CN106544366A - The preparation method of the immunocyte of special sex modification inflammation - Google Patents

The preparation method of the immunocyte of special sex modification inflammation Download PDF

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CN106544366A
CN106544366A CN201611051336.6A CN201611051336A CN106544366A CN 106544366 A CN106544366 A CN 106544366A CN 201611051336 A CN201611051336 A CN 201611051336A CN 106544366 A CN106544366 A CN 106544366A
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immunocyte
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肖洪宝
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Shenzhen Zhongjian Biotechnology Co Ltd
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Abstract

The present invention relates to the preparation method of the immunocyte of species specificity modification inflammation, belongs to biological pharmacy technical field, comprises the steps：It is prepared by the packaging of S100, RET slow viruss：To be packed after RET gene fragment clones to Lentiviral, obtained the slow viruss containing RET genes；The preparation of the immunocyte of S200, RET modification：By the slow viruss containing RET genes, the infection culture in the complete medium containing polybrene together with human immunocyte, obtains the immunocyte of RET genetic modifications.RET genes as inflammation associated antigen genes, are transfected immune cells as carrier by the use of slow viruss by the present invention, and induction stimulates the every immunocyte for modification inflammation, so as to mediate the specific immune response for inflammatory cell.

Description

The preparation method of the immunocyte of special sex modification inflammation
Technical field
The invention belongs to biological pharmacy technical field, in particular, is related to a kind of specific treatment and modification chronic inflammation The preparation method of the immunocyte of disease.
Background technology
Inflammation is a common physiological reaction, when referring to that tissue is subject to wound or pathogen infection to stimulate, the one kind for being excited Innate immune response.Inflammatory reaction starts from the damaged tissues release signal factor, stimulates leukocyte, strengthens cell splitting rate weight Modeling tissue repair is damaged.Reaction terminates in wound healing, and the interior environment of body and external environment reach new balance.Under normal circumstances, Inflammatory reaction be it is favourable to body, but chronic inflammatory disease is harmful to body.It is in chronic inflammatory disease, different due to immunoreation Often, inflammatory reaction may occur in the case of body is int and not terminate in good time.Pro-inflammatory cytokine is persistently present and long The injury tissue of phase, stimulate cellular proliferation reparation.
Long-term chronic inflammatory disease can cause the disorder of human autoimmune's system, and then cause various diseases, such as pollen Disease, atherosclerosiss, rheumatoid arthritiss, pneumonia hepatitis nephritis even cancer.Many bases and clinical research show Inflammation can accelerate about 30 times of DNA mutation, be one of predisposing factor of a clear and definite cancer.Cancer more than 1/5 is by slow Property inflammation causes, and common inflammation switchs to the case of cancer and includes pulmonary carcinoma intestinal cancer hepatocarcinoma gastric cancer.Excessive reparation local inflammation It is the main cause for causing cell transition increment and organ senescence.14,100,000 newly-increased Cancer death diseases were there are about in 2012 in world wide Example, about 2,800,000 people suffer from tumor because of inflammation, and about 1,700,000 people die from tumor caused by inflammation.In developing country, due to population The impact increased sharply with environment, newly-increased inflammatory type tumor can be increased rapidly.
Existing market has some to have the anti-inflammatory drug of potential preventive and therapeutic effect to organ aging and cancer.Such as 2010 Britain 《Lancet》Article point out that the aspirin (75 milligrams) of the few dosage of long-term taking can prevent cardiovascular and cerebrovascular disease, and reduce The cancer mortality of 8%-16%.Although the long-term taking of oral anti-inflammatory drug has some benefits, but is not appropriate for whole people Group, and is often accompanied by allergy, edema, the side effect such as digestive tract hemorrhage.How the suppression of whole body chronic inflammatory disease is effectively carried out for a long time And modification, and the generation of reduction side effect still falls within blank relatively against the background of the prior art as far as possible.
The content of the invention
The technical problem to be solved is：The preparation side of the immunocyte of one species specificity modification inflammation is provided Method, the suppression and modification for effectively carrying out whole body chronic inflammatory disease in solving the problems, such as prior art for a long time still fall within blank relatively.
