Construction method of functional exosome for efficiently loading specific miRNA
阅读说明：本技术 一种高效装载特异性miRNA的功能性外泌体构建方法 (Construction method of functional exosome for efficiently loading specific miRNA ) 是由 李倩坤 胡文治 张翠萍 付小兵 于 2020-12-01 设计创作，主要内容包括：本发明公开了一种高效装载特异性miRNA的功能性外泌体构建方法,为了使外泌体能够更加高效地携带具有特定调控功能的miRNA,以更加精准高效地发挥靶向调控作用,利用MS2噬菌体衣壳蛋白,编辑构建特定miRNA分子的捕捉元件,重编程胎盘间充质干细胞,使其分泌的外泌体高效装载目的miRNA分子,从而递送至组织细胞发挥有效调控作用,为将来实现特异性精准治疗提供一种新的策略。(The invention discloses a method for constructing a functional exosome capable of efficiently loading specific miRNA, which aims to enable the exosome to more efficiently carry miRNA with a specific regulation function so as to more accurately and efficiently play a targeted regulation role, utilizes the capsid protein of an MS2 bacteriophage, edits and constructs a capture element of specific miRNA molecules, reprograms placenta mesenchymal stem cells, enables the secreted exosome to efficiently load target miRNA molecules, thereby delivering the target miRNA molecules to histiocytes to play an effective regulation role, and provides a new strategy for realizing specific precise treatment in the future.)
1. A method for constructing a functional exosome efficiently loading a specific miRNA is characterized by comprising the following steps:
s1, connecting an MS2 protein coding gene with a C1C2 structural domain in an exosome Lactadherin protein by using an MS2 phage capsid protein to construct a C1C2-MS2 lentiviral plasmid;
s2, connecting a pac protein of a locus for riveting with MS2 with a target miRNA to construct a pac-miRNA-pac lentiviral plasmid, wherein two ends of the pac-miRNA-pac lentiviral plasmid are provided with pac loci combined with MS 2;
s3, packaging the two plasmids obtained in the steps S1 and S2 into lentivirus infected mesenchymal stem cells, obtaining a determined stable transfer line through resistant drug screening, reserving the supernatant of the stem cells, and extracting exosomes by using an ultracentrifugation method.
2. The method according to claim 1, wherein the specific process of step S3 is as follows:
s3.1, respectively utilizing a three-plasmid lentivirus packaging system to transfect 293T cells by using a C1C2-MS2 lentivirus plasmid and a pac-miRNA-pac lentivirus plasmid, and packaging to obtain CM lentivirus and p-miRNA-p lentivirus;
s3.2, adding the mesenchymal stem cells PMSCs from the placenta chorion into a culture medium containing stem cells under the aseptic condition, and placing the mixture at the temperature of 37 ℃ and CO2Incubating in an incubator with a volume fraction of 5%; the stem cell culture medium contains 10% of fetal calf serum by mass;
s3.3, infecting PMSCs through CM lentivirus, screening the medicines by using a culture medium containing 1.0ug/mL puromycin after 48 hours, and obtaining a cell line CM-PMSCs stably expressing MS2 after 10-14 days;
s3.4, further infecting the CM-PMSCs with p-miRNA-p lentiviruses, screening the medicaments by using a culture medium containing 600 mug/mL G418 after 48 hours, and obtaining cell lines CM-miRNA-PMSCs for stably expressing the target miRNA after 2 weeks;
s3.5, incubating CM-miRNA-PMSCs by using a stem cell culture medium containing 10% of exosome-free serum in percentage by mass, collecting cell supernatant for 24-48h, and after ultrafiltration and concentration, obtaining the exosome CM-miRNA-Exo efficiently loaded with the target miRNA through an ultracentrifugation method and an exosome extraction kit.
3. The method according to claim 2, characterized in that in step S3.2, the stem cell culture medium is prepared from high-sugar DMEM and DMEM-F12 in a volume ratio of 1: 1 proportion, and contains 100U/mL streptomycin, 10ng/mL fibroblast growth factor and 10ng/mL epidermal growth factor.
