Application of IGF-1 gene modified mesenchymal stem cells in treatment of premature ovarian failure

Abstract

The invention provides an application of a mesenchymal stem cell modified by an IGF-1 (Insulin-like Growth Factor 1) gene in treatment of premature ovarian failure. The invention provides a modified IGF-1mRNA (Insulin-like Growth Factor). The modified IGF-1mRNA comprises a 5'end capping structure (Cap) and a 3 '-polyA tailed structure of an RNA (Ribonucleic Acid) sequence, and encapsulating the modified IGF-1 mRNA (messenger Ribonucleic Acid) by using an ionizable lipid. The invention further provides a preparation method of the liposome based on the ionizable lipid, a method for improving the transfection efficiency of the IGF-1 in-vitro transfected mesenchymal stem cells and the IGF-1 transfected mesenchymal stem cells obtained by adopting the method, MSCs are promoted to continuously and stably express IGF-1, the MSCs can stably secrete factors to promote cell proliferation and differentiation, and the granular cell secretion activity is maintained. The IGF-1 transfected mesenchymal stem cells can be used for preparing a medicine for treating premature ovarian failure, the IGF-1 modified MSCs can assist the premature ovarian failure in recovery, and the technical problem that the treatment effect cannot be improved due to the fact that the dosage of the MSCs is limited in the clinical treatment process can be solved.

Description

Technical Field

[0001] The present invention belongs to the field of medical application of stem cells, and specifically relates to the application of mesenchymal stem cells modified with IGF-1 gene in the treatment of premature ovarian failure. Background Art

[0002] Premature ovarian failure (POF) is one of the common problems and diseases in women before the age of 40, characterized by amenorrhea, low estrogen levels, infertility, high gonadotropin levels, and lack of mature follicles. The etiology of this disease involves some genetic diseases, autoimmune diseases, and environmental factors. There are various methods to treat POF, but with little success. Nowadays, stem cells are used to treat POF, which have the potential for self-repair and regeneration and are effective in treating ovarian failure and infertility. Since mesenchymal stem cells (MSCs) can activate multiple mechanisms at the same time, many researchers believe that MSCs transplantation is the best and most effective method in cell therapy. IGF-1 is an effective FDA-approved treatment for short children, and it has beneficial effects on the entire musculoskeletal system. In addition to its anabolic properties, IGF-1 enhances tissue repair by promoting angiogenesis and reducing inflammation, necrosis, and apoptosis.

[0003] On the one hand, in vitro studies have shown that exogenous IGF-1 factor is an effective stimulator of cell proliferation and differentiation, regulating granulosa cell apoptosis and steroid production during follicular development. IGF-1 has the effect of promoting cell proliferation and preventing cell apoptosis in in vitro cultured follicles. Studies have shown that IGF-1 can not only stimulate the production of vascular endothelial growth factor (VEGF) and progesterone in granulosa cells, but also cooperate with gonadotropins to maintain the secretory activity of granulosa cells. On the other hand, the mechanism of stem cell transplantation for the treatment of premature ovarian failure can be divided into the following three categories. First, the transplanted stem cells can differentiate into oocytes. Second, the transplanted stem cells can differentiate into ovarian cells, mainly including granulosa cells. Third, the transplanted stem cells can secrete a variety of growth factors through paracrine pathways to restore ovarian function. In general, there is no literature reporting the treatment of premature ovarian failure with IGF-1 combined with MSCs, and there is no literature reporting the use of biological regulation to improve the treatment effect of MSCs for premature ovarian failure.

[0004] The application of transient protein expression induced by mRNA goes far beyond infectious disease vaccines. It also has great potential application value in cancer vaccines, protein replacement therapy, and gene editing components for rare genetic diseases. However, naked mRNA itself is extremely unstable and easily degraded by nucleases and self-hydrolysis. LNP encapsulation can protect mRNA from extracellular RNases and assist in the intracellular delivery of mRNA. With the approval of the two new crown vaccines and Onpattro, lipid nanoparticles (LNP) are undoubtedly considered to be the most promising and safest non-viral vectors for the in vivo delivery of exogenous mRNA. Dlin-MC3-DMA, SM102, and AL-0315 are the most preferred ionizable lipids for mRNA delivery.

[0005] At present, the lipid components of classic LNPs usually include four types: ionizable lipids (or cationic lipids), auxiliary lipids, cholesterol, and polyethylene glycol lipids (PEG-lipids). Microfluidic mixing technology is used to prepare nucleic acid lipid nanoparticles. The basic operation process is: 1) Dissolve various lipids in ethanol, dissolve mRNA in acidic water buffer, and quickly mix the two phases. By diluting the ethanol phase, the solubility of lipids is reduced, and lipid nanoparticles are gradually precipitated and solidified in the mixed solution to form lipid nanoparticles, while efficiently encapsulating mRNA. 2) Remove residual ethanol through buffer membrane ultrafiltration or dialysis to neutralize the pH value of the buffer.