Technical proposal that the invention solves the above-mentioned problems is：The preparation side of the immunocyte of one species specificity modification inflammation Method, comprises the steps：
It is prepared by the packaging of S100, RET slow viruss：To be packed after RET gene fragment clones to Lentiviral, Obtain the slow viruss containing RET genes；
The preparation of the immunocyte of S200, RET modification：By the slow viruss containing RET genes and human immunocyte The infection culture in the complete medium containing polybrene together, obtains the immunocyte of RET genetic modifications.
In the preparation method of the immunocyte of the special sex modification inflammation that the present invention is provided, step S100 also includes Following steps：
S101, amplification RET genes：The cDNA that large intestine is extracted expands template as PCR, adds Taq polymerase, using such as Lower RET amplimers enter performing PCR amplification and purification in amplification system：
RET-F:5’-ATGGCGAAGGCGACGTCCGGT-3’
RET-R:5’-TTAACTATCAAACGTGTCCAT-3’；
S102, structure RET Lentivirals：By RET genetic fragments and Lentiviral pLenti6.3/V- After TOPO carries out enzyme link process, slow viruss clone products are obtained；By the slow viruss clone products and E.coli DH5a Competent cell carries out conversion incubation, then by ampicillin Screening Treatment in the culture medium containing ampicillin again Secondary incubation, isolates and purifies recombinant plasmid dna and obtains RET Lentiviral pLenti-RET；
S103, packaging prepare RET Lentivirals：The Lentiviral pLenti-RET, with transfection examination Carry out carrying out packing incubation process with incasing cellss 293FT by which after incubation forms DNA transfection reagent complex after agent mixing, obtain The slow viruss RET-LV of the RET genes must be contained.
In the preparation method of the immunocyte of the special sex modification inflammation that the present invention is provided, human immunocyte is At least one of PBMC, CIK, NK, DC cell.
In the preparation method of the immunocyte of the special sex modification inflammation that the present invention is provided, step S200 also includes Following steps：
The separation and Culture of S201, immunocyte：Human peripheral blood is extracted, using the isolated immunity of lymphocyte separation medium Cell, using collecting immunocyte after the lymph culture fluid culture containing 10% autologous plasma；
S202, RET-LV infection immunity cell：By the slow viruss containing RET genes together with human immunocyte Infection culture in complete medium containing polybrene, the complete medium is the lymph culture containing 10% autologous plasma Liquid.
In the preparation method of the immunocyte of the special sex modification inflammation that the present invention is provided, in step S202, institute State after RET-LV mixed with immunocyte, cell quantity ratio is 20-100:1.
In the preparation method of the immunocyte of the special sex modification inflammation that the present invention is provided, in step S202, institute The concentration that RET-LV is stated with polybrene in immune cells culture medium is 8 μ g/ml.
Implement the present invention, have the advantages that：RET genes as inflammation associated antigen genes, are utilized by the present invention Slow viruss (LV:Lentivirus immune cells are transfected as carrier), induction stimulates the items immunity for modification inflammation Cell, so as to mediate the specific immune response for inflammatory cell or tissue.
Description of the drawings
For the technical scheme being illustrated more clearly that in the embodiment of the present invention, below by to be used needed for embodiment Accompanying drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for ability For the those of ordinary skill of domain, on the premise of not paying creative work, can be attached to obtain others according to these accompanying drawings Figure.
