CN101684456A - Method for amplifying NK cells of human beings under condition of in vitro culture - Google Patents
Method for amplifying NK cells of human beings under condition of in vitro culture Download PDFInfo
- Publication number
- CN101684456A CN101684456A CN200810198913A CN200810198913A CN101684456A CN 101684456 A CN101684456 A CN 101684456A CN 200810198913 A CN200810198913 A CN 200810198913A CN 200810198913 A CN200810198913 A CN 200810198913A CN 101684456 A CN101684456 A CN 101684456A
- Authority
- CN
- China
- Prior art keywords
- cell
- leu
- ser
- ala
- glu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for amplifying NK cells of human beings under the condition of in vitro culture, which is characterized in that a peripheral blood mononuclear cell (PBMC) is taken asthe original culturing material, and the PBMC is cultured together with an engineering cell which is built by adopting the genetic engineering method and is used for stimulating the growth of the NK cell. The built engineering cell which is used for stimulating the growth of the NK cell expresses several cytokines (IL-2, IL-12, IL-15, IL-18, 4-1BB) which can promote the growth of the NK cell on the surface of the K562 cell; after irradiation and inactivation with gamma-rays, the engineering cell is cultured with the PBMC in vitro, as a result, the amplification effect of the stimulation methodin the invention is hundreds of times stronger than that of the currently universal method in which soluble cytokines are added purely to the culture solution; and after cultured for 3 weeks, the non-NK cells in the PBMC are mainly dead and disappeared, the NK cells are proliferated in great quantity, the purity of the NK cells reaches over 96% and the total number of the NK cells is amplified byover 1500 times.
Description
Technical field
The present invention relates to immunology and biology field, be specifically related to effectively to improve the method for a kind of amplifying NK cells of human beings under condition of in vitro culture of the purity of NK cell and killing activity thereof.
Background technology
NK (natural killer, natural killer) cell is the important immunocyte of body, plays an important role in antitumor, anti-virus infection is immune.Because the killing activity of NK cell does not have the MHC restriction, therefore be called natural killer.The target cell of NK cell mainly is tumour cell, virus infected cell, some autologous tissue's cell (as hemocyte), parasite etc., so the NK cell is that body is antitumor, the important component part of anti-infectious immunity.Because these characteristics, the NK cell has a wide range of applications in the cellular immunization treatment.But the content of NK cell seldom in the human peripheral (accounting for lymphocytic 5%-20%), separates NK cell cost costliness from peripheral blood, and isolating NK cell generally all is in the disactivation state, limited its application clinically to a great extent.Studies show that cytokines such as IL-2, IL-12, IL-15, IL-18,4-1BBL play an important role to differentiation and maturation, activation, propagation and the cytotoxic activity that promotes the NK cell.For many years, people attempt adding these cytokines under condition of in vitro culture always, and expectation can make the NK cell obtain a large amount of amplifications.Research report from the past adopts the method that adds cytokine in nutrient solution, and 14-21 days cultivation of peripheral blood lymphocytes (PBMC) process can make tens times of NK cell amplifications, and purity reaches 50-60%.In addition, the K562 cell of employing deactivation or HFWT tumour cell and PBMC cultivate altogether, also can make tens times of NK cell amplifications.But this expanding effect still can not satisfy actual application, generally speaking, still lacks effective amplification in vitro system at present, so the clinical application of NK cell has been subjected to serious restriction.
Summary of the invention
The invention provides a kind of method of amplifying NK cells of human beings, make the NK cell amplification on a large scale under condition of in vitro culture among the human PBMC, effectively improve total amount, purity and the killing activity of NK cell.
The present invention realizes by following technical scheme in the method for amplifying NK cells of human beings under condition of in vitro culture: adopt engineered method to make up a kind of engineering cell of the NK of stimulation cell growth, promptly have promote the growth of NK cell several cytokine-expressings on the film surface of K562 cell, made up the engineering cell of expressing several cytokines.This project cell is through carrying out common cultivation with the human PBMC external after the gammairradiation deactivation, the result shows that this stimulating method is than the at present existing simple expanding effect hundred times eager to excel in whatever one does that adds the soluble cell factor in nutrient solution, cultivation through 3 weeks, non-NK cell among the PBMC is dead basically to disappear, the NK cell is bred in a large number, the NK cell purity has reached more than 96%, and the NK total cellular score has increased more than 1500 times.
It is characterized in that of the method for amplification in vitro NK cells of human beings provided by the invention, adopt gene engineering method to make up special irritation cell, NK cell among the human PBMC is increased under external specific culture condition with in the process of special irritation cell co-cultivation on a large scale.Special irritation cell is meant and adopts engineered method in passage cell surface difference expressing human IL-2, IL-12, IL-15, five kinds of cytokines of IL-18,4-1BBL that the passage cell of expressing these several cytokines can be K562 cell or HFWT cell; Specific culture condition comprises the nutrient solution that preparation is special.
It is characterized in that of the method for amplification in vitro NK cells of human beings provided by the invention in passage cell surface expression human IL-2, IL-12, IL-15, several cytokines of IL-18,4-1BBL, be gene level with the gene of IL-2, IL-12, IL-15, four kinds of cytokines of IL-18 respectively with the gene splicing of one section transmembrane protein.4-1BBL itself contains and strides the film district, thus not with the gene splicing of transmembrane protein, with himself complete gene.Subsequently these genes are cloned into carrier for expression of eukaryon pcDNA3.1 respectively, transfection K 562 cell has made up special irritation cell again.Transmembrane protein is meant behind IL-2, IL-12, IL-15, IL-18 gene and transmembrane protein gene fusion expression, IL-2, IL-12, IL-15, IL-18 albumen can be anchored on a proteinoid in the express cell film outside.
Method at amplifying NK cells of human beings under condition of in vitro culture among the present invention has the following advantages: the amplification times of NK cell has obtained remarkable effect and has improved, than mix solubility IL-2, IL-12, IL-15, IL-18 or the simple high hundred times of K562 merely in nutrient solution; And, having improved the purity of NK cell, the NK cell purity that amplification obtains can reach 96%; Compare with direct isolating NK cell in the peripheral blood, improve 20 times, gamma-interferon expression amount by the NK cell killing activity of the present invention amplification and increase more than 300 times, the NK cell of expressing the NKp46 activation receptor brings up to 65% from 0.31%, differs about 200 times.
Embodiment
The key of amplifying NK cells of human beings is to be starting materials with the human PBMC among the present invention, under given conditions PBMC is cultivated with the irritation cell that adopts engineered method to make up.Specifically introduce among the present invention method below in conjunction with embodiment in the amplification in vitro NK cells of human beings.
Embodiment 1:CD8 α chain is striden the clone of film district gene.
In order at first to stride film district gene fusion to born of the same parents' outside part of these cytokines with CD8 α chain respectively with IL-2, IL-12, IL-15, IL-18 cytokine with the film skin of membranin formal representation at the K562 cell.
Complete CD8 α chain is striden film district gene (CD8tm):
ggatcctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgctaagcggccgc
CD8tm is with two sections following nucleotide chain synthetic:
CD8F：5’tcggatcctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactgg?3’
CD8R：5’atgcggccgcttagcagtaaagggtgataaccagtgacaggagaaggaccccacaagtcccgg?3’
These two sections nucleotide chains have obtained complete CD8 α chain and have striden film district gene by annealing, extension, and the 5 ' end and the 3 ' end of gene have been introduced BamHI and NotI restriction enzyme site respectively.Utilize this two restriction enzyme sites, CD8 α chain is striden the gene clone of film district to the pcDNA3.1+/Neo carrier for expression of eukaryon, the correct back of order-checking is standby.Structure contains CD8 α chain and strides the carrier of film district gene and be called: pcDNA3.1-CD8.
Embodiment 2:IL-2, IL-12, IL-15, the expression of IL-18 gene on the K562 cytolemma.
Adopt the method for RT-PCR from the human PBMC, to extract mRNA respectively, the cDNA of amplification IL-2, IL-12, IL-15, IL-18.
