CN1879892B - Method for establishing human extranodal rhinoid NK/T cell lymphoma animal model - Google Patents
Method for establishing human extranodal rhinoid NK/T cell lymphoma animal model Download PDFInfo
- Publication number
- CN1879892B CN1879892B CN200610020706A CN200610020706A CN1879892B CN 1879892 B CN1879892 B CN 1879892B CN 200610020706 A CN200610020706 A CN 200610020706A CN 200610020706 A CN200610020706 A CN 200610020706A CN 1879892 B CN1879892 B CN 1879892B
- Authority
- CN
- China
- Prior art keywords
- tumor
- cell
- cell lymphoma
- animal model
- human
- 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.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 55
- 208000016937 Extranodal nasal NK/T cell lymphoma Diseases 0.000 title claims abstract description 44
- 238000010171 animal model Methods 0.000 title claims abstract description 44
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 118
- 210000001331 nose Anatomy 0.000 claims description 45
- 210000004027 cell Anatomy 0.000 claims description 37
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 16
- 229930182555 Penicillin Natural products 0.000 claims description 8
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 8
- 229940049954 penicillin Drugs 0.000 claims description 8
- 229960005322 streptomycin Drugs 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000006285 cell suspension Substances 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 230000008520 organization Effects 0.000 claims 1
- 238000011580 nude mouse model Methods 0.000 abstract description 35
- 230000012010 growth Effects 0.000 abstract description 8
- 238000011081 inoculation Methods 0.000 abstract description 8
- 238000002054 transplantation Methods 0.000 abstract description 8
- 239000003814 drug Substances 0.000 abstract description 2
- 230000007547 defect Effects 0.000 abstract 1
- 210000004881 tumor cell Anatomy 0.000 description 37
- 210000001519 tissue Anatomy 0.000 description 33
- 241000699660 Mus musculus Species 0.000 description 31
- 108090000623 proteins and genes Proteins 0.000 description 21
- 108020004414 DNA Proteins 0.000 description 14
- 238000011084 recovery Methods 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 12
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 11
- 238000007901 in situ hybridization Methods 0.000 description 11
- 210000002784 stomach Anatomy 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 238000009396 hybridization Methods 0.000 description 9
- 230000008707 rearrangement Effects 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 230000004709 cell invasion Effects 0.000 description 8
- 210000001165 lymph node Anatomy 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 101150113776 LMP1 gene Proteins 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000011156 evaluation Methods 0.000 description 7
- 210000003734 kidney Anatomy 0.000 description 7
- 210000000056 organ Anatomy 0.000 description 7
- 208000011581 secondary neoplasm Diseases 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 6
- 210000004072 lung Anatomy 0.000 description 6
- 238000007920 subcutaneous administration Methods 0.000 description 6
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 5
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 5
- 108091035242 Sequence-tagged site Proteins 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 231100000433 cytotoxic Toxicity 0.000 description 5
- 230000001472 cytotoxic effect Effects 0.000 description 5
- 210000003194 forelimb Anatomy 0.000 description 5
- 210000003491 skin Anatomy 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- 206010025323 Lymphomas Diseases 0.000 description 4
- 210000002593 Y chromosome Anatomy 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 238000011532 immunohistochemical staining Methods 0.000 description 4
- 230000008595 infiltration Effects 0.000 description 4
- 238000001764 infiltration Methods 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 206010061289 metastatic neoplasm Diseases 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 102100032620 Cytotoxic granule associated RNA binding protein TIA1 Human genes 0.000 description 3
- 101710086368 Cytotoxic granule associated RNA binding protein TIA1 Proteins 0.000 description 3
- 206010015108 Epstein-Barr virus infection Diseases 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 210000003928 nasal cavity Anatomy 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 2
- 102100021260 Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Human genes 0.000 description 2
- 206010061968 Gastric neoplasm Diseases 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 2
- 101000894906 Homo sapiens Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Proteins 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 2
- 206010027336 Menstruation delayed Diseases 0.000 description 2
- 206010028851 Necrosis Diseases 0.000 description 2
- 240000002853 Nelumbo nucifera Species 0.000 description 2
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 2
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000002490 cerebral effect Effects 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 238000007877 drug screening Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 238000002695 general anesthesia Methods 0.000 description 2
- 210000004907 gland Anatomy 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 229960002275 pentobarbital sodium Drugs 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001902 propagating effect Effects 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 210000004872 soft tissue Anatomy 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 210000001835 viscera Anatomy 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 206010004173 Basophilia Diseases 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 108091034120 Epstein–Barr virus-encoded small RNA Proteins 0.000 description 1
- 206010019695 Hepatic neoplasm Diseases 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 208000007452 Plasmacytoma Diseases 0.000 description 1
- 201000000660 Pyloric Stenosis Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 206010040943 Skin Ulcer Diseases 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 108700042082 T-Cell Receptor gamma Genes Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 206010064390 Tumour invasion Diseases 0.000 description 1
- 208000012886 Vertigo Diseases 0.000 description 1
- 206010000059 abdominal discomfort Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002230 centromere Anatomy 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000000028 corpus adiposum pararenale Anatomy 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 1
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 201000006569 extramedullary plasmacytoma Diseases 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000009432 framing Methods 0.000 description 1
- 101150034785 gamma gene Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000001182 human Y chromosome Anatomy 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 231100000019 skin ulcer Toxicity 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
- 231100000889 vertigo Toxicity 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000002689 xenotransplantation Methods 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of biological medicine, and relates to a method for establishing an animal model of human extranodal rhinopathy type NK/T cell lymphoma. The method comprises a, inoculation; b. passage; c. identification and the like, and the main parameters of each step are optimized, thereby overcoming the defects of the prior art. The method has the advantages of short passage time, high success rate of one-time inoculation of each generation, rapid and accurate identification result, and is far superior to the prior art, and the animal model strain of the human extranodal rhinoid NK/T cell lymphoma which can be stably passaged is established. The nude mouse transplantation tumor model established by the method reproduces the important biological characteristics of the human extranodal rhinoid NK/T cell lymphoma, is basically consistent with donor cases in all aspects, has stable growth cycle and good application prospect.
