CN108753726A - A kind of excretion body and its preparation method and application containing ECRG4 mRNA - Google Patents
A kind of excretion body and its preparation method and application containing ECRG4 mRNA Download PDFInfo
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- CN108753726A CN108753726A CN201810594022.3A CN201810594022A CN108753726A CN 108753726 A CN108753726 A CN 108753726A CN 201810594022 A CN201810594022 A CN 201810594022A CN 108753726 A CN108753726 A CN 108753726A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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Abstract
The invention discloses a kind of excretion body and its preparation method and application containing ECRG4 mRNA；The excretion body is mounted with the ECRG4 mRNA for the Hemapoiesis for being overexpressed ECRG4 genes, and it can carry it to recipient cell, to significantly improve the expression of recipient cell ECRG4 mRNA, and then inhibit tumor cell proliferation/growth, the expression of angiogenesis, inflammatory reaction related gene；Meanwhile the compatibility of the excretion body and recipient cell is good, will not cause the immune response and inflammatory reaction of receptor, can significantly reduce the toxic side effect of gene therapy；The excretion physical efficiency significantly inhibits the proliferation growth of tumour cell；There is positive effect to the treatment of tumor disease.
Description
Technical field
The present invention relates to cell biologies, and in particular to a kind of excretion body containing ECRG4 mRNA and its preparation side
Method and application.
Background technology
Oral squamous cell carcinoma（Oral Squamous Cell Carcinoma, OSCC）Seriously threaten human health
Malignant tumour explores the hot spot that safe and efficient therapeutic scheme is medical research.People's Genes of Esophageal Cancer 4（Esophageal
Cancer Related Gene 4, ECRG4) it is to use mRNA differential display techniques, have from what normal person's Esophageal Epithelium was cloned
The tumor suppressor gene of extensive tumor suppression function.ECRG4 is widely distributed in human multiple tissue organ, including the heart, brain, tire
Disk, lung, liver, skeletal muscle, kidney and pancreas etc..In the generation of many tumours such as cancer of the esophagus, colon cancer, breast cancer, lung cancer, kidney
Cheng Zhong, ECRG4 expression quantity are obviously lowered, and the prognosis of its expression and tumour patient is proportionate.Experiments have shown that：Restore
The expression of ECRG4 can inhibit the proliferation of the tumour cells such as gastric cancer, colon cancer, the cancer of the esophagus, liver cancer, glioma and moves in vitro
It moves, and inhibits the growth and transfer of a variety of transplantable tumors in vivo.Therefore, tumour cell ECRG4 is improved by gene transportation techniques
Expression quantity, will have potential therapeutic value.
Excretion body (exosome) is that cell is secreted, the film property corpusculum of diameter about 30-150nm.It is almost all of in human body
Cell can secrete excretion body, and excretion body exists in all body fluid.The forming process of excretion body is as follows：Including in cell
The membrane invagination of body simultaneously selectively wraps up the protein in endochylema, the nucleic acid such as lipid and miRNA, mRNA, DNA, lncRNA
And form film property corpusculum, when inclusion body and when cell membrane fusion, film property corpusculum be released to it is extracellular enter body circulation, the film property is small
Body is excretion body.Excretion body is discharged into recipient cell by being combined with recipient cell, by the content of excretion body, to mediate
Signal between ' cell-ECM ' transmits and the exchange of inhereditary material, and then influences the occurrence and development process of recipient cell disease.
The study found that excretion body enters recipient cell by a kind of efficient mechanism similar to Virus entry cell, and excretion body has
There are good bio distribution and biocompatibility, naturally, stablize, nano-scale, biological barrier can be penetrated, low immunogenicity is gone back
Treatment preparation that is single or being optionally combined can be carried, is that ideal gene transports carrier.
Early-stage study of the present invention finds, the ECRG4 mRNA containing overall length in human serum excretion body, and oral squamous cell carcinomas disease
The expression quantity of ECRG4 mRNA is substantially less than Healthy People in human serum excretion body, prompts ECRG4 mRNA expression in serum excretion body
The reduction of amount and being related closely for tumour improve ECRG4 mRNA in serum excretion body and make with potential tumor suppression
With.But the mRNA of external source is directly entered the nuclease degradation being easy in blood by blood, it is therefore desirable to be transported by means of gene
Send carrier.Currently, it includes viral vectors or non-viral carrier systems that traditional gene, which transports carrier mainly, there is carrier poison
Property, lose target phenomenon, inefficient gene transports, vivo biodistribution half-life short and the defects of cause inflammation and immune response, seriously affect
The safety of gene therapy and validity need to find a kind of more safe and efficient gene transport carrier of exploitation.Due to excretion
Body can cross the good characteristics such as biological barrier, excretion body with natural stabilisation, nano-scale, non-immunogenicity, no cytotoxicity
Huge application value will be had by transporting carrier as gene.
Since excretion body is cell itself secretion, and the signaling molecule in endochylema is selectively wrapped up in forming process,
But its selective mechanism is not known.Therefore, how to be a technological difficulties by the gene for having therapeutic value loading excretion body.It will
Foreign gene is loaded into excretion body, mainly there is following four method：（1）Electroporation：The foreign gene synthesized in vitro is led to
The mode for crossing electroporation imports in excretion body；（2）Multigelation method：After excretion body is mixed with foreign gene multigelation by its
It is packaged into excretion body；（3）Direct transfection method：The external source that will directly be synthesized in vitro using transfection reagent such as Lipofectamine
Gene is transfected into excretion body；（4）Indirect infection protocol：By in exogenous gene cloning to expression vector, transfection excretion body " occurs thin
Born of the same parents " make target gene be overexpressed " occur cell ", the foreign gene being overexpressed in endochylema in the generating process of excretion body just
It is wrapped into excretion body.
First three methods mainly change the physical arrangement of excretion body by certain stimulus, to keep excretion body passive
Foreign gene is loaded, there is the great risks for changing excretion volume morphing structure for this method, and packaging efficiency is limited.And the
The difference of expression way, effect, limiting factor of 4 kinds of methods due to each gene in different cells etc., by different genes
It is loaded into excretion body, the type according to gene, the type of " occur cell " and cultural method, culture environment etc. is needed to carry out specific aim
Selection, is likely to obtain and loads that gene dosage is more, expression stability is good, the excretion body that compatibility is good, applicability is good.
Invention content
It being mounted with the excretion body of a large amount of ECRG4 mRNA the purpose of the present invention is to provide a kind of, and provides the excretion body
Specific preparation method and the related application in gene therapy.A large amount of ECRG4 mRNA are mounted in excretion body of the present invention, and
It can carry it to recipient cell, so as to significantly improve the expression of ECRG4 in recipient cell, and then inhibit tumour cell
The expression of proliferation/growth, angiogenesis, inflammatory reaction related gene；Simultaneously as the compatibility of the excretion body and recipient cell
Very well, the immune response and inflammatory reaction that will not cause receptor, can significantly reduce the toxic side effect of gene therapy；Especially this is outer
Secrete physical efficiency significantly inhibit oral squamous cell carcinomas proliferation growth, it can also be used to inhibit cancer of the esophagus, colon cancer, breast cancer, lung cancer, kidney,
The growth of the tumour cells such as gastric cancer, colon cancer, the cancer of the esophagus, liver cancer, glioma；There is positive effect to oncotherapy.
