CN108753722A - A kind of quick Activiation method of LAK LAK - Google Patents
A kind of quick Activiation method of LAK LAK Download PDFInfo
- Publication number
- CN108753722A CN108753722A CN201810656001.XA CN201810656001A CN108753722A CN 108753722 A CN108753722 A CN 108753722A CN 201810656001 A CN201810656001 A CN 201810656001A CN 108753722 A CN108753722 A CN 108753722A
- Authority
- CN
- China
- Prior art keywords
- enzymolysis
- lak
- quick
- freeze
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
- C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Cell Biology (AREA)
- Water Supply & Treatment (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a kind of quick Activiation methods of LAK LAK, use 1640 culture mediums of RPMI (10%FCS-RPMI 1640) modulation containing 10% fetal calf serum to 2 × 10 cord blood mononuclear cells6/ mL, is inoculated according to total number of cells in the culture vessel of different capabilities, while enzymolysis polypeptide A, the enzymolysis polypeptide C of final concentration of 40-60 μ g/mL or the rIL-2 of enzymolysis polypeptide D and 500IU/mL is added, and is placed in 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours.The quick Activiation method of the present invention is not under the premise of largely using rIL-2, by adding enzymolysis polypeptide A, enzymolysis polypeptide C or enzymolysis polypeptide D in the medium, is quickly activated using only i.e. realization LAK cells for 24 hours.
Description
Technical field
The invention belongs to biological fields, are related to a kind of quick Activiation method of LAK LAK.
Background technology
Immunization therapy is a kind of emerging oncotherapy mode, has been listed in the 4th kind after operation, radiotherapy, chemotherapy
Therapeutic modality gradually plays a significant role in the complex treatment of tumour.2010, U.S. FDA approval was first thin in the world
The Provenge listings of born of the same parents' immunotherapeutic product, fully show value of the cellular immunotherapy in oncotherapy.
LAK (LAK) is in the 1980s by research and development such as Rosenberg, its essence is
IL-2 activation has the NK and T cell for killing tumor activity.In November, 1984, Rosenberg seminar ratified through U.S. FDA, for the first time
LAK cells are used for clinical treatment.The therapy is to metastatic renal cell cancer, melanoma, colon cancer and non-Hodgkin lymphoma
The effect of patient, is more significant.But LAK lethality is not strong, and clinical application needs a large amount of infusions.And its amplification ability is limited, needs
Large dosage application IL-2 while infused cells, so as to cause apparent toxic side effect, most common and most serious poison is secondary to be made
With being capillary leak syndrome, be mainly shown as anasarca and multiple organ dysfunction imbalance, can cause pleural effusions and ascites,
Pulmonary interstitial edema and congestive heart failure.
Currently, LAK cells will pass through in vitro culture at least 3~7 days, long flow path, somewhat expensive is easy to pollute, and clinic is made to answer
With being restricted, therefore the research for being dedicated to simple and efficient Fiber differentiation never stopped.
Invention content
The present invention is directed to overcome the shortage of prior art, a kind of quick activation of LAK LAK is provided
Method.
Technical solution of the present invention is as follows：
A kind of quick Activiation method of LAK LAK, adds rIL-2, is cultivating in the medium
Activator enzymolysis polypeptide A is also added in base.
Preferably, the quick Activiation method the specific steps are：
By RPMI 1640 culture medium (10%FCS-RPMI 1640) of the cord blood mononuclear cells containing 10% fetal calf serum
Modulation is inoculated according to total number of cells in the culture vessel of different capabilities, while final concentration of 40-60 μ are added to 2 × 106/mL
The rIL-2 of the enzymolysis polypeptide A and 500IU/mL of g/mL are placed in 37 DEG C, cultivate for 24 hours under the conditions of 5%CO2；
The preparation method of enzymolysis polypeptide A is：
By the wheat germ powder of degreasing through papain+neutral protease enzymolysis, destroy the enzyme treatment obtains after enzymolysis
The enzymolysis liquid of wheat embryo, after cooling, centrifugation, supernatant is freeze-dried to obtain freeze-dried powder.
By the ultrapure water dissolution of freeze-dried powder, then successively with the ultrafiltration membrane that molecular cut off is 10000u, 7000u to gained
To enzymolysis liquid freeze-dried powder detached, be freeze-dried 10000u~7000u components obtained by ultrafiltration to obtain enzymolysis polypeptide A.
