CN109837306A - Contain the excretion body and its preparation method and application of miRNA-204-5p - Google Patents
Contain the excretion body and its preparation method and application of miRNA-204-5p Download PDFInfo
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Abstract
The invention discloses a kind of excretion bodies and its preparation method and application for containing miRNA-204-5p, it is related to biomedicine technical field, stablize the steady of overexpression tumor suppressor gene miRNA-204-5p by building and turns strain cell, separation and enrichment contain the excretion body of miRNA-204-5p, using excretion body as the drug delivery system of miRNA-204-5p, its therapeutic effect to tumour is played.The excretion body provided by the invention for containing miRNA-204-5p, significantly improves the compatibility with tumour cell, can efficiently discharge into miRNA-204-5p in tumour cell, increases tumour cell to the sensibility of chemotherapeutics, significantly inhibits the proliferation of tumour cell.Pass through situation in analogue body, the parameters such as reagent type used in preferred operations process, quality or concentration, miRNA-204-5p is loaded into naturally in excretion body in the cell, significantly improve miRNA-204-5p and excretion body in the cell contain efficiency, the risk during containing is reduced, the excretion body of preparation has more stability, safety and high efficiency.
Description
Technical field
The present invention relates to biomedicine technical field more particularly to a kind of excretion body for containing miRNA-204-5p and its systems
Preparation Method and application.
Background technique
Tumour is to lead to the dead second largest main cause.With molecular biology theory and technical method (especially
Recombinant DNA technology) rapid development, people can construct various carriers, clone and analysis target gene in laboratory, make disease
Research and treatment are deeply to molecular level, be then born gene diagnosis, gene therapy technology.Gene therapy is managed from gene angle
Solution is that reparation supplement is carried out to the gene of defect, or the method for corresponding to dcc gene with normal functional gene substitution.From controlling
Treat angle and understand be it is a kind of based on inhereditary material is imported to change the gene expression of Patient cells to reach treatment or prevention disease
The New Measure of the target of disease.Therapy of tumor has become the most noticeable research field of tumor biotherapy, be after operation,
The 5th kind of mode after radiotherapy, chemotherapy, immunization therapy, is the useful supplement of oncotherapy.
In recent years, a kind of non-coding RNA molecule being referred to as Microrna (miRNA) has been found to can be used as tumor suppression
Gene or oncogene regulate and control the occurrence and development of cancer.These miRNA length are generally 19 to 24 nucleotide.The function of miRNA
Exception takes part in the occurrence and development of nearly all tumor types.In general, miRNA is integrated to target gene courier by reverse complemental
The 3 '-non-translational regions (3 ' UTR) of RNA (mRNA) carry out negative regulation to gene.It is swollen that miRNA-204 was once reported in a variety of mankind
It is played in tumor and inhibits tumour function, the occurrence and development of hypofunction and kinds of tumors are closely related.For example, miRNA-204 exists
Breast cancer, clear cell carcinoma of kidney, cancer of pancreas, carcinoma of endometrium, glioma, gastric cancer, intrahepatic cholangiocarcinoma and head and neck neoplasm
Middle inhibition growth and/or invasion.In addition, miRNA-204 can be influenced by targeting BCL2 in gastric cancer and neuroblastoma
Chemosensitivity.Early-stage study discovery of the present invention, miRNA-204-5p can inhibit the proliferation and transfer of colorectal cancer in vivo and in vitro,
And colorectal cancer cell apoptosis can be promoted, increase its chemosensitivity to a variety of drugs.These are research shows that miRNA-
204-5p plays a significant role in tumor development, has potential application.
Currently, liposome transfection is the master that the recombinant expression plasmid of miRNA or the mature miRNA sequence of synthesis enter cell
Regulate and control the work that target gene downstream realizes its inhibition tumor cell proliferation, promotes apoptosis after wanting technological means, miRNA to enter cell
With.But its transfection efficiency is unstable using liposome transfection, different iuntercellulars have differences, and lipofectamine
It is more toxic, action time is shorter, and stability is poor.In this regard, people attempt that miRNA is inserted into cell with various viral vectors again
Genome is to solve liposome transfection efficiency.It is threatened however, viral vectors still has the safety of human genome, it may
Cause the hidden danger for being immunoreacted, being integrated into genome and virus that there is series of security questions caused by possibility for bringing back to life toxicity etc.
Endure dispute to the fullest extent always, scientists have obtained the virus table excessively for meeting clinical application requirement by the transformation to virus in recent years
Up to system.
