CN106939298A - A kind of full tumour antigen preparation method of glioma and device - Google Patents
A kind of full tumour antigen preparation method of glioma and device Download PDFInfo
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Abstract
The present invention relates to a kind of full tumour antigen preparation method of glioma and device, comprise the following steps：The Multiplying culture carried out using serum-free medium to the glioma stem cell and glioma non-stem cell；The glioma stem cell of exponential phase and glioma non-stem cell are pre-processed；Pretreated glioma stem cell and glioma non-stem cell are mixed and are positioned in lysis process chamber one horizontal pulse cracking and synchronous centrifugal treating；Supernatant is abandoned, the glioma stem cell and glioma non-stem cell mixed liquor after being handled by more than, which are positioned in lysis process chamber two, carries out the cracking of pulse again, that is, harvests the full tumour antigen of glioma.The present invention uses low frequency and high frequency steep-sided pulse realizes glioma stem cell and glioma non-stem cell treatment by stages and cracking, simple to operate, and cracking more fully, quickly, can obtain more complete glioma tumour antigen.
Description
Technical field
The invention mainly relates to glioma field, more particularly to a kind of full tumour antigen preparation method of glioma
And device.
Background technology
Glioma is derived from neurepithelial tumour, is the most common malignant tumour of encephalic, accounts for whole encephalics
The 40-50% of tumour, problem as oncotherapy high with incidence of disease height, high recurrence rate, fatal rate.Traditional therapy master
If performing the operation with chemicotherapy to improve the existence probability of patient.But the glioma positioned at critical function area is difficult to accomplish to cut entirely
Remove.Radiotherapy is almost the conventional therapy of various glioma, but therapeutic evaluation differs, except medulloblastoma is highly quick to radiotherapy
Outside sense, ependymoma medium sensitivity, other types are insensitive to radiotherapy.Chemotherapeutics is limited to blood-brain barrier and the poison of medicine is secondary
Effect, curative effect is not affirmed still.In recent years, immunization therapy turns into glioma after effective treatment means after operation, chemicotherapy.With
Other modes use in conjunction, specifically there is very strong complementation, and the immune system being damaged to patient can play recovery with rebuilding
Unique curative effect, so as to further prevent the recurrence and transfer of tumour.
Glioma antigen is obtained by carrying out cracking after Multiplying culture to glioma stem cell in the prior art,
Because tradition cracking is main using methods such as multigelation or radiation, cracking is caused can not exclusively to obtain holoantigen information, and
And there is certain microorganism pollution, and animal sources immunogenicity in nutrient solution, therefore cause the glioma obtained to be done
Antigen Stability prepared by cell is poor, and specificity is weaker, so that the therapeutic epidemic disease of the glioma obtained by the antigen
Seedling has that stability is poor, specificity is weaker, kill the defects such as ratio of outflow is not high and immunogenicity is weak.
Cell membrane can preferably hinder the transmission of ion and hydrophilic molecules under normal physiological function situation.But certain
The electric pulse effect of dosage is lower will to occur electroporation phenomenon, and the bilayer lipid membrane of cell, which is temporarily rearranged, forms some quilts
Referred to as the hydrophilic pathway of micropore, enables large hydrophilic molecular to pass through.Electrical conductivity changes during membrane perforation, such as right
The electrical conductivity of sodium ion and potassium ion is typically smaller than 1mS/cm2；When transmembrane voltage exceed film dielectric strength when, electrical conductivity compared with
It will be increased sharply to 1S/cm in short time2, cause cell membrane to hinder after the reduction electric field cancellation of particulate penetrating power, in most cases
Micropore can be closed without being had any impact to cell, and temporary transient micropore occurs in this cell membrane under the effect of electric pulse in short-term
Physical process is referred to as electroporation i.e. invertibity electrical breakdown (REB).Electroporation greatly enhances cell membrane permeability, thus proposes
