Multi-engineered stem cell preparation as well as preparation method and application thereof

Abstract

The invention discloses a multi-engineered stem cell preparation as well as a preparation method and application thereof. The cell membrane of the stem cell is modified by the heparin-lipid conjugate, so that the fate of the stem cell in vivo is remarkably regulated. Meanwhile, cytoplasm of the stem cells is modified by utilizing the developed annular HGF preparation and the annular VEGF preparation, and the stem cells are endowed with high expression and persistent expression of anti-fibrosis growth factors. After intravenous administration, the prepared multi-engineered stem cells can efficiently target the liver, and secreted growth factors can effectively inhibit activation of hepatic stellate cells, reduce fibrosis indexes, relieve inflammatory response and promote repair and regeneration of hepatic tissues. The preparation process is simple, the operability is high, the stem cells are endowed with higher targeting capacity and treatment effect, and a novel scheme with potential is provided for treatment of hepatic fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a multi-engineered stem cell preparation and a preparation method and application thereof. Background Art

[0002] Liver fibrosis is a chronic disease characterized by excessive accumulation of extracellular matrix proteins, which may lead to serious diseases such as cirrhosis and liver cancer. To date, a variety of methods have been proposed for the treatment of liver fibrosis, including lifestyle modification, application of anti-fibrotic drugs, exosome therapy, and stem cell therapy. Among these methods, stem cell therapy has become a research hotspot due to its therapeutic effect, feasibility, and safety. Mesenchymal stem cells are a type of multipotent stromal cells with immunomodulatory and tissue repair capabilities, and are also the most widely used type of stem cells. Studies have shown that mesenchymal stem cells can secrete a large number of bioactive factors to relieve liver inflammation and promote the degradation of extracellular matrix, and are considered to be potential candidates for the treatment of liver fibrosis. Despite significant progress, mesenchymal stem cell-based therapies still face major challenges. On the one hand, stem cell therapies usually use systemic administration, which can cause a large number of cells to be lost in the blood circulation and reduce their retention in the liver. On the other hand, the number of active growth factors secreted by mesenchymal stem cells is limited, the duration is short, and the growth factors secreted by different batches of stem cells have significant differences, further limiting the biological application of stem cell therapies. Therefore, the development of highly effective mesenchymal stem cell-based therapies remains highly anticipated.

[0003] Cell membrane modification technology is a technology that physically or chemically modifies the cell membrane to improve the function of the cell, enhance its targeting or improve its stability in the body. Common modification methods include embedding specific molecules into the cell membrane through liposomes, polymers, nanoparticles or other biomaterials to promote the interaction between cells and specific targets. Genetic engineering technology introduces specific genes such as DNA, mRNA, circular RNA, etc. into cells to modify cell functions to achieve the treatment of specific diseases. Unlike DNA therapy, a significant advantage of circular RNA therapy is that it does not change the genetic characteristics of cells. Compared with linear RNA, circular RNA has a longer half-life, which enables it to show stronger and more persistent expression ability in cells. In addition, the stability of circular RNA in cells enables it to overcome the degradation problem common in traditional RNA therapy, ensuring that it provides stable biological activity during treatment. Therefore, the synergistic use of cell membrane modification technology and genetic engineering technology in the transformation of stem cells can not only enhance the targeting ability of stem cells, but also greatly improve the expression level of stem cells. This type of innovative integration approach is expected to develop a more promising treatment strategy that can precisely act on the diseased areas of liver fibrosis, thereby effectively promoting liver repair and regeneration. Summary of the invention

[0004] Purpose of the invention: The present invention aims to solve the problems of stem cell loss, insufficient secreted growth factors, short duration of action, and inconsistent secretion of growth factors from different batches of stem cells, and provides a multi-engineered stem cell preparation and its preparation method and application.

[0005] In order to solve the above technical problems, the present invention discloses a multi-engineered stem cell preparation and its preparation method and application. The specific technical solution is as follows:

[0006] A method for preparing a multi-engineered stem cell preparation comprises the following steps:

[0007] (1) Preparation of heparin-lipid conjugate: Heparin and a first lipid are coupled by condensation reaction; preferably, heparin is coupled to the first lipid in consideration of its immunomodulatory effect and affinity for the liver. The synthesized heparin-lipid conjugate can be inserted into the cell membrane of stem cells through membrane modification technology to improve their biological fate in vivo.

