CN106906269B - A kind of enzymolysis polypeptide and the purposes for inhibiting Small Cell Lung Cancer stem cell to invade and shift - Google Patents
A kind of enzymolysis polypeptide and the purposes for inhibiting Small Cell Lung Cancer stem cell to invade and shift Download PDFInfo
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Abstract
The invention discloses a kind of enzymolysis polypeptide and for inhibiting, Small Cell Lung Cancer stem cell invades and the purposes of transfer is prepared by the following method using cicada monkey as raw material：It is twisted into meat gruel after cicada monkey is cleaned, is placed in ultra-pure water, alkali protease enzymolysis is added, enzymatic hydrolysis condition is：PH value, 8.3 8.7；Hydrolysis temperature, 53 57 DEG C；Enzymolysis time, 9 11 hours；After enzymolysis, heating makes basic protein enzyme-deactivating, is cooled to room temperature；After cooling, 8000 10000rpm are centrifuged 20 30 minutes, take supernatant, are freeze-dried to obtain freeze-dried powder；By the ultrapure water dissolution of freeze-dried powder, obtained enzymolysis polypeptide is detached with the ultrafiltration membrane of different molecular weight cut off, the enzymolysis polypeptide up to different molecular weight size is lyophilized in ultrafiltration obtained component.Enzymolysis polypeptide provided by the invention is expected to the drug of metastases occur after developing into prevention Patients With Small Cell Carcinoma of The Lung chemotherapy.
Description
Technical field
The invention belongs to field of medicaments, are related to the preparation of polypeptide drugs, and in particular to a kind of enzymolysis polypeptide and for inhibiting
Small Cell Lung Cancer stem cell invades and the purposes of transfer.
Background technology
Lung cancer is the most common malignant tumour in the whole world, and the death rate occupies first of malignant tumour.China 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 as squamous cell carcinoma (SCC), gland cancer (AC) and maxicell
Cancer (LCC).Different lung cancer clinical therapeutic schemes are different, and outcome is also different.Therefore, in order to improve therapeutic effect, lung cancer
It is guidance that treatment mode of the therapeutic strategy from traditional based on by stages, which is changed into histological type and gene mutation,
Individuation, accurately multimodality therapy pattern.Individualized treatment improves treatment and the outcome of lung cancer.
Small Cell Lung Cancer accounts for the 20%-25% of lung cancer sum, sensitive to chemotherapy and radiotherapy, but easily recurrence and hair
5 years survival rates of raw DISTANT METASTASES IN, document report limited stage small cell lung are 7%, and the extensive phase is only 1%.It proposed in recent years
Cancer stem-cell hypothesis think, the minute quantity tumor stem cell to lie dormant in most of tumour be tumor recurrence and transfer
Root.
Biologically active peptide (Biologically active peptides, BAP) is that a kind of having 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 proteolysis polypeptide, microbial fermentation
Metabolising polypeptide, natural biological polypeptide and chemically synthesized polypeptide.
Due to the continuous improvement of Cancer Mortality in recent years, and traditional chemotherapeutics not only toxic side effect it is big and
And therapeutic effect is unsatisfactory, therefore, biologically active peptide is obtained by its active height, Small side effects, advantage with strong points
The research and concern of domestic and international researcher.Extraction has obtained antitumor activity peptide from a variety of biologies at present, wherein
The active peptide of animal origin presents wide foreground.Hsu etc. uses papain and protease XX III to digest tuna respectively
Meat has obtained the two kinds of preferable polypeptide of antitumor activity (Antiproliferative activity of after isolating and purifying
fish protein hydrolysates on human breast cancer cell lines.Process
Biochemistry,2006,41:1217-1222).This two polypeptides have reached 8.1 to the IC50 values of breast cancer cell MCF-7
With 8.8 μM.Leng et al. has successfully been isolated from shell clam pair by SephadexG-25, FPLC and in conjunction with MALDI-TOF
It is 3147u active peptides that stomach cancer cell BGC-823, which has the molecular weight of obvious inhibiting effect, and as a concentration of 4.0 μ g/mL of peptide, its is right
The inhibiting rate of cancer cell has reached 60% (Inhibitory effects of anticancer peptide from
Mercenaria on the BGC-823 cells and several enzymes.FEBS Letters,2005,579:
1187-1190).In addition also obtained natineoplaston out of animal bodies such as mussel, mud blood clam, ascidian, oysters respectively, these all be with
The extraction of natineoplaston is laid a good foundation in animal body afterwards.