Description
Technical field
The invention belongs to biomedicine field, specifically, relate to the method that a kind of people of foundation ties external nose type NK/T cell lymphoma animal model.
Background technology
It is a kind of tumor with the unique form, immunophenotype and biological behaviour that the people ties external nose type NK/T cell lymphoma (human extranodal nasal-type NK/T celllymphoma), pilosity is born in the outer organ or tissue of knot, infect height correlation with Epstein-Barr virus (EBV), but EBV be this lymphadenomatous paathogenic factor or satellite phenomenon do not have clearly final conclusion so far.This tumor is especially higher at Chinese sickness rate in the Asia, and West Europe and north America region are rare.The knot external nose type NK/T cell lymphoma of going to a doctor in Huaxi Hospital Attached to Sichuan Univ Pathology Deparment in past 21 years has 1012 examples, account for all lymphadenomatous 13.83%.Such lymphoma is an invasive tumor, and disease progression is rapid, and five year survival rate only is about 17%.
Be born in the center line face because of this tumor pilosity, how less local biopsy is, remains little except that doing the detection of pathological diagnosis and immunophenotype, and the scarcity that historical facts or anecdotes is tested material is the major obstacle that restriction is furtherd investigate and treated and carry out drug screening this tumor.Up to now, a few knot external nose type NK/T cell lymphoma cell system that is set up by Japanese scholar is only arranged, as HANK1 (Kagami etc., 1998), NK-YS (Tsuchiyama etc., 1998), SNK-6 and SNT-8 (Nagata etc., 2001).Because tumor cell is at In vitro culture, left internal milieu, through the cultivation of going down to posterity for a long time, eliminated the interaction between cells in vivo and the cell, can not be complete the biological behaviour and the self-sow process of simulation knot external nose type NK/T cell lymphoma, also can cause the change of cellular biology of tumor characteristic and hereditism's feature, and then influence the accuracy of experimental result and the reliability of conclusion.
Document " nude mice of people's nose type NK/T cell lymphoma is transplanted and goes down to posterity " discloses the method for building up that a kind of people ties the animal model of external nose type NK/T cell lymphoma in (Chinese pathology magazine in April, 2004), but this method biases toward the universality method that general animal model for tumour is set up of using, and the characteristics of people not being tied external nose type NK/T cell lymphoma are optimized targetedly, as: two anti-concentration are selected, the go down to posterity selection in cycle, the selection of cell concentration and the selection in gene sequence label site etc. when the freeze-stored cell recovery is gone down to posterity, therefore also exist and transplant difficulty, pollute easily, the cycle instability that goes down to posterity (can only be passaged to for the 9th generation at most), be difficult for identifying accurately whether modeling successfully waits weak point, and do not find the advanced tumor lung as yet in the tumor model of this method foundation, the transfer case of internal organs such as kidney is not observed situations such as vertigo moves yet.Simultaneously, do not relate to the content of the kind evaluation aspect of transplanted tumor in this method, still can not confirm modeling success exactly, make this animal model can't be directly used in the research of aspects such as genetic treatment of tumor and drug screening or its use also is restricted.
Therefore, a kind of method that the people ties the animal model of external nose type NK/T cell lymphoma of setting up that can overcome the prior art shortcoming is badly in need of in current this area.
Summary of the invention
The invention provides the method that a kind of people of foundation ties external nose type NK/T cell lymphoma animal model.This method may further comprise the steps:
A, inoculation: choose fresh no downright bad tumor tissue, insert immediately in 1640 culture medium that are added with penicillin and streptomycin, and will be organized in and be cut into about 1mm under the aseptic condition
3The size piece of tissue is seeded to and is in the narcose nude mouse, and the concentration of described penicillin and streptomycin is respectively 100～1000u/ml and 100～1000ug/ml;
B, go down to posterity: after transplanting first, the a method is got the transplanted tumor tissue and is inoculated and go down to posterity set by step after the 25th～28 week, 2nd, 3 generation times in generation were 92～98 days, and the 4th～10 generation time in generation was 28～32 days, will change 18～22 days the cycle of going down to posterity into after reaching for the 10th～15 generation;
C, evaluation.
Wherein, the time of completing steps a is no more than 30 minutes, and the nude mice of being adopted all is that Mus age is the nude mice in 3～4 weeks.
Authentication method described in the step c is to detect with round pcr whether the paraspecific gene sequence label of people site (Sequence Tagged Sites, STS) existence are arranged in the transplanted tumor.
Preferably, when being the male mankind for the tumor individuality, described gene sequence label site is human male gender decision gene sequence label site SY14.
Authentication method described in the inventive method step c can also be to recover for the freeze-stored cell of transplanted tumor and cryopreserved tissue piece to go down to posterity with each, and the recovery propagating method may further comprise the steps:
1), frozen tumor cell or cryopreserved tissue piece being carried out water-bath thaws;
2), with the 1640 culture medium washing that is added with penicillin and streptomycin, the concentration of described penicillin and streptomycin is respectively 100～1000u/ml and 100～1000ug/ml;
3), get 0.5ml～1.0ml cell suspension or 0.5～0.8cm
3Piece of tissue be inoculated in and be in the narcose nude mouse.
The condition that the described water-bath of step 1) is thawed is 37～41 ℃, in 30 seconds; The cell concentration of cell suspension is 10
7～10
8/ ml.
Owing to the human entity tumor tissue is carried out the difficulty of chromosome karyotype analysis, whether set up success in order to confirm model fast and accurately, adopted in the authentication step of the inventive method with whether having in the round pcr detection transplanted tumor and the paraspecific gene sequence label of people site (Sequence Tagged Sites, STS) method of consistent nucleotide sequence existence.This method is easy and simple to handle, expense is cheap, and the result is accurate.In addition, when the tumor donor of modeling is the male mankind, (Sequence Tagged Sites, STS) SY14 then is a kind of good label, can confirm modelling rapidly and accurately not have human male's sex determining gene sequence tagged site of homology with mice.In addition, this method can also detect different tissues, to follow the tracks of the transfer spread condition of transplanted tumor rapidly and accurately.Simultaneously, also comprised the method that each is recovered and go down to posterity for the freeze-stored cell and the cryopreserved tissue piece of transplanted tumor in the authentication step of the inventive method, this method can verify further that animal model is set up and successfully reach stable going down to posterity that recovery propagating method of the present invention can also be applied to the aspects such as foundation of cell culture and cell line simultaneously.In the inventive method the people being tied the operation that the frozen tumor cell of external nose type NK/T cell lymphoma nude mice animal model and the recovery of cryopreserved tissue piece go down to posterity optimizes, the recovery that makes the people tie the freeze-stored cell of the external nose type NK/T cell lymphoma success rate that goes down to posterity has reached 100%, and the general cell concentration that uses of classical way is 10
6/ ml, its recovery success rate is generally 50～100%, has solved the prior art recovery low difficult problem of success rate that goes down to posterity.
