CN107737409A - 450 490nm LED blue lights combine application of the arsenic trioxide in clinical treatment of osteosarcoma - Google Patents

450 490nm LED blue lights combine application of the arsenic trioxide in clinical treatment of osteosarcoma Download PDF

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Publication number
CN107737409A
CN107737409A CN201711139478.2A CN201711139478A CN107737409A CN 107737409 A CN107737409 A CN 107737409A CN 201711139478 A CN201711139478 A CN 201711139478A CN 107737409 A CN107737409 A CN 107737409A
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osteosarcoma
cell
led
group
blue lights
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杨磊
蔡本志
袁野
杜伟杰
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Harbin Engineering University
Harbin Medical University
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Harbin Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/36—Arsenic; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N2005/0658—Radiation therapy using light characterised by the wavelength of light used
    • A61N2005/0662—Visible light
    • A61N2005/0663—Coloured light

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  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Radiology & Medical Imaging (AREA)
  • Pathology (AREA)
  • Chemical & Material Sciences (AREA)
  • Inorganic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

The invention discloses application of the 450 490nm LED blue lights joint arsenic trioxide in clinical treatment of osteosarcoma.The present invention applies Trypan Blue, and cell count and EdU decoration methods detect 450 490nm LED blue lights joint As2O3Influence to osteosarcoma cell growing multiplication, using the influence of scratch experiment and Transwell experiments detection cell migration and invasive ability, the influence to cell nuclear damage is detected using γ H2AX immunofluorescence dyeings.Test result indicates that 450 490nm LED blue lights and As2O3Combination can increase low dose of As2O3The effect of being used alone, toxicity is reduced, and can effectively suppress osteosarcoma cell and grow, breed, migrate and attack, inducing cell nuclear damage, and then alleviate and treat the purpose of osteosarcoma.Clinical practice of the present invention is simple and easy, and medical treatment cost is low, while alleviates patient and treat pain, and new technological means is provided for the treatment of osteosarcoma.