[0008] (2) Preparing a circular RNA preparation: mixing a circular RNA solution with a second lipid solution, and ultrafiltration to obtain a circular RNA preparation; the circular RNA includes an IRES sequence and a target sequence, and the 3' end of the IRES sequence is connected to the 5' end of the target sequence, and the 5' end of the IRES sequence is connected to the 3' end of the target sequence; wherein the target sequence consists of a signal peptide and an anti-fibrotic growth factor; preferably, the mixing is performed by a microfluidic device. More preferably, after the mixing, PBS buffer is added for dilution and then ultrafiltration is performed.

[0009] (3) Preparation of multi-engineered stem cells: Cultivate stem cells until the cell confluence is 70% to 85%, add the circular RNA preparation prepared in step (2) and culture for 4 to 72 hours, then replace with fresh culture medium, add the heparin-lipid conjugate prepared in step (1) and continue culturing for 4 to 72 hours to obtain a multi-engineered stem cell preparation. Preferably, the cell confluence is 80%, wherein in step (1), the heparin has a weight average molecular weight of 10 to 20 kDa; preferably 15 kDa. The first lipid is any one of 1,2-tetradecanoylphosphatidylethanolamine DMPE, 1,2-dipalmitoyl-sn-glyceryl-3-phosphoethanolamine DPPE, 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine DSPE, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine POPE, 1,2-dioleyl-sn-glycerol-3-phosphoethanolamine DOPE, 1-palmitoyl-2-oleoyl-phosphatidylcholine-phosphatidylethanolamine POPC-PE or sphingomyelin; preferably any one of DMPE, DPPE and DSPE; further preferably DSPE.

[0010] Wherein, in step (1), the reaction molar ratio of heparin to the first lipid is 1:1; the reaction temperature of the condensation reaction is 60-70°C, and the reaction time is 12-36 hours, preferably 65°C for 24 hours.

[0011] Wherein, in step (1), the heparin-lipid conjugate has a structure as shown in Formula I:

[0012]

[0013] The blue frame is the first lipid, n is any even number between 10 and 20, and the weight average molecular weight of heparin is 10 to 20 kDa. n is preferably 12, 14 or 16. + The weight average molecular weight of heparin is 15 kDa.

[0014] Wherein, in step (2), the IRES sequence is a CVB3 IRES sequence, and its nucleotide sequence is shown in SEQ ID No. 1.

[0015] The anti-fibrotic growth factor is any one of VEGF and HGF, and the sequences thereof correspond to SEQ ID No. 2 to 3 respectively; that is, the sequence of VEGF is shown in SEQ ID No. 2, and the sequence of HGF is shown in SEQ ID No. 3.

[0016] The sequence of the signal peptide is shown in SEQ ID No.4.

[0017] The circular RNA preparation is any one of circular HGF RNA preparation or circular VEGF RNA preparation or a combination of the two. The circular RNA is named circular VEGF RNA or circular HGF RNA according to the difference of anti-fibrotic growth factors.

[0018] Wherein, in step (2), the circular RNA is prepared according to the following method: the IRES sequence is cut into two segments from the 5' end to the 3' end at the cleavage site, the front segment is IRES segment 2, and the rear segment is IRES segment 1, the 5' end of the target sequence is connected to the 3' end of IRES segment 1, and the 3' end of the target sequence is connected to the 5' end of IRES segment 2 to obtain a linear RNA sequence, and the linear RNA sequence is cyclized to obtain a circular RNA; the cleavage site, from the 5' end to the 3' end, is any one of the 346th, 359th, 369th, 391st, 401st or 453rd site of the IRES sequence. Preferably, the cleavage site is the 391st site, the sequence of IRES segment 1 is shown in SEQ ID No.5, and the sequence of IRES segment 2 is shown in SEQ ID No.6. Preferably, the cyclization is achieved by T4 RNA ligase.

[0019] Wherein, in step (2), the concentration of circular RNA in the circular RNA solution is 0.05-5 μg / μL, and the solvent is citric acid buffer.