Preferably, enzymatic hydrolysis condition is：Through papain (enzyme activity is 800,000 U/g)+neutral proteinase (enzyme activity 20
Ten thousand U/g) in compound proportioning 3:1, pH 6.0,55 DEG C of temperature, substrate mass concentration 40g/L, (100g substrates add 5g to enzyme concentration 5%
Enzyme), digest under conditions of enzymolysis time 8h.
Preferably, 90 DEG C are warming up to after enzymolysis keeps 20min progress destroy the enzyme treatment to obtain the enzymolysis liquid of wheat embryo.
Preferably, centrifugal condition is：9000rpm centrifuges 25min.
Preferably, thin using the ficoll cardiografin layering single core of liquid density-gradient centrifugation method separating umbilical blood from bleeding of the umbilicus
Born of the same parents.
Preferably, cord blood collection method is：Full-term normal delivery healthy newborn bleeding of the umbilicus is acquired using closed collecting method, and
Its parent of antenatal exaination is without genetic disease family history, using the closed blood bag of citrate-phosphate-glucose anti-freezing in tire
Disk acquires before giving birth to from the umbilical cord broken ends of fractured bone far from newborn side；4 DEG C of refrigerators preserve after acquisition, interior for 24 hours to be detached and frozen processing
It is spare.
A kind of quick activation culture medium of LAK LAK, in the RPMI containing 10% fetal calf serum
The rIL-2 of the enzymolysis polypeptide A and 500IU/mL of final concentration of 40-60 μ g/mL are added in 1640 culture mediums.
Application of the above-mentioned culture medium in terms of the quick activation of LAK LAK.
A kind of quick Activiation method of LAK LAK, adds rIL-2, is cultivating in the medium
Activator enzymolysis polypeptide C is also added in base.
Preferably, the quick Activiation method the specific steps are：
By RPMI 1640 culture medium (10%FCS-RPMI 1640) of the cord blood mononuclear cells containing 10% fetal calf serum
Modulation is inoculated according to total number of cells in the culture vessel of different capabilities, while final concentration of 40-60 μ are added to 2 × 106/mL
The rIL-2 of the enzymolysis polypeptide C and 500IU/mL of g/mL are placed in 37 DEG C, cultivate for 24 hours under the conditions of 5%CO2；
The preparation method of enzymolysis polypeptide C is：
By the wheat germ powder of degreasing through papain+neutral protease enzymolysis, destroy the enzyme treatment obtains after enzymolysis
The enzymolysis liquid of wheat embryo, after cooling, centrifugation, supernatant is freeze-dried to obtain freeze-dried powder.
By the ultrapure water dissolution of freeze-dried powder, successively with the ultrafiltration that molecular cut off is 10000u, 7000u, 5000u, 3000u
Film detaches obtained enzymolysis liquid freeze-dried powder, is freeze-dried 5000u~3000u components obtained by ultrafiltration to obtain enzymolysis more
Peptide C.
Preferably, enzymatic hydrolysis condition is：Through papain (enzyme activity is 800,000 U/g)+neutral proteinase (enzyme activity 20
Ten thousand U/g) in compound proportioning 3:1, pH 6.0,55 DEG C of temperature, substrate mass concentration 40g/L, (100g substrates add 5g to enzyme concentration 5%
Enzyme), digest under conditions of enzymolysis time 8h.
Preferably, 90 DEG C are warming up to after enzymolysis keeps 20min progress destroy the enzyme treatment to obtain the enzymolysis liquid of wheat embryo.
Preferably, centrifugal condition is：9000rpm centrifuges 25min.
Preferably, thin using the ficoll cardiografin layering single core of liquid density-gradient centrifugation method separating umbilical blood from bleeding of the umbilicus
Born of the same parents.
Preferably, cord blood collection method is：Full-term normal delivery healthy newborn bleeding of the umbilicus is acquired using closed collecting method, and
Its parent of antenatal exaination is without genetic disease family history, using the closed blood bag of citrate-phosphate-glucose anti-freezing in tire
Disk acquires before giving birth to from the umbilical cord broken ends of fractured bone far from newborn side；4 DEG C of refrigerators preserve after acquisition, interior for 24 hours to be detached and frozen processing
It is spare.
