CN107375557A - Application of the Lemna paucicostata TH b parts in immunotherapy of tumors medicine and human dendritic cell multiplication agent is prepared - Google Patents
Application of the Lemna paucicostata TH b parts in immunotherapy of tumors medicine and human dendritic cell multiplication agent is prepared Download PDFInfo
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Abstract
The invention provides a kind of application of Lemna paucicostata TH b parts in immunotherapy of tumors medicine and BMDC is prepared, belong to technical field of pharmaceutical biotechnology.Lemna paucicostata TH b parts can remarkably promote the propagation of human dendritic cell, improve its phagocytic activity, promote costimulatory molecules CD80, CD86 expression and IL 12 secretion, have certain dose dependent.High concentration Lemna paucicostata can suppress growth of tumour cell.Present invention firstly discovers that low concentration Lemna paucicostata TH b parts can significantly improve the phagocytic activity of BMDC, promote BMDC CD80 and CD86 expression and IL 12 secretion, improve maturing dendritic cell mark CD83 percentage.
Description
Technical field
The present invention relates to a kind of Lemna paucicostata TH-b parts to prepare immunotherapy of tumors medicine and human dendritic is thin
Application in born of the same parents' multiplication agent, belongs to field of medical biotechnology.
Background technology
In recent years, the biological therapy of tumour has become the focus of therapeutic field of tumor research.BMDC
(dendritic cell, DC) is known most strong professional antigen presenting cells (the antigen p of antigen presentation function in vivo
Resentation cell, APC), can absorb all kinds of antigens, the MHC molecule of expressed in abundance, costimulatory molecules, adhesion molecule and
High-caliber TH1 cytokines IL-12 etc..DC by its development degree can be divided into mature dendritic cell (matrer DC,
) and immature dendritic cell (immature DC, imDC) mDC.Im DC express acceptor (such as sweet dew relevant with phagocytosis because high
Saccharide acceptor) there is stronger antigen endocytosis and the costimulatory molecules such as working process ability, low expression CD80, CD83, CD86 and resist
Original offers molecule MHC II, it is impossible to effectively activate complementary T cell and inducing immune tolerance；And mDC is then on the contrary, because of it
Height expression costimulatory molecules and the molecules of MHC II can excite helper T lymphocyte and trigger immune response.BMDC can also be with
Other inherent immunity cell interactions, collaboration play antitumor action, and the DC through all kinds of tumour antigen sensitization can induce body
Produce high-caliber specificity antineoplastic immunity response.Human dendritic cell has become tumor patient adoptive immunotherapy at present
In an important effector cell.As can be seen here, effectively culture BMDC improve its biological function be it is particularly significant and
It is necessary.And surface of dendritic cells costimulatory molecules CD80, CD86 and ripe mark CD83 molecules can directly react the effect
Answer cell function；The phagocytic function of BMDC directly reacts the antigen uptake ability of this effector cell.This research stream
Formula cell art and directly phagocytosis method detection dendritic cell function change, and enzyme exempts from the IL-12 abilities of method detection DC secretions.Further
Verify that three leaf table extract TH-b parts can promote dendritic cell function.
Radix tetrastigme is Vitaceae Tetrastigma, is plant tetratigma hemsleyanum (Tetrastigmahemsleyanum Diels
Et Gilg) dried root, also known as hemsley rockvine root etc., be China endemic plant, be distributed mainly on the Southeast China people
Between common drug.The active ingredient of radix tetrastigme predominantly contains flavonoids, and its is cool in nature, acrid flavour, hardship, nontoxic, has
Clearing heat and detoxicating, promoting blood circulation and stopping pain, dispelling pathogenic wind and eliminating phlegm, promoting blood circulation and stopping pain and other effects, are usually used in hyperpyretic convulsion, and cold in children heating, larynx are itched swollen
Bitterly, the disease such as cough, pneumonia, hepatitis, enteritis, dysentery.Modern pharmacological research shows that Lemna paucicostata has preferably anti-swollen
Knurl, anti-inflammatory, analgesia and refrigeration function etc..According to the literature, radix tetrastigme general flavone to tumour cell have suppress cell propagation and
Apoptosis-induced effect.