The upstream primer of amplification IL-2 is IL-2F:5 ' tcgctagcatgtacaggatgcaactcctg3 ', and downstream primer is IL-2R:5 ' tcggatccagtcagtgttgagatgatgc3 ', has introduced NheI and BamHI restriction enzyme site in the upstream and downstream primer respectively;
The upstream primer of amplification IL-12 is IL-12F:5 ' tagctagcatgtggccccctgggtcagcct 3 ', downstream primer is IL-12R:5 ' tcggatccggaagcattcagatagctc 3 ', has introduced NheI and BamHI restriction enzyme site in the upstream and downstream primer respectively;
The upstream primer of amplification IL-15 is IL-15F:5 ' tcgctagcatgagaatttcgaaaccacatttgag3 ', downstream primer is IL-15R:5 ' tcggatccagaagtgttgatgaacatttggac3 ', has introduced NheI and BamHI restriction enzyme site in the upstream and downstream primer respectively;
The upstream primer of amplification IL-18 is IL-18F:5 ' tcgctagcatggctgctgaaccagtagaagac3 ', downstream primer is IL-18R:5 ' tcggatccgtcttcgttttgaacagtgaacattatag3 ', has introduced NheI and BamHI restriction enzyme site in the upstream and downstream primer respectively;
IL-2, IL-12, IL-15, the IL-18cDNA of amplification is cloned into the upstream that CD8 α chain in the pcDNA3.1-CD8 carrier is striden film district gene after with NheI and BamHI double digestion respectively, distinguish transfection K 562 cell then, G418 screens single clone cell, antibody staining is identified, has obtained respectively at last to express the K562 engineering cell of IL-2, IL-12, IL-15, IL-18 (stimulating the K562 cell of NK cell amplification) to stride form membrane.
Fusion gene (IL-2CD8) sequence that IL-2 and CD8 α chain are striden the film district is:
gctagcatgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacaaacagtgcacctacttcaagttctacaaagaaaacacagctacaa
ctggagcatttactgctggatttacagatgattttgaatggaattaataattacaagaatc
ccaaactcaccaggatgctcacatttaagttttacatgcccaagaaggccacagaactgaaacatcttcagtgtctagaa
gaagaactcaaacctctggaggaagtgctaaatttagctcaaagcaaaaactttcacttaagacccagggacttaatcag
caatatcaacgtaatagttctggaactaaagggatctgaaacaacattcatgtgtgaatatgctgatgagacagcaacca
ttgtagaatttctgaacagatggattaccttttgtcaaagcatcatctcaacactgactggatcctacatctgggcgcccttggccgggacttgtggggtccttctc
ctgtcactggttatcaccctttactgctaagcggccgc
Fusion gene (IL-12CD8) sequence that IL-12 and CD8 α chain are striden the film district is:
gctagcatgtggccccctgggtcagcctcccagccaccgccctcacctgccgcggccacaggtctgcatccagcggctcgccctgt
gtccctgcagtgccggctcagcatgtgtccagcgcgcagcctcctccttgtggctaccctggtcctcctggaccactca
gtttggccagaaacctccccgtggccactccagacccaggaatggttcccatgccttcaccactcccaaaacctgctgagg
gccgtcagcaacatgctccagaaggccagacaaactctagaattttacccttgcacttctgaagagattgatcatgaaga
tatcacaaaagataaaaccagcacagtggaggcctgtttaccattggaattaaccaagaatgagagttgcctaaattcca
gagagacctctttcataactaatgggagttgcctggcctccagaaagacctcttttatgatggccctgtgccttagtagt
atttatgaagacttgaagatgtaccaggtggagttcaagaccatgaatgcaaagcttctgatggatcctaagaggcagat
ctttctagatcaaaacatgctggcagttattgatgagctgatgcaggccctgaatttcaacagtgagactgtgccacaaa
aatcctcccttgaagaaccggatttttataaaactaaaatcaagctctgcatacttcttcatgctttcagaattcgggca
gtgactattgatagagtgatgagctatctgaatgcttccggatcctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgctaagcgg
ccgc
Fusion gene (IL-15CD8) sequence that IL-15 and CD8 α chain are striden the film district is:
gctagcatgagaattcgaaaccacatttgagaagtatttccatccagtgctacttgtgtttacttctaaacagtcattttctaactgaagctggcattcatgtcttcatt
ttgggctgtttcagtgcagggcttcctaaaacagaagccaactgggtgaatgtaataagtgatttgaaaaaaattgaagatcttattcaatctatgcaatattgatgc
tactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggagatgcaagtat
tcatgatacagtagaaaatctgatcatcctagcaaacaacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaa
aaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttctggatcctacatctgggcgcccttggccgggacttgtggggtccttctcctgtca
ctggttatcaccctttactgctaagcggccgc
Fusion gene (IL-18CD8) sequence that IL-18 and CD8 α chain are striden the film district is:
gctagcatggctgctgaaccagtagaagacaattgcatcaactttgtggcaatgaaatttattgacaatacgctttactttatagc
tgaagatgatgaaaacctggaatcagattactttggcaagcttgaatctaaattatcagtcataagaaatttgaatgacc
aagttctcttcattgaccaaggaaatcggcctctatttgaagatatgactgattctgactgtagagataatgcaccccgg
accatatttattataagtatgtataaagatagccagcctagaggtatggctgtaactatctctgtgaagtgtgagaaaat
ttcaactctctcctgtgagaacaaaattatttcctttaaggaaatgaatcctcctgataacatcaaggatacaaaaagtg
acatcatattctttcagagaagtgtcccaggacatgataataagatgcaatttgaatcttcatcatacgaaggatacttt
ctagcttgtgaaaaagagagagacctttttaaactcattttgaaaaaagaggatgaattgggggatagatctataatgtt
cactgttcaaaacgaagacggatcctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactgg
ttatcaccctttactgctaagcggccgc
The expression of embodiment 3:4-1BBL gene on the K562 cytolemma.
4-1BBL itself is exactly a transmembrane protein, does not need to stride film district gene fusion with CD8 α chain, directly is cloned into the pcDNA3.1+/Neo carrier for expression of eukaryon and gets final product.The amplification of 4-1BBL gene is identical with above-mentioned cytokine amplification, also is to adopt the method for RT-PCR to obtain its mRNA from the human PBMC.The upstream primer of amplification 4-1BBL is 4-1BB-F:5 ' tcgctagcatggaatacgcctctgacgcttcac 3 ', downstream primer is 4-1BB-R:5 ' tcggatccttattccgacctcggtgaagggag 3 ', has introduced NheI and BamHI restriction enzyme site in the upstream and downstream primer respectively; The 4-1BBL cDNA of amplification uses NheI and BamHI double digestion rear clone in the pcDNA3.1+/Neo carrier, distinguish transfection K 562 cell then, G418 screens single clone cell, antibody staining is identified, has obtained at last to express the K562 engineering cell of 4-1BBL (stimulating the K562 cell of NK cell amplification) to stride form membrane.Complete 4-1BBL gene is:
gctagcatggaatacgcctctgacgcttcactggaccccgaagccccgtggcctcccgcgccccgcgctcgcgcctgccgcgtact
gccttgggccctggtcgcggggctgctgctgctgctgctgctcgctgccgcctgcgccgtcttcctcgcctgcccctggg
ccgtgtccggggctcgcgcctcgcccggctccgcggccagcccgagactccgcgagggtcccgagctttcgcccgacgat
cccgccggcctcttggacctgcggcagggcatgtttgcgcagctggtggcccaaaatgttctgctgatcgatgggcccct
gagctggtacagtgacccaggcctggcaggcgtgtccctgacggggggcctgagctacaaagaggacacgaaggagctgg
tggtggccaaggctggagtctactatgtcttctttcaactagagctgcggcgcgtggtggccggcgagggctcaggctcc
gtttcacttgcgctgcacctgcagccactgcgctctgctgctggggccgccgccctggctttgaccgtggacctgccacc
cgcctcctccgaggctcggaactcggccttcggtttccagggccgcttgctgcacctgagtgccggccagcgcctgggcg
tccatcttcacactgaggccagggcacgccatgcctggcagcttacccagggcgccacagtcttgggactcttccgggtg
acccccgaaatcccagccggactcccttcaccgaggtcggaataaggatcc
Embodiment 4:NK cells in vitro amplification cultivation.
