CN104017854A - Method for evaluating killing capacity of NKT cells to liver parenchymal cells - Google Patents
Method for evaluating killing capacity of NKT cells to liver parenchymal cells Download PDFInfo
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Abstract
The invention relates to a medical detecting technique and aims to provide a method for evaluating the killing capacity of NKT cells to liver parenchymal cells. The method particularly comprises the following steps of respectively grinding and filtering a liver and a spleen of a small mouse, removing impurities, respectively preparing into a liver lymphocyte suspension and a spleen lymphocyte suspension and respectively sorting NKT cells and dendritic cells through a flow cell sorting technique; adding a liver parenchymal cell strain TLR2 of the small mouse into a culture plate and dyeing by using a CFSE-FITC dye; mixing the treated NKT cell suspension and dendritic cell suspension and adding into the culture plate; collecting cells after culturing for 4 hours, adding PI into the cell mixed solution to dye and detecting by using a machine; if the PI positive proportion is greater than 5% in CFSE<+> cells, the killing capacity of the NKT cells to the liver parenchymal cells is at an overkill level. The method disclosed by the invention has the advantages that the killing effect of the NKT cells in different states on the liver parenchymal cells can be stably and safely detected and technical support is provided for the later scientific research of laboratories.
Description
Technical field
The present invention relates to medical science detection technique, be specially by detecting the apoptosis rate of hepatic parenchymal cells and assess the kill capability of liver NKT cell to hepatic parenchymal cells.
Background technology
Liver is the removing toxic substances organ of animal maximum, major function be synthesize, the albumen such as Fibrinogen, thrombogen; Participate in bio-transformation and metabolism, as bile is synthetic, the conversion of metabolism and foreign organic matter; Participate in processing and the bile excretion of albumen.Hepatic parenchymal cells is multiaspect polygon, is the chief component of liver, and under different physiological conditions, form can change.The fine structure that many complexity are contained in hepatic parenchymal cells the inside: liver cell nuclear, cytoplasm of liver, plastosome, endoplasmic reticulum, lysosome, Golgi apparatus, microsome and drink bubble etc., exercise separately its function.Liver is extremely important to maintaining 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, immunity hepatopathy exceed 100,000,000.For a long time, because hepatopathy pathogenesis is not clear, lack the effectively control section of having, 10%-20% can make 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 are fuzzy, lack clinically at present special, effectively treat target spot and intervention means, can not reverse the process of hepatopathy completely.Liver injury be the common trait of hepatopathy.The major cause that at present known hepatic parenchymal cells necroses is, viral hepatitis, excessive drinking, Autoimmune Disorders.These are because usually causing effector cell that hepatic parenchymal cells is attacked and killed and wounded, and if things go on like this, consequence is hardly imaginable.
NKT (natural killer T) cell is found in 1986, different from (TCR) gene of other T cell antigen receptors, and NKT cell has its unique constitutional features: express φt cell receptor and NK cell receptor 1. simultaneously.Be generally CD4
+with DN NKT (CD4-CD8-) cell.Wherein NK1.1 is the topmost surface markers of NKT cell; 2. compare with other T cell subsets, 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 forms of NKT cell tissue differentiation outside thymus gland, and function is in underdeveloped state.NKT cell not only can be secreted Th1 and Th2 cytokine, also has and CD8 simultaneously
+the target cell effect that kills and wounds that killer T cell (cytotoxic Tlymphocyte, CTL) is identical.NKT cell and disease may have many relations, such as pathogenesis, allergic adjusting, anti-effect and suppress to infect etc.After NKT cell is upset, can secrete a large amount of cytokines and chemokine, performance immunoregulation effect, is one of bridge of contact inherent immunity and acquired immunity.After NKT cell activation, there is NK cell like cell cytotoxic activity, the cell sensitive target cell of solubilized NK, main effects molecule is FasL and IFN-γ.Have and studies show that recently, NKT cell plays a significant role in the pathogenic process of the hepatopathys such as hepatitis, fatty liver, can detect the kill capability of NKT cell to hepatic parenchymal cells, for scientific research and clinical treatment liver related disease, provides valuable help.
Existing mensuration effector cell mainly contains the method for target cell kill capability: form method, enzyme method for releasing, nucleic method, chemoluminescence method.But these methods are used for CD8
+t cell or NK cell kill and wound target cell, not yet propose a kind of method for NKT cell killing ability, and respectively have its own imperfection part in these methods.Form method human factor is larger, and accuracy rate is low; Enzyme discharges and chemoluminescence method method less stable; Although nucleic method Detection accuracy is higher, larger to the potential safety hazard of operation.Therefore it is very necessary, need looking for a kind of alternative way.
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 the method for hepatic parenchymal cells mortality ratio assessment NKT cell to hepatic parenchymal cells kill capability.
