CN106801036B - A kind of biologically active peptide and the method with its external efficient amplification CIK cell - Google Patents
A kind of biologically active peptide and the method with its external efficient amplification CIK cell Download PDFInfo
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- 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
- C12N5/0646—Natural killers cells [NK], NKT cells
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Abstract
The invention discloses a kind of biologically active peptide and with the method for its external efficient amplification CIK cell, method includes: to extract peripheric venous blood separation mononuclearcell, it is placed in the culture bottle of coating biologically active peptide and CD 3-resisting monoclonal antibody in advance with serum free medium culture, IL-2 and serum free medium is only added in later cultivating system, in 37 DEG C, 5%CO2Under the conditions of cultivate 14-21 days；Wherein, the biologically active peptide, with the component for using ultrafiltration retaining molecular weight 3000-1000u after papain enzymolysis, is lyophilized using cicada monkey as raw material to obtain the final product.Biologically active peptide provided by the invention efficiently can induce CIK cell in vitro to expand, and survival rate is high, purity is high, and the CIK cell that amplification in vitro CIK cell method of the present invention obtains kills tumor effect and internal tumor killing effect with outstanding in vitro；Meanwhile the method for the present invention using biologically active peptide cheap and easy to get instead of the recombinanthumanifn-γ and IL-1 α in classical culture protocols, it is at low cost.
Description
Technical field
The invention belongs to field of medicaments, are related to the preparation and application of polypeptide drugs, and in particular to a kind of biologically active peptide and
With the method for its external efficient amplification CIK cell.
Background technique
Lung cancer is the most common malignant tumour in the whole world, and the death rate occupies first of malignant tumour.China's lung cancer morbidity rate present by
Year ascendant trend, average annual growth rate nearly 2%.There are many organization type, cancerous lung tissues according to the difference of Pathologic Characteristics for lung cancer
Type is different, and remedy measures are also different.Classified according to 2004 editions WHO, common cancerous lung tissue histological typing is divided into non-small thin
Born of the same parents' cancer (NSCLC) and small cell carcinoma (SCLC).Non-small cell carcinoma is divided into squamous cell carcinoma (SCC), gland cancer (AC) and maxicell again
Cancer (LCC).Different lung cancer clinical therapeutic schemes are different, and outcome is also different.Therefore, in order to improve therapeutic effect, lung cancer
Treatment mode of the therapeutic strategy from traditional based on by stages is changed into histological type and gene mutation as guidance
Individuation, accurately multimodality therapy mode.Individualized treatment improves treatment and the outcome of lung cancer.
Biologically active peptide (Biologically active peptides, BAP) is that a kind of have the function of special physiological
Small-molecule active substance, such as it is antitumor, antibacterial, anti-hypertension, antiviral and improve immunity, these substances are widely present
In animal, plant and microorganism.Biologically active peptide mainly has 4 kinds of sources, respectively protein biological activity peptides, microorganism hair
Ferment metabolising polypeptide, natural biological polypeptide and chemically synthesized polypeptide.Cicada monkey is also known as cicada tortoise, climbs grasshopper etc., is the maturation of golden cicada
Nymph, protein content is very high, edible and medical value with higher.Applicant has carried out greatly cicada monkey biologically active peptide
Quantity research, it has been found that the biologically active peptide based on cicada monkey of different process preparation obviously inhibits lung adenocarcinoma A549 cell line to increase
It grows (application number 201710109721X) and inhibits the invasion of Small Cell Lung Cancer stem cell and transfer (application number 2017101067746)
Effect.
Immunotherapy of tumors is the main method in tumor biotherapy, and adoptive immunotherapy is immunotherapy of tumors
A kind of effective ways.Cytokine induced kill cell (cytokine-inducedkiller, CIK) has T lymphocyte concurrently
Powerful anti-tumor activity and the non-principal histocompatibility complex (major of natural kill (natural killer, NK) cell
Histocompatibility complex, MHC) restricted the advantages of killing tumor.It can not damage body immune system knot
Under the premise of structure and function, direct killing tumour cell, and body's immunity is adjusted and enhances, in treatment malignant entity tumor
In acquired effect widely accepted.And obtaining the CIK cell that quantity is enough and bioactivity is strong is to obtain well
The basis of curative effect.