A kind of quick activation culture medium of LAK LAK, in the RPMI containing 10% fetal calf serum
The rIL-2 of the enzymolysis polypeptide C and 500IU/mL of final concentration of 40-60 μ g/mL are added in 1640 culture mediums.
Application of the above-mentioned culture medium in terms of the quick activation of LAK LAK.
A kind of quick Activiation method of LAK LAK, adds rIL-2, is cultivating in the medium
Activator enzymolysis polypeptide D is also added in base.
Preferably, the quick Activiation method the specific steps are：
By RPMI 1640 culture medium (10%FCS-RPMI 1640) of the cord blood mononuclear cells containing 10% fetal calf serum
Modulation is inoculated according to total number of cells in the culture vessel of different capabilities, while final concentration of 40-60 μ are added to 2 × 106/mL
The rIL-2 of the enzymolysis polypeptide D and 500IU/mL of g/mL are placed in 37 DEG C, cultivate for 24 hours under the conditions of 5%CO2；
The preparation method of enzymolysis polypeptide D is：
By the wheat germ powder of degreasing through papain+neutral protease enzymolysis, destroy the enzyme treatment obtains after enzymolysis
The enzymolysis liquid of wheat embryo, after cooling, centrifugation, supernatant is freeze-dried to obtain freeze-dried powder.
By the ultrapure water dissolution of freeze-dried powder, then successively with molecular cut off be 10000u, 7000u, 5000u, 3000u with
And the ultrafiltration membrane of 1000u detaches obtained enzymolysis liquid freeze-dried powder, and 3000u~1000u components obtained by ultrafiltration are freezed
It is dried to obtain enzymolysis polypeptide D.
Preferably, enzymatic hydrolysis condition is：Through papain (enzyme activity is 800,000 U/g)+neutral proteinase (enzyme activity 20
Ten thousand U/g) in compound proportioning 3:1, pH 6.0,55 DEG C of temperature, substrate mass concentration 40g/L, (100g substrates add 5g to enzyme concentration 5%
Enzyme), digest under conditions of enzymolysis time 8h.
Preferably, 90 DEG C are warming up to after enzymolysis keeps 20min progress destroy the enzyme treatment to obtain the enzymolysis liquid of wheat embryo.
Preferably, centrifugal condition is：9000rpm centrifuges 25min.
Preferably, thin using the ficoll cardiografin layering single core of liquid density-gradient centrifugation method separating umbilical blood from bleeding of the umbilicus
Born of the same parents.
Preferably, cord blood collection method is：Full-term normal delivery healthy newborn bleeding of the umbilicus is acquired using closed collecting method, and
Its parent of antenatal exaination is without genetic disease family history, using the closed blood bag of citrate-phosphate-glucose anti-freezing in tire
Disk acquires before giving birth to from the umbilical cord broken ends of fractured bone far from newborn side；4 DEG C of refrigerators preserve after acquisition, interior for 24 hours to be detached and frozen processing
It is spare.
A kind of quick activation culture medium of LAK LAK, in the RPMI containing 10% fetal calf serum
The rIL-2 of the enzymolysis polypeptide D and 500IU/mL of final concentration of 40-60 μ g/mL are added in 1640 culture mediums.
Application of the above-mentioned culture medium in terms of the quick activation of LAK LAK.
Advantageous effect：
The quick Activiation method of the present invention is more by adding enzymolysis in the medium under the premise of largely not using rIL-2
Peptide A, enzymolysis polypeptide C or enzymolysis polypeptide D are quickly activated using only i.e. realization LAK cells for 24 hours.
Description of the drawings
Fig. 1 is killing rate (%) of each group LAK cells to stomach cancer cell.
Specific implementation mode
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but the guarantor of the present invention is not limited with this
Protect range.
Embodiment 1：The preparation of polypeptide
1, wheat embryo degreasing
It will be used after wheat embryo degreasing, or directly purchase defatted wheat germ carries out subsequent experimental.Degreasing condition：It carries
Take agent：Petroleum ether (60 DEG C~90 DEG C), bath temperature：65 DEG C, solid-liquid ratio：1:8 (g/mL), degreasing time：4h.
Defatted wheat germ is ground into 80 mesh and obtains the wheat germ powder of degreasing, spare.
