CN110420221A - A kind of natural nano vesica and its preparation method and application - Google Patents
A kind of natural nano vesica and its preparation method and application Download PDFInfo
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- CN110420221A CN110420221A CN201910460219.2A CN201910460219A CN110420221A CN 110420221 A CN110420221 A CN 110420221A CN 201910460219 A CN201910460219 A CN 201910460219A CN 110420221 A CN110420221 A CN 110420221A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/13—Tumour cells, irrespective of tissue of origin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0693—Tumour cells; Cancer cells
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Abstract
The invention discloses a kind of natural nano vesica and its preparation method and application, a kind of natural nano vesica of the invention is preparing the application in regulating cell transfer and TGF-β signal pathway inhibitor.A kind of natural nano vesica of the invention is preparing hepar damnification drug caused by various acute and chronic hepatitis or acute and chronic hepatitis or the application in inhibitor.A kind of application of natural nano vesica of the invention in the drug or inhibitor for preparing HCC primary carcinoma of liver.
Description
Technical field
The present invention relates to biopharmaceutical technologies, and in particular to a kind of natural nano vesica and preparation method thereof and answers
With.
Background technique
The nano grade biological vesica that excretion body is discharged as various kinds of cell (including tumour cell), includes various active cell
The factor, albumen and inhereditary material (mRNA, DNA and miRNA) etc..In this research, it is thin that we have detected different invasion characteristic liver cancer
Effect and molecular mechanism of the excretion body of born of the same parents' strain and normal liver cell origin in epithelial phenotype hepatocellular carcinoma cells EMT, grind
Study carefully as a result, it has been found that, the excretion body of HCC cell origin can promote recipient cell migrate and invasion, inducing receptor cell E-
Cadherin expression is reduced, and Vimentin expression increases and EMT, and the excretion body effect of high invasion HCC cell origin be better than it is low
Invade HCC cell and normal liver cell excretion body.In addition, experiment in vivo equally prompts the high invasion lower invasion of HCC excretion body
HCC excretion body and normal liver cell excretion body promote HCC Lung metastases in nude mouse.In short, our results prompt the outer of the source HCC
The agonist of HCC transfer may be become by from/paracrine mode inducing receptor cell generation EMT by secreting body.
HCC postoperative recurrence and transfer are the main reason for influencing patient's prognosis.Process of the metastases as a multistep,
Epithelial tumour cell obtains interstitial and changes, i.e., Epithelial and stromal conversion (Epithelial mesenchymal occurs for cell
Transition, EMT) be metastases process the first step.The EMT process of interference tumour may be decreased metastases, invasion
Ability brings improvement to the prognosis of HCC patient.It is a large amount of extracellular that more and more research discovery tumour cells can secrete release
The excretion body of vesica (including excretion body), tumour source passes through transhipment multiple biological activities molecule (cell factor, albumen, mRNA
With miRNAs etc.) progress, transfer and the drug resistance etc. of modulate tumor microenvironment tumour.
In recent years, research finds that excretion body can be used as tumor microenvironment regulation person and influence tumor cell invasion and EMT etc..
The intracorporal albumen of excretion can change with lung carcinoma cell character mutation, and apply the outer of the cell origin after TGF-β induction
It secretes body and epithelial phenotype cell co-cultures, it is found that recipient cell form and phenotype marker albumen are changed.High malignancy
Tumour cell may transport the tumour cell of excretion body to low transfer ability, promote its cell Proliferation and vitro invasion, transfer
Deng.The excretion body of the discovery such as Rahman and colleague, high-metastasis tendency lung carcinoma cell and Patients with Advanced Lung Cancer serum origin can lead to
Up-regulation Vimentin expression is crossed, EMT process is mediated to induce non-tumor receptor cell migration, invasion and competence for added value enhancing.
The above result shows that excretion body discharges into extracellular microenvironment by tumour cell, transhipment bioactivity point can be passed through
Son participates in progress and transfer of regulation target cell etc. to neighbouring or distant place target cell.However, current HCC cell origin is outer
It secretes role of the body in the invasion of HCC in vitro and in vivo, transfer to be still not clear, this research attempts to inquire into this problem, and further visits
The excretion body of the HCC cell origin of different invasion characteristics is begged for the presence or absence of difference between the effects and specific molecular mechanism etc..
