. The above results show that the engineered M1-like BMDM with ABT@MP as the "cell backpack", namely ABT@MP@M1, was successfully prepared.

[0092] Example 4 Establishment of pulmonary fibrosis mouse model

[0093] 1. Experimental Materials

[0094] 1. Experimental Animals

[0095] Healthy 6-8 week old male C57BL / 6 mice, weighing 18-20 g, were purchased from the Experimental Animal Center of Air Force Medical University and maintained in an SPF animal room.

[0096] 2. Experimental Equipment

[0097] Cryostat, low-temperature high-speed centrifuge, X-ray irradiation device, electronic balance

[0098] 3. Experimental Reagents

[0099] Anhydrous ethanol, methanol, PBS, 10% tissue fixative, hematoxylin-eosin staining solution, Masson staining kit

[0100] 2. Experimental Methods

[0101] 1. Take 6-8 week old wild type C57BL / 6 male mice, put them in lead suits after anesthesia, protect the other organs and only expose the chest cavity. Irradiate the mouse chest cavity with X-rays at 160kV, 10mA, 1.785Gy / min*11min dose parameters;

[0102] 2. Mice were killed 26 weeks after irradiation, and lung tissues were fixed and paraffin sections were sliced ​​for Masson staining to detect collagen deposition;

[0103] 3. The mice were killed 26 weeks after irradiation, and the lung tissue was homogenized to detect the hydroxyproline (HYP) content.

[0104] 3. Experimental Results

[0105] like Figure 5 As shown in the figure, obvious collagen deposition in lung tissue was observed in the irradiated group after 5 weeks, and the collagen deposition area reached about 15% of the lung tissue after 26 weeks, which was about 3.5 times that of the control group ( Figure 5 B, C). HYP is a unique amino acid in collagen, accounting for about 13% of the total collagen amino acids. Therefore, HYP content is considered an important indicator for evaluating the level of collagen deposition. After 26 weeks of irradiation, the HYP content in the whole lung of the irradiated group was about 90 μg, which was 4.5 times that of the control group ( Figure 5 D). The above conclusions fully demonstrate that the radiation-induced pulmonary fibrosis mouse model was successfully established.

[0106] Example 5 Evaluation of the efficacy of ABT@MP@M1 adoptive therapy in the treatment of pulmonary fibrosis

[0107] 1. Experimental Materials

[0108] 1. Experimental Animals

[0109] Healthy 6-8 week old male C57BL / 6 mice, weighing 18-20 g, were purchased from the Experimental Animal Center of Air Force Medical University and maintained in an SPF animal room.

[0110] 2. Experimental Equipment

[0111] Cell culture box, ultrasonic crusher, fluorescence microscope, cryostat, low-temperature high-speed centrifuge, X-ray irradiation device, small animal CT machine, small animal pulmonary ventilation function tester, electronic balance

[0112] 3. Experimental Reagents

[0113] Fetal bovine serum, DMEM medium, penicillin-streptomycin solution, PBS, cytokine M-CSF, 0.25% trypsin, poly 3-hydroxybutyrate-3-hydroxyhexanoate, ABT-263, dichloromethane, anhydrous ethanol, methanol, PBS, 10% tissue fixative, hematoxylin-eosin staining solution, Masson and Sirius red staining kit

[0114] 2. Experimental Methods

[0115] 1. Obtain M1-like BMDM as described in Example 1;

[0116] 2. Construct ABT@MP@M1 as described in Examples 1-3;

[0117] 3. Establish a mouse model of pulmonary fibrosis as described in Example 4;

[0118] Using healthy mice and non-transplanted model mice as controls, 2×10 6 M1-like BMDM or ABT@MP@M1;

[0119] 4. At 4 weeks after transplantation, i.e. 26 weeks after X-ray irradiation modeling, the lung ventilation function of mice was tested and lung CT was performed in parallel;

[0120] 5. The mice were then killed, and lung tissue collagen staining was performed to detect the clearance of aging ACEⅡ.