To achieve the above object of the invention, the present invention provides a kind of excretion body, which, which is mounted with, is overexpressed ECRG4 bases
The ECRG4 mRNA of the Hemapoiesis of cause；Wherein, the ECRG4 mRNA include complete ECRG4 encoding genes.
The present invention provides a kind of excretion body, which is formed by being overexpressed the cell secretion of ECRG4 genes,
A large amount of ECRG4 mRNA are inside mounted with, it, can be by a large amount of ECRG4 of loading by the superior compatibility of excretion body and recipient cell
MRNA is transported to recipient cell, significantly improves the expression quantity of ECRG4 in recipient cell, so as to significantly inhibit the life of tumour cell
Long and proliferation；And the immune response and inflammatory reaction of receptor will not be caused；Therefore, it is mounted with the excretion body of a large amount of ECRG4 mRNA
Appearance have to the method for exploring gene therapy tumour and greatly facilitate and directive function.
Above-mentioned excretion body, wherein the cell for being overexpressed ECRG4 is ECRG4 genes by a large amount of transcription and translation, because
This ECRG4 gene expression product is more than the cell of normal level.
The ECRG4 mRNA amounts loaded in above-mentioned excretion body are not less than 1000 times of conventional excretion body；ECRG4 in excretion body
The content of mRNA is higher, then better to the humidification of ECRG4 expressions in recipient cell, to the inhibition of tumour cell
Better；The routine excretion body refers to the excretion body for the normal cell secretion for not being overexpressed ECRG4 genes.
Above-mentioned excretion body, wherein preferred, the cell of the overexpression ECRG4 is ECRG4-HEK293 cell lines；It should
The overexpression ECRG4 that cell line can be stablized, to which secretion obtains the excretion body containing a large amount of ECRG4 mRNA.
Wherein, it is preferred that the ECRG4-HEK293 cell lines are prepared by following preparation method：
A, pLVX-IRES-ZsGreen1-ECRG4 plasmid constructions：Total serum IgE is extracted, ECRG4 genes are expanded with reverse transcription PCR, it will
ECRG4 genes are loaded on pLVX-IRES-ZsGreen1 carriers, obtain pLVX-IRES-ZsGreen1-ECRG4 plasmids；
B, the preparation of slow virus：With containing tri- kinds of plasmids of pMD2.G, psPAX2 and pLVX-IRES-ZsGreen1-ECRG4
One mixed liquor transfects HEK293T cells；HEK293T cells after culture transfection, collect culture solution, are carried out to culture solution
Processing obtains the slow virus for being packaged with ECRG4 genes；
C, the foundation of ECRG4-HEK293 cell lines：With the second mixed liquor pair containing slow virus, DMEM culture mediums and polybrene
HEK293 cells are infected；Metainfective HEK293 cells are cultivated, by cell sorting, obtain ECRG4-HEK293
Cell line.
The ECRG4-HEK293 cell lines being prepared by the above method can be continual and steady overexpression ECRG4, cross table
More preferable up to stability, the content higher of ECRG4 mRNA, solves from source containing a large amount of ECRG4 in obtained excretion body
The excretion body of mRNA carrys out source problem, is conducive to extensive, the standardized production of the excretion body containing ECRG4 mRNA.
Wherein, it is preferred that in step A, the method for extracting total serum IgE is：It is extracted and is obtained from human body cell with Trizol methods
Total serum IgE.
Wherein, it is preferred that in step A, when expanding ECRG4 genes with reverse transcription PCR, used ECRG4 primers are
With people's ECRG4 gene orders（GenBank: AF325503.1）For stencil design, sense primer includes XhoI restriction enzyme sites,
Downstream primer includes BamHI restriction enzyme sites.Sense primer base sequence is：5'-
ATGACTCGAGTGCTCGCGCCCCGCCGCCATG-3'；Downstream primer base sequence is：5'-
TCATGGATCCTGTGGCAAGTCATGGTTAGT-3’。
Wherein, the slow virus carrier pLVX-IRES-ZsGreen1 used in step A be include ZsGreen1 coding base
The bi-cistronic vectors of cause.ZsGreen1 is third generation green fluorescent protein, is the highest fluorescin of brightness, is turned convenient for cell
Contaminate monitoring and the airflow classification positive cell of efficiency.PLVX-IRES-ZsGreen1-ECRG4 plasmids are same in the form of bicistronic mRNA
When expression ZsGreen1 fluorescins and ECRG4 genes, the expression of ZsGreen1 fluorescins can be used as destination gene expression
" indicator " indirectly, and ZsGreen1 fluorescins are not merged with destination protein, do not influence the normal expression of destination protein, no
Influence the normal configuration and function of destination protein.
Wherein, it is preferred that in step A, ECRG4 genes are loaded into the method on pLVX-IRES-ZsGreen1 carriers
For：After ECRG4 genes and pLVX-IRES-ZsGreen1 carriers are all carried out double digestion with XhoI and BamHI, connected with T4 DNA
Enzyme is connect to be attached.
Wherein, it is preferred that in step A, with bacterium convert method to pLVX-IRES-ZsGreen1-ECRG4 plasmids into
Row proliferation；Specifically method is：PLVX-IRES-ZsGreen1-ECRG4 plasmids are transformed into Escherichia coli, the positive is screened
Monoclonal colonies are enlarged culture, to which extraction obtains a large amount of pLVX-IRES-ZsGreen1-ECRG4 plasmids.
Wherein, it is preferred that in step B, HEK293T cells are being carried out to transfect preceding 2-6h, are being changed to antibiotic-free
DMEM culture mediums（Containing 10%FBS）HEK293T cells are cultivated, cell can be made to be in better growth conditions, to improve
Transfection.
Wherein, it is preferred that in step B, when being transfected to HEK293T cells with the first mixed liquor, can use
Lipofectamine2000 is transfected；This preferably not only can guarantee higher transfection efficiency, but also can reduce transfection reagent to cell
The influence of vigor.
Wherein, in step B, the pMD2.G and psPAX2 are slow virus assembling plasmid；The pLVX-IRES-
ZsGreen1-ECRG4 is ECRG4 encoding plasmids；Preferably, pMD2.G, psPAX2 and pLVX- in first mixed liquor
The mass ratio of IRES-ZsGreen1-ECRG4 is 2：3：4；In the mass ratio, the transfecting notable increasing of HEK293T cells
Add, obtained virus titer highest is best to the infectious effect of HEK293 cells, and expression quantity is maximum, is filled in obtained excretion body
The ECRG4 mRNA quantity of load dramatically increases.
Wherein, in step B, the transfection to HEK293T cells refer to transfection method enable HEK293T cells by
The process of ECRG4 gene slow-virus infections；Preferably, transfection method includes that lipofection, calcium phosphate transfection method or electricity are worn
One kind in the infection protocol of hole；Most preferably, transfection method is lipofection；By that the preferred of transfection method, can drop
Cytotoxicity existing for low transfection reagent, the growth conditions for making cell keep fit improve viral packaging efficiency.
