CN114107209B - Method for inhibiting THP-1 differentiation and macrophage M1 polarization by exosomes with high expression of miR-146a-5p for non-therapeutic purpose - Google Patents
Method for inhibiting THP-1 differentiation and macrophage M1 polarization by exosomes with high expression of miR-146a-5p for non-therapeutic purpose Download PDFInfo
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Abstract
The invention relates to the technical field of biological information, in particular to a method for inhibiting THP-1 differentiation and macrophage M1 polarization by exosomes with high expression of miR-146a-5p, which comprises the following steps: s1, preparing a high-expression miR-146a-5p exosome; s2, identifying exosomes; s3, culturing cells; s4, co-incubating exosome miR-146a-5p with THP-1 or macrophages; s5, analyzing experimental results, wherein the exosomes with high expression of miR-146a-5p, which are derived from liver cells, are prepared by a chemical transfection method, the actions of the exosomes on THP-1 differentiation and macrophage M1 polarization are explored, and genetic information substances carried by the exosomes derived from liver cells are more easily absorbed by the liver cells, wherein the exosomes obtained by the chemical transfection method have good integrity.
Description
Technical Field
The invention relates to the technical field of biological information, in particular to a method for inhibiting THP-1 differentiation and macrophage M1 polarization by exosomes with high expression of miR-146a-5p for non-therapeutic purposes.
Background
Non-alcoholic fatty liver disease (Non-alcoholic fatty liver disease, NAFLD) has become the chronic liver disease with the highest global prevalence, and studies have shown that 27% of NAFL patients progress to NASH stage, requiring drug therapeutic intervention once NASH is progressed, but no effective drug for specific treatment is currently available.
Research shows that macrophage M1 polarization can aggravate the progress of NASH, exosomes have the characteristics of high biocompatibility, escape lysosome mediated degradation, artificial modification and the like, therefore, the exosomes can be used as transport carriers of miRNAs to carry target miRNAs to specific tissues in a targeted manner to play a role in regulation, early research has proved that miR-146a-5p is a negative regulator of LPS-induced innate immunity and inflammatory response, and miR-146a-5p can inhibit release of inflammatory factors IL-1 beta, IL-6 and TNF-alpha in THP-1 cells after LPS treatment, and therefore, the invention provides a method for inhibiting THP-1 differentiation and macrophage M1 polarization by exosomes which express miR-146a-5p in a non-therapeutic purpose.
Disclosure of Invention
Aiming at the problems in the related art, the invention provides a method for inhibiting THP-1 differentiation and macrophage M1 polarization by exosomes with high expression of miR-146a-5p for non-therapeutic purposes, which aims to overcome the technical problems in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention provides a method for inhibiting THP-1 differentiation and macrophage M1 polarization by exosomes with high expression of miR-146a-5p for non-therapeutic purposes, which comprises the following steps:
s1, preparing a high-expression miR-146a-5p exosome, which specifically comprises the following steps:
(1) Respectively transfecting 50nmol/L miR-146a-5p mimic or negative control into HepG2 cells, and collecting cell culture medium supernatant after 48 hours of transfection;
(2) Obtaining an exosome with high expression of miR-146a-5p and an exosome of a control group by an ultracentrifugation method;
s2, identifying exosomes, specifically:
(1) Taking 20 μl of the extracted exosome solution, observing under a transmission electron microscope, and photographing to record the morphology of exosome;
(2) Taking 20 mu l of the extracted exosome suspension to a new EP tube of 1.5ml, adding 20 mu l of RIPA (note adding cocktail, 50:1), adding 5 XSDS (beta-mercaptoethanol) solution in a ratio of 4:1 (exosome suspension: SDS) after full cleavage, placing in a water bath of 100 ℃ for boiling for 5min, collecting a protein sample for electrophoresis, and detecting the expression of exosome marker protein CD 63;
(3) Isolating total RNA from exosomes, performing reverse transcription on the miRNA into cDNA, and quantifying the expression of miR-146a-5p by taking U6 as an internal reference;
s3, culturing cells;
s4, co-incubating exosome miR-146a-5p with THP-1 or macrophages;
s5, analyzing an experimental result.
