CN108441464B - In-vitro preparation method of islet beta cells - Google Patents
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Abstract
The invention discloses an in vitro preparation method of islet beta cells, which is used for inducing stem cell differentiation by continuous incubation of exosomes and comprises the following steps: (1) extracting exosomes; (2) observing an exosome electron microscope; (3) western blotting analysis of the expression of the exosome markers; (4) inducing iPSCs to differentiate directionally by exosomes; (5) functional verification of beta cells; (6) and (4) carrying out function verification on pancreatic islet beta cell exosome miRNA. The experimental result shows that miRNA plays an important role in the differentiation of iPSCs into beta cells; according to the analysis of experimental data and the function screening and verification of miRNA, the miR-212/132 in the exosome is found to have the function of promoting the directional differentiation of iPSCs to islet beta cells.
Description
Technical Field
The invention relates to the technical field of islet beta cell in-vitro induction, in particular to an islet beta cell source exosome for inducing stem cells to be differentiated into islet beta cells.
Background
Diabetes Mellitus (DM) has become the third most serious disease after tumors, cardiovascular and cerebrovascular diseases threatening human health and affecting people's quality of life, and according to the statistical data published by International Diabetes Federation (IDF) 2015 in 12 months, about 8.8% of adults aged 20-79 worldwide have Diabetes Mellitus, which means about 4.15 billion diabetic patients worldwide. Recent reports from World Health Organization (WHO) at 2016, 4, 6 days, show that this figure rises to 4.22 million, while the number of diabetic patients in china exceeds 1 million, with more than 100 million of type I diabetic patients, leading globally.
Insulin therapy is currently the only method of maintaining the life of diabetic patients, but the use of insulin to treat Type I Diabetes Mellitus (T1 DM) does not prevent its complications. Type I diabetes, also known as insulin-dependent diabetes, is a disease in which the beta cells of the pancreatic islets are damaged and the secretion of insulin is reduced due to abnormal autoimmune response generated by the body, and the incidence rate of the disease is increasing year by year at a rate of 3-5%. At present, no effective means is available for radically treating type I diabetes, and hyperglycemia or hypoglycemia caused by unreasonable insulin supplementation is serious, so that various complications ensue, and the service life of patients is generally shortened.
In order to effectively control blood sugar, prevent diabetic complications and improve the quality of life of diabetic patients and replace insulin secreting cells damaged by the autoimmune system of patients, canadian scholars reported in 2000 a novel islet cell isolation and purification technology and a hormone-free immunosuppressive program (Shapiro, A.M.; Lakey, J.R.; Ryan, E.A.; Korbutt, G.S.; Toth, E.; Warnock, G.L.; Kneteman, N.M.; Rajotte, R.V.7 type diabetes patients used hormone-free immunosuppressants for islet transplantation, a new journal of England medicine), so that the clinical application of adult islet cell transplantation becomes possible and islet cell transplantation is vigorous. However, in the case of islet transplantation, the beta cell amount of 2 × 106 cells per kg of body weight is required by calculating the islet equivalent, and therefore, the first problem of islet transplantation, namely, the source of islet beta cells, is solved, and the vigorous development of stem cell technology brings a new hope for in vitro islet beta cell supply. Differentiation of stem cells into islet beta cells is a rapid way to solve the problem of islet source shortage, and stem cells which have been successfully induced in vitro include embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells, pancreatic stem cells and the like.
Exosome (exosome) is a micro vesicle secreted by cells and with the diameter of about 40-100 nm, contains abundant miRNA, lncRNA, circRNA, mRNA and protein, can participate in substance transduction and signal communication between cells, and has the specificity of maternal cells. At present, the method for directionally differentiating the stem cells into the islet beta cells is mainly a chemical reagent induced differentiation method, which not only influences the activity of the cells, but also has the risk of gene mutation. Considering that a large amount of biological information for cell communication exists in exosome, and the exosome contains specific RNA and protein of cells, the biological function of the exosome is used as a directional induction factor of the stem cells, so that the gene sequence mutation generated by cell stimulation by a chemical reagent is avoided, the activity of the cells after directional differentiation is ensured, and the method is the best induction method for directional differentiation of the stem cells at present.
MicroRNA (miRNA) is a non-coding single-stranded RNA molecule with the fragment length of 20-25 nucleotides, and participates in post-transcriptional gene expression regulation of a biological organism. In recent years, miRNA is found to participate in post-transcriptional regulation of stem cells, influence differentiation and proliferation of the stem cells, regulate expression level of specific miRNA, induce the stem cells to differentiate into specific tissue cells, and determine fate of the stem cells to a certain extent, thereby providing new technology and new ideas for treatment of diseases. The biological information in the exosome secreted by the islet beta cell is similar to the gene instantly expressed by the cell, Claudiane et al reports that the type and abundance of microRNA expressed by the islet beta cell exosome are consistent with the expression of the source cell, and only individual miRNA are different. And islet-specific microRNAs are all expressed in islet beta cell exosomes, such as miRNA-375, miRNA-124, iRNA-212, miRNA-26a, let-7, miRNA-9 and the like (Guay, C.; Menoud, V.; Rome, S.; Regazzi, R. exosome miRNA transmit apoptosis signals and cell communication signals among islet beta cells). Therefore, according to the similarity of the biological information of the exosome and the cell from which the exosome is derived, the application of the exosome as the induction factor of the pluripotent stem cell becomes an efficient and safe induction method. .