Description

450-490nm LED- blue lights combine application of the arsenic trioxide in clinical treatment of osteosarcoma
Technical field
The present invention relates to 450-490nm LED- blue lights joint arsenic trioxide (Arsenic trioxide, As2O3) in bone Application in sarcoma treatment.The invention belongs to medicine technology field.
Background technology
Osteosarcoma is a kind of primary pernicious bone tissue tumour for being apt to occur in children and adolescence, and its invasive ability is strong, Disease development is rapid, and patient's fatal rate is high.At present, the clinical treatment method for Patients with Osteosarcoma mainly has surgical operation, A variety of general treatment measures such as chemotherapy, radiotherapy, PCI, but surgical operation can cause the risk of amputation, and patient is to chemicotherapy The drug resistance and prognosis in osteosarcoma effect extreme difference of medicine are still to treat osteosarcoma and improve the huge of Patients with Osteosarcoma living standard Hang-up.Because various treatment methods all bring irreversible physiology and psychic trauma to patient, so being badly in need of finding and developing The new tool for the treatment of osteosarcoma just seems extremely urgent.
As2O3Though one of most ancient poisonous substance, and the most arsenide of commercial value, also known as arsenic.Chinese scholar is early The 1970s just by As2O3Treatment applied to leukaemia.Lot of documents shows, As2O3To Several Kinds of Malignancy such as liver The treatment of cancer, lung cancer, cervical carcinoma and the cancer of the esophagus all has obvious effect.But As2O3Treatment be a double-edged sword, can produce Serious stress reaction, while can also cause the system lesion of a variety of vitals such as heart and liver.Therefore whether can lead to Crossing use in conjunction others technology is reducing As2O3While side effect, its curative effect can be increased.
Phototherapy is a kind of new treatment method occurred in recent years, and inventor is from the light source of numerous different wave lengths The LED- blue-light sources that wavelength is 450-490nm are filtered out, it is found that superpower LED- blue lights grow tool for osteosarcoma cell There is significant inhibitory action, while inventor has found that more low intensive LED- blue lights illumination effect can increase As2O3Suppress osteosarcoma The effect of cell growth.Further probe into and find 450-490nm LED- blue lights and As2O3Use in conjunction have suppress osteosarcoma Cell propagation, migration, invasion and attack and the effect of induction osteosarcoma cell cell nuclear damage.
Clinical practice of the present invention is simple and easy, can reduce medical treatment cost, mitigates patient and undergos surgery excision and chemicotherapy When caused pain, provide a kind of new technological means for the treatment of osteosarcoma.
The content of the invention
The purpose of the present invention is to probe into the LED- blue lights illumination of 450-490nm wavelength to improve the As of low dosage2O3To osteosarcoma Effect in cell growth, propagation, migration, the inhibition of invasion and attack and inducing cell nuclear damage, to reduce As2O3Pair is made With while, increase its curative effect.
To reach above-mentioned purpose, present invention employs following technological means：
The present invention screens As first by cultured in vitro osteosarcoma cell line U2OS2O3Suitable treatment dosage, that is, use The As of various dose2O3Osteosarcoma cell is handled, is grouped into 0 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM and 10 μM, application station after 24h Expect blue dyeing, count, detect As2O3The influence of cell proliferation.Choose 0.3 μM of As2O3The osteosarcoma cell after 2h is handled, Illumination experiment is carried out using the LED- blue light sources of 450-490nm wavelength, illumination power is 100mW/cm2Light application time is 30min, total intensity of illumination are 180J/cm2, it is thin using Trypan Blue, cell count and EdU dyeing detection osteosarcoma after 24h The situation of intracellular growth propagation；Using the ability of migration and the invasion and attack of scratch experiment and Transwell experiment inspection cells；Using γ-H2AX immunofluorescence dyeings detect nucleus degree of impairment.Test result indicates that 450-490nm wavelength LED- blue lights are combined As2O3For the treatment of osteosarcoma, compared to exclusive use, the suppression osteosarcoma cell with enhancing grows, breeds, migrates, invaded Attack and the effect of inducing cell nuclear damage, and As can be reduced2O3Usage amount, so as to reduce toxic side effect.
Therefore, the LED- blue light sources with 450-490nm wavelength are proposed and are being made based on the studies above basis, the present invention It is standby to use As2O3Treat the application in the assistive device of osteosarcoma.
Wherein, there is the LED- blue light sources and As of 450-490nm wavelength2O3Combination, there is enhancing As2O3Suppress bone and flesh Tumor cell growth, propagation, migration and the effect of invasion and attack.
Wherein, there is the LED- blue light sources and As of 450-490nm wavelength2O3Combination, having reduces As2O3Toxicity Effect.
Wherein, there is the LED- blue light sources and As of 450-490nm wavelength2O3Combination, there is enhancing As2O3Induce bone and flesh The effect of oncocyte cell nuclear damage.
Relative to prior art, the beneficial effects of the invention are as follows：
As2O3As is not only reduced with the LED- blue light illumination use in conjunction with 450-490nm wavelength2O3To body institute band The side effect come, light therapy to a certain extent, alleviate patient due to tight caused by surgical resection and chemicotherapy Damage on the body ＆ mind of weight, and in growth, propagation, the migration for suppressing osteosarcoma cell, invasion and attack and inducing cell Significant effect in terms of nuclear damage, therefore the proposition of the present invention provides a kind of new technological means for the treatment of osteosarcoma.
Brief description of the drawings
Fig. 1 is the As of various dose2O3Influence to U2OS cell growths；
In U2OS osteosarcoma cells, respectively using 0 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM and 10 μM of As2O3Handle 24h Afterwards, Trypan Blue, cell count are carried out；(A) taken pictures under microscope；(B) total viable cell；(C) percentage of dead cells；
Fig. 2 is 0.3 μM of As2O3With 450-490nm LED- blue lights individually and combination to U2OS cell growths, propagation Influence；
Using 0.3 μM of As2O3Osteosarcoma cell is acted on alone or in combination with LED- blue lights, after 24h, carries out trypan blue dye Color and EdU dyeing；(A) taken pictures under microscope；(B) total viable cell；(C) percentage of dead cells；(D) clapped under fluorescence microscope According to；(E) EdU positive rates statistical chart；
Fig. 3 is 0.3 μM of As2O3With 450-490nm LED- blue lights individually and combination to U2OS cell migrations, invasion and attack Influence；
Tested using scratch experiment and Transwell, experiment is divided into following four groups：Control group, 0.3 μM of As2O3Processing Group, LED- blue light illumination 180J/cm2Group and use in conjunction group, respectively in processing 24h, 48h and 72h Microscopic observations and inspection of taking pictures Violet staining is carried out after handling 24h while surveying the transfer ability of cell；(A) taken pictures under microscope；(B) cell migration distance Statistical chart；(C) taken pictures under microscope；(D) crystal violet absorbance statistical chart；
Fig. 4 is 0.3 μM of As2O3With 450-490nm LED- blue lights individually and combination induction U2OS cell nuclear damagies.
Experiment is divided into four groups：Control group, 0.3 μM of As2O3Treatment group, LED- blue light illumination 180J/cm2Group and joint should With group, γ-H2AX immunofluorescence dyeings are carried out after 24h is handled respectively；(A) taken pictures under fluorescence microscope；(B) γ-H2AX sun Property rate statistical chart.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and It is apparent.But embodiment is only exemplary, does not form any restrictions to the scope of the present invention.Those skilled in the art should It should be appreciated that the details and form of technical solution of the present invention can be repaiied without departing from the spirit and scope of the invention Change or replace, but these modifications and replacement are each fallen within protection scope of the present invention.
