CN107970273A - It is a kind of to pass through Chinese medicinal formulae of NKG2D activated NKs and its preparation method and application - Google Patents
It is a kind of to pass through Chinese medicinal formulae of NKG2D activated NKs and its preparation method and application Download PDFInfo
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- CN107970273A CN107970273A CN201810072037.3A CN201810072037A CN107970273A CN 107970273 A CN107970273 A CN 107970273A CN 201810072037 A CN201810072037 A CN 201810072037A CN 107970273 A CN107970273 A CN 107970273A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
- A61K36/258—Panax (ginseng)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
- A61K36/076—Poria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- A61K36/284—Atractylodes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/484—Glycyrrhiza (licorice)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
Abstract
Following Chinese herb is included by Chinese medicinal formulae of NKG2D activated NKs and its preparation method and application, the Chinese medicinal formulae the invention discloses a kind of：Red ginseng, Poria cocos, Rhizoma Atractylodis Macrocephalae and honey-fried licorice root；The red ginseng, Poria cocos, the weight ratio of Rhizoma Atractylodis Macrocephalae and honey-fried licorice root are 2：3：3：1.The Chinese medicinal formulae of the present invention is by increasing NKG2D with activated NK, and after NK cells contact tumour cell, by activated receptor NKG2D tumor cells, the transduction of reactivity signal, produces cytotoxicity, and then killing tumor cell in active cell.
Description
Technical field
The present invention relates to a kind of Chinese medicinal formulae of activated NK, and in particular to a kind of by NKG2D activated NKs
Chinese medicinal formulae and its preparation method and application.
Background technology
Recently as society, economic development and aging of population, the incidence of the most of malignant tumours of China and death
Rate is in rising trend.Treatment to malignant tumour, at present based on surgery surgical radical treatment, chemotherapy or chemoradiotherapy,
Traditional Chinese medicine aids in western medicine.The remedy measures such as operation, chemotherapy or radiotherapy, while tumor tissues are killed, also to body
Normal cell causes the infringement that degree differs, including the suppression and killing to immunocyte.Although traditional Chinese medicine is thin to tumour
The direct killing effect of born of the same parents is not as good as chemotherapeutic, but it effectively reduces postoperative recurrence and transfer, alleviates radiotherapy, the poison pair of chemotherapy is made
With the effect of improving chemicotherapy, improving immunity, extending life cycle etc. has a clear superiority.Traditional Chinese medicine improves patient and exempts from
Although the phenomenon of epidemic disease function is tentatively confirmed, the mechanism wherein contained is still treated further to study.
Natural killer cells (Natural killer cell, abbreviation NK cell) is the important of body inherent immunity system
Effector cell, is the third-largest lymphocyte in addition to T, bone-marrow-derived lymphocyte, is responsible for body disease-resistant poison, antitumor and anti-heterologous
The important task of graft, this group of cell can in the case of from extraneous " sensitization " spontaneous kill body " being not intended to " it is abnormal thin
Born of the same parents, such as tumour cell, are the important compositions of cancer immunosurveillance.Studies have shown that primary carcinoma of liver postoperative patient, takes QI invigorating
Invigorating the spleen side can improve survival, reduce tumor recurrence rate, may be related with increased NK cell numbers in body.QI invigorating
Patients with gastric cancer after radical operation is treated by invigorating the spleenization product side, can significantly improve NK cell numbers, and immune factor interferon-γ (IFN-γ)
With the content of tumor necrosis factor-alpha (TNF-α), formulation of astragalus root can reverse the NK cell killing work(caused by tumour cell
It can suppress.
The NK cells only activated could spread, infiltrate antitumor action is played into tumor tissues.NK cell functions
Play, the signal for suppressing dependent on surface receptor transmission or activating, activated receptor includes：NKG2D in c-type agglutinin receptor,
CD226(Cluster of Differentiation 226)；Nature cell toxin receptor (NCR), wherein mainly have NKp46,
NKp44、NKp30；Cytokine receptor, such as IL-2R, IL-15R, IL-18R, IL-21.
NKG2D is one of most effective activated receptor of NK cell surfaces, can identify substantial amounts of ligand, NK cells contact tumour
After cell, by activated receptor NKG2D tumor cells, the transduction of reactivity signal in active cell, produces cell toxicant
Effect, and then killing tumor cell, form immunosurveillance in vivo.
