CN106520901A - Research method for improving human costimulatory T cell proliferation by means of radix tetrastigme extracts - Google Patents
Research method for improving human costimulatory T cell proliferation by means of radix tetrastigme extracts Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- G01N33/505—Cells of the immune system involving T-cells
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- C12N2503/00—Use of cells in diagnostics
Abstract
The invention relates to a research method for improving human costimulatory T cell proliferation by means of radix tetrastigme extracts, and belongs to the technical field of medicine biology. The research method comprises the following steps that 1, in-vitro induction culture of human costimulatory T cells is conducted by means of the radix tetrastigme extract, wherein an appropriate amount of peripheral anticoagulated blood of a healthy blood donor is taken, mononuclear cells are separated through a lymphocyte separation medium, the cell density is regulated to be 3.5*10<5>/mL through a costimulatory T cell culture medium containing different cell growth factors and stimulatory molecules, a cell culture plate is inoculated with the cells, culture is conducted in a culture box, liquid is replaced regularly, the cell density is regulated to be 5*10<5>/mL, the costimulatory T cells cultured for 7 days are collected, and a monoclonal antibody is added for cell surface mark detection; 2, influences of different radix tetrastigme extracts on human costimulatory T cell growth are detected. According to the method, a reference can be provided for novel medicinal application of the radix tetrastigme extracts in preparation of a human costimulatory T cell proliferator, and an experimental reference can be provided for research and development of tumor immunotherapy drugs.
Description
Technical field
The invention belongs to technical field of pharmaceutical biotechnology, and in particular to a kind of research Lemna paucicostata improves people costimulation T
The method of cell propagation.
Background technology
The invention belongs to technical field of pharmaceutical biotechnology, and in particular to a kind of research Lemna paucicostata improves people costimulation T
The method of cell propagation.
The content of the invention
The invention aims to solve the deficiencies in the prior art, and a kind of research Lemna paucicostata is provided and improves people
The method of costimulation T cell propagation, the research method provides reference for the new medical usage of Lemna paucicostata, can be tumor
Immunotherapy medicaments research and development provide laboratory reference.
The present invention is adopted the following technical scheme that：
The method that research Lemna paucicostata improves people's costimulation T cell propagation, step are as follows：
Step one, the external evoked culture people's costimulation T cell of Lemna paucicostata；
Step 2, the impact that the different Lemna paucicostatas of detection are grown to people's costimulation T cell, records result, adopts
SPSS13.0 softwares carry out statistical procedures, and measurement data is represented using mean ± standard deviation (mean ± SD), compares and adopt between group
Use one factor analysis of variance（One-way ANOVA）, inspection level α=0.05,P≤ 0.05 is statistically significant.
Further, the method that described research Lemna paucicostata improves people's costimulation T cell propagation, wherein step
The external evoked culture people's costimulation T cell of Lemna paucicostata described in is specially：Take the anticoagulant of appropriate healthy blood donor periphery
Blood, lymphocyte separation medium separate mononuclearcell, with the costimulation T cell containing different cell growth factor and stimulation molecule
Culture medium adjustment cell density is 3.5 × 105/ mL, is inoculated in Tissue Culture Plate, and incubator culture is periodically changed liquid, and adjusted
Cell density is 5 × 105/ mL, collects the culture costimulation T cell of 7 days, adds monoclonal antibody to carry out cell surface marker detection.
Further, the method that described research Lemna paucicostata improves people's costimulation T cell propagation, wherein step
Cell growth factor described in one and stimulating factor are CD3mAb, CD28 mAb, IFN-γ, IL-2, IL-1 α, IL-15, IL-18
Or IL-21.
Further, the method that described research Lemna paucicostata improves people's costimulation T cell propagation, wherein step
It is inoculated in one in 6 porocyte culture plates, 5 mL of hole, the incubator is 37 °C, 5%CO2Incubator, it is described periodically to change liquid
It is that liquid 1 time is periodically changed per 2 days half amounts, the CD56 of the monoclonal antibody of the addition for CD3 the and FITC labellings of PerCP-Py5.5 labellings.
Further, the method that described research Lemna paucicostata improves people's costimulation T cell propagation, wherein step
The impact that different Lemna paucicostatas are grown to people's costimulation T cell is detected described in two, specially：Take the culture common thorn of 7 days
Sharp T cell, is made into cell suspension, adds Tissue Culture Plate, if 11 concentration groups, are separately added into costimulation T cell, are subsequently adding
The Lemna paucicostata of variable concentrations, sets 5 multiple holes per group, while setting last dosing thing blank control group, cultivates in incubator
24 h、48 h、72 h；Culture adds 20 μ l of CCK8 before terminating per hole, continues culture, detects each hole light absorption value afterwards, and record
As a result.
Further, the method that described research Lemna paucicostata improves people's costimulation T cell propagation, wherein step
The impact that different Lemna paucicostatas are grown to people's costimulation T cell is detected described in two, specially：Take the culture common thorn of 7 days
Sharp T cell, is made into 5 × 104The cell suspension of/mL, adds 96 porocyte culture plates, and 200 μ l/ holes, if 11 concentration groups, are divided
Jia Ru 5 × 104The costimulation T cell of/mL, 180 μ l/hole, is subsequently adding the Lemna paucicostata of variable concentrations, final concentration
Respectively 0.025,0.05,0. 1,0.2,0.4,0.8,1.6,3.2,6.4,12.8,25.6 μ g/mL, set 5 multiple holes per group,
Set last dosing thing blank control group simultaneously, 24 h, 48 h, 72 h are cultivated in incubator；Culture terminates front 4 hours and adds per hole
Enter 20 μ l of CCK8, continue 4 h of culture and each hole light absorption value is detected at 450 nm of microplate reader, and record result.