Traditional view thinks intercellular AC mode, is the signal of donorcells such as autocrine, paracrine and endocrine
Molecule (such as hormone, cell factor) and receptor in target cell combine, and then play physiological function.Recently research confirms, cell energy
Extracellular vesica-excretion body that tool signal transfer function is enough secreted by special mechanism, conveys signaling molecule, into target cell
And play adjusting function.Excretion body is the extracellular vesica of partial size 30-150nm, can be released by the cell under physiology and pathological state
It puts, is widely present in the body fluid such as saliva, urine, milk.Excretion body can specific enrichment nucleic acid, protein and lipid, and pass
Recipient cell is passed, the physiology and pathologic process of the latter are adjusted.Compared with artificial constructed nano-carrier, excretion body not only has
Small in size, stable structure, the features such as its content can be protected not to be degraded, and because it derives from cell itself, naturally have
The advantages that no cytotoxicity, low immunogenicity, high-biocompatibility is a kind of ideal gene delivery vector.But excretion body
The Selecting Mechanism and Procedure of the substances such as specific enrichment nucleic acid, protein and lipid is unclear, the type of gene, the kind of " cell occurs "
The factors such as class and cultural method, culture environment will affect the combined coefficient for containing the excretion body of gene, stability and effect effect
Fruit, therefore how to be a technological difficulties by the gene for having therapeutic value loading excretion body, and at present carry miRNA-204-5p
Entering excretion body formation stable compound does not have effective method also.
Summary of the invention
In view of the above problems, the present invention provides a kind of excretion body and preparation method thereof for containing miRNA-204-5p
And application, by situation in analogue body, the parameters such as reagent type, quality or concentration used in preferred operations process, thin
It is intracellular that miRNA-204-5p is loaded into naturally in excretion body, significantly improve miRNA-204-5p and the packet of excretion body in the cell
Efficiency is carried, the risk during containing is reduced, the excretion body of preparation has more stability, safety and high efficiency.
To achieve the above object, technical solution provided by the invention are as follows:
The preparation method of the excretion body provided by the invention for containing miRNA-204-5p, comprising:
(1) building of expression vector: the oligonucleotide sequences of synthesis miRNA-204-5p precursor；By miRNA-204-5p
Precursor double stranded oligonucleotide is cloned into construction of expression vector in pCDH carrier；
(2) slow virus is packed: with the containing pCDH-miRNA-204, virus packaging plasmid pA2 and envelope plasmid pMDG2
One mixed liquor transfects HEK-293T cell；HEK-293T cell after culture transfection, collects culture solution, after isolating and purifying
Obtain slow virus；
(3) surely turn the foundation of strain cell: with the second mixed liquor containing polybrene, pCDH-miRNA-204 virus to HEK-
293T cell is transfected；It is cultivated for and obtains HEK-293T after being screened with puromycin surely turning a plant cell；
(4) extraction of excretion body: the cell culture fluid in collection step (3) carries out the separation and Extraction of excretion body, is wrapped
Carry the excretion body of miRNA-204-5p.
The preparation method of the excretion body provided by the invention for containing miRNA-204-5p, it is preferable that in step (1), also
The expression vector is converted including the use of Escherichia coli, extracts through amplification and after purification the expression vector.
The preparation method of the excretion body provided by the invention for containing miRNA-204-5p, it is preferable that first mixed liquor
Middle pCDH-miRNA-204, virus packaging plasmid pA2 and envelope plasmid pMDG2 mass ratio be 4:3:1.
The preparation method of the excretion body provided by the invention for containing miRNA-204-5p, it is preferable that second mixed liquor
The concentration of middle polybrene is 5~8 μ g/ml.
The preparation method of the excretion body provided by the invention for containing miRNA-204-5p, it is preferable that after step (4), also
It is specific to verify excretion body protein CD63 and flotillin2 and internal reference albumen β-actin including being verified to the excretion body
Expression.
The preparation method of the excretion body provided by the invention for containing miRNA-204-5p, it is preferable that after step (4), also
It include: to utilize the excretion body that miRNA-204-5p is contained described in modified with folic acid.
The preparation method preparation of the excretion body provided by the invention that contain miRNA-204-5p provided according to the present invention
Contain the excretion body of miRNA-204-5p.
Application of the excretion body provided by the invention for containing miRNA-204-5p in terms of preparing anti-tumor drug.
The application of the excretion body provided by the invention for containing miRNA-204-5p, it is preferable that described to contain miRNA-204-
The excretion body of 5p is preparing targeted inhibition tumor cell proliferation and/or is increasing on drug of the tumour cell to chemotherapy drug susceptibility
Application.