Electroporation therapy.Then the expendable rupture of cell membrane appearance punctures cell to increase electric pulse dosage, and this is irreversibility electricity
Puncture (IREB), and have been used in tumor cell lysis research
The content of the invention
It is an object of the invention in order to solve in current glioma tumour antigen preparation process, cracking is insufficient,
Seriously polluted the problems such as can not obtain holoantigen information, there is provided a kind of glioma based on pulse breakdown principle by the present invention
Full tumour antigen preparation method and device.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：A kind of full tumour antigen preparation method of glioma
And device, comprise the following steps：
A kind of full tumour antigen preparation method of glioma, comprises the following steps：
Fresh Human Brain Gliomas is cleaned into blood stains with buffer solution, lmm is cut into3The samples of human glioma block of size is standby
With；
Glioma will be obtained in glioma tumor tissue unicellular and carry out Multiplying culture；
Glioma after propagation is unicellular to isolate glioma stem cell and glioma non-stem cell；
The propagation carried out using serum-free medium to the glioma stem cell and glioma non-stem cell
Culture, and obtain glioma stem cell and the glioma non-stem cell of exponential phase；
The glioma stem cell of exponential phase and glioma non-stem cell are positioned over pretreatment chamber one, it is pre-
Pre-processed in process chamber two；
Pretreated glioma stem cell and glioma non-stem cell are mixed and are positioned over lysis process chamber
Enter horizontal pulse cracking and synchronous centrifugal treating in one；
Supernatant is abandoned, the glioma stem cell and glioma non-stem cell mixed liquor after being handled by more than are placed
The cracking of pulse again is carried out in lysis process chamber two, that is, harvests the full tumour antigen of glioma.
On the basis of above-mentioned technical proposal, the present invention can also do following improvement.
Further, it is 1 by number ratio:2-1:5 glioma stem cell and glioma non-stem cell are put respectively
It is placed in pretreatment chamber one and pretreatment chamber two and carries out cracking pretreatment 5-10min；
With CO in the pretreatment chamber one2Atmosphere is filled up completely with, and temperature remains 37 DEG C -45 DEG C, and electric pulse parameter is set
For：Peak value 1-2kV/cm, the μ s of pulsewidth 100, frequency 1-4Hz, pulse number 4-10；
With CO in the pretreatment chamber two2Atmosphere is filled up completely with, and temperature remains 40 DEG C -45 DEG C, and electric pulse parameter is set
For：Peak value 2-5kV/cm, the μ s of pulsewidth 50, frequency 4-8Hz, pulse number 6-10.
The pretreatment chamber one, pretreatment chamber two use CO2Atmosphere is filled up completely with, to prevent outside air during pretreatment
Into causing pollution；Pretreatment chamber one, pretreatment chamber two are using low-frequency pulse to glioma stem cell and glioma
Non-stem cell carries out invertibity and punctured, to lift the transmission of glioma stem cell and glioma non-stem cell cell membrane
Property, reach the purpose for promoting glioma stem cell and the expansion of glioma non-stem cell.While pretreatment chamber one, pre- place
The temperature control in room two is managed, glioma stem cell and glioma non-stem cell cell membrane can be promoted to change, plus
Fast glioma stem cell and glioma non-stem cell expansion process.
Because glioma stem cell is more much larger than glioma non-stem cell volume, itself expansion space is limited,
Therefore pulse frequency will be less than pulse frequency in pretreatment chamber two in pretreatment chamber one of the present invention.
Further, supernatant is abandoned, pretreated glioma stem cell and glioma non-stem cell are mixed
It is positioned in lysis process chamber one and enters horizontal pulse cracking and synchronous centrifugal treating 4.5-10min；
With CO in the lysis process chamber one2Atmosphere is filled up completely with, and temperature setting is：Rise 1 DEG C to 45 per 30s from 37 DEG C
℃；
Electric pulse parameter is set to：20-250V, pulse width 1-20ps, the finger of repetition rate 10Hz-1kHz Independent adjustables
Number decay waveform, its pulse steepness is rise time 90-180ns；
The parameter of noncentricity is：1000r/min rises in 5000r/min, 5min and completed.
Similarly, with CO in lysis process chamber one2Atmosphere is filled up completely with, for avoiding in cracking process by outside contamination；
And promote the glioma stem cell after expanding, glioma non-dry using what stepped temperature hoisting way can be stablized
The metamorphosis of cell film.