[0121] 3. Experimental Results

[0122] To clarify the therapeutic effect of ABT@MP@M1 on pulmonary fibrosis, healthy mice and non-transplanted model mice were used as controls. 2×10 6 M1-like BMDM or ABT@MP@M1. At 4 weeks after transplantation, i.e. 26 weeks after X-ray irradiation modeling, various tests were performed and found:

[0123] 1. Pro-SPC and p16INK4a immunofluorescence staining was performed on frozen sections of lung tissues of mice in each group. Pro-SPC is used to indicate ACEⅡ, and p16INK4a is an important aging-related marker. Figure 6 As shown in the figure, compared with the non-modeling group, the p16INK4a level in the modeling group was significantly increased, indicating that ACEⅡ in fibrotic lung tissue showed an aging phenotype. However, only ABT@MP@M1, but not M1-like BMDM adoptive transplantation alone, could reduce the p16INK4a level. This result shows that ABT@MP@M1 has a better therapeutic effect of efficiently clearing aging ACEⅡ;

[0124] 2. The lung tissues of each group of mice were frozen and sectioned and then stained with Masson and Sirius red. Both stains are used to mark collagen fibers, which are the extracellular matrix that is excessively deposited in pulmonary fibrosis. Figure 7 As shown in the figure, compared with the non-modeling group, the modeling group showed significant extracellular matrix over-deposition. After 4 weeks of adoptive transplantation of M1-like BMDM and ABT@MP@M1, the extracellular matrix over-deposition was significantly reduced, among which the effect of the ABT@MP@M1 adoptive transplantation group was better, confirming that ABT@MP@M1 has a better therapeutic effect of significantly reducing the extracellular matrix over-deposition.

[0125] 3. After lung CT scans were performed on each group of mice, the threshold was automatically divided to distinguish between fibrotic and non-fibrotic lung tissues, and finally a three-dimensional model of lung tissue reconstruction was obtained and the volume of non-fibrotic lung tissue was counted. Figure 8 As shown in the figure, the volume of non-fibrotic lung tissue in the modeling group was significantly reduced compared with that in the non-modeling group. After 4 weeks of adoptive transplantation of M1-like BMDM and ABT@MP@M1, the volume of non-fibrotic lung tissue increased, and the effect of the ABT@MP@M1 adoptive transplantation group was better, indicating that ABT@MP@M1 has a better therapeutic effect in increasing the volume of non-fibrotic lung.

[0126] 4. Each group of mice underwent pulmonary ventilation function tests. Fig. 9 As shown in the figure, compared with the non-modeling group, the tidal volume (TV), minute ventilation (MV) and peak inspiratory flow (PIF) of the modeling group were significantly reduced, while the airway resistance (RAW) was significantly increased, indicating that fibrosis has seriously affected the normal ventilation function of lung tissue. Four weeks after the adoptive transplantation of M1-like BMDM and ABT@MP@M1, the above indicators were significantly restored, among which the MV and PIF of the ABT@MP@M1 adoptive transplantation group were significantly higher than those of the M1-like BMDM adoptive transplantation group, indicating that ABT@MP@M1 has a better therapeutic effect in effectively improving the small lung function of pulmonary fibrosis.

[0127] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. 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.

