CN114292848A - Engineered exosome with high expression of cancer-suppressing miRNA and targeting lung cancer - Google Patents
Engineered exosome with high expression of cancer-suppressing miRNA and targeting lung cancer Download PDFInfo
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Abstract
The invention provides an engineered exosome for high-expression cancer-suppressing miRNA and targeting lung cancer, a preparation method of the engineered exosome, and drug loading and targeting therapy in lung cancer disease treatment by using the engineered exosome. The engineered exosome for high-expression cancer-inhibiting miRNA and targeting lung cancer can stably carry lung targeting alpha 6 beta 1 protein, and simultaneously wraps miRNA for inhibiting growth and transfer of lung cancer: the miR-214 and miR-770 can be transmitted to the lung in a targeted mode and can inhibit the lung cancer, and experimental results show that the produced engineered exosome has the characteristic of targeting the lung cancer, so that the amount of the exosome entering lung tissues is increased, meanwhile, the produced engineered exosome carries high-kurtosis miR-214 and miR-770, the concentrations of miR-214 and miR-770 of the lung tissues are increased, the growth and transfer of the lung cancer can be effectively inhibited, in addition, the engineered stable cell line can be conveniently protected, passage is convenient, and the engineered exosome can be continuously generated.
Description
Technical Field
The invention relates to the technical field of engineered exosomes, in particular to an engineered exosome with high expression of cancer-suppressing miRNA and targeting lung cancer.
Background
The exosome is a micro vesicle with a lipid bilayer membrane structure, has the diameter of 30-150nm, can be secreted by almost all cells, can mediate substance exchange and information exchange among the cells, and researches show that the exosome carries various cancer-promoting genes or cancer-inhibiting genes.
At present, exosomes are often made to carry specific miRNA through electrotransfer or specific transfection reagents to prepare engineered exosomes, but the modes are often disposable, the operation is complex and the price is high, the exosomes cannot be stably carried at a high level and are only used for scientific research, in addition, the exosomes also have targeting performance, the number of exosomes actually transmitted to specific tissue organs is small after the exosomes are usually returned into the body, most of the exosomes enter other organs in the body through blood circulation, the injected exosomes are diluted to a great extent, and the organs needing treatment have no drug accumulation, so that the expected treatment effect cannot be achieved, and therefore, the engineered exosomes which highly express cancer-suppressing miRNA and target lung cancer are provided to solve the problems.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides an engineered exosome for high-expression cancer-suppressing miRNA and targeting lung cancer, the high-expression cancer-suppressing miRNA and lung cancer targeting engineered exosome can infect 293T cells by packaging lentiviruses capable of high-expression integrin alpha 6 beta 1 and lentiviruses capable of high-expression miR-214 and miR-770, and then screening out a lentivirus 293T stable cell line capable of simultaneously expressing alpha 6 beta 1, miR-214 and miR-770 through puromycin, so that the cell membrane of the secreted exosome can stably carry lung targeting alpha 6 beta 1 protein, and simultaneously the miRNA for suppressing the growth and transfer of the lung cancer is packaged: miR-214 and miR-770, thereby obtaining the engineered exosome which can be transmitted to the lung in a targeted manner and can inhibit the lung cancer.
(II) technical scheme
Aiming at the problem that the targeting of exosome is poor, exosome which is actually transmitted to a specific tissue organ is less after being returned to a body often, most exosome enters other organs in the body through blood circulation, the exosome which is equivalent to injection is diluted to a great extent, and the organ needing to be treated does not accumulate drugs, so that the expected treatment effect cannot be achieved, the inventor finds in research that integrin alpha 6 beta 1 has lung targeting property, and the exosome which is highly expressed and carries alpha 6 beta 1 can improve the aggregation of the integrin in the lung, moreover, the alpha 6 beta 1 can be spontaneously packaged on an exosome membrane to resist miRNA related to lung cancer, if miR-302b, miR-214, miR-3180-3p, miR-770, miR-338-3p, miR-let-7e, miR-613 and the like exist in exosomes, it is suggested that the miRNA kurtosis in exosomes can be improved by over-expressing the specific miRNAs.