The technical scheme has the following advantages or beneficial effects:
The excretion body provided by the invention for containing miRNA-204-5p, significantly improves the compatibility with tumour cell, can
Efficiently miRNA-204-5p is discharged into tumour cell, increases tumour cell to the sensibility of chemotherapeutics, significantly inhibits
The proliferation of tumour cell.By situation in analogue body, reagent type, quality or concentration used in preferred operations process etc. is joined
MiRNA-204-5p, is loaded into excretion body naturally in the cell, significantly improves miRNA-204-5p with excretion body thin by number
The risk for containing efficiency, reducing during containing intracellular, the excretion body of preparation have more stability, safety and high efficiency.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, the present invention and its feature, outer
Shape and advantage will become more apparent.Identical label indicates identical part in all the attached drawings.Not deliberately according to than
Example draws attached drawing, it is preferred that emphasis is shows the gist of the present invention.
Fig. 1 is the building schematic diagram for the miRNA-204-5p expression vector that the embodiment of the present invention 1 provides；
Fig. 2 is the transfection schematic diagram for the miRNA-204-5p expression vector that the embodiment of the present invention 1 provides；
Fig. 3 is the result figure for the WesternBlot experimental verification excretion body marker protein that the embodiment of the present invention 1 provides；
Fig. 4 is the morphosis figure for the excretion body observed under the transmission electron microscope that the embodiment of the present invention 1 provides；
Fig. 5 is that the excretion body for containing miRNA-204-5p that the embodiment of the present invention 1 provides absorbs schematic diagram by tumour cell；
Fig. 6 is that the excretion body for containing miRNA-204-5p that the embodiment of the present invention 1 provides inhibits the proliferation of tumour cell to show
It is intended to；
Fig. 7 is clone's shape that the excretion body for containing miRNA-204-5p that the embodiment of the present invention 1 provides inhibits tumour cell
At schematic diagram；
Fig. 8 is that the excretion body for containing miRNA-204-5p that the embodiment of the present invention 1 provides increases tumour cell to chemotherapeutic
Object sensibility schematic diagram；
Fig. 9 is that the excretion body for containing miRNA-204-5p that the embodiment of the present invention 1 provides inhibits various tumour growth signals
Figure.
Figure 10 is the excretion body modified with folic acid schematic diagram for containing miRNA-204-5p that the embodiment of the present invention 1 provides；
Figure 11 is that the partial size for the excretion body that the modified with folic acid front and back that the embodiment of the present invention 1 provides contains miRNA-204-5p shows
It is intended to；
Figure 12 is that the current potential for the excretion body that the modified with folic acid front and back that the embodiment of the present invention 1 provides contains miRNA-204-5p shows
It is intended to；
Figure 13 is to contain the excretion body grain size stability of miRNA-204-5p after the modified with folic acid that the embodiment of the present invention 1 provides
Schematic diagram；
Figure 14 is to contain the excretion bulk potential stability of miRNA-204-5p after the modified with folic acid that the embodiment of the present invention 1 provides
Schematic diagram；
Figure 15 be the embodiment of the present invention 1 provide modified with folic acid after contain miRNA-204-5p excretion body targeting enter
Tumour cell schematic diagram.
Specific embodiment
With reference to the attached drawing in the embodiment of the present invention, technical solution in the embodiment of the present invention carries out clear, complete
Explanation, it is clear that described embodiment is only a part of the embodiments of the present invention, instead of all the embodiments.Therefore,
The range of claimed invention is not intended to limit to the detailed description in the embodiment of the present invention provided in attached drawing below,
But it is merely representative of selected embodiment of the invention.Based on the embodiment of the present invention, those skilled in the art are not making wound
Every other embodiment obtained under the premise of the property made is worked, belongs to protection scope of the present invention.
Embodiment 1:
1, the building and transfection of miRNA-204-5p expression vector
1) using Pubmed database search miRNA-204-5p precursor sequence and according to restriction enzyme site BamH I and EcoR
The protection base of I separately designs the inside and outside circle primer of miRNA-204 precursor.Relevant primer sequence (5 ' to 3 ') is as follows:
5 '-CTGCCTACTATGGATCATCC-3 ' of miRNA-204 precursor outer ring primer forward direction sequence
5 '-AGTTACTGACAGCATTCAC-3 ' of miRNA-204 precursor outer ring primer reverse sequence
MiRNA-204 precursor inner ring primer forward direction sequence
5’-CGCCGATCCGAATTACCCACAGGACAC-3’
MiRNA-204 precursor inner ring primer forward direction sequence
5’-CCGGCATTCGACTTGGAGCCAGATCTA-3’
2) target fragment containing pri-miRNA-204 sequence is obtained using PCR amplification kit.