Beneficial effect of the present invention is: set up the method that the people ties the animal model of external nose type NK/T cell lymphoma, it has overcome animal model that present people ties external nose type NK/T cell lymphoma and has set up all difficult problems in the process, set up and to have stablized the animal model strain system (can reach for 35 generations at least) that the people of going down to posterity ties external nose type NK/T cell lymphoma, generation time short (20 days), and a success ratio of inoculation in per generation reaches 100%, qualification result is accurate rapidly, be much better than prior art. the experiment proved that, the transplanted tumor in nude mice model that uses the inventive method to set up has reproduced the important biomolecule that the people ties external nose type NK/T cell lymphoma and has learned feature, from pathomorphology, immunophenotype, the EBV infection conditions, aspect such as molecular biological characteristics and molecular genetics all with donor case basically identical, growth cycle is stable. not only solved the problem of this tumor research material scarcity, simultaneously owing to simulated this tumor at the intravital growing state of people, research to the evolution of the molecular mechanism of this tumor invasion and tumor, the exploration of treatment means, the screening of medicine and evaluation all have important practical value, have good application prospects.
Description of drawings
Fig. 1 people ties animal model donor primary tumor morphology of external nose type NK/T cell lymphoma and immunophenotype observed result.
Wherein: 1A is morphology; 1B is CD3 ε; 1C is CD8; 1D is CD56; 1E is a Cytotoxic cell proteinase-1; 1F is EBER1/2-ISH.
Fig. 2 people ties the animal model donor secondary tumor morphology of external nose type NK/T cell lymphoma and immunophenotype is observed.Wherein: 2A is morphology; 2B is CD3 ε; 2C is CD56; 2D is a Cytotoxic cell proteinase-1; 2E is EBER1/2-ISH; 2F is LMP1.
Fig. 3 people ties external nose type NK/T cell lymphoma animal model transplanted tumor, shows former 30 generation of generation to the animal model.Wherein: 3A is F1 generation; 3B is F4 generation; 3C is F5 generation; 3D is F10 generation; 3E is F11 generation; 3F is F12 generation; 3G is F15 generation; 3H is F20 generation; 3I is that F20 is for recovery; 3J is F25 generation; 3K is F27 generation; 3L is F30 generation.Each arrow all points to transplanted tumor among the figure, the visible forelimb axillary gland enlargement of the 4th, 5 and 27 generations, the visible local skin diabrosis of the 11st, 12 and 25 generations.
The pcr amplification testing result of Fig. 4 SY14.Wherein M is a standard molecular weight; P is the human normal structure sample of male; C ties external nose type NK/T cell lymphoma tissue samples for the people; 1,5,10,15,20,30 be respectively the 1st, 5,10,15,20,30 generation model the transplanted tumor tissue samples; Mm is the normal mouse tissue samples; B is a blank.
The FISH testing result of Fig. 5 SNK-6.
The FISH testing result of Fig. 6 transplanted tumor.
Fig. 7 FCM detects transplanted tumor HLA-ABC result.Wherein: axis of ordinates (PMT2LOG) expression contrast fluorescence intensity, reflection total cellular score; Axis of abscissas (PMT3LOG) expression HLB-ABC fluorescence intensity; Reflection HLA-ABC positive cell sum.
Fig. 8 people ties animal model transplanted tumor morphology of external nose type NK/T cell lymphoma and immunophenotype.Wherein: 8A is morphology; 8B is CD3 ε; 8C is CD56; 8D is a Cytotoxic cell proteinase-1; 8E is TIA-1; 8F is EBER1/2-ISH; 8D is LMP1.
Fig. 9 people ties external nose type NK/T cell lymphoma animal model transplanted tumor spread condition.Wherein: 9A is a liver; 9B is a lymph node; 9C is a spleen; 9D is a kidney; 9E is a lung; 9F is a peripheral blood.
Figure 10 T
VG/ J
JXThe result that the special primer pcr amplification detects.Wherein M is a standard molecular weight; P is human normal structure sample; 1,5,10,15,20,30 be respectively the 1st, 5,10,15,20,30 generation model the transplanted tumor tissue samples; B is a blank.
The result that Figure 11 detects with VH/JH special primer pcr amplification.Wherein M is a standard molecular weight; P is human normal structure sample; 1,5,10,15,20,30 be respectively the 1st, 5,10,15,20,30 generation model the transplanted tumor tissue samples; B is a blank.
The invention will be further described below in conjunction with the description of accompanying drawing by the specific embodiment, but this is not a limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modification or improvement, only otherwise break away from basic thought of the present invention, all within the scope of the present invention.
The specific embodiment
Embodiment one usefulness the inventive method is set up the animal model that the people ties external nose type NK/T cell lymphoma
1, the people ties the collection and the evaluation of external nose type NK/T cell lymphoma tissue samples
The people ties external nose type NK/T cell lymphoma 1 example in the collection Huaxi Hospital Attached to Sichuan Univ operation case.Detect through pathomorphology inspection, immunophenotype and EBER hybridization in situ technique, make a definite diagnosis to the people and tie external nose type NK/T cell lymphoma.The patient has accepted radiotherapy and chemotherapy (CHOP), and after 2 courses of treatment, local patholoic change disappears, and reaches clinical remission.Go to a doctor once more because of heating, stomach discomfort behind the patient, be diagnosed as stomach Secondary cases knot external nose type NK/T cell lymphoma.Because the pyloric obstruction that carrying out property increases the weight of, the patient is treated surgically.The gastric tumor tissue that excision is chosen in operation is simultaneously planted in nude mice subcutaneous, sees Fig. 1～2.