Material and its source involved by the embodiment of the present invention：
1. main agents：Trypan blue reagent (biosharp companies)；EdU staining kits (Ribobio companies) γ- H2A.X phospho S139 antibody (Abcam companies)；Transwell cells (Corning companies)
2. key instrument：Count Star cell counters；Fluorescence inverted microscope；Inverted microscope
Embodiment 1：As2O3Suppress osteosarcoma cell growth, propagation
1 experimental method
1.1 Trypan Blues detect osteosarcoma cell survival rate
1.2 experiment packets design：
According to As2O3Different concentration for the treatment of is divided into following five groups:0μM；0.1μM；0.3μM；1μM；3 μM and 10 μM.
1.3 experiment detection projects：Trypan Blue, cell count
In U2OS osteosarcoma cells, respectively using 0 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM and 10 μM of As2O3Handle 24h Afterwards, Trypan Blue, cell count are carried out.
1.4 data processing
The result of the present invention is represented using standard deviation ± standard error.Mapped with Graphpad prism 5.0, respectively Correlation between group is weighed with T inspections.
2 observation results
As shown in figure 1, with 0 μM of group ratio, 0.3 μM of group cell state starts to change, but cell quantity is without significant change, with As2O3Dosage increase, cell are gradually rounded, and dead cell rate dramatically increases.Illustrate As2O3The life of osteosarcoma cell can be significantly inhibited Long propagation, with As2O3Dosage increase, the effect for suppressing growth and proliferation of cell is stronger.
The As that 2 0.3 μM of embodiment2O3It is combined with 450-490nm LED- blue lights and suppresses osteosarcoma cell growth, propagation
1 experimental method
1.1 Trypan Blues and EdU dyeing detection osteosarcoma cell survival rates
1.2 experiment packets design：
Experiment is divided into 4 groups：Control group, 0.3 μM of As2O3Treatment group, LED- blue light illumination 180J/cm2Group (illumination power For 100mW/cm2, light application time 30min, total intensity of illumination is 180J/cm2) and use in conjunction group, the selection of use in conjunction group 0.3 μM of As2O3The osteosarcoma cell after 2h is handled, illumination experiment is carried out using the LED- blue light sources of 450-490nm wavelength, Illumination power is 100mW/cm2Light application time is 30min, and total intensity of illumination is 180J/cm2。
1.3 experiment detection projects：Trypan Blue, cell count and EdU dyeing
In U2OS osteosarcoma cells, per component other places after reason 24h, Trypan Blue, cell count and EdU dyes are carried out Color, statistics viable count, dead cell rate and EdU positive rates.
1.4 data processing
The result of the present invention is represented using standard deviation ± standard error.Mapped with Graphpad prism 5.0, respectively Correlation between group is weighed with T inspections.
2 observation results
As shown in Fig. 2 0.3 μM of As2O3Group is used alone compared with control group with LED- blue lights, cell state and cell Number is without significant change, but use in conjunction group viable count significantly reduces, and cell rounding brightens, dead cell rate significantly raise (Fig. 2A- C).Group EdU positive rates are used alone compared with control group without significant change simultaneously, but use in conjunction group EdU positive rates show Writing reduces (Fig. 2 D-E).Illustrate 0.3 μM of As2O3Osteosarcoma cell can be significantly inhibited with the combination of 450-490nm LED- blue lights Growth and propagation.
Embodiment 3：0.3 μM of As2O3It is combined with 450-490nm LED- blue lights and suppresses U2OS cell migrations
1 experimental method
1.1 scratch experiments detect osteosarcoma cell transfer ability
1.2 experiment packets design：
Experiment is divided into 4 groups：Control group, 0.3 μM of As2O3Treatment group, LED- blue light illumination 180J/cm2Group (illumination power For 100mW/cm2, light application time 30min, total intensity of illumination is 180J/cm2) and use in conjunction group, the selection of use in conjunction group 0.3 μM of As2O3The osteosarcoma cell after 2h is handled, illumination experiment is carried out using the LED- blue light sources of 450-490nm wavelength, Illumination power is 100mW/cm2Light application time is 30min, and total intensity of illumination is 180J/cm2。
1.3 scratch experiment
Cut is carried out on U2OS osteosarcoma cells surface using 200 μ l pipette tips, scratch width is consistent as far as possible, every group Distinguish the micro- Microscopic observation of 24h, 48h and 72h after treatment and photograph to record hecatomeral cells migration situation.
1.4 data processing
The result of the present invention is represented using standard deviation ± standard error.Mapped with Graphpad prism 5.0, respectively Correlation between group is weighed with T inspections.
2 observation results
As shown in figures 3 a-b, control group, 0.3 μM of As2O3Treatment group and LED- blue light illumination 180J/cm2Group cut distance It is significantly less than use in conjunction group over time.Illustrate 0.3 μM of As2O3It can show with the combination of 450-490nmLED- blue lights Write and suppress U2OS cell migrations.
Embodiment 4：0.3 μM of As2O3It is combined with 450-490nm LED- blue lights and suppresses osteosarcoma cell invasion and attack
1 experimental method
1.1 experiment packets design：
Experiment is divided into 4 groups：Control group, 0.3 μM of As2O3Treatment group, LED- blue light illumination 180J/cm2Group (illumination power For 100mW/cm2, light application time 30min, total intensity of illumination is 180J/cm2) and use in conjunction group, the selection of use in conjunction group 0.3 μM of As2O3The osteosarcoma cell after 2h is handled, illumination experiment is carried out using the LED- blue light sources of 450-490nm wavelength, Illumination power is 100mW/cm2Light application time is 30min, and total intensity of illumination is 180J/cm2。
1.2Transwell experiment detection osteosarcoma cell invasive abilities
Osteosarcoma cell is with 2 × 105Individual/ml density is seeded in upper chamber, and the culture of low serum is changed to after cell settlement Liquid, hungry 2h, lower room are changed to normal complete culture solution, and upper chamber is handled after being changed to low serum free culture system liquid, and every group is being located respectively Violet staining is carried out after reason 24h and detects its absorbance.
1.3 data processing
The result of the present invention is represented using standard deviation ± standard error.Mapped with Graphpad prism 5.0, respectively Correlation between group is weighed with T inspections.
2 observation results
As shown in Fig. 3 C-D, the color of use in conjunction group violet staining is compared to control group, 0.3 μM of As2O3Treatment group With LED- blue light illumination 180J/cm2Group is shallow, and absorbance significantly reduces.Illustrate 0.3 μM of As2O3With 450-490nm LED- Blue light is combined the invasion and attack that can significantly inhibit osteosarcoma cell.
Embodiment 5：0.3 μM of As2O3Induction osteosarcoma cell nuclear damage is combined with 450-490nm LED- blue lights
1 experimental method
1.1 experiment packets design：Experiment is divided into 4 groups：Control group, 0.3 μM of As2O3Treatment group, LED- blue light illumination 180J/cm2(illumination power is 100mW/cm to group2, light application time 30min, total intensity of illumination is 180J/cm2) and use in conjunction Group, use in conjunction group choose 0.3 μM of As2O3The osteosarcoma cell after 2h is handled, utilizes the LED- blue lights of 450-490nm wavelength Light source carries out illumination experiment, and illumination power is 100mW/cm2Light application time is 30min, and total intensity of illumination is 180J/cm2。
The experiment detection osteosarcoma cell nuclear damage of 1.2 γ-H2AX immunofluorescence dyeings
γ-H2AX immunofluorescence dyeings are carried out after every group of processing 24h：U2OS osteosarcoma cells are seeded in identical density In orifice plate, fluorescence microscopy Microscopic observation and taken pictures after cell settlement.
1.3 data processing
The result of the present invention is represented using standard deviation ± standard error.Mapped with Graphpad prism 5.0, respectively Correlation between group is weighed with T inspections.
2 observation results
As shown in figure 4, control group, 0.3 μM of As2O3Treatment group and LED- blue light illumination 180J/cm2The positive rate of group shows Work is higher than use in conjunction group.Illustrate 0.3 μM of As2O3It can significantly induce osteosarcoma thin with the combination of 450-490nm LED- blue lights Karyon damages.