[0020] The second lipid solution is formed by mixing ionizable lipids, cholesterol, phospholipids and PEG-modified lipids in a molar ratio of 40-60:30-40:5-15:1-2. Preferably, ionizable lipids, cholesterol, phospholipids and PEG-modified lipids are mixed in a molar ratio of 50:38.5:10:1.5. The solvent of the second lipid solution is anhydrous ethanol.

[0021] The ionizable lipids include any one or a combination of C12-200, cKK-E12, OF-02, DODAC, DDAB, DLin-MC3-DMA, DLinkC2DMA, DMRIE, DOSPA, DODAC, DMRIE, DOGS, DODAP, DODMA, DLinDMA, DLenDMA, CLinDMA, DLinDAP, SM-102, ALC-0315 and JK-102-CA; preferably ALC-0315.

[0022] The phospholipids include any one or a combination of 1,2-distearoyl-sn-glycero-3-phosphocholine DSPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol sodium salt DPPG, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine DOPE, DSPE, 1,2-dioleyl-sn-glycero-3-phosphatidylcholine DOPC, POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPC, DPPE, 1,2-dioleoyl-sn-glycero-3-phosphocholine (1'-rac-glycerol DOPG, DMPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine SOPE, cerebrosides, ceramides, diacylglycerols, cerebrosides or sphingomyelins; preferably DSPC. The PEG-modified lipids include any one or a combination of DSPE-PEG1000, DSPE-PEG2000, DMG-PEG1000, DMG-PEG1300, DMG-PEG1500, DMG-PEG1800, DMG-PEG2000, DMG-PEG2200, DMG-PEG2500, DMG-PEG2700, DMG-PEG3000, DMG-PEG3200, DMG-PEG3500, DMG-PEG3700, DMG-PEG4000, DMG-PEG4200, DMG-PEG4500, DMG-PEG4700, DMG-PEG5000 or ceramide-PEG2000, etc.; preferably DMG-PEG2000.

[0023] The mixing volume ratio of the circular RNA solution and the second lipid solution is 1:1 to 10:1, preferably 3:1.

[0024] The ultrafiltration has a molecular weight cut-off of 90-120 kDa, preferably 100 kDa.

[0025] Wherein, in step (3), the stem cells include any one of embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, muscle stem cells, liver stem cells, cardiac stem cells, dental pulp stem cells, adipose stem cells, amniotic membrane stem cells, corneal epithelial stem cells, osteoblastic stem cells, or placental stem cells. Preferably, they are mesenchymal stem cells, and more preferably, they are bone marrow mesenchymal stem cells or umbilical cord mesenchymal stem cells.

[0026] Wherein, in step (3), the concentration of the circular RNA preparation added is 0.05-10 mg / mL (in terms of RNA), preferably 0.1-0.3 mg / mL; the concentration of the heparin-lipid conjugate added is 0.1-10 mg / mL, preferably 1 mg / mL. The circular RNA preparation is any one of a circular HGF RNA preparation or a circular VEGF RNA preparation or a combination of the two; preferably, a combination of a circular HGF RNA preparation and a circular VEGF RNA preparation, and the mass ratio of the circular HGF RNA to the circular VEGF RNA is 1:1.

[0027] In a second aspect, the present invention provides a multi-engineered stem cell preparation prepared by the preparation method described in the first aspect.

[0028] In a third aspect, the present invention provides use of the multi-engineered stem cell preparation described in the second aspect in the preparation of a drug for treating liver fibrosis.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention develops a heparin-lipid conjugate, which modifies the cell membrane of stem cells, giving the stem cells stronger liver targeting, reducing the loss of stem cells in the blood circulation, and promoting more cells to reach the lesion site.

[0031] (2) The present invention utilizes independently designed circular RNA to transform the cytoplasm of stem cells to solve the problems of insufficient secreted growth factors, short duration of action, and inconsistent secretion of growth factors by different batches of stem cells, thereby giving stem cells the ability to highly and continuously express anti-fibrotic growth factors.

[0032] (3) Through the two-step method of engineering stem cells, the stem cells are given powerful liver-targeting capabilities and biological functions. The secreted growth factors can effectively inhibit the activation of hepatic stellate cells, reduce fibrosis indicators, alleviate inflammatory responses, and promote the repair and regeneration of liver tissue, which helps to treat liver-related diseases.