Radix tetrastigme plant is distributed mainly on various regions on the south the Changjiang river, is conventional Chinese herbal medicine among the people, itches in cold, fever, larynx swollen
Bitterly, it is that can reach obvious clinical efficacy that relatively low dose is only needed in the disease such as cough, pneumonia, hepatitis, enteritis, dysentery, and mesh
Preceding researchs many both at home and abroad think that the active ingredient in high concentration Lemna paucicostata can suppress tumor cell proliferation, to tumour
Dosage when cell suppresses is mostly in 10mg/ml or so.This result is not substantially inconsistent with clinical practice dosage.
The content of the invention
(1) technical problems to be solved
It is an object of the invention to provide a kind of Lemna paucicostata TH-b parts in immunotherapy of tumors medicine is prepared
Using the phagocytic activity of human dendritic cell being improved using leaf green grass or young crops extract, so as to prepare killing tumor cell medicine.
(2) technical scheme
In order to solve the above-mentioned technical problem, Lemna paucicostata TH-b parts provided by the invention are controlled in preparation tumour immunity
Treat the application in medicine.
Optionally, the immunotherapy of tumors medicine is melanoma immunotherapy medicaments.
The invention also discloses application of the Lemna paucicostata TH-b parts in human dendritic cell multiplication agent is prepared.
(3) beneficial effect
A kind of application of the Lemna paucicostata TH-b parts provided by the invention in immunotherapy of tumors medicine is prepared, its
With advantages below：First：Present invention firstly discovers that various concentrations radix tetrastigme TH-b extracting sections thing can remarkably promote people
The phagocytic activity of BMDC；Second：Low concentration radix tetrastigme TH-b extracting sections thing can promote BMDC CD80,
CD83, CD86, HLA-DR expression；3rd：The BMDC IL-12 secretion capacities of radix tetrastigme TH-b extracting sections thing induction
Increase.
Brief description of the drawings
Form (400 ×) under the DC micro- aobvious mirror of inversion in cell after Fig. 1 cultures 8 days；
Fig. 2 DC control group × 1000；
Fig. 3 DC phagocytosis spores photo × 1000；
Inhibitory action result figure of Fig. 4 radix tetrastigme TH-b extracts to melanoma cells strain；
Fig. 5 CD80, CD83 Isotype controls；
Fig. 6 control group DCCD80, CD83 streaming result figure (TH-b moiety concentrations：0μg/ml)；
Fig. 7 induction groups DC CD80, CD83 streaming result figure (TH-b moiety concentrations：0.64μg/ml)；
Fig. 8 DC CD86, HLA-DR Isotype control figure；
Fig. 9 control groups DC CD86, HLA-DR streaming result figure (TH-b moiety concentrations：0μg/ml)；
Figure 10 induction groups DC CD86, HLA-DR streaming result figure (TH-b moiety concentrations：0.64μg/ml)；
Figure 11 various concentrations TH-b parts induction DC secretion IL-12 result figures.
Embodiment
With reference to the accompanying drawings and examples, the embodiment of the present invention is described in further detail.Following instance
For illustrating the present invention, but it is not limited to the scope of the present invention.
Radix tetrastigme medicinal material extract separates：
The material of use：Radix tetrastigme dried root is purchased from Ningbo City Yin states pharmaceutical material and drug corporation；80% macroporous absorbent resin
Set chromatograph；Yanaco micro melting point apparatus (thermometer does not correct)；Varian MAT-212 type mass spectrographs；Bruker-
Speckospin AC-600P type NMRs.Thin-layer chromatography silica gel plate and column chromatography silica gel (100~200 mesh, 200~300
Mesh) it is the production of Shandong Yantai Jiang You silica gel development company；(20~80 μm) of Sephadex LH-20 are that Pharmacia companies give birth to
Production；ODS C18 reverse phase silica gels produce for Merck companies；Extraction ethanol；Petroleum ether, ethyl acetate, n-butanol are pharmaceutical grade,
Water is distilled water, and remaining reagent is that analysis is pure.
