CN109847061A - It is a kind of for low temperature plasma activating fluid preparation after storage method - Google Patents
It is a kind of for low temperature plasma activating fluid preparation after storage method Download PDFInfo
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- CN109847061A CN109847061A CN201910208648.0A CN201910208648A CN109847061A CN 109847061 A CN109847061 A CN 109847061A CN 201910208648 A CN201910208648 A CN 201910208648A CN 109847061 A CN109847061 A CN 109847061A
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Abstract
The invention belongs to the preparation of low-temperature plasma activating fluid and technical field of memory, a kind of preparation method and preparation facilities for low temperature plasma activating fluid is disclosed, handling common cell culture complete medium preparation using air source low-temperature plasma device has treatment basal-cell carcinoma, squamous carcinoma, the plasma activating fluid of melanoma ability.The present invention obtains PAL using dielectric barrier discharge device (DBD) processing cell complete culture solution (DMEM), and the rodent ulcer of culture, squamous carcinoma, melanoma cells are handled in vitro after (20 DEG C, 4 DEG C, -20 DEG C) at different temperatures store same time (72h) using PAL obtained, demonstrating rear PAL inhibits the activity of malignant tumour of skin cell to have apparent temperature dependency, it was found that PAL suitable storage temperature.
Description
Technical field
The invention belongs to low temperature plasma activating fluid (plasma activated liquid, PAL) preparation and storage skills
Art field, more particularly to one kind are used for PAL system for after, to the requirement of temperature in storage.
Background technique
Currently, the prior art commonly used in the trade is such that
Low temperature plasma activating fluid development course
Lower temperature plasma technology has gradually been grown up since nearest 10 years, is produced in life science and application field
The new discipline of raw tremendous influence.Low temperature plasma refers to operating temperature at 40 DEG C hereinafter, with traditional high-energy plasma
Body is different, to the effect of biological tissue independent of high-energy, and is to rely on the active material of its generation.Research has been sent out
The existing low temperature plasma property of can choose acts on the specialized hospitals such as bacterium, fungi, virus microorganism；Accelerate blood coagulation and the surface of a wound is promoted to be cured
It closes；And the property of can choose inhibit the hyperplasia of malignant cell.Recently the liquid that research discovery is activated by low temperature plasma
Body, i.e. PAL also have the activity similar with plasma.Since PAL can overcome direct plasma to be merely capable of acting on
The drawbacks of organism surface, the application in future medicine field will more attract attention.But up to the present, low temperature etc. from
The molecular mechanism of daughter and PAL selective depression tumor cell proliferation is still indefinite, and research shows that different substrate liquids (are such as gone
Ionized water, physiological saline, phosphate buffer) or medium (blood plasma, tissue fluid, inoculum etc.) generate PAL life
Object activity has differences.
The storage problem of low temperature plasma activating fluid
Low temperature plasma is mainly used for organism or surgical instrument surface sterilization and sterilizing, promotes blood coagulation, thin to tumour
Born of the same parents' selective inhibitory and gradually be applied to clinical tumor treatment.By low-temperature plasma device (such as common DBD dress
Set) liquid crossed for the treatment of with irradiation, such as: phosphate buffer (PBS), physiological saline and cell culture medium (DMEM) liquid, i.e.,
PAL has same antitumous effect as direct low-temperature plasma jet.The a large amount of active nitrogen oxygen wherein generated
Substance has played main function, and compared to the direct processing mode of plasma device, PAL easily facilitates future and is applied even more extensively
In clinical medicine.But at present for PAL on storing mode, including storage temperature and storage time length etc. determine future
Still lack enough research in some key technologies of clinical application.
Problem of the existing technology is:
PAL whether can because of storage temperature difference and show different antitumous effects.The storage of usual drug
It needs to pay close attention to environment temperature, or even needs to save under certain temperature conditions.And depositing about plasma activating fluid
Storing temperature is at present still without clear parameter.
(2) low-temperature plasma device acts on different substrates, such as PBS, DMEM or deionized water or physiological saline etc.,
Whether its activity can have differences.
(3) whether storage time has an impact PAL activity.PAL storage time has a major impact future clinical application,
The ability for not only needing clear PAL that can guarantee maximum suppression tumor cell proliferation under the conditions of which kind of temperature, it is also necessary to clear
These PAL can save the activity for how long still having anti-tumour cell proliferative.