2, the hydrolysis and purifying of polypeptide
By the wheat germ powder of degreasing through papain (enzyme activity is 800,000 U/g)+neutral proteinase (enzyme activity 20
Ten thousand U/g) in compound proportioning 3:1 (mass ratio), pH 6.0,55 DEG C of temperature, substrate mass concentration 40g/L, 5% (100g of enzyme concentration
Substrate adds 5g enzymes), digest under conditions of enzymolysis time 8h, 90 DEG C are warming up to after enzymolysis, 20min are kept to carry out destroy the enzyme treatments
The enzymolysis liquid of wheat embryo is obtained, after cooling, 9000rpm centrifuges 25min, and supernatant is freeze-dried to obtain freeze-dried powder.
By freeze-dried powder ultrapure water dissolution (1g freeze-dried powders add 10mL ultra-pure waters), then it is with molecular cut off successively
The ultrafiltration membrane of 10000u, 7000u, 5000u, 3000u and 1000u detach obtained enzymolysis liquid freeze-dried powder, will surpass
Filter obtained component freeze-drying (water content≤5%) obtain enzymolysis polypeptide A (10000u~7000u), enzymolysis polypeptide B (7000u~
5000u), enzymolysis polypeptide C (5000u~3000u), enzymolysis polypeptide D (3000u~1000u).
CO is controlled in ultra-filtration process2Pressure make its be less than 0.25MPa.
It is spare by enzymolysis polypeptide A, enzymolysis polypeptide B, enzymolysis polypeptide C, enzymolysis polypeptide D in 4 DEG C of kept dries.
Embodiment 2：The quick Activiation method of LAK cells
One, experiment material
1640 culture mediums of RPMI, 10% fetal calf serum are purchased from Gibco companies.
RIL-2 is purchased from Beijing Fourth Ring biopharmaceutical company.
Two, experimental method
1, cord blood collection and mononuclearcell separation
Using closed collecting method acquisition full-term normal delivery healthy newborn bleeding of the umbilicus 100, and its parent of antenatal exaination is equal
Without genetic disease family history, using the closed blood bag of citrate-phosphate-glucose (CPDA) anti-freezing before placenta is given birth to from
The umbilical cord broken ends of fractured bone is acquired far from newborn side.4 DEG C of refrigerators preserve after acquisition, interior for 24 hours to be detached and frozen processing.
With reference to art technology handbook (Shen Guanxin etc., Immunology Today experimental technique, Hubei science tech publishing house,
Page 1998,172-175), liquid density-gradient centrifugation method separating umbilical blood mononuclearcell is layered using ficoll cardiografin.
2, the quick activation of LAK cells
Regular active：By RPMI 1640 culture medium (10%FCS- of the cord blood mononuclear cells containing 10% fetal calf serum
RPMI 1640) it modulates to 2 × 106/ mL, is inoculated according to total number of cells in the culture vessel of different capabilities, while being added dense eventually
Degree is the rIL-2 of 500IU/mL, is placed in 37 DEG C, 5%CO2Under the conditions of cultivate 72h.
Quickly activation：By RPMI 1640 culture medium (10%FCS- of the cord blood mononuclear cells containing 10% fetal calf serum
RPMI 1640) it modulates to 2 × 106/ mL, is inoculated according to total number of cells in the culture vessel of different capabilities, while being added dense eventually
Degree is enzymolysis polypeptide A, B, C or D of 50 μ g/mL and the rIL-2 of 500IU/mL, is placed in 37 DEG C, 5%CO2CMC model is for 24 hours.
3, tumor cell killing potential is detected
Human stomach cancer cell line MGC-803 freezes for our company, is trained using the RPMI 1640 containing 10% fetal calf serum after recovery
Foster base is placed in 37 DEG C, 5%CO2Secondary culture in incubator takes logarithmic phase to carry out subsequent experimental.
The LAK cells that each group activates are taken as effector cell, using MGC-803 stomach cancer cells as target cell, by 20:1 effect target
Than 96 orifice plates are added in effector cell and target cell respectively, independent target cell and individual effect groups of cells are separately set, every group sets 3 again
37 DEG C, 5%CO are placed in hole2After being cultivated for 24 hours in incubator, add 10 μ L of MTT (5mg/mL) per hole, continues to cultivate 4h, centrifugation is abandoned
Clearly, after adding 150 μ L of dimethyl sulfoxide (DMSO), oscillation to dissolve 10min per hole, light absorption value A is surveyed with microplate reader 570nm, is counted as follows
Calculate killing activity of each group LAK cells to stomach cancer cell.