Further, the step S3 specifically includes:
(1) HepG2 was cultured in DMEM medium supplemented with 10% fetal bovine serum, THP-1 in RPMI-1640 medium containing 10% fetal bovine serum, and both cells were cultured in a 5% CO2 incubator at 37 ℃;
(2) PMA was added to THP-1 cells in suspension culture to a final concentration of 100ng/ml to induce differentiation of THP-1 into macrophages.
Further, the step S4 specifically includes:
(1) Adding the exosomes with high expression of miR-146a-5p or exosomes of a control group into THP-1 or THP-1 differentiated macrophages (the final concentration of the exosomes is 50 mug/ml) for co-incubation for 48 hours;
(2) Taking U6 as an internal reference, quantifying the expression of THP-1 and miR-146a-5p in macrophages; quantifying the expression of MCP-1, TNF-a and IL-6 in macrophages by taking 18S as an internal reference;
(3) Cell samples were collected in 1.5mLEP tubes, after RIPA lysis was complete, 5 XSDS (beta-mercaptoethanol) was added at 4:1 to obtain protein samples, and after electrophoresis, the expression of CD68 in THP-1 cells and CD86 in macrophages was examined.
Further, the step S5 specifically includes:
(1) Compared with a control group, the expression of miR-146a-5p in HepG2 cells of the Mimic-146a-5p group and in exosomes of cell culture supernatants is obviously increased;
(2) After the aposome with high miR-146a-5p expression is incubated with THP-1 or macrophage for 48h, the expression of miR-146a-5p in the THP-1 or macrophage is obviously increased;
(3) The exosomes with high expression of miR-146a-5p can reduce the release of macrophage pro-inflammatory factors MCP-1, TNF-a and IL-6;
(4) The exosomes with high expression of miR-146a-5p can reduce the expression of a macrophage marker CD68 in THP-1 cells;
(5) The exosomes with high expression of miR-146a-5p can reduce the expression of the M1 type macrophage marker CD 86.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention relates to a method for inhibiting THP-1 differentiation and macrophage M1 polarization by exosomes of high-expression miR-146a-5p for non-therapeutic purposes, which is characterized in that exosomes of high-expression miR-146a-5p from liver cells are prepared by a chemical transfection method, and the exosomes of high-expression miR-146a-5p have the effect of inhibiting THP-1 differentiation and macrophage M1 polarization;
(2) The invention relates to a method for inhibiting THP-1 differentiation and macrophage M1 polarization by exosomes of high-expression miR-146a-5p for non-therapeutic purposes, which is characterized in that the exosomes obtained by a method of transfection and ultracentrifugation are good in integrity and high in purity;
(3) The invention relates to a method for inhibiting THP-1 differentiation and macrophage M1 polarization by exosomes with high expression miR-146a-5p for non-therapeutic purposes, wherein genetic information materials carried by exosomes derived from liver cells are easier to be absorbed by the liver cells;
(4) The invention relates to a method for inhibiting THP-1 differentiation and macrophage M1 polarization by exosomes with high expression miR-146a-5p for non-therapeutic purposes, which is an ideal natural drug delivery carrier due to the characteristics of low immunogenicity, low toxicity, high biocompatibility, free crossing of biological barriers, escape of lysosome-mediated degradation, artificial modification and the like;
(5) The invention provides a method for inhibiting THP-1 differentiation and macrophage M1 polarization for exosomes with high expression of miR-146a-5p for non-therapeutic purposes, provides a theoretical basis for treating non-alcoholic steatohepatitis by using exosomes as a tool in the follow-up process, and provides a new direction for treating various liver diseases.
