CN104017854B - A kind ofly assess the method for NKT cell to hepatic parenchymal cells kill capability - Google Patents
A kind ofly assess the method for NKT cell to hepatic parenchymal cells kill capability Download PDFInfo
- Publication number
- CN104017854B CN104017854B CN201410263954.1A CN201410263954A CN104017854B CN 104017854 B CN104017854 B CN 104017854B CN 201410263954 A CN201410263954 A CN 201410263954A CN 104017854 B CN104017854 B CN 104017854B
- Authority
- CN
- China
- Prior art keywords
- cell
- suspension
- liver
- add
- nkt
- 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
- 210000000581 natural killer T-cell Anatomy 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 36
- 230000002440 hepatic effect Effects 0.000 title claims abstract description 32
- 210000004738 parenchymal cell Anatomy 0.000 title claims abstract description 32
- 210000004027 cell Anatomy 0.000 claims abstract description 42
- 210000004185 liver Anatomy 0.000 claims abstract description 38
- 239000006285 cell suspension Substances 0.000 claims abstract description 33
- 239000000725 suspension Substances 0.000 claims abstract description 27
- 210000004443 dendritic cell Anatomy 0.000 claims abstract description 23
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 22
- 210000000952 spleen Anatomy 0.000 claims abstract description 18
- 101000831567 Homo sapiens Toll-like receptor 2 Proteins 0.000 claims abstract description 11
- 102100024333 Toll-like receptor 2 Human genes 0.000 claims abstract description 11
- 230000003393 splenic effect Effects 0.000 claims abstract description 11
- 238000012360 testing method Methods 0.000 claims abstract description 10
- 239000000975 dye Substances 0.000 claims abstract description 9
- 239000012535 impurity Substances 0.000 claims abstract description 8
- 238000001914 filtration Methods 0.000 claims abstract description 7
- 238000005516 engineering process Methods 0.000 claims abstract description 6
- 238000000227 grinding Methods 0.000 claims abstract description 6
- 238000011160 research Methods 0.000 claims abstract description 6
- 238000004043 dyeing Methods 0.000 claims abstract description 5
- 210000003494 hepatocyte Anatomy 0.000 claims abstract description 5
- 238000003306 harvesting Methods 0.000 claims abstract description 3
- 239000000203 mixture Substances 0.000 claims abstract description 3
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 23
- 239000006228 supernatant Substances 0.000 claims description 13
- 239000002504 physiological saline solution Substances 0.000 claims description 10
- 239000003082 abrasive agent Substances 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 239000013592 cell lysate Substances 0.000 claims description 7
- 210000003743 erythrocyte Anatomy 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
- 230000004913 activation Effects 0.000 claims description 6
- 238000005336 cracking Methods 0.000 claims description 6
- 210000001519 tissue Anatomy 0.000 claims description 6
- 230000001413 cellular effect Effects 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000005204 segregation Methods 0.000 claims description 4
- 102000000588 Interleukin-2 Human genes 0.000 claims description 3
- 108010002350 Interleukin-2 Proteins 0.000 claims description 3
- 241000699670 Mus sp. Species 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 230000003511 endothelial effect Effects 0.000 claims description 3
- 238000007667 floating Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 230000002779 inactivation Effects 0.000 claims description 3
- 230000008520 organization Effects 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 238000010186 staining Methods 0.000 claims description 3
- 239000002344 surface layer Substances 0.000 claims description 3
- VQFKFAKEUMHBLV-BYSUZVQFSA-N 1-O-(alpha-D-galactosyl)-N-hexacosanoylphytosphingosine Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCCC(=O)N[C@H]([C@H](O)[C@H](O)CCCCCCCCCCCCCC)CO[C@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O VQFKFAKEUMHBLV-BYSUZVQFSA-N 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 3