Target fragment outer ring PCR system:
PCR program:
98 DEG C, 10s, 59 DEG C, 15s, 72 DEG C, 1min, 5 circulations；
98 DEG C, 10s, 57 DEG C, 15s, 72 DEG C, 1min, 5 circulations；
98 DEG C, 10s, 55 DEG C, 15s, 72 DEG C, 1min, 20 circulations.
Target fragment inner ring PCR system:
PCR program: with outer ring PCR.Product is purified using rapid DNA Product Purification Kit.
3) pCDH carrier and target fragment double digestion
Digestion system:
Digestion condition: 37 DEG C of water-baths, 6h.
Digestion products use purification kit gel extraction after carrying out agarose electrophoresis.
2% gel preparation method of target fragment electrophoresis are as follows: 2g agar+100ml H2O is heated to dissolving, and is added after cooling
EB。
1% gel preparation method of linearized vector electrophoresis are as follows: 1g agar+100ml H2O is heated to dissolving, and adds after cooling
Enter EB.
Deposition condition 120V constant pressure.
After the completion of electrophoresis, fragments gel is received next time in ultraviolet irradiation incision and is weighed, recycled according to plastic recovery kit double
Carrier and target fragment after digestion.
4) it connects
Linked system:
Carrier and the molecular weight ratio of target fragment should be 1:3~10 in system.
Condition of contact: 16 DEG C of low temperature water-baths are stayed overnight.
Wherein, the building schematic diagram of expression vector is as shown in Figure 1.
5) transformed competence colibacillus
From -80 DEG C of taking-up JM109 type E. coli competents, it is placed on ice slowly thawing.
Competence is added in 10 μ L linked systems (or 30ng plasmid), is incubated for 30min on ice, 42 DEG C of water-bath 90s are incubated on ice
Educate 3min.
6) clone is chosen
200 μ L are added in the Escherichia coli converted to be free of in the fluid nutrient medium of ammonia benzyl, 37 DEG C of constant-temperature tables, 80rpm
Concussion, expands 1h in advance.
The bacterium solution 2000rpm expanded in advance is centrifuged 3min.140 μ L supernatants are discarded, are gently blown remaining bacterium solution with liquid-transfering gun
After beating uniformly, scribing line is applied on the solid medium of benzyl containing ammonia, 37 DEG C of overnight incubations.
Bacteria culture media is prepared:
It is dissolved using ultrapure water, after high pressure sterilization, when being naturally cooling to 55 DEG C or so (hand, which is touched, not to scald), ammonia benzyl antibiosis is added
Element.Culture medium can be placed in 4 DEG C and save backup.
7) plasmid amplification
Next day bacterium colony is long to diameter about 1~2mm, provokes bacterium colony using 10 μ L lancet points, the liquid training of 5ml benzyl containing ammonia is added
Base is supported, is placed in 37 DEG C of constant-temperature tables, 200rpm concussion is overnight.
8) plasmid extracts
The bacterium solution 4000rpm of plasmid amplification is centrifuged 5min, and bacterial sediment is said referring to plasmid extraction kit after removing supernatant
Bright book extracts plasmid.
9) digestion verification
Digestion system and condition are same as above, and digestion system carries out agarose gel electrophoresis verifying.It selects digestion products and contains mesh
The correspondence plasmid of segment be sequenced.Sequencing result is compared using DNAMAN software with aim sequence, correct recombinant plasmid
It is expanded, 5) step is same as above to 8).
2, building HEK-293T surely turns a plant cell
1) slow virus is packed
The bioblast transfection method mediated using Lipofectamine 2000 is by recombinant plasmid pCDH-miRNA-204
(using pCDH-GFP plasmid as control empty vectors plasmid), virus packaging plasmid pA2 and envelope plasmid pMDG2 are transfected jointly
HEK-293T cell.
The good HEK-293T cell of the growth conditions of density about 70%, culture solution is changed into without blood in 6cm culture dish
The pure training of the DMEM of cleer and peaceful antibiotic is transfected after being placed in 37 DEG C of incubator culture 30min.
