CN110373428A - A kind of transgene carrier system and its application promoting cell transplantation and gene expression - Google Patents
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Abstract
The present invention discloses a kind of transgene carrier system and its application for promoting cell transplantation and gene expression.The system includes screening-gene system, destination gene expression system and the carrier for carrying above-mentioned two system, and wherein screening-gene system is the silencing system for the gene for inhibiting division or promoting cell death.Compared with prior art, the present invention whether not there is growth vigor to be limited by target gene first；Secondly screening had both occurred to also occur in noble cells level in stem cell levels, therefore genetically modified cell stable all the life can keep or accelerate；The influence that drug toxicity is avoided using drug screening is not needed again；Only inhibit the mutant of division or promotion cell death because having used again, just not carcinogenic risk.
Description
Original application date: 2016-10-08, original applying number: 201610877184.9
A kind of former patent name: transgene carrier system and its application promoting cell transplantation and gene expression
Technical field
The present invention relates to a kind of transgene carrier systems, can especially promote the carrier system of cell transplantation and gene expression
System.
Background technique
Gene therapy establishes the animal bioreactor for being based on adult (dry) cell or animal model needs in receptor disease
There are stable destination gene expression amount and stable destination gene expression positive cell quantity in human or animal's body.But in reality
In but generally occur in individual cells express target gene efficiency decline and express target gene cell proportion decline.
The approach for solving drawbacks described above is to be screened in vivo.Have the method screened in three classes body at present: 1) target gene, which has, makes place
Chief cell persistently divides and promotes the ability of survival, thus in the case where any screening pressure of no application in naturally-occurring body
Screening, but the characteristic of this target gene is not common, that is, and not all target gene all have host can be made thin
Born of the same parents persistently divide and promote the ability of survival；2) in addition to target gene, then a screening-gene is imported simultaneously, which makes host
Cell has the ability (so-called cell grows switch cell growth switches) of the promotion survival of pharmacological dependence, but
This method shows as the cellular level that screening only occurs in short term survival on big animal, so that it cannot persistently keeping enough
The cell of target gene is expressed, therefore limits its use；3) in addition to target gene, then a resistance screening pressure is imported simultaneously
The screening-gene of (usually toxic medicament) causes drug only to remove the cell without gene modification without removing gene modification
Cell, but the system hinders its application because of drug toxicity.4) when use the gene with growth vigor as screening base
Because when, have lead to tumorigenic possibility.If current most study is HOXB4 gene, it is just found to have carcinogenic risk.
Up to the present, can overcome the internal screening system of drawbacks described above yet there are no.
Summary of the invention
It is an object of the invention in view of the deficiencies of the prior art, provide it is a kind of both can be carried out nature screen in vivo thus
The use for avoiding screening drug can be such that screening had not only occurred in stem cell levels again but also the screening system in noble cells level occurs
System can make target gene have the cell number of stable gene expression amount and stable expression target gene in host
Amount, animal bioreactor or animal model for gene therapy, cell therapy, production based on adult (dry) cell provide new
Platform.
Screening system of the present invention includes screening-gene system, destination gene expression system and above-mentioned two system of carrying
Carrier, screening-gene system be promote cell division or promote cell survival gene or its mutant expression system, or
For the silencing system for inhibiting the gene for dividing or promoting cell death, non-induced type, induction type and conditional gene can be divided into and struck
Except type.
The expression system of the gene for promoting cell division or promoting cell survival and its mutant, including starting
Son promotes cell division or promotes the gene or its mutant sequence, transcription terminator of cell survival.
The described gene for promoting cell division or promoting cell survival and its mutant sequence be Survivin, BCL2,
The genes such as BCLxL, MCL1, BCL-W, A1, Boo/DIVA, BCL2L13, BCL2L12 and its mutant sequence.
The bcl2 mutant sequence is the mutant nucleotide sequence that cannot promote cell tumorigenesis, including AAA, S70A,
The mutant such as S69AS70A, S70AT87A, Y28A.These mutant are because remaining anti-apoptotic ability but being reduction of to DNA
The inhibition of repair ability does not influence P53 and induces cell apoptosis and/or eliminate to keep c-Myc stable and promote tumorigenic
Ability, thus can play make to be transferred to the gene cell there is survival advantage in vivo but be unlikely to cause tumour generation,
So being the fabulous candidate of internal screening-gene.
The bcl2 mutant sequence is the BCL2 mutant of the anti-apoptotic ability with enhancing, including EEE, S70E etc.
Mutant.
The gene silencing system of the inhibition division or promotion cell death, including promoter, inhibition divide or promote
The gene order of cell death is the siRNA or shRNA of target sequence or the DNA template sequence and tanscription termination sequence of miRNA
Column；Wherein the DNA template sequence of miRNA can also be inserted in the 3 ' ends or 5 ' ends of target gene in destination gene expression box.This is heavy
Silent system can be the single target spot of individual gene or the multiple target spot silencings of individual gene, be also possible to multiple genes silencing simultaneously.