- 238000013467 fragmentation Methods 0.000 abstract description 2
- 238000006062 fragmentation reaction Methods 0.000 abstract description 2
- 208000019423 liver disease Diseases 0.000 description 10
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- 230000006378 damage Effects 0.000 description 4
- 206010019799 Hepatitis viral Diseases 0.000 description 3
- 206010052428 Wound Diseases 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 230000022534 cell killing Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000000941 bile Anatomy 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 208000019425 cirrhosis of liver Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- 201000001862 viral hepatitis Diseases 0.000 description 2
- 208000022309 Alcoholic Liver disease Diseases 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 description 1
- 208000008964 Chemical and Drug Induced Liver Injury Diseases 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 206010008635 Cholestasis Diseases 0.000 description 1
- 206010072268 Drug-induced liver injury Diseases 0.000 description 1
- 108010039471 Fas Ligand Protein Proteins 0.000 description 1
- 102000015212 Fas Ligand Protein Human genes 0.000 description 1
- 208000004930 Fatty Liver Diseases 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 208000027761 Hepatic autoimmune disease Diseases 0.000 description 1
- 206010019663 Hepatic failure Diseases 0.000 description 1
- 206010019708 Hepatic steatosis Diseases 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- NNJVILVZKWQKPM-UHFFFAOYSA-N Lidocaine Chemical compound CCN(CC)CC(=O)NC1=C(C)C=CC=C1C NNJVILVZKWQKPM-UHFFFAOYSA-N 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091008877 NK cell receptors Proteins 0.000 description 1
- 102000010648 Natural Killer Cell Receptors Human genes 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 108010064129 Thrombogen Proteins 0.000 description 1
- 239000000370 acceptor Substances 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 231100000594 drug induced liver disease Toxicity 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 208000010706 fatty liver disease Diseases 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 229960004194 lidocaine Drugs 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 231100000835 liver failure Toxicity 0.000 description 1
- 208000007903 liver failure Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 210000001589 microsome Anatomy 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 206010036067 polydipsia Diseases 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 210000003370 receptor cell Anatomy 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to medical science detection technique, aim to provide and a kind ofly assess the method for NKT cell to hepatic parenchymal cells kill capability.The method specifically comprises: liver and the spleen of getting mouse, and grinding removes impurity after filtering separately, and after making liver lymphocyte suspension and splenic lymphocyte suspension respectively, respectively by fluidic cell sorting technology sorting NKT cell suspension, dendritic cell; Mouse hepatocytes strain TLR2 is added culture plate, and contaminates CFSE-FITC dyestuff; Culture plate is added after treated NKT cell suspension mixes with cell suspension; Cultivate 4 h before harvest cells, in cell mixture, add PI dyeing, upper machine testing; If there is the situation that the positive ratio of PI in CFSE+ cell is greater than 5%, then illustrate that NKT cell is in Overkill level to hepatic parenchymal cells kill capability.The present invention can stablize and the NKT cell measured safely under different states to the fragmentation effect of hepatic parenchymal cells, for later laboratory science research provides technical support.
Description
Technical field
The present invention relates to medical science detection technique, assess liver NKT cell to the kill capability of hepatic parenchymal cells specifically by the apoptosis rate detecting hepatic parenchymal cells.