Fig. 1 is the flow chart of the preparation method preferred embodiment of the immunocyte of special sex modification inflammation of the invention；
Fig. 2 is the electrophoretic band figure of RET gene amplification products in the embodiment of the present invention；Wherein M represents DNA Ladder；
The electrophoretic band figure of Tu3Shi embodiment of the present invention squadron recombiant plasmid digestion products；Wherein M represents DNA Ladder；1,2,3,4 is designated from four different bacterium grains the DNA for being extracted；
Fig. 4 is the immunostaining of RET protein expression levels in WT-PBMC in embodiment in RET-PBMC and comparative example Figure；
Fig. 5 is RET gene expressions (qRT-PCR) horizontal columns in WT-PBMC in embodiment in RET-PBMC and comparative example Shape figure；
Fig. 6 is the immunoblotting knot of RET protein expression levels in WT-PBMC in embodiment in RET-PBMC and comparative example Fruit is schemed；
Fig. 7 is pierced in LPS after the RET-PBMC, WT-PBMC and HEVEC for being cultivated in embodiment and comparative example respectively is co-cultured Swash the block diagram of lower secreted TNF alpha；
Fig. 8 is pierced in LPS after the RET-PBMC, WT-PBMC and HEVEC for being cultivated in embodiment and comparative example respectively is co-cultured Swash the block diagram of lower secreted adhesion factor ICAM1；
Fig. 9 is pierced in LPS after the RET-PBMC, WT-PBMC and HEVEC for being cultivated in embodiment and comparative example respectively is co-cultured Swash the block diagram of lower secreted interleukin-11 β；
Figure 10 is in LPS after the RET-PBMC, WT-PBMC and HEVEC for being cultivated in embodiment and comparative example respectively is co-cultured Apoptotic ratio under stimulation.
Specific embodiment
Embodiments of the invention are specifically described below in conjunction with accompanying drawing.
Long-term chronic inflammatory disease can induce a series of pathological changes and include cancer by the reparation and propagation for not stopping stimulatory organs Disease.Inflammation is also the one of the main reasons for causing organ failure's body aging simultaneously.The present invention is by immunocyte specificity Transformation with reach allow its suppress and modify inflammation purpose.By the treatment of long-term stage, it is contemplated that effectively can repair Multiple or restraining chronic inflammation, to reduce the speed of the incidence probability and organ senescence of cancer.Autoimmune cell carries out specificity Side effect and repulsive interaction can be preferably minimized after transformation.
The preparation method of the immunocyte of special sex modification inflammation of the invention, comprises the steps：
It is prepared by the packaging of S100, RET slow viruss：To be packed after RET gene fragment clones to Lentiviral, Obtain the slow viruss containing RET genes, i.e. RET-LV；
The preparation of the immunocyte of S200, RET modification：By the slow viruss containing RET genes and human immunocyte The infection culture in the complete medium containing polybrene together, obtains the immunocyte of RET genetic modifications, i.e. RET- immunity is thin Born of the same parents.
RET (receptor tyrosine kinase, receptor type TYR kinases) gene code in above-mentioned steps 100 Albumen be receptor type TYR kinases.RET genes (NCBI Ref Seq:NM_020975.4) fragment length is 3345 bases It is right.On July 13rd, 2016, Veiga-Fernandes existed《It is natural》Illustrate on magazine that RET albumen can regulate and control in mucosal inflammation Signal path.The mice of RET disappearances is highly prone to inflammation, and infection is simultaneously rapid dead.The transgenic mice of expression RET is higher than normal Level fully against these diseases.
Preferred step S100 also comprises the steps：
S101, amplification RET genes：The cDNA that large intestine is extracted expands template as PCR, adds Taq polymerase, using such as Lower RET amplimers enter performing PCR amplification and purification in amplification system：
RET-F:5’-ATGGCGAAGGCGACGTCCGGT-3’
RET-R:5’-TTAACTATCAAACGTGTCCAT-3’；
S102, structure RET Lentivirals：By RET genetic fragments and Lentiviral pLenti6.3/V- After TOPO carries out enzyme link process, slow viruss clone products are obtained；By the slow viruss clone products and E.coli DH5a Competent cell carries out conversion incubation, then by ampicillin Screening Treatment in the culture medium containing ampicillin again Secondary incubation, isolates and purifies recombinant plasmid dna and obtains RET Lentiviral pLenti-RET；
S103, packaging prepare RET Lentivirals：The Lentiviral pLenti-RET, with transfection examination Carry out carrying out packing incubation process with incasing cellss 293FT by which after incubation forms DNA transfection reagent complex after agent mixing, obtain The slow viruss RET-LV of the RET genes must be contained.