Tradition culture CIK cell method needs higher basal cell number, and peripheral blood need to be separated with blood cell separator, is mentioned
Mononuclearcell is taken, cumbersome time-consuming, opportunities for contamination is big, is especially not suitable for allosome blood supply.How simplified culture process, improve CIK
Cell is proliferated efficiency in vitro and cytotoxic activity is a hot issue of basic research, and effective solution of the problem can mention
Application of the high adoptive immunotherapy in immunotherapy of tumors field.
Not yet there are some researches prove cicada monkey biologically active peptides, and CIK cell in vitro proliferation efficiency and cell toxicant can be improved at present
Activity.
Summary of the invention
The purpose of the present invention is to provide a kind of biologically active peptide and with the method for its external efficient amplification CIK cell.
Above-mentioned purpose is achieved by the following technical solution:
A kind of method of amplification in vitro CIK cell extracts peripheric venous blood and separates mononuclearcell, uses serum free medium
Cell suspension is made, is placed in the culture bottle for being coated with anti-human CD3 monoclonal antibody in advance and cultivates, is only mended in later cultivating system
IL-2 and serum free medium are filled, in 37 DEG C, 5%CO2Under the conditions of cultivate 14-21 days, in which: it is anti-to be coated with anti-human CD3 monoclonal
Also while it being coated with biologically active peptide when body, the biologically active peptide is using cicada monkey as raw material, with using after papain enzymolysis
The component of ultrafiltration retaining molecular weight 3000-1000u, is lyophilized to obtain the final product.
Preferably, the peridium concentration of biologically active peptide and anti-human CD3 monoclonal antibody in culture bottle is respectively 20 μ g/mL
With 5 μ g/mL, it is coated within preparatory 24 hours.
Preferably, cell suspension be placed in culture bottle start culture 24 hours after, 1000U/mL is added into cultivating system
IL-2 continue to cultivate, only supplement IL-2 and serum free medium in later cultivating system.
Preferably, the specific preparation method of the biologically active peptide includes the following steps:
It is twisted into meat gruel after cicada monkey is cleaned, is placed in ultrapure water, papain enzymolysis, enzymatic hydrolysis condition are as follows: pH is added
Value, 6.8-7.2；Hydrolysis temperature, 53-57 DEG C；Enzymolysis time, 6-10 hours；
After enzymatic hydrolysis, heating makes Papain enzyme-deactivating, is cooled to room temperature；
After cooling, 8000-10000rpm is centrifuged 20-30 minutes, is taken supernatant, is freeze-dried to obtain freeze-dried powder；
Freeze-dried powder is dissolved with ultrapure water, successively with molecular cut off be respectively 10000u, 5000u, 3000u and
The ultrafiltration membrane of 1000u separates obtained biologically active peptide, collects the component of molecular cut off 3000-1000u, freeze-drying
To obtain the final product.
Preferably, heating makes the method for Papain enzyme-deactivating are as follows: water-bath 8-12 minutes under the conditions of 80-90 DEG C.
Preferably, enzymatic hydrolysis condition are as follows: pH value, 7.0；Hydrolysis temperature, 55 DEG C；Enzymolysis time, 8 hours.
Preferably, centrifugal condition are as follows: 9000rpm is centrifuged 25 minutes.
Preferably, the additive amount of the papain is the 0.4-0.6% of substrate meat gruel weight.
The CIK cell of above method preparation.
Above-mentioned CIK cell is used to prepare the purposes of the drug for the treatment of lung cancer.
Beneficial effects of the present invention:
Biologically active peptide provided by the invention efficiently can induce CIK cell in vitro to expand, and survival rate is high, purity is high, this
The CIK cell that invention amplification in vitro CIK cell method obtains kills tumor effect and internal tumor killing effect with outstanding in vitro；Together
When, the method for the present invention is using biologically active peptide cheap and easy to get instead of the recombinanthumanifn-γ and IL-1 in classical culture protocols
α, it is at low cost.
Detailed description of the invention
Fig. 1 is the proliferative capacity of the CIK cell of two kinds of cultural methods induction；
Fig. 2 is the 21st day CD3+CD56+CD16+ cell subset ratio streaming figure of biologically active peptide Fiber differentiation method；
Fig. 3 is tumour inhibiting rate in the CIK cell body of two kinds of cultural methods induction.
Specific embodiment
Technical solution of the present invention and technical effect is discussed in detail with attached drawing combined with specific embodiments below.It is not specified specific
The experimental method of condition, usually according to normal condition, such as condition described in textbook and experiment guide, or according to manufactory
Condition proposed by quotient is well known within the skill of those ordinarily skilled or is easy to know.