[0033] (4) This engineering method is simple to operate, easy to implement and promote, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0035] Figure 1 It is a synthetic route for three heparin-lipid conjugates: heparin-DMPE, heparin-DPPE, and heparin-DSPE.

[0036] Figure 2 It is a fluorescence image of heparin-lipid conjugate engineered mesenchymal stem cells.

[0037] Figure 3 This is an in vitro image of heparin-lipid conjugate engineered stem cells.

[0038] Figure 4 Diagram for the design and characterization of circular RNA. Figure 4 a is the design route of circular RNA, Figure 4 b is the sequencing result of circular RNA, Figure 4 c is a transmission electron microscopy image of the circular RNA preparation.

[0039] Figure 5 is the expression level of cyclic VEGF preparation and cyclic HGF preparation in vivo.

[0040] Figure 6 It is a fluorescence image of engineered stem cells against fibrosis (in vitro).

[0041] Figure 7 This is a picture of the liver after multi-engineered stem cell therapy

[0042] Figure 8 This is the Sirius Red image after multi-engineered stem cell treatment DETAILED DESCRIPTION

[0043] The above scheme is further described below in conjunction with specific embodiments. It should be understood that these embodiments are used to illustrate the present invention and are not limited to the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

[0044] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0045] In the following examples, the PBS buffer, unless otherwise specified, has an ion concentration of 0.1 M and a pH of 7.2-7.4.

[0046] Example 1 Preparation of heparin-lipid conjugate

[0047] In this example, heparin-lipid conjugates were synthesized. The synthesis steps are as follows: Figure 1 As shown, the details are as follows:

[0048] The lipids involved in this example are 1,2-tetradecanoylphosphatidylethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0049] (1) Heparin with a weight average molecular weight of 15 kDa is completely dissolved in deionized water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) in a 45-fold molar amount based on heparin, and triethanolamine (TEA) in a 25-fold molar excess based on heparin are added to obtain an activated heparin solution.

[0050] (2) The lipid (DMPE, DPPE or DSPE) in an equimolar amount based on heparin was dissolved in 20 mL of isopropanol, and added to the activated heparin solution at 65° C. for 24 hours. After the reaction, the reaction product was added to 10 volumes of cold acetone for precipitation. In order to prepare a pure product, the precipitate was dissolved in distilled water, and the heparin lipid conjugate was obtained by dialysis (molecular weight: 8 kDa), centrifugation and freeze drying. In this example, heparin-DMPE conjugate, heparin-DPPE conjugate and heparin-DSPE conjugate were prepared.

[0051] Example 2 Heparin-lipid conjugate engineered cell membrane

[0052] The steps of engineering cell membrane using the heparin-lipid conjugate prepared in Example 1 are as follows:

[0053] Bone marrow-derived mesenchymal stem cells were added to the cell culture plate (1×10 4 Cells) were added with DMEM medium and cultured at 37°C. After culturing to 80% confluence, the cells were washed, 2 mL of fresh DMEM medium was added, and then FITC-labeled heparin-lipid conjugate (added at a concentration of 1 mg / mL) was added. After continuing to culture for 24 hours, the cells were observed under a fluorescence microscope. The specific groups are as follows:

[0054] Group G1: untreated group, without any substance modification;

[0055] Group G2: heparin-DMPE-modified mesenchymal stem cells;

[0056] Group G3: heparin-DPPE-modified mesenchymal stem cells;

[0057] Group G4: heparin-DSPE-modified mesenchymal stem cells.

[0058] Fluorescence results such as Figure 2As shown, the mesenchymal stem cells in the control group (G1) did not have any fluorescence, while the G4 group exhibited a large area of ​​green fluorescence, indicating the highest engineering ratio.

[0059] Example 3 Validation of the targeting of heparin-lipid conjugate engineered stem cells

[0060] Verification of targeting of engineered stem cells using the heparin-lipid conjugate prepared in Example 1:

[0061] Mesenchymal stem cells were cultured according to the method described in Example 2 until 80% confluence was achieved. The culture medium was replaced, and heparin-lipid conjugate at a concentration of 1 mg / mL was added to each cell culture well. The culture was continued for 24 hours, and the cells were treated with DiD reagent, and the stem cells were collected for administration after trypsin digestion. Male Balb / c mice (6-8 weeks old, 5×10 6 Stem cells / mouse), and three days later, the main organs of the mice (heart, liver, spleen, lung, and kidney) were removed, and the enrichment of stem cells in the body was observed using a mouse imaging device. Figure 3 As shown, the grouping of G1 to G4 is the same as in Example 2. Compared with the control group or other experimental groups, the liver of the G4 group has the strongest fluorescence, indicating that the mesenchymal stem cells modified with heparin-DSPE have a strong liver targeting ability.