Based on the technical scheme, the invention provides an engineered exosome for high-expression cancer-suppressing miRNA and targeting lung cancer, and the exosome can stably carry lung targeting alpha 6 beta 1 protein and wrap miRNA for suppressing growth and transfer of lung cancer.
In specific embodiments, the miRNAs are miR-214 and miR-770.
The invention also aims to provide a preparation method of the engineered exosome for high-expression cancer-suppressing miRNA and targeting lung cancer, which comprises the following steps: 293T is infected by three lentiviruses of packaging overexpression alpha 6 beta 1, miR-214 and miR-770, then stable cell lines of the lentiviruses of the expression alpha 6 beta 1, miR-214 and miR-770 are screened by puromycin, cell supernatants are collected, and engineered exosomes are separated by ultracentrifugation.
The slow virus for packaging the overexpression alpha 6 beta 1, miR-214 and miR-770 constructs gene sequences of the alpha 6 beta 1, miR-214 and miR-770 into a pCDH-CMV-MCS-EF1-copGFP vector multi-cloning site and packages the vector multi-cloning site, and specifically comprises the following steps:
a) plating cells with complete medium, 5X 106Perform a hole on a 10cm plate2A culture dish is used for ensuring that the cell density reaches 70-80% of the fusion degree on the next day;
b) diluting 7 mu g of recombinant shuttle plasmid +7 mu g of paspax2+7 mu g of pMD2.G in 800 mu L of serum-free culture medium;
c) diluting 63ul PEI solution by 800 mul of serum-free culture medium;
d) mixing the plasmid and PEI, incubating for 10min at room temperature, and supplementing 3ml of serum-free culture medium after the incubation time is up;
e) sucking out the original culture medium from the culture dish, and slowly adding the mixture of the plasmids and the PEI;
f) culturing in a carbon dioxide incubator at 37 ℃ for 6 h;
g) after 6h, removing the culture medium containing the DNA-PEI complex, and replacing the culture medium with a normal complete culture solution to continue culturing for 40-48 h;
h) collecting cell supernatant 48h after transfection;
i) centrifuging at 4 deg.C and 3000rpm for 10min to remove cell debris;
j) the supernatant was filtered through a 0.45 μm filter into a 40mL ultracentrifuge tube;
k) adding 2ml of 20% sucrose solution and 10ml of filtered supernatant into a centrifuge tube, and centrifuging at 4 ℃ for 2h at 100000 g;
l) absorbing and discarding the supernatant, standing the tube for 1-2min, and absorbing residual liquid;
m) adding 0.5ml of DMEM medium to dissolve the lentivirus precipitate;
n) filtering the viral resuspension through a 0.22 μm filter;
o) the concentrated virus suspension was aliquoted into 100. mu.L portions, stored in finished tubes, frozen quickly with crushed dry ice and stored at-80 ℃.
The puromycin screening expression alpha 6 beta 1, miR-214 and miR-770 lentivirus stable cell line is used for screening virus-infected cells through puromycin to obtain a monoclonal stable cell line, and the method specifically comprises the following steps:
a) culturing cells and adjusting the state, wherein a DEME culture medium is used as a culture medium and 10% fetal calf serum is added;
b) preparing 1mg/mL mother liquor from Puromycin, diluting the mother liquor with a complete culture medium in a 24-well plate, adding Puromycin according to working concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mu g/mL, and putting the mother liquor into an incubator for culture;
c) observing the growth condition of the cells on the second day and the third day, taking the lowest concentration of killing all the cells after the observation for three days as the screening concentration, and continuously screening the stable transformants by using Puromycin with the working concentration;
d) taking out the slow virus liquid frozen at-80 ℃, and thawing in ice bath for later use;
e) diluting the virus solution with culture medium to add 10 virus particles per well7The optimal MOI value can be found by literature reference and best pre-experiment, and the solution is changed into 2mL of culture medium after the virus infection is carried out for 6 hours; at the same time, uninfected control cells are made;
f) 48 hours after infection, changing the culture medium into a screening culture medium containing Puromycin concentration obtained in a drug sensitive experiment, and observing the cell state every day;
g) 3-5 days after the addition of the selection medium, observing that the cells in the control group have died, at which time the medium is changed to a maintenance medium of 0.1. mu.g per mL of Puromycin, and the culture is continued, observing every day;
h) after 2-3 days of culture, the cells are continuously expanded and cultured;
i) after about 15 days of drug screening, cells were collected and the expression of the target gene was detected using Q-PCR;
j) and (5) freezing and storing the stable cell strain.