Solve the difficulty and solution of the invention of above-mentioned technical problem:
(1) PAL future is widely applied in clinic, and storage is also similar to that drug, needs specific environment temperature.How
Solving PAL storage temperature condition is to influence one of the key link of its following clinical application.The variation range of environment temperature compares
Greatly, for it is clear whether temperature PAL storage in its anti-tumor activity is had an impact, the present invention select 3 it is most representative
Storage temperature (20 DEG C, 4 DEG C, -20 DEG C) is analyzed as target storage temperature.
PAL future clinical be widely applied also must clear storage time on its active influence.Whether PAL is with storage
Whether the extension of time, activity can also change, up to the present these problems are not also come to a conclusion clearly.To avoid storing
Time, the present invention selected the PAL obtained after DBD device processing DMEM90s different temperatures in above-mentioned 3 on PAL active influence
72h is stored under condition (20 DEG C, 4 DEG C, -20 DEG C), then horizontal to same room temperature in 26 DEG C of ambient temperature conditions rewarming.In this base
The effect disquisition that PAL inhibits tumour cell is carried out on plinth.
(1) present invention selects 3 kinds of common skin malignant cells as representing, and carries out the effect that PAL inhibits tumour cell
Fruit research.Because skin neoplasin betides the outermost layer of human body tissue, it is easiest to realize the treatment of PAL direct injection.
Solve the meaning of above-mentioned technical problem:
The same, storage of the different environment temperatures to PAL is required similar to condition of the drug to storage condition temperature and humidity
Deposit and transport all generation significant impacts.Meaning of the present invention is the extensive use for PAL on future clinical, stores temperature to it
It spends and the influence of tumor cell proliferation is being inhibited to establish necessary standard.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of preparation methods and storage temperature for PAL.This
It is right after same time stores under different ambient temperature conditions after invention low temperature plasma active cell culture solution
The influence of the inhibitory effects on proliferation of Skin Tumor Cells.Specific PAL is respectively by storing 72h under the conditions of 20 DEG C, 4 DEG C, -20 DEG C
Afterwards, the source of people basal-cell carcinoma (TE354T) of in vitro culture, squamous carcinoma (A431) and melanoma (A375) cell are showed respectively
The inhibitory effect gradually increased out.
The invention is realized in this way a kind of preparation method for PAL includes:
Using cell culture complete medium (DMEM) as substrate, DMEM is handled using low-temperature plasma device (DBD).It connects
Behind energization source, DBD device launches plasma jet, sterile 30,60,90,120 and of DMEM of plasma jet processing
After 180s, PAL is obtained；And it is respectively placed in and stores 72h under 20 DEG C, 4 DEG C, -20 DEG C of environmental conditions.
Further, the PAL of same time (72h) is stored under 20 DEG C, 4 DEG C, -20 DEG C of environmental conditions respectively respectively to substrate
Cell cancer (TE354T), squamous carcinoma (A431) and melanoma (A375) are handled, processing method are as follows: above-mentioned difference storage temperature
Under PAL under conditions of environment temperature is 26 DEG C rewarming, take after rewarming target cell that above-mentioned each 2ml of PAL is added in constant temperature respectively
2h is cultivated in case, is then siphoned away, and is added the not plasma-treated DMEM culture medium of 2ml and is cultivated for 24 hours in insulating box.
Treated target cell carries out the detection of mtt assay cell viability and apoptosis detection, whether analyzes PAL with storage
Whether the difference of temperature, antitumous effect symbolize otherness.
As a result, it has been found that in the case where storage time is equal to situation, 3 kinds of representative storage temperature (20 DEG C, 4 provided by the invention
DEG C, -20 DEG C) in, it is -20 DEG C that PAL, which inhibits the Optimum storage temperature of tumour cell, prompts the experiment condition that provides in this patent
Under, -20 DEG C of storage temperature is optimal PAL storage temperature.
Another object of the present invention is to provide the standby generating device of PAL system, described device packet are used for described in a kind of implementation
It includes: direct current high voltage pulses power supply, copper rod array and two steady resistances.
Copper rod array forms and connects direct current high voltage pulses by more copper rods, and outside is wrapped by insulation crust, internal parallel
Several professional thin copper bars are placed as lattice electrode and connect direct current high voltage pulses power supply, two battle array leakage resistance limitation electric currents make to generate
Plasma temperature at 20 DEG C~30 DEG C.