[0062] Example 4 Preparation of circular RNA

[0063] This example designs, verifies and characterizes the corresponding preparations of circular RNA, and the specific steps are as follows:

[0064] (1) Design of circular RNA: The circular RNA comprises a CVB3 IRES sequence (as shown in SEQ ID No. 1) and a target sequence (including a signal peptide and an anti-fibrotic growth factor), and the 3' end of the CVB3 IRES sequence is connected to the 5' end of the target sequence, and the 5' end of the CVB3 IRES sequence is connected to the 3' end of the target sequence to obtain a circular RNA; the specific design method is as follows: first, a linear RNA is designed and synthesized, and then a circular RNA is formed by cyclization, wherein the linear RNA is cut into two segments from the 5' end to the 3' end at the cleavage site of the IRES sequence, the front segment is IRES segment 2, and the rear segment is IRES segment 1, the 5' end of the target sequence is connected to the 3' end of the IRES segment 1, and the 3' end of the target sequence is connected to the 5' end of the IRES segment 2 to obtain a linear RNA sequence. Specifically, the linear RNA sequence is connected in the following connection order: CVB3 IRES segment 1 (sequence shown in SEQ ID No. 5), signal peptide SP (sequence shown in SEQ ID No.4), an anti-fibrotic growth factor sequence (VEGF or HGF, the sequence is shown in SEQ ID No.2 or 3 respectively) and CVB3 IRES fragment 2 (the sequence is shown in SEQ ID No.6), and the specific connection is as follows Figure 4 a. The designed sequence was delivered to Suzhou Anshengda Company for synthesis and introduced into Escherichia coli. After testing, the 391st IRES cleavage site had a good cyclization effect from the 5' end to the 3' end. This embodiment designed two circular RNAs, including circular VEGF RNA and circular HGF RNA.

[0065] (2) Validation of circular RNA: Circular RNA is prepared through bacterial culture, plasmid extraction, enzyme digestion, transcription, purification, and circularization, and sequencing is performed to confirm the success of circularization. Figure 4 b. The specific experimental steps are as follows:

[0066] ① Bacterial culture: Prepare 100mL of LB medium, add to a conical flask, and sterilize at 121℃ for 20min. After the medium cools down, add 10μL of the bacteria synthesized by Suzhou Anshengda Company and the corresponding ampicillin antibiotic (working concentration: 50μg / mL), and shake the conical flask at a speed of 300r / min for 16 hours.

[0067] ②Plasmid extraction and enzyme digestion: Extract and digest the plasmid according to the steps of the large extraction kit (No.: DC202, purchased from Nanjing Novozymes Co., Ltd.) and XhoI enzyme digestion kit (No.: 15034ES76, purchased from Shanghai Yisheng Biotechnology).

[0068] ③Transcription: Take a 1.5mL centrifuge tube and add 2μL each of T7 RNA polymerase, buffer solution, UTP, GTP, CTP and ATP. Add 1μg of the plasmid extracted in step ② to the centrifuge tube, and add double distilled water to make the solution 20μL. Use a pipette to gently mix the components and incubate at 37℃ for 4 hours. After the incubation, add 1μL of DNase I to the reaction system and incubate at 37℃ for 15min to digest the transcribed DNA template.

[0069] ④Purification: Take 20μL of the transcribed solution, add 30μL of 7.5M lithium chloride solution, mix well, and then add 30μL of RNase Free H2O. After oscillation, place at -20℃ for at least 30min, centrifuge at 15000rpm for 15min, and discard the supernatant. Add pre-cooled 70% ethanol to a 1.5mL EP tube, mix well, centrifuge, repeat three times, and re-dissolve in RNase Free Water.