2, laboratory animal
BALB/c (nu/nu) nude mice, Mus age was 3～4 weeks, body weight 15～20 grams, male female dual-purpose is raised in the SPF barrier system, is provided by Sichuan University's West China animal center.
3, tumor tissues is transplanted
Corrective surgery is chosen the gastric tumor tissue of excision simultaneously, the vivo tumor fresh, that matter is tender organized to insert immediately in Hank ' the s liquid clean, put into then and be added with penicillin and streptomycin (its concentration is respectively the PRMl RPMI-1640 of 100～1000u/ml and 100～1000ug/ml), and will be organized in and be cut into about 1mm * 1mm * 1mm size piece of tissue under the aseptic condition, make nude mice be in narcotism with 0.6% pentobarbital sodium lumbar injection, it is subcutaneous rapidly tumor tissues to be seeded to nude mice forelimb axillary fossa then.
4, transplanted tumor is observed and is gone down to posterity between Mus first
Day by day observe nude mice whole body situation after the inoculation, transplantation site has or not infection, and transplanted tumor has or not growth or spontaneous regression, and transplantation site skin has or not diabrosis and lymph node to have or not enlargement etc.After finding that transplanted tumor begins growth, measure the size of tumor weekly.
When surpassing 1cm, the diameter of transplanted tumor goes down to posterity between the row Mus: under the pentobarbital sodium lumbar injection general anesthesia 0.6%, tumor is got in operation, be cut into about 1mm * 1mm * 1mm piece of tissue, nude mice (Mus 3～4 weeks of age) the forelimb axillary fossa of planting under the general anesthesia state is subcutaneous, same this tumor bearing nude mice whole body situation that goes down to posterity of observing, transplantation site has or not infection, and transplanted tumor has or not growth or spontaneous regression, and transplantation site skin has or not diabrosis and lymph node to have or not enlargement etc.Measure the size of tumor weekly.
5, go down to posterity between transplanted tumor observation and Mus
In former generation,, transplanted tumor reached for 12 weeks incubation period, and tumor begins growth after three months, and behind the slow trophophase through 10 weeks, tumor is grown to 1.38 * 1.0 * 1.1cm gradually
3, the 28th week went down to posterity.2nd, 3 generation times in generation were 92～98 days, and growth cycle shortens and stable gradually after the 4th generation, and the 4th～10 generation time in generation was 28～32 days, after the 10th generation, went down to posterity about about 20 day week, existing transplanted tumor has been stable reach the 35th generation (see figure 3).
6, inoculation result
Success ratio of inoculation: 10 of the 32nd pickup kinds, success rate 100%.
Embodiment two people tie the external nose type NK/T cell lymphoma freeze-stored cell recovery experiment of going down to posterity:
Get the frozen tumor cell in the 5th generation, 6 generations, 8 generations, 10 generations, 16 generations, 18 generations, 20 generations, 25 generations and 30 generations of the animal model that embodiment one sets up.Respectively above-mentioned freeze-stored cell is thawed rapidly (in 30 seconds) in 37～41 ℃ of water-baths, then in superclean bench with the 1640 culture medium washed cells 2～3 times that are added with penicillin and streptomycin, adjust cell concentration to 10 then
7-8/ ml, getting 0.5ml～1.0ml, to be inoculated in Mus be that the healthy nude mice forelimb axillary fossa in 3～4 weeks is subcutaneous age.Attention will be finished the operation of going down to posterity in 30 minutes.
The result as seen, the recovery success rate is 100%, recovery transplanted tumor in nude mice and former source tumor growth cycle and the similar (see figure 3) of lotus tumor situation.And the general cell concentration that uses of classical way is 10
6/ ml, its recovery success rate is generally 50～100%.
Embodiment three people tie the evaluation of external nose type NK/T cell lymphoma animal model
(1) certified variety
1 animal model transplanted tumor kind is identified
1.1 human source gene is identified
Because of people and musculus cdna group height homology, and animal model transplanted tumor tumor tissues derives from patient male.The query gene storehouse found that: (Sequence Tagged Sites, STS) SY14 and mice do not have homology to human male's sex determining gene sequence tagged site.So,, then confirm the modelling success as the existence of human source gene SY14 in the proof animal model transplanted tumor with having or not the SY14 gene in the round pcr detection transplanted tumor.
1.2 fluorescence in situ hybridization (FISH) technology for detection people Y chromosome
The probe that this research is adopted is JAB-Y (DYZ1) (Yq12 SatelliteIII) dna probe (Sino-U.S.'s cooperation Shanghai collection likes that heredity and sterile diagnosis and treatment center provide) of the direct labelling of Spectrum Orange fluorescein.The hybridization site of probe is the Y chromosome centromere, is labeled as redness.With the positive contrast of SNK-6 (people's lymphoma extranodal NK/Tcell cell strain derives from the male), with ddH
2O replaces the probe mixed liquor to make blank.Adopt OLYMPUSMICROSCOPE-AX80 fluorescence microscope result, exciting light is blue and red.Fluorescence signal when gathering exciting light for blue and redness respectively by computer under the same visual field, machine is synthetic automatically as calculated again, just can see red and blue-fluorescence signal simultaneously in a visual field.Seeing the red fluorescence signal in female transplanted tumor in nude mice tumor cell is in the provable animal model transplanted tumor people's Y chromosome to be arranged.
1.3 (flow cytometer FCM) detects human leucocyte differentiation antigen (HLA-ABC) and carries out according to a conventional method flow cytometry.
2 PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM and immunophenotype detect
Immunohistochemical staining adopts SP (streptavidin-biotin immunoperoxidase) method.Used first antibody title, clone number, source and specificity see Table 1.
Carry out Flow cytometry with fluorescein-labeled antibody CD45RO, CD5, CD1a, CD7, CD117, CD34 simultaneously.