The content of the invention
Pass through Chinese medicinal formulae of NKG2D activated NKs and preparation method thereof and use the object of the present invention is to provide a kind of
On the way, the Chinese medicinal formulae is by increasing NKG2D with activated NK, and then strengthens NK tumor cells, produces cytotoxicity,
And then killing tumor cell.
In order to achieve the above object, the present invention provides a kind of Chinese medicinal formulae by NKG2D activated NKs, the Chinese medicine
Prescription includes following Chinese herb：Red ginseng, Poria cocos, Rhizoma Atractylodis Macrocephalae and honey-fried licorice root；The red ginseng, Poria cocos, Rhizoma Atractylodis Macrocephalae and honey-fried licorice root
Weight ratio is 2：3：3：1.
The Chinese medicinal formulae includes following Chinese herb：6 grams of red ginseng, 9 grams of Poria cocos, 9 grams of Rhizoma Atractylodis Macrocephalae, 3 grams of honey-fried licorice root.
The Chinese medicinal formulae is soaked by the way that red ginseng, Poria cocos, Rhizoma Atractylodis Macrocephalae and honey-fried licorice root are added water, and heating and refluxing extraction, is carried
Liquid is taken, is filtered, decompression, is evaporated to medicinal extract, is dried under reduced pressure into dry powder and prepares.
The water adds 6 times of the weight that volume is Chinese herb.
The temperature of the heating is 90 DEG C.
Present invention also offers a kind of preparation method of the Chinese medicinal formulae by NKG2D activated NKs, this method includes：
Red ginseng, Poria cocos, Rhizoma Atractylodis Macrocephalae and honey-fried licorice root are added into water immersion, heating and refluxing extraction, obtains extracting solution, filters, and decompression, is evaporated to leaching
Cream, is dried under reduced pressure into dry powder, obtains Chinese medicinal formulae；The red ginseng, Poria cocos, the weight ratio of Rhizoma Atractylodis Macrocephalae and honey-fried licorice root are 2：3：3：1.
The water adds 6 times of the weight that volume is Chinese herb；The temperature of the heating is 90 DEG C.
The number of the heating and refluxing extraction is three times, extracting solution three times to be merged and is filtered together.
Present invention also offers a kind of purposes of the Chinese medicinal formulae described in basis in activated NK.
Present invention also offers a kind of purposes of the Chinese medicinal formulae described in basis in the medicine of immunotherapy of tumors is prepared.
Preferably, purposes of the Chinese medicinal formulae in the medicine for preparing colon cancer immunization therapy.NKG2D is that NK cell surfaces most have
One of activated receptor of effect, after identifying that substantial amounts of ligand, NK cells contact tumour cell, is known by activated receptor NKG2D
Other tumour cell, the transduction of reactivity signal in active cell, produces cytotoxicity, and then killing tumor cell, in vivo
Form immunosurveillance.
The tumour is colon cancer.
The present invention by Chinese medicinal formulae of NKG2D activated NKs and its preparation method and application, it is with following excellent
Point：
The Chinese medicinal formulae of the present invention after NK cells contact tumour cell, is passed through by increasing NKG2D with activated NK
Activated receptor NKG2D tumor cells, the transduction of reactivity signal in active cell, produces cytotoxicity, and then kills
Tumour cell, forms immunosurveillance in vivo；
The Chinese medicinal formulae of the present invention can significantly increase the NK cells of expression NKG2D, Ca2+Concentration and CD107a+Expression
It can increase, show NK cell activations, and with concentration according to lazyness；
The Chinese medicinal formulae of the present invention has obvious action to colon cancer, and it is thin to colon cancer LOVO to dramatically increase NK cells
The lethal effect of born of the same parents.
Brief description of the drawings
Fig. 1 is that the Chinese medicinal formulae of the present invention expresses people's primary NK cells in the influence figure of NKG2D.
Fig. 2 is the Chinese medicinal formulae of the present invention to people's primary NK cells intracellular Ca2+The influence figure of concentration.
Fig. 3 is the Chinese medicinal formulae of the present invention to CD107a in people's primary NK cells+The influence figure of NK cell number ratios.