[0070] ⑤ Circularization: Use T4 RNA ligase kit (No.: M0239, purchased from NEB) to connect the two ends of RNA. For details, refer to the instructions of the kit: Mix 2μL RNA, 2μL buffer, 1μL T4 RNA ligase and 15μL double distilled water, and react at 25℃ for 1h. Add RNase R to remove the linear RNA precursor to obtain circular RNA.

[0071] (3) Characterization of circular RNA preparations: Use a microfluidic device (aqueous phase: organic phase flow rate = 3:1, total flow rate 12mL / min) to mix four lipid solutions (150μL organic phase, the solvent is anhydrous ethanol, the molar ratio of ALC-0315 lipid, cholesterol, DSPC and DMG-PEG2000 lipid is 50:38.5:10:1.5) and a citric acid buffer containing circular RNA (450μL aqueous phase, wherein the concentration of the citric acid buffer is 25mM, pH is 4.0, circular RNA = 50μg). After mixing, add 10 times the volume of PBS to dilute the nanopreparation to evenly disperse it. Centrifuge through a 100kDa ultrafiltration tube to finally obtain a concentrated circular RNA preparation (0.1mg / mL). The morphology and size of the circular RNA preparation were observed using a transmission electron microscope. Figure 4 As shown in c, the circular RNA preparation has a uniform circular shape and is approximately 100 nm in size.

[0072] Example 5 Circular RNA Engineering Cytoplasm

[0073] The steps of engineering cytoplasm using the circular RNA prepared in Example 4 are as follows:

[0074] Mesenchymal stem cells were cultured according to the method described in Example 2 until 80% confluence was achieved. Fresh culture medium was replaced and cyclic VEGF RNA preparation (RNA = 2 μg, LNP-cirVEGF group) or cyclic HGF RNA preparation (RNA = 2 μg, LNP-cirHGF group) was added. After 24 hours of culture, 100 μL of culture medium was taken for testing every day, and the same amount of culture medium was added to continue the culture. At the same time, three control groups were set up, namely:

[0075] Untreated group: No treatment was done after cell culture, i.e. no preparation was added;

[0076] Linear VEGF mRNA preparation group (LNP-linVEGF): RNA = 2 μg;

[0077] Linear HGF mRNA preparation group (LNP-linHGF): RNA=2 μg.

[0078] Elisa method was used for detection. The results are as follows Figure 5 As shown, after being transformed with cyclic VEGF RNA preparations or cyclic HGF RNA preparations, the expression level of mesenchymal stem cells was much higher than that of the untreated group or the linear mRNA preparation group, and the expression could last for nearly 13 days.

[0079] Example 6 Validation of circular RNA anti-fibrosis in vitro

[0080] The validation steps of in vitro anti-fibrosis are as follows:

[0081] Mesenchymal stem cells were cultured according to the method described in Example 2 until the confluence reached 80%. The culture medium was replaced, and the cyclic VEGF RNA preparation, cyclic HGF RNA preparation, or a mixture of the cyclic VEGF RNA preparation and the cyclic HGF RNA preparation prepared in Example 4 at a mass ratio of 1:1 (RNA = 2 μg) was added, and the culture was continued for 48 hours to obtain an MSCs culture medium (culture medium 1).

[0082] Hepatic stellate cells (HSC-T6) were added to the cell culture plate (1×10 4 Cells) were added with DMEM medium and cultured at 37°C. After culturing for 24 hours, hepatic stellate cells were activated with 10 nM TGF-β1, and HSC-T6 were incubated with 2 mL of the culture medium 1 collected above (incubated at 37°C for 24 hours). The nuclei and actin α (α-SMA) were stained and observed under a fluorescence microscope. The grouping of this example is as follows:

[0083] Group G1: HSC-T6 obtained without any treatment and only according to the above culture method;

[0084] Group G2: HSC-T6 activated by TGF-β1;

[0085] Group G3: HSC-T6 treated with TGF-β1 and MSCs culture medium (without circular RNA engineering);

[0086] Group G4: HSC-T6 were treated with TGF-β1 and circular RNA engineered MSCs culture medium. The circular RNA engineering described here is: a mixture of circular VEGF RNA preparation and circular HGF RNA preparation in a mass ratio of 1:1.