The invention further provides a drug loading and targeted therapy in the treatment of lung cancer by using the engineered exosome.
(III) advantageous effects
Compared with the prior art, the invention provides an engineered exosome with high expression of cancer-suppressing miRNA and targeting lung cancer, and has the following beneficial effects:
1) the engineered stable cell line can preserve seeds, is convenient to passage and can continuously generate engineered exosomes;
2) the produced engineered exosome has the characteristic of targeting lung cancer, and the amount of exosome entering lung tissues is increased;
3) the produced engineered exosome carries high-kurtosis miR-214 and miR-770, so that the concentrations of miR-214 and miR-770 in lung tissues are improved, and the growth and the transfer of lung cancer can be effectively inhibited.
Drawings
FIG. 1 is a diagram showing the construction of the α 6 β 1 gene sequence into the pCDH-CMV-MCS-EF1-copGFP vector in the present invention;
FIG. 2 is a schematic flow diagram of the preparation of engineered exosomes of the present invention;
FIG. 3 is a diagram of the engineered exosome mode containing alpha 6 beta 1, miR-214 and miR-770 prepared by the invention;
FIG. 4 is a fluorescence diagram of the screening of the engineered 293T stable cell line expressing alpha 6 beta 1, miR-214 and miR-770 of the invention;
FIG. 5 is a diagram showing the results of Zetaview detection of exosome size and concentration according to the present invention;
FIG. 6 is a schematic diagram of the detection of exosome morphology by a projection electron microscope of the present invention;
FIG. 7 is a schematic diagram showing the results of western blot detection of the expression of an exosome marker and integrin α 6 β 1 of the present invention;
FIG. 8 is a diagram illustrating the results of detecting the expression level of miRNA in the engineered cells and exosomes according to the present invention;
FIG. 9 is a schematic diagram showing the results of detecting the effect of the engineered exosomes of the present invention in inhibiting the growth and metastasis of lung cancer cells;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In one aspect of the invention, the invention provides an engineered exosome for highly expressing cancer-suppressing miRNA and targeting lung cancer, the exosome can stably carry lung targeting alpha 6 beta 1 protein and simultaneously wrap miRNA for suppressing growth and transfer of lung cancer, wherein the miRNA is miR-214 and miR-770.
The integrin alpha 6 beta 1 has lung targeting property, high expression and alpha 6 beta 1-carrying exosomes can improve the aggregation of the exosomes in the lung, and the alpha 6 beta 1 can be spontaneously packaged on exosome membranes to resist lung cancer related miRNAs such as miR-302b, miR-214, miR-3180-3p, miR-770, miR-338-3p, miR-let-7e, miR-613 and the like existing in exosomes, so that the possibility of improving miRNA kurtosis in the exosomes by over-expressing the specific miRNAs is suggested.
Based on this, please refer to fig. 1, a lentivirus capable of highly expressing integrin α 6 β 1 and lentiviruses capable of highly expressing miR-214 and miR-770 are packaged, the lentivirus has a region containing 5 'LTR and 3' LTR, and the constructed target sequence can be inserted into a host cell (293T) genome, so as to realize long-term stable expression in the host cell, in addition, the lentivirus carries a puromycin resistance gene (PuroR), a lentivirus 293T stable cell line capable of simultaneously expressing α 6 β 1, miR-214 and miR-770 is screened out through puromycin, so that the secreted exosome cell membrane thereof can stably carry lung targeting α 6 β 1 protein, and simultaneously, miRNA for inhibiting lung cancer growth and metastasis is wrapped: miR-214 and miR-770, thereby obtaining an engineered exosome capable of being transmitted to lung in a targeted mode and inhibiting lung cancer.