Killing rate (%)=[1- (experimental group A values-individual effect cell A values)/independent target cell A values] × 100%
4, statistical procedures
Data are indicated using mean value ± standard deviation, are examined with SPSS17.0 statistical softwares row t, P<0.05 has statistics
Meaning.
Three, experimental result
Each group LAK cells are to the killing rate of stomach cancer cell as shown in table 1 and Fig. 1.Should the experimental results showed that, the present invention is quick
Activiation method quickly activates under the premise of largely not using rIL-2 using only i.e. realization LAK cells for 24 hours.
Killing rate of the 1 each group LAK cells of table to stomach cancer cell
| Group | Killing rate (%) |
| Regular active | 17.4±5.1 |
| Enzymolysis polypeptide A | 45.7±6.2 |
| Enzymolysis polypeptide B | 19.8±5.7 |
| Enzymolysis polypeptide C | 48.5±6.4 |
| Enzymolysis polypeptide D | 43.1±6.0 |
Embodiment 3：The quick Activiation method of LAK cells
One, experiment material
1640 culture mediums of RPMI, 10% fetal calf serum are purchased from Gibco companies.
RIL-2 is purchased from Beijing Fourth Ring biopharmaceutical company.
Two, experimental method
1, cord blood collection and mononuclearcell separation
Using closed collecting method acquisition full-term normal delivery healthy newborn bleeding of the umbilicus 100, and its parent of antenatal exaination is equal
Without genetic disease family history, using the closed blood bag of citrate-phosphate-glucose (CPDA) anti-freezing before placenta is given birth to from
The umbilical cord broken ends of fractured bone is acquired far from newborn side.4 DEG C of refrigerators preserve after acquisition, interior for 24 hours to be detached and frozen processing.
With reference to art technology handbook (Shen Guanxin etc., Immunology Today experimental technique, Hubei science tech publishing house,
Page 1998,172-175), liquid density-gradient centrifugation method separating umbilical blood mononuclearcell is layered using ficoll cardiografin.
2, the quick activation of LAK cells
By RPMI 1640 culture medium (10%FCS-RPMI 1640) of the cord blood mononuclear cells containing 10% fetal calf serum
It modulates to 2 × 106/ mL, is inoculated according to total number of cells in the culture vessel of different capabilities, while final concentration of 40 μ g/ are added
The rIL-2 of enzymolysis polypeptide A, C, D and 500IU/mL of mL are placed in 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours.
3, tumor cell killing potential is detected
Human stomach cancer cell line MGC-803 freezes for our company, is trained using the RPMI 1640 containing 10% fetal calf serum after recovery
Foster base is placed in 37 DEG C, 5%CO2Secondary culture in incubator takes logarithmic phase to carry out subsequent experimental.
The LAK cells that each group activates are taken as effector cell, using MGC-803 stomach cancer cells as target cell, by 20:1 effect target
Than 96 orifice plates are added in effector cell and target cell respectively, independent target cell and individual effect groups of cells are separately set, every group sets 3 again
37 DEG C, 5%CO are placed in hole2After being cultivated for 24 hours in incubator, add 10 μ L of MTT (5mg/mL) per hole, continues to cultivate 4h, centrifugation is abandoned
Clearly, after adding 150 μ L of dimethyl sulfoxide (DMSO), oscillation to dissolve 10min per hole, light absorption value A is surveyed with microplate reader 570nm, is counted as follows
Calculate killing activity of each group LAK cells to stomach cancer cell.
Killing rate (%)=[1- (experimental group A values-individual effect cell A values)/independent target cell A values] × 100%
4, statistical procedures
Data are indicated using mean value ± standard deviation, are examined with SPSS17.0 statistical softwares row t, P<0.05 has statistics
Meaning.
Three, experimental result
The experimental results showed that when the final concentration of 40 μ g/mL of enzymolysis polypeptide A, C, D, the quick LAK cells pair of activation for 24 hours
The killing rate of MGC-803 stomach cancer cells is respectively (35.3 ± 5.5) %, (39.5 ± 5.9) %, (33.8 ± 5.3) %.
Embodiment 4：The quick Activiation method of LAK cells
One, experiment material
1640 culture mediums of RPMI, 10% fetal calf serum are purchased from Gibco companies.