A: plasmid pCDH-miRNA-2044.4 μ g+ packaging plasmid pA23.3 μ g+ envelope plasmid pMDG21.3 μ g+DMEM
1.5mL is mixed and is stood；
200020 μ L+DMEM of B:Lipofectamine, 500 μ L is mixed and is stood 5min；
Both A, B are mixed, 20min is placed at room temperature for, is slowly added dropwise in HEK-293T Tissue Culture Dish.After 8h, replacement
20% serum complete medium 4ml, continues to cultivate.Fluorescence microscopy microscopic observation fluorescence intensity judges transfection efficiency after 48h.GFP
Group fluorescence is very strong, 95% or more positive rate.After 72h, culture medium is collected, 3000rpm room temperature is centrifuged 5min, and supernatant passes through 0.45 μm
Filter membrane filters, the packing of 500 μ l/ pipes, and virus can be directly used for infection or -80 DEG C of preservations.
The preparation method for the excretion body for containing miRNA-204-5p that the embodiment of the present invention 1 provides, by carrying out slow virus
Packaging process, so that target gene preservation stable in host cell and duplication, as the duplication of host cell guarantees purpose
Gene is not lost, and heredity can be stablized.
2) slow-virus infection building HEK-293T surely turns a plant cell
By the good HEK-293T cell of growth conditions, 12 orifice plates, cell density about 30% are inoculated in, cell should disperse
It is even, avoid growth effect efficiency of infection in blocks.The complete culture containing polybrene (final concentration of 6 μ g/ml) is separately added into each hole
Base after 30min, is added 500 μ LpCDH-miRNA-204 viruses and carries out cell infection (using pCDH-GFP as blank control group),
Fresh complete medium is changed after overnight.Observe fluorescence intensity after 48h, positive rate is close to 100%, wherein miRNA-204-5p table
Up to carrier transfection schematic diagram as shown in Figure 2.It, will be above-mentioned since pCDH carrier itself has the screening site of puromycin
Cell is cultivated for and obtains HEK-293T after being screened with puromycin surely turning a plant cell.
3, excretion body separation and Extraction and verifying
Collect HEK-293T cell culture fluid, at centrifugal force 2000*g, 4 DEG C be centrifuged 35 minutes, take supernatant in centrifugal force
Under 10000*g, 4 DEG C be centrifuged 60 minutes, then take supernatant at centrifugal force 110000*g, 4 DEG C be centrifuged 60 minutes, the PBS with 4 DEG C is molten
Liquid washing precipitating, obtains miRNA-204-5p excretion body.The albumen for extracting HEK-293T cell and its excretion body respectively, passes through
The expression of Western Blot experimental verification CD63, flotillin2, internal reference albumen β-actin, Western Blot are tested
Demonstrate,prove the result figure of excretion body marker protein as shown in Figure 3.Excretion can be observed through transmission electron microscope analysis in isolated excretion body sample
The classical saucer spline structure of body, the morphosis figure for the excretion body observed under transmission electron microscope is as shown in Figure 4.
The preparation method for the excretion body for containing miRNA-204-5p that the embodiment of the present invention 1 provides, by selecting excretion body
Target gene is imported to the carrier of cell, on the one hand, excretion body as natural lipid bilayer vesica, have it is small in size,
The characteristic of stable structure can avoid removing of the miRNA in blood circulation, extend the residence time, enhance miRNA-204-5p pairs
The therapeutic effect of colorectal cancer；On the other hand, excretion body naturally has no cytotoxicity, low exempts from due to deriving from cell itself
Epidemic focus, can pass through the advantages that blood-brain barrier at high-biocompatibility, therefore toxic side effect is small, is not easy to be removed by immune system, easily
In popularization and application, the function and effect of target gene are improved.
The preparation method for the excretion body for containing miRNA-204-5p that the embodiment of the present invention 1 provides, passes through analogue body inside information
Condition, the parameters such as reagent type, quality or concentration used in preferred operations process, in the cell by miRNA-204-5p nature
Be loaded into excretion body, significantly improve miRNA-204-5p and excretion body in the cell contain efficiency, during reduction contains
Risk, the excretion body of preparation has more stability, safety and high efficiency.