Currently, lacking effective one kind, high yield and the specific natural nano vesica of effect and its preparation method and application.
Summary of the invention
The object of the present invention is to provide the natural nanos for extracting liver cell secretion effective, high yield under a kind of normal temperature state
Vesica and its preparation method and application.
The purpose of the present invention is what is realized by following technical proposal: a kind of natural nano vesica of the invention is adjusted in preparation
Control the application in cell transfer and TGF-β signal pathway inhibitor.
A kind of natural nano vesica of the invention is preparing the damage of liver caused by various acute and chronic hepatitis or acute and chronic hepatitis
Application in vulnerary object or inhibitor.
A kind of application of natural nano vesica of the invention in the drug or inhibitor for preparing HCC primary carcinoma of liver.
Further, the cell is the excretion body in HCC Primary hepatic carcinoma cell source.
Further, the excretion body in the HCC Primary hepatic carcinoma cell source promotes internal HCC invasion, transfer.
Further, the excretion body body in the HCC Primary hepatic carcinoma cell source can be by transporting fibroblast
Growth factor FGF-2, vascular endothelial growth factor VEGF and epidermal growth factor EGF activated receptor cell influence cell growth, move
It moves.
Further, the excretion body in the HCC Primary hepatic carcinoma cell source is transported in it by certainly/paracrine mode
The some biological activity factor change tumor microenvironment molecule constitute, modulate tumor EMT occur.The HCC Primary Hepatic
The excretion body of cancer cells generates or the targeted drug of the inhibitor of release and TGF-β, HGF and VEGF biomolecule,
Help is provided to HCC patient's prognosis is improved.
The preparation method of natural nano vesica of the present invention, includes the following steps: the extraction of (1) excretion body: patient
The extraction of excretion body uses Exoquick TM Kit (System Biosciences, USA) method in serum；
(2) the excretion body that transmission electron microscope tem observation extracts: the excretion body precipitating that serum extracts is mixed through PBS solution
Outstanding, the excretion liquid solution for taking 5-20 μ l to obtain is 5-20 minutes fixed through 1% glutaraldehyde, and purified water rinses, then by 2-10 μ l
Suspension the copper mesh in overlay film is added dropwise, being stored at room temperature 10-30 minute parches it, and drop 10-50 μ l phosphotungstic acid is in negative on copper mesh
Dye 5 minutes, in transmission electron microscope observation and is taken pictures with 80KV voltage；
(3) immunofluorescence: the CM-DIL staining reagent of 2-10 μ L is added in the excretion body suspension of 100 μ L, at room temperature
It is incubated for 10-30min；Through centrifugation, again mark, Fibronectin coating, creep plate, cleaning, it is fixed after use fluorescence co-focusing micro-
Sem observation specimen staining situation is simultaneously taken pictures；
(4) Xenografts in nude mice model: the cell suspension of certain volume is drawn using asepsis injector, then by 10-
200 μ L pallium cell injections are subcutaneous in nude mice armpit, and 75% alcohol cotton stick wipes sepage at pin hole；To all nude mices of experimental endpoints
It takes off neck after 5% chloral hydrate anesthesia to put to death, scissors removes subcutaneous tumor, and measurement gross tumor volume, balance weighing are organized through poly
It is spare after formaldehyde is fixed, natural nano vesica is made.
Further, in step (1), the step of Exoquick TM Kit (System Biosciences, USA) method
Rapid as follows: 50-500 μ l serum is centrifuged 10-30 minutes removal cells through 1000-5000g and fragment, supernatant move to another cleaning and go out
In tube, Exoquick TM Kit reagent 20-100 μ l is added and turns upside down mixings, 4 DEG C of incubations, 1500g is centrifuged 10-30 and divides
Clock, the visible rice white precipitating of tube bottom, removes supernatant and 1500g is centrifuged 5-30 minutes again, carefully remove supernatant fluid, according to
Precipitating is resuspended in subsequent use PBS buffer solution or RNA lysate.It extracts gained precipitating and describes method progress thoroughly by prior document
Radio sem observation.