In conclusion advantages of the present invention and good effect are as follows:
1. conclusion derives from careful research
For inquire into different storage temperature whether can the proliferative capacity of inhibition tumour cell to PAL have an impact, this hair
It is bright using plasma device (DBD) processing DMEM (30,60,90,120,180) s after, the PAL of acquisition be separately stored in 20 DEG C,
Under the conditions of 4 DEG C, -20 DEG C, after 72h again under 26 DEG C of environmental conditions natural rewarming to environment temperature.Reuse 26 DEG C it is above-mentioned swash
Liquid living is respectively to common skin neoplasms such as human skin basal-cell carcinoma (TE354T), squamous carcinoma (A431) and melanomas (A375)
Cell is handled, to obtain the storage temperature that best PAL inhibits tumor cell proliferation.
The present invention is stored after same time to skin kinds of tumor using the PAL of preparation under the conditions of 20 DEG C, 4 DEG C, -20 DEG C
Having a certain difference property of Cell killing efficacy.
The present invention inhibits the effect of tumor cell proliferation to have apparent temperature difference using the DMEM activating fluid of the preparation
It is anisotropic.In the case where storage time is equal to situation, the PAL under -20 DEG C of conditions of storage inhibits activity of tumor cells most strong.
Have the effect a large number of studies show that the selective inhibition tumour cell confirmation of PAL at present, and to normal cell
Influence is smaller, therefore it has very big potentiality in following clinically using.Specifying storage temperature can on the active influence of PAL
It lays a solid foundation for it in following extensive utilization clinically.
Creative utilization air-source low-temperature plasma device (DBD) of the present invention handles common cell culture medium DMEM preparation
PAL with treatment basal-cell carcinoma (TE354T), squamous carcinoma (A431), melanoma (A375) ability.Low temperature plasma does not have
There is specific dosage unit, uses the processing time as Dose standard at present in the world.The present invention handles DMEM using CAP device
Complete medium 90s is obtained activating fluid and is stored up using activating fluid obtained by (20 DEG C, 4 DEG C, -20 DEG C) at different temperatures
Basal-cell carcinoma (TE354T), squamous carcinoma (A431), melanoma (A375) are handled after depositing same time, specify PAL
The lower preservation for inhibiting proliferative activity o f tumor to it of storage temperature it is more advantageous.
Detailed description of the invention
Fig. 1 is the preparation method flow chart provided in an embodiment of the present invention for PAL.
Fig. 2 mtt assay detects the activity that PAL inhibits malignant tumour of skin cell.It is different storages provided in an embodiment of the present invention
Deposit the effect picture of the PAL inhibition malignant tumour of skin cell Proliferation under the conditions of temperature.
Fig. 3 Flow Cytometry detects PAL induced skin malignant tumor cell apoptosis.Be it is provided in an embodiment of the present invention not
With the PAL inducing malignant tumor cell apoptosis under the conditions of storage temperature, (target cell used herein is basal-cell carcinoma and squama
Cancer) effect picture.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
PAL whether can because of storage temperature difference and show different antitumous effects, the prior art lacks clinical
Some significant datas of application.
To solve the above problems, below with reference to concrete scheme, the present invention is described in detail.
As shown in Figure 1, the preparation method provided in an embodiment of the present invention for PAL is for basal-cell carcinoma
(TE354T), the preparation for the PAL that squamous carcinoma (A431), melanoma (A375) are treated and storage method.
For low temperature plasma activating fluid the preparation method comprises the following steps: using DMEM complete medium as substrate matchmaker described in 1.
It is situated between, handles DMEM using air source low-temperature plasma device (DBD), obtain DMEM activating fluid.Specially low temperature plasma
After device powers on, CAP device launches plasma jet, and plasma jet handles sterile DMEM respectively and cultivates completely
After 30,60,90,120 and 180s of base, obtain low temperature plasma activating fluid (PAL), respectively environment temperature be 20 DEG C, 4 DEG C ,-
72h is stored under 20 DEG C of environmental conditions.
2. by (20 DEG C, 4 DEG C, -20 DEG C) at different temperatures storage same time, (this research is using storage time
Low temperature plasma activating fluid (CAP-DMEM) 72h) is respectively to basal-cell carcinoma (TE354T), squamous carcinoma (A431), melanin
Tumor (A375) is handled, processing method are as follows: the condition that the PAL in above-mentioned 3 under different storage temperatures is 26 DEG C in environment temperature
Lower rewarming takes above-mentioned 3 kinds of common skins pernicious respectively when the PAL under the conditions of above-mentioned 3 kinds different storage temperatures is restored to 26 DEG C
Tumour cell is added 2mlPAL and cultivates 2h in insulating box, then siphons away PAL, adds 2ml DMEM complete medium in constant temperature
It is cultivated for 24 hours in case.Treated cell carries out MTT viability examination, with clear PAL whether with storage temperature difference, resist
Tumor effect can also symbolize otherness, i.e. temperature dependency.See Fig. 2 and Fig. 3.