The inhibition division promotes the gene order of cell death for the rush apoptosis of bcl2 family and other families
Gene order, including Bax, Bak, Bok/Mtd, Bcl-xs, Bcl-g/Bcl2L14, Bfk/Bcl2L15, Bid, Bad, Bik/
The gene orders such as Nbk, Hrk, Bim/Bod, Bmf, Mule/ARF-BP3, Nix/Bnip3, Puma, Noxa.
The induction type screening-gene system, promoter therein are inducible promoter.
The conditional gene knockout type screening-gene system, screening-gene system two sides include that conditional gene strikes
The particular sequence (such as LOXP or FRT sequence) removed, so that conditionity removes screening-gene system.
Above-mentioned screening-gene system can guarantee that target gene system is expressed to high-efficient and lasting in target cell or target organ.
The carrier is viral vectors and non-virus carrier, and wherein viral vectors includes retrovirus, slow virus, gland
One of virus, adeno-associated virus (adeno-associated virus, AAV) etc..
The present invention by the introducing of screening-gene system, using be overexpressed promote cell division or promote cell survival gene or
Fall to inhibit division using silencing or promote cell death, so that turning have the cell of screening-gene system Natural Selection process in vivo
Middle acquisition advantage, so that turning there is the cell of screening-gene system to obtain stable quantity, while it is also possible that with screening base
Because the expression of (therapeutic) target gene of coupling expression increases, make gene, cell therapy and moving based on adult (dry) cell
The production of object bioreactor or animal model becomes easy success.If using the sieve of induction type and conditional gene knockout type
Genic system is selected, then can be eliminated after the completion of screening caused by the overexpression or silencing of screening-gene to the permanent of host cell
It influences.
It is a further object to provide above system, production animal bioreactor, production humanization mouse,
Application in gene therapy, cell therapy.
Compared with prior art, the present invention possessed advantage, feature or good effect, illustrate the defect of the prior art:
As described in the background art, defect of the existing technology mainly has: 1) when target gene that is therapeutic or being transferred to
When itself has growth or survival advantage, it can make to obtain advantage in the spontaneous competition in vivo of host cell and have cell in vivo
Stable quantity, but having the target gene of growth or survival advantage so after all is minority after all, when target gene does not have
When standby such ability, it is possible to stable genetically modified cell quantity cannot be obtained in vivo, so that gene, cell be made to control
It treats or animal bioreactor or animal model of the production based on adult (dry) cell fails；2) when the screening-gene of importing is energy
Make host cell that there is ability (the so-called cell growth switch cell growth of the promotion survival of pharmacological dependence
Switches) when gene, experiment discovery above shows as the cellular level that screening only occurs in short term survival in the application of big animal,
Namely there is no in stem cell levels, so that it cannot enough genetically modified cells are persistently kept, because short term survival is thin
Born of the same parents are non-stem cell, and self-renewal capacity is weak, therefore are easy to exhaust, when the short term survival cell depleting of gene modification
(exhaust) after, genetically modified cell just can not be obtained again, therefore limits its use；3) when one resistance screening pressure of importing
When the screening-gene of power (usually toxic medicament), the not cell by gene modification need to be removed using drug, but the system
Its application is hindered because of drug toxicity；4) when using the gene with growth vigor as screening drug, research at present is most
Mostly is HOXB4 gene, but is found to have carcinogenic risk.
Compared with prior art, the present invention whether not there is growth vigor to be limited by target gene first；Secondly it screens
Both occurred to also occur in noble cells level in stem cell levels, therefore genetically modified cell can stable holding all the life or increase number
Amount；The influence that drug toxicity is avoided using drug screening is not needed again；Only promote cell survival but not because having used again
Promote the mutant of cell mutation, just not carcinogenic risk, for example uses the mutant aaa or S70A etc. of bcl2.Therefore exist
There is very big answer in the production of gene, cell therapy and animal bioreactor or animal model based on adult (dry) cell
With value.