Table 1 immunophenotype detects used antibody and specificity catalog thereof
* be polyclonal antibody
3 animal model transplanted tumor Epstein-Barr virus detect
3.1EB the virus in situ hybridization technique detects
Method one: the EBER1/2 oligonucleotide probe with the FITC labelling is made in situ hybridization on paraffin section, and the anti-FITC antibody of bridge joint alkali phosphatase enzyme mark is that alkaline phosphatase substrate detects hybridization signal with NBT/BCIP.With the male nose NK/T of the known EBER1/2 cell lymphoma paraffin-embedded tissue positive comparison film of cutting into slices, it is blank that DEPC water substitutes the hybridization solution that contains probe.
Method two: the EBER1/2 oligonucleotide probe with digoxigenin labeled is made in situ hybridization on paraffin section, is one anti-with the mouse-anti DigiTAb, uses the SP method, DAB color developing detection hybridization signal.Contrast is set up with method one.
The in situ hybridization signal framing is in nucleus.The employing method for the moment, positive hybridization signal is a hyacinthine; When adopting method two, positive hybridization signal is pale brown color.
3.2EB virus lays dormant memebrane protein LMP1 expresses
The LMP1 monoclonal antibody is DAKO company product (DAKO-M0897, clone number be CS1-4).Adopt the SP method, the LMP1 positive signal is positioned at cell membrane and endochylema.
4 animal model transplanted tumor molecular biological characteristics detect
4.1T cell receptor (TCR) γ gene rearrangement is analyzed
Adopt T
VG/ J
JXPrimer (T
VGPrimer: 5 '-AGGGTTGTGTTGGAATCAGG-3 ', see SEQ ID No.4; J
JXPrimer: 5 '-CGTCGACAACAAGTGTTGTTCCAC-3 ', see SEQID No.5) carry out PCR, detect TCR γ gene rearrangement.
4.2 heavy chain immunoglobulin (IgH) gene rearrangement analysis
Employing VH/JH primer (the VH primer: 5 '-CTGTCGACACGGCCGTGTA TTACTG-3 ', see SEQ ID No.6; The JH primer: 5 '-AACTGCAGAGGAGACGGTGACC-3 ', see SEQ ID No.7) carry out PCR, detect IgH gene rearrangement.
(2) experimental result
1 transplanted tumor in nude mice the race identify
1.1 human source gene is identified
Auele Specific Primer with human sequence tagged site SY14:
Forward primer: 5 '-CGCCAGGGTTTTCCCAGTCACG-3 ' (SEQ ID No.2);
Downstream primer: 5 '-GAGCGGATAACAATTTCACACAGG-3 ' (SEQ ID No.3);
With the positive contrast of male's reactive hyperplasia of lymph node case (P), the male nude mouse normal structure is done negative control, ddH
2It is blank that O replaces template DNA (B), and donor tissue (C) and the 1st, 5,10,15,20 and 30 generation transplanted tumor in nude mice DNA are carried out pcr amplification, and (the amplified production clip size is 472～500bp) specific band to have occurred.
The dna sequencing result (sees Table 2 with human sequence's label site SY14 sequence (SEQ ID No.1) is consistent, Query represents to require the sequence of comparison, Sbjct represents known array in the gene bank), illustrate to have human source gene (agarose gel electrophoresis the results are shown in Figure 4) in the transplanted tumor.
Order-checking of table 2SY14PCR product and gene bank sequence comparative result
Query?2 AGCTCTTCCTTCCTTTGCACTGAAAGCTGTAACTCTAAGTATCAGTGTGAAACGGGAGAA 61
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct?104?AGCTCTTCCTTCCTTTGCACTGAAAGCTGTAACTCTAAGTATCAGTGTGAAACGGGAGAA?163
Query?62 AACAGTAAAGGCANGTCCAGGATAGAGTGAAGCGACCCATGAACGCATTCATCGTGTGG 121
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct?164?AACAGTAAAGGCAACGTCCAGGATAGAGTGAAGCGACCCATGAACGCATTCATCGTGTGG?223
Query?122?TCTCGCGATCAGAGGCGCAAGATGGCTCTAGAGAATCCCAGAATGCGAAACTCAGAGATC?181
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct?224?TCTCGCGATCAGAGGCGCAAGATGGCTCTAGAGAATCCCAGAATGCGAAACTCAGAGATC?283
Query?182?AGCAAGCAGCTGGGATACCAGTGGAAAATGCTTACTGAAGCCGAAAAATGGCCATTCTTC?241
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct?284?AGCAAGCAGCTGGGATACCAGTGGAAAATGCTTACTGAAGCCGAAAAATGGCCATTCTTC?343
Query?242?CAGGAGGCACAGAAATTACAGGCCATGCACAGAGAGAAATACCCGAATTATAAGTATCGA?301
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct?344?CAGGAGGCACAGAAATTACAGGCCATGCACAGAGAGAAATACCCGAATTATAAGTATCGA?403
Query?302?CCTCGTCGGAAGGCGAAGATGCTGCCGAAGAATTGCAGTTTGCTTCCCGCAGATCCCGCT?361
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct?404?CCTCGTCGGAAGGCGAAGATGCTGCCGAAGAATTGCAGTTTGCTTCCCGCAGATCCCGCT?463
Query?362?TCGGTACTCTGCAGCGAAGTGCAACTGGACAACAGGTTGTACAGGGATGACTGTACGAAA?421
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct?464?TCGGTACTCTGCAGCGAAGTGCAACTGGACAACAGGTTGTACAGGGATGACTGTACGAAA?523
Query?422?GCCACACACTCAAGAATGGAGCACCAGC?449
||||||||||||||||||||||||||||
Sbjct?524GCCACACACTCAAGAATGGAGCACCAGC?551
1.2 fluorescence in situ hybridization (FISH) technology for detection people Y chromosome
Blank: do not have any hybridization fluorescent signal; Positive control: red color visible fluorescence (see figure 5) in the SNK-6 cell strain tumor cell; The people ties in the transplanted tumor in nude mice tumor cell of external nose type NK/T cell lymphoma also red color visible fluorescence, and the prompting transplanted tumor in nude mice derives from people's (see figure 6) really.
1.3 Flow cytometry human leucocyte differentiation antigen (HLA-ABC) result
The HLA-ABC positive cell number accounts for 1.3%, can think HLA-ABC (+) (see figure 7).