A kind of natural nano vesica as made from the method for the invention.
The utility model has the advantages that a kind of natural nano vesica of the invention is preparing regulating cell transfer and the suppression of TGF-β signal path
Application in preparation.A kind of natural nano vesica of the invention is preparing liver caused by various acute and chronic hepatitis or acute and chronic hepatitis
Application in dirty damage medicine or inhibitor.A kind of natural nano vesica of the invention is in the drug for preparing HCC primary carcinoma of liver
Or the application in inhibitor.
Detailed description of the invention
It is further illustrated below in conjunction with attached drawing, in attached drawing:
Fig. 1 is the map of the nano grade biological vesica of different cell extractions of the invention；Electronic Speculum observation shows that vesica is dispersed in point
Cloth, rounded, diameter about 30-150nm or so.
Fig. 2 is the map that discovery nano grade biological vesica internalization enters cell under fluorescence microscope of the invention；Through special
The biological vesica of dyeing is melted film effect and is entered in cell cytosol, is gathered in around nucleus.
Fig. 3 is that the present invention applies nano grade biological vesica, can promote the map of nude mouse tumor Lung metastases.Injection of exogenous is raw
The incidence of the nude mice of object vesica, neoplasm lung metastasis is significantly raised.
Specific embodiment
By following embodiment, present invention be described in more detail, but should be noted that the scope of the present invention is not implemented by these
Any restrictions of example.
Embodiment 1
A kind of natural nano vesica of the invention prepare regulating cell transfer and TGF-β signal pathway inhibitor in answer
With.
A kind of natural nano vesica of the invention is preparing the damage of liver caused by various acute and chronic hepatitis or acute and chronic hepatitis
Application in vulnerary object or inhibitor.
A kind of application of natural nano vesica of the invention in the drug or inhibitor for preparing HCC primary carcinoma of liver.
The cell is the excretion body in HCC Primary hepatic carcinoma cell source.
The excretion body in the HCC Primary hepatic carcinoma cell source promotes internal HCC invasion, transfer.
The excretion body body in the HCC Primary hepatic carcinoma cell source can by transhipment fibroblast growth factor FGF,
Vascular endothelial growth factor VEGF and epidermal growth factor EGF activated receptor cell influence cell growth, migration.
The excretion body in the HCC Primary hepatic carcinoma cell source transports the life of the part in it by certainly/paracrine mode
The molecule that object active factors change tumor microenvironment is constituted, and modulate tumor EMT occurs.
The excretion body in the HCC Primary hepatic carcinoma cell source generates or the inhibitor and TGF-β, HGF of release
And the targeted drug of VEGF biomolecule, help is provided to HCC patient's prognosis is improved.
The preparation method of natural nano vesica of the present invention, includes the following steps: the extraction of (1) excretion body: patient
The extraction of excretion body uses Exoquick TM Kit (System Biosciences, USA) method in serum；Exoquick
The step of TM Kit (System Biosciences, USA) method is as follows: 500 μ l serum are centrifuged 10-30 minutes through 1000g and go
Except cell and fragment, supernatant is moved in another clean sterile tube, and addition 80 μ l of Exoquick TM Kit reagent turns upside down mixed
Even, 4 DEG C of incubations, 1500g is centrifuged 30 minutes, and the visible rice white precipitating of tube bottom removes supernatant and 1500g is centrifuged 5 minutes again, small
The heart removes supernatant fluid, and precipitating is resuspended according to subsequent use PBS buffer solution or RNA lysate.Gained precipitating is extracted by previous
Document describes method and carries out transmission electron microscope observing.
