CN107916253A - A kind of preparation method of mescenchymal stem cell for expressing people source immuno-stimulator LIGHT and obtained MSC L cells - Google Patents
A kind of preparation method of mescenchymal stem cell for expressing people source immuno-stimulator LIGHT and obtained MSC L cells Download PDFInfo
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Abstract
The invention discloses a kind of preparation method for the mescenchymal stem cell for expressing people source immuno-stimulator LIGHT, include the following steps：Separating mesenchymal stem cell, and carry out original cuiture and secondary culture；By building reverse transcription and slow virus carrier pMIGR3 hLIGHT, pCDHEF hLIGHT plasmids, human LIGHT gene is transferred to mescenchymal stem cell structure MSC L cells, which is the mescenchymal stem cell of expression people source immuno-stimulator LIGHT.The MSC L cells prepared the invention further relates to a kind of above method and its application.The present invention is using umbilical cord as MSC source, human LIGHT gene is transferred to MSC, LIGHT is transported to tumor tissues by MSC, the MSC of LIGHT modifications can change tolerance status of the host to tumour by producing the immune response of strong antitumor cell and tumour correlation interstitial cell, so as to effectively control tumour, the present invention can be prepared on a large scale, for the treatment of a variety of solid malignants and cancer prevention.
Description
Technical field
The present invention relates to biological immune treatment technology field, more particularly to a kind of expression people source immuno-stimulator LIGHT
Mescenchymal stem cell preparation method and obtained MSC-L cells and its application.
Background technology
Tumor tissues are except containing tumour cell, also containing substantial amounts of tumor stroma, tumor stroma be often tumour wantonly
The accomplice of expansion.First, tumor stroma provides the microenvironment of stabilization for tumour progression, such as one of barrier, can interfere with curative
Thing is efficiently entering.Moreover, tumor stroma can by directly contact or secrete cytokines promote tumor proliferation.In addition, base
Cell plastid also suppresses host immune, induction tumour immunity tolerance.These find prompt tumor stroma to tumor development to close weight
Will.There is researcher's discovery, only destroying tumor stroma can make tumour be well controlled, and can at least keep tumour and host
It is in a relatively stable equilibrium state.
For most of tumor tissues, tumor stroma often such as one of barrier, can interfere with medicine and be efficiently entering,
It is the accomplice that tumour is expanded wantonly.Moreover, stroma cell usually suppresses host immune, induction tumour immunity tolerance.In clinical work
In work, it has been found that tumour cell often finally overcomes the immune system of host and host is withered away.Therefore, change tumour to exempt from
The microenvironment of epidemic disease inhibition is most important to effectively extending patient survival.The tumour for overcoming targeting conveying antitumor drug to run into
Immune-suppression problems, even change the microenvironment of tumour immunity inhibition, and how to be reversed and exempted from by immuno-stimulator
Epidemic disease inhibition microenvironment, it is then urgently to be resolved hurrily the problem of targeting anti-tumor.
Due to the randomness of tumor tissues, traditional means of administration is often difficult to effectively reach inside tumor tissues.Between
It is mesoblastic a kind of how competent thin that mesenchymal stem cells (mesenchymal stem cells, MSCS) are derived from mesoderm growing early stage
Born of the same parents, are present in marrow, peripheral blood, bleeding of the umbilicus, cancellous bone, adipose tissue synovial membrane and umbilical cord, have very strong immunoregulation effect, right
Tumour immunity tolerance plays an important role.MSCs has low-level MHC- I to express under quiescent condition, and does not usually express MHC- II
And costimulating factor CD80, CD86, CD40, the missing of costimulating factor often lead to T cell incapability, therefore the antigen of MSCs
Presentation ability and the ability of further activating immune cell are very weak.In addition, MSCs can also be by directly contacting or passing through
Discharge cell factor and suppress T cell propagation.Except that can suppress T cell, someone proves that MSCs can be by directly contacting and discharging
The mode of cell factor suppresses B cell activation and antibody-secreting.In addition, MSCs can also suppress NK cells and dendron shape is thin
The function of born of the same parents (DC), suppresses TNF secretion-α and promotes IL-10 to secrete, it is obvious that this will further result in the secretion of Th1 cells
IFN-γ is reduced, and promotes Th2 to secrete IL-4, so as to weaken antineoplastic immune, still more immature DC in itself exempts from tumour
Epidemic disease tolerance plays an important role.It is therefore seen that MSCs has stronger immune negative regulation ability to host.
Research finds that MSCs can be to inflammatory tissue specificity chemotactic, and tumor tissues are considered as then that a kind of can not cure
Chronic inflammation, it is desirable to deliver antitumor drug by MSCs to be acted on up to targeting anti-tumor.Many researchs will simply carry
The MSCs of medicine mixes or is expelled in vitro xenograft tumor animal model with tumour cell, and have ignored MSCs have in itself it is stronger
Immunosuppressive action, it can provide the protection place for avoiding immune attack for tumour cell, very likely tumour will be induced to exempt from
The generation of epidemic disease tolerance.And have researches show that MSCS is the important sources of stromal cells, MSCs is to tumor tissues chemotactic
Tumor proliferation provides suitable microenvironment, by contact with each other with tumour cell or by secrete growth factor and chemotactic because
Son, promotes tumor proliferation even to shift, can also be swollen by being divided into the further promotion of tumor vessel pericyte, fibroblast
Knurl is grown.Therefore, antitumor drug is conveyed using MSCs as carrier, often reduces the anti-tumor effect of relative medicine.It is existing
Report that this method can be by tumor regression to a certain extent, but seldom have been reported that how to transform MSCs carrys out reversing tumor in itself
Suppress microenvironment.