The preparation of 1 biologically active peptide of embodiment
One, experimental material
1, instrument reagent
Ultrafiltration cup and ultrafiltration membrane are purchased from Shanghai and rub fast scientific equipment Co., Ltd；
Papain (1:800U/mg) is purchased from Guangzhou Qi Yun Bioisystech Co., Ltd.
2, laboratory sample
The upper fresh cicada monkey set of being just unearthed is collected, saves in water, is frozen in -20 DEG C.Using it is preceding in room temperature condition from
So thaw.
Two, experimental method and result
1, enzyme solution
It is twisted into meat gruel after cicada monkey is cleaned, 10g meat gruel is taken to be placed in 50mL ultrapure water, papain is added and carries out enzyme
Solution, the additive amount of papain are the 0.5% of substrate meat gruel weight, enzymatic hydrolysis condition are as follows: pH value, 7.0；Hydrolysis temperature, 55 DEG C；
Enzymolysis time, 8 hours；
After enzymatic hydrolysis, makes Papain enzyme-deactivating within water-bath 10 minutes under the conditions of 85 DEG C, be cooled to room temperature；
After cooling, 9000rpm is centrifuged 25 minutes, is taken supernatant, is freeze-dried to obtain freeze-dried powder.
2, purification process
Freeze-dried powder ultrapure water is dissolved into (1g freeze-dried powder adds 10mL ultrapure water), is then successively with molecular cut off
The ultrafiltration membrane of 10000u, 5000u, 3000u and 1000u separate obtained biologically active peptide, collect retention molecule
Measure the component of 3000-1000u, freeze-drying (water content≤5%) to obtain the final product.
CO is controlled in ultra-filtration process2Pressure make its be less than 0.25MPa.
In vitro culture, amplification, survival rate measurement and the immunophenotype detection of 2 CIK cell of embodiment
One, experimental method
1, the in vitro culture of CIK cell, amplification and survival rate measurement
It extracts 30 healthy human peripheric venous bloods and amounts to 150mL, it is thin to extract the single core of peripheral blood with lymphocyte separation medium
Born of the same parents, are made cell suspension with KBM551 serum free medium after PBS washing, and adjustment cell concentration is 1 × 105/ mL, is divided into 6
Part, respectively with biologically active peptide Fiber differentiation method of the present invention and tradition Fiber differentiation method culture, every kind 3 parts of method operation repetitive.
Biologically active peptide Fiber differentiation method: culture 24 hours before, with containing 20 μ g/mL biologically active peptides and 5 μ g/mL it is anti-human
The ice PBS 250mL of CD3 monoclonal antibody is placed in culture bottle, and 4 DEG C overnight；After 24 hours, PBS is discarded, above-mentioned cell is added
Suspension 250mL, in 37 DEG C, 5%CO2Under the conditions of start to cultivate.After culture 24 hours, add 1000U/mL's into cultivating system
IL-2 continues to cultivate, and IL-2 and serum free medium are only supplemented in later cultivating system.
Traditional Fiber differentiation method: before culture 24 hours, with the ice PBS containing the anti-human CD3 monoclonal antibody of 5 μ g/mL
250mL is placed in culture bottle, and 4 DEG C overnight；After 24 hours, PBS is discarded, above-mentioned cell suspension 250mL is added, in 37 DEG C, 5%
CO2Under the conditions of start to cultivate.The culture same day adds the IL- of the recombinanthumanifn-γ of 1000U/mL, 500U/mL into cultivating system
The IL-2 of 1 α and 1000U/mL continues to cultivate, and IL-2 and serum free medium are only supplemented in later cultivating system.
Cell survival rate is detected with Trypan Blue within every 3 days, record cell absolute number.
2, immunophenotype detects
The the 0th, 7,14,21 day sample 6mL of culture is taken respectively, is washed 2 times after centrifugation with PBS, according to BD company flow cytometer detection
The specification dying operation of kit, and using CD3+, CD3+CD4+, CD3+CD8+, CD3 in flow cytometer measurement culture solution
The percentage of+CD16+CD56+ cell.