Technical Field
The invention relates to the field of biomedical materials and cell molecule therapy, in particular to a method for constructing a functional exosome efficiently loaded with specific miRNA.
Background
With the rapid development of cellular molecular therapy, exosome therapy has become a hot spot for research in the field of biomedical science, and has received extensive attention. The exosome is a member of extracellular secretion vesicles, can be used as a transport carrier, carries components such as specific proteins and RNA in cells, is absorbed by fusion to a target cell membrane or endocytosis of the target cell, directly delivers molecules such as RNA into the target cell, and plays an effective regulation and control role on receptor cell tissues. Compared with stem cell therapy, the exosome derived from the stem cells not only contains various effective components secreted by the stem cells, but also can avoid transient rejection reaction brought by cell surface antigens derived from heterogenous sources, and is safer in clinical therapy application.
MicroRNA (miRNA) is an endogenous, small RNA of about 20-24 nucleotides in length that has a number of important regulatory roles within the cell. It is speculated that mirnas regulate one third of human genes and play a great role in cell differentiation, biological development and disease development. However, the single miRNA molecule has poor stability in vitro and needs to enter the inside of the cell to exert a regulatory effect, and the exosome can be used as a vector of miRNA to effectively deliver miRNA from cell to cell. Because the extracellular body carries limited intracellular protein and RNA quantity, complex components and low efficiency of specific action. In the traditional method, miRNA is over-expressed in cells simply by means of liposome transfection or electrotransfection and the like, miRNA molecules are still carried by means of the self-assembly capacity of exosome, and the loading efficiency is still limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for constructing a functional exosome for efficiently loading a specific miRNA.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for constructing a functional exosome efficiently loaded with specific miRNA comprises the following steps:
s1, connecting an MS2 protein coding gene with a C1C2 structural domain in an exosome Lactadherin protein by using an MS2 phage capsid protein to construct a C1C2-MS2(CM) lentiviral plasmid;
s2, linking a pac protein of a locus for riveting with MS2 with a target miRNA to construct a pac-miRNA-pac (p-miRNA-p) lentiviral plasmid, wherein both ends of the pac-miRNA-pac (p-miRNA-p) lentiviral plasmid have pac loci combined with MS 2;
s3, packaging the two plasmids obtained in the steps S1 and S2 into lentivirus infected mesenchymal stem cells, obtaining a determined stable transfer line through resistant drug screening, reserving the supernatant of the stem cells, and extracting exosomes by using an ultracentrifugation method.
Further, the specific process of step S3:
s3.1, respectively utilizing a three-plasmid lentivirus packaging system to transfect 293T cells by using a C1C2-MS2 lentivirus plasmid and a pac-miRNA-pac lentivirus plasmid, and packaging to obtain CM lentivirus and p-miRNA-p lentivirus;
s3.2, adding the mesenchymal stem cells PMSCs from the placenta chorion into a culture medium containing stem cells under the aseptic condition, and placing the mixture at the temperature of 37 ℃ and CO2Incubating in an incubator with a volume fraction of 5%; the stem cell culture medium contains 10% of fetal calf serum by mass;
s3.3, infecting PMSCs through CM lentivirus, screening the medicines by using a culture medium containing 1.0ug/mL puromycin after 48 hours, and obtaining a cell line CM-PMSCs stably expressing MS2 after 10-14 days;
s3.4, further infecting the CM-PMSCs with p-miRNA-p lentiviruses, screening the medicaments by using a culture medium containing 600 mug/mL G418 after 48 hours, and obtaining cell lines CM-miRNA-PMSCs for stably expressing the target miRNA after 2 weeks;
s3.5, incubating CM-miRNA-PMSCs by using a stem cell culture medium containing 10% of exosome-free serum in percentage by mass, collecting cell supernatant for 24-48h, and after ultrafiltration and concentration, obtaining the exosome CM-miRNA-Exo efficiently loaded with the target miRNA through an ultracentrifugation method and an exosome extraction kit.