Fresh healthy human PBMC is divided into two parts, a copy of it is used for the content and the surface markers of the preceding NK cell of flow cytometry analysis amplification, another part adds in 24 well culture plates, 30,000 cells in every hole, add simultaneously equivalent through 10, the K562 engineering cell of the gamma-rays deactivation of 000rad (express the K562 engineering cell of IL-12, IL-15, IL-18,4-1BBL, or the K562 engineering cell of IL-2, IL-12, IL-15, IL-18,4-1BBL).37 ℃ of carbonic acid gas incubators are cultivated, and the liquid in the hole was carried out half amount change liquid, amplification cultivation 21 days in per three days.Nutrient solution is to contain the RPMI1640 of 10% calf serum, the IL-2 of 100IU/ml.Content and surface markers with NK cell in the cell of flow cytometry analysis amplification cultivation.With the special marking of CD56+CD3-as the NK cell.The result shows that the preceding content of NK cell in PBMC of amplification is 6%, after 21 days amplification, the purity of NK cell ' is expressed the K562 engineering cell of IL-12, IL-15, IL-18,4-1BBL ', and stimulating group is 95%, and ' expressing the K562 engineering cell of IL-2, IL-12, IL-15, IL-18,4-1BBL ' stimulating group is 93%.The quantity of NK cell from increase first 30,000/hole after the amplification 5,000 ten thousand and 5.5 thousand ten thousand, increased 1666 times and 1833 times respectively.
The experiment of embodiment 5:NK killing activity
The NK cell of the NK cell of ' expressing the K562 engineering cell of IL-12, IL-15, IL-18,4-1BBL ' stimulating group amplification and the amplification of ' expressing the K562 engineering cell of IL-2, IL-12, IL-15, IL-18,4-1BBL ' stimulating group, with the K562 cytosis, observe the kill rate of NK cell respectively to the K562 cell.NK cell (effector cell) is 5: 2 with the ratio of K562 cell (target cell).37 ℃ of carbonic acid gas incubators of effector cell and target cell are hatched after 4 hours and to be added methyl-azoles salt (methy thiazolyl tetrazolium, M TT) and hatched 4 hours again, and the 570nm wavelength is surveyed also calculating kill rate of OD value.Kill rate (%)=(1-(effector cell adds the OD value-effector cell hole OD value in target cell hole)/target cell hole OD value) * 100%; The kill rate of the NK cell of ' expressing the K562 engineering cell of IL-12, IL-15, IL-18,4-1BBL ' stimulating group amplification is 80, and the kill rate of the NK cell of ' expressing the K562 engineering cell of IL-2, IL-12, IL-15, IL-18,4-1BBL ' stimulating group amplification is 85%.
The tabulation of specification sheets unitary part nucleotides sequence
＜110〉Jiangmen Luosen Bio-Pharmaceutical Co., Ltd.
＜120〉a kind of method of amplifying NK cells of human beings under condition of in vitro culture
<130>
<140>
<141>
<150>
<151>
<160>18
<170>
<210>1
<211>
<212>DNA
＜213〉CD8 α chain is striden the film district
<220>
<221>CDS
<222>(1)...(78)
<223>
<400>1
gga?tcc?tac?atc?tgg?gcg?ccc?ttg?gcc?ggg?act?tgt?ggg?gtc?ctt 45
Gly?Ser?Tyr?Ile?Trp?Ala?Pro?Leu?Ala?Gly?Thr?Cys?Gly?Val?Leu
1 5 10 15
ctc?ctg?tca?ctg?gtt?atc?acc?ctt?tac?tgc?taa?gcg?gcc?gc 86
Leu?Leu?Ser?Leu?Val?Ile?Thr?Leu?Tyr?Cys?＊
20 25
<210>2
<211>
<212>DNA
<213>CD8F
<220>
<221>
<222>
<223>
<400>2
tcggatccta?catctgggcg?cccttggccg?ggacttgtgg?ggtccttctc?ctgtcactgg 60
<210>3
<211>
<212>DNA
<213>CD8R
<220>
<221>
<222>
<223>
<400>3
atgcggccgc?ttagcagtaa?agggtgataa?ccagtgacag?gagaaggacc?ccacaagtcc?60
cgg 63
<210>4
<211>
<212>DNA
<213>IL-2F
<220>
<221>
<222>
<223>
<400>4
tcgctagcat?gtacaggatg?caactcctg 29
<210>5
<211>
<212>DNA
<213>IL-2R
<220>
<221>
<222>
<223>
<400>5
tcggatccag?tcagtgttga?gatgatgc 29
<210>6
<211>
<212>DNA
<213>IL-12F
<220>
<221>
<222>
<223>
<400>6
tagctagcat?gtggccccct?gggtcagcct 30
<210>7
<211>
<212>DNA
<213>IL-12R
<220>
<221>
<222>
<223>
<400>7
tcggatccgg?aagcattcag?atagctc 27
<210>8
<211>
<212>DNA
<213>IL-15F
<220>
<221>
<222>
<223>
<400>8
tcgctagcat?gagaatttcg?aaaccacatt?tgag 34
<210>9
<211>
<212>DNA
<213>IL-15R
<220>
<221>
<222>
<223>
<400>9
tcggatccag?aagtgttgat?gaacatttgg?ac 32
<210>10
<211>
<212>DNA
<213>IL-18F
<220>
<221>
<222>
<223>
<400>10
tcgctagcat?ggctgctgaa?ccagtagaag?ac 32
<210>11
<211>
<212>DNA
<213>IL-18R
<220>
<221>
<222>
<223>
<400>11
tcggatccgt?cttcgttttg?aacagtgaac?attatag 37
<210>12
<211>
<212>DNA
<213>IL-2CD8
<220>
<221>
<222>(7)...(543)
<223>
<400>12
gct?agc?atg?tac?agg?atg?caa?ctc?ctg?tct?tgc?att?gca?cta?agt 45
Met?Tyr?Arg?Met?Gln?Leu?Leu?Ser?Cys?Ile?Ala?Leu?Ser
1 5 10
ctt?gca?ctt?gtc?aca?aac?agt?gca?cct?act?tca?agt?tct?aca?aag 90
Leu?Ala?Leu?Val?Thr?Asn?Ser?Ala?Pro?Thr?Ser?Ser?Ser?Thr?Lys
15 20 25
aaa?aca?cag?cta?caa?ctg?gag?cat?tta?ctg?ctg?gat?tta?cag?atg 135
Lys?Thr?Gln?Leu?Gln?Leu?Glu?His?Leu?Leu?Leu?Asp?Leu?Gln?Met
30 35 40
att?ttg?aat?gga?att?aat?aat?tac?aag?aat?ccc?aaa?ctc?acc?agg 180
Ile?Leu?Asn?Gly?Ile?Asn?Asn?Tyr?Lys?Asn?Pro?Lys?Leu?Thr?Arg
45 50 55
atg?ctc?aca?ttt?aag?ttt?tac?atg?ccc?aag?aag?gcc?aca?gaa?ctg 225
Met?Leu?Thr?Phe?Lys?Phe?Tyr?Met?Pro?Lys?Lys?Ala?Thr?Glu?Leu
60 65 70
aaa?cat?ctt?cag?tgt?cta?gaa?gaa?gaa?ctc?aaa?cct?ctg?gag?gaa 270
Lys?His?Leu?Gln?Cys?Leu?Glu?Glu?Glu?Leu?Lys?Pro?Leu?Glu?Glu
75 80 85
gtg?cta?aat?tta?gct?caa?agc?aaa?aac?ttt?cac?tta?aga?ccc?agg 315
Val?Leu?Asn?Leu?Ala?Gln?Ser?Lys?Asn?Phe?His?Leu?Arg?Pro?Arg
90 95 100
gac?tta?atc?agc?aat?atc?aac?gta?ata?gtt?ctg?gaa?cta?aag?gga 360
Asp?Leu?Ile?Ser?Asn?Ile?Asn?Val?Ile?Val?Leu?Glu?Leu?Lys?Gly
105 110 115