Wherein, it is preferred that the preparation method of the first mixed liquor in step B includes：It is 10 ~ 30 to take volume ratio：1
Opti-MEM culture mediums are uniformly mixed so as to obtain solution a with lipofectamine 2000；It takes and is cultivated with the Opti-MEM of solution a moderates
Base, is added plasmid pMD2.G, psPAX2 and pLVX-IRES-ZsGreen1-ECRG4, and three kinds of plasmid mass ratioes are 2：3：4, mixing
Obtain solution b；Solution a and solution b mixings are both obtained into the first mixed liquor；By preferred, the transfecting of the first mixed liquor can be improved
And stability.
Wherein, the culture solution described in step B refers to removing cell after the completion of cell is cultivated in the medium and cell being broken
Remaining liquid after piece is the culture solution for including slow virus.
Wherein, it is preferred that the HEK293T cell culture processes after the transfection in step B include：After transfection
It after HEK293T cell culture 6-12h, uses DMEM culture mediums instead and is cultivated, continue to cultivate 30-48h completion cultures；By excellent
Choosing, the HEK293T cell growth states after transfection are more preferable.
Wherein, it is preferred that in step B, the centrifuging temperature of culture solution is 0-8 DEG C, centrifugal speed 1500-2500g, centrifugation
Time is 5-10min；Most preferably, centrifuging temperature is 4 DEG C, centrifugal speed 2000g, centrifugation time 7min；By to from
Heart condition it is preferred, cell and cell fragment remaining in culture solution can be removed, so as to get virus liquid purity higher, infection
Effect is more preferable.
Wherein, it is preferred that in step C, a concentration of 6-10 μ g/ml of polybrene in the second mixed liquor；Most preferred concentration
For 8 μ g/ml, preferred coacervation amine concentration can reduce the cytotoxicity of polybrene, significantly improve the efficiency of infection of virus；Coacervation
Amine concentration is excessively high, will increase the toxicity of cell, and can influence the vigor of cell, or even cause cell mortality；Polybrene is dense
Spend low, improving the effect of viral efficiency of infection will significantly reduce.
Wherein, it is preferred that in step C, 2 subinfections are carried out to HEK293 cells with the second mixed liquor；By 2 subinfections,
Viral efficiency of infection can significantly be improved；Efficiency of infection is high, then positive cell ratio is high, and the time of sorting is short, efficient, right
The influence of cell viability is small, can just make the overexpression best results of ECRG4 in HEK293 cells, ECRG4 in the excretion body of secretion
The content of mRNA is maximum.
Wherein, it is preferred that in step C, each metainfective HEK293 cells will also be centrifuged before being cultivated
Processing, centrifugal speed 1800rpm, centrifuging temperature is room temperature, centrifugation time 45min；Centrifugal treating contributes to virion
It is combined with cell surface, can further significantly improve the efficiency of infection of virus.
Wherein, it is preferred that before carrying out the 2nd subinfection to HEK293 cells, will pass through metainfective for the first time
HEK293 cells cultivate 12-24h in the culture medium without virus；By culture, it is thin to HEK293 that the first subinfection can be repaired
Influence, makes to restore vigorous growth conditions by the HEK293 cells of the first subinfection, to the effect of the second subinfection caused by born of the same parents
Rate, which improves, has remarkable result.
Wherein, it is preferred that step（C）In, the sorting of the cell refer to sort to obtain GFP using flow cytometry it is green
The summation of the cell of color fluorescent positive, the GFP gfp positive cells is ECRG4-HEK293 cell lines.
Wherein, it is preferred that step B, the condition of culture of cell is in C：37 DEG C of temperature, CO2A concentration of 5%, by preferred,
Cell can keep the speed of growth and physiological status of normal table in incubation.
Wherein, in step B and C, the culture medium for carrying out secondary culture refers to that can be carried for the growth and maintenance of HEK293 cells
For the culture medium of required nutrition；Preferably, the culture medium includes one kind in DNEM or RPMI-1640, most preferably, institute
Culture medium be DNEM（Containing 10% FBS, 1% mycillin is dual anti-）；Pass through high cell growth speed that is preferred, cultivating, shape
State is normal, and transfection efficiency is high.
To achieve the above object of the invention, further, the present invention also provides a kind of excretion body containing ECRG4 mRNA
Preparation method, include the following steps：
1, the cell for being overexpressed ECRG4 is prepared；
2, the cell for being overexpressed ECRG4 is cultivated in the DMEM of serum-free, culture solution is collected after the completion of culture；
3, the culture solution for being collected into step 2 carries out separating-purifying, obtains the excretion body containing ECRG4 mRNA.
The preparation method of the above-mentioned excretion body containing ECRG4 mRNA, can be in the cell using the cell for being overexpressed ECRG4
Transcription generates a large amount of ECRG4 mRNA, and can actively be loaded into ECRG4 mRNA when the cell membrane invagination of the cell forms excretion body
Principle；By the culture of the cell to being overexpressed ECRG4, making cell, largely secretion contains in growth and breeding
In the excretion body to culture medium of ECRG4 mRNA；Again through separating-purifying, the excretion body for being mounted with a large amount of ECRG4 mRNA is obtained；It should
Preparation method is easy to operate, reliable and stable, is suitble to extensive, standardized production.
The preparation method of the above-mentioned excretion body containing ECRG4 mRNA, wherein it is preferred, it is overexpressed ECRG4's in step 2
Cell culture is to density 80%, after cleaning 2 times with PBS, then changes the DMEM culture mediums of serum-free into；Preferred operation can remove residual
Excretion body in the culture medium serum stayed improves the purity of the excretion body of cell secretion.
Wherein, it is preferred that the cell of overexpression ECRG4 incubation time in the DMEM culture mediums of serum-free is in step 2
36-60h；Most preferably, incubation time 48h；Incubation time is too short, and the excretion body quantity of Hemapoiesis is few, detaches low output,
Incubation time is long, and cell viability can be substantially reduced, and cell death increases, and has more cell fragments and cell death phase
The small molecule structure of pass is secreted into culture medium, and the quality of excretion body and purity is made to decline.
The preparation method of the above-mentioned excretion body containing ECRG4 mRNA, wherein it is preferred, in step 3, culture solution is divided
Include from method of purification：A, culture solution under conditions of 4 DEG C, 200g is centrifuged into 10min, takes supernatant spare；B, it will be obtained in a
Supernatant at 4 DEG C, 10min is centrifuged under conditions of 3000g, takes supernatant spare；C, by the supernatant obtained in b at 4 DEG C,
30min is centrifuged under conditions of 10000g, takes supernatant spare；D, by the supernatant obtained in c at 4 DEG C, 100000-160000g
Under conditions of centrifuge 70-120min, take precipitation it is spare；E, PBS is added in the precipitation obtained in d, precipitation is resuspended, and at 4 DEG C,
70-120min is centrifuged under conditions of 100000-160000g, is precipitated as the excretion body containing ECRG4 mRNA；Wherein, most preferably
, step d, centrifugal speed is 120000g, centrifugation time 90min in e；It is dead in removable cell culture medium by preferred
Cell, cell fragment and high molecular weight protein particle, obtained excretion body purity higher.
The preparation method of the above-mentioned excretion body containing ECRG4 mRNA, wherein preferred, the culture of cell in step 2,3
Condition is：37 DEG C of temperature, CO2A concentration of 5%, by preferred, cell can keep the speed of growth of normal table in incubation
And physiological status.