(2) the excretion body that transmission electron microscope tem observation extracts: the excretion body precipitating that serum extracts is mixed through PBS solution
Outstanding, the excretion liquid solution for taking 20 μ l to obtain fixes 20 minutes through 1% glutaraldehyde, and purified water rinses, and then drips the suspension of 9 μ l
It is added in the copper mesh of overlay film, being stored at room temperature 20 minutes parches it, drips 10 μ l phosphotungstic acids in negative staining 5 minutes on copper mesh, with 80KV
Voltage is in transmission electron microscope observation and takes pictures；
(3) immunofluorescence: the CM-DIL staining reagent of 2 μ L is added in the excretion body suspension of 100 μ L, is incubated at room temperature
30min；Through centrifugation, again marks, Fibronectin coating, creep plate, cleaning, observed using confocal microscope after fixation
Specimen staining situation is simultaneously taken pictures；
(4) Xenografts in nude mice model: the cell suspension of certain volume is drawn using asepsis injector, then by 10 μ L
Pallium cell injection is subcutaneous in nude mice armpit, and 75% alcohol cotton stick wipes sepage at pin hole；To all nude mices of experimental endpoints through 5%
It takes off neck after chloral hydrate anesthesia to put to death, scissors removes subcutaneous tumor, and measurement gross tumor volume, balance weighing are organized through paraformaldehyde
It is spare after fixation, natural nano vesica is made.
Statistical method: it is analyzed using SPSS18.0 statistical software.Using chi-square criterion, t is examined or one-way analysis of variance
Each group difference is detected, correlation detection is analyzed using Spearman, is that difference is statistically significant with P < 0.05.
A kind of natural nano vesica as made from the method for the invention.
Embodiment 2
Embodiment 2 the difference from embodiment 1 is that:
The preparation method of natural nano vesica of the present invention, includes the following steps: the extraction of (1) excretion body: patient
The extraction of excretion body uses Exoquick TM Kit (System Biosciences, USA) method in serum；Exoquick
The step of TM Kit (System Biosciences, USA) method is as follows: 50 μ l serum are centrifuged removal in 20 minutes carefully through 5000g
Born of the same parents and fragment, supernatant move in another clean sterile tube, and 20 μ l of Exoquick TM Kit reagent is added and turns upside down mixing, and 4
It DEG C is incubated for, 1500g is centrifuged 20 minutes, tube bottom visible rice white precipitating, removal supernatant and 1500g centrifugation 20 minutes again, carefully
Supernatant fluid is removed, precipitating is resuspended according to subsequent use PBS buffer solution or RNA lysate.Gained precipitating is extracted to press first above
It offers description method and carries out transmission electron microscope observing.
In step (2), the excretion body of transmission electron microscope tem observation extraction: the excretion body that serum extracts precipitates warp
PBS solution is suspended, and the excretion liquid solution for taking 5 μ l to obtain fixes 10 minutes through 1% glutaraldehyde, and purified water rinses, then by 2 μ l
Suspension be added dropwise overlay film copper mesh, being stored at room temperature 10 minutes parches it, drop 40 μ l phosphotungstic acids divide in negative staining 5 on copper mesh
Clock in transmission electron microscope observation and is taken pictures with 80KV voltage；
In step (3), the CM-DIL staining reagent of 10 μ L immunofluorescence: is added to the excretion body suspension of 100 μ L
In, it is incubated for 20min at room temperature；Through centrifugation, again marks, Fibronectin coating, creep plate, cleaning, is copolymerized using fluorescence after fixation
Focusing microscope observation specimen staining situation is simultaneously taken pictures；
In step (4), Xenografts in nude mice model: drawing the cell suspension of certain volume using asepsis injector,
Then 200 μ L pallium cell injections are subcutaneous in nude mice armpit, 75% alcohol cotton stick wipes sepage at pin hole；To experimental endpoints institute
There is nude mice to take off neck after 5% chloral hydrate anesthesia to put to death, scissors removes subcutaneous tumor, measurement gross tumor volume, balance weighing, tissue
It is spare after paraformaldehyde is fixed, natural nano vesica is made.
Embodiment 3
Embodiment 3 the difference from embodiment 1 is that:
The preparation method of natural nano vesica of the present invention, includes the following steps: the extraction of (1) excretion body: patient
The extraction of excretion body uses Exoquick TM Kit (System Biosciences, USA) method in serum；Exoquick
The step of TM Kit (System Biosciences, USA) method is as follows: 300 μ l serum are centrifuged 10 minutes through 4000g and remove
Cell and fragment, supernatant move in another clean sterile tube, and addition 100 μ l of Exoquick TM Kit reagent turns upside down mixed
Even, 4 DEG C of incubations, 1500g is centrifuged 10 minutes, and the visible rice white precipitating of tube bottom removes supernatant and 1500g is centrifuged 30 minutes again,
Careful removal supernatant fluid, is resuspended precipitating according to subsequent use PBS buffer solution or RNA lysate.Gained precipitating is extracted by first
Preceding document describes method and carries out transmission electron microscope observing.