LIGHT is a kind of very strong immuno-stimulator in tumor necrosis factor superfamily (TNFSF), it can be in tumor group
Knit the strong antineoplastic immune of excitation.As other family members, it can be activated through induction.The T cell of periphery tranquillization
After MPA and PHA Co stituations, its expression significantly raises, and shows height tune of the expression by immunocyte of LIGHT
Section, and played a role in the adjusting of immunocyte.LIGHT plays different effects by its 3 acceptors.For at the same time
Expression has the tumour cell (eg.MDA-MB-231) of LT β R and TR2/HVEM, and LIGHT can induce its apoptosis；And to only expressing
The T cell of HVEM acceptors then stimulates its hyperplasia.In addition, LIGHT provides T cell activation costimulatory signal, its costimulation effect
Degree it is suitable with B7-CD28 signal transduction paths and independent of CD28.
Constructive expression LIGHT can make the high expression CCL21 chemotactic factor (CF)s of lymphoid tissue, and the latter oozes out T cell from blood vessel
And go back to the nest and play an important role.Recent studies have shown that LIGHT especially plays a crucial role antineoplastic immune immune activation.
For example, LIGHT can be promoted into a large amount of T cells to tumor tissues chemotactic in expression of tumor tissue.Moreover, because there is depositing for LIGHT
It is not to be induced into immune tolerance after, T cell enters tumor tissues, but is greatly activated, and thereby obtains tumour
To remove.Obviously, activation of the LIGHT to immunocyte under tumor microenvironment serves vital.Therefore, inventor intends
LIGHT genes are transferred to MSCs structure MSC-L cells, to observe whether MSC-L cells can be transported to tumor tissues by LIGHT,
Tumour immunity is resistant to break host by varying tumor microenvironment.
But the structure of above-mentioned MSC-L cells pair still suffers from problems with：
1) easily and efficiently method how is chosen, makes the MSCs activity of acquisition good, purity is high.Separation MSCs's has efficacious prescriptions
Method has an adherent screening method, enzymic digestion centrifugal process and carries out airflow classification or immunomagnetic beads according to the relatively special phenotype of cell
Sorting.The methods of enzymic digestion centrifugation and fluidic cell sort, although the cell obtained can be relatively pure, often because repeatedly
Interminable operating process, may make cell damage；In addition cell density is diluter after sorting, it is impossible to obtains and divides by other cells
Secreting the cell factor of release stimulates, and growth is often relatively slower；
2) MSCs of original cuiture is easy to aging, is often difficult to expand afterwards to certain generation, therefore need to improve mesenchyma
The multiplication capacity of stem cell simultaneously avoids cell senescence at the same time, with obtain the material of enough research and avoid between generation because
Senile cause and the non-specific difference brought；
3) MSCs can specificity to tumor tissues chemotactic, therefore be used as carrier conveying antitumor activity molecule to up to
Acted on to targeting anti-tumor.Due to itself having very strong immunosuppressive action, and tumor tissues or even tumor patient are often
Immunosuppressive condition is also at, how to change the microenvironment of tumor patient particularly tumor by local histogenic immunity inhibition；
4) can LIGHT is transferred to after MSCs stablize expression, and how is Proliferation, Differentiation ability, if has Tumor formation；
5) after LIGHT being transferred to MSCs, LIGHT could be targeted input tumor tissues by MSCs, and be produced strong anti-
Tumour immunity；
6) tumour growth is suppressed can generally occur in a case where：Tumor cell proliferation is obstructed；Tumour cell occurs
Tune is died；Tumor Angiongesis is destroyed；Antineoplastic immune activates, and body has intact immune surveillance function.It need to determine any
Antitumor mechanism works in the anti-tumor effect that MSC-L is mediated.
The content of the invention
The defects of it is an object of the invention to overcome in the prior art, there is provided one kind expression people source immuno-stimulator
The preparation method of the mescenchymal stem cell of LIGHT, in the MSC-L cells obtained by the preparation method, LIGHT is transported to swollen by MSC
Tumor tissue, the MSC of LIGHT modifications can be by producing the immune of strong antitumor cell and tumour correlation interstitial cell
React and change tolerance status of the host to tumour, so as to effectively control tumour.
To achieve the above object, the present invention adopts the following technical scheme that：
First purpose of the present invention is to provide a kind of mescenchymal stem cell for expressing people source immuno-stimulator LIGHT
Preparation method, it includes the following steps：
(1) separating mesenchymal stem cell, and carry out original cuiture and secondary culture；
(2) retrovirus and slow virus of the structure with LIGHT genes；
(3) retrovirus with LIGHT genes or slow virus prepared step (2) is transferred to what step (1) obtained
Mescenchymal stem cell, builds MSC-L cells.The present invention has also carried out antitumor action effect test to the MSC-L cells of preparation,
Anti-tumor experiment result etc. in Mice Body including MSC-L cells.
In order to further optimize above-mentioned preparation method, technical measures of the invention further include：
Further, the separation method of the separating mesenchymal stem cell in the step 1) includes adherent screening method, enzyme disappears
Change method and airflow classification or immunological magnetic bead sorting are carried out according to the relatively special phenotype of cell；Further, the step
It is rapid 1) in the separation method of separating mesenchymal stem cell be adherent screening method, stationary culture is relative to other methods such as streaming
Phenotype sorts, and is one simple and efficiently separate method.
Further, the mescenchymal stem cell of the step 1) be from marrow, peripheral blood, cancellous bone, adipose tissue,
The mescenchymal stem cell of bleeding of the umbilicus or umbilical cord.