Further, in step S3.2, the stem cell culture medium is prepared from high-glucose DMEM and DMEM-F12 in a volume ratio of 1: 1 proportion, and contains 100U/mL streptomycin, 10ng/mL fibroblast growth factor and 10ng/mL epidermal growth factor.
The invention has the beneficial effects that: in order to enable exosomes to carry miRNA with a specific regulation function more efficiently and to play a targeted regulation role more accurately and efficiently, the invention utilizes the capsid protein of the MS2 phage to edit and construct a capture element of specific miRNA molecules and reprograms mesenchymal stem cells of placenta to enable exosomes secreted by the exosomes to load target miRNA molecules efficiently, so that the exosomes are delivered to histiocytes to play an effective regulation role, and a new strategy is provided for realizing specific precise treatment in the future.
Drawings
FIG. 1 is a schematic diagram of construction of an engineered miR-146a exosome expressed efficiently in example 2 of the present invention;
FIG. 2 is a schematic diagram of the loading efficiency and functional verification results of CM-miR146a-Exo in example 2 of the present invention.
FIG. 3 is a schematic diagram of the results of the healing effect of CM-miR146a-Exo on the wound of a diabetic mouse in example 2 of the invention.
FIG. 4 is a schematic diagram of the network analysis of inflammatory-related proteins by CM-miR146a-Exo acting on the sequencing of wound tissue transcriptome of diabetic mice in example 2 of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, and it should be noted that the present embodiment is based on the technical solution, and the detailed implementation and the specific operation process are provided, but the protection scope of the present invention is not limited to the present embodiment.
Example 1
A method for constructing a functional exosome efficiently loaded with specific miRNA comprises the following steps:
s1, connecting an MS2 protein coding gene with a C1C2 structural domain in an exosome Lactadherin protein by using an MS2 phage capsid protein to construct a C1C2-MS2(CM) lentiviral plasmid;
s2, linking a pac protein of a locus for riveting with MS2 with a target miRNA to construct a pac-miRNA-pac (p-miRNA-p) lentiviral plasmid, wherein both ends of the pac-miRNA-pac (p-miRNA-p) lentiviral plasmid have pac loci combined with MS 2;
s3, packaging the two plasmids obtained in the steps S1 and S2 into lentivirus infected mesenchymal stem cells, obtaining a determined stable transfer line through resistant drug screening, reserving the supernatant of the stem cells, and extracting exosomes by using an ultracentrifugation method.
Further, the specific process of step S3 is:
s3.1, a CM lentivirus plasmid and a p-miRNA-p lentivirus plasmid are packaged by a three-plasmid lentivirus packaging system through transfecting 293T cells to obtain a CM lentivirus and a p-miRNA-p lentivirus;
s3.2, adding mesenchymal stem cells (PMSCs) from placenta chorion into stem cell culture medium containing 10% fetal calf serum under aseptic condition, placing at 37 deg.C and 5% CO2Culturing in an incubator;
s3.3, infecting PMSCs through CM lentivirus, screening the medicines by using a culture medium containing 1.0ug/mL puromycin after 48 hours, and obtaining a cell line CM-PMSCs stably expressing MS2 after 10-14 days;
s3.4, further infecting the CM-PMSCs with p-miRNA-p lentiviruses, screening the medicaments by using a culture medium containing 600 mug/mL G418 after 48 hours, and obtaining cell lines CM-miRNA-PMSCs for stably expressing the target miRNA after 2 weeks;
s3.5, incubating CM-miRNA-PMSCs by using a 10% exosome serum-free stem cell culture medium, collecting cell supernatant after 24-48 hours, and obtaining an exosome CM-miRNA-Exo efficiently loading the target miRNA through an ultracentrifugation method and an exosome extraction kit after ultrafiltration concentration.
Further, in step S3.2, the stem cell culture medium is prepared from high-glucose DMEM and DMEM-F12 in a volume ratio of 1: 1 proportion, and contains 100U/mL streptomycin, 10ng/mL fibroblast growth factor and 10ng/mL epidermal growth factor.