[0006] At present, most of the commercial in vitro cell lipid transfection reagents are cationic liposomes. The in vitro transfection process is roughly as follows: 1) Dilute the mRNA with Buffer and mix thoroughly to make an mRNA diluent. 2) Slowly add the transfection reagent (ensuring that the volume ratio of mRNA diluent: transfection reagent is between 3:1 and 5:1), mix thoroughly and evenly to make a transfection complex. 3) Dilute the transfection complex with serum-free culture medium, incubate at room temperature for a certain period of time, and then add it to the culture medium for cell transfection. Its transfection efficiency for different cells is inconsistent and there is generally a certain degree of cell transfection toxicity.

[0007] Therefore, there is an urgent need to provide a method that can simplify the liposome preparation process for encapsulating IGF-1 mRNA and reduce the construction cost, while providing an IGF-1 in vitro transfection mesenchymal stem cell transfection system that can improve the safety of use and in vitro transfection efficiency, and solve the recovery process of premature ovarian failure and solve the technical problem that the treatment effect cannot be improved due to the limited dosage of MSCs during clinical treatment. Summary of the invention

[0008] In order to simplify the traditional LNP lipid preparation process, reduce the cost of vector construction, and at the same time improve the clinical use safety and in vitro transfection efficiency of the constructed vector components, the present invention independently developed an LNP-based in vitro liposome transfection method with a transfection efficiency of up to 99%, high biosafety, and simple operation. The in vitro transfection method can promote MSCs to efficiently and stably express IGF-1. It is a new non-viral vector method for LNP in vitro delivery, which is used for in vitro transfection of MSCs and greatly improves the in vitro transfection efficiency of current commercial kits.

[0009] The first object of the present invention is to provide a modified IGF-1, wherein the modified IGF-1 is the nucleotide sequence shown in SEQ ID NO.1, or the complementary sequence of the nucleotide sequence shown in SEQ ID NO.1.

[0010] SEQ ID NO.1 is:

[0011] (SEQ ID NO.1).

[0012] Furthermore, the modified IGF-1 sequence is obtained by the following modification:

[0013] The nucleotide sequence of IGF-1 before modification (NCBI Reference Sequence: NM_000618.5) is:

[0014]

[0015] The 5'-untranslated region, coding region, and 3'-untranslated region of the IGF-1 sequence shown in SEQ ID NO. 2 were modified respectively to construct a modified 5'-untranslated region (UTR)-modified TNFR1 mRNA sequence coding region-modified 3'-untranslated region (UTR), and a poly-A tail was added to the 3'-untranslated region (UTR);

[0016] Specifically, the IGF-1 coding region is modified by optimizing the gene sequence by designing the coding region sequence shown at 183 to 644 bp of SEQ ID NO.2 without changing its amino acid sequence through mammalian cell codon preference. The specific operation method can be optimized online at https: / / www.vectorbuilder.cn to select the sequence with the highest codon adaptation index (CAI). The optimized coding region sequence is:

[0017] atgggcaaaatttccagcctgcccacacagctgtttaaatgttgcttttgcgactttctgaaggtgaagatgcacaccatgtcctccagccacctgttttatctggccctgtgcct gctgaccttcacatctagcgccacagccggacctgagactctgtgtggcgccgagctggtggatgccctgcagttcgtgtgtggcgacaggggcttctacttcaacaaacccacagg ctacggcagcagctcccggcgcgccccccagaccggaattgtggacgagtgttgcttccggtcctgtgacctgaggaggctggaaatgtactgtgcccccctgaagcctgccaaga gcgcccgcagcgtgcgggctcagaggcacaccgacatgcccaaaacccagaaggaggtgcatctgaagaatgccagccggggatctgccggcaataagaactaccggatgtaa(SEQ ID NO.3);

[0018] The 5'-untranslated region is modified by replacing the 5'-untranslated region shown at bp 1 to 182 of SEQ ID NO.2 with a nucleotide sequence shorter than 100 bp to avoid the risk of mRNA degradation; in a certain embodiment, the nucleotide sequence shorter than 100 bp is taatacgactcactatagggagatcagagagaaaagaagagtaagaagaaatataagagccaccgccacc (SEQ ID NO.4)

[0019] The 3'-untranslated region modification method is to select globin as the 3'-untranslated region shown at 645 to 6633 bp of SEQ ID NO.2, and the sequence is:

[0020] gctgccttctgcggggcttgccttctggccatgcccttcttctctcccttgcacctgtacctcttggtctttgaataaagcctgagtaggaaggc ggccgctcgagcatgcatctaga (SEQ ID NO. 5);

[0021] The sequence to add a poly-A tail at the end is:

[0022] aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaatgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO. 6).