Disclosure of Invention
The invention aims to provide a safe and efficient method for differentiating islet beta cells in vitro.
The technical scheme adopted by the invention is as follows:
an in vitro preparation method of islet beta cells, which is a method for inducing stem cell differentiation by continuous incubation of exosomes.
The invention relates to an in vitro preparation method of islet beta cells, wherein the stem cells are human iPSCs; the source of the exosomes is mature islet beta cells.
The invention discloses an in vitro preparation method of islet beta cells, wherein a factor for generating functions in exosomes is miR-212/132.
The in vitro preparation method of the islet beta cells comprises the following steps:
(1) exosome extraction
Serum in a culture medium is replaced by a serum substitute, human beta cells are cultured in vitro, culture supernatant is collected every two days in the cell culture process, and exosomes in the cell culture medium are separated by an ultracentrifugation method;
(2) electron microscope observation of exosomes
(3) Western blotting analysis of the expression of exosome markers
(4) Exosome induced directional differentiation of iPSCs
Firstly, the concentration of the collected exosomes is calculated by adopting a BCA method of protein, the collected exosomes are adopted to directionally induce iPSCs, the exosome inducing dose is calculated according to the protein concentration, and 0.2 micrograms of exosomes needs to be added in each microliter of culture medium;
adding the cells into an iPSCs culture medium for continuous induction for 10 days, and carrying out primary analysis on the expression of the beta cell marker on the differentiated cells, wherein the primary analysis comprises immunofluorescence and flow cytometry analysis on the expression of the beta cell marker;
(5) beta cell functional validation
Firstly, respectively adopting two stimulation solutions of glucose with different concentrations, namely a stimulation solution A5.5mM and a stimulation solution B20.5 mM; the method comprises the following specific operations: discarding the original inducing solution, washing with PBS for 2 times, adding stimulating solution without serum, collecting culture solution after 2h, and detecting the secretory expression of insulin by ELISA method;
② respectively collect 3 × 106Injecting the differentiated β cells under the kidney capsule of a 4-week-old immunodeficiency mouse, injecting glucose (+) -D-glucose into the abdominal cavity according to the weight of 2g/kg after 2 weeks, respectively detecting the level of human insulin in the serum of the mouse by using a high-sensitivity ELISA kit 0min before injection and 30min after injection, and comparing the change of the expression quantity;
(6) functional verification of islet beta cell exosome miRNA
Verifying the function of miRNA in exosome in beta cell differentiation, carrying out shRNA design on Dicer enzyme, screening effective shRNA to knock down Dicer expression, blocking effective shearing of pre-miRNA, and reducing the expression quantity of format miRNA; screening effective shRNA, infecting iPSCs through a lentiviral vector, carrying out exosome incubation method to directionally induce the differentiation of the shRNA, and analyzing the change of the differentiation efficiency of beta cells by flow cytometry; wherein the shRNA sequence is shown as SEQ ID No. 1 in the sequence table;
and secondly, miRNA microarray is used for detecting the expression of miRNA in iPSCs, beta cells and exosomes thereof, the expression characteristics of miRNA in the two cells and exosomes thereof are analyzed in a comparison manner, real time-PCR is used for verifying the experimental result of the miRNA microarray, and the specific miRNA which has obvious analysis difference and is related to islet development is analyzed.
The invention relates to an in vitro preparation method of islet beta cells, wherein the source of exosomes is mature islet beta cells.
According to the in vitro preparation method of the islet beta cells, miR-212/132 in the exosome has the function of promoting directional differentiation of iPSCs to the islet beta cells.
The method for preparing the islet beta cells in vitro comprises the following steps of (1), under the environment of 4 ℃, 300g 10min and 2000g 20min of cell culture supernatant to be extracted, discarding precipitates, removing cells, then 10,000g30min, discarding precipitates, removing subcellular components, then 10,000g 60min, discarding supernatant, and finally obtaining precipitates, namely exosomes.