Further, the mescenchymal stem cell of the step 1) is the mescenchymal stem cell from bleeding of the umbilicus or umbilical cord；More
Further, the mescenchymal stem cell of the step 1) is the mescenchymal stem cell from umbilical cord.Bleeding of the umbilicus and umbilical cord are MSCS
Optimal source, because they can be readily available by Noninvasive means, and the risk of virus pollution is low, can also be cold
Freeze row autotransplantation after preserving.However, the culture success ratio of bleeding of the umbilicus MSC is not high, there is research to think only 6%, and umbilical cord MSCS
Culture success ratio up to 100%.In addition, MSC is separated from bleeding of the umbilicusS, waste hematopoietic stem/progenitor therein, therefore, navel
Band MSCSJust become important sources.
Further, in the step 2) retrovirus and slow virus of the structure with LIGHT genes the step of wrap
Include：
1) retrovirus and slow virus carrier pMIGR3-hLIGHT, pCDHEF-hLIGHT plasmid are built, and is had
Select the expression plasmid of antibiotic；
2) by the pMIGR3-hLIGHT plasmids of step 1) structure or corresponding plasmid and packaging plasmid with selection antibiotic
PCL-Ampho is transfected to 293X cells, the retrovirus of production expression hLIGHT；
3) by the pCDHEF-hLIGHT plasmids of step 1) structure or corresponding plasmid and packaging plasmid with selection antibiotic
VSV-g, RSV-REV, pMDL g/p RRE cotransfections to 293X cells, the slow virus of production expression hLIGHT.Further,
The concrete operation step of retrovirus and slow virus of the above-mentioned structure with LIGHT genes includes：
1) structure of reverse transcription and slow virus carrier pMIGR3-hLIGHT, pCDHEF-hLIGHT plasmid；
RNA is extracted from human PBMC first, cDNA is obtained using reverse transcription reagent box, designs primer, draw respectively at both ends
Entering EcoRI and BamHI restriction enzyme sites, while kozak sequences are introduced at 5 ' ends, PCR amplification obtains people's light full-length genes,
EcoRI and BamHI digestions, pMIGR3 retroviral vectors and pCDHEF slow virus carriers are building up to by T4 ligases, are carried
Body is correct by sequence verification.PMIGR3-blasticidin, pMIGR3- are cloned into respectively again by correct fragment is sequenced
Puro, pCDHEF-blasticidin, pCDHEF-puro carrier obtain the expression plasmid for having selection antibiotic；
2) the retrovirus production of hLIGHT is expressed：By retroviral plasmid pMIGR3-hLIGHT (or corresponding band choosings
Select the plasmid of antibiotic) and packaging plasmid pCL-Ampho according to 1:1 quality is than cotransfection 293X cells, and 48 after transfection, 72 is small
When collect viral supernatants, with 0.45uM filters filter, packing be stored in -80 degree；
3) lentivirus production of hLIGHT is expressed：By slow virus plasmid pCDHEF-hLIGHT (or corresponding band selection antibiotic
Plasmid) and packaging plasmid VSV-g, RSV-REV, pMDLg/p RRE according to 2:1:1:1 ratio cotransfection to 293X cells,
48 after transfection, 72 collect viral supernatants when small, are filtered with 0.45uM interest rates device, and packing is stored in -80 degree；
Further, include in the step 3) the step of structure MSC-L cells：
A) LIGHT is imported into mescenchymal stem cell：The mescenchymal stem cell that step (1) obtains is placed in culture medium, is added
Enter retrovirus with LIGHT genes or the slow virus of step (2) preparation, cultivated, obtained between infecting after LIGHT
Mesenchymal stem cells；
B) expression of hLIGHT in MSC is detected：Using the sense obtained in dye solution and LIGHT antibody incubation steps a)
The mescenchymal stem cell after LIGHT is contaminated, and detects LIGHT positive MSC cells；
C) enrichment of MSC positive cells：Mescenchymal stem cell of the sorting more than 95%LIGHT positive cells.
Further, the step of above-mentioned structure MSC-L cells include：
A) LIGHT is imported into MSC：Take 2 × 105MSC cells add 1ml fresh cultures in 6 orifice plates, add above-mentioned
1.5ml retrovirus or slow virus, add polybrene to final concentration of 10ug/ml, when 16-24 is small after discard in culture
Clearly, fresh culture is added；
B) expression of hLIGHT in MSC is detected：Metainfective cell is digested using pancreatin and control obtains for infection cell
Single cell suspension, takes 1 × 105Cell is resuspended to 100ul FACS dye solutions, adds 0.2ugLIGHT antibody, 4 degree of incubations
30 minutes, 0.5mlFACS dye solutions are added, 1500RPM is centrifuged 3 minutes, supernatant discarding, repeats to add 0.5mlFACS dyes
Color buffer solution, 1500RPM are centrifuged 3 minutes, and supernatant discarding, cell is resuspended to 100ul FACS dye solutions, add 50ng
Anti-hFc-APC antibody, 4 degree are incubated 30 minutes, add 0.5mlFACS dye solutions, and 1500RPM is centrifuged 3 minutes, discarded
Supernatant, is resuspended to 300ulFACS dye solutions, positive (APC passages) using Cytoflex flow cytomeries LIGHT
MSC cells；
C) enrichment of MSC positive cells：After metainfective cell is dyed by the above method, sorted by fluidic cell
Obtain being more than 95%LIGHT positive cells；Or obtain being more than 95%LIGHT expression cells by antibiotic-screening.
Further, the step of MSC-L antitumor actions experiment includes：
1) by 1x106Raji tumor cell injections inject 1x10 on the 11st day to immune deficiency NSG mouse6PBMC, the 12nd day
Use 1x106Control MSC and MSC-hLIGHT is subcutaneously injected into around tumour, measures a tumor size every three days, records tumour
Growth curve；
2) by 1x106B16-OVA tumor cell injections perfect B6 mouse to immune, and the 10th day by 1x106Compare MSC and
MSC-hLIGHT is subcutaneously injected into around tumour, measures a tumor size every three days, records tumor growth curve.