Cicada monkey is also known as cicada tortoise, climbs grasshopper etc., is the ripe nymph of golden cicada, and protein content is very high, has higher food
With and medical value.However, the invasion of Small Cell Lung Cancer stem cell are purified and inhibited for the extracting and developing of cicada monkey polypeptide at present
It is rarely reported with the research in terms of transfer.
Invention content
The purpose of the present invention is to provide a kind of enzymolysis polypeptide and for inhibiting Small Cell Lung Cancer stem cell to invade and shift
Purposes.
Above-mentioned purpose is achieved by the following technical solution：
A kind of enzymolysis polypeptide is prepared by the following method using cicada monkey as raw material：
It is twisted into meat gruel after cicada monkey is cleaned, is placed in ultra-pure water, alkali protease enzymolysis is added, enzymatic hydrolysis condition is：pH
Value, 8.3-8.7；Hydrolysis temperature, 53-57 DEG C；Enzymolysis time, 9-11 hours；
After enzymolysis, heating makes basic protein 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；
By the ultrapure water dissolution of freeze-dried powder, obtained enzymolysis polypeptide is divided with the ultrafiltration membrane of different molecular weight cut off
From ultrafiltration obtained component to be lyophilized to the enzymolysis polypeptide up to different molecular weight size.
Preferably, the ultrafiltration membrane of the different molecular weight cut off include molecular cut off be respectively 10000u, 5000u,
The ultrafiltration membrane of 3000u and 1000u.
Preferably, the molecular weight ranges of enzymolysis polypeptide are 3000-5000u.
Preferably, the molecular weight ranges of enzymolysis polypeptide are 1000-3000u.
Preferably, heating makes the method for basic protein enzyme-deactivating be：Water-bath 8-12 minutes under the conditions of 80-90 DEG C.
Preferably, heating makes the method for basic protein enzyme-deactivating be：The water-bath 10 minutes under the conditions of 85 DEG C.
Preferably, enzymatic hydrolysis condition is：PH value, 8.5；Hydrolysis temperature, 55 DEG C；Enzymolysis time, 10 hours.
Preferably, centrifugal condition is：9000rpm is centrifuged 25 minutes.
Preferably, the additive amount of the alkali protease is the 0.4-0.5% of substrate meat gruel weight.
Application of the above-mentioned enzymolysis polypeptide in there is the drug of metastases after preparing prevention Patients With Small Cell Carcinoma of The Lung chemotherapy.
Beneficial effects of the present invention：
Enzymolysis polypeptide provided by the invention can effectively inhibit Small Cell Lung Cancer NCI-H446 cell line lung cancer stem-like cells
Cell invasion and transfer, toxicological experiment research have shown that enzymolysis polypeptide without obvious toxic-side effects.Cancer stem-cell hypothesis thinks, big
The minute quantity tumor stem cell to lie dormant in Partial tumors is the root of tumor recurrence and transfer, therefore, enzyme provided by the invention
Solution polypeptide is expected to the drug of metastases occur after developing into prevention Patients With Small Cell Carcinoma of The Lung chemotherapy.
Description of the drawings
Fig. 1 is influence of the enzymolysis polypeptide to the transfer of human small cell lung carcinoma NCI-H446 cell line lung cancer stem cells, invasion.