tct?gaa?aca?aca?ttc?atg?tgt?gaa?tat?gct?gat?gag?aca?gca?acc 405
Ser?Glu?Thr?Thr?Phe?Met?Cys?Glu?Tyr?Ala?Asp?Glu?Thr?Ala?Thr
120 125 130
att?gta?gaa?ttt?ctg?aac?aga?tgg?att?acc?ttt?tgt?caa?agc?atc 450
Ile?Val?Glu?Phe?Leu?Asn?Arg?Trp?Ile?Thr?Phe?Cys?Gln?Ser?Ile
135 140 145
atc?tca?aca?ctg?act?gga?tcc?tac?atc?tgg?gcg?ccc?ttg?gcc?ggg 495
Ile?Ser?Thr?Leu?Thr?Gly?Ser?Tyr?Ile?Trp?Ala?Pro?Leu?Ala?Gly
150 155 160
act?tgt?ggg?gtc?ctt?ctc?ctg?tca?ctg?gtt?atc?acc?ctt?tac?tgc 540
Thr?Cys?Gly?Val?Leu?Leu?Leu?Ser?Leu?Val?Ile?Thr?Leu?Tyr?Cys
165 170 175
taa?gcg?gcc?gc 548
＊
<210>13
<211>
<212>DNA
<213>IL-12CD8
<220>
<221>
<222>(7)...(843)
<223>
<400>13
gct?agc?atg?tgg?ccc?cct?ggg?tca?gcc?tcc?cag?cca?ccg?ccc?tca 45
Met?Trp?Pro?Pro?Gly?Ser?Ala?Ser?Gln?Pro?Pro?Pro?Ser
1 5 10
cct?gcc?gcg?gcc?aca?ggt?ctg?cat?cca?gcg?gct?cgc?cct?gtg?tcc 90
Pro?Ala?Ala?Ala?Thr?Gly?Leu?His?Pro?Ala?Ala?Arg?Pro?Val?Ser
15 20 25
ctg?cag?tgc?cgg?ctc?agc?atg?tgt?cca?gcg?cgc?agc?ctc?ctc?ctt 135
Leu?Gln?Cys?Arg?Leu?Ser?Met?Cys?Pro?Ala?Arg?Ser?Leu?Leu?Leu
30 35 40
gtg?gct?acc?ctg?gtc?ctc?ctg?gac?cac?ctc?agt?ttg?gcc?aga?aac 180
Val?Ala?Thr?Leu?Val?Leu?Leu?Asp?His?Leu?Ser?Leu?Ala?Arg?Asn
45 50 55
ctc?ccc?gtg?gcc?act?cca?gac?cca?gga?atg?ttc?cca?tgc?ctt?cac 225
Leu?Pro?Val?Ala?Thr?Pro?Asp?Pro?Gly?Met?Phe?Pro?Cys?Leu?His
60 65 70
cac?tcc?caa?aac?ctg?ctg?agg?gcc?gtc?agc?aac?atg?ctc?cag?aag 270
His?Ser?Gln?Asn?Leu?Leu?Arg?Ala?Val?Ser?Asn?Met?Leu?Gln?Lys
75 80 85
gcc?aga?caa?act?cta?gaa?ttt?tac?cct?tgc?act?tct?gaa?gag?att 315
Ala?Arg?Gln?Thr?Leu?Glu?Phe?Tyr?Pro?Cys?Thr?Ser?Glu?Glu?Ile
90 95 100
gat?cat?gaa?gat?atc?aca?aaa?gat?aaa?acc?agc?aca?gtg?gag?gcc 360
Asp?His?Glu?Asp?Ile?Thr?Lys?Asp?Lys?Thr?Ser?Thr?Val?Glu?Ala
105 110 115
tgt?tta?cca?ttg?gaa?tta?acc?aag?aat?gag?agt?tgc?cta?aat?tcc 405
Cys?Leu?Pro?Leu?Glu?Leu?Thr?Lys?Asn?Glu?Ser?Cys?Leu?Asn?Ser
120 125 130
aga?gag?acc?tct?ttc?ata?act?aat?ggg?agt?tgc?ctg?gcc?tcc?aga 450
Arg?Glu?Thr?Ser?Phe?Ile?Thr?Asn?Gly?Ser?Cys?Leu?Ala?Ser?Arg
135 140 145
aag?acc?tct?ttt?atg?atg?gcc?ctg?tgc?ctt?agt?agt?att?tat?gaa 495
Lys?Thr?Ser?Phe?Met?Met?Ala?Leu?Cys?Leu?Ser?Ser?Ile?Tyr?Glu
150 155 160
gac?ttg?aag?atg?tac?cag?gtg?gag?ttc?aag?acc?atg?aat?gca?aag 540
Asp?Leu?Lys?Met?Tyr?Gln?Val?Glu?Phe?Lys?Thr?Met?Asn?Ala?Lys
165 170 175
ctt?ctg?atg?gat?cct?aag?agg?cag?atc?ttt?cta?gat?caa?aac?atg 585
Leu?Leu?Met?Asp?Pro?Lys?Arg?Gln?Ile?Phe?Leu?Asp?Gln?Asn?Met
180 185 190
ctg?gca?gtt?att?gat?gag?ctg?atg?cag?gcc?ctg?aat?ttc?aac?agt 630
Leu?Ala?Val?Ile?Asp?Glu?Leu?Met?Gln?Ala?Leu?Asn?Phe?Asn?Ser
195 200 205
gag?act?gtg?cca?caa?aaa?tcc?tcc?ctt?gaa?gaa?ccg?gat?ttt?tat 675
Glu?Thr?Val?Pro?Gln?Lys?Ser?Ser?Leu?Glu?Glu?Pro?Asp?Phe?Tyr
210 215 220
aaa?act?aaa?atc?aag?ctc?tgc?ata?ctt?ctt?cat?gct?ttc?aga?att 720
Lys?Thr?Lys?Ile?Lys?Leu?Cys?Ile?Leu?Leu?His?Ala?Phe?Arg?Ile
225 230 235
cgg?gca?gtg?act?att?gat?aga?gtg?atg?agc?tat?ctg?aat?gct?tcc 765
Arg?Ala?Val?Thr?Ile?Asp?Arg?Val?Met?Ser?Tyr?Leu?Asn?Ala?Ser
240 245 250
gga?tcc?tac?atc?tgg?gcg?ccc?ttg?gcc?ggg?act?tgt?ggg?gtc?ctt 810
Gly?Ser?Tyr?Ile?Trp?Ala?Pro?Leu?Ala?Gly?Thr?Cys?Gly?Val?Leu
255 260 265
ctc?ctg?tca?ctg?gtt?atc?acc?ctt?tac?tgc?taa?gcg?gcc?gc 851
Leu?Leu?Ser?Leu?Val?Ile?Thr?Leu?Tyr?Cys?＊
270 275
<210>14
<211>
<212>DNA
<213>IL-15CD8
<220>
<221>
<222>(7)...(570)
<223>
<400>14
gct?agc?atg?aga?att?tcg?aaa?cca?cat?ttg?aga?agt?att?tcc?atc 45
Met?Arg?Ile?Ser?Lys?Pro?His?Leu?Arg?Ser?Ile?Ser?Ile
1 5 10
cag?tgc?tac?ttg?tgt?tta?ctt?cta?aac?agt?cat?ttt?cta?act?gaa 90
Gln?Cys?Tyr?Leu?Cys?Leu?Leu?Leu?Asn?Ser?His?Phe?Leu?Thr?Glu
15 20 25
gct?ggc?att?cat?gtc?ttc?att?ttg?ggc?tgt?ttc?agt?gca?ggg?ctt 135
Ala?Gly?Ile?His?Val?Phe?Ile?Leu?Gly?Cys?Phe?Ser?Ala?Gly?Leu
30 35 40
cct?aaa?aca?gaa?gcc?aac?tgg?gtg?aat?gta?ata?agt?gat?ttg?aaa 180
Pro?Lys?Thr?Glu?Ala?Asn?Trp?Val?Asn?Val?Ile?Ser?Asp?Leu?Lys
45 50 55
aaa?att?gaa?gat?ctt?att?caa?tct?atg?cat?att?gat?gct?act?tta 225
Lys?Ile?Glu?Asp?Leu?Ile?Gln?Ser?Met?His?Ile?Asp?Ala?Thr?Leu
60 65 70
tat?acg?gaa?agt?gat?gtt?cac?ccc?agt?tgc?aaa?gta?aca?gca?atg 270
Tyr?Thr?Glu?Ser?Asp?Val?His?Pro?Ser?Cys?Lys?Val?Thr?Ala?Met
75 80 85
aag?tgc?ttt?ctc?ttg?gag?tta?caa?gtt?att?tca?ctt?gag?tcc?gga 315
Lys?Cys?Phe?Leu?Leu?Glu?Leu?Gln?Val?Ile?Ser?Leu?Glu?Ser?Gly
90 90 100
gat?gca?agt?att?cat?gat?aca?gta?gaa?aat?ctg?atc?atc?cta?gca 360
Asp?Ala?Ser?Ile?His?Asp?Thr?Val?Glu?Asn?Leu?Ile?Ile?Leu?Ala
105 110 115
aac?aac?agt?ttg?tct?tct?aat?ggg?aat?gta?aca?gaa?tct?gga?tgc 405
Asn?Asn?Ser?Leu?Ser?Ser?Asn?Gly?Asn?Val?Thr?Glu?Ser?Gly?Cys
120 125 130
aaa?gaa?tgt?gag?gaa?ctg?gag?gaa?aaa?aat?att?aaa?gaa?ttt?ttg 450
Lys?Glu?Cys?Glu?Glu?Leu?Glu?Glu?Lys?Asn?Ile?Lys?Glu?Phe?Leu
135 140 145
cag?agt?ttt?gta?cat?att?gtc?caa?atg?ttc?atc?aac?act?tct?gga 495
Gln?Ser?Phe?Val?His?Ile?Val?Gln?Met?Phe?Ile?Asn?Thr?Ser?Gly
150 155 160
tcc?tac?atc?tgg?gcg?ccc?ttg?gcc?ggg?act?tgt?ggg?gtc?ctt?ctc 540
Ser?Tyr?Ile?Trp?Ala?Pro?Leu?Ala?Gly?Thr?Cys?Gly?Val?Leu?Leu
165 170 175
ctg?tca?ctg?gtt?atc?acc?ctt?tac?tgc?taa?gcg?gcc?gc 578
Leu?Ser?Leu?Val?Ile?Thr?Leu?Tyr?Cys?＊
180 185
<210>15
<211>
<212>DNA
<213>IL-18CD8
<220>
<221>
<222>(7)...(663)
<223>
<400>15
gct?agc?atg?gct?gct?gaa?cca?gta?gaa?gac?aat?tgc?atc?aac?ttt 45
Met?Ala?Ala?Glu?Pro?Val?Glu?Asp?Asn?Cys?Ile?Asn?Phe
1 5 10
gtg?gca?atg?aaa?ttt?att?gac?aat?acg?ctt?tac?ttt?ata?gct?gaa 90
Val?Ala?Met?Lys?Phe?Ile?Asp?Asn?Thr?Leu?Tyr?Phe?Ile?Ala?Glu
15 20 25
gat?gat?gaa?aac?ctg?gaa?tca?gat?tac?ttt?ggc?aag?ctt?gaa?tct 135
Asp?Asp?Glu?Asn?Leu?Glu?Ser?Asp?Tyr?Phe?Gly?Lys?Leu?Glu?Ser
30 35 40
aaa?tta?tca?gtc?ata?aga?aat?ttg?aat?gac?caa?gtt?ctc?ttc?att 180