Further, the method that above-mentioned research Lemna paucicostata improves people's costimulation T cell propagation, wherein step
Lemna paucicostata described in two be decocting in water desaccharide position Th-w3, i.e., the Radix Apioris Fortunei (Radix Lespedezae Buergeri) medical material decocting in water Jing after organic solvent extraction
Afterwards, after vacuum distillation gained dehydrate.
To sum up, main approaches of the invention are：By separating mononuclearcell in healthy human peripheral blood, with containing
CD3mAb, CD28 mAb and cytokine：The inducing culture such as IFN-γ, IL-2, IL-1 α, IL-15, IL-18 and IL-21 is pierced altogether
Sharp T cell.The culture costimulation T cell of the 7th day is collected, using the Lemna paucicostata effect costimulation T cell of variable concentrations
After 24h, 48h and 72h, CCK-8 methods detection people's costimulation T cell rate of increase；Mtt assay detection Radix Apioris Fortunei (Radix Lespedezae Buergeri) is extracted to cancer of pancreas SW-
The impact of 1990 growths；Flow cytometry（FCM）Costimulation T cell perforin before and after detection Lemna paucicostata effect
（PFP）, granzyme B (GrB), the expression of CD107a；Lactic acid dehydrogenase（LDH）Method for releasing determines Lemna paucicostata to costimulation
It is active that T cell kills cancer of pancreas SW-1990；Before and after Western blot detections are drug-induced, costimulation T cell extracellular signal is adjusted
Section kinases（ERK1/2）Protein expression.Drug-induced cancer of pancreas SW-1990 in front and back is carried out with transwell chambers cells
Mobility is detected.Cancer of pancreas SW-1990 growth fusion situations after the observation Lemna paucicostata induction of cut Healing Experiments.
Compared with prior art, beneficial effects of the present invention：
Research shows that Lemna paucicostata can be obviously promoted costimulation T cell propagation, when concentration is 0.2mg/L, cell proliferation rate
Compare matched group（Lemna paucicostata concentration：0 mg/L）High by 46%, both compare significant difference（P＜ 0.05）.In Radix Apioris Fortunei (Radix Lespedezae Buergeri)
When extract is 0.2mg/L, induction costimulation T cell is to target cell SW-1990 killing activity highests（69.8%）, with matched group
（52.1%）Comparing difference is statistically significant（P＜ 0.05）.Jing containing Lemna paucicostata induction after costimulation T cell PFP,
GrB, CD107a expression is significantly higher than matched group（P<0.05）.Costimulation T cell its ERK1/2 albumen after Radix Apioris Fortunei (Radix Lespedezae Buergeri) effect 48h
Expression has been raised compared with matched group, and in 1.6-0.02mg/L, ERK1/2 protein expressions are higher than matched group to concentration（P<0.05）.
Jing concentration be 50,12.5,3.2,0.8,0.2mg/L Radix Apioris Fortunei (Radix Lespedezae Buergeri)s induction 48h after, cancer of pancreas SW-1990 pass through transwell apertures
Cell number is minimum when being 12.5 mg/L with Radix Apioris Fortunei (Radix Lespedezae Buergeri) concentration, the fusion rate of cancer of pancreas SW-1990 with minimum during 3.2mg/L, it is and right
It is more statistically significant according to organizing（P<0.05）.High concentration Lemna paucicostata can suppress growth of tumour cell.Therefore the application
New medical usage that can be for Lemna paucicostata in people's costimulation T cell multiplication agent is prepared provides reference, can exempt from for tumor
The research and development of epidemic disease medicine provide laboratory reference.
Description of the drawings
Fig. 1：Cell growth condition under inverted microscope before costimulation T cell culture（× 400 times）；
Fig. 2：Cell growth condition under inverted microscope after costimulation T cell culture（× 400 times）；
Fig. 3：PBMC streaming results before culture；
Fig. 4：The streaming result of costimulation T cell after culture；
Fig. 5：Lemna paucicostata（Th-w3）The costimulation T cell rate of increase after induction；
Fig. 6：Lemna paucicostata（TH-W3）Inhibitory action to cancer of pancreas SW-1990 cell strains；
Fig. 7：0.16 μ g/ml Lemna paucicostatas（TH-W3）Costimulation T cell granzyme, perforin and CD107a after induction
Expression；
Fig. 8：Killing activity of the costimulation T cell of Lemna paucicostata Th-w3 inductions to SW-1990；
Fig. 9：The expression of costimulation T cell ERK1/2 after Lemna paucicostata Th-w3 induction 48h；
Figure 10：Costimulation T cell ERK1/2 gray scale scanning after Lemna paucicostata Th-w3 induction 48h；
Figure 11：SW-1990 cell fusion results after 0.16 μ g/ml Lemna paucicostatas TH-W3 inductions；
Figure 12：After 0.16 μ g/ml Lemna paucicostatas TH-W3 inductions, cancer of pancreas SW-1990 cells pass through rate.
Specific embodiment
With reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and should not be regarded as limiting this
Bright scope.