Specific implementation mode
Technical scheme of the present invention and technique effect is discussed in detail with reference to specific embodiments and the drawings.It is not specified specific
The experimental method of condition, usually according to normal condition, such as the 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 enzymolysis polypeptide of embodiment
One, experiment material
1, instrument reagent
Ultrafiltration cup and ultrafiltration membrane are purchased from Shanghai and rub fast science equipment Co., Ltd；
Alkali protease 37017 (1:2.5U/g) it is purchased from Novozymes Company.
2, laboratory sample
The upper fresh cicada monkey set of being just unearthed is collected, preserves in water, is frozen in -20 DEG C.Using it is preceding in room temperature condition from
So thaw.
Two, experimental method
1, enzyme solution
It is twisted into meat gruel after cicada monkey is cleaned, 10g meat gruels is taken to be placed in 50mL ultra-pure waters, alkali protease is added and carries out enzyme
Solution, the additive amount of alkali protease are the 0.45% of substrate meat gruel weight, and enzymatic hydrolysis condition is：PH value, 8.5；Hydrolysis temperature, 55
℃；Enzymolysis time, 10 hours；
After enzymolysis, under the conditions of 85 DEG C water-bath make basic protein enzyme-deactivating within 10 minutes, 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
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, 5000u, 3000u and 1000u detach obtained enzymolysis polypeptide, by ultrafiltration obtained component
(water content≤5%) is freeze-dried up to the enzymolysis polypeptide of different molecular weight size.
CO is controlled in ultra-filtration process2Pressure make its be less than 0.25MPa.
Three, experimental result
The enzymolysis polypeptide of five different molecular weight ranges is obtained, molecular weight ranges are respectively：
Enzymolysis polypeptide 1, molecular weight are 10000u or more；
Enzymolysis polypeptide 2, molecular weight is between 10000u and 5000u；
Enzymolysis polypeptide 3, molecular weight is between 5000u and 3000u；
Enzymolysis polypeptide 4, molecular weight is between 3000u and 1000u；
Enzymolysis polypeptide 5, molecular weight are 1000u or less.
The influence that 2 enzymolysis polypeptide of embodiment is invaded Small Cell Lung Cancer NCI-H446 cell line lung cancer stem cells and shifted
(Chen Gongrang etc., casticin inhibit Small Cell Lung Cancer to the operation of the present embodiment experimental method reference literature method
NCI-H446 cell line lung cancer stem cell-like cells are invaded and the research of transfer, Central-South pharmacy in the October, 2014 of volume 12 the 10th
Phase), and use casticin disclosed in the document as positive drug.
One, experimental method
1, the culture and passage of human small cell lung carcinoma NCI-H446 cell lines
Human small cell lung carcinoma NCI-H446 cell lines are purchased from terrestrial Science and Technology Ltd. of Shanghai Austria.With containing 10% tire ox blood
Clearly, the DMEM in high glucose culture medium of 100U/mL penicillin and 100U/mL streptomysins, in 37 DEG C, 5%CO2Saturated humidity under the conditions of
Culture.Cell adherent growth, about 1-2d cover with bottom of bottle 90%.After 0.25% trypsin digestion, renewed vaccination to new culture
Bottle, continues to cultivate, and logarithmic growth phase cell is for testing.
2, human small cell lung carcinoma NCI-H446 cell lines lung cancer stem cell is cultivated
Logarithmic growth phase NCI-H446 cell line cells, are digested to single cell suspension, and 800rpm centrifugations 5min abandons supernatant
Liquid is added PBS and washs 2 times, supernatant abandoned after centrifuging again, with 2 × 104A/hole, which is inoculated in, ultralow sticks 6 hole cell culture
Plate, and be added 2mL stem cells conditioned medium (addition 100IU/mL benzyl penicillins, 100 μ g/mL streptomysins, 20ng/mL EGF,
The DMEM/ of 20ng/mL bFGF, 2% without the B27 additives of vitamin A, 0.4% bovine serum albumin(BSA), 5 μ g/mL insulin
F12 culture mediums) suspend culture.1mL stem cell conditioned mediums are added every 2d.About 5d, the people that proliferation forms 60 μm of sizes are small
Cell lung cancer NCI-H446 cell line lung cancer stem cells.