[0023] The second object of the present invention is to provide a modified IGF-1 mRNA, which is obtained by transcribing the aforementioned modified IGF-1 sequence and adding a cap at the 5' end.

[0024] Furthermore, the modified IGF-1 nucleotide sequence obtained by transcription is shown as SEQ ID NO.7.

[0025] uaauacgacucacuauagaggggagaucagagagaaaagaagaguaagaagaaauauaagagccaccgccaccaugggcaaaauuuccagccugcccacacagcuguuuaaauguugcuuuugcgacuuucugaaggugaagaugcaccauguccuccagccaccuguuuuaucuggcccugugccugcuga ccuucacaucuagcgccacagccggaccugagacucuguguggcgccgagcugguggaugcccugcaguucguguguggcgacaggggcuucuacuucaacaaacccacaggcuacggcagcagcucccggcgcgccccccagaccggaauuguggacgagugugcuuccgguccugugaccugaggaggcug gaaauguacugccccccugaagccugccaagagcgcccgcagcgugcgggcucagaggcacaccgacaugcccaaaacccagaaggaggugcaucugaagaaugccagccggggaucugccggcaauaagaacuaccggauguaagcugccuucugcggggcuugccuucuggccaugcccuucuucuccc cuugcaccuguaccuuggucuuugaauaaagccugaguaggaaggcggccgcucgagcaugcaucuagaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

[0026] The third object of the present invention is to provide a liposome based on ionizable lipids, wherein the liposome is an ionizable lipid that encapsulates the aforementioned modified IGF-1 mRNA, the ionizable lipid is a combination of SM102, cholesterol, DSPC, and DMG-PEG2000, and the molar ratio of SM102, cholesterol, DSPC, and DMG-PEG2000 is 40-50:38.5:10:1.5-3; the N / P ratio of the ionizable lipid to the modified IGF-1 mRNA is 4-6:1.

[0027] The fourth object of the present invention is to provide a method for preparing the aforementioned ionizable lipid-based liposomes, which is simple and rapid, does not require incubation, and can be used immediately after vortex mixing. The preparation method comprises the following steps:

[0028] (1) SM102, cholesterol, DSPC, and DMG-PEG2000 were mixed according to the prescribed amount and dissolved in anhydrous ethanol to form the organic phase;

[0029] (2) taking the modified IGF-1 mRNA according to the N / P ratio and dissolving it in acetic acid-sodium acetate buffer at pH 5.5 as the aqueous phase;

[0030] (3) adding the organic phase (comprising the liposomes) to the aqueous phase (comprising the modified IGF-1 mRNA) under vortexing conditions, and then continuously vortexing to complete the encapsulation, thereby obtaining the ionizable lipid-based liposomes.

[0031] Furthermore, the vortexing time is 20-25 seconds, and the vortexing is continued for 5-10 seconds after the dropwise addition is completed.

[0032] A fifth object of the present invention is to provide a method for improving the transfection efficiency of IGF-1 in vitro transfection of mesenchymal stem cells, the method comprising the following steps:

[0033] (1) adding the aforementioned ionizable lipid-based liposomes to a serum-free medium (opti-MEM), wherein the mass volume ratio of the ionizable lipid-based liposomes to the serum-free medium is 2-6 μg:940-980 μl, to form a transfection complex;

[0034] (2) culturing the mesenchymal stem cells until the cell confluence reaches 70%, discarding the old culture medium, washing with PBS, and then adding the transfection complex described in step (2);

[0035] (3) Mesenchymal stem cells were transfected in vitro after incubation.

[0036] The sixth object of the present invention is to provide mesenchymal stem cells transfected with IGF-1.

[0037] Furthermore, the mesenchymal stem cells transfected with IGF-1 are obtained by transfection using the aforementioned transfection method.

[0038] The seventh object of the present invention is to provide the use of the aforementioned modified IGF-1 or the aforementioned modified IGF-1 mRNA or the aforementioned ionizable lipid-based liposome or the aforementioned preparation method or the aforementioned method or mesenchymal stem cells transfected with IGF-1 in the preparation of a drug for treating premature ovarian failure.