Fig. 4 is that the Chinese medicinal formulae of the present invention kills people's primary NK cells in the influence figure of colon cancer cell.
Embodiment
Technical scheme is described further below in conjunction with drawings and examples.
A kind of Chinese medicinal formulae by NKG2D activated NKs, the Chinese medicinal formulae include following Chinese herb：Red ginseng,
Poria cocos, Rhizoma Atractylodis Macrocephalae and honey-fried licorice root, the red ginseng, Poria cocos, the weight ratio of Rhizoma Atractylodis Macrocephalae and honey-fried licorice root are 2：3：3：1.
The dosage of above-mentioned Chinese medicine preparation clinically is：6 grams of red ginseng, 9 grams of Poria cocos, 9 grams of Rhizoma Atractylodis Macrocephalae, 3 grams of honey-fried licorice root.
The concocting method of the Chinese medicinal formulae, it is specific as follows：
6 grams of red ginseng, 9 grams of Poria cocos, 9 grams of Rhizoma Atractylodis Macrocephalae, 3 grams of honey-fried licorice root are taken by above-mentioned parts by weight, adds the water immersion of 6 times of volumes,
Refluxing extraction, 90 DEG C are heated to reflux 3 times, the 1st refluxing extraction 2h, the 2nd time and the 3rd time, each refluxing extraction 1h.Merge 3 times
Extracting solution, is filtered, and decompression, is evaporated to medicinal extract, is dried under reduced pressure into dry powder, every 1 gram of crude drug obtains 0.32 gram of extract.This preparation side
The Chinese medicinal formulae that method obtains can be with activated NK.
By the Chinese medicinal formulae of above-mentioned preparation, quantitatively weigh, ultraviolet radiation sterilization, sterile distilled water dissolving, 4 DEG C of refrigerators are protected
Deposit it is spare, to carry out following related Chinese medicinal formulae effect experiments.
1st, the influence that Chinese medicinal formulae activates NK cell receptors
By people's primary NK cells according to 10 × 104The cell of a/mL concentration, per hole 2mL, is inoculated in 6 orifice plates, is put into 37
DEG C, 5%CO2After cultivating 24h in incubator, SJZ (for the numbering of the Chinese medicinal formulae of the present invention) (0.5mg/ of various concentrations is added
ML, 1mg/mL, 1.5mg/mL, 2mg/mL, 2.5mg/mL), after acting on 24h, 0.25% Trypsin Induced collects cell,
1000rpm centrifuges 5min, abandons culture medium, adds 1mL PBS featheriness, centrifuges 5min under 1000rpm rotating speeds, use NK cell tables
Face activated receptor FITC-NKG2D antibody (life technologies, article No.：521927) FCM Flow cytometries, are passed through
The expression of NK cell surface activation acceptors NKG2D.
As shown in Figure 1, for Chinese medicinal formulae of the invention people's primary NK cells are expressed with the influence figure of NKG2D, CTRL in figure
Control group is represented, that is, is added and physiological saline isometric SJZ.It will be seen from figure 1 that Chinese medicinal formulae energy activated receptor NKG2D,
And with the increase of Chinese medicinal formulae concentration, facilitation is more notable, and under Chinese medicinal formulae 2.5mg/mL concentration, the primary NK of people is thin
The cell of cellular expression NKG2D accounts for 41.86%, is 3.46 times of control group.
2nd, Chinese medicinal formulae is to NK cell intracellulars Ca2+The influence of concentration
By people's primary NK cells according to 10 × 104The cell of a/mL concentration, per hole 2mL, is inoculated in 6 orifice plates, 37 DEG C,
5%CO2In incubator cultivate 24h after, add various concentrations SJZ (0.5mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL,
2.5mg/mL), after acting on 24h, 0.25% Trypsin Induced collects cell, and 1000rpm centrifugation 5min, abandon culture medium, add
1mL PBS featheriness, 5min is centrifuged under 1000rpm rotating speeds, uses Fluo-4 (Invitrogen, article No.：F14200), pass through
Ca intracellular FCM Flow cytometries NK2+Concentration.