Drawings
FIG. 1 is the external body form observed by a transmission electron microscope in example 1;
FIG. 2 shows the protein level of the exosome marker CD63 of example 1;
FIG. 3 is a graph showing the expression levels of miR-146a-5p in the exosomes over-expressing miR-146a-5p in example 1 and in the exosomes of the control group;
FIG. 4 is the expression of miR-146a-5p in THP-1 cells following transfer into exosomes highly expressing miR-146a-5p in example 1;
FIG. 5 is the expression of miR-146a-5p in macrophages following the transfer of an exosome highly-expressed miR-146a-5p in example 1;
FIG. 6 is a graph showing the change in protein levels of CD68 in THP-1 cells following transfer into exosomes highly expressing miR-146a-5p in example 1;
FIG. 7 is a graph showing the change in protein levels of CD86 in macrophages following the transfection of high miR-146a-5 p-expressing exosomes in example 1;
FIG. 8 shows the expression of pro-inflammatory factors MCP-1, TNF-a, IL-6 in macrophages following the transformation of exosomes with high expression miR-146a-5p in example 1.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A method for inhibiting the polarization of THP-1 differentiated macrophage M1 by exosomes highly expressing miR-146a-5p, comprising the following steps:
s1, preparing a high-expression miR-146a-5p exosome, which specifically comprises the following steps:
(1) HepG2 cells were inoculated into 10cm cell culture dishes (inoculation density 50%), after 12h of cell growth, 50nmol/L miR-146a-5p mimic or negative control was transfected into HepG2 cells respectively using RiboFECTTMCP Reagent, and after 48h of transfection, cell culture supernatant was collected for exosome preparation;
(2) The preparation method of the cell culture medium exosome comprises the following specific preparation methods:
1) Subjecting the cell culture medium to fractional centrifugation at 300g and 2000g for 20min to eliminate large dead cells and cell debris, removing sediment after each centrifugation, and reserving the supernatant for the next centrifugation;
2) Centrifuging the obtained supernatant at 20000g for 30min to remove larger microvesicles;
3) Centrifuging the obtained supernatant at 100,000g for 70min to collect exosome precipitate, washing the exosome precipitate with 6ml PBS once, centrifuging at 100,000g for 70min to obtain exosome with high expression of miR-146a-5p and exosome of control group;
s2, identifying exosomes, specifically:
(1) Transmission electron microscope: placing 20 μl of the extracted exosome solution on a copper grid, carrying out negative dyeing for 5min with 3% phosphotungstic acid aqueous solution, drying the grid under an incandescent lamp for 10min, placing under a transmission electron microscope for observation, and photographing to record the morphology of the exosome;
(2) Exosome marker protein detection: separately taking 20 μl of the extracted exosome suspension to a new EP tube of 1.5ml, adding 20 μl RIPA (note adding cocktail, 50:1), lysing for 30min at 4deg.C, not centrifuging, taking 2.5 μl of BCA, adding 5SDS (beta-mercaptoethanol) solution to the rest sample at a ratio of 4:1 (exosome suspension: SDS), boiling in 100deg.C water bath for 5min, electrophoresis using 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), transferring proteins to polyvinylidene fluoride (PVDF) membrane after electrophoresis, blocking membrane with 5% BSA at room temperature for 1h, then incubating overnight at 4deg.C with primary antibody CD63 (Santacruzsc-5275), and detecting protein expression of CD 63;
(3) And (3) detecting the expression quantity of miR-146a-5p in the exosome: total RNA was isolated from exosomes with Trizol (RNAexProReagent, AG), and miRNA was reverse transcribed to cDNA using the mircutePlusmiRNA first strand cDNA kit (TIANGEN, KR 211), and miR-146a-5p expression was quantified using the mircutePlusmiRNAqPCRKit (TIANGEN, FP 411) kit, with U6 as an internal reference;
s3, culturing cells;
s4, co-incubating exosome miR-146a-5p with THP-1 or macrophages;
s5, analyzing an experimental result.