Lys?Leu?Ser?Val?Ile?Arg?Asn?Leu?Asn?Asp?Gln?Val?Leu?Phe?Ile
45 50 55
gac?caa?gga?aat?cgg?cct?cta?ttt?gaa?gat?atg?act?gat?tct?gac 225
Asp?Gln?Gly?Asn?Arg?Pro?Leu?Phe?Glu?Asp?Met?Thr?Asp?Ser?Asp
60 65 70
tgt?aga?gat?aat?gca?ccc?cgg?acc?ata?ttt?att?ata?agt?atg?tat 270
Cys?Arg?Asp?Asn?Ala?Pro?Arg?Thr?Ile?Phe?Ile?Ile?Ser?Met?Tyr
75 80 85
aaa?gat?agc?cag?cct?aga?ggt?atg?gct?gta?act?atc?tct?gtg?aag 315
Lys?Asp?Ser?Gln?Pro?Arg?Gly?Met?Ala?Val?Thr?Ile?Ser?Val?Lys
90 95 100
tgt?gag?aaa?att?tca?act?ctc?tcc?tgt?gag?aac?aaa?att?att?tcc 360
Cys?Glu?Lys?Ile?Ser?Thr?Leu?Ser?Cys?Glu?Asn?Lys?Ile?Ile?Ser
105 110 115
ttt?aag?gaa?atg?aat?cct?cct?gat?aac?atc?aag?gat?aca?aaa?agt 405
Phe?Lys?Glu?Met?Asn?Pro?Pro?Asp?Asn?Ile?Lys?Asp?Thr?Lys?Ser
120 125 130
gac?atc?ata?ttc?ttt?cag?aga?agt?gtc?cca?gga?cat?gat?aat?aag 450
Asp?Ile?Ile?Phe?Phe?Gln?Arg?Ser?Val?Pro?Gly?His?Asp?Asn?Lys
135 140 145
atg?caa?ttt?gaa?tct?tca?tca?tac?gaa?gga?tac?ttt?cta?gct?tgt 495
Met?Gln?Phe?Glu?Ser?Ser?Ser?Tyr?Glu?Gly?Tyr?Phe?Leu?Ala?Cys
150 155 160
gaa?aaa?gag?aga?gac?ctt?ttt?aaa?ctc?att?ttg?aaa?aaa?gag?gat 540
Glu?Lys?Glu?Arg?Asp?Leu?Phe?Lys?Leu?Ile?Leu?Lys?Lys?Glu?Asp
165 170 175
gaa?ttg?ggg?gat?aga?tct?ata?atg?ttc?act?gtt?caa?aac?gaa?gac 585
Glu?Leu?Gly?Asp?Arg?Ser?Ile?Met?Phe?Thr?Val?Gln?Asn?Glu?Asp
180 185 190
gga?tcc?tac?atc?tgg?gcg?ccc?ttg?gcc?ggg?act?tgt?ggg?gtc?ctt 630
Gly?Ser?Tyr?Ile?Trp?Ala?Pro?Leu?Ala?Gly?Thr?Cys?Gly?Val?Leu
195 200 205
ctc?ctg?tca?ctg?gtt?atc?acc?ctt?tac?tgc?taa?gcg?gcc?gc 671
Leu?Leu?Ser?Leu?Val?Ile?Thr?Leu?Tyr?Cys?＊
210 215
<210>16
<211>
<212>DNA
<213>4-1BB-F
<220>
<221>
<222>
<223>
<400>16
tcgctagcat?ggaatacgcc?tctgacgctt?cac 33
<210>17
<211>
<212>DNA
<213>4-1BB-R
<220>
<221>
<222>
<223>
<400>17
tcggatcctt?attccgacct?cggtgaaggg?ag 32
<210>18
<211>
<212>DNA
<213>4-1BB-R
<220>
<221>
<222>(7)...(624)
<223>
<400>18
gct?agc?atg?gaa?tac?gcc?tct?gac?gct?tca?ctg?gac?ccc?gaa?gcc 45
Met?Glu?Tyr?Ala?Ser?Asp?Ala?Ser?Leu?Asp?Pro?Glu?Ala
1 5 10
ccg?tgg?cct?ccc?gcg?ccc?cgc?gct?cgc?gcc?tgc?cgc?gta?ctg?cct 90
Pro?Trp?Pro?Pro?Ala?Pro?Arg?Ala?Arg?Ala?Cys?Arg?Val?Leu?Pro
15 20 25
ccc?gac?gat?ccc?gcc?ggc?ctc?ttg?gac?ctg?cgg?cag?ggc?atg?ttt 135
Pro?Asp?Asp?Pro?Ala?Gly?Leu?Leu?Asp?Leu?Arg?Gln?Gly?Met?Phe
30 35 40
gcg?cag?ctg?gtg?gcc?caa?aat?gtt?ctg?ctg?atc?gat?ggg?ccc?ctg 180
Ala?Gln?Leu?Val?Ala?Gln?Asn?Val?Leu?Leu?Ile?Asp?Gly?Pro?Leu
45 50 55
agc?tgg?tac?agt?gac?cca?ggc?ctg?gca?ggc?gtg?tcc?ctg?acg?ggg 225
Ser?Trp?Tyr?Ser?Asp?Pro?Gly?Leu?Ala?Gly?Val?Ser?Leu?Thr?Gly
60 65 70
ggc?ctg?agc?tac?aaa?gag?gac?acg?aag?gag?ctg?gtg?gtg?gcc?aag 270
Gly?Leu?Ser?Tyr?Lys?Glu?Asp?Thr?Lys?Glu?Leu?Val?Val?Ala?Lys
75 80 85
gct?gga?gtc?tac?tat?gtc?ttc?ttt?caa?cta?gag?ctg?cgg?cgc?gtg 315
Ala?Gly?Val?Tyr?Tyr?Val?Phe?Phe?Gln?Leu?Glu?Leu?Arg?Arg?Val
90 95 100
gtg?gcc?ggc?gag?ggc?tca?ggc?tcc?gtt?tca?ctt?gcg?ctg?cac?ctg 360
Val?Ala?Gly?Glu?Gly?Ser?Gly?Ser?Val?Ser?Leu?Ala?Leu?His?Leu
105 110 115
cag?cca?ctg?cgc?tct?gct?gct?ggg?gcc?gcc?gcc?ctg?gct?ttg?acc 405
Gln?Pro?Leu?Arg?Ser?Ala?Ala?Gly?Ala?Ala?Ala?Leu?Ala?Leu?Thr
120 125 130
gtg?gac?ctg?cca?ccc?gcc?tcc?tcc?gag?gct?cgg?aac?tcg?gcc?ttc 450
Val?Asp?Leu?Pro?Pro?Ala?Ser?Ser?Glu?Ala?Arg?Asn?Ser?Ala?Phe
135 140 145
ggt?ttc?cag?ggc?cgc?ttg?ctg?cac?ctg?agt?gcc?ggc?cag?cgc?ctg 495
Gly?Phe?Gln?Gly?Arg?Leu?Leu?His?Leu?Ser?Ala?Gly?Gln?Arg?Leu
150 155 160
ggc?gtc?cat?ctt?cac?act?gag?gcc?agg?gca?cgc?cat?gcc?tgg?cag 540
Gly?Val?His?Leu?His?Thr?Glu?Ala?Arg?Ala?Arg?His?Ala?Trp?Gln
165 170 175
ctt?acc?cag?ggc?gcc?aca?gtc?ttg?gga?ctc?ttc?cgg?gtg?acc?ccc 585
Leu?Thr?Gln?Gly?Ala?Thr?Val?Leu?Gly?Leu?Phe?Arg?Val?Thr?Pro
180 185 190
gaa?atc?cca?gcc?gga?ctc?cct?tca?ccg?agg?tcg?gaa?taa?gga?tcc 630
Glu?Ile?Pro?Ala?Gly?Leu?Pro?Ser?Pro?Arg?Ser?Glu?＊
195 200 205
Claims (5)
1, a kind of method of amplification in vitro NK cells of human beings is characterized in that with human peripheral blood single nucleus cell (PBMC) be original cultivated material, with the engineering cell co-cultivation of the stimulation NK cell growth of adopting engineered method to make up.
2, method according to claim 1 is characterized in that stimulating the engineering cell of NK cell growth, promotes some kinds of cytokine-expressings that the NK cell grows on passage cell film surface having.
3, method according to claim 2 is characterized in that cytokine at passage cell film surface expression comprises IL-2, IL-12, IL-15, IL-18, five kinds of albumen of 4-1BBL separately or co expression, and passage cell comprises K562 cell or HFWT cell.
4, method according to claim 3, it is characterized in that on the passage cell film expressing human IL-2, IL-12, IL-15, IL-18, five kinds of albumen of 4-1BBL be gene level with IL-2, IL-12, four kinds of proteic genes of IL-15, IL-18 respectively with the gene splicing of one section transmembrane protein; 4-1BBL itself contains and strides the film district, thus not with the gene splicing of transmembrane protein, with himself complete gene; Subsequently these genes are cloned into carrier for expression of eukaryon respectively, independent or mixing transfection passage cell makes up irritation cell.