The invention will be further described combined with specific embodiments below.
The embodiment of the present invention uses cell culture complete medium (dulbecco's modified eagle medium, DMEM)
To prepare PAL.DMEM is a kind of culture medium containing a variety of amino acid and glucose, be widely used in production of vaccine and it is various just
Cell culture and single cell culture for virus host cells.
PAL system Preparation Method provided in an embodiment of the present invention for malignant tumour of skin cell therapy, is trained completely with DMEM
Base is supported as substrate medium, DMEM is activated using DBD, to obtain PAL.And utilize PAL obtained at different temperatures (20
DEG C, 4 DEG C, -20 DEG C) storage same time (72h) after to basal-cell carcinoma (TE354T), squamous carcinoma (A431), melanoma
(A375) it is handled, specifying PAL has apparent temperature dependency to inhibiting tumour cells effect.
Application of the invention is further described below with reference to concrete analysis.
In as shown in Figure 1, it is that air-source low-temperature plasma device is made of three parts that the present invention, which prepares basis: straight
Flow high-voltage pulse power source, copper rod array and steady resistance.Copper rod array is the device electrode part, by the copper of 105 diameter 1mm
Stick forms and connects direct current high voltage pulses, outside wrapped by insulation crust, internal parallel places several professional thin copper bar (diameters
Direct current high voltage pulses power supply is connected as lattice electrode for 1mm), battle array leakage resistance limitation electric current makes the plasma temperature generated control
At 20 DEG C~30 DEG C or so.
After powering on, the sterile DMEM complete medium 90s of DBD jet stream processing can be obtained PAL, then by preparation
PAL stores 72h, then the rewarming in 26 DEG C of water-baths under 20 DEG C, 4 DEG C, -20 DEG C of ambient temperature conditions respectively, after rewarming
PAL respectively to the tumour cells such as human skin basal-cell carcinoma (TE354T), squamous carcinoma (A431) and melanoma (A375) into
Row processing, then carries out Activity determination to processed cell, to obtain optimal storage temperature condition.
PAL under the conditions of Fig. 2 difference storage temperature inhibits the effect of malignant tumour of skin cell Proliferation.
Wherein abscissa represents the storage temperature of PAL (CAP-DMEM), and ordinate represents cell survival rate.CO is control
Group is that sterile DMEM complete medium carries out above-mentioned processing to four kinds of cells immediately after low temperature plasma irradiates 90s.
As the result is shown with the raising of plasma activating fluid storage temperature, the survival rate of four kinds of cells is gradually increasing, shows MTT
PAL to the fragmentation effect of cell is gradually decreased as storage temperature increases.
The present invention irradiates sterile DMEM complete medium 90s acquisition PAL using plasma beam (DBD device) in experiment,
Then 72h is stored under the conditions of 20 DEG C, 4 DEG C, -20 DEG C, the PAL after storing 72h is after rewarming respectively to basal-cell carcinoma
(TE354T), squamous carcinoma (A431), melanoma (A375) are handled, while carrying out phase to Human keratinocytes (Hacat)
Normal cell controls are used as with processing.Processing method are as follows: the PAL after 2ml rewarming is added cultivates 2h in insulating box, then siphons away
2mlDMEM complete medium is added to cultivate in insulating box for 24 hours, MTT viability examination finally is carried out to treated cell.
As a result the activity of visible gradually decreasing with PAL storage temperature, MTT three kinds of tumour cells as the result is shown is gradually
Rise, shows PAL to three kinds of malignant tumour of skin cells (basal-cell carcinoma (TE354T), squamous carcinoma (A431) and melanoma
(A375)) fragmentation effect gradually weakens, it was demonstrated that PAL has apparent temperature dependency to storage temperature, in this experiment
PAL under the 3 kinds of different temperatures provided save has different inhibition activity of tumor cells abilities, under -20 DEG C of conditions preservations
PAL inhibits the activity of tumour cell most strong.
Further to confirm the above results, this team uses Flow Cytometry detection PAL to inhibit tumor cell proliferation again
Ability.Using basal-cell carcinoma and squamous cell carcinoma as target cell.
Fig. 3 is basal-cell carcinoma (TE354T) and squamous cell carcinoma (A431) streaming apoptosis testing result.