Embodiment 1：Radix Apioris Fortunei (Radix Lespedezae Buergeri) medicinal material extract separating experiment situation
Material Radix Apioris Fortunei (Radix Lespedezae Buergeri) dried root is purchased from Ningbo City Yin states pharmaceutical material and drug corporation；80% macroporous adsorbent resin tree chromatograph；
Yanaco micro melting point apparatus（Thermometer is not corrected）；Varian MAT-212 type mass spectrographs；Bruker-speckospin
AC-600P type nuclear magnetic resonance analyser.Thin layer chromatography silica gel plate and column chromatography silica gel（100 ~ 200 mesh, 200 ~ 300 mesh）It is Shandong cigarette
The production of Taijiang friend's silica gel development company；Sephadex LH-20（20~80 µm）Produce for Pharmacia companies；ODS C18 are anti-
Phase silica gel is produced for Merck companies；Extraction ethanol；Petroleum ether, ethyl acetate, n-butyl alcohol are pharmaceutical grade, and water is distilled water, its
It is pure that remaining reagent is analysis.
Extracting solution is grouped：Extracting solution is divided into into 9 groups according to preparation selected when extracting, is respectively designated as：TH-t、TH-
P, TH-a, TH-b, TH-w, TH-w1, TH-w2, TH-w3 and TH-w4
The extracting method of each group Lemna paucicostata：Lemna paucicostata extraction reference literature Yang Yang. Salvia przewalskii and headdress flower
Knotweed chemical constitution study. The 2nd Army Medical College master thesis. Shanghai：The 2nd Army Medical College, 2009 ］ isolate and purify and obtain,
Jing high performance liquid chromatography（HPLC）Check.
TH-t parts：Take Radix Apioris Fortunei (Radix Lespedezae Buergeri) and be dried medical material 200g, plus after 5 times of 80% ethanol waters of volume soak 24h, heat back
Stream is extracted, totally 3 times, each 40min, united extraction liquid, is filtered, and recovered under reduced pressure is evaporated, and obtains total extract TH-t 21.6g, yield
10.8%。
TH-p parts：This total extraction is taken, add water suspension, and the petroleum ether of 10 times of volumes is respectively adopted（Water saturation）Carry out substep
Extraction.Respectively obtain petroleum ether part TH-p 0.15g, yield 0.075%.
TH-a parts：Ethyl acetate extract TH-a 0.58g, yield 0.29%.
TH-b parts：N-butanol portion TH-b 2g, yield 1%.
TH-w parts：And water position TH-w 18.9g, yield 9.45%.
TH-w1 parts：Water intaking position carries out macroporous adsorbent resin chromatography, first carries out eluting using pure water, removes sugar, then
Using 10%, 30%, 60%, 95% ethanol water difference successively eluting.Wherein, 10% ethanol water elution solution decompression is reclaimed and is evaporated
After obtain water position desaccharide flow point TH-w1 0.14g, yield 0.07%.
TH-w2 parts：Water intaking position carries out macroporous adsorbent resin chromatography, first carries out eluting using pure water, removes sugar, then
Using 10%, 30%, 60%, 95% ethanol water difference successively eluting.Wherein, 30% ethanol aqueous strip solution obtains water after being evaporated
Position desaccharide flow point TH-w1 0.3g, yield 0.15%.
TH-w3 parts：Recovered under reduced pressure after the above-mentioned Radix Apioris Fortunei (Radix Lespedezae Buergeri) medical material decocting in water Jing after organic solvent extraction is evaporated, is obtained
2.1 grams of dehydrate.
TH-w4 parts：The above-mentioned Radix Apioris Fortunei (Radix Lespedezae Buergeri) medical material recovered under reduced pressure Jing after organic solvent extraction is evaporated into dehydration, is reused
10%th, 30%, 60%, 95% ethanol water desaccharide successively respectively, wherein, obtain 10% ethanol water position desaccharide flow point.