[0087] The results are as follows Figure 6 As shown, compared with the G1-G3 groups, the circular RNA-engineered MSCs culture medium significantly reduced the expression of α-SMA (G4 group), mainly because the culture medium contained a large amount of anti-fibrotic growth factors. The green in the figure is α-SMA and the blue is the cell nucleus.

[0088] Example 7 Multi-engineered stem cells for the treatment of liver fibrosis

[0089] Preparation of multi-engineered MSCs modified with heparin-lipid conjugates (specifically heparin-DSPE conjugates) and two circular RNA formulations:

[0090] First, 1×10 5 Mesenchymal stem cells were placed in a cell culture flask until they reached 80% confluence. The culture medium was replaced, and cyclic VEGF RNA preparation (RNA 0.1 mg / mL) and cyclic HGF RNA preparation (RNA 0.1 mg / mL) were added. After 12 hours of culture, the culture medium was replaced, and heparin-DSPE conjugate (1 mg / mL) was added. After 12 hours of culture at 37°C, the cells were washed, digested, centrifuged, and collected to obtain multi-engineered stem cells for subsequent injection.

[0091] The experimental steps for multi-engineered stem cell therapy for liver fibrosis are as follows:

[0092] Male Balb / c mice aged 6-8 weeks were randomly divided into five groups (n=6): G1, G2, G3, G4 and G5.

[0093] To establish the liver fibrosis model, Balb / c mice in the G2-G5 groups were intraperitoneally injected with 20% v / v CCl4 olive oil solution (injection dose: 0.75 mL / kg), while the G1 group received only the same volume of olive oil injection twice a week for five weeks. In the third week, mice in the G3, G4, and G5 groups received MSCs (5×10 6 The G1 and G2 groups received an equal volume of PBS. The specific groupings are as follows:

[0094] G1 (control group): healthy mice;

[0095] G2 (model group): mice with liver fibrosis;

[0096] G3: Heparin-DSPE conjugate-modified MSCs for the treatment of liver fibrosis mice (modification method refers to Example 2);

[0097] G4: Two circular RNA preparations modified MSCs to treat liver fibrosis mice (the modification method refers to Example 5, the circular VEGF RNA preparation and the circular HGF RNA preparation are modified at the same time, and the dosage of both preparations is 0.1 mg / mL of RNA);

[0098] G5: Treatment of mice with liver fibrosis with multi-engineered MSCs modified with heparin-lipid conjugate (heparin-DSPE) and two circular RNA formulations.

[0099] Five weeks after injection, the livers of mice in different experimental groups were collected, sliced, and stained. Figure 7 As shown, the livers in group G2 had the most obvious fibrotic appearance, while the appearance of the livers in group G5 tended to that of the healthy livers in group G1. Figure 8 Sirius red staining also confirmed this conclusion. The G2 group showed obvious red fibrous lines, and the fibrosis lines in the G3, G4, and G5 groups gradually disappeared, indicating that liver fibrosis was significantly improved.

[0100] The present invention provides a multi-engineered stem cell preparation and its preparation method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing a multi-engineered stem cell preparation, characterized in that: The steps include: (1) preparing a heparin-lipid conjugate: coupling heparin and a first lipid through a condensation reaction; (2) preparing a circular RNA preparation: mixing a circular RNA solution with a second lipid solution, and obtaining a circular RNA preparation by ultrafiltration; the circular RNA comprises an IRES sequence and a target sequence, and the 3' end of the IRES sequence is connected to the 5' end of the target sequence, and the 5' end of the IRES sequence is connected to the 3' end of the target sequence, to obtain the circular RNA; Wherein, the target sequence consists of a signal peptide and an anti-fibrotic growth factor; (3) Preparing multi-engineered stem cells: culturing the stem cells until the cell confluence is 70% to 85%, adding the circular RNA preparation prepared in step (2) and culturing for 4 to 72 hours, then replacing the culture medium with fresh culture medium, adding the heparin-lipid conjugate prepared in step (1) and continuing to culture for 4 to 72 hours to obtain a multi-engineered stem cell preparation.

2. The preparation method according to claim 1, characterized in that: In step (1), the heparin has a weight average molecular weight of 10 to 20 kDa; The first lipid is any one of 1,2-tetradecanoylphosphatidylethanolamine, 1,2-dipalmitoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, 1,2-dioleyl-sn-glycerol-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine or sphingomyelin.