The method for preparing the islet beta cells in vitro, provided by the invention, comprises the following steps of (2):
firstly, fixing a specimen: fixing the exosome with 2.5% glutaraldehyde at 4 ℃ for 2h, transferring into a penicillin vial, and rinsing with PBS for 3 times at 4 ℃ for 5min each time; fixing with osmium tetrachloride at 4 deg.C for 30min, and rinsing with PBS for 3 times;
and (2) dehydrating: 50% acetone solution for 10min for 1 time; 70% acetone solution for 10min for 1 time; 90% acetone solution for 10min for 2 times; 100% acetone solution for 10min for 3 times;
③ soaking: absorbing the dehydrating agent in the bottle, adding 3ml of pure acetone-EP 0N812 embedding agent, standing at room temperature for 30min, discarding the diluted embedding agent, adding 1ml of pure embedding agent, and standing at room temperature for 2 h;
embedding: transferring the cell block mass into the center of the bottom of the capsule membrane block hole, filling with a mixed embedding medium, and baking in an oven at 60 ℃ for 24 hours to solidify the cell block mass into a hard block;
repairing blocks: arranging the embedding block on a special fixture, finishing embedding by using a single-edge knife and marking;
preparing a semi-thin slice: cutting the repaired embedded block into a semi-thin section with the thickness of l mum on an ultrathin slicer; soaking clean glass slide in the mixture of 1% gelatin and 1% chromium alum, taking out, and drying by heating to 60 deg.C on a baking sheet machine; adding a drop of distilled water on the slide, transferring the semi-thin slice into the water drop by using a pair of tweezers, and heating the semi-thin slice on a piece baking machine to flatten and dry the semi-thin slice; then dripping a methylene blue solution, dyeing for 30s at 60 ℃, washing with water, and baking; observing the image of the ultrathin section under a microscope, determining the position of the ultrathin section and marking;
preparing ultrathin slices: preparing 0.45 percent Formvar solution by using chloroform, vertically immersing a clean glass slide into the solution, and immediately taking out the glass slide, wherein a layer of film is formed on the surface of the glass slide; cutting the periphery of the film by a blade, and immersing the film in a water tank filled with water to separate the film from the glass slide; carefully placing the copper mesh on the film and covering the copper mesh and the sealing film on the film, and taking out the copper mesh and the supporting film; fixing the embedding block on an ultrathin slicer, adjusting the knife distance, cutting an ultrathin slice with the thickness of 50nm, sticking the ultrathin slice on one side of a copper net with a supporting film, and storing the ultrathin slice in a drying vessel to be dyed;
electron dyeing: vertically clamping the copper mesh loaded with the slices on a rubber plate and putting the rubber plate into a plate, dropwise adding 1 drop of uranyl acetate staining solution to one side of each slice, and staining for 5-10 min at room temperature; taking out the rubber plate, washing the rubber plate with double distilled water, slicing, sucking dry filter paper, putting the rubber plate into a plate, adding 1 drop of lead for dyeing, and dyeing for 4-10 min at room temperature; washing, sucking and drying for later use;
ninthly, observing and taking pictures through a transmission electron microscope, and showing a typical exosome result under the transmission electron microscope.
The method for preparing the islet beta cells in vitro, provided by the invention, comprises the following steps of (3):
firstly, extracting and quantifying protein, adding 50 mu l of RIPA lysate into exosome obtained by centrifugation, carrying out lysis for 30min, then carrying out centrifugation for 10min at 12000rmp and 4 ℃, and collecting supernatant; reading the value at 570nm of an enzyme labeling instrument according to the operation of a BCA protein quantitative kit (Beijing-Plumeria biological company); taking A570 as a vertical coordinate and the concentration of a standard substance as a horizontal coordinate to make a standard curve, calculating the concentration of a protein sample from the standard curve, multiplying the concentration by a dilution multiple to calculate the concentration of a sample stock solution, sucking a proper amount of sample, adding 5 xSDS loading buffer, boiling for 10min at 100 ℃, subpackaging and freezing;
② SDS-polyacrylamide gel electrophoresis
The separation gel and lamination gel were formulated as follows:
loading 50 μ g total protein/lane, performing electrophoresis at 80V for 20min, applying voltage to 160V, and continuing electrophoresis for about 60min until bromophenol blue reaches 2cm from the bottom of gel plate; roughly cutting gel according to the range shown by the pre-dyed protein molecule standard;
③ transferring film
Under the voltage of 100V, carrying out constant-pressure wet rotation for 100min, and transferring the protein to a PVDF membrane; ponceau stains the membrane after electrotransformation, a clear protein band can be seen at the moment, the PVDF membrane is finely cut according to the range shown by the pre-staining protein molecular standard, and finally ponceau is washed away;
antibody incubation, color development and data analysis
And (3) sealing: incubating the electroporated PVDF membrane for 1h at room temperature by using 5% skimmed milk powder sealing solution;
primary antibody incubation: incubation with CD9, CD63, CD81, and Hsp70 antibody antibodies, respectively, overnight at 4 ℃;
washing: washing with TBST for 10min × 3 times;
and (3) secondary antibody incubation: incubating for 2h at room temperature by using HRP-coupled goat anti-rabbit IgG, goat anti-mouse IgM and goat anti-mouse IgG respectively;
washing: TBST washing, 10min is multiplied by 3 times;
color development: developing with chemiluminescence kit SuperSignal West Pico kit at room temperature for about 5min, developing with film in dark room, and taking picture after fixing.