Table 1 is each composition extracting method of Radix Apioris Fortunei (Radix Lespedezae Buergeri) and acquisition amount
| Numbering | Position | Packet | Quality | Actual weight | Yield |
| 1 | 80% ethanol total extract（Ethanol DMS0） | TH-t | 21.5g | 0.55g | 10.8% |
| 2 | Petroleum ether part (DMS0) | TH-p | 0.15g | 0.05g | 0.075% |
| 3 | Ethyl acetate extract (DMS0) | TH-a | 0.58g | 0.58g | 0.29% |
| 4 | N-butanol portion (H2O alcohol) | TH-b | 2g | 1.4g | 1% |
| 5 | Water position（H2O） | TH-w | 18.9g | 0.04g | 9.45% |
| 6 | Water position desaccharide flow point（H2O） | TH-w1 | 0.15g | 0.15g | 0.07% |
| 7 | Flow point after the desaccharide of water position（H2O） | TH-w2 | 2.06 | 0.3g | |
| 8 | Decocting in water thing（Desaccharide） | TH-w3 | 0.6 g | ||
| 9 | Decocting in water thing（Containing sugar） | TH-w4 | 2.1 g |
Embodiment 2：The impact experiment that Lemna paucicostata is bred to costimulation T cell
Experiment material：Serum-free medium, costimulation T cell culture medium（Zhejiang Beaune bio tech ltd product）；Four
Methyl- azoles indigo plant (methlthiazolyl tetrazolium, MTT) and dimethyl sulfoxide (dimethyl s μ lfoxide
DMSO) (Sigma companies)；Lymphocyte separation medium (Chinese Academy of Sciences's hematopathy institute)；IL-2, IL-15 and IL-21
（Xiamen Te Bao biotech firms）；INF- γ are purchased from Abender companies；Trypsin, RPMl1640, calf serum (are purchased from
GIBCO companies)；People's AB serum (Xuzhou City blood station)；Enter CD56 monoclonal antibodies, the PE of CD3, FITC labelling of PerCP-Py5.5 labellings
The perforin of labelling（Pore-forming protein, PFP), granzyme B (Granzyme B, GrB), APC labellings
CD107a （Biology company limited of connection section）；Lactic acid dehydrogenase (Lactate dehydrogenase, LDH) test kit (Japanese generation
Promise clinical diagnosises product Co., Ltd.)；Encore automatic biochemistry analyzers（Beijing Xi Yake Technology Co., Ltd.；Fluorescence is inverted
Microscope TE2000-S (Japanese Nikon companies)；Flow cytometer (U.S. company BD)；Flow cytometry analysis software Cell
Quest, each analyzing specimen cell number >=l × 104 。
The culture of costimulation T cell and identification：10 ml of healthy blood donor periphery anticoagulation is taken, lymphocyte separation medium is separated
Mononuclearcell, with the costimulation T cell culture medium adjustment cell density containing different cell growth factor and stimulation molecule be
3.5×105/ mL, is inoculated in 6 porocyte culture plates, 5 ml of hole, in 37 °C, 5%CO2Incubator culture, changes per 2 days half amounts
Liquid 1 time, and cell density is adjusted for 5 × 105/ mL, collects the culture costimulation T cell of 7 days, adds PerCP-Py5.5 labellings
The CD56 monoclonal antibodies of CD3, FITC labelling carry out cell surface marker detection.
The impact that the different Lemna paucicostatas of CCK8 methods detection are grown to people's costimulation T cell：Take the culture costimulation of 7 days
T cell is made into 5 × 104The cell suspension of/mL, adds 96 porocyte culture plates, 200 μ l/ holes.Experimental group：11 concentration are set altogether
Group, is separately added into 5 × 104The costimulation T cell of/mL, 180 μ l/hole.It is subsequently adding the Lemna paucicostata of variable concentrations
（Final concentration is respectively 0.025,0.05,0. 1,0.2,0.4,0.8,1.6,3.2,6.4,12.8,25.6 μ g/ml）, 5 are set per group
Individual multiple holes, while setting last dosing thing blank control group：24 h, 48 h, 72 h are cultivated in incubator.Culture terminate first 4 it is little
When 20 μ l of CCK8 are added per hole, continue 4 h of culture and detect each hole light absorption value after 450 nm of microplate reader places（OD）Record result.
The rate of increase（%）=（Experiment OD values）—（Control OD values）/（Control OD values）×100%.
Statistical method：Statistical procedures are carried out using SPSS13.0 softwares, measurement data uses mean ± standard deviation (mean
± SD) represent, compare between group and adopt one factor analysis of variance（One-way ANOVA）, inspection level α=0.05,P≤ 0.05 has
Statistical significance.
As a result it is as follows：
Costimulation T cell is identified：Under costimulation T cell culture back to front microscope, cell growth condition is shown in Fig. 1 and Fig. 2.Training
CD3 in peripheral blood PBMC before supporting-CD56+For 4.73%, costimulation T cell CD3 during 10 d is cultivated-CD56+Ratio is up to
38.85%, see Fig. 3 and Fig. 4.
Each stage Lemna paucicostata is as follows to costimulation T cell growth effect：
Find in preliminary experiment, each stage Lemna paucicostata that extracts has larger difference to costimulation T cell growth effect, in medicine
Under thing concentration the same terms, with decocting in water desaccharide position（Th-w3）When promote costimulation T cell growth activity it is most strong；Total extract
（Th-t）, petroleum ether part（Th-p）Take second place with water position desaccharide flow point.As can be seen from Fig. 5, Lemna paucicostata（Th-w3）It is right
The growth of costimulation T cell has time dependence, and the drug-induced time is longer, promotes cell to breed more obvious, and drug level exists
Promote cell propagation during 0.025-0.02 μ g/ml most substantially, concentration is higher than propagation capable of inhibiting cell after 0.8 μ g/ml.Different doses
Comparing between group and different time group and group has significant difference（p＜ 0.05）.