3, human small cell lung carcinoma NCI-H446 cell lines lung cancer stem cell secondary culture
Above-mentioned diameter is collected up to 60 μm of lung cancer stem cell, 500rpm centrifuges 5min, supernatant abandoned, after washing 1 time with PBS
0.05% trypsin digestion 3min of 1mL are added.It is terminated and is digested with stem cell conditioned medium, liquid-transfering gun is used in combination to blow and beat to list
Cell suspension counts.With every hole 2 × 104A cell be reinoculated on it is ultralow stick in 6 porocyte culture plates, it is dry thin that 2mL is added
Born of the same parents' conditioned medium, which suspends, to be cultivated.3rd generation lung cancer stem cell is used for aftermentioned experiment.
4, phycoerythrin (PE) fluorescent marker CD133 antibody flow cytometries
Human small cell lung carcinoma NCI-H446 cell line cells and the 3rd generation lung cancer stem cell are collected, single cell suspension is digested to.
10min is fixed with 1mL4% paraformaldehydes, 400 μ L 0.5%BSA buffer solutions are added and are resuspended.It sets in 4 DEG C of refrigerators and closes 10min,
It counts and adjusts cell density to 5 × 105A/200 μ L, are sub-packed in EP pipes.It is separately added into the anti-human CD133 of 2 μ LPE labels
The anti-igg 2b isotype control Abs of antibody and 2 μ L PE labels, are placed in 4 DEG C of refrigerators and are protected from light overnight incubation.13000rpm is centrifuged
10s is simultaneously cleaned 1 time with physiological saline, and flow cytomery average fluorescent strength value and CD133+ cells hundred are used after blowing and beating uniformly
Divide rate.Often pipe counts 1 × 105A, experiment is repeated 3 times.
5, the transfer of human small cell lung carcinoma NCI-H446 cell line cells and lung cancer stem cell, invasive ability
Using the cells Transwell, (Transwell that Matrigel uses BD biosciences to spread glue is small for experiment
Room).Cell culture cell bottom is the makrolon miillpore filter in 8 μm of apertures, and upper chamber is separately added into 200 μ L of DMEM culture mediums
(cell line cells of NCI-H446 containing Small Cell Lung Cancer, 2 × 105A/mL) and 200 μ L of DMEM/F12 culture mediums (contain the 3rd generation lung cancer
Stem cell, 2 × 105A/mL and 0.1%BSA).The corresponding 500 μ L that are added contain 10% tire ox blood respectively for room under the cells Transwell
Clear DMEM culture mediums and the stem cell conditioned medium containing 10% fetal calf serum.It sets in 37 DEG C of incubators, is incubated 48h.It takes out
The cells Transwell are washed 2 times with PBS, and cotton balls wipes the cell that upper surface is not shifted.With the 4 DEG C of fixations of 0.5mL4% paraformaldehydes
20min, is added 0.1% violet staining 20min of 0.5mL, and PBS is washed 2 times, observed under inverted microscope.Randomly select 5 400
Times visual field counts in the visual field relative number of transfer and invasion cell to indicate transfer and the invasive ability of tumour cell, tests
It is repeated 3 times.