Example 2
This example provides a method for constructing a functional exosome with anti-inflammatory effect, which efficiently carries miRNA-146a, based on the method described in example 1.
A large number of researches show that miRNA-146a plays an important role in regulating and controlling inflammatory response. IRAK is a classical downstream target gene of miR-146a and is also a key regulatory factor of an NF-kB pathway, and miR-146a can inhibit activation of the NF-kB pathway by regulating IRAK 1. Therefore, in the embodiment, the engineered exosomes act on epidermal cells, the expression level of IRAK1 in the epidermal cells is detected, the anti-inflammatory effect of the CM-miR146a functional exosomes (CM-miR146a-Exo) is verified, the healing effect of the CM-miR146a-Exo on inflammatory wounds of diabetic mice is observed, and differential gene expression and related inflammatory proteins and signal pathways are analyzed through wound tissue transcriptome sequencing.
The specific process is as follows:
1. constructing pac-pre-miR-146a-pac lentivirus plasmid: inquiring the gene sequence of the miR-146a precursor, constructing a pre-miR-146a lentiviral plasmid of the miRNA-146a precursor, wherein two sides of the pre-miR-146a lentiviral plasmid are respectively provided with a group of pac anchoring sites combined with MS2 to form a pac-pre-miR-146a-pac (p-miR146a-p) lentiviral plasmid, as shown in figure 1A. FIG. 1A is a schematic of the construction of engineered exosomes: by reprogramming CM and p-miR146a-p lentivirus plasmid genes, the exosome derived from the stem cell is enabled to efficiently carry miR-146a targeted regulatory molecules.
2. 293T cells were cultured to package lentivirus: 293T cells were collected by trypsinization and plated on a 35mm dish using appropriate complete media to achieve a cell fusion area of more than 80% of the total area of the dish. Place the cells in a solution containing 5% CO2And incubating for 8-24h in an incubator at 37 ℃, starting transfection when the cells are completely attached to the wall, and changing the solution 2h before transfection.
3. Packaging p-miR146a-p lentivirus: p-miR146a-p lentiviral plasmid was transfected into 293T cells with a viral packaging plasmid Mix:1 μ G/μ l (Mix ═ pMDL: VSV-G: REV ═ 5:3:2) via lipo3000 using a three plasmid system. After 6h the plasmid transfection medium was removed, 2.5mL of complete medium pre-heated at 37 ℃ was added and the cells were placed in the incubator for further incubation.
4. Collecting p-miR146a-p lentivirus supernatant: after 48h, the supernatant containing the lentivirus can be collected, centrifuged at 1500rpm for 5 minutes (min), and 2mL of the lentivirus supernatant can be collected generally and can be directly used for infection of secretion cells of exosomes, or subpackaged and stored at-80 ℃.
p-miR146a-p lentivirus infection: CM-PMSCs (obtained as described in example 1) were plated on 6-well plates with an optimal density of 30-70%. Adding collected p-miR146a-p lentivirus supernatant to infect CM-PMSCs, changing to a normal stem cell culture medium after 12h, adding a culture medium containing 600 mug/mL G418 to perform drug screening on cells after 48h, and obtaining a cell line (CM-miR146a-PMSCs) for stably expressing miR-146a after 2 w.
6. Extracting an exosome for efficiently expressing miR-146 a: and (2) incubating CM-miR146a-PMSCs by using a 10% exosome serum-free stem cell culture medium, collecting cell supernatant after 24-48 hours, and obtaining an exosome CM-miR146a-Exo efficiently expressing miR-146a by an ultracentrifugation method and an exosome extraction kit after ultrafiltration concentration. Morphology of engineered exosomes derived from placental mesenchymal stem cells was observed by SEM and particle size analysis was performed on exosomes (as shown in fig. 1B, 1C). From fig. 1B, it can be seen that the engineered exosomes derived from placental mesenchymal stem cells were observed by transmission electron microscopy as discoid vesicles. From FIG. 1C, it can be seen that the engineered exosomes have particle size detection diameters of 70-120 nm. From FIG. 1D, Western blot identifies that the marker proteins CD63, CD9 and TSG101 specific to the surface of the exosome are positive in expression and the endoplasmic reticulum specific molecule Calnexin is negative in expression.