Claims

1. A method for preparing engineered multipotent macrophages for the treatment of pulmonary fibrosis, characterized in that: Including the acquisition of M1-like BMDM, the preparation of ABT@MP and the construction of ABT@MP@M1; M1-like BMDM acquisition includes Step 1: strip the bone of the male mouse, flush out the bone marrow cells, and culture them in DMEM containing FBS; Step 2: Change the medium and add induction factors every 3 days, and obtain BMDM after 7 days; add stimulatory factors to the above BMDM, stimulate for 24 hours, and finally obtain M1-like BMDM; The preparation of ABT@MP includes Step 1: poly 3-hydroxybutyrate-3-hydroxyhexanoate and ABT-263 are dissolved in dichloromethane as the oil phase; Step 2: The sodium cholate aqueous solution is filtered through a filter to remove impurities and used as the aqueous phase; Step 3: The oil phase was added dropwise to the water phase under ice bath ultrasonic conditions to form an O / W emulsion, and ultrasonication was continued for 15 min to reduce and stabilize the emulsion droplets; Step 4: After the ultrasound is finished, the organic phase is removed by evaporation under reduced pressure, so that the poly 3-hydroxybutyrate-3-hydroxyhexanoate aggregates and solidifies into micron particles (MP); Step 5: Place the system obtained in step 4 in an ultrafiltration tube, centrifuge at 3000 rpm for 15 min, discard the filtrate, and add fresh PBS to the retentate; Step 6: Repeat step 5 to remove sodium cholate and collect ABT@MP microparticles; The construction of ABT@MP@M1 includes Step 1: Add EDC and NHS to the ABT@MP solution in sequence and shake to activate the carboxyl group; Subsequently, DSPE was added thereto and stirred continuously at room temperature to achieve amide bond cross-linking; Step 2: The system obtained in step 1 is placed in a dialysis bag to remove residual EDC and NHS to obtain DSPE@ABT@MP; Step 3: Co-incubate DSPE@ABT@MP with M1-like BMDM, and use the lipid spear effect of DSPE to fix ABT@MP as a "cell backpack" on the cell membrane surface of M1-like BMDM; Step 4: Wash the cells with PBS to remove free DSPE@ABT@MP and assemble ABT@MP@M1.

2. The method for preparing engineered multipotent macrophages for treating pulmonary fibrosis according to claim 1, characterized in that: The bone tissue of male mice was the femur and tibia of 6-8 week old C57BL / 6 male mice. Bone marrow cells were flushed out with sterile PBS, and red blood cells were lysed and resuspended in DMEM containing 10% FBS and 25 ng / mL M-CSF for culture.

3. The method for preparing engineered multipotent macrophages for treating pulmonary fibrosis according to claim 1, characterized in that: The induction factor is 25 ng / mL of M-CSF, and the stimulatory factor is 20 ng / mL of INF-γ.

4. The method for preparing engineered multipotent macrophages for treating pulmonary fibrosis according to claim 1, characterized in that: FACS was used to detect CD11b and F4 / 80 to verify the BMDM identity of the obtained cells; qRT-PCR was used to detect M1 polarization markers to verify the M1-like macrophage identity of the obtained cells, and the cell purity was higher than 97%.

5. The method for preparing engineered multipotent macrophages for treating pulmonary fibrosis according to claim 1, characterized in that: Poly 3-hydroxybutyric acid-3-hydroxyhexanoate is 50 mg, ABT-263 is 1 mg, and dichloromethane is 1 mL.

6. The method for preparing engineered multipotent macrophages for treating pulmonary fibrosis according to claim 1, characterized in that: The sodium cholate aqueous solution was 20 mL 0.1% (w / v) and was filtered through a 0.22 μm filter to remove impurities; the molecular weight cutoff of the ultrafiltration tube was 3.5 KD.

7. The method for preparing engineered multipotent macrophages for treating pulmonary fibrosis according to claim 1, characterized in that: The particle size of ABT@MP was about 4 μm under light microscopy and transmission electron microscopy.

8. The method for preparing engineered multipotent macrophages for treating pulmonary fibrosis according to claim 1, characterized in that: EDC is 20 mg, NHS is 20 mg, the pH of ABT@MP solution is 6.0, the concentration of DSPE is 1 mg / mL, and the dosage is 1 mL.

9. The method for preparing engineered multipotent macrophages for treating pulmonary fibrosis according to claim 1, characterized in that: The modification efficiency of ABT@MP fixed on the surface of M1-like BMDM was detected by imaging flow cytometry, which was higher than 80% for the treatment of pulmonary fibrosis.

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