Detailed description of the invention
Fig. 1 is the retroviral vector MIGR1-S70A plasmid containing Bcl-2 mutant S70A used by embodiment 1
Schematic diagram, wherein each element is as follows:
Fig. 2 is the GFP positive cell that embodiment 1 transplants gene modification bone marrow cell mouse peripheral blood leucocyte and red blood cell
Percentage changes with time.Donor bone marrow cell GFP positive cell percentage, 1.5,2,2.5,6 generations before " preceding " representative is transplanted
1.5,2,2.5,6 months after table transplanting, using the data of three independent experiments；
Fig. 3 is the Feline Immunodeficiency disease of self inactivation used by embodiment 2 containing bcl2 mutant S70A
Viral (FIV) slow virus carrier plasmid schematic diagram, wherein each element is as follows:
Fig. 4 is the Recipient mice peripheral white blood cells GFP positive cell ratio of 5-FU Nonmyeloablative pretreatment in embodiment 3
Example.Recipient mice presses the 5-Fluorouracil intravenously administrable of 150 mg/kg weight for 5 days before transplantation, and transplanting is through reversing after 5 days
Record the Sca-1 positive bone marrow cells of virus infection, six months Flow cytometry peripheral white blood cells GFP positive cells after transplanting
Ratio.
Fig. 5 is that the GFP of transplanting gene modification bone marrow cell mouse peripheral blood leucocyte and red blood cell is positive thin in embodiment 4
Born of the same parents' percentage changes with time.Donor bone marrow cell GFP positive cell percentage before " preceding " representative is transplanted, 1.5,2,2.5,6
It represents after transplanting 1.5,2,2.5,6 months, using the data of three independent experiments.
Fig. 6 is the retroviral vector containing Bax miRNA and Bak miRNA expressed sequence used by embodiment 7
Plasmid schematic diagram, wherein each element is as follows:
| Element | Function |
| 5’LTR | Include retrovirus MSCV promoter, starting transcription |
| Ψ+ | Packaging signal |
| GFP | The cDNA of encoding reporter gene green fluorescent protein |
| miRNA(Bax) | For the miRNA template sequence of Bax |
| miRNA(Bak) | For the miRNA template sequence of Bak |
| 3’LTR | Tanscription termination |
| Ori | Replication origin in Col E1 |
| AMPr | Ampicillin resistance gene |
；
Fig. 7 is expression of the GFP albumen in red blood cell in 8 animal blood bioreactor of embodiment.The road A is MIGR1-
Bcl2 mouse red blood cell lysate electrophoresis road, the road B are control (uninfecting virus bone marrow transplanted mice) mouse red blood cell lysate
Electrophoresis road, the road M are molecular weight marker road.
Fig. 8 is pCDH-Bcl2-MSCV slow virus carrier plasmid schematic diagram used by embodiment 9, wherein each element is such as
Under:
Fig. 9 is inducing expression Bcl-2 mutant S70A of all elements on a carrier used by embodiment 11
FIV slow virus carrier plasmid schematic diagram, wherein each element is as follows:
Figure 10 be embodiment 11 in, by Dox handle 1.5 months after, peripheral white blood cells, bone marrow cell, lin-
sca1highckithighGFP positive cell rate in bone marrow cell (LSK cell), and after removing Dox 4.5 months, 8.5 months
Afterwards, peripheral blood GFP positive percentage.
Figure 11 is the GFP positive cell percentage that embodiment 12 transplants gene modification bone marrow cell mouse peripheral blood leucocyte
It changes with time.Donor bone marrow cell GFP positive cell percentage before " preceding " representative is transplanted, after 1.5,3,6 represent transplanting
1.5, percentage variation of 3,6 months gene modification blood cells after screening in vivo.
Specific embodiment
The present invention is further analyzed combined with specific embodiments below.
Internal screening system of the embodiment 1. using BCL2 mutant S70A as screening-gene
In the present embodiment 1, using the mutant S70A of BCL2 as screening-gene, while retroviral vector is utilized
MIGR1 is as carrier.As previously mentioned, S70A remains the anti-apoptotic ability of part BCL2, but eliminates it and promote tumor ability, because
This is particularly suitable as internal Natural Selection gene.
It (1) is MIGR1-S70A plasmid, as shown in Figure 1 before S70A being inserted into the IRES of MIGR1.
(2) Retroviral supernatant is prepared with MIGR1-S70A plasmid and incasing cells BOSC23.Specifically, will
Improvement Du Shi Eagle's medium (Dulbecco's Modified of the BOSC23 cell culture in 6 porocyte culture plates
Eagle's Medium, DMEM) complete medium (is added to 10% fetal calf serum, 100 units/ml penicillin, 100 μ g/ml chains
Mycin, 2mM L-Glutamine etc.) in (37 DEG C, 5 ﹪ CO2), when 90 ﹪ of cell converges, with containing 5 ﹪ FBS, be free of antibiotic
DMEM fresh culture replace old culture medium, every hole adds 1.5ml culture medium, is put into 37 DEG C, 5 ﹪ CO incubators are spare.It takes
Two 1.5ml centrifuge tubes, the 250 μ l of OPTI-MEM culture medium of preparatory 37 DEG C of heating is added in every pipe, then in a wherein Guan Zhongjia
Enter the MIGR1-S70A plasmid that 2.5 purified μ g are built, mixes, room temperature 5 minutes；It is added in another pipe simultaneously
2000 liposome of Lipofectamine, 5 μ l is mixed, room temperature 5 minutes.Two pipe contents are added together mixing, room temperature
It 20 minutes, is then added in cell culture well, jiggles uniformly, be subsequently placed in 37 DEG C, 5 ﹪ CO2Culture, with new after 6 hours
Fresh complete DMEM culture medium (is added to 10% fetal calf serum, 100 units/ml penicillin, 100 μ g/ml chains in DMEM culture medium
Mycin, 2mM L-Glutamine etc.) old culture medium is displaced, supernatant is collected after being further cultured for about 30 hours, 500g is centrifuged 5 points
Clock, takes supernatant, and -80 degree storages are spare.