[0039] The technical solution of the present invention has the following beneficial effects compared with the prior art:

[0040] The present invention designs the mRNA sequence of IGF-1 for the first time and optimizes it to obtain modified IGF-1 mRNA (SEQ ID NO.1). Modification methods such as capping and tailing can effectively reduce the degradation rate of in vitro synthesized IGF-1 mRNA and significantly improve the translation efficiency of IGF-1 mRNA.

[0041] Based on the modified IGF-1 mRNA, the present invention also provides a novel in vitro efficient transfection method of non-viral vectors, which promotes MSCs to continuously and stably express IGF-1.

[0042] The present invention introduces exogenous IGF-1 gene into MSCs for the first time, that is, transfects the cytokine IGF-1 gene into MSCs, so that MSCs can stably secrete factors to promote cell proliferation and differentiation and maintain the secretion activity of granulocytes.

[0043] The present invention discovered for the first time that IGF-1 gene-modified mesenchymal stem cells have a significant effect in enhancing the efficacy of premature ovarian failure, providing a new method for treating premature ovarian failure. The use of IGF-1-modified MSCs can assist in the recovery process of premature ovarian failure and solve the technical problem of being unable to enhance the therapeutic effect due to the limited dosage of MSCs during clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 .IVT preparation of IGF-1 mRNA; (A) agarose gel electrophoresis to detect plasmid enzyme digestion; (B) denaturing gel electrophoresis to detect the quality of synthesized mRNA; (C) denaturing gel electrophoresis to detect the quality of tailed mRNA.

[0045] Figure 2 .A research idea for an in vitro transfection method of liposomes based on ionizable lipids (the dotted box indicates the research point).

[0046] Figure 3 .Gel electrophoresis of SM1-SM6 LNP liposome formulation.

[0047] Figure 4 .SM1-SM6 LNP liposome formulation 2 / 4 / 6μg transfection 24h green fluorescence expression.

[0048] Figure 5 . Quantification of transfection efficiency of SM1-SM6 LNP liposome formulations 2 / 4 / 6μg 24h.

[0049] Figure 6 .TransIT-mRNAKit MSC (positive control group) transfection green fluorescence expression and efficiency quantification, among which, Figure 6 A is the green fluorescence expression graph; Figure 6 B is the quantitative graph of transfection green fluorescence efficiency.

[0050] Figure 7 .MSCs secrete IGF-1 after SM5 was transfected with IGF-1.

[0051] Figure 8 HE staining of ovarian tissue after in situ injection of MSCs and IGF-1-MSCs; (A) blank control group; (B) premature ovarian failure group; (C) MSCs injection group; (D) IGF-1-MSCs injection group. DETAILED DESCRIPTION

[0052] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0053] Example 1 Preparation and Quality Control of IGF-1 mRNA

[0054] The IGF-1 mRNA sequence was designed and optimized, plasmids were constructed, plasmids were linearized, capping transcription (Invitro transcription, IVT) was performed in vitro, and large-scale purification of mRNA was carried out. The optimized IGF-1 mRNA sequence and plasmid vector construction were commissioned to Nanjing Qingke Biotechnology Co., Ltd.

[0055] IVT mainly uses a linear modified IGF-1 DNA sequence as a template to prepare mRNA. The modified IGF-1 sequence is shown in SEQ ID NO.1, and the IGF-1 mRNA sequence is shown in SEQ ID NO.7. It can be directly synthesized or prepared through the following process steps:

[0056] 1) Plasmid linearization: Take 5 μg of the plasmid containing the modified IGF-1 sequence (SEQ ID NO.1), the plasmid vector is pcDNA3.1(+), add 3 μl of EcoRⅠ endonuclease, 15 μl of EcoRⅠ endonuclease buffer and double distilled water to a final system of 300 μl, and incubate at 37°C for 1 h;

[0057] 2) Purification of linearized plasmid: Use FastPure Gel DNA Extraction Mini Kit to purify the linearized plasmid. Take 50 μl linearization system as an example. Add 50 μl RNase-free water to each system, then add 500 μl Buffer GDP, mix well and transfer to the adsorption column. Centrifuge and discard the filtrate. Then add eluent and collect the eluate, which is the purified linear plasmid. Finally, use gel electrophoresis to determine the size and integrity of the product before using it in subsequent in vitro transcription experiments.

[0058] 3) In vitro transcription and capping: Use HiSynth T7 Co-transcription RNASynthesis Kit for in vitro capping and co-transcription, take 5 μg of linear plasmid and add it into an appropriate amount of nuclease-free water, then add 10 μl of 10× reaction buffer, 10 μl of 2×NTP / cap analog and 5 μl of T7 RNA Polymerase in sequence, mix well and incubate at 37°C for 3 hours to obtain the capped mRNA shown in SEQ ID NO.7;

[0059] 4) mRNA purification: use Use RNACleanup Kit for purification. Add 200 μl of concentrated binding buffer in the kit to every 100 μl, mix well, then add 0.1-1 ml of anhydrous ethanol and mix well. Centrifuge at high speed to pass through the column and discard the filtrate. Wash twice with 1 ml of washing buffer by high-speed centrifugation, dissolve the capped mRNA with 100 μl of elution buffer, elute by high-speed centrifugation, and collect the filtrate.