2 animal model PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM, immunophenotype and Epstein-Barr virus testing result
2.1 macropathology performance
It is subcutaneous that transplanted tumor in nude mice grows in transplantation site, at subcutaneous formation enclosed mass, and oval or irregular shape, owe clearly on the border, canescence, the greyish white bois de rose of tangent plane, matter are tender, are flesh of fish shape.11st,, there was the local skin diabrosis in 12 and 25 generations, the visible forelimb axillary gland enlargement of the 4th, 5 and 27 generations.Compare with normal nude mice, the tumor bearing nude mice liver is grown up not obvious, and all tumor bearing nude mices are all grown up with the spleen volume, and lung, kidney and brain naked eyes show no obvious abnormalities (see figure 3).
2.2 tissue pathologies change
Change basically identical down with the mirror of donor nose primary tumor, stomach secondary tumor specimen, promptly on the background of coagulation necrosis and inflammatory cell infiltration in various degree, tumor cell fills the air distribution, between matter few, cell median size～medium bigger than normal, circular or oval, endochylema is few, lightly dye or slightly have a liking for acid, nucleus is big, rounded, oval or irregular shape, nuclear chromatin is speckle shape uniform distribution, as seen the part cell has 1～2 basophilia kernel, and karyokinesis resembles 3～10/HPF, visible pathologic mitosis (see figure 8).As seen tumor cell invasion blood vessel phenomenon.Tumor is involved surrounding soft tissue and skin, causes skin ulcer to form.As seen tumor cell is invaded neurological phenomena.
Late period, the tumorigenic whole body diffusion of mice with tumor, all visible tumor cell invasion (see figure 9) in nude mice peripheral blood, liver, spleen, lymph node, kidney and the lung.Except that the 28th generation, all the other mice with tumor livers are all involved, and mainly show as the portal area tumor cell invasion, also visible tumor cell invasions in the sinus hepaticus.The 4th～7,10,11,15,20～21 generations, and the 25th～27 generation mice with tumor lymph node in tumor cell invasion is arranged, account for 40%, mainly show as lymph node structure destruction in various degree, pantomorphic medium tumor cell bigger than normal soaks in flakes.100% mice with tumor has spleen in various degree to get involved, and tumor cell mainly soaks in the white pulp district, also can see the inequality tumor cell invasion in the red pulp district simultaneously.The 14th～25,27 and 29 generation mice with tumor (totally 13 generations) have kidney to involve, and account for 81%, mainly show as tumor cell and be the kitchen range infiltration in matter and the perirenal fat tissue between kidney.14th, 15,17～25 generations and the 27th generation mice with tumor (totally 12 generations) have pneumonopathy to become, and account for 86%, mainly show as brief summary nodular focus in the interstitial lung, and be still clear with the surrounding tissue boundary, and visible tumor cell exists in the little blood vessel that has.To 12 generation mice with tumor cerebral morphology observed result find that all mice with tumor cerebral tissue there is no tumor cell invasion.
2.3 immunohistochemical staining result
Source case and transplanted tumor in nude mice tumor cell immunophenotype and Epstein-Barr virus testing result see Table 3.
Table 3 donor case and animal model tumor tissues immunophenotype and Epstein-Barr virus testing result
Annotate :+: positive-: negative ±: suspicious ND: do not do
Seen by table 3: the tumor cell of orthotopic transplantation tumor and infiltration in each organ of lotus Mus tumor, tissue all is CD3 ε (+), CD3 (-), CD45RO (+), CD7 (-), CD8 (+), CD56/CD57 (+), CD20 (-), Cytotoxic cell proteinase-1 (+) and TIA-1 (+), and prompting has identical immunophenotype (Fig. 8) with donor nasal cavity primary tumor, stomach metastatic tumor tumor cell.
CD1a is in transplanted tumor and involve a few cell expression that is positive is arranged in the tumor of organ, and is similar to donor case stomach metastatic tumor tumor cell.CD5 is in transplanted tumor and involve a few cell expression that is positive is arranged in the tumor cell of organ, and the CD5 expression that is negative in the donor case stomach metastatic tumor.
Ki-67 is Ki-67 positive rate height in the animal model tumor cell, reaches 60～90%.
2.4 Flow cytometry result
The positive mark: the CD5 positive cell number accounts for 3.5%, the CD1a positive cell number accounts for 1.4%.
Negative marker: CD45RO, CD7, CD117 and CD34 positive cell number percentage ratio are respectively 0,0.3,0 and 0.1.
2.5EB virus detects
People's lymphoma extranodal NK/Tcell animal model Epstein-Barr virus testing result sees Table 3. and is seen by table 3, the EBER1/2 hybridization in situ technique detects and shows that the tumor cell of transplanted tumor is EBER1/2 (+) (the results are shown in Figure 8), the tumor cell that shows transplanted tumor with the identical .LMP1 immunohistochemical staining of testing result of donor case and involve organ is positive expression, with the testing result identical (the results are shown in Figure 8) of donor case.
3.T cell receptor (TCR) and heavy chain immunoglobulin (IgH) gene rearrangement analysis
3.1T cell receptor (TCR) gene rearrangement analysis
Use T
VG/ J
JXPrimer is with the positive contrast of human T lymphocyte's leukemia cell line Jurkat (P), ddH
2It is blank (B) that O replaces template DNA, donor nose primary tumor (1), stomach secondary tumor (2～3) and transplanted tumor in nude mice DNA (4～8) are carried out pcr amplification, donor case nose primary tumor, stomach secondary tumor and transplanted tumor in nude mice all do not amplify specific band as a result, promptly do not detect clone's property rearrangement (see figure 10) of TCR-gamma receptor gene.
3.2 resetting, heavy chain immunoglobulin (IgH) acceptor gene analyzes
Use the VH/JH primer, with the positive contrast of people's extramedullary plasmacytoma (P), ddH
2It is blank (B) that O replaces template DNA, donor case nose primary tumor (1), stomach secondary tumor (2～3) and transplanted tumor in nude mice DNA (4～8) are carried out pcr amplification, donor case nose primary tumor, stomach secondary tumor and transplanted tumor in nude mice all do not amplify specific band as a result, promptly do not detect clone's property rearrangement (seeing Figure 11) of IgH acceptor gene.