Embodiment 3：Lemna paucicostata is to pancreatic cancer cell SW-1990 inhibition tests
Experiment material：Serum-free medium（Zhejiang Beaune bio tech ltd product）；Methyl thiazoly tetrazolium assay
(Sigma is public for (methlthiazolyl tetrazolium, MTT) and dimethyl sulfoxide (dimethyl s μ lfoxide DMSO)
Department)；Lymphocyte separation medium (Chinese Academy of Sciences's hematopathy institute)；Lactic acid dehydrogenase (Lactate dehydrogenase,
LDH) test kit (Japanese Shino Test Corp.)；Encore automatic biochemistry analyzers（Beijing Xi Yake technologies
Company limited；Fluorescence inverted microscope TE2000-S (Japanese Nikon companies)；Flow cytometer (U.S. company BD)；Streaming is thin
Born of the same parents instrument analysis software Cell Quest, each analyzing specimen cell number >=l × 104 。
The impact that Lemna paucicostata is grown to pancreatic cancer cell SW-1990 strains：Trophophase take the logarithm to pancreatic cancer cell
SW-1990 cells are made into 3 × 107The cell suspension of/L adds 96 orifice plates, per 180 μ l of hole, 5 multiple holes is set per group, while setting the moon
Property matched group；Culture plate is put into into 37 DEG C, 50mL/L CO2After 24 h of incubator culture, experimental group be separately added into variety classes and
The Lemna paucicostata of concentration（Concentration is respectively 0.05,0.1,0.2,0.4,0.8,1.6,3.2,6.25,12.5,25,50,100
μg/ml）, while setting the matched group of not dosing.In 37 DEG C, 50mL/L CO272 h are cultivated in incubator；Before culture terminates, 4 is little
When add MTT 20 μ l/ holes, continue supernatant discarded after 4 h of culture, add 150 μ l of DMSO, praise first and be completely dissolved, in enzyme
The each hole light absorption value of detection at mark instrument 495nm（OD）Record result.Cell inhibitory rate IR（%）=（Control OD values）—（Experiment OD values）/
（Control OD）×100%.Identical experiment is repeated 3 times, and averages.
Statistical method：Statistical procedures are carried out using SPSS13.0 softwares, measurement data uses mean ± standard deviation (mean
± SD) represent, compare between group and adopt one factor analysis of variance（One-way ANOVA）, inspection level α=0.05,P≤ 0.05 has
Statistical significance.
As a result it is as follows：
Pancreatic cancer cell SW-1990 is carried out into induction experiment with Lemna paucicostata, Jing after induction 72 hours, the SANYE of high concentration
Blue or green extract has inhibitory action to three kinds of tumor cells, as the increase of drug level suppresses more obvious.But, each concentration group
To inhibiting tumour cells concentration in more than 6.25 μ g/ml, less than the equal unrestraint phenomenon of each group during 1.6 μ g/ml.Radix Apioris Fortunei (Radix Lespedezae Buergeri) is extracted
Thing is shown in Fig. 6 to the suppression ratio of SW-1990 cells.
Embodiment 4：Expression and killing of the Lemna paucicostata to perforin, CD107a and granzyme B in costimulation T cell
Activity influence is tested
Experiment material：Serum-free medium, costimulation cell T culture medium（Zhejiang Beaune bio tech ltd product）；Four
Methyl- azoles indigo plant (methlthiazolyl tetrazolium, MTT) and dimethyl sulfoxide (dimethyl s μ lfoxide
DMSO) (Sigma companies)；Lymphocyte separation medium (Chinese Academy of Sciences's hematopathy institute)；IL-2, IL-15 and IL-21
（Xiamen Te Bao biotech firms）；INF- γ are purchased from Abender companies；Trypsin, RPMl1640, calf serum (are purchased from
GIBCO companies)；People's AB serum (Xuzhou City blood station)；Enter CD56 monoclonal antibodies, the PE of CD3, FITC labelling of PerCP-Py5.5 labellings
The perforin of labelling（Pore-forming protein, PFP), granzyme B (Granzyme B, GrB), APC labellings
CD107a （Biology company limited of connection section）；Lactic acid dehydrogenase (Lactate dehydrogenase, LDH) test kit (Japanese generation
Promise clinical diagnosises product Co., Ltd.)；Encore automatic biochemistry analyzers（Beijing Xi Yake Technology Co., Ltd.；Fluorescence is inverted
Microscope TE2000-S (Japanese Nikon companies)；Flow cytometer (U.S. company BD)；Flow cytometry analysis software Cell
Quest, each analyzing specimen cell number >=l × 104 。
The culture of costimulation T cell and identification：10 ml of healthy blood donor periphery anticoagulation is taken, lymphocyte separation medium is separated
Mononuclearcell, with containing different cytokines and stimulation molecule costimulation T cell culture medium adjustment cell density be 3.5 ×
105/ mL, is inoculated in 6 porocyte culture plates, 5 ml of hole, in 37 °C, 5%CO2Incubator culture, changed liquid 1 time per 2 days half amounts,
And cell density is adjusted for 5 × 104/ mL, the collection culture costimulation T cell of 7 days, the CD3 of addition PerCP-Py5.5 labellings,
The CD56 monoclonal antibodies of FITC labellings carry out cell surface marker detection.
LDH method for releasing detects Lemna paucicostata（TH-W3）The costimulation T cell of induction is to SW-1990 killing activities：Take
Jing variable concentrations（Respectively 0.01,0.02,0.04,0.08,0.16,0.32,0.64,1.28,2.56,5.12,10.24 μ g/
ml）Lemna paucicostata（Th-w3）The costimulation T cell after 48 h is induced to be made into 2 × 109/ L, action effect cell；Logarithm is given birth to
Long-term pancreatic cancer cell SW-1990 is made into 2 × 108/ L, as target cell；Effect target cell is respectively taken into 0.5 ml mixed in equal amounts
（Effect target ration=10:1）, while arrange being not added with Lemna paucicostata for matched group.Put 37 °C, 5%CO2It is incubated in incubator
After 6 h, gently mix, re-suspended cell, 1500 r/min are centrifuged 10 min, collect supernatant.LDH test kits by specification is required
Operation, under 340 nm wavelength, Encore automatic biochemistry analyzers determine the active unit (U/L) of LDH.Often detect that sample sets 3 again
Pipe, repeats 3 times, averages.
