CN107281214A - Hydrogen molecule liquid/gas is preparing the application in suppressing tumor stem cell medicine - Google Patents
Hydrogen molecule liquid/gas is preparing the application in suppressing tumor stem cell medicine Download PDFInfo
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Abstract
Hydrogen molecule liquid/gas is preparing the application in suppressing tumor stem cell medicine, belongs to preparation and suppresses tumor stem cell technical field of pharmaceuticals.Liquid/gas containing hydrogen molecule is used to prepare and suppresses tumor stem cell medicine, significantly inhibit one-tenth knurl ability in tumour body, so that the invasive ability of tumour cell is remarkably decreased, tumor stem cell surface markers CD133 expression is further significantly inhibited, and promotes the expression of differentiation marker.
Description
Technical field
The invention belongs to prepare to suppress tumor stem cell technical field of pharmaceuticals, and in particular to hydrogen molecule liquid/gas is in system
The standby application suppressed in tumor stem cell medicine.
Technical background
In recent years, as research finds that tumour is not to be made up of homogeneous tumour cell, the different cell of intra-tumor
With different propagation, infiltration and transfer ability, with very strong heterogeneity.Wherein sub-fraction cell have self-renewing,
The potential of infinite multiplication and Multidirectional Differentiation, this part cell is referred to as tumor stem cell (cancer stem cells, CSC), its
Tumor stem cell (CSC) has the ability of self-renewing and differentiation, is to form tumour and start the initial thin of tumour fast breeding
Born of the same parents, can also produce new tumour cell formation complete tumors by constantly breaking up.Tumor stem cell be also considered as resistance and
The arch-criminal of recurrence, find the effective medicine for suppressing glioma stem cells has turned into the heat that researcher and clinician pay close attention to
Point.
Hydrogen is minimum, the most abundant element of molecular weight in the world, accounts for the 90% of whole mass of the universe.Hydrogen is a kind of nothing
The tasteless diatomic gas of color.Current hydrogen is widely studied as clean energy resource gas, until the too field of Japanese scholars in 2007
The research group of professor Cheng Nan leader finds that the hydrogen of animal breath 2% can effectively remove free radical, significantly improves cerebral ischemia
Reperfusion injury, the characteristic of hydrogen selective antioxidation causes the biological function of hydrogen molecule gradually to receive significant attention,
It is a variety of due to disease caused by oxidative damage, such as Parkinson that increasing Clinical and experimental study shows that hydrogen molecule can be treated
Disease, diabetes, metabolic syndrome, mitochondrial disease etc..Hydrogen is used to suppress the research of tumour, in fact early in Dole in 1975 etc.
The article using hydrogen treating malignant mela noma has been delivered on science, but has been the hydrogen using 8 atmospheric pressure of breathing
Gas, because breathing high pressure hydrogen has certain danger, it is difficult to be widely adopted.Saitoh etc. is electrolysed water process people's tongue using hydrogen-rich
Cancer cell HSC-4, it was demonstrated that can significantly reduce tumor cell clone Forming ability in cellular level hydrogen molecule.Up to the present,
Whether hydrogen or hydrogen rich water can suppress tumor stem cell still has no relevant report at present.
The research of the present invention finds that hydrogen can significantly inhibit glioma and the multiplication capacity of other tumor spheres,
Breathing hydrogen and hydrogen rich water can significantly inhibit one-tenth knurl ability in the glioma body in people and rat source, and hydrogen and hydrogen rich water
The invasive ability of processing descendant and rat Glioma cells is remarkably decreased, it has further been found that hydrogen and hydrogen rich water can be significantly inhibited
Tumor stem cell surface markers CD133 expression, and promote the expression of differentiation marker.
The content of the invention
Prepared by the purpose of the present invention can significantly inhibit the medicine of people and rat tumor stem cell sphere.Hydrogen will especially be contained
The liquid/gas of molecule, which is used to prepare, suppresses brain tumor tumor stem cell and other tumor stem cell medicines, significantly inhibits tumour body
Interior one-tenth knurl ability, further significantly inhibits tumor stem cell surface markers CD133 expression, and promotes the expression of differentiation marker,
And the invasive ability of tumour cell is remarkably decreased.
Liquid containing hydrogen molecule is used to prepare the application for suppressing tumor stem cell medicine.
Liquid containing hydrogen molecule is hydrogen-rich solution, and the solvent of the hydrogen-rich solution contains water, preferably (included but is not limited to)
Hydrogen is dense in the drinkable aqueous solution such as pure water, physiological saline, milk, the acidic aqueous solution of organic acid, beverage, hydrogen-rich solution
Spend for hydrogen molecule saturation, be 15-30 DEG C, the concentration range that is prepared under 1 atmospheric pressure or by the other method such as pressurize not
Less than 100 μm ol/L, upper concentration is supersaturation concentration of the hydrogen in water.