It is to be noted that the different lentiviruses are named: lv-alpha 6 beta 1, Lv-miR-214 and Lv-miR-770.
Therefore, referring to fig. 2-3, in another aspect of the present invention, the present invention further provides a method for preparing an engineered exosome for highly expressing cancer-suppressing miRNA and targeting lung cancer, the method comprises the following steps: 293T is infected by three lentiviruses of packaging overexpression alpha 6 beta 1, miR-214 and miR-770, then stable cell lines of the lentiviruses of the expression alpha 6 beta 1, miR-214 and miR-770 are screened by puromycin, cell supernatants are collected, and engineered exosomes are separated by ultracentrifugation.
The slow virus for packaging the over-expression alpha 6 beta 1, miR-214 and miR-770 constructs gene sequences of the alpha 6 beta 1, miR-214 and miR-770 into a pCDH-CMV-MCS-EF1-copGFP vector multiple cloning site and packages the vector multiple cloning site, and specifically comprises the following steps:
a) plating cells with complete medium, 5X 106Perform a hole on a 10cm plate2A culture dish is used for ensuring that the cell density reaches 70-80% of the fusion degree on the next day;
b) diluting 7 mu g of recombinant shuttle plasmid +7 mu g of paspax2+7 mu g of pMD2.G in 800 mu L of serum-free culture medium;
c) diluting 63ul PEI solution by 800 mul of serum-free culture medium;
d) mixing the plasmid and PEI, incubating for 10min at room temperature, and supplementing 3ml of serum-free culture medium after the incubation time is up;
e) sucking out the original culture medium from the culture dish, and slowly adding the mixture of the plasmids and the PEI;
f) culturing in a carbon dioxide incubator at 37 ℃ for 6 h;
g) after 6h, removing the culture medium containing the DNA-PEI complex, and replacing the culture medium with a normal complete culture solution to continue culturing for 40-48 h;
h) collecting cell supernatant 48h after transfection;
i) centrifuging at 4 deg.C and 3000rpm for 10min to remove cell debris;
j) the supernatant was filtered through a 0.45 μm filter into a 40mL ultracentrifuge tube;
k) adding 2ml of 20% sucrose solution and 10ml of filtered supernatant into a centrifuge tube, and centrifuging at 4 ℃ for 2h at 100000 g;
l) absorbing and discarding the supernatant, standing the tube for 1-2min, and absorbing residual liquid;
m) adding 0.5ml of DMEM medium to dissolve the lentivirus precipitate;
n) filtering the viral resuspension through a 0.22 μm filter;
o) the concentrated virus suspension was aliquoted into 100. mu.L portions, stored in finished tubes, frozen quickly with crushed dry ice and stored at-80 ℃.
In addition, the puromycin screening expression alpha 6 beta 1, miR-214 and miR-770 lentivirus stable cell line is obtained by screening virus-infected cells through puromycin to obtain a monoclonal stable cell line, and the method specifically comprises the following steps:
a) culturing cells and adjusting the state, wherein a DEME culture medium is used as a culture medium and 10% fetal calf serum is added;
b) preparing 1mg/mL mother liquor from Puromycin, diluting the mother liquor with a complete culture medium in a 24-well plate, adding Puromycin according to working concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mu g/mL, and putting the mother liquor into an incubator for culture;
c) observing the growth condition of the cells on the second day and the third day, taking the lowest concentration of killing all the cells after the observation for three days as the screening concentration, and continuously screening the stable transformants by using Puromycin with the working concentration;
d) taking out the slow virus liquid frozen at-80 ℃, and thawing in ice bath for later use;
e) diluting the virus solution with culture medium to add 10 virus particles per well7The optimal MOI value can be found by literature reference and best pre-experiment, and the solution is changed into 2mL of culture medium after the virus infection is carried out for 6 hours; at the same time, uninfected control cells are made;
f) 48 hours after infection, changing the culture medium into a screening culture medium containing Puromycin concentration obtained in a drug sensitive experiment, and observing the cell state every day;
g) 3-5 days after the addition of the selection medium, observing that the cells in the control group have died, at which time the medium is changed to a maintenance medium of 0.1. mu.g per mL of Puromycin, and the culture is continued, observing every day;
h) after 2-3 days of culture, the cells are continuously expanded and cultured;
i) after about 15 days of drug screening, cells were collected and the expression of the target gene was detected using Q-PCR;
j) and (5) freezing and storing the stable cell strain.