For technical solution problem, the invention provides following technical scheme:
The method of a kind of NKT of assessment cell to hepatic parenchymal cells kill capability is provided, and is to assess the kill capability of liver NKT cell to hepatic parenchymal cells by detecting the apoptosis rate of hepatic parenchymal cells, specifically comprises:
(1) get the Mouse Liver parenchyma strain TLR2 processing of recovering, pass after a generation standby;
(2) get liver and the spleen of C57/B6 strain wild-type healthy mice, removing foreign matter after each self-grind filters, and make respectively liver lymphocyte suspension and splenic lymphocyte suspension;
(3) by fluidic cell sorting technology, from liver lymphocyte suspension, sub-elect the NKT cell of liver, make NKT cell suspension; The IL-2 activation culture that adds 10ul;
(4) by fluidic cell sorting technology, from splenic lymphocyte suspension, sub-elect the dendritic cell of spleen, make dendritic cell suspension; The a-GalCer thorn activating signal activation that adds 2ul, is then placed under ray and irradiates and make, after the sex change of dendritic cell inactivation, to make cell suspension;
(5) step (1) small mouse hepatic parenchymal cells strain TLR2 is added to culture plate, and dye CFSE-FITC dyestuff; After being mixed with cell suspension in step (4), NKT cell suspension treated in step (3) adds the culture plate in step (1); Cultivate collecting cell after 4 hours, 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%, illustrate NKT cell to hepatic parenchymal cells kill capability in Overkill level.
In the present invention, described step (2) specifically refers to:
The liver of mouse, spleen tissue are carried out respectively to following processing:
Get each of mouse liver, spleen, shred and with grinding rod, on filter screen, grind and be filtered to centrifuge tube afterwards, physiological saline washes away cell debris and endothelial tissue, removes unnecessary supernatant in centrifuge tube, abrasive material bottom staying;
To the Percoll cellular segregation liquid that adds 33% mass percent of 20ml in the centrifuge tube of 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;
To the centrifuge tube that spleen tissue abrasive material is housed, directly add 1ml mouse red blood cell lysate, 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:
Turn centrifugal treating liver lymphocyte suspension abandoning supernatant after 5 minutes with 1200, add respectively one milliliter of PBS to remove unnecessary impurity 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:
Turn centrifugal treating splenic lymphocyte suspension sorting abandoning supernatant after 5 minutes with 1200, add respectively one milliliter of PBS to remove unnecessary impurity 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, the gamma-rays that described in step (4), ray is 100Gy.
In the present invention, in described step (5), testing process is specifically:
To being equipped with 2 * 10
5in the culture plate of/ml TLR2 cell strain, add the staining fluid of CFSE-FITC of 10uM to covering cell surface layer, dye 15 minutes; Abandoning supernatant, washes three times with PBS; And in TLR2 cell strain: NKT cell=1: 10 ratio adds the described NKT cell suspension of step (3); Add again the described dendritic cell suspension of step (4), make the number equivalent of dendritic cell and NKT cell; In 37 ℃ of cell culture incubators, hatch after 4 hours, catch PI dyestuff, machine testing in taking-up.
Compared with prior art, beneficial effect of the present invention is:
Because stable feature and the method for flow cytometry have been avoided the security hidden troubles such as isotropic substance, thereby the present invention can stablize and measure safely NKT cell under the different states fragmentation effect to hepatic parenchymal cells, for laboratory scientific research later 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
Below in conjunction with concrete enforcement, further set forth the present invention, should be understood that following examples are only not used in and limit the scope of the invention for the present invention is described.
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 relatively employing Student t test of each sample average analyzes.
Tissue Culture Plate and culturing bottle are all purchased from Corning, and cell culture medium and serum are all purchased from Gibco, scissors, tweezers etc. are organized abrasive tool, phosphate buffered saline buffer (PBS) and 0.9% physiological saline, lignocaine narcotic 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 Molecular Probes, 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 Beckman coulter, flow cell sorter is purchased from BD.
The manufacturer of C57/B6 strain wild-type mice is this Leco Corp. of Shanghai.Mouse Liver parenchyma strain TLR2, mouse liver and spleen provide by the attached First Hospital of Zhejiang University.
Utilize the method steps that detects hepatic parenchymal cells apoptosis rate reaction NKT cell killing ability, comprise the following steps:
(1) in advance from-80 ℃ of refrigerators, get the Mouse Liver parenchyma strain TLR2 processing of recover, standby after a biography generation.
(2) liver of mouse, spleen tissue are carried out respectively to following processing:
Get each of mouse liver, spleen, shred and with grinding rod, on filter screen, grind and be filtered to centrifuge tube afterwards, physiological saline washes away impurity (cell debris, endothelial tissue);
To the Percoll cellular segregation liquid that adds 33% mass percent of 20ml in the centrifuge tube of 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, obtains liver lymphocyte suspension;
To the centrifuge tube that spleen tissue abrasive material is housed, directly add 1ml mouse red blood cell lysate, 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:
Turn centrifugal treating abandoning supernatant after 5 minutes with 1200, add respectively one milliliter of PBS to remove unnecessary impurity through strainer filtering; In liver lymphocyte suspension, add anti-NK1.1-FITC antibody and each 1ul of anti-TCR-β-APC antibody, in splenic lymphocyte suspension, add anti-CD11c-PE antibody 1ul, hatch respectively upper machine sorting after 30 minutes, 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;
In dendritic cell suspension after sorting, add a-GalCer thorn activating signal activation.