Background technology
Liver is the maximum removing toxic substances organ of animal, and major function is the albumen such as synthesis, Fibrinogen, thrombogen; Participate in bio-transformation and metabolism, as the conversion of bile synthesis, metabolism and foreign organic matter; Participate in processing and the bile excretion of albumen.Hepatic parenchymal cells is multiaspect polygon, and be the chief component of liver, under different physiological conditions, form can change.Fine structure containing many complexity inside hepatic parenchymal cells: liver cell nuclear, cytoplasm of liver, plastosome, endoplasmic reticulum, lysosome, Golgi apparatus, microsome and drink bubble etc., exercises its function separately.Liver is extremely important to maintenance HUMAN HEALTH, once liver cell generation degeneration necrosis, is very large to the harm of life.Hepatic diseases is that a class is in a bad way, clinical symptom complicated, case fatality rate is high, the disease of serious harm global human health.Hepatopathy comprises the hepatic diseases such as viral hepatitis, liver cirrhosis, primary hepatocarcinoma, autoimmune liver disease, alcoholic liver disease, drug-induced liver disease, cholestatic liver disease.China is hepatitis big country, and the patients such as China's PI virus hepatitis and fatty, Alcoholic, Drug, immunological liver diseases exceed 100,000,000.For a long time, because hepatopathy pathogenesis is not clear, lack the effectively control section of having, 10%-20% can be in progress as severe liver diseases such as liver failure, liver cirrhosis, liver cancer, and the state of an illness is dangerous, case fatality rate is high, serious harm people ' s health and social development.Because hepatopathy pathogenesis and influence factor fuzzy, lack special, effective therapy target and intervention means at present clinically, the process of hepatopathy can not be reversed completely.Liver injury be the common trait of hepatopathy.The major cause that hepatic parenchymal cells known at present necroses is, viral hepatitis, excessive drinking, Autoimmune Disorders.These are because usually causing effector cell to attack hepatic parenchymal cells and killing and wounding, and if things go on like this, consequence is hardly imaginable.
NKT (natural killer T) cell is found in 1986, and different from (TCR) gene of other T cell antigen acceptors, NKT cell has the constitutional features of its uniqueness: 1. express φt cell receptor and NK cell receptor simultaneously.Be generally CD4
+with DNNKT (CD4-CD8-) cell.Wherein NK1.1 is the topmost surface markers of NKT cell; 2., compared with other T cell subgroup, NKT cell has unique restricted TCR expression library; 3. accept the lipid antigen of CD1d submission, in play a role.6 days viviparous initial stages before thymus gland is formed of NKT cell are in the outer tissue differentiation of thymus gland, and function is in underdeveloped state.NKT cell not only can secrete Th1 and Th2 cytokine, also has and CD8 simultaneously
+what killer T cell (cytotoxicTlymphocyte, CTL) was identical kills and wounds target cell effect.NKT cell and disease may have many relations, and pathogenesis such as, allergic adjustment, anti-effect and suppression are infected.After NKT cell is upset, can secrete a large amount of cytokines and chemokine, play immunoregulation effect, be one of bridge of contact inherent immunity and acquired immunity.Have NK cell-like cell cytotoxic activity after NKT cell activation, the cell sensitive target cell of solubilized NK, main effects molecule is FasL and IFN-γ.Existing research display recently, NKT cell plays a significant role in the pathogenic process of the hepatopathy such as hepatitis, fatty liver, can detect the kill capability of NKT cell to hepatic parenchymal cells, provides valuable help for scientific research and clinical treatment liver related disease.
The method of existing mensuration effector cell to target cell kill capability mainly contains: waveforms method, enzyme release assay, nucleic method, chemoluminescence method.But these methods are used for CD8
+t cell or NK cell killing and wounding target cell, not yet propose a kind of method for NKT cellkilling capacity, and respectively have its own imperfection part in these methods.Waveforms method human factor is comparatively large, and accuracy rate is low; Enzyme r e lease and chemoluminescence method method less stable; Although nucleic method Detection accuracy is higher, larger to the potential safety hazard of operation.Therefore, a kind of alternative way need be looked for be very necessary.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes deficiency of the prior art, provides a kind of by detecting hepatic parenchymal cells mortality ratio assessment NKT cell to the method for hepatic parenchymal cells kill capability.