RIL-2 is purchased from Beijing Fourth Ring biopharmaceutical company.
Two, experimental method
1, cord blood collection and mononuclearcell separation
Using closed collecting method acquisition full-term normal delivery healthy newborn bleeding of the umbilicus 100, and its parent of antenatal exaination is equal
Without genetic disease family history, using the closed blood bag of citrate-phosphate-glucose (CPDA) anti-freezing before placenta is given birth to from
The umbilical cord broken ends of fractured bone is acquired far from newborn side.4 DEG C of refrigerators preserve after acquisition, interior for 24 hours to be detached and frozen processing.
With reference to art technology handbook (Shen Guanxin etc., Immunology Today experimental technique, Hubei science tech publishing house,
Page 1998,172-175), liquid density-gradient centrifugation method separating umbilical blood mononuclearcell is layered using ficoll cardiografin.
2, the quick activation of LAK cells
By RPMI 1640 culture medium (10%FCS-RPMI 1640) of the cord blood mononuclear cells containing 10% fetal calf serum
It modulates to 2 × 106/ mL, is inoculated according to total number of cells in the culture vessel of different capabilities, while final concentration of 60 μ g/ are added
The rIL-2 of enzymolysis polypeptide A, C, D and 500IU/mL of mL are placed in 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours.
3, tumor cell killing potential is detected
Human stomach cancer cell line MGC-803 freezes for our company, is trained using the RPMI 1640 containing 10% fetal calf serum after recovery
Foster base is placed in 37 DEG C, 5%CO2Secondary culture in incubator takes logarithmic phase to carry out subsequent experimental.
The LAK cells that each group activates are taken as effector cell, using MGC-803 stomach cancer cells as target cell, by 20:1 effect target
Than 96 orifice plates are added in effector cell and target cell respectively, independent target cell and individual effect groups of cells are separately set, every group sets 3 again
37 DEG C, 5%CO are placed in hole2After being cultivated for 24 hours in incubator, add 10 μ L of MTT (5mg/mL) per hole, continues to cultivate 4h, centrifugation is abandoned
Clearly, after adding 150 μ L of dimethyl sulfoxide (DMSO), oscillation to dissolve 10min per hole, light absorption value A is surveyed with microplate reader 570nm, is counted as follows
Calculate killing activity of each group LAK cells to stomach cancer cell.
Killing rate (%)=[1- (experimental group A values-individual effect cell A values)/independent target cell A values] × 100%
4, statistical procedures
Data are indicated using mean value ± standard deviation, are examined with SPSS17.0 statistical softwares row t, P<0.05 has statistics
Meaning.
Three, experimental result
The experimental results showed that when the final concentration of 60 μ g/mL of enzymolysis polypeptide A, C, D, the quick LAK cells pair of activation for 24 hours
The killing rate of MGC-803 stomach cancer cells is respectively (50.7 ± 5.8) %, (55.2 ± 6.1) %, (48.9 ± 5.7) %.
Embodiment 5：LAK cells quickly activate culture medium
A kind of LAK cells quickly activate culture medium, are added in 1640 culture mediums of RPMI containing 10% fetal calf serum dense eventually
Spend enzymolysis polypeptide A, the enzymolysis polypeptide C of 40-60 μ g/mL or the rIL-2 of enzymolysis polypeptide D and 500IU/mL.
The preparation method of enzymolysis polypeptide A, enzymolysis polypeptide C or enzymolysis polypeptide D is：
By the wheat germ powder of degreasing through papain (enzyme activity is 800,000 U/g)+neutral proteinase (enzyme activity 20
Ten thousand U/g) in compound proportioning 3:1 (mass ratio), pH 6.0,55 DEG C of temperature, substrate mass concentration 40g/L, enzyme concentration 5%, enzymolysis
It is digested under conditions of time 8h, 90 DEG C is warming up to after enzymolysis, 20min progress destroy the enzyme treatment is kept to obtain the enzyme of wheat embryo
Liquid is solved, after cooling, 9000rpm is centrifuged 25 minutes, is taken supernatant, is freeze-dried to obtain freeze-dried powder.