One kind prepares suppression tumor stem cell medicine, it is characterised in that medicine is hydrogen-rich solution, and the hydrogen-rich solution contains
Water, preferably (includes but is not limited to) the acid water-soluble of the aqueous solution, milk, organic acid that pure water, physiological saline or be available for is drunk
The concentration of hydrogen is hydrogen molecule saturation in the drinkable aqueous solution such as liquid, beverage, hydrogen-rich solution, is 15-30 DEG C, 1 air press strip
The concentration range prepared under part or by other methods such as pressurizations is not less than 100 μm of ol/L, and upper concentration is mistake of the hydrogen in water
Saturated concentration.
It is oral medicine, injection medicine or bathing medication to suppress tumor stem cell medicine.
Gas containing hydrogen molecule is used to prepare the application for suppressing tumor stem cell medicine.
Gas containing hydrogen molecule is the mixed gas containing the one or more of and hydrogen in oxygen, air, vapor,
The volumn concentration of hydrogen is 2%-80% or High Pressure Hydrogen in gas containing hydrogen molecule, and preferred volume percentage composition is 20-
70%；
One kind prepares suppression tumor stem cell medicine, it is characterised in that medicine is the gas containing hydrogen molecule, for containing aerobic
One or more and the mixing of hydrogen in gas, air, vapor, the volumn concentration of hydrogen is 2%-80% or high pressure
Hydrogen, preferred volume percentage composition is 20-70%.
Suppress gas Sucked medicine of the tumor stem cell medicine for breathing.
The hydrogen molecule liquid/gas of the present invention is preparing the application in suppressing tumor stem cell medicine, wherein hydrogen molecule energy
Significantly inhibit one-tenth knurl ability in tumour body so that the invasive ability of tumour cell is remarkably decreased, and further significantly inhibits Tumor Stem
Cell surface marker CD133 expression, and promote the expression of differentiation marker.
Brief description of the drawings
Fig. 1 hydrogen treats group and contrast groups suppress the comparison figure of in-vivo tumour volume, and hydrogen treat group significantly inhibits colloid
Breed in knurl body, wherein, figure A is that experimental animal drinks hydrogen rich water and control group result in kind, and figure B is that experimental animal breathes hydrogen
Group and control group result；
Fig. 2 hydrogen treats group and contrast groups suppress the comparison figure of the propagation of tumor spheres, and hydrogen treat group significantly presses down
The tumor spheres propagation of kinds of tumor cells processed；Wherein, figure A is rat and brain glioblastoma cell C6 and the U87 tumour of people
The cellular morphology result and statistical chart of ball formation experiment, figure B form result for the tumour ball of HepG2, PA-1 and Hs38.T cell
Statistics.
Fig. 3 hydrogen treats group and contrast groups suppress the comparison figure of stem cell labeling CD133 expression, and hydrogen treat group is notable
Suppress stem cell labeling CD133 expression；
The immunofluorescence figure that Fig. 4 hydrogen treats group and contrast groups suppress tumor stem cell mark CD133 expression compares, and exempts from
Epidemic disease fluorescence display hydrogen treat group significantly inhibits the expression that tumor stem cell marks CD133, promotes differentiation marker GFAP expression；
Fig. 5 hydrogen treats group and contrast groups suppress the comparison figure of glioma migration invasive ability, and hydrogen treat group significantly presses down
Glioma migration invasive ability processed.
Embodiment
It is below the experimental data explanation present invention using following examples, but the technical scope of the present invention is not limited only to this,
Various changes and modification can be carried out to the present invention in coordinated fashion in the case where not departing from present subject matter and scope.
Embodiment 1：
(1) in cell culture hydrogeneous culture medium preparation
1, after magnesium rod autoclaving, it is positioned in cell culture medium, hydrogen concentration reaches saturation in culture medium two days later, can
For using.
2, the 100% volume ratio pure hydrogen produced using electrolysis water is passed through in culture medium certain time (50mL 30min),
Now hydrogen concentration reaches saturation in culture medium, available.
(2) in animal Brain Glioma Model hydrogen group processing
1, breathe in hydrogen group animal model, hydrogen is produced by electrolysis water, by hydrogen and sky containing 60% volume ratio
Oxygen mixture is passed through in terrarium, for animal breath.