The method for preparing the islet beta cells in vitro, provided by the invention, comprises the following specific steps of (4) immunofluorescence and flow cytometry analysis:
immunofluorescence: after preparing the cell climbing tablet, fixing the cell climbing tablet by 4% paraformaldehyde for 15min, and washing the cell climbing tablet for 3 times and 5min each time by using PBS buffer solution; treating with 0.5% TritonX-100 for 15min, and washing with PBS buffer solution for 3 times; sealing with goat serum at 37 deg.C for 1 hr; dropwise adding diluted primary antibody Pdx1 and proinsulin, placing in a wet box, and incubating overnight at 4 ℃; washing with PBS buffer for 3 times, dripping diluted FITC-labeled secondary antibody IgG 5min each time, placing in a wet box, and keeping away from light at 37 deg.C for 60 min; washing with PBS for 5min for 3 times, washing with distilled water for 1 time, sealing with 50% buffer glycerol, and observing under laser confocal microscope;
flow cytometry analysis: digesting cells conventionally, collecting the cells by centrifugation, washing the cells for 3 times by PBS, adding a proinsulin antibody diluted by 1% BSA, incubating for 1h at 37 ℃, washing the cells for 3 times by PBS, adding a FITC-labeled secondary antibody diluted by PBS, incubating for 1h at 37 ℃, washing the cells for 3 times by PBS, collecting the cells by centrifugation, filtering by a 70-micron screen, and analyzing the cell expression level on a flow cytometer.
Compared with the prior art, the invention has the outstanding effects that:
(1) in the method, the source of the islet beta cells is induced pluripotent stem cells. By verifying the function of miRNA in exosome in beta cell differentiation, shRNA design is carried out on Dicer enzyme, effective shRNA knockdown Dicer expression is screened, effective shearing of pre-miRNA is blocked, and the expression quantity of format miRNA is reduced. Screening effective shRNA, infecting iPSCs through a lentiviral vector, performing exosome incubation method to directionally induce differentiation, analyzing beta cell differentiation efficiency change by flow cytometry, and achieving the purpose of verifying the function of exosome miRNA in the directional differentiation of iPSCs, wherein the result shows that miRNA plays an important role in the differentiation of iPSCs into beta cells.
(2) The miRNA microarray is used for detecting the expression of miRNA in iPSCs, beta cells and exosomes thereof, the expression characteristics of miRNA in the two cells and exosomes thereof are contrastively analyzed, real time-PCR is used for verifying the experimental result of the miRNA microarray, specific miRNA which has obvious difference and is related to islet development is analyzed, miR-212/132 in exosomes is found to have the function of promoting the iPSCs to directionally differentiate into islet beta cells according to the analysis of experimental data and the function screening and verification of miRNA,
the method for preparing islet beta cells in vitro according to the present invention is further described below with reference to the accompanying drawings and the specific examples.
Drawings
FIG. 1 shows morphological observations of exosomes derived from iPSC and beta cells and protein marker expression;
FIG. 2 shows immunofluorescence analysis of differentiated beta cell gene expression;
FIG. 3 is a graph demonstrating differentiated beta cell function in vitro and in vivo;
FIG. 4 is a diagram of shRNA sequences and lentiviral vectors;
FIG. 5 shows that shRNA affects Dicer expression and affects exosomes to induce beta cell differentiation;
FIG. 6 shows the effect of miRNA on directed differentiation of iPSCs into beta cells;
FIG. 7 is a technical flowchart of the islet beta cell-derived exosome induced directional differentiation of iPSCs into beta cells;
FIG. 8 is a flow chart of exosome miRNA screening and miR-212/132 functional validation technology.
Detailed Description
As shown in fig. 7, an in vitro preparation method of islet beta cells comprises the following steps:
1. exosome extraction
The method comprises the steps of replacing Serum in a culture medium with a Serum substitute (Serum Replacement), culturing human beta cells in vitro, collecting culture supernatant every two days in the cell culture process, separating exosomes in the cell culture medium by using an ultracentrifugation method, namely 10ml of cell culture supernatant to be extracted, discarding precipitates and removing cells in an environment of 4 ℃ for 300g 10min and 2000g 20min, then discarding the precipitates and removing subcellular components for 10,000g30min, discarding the supernatant, and finally obtaining precipitates which are exosomes.
2. Electron microscope observation of exosomes
(1) Fixing a specimen: exosomes were fixed with 2.5% glutaraldehyde at 4 ℃ for 2h, transferred to penicillin vials, and rinsed 3 times with PBS at 4 ℃ for 5min each. Then fixed with osmium tetrachloride at 4 ℃ for 30min, followed by rinsing 3 times with PBS.
(2) And (3) dehydrating: 50% acetone solution for 10min for 1 time; 70% acetone solution for 10min for 1 time; 90% acetone solution for 10min for 2 times; 100% acetone solution, 10min, 3 times.
(3) Soaking: removing the dehydrating agent in the bottle by suction, adding 3ml of pure acetone-EP 0N812 embedding agent, standing at room temperature for 30min, discarding the diluted embedding agent, adding 1ml of pure embedding agent, and standing at room temperature for 2 h.
(4) Embedding: and transferring the cell block mass into the center of the bottom of the capsule membrane block hole, filling the mixed embedding medium, and baking the cell block mass in an oven at 60 ℃ for 24 hours to solidify the cell block mass into a hard block.
(5) Block repairing: the embedded block was placed on a special fixture, trimmed with a single-edged knife and marked.