In step (2), the excretion body of transmission electron microscope tem observation extraction: the excretion body that serum extracts precipitates warp
PBS solution is suspended, and the excretion liquid solution for taking 15 μ l to obtain fixes 5 minutes through 1% glutaraldehyde, and purified water rinses, then by -10
The copper mesh in overlay film is added dropwise in the suspension of μ l, and being stored at room temperature 30 minutes parches it, and 50 μ l phosphotungstic acids of drop divide in negative staining 5 on copper mesh
Clock in transmission electron microscope observation and is taken pictures with 80KV voltage；
In step (3), immunofluorescence: the CM-DIL staining reagent of 8 μ L is added in the excretion body suspension of 100 μ L,
It is incubated for 10min at room temperature；Through centrifugation, again mark, Fibronectin coating, creep plate, cleaning, it is fixed after use fluorescence co-focusing
Micro- sem observation specimen staining situation is simultaneously taken pictures；
In step (4), Xenografts in nude mice model: drawing the cell suspension of certain volume using asepsis injector,
Then 160 μ L pallium cell injections are subcutaneous in nude mice armpit, 75% alcohol cotton stick wipes sepage at pin hole；To experimental endpoints institute
There is nude mice to take off neck after 5% chloral hydrate anesthesia to put to death, scissors removes subcutaneous tumor, measurement gross tumor volume, balance weighing, tissue
It is spare after paraformaldehyde is fixed, natural nano vesica is made.
Test example 1
1. excretion body extracts and identification by morphological characters
Select normal liver cell (LO2) and two plants of HCC cell lines (MHCC-97H and MHCC-97L) cells and supernatant
Excretion body is extracted, wherein MHCC-97H cell metastatic potential with higher.According to document report before, we using hypervelocity from
Heart method extracts the excretion body in different cells and supernatants, and application transmission electron microscope, granularmetric analysis, marker protein detection etc. are ground
Study carefully excretion volume morphing and feature.As a result, it has been found that the excretion body of different cell origins is in vesica shape, diameter about 30-150nm, expression mark
Will PROTEIN C D9 and CD63.Fig. 1 is the map of the nano grade biological vesica of different cell extractions of the invention；Different liver cells
The map of the nano grade biological vesica of extraction；Electronic Speculum observation shows that vesica is dispersed in distribution, rounded, diameter about 30-150nm.
In addition, we mark excretion body using green fluorescence reagent PKH-67, inquire into whether it can be absorbed by recipient cell
Internalization.The excretion body in the source MHCC-97H of fluorescence microscope result prompt green fluorescence PKH-67 label can enter HCC cell
It is HepG2 intracellular, is gathered in the nucleus week of DAPI dyeing.
Fig. 2 is the map that discovery nano grade biological vesica internalization enters cell under fluorescence microscope of the invention；
2.HCC cell excretion body promotes cell migration
In order to inquire into the excretion body of HCC cell origin to recipient cell migration, the influence of invasive ability.We apply scratch
The excretion body of Healing Experiments detection discovery MHCC-97L and MHCC-97H cell origin promotes recipient cell HepG2 migration, and high
The excretion body effect for invading the source characteristic MHCC-97H is stronger.The excretion body group in the Transwell result prompt source MHCC-97H
Positive stained cells number increased significantly, and prompts the excretion body of HCC cell origin to can promote HepG2 and invades, and the excretion in the source LO2
It is unobvious that body promotees cell invasion ability.