Two, experimental result
1, the proliferation activity and survival rate of two methods induction CIK cell
As shown in Figure 1, biologically active peptide Fiber differentiation method and the CIK cell of traditional Fiber differentiation method culture show it is non-
Often high proliferation activity, when cultivating the 21st day, CIK cell quantity reaches peak, the training of bioactivity inducing peptide in cultivating system
The proliferation activity of CIK cell is apparently higher than traditional Fiber differentiation method in the method for supporting, and when cultivating the 21st day, the former is at CIK cell quantity
2.1 times of the latter's CIK cell quantity have statistical difference (P < 0.05).
The CIK cell survival rate of biologically active peptide Fiber differentiation method and traditional Fiber differentiation method is detected, is respectively within the 15th day
(97.65 ± 1.24) % and (94.36 ± 1.68) %, be respectively within the 21st day (95.84 ± 1.18) % and (90.22 ±
2.15) %, the survival rate of CIK cell is apparently higher than tradition to survival rate all in 90% or more, biologically active peptide Fiber differentiation method
Fiber differentiation method.
2, the variation of two methods induction CIK cell immunophenotype
As shown in table 1, healthy human peripheral blood lymphocyte through induction and amplification in vitro culture after, CD3+, CD3+CD8+,
CD3+CD56+CD16+ cell proportion obviously increases before relatively cultivating, and the ratio of CD3+CD4+ and CD3+CD4+ and CD3+CD8+ are then bright
Aobvious to reduce, i.e., killer T cell ratio increases, and helper T lymphocyte is reduced.Surface markers are the cell quilt of CD3+CD56+CD16+
It is considered the main effects cell of CIK cell, it is amplifiable to 2000 times or more in amplification procedure in vitro.Fig. 2 streaming result figure
For the ratio of the 21st day CD3+CD56+CD16+ cell subset of biologically active peptide Fiber differentiation method.
1 two methods of table induction CIK cell in each subset proportions with cultivated days variation (n=3 takes mean value)
The above results show in two kinds of cultural methods that killer T cell ratio increases, and helper T lymphocyte is reduced.
3 CIK cell in vitro of embodiment kills ratio of outflow measurement
One, experimental method
1, tumor cell culture
RPMI-1640 culture medium of the human lung carcinoma cell line A549 containing 10% calf serum, in 37 DEG C, 5%CO2Under the conditions of
Routine culture, passage.With 0.25% trypsin digestion 3 minutes when passage, cell suspension is made with culture solution piping and druming, by required
Concentration inoculation.When being mixed with CIK cell, as target cell.
2, mtt assay measurement CIK cell in vitro kills ratio of outflow (%)
The human lung carcinoma cell line A549 of logarithmic growth phase is selected, adjustment cell concentration is 8 × 104/ mL, every hole in 96 orifice plates
100 μ L, 37 DEG C, 5%CO are added24h is cultivated in incubator.The CIK cell for taking culture the 15th day is effector cell, by effect target ratio
CIK cell is added in 20:1 and 40:1, and laying effect cell killing group, effector cell's control group, tumour cell control group, every group sets 6
A multiple holes.4h is further cultured for after 40 μ L of 2.5mg/mL MTT solution is added after co-incubation 48h.Supernatant is abandoned in centrifugation, and 150 μ L are added
DMSO solution concussion mixes, and microplate reader selects 570nm wavelength, measures each hole OD value (optical density, OD), counts
Ratio of outflow, calculation formula are killed in calculation are as follows:
Kill ratio of outflow (%)=1- [(killing group OD value-effector cell control group OD value)/tumour cell control group OD value] ×
100%.
Two, experimental result
As shown in table 2, biologically active peptide Fiber differentiation method and the CIK cell of traditional Fiber differentiation method culture are shown non-
Chang Qiang's kills tumor activity in vitro, and the CIK cell of biologically active peptide Fiber differentiation method culture is killed tumor activity in vitro and is significantly better than
The CIK cell of traditional Fiber differentiation method culture kills tumor activity in vitro, there is statistical difference (P < 0.05).
The CIK cell in vitro of 2 two methods of table induction kills ratio of outflow and compares (%)
Upper table statistics indicate that, the CIK cell of biologically active peptide Fiber differentiation method culture is killed tumor activity and is significantly better than in vitro
The CIK cell of traditional Fiber differentiation method culture kills tumor activity in vitro.