Extracting method：
1) extract solution is grouped：
Extract solution is divided into 9 groups by selected preparation during according to extraction, is respectively designated as：TH-t、TH-p、TH-a、TH-b、
TH-w, TH-w1, TH-w2, TH-w3 and TH-w4.
2) extracting method of each group Lemna paucicostata
Lemna paucicostata extracts reference literature [Yang Yang Salvia przewalskii Maxims and polygonum capitatum chemical constitution study the second medical officers of
University master thesis Shanghai：The 2nd Army Medical College, 2009] isolate and purify to obtain, examined through high performance liquid chromatography (HPLC)
Look into.
3) TH-t parts：Radix tetrastigme is taken to dry medicinal material 200g, after adding the 5 times of ethanol water of volume 80% immersion 24h, heating
Refluxing extraction, totally 3 times, each 40min, merge extract solution, filtering, be recovered under reduced pressure and be evaporated, obtain total extract TH-t 21.6g, obtain
Rate 10.8%.
4) TH-p parts：Take this always to extract, add water to be suspended, the petroleum ether (water saturation) that 10 times of volumes are respectively adopted is divided
Step extraction.Respectively obtain petroleum ether part TH-p 0.15g, yield 0.075%.
5) TH-a parts：Ethyl acetate extract TH-a 0.58g, yield 0.29%.
6) TH-b parts：N-butanol portion TH-b 2g, yield 1%.
7) TH-w parts：And water position TH-w 18.9g, yield 9.45%.
8) TH-w1 parts：Water intaking position carries out macroporous adsorbent resin chromatography, is first eluted using pure water, removes sugar,
10%, 30%, 60%, 95% ethanol water is reused to elute successively respectively.Wherein, 10% ethanol water elution solution decompression returns
Receipts obtain water position desugar flow point TH-w1 0.14g, yield 0.07% after being evaporated.
9) TH-w2 parts：Water intaking position carries out macroporous adsorbent resin chromatography, is first eluted using pure water, removes sugar,
10%, 30%, 60%, 95% ethanol water is reused to elute successively respectively.Wherein, after 30% ethanol aqueous strip solution is evaporated
Obtain water position desugar flow point TH-w1 0.3g, yield 0.15%.
10) TH-w3 parts：The above-mentioned radix tetrastigme medicinal material after organic solvent extracts is evaporated with being recovered under reduced pressure after boiling,
Obtain 2.1 grams of dehydrate.
11) TH-w4 parts：The above-mentioned radix tetrastigme medicinal material after organic solvent extracts is evaporated dehydration with being recovered under reduced pressure, then
Using the desugar successively respectively of 10%, 30%, 60%, 95% ethanol water, wherein, obtain 10% ethanol water position desugar stream
Point.
As a result it is as shown in table 1：
The Different Extraction Method of table 1 extracts the result of radix tetrastigme
Embodiment 1：The phagocytic activity of various concentrations radix tetrastigme TH-b extracting section thing inducing dendritic shape cells
1) experiment material：
Serum free medium and BMDC culture medium (Zhejiang Beaune bio tech ltd product)；Dimethyl is sub-
Sulfone (dimethyl s μ lfoxide DMSO) (Sigma companies)；Lymphocyte separation medium (Chinese Academy of Sciences's blood disease research
Institute)；RhIL-4, GM-CSF (Xiamen Te Bao biotech firms)；INF- γ are purchased from Abender companies；Trypsase, RPMl1640
(being purchased from GIBCO companies)；HLA-DR monoclonal antibodies (the connection of CD80, CD86 and PerCP mark of FITC CD83, PE mark
Biology Co., Ltd of section)；Fluorescence 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。
2) BMDC culture and identification；
Take healthy blood donor (at 18~25 years old age, to ratify and sign informed consent form) periphery through Hospital Ethical Committee to resist
Blood coagulation 20ml, lymphocyte separation medium separation mononuclearcell, with DC Serum-free complete mediums adjustment cell concentration be 2.0 ×
109/ L, 6 porocyte culture plates are added, per hole 3ml.Put 37 DEG C, 5%CO2Incubator is incubated 2h, removes suspension cell and cell training
Supernatant is supported, retains adherent cell.Add rhIL-4 containing 50ng/ml, 200ng/ml GM-CSF BMDC culture
Base, in 37 DEG C, 5%CO2Incubator culture, measured with BMDC culture medium half within every 2 days and change liquid 1 time, to the 6th day after add
γ-IFN to 1000U/ml, LPS to 10ng/ml is added at the 7th day, cultivate the 8th day and collect cell, carry out surface of dendritic cells
Mark detection is (see accompanying drawing 1).