For technical solution problem, the invention provides following technical scheme:
There is provided a kind of and assess the method for NKT cell to hepatic parenchymal cells kill capability, being that the apoptosis rate by detecting hepatic parenchymal cells assesses liver NKT cell to the kill capability of hepatic parenchymal cells, specifically comprising:
(1) get mouse hepatocytes strain TLR2 and carry out recovery process, for subsequent use after passing a generation;
(2) get liver and the spleen of C57/B6 strain wild-type healthy mice, rear removing foreign matter is filtered in grinding separately, and makes liver lymphocyte suspension and splenic lymphocyte suspension respectively;
(3) from liver lymphocyte suspension, sub-elected the NKT cell of liver by fluidic cell sorting technology, make NKT cell suspension; Add the IL-2 activation culture of 10ul;
(4) sub-elected the dendritic cell of spleen by fluidic cell sorting technology from splenic lymphocyte suspension, make dendritic cell suspension; Add 2ul a-GalCer stimulate activation, be then placed under ray irradiate make the sex change of dendritic cell inactivation after, make cell suspension;
(5) step (1) small mouse hepatic parenchymal cells strain TLR2 is added culture plate, and contaminate CFSE-FITC dyestuff; The culture plate in step (1) is added after being mixed with cell suspension in step (4) by NKT cell suspension treated in step (3); Cultivate 4 h before harvest cells, in cell mixture, add PI dyeing, upper machine testing; If there is CFSE
+the situation that in cell, the positive ratio of PI is greater than 5%, then illustrate that NKT cell is in Overkill level to hepatic parenchymal cells kill capability.
In the present invention, described step (2) specifically refers to:
Respectively following process is carried out to the liver of mouse, spleen tissue:
Get each one of mouse liver, spleen, shred rear grinding rod and grind on filter screen and be filtered to centrifuge tube, physiological saline washes away cell debris and endothelial tissue, unnecessary supernatant in removing centrifuge tube, leaves bottom abrasive material;
In the centrifuge tube that liver organization abrasive material is housed, add the Percoll cellular segregation liquid of 33% mass percent of 20ml, 2000 leave the heart removes upper strata floating matter and unnecessary supernatant after 18 minutes; Add 1ml mouse red blood cell lysate, cracking, after one minute, adds physiological saline to 10ml termination reaction, and PBS washes and obtains liver lymphocyte suspension for one time;
Directly add 1ml mouse red blood cell lysate to the centrifuge tube that spleen tissue abrasive material is housed, cracking, after one minute, adds physiological saline to 10ml termination reaction, and PBS washes one time and obtains splenic lymphocyte suspension.
In the present invention, in described step (3), the preparation process of NKT cell suspension specifically comprises:
With 1200 turns of centrifugal treating liver lymphocyte suspensions abandoning supernatant after 5 minutes, add one milliliter of PBS respectively and remove undesired impurities through strainer filtering; In suspension, add anti-NK1.1-FITC antibody and each 1ul of anti-TCR-β-APC antibody, hatch upper machine sorting after 30 minutes, obtain the NKT cell suspension of liver.
In the present invention, in described step (4), the preparation process of dendritic cell suspension specifically comprises:
With 1200 turns of centrifugal treating splenic lymphocyte suspensions sorting abandoning supernatant after 5 minutes, add one milliliter of PBS respectively and remove undesired impurities through strainer filtering; In suspension, add anti-CD11c-PE antibody 1ul, hatch upper machine sorting after 30 minutes, obtain the dendritic cell suspension of spleen.
In the present invention, described in step (4), ray is the gamma-rays of 100Gy.
In the present invention, in described step (5), testing process is specifically:
To being equipped with 2 × 10
5in the culture plate of/mlTLR2 cell strain, adding the staining fluid of the CFSE-FITC of 10uM to covering cell surface layer, dyeing 15 minutes; Abandoning supernatant, washes three times with PBS; And in TLR2 cell strain: NKT cell=1: the ratio of 10 adds the described NKT cell suspension of step (3); Add the described dendritic cell suspension of step (4) again, make the number equivalent of dendritic cell and NKT cell; After 37 DEG C of cell culture incubators hatch 4 hours, catch PI dyestuff, machine testing in taking-up.