(3) the conventional front and back thigh bone from male 8 week old C57BL/6 mouse takes out marrow, according to operation instruction Sca-1 magnetic
Pearl separating kit isolates Sca-1 positive bone marrow cells, and be incubated at added with 50 nanograms/milliliter stem cell factors (SCF),
20 nanograms/milliliter interleukin-13s, 50 nanograms/milliliter interleukin 6s, 15% fetal calf serum, 2 mM/ls of glutamine, 0.1 mmoles
The height sugar improvement Du Shi Eagle's medium of that/liter nonessential amino acid, 1% penicillin/streptomycin (100x, Gibco)
(Dulbecco's Modified Eagle's Medium, DMEM), with the Retroviral supernatant prepared after 48 hours
Virus infection is carried out, infection method is centrifugation infection method, i.e., adds the good disease containing 6 micrograms per millilitre polybrene in cell
Malicious supernatant, then 900g is centrifuged 45 minutes on centrifuge, carries out within continuous two days centrifugation infection twice.It is changed after second subinfection
The upper fresh complete medium containing cell factor, is cultivated 24 hours.Fraction flow cytometer is taken to measure GFP positive cell
Content expression, remaining is for transplanting.
(4) female 8 week old C57BL/6 mouse are irradiated by 9Gy 137Cs source radioactive ray before transplanting, are injected and are walked from tail vein
Suddenly the Sca-1 positive bone marrow cells that (3) have been infected.Recipient mice carries out nursing 1 month with antibiotic water after transplanting, then
It is fed with light water.
Tail vein takes peripheral white blood cells and red blood cell flow cytometer to measure 1.5 after transplanting, after 2,2.5,6 months
GFP positive cell.Compared with before transplanting, GFP positive cell ratio rises at any time, rises to 90% or so or more always
(see Fig. 2).(containing GFP gene but not having S70A gene) is compareed because the not expression of S70A, GFP positive cell ratio is at any time
Between and decline (see Fig. 2).These results illustrate that S70A can improve the ratio of genetically modified cell.Simultaneously compared with the control, it represents
The average fluorescent strength (MFI) of the GFP of transgene expression intensity is also that S70A mouse significantly improves than control mice, explanation
S70A also promotes the expression quantity of modifier.Valuable is the gene GFP that the expression of S70A mouse energy Final height is transferred to, therefore right
It is especially valuable in the gene therapy for needing persistent high efficiency to express transgene.
Then prove that gene modification occurs in candidate stem cell level by testing.It detects within first after the transfer 6 months
The marrow and peripheral blood GFP positive cell ratio of transplant recipient mouse, discovery GFP positive cell ratio donorcells before transplanting
44.52% rise in 93.15 ± 2.64% in bone marrow cell (p < 0.0001) or marrow and risen in LSK cell
(LSK cell is generally acknowledged primary hematopoietic cell, is that Lin is negative, Sca-1 is positive, c- for 83.68 ± 3.33% (p < 0.0001)
Kit positive cell)；And control mice GFP positive cell ratio drops to marrow from 31.36% transplanted in preceding donorcells
Drop to 0.39 ± 0.29% (p < 0.0001) in 5.39 ± 3.55% (p < 0.0001) or LSK cell in cell.?
The low explanation internal screening in marrow of GFP positive cell ratio ratio not only occurs on stem cell levels in LSK cell, also occurs
It is horizontal in the hemopoietic forebody cell that is breaking up, because the cell broken up needs to rely on anti-apoptotic molecule more come under surviving
Come, and stem cell is because be mostly in quiescent condition, their survival is lower to the dependence of anti-apoptotic molecule.Then we
It has carried out within 6 months after first set grafting second to transplant, has carried out full bone-marrow transplantation and GFP+LSK cell transplantation respectively.Knot
Contain 93.23 ± 2.33% and 99.54 ± 0.13% respectively in the Recipient mice peripheral red blood cells of fruit second of transplanting of discovery
GFP positive cell, illustrate that genetically modified cell can be stablized and be transferred in second of transplant recipient mouse.These result explanations
The internal screening not only occurs to also occur in noble cells level in candidate stem cell level.