3) detection that radix tetrastigme TH-b extracting sections thing influences on BMDC phagocytic activity:
Take the BMDC of culture 5 days to be made into 5 × 104/mL cell suspension, add 24 porocyte culture plates, 1ml/
Hole.Experimental group：5 concentration groups are set altogether, and being separately added into the radix tetrastigme TH-b extracting sections things of various concentrations, (final concentration is respectively
0.001st, 0.01,0.1,1.0,10.0 μ g/ml) every group set 3 multiple holes, while set last dosing thing blank control group：Trained in cell
Support and add the μ l/ holes (1 × 108/ml of spore density) of candida albicans spore 10 after 48h is cultivated in case, continue to be incubated 4h, collect cell and be made
Smear, cell dyeing is carried out according to a conventional method with Wright's stain after natural drying.Observed and taken the photograph with oil mirror after natural drying
Piece.
4) statistical method:
Statistical procedures are carried out using SPSS13.0 softwares, measurement data uses mean ± standard deviation (mean ± SD) table
Show, compare between group using one-way analysis of variance (One-way ANOVA), inspection level α=0.05, there is statistics P≤0.05
Meaning.
5) result:
TH-b part stages Lemna paucicostata has considerable influence to BMDC phagocytic activity, is in drug concentration
1.0 μ g/ml time phagocytic activities are most strong.As a result (see accompanying drawing 2,3).
It can be seen that the TH-b part stages Lemna paucicostata of low dosage can significantly improve BMDC phagocytic activity.
Embodiment 2：Suppression of the Lemna paucicostata to melanoma strain is tested
1) experiment material：
Melanoma cell strain (is purchased from Shanghai cell institute of the Chinese Academy of Sciences, the passage of this room preserves)；Serum free medium (Zhejiang
Jiang Bona bio tech ltd product)；Methyl thiazoly tetrazolium assay (methlthiazolyltetrazolium, MTT) and two
Methyl sulfoxide (dimethyl s μ lfoxideDMSO) (Sigma companies)；Lymphocyte separation medium (grind by Chinese Academy of Sciences's blood disease
Study carefully institute)；ST-360 ELIASAs, purchased from Shanghai Kehua Experimental System Co., Ltd.；Encore automatic biochemistry analyzers (the uncommon Asia in Beijing
Gram Technology Co., Ltd.)；Fluorescence 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.
2) influence that TH-b parts Lemna paucicostata grows to melanoma cell strain：
Take the logarithm growth period melanoma cells (M12) be made into respectively 3 × 107/L cell suspension add 96 orifice plates,
Per the μ l of hole 180, every group sets 5 multiple holes, while sets negative control group；Culture plate is put into 37 DEG C, 50mL/L CO2Incubator is trained
Support 24h after, experimental group be separately added into various concentrations TH-b part stages Lemna paucicostata 0.25,1,4,16,31.5,125,
500th, 2000 μ g/ml), while set the control group of not dosing.In 37 DEG C, 50mL/L CO272h is cultivated in incubator；Tied in culture
Add the μ l/ holes of MTT 20 within 4 hours before beam, continue supernatant discarding after culture 4h, add the μ l of DMSO 150, praise first and be completely dissolved,
Each hole light absorption value (OD) record result is detected at ELIASA 495nm.Cell inhibitory rate IR (%)=(control OD values)-(experiment
OD values)/(control OD) × 100%.Identical experiment is repeated 3 times, and is averaged.