(5) dendritic cell suspension activated in step (4) is placed under the gamma-rays of 100Gy and irradiates, make to make cell suspension after dendritic cell inactivation;
(6) be equipped with 2 * 10
5the culture plate of/ml TLR2 cell strain, adds the staining fluid of 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 again the described dendritic cell suspension of step (4), make the number equivalent of dendritic cell and NKT cell; In 37 ℃ of cell culture incubators, hatch after 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%, illustrate NKT cell to hepatic parenchymal cells kill capability in Overkill level.
Application example:
This embodiment adopts the operation steps of specific embodiment, and in step 5, the concrete stimulator that adds dendritic cell is Salmonellas and streptococcic antigen.After testing, with the NKT cell that the post-stimulatory dendritic cell of a-GalCer of 2ul offers to activate, to the kill rate of TLR2 cell, it was 18.9% (result as shown in Figure 1).
Based on this, analyze conclusion, can to hepatic parenchymal cells kill capability, provide quantitative basis to NKT cell in scientific research, and then NKT causes the mechanism of damage that investigative technique is provided to liver.
Claims (6)
1. assessing the method for NKT cell to hepatic parenchymal cells kill capability, it is characterized in that, is that the mortality ratio that detects hepatic parenchymal cells by flow cytometer is assessed the kill capability of liver NKT cell to hepatic parenchymal cells, specifically comprises:
(1) get the Mouse Liver parenchyma strain TLR2 processing of recovering, pass after a generation standby;
(2) get liver and the spleen of C57/B6 strain wild-type healthy mice, after each self-grind filters, remove impurity, and make respectively liver lymphocyte suspension and splenic lymphocyte suspension;
(3) by fluidic cell sorting technology, from liver lymphocyte suspension, sub-elect the NKT cell of liver, make NKT cell suspension, and add the IL-2 activation culture of 10ul;
(4) by fluidic cell sorting technology, from splenic lymphocyte suspension, sub-elect the dendritic cell of spleen, make dendritic cell suspension; Add α-GalCer of 2ul to sting activating signal activation, be then placed under ray and irradiate and make, after the sex change of dendritic cell inactivation, to make cell suspension;
(5) step (1) small mouse hepatic parenchymal cells strain TLR2 is added to culture plate, and dye CFSE-FITC dyestuff; After being mixed with cell suspension in step (4), NKT cell suspension treated in step (3) adds the culture plate in step (1); Cultivate collecting cell after 4 hours, 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%, illustrate NKT cell to hepatic parenchymal cells kill capability in Overkill level.
2. method according to claim 1, is characterized in that, described step (2) specifically refers to:
The liver of mouse, spleen tissue are carried out respectively to following processing:
Get each of mouse liver, spleen, shred and with grinding rod, on filter screen, grind and be filtered to centrifuge tube afterwards, physiological saline washes away cell debris and endothelial tissue; Remove unnecessary supernatant in centrifuge tube, leave bottom abrasive material;
The Percoll cellular segregation liquid that adds 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;
To the centrifuge tube that spleen tissue abrasive material is housed, directly add 1ml mouse red blood cell lysate, 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:
Turn centrifugal treating liver lymphocyte suspension abandoning supernatant after 5 minutes with 1200, add respectively one milliliter of PBS to remove unnecessary impurity 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:
Turn centrifugal treating splenic lymphocyte suspension sorting abandoning supernatant after 5 minutes with 1200, add respectively one milliliter of PBS to remove unnecessary impurity 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, the gamma-rays that described in step (4), ray is 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/ml TLR2 cell strain, add the staining fluid of CFSE-FITC of 10uM to covering cell surface layer, dye 15 minutes; Abandoning supernatant, washes three times with PBS; And in TLR2 cell strain: NKT cell=1: 10 ratio adds the described NKT cell suspension of step (3); Add again the described dendritic cell suspension of step (4), make the number equivalent of dendritic cell and NKT cell; In 37 ℃ of cell culture incubators, hatch after 4 hours, catch PI dyestuff, machine testing in taking-up.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110196219A (en) * | 2019-05-16 | 2019-09-03 | 扬州大学 | One kind being based on Flow cytometry chicken spleen CD8+The method of T cell specific killing action |
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| 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 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110196219A (en) * | 2019-05-16 | 2019-09-03 | 扬州大学 | One kind being based on Flow cytometry chicken spleen CD8+The method of T cell specific killing action |
| 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 |
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