Lysis process chamber one is using steep-sided pulse to the glioma stem cell after expansion and glioma non-stem cell
Irreversible electrical breakdown is carried out, the quick released antigen material of glioma stem cell can be promoted, while irreversible pulse can
To inactivate polluted bacteria, pollution sources are thoroughly eliminated.
When carrying out steep-sided pulse cracking, centrifugal treating is synchronously carried out, this operation can with accelerans glioma stem cells and
The cracking of glioma non-stem cell so that antigenic substance is fully discharged with merging, and centrifuge synchronous carry out can be to prevent
Only during pulse breakdown bubble accumulation, occur gas breakdown cause device to damage.
Centrifugal rotational speed is risen in 5000r/min, 5min by 1000r/min and completed simultaneously, and this, which is set, make it that rotating speed is steady
Lifting, is conducive to keeping the uniformity in cracking process.
Further, supernatant is abandoned, the glioma stem cell and glioma non-stem cell after being handled by more than mix
Conjunction liquid, which is positioned in lysis process chamber two, carries out the cracking 20-30min of pulse again, until pH value fluctuating range is less than in mixed liquor
0.05；
Wherein, with CO in the lysis process chamber two2Atmosphere is filled up completely with, and temperature remains 37 DEG C；
Electric pulse parameter is set to：Peak value 1-2kV/cm, the μ s of pulsewidth 100, frequency 1-4Hz, pulse number 4-10.
Again low is carried out to the mixed liquor after the processing of high frequency steep-sided pulse using low-frequency pulse in the lysis process chamber two
Frequency handle, it is therefore an objective to so that in mixed liquor antigenic information further release.Because cell cracking h substance can cause mixing
Liquid pH numerical value changes, therefore pH value fluctuating range is with reference to foundation to the present invention using in mixed liquor, and judgement mixed liquor, which is cracked, is
It is no cmpletely.
The present invention solves above technical problem and also provides a kind of glioma full tumour antigen preparation facilities, including：It is micro-
Type controller and the power module being connected with the microcontroller, control module, pretreatment module, detection feedback module,
Processing module is cracked, the pretreatment module is connected with pretreatment chamber one, pretreatment chamber two, and the detection feedback module is connected with
Temperature detection sensor group, pH detection sensors, total reflection air-foam detector, the cracking processing module are connected with cracking processing
Room one, lysis process chamber two, sedimentator, temperature controller, vacuumize process device.
The microcontroller is as present apparatus control centre, and the coordination for whole device is controlled, the power module
For the power supply of the present apparatus, the control module is used for the operational control of whole device, and the pretreatment module is used for god
Expansion process and sterilization processing through glioma stem cells and glioma non-stem cell, the cracking processing module are used for god
It is prepared by cracking and antigen through glioma stem cells and glioma non-stem cell.
Further, in the pretreatment chamber one, pretreatment chamber two, lysis process chamber one, lysis process chamber two on set respectively
Impulse generator is equipped with, the impulse generator is provided with porous nano electrode, and the porous nano electrode is arranged at pretreatment
Room one, pretreatment chamber two, lysis process chamber one, inside lysis process chamber.
The impulse generator is used for the generation of cycle low frequency or high frequency steep-sided pulse, and the porous nano electrode is due to porous
Structural table area is larger, and the electric field radiation scope that it is produced is wider, is conducive to pre-processing and cracks the lifting of the efficiency handled.
Further, it is respectively arranged with the pretreatment chamber one, pretreatment chamber two, lysis process chamber one, lysis process chamber
Temperature sensor.The temperature sensor is used for pretreatment chamber one, pretreatment chamber two, lysis process chamber one, lysis process chamber two
The collection of middle temperature data, and gathered data is sent to detection feedback module be amplified, filter, the processing such as numerical value conversion,
Finally it is sent to microcontroller to be analyzed, and trip temperature is entered by temperature controller and controls in real time.