3) statistical method:
Statistical procedures are carried out using SPSS13.0 softwares, measurement data uses mean ± standard deviation (mean ± SD) table
Show, compare between group using one-way analysis of variance (One-way ANOVA), inspection level α=0.05, there is statistics P≤0.05
Meaning.
3rd, result:
Melanoma is subjected to Fiber differentiation with Lemna paucicostata (TH-b parts) respectively, it is high after induction 72 hours
The TH-b melanoma cells of concentration have inhibitory action, as the increase of drug concentration suppresses more obvious.But each concentration group pair
Inhibiting tumour cells depth is more than 500 μ g/ml, each group equal unrestraint phenomenon during less than 32 μ g/ml.TH-b parts radix tetrastigme
Inhibiting rate of the extract to tumour cell (see accompanying drawing 4).
It can be seen that the TH-b parts Lemna paucicostata of low dosage has significant suppression to the TH-b melanoma cells of high concentration
Effect.
Embodiment 3：Radix tetrastigme TH-b extracting sections thing marks CD80, CD83, CD86 and HLA- to dendritic cell phenotypic
DR influence
1) experiment material：
Serum free medium and BMDC culture medium (Zhejiang Beaune bio tech ltd product)；Dimethyl is sub-
Sulfone (dimethyl s μ lfoxide DMSO) (Sigma companies)；Lymphocyte separation medium (Chinese Academy of Sciences's blood disease research
Institute)；RhIL-4, GM-CSF (Xiamen Te Bao biotech firms)；INF- γ are purchased from Abender companies；Trypsase, RPMl1640
(being purchased from GIBCO companies)；HLA-DR monoclonal antibodies (the connection of CD80, CD86 and PerCP mark of FITC CD83, PE mark
Biology Co., Ltd of section)；Fluorescence 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.
2) BMDC culture and identification:
Take healthy blood donor (at 18~25 years old age, to ratify and sign informed consent form) periphery through Hospital Ethical Committee to resist
Blood coagulation 20ml, lymphocyte separation medium separation mononuclearcell, with DC Serum-free complete mediums adjustment cell concentration be 2.0 ×
109/L, 6 porocyte culture plates are added, per hole 3ml.Put 37 DEG C, 5%CO2Incubator is incubated 2h, removes suspension cell and cell
Culture supernatant, retain adherent cell.Add rhIL-4 containing 50ng/ml, 200ng/ml GM-CSF BMDC training
Base is supported, in 37 DEG C, 5%CO2Incubator culture, measured with BMDC culture medium half within every 2 days and change liquid 1 time, to the 6th day after add
γ-IFN to 1000U/ml, LPS to 10ng/ml is added at the 7th day, cultivate the 8th day and collect cell, carry out surface of dendritic cells
Mark detection.
3) flow cytomery BMDC CD80, CD83, CD86 and HLA-DR are expressed:
Successful BMDC will be cultivated and be made into the cell suspension that concentration is 1.0 × 105/ml, be inoculated in the culture of 6 holes
Plate, per hole 3ml, it is respectively the and of 0 μ g/ml, 39,9.76,2.44,0.62,0.15,0.039,0.01 then to add final concentration
0.0025 Lemna paucicostata (TH-b parts), every group of 3 multiple holes.In 37 DEG C, 5%CO248h is cultivated in incubator.Collect
Cell simultaneously washs 2 times with phosphate buffer (PBS), often pipe add anti-FITC CD83, PE mark CD80, CD86 and
The HLA-DR monoclonal antibodies of PerCP marks, room temperature lucifuge is incubated 15min, after PBS washings, is resuspended in 0.5ml PBS solution
In, respectively with Flow cytometry CD80, CD83, CD86 and HLA-DR expression.