S70A mouse is white other than spleen and peripheral white blood cells are overturned than compareing big and 1-2 times more, CD4/CD8 ratio
Various types of cells subgroup illustrates that S70A has no substantial effect on hematopoiesis compared with the control without changing significantly in cell.In addition to
Transplant recipient mouse and second of transplanting mouse are observed, and do not have to see the blood such as transplant recipient mouse leukaemia
System tumor disease illustrates that S70A is the screening-gene of a safety.
This system can make the cell of external source transgene express to persistent high efficiency the base being transferred in transplant recipient animal body
Cause, and vicious transformation will not also occur for transgenic cell, therefore be the ideal carrier system of gene therapy.
Embodiment 2. is based on inducible system and slow virus carrier using Bcl-2 mutant S70A as the internal of screening-gene
Screening system
Slow virus has the characteristic more safer than retrovirus.Therefore with itself inactivation type Feline Immunodeficiency virus
To repeat the research of embodiment 1 (Fig. 3, empty viral vectors come from SBI company).The incasing cells that system virus uses is that 293T is thin
Born of the same parents will also be transferred to another two packaging plasmid (pFIV-34N, pVSV-G) other than slow virus carrier in 293T cell.Slow disease
Malicious packing method, bone marrow cell obtain, virus infection and cell transplantation are the same as embodiment 1.
6 months after transplanting, S70A of the discovery based on slow virus can increase GFP positive cell ratio, but unlike reverse transcription
Viral S70A is such, and slow virus S70A can only increase GFP positive cell ratio to a lower level, and about 30% or so.
By comparing the fluorescence intensity level that GFP in transplant recipient mouse bone marrow cells is expressed, the donor mice bone of discovery slow virus S70A infection
The fluorescence intensity level (MFI) of GFP positive cell is 10.57 in myelocyte, and the donor of significantly lower than retroviral infection is small
The fluorescence intensity level 96.07 of GFP positive cell in mouse marrow, illustrates that the expression of slow virus S70A is significantly lower than retrovirus
The expression of S70A, that this may drive S70A and GFP expression in slow virus is internal promoter MSCV, and retrovirus
Promoter be retrovirus LTR promoter, the latter is strong than the former, thus result in slow virus S70A can only make GFP sun
Property cell proportion reaches 30% or so.Because the expression of S70A and GFP depends on being integrated into the virus in chromosomal DNA
Copy number and promoter intensity, therefore GFP cell proportion height should can be by using different promoter (including induction type
Promoter) and different virus infection times quantity (MOI) controlled.Based on this as a result, lasting and low-level S70A is expressed
Seem that the internal screening be necessary.Other results (including hematopoiesis influences, whether screening occurs in stem cell levels etc.) with
Retrovirus in embodiment 1 is similar.It is worth noting that (being observed for the first time and in second of transplant recipient mouse
6-24 months) do not find the hematopoietic system cancers disease such as leukaemia, it is safe for illustrating that this method is applied to gene therapy etc..
It is pre-processed before the non-clear marrow transplanting that embodiment 3. is handled by 5-FU
Transplant recipient patient tends not to the clear marrow of radiation exposure using lethal dose in clinical application, thus with drug into
The whether feasible non-clear marrow transplanting pre-treatment of row is very crucial.150 mgs/kg of bodies are injected to Recipient mice before transplantation
The 5-Fluorouracil (5-FU) of weight, transplanting find that the pretreatment is enough to make retrovirus MIGR1-S70A infection after 6 months
Bone marrow cell transplantation success (Fig. 4), therefore this method is very suitable to clinical application.
Internal screening system of the embodiment 4. using BCL2 mutant EEE as screening-gene, studies EEE mutant
Method is with embodiment 1, as a result also (Fig. 5) similar to Example 1, but finds the hair of the neoplastic hematologic disorders diseases such as leukaemia
It is raw.Illustrate EEE under certain condition and also can be used as the screening-gene of internal screening system.
The preparation of humanization mouse of the embodiment 5. based on CD34 cell
The preparation of MIGR1-S70A retrovirus is substantially with embodiment 1, and only BOSC23 Viral packaging cell changes into
PT67 incasing cells, remaining step is viral with the preparation of embodiment 1, and the virus prepared can infect human body cell.It prepares
Virus is saved backup in -80 DEG C.