By freeze-dried powder ultrapure water dissolution (1g freeze-dried powders add 10mL ultra-pure waters), then it is with molecular cut off successively
The ultrafiltration membrane of 10000u, 7000u, 5000u, 3000u and 1000u detach obtained enzymolysis liquid freeze-dried powder, will surpass
Filter obtained component freeze-drying (water content≤5%) obtain enzymolysis polypeptide A (10000u~7000u), enzymolysis polypeptide C (5000u~
3000u), enzymolysis polypeptide D (3000u~1000u).
The quick Activiation method of the present invention is more by adding enzymolysis in the medium under the premise of largely not using rIL-2
Peptide A, enzymolysis polypeptide C or enzymolysis polypeptide D are quickly activated using only i.e. realization LAK cells for 24 hours.
Above-described embodiment is intended to specifically introduce essentiality content of the present invention, but protection scope of the present invention is confined to this specifically
Embodiment.
Claims (9)
1. a kind of quick Activiation method of LAK LAK, adds rIL-2, feature exists in the medium
In：Also addition activator enzymolysis polypeptide A in the medium.
2. quick Activiation method according to claim 1, which is characterized in that the specific steps are：
1640 culture mediums of RPMI (10%FCS-RPMI 1640) containing 10% fetal calf serum are used to modulate cord blood mononuclear cells
To 2 × 106/ mL, is inoculated according to total number of cells in the culture vessel of different capabilities, while final concentration of 40-60 μ g/mL are added
Enzymolysis polypeptide A and 500IU/mL rIL-2, be placed in 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours；
The preparation method of enzymolysis polypeptide A is：
By the wheat germ powder of degreasing through papain+neutral protease enzymolysis, destroy the enzyme treatment obtains wheat after enzymolysis
The enzymolysis liquid of plumule, after cooling, centrifugation, supernatant is freeze-dried to obtain freeze-dried powder.
By the ultrapure water dissolution of freeze-dried powder, then successively with the ultrafiltration membrane that molecular cut off is 10000u, 7000u to obtained
Enzymolysis liquid freeze-dried powder is detached, and is freeze-dried 10000u~7000u components obtained by ultrafiltration to obtain enzymolysis polypeptide A.
3. quick Activiation method according to claim 2, which is characterized in that enzymatic hydrolysis condition is：Through papain (enzyme activity
Power is 800,000 U/g)+neutral proteinase (enzyme activity is 200,000 U/g) it is compound proportioning 3:1, pH 6.0,55 DEG C of temperature, substrate matter
It is digested under conditions of amount concentration 40g/L, enzyme concentration 5% (100g substrates add 5g enzymes), enzymolysis time 8h.
4. quick Activiation method according to claim 2, it is characterised in that：90 DEG C of holdings are warming up to after enzymolysis
20min carries out destroy the enzyme treatment and obtains the enzymolysis liquid of wheat embryo.
5. quick Activiation method according to claim 2, which is characterized in that centrifugal condition is：9000rpm centrifuges 25min.
6. quick Activiation method according to claim 2, it is characterised in that：Liquid-tight degree is layered using ficoll cardiografin
Gradient centrifugation separating umbilical blood mononuclearcell from bleeding of the umbilicus.
7. quick Activiation method according to claim 6, which is characterized in that cord blood collection method is：Using closed receipts
Collection method acquires full-term normal delivery healthy newborn bleeding of the umbilicus, and antenatal exaination its parent is without genetic disease family history, using lemon
The closed blood bag of lemon acid-phosphate-dextrose anti-freezing acquires before placenta is given birth to from the umbilical cord broken ends of fractured bone far from newborn side；After acquisition
4 DEG C of refrigerators preserve, for 24 hours in be detached and frozen processing spare.
8. the quick activation culture medium of LAK LAK a kind of, it is characterised in that：Containing 10% fetal calf serum
1640 culture mediums of RPMI in add final concentration of 40-60 μ g/mL enzymolysis polypeptide A and 500IU/mL rIL-2.