4) statistical method:
Statistical procedures are carried out using SPSS13.0 softwares, measurement data uses mean ± standard deviation (mean ± SD) table
Show, compare between group using one-way analysis of variance (One-way ANOVA), inspection level α=0.05, there is statistics P≤0.05
Meaning.
5) result:BMDC CD80, CD83, CD86 and HLA-DR result of TH-b inductions:
Compared with 0 μ g/ml control groups, 39,9.76,2.44,0.62,0.15,0.039,0.01 and 0.0025 μ g/ml's
After TH-b effect BMDCs 48h, its CD80, CD83, CD86 and HLA-DR expression are significantly raised.TH-b concentration is 0.62
BMDC CD80, CD83, CD86 and HLA-DR expression rate induced during μ g/ml reaches peak, respectively 88.56% ±
8.16%th, 86.75% ± 7.59%, 99.34% ± 8.24%, 96.15% ± 8.27%, it is significantly higher than before induction (control group)
71.38% ± 7.31%, 43.61% ± 4.29%, 93.41% ± 8.34%, 79.26% ± 5.12%, be and concentration surpasses
All DC surface markers expression rates have declined when crossing 9.76 μ g/ml, as a result (see accompanying drawing 5 to 10).
Very low dosage radix tetrastigme TH-b extracting sections thing can be obviously promoted human dendritic cell CD80, CD86, HLA-DR and
CD83 expression, promote the activation of peripheral blood lymphocyte and improve multiplication capacity.
Embodiment 4：Radix tetrastigme TH-b extracting sections thing secretes IL-12 (interleukin 12) shadow to BMDC
Ring
1) experiment material:
Serum free medium and BMDC culture medium (Zhejiang Beaune bio tech ltd product)；Dimethyl is sub-
Sulfone (dimethyl s μ lfoxide DMSO) (Sigma companies)；Lymphocyte separation medium (Chinese Academy of Sciences's blood disease research
Institute)；RhIL-4, GM-CSF (Xiamen Te Bao biotech firms)；INF- γ are purchased from Abender companies；Trypsase, RPMl1640
(being purchased from GIBCO companies)；HLA-DR monoclonal antibodies (the connection of CD80, CD86 and PerCP mark of FITC CD83, PE mark
Biology Co., Ltd of section)；Fluorescence 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.
2) BMDC culture and identification:
Take healthy blood donor (at 18~25 years old age, to ratify and sign informed consent form) periphery through Hospital Ethical Committee to resist
Blood coagulation 20ml, lymphocyte separation medium separation mononuclearcell, with DC Serum-free complete mediums adjustment cell concentration be 2.0 ×
109/L, 6 porocyte culture plates are added, per hole 3ml.Put 37 DEG C, 5%CO2Incubator is incubated 2h, removes suspension cell and cell
Culture supernatant, retain adherent cell.Add rhIL-4 containing 50ng/ml, 200ng/ml GM-CSF BMDC training
Base is supported, in 37 DEG C, 5%CO2Incubator culture, measured with BMDC culture medium half within every 2 days and change liquid 1 time, to the 6th day after add
γ-IFN to 1000U/ml, LPS to 10ng/ml is added at the 7th day, cultivate the 8th day and collect cell, carry out surface of dendritic cells
Mark detection.
3) DC secretes IL-12 sample extracting methods:
Successful BMDC will be cultivated and be made into the cell suspension that concentration is 1.0 × 105/ml, be inoculated in the culture of 6 holes
Plate, per hole 3ml, it is respectively the and of 0 μ g/ml, 39,9.76,2.44,0.62,0.15,0.039,0.01 then to add final concentration
0.0025 Lemna paucicostata (TH-b parts), every group of 3 multiple holes.In 37 DEG C, 5%CO248h is cultivated in incubator.Collect
Cells and supernatant carries out IL-12 detections.
5) IL-12 reagent test methods:
The preparation of IL-12ELISA kits
1. 30min takes out IL-12ELISA kits from refrigerator in advance, balance to room temperature.