Preferred step S200 also comprises the steps：
The separation and Culture of S201, immunocyte：Human peripheral blood is extracted, using the isolated immunity of lymphocyte separation medium Cell, using collecting immunocyte after the lymph culture fluid culture containing 10v/v% autologous plasmas；
S202, RET-LV infection immunity cell：By the slow viruss containing RET genes together with human immunocyte Infection culture in complete medium containing polybrene, the complete medium are the training of the lymph containing 10v/v% autologous plasmas Nutrient solution (Takara).After the RET-LV is mixed with immunocyte, cell quantity ratio is 20-100:1.The RET-LV with exempt from In epidemic disease cell culture medium, the concentration of polybrene is 8 μ g/ml.
Human immunocyte according to the present invention includes that (cytokine induction is killed for PBMC (peripheral blood lymphocytes), CIK Hinder cell), NK (natural killer cell), DC (dendritic cell).The present invention be RET genes are proceeded to slow virus carrier it is above-mentioned each In anthropoid immunocyte, the immunocyte of special sex modification inflammation is prepared in induction.First illustrate that specificity is repaiied by taking PBMC as an example The detailed process of the preparation of the immunocyte of decorations inflammation.So that specificity can be accordingly obtained during CIK, NK, DC cell replacement PBMC The cytokine induced kill cell RET-CIK of modification inflammation, the natural killer cell RET-NK of special sex modification inflammation, spy The dendritic cell RET-DC of opposite sex modification inflammation.
Embodiment
The preparation of the peripheral blood lymphocytes (RET-PBMC) of special sex modification inflammation, comprises the steps：
1. it is prepared by the packaging of the slow viruss (RET-LV) containing RET genes
The amplification of 1.1RET genetic fragments
Genes of interest RET (NCBI Ref Seq:NM_020975.4) fragment length is 3345 base pairs.To there is RET The large intestine epidermis cell cDNA of gene is template, carries out polymerase chain reaction PCR expansion with the primer RET-F and RET-R of design Increase.PCR reaction systems are as follows:DNA profiling 20ng, 100nM RET-F, 100nM RET-R, 0.5 μ l of Taq polymerase, buffer 5 μ l, 10mM dNTP, distilled water to 50 μ l.PCR reaction conditions are as follows：95 degree 5 minutes, (94 degree 30 seconds, 57 degree 30 seconds, 72 degree 45 Second), 2-34 circulations, 72 degree 10 minutes, 4 degree preserve. primer sequence is：
RET-F:5’-ATGGCGAAGGCGACGTCCGGT-3’
RET-R:5’-TTAACTATCAAACGTGTCCAT-3’
The purification of 1.2RET genetic fragments
After PCR RET amplified production Jing agarose gel electrophoresiies detection, it is seen that a specific band, length in 3-4k, As shown in Fig. 2 in the same size with expection, it was demonstrated that obtain correct genes of interest by PCR method.It is clear by what is obtained at 3-4k The shearing of clear band, and with Promega companiesSV Gel and PCR Clean-Up System carry out glue and return Receive, purification DNA product.
The structure of 1.3RET slow virus carriers
Slow viruss pLenti6.3/Expression vector is bought from Invitrogen companies.The RET that PCR is generated (20-25 DEG C) is attached acquisition clone products (restructuring matter to genetic fragment at room temperature with pLenti6.3/V-TOPO carriers Grain).Recombiant plasmid carries out conversion incubation in E.coli DH5a competent cells (Promega), then is sieved by ampicillin Choosing is processed and is incubated in the culture medium containing ampicillin again.Isolate and purify recombinant plasmid dna and with SpeI and EcoRV Carry out enzyme action identification.Whether analytical electrophoresises band is judging plasmid as recombiant plasmid.If there is clear band (such as Fig. 3 at 3kb It is shown), then illustration purpose gene RET is successfully plugged into pLenti6.3/It is in carrier, sick slowly so as to obtain RET- Malicious expression vector.