[0060] 5) Final purification of mRNA: Add 0.5 ml of clearing binding buffer, mix well, then add 0.5 ml of anhydrous ethanol and mix well, centrifuge at high speed to pass through the column and discard the filtrate; after washing twice with 1 ml of washing solution by high-speed centrifugation, add 30 μl of preheated nuclease-free water to the center of the central filter membrane of the filter column to dissolve the capped mRNA and elute by high-speed centrifugation, collect the filtrate to complete the preparation of IGF-1 mRNA with high translation stability.

[0061] Plasmid digestion was detected by agarose gel electrophoresis, and the quality of synthesized mRNA was detected by denaturing gel electrophoresis. Figure 1 The product bands in lane 2 after linearization, transcription, co-capping and tailing are clear and meet the target product size, where A is the IGF-1 plasmid after enzyme digestion, the product size is 7196bp, and B is the modified IGF-1 mRNA after in vitro capping and transcription, the product size is 776nt, which is consistent with the size of the corresponding sequence of SEQ ID NO.1, indicating that the IVT preparation of IGF-1 mRNA is successful and meets the experimental requirements.

[0062] Example 2 A self-developed LNP-based liposome in vitro transfection method

[0063] The LNP components in Moderna mRNA1273 vaccine include ionizable lipids (SM102), cholesterol (Cholesterol), zwitterionic auxiliary phospholipids (DSPC), and PEGylated lipids (DMG-PEG2000).

[0064] The present invention uses the above four lipids to carry out research on optimizing the in vitro transfection method, and uses SM102, cholesterol, DSPC, and DMG-PEG2000 in a molar ratio of 35%-50%, 15%-40%, 10%-15%, and 1%-3%, respectively, and cross-optimizes 6 optimal lipid ratios and proportions for in vitro transfection (Table 1) by N / P 4:1-10:1. The buffer in the transfection process is an acetic acid-sodium acetate buffer with a pH of 5.5. The transfection incubation time is 10 minutes.

[0065] The steps of LNP liposome preparation are as follows:

[0066] 1. SM102, cholesterol, DSPC, and DMG-PEG2000 were dissolved in anhydrous ethanol at different molar ratios as the organic phase; the lipid components and proportions are shown in Table 1 below.

[0067] 2. According to the N / P calculation, the modified IGF-1 mRNA shown in SEQ ID NO.7 was dissolved in an acetic acid-sodium acetate buffer at pH 5.5 as the aqueous phase; N / P is specifically shown in Table 1 below;

[0068] Table 1 Lipid components and ratios

[0069]

[0070] 3. Use a pipette to add the organic phase prepared in step 1 under vortexing conditions of the aqueous phase (time is about 20 seconds). After adding, continue vortexing for 5 seconds to complete the encapsulation and obtain the ionizable lipid-based liposomes.

[0071] 4. Gel electrophoresis experiment:

[0072] Detect encapsulation by gel electrophoresis blocking experiment: Incubate the mRNA-LNP liposome solution with a small amount of PBS solution or Triton X-100, and perform agarose gel electrophoresis blocking experiment:

[0073] a) Weigh 2 g of agarose and dissolve it in 90 mL of nuclease-free water. Heat in a microwave oven until completely dissolved. Transfer the solution to a 50-60°C water bath until equilibrium is reached.

[0074] b) In a fume hood, add 10 mL NorthernMax TM Add 10× denaturing gel buffer to the balanced agarose solution and mix well;

[0075] c) Pour the agarose solution into a gel casting tray in a fume hood and allow it to solidify. The gel should be 0.6 cm thick for efficient transfer.

[0076] d) After the gel hardens, remove it and set aside for later use.TM Electrophoresis was performed in running buffer;

[0077] e) taking appropriate amounts of the mRNA-LNP-loaded liposome formulations for electrophoresis loading, and incubating the formulations with Triton X-100 for 10 min to lyse the mRNA-LNP;

[0078] f) incubating the above sample in a metal bath at 65° C. for 15 minutes to denature the RNA secondary structure;

[0079] g) Spin down the sample in a microcentrifuge and place on ice for 2 min;

[0080] h) Add the sample to the denaturing formaldehyde agarose gel using an RNase-free pipette tip;

[0081] i) Voltage 120mV, current 100mA, time 30min;

[0082] j) After electrophoresis, observe the results in a gel imaging analyzer and take photos.