Second object of the present invention is to provide expression people source immuno-stimulator LIGHT's prepared by a kind of above method
Mescenchymal stem cell (MSC-L cells).
Third object of the present invention is to provide a kind of preparation containing above-mentioned MSC-L cells；Further, the preparation
Further include medicinal diluent or excipient.
Fourth object of the present invention is to provide a kind of above-mentioned MSC-L cells in treatment or prevention tumour medicine is prepared
Using.Further, the tumour includes liver cancer, lung cancer, kidney, cancer of the esophagus, stomach cancer, colorectal cancer, the cancer of the brain, breast cancer, bladder
Cancer, cancer of pancreas, nasopharyngeal carcinoma, lymthoma, melanoma.
In MSC-L cells prepared in the present invention, for MSC as transport agent, LIGHT is transported to tumor tissues by it.
It will be appreciated that technical scheme can be adjusted correspondingly according to different pumped (conveying) mediums, such as using MSC as one
A transport agent, prepares MSC-41BBL, MSC-IFN and MSC-anti-PDL1 etc., it is respectively used to using same method
Convey 41BBL, IFN and anti-PDL1 etc..
Compared with prior art, the present invention is had the advantages that using above-mentioned technical proposal：
The present invention is using umbilical cord as MSCSSource, it is with many advantages：1. umbilical cord has more sufficient source, collect and hold
Easily, donor is limited without any damage from any ethics and legal principle；2. umbilical cord is protected by placental barrier, its component is sick
Poison, the probability of germ contamination are low；3. umbilical cord MSCSImmunogenicity it is lower, the HLA distribution type being resistant to a greater extent is not inconsistent；④
Umbilical cord includes abundant stem cell；5. umbilical cord MSCSThe external doubling time is short, and cloning efficiency is high, and amplification ability is strong.Therefore, navel
Band is used as MSCSSource, had broad application prospects in gene therapy.
There is MSC the innate immunity to exempt characteristic, the potential broken up to the ability and polyphyly of tumor cell migration, become
A kind of antitumor biological agent.Tumor necrosis factor superfamily member LIGHT is a kind of very strong immuno-stimulator, its
While inducing apoptosis of tumour cell, can also costimulation T cell activation, have potential oncotherapy value.By LIGHT genes
MSC is transferred to, LIGHT is transported to tumor tissues by MSC, and the MSC of LIGHT modifications can be by producing strong antitumor cell
And tumour correlation interstitial cell immune response and change tolerance status of the host to tumour, so as to effectively control tumour.This
Invention can be prepared on a large scale, for the treatment of a variety of solid malignants and cancer prevention.
Brief description of the drawings
Fig. 1 shows the positive test symbol of MSC-L cells expression LIGHT；
Fig. 2 represents the positive-selecting result of MSC-L cells expression LIGHT；
Fig. 3 is MSC-L cellular morphologies figure and its cell state figure under 10 × times mirror；
Fig. 4 represents the testing result of MSC-L cell surface expressions；
Fig. 5 represents MSC-L anti-tumor experiment results in humanization Mice Body；
Fig. 6 represents MSC-L interior anti-tumor experiment results in B6 Mice Bodies.
Embodiment
A kind of preparation method for the mescenchymal stem cell for expressing immuno-stimulator LIGHT of provider of the present invention, it includes
Following steps：1) separating mesenchymal stem cell, and carry out original cuiture and secondary culture；2) structure is inverse with LIGHT genes
Retroviral and slow virus；3) LIGHT retrovirus or slow virus are transferred to MSCs, build MSC-L cells and to its surface
Expression is detected；4) antitumous effect experiment is carried out to the MSC-L cells of preparation.Prepared the invention further relates to the above method
MSC-L cells and its application.
With reference to the accompanying drawings and examples, the embodiment of the present invention is further described.Following embodiments are only
For clearly illustrating technical scheme, and it is not intended to limit the protection scope of the present invention and limits the scope of the invention.
Embodiment 1
The present embodiment is source using umbilical cord as MSCs, prepares a kind of express between people source immuno-stimulator LIGHT
The preparation method of mesenchymal stem cells, as depicted in figs. 1 and 2, it is comprised the following steps that：
(1) adherent screening method separating mesenchymal stem cell is used, and carries out original cuiture and secondary culture, it includes following
Step：
A) in vitro umbilical cord is submerged in PBS or physiological saline and is cut to 0.25- with normal saline flushing to no clot
0.35cm3 fragments, 2500-3000rpm centrifugations 1min obtain umbilical cord tissue block precipitation, abandon supernatant；
B) II isometric Collagenase Type is added to umbilical cord tissue block precipitation, piping and druming is uniform, and sealing is placed in 37 DEG C of shaking table 20-
40min, stands, liquid-transfering gun is collected the II Collagen Type VI enzyme liquid containing MSC and is transferred in MEM nutrient solutions, 2500- after blowing and beating uniformly
3000rpm centrifuges 3min, abandons supernatant, and cell is resuspended with complete medium, is placed in 37 DEG C, 5%CO2 incubators, remaining umbilical cord group
Knit block and be transferred to step c)；
C) isometric mixing enzyme solutions are added to remaining umbilical cord tissue block and carries out secondary digestion 20-40min, to umbilical cord group
Block residue 30-40% volumes to be knitted, are transferred in the lump in the complete medium in step b), piping and druming is uniform, with 0.5~1 ×
106cells/cm2Density kind in T75 blake bottles, 37 DEG C, 5%CO2Incubator 36-48h, wherein mixing enzyme solutions are by 0.05-
0.1% II Collagenase Type solution and 0.05-0.1% Type I collagen enzyme solutions are with volume ratio 6-8：2-4 uniformly mixes composition；
D) old culture medium is absorbed, is changed to containing 10%FBS, 1% dual anti-, 30,000 U unit gentamicins improved culture mediums
Continue to cultivate 48-72h；
E) gently rock blake bottle and remove tissue and floating cells, after PBS or physiological saline rinse again, add tryptose
37 DEG C of digestion of enzyme add improved culture medium and terminate digestion, suction pipe piping and druming blake bottle bottom, is transferred to 70~80% cell detachments
Centrifuge tube, 2500-3000rpm centrifugation 3-5min, abandons supernatant, is resuspended and counts, according to 3000~5000cells/cm2Density kind
Passed in blake bottle；
F) liquid is changed once within every 3 days, culture treats that cell length to 70~80% degrees of fusion, repeat step (5), passage is expanded for 3-5 days
Increase to the 5th or the 6th generation, you can obtain enough mesenchymal cells and be used to test or freeze.