2. 20 times of concentrated cleanings in IL-12ELISA kits are taken out, concussion on earthquake instrument is until crystallization is complete
Melt.Concentrated cleaning 10ml is taken to add in 500ml indigo plant cover glass bottles with micro sample-adding rifle, with 100ml graduated cylinders at pure water meter
Measure two pure water 190ml to add in blue lid bottle, changed bottle cap and turn upside down and shake up, be made into the cleaning solution 200ml of 20 times of dilutions, make
Upper mark, it is standby to be put in normal temperature.
3. the solution of standard items is prepared, freeze and dilution 0.5ml is added in standard items, it is 4000pg/ml to be configured to concentration
Stoste, thoroughly dissolving, stand 15min be well mixed, as needed dilution, be made into following:The concentration of gradient：62.5、125、
250、500、1000、2000、4000pg/ml。
The specific dilution process of standard items is as follows：
A. 6 clean 1.5ml centrifuge tubes are taken, mark A1-A6 respectively；
B. 250ul 4000pg/ml standard solutions are added in A1 with micro sample-adding rifle, then standard items is added in A1
Dilution 250ul, and be well mixed；
C. take 250ul to be added in A2 in A1, then add standard dilutions 250ul in A2 and mix；
D. take 250ul to be added in A3 in A2, then add standard dilutions 250ul in A3 and mix；
E. take 250ul to be added in A4 in A3, then add standard dilutions 250ul in A4 and mix；
F. take 250ul to be added in A5 in A4, then add standard dilutions 250ul in A5 and mix；
G. take 250ul to be added in A6 in A5, then add standard dilutions 250ul in A6 and mix；
H. 250ul is taken to give up in A6.Now standard items IL-12 concentration is respectively：4000pg/ml、2000pg/ml、
1000pg/ml、500pg/ml、250pg/ml、125pg/ml、62.5pg/ml。
4. the preparation of biotinylated antibody working solution, by 1:100 ratio biotinylation diluted biotinylation
Antibody.Total dosage of working solution is prepared, it is necessary to 100~200ul of polygamy, best matching while using by 100ul/ holes.
5. the preparation of enzyme conjugates working solution：By 1:The enzyme that 100 ratio enzyme combination diluent dilutes concentration combines
Thing.Best matching while using.
6) IL-12 vitality tests step
The concrete operation step of DCs secrete cytokines IL-12 vitality tests is as follows：
1. the cell supernatant for being stored in -80 ° of refrigerators is taken out, thaw at room temperature.Centrifuged in 4 ° of refrigerated centrifuges,
13000 revs/min 5 minutes, further remove sample in may residual impurity, reduce the influence to experimental result.By sample with
1:2 dilutions, mark are standby；
2. prepare various solution according to above-mentioned preparation；
3. by the quantity of standard items and testing sample, to determine lath quantity, wherein standard items set 1 multiple holes, and wrap
Include 1 hole blank control.Each 1 hole of supernatant sample, and with 1:Each 1 hole of sample after 2 dilutions, then including blank control, share 32
Individual hole.The standard solution of 100ul various concentrations and testing sample are added into respective aperture respectively, and set duplicate hole [30].With envelope
Plate gummed paper seals reacting hole, and 37 DEG C of incubators are incubated 90min；
4. be incubated completion, ELISA Plate is taken out, shrouding gummed paper is carefully made known, pats dry liquid on filter paper, then in every hole
The cleaning solution 350ul diluted in advance is filled it up with, most liquid is got rid of after standing 30 seconds, then is patted dry on filter paper, washes ELISA Plate 4 repeatedly
It is secondary；
5. the biotinylated antibody working solution (100ul/ holes) prepared in advance is added after patting dry.Sealed instead with shrouding gummed paper
Ying Kong, 37 DEG C of incubators are incubated 60min；
6. lavation buffer solution board-washing 4 times, add enzyme conjugates working solution (100ul/ holes).Reaction is sealed with shrouding gummed paper
Hole, 37 DEG C of incubators are incubated 30min.Each 50ul of enzyme marking reagent is added in A1-A10, B2-B5 hole, shrouding film is posted, is put into 37 DEG C and incubates
Educate in case and be incubated 30 minutes；
7. board-washing 4 times.Developer is added by the amount in 100ul/ holes, lucifuge gently shakes, and 37 DEG C of incubators are incubated 20 minutes, together
When open ELIASA preheating, and set parameter；
8. after the completion of being incubated, taking out coating plate, terminate liquid is added by the amount in 100ul/ holes, color is become by indigo plant after being well mixed
Huang, after being returned to zero with blank, OD values are determined in 5min at once.As a result it is as follows：Standard items 1.513,1.167；0.637、0.743；
0.429、0.467；0.300、0.336；0.234、0.232；0.215、0.200；0.184、0.186；0.183、0.182.Detection
Sample 0.216,0.205,0.273,0.242,0.242,0.244,0.269,0.214,0.183；Result 0.168 after dilution,
0.172、0.182、0.215、0.194、0.209、0.179、0.163。
Preliminary result prompting is not required to sample dilutions by kit operation, and measured result is in standard curve range.Therefore
Sample need not dilute, then to repeat to test to reduce error.