9. application of the culture medium according to any one of claims 8 in terms of the quick activation of LAK LAK.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810656001.XA CN108753722A (en) | 2018-06-23 | 2018-06-23 | A kind of quick Activiation method of LAK LAK |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810656001.XA CN108753722A (en) | 2018-06-23 | 2018-06-23 | A kind of quick Activiation method of LAK LAK |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108753722A true CN108753722A (en) | 2018-11-06 |
Family
ID=63976496
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810656001.XA Withdrawn CN108753722A (en) | 2018-06-23 | 2018-06-23 | A kind of quick Activiation method of LAK LAK |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108753722A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168124A (en) * | 2010-12-29 | 2011-08-31 | 山东广明实业有限公司 | Method for preparing wheat germ active polypeptide by composite enzyme and alkaline combination method |
| CN104313094A (en) * | 2014-10-21 | 2015-01-28 | 河北子丰生物科技有限公司 | Composite enzymatic hydrolysis method of protein in degreased plant meal |
| CN107163139A (en) * | 2017-04-24 | 2017-09-15 | 华南理工大学 | The method that immunoglobulin and immune-active peptides are prepared by raw material of defatted wheat germ |
-
2018
- 2018-06-23 CN CN201810656001.XA patent/CN108753722A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168124A (en) * | 2010-12-29 | 2011-08-31 | 山东广明实业有限公司 | Method for preparing wheat germ active polypeptide by composite enzyme and alkaline combination method |
| CN104313094A (en) * | 2014-10-21 | 2015-01-28 | 河北子丰生物科技有限公司 | Composite enzymatic hydrolysis method of protein in degreased plant meal |
| CN107163139A (en) * | 2017-04-24 | 2017-09-15 | 华南理工大学 | The method that immunoglobulin and immune-active peptides are prepared by raw material of defatted wheat germ |
Non-Patent Citations (3)
| Title |
|---|
| WENJIA WU等: "Enzymatic preparation of immunomodulatory hydrolysates from deffated wheat germ(Triticum vulgare) globulin", 《INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY》 * |
| 宋岩峰等: "淋巴因子激活杀伤细胞的快速激活及其临床应用", 《中华妇产科杂志》 * |
| 徐从高等: "IL-12抗肿瘤活性的研究进展", 《国外医学肿瘤学分册》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111670997B (en) | Preparation method of immunity-enhancing compound protein peptide enzymatic hydrolysate, immunity-enhancing compound protein peptide beverage and preparation method thereof | |
| CN1943425B (en) | Fishy smell removing method for spirulina princeps | |
| CN104762358B (en) | Quick preparation method of mussel protein antihypertensive peptide | |
| CN103565839B (en) | The separating and extracting process of one boar Placenta extract | |
| CN102766670A (en) | Preparation method of oyster polypeptide powder | |
| CN101756300A (en) | Chlorella cell wall disruption method | |
| CN106047974B (en) | It is a kind of with anti-oxidant, anti-fatigue active hippocampus polypeptide and its preparation process | |
| CN111154824A (en) | Industrial production method for obtaining spirulina antioxidant oligopeptide through high-concentration two-step enzymolysis | |
| CN110950925A (en) | ACE inhibitory peptide with function of repairing nutrition activating cells and preparation method thereof | |
| CN104223115B (en) | The new application of scale collagen polypeptide | |
| CN107988301A (en) | A kind of preparation method and applications of chick-pea bean cotyledon polypeptide | |
| JP5654666B2 (en) | Method for producing fermented natural products | |
| CN110693007A (en) | Probiotics compound nutrient for inhibiting gynecological inflammation and promoting cell metabolism and preparation method thereof | |
| CN105687036B (en) | Bletilla skin-protection product and preparation method thereof | |
| Wu et al. | Extraction, functionality, and applications of Chlorella pyrenoidosa protein/peptide | |
| WO2014194707A1 (en) | Calculus enzyme and preparation method therefor | |
| CN108753722A (en) | A kind of quick Activiation method of LAK LAK | |
| CN108753720A (en) | A kind of method of quick activation LAK LAK | |
| CN108753721A (en) | The Activiation method of LAK LAK | |
| CN108753719B (en) | A method of the LAK LAK of derived from cord blood is improved to tumor-killing power | |
| CN112675285A (en) | Cervical cancer treatment medicine prepared from wood frog extract and preparation method thereof | |
| CN106834404A (en) | Enzymolysis polypeptide and application thereof in preparing medicament for treating lung adenocarcinoma | |
| CN104945471B (en) | Mussel protein antihypertensive peptide | |
| CN109369781B (en) | Eucheuma anti-oxidation tetrapeptide and application thereof | |
| CN106509575A (en) | Genaoduotai solid beverage preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20181106 |
|
| WW01 | Invention patent application withdrawn after publication |