Compared with prior art, beneficial effect of the present invention is:
Due to flow cytometry stable feature and this method avoid the security hidden troubles such as isotropic substance, thus the present invention can stablize and the NKT cell measured safely under different states to the fragmentation effect of hepatic parenchymal cells, for later laboratory science research provides technical support.
Accompanying drawing explanation
Fig. 1 is after the NKT cell under different states kills and wounds hepatic parenchymal cells, the fatality ratio streaming figure of hepatic parenchymal cells.
Fig. 2 is after the NKT cell under different states kills and wounds hepatic parenchymal cells, the fatality ratio column statistical study figure of hepatic parenchymal cells.
Specific implementation method
Set forth the present invention further below in conjunction with concrete enforcement, should be understood that following examples are only not used in for illustration of the present invention and limit the scope of the invention.
Experimental technique in the embodiment of the present invention, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
Statistical method: adopt the analysis of SPSS19.0 statistical analysis software, the comparison of each sample average adopts Studentttest to analyze.
Tissue Culture Plate and culturing bottle are all purchased from Corning, and cell culture medium and serum are all purchased from Gibco, scissors, the tissue grinder instruments such as tweezers, phosphate buffered saline buffer (PBS) and 0.9% physiological saline, lidocaine anesthesia medicine is all from attached First Hospital medical professionals section of Zhejiang University, Percoll cellular segregation liquid is purchased from Zhejiang University's goods and materials, a-GalCer is purchased from ENZO, centrifuge tube is purchased from Axegen, CFSE-FITC dyestuff is purchased from MolecularProbes, PI dyestuff is purchased from eBioscience, anti-NK1.1-FITC mouse antibodies, anti-TCR-β-APC mouse antibodies, anti-CD11c-PE mouse antibodies and mouse red blood cell lysate are all purchased from BD,-80 degree refrigerator whizzers are purchased from Thermo, the LB culture medium powder of microbial culture is biochemical purchased from raw work, bacteriological incubator is purchased from Shanghai good fortune agate, flow cytometer is purchased from Beckmancoulter, flow cell sorter is purchased from BD.
The manufacturer of C57/B6 strain wild-type mice is this Leco Corp. of Shanghai.Mouse hepatocytes strain TLR2, mouse liver and spleen provide by the attached First Hospital of Zhejiang University.
Utilize the method steps detecting hepatic parenchymal cells apoptosis rate reaction NKT cellkilling capacity, comprise the following steps:
(1) get mouse hepatocytes strain TLR2 from-80 DEG C of refrigerators in advance and carry out recovery process, for subsequent use after passing a generation.
(2) respectively following process is carried out to the liver of mouse, spleen tissue:
Get each one of mouse liver, spleen, shred rear grinding rod and grind on filter screen and be filtered to centrifuge tube, physiological saline washes away impurity (cell debris, endothelial tissue);
In the centrifuge tube that liver organization abrasive material is housed, add the Percoll cellular segregation liquid of 33% mass percent of 20ml, 2000 leave the heart removes upper strata floating matter and unnecessary supernatant after 18 minutes; Add 1ml mouse red blood cell lysate, cracking, after one minute, adds physiological saline to 10ml termination reaction, obtains liver lymphocyte suspension;
Directly add 1ml mouse red blood cell lysate to the centrifuge tube that spleen tissue abrasive material is housed, cracking, after one minute, adds physiological saline to 10ml termination reaction, obtains splenic lymphocyte suspension.
(3) two kinds of suspensions in step (2) are handled as follows respectively:
With 1200 turns of centrifugal treating abandoning supernatant after 5 minutes, add one milliliter of PBS respectively and remove undesired impurities through strainer filtering; Anti-NK1.1-FITC antibody and each 1ul of anti-TCR-β-APC antibody is added in liver lymphocyte suspension, anti-CD11c-PE antibody 1ul is added in splenic lymphocyte suspension, hatch upper machine sorting after 30 minutes respectively, obtain the NKT cell suspension of liver and the dendritic cell suspension of spleen.