2, drink in hydrogen rich water animal model, experimental animal drinks the hydrogen rich water containing saturated concentration hydrogen molecule.Hydrogen rich water
Prepare and sealing preserve in common drinking water is poured into by electrolysis water generation hydrogen high pressure.
(3) encephalic glioma in situ compares into knurl model treatment
Eight week old male Wistar rats (170-180g) are normally fed one week.It is fixed using solid after chloral hydrate anesthesia
The C6 cells of culture are injected rat brain caudate nucleus area by position instrument, and particular location is x=3.5mm, y=0.5mm, z=-5.0mm.
Open after cranium, C6 cell suspensions inject rat with 1 μ L/min flow, cell suspension volume is that 10 μ L injection cell quantities are
4.8x106 individual every rat.After experiment, rat, which is randomly divided into three groups, includes normal culture group, hydrogen rich water group and breathing hydrogen group,
Hydrogen rich water group and breathing hydrogen group are using the method for (2), and normally culture group, hydrogen rich water group and the design sketch for breathing hydrogen group, are shown in
Accompanying drawing 1.
(4) tumor spheres are formed
1, it is collected by centrifugation after tumour cell digestion.
2, cell is resuspended in PBS, after counting, adds 1.5 × 104Individual cell to 5mL tumor stem cells culture medium (divides
Not Cai Yong hydrogeneous culture medium and common not hydrogeneous culture medium be compared, prepared by hydrogeneous culture medium sees step (1), and Tumor Stem is thin
Born of the same parents' culture medium uses DMEM/F12, wherein adding 20ng/mL EGF, 20ng/mL bFGF and 50x B27.
3, after cell culture 14 days, tumor spheres are formed, accompanying drawing 2 is seen.
(5) cell flow cytometer detection tumour cell CD133 expression
1, the glioma cell ball according to hydrogen and the control group processing formed in step (4) is taken, into unicellular after dispelling
Suspension, by 4 × 105Individual often pipe packing.
2, PBS wash once, 300g centrifugations 10min；Abandon supernatant.
Cell is resuspended in 3,100 μ l PBS, adds primary antibody CD133-FITC (Biobyt 1：200), if a pipe is straight labeling antibody
PE-isotype blank controls a, pipe is straight labeling antibody FITC-isotype blank controls, 4 DEG C of shaking tables, lucifuge reaction 40min.
4, PBS 3 times, each 300g centrifuges 10min, and 1% paraformaldehyde is fixed, 4 DEG C of placements, flow cytometer inspection
Survey, see accompanying drawing 3.
(6) cellular immunofluorescence is detected
1, when cell is bred to 70%, digestion centrifugation is resuspended after cell count, takes 1 × 105 cell to be seeded to 24 holes thin
In born of the same parents' culture plate, normal adhere-wall culture changes hydrogeneous culture medium and common not hydrogeneous medium culture respectively after 24 hours, hydrogeneous
Culture medium prepare see step (1).
2, the tumour cell of adhere-wall culture is taken, culture medium is suctioned out, is washed with PBS three times, note that cell can not be blown and beaten when washing, plus
Enter 4% paraformaldehyde and fix 15min；
3, fixer is suctioned out, is washed with PBS 3 times, each 5min, 0.1%Triton room temperatures rupture of membranes 10 minutes, PBS is washed 3 times,
Each 5min；
4,10% lowlenthal serum room temperature closing 30min；
5, suction out closing serum, it is not necessary to wash, be directly added into primary antibody：Rabbit-anti people CD133 ((Biobyt 1：200), GFAP
(SAB 1:200) negative control only adds PBS, is incubated overnight in 4 DEG C of refrigerators；
6, the sheep anti-Mouse secondary antibody of rhodamine mark goat-anti rabbit secondary antibody or FITC marks is added after PBS 3 times, in room temperature
It is incubated 1h, PBS 3 times；
7, DAPI is finally marked, is incubated at room temperature after 20min, PBS in fluorescence microscopy Microscopic observation, takes pictures, see accompanying drawing
4。
(7) Matrigel Matrigels
1, prepare invasion and attack cell：Matrigel glue can solidify quickly at normal temperatures, therefore, and it is rearmounted from -20 DEG C of taking-ups
Melt in 4 DEG C, and whole process is all placed in operating on ice.(1 is diluted with serum free medium：5~1：8) after, 400 μ l are taken to be taped against
On Transwell films, Transwell cells are placed in 6 orifice plates, placing 3h in 37 DEG C of incubators solidifies it.