Wherein, step 2, the culture solution described in 3 refer to removing remaining liquid after cell by after the completion of cell culture.
Further, the application of the present invention also provides a kind of excretion body containing ECRG4 mRNA；The excretion body exists
The application in tumour is treated, which significantly inhibits proliferation and the growth of tumour cell, has to the treatment of tumour positive
Effect.
Excretion body containing ECRG4 mRNA can be used for treating oral squamous cell carcinomas, it can also be used to colon cancer, breast cancer, lung cancer,
The treatment of kidney, gastric cancer, colon cancer, the cancer of the esophagus, liver cancer or glioma.
Compared with prior art, beneficial effects of the present invention：
1, excretion body of the present invention is secreted by being overexpressed the cell of ECRG4 genes, is inside mounted with more than conventional excretion body
ECRG4 mRNA, and a large amount of ECRG4 mRNA of loading can be transported to recipient cell, can significantly improved in recipient cell
The expression quantity of ECRG4.
2, excretion body of the present invention prepare overexpressing cell during, by pMD2.G, psPAX2 and pLVX-
The superinfection preferred, to cell preferred, to coacervation amine concentration of the mass ratio of IRES-ZsGreen1-ECRG4, can be significantly
The ECRG4 mRNA amounts loaded in excretion body are improved, the ECRG4 mRNA loaded in excretion body is made to reach the 1000 of conventional excretion body
Times or more, it is notable to the inhibiting effect of growth of tumour cell and proliferation.
3, the overexpression ECRG4 that the ECRG4-HEK293 cells that the present invention is prepared can be continual and steady, obtained excretion
The content of ECRG4 mRNA is high in body, and solve excretion body of the present invention from source carrys out source problem, is conducive to excretion of the present invention
Extensive, the standardized production of body, and extend to loading, transport and the scale of the gene that other have treatment potentiality, standard
Metaplasia is produced.
4, excretion body of the present invention can be used for treating tumor disease, can significantly inhibit proliferation and the growth of tumour cell, to swollen
The treatment of tumor disease has positive effect.
Description of the drawings：
Fig. 1 is ECRG4 genetic test figures（（A）100bp ladder indicate DNA molecular amount marker；ECRG4 Frag are indicated
ECRG4 partial gene fragments；ECRG4 mRNA indicate ECRG4 encoding genes；（B）Controls indicates Healthy Human Serum excretion
ECRG4 mRNA expressions in body；Patients indicates ECRG4 mRNA tables in oral squamous cell carcinoma patients serum's excretion body
Up to level）.
Fig. 2 is the qualification result figure of the HEK293 cell lines after being infected in the embodiment of the present invention 1 and comparative example 1（（A）For
Real-time quantitative PCR result statistical chart,（B）For Western blot result figures；ECRG4 HEK293 are indicated in embodiment 1
ECRG4-HEK293 cell lines, Vector HEK293 indicate Vector-HEK293 cell lines in comparative example 1）.
Fig. 3 is the form and droplet measurement result figure of the excretion body prepared in the embodiment of the present invention 1（（A）For transmission electron microscope
The excretion volume morphing structure chart of lower observation；（B）For nano particle follow-up analysis technology（NTA）The excretion body particle diameter distribution of detection
Figure）.
Fig. 4 is the feature testing result figure for the excretion body being prepared in the embodiment of the present invention 1 and comparative example 1；（（A）Outside
Secrete the significant albumen western blot testing result figures of body；（B）Indicate excretion body in ECRG4 mRNA content detections as a result,
ECRG4-Exo indicates that the excretion body being prepared in embodiment 1, Vector-Exo indicate the excretion being prepared in comparative example 1
Body）.
Fig. 5 is that TCA8113 absorbs the result figure after the excretion body prepared in the embodiment of the present invention 1（DAPI indicates nucleus；
ECRG4-Exo indicates the excretion body extracted in embodiment 1；α-Actin indicate cell actin；Merge indicates fluorescence superposition
Design sketch）.
Fig. 6 is intracellular ECRG4 genes in the TCA8113 cellular uptakes embodiment of the present invention 1 and comparative example 1 after excretion body
The detection figure of expression quantity（ECRG4-Exo indicates that the excretion body that TCA8113 cells are added in embodiment 1, Vector-Exo indicate
The excretion body in comparative example 1 is added in TCA8113 cells）.
Fig. 7 is that excretion body inhibits TCA8113 cell Proliferation testing result figures in the embodiment of the present invention 1 and comparative example 1（（A）
Indicate the TCA8113 cell cycle testing results that excretion body in comparative example 1 is added；（B）Indicate to be added excretion body in embodiment 1
TCA8113 cell cycle testing results；（C）After two kinds of excretion bodies are added, with the TCA8113 Cell proliferation results of CCK8 detections
Statistical chart）.
Fig. 8 is that excretion body inhibits A549 cell Proliferation testing result figures in the embodiment of the present invention 1 and comparative example 1（（A）It indicates
The A549 cell cycle testing results of excretion body in comparative example 1 are added；（B）Indicate that the A549 that excretion body in embodiment 1 is added is thin
Born of the same parents' cycle detection result；（C）After two kinds of excretion bodies are added, with the A549 Cell proliferation results statistical charts of CCK8 detections）.
Fig. 9 is the reality that excretion body inhibits the intracellular related gene expressions of TCA8113 in the embodiment of the present invention 1 and comparative example 1
When quantitative PCR detection result figure（ECRG4-Exo indicates the TCA8113 cells that excretion body in embodiment 1 is added；Vector-Exo
Indicate the TCA8113 cells that the excretion body of comparative example 1 is added）.
Figure 10 be vaccinated with using in the embodiment of the present invention 1 and comparative example 1 the processed TCA8113 cells of excretion body it is naked
The upgrowth situation comparison diagram of mouse（（A）For nude mice average weight growth curve during experiment；（B）It is averagely taken the photograph for nude mice during experiment
Appetite statistical chart；ECRG4-Exo indicates to be vaccinated with the nude mice using the processed TCA8113 cells of excretion body in embodiment 1；
Vector-Exo indicates to be vaccinated with the nude mice using the processed TCA8113 cells of excretion body in comparative example 1）.
Figure 11 is that excretion body inhibits transplanted tumor in nude mice growth result figure in the embodiment of the present invention 1 and comparative example 1（（A）To connect
The tumor bearing nude mice using the processed TCA8113 cells of excretion body in embodiment 1 is planted；（B）It is used in comparative example 1 to be vaccinated with
The tumor bearing nude mice of the processed TCA8113 cells of excretion body；（C）For nude mouse tumor growth curve statistical chart；（D）It is to be shelled after 24 days
From transplanted tumor in nude mice；（E）For nude mice model tumor weight statistical chart；ECRG4-Exo expressions are vaccinated with using excretion in embodiment 1
The nude mice of the processed TCA8113 cells of body, Vector-Exo expressions are vaccinated with processed using excretion body in comparative example 1
The nude mice of TCA8113 cells）.