(6) Preparing a semi-thin slice: the trimmed embedded blocks were cut into a microtome to give half-thin sections of thickness l μm. A clean glass slide is taken and immersed into a mixed solution of 1% gelatin and 1% chrome alum, and the glass slide is taken out and placed on a baking sheet machine to be heated to 60 ℃ for drying. A drop of distilled water was added to the slide, and the half thin section was transferred to the drop of distilled water with tweezers, and then heated on a baking machine to flatten and dry it. Then, methylene blue solution is added dropwise, and the mixture is dyed at 60 ℃ for 30s, washed with water and dried by baking. And observing the image of the ultrathin section under a microscope, and determining and marking the position of the ultrathin section.
(7) Preparing an ultrathin slice: a0.45 percent Formvar solution is prepared by chloroform, a clean glass slide is vertically immersed in the solution and immediately taken out, and a layer of thin film is formed on the surface of the glass slide. The periphery of the film is cut by a blade and immersed into a water tank filled with water to separate the film from the glass slide. The copper mesh was carefully placed on the film and covered with a sealing film, and the copper mesh and the supporting film were taken out together. Fixing the embedding block on an ultrathin slicer, adjusting the knife distance, cutting an ultrathin section with the thickness of 50nm, sticking the ultrathin section on one side of a copper net with a supporting film, and storing the ultrathin section in a drying vessel to be dyed.
(8) Electronic dyeing: and vertically clamping the copper mesh loaded with the slices on a rubber plate and putting the rubber plate into a plate, dropwise adding 1 drop of uranyl acetate staining solution to one side of each slice, and staining for 5-10 min at room temperature. And taking out the rubber plate, washing the rubber plate with double distilled water, slicing, sucking dry filter paper, putting the rubber plate into a plate, adding 1 drop of lead for dyeing, and dyeing for 4-10 min at room temperature. Washing with water, sucking, and air drying.
(9) The transmission electron microscope is used for observation and photographing, and typical exosome results can be seen under the transmission electron microscope, as shown in figure 1.
Western blotting analysis of the expression of exosome markers
(1) Extracting and quantifying protein, adding 50 μ l RIPA lysate into the exosome obtained by centrifugation, performing lysis for 30min, then performing centrifugation for 10min at 12000rmp and 4 ℃, and collecting supernatant; the data were read at 570nm using a microplate reader according to the BCA protein quantification kit (Beijing-Plumeria Biotech). Taking A570 as ordinate and the concentration of the standard substance as abscissa to make a standard curve, calculating the concentration of the protein sample from the standard curve, multiplying the concentration by the dilution factor to calculate the concentration of the original sample solution, sucking a proper amount of sample, adding 5 xSDS sample buffer, boiling for 10min at 100 ℃, subpackaging and freezing.
(2) SDS-polyacrylamide gel electrophoresis
The separation gel and lamination gel were formulated as follows:
loading 50 μ g total protein/lane, electrophoresis at 80V for 20min, applying voltage to 160V, and continuing electrophoresis for about 60min until bromophenol blue reaches 2cm from the bottom of the gel plate. The gel was roughly cut as indicated by the prestained protein molecular standards.
(3) Rotary film
And (3) carrying out constant-pressure wet rotation for 100min under the voltage of 100V, and transferring the protein to the PVDF membrane. Ponceau stains the electrotransformed membrane, at which time a clear protein band is visible, fine cuts the PVDF membrane to the extent indicated by the pre-stained protein molecular standards, and finally washes off ponceau.
(4) Antibody incubation, visualization and data analysis
And (3) sealing: the electroporated PVDF membrane was incubated with 5% skim milk (Tris buffersaline, TBS in 0.05M, pH 7.4) blocking solution at room temperature for 1 h.
Primary antibody incubation: the cells were incubated overnight at 4 ℃ with CD9(1:400), CD63(1:600), CD81(1:500) and Hsp70 (1:500) antibodies, respectively.
Washing: washing with TBST (TBS supplemented with 0.1% Tween-20) for 10min X3 times.
And (3) secondary antibody incubation: the cells were incubated with HRP-conjugated goat anti-rabbit IgG (1:10000), goat anti-mouse IgM (1:3000) and goat anti-mouse IgG (1:10000), respectively, at room temperature for 2 h.
Washing: TBST washing, 10min × 3 times.
Color development: development was carried out for about 5min at room temperature using a chemiluminescence kit SuperSignal West Pico kit (Thermo Scientific, USA), developed on a film in a dark room, and photographed after fixation. The results are shown in FIG. 1.
4. Exosome induced directional differentiation of iPSCs
(1) The collected exosomes are used for calculating the concentration by using a BCA method of protein, the collected exosomes are used for carrying out directional induction on the iPSCs, the exosome induction dose is calculated according to the protein concentration, and 0.2 micrograms of exosomes are required to be added in each microliter of culture medium;
(2) adding the cells into an iPSCs culture medium for continuous induction for 10 days, and carrying out primary analysis on the expression of the beta cell marker on the differentiated cells, wherein the primary analysis comprises immunofluorescence and flow cytometry analysis on the expression of the beta cell marker, and the method comprises the following steps:
immunofluorescence: after preparing the cell climbing tablet, fixing the cell climbing tablet by 4% paraformaldehyde for 15min, and washing the cell climbing tablet for 3 times and 5min each time by using PBS buffer solution; treating with 0.5% TritonX-100 for 15min, and washing with PBS buffer solution for 3 times; sealing with goat serum at 37 deg.C for 1 hr; dropwise adding diluted primary antibody Pdx1(1:200) and proinsulin (1:300), placing in a wet box, and incubating overnight at 4 ℃; washing with PBS buffer for 3 times, dripping diluted FITC-labeled secondary antibody IgG (1:2000) 5min each time, placing in a wet box, and keeping away from light at 37 deg.C for 60 min; washing with PBS for 5min for 3 times, washing with distilled water for 1 time, sealing with 50% buffered glycerol, and observing under laser confocal microscope. The results are shown in FIG. 2.