3. The preparation method according to claim 2, characterized in that: In step (1), the heparin-lipid conjugate has a structure as shown in Formula I: Among them, the blue frame is the first lipid, n is any even number between 10 and 20; the weight average molecular weight of heparin is 10 to 20 kDa.

4. The preparation method according to claim 1, characterized in that: In step (1), the reaction molar ratio of heparin to the first lipid is 1:1; the reaction temperature of the condensation reaction is 60-70° C., and the reaction time is 12-36 hours.

5. The preparation method according to claim 1, characterized in that: In step (2), the IRES sequence is a CVB3IRES sequence, and its nucleotide sequence is shown in SEQ ID No. 1; The anti-fibrotic growth factor is any one of VEGF and HGF, and the nucleotide sequences thereof correspond to SEQ ID No. 2 to 3 respectively; The signal peptide, the nucleotide sequence of which is shown in SEQ ID No.4; The circular RNA preparation is any one of a circular HGF RNA preparation or a circular VEGF RNA preparation or a combination of the two.

6. The preparation method according to claim 1, characterized in that: In step (2), the circular RNA is prepared according to the following method: the IRES sequence is cut into two segments from the 5' end to the 3' end at the cleavage site, the front segment is IRES segment 2, and the rear segment is IRES segment 1, the 5' end of the target sequence is connected to the 3' end of IRES segment 1, and the 3' end of the target sequence is connected to the 5' end of IRES segment 2 to obtain a linear RNA sequence, and the linear RNA sequence is cyclized to obtain a circular RNA; The target sequence is obtained by connecting the 3' end of the signal peptide and the 5' end of the anti-fibrotic growth factor; The cleavage site, from the 5' end to the 3' end, is any one of the 346th, 359th, 369th, 391st, 401st or 453rd sites of the IRES sequence.

7. The preparation method according to claim 1, characterized in that: In step (2), the concentration of circular RNA in the circular RNA solution is 0.05-5 μg / μL, and the solvent is citric acid buffer; The second lipid solution is prepared by mixing ionizable lipid, cholesterol, phospholipid and PEG-modified lipid in a molar ratio of 40-60:30-40:5-15:1-2; The ionizable lipids include any one or a combination of C12-200, cKK-E12, OF-02, DODAC, DDAB, DLin-MC3-DMA, DLinkC2DMA, DMRIE, DOSPA, DODAC, DMRIE, DOGS, DODAP, DODMA, DLinDMA, DLenDMA, CLinDMA, DLinDAP, SM-102, ALC-0315 and JK-102-CA; The phospholipids include 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol sodium salt, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, Any one or a combination of alcoholamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, cephalin, ceramide, diacylglycerol, cerebroside or sphingomyelin; The PEG-modified lipid includes any one or a combination of DSPE-PEG1000, DSPE-PEG2000, DMG-PEG1000, DMG-PEG1300, DMG-PEG1500, DMG-PEG1800, DMG-PEG2000, DMG-PEG2200, DMG-PEG2500, DMG-PEG2700, DMG-PEG3000, DMG-PEG3200, DMG-PEG3500, DMG-PEG3700, DMG-PEG4000, DMG-PEG4200, DMG-PEG4500, DMG-PEG4700, DMG-PEG5000 or ceramide-PEG 2000; The mixing volume ratio of the circular RNA solution and the second lipid solution is 1:1 to 10:1; The ultrafiltration has a molecular weight cut-off of 90 to 120 kDa.

8. The preparation method according to claim 1, characterized in that: In step (3), the stem cells include any one of embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, muscle stem cells, liver stem cells, cardiac stem cells, dental pulp stem cells, adipose stem cells, amniotic membrane stem cells, corneal epithelial stem cells, osteoblastic stem cells or placental stem cells.

9. The preparation method according to claim 1, characterized in that: In step (3), the concentration of the circular RNA preparation added is 0.05 to 10 mg / mL; the concentration of the heparin-lipid conjugate added is 0.1 to 10 mg / mL.

10. The multi-engineered stem cell preparation prepared by the preparation method according to any one of claims 1 to 9.

11. Use of the multi-engineered stem cell preparation according to claim 10 in the preparation of a drug for treating liver fibrosis.

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