Further, the lysis process chamber one is fixedly installed on sedimentator, for realize high frequency steep-sided pulse cracking with from
The synchronization process of the heart, is conducive to acceleration cracking and the bubble breakdown of glioma stem cell and glioma non-stem cell
Occur.
Further, the total reflection air-foam detector is arranged inside lysis process chamber one, and the pH detection sensors are set
It is placed in inside lysis process chamber two, due to being easy to produce ionized gas, the total reflection bubble detection during high frequency breakdown
Device utilizes total reflection principle for the data acquisition to bubble yield in lysis process chamber one, and gathered data is sent into inspection
The processing such as feedback module is amplified, filtered, numerical value is changed are surveyed, microcontroller is finally sent to and is analyzed, if bubble stream
Amount reaches preset value, and microcontroller starts vacuumize process device and carries out pumping pressurized treatments so that gas in cracking mixed liquor
It is excluded.The pH detection sensors are used for the collection of pH numerical value changes in lysis process chamber two, and gathered data is sent to
The processing such as detection feedback module is amplified, filtered, numerical value conversion, are finally sent to microcontroller and are analyzed, if cracking
Mixed liquor pH value fluctuating range is less than 0.05pH in process chamber two, and microcontroller judges that whole cracking is completed.
Further, the vacuumize process device is connected by tracheae with lysis process chamber one, for realizing lysis process chamber
Pumping process in one.
The beneficial effects of the invention are as follows：
The present invention realizes glioma stem cell and glioma non-stem cell point using low frequency and high frequency steep-sided pulse
Phase process and cracking, simple to operate, cracking more fully, quickly, can obtain more complete glioma tumour antigen.
Brief description of the drawings
A kind of full tumour antigen preparation facilities structure chart of glioma of Fig. 1 present invention.
Embodiment
The principle and feature of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
The preparation of the full tumour antigen of glioma
Embodiment 1
Fresh Human Brain Gliomas is cleaned into blood stains with buffer solution, lmm is cut into3The samples of human glioma block of size is standby
With；Glioma will be obtained in glioma tumor tissue unicellular and carry out Multiplying culture；Neuroglia after propagation
Matter knurl is unicellular to isolate glioma stem cell and glioma non-stem cell；Using serum-free medium to the god
The Multiplying culture carried out through glioma stem cells and glioma non-stem cell, and obtain the glioma of exponential phase
Stem cell and glioma non-stem cell；It is 1 by number ratio:2 glioma stem cell and glioma are non-dry thin
Born of the same parents, which are respectively placed in pretreatment chamber one and pretreatment chamber two, carries out cracking pretreatment 5-10min；In the pretreatment chamber one with
CO2Atmosphere is filled up completely with, and temperature remains 37 DEG C, and electric pulse parameter is set to：Peak value 1kV/cm, the μ s of pulsewidth 100, frequency 1Hz,
Pulse number 10；With CO in the pretreatment chamber two2Atmosphere is filled up completely with, and temperature remains 40 DEG C, and electric pulse parameter is set to：
Peak value 2kV/cm, the μ s of pulsewidth 50, frequency 8Hz, pulse number 10.Supernatant is abandoned, by pretreated glioma stem cell
It is positioned over the mixing of glioma non-stem cell in lysis process chamber one and enters horizontal pulse cracking and synchronous centrifugal treating 4.5-
10min；With CO in the lysis process chamber one2Atmosphere is filled up completely with, and temperature setting is：Rise 1 DEG C to 45 per 30s from 37 DEG C
℃；Electric pulse parameter is set to：250V, pulse width 20ps, the exponential damping waveform of repetition rate 1kHz Independent adjustables, its arteries and veins
Rush steepness i.e. rise time 180ns；The parameter of noncentricity is：1000r/min rises in 5000r/min, 5min and completed.