6, influence of the enzymolysis polypeptide to the transfer of human small cell lung carcinoma NCI-H446 cell line lung cancer stem cells, invasion
Lung cancer stem cell is grouped：
Control group：The 3rd generation lung cancer stem cell is anticipated with 1%DMSO for 24 hours；
Positive drug group：Anticipated for the 3rd generation with casticin (3 μM, be dissolved in the stem cell media containing 1%DMSO)
Lung cancer stem cell is for 24 hours；
3 low dose group group of enzymolysis polypeptide：With enzymolysis polypeptide 3 (5 μ g/mL, be dissolved in the stem cell media containing 1%DMSO)
Anticipate the 3rd generation lung cancer stem cell for 24 hours；
3 high dose group group of enzymolysis polypeptide：With enzymolysis polypeptide 3, (10 μ g/mL, are dissolved in the stem cell media containing 1%DMSO
In) anticipate the 3rd generation lung cancer stem cell for 24 hours；
4 low dose group group of enzymolysis polypeptide：With enzymolysis polypeptide 4, (80 μ g/mL, are dissolved in the stem cell media containing 1%DMSO
In) anticipate the 3rd generation lung cancer stem cell for 24 hours；
4 high dose group group of enzymolysis polypeptide：With enzymolysis polypeptide 4, (160 μ g/mL, are dissolved in the stem cell media containing 1%DMSO
In) anticipate the 3rd generation lung cancer stem cell for 24 hours.
Using the cells Transwell, (Transwell that Matrigel uses BD biosciences to spread glue is small for experiment
Room).Cell culture cell bottom is the makrolon miillpore filter in 8 μm of apertures, and upper chamber is separately added into 200 μ L of DMEM culture mediums
(cell line cells of NCI-H446 containing Small Cell Lung Cancer, 2 × 105A/mL) and 200 μ L of DMEM/F12 culture mediums (containing pretreated
3rd generation lung cancer stem cell, 2 × 105A/mL and 0.1%BSA).The corresponding 500 μ L that are added contain respectively for room under the cells Transwell
The DMEM culture mediums of 10% fetal calf serum and stem cell conditioned medium containing 10% fetal calf serum.It sets in 37 DEG C of incubators, incubates
Educate 48h.It takes out the cells Transwell to be washed 2 times with PBS, cotton balls wipes the cell that upper surface is not shifted.With 0.5mL4% poly first
The fixed 20min of 4 DEG C of aldehyde, is added 0.1% violet staining 20min of 0.5mL, and PBS is washed 2 times, observed under inverted microscope.At random
5 400 times of visuals field are chosen, is shifted in the statistics visual field and indicates enzymolysis polypeptide to people's cellule lung with the relative number of invasion cell
The inhibiting effect of the transfer of cancer NCI-H446 cell line lung cancer stem cells, invasion.
Two, experimental result
1, the morphological feature of human small cell lung carcinoma NCI-H446 cell line cells and lung cancer stem cell
Human small cell lung carcinoma NCI-H446 cell line cells are in fusiformis adherent growth, and lung cancer stem cell presentation typical case is non-to stick
Three-dimensional globular, it is consistent with document report.
2, PE fluorescent markers CD133 antibody flow cytometries
The anti-human CD133 antibody Flow cytometries of mouse of PE fluorescent markers the result shows that, human small cell lung carcinoma NCI-
The CD133+ cell percentages of H446 cell line cells are (4.26 ± 2.41) %；Human small cell lung carcinoma NCI-H446 cell line lungs
The CD133+ cell percentages of cancer stem cell are (16.48 ± 4.96) %.The difference of CD133+ cell percentages has system between two groups
Meter learns meaning (P ＜ 0.05).The result shows that human small cell lung carcinoma NCI-H446 cell line lung cancer stem cell height expresses stem cell table
Face marker CD133+ has tumor stem cell characteristic, consistent with document report.
3, the comparison of human small cell lung carcinoma NCI-H446 cell line cells and lung cancer stem cell transfer and invasive ability
Transwell the result shows that, human small cell lung carcinoma NCI-H446 cell line lung cancer stem cells compare Small Cell Lung Cancer
NCI-H446 cell line cells have higher external transfer and invasive ability, are shown in Table 1.Statistically significant (the P of 2 group differences
＜ 0.05).