[0083] A non-lysate group (without adding Triton X-100) was set up, and the remaining operations were the same as those of the above-mentioned lysate group except that step e) was not performed.

[0084] like Figure 3 The results of gel electrophoresis of the carriers of each component ratio showed that compared with the unlysed group, the washed nucleic acid electrophoresis bands were visible after Triton X-100 lysis, which indicated that the liposomes formed by LNPs of each ratio had good encapsulation.

[0085] 5. Cell transfection experiment:

[0086] a) One day before transfection, 1×10 5 MSCs were seeded into 12-well plates with viable cells / well for culture;

[0087] b) taking 20 / 40 / 60 μl of the mRNA-LNP liposomes shown in Table 1, i.e. 2, 4, and 6 μg, respectively, and adding 980 / 960 / 940 μl of serum-free culture medium (Opti-MEM, manufacturer: Shanghai Yuanpei Biotechnology Co., Ltd., specification 500 ml / bottle) to form 1 ml of transfection complex;

[0088] c) Observe that the MSCs cell confluence in the 12-well plate is about 70%. Discard the old culture medium before adding the sample, wash twice with PBS, and then add 1 ml of the transfection complex after the above SM1-SM6 incubation to the well plate;

[0089] d) After the cells were incubated for 4 hours after transfection, the supernatant transfection complex was discarded, 2 ml of PBS was added to each well and the plate was gently shaken to allow the PBS to fully rinse the plate. After washing twice, 1 ml of fresh MSCs complete medium (manufacturer: German HELIOS, specification 500 ml / bottle) was added and the plate was placed in a 37°C CO2 incubator for further incubation and observation for 24 hours.

[0090] The cells were observed under a fluorescence microscope 24 hours after transfection. The fluorescence of cells transfected with different doses of SM1-SM6 were as follows Figure 4 The flow cytometry quantitative transfection efficiency is as follows Figure 5 As shown. In this experiment, the MSCs that were not transfected were the negative control group, i.e., Negative control. The group transfected with the positive kit was the positive control group, i.e., Positive Control. As the transfection dose of SM1-SM6 increased within the range of 2, 4, and 6 μg, the transfection efficiency of the LNP liposomes in the SM1-SM6 group also showed an upward trend. However, the transfection efficiency of each group was above 90%, which was better than the existing commercial MSC in vitro transfection kit TranIT-mRNA Kit, which was only 70% (see Figure 6 ), therefore, the new LNP-based liposome in vitro transfection method not only has a simple construction process, but also has the advantage of greatly increasing the transfection efficiency.

[0091] 6. SM5 LNP liposome ratio delivery of IGF-1 mRNA transfection into MSCs

[0092] a) according to the transfection efficiency of SM1-SM6 above, the highest group SM5 LNP liposome ratio was selected for IGF-1 delivery to MSCs;

[0093] b) The protein secretion expression of IGF-1 after in vitro transfection of MSCs was quantitatively detected using ELISA kits at 0, 24, 48, 72, 96, and 120 hours after transfection, such as Figure 7 As shown in the figure, compared with MSC, the expression of IGF-1 in MSC+IGF-1 was significantly increased, indicating that the liposome and in vitro transfection method of the present invention can promote MSCs to express IGF-1 efficiently and stably; Example 3 Experiment on the treatment of premature ovarian failure with IGF-1-MSCs at the animal level

[0094] The rat model of premature ovarian failure was established by inducing the chemotherapy drug cyclophosphamide (CTX). MSCs were used as a control to investigate the ability of gene-empowered stem cells IGF-1-MSCs to improve premature ovarian failure (POF).

[0095] The experimental method is:

[0096] 1. Establishment of POF rat model: After 1 week of adaptive feeding of rats, the estrous cycle was observed by vaginal smear method, and 30 healthy female rats with normal estrous cycle were selected. They were randomly divided into control group (n=10) and premature aging model group (n=20). The rats in the model group were injected with 0.35mL of ovarian antigen subcutaneously at multiple points. The rats were exposed to 100mg / kg / d for 10 days, and the premature ovarian failure model was established one week after drug withdrawal. After CTX injection, the estrous cycle of rats was observed for 2 consecutive weeks, and the changes in E2 (17-estradiol), P4 (progesterone) and AMH (anti-Müllerian hormone) levels in the blood, as well as the morphological observation of ovarian sections, were combined to comprehensively evaluate the premature ovarian failure.