5, method according to claim 4 when it is characterized in that stimulating the NK cell amplification, is used a kind of engineering cell of the some kinds of cytokines of expression of some kinds of gene co-transfections, or uses the combination of the engineering cell of the two or more at least expression single cell factors.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810198913A CN101684456A (en) | 2008-09-28 | 2008-09-28 | Method for amplifying NK cells of human beings under condition of in vitro culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810198913A CN101684456A (en) | 2008-09-28 | 2008-09-28 | Method for amplifying NK cells of human beings under condition of in vitro culture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101684456A true CN101684456A (en) | 2010-03-31 |
Family
ID=42047833
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810198913A Pending CN101684456A (en) | 2008-09-28 | 2008-09-28 | Method for amplifying NK cells of human beings under condition of in vitro culture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101684456A (en) |
Cited By (27)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174522A (en) * | 2011-02-24 | 2011-09-07 | 杭州师范大学 | Preparation method of protein 4-1BBL |
| CN102268405A (en) * | 2011-07-06 | 2011-12-07 | 安徽省立医院 | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof |
| CN102994449A (en) * | 2012-12-13 | 2013-03-27 | 上海柯莱逊生物技术有限公司 | Method for in-vitro amplification of NK cells |
| CN103484429A (en) * | 2013-09-28 | 2014-01-01 | 青岛麦迪赛斯生物科技有限公司 | Method for preparing NK (natural killer) cell |
| CN103937747A (en) * | 2014-03-19 | 2014-07-23 | 成都康景生物科技有限公司 | Recombinant multi-stimulus molecular cell, preparation method and application in NKT cell amplification thereof |
| CN105567634A (en) * | 2016-01-27 | 2016-05-11 | 上海润泉生物技术有限公司 | Culture medium and method for NK cell expansion in vitro |
| CN105838677A (en) * | 2016-05-20 | 2016-08-10 | 深圳市科晖瑞生物医药有限公司 | Method for high-efficient expansion and cryopreservation of NK (Natural Killer) cells and application of method |
| CN106222141A (en) * | 2016-10-17 | 2016-12-14 | 湖南丰晖生物科技有限公司 | NK cell culture fluid and cell culture processes |
| CN107177548A (en) * | 2017-06-26 | 2017-09-19 | 杭州中赢生物医疗科技有限公司 | A kind of cultivating system of amplification in vitro lymphocyte and amplification method and application |
| CN107760648A (en) * | 2010-07-13 | 2018-03-06 | 人类起源公司 | Produce the method for NK, thus obtained cell colony and application thereof |
| CN107779434A (en) * | 2016-08-30 | 2018-03-09 | 天津市康婷生物工程有限公司 | The experimental method of efficient amplification Human peripheral blood NK cells |
| WO2018182511A1 (en) * | 2017-03-27 | 2018-10-04 | National University Of Singapore | Stimulatory cell lines for ex vivo expansion and activation of natural killer cells |
| CN108823171A (en) * | 2018-07-24 | 2018-11-16 | 武汉赛云博生物科技有限公司 | A kind of method of genetically engineered cell and external efficient amplification NK cell |
| WO2018213731A1 (en) * | 2017-05-18 | 2018-11-22 | Modernatx, Inc. | Polynucleotides encoding tethered interleukin-12 (il12) polypeptides and uses thereof |
| CN109762785A (en) * | 2018-12-10 | 2019-05-17 | 吴江近岸蛋白质科技有限公司 | A kind of efficient application is in the method for the non-trophoderm in vitro culture of NK cells of human beings |
| CN109844099A (en) * | 2016-07-25 | 2019-06-04 | 美国政府(由卫生和人类服务部的部长所代表) | Generate the method and application method of modified natural killer cells |
| US10428305B2 (en) | 2014-05-15 | 2019-10-01 | National University Of Singapore | Modified natural killer cells that express IL15 and uses thereof |
| CN111349601A (en) * | 2020-01-14 | 2020-06-30 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Method for efficient in-vitro amplification culture of natural killer cells with strong killing power |
| CN112029720A (en) * | 2020-08-24 | 2020-12-04 | 海南优尼科尔生物科技有限公司 | Construction method of human peripheral blood NK cell bank |
| CN112391349A (en) * | 2019-08-19 | 2021-02-23 | 刘韬 | Trophoblast cell strain, preparation method thereof and method for in vitro induced amplification of NK cells |
| CN112538462A (en) * | 2020-12-25 | 2021-03-23 | 上海纳米技术及应用国家工程研究中心有限公司 | Cell membrane for rapid amplification of NK cells and application thereof |
| CN112852870A (en) * | 2021-01-13 | 2021-05-28 | 北京鼎成肽源生物技术有限公司 | Application of IL-18 gene fusion H2Kk gene in preparation of cell for enhancing cellular immunity, cell and preparation method |
| US11141436B2 (en) | 2019-03-05 | 2021-10-12 | Nkarta, Inc. | Immune cells engineered to express CD19-directed chimeric antigen receptors and uses thereof in immunotherapy |
| WO2021251707A1 (en) * | 2020-06-09 | 2021-12-16 | 사회복지법인 삼성생명공익재단 | Genetically-modified cell line for nk cell activation and amplification, and use thereof |
| WO2021251708A1 (en) * | 2020-06-09 | 2021-12-16 | 사회복지법인 삼성생명공익재단 | Genetically manipulated cell strain for activating and amplifying nk cells and use thereof |
| US11365236B2 (en) | 2017-03-27 | 2022-06-21 | Nkarta, Inc. | Truncated NKG2D chimeric receptors and uses thereof in natural killer cell immunotherapy |
| EP4003379A4 (en) * | 2019-07-31 | 2023-08-30 | Nkarta, Inc. | Methods and compositions for enhanced expansion and cytotoxicity of natural killer cells |
-
2008
- 2008-09-28 CN CN200810198913A patent/CN101684456A/en active Pending
Cited By (54)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828727A (en) * | 2010-07-13 | 2018-03-23 | 人类起源公司 | Produce the method for NK, thus obtained cell colony and application thereof |
| CN107760648A (en) * | 2010-07-13 | 2018-03-06 | 人类起源公司 | Produce the method for NK, thus obtained cell colony and application thereof |
| CN102174522A (en) * | 2011-02-24 | 2011-09-07 | 杭州师范大学 | Preparation method of protein 4-1BBL |