Embodiment 2
Knurl experiment is killed in experiment one in vitro
Target cell (neuroglial cytoma) is inoculated in 96 orifice plates after cultivating 100h in (three wells), incubator and uses silk
Rimocidin C processing.It is 1 by effect target ratio to take the effector cell after co-culturing:1、5:1、10:1、20:1、40:L is added in 96 orifice plates,
Cultivate and incubated after 20h with adding lml after KRP buffereds containing 37 DEG C of 0.5 μ C/ml [methyl -3H]-thymidine in incubator
Educate 12-18 hours, 10 stopped reactions are got express developed with the PBS of the glucose containing 10mmol/L of precooling.Take cell pyrolysis liquid,
Gamma-5500 liquid flashing countings determine umber of pulse (cpm) per minute.
According to formula killing rate (%)=[target control group cpm mono- (the effect control group cpm of effect target group cpm mono-)/target control group
Cpm] × 100% calculate killing rate.
As a result：Experimental group has stronger killing ability compared with control group.Effect target compares 60:When 1, experimental group vaccine can be killed
The glioma non-stem cell like cell of 86.91% scholar 6.79% and the stem cell-like cell of 80.82% scholar 6.36%, and control group
It is only capable of the killing glioma non-stem cell like cell of 25.7% scholar 7.25% and the stem cell-like cell of 50.15% scholar 4.69%.
Embodiment 3
Knurl experiment is killed in experiment two in vivo
Experimental procedure：Tumor-bearing mice is randomly divided into three groups：Experimental group, control group and blank group, blank group root of the tail portion skin
Lower injecting normal saline；Existing glioma therapeutic vaccine (10 is subcutaneously injected in control group root of the tail portion5Individual/mL, 0.3mL)；
The glioma therapeutic vaccine (10 in the inventive embodiments 1 is subcutaneously injected in experimental group root of the tail portion5Individual/mL, 0.3mL) epidemic disease
Seedling；Observe the life cycle of each group mouse in 50d.
As a result：Found through analysis life cycle:The median survival interval of each experimental group rat is respectively:Control group 18d days, it is real
It is 33d blank control groups 6d to test group.
Experimental animal peripheral blood IFN-γ testing result:Each group rat peripheral blood IFN-γ concentration is respectively:Existing vaccine
120.12 scholar 2.13pg/ml of group, the scholar 6.95pg/ml of experimental group 181.76, blank group is 78.98 scholar 5.74pg/ml.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modifications, equivalent substitutions and improvements made etc. should be included within the scope of the present invention.
Claims (10)
1. a kind of full tumour antigen preparation method of glioma, it is characterised in that comprise the following steps：
Fresh Human Brain Gliomas is cleaned into blood stains with buffer solution, lmm is cut into3The samples of human glioma block of size is standby；
Glioma will be obtained in glioma tumor tissue unicellular and carry out Multiplying culture；
Glioma after propagation is unicellular to isolate glioma stem cell and glioma non-stem cell；
The Multiplying culture carried out using serum-free medium to the glioma stem cell and glioma non-stem cell,
And obtain glioma stem cell and the glioma non-stem cell of exponential phase；
The glioma stem cell of exponential phase and glioma non-stem cell are positioned over pretreatment chamber one, pretreatment
Pre-processed in room two；
Pretreated glioma stem cell and glioma non-stem cell are mixed and are positioned in lysis process chamber one
Enter horizontal pulse cracking and synchronous centrifugal treating；
Supernatant is abandoned, the glioma stem cell and glioma non-stem cell mixed liquor after being handled by more than are positioned over and split
The cracking of pulse again is carried out in solution process chamber two, that is, harvests the full tumour antigen of glioma.
2. the full tumour antigen preparation method of a kind of glioma according to claim 1, it is characterised in that described to locate in advance
Glioma stem cell after reason and the mixing of glioma non-stem cell, which are positioned over, enters horizontal pulse in lysis process chamber one and splits
Solution and synchronous centrifugal treating are implemented as follows：
It is 1 by number ratio:2-1:5 glioma stem cell and glioma non-stem cell are respectively placed in pretreatment chamber
One and pretreatment chamber two in carry out cracking pretreatment 5-10min；
With CO in the pretreatment chamber one2Atmosphere is filled up completely with, and temperature remains 37 DEG C -45 DEG C, and electric pulse parameter is set to：Peak
Value 1-2kV/cm, the μ s of pulsewidth 100, frequency 1-4Hz, pulse number 4-10；
With CO in the pretreatment chamber two2Atmosphere is filled up completely with, and temperature remains 40 DEG C -45 DEG C, and electric pulse parameter is set to：Peak
Value 2-5kV/cm, the μ s of pulsewidth 50, frequency 4-8Hz, pulse number 6-10.