(2) retrovirus and slow virus of the structure with LIGHT genes；
1) structure of reverse transcription and slow virus carrier pMIGR3-hLIGHT, pCDHEF-hLIGHT plasmid；First from people
RNA is extracted in PBMC, cDNA is obtained using reverse transcription reagent box, designs primer, EcoRI and BamHI enzymes are introduced respectively at both ends
Enzyme site, while kozak sequences are introduced at 5 ' ends, PCR amplification obtains people's light full-length genes, EcoRI and BamHI digestions, lead to
Cross T4 ligases and be building up to pMIGR3 retroviral vectors and pCDHEF slow virus carriers, carrier is correct by sequence verification.
PMIGR3-blasticidin, pMIGR3-puro, pCDHEF- are cloned into respectively again by correct fragment is sequenced
Blasticidin, pCDHEF-puro carrier obtain the expression plasmid for having selection antibiotic；
2) the retrovirus production of hLIGHT is expressed：By retroviral plasmid pMIGR3-hLIGHT (or corresponding band choosings
Select the plasmid of antibiotic) and packaging plasmid pCL-Ampho according to 1:1 quality is than cotransfection 293X cells, and 48 after transfection, 72 is small
When collect viral supernatants, with 0.45uM filters filter, packing be stored in -80 degree；
3) lentivirus production of hLIGHT is expressed：By slow virus plasmid pCDHEF-hLIGHT (or corresponding band selection antibiotic
Plasmid) and packaging plasmid VSV-g, RSV-REV, pMDLg/p RRE according to 2:1:1:1 ratio cotransfection to 293X cells,
48 after transfection, 72 collect viral supernatants when small, are filtered with 0.45uM interest rates device, and packing is stored in -80 degree；
(3) retrovirus with LIGHT genes will be prepared in step (2) or slow virus is transferred to step (1) acquisition
Mescenchymal stem cell, build MSC-L cells；
A) LIGHT is imported into MSC：Go 2 × 105MSC cells add 1ml fresh cultures in 6 orifice plates, add above-mentioned
1.5ml retrovirus or slow virus, add polybrene to final concentration of 10ug/ml, when 16-24 is small after discard in culture
Clearly, fresh culture is added；
B) expression of hLIGHT in MSC is detected：Metainfective cell is digested using pancreatin and control obtains for infection cell
Single cell suspension, takes 1 × 105Cell is resuspended to 100ul FACS dye solutions, adds 0.2ugLIGHT antibody, 4 degree of incubations
30 minutes, 0.5mlFACS dye solutions are added, 1500RPM is centrifuged 3 minutes, supernatant discarding, repeats to add 0.5mlFACS dyes
Color buffer solution, 1500RPM are centrifuged 3 minutes, and supernatant discarding, cell is resuspended to 100ul FACS dye solutions, add 50ng
Anti-hFc-APC antibody, 4 degree are incubated 30 minutes, add 0.5mlFACS dye solutions, and 1500RPM is centrifuged 3 minutes, discarded
Supernatant, is resuspended to 300ulFACS dye solutions, positive (APC passages) using Cytoflex flow cytomeries LIGHT
MSC cells, its testing result are as shown in Figure 1；
C) enrichment of MSC positive cells：After metainfective cell is dyed by the above method, sorted by fluidic cell
Obtain being more than 95%LIGHT positive cells；Or obtain being more than 95%LIGHT expression cells by antibiotic-screening, its sort or
The selection result is as shown in Figure 2.
Embodiment 2
The present embodiment is the research of the inside and outside migration characteristic of the MSC-L cells prepared.
The present embodiment has carried out measure of the LIGHT in MSC-L cell surface expressions, it specifically includes following steps：
(1) MSC-L is collected, cell total amount is less than 106；
(2) 1700rpm centrifuges 3min, and is resuspended with FACS buffer solution.Then this step is repeated once.
(3) 50U 2.4G2 are added, are incubated at room temperature 1-10min.LT β R-Ig lug are added, are incubated at room temperature 15-30min.
(4) 1 milliliter of FACS buffer solution is added, is gently mixed, 1700rpm centrifugations, abandon supernatant.
(5) anti-mouse IgG2-PE lug are added, are incubated at room temperature 15-20min.
(6) FACS buffer solution washing, 1700rpm centrifugations are added.
(7) supernatant is abandoned.Add 300ulFACS and gently mix sentence.Upper machine analysis.