(4) the NKT cell suspension after sorting adds the IL-2 activation culture of 10ul;
Add a-GalCer in dendritic cell suspension after sorting and stimulate activation.
(5) irradiate under dendritic cell suspension activated in step (4) being placed in the gamma-rays of 100Gy, after making dendritic cell inactivation, make cell suspension;
(6) 2 × 10 are being equipped with
5the culture plate of/mlTLR2 cell strain, adds the staining fluid of the CFSE-FITC of 10uM to covering cell surface layer, dyes 15 minutes; Abandoning supernatant, washes three times with PBS; And in TLR2 cell (target cell): the ratio of NKT cell (effector cell)=1: 10 adds the described NKT cell suspension of step (3), add the described dendritic cell suspension of step (4) again, make the number equivalent of dendritic cell and NKT cell; After 37 DEG C of cell culture incubators hatch 4 hours, catch PI dyestuff, machine testing in taking-up;
If there is CFSE
+the situation that in cell, the positive ratio of PI is greater than 5%, then illustrate that NKT cell is in Overkill level to hepatic parenchymal cells kill capability.
Application example:
This embodiment adopts the operation steps of specific embodiment, and the stimulator adding dendritic cell concrete in step 5 is Salmonellas and streptococcic antigen.After testing, be 18.9% (result as shown in Figure 1) with the NKT cell that the post-stimulatory dendritic cell of the a-GalCer of 2ul offers to activate to the kill rate of TLR2 cell.
Based on this analysis conclusion, quantitative basis can be provided to NKT cell in scientific research to hepatic parenchymal cells kill capability, and then NKT causes the mechanism of damage to provide investigative technique to liver.
Claims (6)
1. assess the method for NKT cell to hepatic parenchymal cells kill capability, it is characterized in that, be assess liver NKT cell to the kill capability of hepatic parenchymal cells by the mortality ratio of flow cytomery hepatic parenchymal cells, specifically comprise:
(1) get mouse hepatocytes strain TLR2 and carry out recovery process, for subsequent use after passing a generation;
(2) get liver and the spleen of C57/B6 strain wild-type healthy mice, grinding removes impurity after filtering separately, and makes liver lymphocyte suspension and splenic lymphocyte suspension respectively;
(3) from liver lymphocyte suspension, sub-elected the NKT cell of liver by fluidic cell sorting technology, make NKT cell suspension, and add the IL-2 activation culture of 10ul;
(4) sub-elected the dendritic cell of spleen by fluidic cell sorting technology from splenic lymphocyte suspension, make dendritic cell suspension; Add 2ul α-GalCer stimulate activation, be then placed under ray irradiate make the sex change of dendritic cell inactivation after, make cell suspension;
(5) step (1) small mouse hepatic parenchymal cells strain TLR2 is added culture plate, and contaminate CFSE-FITC dyestuff; The culture plate in step (1) is added after being mixed with cell suspension in step (4) by NKT cell suspension treated in step (3); Cultivate 4 h before harvest cells, in cell mixture, add PI dyeing, upper machine testing; If there is CFSE
+the situation that in cell, the positive ratio of PI is greater than 5%, then illustrate that NKT cell is in Overkill level to hepatic parenchymal cells kill capability;
The method is used for scientific research object.
2. method according to claim 1, is characterized in that, described step (2) specifically refers to:
Respectively following process is carried out to the liver of mouse, spleen tissue:
Get each one of mouse liver, spleen, shred rear grinding rod and grind on filter screen and be filtered to centrifuge tube, physiological saline washes away cell debris and endothelial tissue; Unnecessary supernatant in removing centrifuge tube, leaves bottom abrasive material;
Add the Percoll cellular segregation liquid of 33% mass percent of 20ml to the centrifuge tube that liver organization abrasive material is housed, 2000 leave the heart removes upper strata floating matter and unnecessary supernatant after 18 minutes; Add 1ml mouse red blood cell lysate, cracking, after one minute, adds physiological saline to 10ml termination reaction, and PBS washes and obtains liver lymphocyte suspension for one time;
Directly add 1ml mouse red blood cell lysate to the centrifuge tube that spleen tissue abrasive material is housed, cracking, after one minute, adds physiological saline to 10ml termination reaction, and PBS washes one time and obtains splenic lymphocyte suspension.