Flow cytometry analysis: digesting cells by a conventional method, collecting the cells by centrifugation, washing the cells for 3 times by PBS, adding a proinsulin antibody (1:200) diluted by 1% BSA, incubating for 1h at 37 ℃, washing the cells for 3 times by PBS, adding a FITC-labeled secondary antibody (1:200) diluted by PBS, incubating for 1h at 37 ℃, washing the cells for 3 times by PBS, collecting the cells by centrifugation, filtering by a 70-micron screen, and analyzing the cell expression level on a flow cytometer. The results are shown in FIG. 6.
5. Beta cell functional validation
(1) To verify the ability of induced differentiated beta cells to release insulin, two different concentrations of glucose were used as stimulus, stimulus A (5.5mM) and stimulus B (20.5mM), respectively. The method comprises the following specific operations: abandoning the original inducing solution, washing with PBS for 2 times, adding stimulating solution without serum, collecting culture solution after 2h, and detecting the secretory expression of insulin by ELISA method.
(2) Respectively collecting 3 × 106The differentiated β cells were injected under the kidney capsule of 4-week-old immunodeficient mice (SCID mouse). The (+) -D-glucose was intraperitoneally injected at 2g/kg body weight 2 weeks later, and the human insulin level in the serum of the mice was measured using a highly sensitive ELISA kit before (0min) and after (30min) injection, respectively, and the results were compared, as shown in FIG. 3.
6. Functional verification of islet beta cell exosome miRNA (microribonucleic acid) as shown in figure 8
(1) Verifying the function of miRNA in exosome in beta cell differentiation, carrying out shRNA design on Dicer enzyme, screening effective shRNA to knock down Dicer expression, blocking effective shearing of pre-miRNA, and reducing the expression quantity of format miRNA. Screening effective shRNA, infecting iPSCs (shRNA sequence and vector diagram are shown in figure 4) through a lentiviral vector, carrying out exosome incubation method to directionally induce differentiation, analyzing beta cell differentiation efficiency change through flow cytometry, achieving the purpose of verifying the function of exosome miRNA in iPSCs directional differentiation, and showing that miRNA plays an important role in iPSCs differentiation into beta cells through results shown in figure 6. Wherein the shRNA sequence is shown as SEQ ID No. 1 in the sequence table.
(2) The miRNA microarray is used for detecting the expression of miRNA in iPSCs, beta cells and exosomes thereof, the expression characteristics of miRNA in the two cells and exosomes thereof are contrastively analyzed, real time-PCR is used for verifying the experimental result of the miRNA microarray, specific miRNA which has obvious difference and is related to pancreatic islet development is analyzed, and miR-212/132 in exosomes is found to have the function of promoting the iPSCs to directionally differentiate into pancreatic islet beta cells according to the data analysis of the research and the function screening and verification of the miRNA, and the result is shown in figure 7.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Sequence listing
<110> Jining medical college
<120> in vitro preparation method of islet beta cells
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<213> Artificial Sequence (Artificial Sequence)
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Claims (5)
1. An in vitro preparation method of islet beta cells is characterized in that: the method is a method for inducing stem cell differentiation by continuous exosome incubation; the stem cells are human iPSCs; the source of the exosome is mature islet beta cells; the factor for generating functions in the exosome is miR-212/132; the miR-212/132 has the function of promoting the directional differentiation of iPSCs to islet beta cells;
the method comprises the following steps:
(1) exosome extraction
Serum in a culture medium is replaced by a serum substitute, human islet beta cells are cultured in vitro, culture supernatant is collected every two days in the cell culture process, and exosomes in the cell culture medium are separated by an ultracentrifugation method;
(2) electron microscope observation of exosomes
(3) Western blotting analysis of the expression of exosome markers
(4) Exosome induced directional differentiation of iPSCs
Firstly, the concentration of the collected exosomes is calculated by adopting a BCA method of protein, the collected exosomes are adopted to directionally induce iPSCs, the exosome inducing dose is calculated according to the protein concentration, and 0.2 micrograms of exosomes needs to be added in each microliter of culture medium;
adding the cells into an iPSCs culture medium for continuous induction for 10 days, and carrying out primary analysis on the expression of the beta cell marker on the differentiated cells, wherein the primary analysis comprises immunofluorescence and flow cytometry analysis on the expression of the beta cell marker;
(5) beta cell functional validation
Firstly, respectively adopting two stimulation solutions of glucose with different concentrations, namely a stimulation solution A of 5.5mM and a stimulation solution B of 20.5 mM; the method comprises the following specific operations: discarding the original inducing solution, washing with PBS for 2 times, adding stimulating solution without serum, collecting culture solution after 2h, and detecting the secretory expression of insulin by ELISA method;
② respectively collect 3 × 106Injecting the differentiated β cells under the kidney capsule of an immunodeficiency mouse with the age of 4 weeks, injecting D- (+) -glucose into the abdominal cavity according to the weight of 2g/kg after 2 weeks, respectively detecting the level of human insulin in the serum of the mouse by using a high-sensitivity ELISA kit 0min before injection and 30min after injection, and comparing the change of the expression level;
(6) functional verification of islet beta cell exosome miRNA
Verifying the function of miRNA in exosome in beta cell differentiation, carrying out shRNA design on Dicer enzyme, screening effective shRNA to knock down Dicer expression, blocking effective shearing of pre-miRNA, and reducing the expression quantity of format miRNA; screening effective shRNA, infecting iPSCs through a lentiviral vector, carrying out exosome incubation method to directionally induce the differentiation of the shRNA, and analyzing the change of the differentiation efficiency of beta cells by flow cytometry; wherein the shRNA sequence is shown as SEQ ID No. 1 in the sequence table;
and secondly, miRNA microarray is used for detecting the expression of miRNA in iPSCs, beta cells and exosomes thereof, the expression characteristics of miRNA in the two cells and exosomes thereof are analyzed in a comparison manner, real time-PCR is used for verifying the experimental result of the miRNA microarray, and the specific miRNA which has obvious analysis difference and is related to islet development is analyzed.