By identifying the screened stable cells, as shown in fig. 4, the cells infected with the virus express GFP protein and therefore have green fluorescence, so that almost all the cells express GFP protein, and thus the success of screening the stable cell line of the lentivirus 293T can be determined.
Meanwhile, the engineered exosomes prepared by the method are identified:
1) exosome concentration and size were measured by Zetaview as follows:
a) extracting supernatant exosomes of the engineered cells by an ultracentrifugation method;
b) diluting exosomes to appropriate concentrations;
c) and (4) sucking the diluted exosome, loading the exosome to a Zetaview nanoparticle tracer, and detecting the concentration and the size of the exosome.
The results are shown in FIG. 5, and Zetaview assay shows that exosome concentration is 2.1X 1012Size (. times.50) of 123.8nm per ml, indicating that exosomes were obtained from the extraction and the concentration was relatively high.
2) Detecting the form of the exosome through a projection electron microscope, wherein the method comprises the following steps:
a) sucking 10 mu L of the exosome sample resuspended in PBS, dropwise adding the exosome sample on a copper net for precipitation for 1min, and sucking the supernatant by filter paper;
b) dripping 10 μ L uranyl acetate (phosphotungstic acid) on copper net for precipitation for 1min, and removing the supernatant with filter paper;
c) rinsing with PBS for 5min for 3 times;
d) dripping 10 μ L uranyl acetate (phosphotungstic acid) on copper net for precipitation for 1min, and removing the supernatant with filter paper;
e) drying for several minutes at normal temperature;
f)80kv is subjected to electron microscope detection imaging;
g) transmission electron microscopy (FEI Tecnai Spirit TEM T12) imaging results were obtained.
The detection result is shown in fig. 6, and the exosome-shaped particles detected by a projection electron microscope are in a saucer-shaped standard exosome shape and have the size of about 100 nm.
3) The expression of the exosome markers and the integrin alpha 6 beta 1 was detected by western blot, the method was as follows:
a) adding protein lysate to crack exosome, centrifuging at 12000rpm for 15min, and taking protein supernatant;
b) determining the sample protein concentration using a BCA protein concentration assay kit;
c) determining the loading amount according to the concentration of the sample, ensuring that the total protein loading amount of each sample is 50 mug, adding a proper amount of 5 Xprotein loading buffer solution into the protein sample, and carrying out boiling water bath at 95-100 ℃ for 5 min;
d) preparing SDS-PAGE protein separation gel and concentrated gel, inserting the gel into an electrophoresis comb, and pulling out the comb after the gel is solidified at room temperature;
e) performing constant-pressure electrophoresis according to the concentration glue of 80V and the separation glue of 120V until bromophenol blue reaches the lower edge of the gel plate;
f) transferring a membrane, preparing membrane transferring filter paper and a PVDF membrane, activating the PVDF membrane with methanol for 3min before use, placing a membrane transferring sandwich structure according to the directions of a positive electrode and a negative electrode, sequentially arranging membrane transferring sponge, 3 layers of filter paper, the PVDF membrane, glue, 3 layers of filter paper and membrane transferring sponge from the positive electrode to the negative electrode, removing bubbles in each layer in the placing process, transferring the membrane at a constant current of 300mA, and adjusting the membrane transferring time according to the molecular weight of a target protein;
g) and (3) incubating the antibody, adding the transferred membrane into a confining liquid for warm confining for 1h, removing the confining liquid, adding a primary antibody diluted by a primary antibody diluent at 4 ℃ overnight (the concentration range of the primary antibody is 0.5-2ug/ml), washing with TBST for three times, 5min each time, adding a secondary antibody diluted by a secondary antibody diluent, incubating for 30min at room temperature, washing with TBST for four times, 5min each time, in a shaking bed at room temperature, wherein the dilution ratio of the secondary antibody is 1: 10000) (ii) a
h) Chemiluminescence detection was performed by dropping a freshly prepared ECL mixed solution (a: B ═ 1:1) onto the protein surface side of the membrane, exposing in a dark room, adjusting the exposure conditions according to the different light intensities, developing, and fixing.