Human cord blood comes from healthy donors, and mononuclearcell is obtained by Ficoll-Hyaque density centrifugation.CD34+ is thin
Born of the same parents are enriched with according to operation manual miniMACS (Miltenyi Biotec, Gladbach, Germany).1 × 10e5 is rich
The CD34+ cell culture of collection is in culture medium [DMEM in high glucose culture medium (Gibco, Grand Island, NY, USA), 15% tire ox
Serum (embryonic stem cell is dedicated, Gibco, Auckland, NZ), 2mM L-Glutamine (Gibco, Grand Island, NY,
), USA 0.1mM nonessential amino acid (Gibco, Grand Island, NY, USA), 1 μM of hydrocortisone, 0.1 μM of β-sulfydryl second
Alcohol, 1% penicillin/streptomycin (100x, Gibco, Grand Island, NY, USA)] in addition addition 100ng/ml stem cell
The factor (rhSCF, from R&D Systems Inc.), 100ng/mL Flt3 ligand (Flt3-L, from R&D Systems
), Inc. 50ng/mL thrombopoietin (Tpo, from R&D Systems Inc.).After culture 48 hours, cell PBS
Wash one time, be resuspended in the retrovirus S70A supernatant containing SCF, Flt3-L and Tpo again, be transferred to through
In 24 orifice plates of retronectin (Takara) cladding, 900g is centrifuged 45 minutes, is then incubated for 2 hours at 33 DEG C.Viral supernatants
It is cultivated 48 hours with the fresh culture medium containing SCF, Flt3-L and Tpo, then at 37 DEG C.Then cell uses pancreas enzyme -EDTA
Digestion takes fraction flow cytometer to detect GFP expression, remaining is for transplanting into immunodeficient mouse.
NSG (NOD-SCID IL-2 receptor gamma null) mouse more can be successfully moved than NOD/SCID mouse
Plant the mouse of mankind CD34 hematopoietic cell.8 week old NSG mouse are first through sublethal dose ray (2.4Gy；The source 137Cs) irradiation, so
Pass through CD34 cell of the 1 × 10e5 of tail vein injection through virus infection afterwards.10 weeks after transplanting, Flow cytometry transplants mouse
The content of people CD45 cell in peripheral blood.With control group (only expressing the mouse of the CD34 cell transplantation of the virus infection of GFP) phase
Reach 55.64 ± 4.37% than, S70A mouse source of people GFP positive cell (CD45+GFP+), total humanizing cells are up to 66.21
± 4.89%；And control mice humanization GFP positive cell be 9.68 ± 3.33%, total humanizing cells be 37.65 ±
2.79%, illustrate that S70A promotes humanizing cells' ratio of humanization mouse.
Embodiment 6. is based on the foundation of the humanization mouse of human peripheral blood single nucleus cell (PBMC)
The slow virus carrier that uses, virus preparation are the same as embodiment 2.
Peripheral blood is obtained from Healthy People, with PBS buffer solution by 1: 2 dilution, is laid on Ficoll separating liquid surface, 1 700 turns/
The cellular layer at separating liquid interface, as human peripheral blood single nucleus cell (PBMC), PBS washing 3 are drawn in minute centrifugation 30 minutes
It is secondary, it is resuspended in the slow virus supernatant containing 8 mcg/mls cohesion amine, is transferred to 6 through retronectin (Takara) cladding
In orifice plate, 900g be centrifuged 50 minutes, remove viral supernatants, with contain 15% fetal calf serum, 100 units per ml penicillin, 100
Mcg/ml streptomysin, 2mM L-Glutamine, 100 nanograms/milliliter interleukin-22s 1640 culture mediums be resuspended cell, culture 24
Hour, then superinfection 1 time is further cultured in 1640 culture mediums 24 hours, and flow cytometer carries out GFP positive cell point
Choosing.Human peripheral blood single nucleus cell 20 × 10e6/ of the 8 week old NSG mouse through the well-graded GFP positive of tail vein injection is only.It moves
It takes within 4 weeks after plant mouse peripheral blood to detect people CD45 cell, finds the CD45 positive cell of FIV-S70A mouse ratio FIV-GFP mouse
Ratio is by significantly improving (65.44 ± 5.35%vs 36.83 ± 3.95%, p < 0.01).
Embodiment 7. is to promote apoptogene as the internal screening system of screening-gene
In the present embodiment, Bax the and Bak gene expression by the way of expressing miRNA in silencing target cell.Structure first
Carrier is built up, each element arrangements are as shown in Figure 6.The cDNA sequence that Bax and Bak that ethnic group belongs to are obtained from GENEBANK, as setting
Count the template of miRNA.Selection goes out miRNA template sequence (including 5 ' using the Photographing On-line software design of invitrogen company
MiR flanking sequence, the antisense strand with BAX or bak gene complete complementary, the loop ring of 19 nucleotide, two nucleotide deletions
Positive-sense strand, followed by 3 ' miR flanking sequences).As shown in figure 5, the MSCV promoter in the LTR of retrovirus drives
GFP gene and the miRNA for BAX gene and the miRNA expression for bak gene.It is high in order to filter out silence efficiency
MiRNA template sequence transiently transfects HEK293 cell with the vector plasmid built, goes out the GFP positive through flow cytometric sorting
Cell expresses quilt with Bax the and Bak expression of western blot testing inspection cell with the Bax or Bak greater than 85%
The miRNA template sequence of inhibition is as effective miRNA template sequence, the miRNA mould of effective BAX and BAK that this is filtered out
Plate sequence entrusts the synthesis of DNA Synesis Company, and sequence series connection as shown in Figure 5 is inserted into the downstream of GFP, completes the building of carrier.