The step S3 specifically includes:
(1) Purchasing a human liver cancer cell line HepG2 and a monocyte cell line THP-1, culturing the HepG2 in a DMEM medium added with 10% fetal bovine serum, culturing the THP-1 in an RPMI-1640 medium containing 10% fetal bovine serum, and culturing both cells in a 5% CO2 incubator at 37 ℃;
(2) THP-1 differentiation: PMA was added to THP-1 cells cultured in suspension to a final concentration of 100ng/ml to induce differentiation of THP-1 into macrophages, after 48 hours of induction, the medium was removed, and the cells were washed 2 times with PBS, and differentiation of THP-1 cells was observed under a microscope to obtain fully differentiated macrophages.
The step S4 specifically includes:
(1) Exosomes miR-146a-5p co-incubate with THP-1 or macrophages: adding the exosomes with high expression of miR-146a-5p or exosomes of a control group into THP-1 or THP-1 differentiated macrophages (the final concentration of the exosomes is 50 mug/ml) for co-incubation for 48 hours;
(2) Detection of the expression levels of miR-146a-5p in THP-1 cells and miR-146a-5p in macrophages, MCP-1, TNF-a and IL-6: collecting cells, isolating total RNA from the cells with Trizol (RNAexProReagent, AG), reverse-transcribing miRNA into cDNA using a miRcutePlusmiRNA first strand cDNA kit (TIANGEN, KR 211), and quantifying miR-146a-5p expression using a miRcutePlusmiRNAqPCRK (TIANGEN, FP 411) kit with U6 as an internal reference; reverse transcription of mRNA into cDNA using PrimeScript TMRT kit, quantification of MCP-1, TNF-a, IL-6 expression using SYBRGreenPCR kit, 18S as reference;
the QPCR primer sequences were:
(3) Detection of macrophage marker protein CD68 in THP-1 cells and M1 type macrophage marker protein CD86 in macrophages: cell samples were collected in 1.5mLEP tubes, washed 2 times with PBS, added with 100ul RIPA (containing 1. Mu. Protease inhibitor and 1. Mu. Phosphatase inhibitor), blown to complete digestion of the cells with a gun, after Votext shaking, the tubes were placed in a 4 degree decolorizing shaker for full lysis 30min, after Votext shaking, centrifuged at 12000RPM for 30min with a 4 degree centrifuge, the supernatant was extracted, added with 5 XSDS (containing beta-mercaptoethanol) at 4:1, then metal cooked at 100℃for 3-5min, and each sample was electrophoresed with 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membrane, the membrane was blocked with 5% BSA for 1h at room temperature, and then incubated with primary antibody CD86 (Santacruzsc-28347) overnight at 4℃to examine the protein expression of CD68 and CD 86.
The step S5 specifically comprises the following steps:
(1) Compared with a control group, the expression of miR-146a-5p in the exosomes of the cell culture supernatant of the Mimic-146a-5p group is obviously increased;
(2) After the exosomes with high miR-146a-5p expression are incubated with the macrophages for 48 hours, the miR-146a-5p expression in the macrophages is obviously increased;
(3) The exosomes with high expression of miR-146a-5p can reduce the release of pro-inflammatory factors MCP-1, TNF-a and IL-6.
(4) The exosomes with high miR-146a-5p expression can reduce the expression of macrophage marker CD68 in THP-1 cells
(5) The exosomes with high expression of miR-146a-5p can reduce the expression of the M1 type macrophage marker CD 86.
The invention prepares the exosome of the over-expression miR-146a-5p, and incubates the exosome with THP-1 or macrophage, and experimental results prove that the exosome miR-146a-5p can inhibit THP-1 differentiation and macrophage M1 polarization, and can reduce release of macrophage pro-inflammatory factors MCP-1, TNF-alpha and IL-6.