| CN102174522B (en) * | 2011-02-24 | 2013-11-06 | 杭州师范大学 | Preparation method of protein 4-1BBL |
| CN102268405A (en) * | 2011-07-06 | 2011-12-07 | 安徽省立医院 | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof |
| CN102994449A (en) * | 2012-12-13 | 2013-03-27 | 上海柯莱逊生物技术有限公司 | Method for in-vitro amplification of NK cells |
| CN103756963A (en) * | 2012-12-13 | 2014-04-30 | 上海柯莱逊生物技术有限公司 | Method used for in vitro proliferation of NK cells |
| CN103756963B (en) * | 2012-12-13 | 2019-10-29 | 上海柯莱逊生物技术有限公司 | A kind of method of amplification in vitro NK cell |
| CN103484429A (en) * | 2013-09-28 | 2014-01-01 | 青岛麦迪赛斯生物科技有限公司 | Method for preparing NK (natural killer) cell |
| CN103937747A (en) * | 2014-03-19 | 2014-07-23 | 成都康景生物科技有限公司 | Recombinant multi-stimulus molecular cell, preparation method and application in NKT cell amplification thereof |
| CN103937747B (en) * | 2014-03-19 | 2016-06-22 | 成都康景生物科技有限公司 | Restructuring stimulates molecular cell and preparation method thereof and the application in NKT cell amplification |
| US10428305B2 (en) | 2014-05-15 | 2019-10-01 | National University Of Singapore | Modified natural killer cells that express IL15 and uses thereof |
| US10774311B2 (en) | 2014-05-15 | 2020-09-15 | National University Of Singapore | Natural killer cells modified to express membrane-bound interleukin 15 and uses thereof |
| US11560548B2 (en) | 2014-05-15 | 2023-01-24 | National University Of Singapore | Immune cells expressing membrane-bound interleukin 15 (mbIL15) and uses thereof |
| CN105567634A (en) * | 2016-01-27 | 2016-05-11 | 上海润泉生物技术有限公司 | Culture medium and method for NK cell expansion in vitro |
| CN110358736B (en) * | 2016-05-20 | 2023-07-07 | 杭州朔溪生物医药有限公司 | Modified K562 cells, preparation method thereof and NK cell culture composition |
| CN105838677A (en) * | 2016-05-20 | 2016-08-10 | 深圳市科晖瑞生物医药有限公司 | Method for high-efficient expansion and cryopreservation of NK (Natural Killer) cells and application of method |
| CN110358736A (en) * | 2016-05-20 | 2019-10-22 | 杭州优善生物科技有限公司 | A kind of K562 cell of modification, preparation method and NK cell culture compositions |
| CN109844099B (en) * | 2016-07-25 | 2024-01-02 | 美国政府(由卫生和人类服务部的部长所代表) | Methods of producing modified natural killer cells and methods of use |
| CN109844099A (en) * | 2016-07-25 | 2019-06-04 | 美国政府(由卫生和人类服务部的部长所代表) | Generate the method and application method of modified natural killer cells |
| CN107779434A (en) * | 2016-08-30 | 2018-03-09 | 天津市康婷生物工程有限公司 | The experimental method of efficient amplification Human peripheral blood NK cells |
| CN106222141A (en) * | 2016-10-17 | 2016-12-14 | 湖南丰晖生物科技有限公司 | NK cell culture fluid and cell culture processes |
| JP2020516238A (en) * | 2017-03-27 | 2020-06-11 | ナショナル ユニヴァーシティ オブ シンガポール | Stimulator cell line for ex vivo expansion and activation of natural killer cells |
| CN110494557A (en) * | 2017-03-27 | 2019-11-22 | 新加坡国立大学 | For expanding the stimulation cell line with Activated NK Cells in vitro |
| KR20190133732A (en) * | 2017-03-27 | 2019-12-03 | 싱가포르국립대학교 | Stimulating Cell Lines for EX VIVO Expansion and Activation of Natural Killer Cells |
| JP7270253B2 (en) | 2017-03-27 | 2023-05-10 | ナショナル ユニヴァーシティ オブ シンガポール | Stimulatory cell lines for ex vivo expansion and activation of natural killer cells |
| US11896616B2 (en) * | 2017-03-27 | 2024-02-13 | National University Of Singapore | Stimulatory cell lines for ex vivo expansion and activation of natural killer cells |
| US11365236B2 (en) | 2017-03-27 | 2022-06-21 | Nkarta, Inc. | Truncated NKG2D chimeric receptors and uses thereof in natural killer cell immunotherapy |
| WO2018182511A1 (en) * | 2017-03-27 | 2018-10-04 | National University Of Singapore | Stimulatory cell lines for ex vivo expansion and activation of natural killer cells |
| KR102624509B1 (en) * | 2017-03-27 | 2024-01-12 | 싱가포르국립대학교 | Stimulatory cell lines for EX VIVO expansion and activation of natural killer cells |
| US11873327B2 (en) | 2017-05-18 | 2024-01-16 | Modernatx, Inc. | Polynucleotides encoding tethered interleukin-12 (IL12) polypeptides and uses thereof |
| WO2018213731A1 (en) * | 2017-05-18 | 2018-11-22 | Modernatx, Inc. | Polynucleotides encoding tethered interleukin-12 (il12) polypeptides and uses thereof |
| AU2018270111B2 (en) * | 2017-05-18 | 2022-07-14 | Modernatx, Inc. | Polynucleotides encoding tethered interleukin-12 (IL12) polypeptides and uses thereof |
| US11421011B2 (en) | 2017-05-18 | 2022-08-23 | Modernatx, Inc. | Polynucleotides encoding tethered interleukin-12 (IL12) polypeptides and uses thereof |
| CN107177548B (en) * | 2017-06-26 | 2021-01-05 | 杭州中赢生物医疗科技有限公司 | Culture system for in vitro lymphocyte amplification, amplification method and application |
| CN107177548A (en) * | 2017-06-26 | 2017-09-19 | 杭州中赢生物医疗科技有限公司 | A kind of cultivating system of amplification in vitro lymphocyte and amplification method and application |
| CN108823171A (en) * | 2018-07-24 | 2018-11-16 | 武汉赛云博生物科技有限公司 | A kind of method of genetically engineered cell and external efficient amplification NK cell |
| CN109762785B (en) * | 2018-12-10 | 2022-06-24 | 苏州近岸蛋白质科技股份有限公司 | Method for efficiently applying to in-vitro culture of human NK cell non-trophoblast |
| CN109762785A (en) * | 2018-12-10 | 2019-05-17 | 吴江近岸蛋白质科技有限公司 | A kind of efficient application is in the method for the non-trophoderm in vitro culture of NK cells of human beings |