As shown in Figure 3 and Figure 4, it is the result shows that LIGHT can be effectively in MSCs surface expressions for the flow cytometer detection result；
The MSC cells of LIGHT are expressed, appoints the characteristic for so possessing mescenchymal stem cell, still there is very strong propagation and differentiation capability, its
Form still keeps fusiformis under light microscopic；Cell surface expression CD73, CD90, CD105 (flow cytometer detection>95%), do not express CD45,
CD34, CD14 or CD11b, CD79 α or CD19, HLA-DR (flow cytometer detection≤2%)；MSC-L possesses into fat Osteoblast Differentiation ability.
The present embodiment has also carried out external migration in MSCs and MSC-L bodies and has tested, it includes：
1.Transwell is tested
(1) the TUBO cells of 0.7ml are pressed 1 × 10 in the previous day5The density of/ml is seeded to the lower room of Transwell, and 37
DEG C 5% CO2gas incubator is incubated overnight；
(2) the next morning, 37 DEG C of water-bath warm bath BSA liquid and complete medium；
(3) pancreatin of the MSC and MSC-L containing EDTA is digested, is neutralized with the complete medium of 5-10ml；
(4) 1000rpm is centrifuged 5 minutes, is abandoned supernatant, is washed once with BSA liquid, and it is 1 to adjust cell density with BSA liquid after resuspension
×105/ ml, each Transwell holes add about 0.5ml.Pay attention to：When Transwell cells are added 24 orifice plate,
Transwell films and the nutrient solution of lower room is kept to be in contact；
(5) by Transwell plates be placed on 37 DEG C 5% CO2gas incubator be incubated 4-5 it is small when；
(6) Transwell cells are taken out, be inverted, gently immersed in PBS for several times, remove the cell not attached；
(7) 10% formalin is then immersed in, 10 minutes is fixed, is washed once with PBS；
(8) HE is dyed 30 minutes, or overnight.Then gently washed several times with water, make clear background；
(9) cell not migrated is gently removed , Liao with cotton swab to do；
(10) film is scaled off along around cell with blade, be placed in downward on slide, dripping confining liquid makes its covering；
(11) number cell under high power lens, the general 5-10 high power field of view of number.
2. vivo migration is tested
(1) separating digesting TUBO, by 4 × 105Cell is resuspended in 200ulPBS, and the left veutro for injecting mouse is subcutaneous；
When tumour length to 3 × 3mm, injection 1 × 10 after (2) 10 days6With the MSC-L that PKH26 is marked to the right veutro skin of mouse
Under；
(3) after two weeks, tumor tissues are taken out, whether living imaging system observation MSC-L can migrate to tumor locus；
(4) then, then by tumor tissues frozen section (5-10um) is made, fluorescence microscopy Microscopic observation MSC-L is in tumor group
The position knitted.
By observing the inside and outside migration characteristic of MSC-L, show that MSC-L has the relatively special chemotactic to tumor tissues
Ability, LIGHT can be targeted input tumour by MSCs, and LIGHT has not significant impact the external transfer ability of MSCs,
MSC-L moves to the periphery that tumor tissues are predominantly located at tumor tissues afterwards, is conducive to the antineoplastic immune of immune cell activated
Act on and then suppress tumour growth.
Embodiment 3
The present embodiment is the research of the antitumor action of the MSC-L cells of preparation, it includes following experimental procedure：
1) MSC-L can control tumour growth with efficient targeting
1.1) the preventative antitumor models of MSC-L
(1) cell is collected when MSCs and MSC-L long is to 70% degree of converging；
(2) washed one time, counted with PBS, adjustment cell to suitable concentration；
(3) by the 1 × 10 of the PBS of 200ul or 200ul6MSC and MSC-L subcutaneous vaccinations are on the left of BALB/c mouse；
After (4) 2 weeks, 200u14 × 10 are inoculated with right side of mice5TUBO cells；
(5) hereafter every the growing state of 2-3 days observation tumours.
1.2) MSC-L therapeutic anti-tumours model
(1) by the 4 × 10 of 200ul5TUBO cell inoculations are subcutaneous to mouse left side；
After (2) 7 days, the MSCs and MSC-L of the long cell to 70% degree of converging are collected, is washed, is counted, adjustment cell arrives
Suitable concentration.
(3) by the 1 × 10 of the PBS of 200ul or 200ul6MSC-L subcutaneous vaccinations are subcutaneous to right side of mice；
(4) tumour growth situation was observed every 2-3 days.
Above-mentioned experimental result effectively can prevent and suppress tumour growth for MSC-L cells.
2) MSC-L controls the antineoplastic immune that tumour growth depends on LIGHT to activate
Carried out using the preventative antitumor models of the MSC-L of above-mentioned preparation and MSC-L therapeutic anti-tumours model thin in vivo
Born of the same parents knock out and the experiment of LIGHT activity blocks.
2.1) production and purifying of anti-CD4 (GK1.5) and CD8 (2.43) T cell antibody
(1) Pristane of 0.3ml is injected into 6-8 weeks nude mice abdominal cavity；
(2) 200 microlitres of 2X10 are injected after 7-10 days6Hybridoma (GK1.5 and 2.43)；
After (3) 7 days, ascites situation is observed daily；
(4) after ascites grows, abdominal cavity is inserted into 16-18 syringe needles, takes ascites；
(5) ascites 4000rpm is centrifuged 30 minutes, takes supernatant.
(6) antibody purification, packing storage.
2.2) experiment is knocked out in cd4 t cell and cd8 t cell body
(1) it is cd4 t cell in knock-out mice body, anti-CD 4 antibodies (GKl.S) 500ug is injected into mouse peritoneal, continuous 3
My god.