The killing activity of costimulation T cell=（Determine pipe LDH units-effector lymphocyte's Spontaneous release LDH pipes）/（Target cell
Maximum release LDH pipe-target cell Spontaneous releases LDH pipes）×100%.
The expression of perforin and granzyme B and CD107a in flow cytomery costimulation T cell：Will be culture successful
Costimulation T cell is made into concentration for 1.0 × 105The cell suspension of/ml, is inoculated in 6 well culture plates, per hole 3ml, is subsequently adding end
Concentration is respectively the Lemna paucicostata of 0.01,0.04,0.16,0.64,2.56 μ g/ml（TH-W3）, per group of 3 multiple holes.In 37
DEG C, cultivate 48h in 5%CO2 incubators.Collect cell and use phosphate buffer（PBS）Washing 2 times, often pipe addition CD56-
20 μ l of FITC antibody, lucifuge incubation 30min.100 μ l fixative room temperatures lucifuges incubation 15min, PBS is added to wash 1 time.Often manage
Plus 100 μ l rupture of membranes liquid, 100 μ l anti-Perforin antibody and anti-granzyme B-PE antibody and APC labellings
CD107a antibody, room temperature lucifuge incubation 15min, after PBS washings, is resuspended in the PBS solution of 0.5ml, uses fluidic cell respectively
The expression of art detection perforin, granzyme B and CD107a.
Statistical method：Statistical procedures are carried out using SPSS13.0 softwares, measurement data uses mean ± standard deviation (mean
± SD) represent, compare between group and adopt one factor analysis of variance（One-way ANOVA）, inspection level α=0.05,P≤ 0.05 has
Statistical significance.
As a result it is as follows：
Lemna paucicostata（TH-W3）Impact result to granzyme B, perforin expression in costimulation T cell：With 0 μ g/ml couple
Compare according to group, concentration is respectively the Lemna paucicostata of 0.01,0.04,0.16,0.64,2.56 μ g/ml（TH-W3）Effect is pierced altogether
After sharp T cell 48h, its perforin and granzyme B positive expression are significantly raised, in 0.1 μ g/ml ~ 1.6 μ g/ml concentration, tool
There is certain dose dependent.Wherein, the perforin of 0.2 μ g/ml Lemna paucicostata groups, granzyme B and CD107a positive table
Peak, respectively 90.48% ± 2.31%, 69.66% ± 1.29% and 93.37% ± 5.11% are reached up to rate, and concentration exceedes
During 6.25 μ g/ml, perforin and granzyme B positive expression rate have declined, and as a result see Fig. 7.
Lemna paucicostata（TH-W3）The costimulation T cell of induction is as follows to SW-1990 killing activities：
Experimental result shows that concentration is 0.64～0.02 μ g/ml Lemna paucicostatas（TH-w3）Costimulation T after effect 72h is thin
Born of the same parents' cell is significantly higher than matched group to the killing activity of pancreatic cancer cell SW-1990, and difference is statistically significant（P＜ 0.05）.
In 0.16 μ g/ml, killing activity reaches and is up to 56.21%, with matched group（42.7%）Comparing difference is statistically significant（P＜
0.05）.When Radix Apioris Fortunei (Radix Lespedezae Buergeri) concentration is higher than 2.56 μ g/ml, killing activity increases with concentration and is gradually lowered, when concentration is up to 2.56-
During 10.32 μ g/ml, killing activity of the costimulation T cell to tumor cell can be suppressed（P ＜ 0.05）, as a result see Fig. 8.
Embodiment 5：Lemna paucicostata is to costimulation T cell ERK1/2 protein expression assay
Experiment material：Serum-free medium and costimulation T cell culture medium（Zhejiang Beaune bio tech ltd product）；Four
Methyl- azoles indigo plant (methlthiazolyl tetrazolium, MTT) and dimethyl sulfoxide (dimethyl s μ lfoxide
DMSO) (Sigma companies)；Lymphocyte separation medium (Chinese Academy of Sciences's hematopathy institute)；IL-2, IL-15 and IL-21
（Xiamen Te Bao biotech firms）；INF- γ are purchased from Abender companies；People's AB serum (Xuzhou City blood station)；FITC and PE labellings
The CD56 of CD3, FITC labelling（Biology company limited of connection section）；GAPDH, Bcl-2 are purchased from Abcam.；Rectilinear electrophoretic apparatuss, electricity
Transfer groove（Beijing Xi Yake Technology Co., Ltd.）；BCA determination of protein concentration test kits（Green skies biotechnology research institute）.
The culture of costimulation T cell and identification：10 ml of healthy blood donor periphery anticoagulation is taken, lymphocyte separation medium is separated
Mononuclearcell, with containing cell growth factor and stimulation molecule costimulation T cell culture medium adjustment cell density be 3.5 ×
105/ mL, is inoculated in 6 porocyte culture plates, 5 ml of hole, in 37 °C, 5%CO2Incubator culture, changed liquid 1 time per 2 days half amounts,
And cell density is adjusted for 5 × 105/ mL, the collection culture costimulation T cell of 7 days, the CD3 of addition PerCP-Py5.5 labellings,
The CD56 monoclonal antibodies of FITC labellings carry out cell surface marker detection.