[0097] The experiment was divided into ovarian in situ injection group (n=12), POF blank control group (n=4) and blank control group (n=4). Two weeks after ovarian antigen injection, 12 rats with successful modeling in the model group were selected as the ovarian in situ injection group for MSC (6 rats) transplantation and IGF-1-MSCs (6 rats) transplantation; 4 rats with successful modeling in the model group were selected as the POF control group. The blank control group was the same group as the blank control group for tail vein injection. The blank control group and the POF group were not treated in any way and maintained normal feeding.

[0098] 2. MSCs in situ transplantation therapy: After the frozen P5 cells were recovered, the cells were gently washed with saline to make a cell suspension and adjusted to 1.6×10 7 / mL, and the cell suspension should be prepared before use, stored in an environment of 37°C, and used within 2 hours. The unilateral ovary of the rat was treated by in situ injection of MSCs and IGF-1-MSCs. Sodium phenobarbital was injected into each rat at a dose of 60 mg / kg by intraperitoneal injection. After 15 minutes, when the rat was completely anesthetized, the rat was fixed on the animal operating table with the abdomen facing up. The skin was disinfected with 75% alcohol. Pinch the skin of the lower abdomen of the rat with ophthalmic forceps, make a small cut horizontally with ophthalmic scissors, bluntly separate with ophthalmic forceps, cut the fascia, drag out the fat pad, and fix the ovary with forceps after determining the position of the ovary. Then, use a microsyringe to inject 25μL (4×10 5 cells)MSCs (MSC and IGF-1-MSCs group).

[0099] 3. Estrous cycle examination: The normal estrus cycle of rats includes the following 4 consecutive stages: proestrus, estrus, metestrus and diestrus. The stage is determined by the presence or absence of leukocytes, keratinized epithelial cells and nucleated epithelial cells. Vaginal cells were washed with saline and transferred to a slide, air-dried, and stained with Giemsa dye solution. After the third treatment, the rats were observed for 2 weeks every day.

[0100] 4. Animal treatment and sampling: After 3 weeks of treatment, the rats were eyeballed and blood was collected. After the blood was allowed to stand for 30 minutes, it was centrifuged at 4000r / min for 10 minutes, and at least 0.5mL of upper serum was collected. It was stored at -20℃ until the estrogen concentration in the serum was determined. After blood collection, the rats in each group were anesthetized, killed and dissected. Fresh ovarian tissue was taken and fixed in tissue fixative (4% paraformaldehyde) for making tissue sections. Among them, the sections made in the ovarian in situ injection group were divided into MSCs in situ transplantation injection side, IGF-1-MSCs group, and MSCs in situ transplantation non-injection side.

[0101] 5. Ovarian follicle counting and morphological analysis: Continuous sections were made with a thickness of 5 μm per slice, and one slice was taken every 20 μm. 20 slices were taken from each tissue, and after HE staining, they were observed under an optical microscope. Follicles were graded according to the characteristics of each grade of follicles according to the following standards.

[0102] (1) Primordial follicles: The oocyte is surrounded by a single layer of flat granulosa cells.

[0103] (2) Primary follicles: A complete, enlarged oocyte with a visible nucleus surrounded by one or more layers of cuboidal granulosa cells. The follicle is surrounded by a connective tissue membrane.

[0104] (3) Secondary follicles: They have two or three layers of cubic granulosa cells without an antral cavity. Early antral follicles contain newly emerged antral cavities and form a granulosa layer. The inner wall of the follicle has densely arranged follicular cells. The follicle membrane is divided into two layers: inner and outer layers.

[0105] (4) Mature follicles: with obvious cumulus and larger follicular cavity. With clear cumulus granulosa cell layer. The inner membrane cells of the follicle are polygonal, with tiny capillaries visible; the outer membrane cells are spindle-shaped, located in the outermost layer, and the boundary with the surrounding connective tissue is not obvious.

[0106] (5) Atretic follicles: The oocyte structure is incomplete, the follicle wall collapses, the granulosa cell layer is loose or even disappears, and the zona pellucida shrinks.

[0107] The results are as follows Figure 8 The ovaries of healthy rats in the control group contained a large number of healthy follicles at various stages, including primordial follicles, primary follicles, secondary follicles and mature follicles ( Figure 8A), while the ovaries of rats with premature ovarian failure were atrophic, with fewer growing follicles and more atretic follicles. Corpora lutea were visible, but there were few mature follicles. The number of follicles at all stages was also significantly reduced ( Figure 8 B). In contrast, the ovarian structures of rats injected with MSCs and IGF-1-MSCs were significantly improved, and the ovarian structures of rats injected with IGF-1-MSCs were better. The ovarian structures were relatively complete and well developed, with follicles of all levels visible, and more corpora lutea and mature large follicles ( Figure 8 C and Figure 8 D).