(2) it is cd8 t cell in knock-out mice body, anti-CD8 antibody (2.43) 500ug is injected into mouse peritoneal, continuous 3
My god.
(3) situation is knocked out by flow cytomery lymphocyte.
2.3) blocking experiment in LIGHT active bodies
In order to block LIGHT activity in Mice Body, the LT β R-Ig injection mouse peritoneals of 100ug, connect note 3 days.
2.3.1) frozen section embedding
(1) keep the tissue to be embedded fresh as far as possible, the fragment of tissue of bloodstain and onrelevant is washed with PBS and is abandoned, and uses paper
Towel sops up excessive moisture；
(2) OCT is added dropwise to embedding mould, tissue is placed in mould, make to be adhered to mould bottom；
(3) the OCT coverings tissue to be embedded is continuously added；
(4) aluminium foil is gently contacted into liquid nitrogen, makes gradually to freeze whole module.It is careful not to too soon, no person can burst；
(5) and then freezing microtome section is transferred to, or -8 DEG C preserve.
2.3.2) immunohistochemistry
(1) tumor tissues are collected, it are fixed with 10% formalin, paraffin embedding, and make HE dyeing；
(2) it is detection tumor proliferation, does Ki67 dyeing；
(3) situation, by specification operation TUNEL are died for detection tumour tune；
(4) it is detection Tumor angiogenesis situation and tumor vaccine cells distribution, tumor tissues is embedded with OCT；
(5) frozen section is done, with the corresponding T lymphocytes distribution situation of antibody test of the anti-CD4 and CD8 of PE couplings, is used
The 1 antibody test Tumor angiogenesis situation of AntiCD3 McAb of biotinylated goat anti-mouse.
2.3.3) ELISPOT detects y-IFN operating procedures
Coating (operates) in super-clean bench：
(1) coated antibody and 70% ethanol solution are prepared；
(2) ethanol of 100ul70% is added into PVDF plate holes, is incubated at room temperature 2-10 minutes；
(3) remove ethanol, washed 4 times with the sterile PBS of 130ul；
(4) 100ul coated antibodies are added；
(5) PVDF plates are moved into 4 DEG C of overnight incubations；
Block non-specific binding and cell activation (being operated in super-clean bench)：
(1) remove coated antibody, washed once with sterile PBS；
(2) the sterile blocking buffer solutions of 150ul are added, when incubation at room temperature 2 is small；
(3) remove blocking buffer solution, washed 4 times with sterile PBS 130ul/ holes；
(4) reacting cells suspension 4xl0 is prepared6/ ml, 100ul is added per hole, pays attention to being added to hole center；
(5) prepare antigen or antigen presenting cell, diluted with ELISPOT medium.When by the use of tumour cell as anti-
Original, by tumour cell:Lymphocyte 1:10 not amount adds.Add 2 times of concentration in 100ul/ holes antigenic solution or 4 ×
105The tumour cell of/ml.Control group, adds the culture that 100ul ELISPOT nutrient solutions or 100ul contain 4ug/ml ConA
Liquid.
(6) 37 DEG C of 5%CO2When incubator incubation 24 is small, it is careful not to mobile or tilts.
Detection：
(1) cell is discarded, by PVDF plate left-hand threads on paper handkerchief；
(2) 150ul lavation buffer solutions are added, 4 DEG C are incubated 10 minutes；
(3) detection antibody is prepared；
(4) washed 3 times with 150ul lavation buffer solutions；
(5) 100ul detection antibody is added, when incubation at room temperature 2 is small；
(6) detection antibody is discarded, is washed 4 times with the washcoated buffer solutions of 150ul；
(7) 100ul streptavidin-HRP are added, when incubation at room temperature 2 is small；
(8) streptavidin-HRP is discarded, is washed 3 times with 150ul lavation buffer solutions；
(9) by plate left-hand thread on paper handkerchief；
(10) 50ulAEC substrates are added per hole；
(11) it is incubated at room temperature 5 minutes；
(12) washed 6-7 times with miHQ, until clear background；
(13) plate is buckled on paper handkerchief；
(14) lucifuge is secretly done, and is counted.
This step is further included verifies that MSC-L can cooperate with tumor vaccine to remove the related of tumour and try by normal experiment means
Test step, including the amplification and purifying of adenovirus vaccine, the extensive amplification of last wheel and cesium chloride density gradient centrifugation are pure
Change, indirect cell ferment connection immunization experiment (indirect cellular ELISA) detects the anti-MSC antibody of mice serum, passes through stream
The special cracking MSCs effects of the monochromatic detection lymphocyte of formula cell instrument.
It can be drawn by above-mentioned experiment：The antitumor action of MSC-L is primarily not by suppressing tumor proliferation, angiogenesis
And promote the approach such as to die tumour week, and it is to rely on the activation of LIGHT signal paths.MSC-L can not only activate specificity
Antitumor cell it is immune, but also the immune of antitumor interstitial cell can be activated.As it can be seen that MSC-L can be by effective
Destroy the microenvironment that tumour is depended on for existence and reach effective antitumor effect, therefore MSC-L and tumor vaccine combination have well
Synergistic antitumor acts on.
Embodiment 4
The present embodiment has carried out real into knurl in humanization Mice Body respectively in order to verify in MSC-L bodies into the possibility of knurl
Test and tested with B6 Mice Bodies into knurl.Both test procedure difference are as follows：
1.MSC-L is tested in humanization Mice Body into knurl；
By 1x106Raji tumor cell injections inject 1x10 on the 11st day to immune deficiency NSG mouse6PBMC, the 12nd day use
1x106Control MSC and MSC-hLIGHT is subcutaneously injected into around tumour, measures a tumor size, record tumour life every three days
Long curve；The results are shown in Figure 5 for its experiment.