3. method according to claim 1, is characterized in that, in described step (3), the preparation process of NKT cell suspension specifically comprises:
With 1200 turns of centrifugal treating liver lymphocyte suspensions abandoning supernatant after 5 minutes, add one milliliter of PBS respectively and remove undesired impurities through strainer filtering; In suspension, add anti-NK1.1-FITC antibody and each 1ul of anti-TCR-β-APC antibody, hatch upper machine sorting after 30 minutes, obtain the NKT cell suspension of liver.
4. method according to claim 1, is characterized in that, in described step (4), the preparation process of dendritic cell suspension specifically comprises:
With 1200 turns of centrifugal treating splenic lymphocyte suspensions sorting abandoning supernatant after 5 minutes, add one milliliter of PBS respectively and remove undesired impurities through strainer filtering; In suspension, add anti-CD11c-PE antibody 1ul, hatch upper machine sorting after 30 minutes, obtain the dendritic cell suspension of spleen.
5. method according to claim 1, is characterized in that, described in step (4), ray is the gamma-rays of 100Gy.
6. method according to claim 1, is characterized in that, in described step (5), testing process is specifically:
To being equipped with 2 × 10
5in the culture plate of/mlTLR2 cell strain, adding the staining fluid of the CFSE-FITC of 10uM to covering cell surface layer, dyeing 15 minutes; Abandoning supernatant, washes three times with PBS; And in TLR2 cell strain: NKT cell=1: the ratio of 10 adds the described NKT cell suspension of step (3); Add the described dendritic cell suspension of step (4) again, make the number equivalent of dendritic cell and NKT cell; After 37 DEG C of cell culture incubators hatch 4 hours, catch PI dyestuff, machine testing in taking-up.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410263954.1A CN104017854B (en) | 2014-06-15 | 2014-06-15 | A kind ofly assess the method for NKT cell to hepatic parenchymal cells kill capability |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410263954.1A CN104017854B (en) | 2014-06-15 | 2014-06-15 | A kind ofly assess the method for NKT cell to hepatic parenchymal cells kill capability |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104017854A CN104017854A (en) | 2014-09-03 |
| CN104017854B true CN104017854B (en) | 2016-03-30 |
Family
ID=51434849
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410263954.1A Expired - Fee Related CN104017854B (en) | 2014-06-15 | 2014-06-15 | A kind ofly assess the method for NKT cell to hepatic parenchymal cells kill capability |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104017854B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110196219B (en) * | 2019-05-16 | 2022-06-03 | 扬州大学 | Chicken spleen CD8 detection based on flow cytometry+Method for specific killing of T cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676453A (en) * | 2011-03-17 | 2012-09-19 | 中国医学科学院肿瘤研究所 | Method for culturing natural killer (NK) and/or natural killer T (NKT) cells |
| CN103756961A (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 induced amplification of NKT cells |
| CN103834614A (en) * | 2014-03-12 | 2014-06-04 | 甘肃中科生物科技有限公司 | Preparation method for monkshood polysaccharide-induced nature killer T (NKT) cell proliferation and application thereof |
-
2014
- 2014-06-15 CN CN201410263954.1A patent/CN104017854B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676453A (en) * | 2011-03-17 | 2012-09-19 | 中国医学科学院肿瘤研究所 | Method for culturing natural killer (NK) and/or natural killer T (NKT) cells |
| CN103756961A (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 induced amplification of NKT cells |