In order to ensure the structural integrity of exosome membranes, the exosome with high expression of miR-146a-5p from liver cells is prepared by a method of transfection and ultracentrifugation, and the invention proves that the exosome from liver cells is taken as a carrier to carry miR-146a-5p, so that THP-1 differentiation and macrophage M1 polarization can be inhibited, and the release of pro-inflammatory factors is reduced. The research provides theoretical basis for the subsequent treatment of nonalcoholic steatohepatitis by using exosomes as tools, and simultaneously provides a new direction for the treatment of various liver diseases.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (2)
1. A method for inhibiting THP-1 differentiation and macrophage M1 polarization in a non-therapeutic, high miR-146a-5 p-expressing exosome comprising the steps of:
s1, preparing a high-expression miR-146a-5p exosome, which specifically comprises the following steps:
(1) Inoculating HepG2 cells into a 10cm cell culture dish, after growing the cells for 12 hours, respectively transfecting 50nmol/L miR-146a-5p mimic or negative control into the HepG2 cells by using RiboFECTTMCP Reagent, and collecting cell culture medium supernatant after 48 hours of transfection for exogenesis;
(2) Subjecting the cell culture medium to fractional centrifugation at 300g and 2000g for 20min to eliminate large dead cells and cell debris, removing sediment after each centrifugation, and reserving the supernatant for the next centrifugation; centrifuging the obtained supernatant at 20000g for 30min to remove larger microvesicles; centrifuging the obtained supernatant at 100,000g for 70min to collect exosome precipitate, washing the exosome precipitate with 6ml PBS for one time, and centrifuging at 100,000g for 70min to obtain exosome with high expression of miR-146a-5p and exosome of control group;
s2, identifying exosomes, specifically:
(1) Taking 20 μl of the extracted exosome solution, observing under a transmission electron microscope, and photographing to record the morphology of exosome;
(2) Taking 20 mu l of the extracted exosome suspension to a new EP tube of 1.5ml, adding 20 mu l of RIPA, adding 5 XSDS solution according to a ratio of 4:1 after full cleavage, placing in a water bath of 100 ℃ for boiling for 5min, collecting a protein sample for electrophoresis, and detecting the expression condition of exosome marker protein CD 63;
(3) Isolating total RNA from exosomes, performing reverse transcription on the miRNA into cDNA, and quantifying the expression of miR-146a-5p by taking U6 as an internal reference;
s3, culturing cells: hepG2 was cultured in DMEM medium supplemented with 10% fetal bovine serum, THP-1 in RPMI-1640 medium containing 10% fetal bovine serum, and both cells were cultured in a 5% CO2 incubator at 37 ℃; PMA was then added to suspension-cultured THP-1 cells to a final concentration of 100ng/ml to induce differentiation of THP-1 into macrophages;
s4, co-incubation of exosome miR-146a-5p with THP-1 or macrophages: adding an exosome with high miR-146a-5p expression or a control exosome into THP-1 or THP-1 differentiated macrophages for co-incubation for 48h; taking U6 as an internal reference, quantifying the expression of THP-1 and miR-146a-5p in macrophages; quantifying the expression of MCP-1, TNF-a and IL-6 in macrophages by taking 18S as an internal reference; collecting cell samples in a 1.5mLEP tube, adding 5 xSDS at a ratio of 4:1 after RIPA is fully cracked to obtain protein samples, and detecting the expression of CD68 in THP-1 cells and CD86 in macrophages after electrophoresis;
s5, analyzing an experimental result.
2. The method of claim 1, wherein step S5 comprises the steps of:
(1) Compared with a control group, the expression of miR-146a-5p in HepG2 cells of the Mimic-146a-5p group and in exosomes of cell culture supernatants is obviously increased;
(2) After the aposome with high miR-146a-5p expression is incubated with THP-1 or macrophage for 48h, the expression of miR-146a-5p in the THP-1 or macrophage is obviously increased;
(3) The exosomes with high expression of miR-146a-5p can reduce the release of macrophage pro-inflammatory factors MCP-1, TNF-a and IL-6;
(4) The exosomes with high expression of miR-146a-5p can reduce the expression of a macrophage marker CD68 in THP-1 cells;
(5) The exosomes with high expression of miR-146a-5p can reduce the expression of the M1 type macrophage marker CD 86.
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