Wherein abscissa represents the different storage temperature of PAL, and ordinate represents apoptosis rate.Control group is sterile DMEM
Complete medium carries out above-mentioned processing to tumour cell immediately after low temperature plasma irradiates 90s.As the result is shown with storage
Deposit the raising of temperature, the apoptosis rate of two kinds of tumour cells is gradually reduced, show PAL to the fragmentation effect of tumour cell with
The raising of storage temperature and gradually weaken.
The processing mode of two kinds of cells is identical as above-mentioned processing step, and control group is sterile DMEM complete medium through too low
Tumour cell is handled immediately after isothermal plasma irradiation 90s.As a result visible gradually rising with PAL storage temperature,
The apoptosis rate of two kinds of tumour cells is gradually reduced, and shows that PAL is with storage temperature liter to the fragmentation effect of tumour cell
It is high and be gradually reduced.Plasma activating fluid in the case where 3 kinds of different temperatures save has different inhibition activity of tumor cells
Ability, and the result shows that the PAL under -20 DEG C of conditions save inhibits the ability of growth of tumour cell most strong.Demonstrate PAL activity guarantor
Depositing has apparent correlation to storage temperature, and storage temperature is lower, is more conducive to PAL and inhibits tumor cell proliferation biological activity
Preservation.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (5)
1. a kind of storage method for low temperature plasma activating fluid, which is characterized in that described to swash for low temperature plasma
The preparation of liquid living and storage method include:
Using cell culture complete medium DMEM as substrate, DMEM is handled using low-temperature plasma device DBD, is powered on
Afterwards, DBD device launches plasma jet, after the sterile DMEM30s~180s of plasma jet processing, obtain low temperature etc. from
Daughter activating fluid PAL；And it is respectively placed in and stores 72h under 20 DEG C, 4 DEG C, -20 DEG C of environmental conditions.
2. being used for the preparation method of PAL as described in claim 1, which is characterized in that the sterile DMEM of plasma jet processing
After 90s, low temperature plasma activating fluid PAL is obtained.
3. being used for the preparation method of PAL as described in claim 1, which is characterized in that respectively in 20 DEG C, 4 DEG C, -20 DEG C of environment
Under the conditions of store the low temperature plasma activating fluid of identical 72h respectively to basal-cell carcinoma (TE354T), squamous carcinoma (A431) and black
Melanoma (A375) is handled；Processing method are as follows: the condition that the PAL under above-mentioned difference storage temperature is 26 DEG C in environment temperature
Lower rewarming when the PAL under the conditions of different storage temperatures is restored to 26 DEG C, takes malignant tumour of skin cell to be added above-mentioned respectively
Each 2ml of PAL cultivates 2h in insulating box, then siphons away PAL, adds the not plasma-treated DMEM culture medium of 2ml and exists
It is cultivated for 24 hours in insulating box.
4. being used for the preparation method of low temperature plasma activating fluid as described in claim 1, which is characterized in that in storage time
In equivalent situation, the Optimum storage temperature of the inhibition tumour cell of plasma activating fluid is -20 DEG C.
5. a kind of preparation facilities implemented described in claim 1 for the preparation method of low temperature plasma activating fluid, feature
It is, the preparation facilities includes: direct current high voltage pulses power supply, copper rod array and two steady resistances.
Copper rod array forms and connects direct current high voltage pulses by more copper rods, and outside is wrapped by insulation crust, and internal parallel is placed
Several profession thin copper bars connect direct current high voltage pulses power supply as lattice electrode, two battle array leakage resistances limitation electric currents make generation etc.
Ion temperature is at 20 DEG C~30 DEG C.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113521282A (en) * | 2021-07-12 | 2021-10-22 | 安徽医科大学第二附属医院 | Preparation method of low-temperature plasma CAP activating solution for hemangioma treatment |
-
2019
- 2019-03-19 CN CN201910208648.0A patent/CN109847061A/en active Pending
Non-Patent Citations (3)
| Title |
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| JIN SHEN等: ""Bactericidal Effects against S. aureus and Physicochemical Properties of Plasma Activated Water stored at different temperatures"", 《SCIENTIFIC REPORTS》 * |
| NAGENDRA KUMAR KAUSHIK等: ""Biological and medical application of plasma-activated media, water and solutions"", 《BIOLOGICAL CHEMISTRY》 * |
| 项良剑: ""低温常压等离子体对不同亚型乳腺癌细胞的影响及机理研究"", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113521282A (en) * | 2021-07-12 | 2021-10-22 | 安徽医科大学第二附属医院 | Preparation method of low-temperature plasma CAP activating solution for hemangioma treatment |
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