The preparation method is the same as that of Example 1 for virus, and only incasing cells changes PT67 cell by BOSC23, so as to packaged retrovirus energy
Infect human archeocyte, the virus prepared be stored in -80 DEG C it is spare.
The acquisition of human cord blood CD 34 cell, culture, virus infection, transplantation method are the same as embodiment 5.Compared with the control group,
The humanizing cells of BAX-BAK miRNA mouse are higher (70.43 ± 6.46%vs35.65 ± 4.13%), illustrate silencing BAX
Promote humanizing cells that there is internal competitive advantage with BAK, increases its ratio greatly.
The foundation of 8. animal blood bioreactor of embodiment
Mouse blood bioreactor is established with the MIGR1-Bcl2 plasmid and C57BL/6 mouse of embodiment 1.Method and
Process is the same as embodiment 1.
6 months after transplanting, red blood cell is isolated with Ficoll-paque density centrifugation.The cold PBS of the red blood cell isolated
Wash three times, be then resuspended in be added to protease inhibitors cell pyrolysis liquid (50mM Tris-HCl [pH 8.0], 0.5%
NP-40；1mM EDTA；150mM sodium chloride；10% glycerol；1mM sodium vanadate；50mM sodium fluoride；10mM sodium pyrophosphate；1mM β mercapto
Base ethyl alcohol) in, it is cracked at a temperature of 4 degree.Extracting solution is centrifuged 10 minutes through 14000 revs/min after cracking.It is centrifuged supernatant and carries out ten
Sodium dialkyl sulfate polyacrylamide gel electrophoresis.Gel is through coomassie brilliant blue staining, as a result, it has been found that the red blood cell of transplanting mouse
In have 1% GFP albumen (Fig. 7) of about hemoglobin content, explanation also quickly can successfully build up expression external source egg with the method
White animal blood bioreactor.
The foundation of 9. animal mammary gland bioreactor of embodiment
In order to establish galactophore biological reactor, using slow virus pCDH-MSCV (being purchased from SBI company), we are mountain herein
Sheep Bcl2 (wild type) gene is placed in the downstream T2A, and GFP is placed in the upstream T2A, while secretion signal is connected before GFP
(kappa secretory signal sequence: ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCAC T
GGTGACGGT), see Fig. 8, which is named as pCDH-Bcl2-MSCV.Virus preparation: 293T/17 cell inoculation is in 10 centimetres
In Tissue Culture Dish, the cells such as second day have 90% converge after with 2000 liposome of lipofectamine transiently transfect 18 micrograms
PCDH-Bcl2-MSCV and 12 microgram packaging plasmid mixed liquors.It (is trained in DMEM with fresh complete DMEM culture medium within 6 hours after transfection
Support in base and be added to 10% fetal calf serum, 100 units/ml penicillin, 100 μ g/ml streptomysins, 2mM L-Glutamine etc.) it sets
Old culture medium is changed, collects supernatant after being further cultured for about 30 hours, 500g is centrifuged 5 minutes, takes supernatant, -80 DEG C of storages are spare.
The polybrene (polybrene) of 80 mcg/mls is added in viral supernatants, with syringe (No. 22 syringe needles) from female
The first retrovirus for adding cohesion amine of goat dairy squeezes into goat mammary gland.Goat muscle at 1,3,5,7,9,11,13 day is infused
Penetrate the estradiol of 0.25 mg/kg weight and the progesterone of 0.75 mg/kg weight, virus liquid respectively injection hormone 3,
5,7,9,11,13 days injection mammary gland.Milk is collected after lactation, -80 storages are spare.
The expression quantity of the milk of collection GFP green fluorescent protein quantification kit detection GFP, initial 3 days as the result is shown
GFP expression it is higher, reach 102.34 ± 25.54 nanograms/milliliters, behind decrease but stablize within several days expression terminate to lactation,
Illustrate that mammary gland animal bioreactor is successfully built up.
Application of 10. present system of embodiment in adoptive immunotherapy tumour
The slow virus carrier that uses, virus preparation are the same as embodiment 2.