The excretion body for containing miR-204-5p can efficiently discharge into miRNA-204-5p in tumour cell, increase swollen
Oncocyte significantly inhibits the proliferation of tumour cell to the sensibility of chemotherapeutics.Its specific function and effect is as follows:
(1) the excretion body for containing miRNA-204-5p can efficiently enter tumour cell
Respectively by HCT116-GFP/miRNA-204, the excretion body and Colon and rectum of HEK-293T-GFP/miRNA-204 cell
Cancer cell co-cultures.It collects colorectal cancer cell RNA afterwards for 24 hours, carries out reverse transcription PCR and fluorescence real-time quantitative PCR detection.With
After HCT116-miRNA-204 excretion body co-cultures the expression of miRNA-204-5p increased separately (31.01 ± 1.60) times and
(29.44 ± 2.93) times, the expression of miRNA-204-5p increases separately after co-culturing with 293T-miRNA-204 excretion body
(30.8 ± 4.65) again with (41.97 ± 6.23) times (P is < 0.001), above-mentioned test result is as shown in Figure 5.Result above table
Bright, the excretion body for containing miRNA-204-5p after modification can efficiently enter tumour cell.
(2) the excretion body for containing miRNA-204-5p inhibits the proliferation of tumour cell
The excretion body colorectal cancer cell of 293T-miRNA-204 cell is co-cultured respectively, detects it using CCK-8 method
OD450 value, and draw growth curve, the results showed that the excretion body of 293T-miRNA-204 cell can inhibit colorectal cancer cell
It is proliferated (Fig. 6).Colorectal cancer cell after we co-culture excretion body simultaneously carries out colony formation, as shown in fig. 7, knot
After fruit shows the excretion body co-cultivation of LoVo cell and 293T-miRNA-204 cell, clone's number is that (111.00 ± 17.28) are a,
Control group is that (189.66 ± 23.01) are a (P < 0.001).HCT116 cell and the excretion body of 293T-miRNA-204 cell are trained altogether
After supporting, clone's number is that (160.00 ± 8.60) are a, and control group is that (240.66 ± 24.28) are a (P < 0.001), result above table
Bright, the excretion body of 293T-miRNA-204 cell can inhibit the proliferation of colorectal cancer cell.
(3) the excretion body for containing miRNA-204-5p increases tumour cell to chemotherapy drug susceptibility
Using the excretion body of 293T-miRNA-204 cell respectively with the co-cultivation of colorectal cancer cell, fluorouracil is added
After (final concentration of 6 μ g/mL) acts on 48h, collects cell and carry out Flow cytometry apoptosis.As shown in figure 8, with control group phase
Than after co-culturing with the excretion body of 293T-miRNA-204, colorectal cancer cell Apoptosis is dramatically increased.Result above table
Bright, excretion body miRNA-204-5p can promote the apoptosis of colorectal cancer cell.
(4) the excretion body for containing miRNA-204-5p inhibits the proliferation of other tumour cells
Recently many research discovery miRNA-204-5p can inhibit the proliferation of different type tumour cell, such as breast cancer, stomach
Cancer etc. shows that miRNA-204-5p is the cancer suppressorfactor of a wide spectrum.We test the packet after detection modification by CCK-8 herein
Carry Proliferation Ability of the excretion body to other types tumour cell (breast cancer, gastric cancer, lung cancer, glioma) of miRNA-204-5p
Effect.As shown in figure 9, excretion body miRNA-204-5p can inhibit breast cancer cell (MDB-231 and MCF-7), stomach cancer cell
(SGC-7901), lung carcinoma cell (A549), the proliferative capacity of brain glioblastoma cell (U251), result above further demonstrate that excretion
Body miRNA-204-5p can be used as a kind of oncotherapy means of wide spectrum.
4, modified with folic acid and the identification of the excretion body of miRNA-204-5p are contained
1) excretion body (FA-PEG-exo) preparation method for targeting folacin receptor modified with DSPE-PEG-FA is as follows:
10 μ L excretion bodies (3 μ g albumen/μ L) are diluted to 900 μ L with sterile PBS buffer (pH7.2), are then added 100 μ L's
DSPE-PEG-FA (50 μ g/ml) is to form new blend.By solution at 37 DEG C oscillation incubation 48 hours.
2) excessive free DSPE-PEG-FA is removed by size exclusion chromatography using Sephadex G25 column, purified
FA-PEG-exosomes.Contain the excretion body modified with folic acid schematic diagram of miRNA-204-5p as shown in Figure 10.
3) FA-PEG-exosomes purified analyzes hydrodynamic diameter and zeta electricity by dynamic light scattering (DLS)
Position, modified with folic acid front and back contain the partial size schematic diagram of the excretion body of miRNA-204-5p as shown in Figure 11, packet before and after modified with folic acid
Carry the current potential schematic diagram of the excretion body of miRNA-204-5p as shown in Figure 12.The excretion body grain of miRNA-204-5p is contained after modification
The excretion bulk potential stability schematic diagram that diameter stability schematic diagram contains miRNA-204-5p as shown in Figure 13, after modification is shown in Figure 14
Shown, the excretion body partial size and current potential that -20 DEG C are stored in it can be seen from Figure 13 and Figure 14 change over time less.