Figure 12 is the HEK293 cell fluorescence figures after slow-virus infection in the embodiment of the present invention（（A）For slow disease in embodiment 1
The metainfective HEK293 cell fluorescences figure of poison；（B）To use the metainfective HEK293 cells of 1 subinfection method of slow virus in embodiment 2
Fluorescent figure；（C）For HEK293 cell fluorescence figures in embodiment 3；（D）For HEK293 cell fluorescence figures in embodiment 4, DAPI is indicated
Nucleus, Ecrg4 indicate that the positive cell that ECRG4 is overexpressed, Merge indicate the design sketch of fluorescence superposition）.
Figure 13 is the HEK293 cell Flow cytometries after slow-virus infection in Example 1 and Example 2 of the present invention
Figure（（A）For the HEK293 cell flow cytometer detection figures after slow-virus infection in embodiment 1；（B）For slow-virus infection 1 in embodiment 2
HEK293 cell flow cytometer detection figures after secondary, P2 indicate the positive cell that ECRG4 is overexpressed）.
Specific implementation mode
The present invention is described in further detail With reference to embodiment.But this should not be interpreted as to the present invention
The range of above-mentioned theme is only limitted to embodiment below, all models that the present invention is belonged to based on the technology that the content of present invention is realized
It encloses.
Embodiment 1
1, pLVX-IRES-ZsGreen1-ECRG4 plasmid constructions：Human umbilical vein endothelial cell total RNA is extracted with Trizol methods, is used
Reverse transcription PCR expands ECRG4 genes, and ECRG4 genes and pLVX-IRES-ZsGreen1 carriers are all carried out with XhoI and BamHI
Double digestion and after being connected with T4 DNA ligases, is transformed into Escherichia coli, and screening positive clone extracts plasmid, with double digestion and
DNA sequencing is identified.Wherein ECRG4 primers are with people's ECRG4 gene orders（GenBank: AF325503.1）For stencil design
, sense primer includes XhoI restriction enzyme sites, and downstream primer includes BamHI restriction enzyme sites.Sense primer base sequence is：5'-
ATGACTCGAGTGCTCGCGCCCCGCCGCCATG-3'；Downstream primer base sequence is：5'-
TCATGGATTCTGTGGCAAGTCATGGTTAGT-3’。
2, cell prepares：Recovery HEK293T cells, with the DMEM culture mediums containing 10% fetal calf serum, 1% mycillin（Letter
Claim DMEM culture mediums）In 37 DEG C, 5% CO2Constant incubator culture, passage 1-2 time after, be inoculated with 0.8 × 106A cell is to 6cm
Culture dish, secondary daily lipofectamine2000 transfections, packs slow virus, and 2h is changed to the DMEM cultures of antibiotic-free before transfecting
Base；
3, prepared by slow virus：Prepare the solution a of the lipofectamine2000 of Opti-MEM and 18 μ l comprising 450 μ l；It prepares
Include the Opti-MEM of 450 μ l, the plasmid pMD2.G, the plasmid pLVX-IRES- of the plasmid psPAX2 of 3 μ g and 4 μ g of 2 μ g
The solution b of ZsGreen1-ECRG4；Solution b is added in solution a, soft mixing obtains the first mixed liquor, after being stored at room temperature 5min,
In the HEK293T cells that first mixed liquor addition step 1 is obtained, 37 DEG C are placed in, 5%CO2Constant incubator in, used after 6h
PBS is rinsed 2 times, is changed to DMEM culture mediums, is collected culture night after 36h, centrifuged under conditions of temperature is 4 DEG C, speed is 2000g
7min, it is virus liquid to take supernatant；
4, the foundation of ECRG4-HEK293 cell lines：Recovery HEK293 cells, with containing 10% fetal calf serum, 1% mycillin
DMEM culture mediums（Abbreviation DMEM culture mediums）In 37 DEG C, 5% CO2Constant incubator culture, passage 1-2 time after, be inoculated with 0.8 ×
106A cell is to 6cm culture dishes；Next day, the virus liquid that step 2 is obtained is with DMEM culture mediums by 3：1 volume ratio mixing is equal
After even, and polybrene is added, makes a concentration of 8 μ g/ml of polybrene, obtain the second mixed liquor；Second mixed liquor is added to
In HEK293 cells, 45min is centrifuged under conditions of 1800rpm, 37 DEG C, is changed to DMEM culture mediums after cultivating 12h, is continued to cultivate
The second mixed liquor is added in 12h again in HEK293 cells, and 45min is centrifuged under conditions of 1800rpm, 37 DEG C, cultivates 12h
After be changed to DMEM culture mediums, continue cultivate 48h after carry out flow cytometry sorting, obtain ECRG4-HEK293 cell lines；
5, the generation of excretion body：ECRG4-HEK293 cell lines are inoculated in 10cm culture dishes, quantity is 2.2 × 106, culture is extremely
Cell density about 80% cleans cell 2 times with PBS, changes the DMEM culture mediums of serum-free into, and culture solution is collected after cultivating 48h；
6, the extraction of excretion body：The culture solution that step 4 is collected into centrifuges 10min at 4 DEG C under conditions of 200g, take supernatant
It is spare；By obtained supernatant at 4 DEG C, 10min is centrifuged under conditions of 3000g, takes supernatant spare；Obtained supernatant is existed
4 DEG C, 30min is centrifuged under conditions of 10000g, takes supernatant spare；By obtained supernatant under conditions of 4 DEG C, 120000g
90min is centrifuged, takes precipitation spare；In obtained precipitation be added PBS be resuspended precipitation, and under conditions of 4 DEG C, 120000g from
Heart 90min, precipitation is the excretion body containing ECRG4 mRNA, and is named as ECRG4-Exo.
7, the preservation of excretion body：Appropriate PBS is added in excretion body to be resuspended, it is quantitative with BCA albumen concentration detection kits,
PBS volumes are the 1% of initial culture volume, -80 DEG C of preservations.
Embodiment 2
1, pLVX-IRES-ZsGreen1-ECRG4 plasmid constructions：Concrete operations are same as Example 1.
2, cell prepares：Concrete operations are same as Example 1.
3, prepared by slow virus：Concrete operations are same as Example 1.
4, the foundation of ECRG4-HEK293 cell lines：Recovery HEK293 cells, with containing 10% fetal calf serum, 1% mycillin
DMEM culture mediums（Abbreviation DMEM culture mediums）In 37 DEG C, 5% CO2Constant incubator culture, passage 1-2 time after, be inoculated with 0.8
×106A cell is to 6cm culture dishes；Next day, the virus liquid that step 2 is obtained is with DMEM culture mediums by 3：1 volume ratio mixing
After uniformly, and polybrene is added, makes a concentration of 8 μ g/ml of polybrene, obtain the second mixed liquor；Second mixed liquor is added to
In HEK293 cells, 45min is centrifuged under conditions of 1800rpm, 37 DEG C, is changed to DMEM culture mediums after cultivating 12h, is continued to cultivate
Fluirescence observation and flow cytometry sorting are carried out after 48h；
5, the generation of excretion body：Concrete operations are same as Example 1.
6, the extraction of excretion body：Concrete operations are same as Example 1.
7, the preservation of excretion body：Concrete operations are same as Example 1.