2. The method for producing islet beta cells according to claim 1, wherein: in the step (1), 10ml of cell culture supernatant to be extracted is subjected to precipitation removal to remove cells, 10,000g of the cell culture supernatant is subjected to 30min, 10,000g of the cell culture supernatant is subjected to precipitation removal to remove subcellular components, 10,000g of the cell culture supernatant is subjected to 60min, and the supernatant is removed, so that the precipitate is an exosome.
3. The method for producing islet beta cells according to claim 2, wherein: the step (2) specifically comprises:
firstly, fixing a specimen: fixing the exosome with 2.5% glutaraldehyde at 4 ℃ for 2h, transferring into a penicillin vial, and rinsing with PBS for 3 times at 4 ℃ for 5min each time; fixing with osmium tetrachloride at 4 deg.C for 30min, and rinsing with PBS for 3 times;
and (2) dehydrating: 50% acetone solution for 10min for 1 time; 70% acetone solution for 10min for 1 time; 90% acetone solution for 10min for 2 times; 100% acetone solution for 10min for 3 times;
③ soaking: absorbing the dehydrating agent in the bottle, adding 3ml of pure acetone-EPON 812 embedding agent, standing at room temperature for 30min, discarding the diluted embedding agent, adding 1ml of pure embedding agent, and standing at room temperature for 2 h;
embedding: transferring the cell block mass into the center of the bottom of the capsule membrane block hole, filling with a mixed embedding medium, and baking in an oven at 60 ℃ for 24 hours to solidify the cell block mass into a hard block;
repairing blocks: arranging the embedding block on a special fixture, finishing embedding by using a single-edge knife and marking;
preparing a semi-thin slice: cutting the repaired embedded block into a semi-thin section with the thickness of l mum on an ultrathin slicer; soaking clean glass slide in the mixture of 1% gelatin and 1% chromium alum, taking out, and drying by heating to 60 deg.C on a baking sheet machine; adding a drop of distilled water on the slide, transferring the semi-thin slice into the water drop by using a pair of tweezers, and heating the semi-thin slice on a piece baking machine to flatten and dry the semi-thin slice; then dripping a methylene blue solution, dyeing for 30s at 60 ℃, washing with water, and baking; observing the image of the ultrathin section under a microscope, determining the position of the ultrathin section and marking;
preparing ultrathin slices: preparing 0.45 percent Formvar solution by using chloroform, vertically immersing a clean glass slide into the solution, and immediately taking out the glass slide, wherein a layer of film is formed on the surface of the glass slide; cutting the periphery of the film by a blade, and immersing the film in a water tank filled with water to separate the film from the glass slide; carefully placing the copper mesh on the film and covering the copper mesh and the sealing film on the film, and taking out the copper mesh and the supporting film; fixing the embedding block on an ultrathin slicer, adjusting the knife distance, cutting an ultrathin slice with the thickness of 50nm, sticking the ultrathin slice on one side of a copper net with a supporting film, and storing the ultrathin slice in a drying vessel to be dyed;
electron dyeing: vertically clamping the copper mesh loaded with the slices on a rubber plate and putting the rubber plate into a plate, dropwise adding 1 drop of uranyl acetate staining solution to one side of each slice, and staining for 5-10 min at room temperature; taking out the rubber plate, washing the rubber plate with double distilled water, slicing, sucking dry filter paper, putting the rubber plate into a plate, adding 1 drop of lead for dyeing, and dyeing for 4-10 min at room temperature; washing, sucking and drying for later use;
ninthly, observing and taking pictures through a transmission electron microscope, and showing a typical exosome result under the transmission electron microscope.