As shown in Fig. 2, be the present invention Chinese medicinal formulae to people's primary NK cells intracellular Ca2+The influence figure of concentration, in figure
CTRL represents control group, that is, adds and physiological saline isometric SJZ, positive controls of the IL2 as NK cell activations.From Fig. 2
As can be seen that Chinese medicinal formulae can cause people's primary NK cells intracellular Ca2+Concentration increase, and with the increasing of Chinese medicinal formulae concentration
Add, promote Ca2+ influx effect more notable, to indicate NK cell activations.
3rd, influence of the Chinese medicinal formulae to NK cells CD107a
By people's primary NK cells according to 10 × 104The cell of a/mL concentration, per hole 2mL, is inoculated in 6 orifice plates, is put into 37
DEG C, 5%CO2In incubator cultivate 24h after, add various concentrations SJZ (0.5mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL,
2.5mg/mL), after acting on 24h, 0.25% Trypsin Induced collects cell, and 1000rpm centrifugation 5min, abandon culture medium, add
1mL PBS (phosphate buffer saline, phosphate buffered saline solution) featheriness, 5min is centrifuged under 1000rpm rotating speeds,
Use NK cell surface markers PE (Phycoerythrin, phycoerythrin)-CD107a antibody (eBioscience, article No.：
E14570-105), NK cell surface markers CD107a is detected by flow cytometry (Flow Cytometry, FCM)+Expression
Situation, to indicate NK cell activation situations.
As shown in figure 3, be the present invention Chinese medicinal formulae to CD107a in people's primary NK cells+The shadow of NK cell number ratios
Figure is rung, CTRL represents control group in figure, that is, adds and physiological saline isometric SJZ.The NK of CD107a molecule positive expressions is thin
Born of the same parents can represent the NK cells with killing activity, from figure 3, it can be seen that CD107a+NK cells have concentration dependent to SJZ,
With the increase of concentration, CD107a+NK cell proportions are being continuously increased, under Chinese medicinal formulae 2.5mg/mL concentration, facilitation
Most significantly, CD107a+Expression from the 7.9% of control group increase be 81.31%.
4th, the influence of Chinese medicinal formulae NK cell killings colon cancer cell
NK cells are detected to colon carcinoma cell line LOVO killing activities, NK cells using co-culture system：LOVO cells (1
×105/ mL) ratio be 10:1, take NK cells to be added to each 100 μ L of LOVO cells in 96 orifice plates；Add SJZ (2.5mg/
ML), after acting on 24h, the NK cells of suspension are sucked, add 100 μ L PBS (phosphate buffer saline, phosphoric acid buffers
Salting liquid) wash LOVO cells once, 10 μ L CCK8 (MedChemExpress, article No.s are added per hole：HY-K0301, for surveying
Determine number of viable cells in cell Proliferation or toxicity test), 37 DEG C culture 2 it is small when after, 490nm absorbance detections are each in microplate reader
Hole absorbance (OD values), judges ability of cell proliferation according to OD values.
As shown in figure 4, for Chinese medicinal formulae of the invention people's primary NK cells are killed with the influence figure of colon cancer cell, in figure
CTRL represents control group, that is, adds and physiological saline isometric SJZ.It is from fig. 4, it can be seen that dense in Chinese medicinal formulae 2.5mg/mL
Under degree, lethal effect of the NK cells to colon cancer LOVO cells can be dramatically increased, the LOVO Leukopenias of survival to control group
58%.
In conclusion the present invention by Chinese medicinal formulae of NKG2D activated NKs and its preparation method and application, in this
Prescription agent strengthens NK tumor cells by increasing NKG2D with activated NK, produces cytotoxicity, and then kill
Hinder tumour cell.
Although present disclosure is discussed in detail by above preferred embodiment, but it should be appreciated that above-mentioned
Description is not considered as limitation of the present invention.After those skilled in the art have read the above, for the present invention's
A variety of modifications and substitutions all will be apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Claims (10)
1. a kind of Chinese medicinal formulae by NKG2D activated NKs, it is characterised in that the Chinese medicinal formulae includes following Chinese medicine former material
Material：Red ginseng, Poria cocos, Rhizoma Atractylodis Macrocephalae and honey-fried licorice root；The red ginseng, Poria cocos, the weight ratio of Rhizoma Atractylodis Macrocephalae and honey-fried licorice root are 2：3：3：1.