7) statistical method:
Statistical procedures are carried out using SPSS13.0 softwares, measurement data uses mean ± standard deviation (mean ± SD) table
Show, compare between group using one-way analysis of variance (One-way ANOVA), inspection level α=0.05, there is statistics P≤0.05
Meaning.
8) result:The BMDC secretion IL-12 results of TH-b inductions
2.44,0.62,0.15, the 0.039 and 0.01 μ g/ml TH-b effect BMDCs compared with 0 μ g/ml control groups
After 48h, the IL-12 that it is secreted is significantly raised.The BMDC secretion that TH-b concentration induces in 0.62 μ g/ml (TH-t)
IL-12 highests, reach 526.3pg/ml, be significantly higher than the 260.1pg/ml of control group, and TH-b concentration is more than 2.44 μ g/ml
When, the IL-12 of DC secretions is begun to decline, and when TH-b is at concentrations up to 39 μ g/ml, the IL-12 contents of the DC secretions of induction only have
107.4pg/ml, on the contrary less than control group.As a result (see accompanying drawing 11).
Very low dosage radix tetrastigme TH-b extracting sections thing can be obviously promoted BMDC secretion IL-12, strengthen dendron shape
The phagocytic activity of cell.
In summary, the present invention extracts separation and identification and analysis by being carried out to the composition of radix tetrastigme, finds to use very low dose
Measure radix tetrastigme TH-b extracting section things, can be obviously promoted human dendritic cell CD80, CD86, H LA-DR and CD83 expression with
And IL-12 secretion, promote the activation of peripheral blood lymphocyte and improve multiplication capacity, and the phagocytosis of BMDC can also be strengthened
Ability, and preferential TH-b concentration is 0.62 μ g/ml-1.0 μ g/ml.
The preferred embodiments of the present invention are the foregoing is only, not thereby limit the scope of patent protection of the present invention, it is all
It is the equivalent structure transformation made with description of the invention and accompanying drawing content, is directly or indirectly used in other related technologies
Field, similarly include within the scope of the present invention.
Claims (3)
1. application of the Lemna paucicostata TH-b parts in immunotherapy of tumors medicine is prepared.
2. application of the Lemna paucicostata TH-b parts as claimed in claim 1 in immunotherapy of tumors medicine is prepared, its
It is characterised by, the immunotherapy of tumors medicine is melanoma immunotherapy medicaments.
3. application of the Lemna paucicostata TH-b parts in human dendritic cell multiplication agent is prepared.
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| CN108888685A (en) * | 2018-07-26 | 2018-11-27 | 杭州金域医学检验所有限公司 | Purposes and its action method of the position Lemna paucicostata TH-w to staphylococcus aureus |
| CN108888685B (en) * | 2018-07-26 | 2021-12-10 | 杭州金域医学检验所有限公司 | Application of Tetrastigma hemsleyanum Diels et Gilg extract TH-w part to staphylococcus aureus and action method thereof |
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