It is found through experiment that：2 step 4 of embodiment handles HEK293 cells only with 1 viral infection, metainfective
HEK293 cell positives cell proportion about 54.8%, is shown in Figure 12（B）And Figure 13（B）, HEK293 cell positive cells in embodiment 1
Ratio is about 97.2%, sees Figure 12（A）And Figure 13（A）.Embodiment 2 is using the method for 1 virus infection processing, the positive of cell
Rate significantly reduces, and the expression quantity of ECRG4 is unfavorable for the airflow classification of cell and proposing for excretion body also below embodiment 1
It takes.Therefore, efficiency of infection can greatly be improved using 2 subinfection methods, accelerates the foundation of stable cell line, improve HEK293
The expression quantity of ECRG4 mRNA and protein in cell, to considerably increase the content of ECRG4 mRNA in excretion body.
Embodiment 3
1, pLVX-IRES-ZsGreen1-ECRG4 plasmid constructions：Concrete operations are same as Example 1.
2, cell prepares：Concrete operations are same as Example 1.
3, prepared by slow virus：Prepare the solution a of the lipofectamine2000 of Opti-MEM and 18 μ l comprising 450 μ l；
Prepare the Opti-MEM, the plasmid pMD2.G, the plasmid pLVX-IRES- of the plasmid psPAX2 of 2 μ g and 2 μ g of 2 μ g for including 450 μ l
The solution b of ZsGreen1-ECRG4；Solution b is added in solution a, soft mixing obtains the first mixed liquor, after being stored at room temperature 5min,
In the HEK293T cells that first mixed liquor addition step 1 is obtained, 37 DEG C are placed in, 5%CO2Constant incubator in, used after 6h
PBS is rinsed 2 times, is changed to DMEM culture mediums, is collected culture night after 36h, centrifuged under conditions of temperature is 4 DEG C, speed is 2000g
7min, it is virus liquid to take supernatant；
4, the foundation of ECRG4-HEK293 cell lines：Concrete operations are same as Example 1.
5, the generation of excretion body：Concrete operations are same as Example 1.
6, the extraction of excretion body：Concrete operations are same as Example 1.
7, the preservation of excretion body：Concrete operations are same as Example 1.
It is found through experiment that：In embodiment 3, pMD2.G, psPAX2 and pLVX-IRES- in first mixed liquor of step 3
The mass ratio of ZsGreen1-ECRG4 is not preferred ratio, in the virus liquid being prepared carry ECRG4 genes slow virus compared with
Few, the infectious of virus liquid reduces, after infecting HEK293 cells, positive cell rate（Positive cell ratio is less than 30%）And
The expression quantity of ECRG4 is substantially lower than embodiment 1 in HEK293 cells（Positive cell ratio is about 97.2%）, see Figure 12（A）,
（C）, the useful load of ECRG4 genes in excretion body is impacted.Therefore, using preferred pMD2.G, psPAX2 and pLVX-
The mass ratio of IRES-ZsGreen1-ECRG4 can greatly improve the titre of ECRG4 viruses, increase HEK293 cells
The overexpression effect of efficiency of infection and ECRG4, to which the content of ECRG4 mRNA in the excretion body of secretion is maximum.
Embodiment 4
1, pLVX-IRES-ZsGreen1-ECRG4 plasmid constructions：Concrete operations are same as Example 1.
2, cell prepares：Concrete operations are same as Example 1.
3, prepared by slow virus：Concrete operations are same as Example 1.
4, the foundation of ECRG4-HEK293 cell lines：Recovery HEK293 cells, with containing 10% fetal calf serum, 1% mycillin
DMEM culture mediums（Abbreviation DMEM culture mediums）In 37 DEG C, 5% CO2Constant incubator culture, passage 1-2 time after, be inoculated with 0.8
×106A cell is to 6cm culture dishes；Next day, the virus liquid that step 2 is obtained is with DMEM culture mediums by 3：1 volume ratio mixing
After uniformly, and polybrene is added, makes a concentration of 12 μ g/ml of polybrene, obtain the second mixed liquor；Second mixed liquor is added to
In HEK293 cells, 45min is centrifuged under conditions of 1800rpm, 37 DEG C, is changed to DMEM culture mediums after cultivating 12h, is continued to cultivate
The second mixed liquor is added in 12h again in HEK293 cells, and 45min is centrifuged under conditions of 1800rpm, 37 DEG C, cultivates 12h
After be changed to DMEM culture mediums, continue cultivate 48h after carry out fluoroscopic examination；
5, the generation of excretion body：Concrete operations are same as Example 1.
6, the extraction of excretion body：Concrete operations are same as Example 1.
7, the preservation of excretion body：Concrete operations are same as Example 1.
It is found through experiment that：In embodiment 4, the concentration of polybrene is not optium concentration in second mixed liquor of step 4, with implementation
Example 1 is compared, and the second mixed liquor increases the toxicity of cell, and HEK293 cell deaths are more, and the infectious of virus liquid reduces.Cause
This, the ratio of positive cell in obtained ECRG4-HEK293 cell lines（Positive cell is less than 30%）Considerably less than embodiment 1
（Positive cell ratio about 97.2%）, it is unfavorable for airflow classification and the extraction of excretion body of positive cell.Therefore, using preferred poly-
Solidifying amine concentration can greatly improve the efficiency of infection of the second mixed liquor, to make the overexpression of ECRG4 in HEK293 cells imitate
Fruit is best, the content bigger of ECRG4 mRNA in the excretion body of secretion.
Comparative example 1
1, cell prepares：Recovery HEK293T cells, with the DMEM culture mediums containing 10% fetal calf serum, 1% mycillin（Referred to as
DMEM culture mediums）In 37 DEG C, 5% CO2Constant incubator culture, passage 1-2 time after, be inoculated with 0.8 × 106A cell is trained to 6cm
Support ware, secondary daily lipofectamine2000 transfections；2h is changed to the DMEM culture mediums of antibiotic-free before transfection；
2, prepared by slow virus：Prepare the solution a of the lipofectamine2000 of Opti-MEM and 18 μ l comprising 450 μ l；It prepares
Include the Opti-MEM of 450 μ l, the plasmid pMD2.G, the plasmid pLVX-IRES- of the plasmid psPAX2 of 3 μ g and 4 μ g of 2 μ g
The solution b of ZsGreen1；Solution b is added in solution a, soft mixing obtains the first mixed liquor, after being stored at room temperature 5min, by first
Mixed liquor is added in the HEK293T cells that step 1 obtains, and is placed in 37 DEG C, 5%CO2Constant incubator in, rinsed with PBS after 6h
2 times, DMEM culture mediums are changed to, culture solution is collected after 36h, centrifuged 7min under conditions of temperature is 4 DEG C, speed is 2000g, take
Supernatant is virus liquid；
3, the foundation of Vector-HEK293 cell lines：Recovery HEK293 cells, with containing 10% fetal calf serum, 1% mycillin
DMEM culture mediums（Abbreviation DMEM culture mediums）In 37 DEG C, 5% CO2Constant incubator culture, passage 1-2 time after, be inoculated with 0.8 ×
106A cell is to 6cm culture dishes；Next day, the virus liquid that step 2 is obtained is with DMEM culture mediums by 3：1 volume ratio mixing is equal
After even, and polybrene is added, makes a concentration of 8 μ g/ml of polybrene, obtain the second mixed liquor；Second mixed liquor is added to
In HEK293 cells, 45min is centrifuged under conditions of 1800rpm, 37 DEG C, is changed to DMEM culture mediums after cultivating 12h, is continued to cultivate
The second mixed liquor is added in 12h again in HEK293 cells, and 45min is centrifuged under conditions of 1800rpm, 37 DEG C, cultivates 12h
After be changed to DMEM culture mediums, continue cultivate 48h after carry out flow cytometry sorting, obtain Vector-HEK293 cell lines；
4, the generation of excretion body：Vector-HEK293 cells are inoculated in 10cm culture dishes, quantity is 2.2 × 106, culture is extremely
Cell density about 80% cleans cell 2 times with PBS, changes the DMEM culture mediums of serum-free into, and culture medium is collected after cultivating 48h；
5, the extraction of excretion body：The culture medium that step 4 is collected into is centrifuged into 10min under conditions of 4 DEG C, 200g, takes supernatant
It is spare；Obtained supernatant is centrifuged into 10min under conditions of 4 DEG C, 3000g, takes supernatant spare；Obtained supernatant is existed
4 DEG C, centrifuge 30min under conditions of 10000g, take supernatant spare；By obtained supernatant under conditions of 4 DEG C, 120000g
90min is centrifuged, takes precipitation spare；In obtained precipitation be added PBS be resuspended precipitation, and under conditions of 4 DEG C, 120000g from
Heart 90min, precipitation are excretion body（Vector-Exo）.