Tumour inhibiting rate measures in 4 CIK cell body of embodiment
One, experimental method
The A549 cell 5 × 10 of logarithmic growth phase is in the right axillary subcutaneous injection of BALB/c nude mice6A, volume is
0.2ml/ is only.The similar nude mice of selection lotus knurl size is randomly divided into saline control group and CIK cell treatment group within 16th day.It controls
Treatment group is in the CIK cell 1 × 10 of locally injected into tumor culture the 15th day7A/only, volume is 0.2ml/, and control group is in swollen
Tumor locally injecting physiological saline 0.2ml/ is only；Weekly treatment 1 time, continuous treatment 3 times.Every 7d is surveyed with vernier caliper since treatment
The maximum for measuring tumour indulges diameter (a) and maximum transverse diameter (b) 1 time, gross tumor volume=0.5 × a × b2, CIK is calculated according to following formula
Tumour inhibiting rate in cell body: tumour inhibiting rate (%)=(control group gross tumor volume-treatment group tumors volume)/control group gross tumor volume ×
100%.
Two, experimental result
As shown in table 3 and figure 3, the equal table of CIK cell of biologically active peptide Fiber differentiation method and traditional Fiber differentiation method culture
Reveal very strong internal antitumor activity, and the internal antitumor activity of the CIK cell of biologically active peptide Fiber differentiation method culture is aobvious
The internal antitumor activity for writing the CIK cell better than traditional Fiber differentiation method culture, there is statistical difference (P < 0.05).
Tumour inhibiting rate compares (%) in the CIK cell body of 3 two methods of table induction
Upper table statistics indicate that, the internal antitumor activity of the CIK cell of biologically active peptide Fiber differentiation method culture is significantly better than
The internal antitumor activity of the CIK cell of traditional Fiber differentiation method culture.
Biologically active peptide provided by the invention efficiently can induce CIK cell in vitro to expand, and survival rate is high, purity is high, this
The CIK cell that invention amplification in vitro CIK cell method obtains kills tumor effect and internal tumor killing effect with outstanding in vitro；Together
When, the method for the present invention is using biologically active peptide cheap and easy to get instead of the recombinanthumanifn-γ and IL-1 in classical culture protocols
α, it is at low cost.
Claims (2)
1. a kind of method of amplification in vitro CIK cell extracts peripheric venous blood and separates mononuclearcell, trained with KBM551 serum-free
Cell suspension is made in feeding base, is placed in the culture bottle for being coated with anti-human CD3 monoclonal antibody in advance and cultivates, in later cultivating system
Only supplement IL-2 and serum free medium, in 37 DEG C, 5% CO2Under the conditions of cultivate 14-21 days, it is characterised in that: coating it is anti-human
It is gone back when CD3 monoclonal antibody while being coated with biologically active peptide, the biologically active peptide uses Papain using cicada monkey as raw material
The component that ultrafiltration retaining molecular weight 3000u-1000u is used after enzyme enzymatic hydrolysis, is lyophilized to obtain the final product；
Wherein:
The peridium concentration of biologically active peptide and anti-human CD3 monoclonal antibody in culture bottle is respectively 20 μ g/mL and 5 μ g/mL, in advance
It is coated within first 24 hours；
Cell suspension be placed in culture bottle start culture 24 hours after, added into cultivating system 1000 U/mL IL-2 continue
It cultivates, IL-2 and serum free medium is only supplemented in later cultivating system；
The biologically active peptide is prepared by the following method:
It is twisted into meat gruel after cicada monkey is cleaned, is placed in ultrapure water, papain enzymolysis, enzymatic hydrolysis condition is added are as follows: pH value,
7.0；Hydrolysis temperature, 55 DEG C；Enzymolysis time, 8 hours；The additive amount of papain is the 0.5% of substrate meat gruel weight；
After enzymatic hydrolysis, heating makes Papain enzyme-deactivating, is cooled to room temperature；
After cooling, 9000rpm is centrifuged 25 minutes, is taken supernatant, is freeze-dried to obtain freeze-dried powder；
Freeze-dried powder is dissolved with ultrapure water, is respectively successively 10000u, 5000u, 3000u and 1000u with molecular cut off
Ultrafiltration membrane separates obtained polypeptide, collects the component of molecular cut off 3000u-1000u, is lyophilized to obtain the final product.
2. the method for amplification in vitro CIK cell according to claim 1, which is characterized in that heating makes papain go out
Method living are as follows: the water-bath 10 minutes under the conditions of 85 DEG C.
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