1 human small cell lung carcinoma NCI-H446 cell line cells of table and the transfer of lung cancer stem cell and invasive ability compare
4, influence of the enzymolysis polypeptide to the transfer of human small cell lung carcinoma NCI-H446 cell line lung cancer stem cells, invasion
Transwell the experimental results showed that, the enzymolysis polypeptide of various concentration is to human small cell lung carcinoma NCI-H446 cell lines
The external transfer of lung cancer stem cell and invasive ability have significant inhibiting effect, and compared with the control group, difference has statistics meaning
Adopted (P ＜ 0.05), the results are shown in Table 2 and Fig. 1.
The influence that 2 enzymolysis polypeptide of table shifts human small cell lung carcinoma NCI-H446 cell line lung cancer stem cells, invades
The above results prove that enzymolysis polypeptide can significantly inhibit human small cell lung carcinoma NCI-H446 cell line lung cancer stem cells
Transfer and invasion, and molecular weight ranges be 3000-5000u the enzymolysis polypeptide enzymolysis of 1000-3000u ranging from than molecular weight
The inhibiting effect that polypeptide shifts human small cell lung carcinoma NCI-H446 cell line lung cancer stem cells and invades is stronger.
The safety evaluatio of 3 enzymolysis polypeptide of embodiment
Kunming mouse is taken, weight 25-31g is random to be grouped, and does not inject using tail vein injection and enzymolysis polypeptide and carries out poison
Property contrast experiment research.The result shows that enzymolysis polypeptide 3 and enzymolysis polypeptide 4 continuously inject 45 under the up to dosage of 600mg/kg
It, without any side effects, the heart, liver, kidney, lung anatomic observation are without exception, and weight has increase.Experimental result is shown in Table 3.
3 enzymolysis polypeptide toxicity test of table
Above-described embodiment proves that enzymolysis polypeptide provided by the invention can effectively inhibit Small Cell Lung Cancer NCI-H446 cells
Being the invasion of lung cancer stem cell-like cell and transfer, toxicological experiment research has shown that enzymolysis polypeptide without obvious toxic-side effects.Tumor Stem is thin
Born of the same parents' theory thinks that the minute quantity tumor stem cell to lie dormant in most of tumour is the root of tumor recurrence and transfer, therefore,
Enzymolysis polypeptide provided by the invention is expected to the drug of metastases occur after developing into prevention Patients With Small Cell Carcinoma of The Lung chemotherapy.
Claims (2)
1. a kind of enzymolysis polypeptide is preparing the invasion of inhibition Small Cell Lung Cancer NCI-H446 cell line lung cancer stem cell-like cells and is turning
Application in the drug of shifting, which is characterized in that the enzymolysis polypeptide is prepared by the following method：
It is twisted into meat gruel after cicada monkey is cleaned, is placed in ultra-pure water, alkali protease enzymolysis is added, enzymatic hydrolysis condition is：PH value,
8.5；Hydrolysis temperature, 55 DEG C；Enzymolysis time, 10 hours；The additive amount of alkali protease is the 0.45% of substrate meat gruel weight；
After enzymolysis, heating makes basic protein 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；
It is respectively successively 10000u, 5000u, 3000u and 1000u with molecular cut off by the ultrapure water dissolution of freeze-dried powder
Ultrafiltration membrane detaches obtained polypeptide, collects the component of molecular cut off 5000u-3000u or 3000u-1000u, freezes
It does to obtain the final product.
2. application according to claim 1, which is characterized in that heating makes the method for basic protein enzyme-deactivating be：At 85 DEG C
Under the conditions of water-bath 10 minutes.
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| Cytotoxic activities of vearious fractions extracted from some pharmaceutical insect relatives.;Yao Ge Huang et.al;《Archives of pharmacal research》;19970430;1-15页 * |
| 蚱蝉若虫蛋白在食品及药品中的应用;王翻艳等;《陕西林业科技》;20121231;第100页左栏2 * |
| 蝉蛹蛋白质的提取及酶解物抗氧化活性测定;宋少华;《中国优秀硕士学位论文全文数据库》;20120915(第9期);第11页第2节，第40页3.5节,第53-54页3.3.2节和表1 * |
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