The change of costimulation T cell p-ERK expression before and after Westernblot detection Lemna paucicostata Th-w3 effects：Take
The culture costimulation T cell of 7 days, is made into 5 × 108/ L cell suspension, is inoculated in 6 orifice plates respectively, and 3 ml/ holes add Radix Apioris Fortunei (Radix Lespedezae Buergeri)
Extract Th-w3（Final concentration is respectively to 0.01,0.04,0.16,0.64,2.56 μ g/ml）, while arrange being not added with Th-w3 controls
Group.After continuing 48 h of culture, plus 500 μ l cell lysis of cell pyrolysis liquid, extract albumen, Forlin detection protein concentrations, SDS-
Polyacrylamide gel electrophoresis separation, transferring film, 2 h of closing, an anti-incubation：Add freshly prepared 1:The anti-human p- of 500 dilution Mus
ERK1/2 antibody, is placed in 4 °C of refrigerator overnights；Two anti-incubations：Add 1:The anti-Mus IgG of alkali phosphatase enzyme mark horse of 1000 dilutions,
Filter membrane is immersed into NBT/BCIP nitrite ions, band is taken out after terminating reaction and is dried, the upper machine scannings of ODSEEY.With Image J images
Analysis software is analyzed.
Statistical method：Statistical procedures are carried out using SPSS13.0 softwares, measurement data uses mean ± standard deviation (mean
± SD) represent, compare between group and adopt one factor analysis of variance（One-way ANOVA）, inspection level α=0.05,P≤ 0.05 has
Statistical significance.
As a result it is as follows：
When three, green grass or young crops extract Th-w3 is expressed to costimulation T cell ERK1/2：Variable concentrations Lemna paucicostata Th-w3 acts on 48h
CD3AK cells afterwards its ERK1/2 protein expressions has been raised compared with matched group, concentration in 0.64-0.01 μ g/ml, ERK1/
2 protein expressions are above matched group, and most obvious with 0.16 μ g/m, difference is statistically significant（P＜ 0.05）, as a result see Fig. 9,
10
Embodiment 6：Lemna paucicostata（Th-w3）SW-1990 cell strains locomotivity is tested
Experiment material：Serum-free medium（Zhejiang Beaune bio tech ltd product）；Methyl thiazoly tetrazolium assay
(methlthiazolyl tetrazolium, MTT) and dimethyl sulfoxide (DMSO) (Sigma companies)；24 hole transweU
Plate (8 μm of aperture, 6.5 mm of film diameter) and 6 porocyte culture plates (Coming companies)；Lymphocyte separation medium (Chinese section
Institute's hematopathy institute).
Cell migration assay：Empirically design is divided into matched group and Lemna paucicostata Th-w3（Concentration be 0.01,0.04,
0.16、0.64、2.56μg/ml）Intervention group.Take the logarithm the SW-1990 cells of trophophase, adjustment density is 1.0 × 108/ L, inhales
Take 200 μ L cell suspension and add 24 holes migration cell（transwell chambers）Upper room, lower room is slowly added to 500 μ L
Culture medium, 37 DEG C, 50mL/L CO248 h are incubated in incubator, cell RPMI1640 are taken out and is washed 3 times, wiped with cotton balls
The cell in upper room face, lower room face dehydrated alcohol fix 10 min, after PBS rinsings with 0.1% 10 min of violet staining, PBS
Rinsing 3 times.Cell of the counted under microscope through transwell apertures, observes 5 visuals field and takes pictures, and experiment is repeated 3 times.
Invasion and attack suppression ratio（IR）=（1- experimental grouies migrating cell number/matched group migrating cell number）×100%.
Cut Healing Experiments：By RPMIs of the pancreatic cancer cell SW-1990 of exponential phase containing 10% hyclone
1640 culture medium culturings, in adding 6 porocyte culture plates, when the fusion of SW-1990 cell growths reaches 90%, with 200 μ L's
Pipettor gun head, in the y direction cut in culture plate central authorities per hole, sucks former culture medium, is washed 2 times with RPMI1640 culture medium,
Remove the floating cells in cut area.The 1640 culture medium culturing 5ml of RPMI of 10% hyclone are added per hole, is then respectively adding
Final concentration is respectively the Lemna paucicostata Th-w3 intervention groups of 0.01,0.04,0.16,0.64,2.56 μ g/ml, while set being not added with
The matched group of medicine, 37 DEG C, 50mL/L CO2It is incubated in constant incubator, it is aobvious respectively at being inverted in culture 0,24,48 h
Observation of cell fusion growth situation take pictures under micro mirror, 3 multiple holes are set per group, experiment is repeated 3 times.
Statistical method：Statistical procedures are carried out using SPSS13.0 softwares, measurement data uses mean ± standard deviation (mean
± SD) represent, compare between group and adopt one factor analysis of variance（One-way ANOVA）, inspection level α=0.05,P≤ 0.05 has
Statistical significance.
As a result it is as follows：
Cut Healing Experiments：Jing concentration is the Lemna paucicostata Th-w3 inductions of 0.01,0.04,0.16,0.64,2.56 μ g/ml
After 48h, the fusion rate of cancer of pancreas SW-1990 compares statistically significant with matched group with low during 0.16 μ g/ml.Other Chinese are yellow
The SW-1990 fusion rate of a kind of reed mentioned in ancient books concentration group induction is also low compared with matched group.As a result see Figure 11.