The results are shown in fig. 7, Western blot detects 293 engineered cells and exosome markers CD9, TSG101, and α 6 β 1 in the exosome proteins, and shows that exosome markers CD9, TSG101 are detected, and α 6 β 1 is expressed in 293T cells, which are also present in exosomes.
4) Detecting the expression quantity of miRNA in the engineered cells and the exosomes, wherein the method comprises the following steps:
cell and exosome RNA are extracted, miR-214 and miR-770 expression amounts are detected by a fluorescence quantitative kit (SYBR Green real PCR Master Mix), and the result is shown in FIG. 8, and shows that miR-214 in engineered 293T cells is overexpressed by 3700 times, miR-770 is overexpressed by 2500 times, miR-214 in engineered exosomes is overexpressed by 220 times and miR-770 is overexpressed by 410 times compared with cells in a control group.
5) The method for detecting the effect of the engineered exosome on inhibiting the growth and metastasis of the lung cancer cells comprises the following steps:
a) PKH26 staining of the engineered exosomes;
b) making an A549 cell slide;
c) incubating the exosomes dyed by PKH26 with A549 lung cancer cells, and observing the condition that the exosomes enter the cells through a laser confocal microscope after 6 hours;
d) detecting the cell transfer condition after the exosome co-incubation cells through a transwell experiment;
e) the dynamic growth of A549 cells after the exosome co-incubation at 0, 12, 24, 48 and 72h was tested by CCK 8.
The detection result is shown in fig. 9, and the result shows that the exosome stained by PKH26 has red fluorescence, the a549 cell nucleus has blue light after being stained by DAPI, and after the A549 cell is incubated by the stained exosome, the detection of a laser confocal microscope shows that the engineered exosome can enter the lung cancer cell (A), the non-engineered 293T cell exosome is used for incubating an A549 lung cancer cell line as a control group (control group Exo), the engineered 293T cell exosome is used as an experimental group (experimental group Exo), the cell metastasis condition is detected after the co-incubation of the cells, and the result shows that the metastasis of the lung cancer cell is obviously inhibited (B, C), the dynamic growth conditions of A549 cells 0, 12, 24, 48 and 72h after the exosome co-incubation are detected by CCK8, the results show that the cell growth of the engineered exosome incubation is obviously inhibited, and the engineered exosome can enter the lung cancer cell and inhibit the growth and metastasis of the lung cancer according to the judgment of the results.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. An engineered exosome for high-expression cancer-suppressing miRNA and targeting lung cancer is characterized in that the exosome can stably carry lung targeting alpha 6 beta 1 protein and simultaneously wraps miRNA for suppressing growth and transfer of lung cancer.
2. The engineered exosome according to claim 1, wherein the mirnas are miR-214 and miR-770.
3. Use of an engineered exosome according to any one of claims 1-2 in the preparation of a medicament for inhibiting lung cancer.
4. A method for the preparation of an engineered exosome according to any one of claims 1-2, comprising the following steps: 293T is infected by three lentiviruses of packaging overexpression alpha 6 beta 1, miR-214 and miR-770, then stable cell lines of the lentiviruses of the expression alpha 6 beta 1, miR-214 and miR-770 are screened by puromycin, cell supernatants are collected, and engineered exosomes are separated by ultracentrifugation.