It is prepared by the packaging of 1.4RET-LV
First day：Take 5x106Individual 293FT cells (Invitrogen), are resuspended in the complete of preheating after centrifugation after abandoning supernatant In culture medium (DMEM+10%FBS+2mM L-glutaminate+0.1mM non essential amino acid+1mM Sodium Pyruvate+1%P/S).Weight Outstanding cell is inoculated in 10cm culture dishs, in 37 DEG C of 5%CO2Overnight incubation in incubator.
Second day：500 μ l serum-frees are added in centrifuge tube 1Culture fluid (Invitrogen), 9 μ g ViraPowerTMThe RET- slow viruss plasmids being purified in packaging plasmid mixture (Invitrogen) and 3 μ g steps 1.3, softly Mixing for standby use.Add 500 μ l serum-frees in centrifuge tube 2Culture fluid and 36 μ l 2000, it is incubated at room temperature 5 minutes after soft mixing.By two centrifuge tubes soft mix homogeneously, it is incubated at room temperature 30 minutes, obtains2000 mixture.After 30 points, by what is obtained2000 Mixture is slowly added to be vaccinated with first day in the culture dish of 293FT cells, and gently rocking back and forth makes mixture uniformly incorporate training In nutrient solution, overnight incubation afterwards.
3rd day：Culture fluid is discarded in morning (after transfection RET- slow viruss plasmid 16-24 hours), 5-6ml DMEM are added Complete culture solution, in 37 DEG C of 5%CO248-72 hours are incubated in incubator.
5th day or the 6th day：By the culture fluid in culture dish with subpackage after 0.44 μm of membrane filtration it is frozen with -80 DEG C.Together When with crystal violet staining assay detect slow viruss titre.
2.RET-LV slow virus infection PBMC
The separation and Culture of 2.1 peripheral blood lymphocytes PBMC
Extract healthy volunteer peripheral blood 50ml, anticoagulant heparin, room temperature centrifugation (700g, 20 points)；Upper plasma is drawn, is put 56 DEG C, 30 points in water-bath.Then, after 4 DEG C static 15 points, it is centrifuged (900g, 30 points), takes 4 DEG C of autologous plasma and save backup.
Above-mentioned 700g is taken, 20 separate heart rear lower cell component, add D-PBS to 50ml, mix, be added slowly to 2 dresses In having the 50ml centrifuge tubes of 20ml human lymphocyte separating liquids, room temperature centrifugation (800g, 15 points).Tunica albuginea confluent monolayer cells are drawn, is added To in the 50ml centrifuge tubes of the RPMI1640 equipped with 5ml.RPMI1640 is added to be 50ml to cumulative volume, be centrifuged (600g, 10 points Clock), abandon supernatant.50ml RPMI are added again, are centrifuged (600g, 10 minutes), are abandoned supernatant.Final cell is resuspended in containing 10% In the lymph culture fluid of autologous plasma (1000IU IL2/ml), in subpackage and six orifice plates.1x106/ hole, 2ml/ holes.In incubator Overnight incubation.Mend half liquid within the 3rd day first day after overnight.Collect PBMC cell countings, PBS centrifuge washing cells within 5th day (1500rpm, 10 minutes), is resuspended in the lymph culture fluid containing 10% autologous plasma, is inoculated in 6 orifice plates, 1x106/ hole, 2ml/ holes.
2.2RET-LV infection of PBMCs
6th day：Thaw a pipe slow viruss solution, with lymph culture fluid (contain 10% autologous plasma) be diluted with Finally give optimal MOI (final volume reduces as far as possible to improve jamming effectiveness).Discard in six orifice plate cells of inoculation in the 5th day Culture fluid, with pipet by the slow viruss solution after above-mentioned dilution gently blow and beat mixing after add in six orifice plates.Add in hole The polybrene (Polybrene, Sigma) of final concentration of 8 μ g/ml, mix homogeneously.37 DEG C are placed in, 5%CO2It is incubated in incubator Overnight.