3. the full tumour antigen preparation method of a kind of glioma according to claim 1, it is characterised in that the step will
Pretreated glioma stem cell and the mixing of glioma non-stem cell are positioned in lysis process chamber one and carry out arteries and veins
Avulsion solution and synchronous centrifugal treating are implemented as follows：
Supernatant is abandoned, pretreated glioma stem cell and glioma non-stem cell are mixed and are positioned at cracking
Enter horizontal pulse cracking and synchronous centrifugal treating 4.5-10min in reason room one；
With CO in the lysis process chamber one2Atmosphere is filled up completely with, and temperature setting is：Rise 1 DEG C to 45 DEG C per 30s from 37 DEG C；
Electric pulse parameter is set to：20-250V, pulse width 1-20ps, the index of repetition rate 10Hz-1kHz Independent adjustables decline
Cut waveform, its pulse steepness is rise time 90-180ns；
The parameter of noncentricity is：1000r/min rises in 5000r/min, 5min and completed.
4. the full tumour antigen preparation method of a kind of glioma according to claim 1, it is characterised in that the step is abandoned
Supernatant, glioma stem cell and glioma non-stem cell mixed liquor after being handled by more than are positioned over cracking processing
The cracking of pulse again is carried out in room two, that is, harvests being implemented as follows for the full tumour antigen of glioma：
Supernatant is abandoned, the glioma stem cell and glioma non-stem cell mixed liquor after being handled by more than are positioned over and split
The cracking 20-30min of pulse again is carried out in solution process chamber two, until pH value fluctuating range is less than 0.05 in mixed liquor；
Wherein, with CO in the lysis process chamber two2Atmosphere is filled up completely with, and temperature remains 37 DEG C；
Electric pulse parameter is set to：Peak value 1-2kV/cm, the μ s of pulsewidth 100, frequency 1-4Hz, pulse number 4-10.
5. a kind of full tumour antigen preparation facilities of glioma, it is characterised in that including：Microcontroller and with it is described micro-
The power module of type controller connection, control module, pretreatment module, detection feedback module, cracking processing module, the pre- place
Reason module is connected with pretreatment chamber one, pretreatment chamber two, and the detection feedback module is connected with temperature detection sensor group, pH inspections
Survey sensor, total reflection air-foam detector, the cracking processing module be connected with lysis process chamber one, lysis process chamber two, from
Heart device, temperature controller, vacuumize process device.
6. a kind of full tumour antigen preparation facilities of glioma according to claim 5, it is characterised in that the pretreatment
Impulse generator, the pulse hair are respectively arranged with room one, pretreatment chamber two, lysis process chamber one, lysis process chamber two
Raw device is provided with porous nano electrode, and the porous nano electrode is arranged at pretreatment chamber one, pretreatment chamber two, lysis process chamber
First, inside lysis process chamber.
7. a kind of full tumour antigen preparation facilities of glioma according to claim 5, it is characterised in that the pretreatment
Temperature sensor is respectively arranged with room one, pretreatment chamber two, lysis process chamber one, lysis process chamber two.
8. the full tumour antigen preparation facilities of a kind of glioma according to claim 5, it is characterised in that at the cracking
Reason room one is fixedly installed on sedimentator.
9. a kind of full tumour antigen preparation facilities of glioma according to claim 5, it is characterised in that the total reflection
Air-foam detector is arranged inside lysis process chamber one, and the pH detection sensors are arranged inside lysis process chamber two.
10. the full tumour antigen preparation facilities of a kind of glioma according to claim 5, it is characterised in that described to take out true
Empty processor is connected by tracheae with lysis process chamber one.
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| CN101085391A (en) * | 2007-06-29 | 2007-12-12 | 重庆大学 | Device for inducing tumor cell apoptosis by high-voltage nanosecond pulse |
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