| US11141436B2 (en) | 2019-03-05 | 2021-10-12 | Nkarta, Inc. | Immune cells engineered to express CD19-directed chimeric antigen receptors and uses thereof in immunotherapy |
| US11154575B2 (en) | 2019-03-05 | 2021-10-26 | Nkarta, Inc. | Cancer immunotherapy using CD19-directed chimeric antigen receptors |
| US11253547B2 (en) | 2019-03-05 | 2022-02-22 | Nkarta, Inc. | CD19-directed chimeric antigen receptors and uses thereof in immunotherapy |
| EP4003379A4 (en) * | 2019-07-31 | 2023-08-30 | Nkarta, Inc. | Methods and compositions for enhanced expansion and cytotoxicity of natural killer cells |
| CN112391349A (en) * | 2019-08-19 | 2021-02-23 | 刘韬 | Trophoblast cell strain, preparation method thereof and method for in vitro induced amplification of NK cells |
| CN112391349B (en) * | 2019-08-19 | 2023-05-23 | 刘韬 | Trophoblast cell strain, preparation method thereof and method for in-vitro induction amplification of NK cells |
| CN111349601A (en) * | 2020-01-14 | 2020-06-30 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Method for efficient in-vitro amplification culture of natural killer cells with strong killing power |
| CN115461450A (en) * | 2020-06-09 | 2022-12-09 | 社会福祉法人三星生命公益财团 | Genetically modified cell lines for activating and amplifying NK cells and uses thereof |
| WO2021251708A1 (en) * | 2020-06-09 | 2021-12-16 | 사회복지법인 삼성생명공익재단 | Genetically manipulated cell strain for activating and amplifying nk cells and use thereof |
| WO2021251707A1 (en) * | 2020-06-09 | 2021-12-16 | 사회복지법인 삼성생명공익재단 | Genetically-modified cell line for nk cell activation and amplification, and use thereof |
| JP7477120B2 (en) | 2020-06-09 | 2024-05-01 | サムスン ライフ パブリック ウェルフェア ファウンデーション | Genetically engineered cell lines for NK cell activation and expansion and uses thereof |
| CN112029720A (en) * | 2020-08-24 | 2020-12-04 | 海南优尼科尔生物科技有限公司 | Construction method of human peripheral blood NK cell bank |
| CN112538462B (en) * | 2020-12-25 | 2023-02-14 | 上海纳米技术及应用国家工程研究中心有限公司 | Cell membrane for rapid amplification of NK cells and application thereof |
| CN112538462A (en) * | 2020-12-25 | 2021-03-23 | 上海纳米技术及应用国家工程研究中心有限公司 | Cell membrane for rapid amplification of NK cells and application thereof |
| CN112852870A (en) * | 2021-01-13 | 2021-05-28 | 北京鼎成肽源生物技术有限公司 | Application of IL-18 gene fusion H2Kk gene in preparation of cell for enhancing cellular immunity, cell and preparation method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101684456A (en) | Method for amplifying NK cells of human beings under condition of in vitro culture | |
| JP7292213B2 (en) | Compositions and methods for gene editing in T cells using CRISPR/CPF1 | |
| Assarsson et al. | CD8+ T cells rapidly acquire NK1. 1 and NK cell-associated molecules upon stimulation in vitro and in vivo | |
| CN103756964B (en) | A kind of efficient amplification CD3 -cD56 +the method of natural killer cell culture systems | |
| DE69334062T2 (en) | In vitro activation of cytotoxic T cells | |
| Stoiber et al. | TYK2 is a key regulator of the surveillance of B lymphoid tumors | |
| CN105008521B (en) | The method for adjusting the immunoregulation effect of stem cell | |
| CN105567634A (en) | Culture medium and method for NK cell expansion in vitro | |
| CN109715171A (en) | Genome editor's enhancer | |
| CN101801374B (en) | An agent for differentiating hematopoietic stem cell into natural killer cell comprising YC-1 or IL-21 and a method of differentiating hematopoietic stem cell into natural killer cell using thereof | |
| CN105848484A (en) | Polyclonal [Gamma] [delta] T cells for immunotherapy | |
| JP2002516562A (en) | Modified rapid expansion method for in vitro expansion of T lymphocytes ("modified REM") | |
| Carlyle et al. | Natural killer cell development and function precede αβ T cell differentiation in mouse fetal thymic ontogeny | |
| CN110055223A (en) | A kind of preparation method and applications of the immunodeficient animals of B2m genetic modification | |
| Rahat et al. | Cultivation of bacteria-free Hydra viridis: missing budding factor in nonsymbiotic hydra | |
| BR112020014803A2 (en) | MODIFICATION OF GENOMIC LOCIES RELATED TO THE IMMUNE SYSTEM USING CRISPR NICKASES PAIRED RIBONUCLEOPROTEINS | |
| MORGAN et al. | Alloantigen-dependent endothelial phenotype and lymphokine mRNA expression in rejecting murine cardiac allografts | |
| KR860000900B1 (en) | Producing method of human luteinizing hormone | |
| JPH07504437A (en) | Use of interleukin-10 to suppress graft-versus-host disease | |
| Seif et al. | Long-term protection from syngeneic acute lymphoblastic leukemia by CpG ODN-mediated stimulation of innate and adaptive immune responses | |
| CN109929875A (en) | A kind of construction method of LAG3 gene humanized animal's model and its application | |
| CN112251463B (en) | Construction method of CD73 humanized mouse model | |
| Song et al. | CAR-T cell expansion platforms yield distinct T cell differentiation states | |
| KR20210152935A (en) | Genetically engineered cell line for activating and expanding natural killer cells, and uses thereof | |
| CN112841458A (en) | Specific feed for feeding springtail and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| DD01 | Delivery of document by public notice |
Addressee: Jiangmen Luosen Bio-Pharmaceutical Co., Ltd. Document name: Notification that Application Deemed to be Withdrawn |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20100331 |