7th day：The culture fluid containing slow viruss in six orifice plates is discarded, fresh complete lymph culture fluid is added, is placed in 37 DEG C, 5%CO2After being incubated 2-3 days in incubator, the PBMC cells (RET-PBMC) of RET genetic modifications are obtained.
The detection of RET expressions in 2.3PBMC
RNA extracts detection：PBMC cultures the collect 1x10 after 9-10 days respectively6Individual ripe WT-PBMC, RET-PBMC Cell, using RNA extracts kits (Aurum Total RNA Mini Kit, BioRad) propose two kinds of cells total serum IgE simultaneously Used iScriptTMCDNA syntheses Kit (BioRad) reverse transcriptions are total cDNA.QRT-PCR detects RET levels：Profit WithGreen (Biorad) test kits carry out the horizontal detection of RET genes to the total cDNA for extracting.Cell protein is carried Take detection：PBMC cultures the collected 1x10 after 9-10 days6Individual ripe WT-PBMC, RET-PBMC cell lysis and 300 μ l In RIPA lysates.Albumen sample-loading buffer is added in the sample, and 95 DEG C prepare protein sample in ten minutes. sample loading and 6% Albumin glue, runs SDS-PAGE and western-blot (immunoblotting) to detect RET protein levels.
Comparative example
The preparation of unmodified peripheral blood lymphocytes (WT-PBMC), comprises the steps：Conventionally cultivate PBMC cells, and with not with RET empty slow virus carrier pLenti6.3/V-TOPO carry out virus pack infection (step and 1.4 is identical), obtain WT-PBMC.
Correlation properties are tested
The relevant cell that culture in above-described embodiment and comparative example is obtained is tested as follows：
1.RET genes and protein expression level
The PBMC cells that the RET-LV and empty carrier slow-virus transfection for obtaining is cultivated in embodiment and comparative example (take identical The PBMC cells of cultivation stage culture 9-10), the cell after RET slow viruss is infected with immunostaining RET albumen visible (Fig. 4) Most of dye is green.The expression of RET genes in WT-PBMC and RET-PBMC is detected with qRT-PCR methods.The RET genes Expression experimental result see Fig. 5, wherein * * P values are less than 0.05, and significant difference is notable.Cultivate in embodiment and comparative example (PBMC for taking identical cultivation stage culture 9-10 is thin for the RET-LV for obtaining and the PBMC cells of empty carrier slow-virus transfection Born of the same parents) RET protein expression levels in WT-PBMC and RET-PBMC are detected with western blot (immunoblotting) method.Experiment knot Fruit is as shown in fig. 6, the band of the sample RET-PBMC of virus infection is significantly stronger than WT-PBMC.From Fig. 4, Fig. 5, Fig. 6, In RET-PBMC cells, RET genes and protein expression level are all remarkably higher than WT-PBMC.
The suppression that 2.RET-PBMC is reacted to cellular inflammation
Reference examples WT-PBMC and embodiment RET-PBMC respectively with Human umbilical vein endothelial cells HUVEC co-culture of cells with In RPMI1640 culture fluid, six orifice plates are laid on, altogether 1x106Cells/well, 2ml.Lipopolysaccharide LPS (Lipopolysaccharide, Sigma) can stimulate epidermis cell inflammatory reaction release inflammatory factor to finally result in apoptosis. HUVEC under by comparing LPS and stimulating, the inflammatory reaction of WT-PBMC+HUVEC or RET-PBMC+HUVEC cells is judging Whether RET-PBMC has inhibition to inflammation.The LPS of 20-1000ng/ml is added in culture fluid, it is soft to mix.Incubation 6- The secretion situation of the cellular inflammation factor, including tumor necrosis factor are detected after 24 hours with ELISA kit (Bio-source) TNF-α, interleukin I L-1, the concentration of cell adhesion molecules ICAM1.The RET-PBMC for being cultivated in embodiment and comparative example respectively, The TNF alpha secreted in the case where LPS stimulates after WT-PBMC and HEVEC co-cultivation, adhesion factor ICAM1, Adhesion factor ICAM1's measures result respectively as shown in Fig. 7, Fig. 8, Fig. 9.The secretion situation of inflammatory factor is in HUVEC, WT- Taper off in PMBC-HUVEC, RET-PBMC-HEVEC cell.Above-mentioned cell LPS stimulation under, with Annexin V-FITC morning Phase apoptosis detection kit (BD) detects the cell percentage of apoptosis.Measure result as shown in Figure 10.As shown in Figure 10, with RET- The apoptosis-induced ratio of the HUVEC that PBMC is cultivated altogether will be significantly lower than HUVEC cells, secondly co-culture less than PBMC-HUVEC Cell, and significant level has been reached each other.*P<0.5, * * P<0.05, * * * P<0.01.To sum up test result indicate that The inflammation and apoptosis that RET-PBMC is induced in HUVEC cells to LPS has obvious inhibitory action.