[0108] Follicle classification was further used for follicle counting and statistical comparison (Table 2). The results showed that compared with the healthy rats in the blank control group, there was no significant difference in the number of follicles at all levels in the rats in the MSCs in situ injection group; compared with the premature ovarian failure control group, the number of primary follicles, mature follicles and corpora lutea in the rats in the MSCs in situ injection group increased significantly, and the number of follicles at all levels in the ovaries of the rats in the IGF-1-MSCs in situ injection group increased more significantly, and the effect of improving premature ovarian failure was better.

[0109] Table 2. HE staining of ovarian tissue after in situ injection of MSCs / IGF-1-MSCs (n=12)

[0110]

[0111]

[0112] Note: Mean+SD, *Compared with the model group, P<0.05; **Compared with the model group, P<0.01.

[0113] 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 scope of protection of the present invention.

Claims

1. A modified IGF-1, characterized in that The modified IGF-1 is the nucleotide sequence shown in SEQ ID NO.1, or the complementary sequence of the nucleotide sequence shown in SEQ ID NO.

1.

2. The modified IGF-1 according to claim 1, characterized in that The modified IGF-1 sequence is obtained by the following modification: The 5'-untranslated region, coding region, and 3'-untranslated region of the IGF-1 sequence shown in SEQ ID NO. 2 were modified respectively to construct a modified 5'-untranslated region (UTR)-modified TNFR1 mRNA sequence coding region-modified 3'-untranslated region (UTR), and a poly-A tail was added to the 3'-untranslated region (UTR); The nucleotide sequence of the modified IGF-1 sequence coding region is shown in SEQ ID NO.3; The modified 5'-untranslated region nucleotide sequence is shown in SEQ ID NO.4; The modified 3'-untranslated region nucleotide sequence is shown in SEQ ID NO.5; The sequence of the poly-A tail is shown in SEQ ID NO.

6.

3. A modified IGF-1 mRNA, characterized in that The modified IGF-1 sequence of claim 1 is transcribed and capped at the 5' end to obtain the modified IGF-1 sequence.

4. The modified IGF-1 mRNA according to claim 3, characterized in that The modified IGF-1 nucleotide sequence obtained by transcription is shown in SEQ ID NO.

7.

5. A liposome based on ionizable lipids, characterized in that The liposome is an ionizable lipid that encapsulates the modified IGF-1 mRNA according to claim 3 or 4, and the ionizable lipid is a combination of SM102, cholesterol, DSPC, and DMG-PEG2000, and the molar ratio of SM102, cholesterol, DSPC, and DMG-PEG2000 is 40-50:38.5:10:1.5-3; the N / P ratio of the ionizable lipid to the modified IGF-1 mRNA is 4-6:

1.

6. The method for preparing liposomes based on ionizable lipids according to claim 5, characterized in that: The preparation method comprises the following steps: (1) SM102, cholesterol, DSPC, and DMG-PEG2000 were mixed according to the prescribed amount and dissolved in anhydrous ethanol to form the organic phase; (2) taking the modified IGF-1 mRNA according to the N / P ratio and dissolving it in acetic acid-sodium acetate buffer at pH 5.5 as the aqueous phase; (3) adding the organic phase to the aqueous phase under vortexing conditions, continuing vortexing after the addition, and incubating at room temperature to complete encapsulation and obtain the ionizable lipid-based liposomes.

7. The preparation method according to claim 6, characterized in that: The vortexing time in step (3) is 20-25 seconds, and the vortexing time after the addition is continued is 5-10 seconds.

8. A method for improving the transfection efficiency of IGF-1 in vitro transfection of mesenchymal stem cells, characterized in that: The method comprises the following steps: (1) adding the ionizable lipid-based liposomes according to claim 5 to a serum-free medium, wherein the mass volume ratio of the ionizable lipid-based liposomes to the serum-free medium is 2-6 μg:940-980 μl, to form a transfection complex; (3) culturing the mesenchymal stem cells until the cell confluence reaches 70%, discarding the old culture medium, washing with PBS, and then adding the transfection complex described in step (2); (4) Mesenchymal stem cells were transfected in vitro after incubation.

9. Mesenchymal stem cells transfected with IGF-1, characterized in that: The method according to claim 8 is used for transfection.

10. Use of the modified IGF-1 according to claim 1 or 2, or the modified IGF-1 mRNA according to claim 3 or 4, or the liposome based on ionizable lipids according to claim 5, or the preparation method according to claim 6 or 7, or the method according to claim 8, or the mesenchymal stem cells transfected with IGF-1 according to claim 9 in the preparation of a drug for treating premature ovarian failure.

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