3.HCC cell excretion body inducing cell EMT
After observing that the excretion body of HCC cell origin can promote recipient cell migration, invasion, we apply Western blot
Method has detected the variation of EMT marker protein, as a result, it has been found that the excretion body with MHCC-97L and MHCC-97H cell origin is trained altogether
After supporting, recipient cell HepG2 epithelial phenotype marker protein E-cadherin expression is in reduction trend, and interstitial phenotype Vimentin
Albumen is in increase trend.We also apply immunofluorescence method to have detected the excretion body of recipient cell HepG2 Yu HCC cell origin
The situation of change of cell phenotype albumen after co-cultivation 48h, after discovery is using the excretion body of exogenous MHCC-97H cell origin, E-
Cadherin expression quantity reduces, and Vimentin expression increases.
4.HCC cell excretion body active cell TGF-β access
One of the most important cell factor of EMT process occurs as tumour for TGF-β, we detect using ELISA method
The horizontal variation of TGF-β in recipient cell supernatant.We have found that using exogenous MHCC-97L and MHCC-97H cell origin
Excretion body after, TGF-β content in HepG2 cells and supernatant is in a degree of up-regulation trend.In addition, we pass through
Western blotting method has detected the phosphorylation activity form of receptor activation type Smad2 and Smad3, and discovery is outer in application
After the excretion body of source property HCC cell origin, the phosphorylation activity form of Smad2 and Smad3 is activated in receptor HepG2 cell
(Fig. 3) prompts the excretion body of HCC cell origin to result in TGF-β signal pathway activated.
For the effect of cell factor TGF-β in clear excretion body, our application cell factor TGF-β and TGF-β inhibit
After agent LY2109761 handles MHCC-97L cell 72h respectively, the excretion body discharged under MHCC-97L cell different conditions is obtained,
Then related experiment detection is carried out again.Scratch Healing Experiments discovery, using the excretion body in TGF-β group source can be obviously promoted by
The migration of body cell HepG2, and apply the excretion body rush cell of MHCC-97L cell origin after TGF-β inhibitor LY2109761
Mobility is substantially reduced compared with TGF-β group cell.The MHCC-97L that the detection discovery of Transwell method is handled using TGF-β group
The excretion body group of cell origin is significantly more compared with the positive cell number that control group and LY2109761 group penetrate hole.
5.HCC cell excretion body influences the variation of cell phenotype albumen by TGF-β
By Western blot method have detected separate sources excretion body handle recipient cell HepG2 after cell phenotype
The situation of change of marker protein, the excretion body in the source MHCC-97L that discovery is induced using TGF-β, compared with control group E-cadherin
Expression reduces, and Vimentin expression increases, and applies the source MHCC-97L of TGF-β inhibitor LY2109761 processing
The effect of excretion body group obvious weaken.Immunofluorescence results find that compared with TGF-β group, LY2109761 group promotees cell phenotype egg
Leucismusization effect is obvious to be weakened.
The excretion body in 6.HCC Primary hepatic carcinoma cell source promotes internal HCC invasion, transfer.
Nude mice hepatocyte in situ Transplanted tumor model and the excretion body tail vein injection processing through different cell origins are established, is led to
It crosses HE colouring method and has detected nude mice lung tissue cancer embolus and transfer stove situation.As a result, it has been found that the source MHCC-97L and MHCC-97H
Excretion body group Pulmonary metastasis focuses nude mice quantity compared with PBS injection group and LO2Excretion body group increases, and increased resistance invasion attacks the excretion body in source
Group rate of transform highest.Fig. 3 is that the present invention applies nano grade biological vesica, can promote the map of nude mouse tumor Lung metastases.
In addition, we have detected the E-cadherin of different disposal group nude mice liver neoplasm using the method for immunohistochemistry
Expression, as a result, it has been found that the excretion body group E-cadherin expression of HCC cell origin is compared with control group and LO2Excretion body group is in
Down regulation trend, and the excretion body group E-cadherin down regulation trend in the high invasion source HCC cell MHCC-97H becomes apparent from.In addition,
Because in the excretion body in the source HCC of Western blot result prompt before, there may be the enrichments of VEGF, we have detected liver
As a result the expression of VEGF in tumor tissues prompts vegf expression water in MHCC-97L and MHCC-97H excretion body group liver tumour tissue
It is flat to be higher than control group and LO2 excretion body group.