| CN103834614A (en) * | 2014-03-12 | 2014-06-04 | 甘肃中科生物科技有限公司 | Preparation method for monkshood polysaccharide-induced nature killer T (NKT) cell proliferation and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| NKT细胞及肝脏疾病关系的研究进展;郭芳等;《现代医药卫生》;20060130;第22卷(第02期);全文 * |
| NKT细胞在肝癌组织中的分布状况与肝癌局部免疫的研究;邱大鹏等;《中国临床医学》;20040825;第13卷(第04期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104017854A (en) | 2014-09-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lumeng et al. | Aging is associated with an increase in T cells and inflammatory macrophages in visceral adipose tissue | |
| O'Rourke et al. | Systemic inflammation and insulin sensitivity in obese IFN-γ knockout mice | |
| Roth et al. | Susceptibility to programmed cell death in T-lymphocytes from septic patients: a mechanism for lymphopenia and Th2 predominance | |
| KR20150129726A (en) | Cell repopulated collagen matrix for soft tissue repair and regeneration | |
| Jules et al. | In vitro investigation of the roles of the proinflammatory cytokines tumor necrosis factor-α and interleukin-1 in murine osteoclastogenesis | |
| Ahmed et al. | Granisetron and carvedilol can protect experimental rats againstadjuvant-induced arthritis | |
| Margaryan et al. | sFasL-mediated induction of neutrophil activation in patients with type 2 diabetes mellitus | |
| Holan et al. | The impact of morphine on the characteristics and function properties of human mesenchymal stem cells | |
| Machalińska et al. | Stem cells are mobilized from the bone marrow into the peripheral circulation in response to retinal pigment epithelium damage—A pathophysiological attempt to induce endogenous regeneration | |
| Sun et al. | A novel mechanism of tumor-induced thymic atrophy in mice bearing H22 hepatocellular carcinoma | |
| CN104017854B (en) | A kind ofly assess the method for NKT cell to hepatic parenchymal cells kill capability | |
| Hong et al. | Effects of interleukin 6 and tumor necrosis factor-α on the proliferation of porcine theca interna cells: Possible role of these cytokines in the pathogenesis of polycystic ovary syndrome | |
| Qiu et al. | Regulatory B10 cells play a protective role in severe acute pancreatitis | |
| Sebti et al. | The LPS/D-galactosamine-induced fulminant hepatitis model to assess the role of ligand-activated nuclear receptors on the NLRP3 inflammasome pathway in vivo | |
| Fouda et al. | A fluorescence-based lymphocyte assay suitable for high-throughput screening of small molecules | |
| Kaleta et al. | Sildenafil, a phosphodiesterase type 5 inhibitor, downregulates osteopontin in human peripheral blood mononuclear cells | |
| Yanuhar et al. | The exposure immunogenic protein of viral nervous necrotic on Humpback grouper that influences to proliferation and expression of immune cells (interferon [gamma] and NF-Kb cell) | |
| Guo et al. | Roles of monocyte chemotactic protein 1 and nuclear factor-κB in immune response to spinal tuberculosis in a New Zealand white rabbit model | |
| CN106267425A (en) | AIDS immunoadsorption therapy instrument | |
| Simon et al. | A primary human trophoblast model to study the effect of inflammation associated with maternal obesity on regulation of autophagy in the placenta | |
| Usharauli et al. | The JAM Test and its daughter P-JAM: simple tests of DNA fragmentation to measure cell death and stasis | |
| Yaguchi et al. | DNA fragmentation and detachment of enterocytes induced by anti-CD3 mAb-activated intraepithelial lymphocytes | |
| Jiang et al. | An optimal host response to a bacterium may require the interaction of leukocytes and resident host cells | |
| Zhang et al. | MyD88‐dependent pathway is essential for the innate immunity to Enterocytozoon bieneusi | |
| EP3311166B1 (en) | Cancer diagnostic |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160330 Termination date: 20190615 |