2, inoculating cell：After experiment is centrifuged with cell dissociation, hydrogen group and control group use hydrogeneous serum free medium respectively
Commonly not hydrogeneous serum free medium is resuspended, and prepared by hydrogeneous culture medium sees step (1).It is thin in small indoor addition 1ml tumours
Born of the same parents' suspension, cell number is 3 × 105~6×105(selecting suitable cell number according to tumor cell invasion ability).Small outdoor, hydrogen group
Hydrogeneous culture medium and common not hydrogeneous culture medium of the 3ml containing 1.5% hyclone are separately added into control group.
3, cultivate cell：24~48h of cellar culture (depending on tumor cell invasion ability).
4, fixed and dyeing：Transwell cells are taken out, PBS is washed once, and the cell on microporous barrier upper strata is wiped with cotton swab,
It is fixed with 1% crystal violet-formalin and dyes 20~30min, suck dyeing liquor, is then slowly washed away with running water residual
Dyeing liquor is stayed, is air-dried.
5, microscopy：Taken pictures under inverted microscope, each 5 visuals field of sample count carry out statistical analysis after counting, seen
Accompanying drawing 5.
Claims (10)
1. the liquid containing hydrogen molecule is used to prepare the application for suppressing tumor stem cell medicine.
2. according to the application described in claim 1, it is characterised in that the liquid containing hydrogen molecule is the hydrogen-rich aqueous solution, the hydrogen-rich
The solvent of solution contains water, the preferably drinkable water such as pure water, physiological saline, milk, the acidic aqueous solution of organic acid, beverage
The concentration of hydrogen is hydrogen molecule saturation in solution, hydrogen-rich solution, is 15-30 DEG C, under 1 atmospheric pressure or by pressurizeing etc. other
Concentration range prepared by method is not less than 100 μm of ol/L, and upper concentration is supersaturation concentration of the hydrogen in water.
3. one kind prepares suppression tumor stem cell medicine, it is characterised in that medicine is hydrogen-rich solution, the hydrogen-rich solution contains water,
It is preferred that acidic aqueous solution, beverage of the aqueous solution, milk, organic acid that pure water, physiological saline or be available for is drunk etc. are drinkable
The concentration of hydrogen is hydrogen molecule saturation in the aqueous solution, hydrogen-rich solution, is 15-30 DEG C, under 1 atmospheric pressure or by pressurizeing etc. it
Concentration range prepared by other party method is not less than 100 μm of ol/L, and upper concentration is supersaturation concentration of the hydrogen in water.
4. suppress tumor stem cell medicine according to the preparation described in claim any one of 1-3, it is characterised in that suppress Tumor Stem
Cell drug is oral medicine, injection medicine or bathing medication.
5. the gas containing hydrogen molecule is used to prepare the application for suppressing tumor stem cell medicine.
6. according to the application described in claim 5, it is characterised in that the gas containing hydrogen molecule is to be steamed containing oxygen, empty gas and water
The volumn concentration of hydrogen is 2%- in one or more and the mixed gas of hydrogen in gas, the gas containing hydrogen molecule
80%；Or the gas containing hydrogen molecule is High Pressure Hydrogen.
7. according to the application described in claim 6, it is characterised in that the volumn concentration of hydrogen in the gas containing hydrogen molecule
For 20-70%.
8. one kind prepares suppression tumor stem cell medicine, it is characterised in that medicine is the gas containing hydrogen molecule, for containing aerobic
One or more and the mixing of hydrogen in gas, air, vapor, the volumn concentration of hydrogen is 2%-80%；Or be height
Press hydrogen.
9. according to the medicine of claim 8, it is characterised in that the volumn concentration of hydrogen is 20-70%.
10. suppress tumor stem cell medicine according to the preparation described in claim any one of 8-9, it is characterised in that suppress tumour
Stem cell drugs are the gas Sucked medicine of breathing.
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Cited By (3)
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| CN109876008A (en) * | 2019-03-13 | 2019-06-14 | 深圳大学 | A kind of drug and its preparation method and application for oncotherapy |
| CN110464736A (en) * | 2019-09-06 | 2019-11-19 | 深圳市罗湖区人民医院 | A kind of application of composition, hydrogen physiological saline for treating bladder cancer |
| CN111004830A (en) * | 2019-12-12 | 2020-04-14 | 上海交通大学 | Method for releasing hydrogen influenced by hydrogen on cells in cell experiment |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109876008A (en) * | 2019-03-13 | 2019-06-14 | 深圳大学 | A kind of drug and its preparation method and application for oncotherapy |
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| CN111004830A (en) * | 2019-12-12 | 2020-04-14 | 上海交通大学 | Method for releasing hydrogen influenced by hydrogen on cells in cell experiment |
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