Folacin receptor is not only widely distributed in normal tissue, but also is distributed in tumor tissues, the difference lies in that most tumors
The quantity of cell folate receptor is with active considerably beyond normal cell.Folacin receptor is distributed not in normal cell and tumour cell
Together, drug administration carrier is exactly passed through to folacin receptor mediated, the theoretical basis of targeting importing tumour cell in conjunction with folate ligand.This
Inventive embodiments 1 are by carrying out modified with folic acid to the excretion body for containing miRNA-204-5p, by being delivered to for excretion body selectivity
Tumour cell plays inhibiting effect of the miRNA-204-5p to tumour cell and the sensibility to chemotherapeutics, improves purpose base
The functioning efficiency of cause enhances antitumor effect.
Excretion prepared by the preparation method of the excretion body for containing miRNA-204-5p provided using the embodiment of the present invention 1
Body can significantly improve the compatibility with tumour cell after modified with folic acid.As shown in figure 15, it after modified with folic acid, contains
The excretion physical efficiency of miRNA-204-5p more efficiently enters tumour cell.
In conclusion the excretion body and its preparation method and application provided by the invention for containing miRNA-204-5p, passes through
The excretion for surely turning strain cell, separation and enrichment miRNA-204-5p for being overexpressed tumor suppressor gene miRNA-204-5p is stablized in building
Body plays its therapeutic effect to tumour using excretion body as the drug delivery system of miRNA-204-5p；It is provided by the invention
Contain the excretion body of miRNA-204-5p, improve the compatibility with tumour cell, can efficiently by miRNA-204-5p discharge into
Enter intracellular, increase tumour cell to the sensibility of chemotherapeutics, plays miRNA-204-5p to the inhibiting effect of tumour cell,
The proliferation and growth for significantly inhibiting tumour cell have the function of the treatment of tumor disease positive；It is provided by the invention to contain
The preparation method of the excretion body of miRNA-204-5p, by situation in analogue body, reagent type used in preferred operations process,
MiRNA-204-5p, is loaded into excretion body naturally in the cell, significantly improves miRNA- by the parameters such as quality or concentration
The risk for containing efficiency, reducing during containing of 204-5p and excretion body in the cell, the excretion body of preparation is with more stabilization
Property, safety and high efficiency.
It should be appreciated by those skilled in the art that those skilled in the art combine the prior art and above-described embodiment can be real
The existing change case, it will not be described here.Such change case does not affect the essence of the present invention, and it will not be described here.
Presently preferred embodiments of the present invention is described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, devices and structures not described in detail herein should be understood as gives reality with the common mode in this field
It applies；Anyone skilled in the art makes many possible changes and modifications not departing from technical solution of the present invention, or
Equivalent example modified to equivalent change, this is not affected the essence of the present invention.Therefore, all without departing from skill of the present invention
The content of art scheme, according to the technical essence of the invention any simple modification made to the above embodiment, equivalent variations and repair
Decorations, all of which are still within the scope of protection of the technical scheme of the invention.
Claims (9)
1. a kind of preparation method for the excretion body for containing miRNA-204-5p characterized by comprising
(1) building of expression vector: the oligonucleotide sequences of synthesis miRNA-204-5p precursor；By miRNA-204-5p precursor
Double stranded oligonucleotide is cloned into construction of expression vector in pCDH carrier；
(2) slow virus is packed: mixed with first containing pCDH-miRNA-204, virus packaging plasmid pA2 and envelope plasmid pMDG2
Liquid is closed to transfect HEK-293T cell；HEK-293T cell after culture transfection, collects culture solution, obtains after isolating and purifying
Slow virus；
(3) surely turn the foundation of strain cell: with the second mixed liquor containing polybrene, pCDH-miRNA-204 virus to HEK-293T
Cell is transfected；It is cultivated for and screens to obtain HEK-293T with puromycin surely to turn a plant cell；
(4) extraction of excretion body: the cell culture fluid in collection step (3) carries out separation and Extraction to excretion body, is contained
The excretion body of miRNA-204-5p.
2. containing the preparation method of the excretion body of miRNA-204-5p as described in claim 1, which is characterized in that in step
(1) in, further include converting the expression vector using Escherichia coli, extract through amplification and after purification the expression vector.