Embodiments of the invention are described above in conjunction with accompanying drawing, but be the invention is not limited in above-mentioned concrete Embodiment, above-mentioned specific embodiment are only schematic, rather than restricted, one of ordinary skill in the art Under the enlightenment of the present invention, in the case of without departing from present inventive concept and scope of the claimed protection, can also make a lot Form, these are belonged within the protection of the present invention.

Claims (6)

1. a species specificity modifies the preparation method of the immunocyte of inflammation, it is characterised in that comprise the steps：
It is prepared by the packaging of S100, RET slow viruss：To be packed after RET gene fragment clones to Lentiviral, obtained Slow viruss containing RET genes；
The preparation of the immunocyte of S200, RET modification：By the slow viruss containing RET genes together with human immunocyte In the complete medium containing polybrene, infection culture, obtains the immunocyte of RET genetic modifications.
2. the preparation method of the immunocyte of special sex modification inflammation according to claim 1, it is characterised in that the step Rapid S100 also comprises the steps：
S101, amplification RET genes：The cDNA that large intestine is extracted expands template as PCR, adds Taq polymerase, using as follows RET amplimers enter performing PCR amplification and purification in amplification system：
RET-F:5’-ATGGCGAAGGCGACGTCCGGT-3’
RET-R:5’-TTAACTATCAAACGTGTCCAT-3’；
S102, structure RET Lentivirals：By RET genetic fragments and Lentiviral pLenti6.3/V-TOPO After carrying out enzyme link process, slow viruss clone products are obtained；The slow viruss clone products are experienced with E.coli DH5a State cell carries out conversion incubation, then is incubated in the culture medium containing ampicillin again by ampicillin Screening Treatment Educate, isolate and purify recombinant plasmid dna and obtain RET Lentiviral pLenti-RET；
S103, packaging prepare RET Lentivirals：The Lentiviral pLenti-RET, it is mixed with transfection reagent Carry out carrying out packing incubation process with incasing cellss 293FT by which after incubation forms DNA transfection reagent complex after conjunction, contained There is the slow viruss RET-LV of the RET genes.
3. the preparation method of the immunocyte of special sex modification inflammation according to claim 1, it is characterised in that the step In rapid S200, human immunocyte is at least one of PBMC, CIK, NK, DC cell.
4. the preparation method of the immunocyte of special sex modification inflammation according to claim 1, it is characterised in that the step Rapid S200 also comprises the steps：
The separation and Culture of S201, immunocyte：Human peripheral blood is extracted, it is thin using the isolated immunity of lymphocyte separation medium Born of the same parents, using collecting immunocyte after the lymph culture fluid culture containing 10% autologous plasma；
S202, RET-LV infection immunity cell：The slow viruss containing RET genes are being contained together with human immunocyte Infection culture in the complete medium of polybrene, the complete medium is the lymph culture fluid containing 10% autologous plasma.
5. the preparation method of the immunocyte of special sex modification inflammation according to claim 4, it is characterised in that the step In rapid S202, after the RET-LV is mixed with immunocyte, cell quantity ratio is 20-100:1.
6. the preparation method of the immunocyte of special sex modification inflammation according to claim 4, it is characterised in that the step In rapid S202, the RET-LV is 8 μ g/ml with the concentration of polybrene in immune cells culture medium.
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