4. The method for producing islet beta cells according to claim 3, wherein: the step (3) specifically comprises:
(1) extracting and quantifying protein, adding 50 μ l RIPA lysate into the exosome obtained by centrifugation, performing lysis for 30min, then performing centrifugation for 10min at 12000rmp and 4 ℃, and collecting supernatant; reading the numerical value at 570nm of an enzyme labeling instrument according to the operation of the BCA protein quantitative kit instruction; taking A570 as a vertical coordinate and the concentration of a standard substance as a horizontal coordinate to make a standard curve, calculating the concentration of a protein sample from the standard curve, multiplying the concentration by a dilution multiple to calculate the concentration of a sample stock solution, sucking a proper amount of sample, adding 5 xSDS loading buffer, boiling for 10min at 100 ℃, subpackaging and freezing;
(2) SDS-polyacrylamide gel electrophoresis
The separation gel and lamination gel were formulated as follows:
loading 50 μ g total protein/lane, performing electrophoresis at 80V for 20min, applying voltage to 160V, and continuing electrophoresis for 60min until bromophenol blue reaches 2cm from the bottom of gel plate; roughly cutting gel according to the range shown by the pre-dyed protein molecule standard;
(3) rotary film
Under the voltage of 100V, carrying out constant-pressure wet rotation for 100min, and transferring the protein to a PVDF membrane; ponceau stains the membrane after electrotransformation, a clear protein band can be seen at the moment, the PVDF membrane is finely cut according to the range shown by the pre-staining protein molecular standard, and finally ponceau is washed away;
(4) antibody incubation, visualization and data analysis
And (3) sealing: incubating the electroporated PVDF membrane for 1h at room temperature by using 5% skimmed milk powder sealing solution;
primary antibody incubation: incubation with CD9, CD63, CD81, and Hsp70 antibody antibodies, respectively, overnight at 4 ℃;
washing: washing with TBST for 10min × 3 times;
and (3) secondary antibody incubation: incubating for 2h at room temperature by using HRP-coupled goat anti-rabbit IgG, goat anti-mouse IgM and goat anti-mouse IgG respectively;
washing: TBST washing, 10min is multiplied by 3 times;
color development: developing with chemiluminescence kit SuperSignal West Pico kit at room temperature for 5min, developing with film in dark room, and taking pictures after fixation.
5. The method for producing islet beta cells according to claim 4, wherein: in the step (4), the specific methods of immunofluorescence and flow cytometry analysis are as follows:
immunofluorescence: after preparing the cell climbing tablet, fixing the cell climbing tablet by 4% paraformaldehyde for 15min, and washing the cell climbing tablet for 3 times and 5min each time by using PBS buffer solution; treating with 0.5% TritonX-100 for 15min, and washing with PBS buffer solution for 3 times; sealing with goat serum at 37 deg.C for 1 hr; dropwise adding diluted primary antibody Pdx1 and proinsulin, placing in a wet box, and incubating overnight at 4 ℃; washing with PBS buffer for 3 times, dripping diluted FITC-labeled secondary antibody IgG 5min each time, placing in a wet box, and keeping away from light at 37 deg.C for 60 min; washing with PBS for 5min for 3 times, washing with distilled water for 1 time, sealing with 50% buffer glycerol, and observing under laser confocal microscope;
flow cytometry analysis: digesting cells conventionally, collecting the cells by centrifugation, washing the cells for 3 times by PBS, adding a proinsulin antibody diluted by 1% BSA, incubating for 1h at 37 ℃, washing the cells for 3 times by PBS, adding a FITC-labeled secondary antibody diluted by PBS, incubating for 1h at 37 ℃, washing the cells for 3 times by PBS, collecting the cells by centrifugation, filtering by a 70-micron screen, and analyzing the cell expression level on a flow cytometer.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468642A (en) * | 2013-09-23 | 2013-12-25 | 山西大学 | Method for separating exosome from cell culture medium |
| KR101524079B1 (en) * | 2014-12-03 | 2015-06-04 | 서울대학교산학협력단 | Method for inducing differentiation of adult cells into insulin producing cells using exosome |
-
2018
- 2018-06-04 CN CN201810561869.1A patent/CN108441464B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468642A (en) * | 2013-09-23 | 2013-12-25 | 山西大学 | Method for separating exosome from cell culture medium |
| KR101524079B1 (en) * | 2014-12-03 | 2015-06-04 | 서울대학교산학협력단 | Method for inducing differentiation of adult cells into insulin producing cells using exosome |
Non-Patent Citations (3)
| Title |
|---|
| Human pluripotent stem cell differentiation to functional pancreatic cells for diabetes therapies: Innovations, challenges and future directions;Jacobson et al.;《Journal of Biological Engineering》;20171231;第11卷(第1期);第1页右栏第2段，第10页右栏第2段 * |
| New emerging tasks for microRNAs in the control of β-cell activities;Claudiane et al.;《Biochim Biophys Acta》;20160510;第1861卷(第12期);第2122页第3节至第2123页第4.1节 * |
| 小鼠胰岛微血管内皮细胞外泌体的提取和鉴定方法;肖芳等;《山东大学学报》;20150131;第53卷(第1期);第7页第1.2节至第8页第2.2节 * |
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