5. The method for preparing the engineered exosome according to claim 4, wherein the slow virus over-expressing the alpha 6 beta 1, the miR-214 and the miR-770 is packaged to construct the alpha 6 beta 1, miR-214 and miR-770 gene sequences into the pCDH-CMV-MCS-EF1-copGFP vector multi-cloning sites and then packaged, and the method specifically comprises the following steps:
a) plating cells with complete medium, 5X 106Perform a hole on a 10cm plate2A culture dish is used for ensuring that the cell density reaches 70-80% of the fusion degree on the next day;
b) diluting 7 mu g of recombinant shuttle plasmid +7 mu g of paspax2+7 mu g of pMD2.G in 800 mu L of serum-free culture medium;
c) diluting 63ul PEI solution by 800 mul of serum-free culture medium;
d) mixing the plasmid and PEI, incubating for 10min at room temperature, and supplementing 3ml of serum-free culture medium after the incubation time is up;
e) sucking out the original culture medium from the culture dish, and slowly adding the mixture of the plasmids and the PEI;
f) culturing in a carbon dioxide incubator at 37 ℃ for 6 h;
g) after 6h, removing the culture medium containing the DNA-PEI complex, and replacing the culture medium with a normal complete culture solution to continue culturing for 40-48 h;
h) collecting cell supernatant 48h after transfection;
i) centrifuging at 4 deg.C and 3000rpm for 10min to remove cell debris;
j) the supernatant was filtered through a 0.45 μm filter into a 40mL ultracentrifuge tube;
k) adding 2ml of 20% sucrose solution and 10ml of filtered supernatant into a centrifuge tube, and centrifuging at 4 ℃ for 2h at 100000 g;
l) absorbing and discarding the supernatant, standing the tube for 1-2min, and absorbing residual liquid;
m) adding 0.5ml of DMEM medium to dissolve the lentivirus precipitate;
n) filtering the viral resuspension through a 0.22 μm filter;
o) the concentrated virus suspension was aliquoted into 100. mu.L portions, stored in finished tubes, frozen quickly with crushed dry ice and stored at-80 ℃.
6. The preparation method of the engineered exosome according to claim 4, wherein the puromycin screening expression α 6 β 1, miR-214 and miR-770 lentivirus stable cell line is obtained by screening virus-infected cells through puromycin to obtain a monoclonal stable cell line, and the preparation method specifically comprises the following steps:
a) culturing cells and adjusting the state, wherein a DEME culture medium is used as a culture medium and 10% fetal calf serum is added;
b) preparing 1mg/mL mother liquor from Puromycin, diluting the mother liquor with a complete culture medium in a 24-well plate, adding Puromycin according to working concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mu g/mL, and putting the mother liquor into an incubator for culture;
c) observing the growth condition of the cells on the second day and the third day, taking the lowest concentration of killing all the cells after the observation for three days as the screening concentration, and continuously screening the stable transformants by using Puromycin with the working concentration;
d) taking out the slow virus liquid frozen at-80 ℃, and thawing in ice bath for later use;
e) diluting the virus solution with culture medium to add 10 virus particles per well7The optimal MOI value can be found by literature reference and best pre-experiment, and the solution is changed into 2mL of culture medium after the virus infection is carried out for 6 hours; at the same time, uninfected control cells are made;
f) 48 hours after infection, changing the culture medium into a screening culture medium containing Puromycin concentration obtained in a drug sensitive experiment, and observing the cell state every day;
g) 3-5 days after the addition of the selection medium, observing that the cells in the control group have died, at which time the medium is changed to a maintenance medium of 0.1. mu.g per mL of Puromycin, and the culture is continued, observing every day;
h) after 2-3 days of culture, the cells are continuously expanded and cultured;
i) after about 15 days of drug screening, cells were collected and the expression of the target gene was detected using Q-PCR;
j) and (5) freezing and storing the stable cell strain.
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| CN114540307A (en) * | 2022-02-16 | 2022-05-27 | 多莱泌生物科技(武汉)有限公司 | Preparation method of liver-targeting engineered exosome |
| CN115074333A (en) * | 2022-06-30 | 2022-09-20 | 宁夏医科大学 | Preparation method and application of miR-206-loaded tumor cell-derived exosome |
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