7. Detecting the loading efficiency of the engineered exosome with miR-146 a: and detecting the expression quantity of miR-146a in an exosome after PMSCs are infected by miR-146a and CM lentivirus through QRT-pcr. The results show that the relative expression amount of miR-146a in the CM-miR146a-Exo group is remarkably increased and is nearly ten-fold higher than that in the exosome miR146a-Exo group which simply over-expresses miR-146a (as shown in figure 2A). As can be seen from FIG. 2, the relative expression level of miR-146a in the CM-miR146a-Exo group is remarkably increased, and compared with the miR-146a group which is only over-expressed, the relative expression level of miR-146a is increased by nearly ten times.
Validation of CM-miR146a functional exosome targeting anti-inflammatory function: the miR-146a has an inhibition and regulation effect on a downstream target gene IRAK 1. IRAK1 is a key regulator of NF-kB pathway, and miR146a can inhibit activation of NF-kB pathway by regulating IRAK 1. By engineering exosomes to act on epidermal cells, the inhibition level of miR-146a on downstream target gene IRAK1 is detected by using a dual-luciferase experiment, so that the anti-inflammatory function of the exosomes is verified. The result shows that after the exosome which simply over-expresses miR-146a acts on the epidermal cell, the relative fluorescence value of IRAK1 is reduced by 38%, and the relative fluorescence value of IRAK1 in the cell which acts on CM-miR146a-Exo is obviously reduced by 81%, which indicates that CM-miR146a-Exo obviously inhibits the expression of the downstream inflammatory factor IRAK1 (as shown in figure 2B). As can be seen from FIG. 2B, the dual-luciferase experiment shows that the relative fluorescence value of IRAK1 of epidermal cells acted by CM-miR146a-Exo is remarkably reduced, which indicates that the inhibitory effect of CM-miR146a-Exo on IRAK1 is the most remarkable.
The effect of CM-miR146a functional exosomes on the inflammatory wound surface of diabetic mice: constructing a full-layer skin wound surface with the diameter of 1cm on the back of a diabetic mouse, respectively acting the wound surface on a blank control and different exosome components, observing the healing condition of the wound surface of each group, and carrying out statistical analysis on the residual wound surface area and the wound surface closure rate of each group. The results are shown in FIG. 3A, B, C. From fig. 3A, it can be seen that the general healing conditions of diabetic wounds at day 14 and day 21 are substantially improved, and compared with other control groups, the CM-miR146a-Exo group has the advantages that the wound healing speed is remarkably increased, and the effect of promoting wound repair is achieved. Fig. 3B shows that the residual area of the wound surface in CM-miR146a-Exo group is significantly smaller than that in the wound surface in the contemporary control group through statistical analysis of the residual wound surface area in each group. As can be seen from fig. 3C, the CM-miR146a-Exo group acted on the wound surface faster than the diabetic wound surface control group, and the wound surface healing rate was (original wound surface area-residual wound surface area)/original wound surface area × 100%.
Analysis of inflammatory-related proteins of CM-miR146a functional exosomes acting on sequencing of diabetic wound tissue transcriptome: differential gene expression and related inflammatory proteins and signal pathways involved in regulation are analyzed through transcriptome sequencing of wound tissues. As shown in figure 4, the results show that in skin wound tissues acted by CM-miR146a-Exo, the expression levels of inflammatory related proteins IL-1a, IL-1B, IL-11, TNF, NF-kB 1, Rela and the like are reduced, and related inflammatory regulation and control channels are inhibited. The CM-miR146a functional exosome is shown to have a remarkable anti-inflammatory effect.
Various corresponding changes and modifications can be made by those skilled in the art based on the above technical solutions and concepts, and all such changes and modifications should be included in the protection scope of the present invention.