Using 8 week old health BALB/C female mices, 3% coloured glaze guanidine-acetic acid sodium 2 milliliters/mouse of culture medium is injected intraperitoneally, and 3 days
Afterwards, peritoneal macrophage is collected with 4 DEG C of D-Hanks liquid lavages, be suspended from containing 15% fetal calf serum, 100 units per ml moulds
Element, 100 mcg/ml streptomysins, 2mM L-Glutamine 1640 culture mediums in cultivate, after 3hr, wash away non-adherent cell,
Remaining attached cell mixes culture medium in above-mentioned 1640 culture medium and virus liquid 1:2 ratio, and 1000 units/milli is added
It is micro- to rise granulocyte-macrophage colony stimutaing factor (GM-CSF), the gamma interferon (IFN-γ) of 200 units per mls and 6
Grams per milliliter agglomerates amine, cultivates cell 48 hours, and centre changes the liquid once, and pancreatin digestion, it is positive thin that selected by flow cytometry apoptosis goes out GFP
Born of the same parents, it is spare.
BALB/C mice is inoculated 5 × 10e5 S180 tumour cell on the right side of stomach wall, thin in inoculated tumour after 6 hours
Born of the same parents position is subcutaneously injected into well-graded macrophage, every once three days, totally 3 times, and 1 × 10e6 cell every time.Observe tumour
Growing state, measure diameter of tumor, calculate gross tumor volume.Compared with the macrophage of infection FIV-GFP virus, FIV- is infected
The macrophage of S70A virus can effectively inhibit tumour growth (tumor control rate: 66.73 ± 9.53%vs 31.44 ±
7.37%, p < 0.05).
Induction screening system of the embodiment 11. using BCL2 mutant S70A as screening-gene
In order to avoid the constructive expression (constitutive expression) of S70A, inducible system has been used.This is lured
The skeleton that guiding systems use is that the Feline Immunodeficiency disease of self inactivation is malicious (SIN FIV slow virus), and tetracycline is added
The original part of induction, forms the carrier of an All-in-One, and vector construction is shown in schematic diagram 9.Vector construction, slow virus preparation, marrow
SCA-1 cell enrichment, virus infection, stem cell transplantation are the same as embodiment 2.Before transplanting mouse feed containing 1000 mg/litre Dox and
The water of 1% glucose continues to feed 45 days after transplanting.Flow cytometry is shown, after Dox is handled 1.5 months, outside
All blood leukocytes, bone marrow cell, lin-sca1highckithighBefore GFP positive cell rate is than transplanting in bone marrow cell (LSK cell)
It increases (Figure 10) more than one times.After removing Dox 4.5 months, to decline when GFP positive cell ratio has just removed Dox, remove Dox
After 8.5 months, decline eases up (Figure 10).It is feasible that these results, which illustrate that the inducible system is applied to gene therapy, is that can protect
Hold persistent transgene expression.
Induction screening system of the embodiment 12. using Bcl-xL as screening-gene
Test method has only with embodiment 1 with the S70A on Bcl-xL replacement plasmid.As a result, it has been found that Bcl-xL can also make
GFP positive cell ratio increases, and sees Figure 11.Illustrate Bcl-xL also and can be used as the screening-gene of internal screening system.
Above-described embodiment is not for limitation of the invention, and the present invention is not limited only in the animal of above-described embodiment
Using also comprising the application of the mankind, as long as meeting, the present invention claims all belonged to the scope of protection of the present invention.
Claims (3)
1. a kind of transgene carrier system for promoting cell transplantation and gene expression, which includes screening-gene system, purpose
Gene expression system and carrier for carrying above-mentioned two system, it is characterised in that screening-gene system is to inhibit division or promote
The silencing system of the gene of cell death including promoter, inhibits division or promotes the gene order of cell death to be target sequence
SiRNA or shRNA or miRNA DNA template sequence and transcription terminator；The wherein DNA template sequence of miRNA
3 ' the ends or 5 ' ends of target gene in destination gene expression box can be inserted in；The base of the inhibition division or promotion cell death
Because of the gene order that sequence is bcl2 family and the rush apoptosis of other families, including Bax, Bak, Bok/Mtd, Bcl-xs, Bcl-
g/Bcl2L14、Bfk/Bcl2L15、Bid、Bad、Bik/Nbk、Hrk、Bim/Bod、Bmf、Mule/ARF-BP3、Nix/Bnip3、
The gene orders such as Puma, Noxa.
2. the system as claimed in claim 1, it is characterised in that silencing system can be the single target spot of individual gene or single base
Because of multiple target spot silencings, it is also possible to multiple genes silencing simultaneously.
3. the system as claimed in claim 1, it is characterised in that screening-gene system uses induction type or conditional gene knockout
Type, screening-gene alternative carry out overexpression or silencing, avoid the permanent influence on host cell.
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