Migration experimental result：Jing concentration is the Lemna paucicostata Th-w3 of 0.01,0.04,0.16,0.64,2.56 μ g/ml
After induction 48h, cancer of pancreas SW-1990 is by transwell pore cells number with Lemna paucicostata Th-w3 concentration as 0.16 μ
It is minimum during g/ml；In 0.01-2.56 μ g/ml, cell passes through rate also below matched group to concentration, each concentration Th-w3 induction group SW-
1990 cells are compared through rate with matched group significant difference.The invasion and attack of the Colorimetric results conversion of eluent after dyeing
Suppression ratio：Drug-induced each group mobility is below matched group.As a result see Figure 12.
It should be understood by those skilled in the art that, the present invention is not restricted to the described embodiments, above-described embodiment and explanation
Merely illustrating the principles of the invention described in book, without departing from the spirit and scope of the present invention, the present invention also has
Various changes and modifications, these changes and improvements are both fallen within scope of the claimed invention.The claimed scope of the invention
By appending claims and its equivalent thereof.
Claims (7)
1. the method that Lemna paucicostata improves people's costimulation T cell propagation is studied, it is characterised in that：Step is as follows：
Step one, the external evoked culture people's costimulation T cell of Lemna paucicostata；
Step 2, the impact that the different Lemna paucicostatas of detection are grown to people's costimulation T cell, records result, adopts
SPSS13.0 softwares carry out statistical procedures, and measurement data is represented using mean ± standard deviation, compared using single factor test side between group
Difference is analysed, inspection level α=0.05,P≤ 0.05 is statistically significant.
2. the method that research Lemna paucicostata according to claim 1 improves people's costimulation T cell propagation, its feature exist
In：The external evoked culture people's costimulation T cell of Lemna paucicostata described in step one is specially：Take outside appropriate healthy blood donor
All anticoagulations, lymphocyte separation medium separate mononuclearcell, with the common thorn containing different cell growth factor and stimulation molecule
Sharp T cell culture medium adjustment cell density is 3.5 × 105/ mL, is inoculated in Tissue Culture Plate, and incubator culture is periodically changed
Liquid, and cell density is adjusted for 5 × 105/ mL, collects the culture costimulation T cell of 7 days, adds monoclonal antibody to carry out cell surface mark
Will is detected.
3. the method that research Lemna paucicostata according to claim 2 improves people's costimulation T cell propagation, its feature exist
In：Cell growth factor described in step one and stimulating factor are CD3mAb, CD28 mAb, IFN-γ, IL-2, IL-1 α, IL-
15th, IL-18 or IL-21.
4. the method that research Lemna paucicostata according to claim 2 improves people's costimulation T cell propagation, its feature exist
In：It is inoculated in step one in 6 porocyte culture plates, 5 mL of hole, the incubator is 37 °C, 5%CO2Incubator, it is described fixed
It is that liquid 1 time is periodically changed per 2 days half amounts that phase changes liquid, and the monoclonal antibody of the addition is CD3 the and FITC labellings of PerCP-Py5.5 labellings
CD56。
5. the method that research Lemna paucicostata according to claim 1 improves people's costimulation T cell propagation, its feature exist
In：The impact that different Lemna paucicostatas are grown to people's costimulation T cell is detected described in step 2, specially：Take culture 7 days
Costimulation T cell, be made into cell suspension, add Tissue Culture Plate, if 11 concentration groups, are separately added into costimulation T cell, so
The Lemna paucicostata of variable concentrations is added afterwards, and 5 multiple holes are set per group, while last dosing thing blank control group is set, in incubator
24 h of middle culture, 48 h, 72 h；Culture adds 20 μ l of CCK8 before terminating per hole, continues culture, detects each hole light absorption value afterwards,
And record result.
6. the method that research Lemna paucicostata according to claim 5 improves people's costimulation T cell propagation, its feature exist
In：The impact that different Lemna paucicostatas are grown to people's costimulation T cell is detected described in step 2, specially：Take culture 7 days
Costimulation T cell, be made into 5 × 104The cell suspension of/mL, adds 96 porocyte culture plates, 200 μ l/ holes, if 11 concentration
Group, is separately added into 5 × 104The costimulation T cell of/mL, 180 μ l/hole, is subsequently adding the Lemna paucicostata of variable concentrations,
Final concentration is respectively 0.025,0.05,0. 1,0.2,0.4,0.8,1.6,3.2,6.4,12.8,25.6 μ g/mL, and 5 are set per group
Multiple holes, while setting last dosing thing blank control group, cultivate 24 h, 48 h, 72 h in incubator；Culture terminates front 4 hours
20 μ l of CCK8 are added per hole, are continued 4 h of culture and each hole light absorption value are detected at 450 nm of microplate reader, and record result.
7. the method that the research Lemna paucicostata described in claim 6 improves people's costimulation T cell propagation, it is characterised in that：
Lemna paucicostata described in step 2 is decocting in water desaccharide position Th-w3, i.e., the Radix Apioris Fortunei (Radix Lespedezae Buergeri) medical material Jing after organic solvent extraction is used
After decocting in water, the dehydrate of gained after vacuum distillation.
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| CN104651310A (en) * | 2015-02-28 | 2015-05-27 | 杭州中德贝尔生物科技有限公司 | NK cell in-vitro induced amplification method |
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