HCC is in progress and shifts as different kinds of molecules mechanism regulating as a result, current about HCC progress, transfer process Central Plains
How tumor of swelling is not fully understood with part and distant place cellular communication this process.Excretion body is as multiple biological activities
The transport agent of molecule, more and more evidences show excretion body by transport function molecule to tumor microenvironment even at a distance
A plurality of types of cells play multiple regulating and controlling effect in tumour progression, invasion, angiogenesis and transfer process.For example, tumour
The excretion body of cell origin can pass through transhipment fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and table
The activated receptors cells such as skin growth factor (EGF).Previously the study found that the excretion body in tumour source can chemotactic endothelial cell.I
The study found that the excretion body of HCC cell origin can influence thin by delivering transforming growth factor β (TGF-β) to recipient cell
Intracellular growth, migration etc..
EMT is by promoting intercellular adhesion to lose, migrating cell, invasive ability influence metastases, as thin
The forfeiture of the key protein E-cadherin of Intercellular Adhesion is one of major reason.In our current research, it is observed that by
After body HCC cell and the excretion body of MHCC-97L and MHCC-97H cell origin co-culture, the decline of E-cadherin expression,
And interstitial phenotypic proteins Vimentin is increased, and prompts receptor HCC cell that EMT has occurred.The excretion body of HCC cell origin may lead to
Crossing autocrine/paracrine mode drives cell phenotype to change, and promotes HCC progress and transfer.It is similar to our results of study, document
The excretion body in report melanoma cells source equally can promote myeloid progenitor phenotypic alternation to before shifting.Outside tumour source
Secreting body can excite smooth muscle progenitor cells to rush tumour cell Phenotypic Change.The excretion body in breast cancer and prostate cancer source can induce
Myofibroblast character mutation.A variety of different types of cells can be thin by tumour in the above result of study prompt tumor microenvironment
The excretion body in born of the same parents source influences, and promotes Lymphocytic phenotype, this may be different type recipient cell to tumor transformation or migration,
One of the mechanism of invasive ability enhancing.
Document report, many A signal pathways, which may be activated, influences melanoma EMT (such as MAPK, NF-KB, P13K/AKT
Deng).And soluble cytokine TGF-β family is as one of most important factor in EMT inducer, TGF-β receptor and downstream
The activation of Smad access can start including Snail family, ZEB family and Twist family transcription factor, inducing cell reprogramming,
Finally make the primary tumor tumour cell of epithelial phenotype obtain the characteristic of interstitial cell and obtain the ability of invasion cell epimatrix,
Trigger point as metastases.We have discovered that the excretion body of HCC cell origin is by influencing receptor HCC cell TGF-β
Signal path promotes its invasion, transfer ability enhancing, moreover, the excretion body effect of the HCC cell origin of high invasion characteristic is more
By force.In short, the excretion body in result above prompt tumour cell source can turn similar oncogenic molecules from the mode of/paracrine
Other tumour cells or normal subject cell are moved on to, recipient cell is promoted to obtain tumor invasion characteristic.When base occurs for part cell
When because of mutation, the excretion of secretion release, which is known from experience, is transported to other recipient cells for this abrupt information by melting film effect, into
And cause recipient cell protooncogene activation, anti-apoptotic genes expression expression and the enhancing of anchoring independent form growth ability.
In addition to the above biotic factor has an impact tumour EMT, the equally possible mistake for participating in tumour EMT of some miRNA
Journey, document report inhibit the expression of miR-191 that may block tumour EMT process, reduce tumor cell invasion and transfer ability.
MiR-23a can be by targeting CAM 120/80 modulate tumor EMT.Let-7a, which is also studied, confirms that it can be by targeting specific egg
White such as LIN28B and HMGA2 participates in tumor migration, invasion and EMT process.Our seminars are quasi- in the future to inquire into Partial key miRNA
Effect and mechanism in the excretion body induction tumour cell EMT of different invasion characteristic HCC cell origins etc., further clarify outer
Secrete body as biotic factor transport vehicle HCC progress, transfer in role and effect.