2.MSC-L is tested in B6 Mice Bodies into knurl；
By 1x106B16-OVA tumor cell injections perfect B6 mouse to immune, and the 10th day by 1x106Compare MSC and MSC-
HLIGHT is subcutaneously injected into around tumour, measures a tumor size every three days, records tumor growth curve；Its experimental result is such as
Shown in Fig. 6.
As shown in Figure 5, in being tested in MSC-L humanizations Mice Body into knurl, no an example mouse grows lung knurl, it will be appreciated from fig. 6 that
In being tested in B6 Mice Bodies into knurl, gross tumor volume increasess slowly, should be the result shows that MSC-L can keep stable multiplication characteristic.Cause
This, MSC-L specific can be migrated also like MSCs is the same to tumor tissues, it will be used as a suitable carrier, LIGHT is targeted
Tumor tissues are conveyed into, so that specific evoke antineoplastic immune, and MSC-L has very strong multiplication capacity.Therefore,
LIGHT genes can be also incremented by because of the propagation of MSC-L, this will make local anti-lung knurl be immunized to obtain a degree of amplification
Effect.
Embodiment 5
The present embodiment is the clinical research of the MSC-L cells prepared.
Patient, recurs after the full resection operation of carcinoma of urinary bladder, and larger transfer occurs in pelvic cavity periphery recurrent tumor infiltration lesion, rectum
Stove, and show Bone tumour.Row rectum lesion resection is performed the operation, and coordinates MSC-L cells to feed back 10 times after operation, each midfeather 1
Week, MSC-L cell quantities are 2 × 107/ time, and combine a variety of treatments such as immunocyte, PD1 antibody.
After MSC-L cells are fed back, there are 38.5 DEG C or so low-heat in patient, 24 it is interior when small voluntarily alleviate, it is bad anti-without other
Should.
After treatment more than April, patient recovers good, and vital sign is normal, and the development of PET bellies shows multiple primary lymphedema after peritonaeum
Section, but FDG metabolism is not high, and pelvic cavity etc. has no that radioactive uptake increases stove extremely.
The embodiment shows, MSC-L cells prepared by the present invention have patient certain clinical efficacy, and side reaction compared with
It is small, it can effectively control tumour.
The specific embodiment of the present invention is described in detail above, but it is only used as example, and the present invention is not intended to limit
In particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and to replace
In generation, is also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and repair
Change, all should be contained within the scope of the invention.
Claims (10)
1. a kind of preparation method for the mescenchymal stem cell for expressing people source immuno-stimulator LIGHT, it is characterised in that including such as
Lower step：
(1) separating mesenchymal stem cell, and carry out original cuiture and secondary culture；
(2) retrovirus and slow virus of the structure with LIGHT genes；
(3) retrovirus with LIGHT genes or slow virus prepared step (2) fills between being transferred to step (1) acquisition
Matter stem cell, builds MSC-L cells.
2. preparation method according to claim 1, it is characterised in that the separating mesenchymal stem cell in the step (1)
Separation method include adherent screening method, enzyme digestion and airflow classification carried out according to the relatively special phenotype of cell or is exempted from
Epidemic disease magnetic bead sorting.
3. preparation method according to claim 1, it is characterised in that the mescenchymal stem cell of the step (1) is source
In the mescenchymal stem cell of marrow, peripheral blood, cancellous bone, adipose tissue, bleeding of the umbilicus or umbilical cord.
4. preparation method according to claim 3, it is characterised in that the mescenchymal stem cell of the step (1) is source
In bleeding of the umbilicus or the mescenchymal stem cell of umbilical cord.
5. preparation method according to claim 1, it is characterised in that step (2) structure is inverse with LIGHT genes
The step of Retroviral and slow virus, includes：
1) retrovirus and slow virus carrier pMIGR3-hLIGHT, pCDHEF-hLIGHT plasmid are built, and acquisition has selection
The expression plasmid of antibiotic；
2) by the pMIGR3-hLIGHT plasmids of step 1) structure or corresponding plasmid and packaging plasmid pCL- with selection antibiotic
Ampho is transfected to 293X cells, the retrovirus of production expression hLIGHT；
3) by the pCDHEF-hLIGHT plasmids of step 1) structure or corresponding band select antibiotic plasmid and packaging plasmid VSV-g,
RSV-REV, pMDL g/p RRE cotransfections to 293X cells, the slow virus of production expression hLIGHT.
6. preparation method according to claim 1, it is characterised in that in the step (3) the step of structure MSC-L cells
Including：
A) LIGHT is imported into mescenchymal stem cell：The mescenchymal stem cell that step (1) obtains is placed in culture medium, adds step
Suddenly the retrovirus with LIGHT genes or slow virus that prepared by (2), are cultivated, obtain the mesenchyma after infecting LIGHT
Stem cell；
B) expression of hLIGHT in MSC is detected：Using the infection obtained in dye solution and LIGHT antibody incubation steps a)
Mescenchymal stem cell after LIGHT, and detect LIGHT positive MSC cells；
C) enrichment of MSC positive cells：Mescenchymal stem cell of the sorting more than 95%LIGHT positive cells.
7. a kind of expression people source immuno-stimulator LIGHT's prepared according to method according to any one of claims 1 to 6
Mescenchymal stem cell.
A kind of 8. preparation of the mescenchymal stem cell of the expression people source immuno-stimulator LIGHT containing described in claim 7.
9. preparation according to claim 8, it is characterised in that the preparation further includes medicinal diluent or excipient.
10. a kind of mescenchymal stem cell of expression people source immuno-stimulator LIGHT as claimed in claim 7 is preparing treatment
Or the application in prevention tumour medicine.
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