(3) interpretation
The strain people that the inventive method is set up ties the animal model of external nose type NK/T cell lymphoma, can reproduce the important biomolecule feature that the people ties external nose type NK/T cell lymphoma, from aspects such as histopathology, immunophenotype, ebv infection situation and molecular biological characteristics all with donor case basically identical, concrete manifestation is as follows:
(1) pathomorphology: see in the transplanted tumor pathological tissues that the medium tumor cell bigger than normal of pleomorphism is diffusivity hypertrophy and infiltration in the soft tissue under epidermis, companion's coagulation necrosis in various degree, and visible tumor cell invasion blood vessel phenomenon.Transplanted tumor has similar pathomorphology performance to donor stomach secondary tumor and nasal cavity primary tumor, and the tumor cell of transplanted tumor is abundanter, and heteromorphism is bigger, and mesenchyma stroma of tumors still less.In late period, the diffusion of organs such as peripheral blood, liver, spleen, lymph node, kidney and lung and tissue appears in mice with tumor, and the form of tumor cell is also similar to the orthotopic transplantation tumor.
(2) immunophenotype: transplanted tumor and the tumor cell that involves organ are CD3 ε (+), CD3 (-), CD45RO (+), CD7 (-), CD8 (+), CD56/CD57 (+), CD20 (-), Cytotoxic cell proteinase-1 (+) and TIA-1 (+), and showing has identical immunophenotype with donor case people nasal cavity primary tumor, stomach metastatic tumor tumor cell.Ki-67 positive rate height in the tumor cell of animal model.
(3) EBV infects: adopt EBER1/2 in situ hybridization and Epstein-Barr virus latent membrane protein LMP1 immunohistochemical staining to detect transplanted tumor tumor cell EBV and infect.
(4) molecular biological characteristics: the TCR γ of transplanted tumor and IgH gene rearrangement detect and are the embryonal system configuration, promptly do not detect TXi Baoshouti and immunoglobulin heavy chain gene and reset.
(5) kind source: there is human specific sequence label site SY14 in transplanted tumor, and does not have this sequence tagged site in the mice genome.Fluorescence in situ hybridization (FISH) technology for detection is found to contain human Y chromosome in the transplanted tumor in nude mice tumor cell.Transplanted tumor in nude mice is expressed HLA-ABC simultaneously, and above-mentioned three kinds of methods have proved that from different perspectives this transplanted tumor derives from the mankind.
By the foregoing description as can be known, adopt model that the inventive method sets up growth cycle after the 10th～15 generation stable, the cycle of going down to posterity is about 20 days, than the existing method weak point that goes down to posterity; Success ratio of inoculation and the recovery success rate that goes down to posterity is 100%, is higher than existing method; Model goes down to posterity can stablize at least and reached for the 35th generation, 9 generations that also are better than prior art. confirmed that by the kind evaluation this transplanted tumor derives from the mankind, and its pathomorphology, immunophenotype, ebv infection and molecular biological characteristics etc. are all consistent with the donor case. in addition, this people ties external nose type NK/T cell lymphoma animal model through a series of evaluations, meet xenotransplantation tumor animal model and set up successful main standard, be that the ideal people of a strain ties external nose type NK/T cell lymphoma animal model. the inventive method has solved prior art and has transplanted difficulty, pollute easily, the cycle instability goes down to posterity, being difficult to occur the advanced tumor internal organs shifts, be difficult to model is identified etc. weak point to have good application prospects.
The above more excellent specific embodiment is that the present invention is further illustrated, but be not limitation of the scope of the invention, those skilled in the art are according to basic thought of the present invention, can make various modification or improvement, only otherwise break away from basic thought of the present invention, all within the scope that spirit of the present invention and claims of being enclosed define.
A kind of people of foundation ties the method .ST25 of external nose type NK/T cell lymphoma animal model
SEQUENCE?LISTING
＜110〉Huaxi Hospital Attached to Sichuan Univ
＜120〉a kind of people of foundation ties the method for external nose type NK/T cell lymphoma animal model
<130>A060058
<160>7
<170>PatentIn?version?3.2
<210>1
<211>448
<212>DNA
<213>Homo?sapiens
<220>
＜223〉human male's sex determining gene sequence tagged site SY14
<400>1
agctcttcct?tcctttgcac?tgaaagctgt?aactctaagt?atcagtgtga?aacgggagaa 60
aacagtaaag?gcaacgtcca?ggatagagtg?aagcgaccca?tgaacgcatt?catcgtgtgg 120
tctcgcgatc?agaggcgcaa?gatggctcta?gagaatccca?gaatgcgaaa?ctcagagatc 180
agcaagcagc?tgggatacca?gtggaaaatg?cttactgaag?ccgaaaaatg?gccattcttc 240
caggaggcac?agaaattaca?ggccatgcac?agagagaaat?acccgaatta?taagtatcga 300
cctcgtcgga?aggcgaagat?gctgccgaag?aattgcagtt?tgcttcccgc?agatcccgct 360
tcggtactct?gcagcgaagt?gcaactggac?aacaggttgt?acagggatga?ctgtacgaaa 420
gccacacact?caagaatgga?gcaccagc 448
<210>2
<211>22
<212>DNA
<213>Artificial
<220>
＜223〉SY14 specificity forward primer
<400>2
cgccagggtt?ttcccagtca?cg 22
<210>3
<211>24
<212>DNA
<213>Artificial
<220>
＜223〉SY14 specificity downstream primer
<400>3
gagcggataa?caatttcaca?cagg 24
<210>4
<211>20
<212>DNA
<213>Artificial
<220>
＜223〉TVG primer
<400>4
agggttgtgt?tggaatcagg 20
<210>5
A kind of people of foundation ties the method .ST25 of external nose type NK/T cell lymphoma animal model
<211>24
<212>DNA
<213>Artificial
<220>
＜223〉JJX primer
<400>5
cgtcgacaac?aagtgttgtt?ccac 24
<210>6
<211>25
<212>DNA
<213>Artificial
<220>
＜223〉VH primer
<400>6
ctgtcgacac?ggccgtgtat?tactg 25
<210>7
<211>22
<212>DNA
<213>Artificial