In short, based on current we have found that the excretion body of HCC cell origin can be transported in it by certainly/paracrine mode
The molecule that some biological activity factor changes tumor microenvironment is constituted, and modulate tumor EMT occurs, accordingly can reasonable expectation excretion body
The targeted drug of the biomolecule such as the inhibitor and TGF-β, HGF and VEGF of generation or release, may be to improvement HCC patient
Prognosis provides help.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, the present invention
Claimed range is delineated by the appended claims, the specification and equivalents thereof from the appended claims.
Claims (10)
1. a kind of natural nano vesica is preparing the application in regulating cell transfer and TGF-β signal pathway inhibitor.
2. a kind of natural nano vesica is preparing hepar damnification drug or suppression caused by various acute and chronic hepatitis or acute and chronic hepatitis
Application in preparation.
3. a kind of application of natural nano vesica in the drug or inhibitor for preparing HCC primary carcinoma of liver.
4. application according to claim 1 or 3, it is characterised in that: the cell is HCC Primary hepatic carcinoma cell source
Excretion body.
5. application according to claim 4, it is characterised in that: the excretion body in the HCC Primary hepatic carcinoma cell source
Promote internal HCC invasion, transfer.
6. application according to claim 4, it is characterised in that: the excretion body in the HCC Primary hepatic carcinoma cell source
Body can by transhipment fibroblast growth factor FGF, vascular endothelial growth factor VEGF and epidermal growth factor EGF activation by
Body cell influences cell growth, migration.
7. application according to claim 4, it is characterised in that: the excretion body in the HCC Primary hepatic carcinoma cell source
The molecule composition that some biological activity factor in it changes tumor microenvironment, modulate tumor are transported by certainly/paracrine mode
EMT occurs.
8. the preparation method of natural nano vesica described in claim 1, it is characterised in that include the following steps:
(1) extraction of excretion body: the extraction of excretion body uses Exoquick TM Kit (System in patients serum
Biosciences, USA) method；
(2) the excretion body that transmission electron microscope tem observation extracts: the excretion body precipitating that serum extracts is suspended through PBS solution, takes
The excretion liquid solution that 5-20 μ l is obtained is 5-20 minutes fixed through 1% glutaraldehyde, and purified water rinses, then by the suspension of 2-10 μ l
The copper mesh in overlay film is added dropwise, being stored at room temperature 10-30 minutes parches it, and drop 10-50 μ l phosphotungstic acid divides in negative staining 5 on copper mesh
Clock in transmission electron microscope observation and is taken pictures with 80 KV voltages；
(3) immunofluorescence: the CM-DIL staining reagent of 2-10 μ L is added in the excretion body suspension of 100 μ L, is incubated at room temperature
10-30min；Through centrifugation, again marks, Fibronectin coating, creep plate, cleaning, seen using confocal microscope after fixation
It examines specimen staining situation and takes pictures；
(4) Xenografts in nude mice model: the cell suspension of certain volume is drawn using asepsis injector, then by 10-200 μ L
Pallium cell injection is subcutaneous in nude mice armpit, and 75% alcohol cotton stick wipes sepage at pin hole；To all nude mices of experimental endpoints through 5%
It takes off neck after chloral hydrate anesthesia to put to death, scissors removes subcutaneous tumor, and measurement gross tumor volume, balance weighing are organized through paraformaldehyde
It is spare after fixation, natural nano vesica is made.
9. the preparation method of natural nano vesica according to claim 8, it is characterised in that: in step (1),
The step of Exoquick TM Kit (System Biosciences, USA) method is as follows: 50-500 μ l serum is through 1000-
5000g is centrifuged 10-30 minutes removal cells and fragment, and supernatant moves in another clean sterile tube, and Exoquick TM Kit is added
Reagent 20-100 μ l turns upside down mixing, and 4 DEG C of incubations, 1500g is centrifuged 10-30 minute, and the visible rice white of tube bottom precipitates, in removal
1500g is centrifuged 5-30 minutes clearly and again, supernatant fluid is carefully removed, according to subsequent use PBS buffer solution or RNA lysate
Precipitating is resuspended.It extracts gained precipitating and describes method progress transmission electron microscope observing by prior document.
10. natural nano vesica made from a kind of method as described in claim 8 or 9.
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