2. the Chinese medicinal formulae according to claim 1 by NKG2D activated NKs, it is characterised in that the Chinese medicinal formulae
Include following Chinese herb：6 grams of red ginseng, 9 grams of Poria cocos, 9 grams of Rhizoma Atractylodis Macrocephalae, 3 grams of honey-fried licorice root.
3. the Chinese medicinal formulae according to claim 2 by NKG2D activated NKs, it is characterised in that the Chinese medicinal formulae
Soaked by the way that red ginseng, Poria cocos, Rhizoma Atractylodis Macrocephalae and honey-fried licorice root are added water, heating and refluxing extraction, obtains extracting solution, filters, and depressurizes, evaporation
To medicinal extract, it is dried under reduced pressure into dry powder and prepares.
4. the Chinese medicinal formulae according to claim 3 by NKG2D activated NKs, it is characterised in that the water adds
Enter 6 times of the weight that volume is Chinese herb.
5. the Chinese medicinal formulae according to claim 4 by NKG2D activated NKs, it is characterised in that the heating
Temperature be 90 DEG C.
6. the preparation method of a kind of Chinese medicinal formulae by NKG2D activated NKs, it is characterised in that this method includes：Will be red
Ginseng, Poria cocos, Rhizoma Atractylodis Macrocephalae and honey-fried licorice root add water immersion, and heating and refluxing extraction, obtains extracting solution, filter, and decompression, is evaporated to medicinal extract,
Dry powder is dried under reduced pressure into, obtains Chinese medicinal formulae；
The red ginseng, Poria cocos, the weight ratio of Rhizoma Atractylodis Macrocephalae and honey-fried licorice root are 2：3：3：1.
7. the preparation method of the Chinese medicinal formulae according to claim 6 by NKG2D activated NKs, it is characterised in that
The water adds 6 times of the weight that volume is Chinese herb；The temperature of the heating is 90 DEG C.
A kind of 8. purposes of the Chinese medicinal formulae in 1-5 according to claim described in any one in activated NK.
A kind of 9. use of the Chinese medicinal formulae in the medicine for preparing treatment tumour in 1-5 according to claim described in any one
On the way.
10. purposes of the Chinese medicinal formulae according to claim 9 in the medicine for preparing treatment tumour, it is characterised in that institute
The tumour stated is colon cancer.
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Citations (5)
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|---|---|---|---|---|
| CN102370801A (en) * | 2011-10-20 | 2012-03-14 | 颜景臣 | Medicine for treating stomach cancers |
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| CN103041312A (en) * | 2013-01-10 | 2013-04-17 | 南京海昌中药集团有限公司 | Traditional Chinese medicine composition with antineoplastic effect and preparation method and application thereof |
| CN103656570A (en) * | 2013-11-26 | 2014-03-26 | 青岛安信医疗器械有限公司 | Chinese medicine liquid for treating stomach tumor and preparation method thereof |
| CN107303384A (en) * | 2016-04-21 | 2017-10-31 | 张东 | It is a kind of to have medicable Chinese medicine composition and its derivative and product to a variety of symptoms |
-
2018
- 2018-01-25 CN CN201810072037.3A patent/CN107970273A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102370801A (en) * | 2011-10-20 | 2012-03-14 | 颜景臣 | Medicine for treating stomach cancers |
| CN102552524A (en) * | 2012-01-11 | 2012-07-11 | 上海中医药大学附属普陀医院 | Traditional Chinese medicine composition for inhibiting residual tumor focus as well as preparation method and application thereof |
| CN103041312A (en) * | 2013-01-10 | 2013-04-17 | 南京海昌中药集团有限公司 | Traditional Chinese medicine composition with antineoplastic effect and preparation method and application thereof |
| CN103656570A (en) * | 2013-11-26 | 2014-03-26 | 青岛安信医疗器械有限公司 | Chinese medicine liquid for treating stomach tumor and preparation method thereof |
| CN107303384A (en) * | 2016-04-21 | 2017-10-31 | 张东 | It is a kind of to have medicable Chinese medicine composition and its derivative and product to a variety of symptoms |
Non-Patent Citations (1)
| Title |
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| 杜锦芳等: "十全大补汤对结肠癌原发瘤切除后转移瘤生长及NK细胞的影响", 《上海中医药杂志》 * |
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