3. containing the preparation method of the excretion body of miRNA-204-5p as described in claim 1, which is characterized in that described first
PCDH-miRNA-204 in mixed liquor, virus packaging plasmid pA2 and envelope plasmid pMDG2 mass ratio be 4:3:1.
4. containing the preparation method of the excretion body of miRNA-204-5p as described in claim 1, which is characterized in that described second
The concentration of polybrene is 5~8 μ g/ml in mixed liquor.
5. containing the preparation method of the excretion body of miRNA-204-5p as described in claim 1, which is characterized in that in step
It (4) further include being verified to the excretion body after, it is specific to verify excretion body protein CD63, flotillin2 and internal reference albumen
The expression of β-actin.
6. containing the preparation method of the excretion body of miRNA-204-5p as described in claim 1, which is characterized in that in step
(4) after, further includes: utilize the excretion body for containing miRNA-204-5p described in modified with folic acid.
7. prepared by a kind of preparation method of excretion body for containing miRNA-204-5p as described in claim 1~6 any one
The excretion body for containing miRNA-204-5p.
8. a kind of excretion body answering in terms of preparing anti-tumor drug for containing miRNA-204-5p as claimed in claim 7
With.
9. containing the application of the excretion body of miRNA-204-5p as claimed in claim 8, which is characterized in that described to contain
The excretion body of miRNA-204-5p is preparing targeted inhibition tumor cell proliferation and/or is increasing tumour cell to chemotherapy medicament sensitive
Application on the drug of property.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110343750A (en) * | 2019-06-06 | 2019-10-18 | 宽盈医疗科技(上海)有限公司 | For detecting reference gene and its application of excretion body amplifying nucleic acid expression |
| CN110530964A (en) * | 2019-09-05 | 2019-12-03 | 江南大学 | A kind of markers characteristic of milk excretion body and the method for identifying milk excretion body |
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| CN113528526A (en) * | 2021-07-15 | 2021-10-22 | 呈诺再生医学科技(珠海横琴新区)有限公司 | MiRNA drug delivery system with exosome as carrier and application thereof |
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| 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 |
| CN115804847A (en) * | 2022-07-26 | 2023-03-17 | 四川省医学科学院·四川省人民医院 | PH/hydrogen peroxide/MMP 9 time-ordered response microsphere, exosome-carrying biological carrier and application |
| WO2023098101A1 (en) * | 2021-12-03 | 2023-06-08 | 温州医科大学 | Exosome secreted by lncrna gene modified cell and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104411339A (en) * | 2012-06-21 | 2015-03-11 | 北京命码生科科技有限公司 | Microparticle comprising functional microRNA/siRNA and application thereof |
| CN105505876A (en) * | 2014-09-26 | 2016-04-20 | 上海市肿瘤研究所 | XL-2 human lung cancer highly metastatic cell line and applications |
| CN106692984A (en) * | 2016-12-08 | 2017-05-24 | 武汉大学 | Tumor-targeted delivery carrier based on cell-derived micro-vacuoles, preparation method and application |
| CN108753726A (en) * | 2018-06-11 | 2018-11-06 | 西南医科大学 | A kind of excretion body and its preparation method and application containing ECRG4 mRNA |
-
2019
- 2019-03-20 CN CN201910211056.4A patent/CN109837306A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104411339A (en) * | 2012-06-21 | 2015-03-11 | 北京命码生科科技有限公司 | Microparticle comprising functional microRNA/siRNA and application thereof |
| CN105505876A (en) * | 2014-09-26 | 2016-04-20 | 上海市肿瘤研究所 | XL-2 human lung cancer highly metastatic cell line and applications |
| CN106692984A (en) * | 2016-12-08 | 2017-05-24 | 武汉大学 | Tumor-targeted delivery carrier based on cell-derived micro-vacuoles, preparation method and application |
| CN108753726A (en) * | 2018-06-11 | 2018-11-06 | 西南医科大学 | A kind of excretion body and its preparation method and application containing ECRG4 mRNA |
Non-Patent Citations (2)
| Title |
|---|
| BOK SIL HONG ET AL.: "Tumor Suppressor miRNA-204-5p Regulates Growth, Metastasis, and Immune Microenvironment Remodeling in Breast Cancer", CANCER MED ., vol. 79, no. 7, pages 2 - 15 * |
| REYHANEH LAHMY ET AL.: "MiRNA-375 promotes beta pancreatic differentiation in human induced pluripotent stem (hiPS) cells", MOL BIOL REP, vol. 41, no. 4, pages 2057 * |
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