<220>
＜223〉JH primer
<400>7
aactgcagag?gagacggtga?cc 22
Claims (1)
1. one kind prepares and sets up the method that the people ties the required organization material of external nose type NK/T cell lymphoma animal model, it is characterized in that: may further comprise the steps:
When handling frozen tumor tissue, in advance water-bath is thawed, and the condition that described water-bath is thawed is 37～41 ℃, and the time is in 30 seconds, tumor tissue is inserted in 1640 culture medium that are added with penicillin and streptomycin then, and will be organized in and be cut into about 1mm under the aseptic condition
3Size piece of tissue or be treated to cell concentration 10
7～10
8The cell suspension of/ml, the concentration of described penicillin are that the concentration of 100～1000u/ml, streptomycin is 100～1000ug/ml, promptly; The above-mentioned steps deadline is no more than 30 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610020706A CN1879892B (en) | 2006-04-13 | 2006-04-13 | Method for establishing human extranodal rhinoid NK/T cell lymphoma animal model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610020706A CN1879892B (en) | 2006-04-13 | 2006-04-13 | Method for establishing human extranodal rhinoid NK/T cell lymphoma animal model |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1879892A CN1879892A (en) | 2006-12-20 |
| CN1879892B true CN1879892B (en) | 2010-05-12 |
Family
ID=37518389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200610020706A Expired - Fee Related CN1879892B (en) | 2006-04-13 | 2006-04-13 | Method for establishing human extranodal rhinoid NK/T cell lymphoma animal model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1879892B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876632B (en) * | 2012-10-17 | 2014-07-02 | 浙江省肿瘤医院 | Human NK/T cell line |
| CN106520932A (en) * | 2016-10-18 | 2017-03-22 | 绍兴金箓生物技术有限公司 | Detection method for detecting NK/T cell lymphoma copy number variation |
| CN108841869B (en) * | 2018-05-31 | 2022-05-06 | 上海交通大学医学院附属瑞金医院 | Construction method and application of zebra fish NK/TCL tumor model |
| CN108835051A (en) * | 2018-06-01 | 2018-11-20 | 郑州大学第附属医院 | Utilize immune deficiency nude mice model constructed by lymphoma cell strain |
| CN108753831A (en) * | 2018-06-01 | 2018-11-06 | 郑州大学第附属医院 | Utilize the immunodeficient mouse model constructed by NK/T lymphoma cell strains |
| CN114304061B (en) * | 2020-09-30 | 2023-08-18 | 复旦大学 | Method for establishing NK/T lymphoma mouse model |
-
2006
- 2006-04-13 CN CN200610020706A patent/CN1879892B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| 何妙侠 等.人鼻型NK/T细胞淋巴瘤的裸鼠移植与传代.中华病理学杂志33 2.2004,33(2),第158-159页. |
| 何妙侠 等.人鼻型NK/T细胞淋巴瘤的裸鼠移植与传代.中华病理学杂志33 2.2004,33(2),第158-159页. * |
| 何妙侠 等.人鼻型NK/T细胞淋巴瘤表达谱基因芯片的筛查研究.中华病理学杂志34 7.2005,34(7),第426-427页. |
| 何妙侠 等.人鼻型NK/T细胞淋巴瘤表达谱基因芯片的筛查研究.中华病理学杂志34 7.2005,34(7),第426-427页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1879892A (en) | 2006-12-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1879892B (en) | Method for establishing human extranodal rhinoid NK/T cell lymphoma animal model | |
| CN102333891A (en) | Single cell gene expression for diagnosis, prognosis and identification of drug targets | |
| Roelofs et al. | Breast tumour organoids: promising models for the genomic and functional characterisation of breast cancer | |
| CN104593372B (en) | A kind of aptamer, kit and method for detecting Pancreatic neoplasms | |
| CN105296430B (en) | A kind of human colon cancer cells system DXH-1 and its application | |
| CN109609460A (en) | A kind of human glioma cell line and its method for building up and application | |
| CN101982178A (en) | Method for building mouse model for human colon carcinoma xenograft | |
| CN113142135A (en) | Construction method of digestive tract tumor PDX model and standardized model library | |
| CN102719403B (en) | A kind of human pancreas's adenosquamous carcinoma clone and establishment method thereof and application | |
| CN106754719B (en) | Inoculation liquid composition and method for constructing malignant pleural effusion source xenograft tumor animal model by using same | |
| CN104694476A (en) | Human non-small cell lung cancer cell line, and establishment method and application thereof | |
| CN102067828A (en) | Highly metastatic model of human melanoma, cell subline, creation methods, and dynamic detection of metastasis | |
| CN105873639A (en) | Autologous tumor vaccines and methods | |
| CN108913796A (en) | For detecting Green Fluorescent Protein gene egfp specific primer, kit and the method for green muscardine fungus plant endogenesis | |
| Alexandrova et al. | Cell cultures as model systems in breast cancer research | |
| CN102329775B (en) | A kind of human pancreatic cancer cell to gemcitabine resistance and application thereof | |
| CN105696087A (en) | Lung cancer PDX standardized model library | |
| Menen et al. | A rapid imageable in vivo metastasis assay for circulating tumor cells | |
| CN110106150A (en) | A kind of preparation method and application of synovial sarcoma cells system hSS-005R | |
| CN105255832A (en) | Human bile duct cancer cell line and applications thereof | |
| CN109182340A (en) | A kind of construction method of OX40 gene modification humanized animal's model and application | |
| CN109008958A (en) | Intestinal flora research method based on fecal filtration and transplantation | |
| CN102168085A (en) | SiRNA for inhibiting expression of miR-130b gene and expression vector and application of siRNA or/and expression vector to preparation of medicament for improving treatment effect of liver cancer | |
| US20210395696A1 (en) | Isolated nasopharyngeal carcinoma cells and derivatives prepared thereof | |
| CN112094890A (en) | Method for quantitatively evaluating proliferation of tumor cells transplanted into zebra fish embryo |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100512 Termination date: 20150413 |
|
| EXPY | Termination of patent right or utility model |