6, the preservation of excretion body：Appropriate PBS is added in excretion body to be resuspended, it is quantitative with BCA albumen concentration detection kits,
PBS volumes are the 1% of initial culture volume, -80 DEG C of preservations.
Experimental example：
（1）ECRG4-HEK293 cell lines and ECRG4-Exo identifications
Respectively with the ECRG4-HEK293 and Vector- established in real-time quantitative PCR and western blot detection embodiments 1
HEK293 cell ECRG4 mRNA and protein expression level.The results show that ECRG4-HEK293 cell ECRG4 mRNA expression
Amount is the 7.8 × 10 of Vector-HEK293 cells5Times, see Fig. 2 A.And it is detected in ECRG4-HEK293 cell pyrolysis liquids
Clear, dense ECRG4 protein bands, molecular weight about 17kDa, and the Vector-HEK293 cell pyrolysis liquids of phase homogenous quantities
ECRG4 protein bands are not detected, see Fig. 2 B.It is indicated above that the ECRG4-HEK293 cell lines established in embodiment 1,
All there is strong overexpression in ECRG4 mRNA and protein, loading ECRG4 genes for excretion body has prepared good premise item
Part.
ECRG4-HEK293 the and Vector-HEK293 cells for taking equivalent, cultivate 48h in plasma-free DMEM medium,
It collects culture medium and extracts excretion body, transmission electron microscope results are shown, excretion body is in typical " cup-shaped " imitated vesicle structure, and grain size mainly collects
In in 93nm, see Fig. 3.Western blot are the results show that the expression quantity of the positive marker CD9 and Alix of excretion body is apparent
Higher than cell pyrolysis liquid, the expression quantity of the negative marker GRP94 and Calnexin of excretion body is substantially less than cell pyrolysis liquid,
See Fig. 3, also, ECRG4 mrna expression amounts are 1162 times of Vector-Exo in ECRG4-Exo, see Fig. 4.It is indicated above implementation
The ECRG4-Exo excretion bodies extracted in example 1 have the morphosis of typical excretion body, and are loaded with high-caliber ECRG4 bases
Cause.
（2）Excretion body transports the experiment detection of ECRG4 mRNA：
Cell Tracker are added in the ECRG4-Exo being prepared in embodiment 1TM CM-DiI (Thermo, C7000), eventually
1 μ g/ml of concentration, 37 DEG C of incubation 30min, 4 DEG C are placed 15min, are transferred in ultracentrifugation pipe, 4 DEG C of 120000g centrifugations
90min abandons supernatant, and precipitation is dissolved in PBS solution, is transferred to ultracentrifugation pipe, and 4 DEG C of 120000g centrifuge 90min, abandon supernatant, sink
Shallow lake is dissolved in PBS solution, and -80 DEG C save backup；TCA8113 cell culture mediums are the RPMI-1640 containing 10%FBS, and TCA8113 is thin
Born of the same parents are seeded on cell climbing sheet, density about 60%, overnight incubation in 6 orifice plates, and the ECRG4-Exo of 50 μ g is added, and cultivate 12h；It takes out
Cell climbing sheet fixes 15min with 4% paraformaldehyde room temperature, and PBS cleans 5min, and the absolute methanol of precooling, -20 DEG C of placements are added
20min, PBS clean 5min, and 5% skim milk room temperature closes 1h, and anti-actin primary antibodies are added（Sigma）4 DEG C were incubated
Night, PBS clean 5min, are repeated 3 times, and green fluorescence secondary antibody is added（Abcam）, it is incubated at room temperature 1h, PBS cleans 5min, repeats 3
It is secondary, 1% nucleus dyestuff Hoechst is added, is incubated at room temperature 10min, PBS cleans 5min, is repeated 3 times, mountant mounting, fluorescence
Microscope detects.The results show that the ECRG4-Exo of red fluorescence label can enter recipient cell by TCA8113 cell membranes
Born of the same parents are mainly distributed in endochylema, are centered around around nucleus, see Fig. 5.
The ECRG4 genes of carrying are transported to TCA8113 cells in order to further confirm that ECRG4-Exo excretion physical efficiencys are no,
ECRG4-Exo and Vector-Exo are added to TCA8113 cells by the present invention respectively, and cell is collected after 12h, carry out real-time quantitative
PCR is detected.The results show that ECRG4-Exo（Embodiment 1）The expression of ECRG4 mRNA in the TCA8113 cells of processing group
It is Vector-Exo（Comparative example 1）29.48 times of processing group are shown in Fig. 6, fully demonstrate ECRG4 mRNA and are transported by excretion body
Recipient cell is arrived.
（3）ECRG4-Exo inhibits Human Tongue Carcinoma Lines TCA8113（Oral squamous cell carcinomas）Cell proliferation experiment detects：
TCA8113 cell inoculations are in 96 orifice plates, and cell quantity is 2000/hole, and for 24 hours, 2 μ g are added in experimental group for culture
ECRG4-Exo（Embodiment 1）, 2 μ g Vector-Exo of control group addition（Comparative example 1）, continue to cultivate 48h, 10 μ l be added per hole
CCK-8 solution, 37 DEG C of culture 1h, detects 450nm absorbances, and SPSS carries out data statistic analysis.By TCA8113 cell inoculations
In 6 orifice plates, quantity is 0.3 × 106/ hole after overnight incubation, after the DMEM culture medium synchronizing cultures 12h for changing serum-free into, is changed
At DMEM culture mediums, while experimental group（3 holes）20ug ECRG4-Exo, control group is added（3 holes）20 μ g Vector- are added
Exo continues culture for 24 hours.Trypsin digestion and cell, PBS cleanings are primary, and with 70% ethyl alcohol, 4 DEG C of fixations are stayed overnight, cell PBS
500 μ l PI/RNase dye liquors are added in cleaning 2 times（BD 550825）, it is detected with flow cytometer.
The results show that compared with the control group, ECRG4-Exo processing group TCA8113 cell proliferation rates have dropped 58%, and
And G0/G1 phase cell quantities are apparently higher than control group（ECRG4-Exo groups 63.97%, control group 54.34%), S phase cell quantities are low
In control group（ECRG4-Exo groups 24.16%, control group 29.23%), see Fig. 7, shows that ECRG4-Exo has blocked TCA8113 cells
The process of G0/G1 → S phases.In order to more fully confirm the effect of ECRG4-Exo, the identical test method of the present invention,
It is further demonstrated in human lung cancer cell A549, ECRG4-Exo has delayed the process of A549 cell S → G2 phases（ECRG4-Exo groups
11.83%, control group 16.04%), eventually leading to A549 cell proliferation rates reduces by 42%, sees Fig. 8.Experiment in vitro is in cellular level
Fully show that ECRG4-Exo effectively inhibits the proliferation of tumour cell.
（3）The Inhibition test of inflammation and angiogenesis related gene expression：
By TCA8113 cell inoculations in 12 orifice plates, density 70%, after overnight incubation, experimental group（6 holes）20ug ECRG4- are added
Exo, control group（6 holes）20 μ g Vector-Exo are added, continue culture for 24 hours, collect cell, extracts total serum IgE, uses real-time quantitative
PCR 2-δδCTMethod detects cell Proliferation, angiogenesis and inflammation-related gene NF- κ B p50, IL-1b, IL-6, MCP1,
The expression of CDK4, VEFGA, are shown in Fig. 9.The results show that ECRG4-Exo significantly suppresses TCA8113 cell NF- κ B p50, IL-
The expression of 1b, IL-6, MCP1, CDK4, VEFGA.
（4）Transplanted tumor in nude mice Cell suppression test：
In order to further verify ECRG4-Exo effects in vivo, the present invention uses ECRG4-Exo and Vector-Exo pairs respectively
TCA8113 cells are pre-processed, and equal cell is then taken to inoculate nude mice, replicate Transplanted tumor model, and concrete operations are as follows.
For TCA8113 cells kind in 10cm culture dishes, culture to density is 80-90%, and 400 μ g ECRG4- are added in experimental group
400 μ g Vector-Exo are added in Exo, control group, continue culture for 24 hours, 10 are diluted to PBS after trypsin digestion7A/ml.
6 monthly age male BALB/C immunodeficiency type nude mices are randomly divided into 2 groups, every group 6.Two groups of cells are seeded in nude mice right fore respectively
Dorsal sc, quantity 106A/only.The the 3rd, 6,9 day after cell inoculation, 100 μ g of experimental group nude mice tail vein injection
ECRG4-Exo, 100 μ g Vector-Exo of nude mice of control group tail vein injection, co-injection is three times.The 2nd after nude inoculation cell
It starts to use vernier caliper measurement tumour longest diameter（L）, shortest diameter（W）, calculate gross tumor volume（V）, V=L × W2× 0.5, together
Shi Jilu feed consumptions and nude mice weight, every 2 day entry 1 time, until the 24th day, nude mice is put to death, tumour is removed, weighs.Number
" mean+SD " indicates according to this, and independent sample T check analyses are carried out with SPSS19.0 softwares.
Two groups of nude mices of statistical result showed average weight and food ration during experiment do not have notable difference, see Figure 10, but
Tumor size difference is apparent.At the 8th, 14,16,20,22,24 day, ECRG4-Exo group gross tumor volumes were substantially less than Vector-
Exo groups (p<0.05), see Figure 11.After nude mice is put to death, tumour is removed, experimental group tumor size is significantly less than control group, passes through
Statistical analysis, experimental group tumor weight be substantially less than control group (p<0.05), see Figure 11.The present invention passes through external and internal examination
Test, respectively from molecular level, cellular level and integral level fully confirm ECRG4-Exo significantly inhibit Dental clinic and
The growth of tumour, and this effect inhibits neonate tumour blood vessel and inflammatory reaction phase mainly by inhibiting tumor cell proliferation
Correlation gene expresses to realize.
Claims (10)
1. a kind of excretion body, which is characterized in that be mounted with the ECRG4 mRNA for the Hemapoiesis for being overexpressed ECRG4 genes.
2. excretion body according to claim 1, which is characterized in that the amount of the ECRG4 mRNA of loading is outer not less than conventional
Secrete body 1000 times.
3. excretion body according to claim 1, which is characterized in that the cell of the overexpression ECRG4 is ECRG4-
HEK293 cell lines.
4. excretion body according to claim 3, which is characterized in that the ECRG4-HEK293 cell lines are by following system
What Preparation Method was prepared：
A, pLVX-IRES-ZsGreen1-ECRG4 plasmid constructions：Total serum IgE is extracted, ECRG4 genes are expanded with reverse transcription PCR, it will
In ECRG4 gene clonings to pLVX-IRES-ZsGreen1 carriers, pLVX-IRES-ZsGreen1-ECRG4 plasmids are obtained；
B, the preparation of slow virus：With containing tri- kinds of plasmids of pMD2.G, psPAX2 and pLVX-IRES-ZsGreen1-ECRG4
One mixed liquor transfects HEK293T cells；HEK293T cells after culture transfection, collect culture solution, are carried out to culture solution
Processing obtains the slow virus for being packaged with ECRG4 genes；
C, the foundation of ECRG4-HEK293 cell lines：With second containing ECRG4 genes slow virus, DMEM culture mediums and polybrene
Mixed liquor infects HEK293 cells；Metainfective HEK293 cells are cultivated, by cell sorting, are obtained
ECRG4-HEK293 cell lines.
5. excretion body according to claim 4, which is characterized in that in step A, pMD2.G, psPAX2 in the first mixed liquor
Mass ratio with tri- kinds of plasmids of pLVX-IRES-ZsGreen1-ECRG4 is 2：3：4.
6. excretion body according to claim 4, which is characterized in that in step B, polybrene is a concentration of in the second mixed liquor
6-10μg/ml。
7. excretion body according to claim 4, which is characterized in that in step C, with the second mixed liquor to HEK293 cells into
2 subinfection of row.
8. excretion body according to claim 4, which is characterized in that in step C, the 2nd sense is being carried out to HEK293 cells
Before dye, 12-24h will be cultivated in the DMEM culture mediums containing 10% FBS by the HEK293 cells of the first subinfection.
9. a kind of preparation method of the excretion body as described in claim any one of 1-3, which is characterized in that include the following steps：
（1）Prepare the cell for being overexpressed ECRG4；
（2）The cell for being overexpressed ECRG4 is cultivated in the DMEM of serum-free, after the completion of culture, collects culture solution；
（3）By step（2）The culture solution being collected into carries out separating-purifying, obtains the excretion body containing ECRG4 mRNA.
10. a kind of application of excretion body as described in claim any one of 1-8 in preparing the drug for treating tumor disease.
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Effective date of registration: 20240808 Address after: B-6, 1st Floor, No. 6, No. 33 Shulu Avenue, Longmatan District, Luzhou City, Sichuan Province 646000 Patentee after: Sichuan Exosome Home Technology Co.,Ltd. Country or region after: China Address before: No.1, Section 1, Xianglin Road, Longmatan District, Luzhou City, Sichuan Province Patentee before: SOUTHWEST MEDICAL University Country or region before: China |
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