Claims (4)

1. the LED- blue light sources with 450-490nm wavelength use As in preparation2O3Treat answering in the assistive device of osteosarcoma With.
2. application as claimed in claim 1, it is characterised in that there is the LED- blue light sources and As of 450-490nm wavelength2O3 Combination, there is enhancing As2O3Suppress the effect of osteosarcoma cell growth, propagation, migration and invasion and attack.
3. application as claimed in claim 1, it is characterised in that there is the LED- blue light sources and As of 450-490nm wavelength2O3 Combination, having reduces As2O3The effect of toxicity.
4. application as claimed in claim 1, it is characterised in that there is the LED- blue light sources and As of 450-490nm wavelength2O3 Combination, there is enhancing As2O3Induce the effect of osteosarcoma cell cell nuclear damage.
CN201711139478.2A 2017-11-16 2017-11-16 450 490nm LED blue lights combine application of the arsenic trioxide in clinical treatment of osteosarcoma Pending CN107737409A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101185793A (en) * 2007-11-30 2008-05-28 徐岩 Application of chip integration LED in treating photo-power tumor and treating equipment
CN106512230A (en) * 2016-12-08 2017-03-22 吉林大学 Blue light-all-transretinoic acid-nano-diamond synergistic treatment device for treating leukemia

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101185793A (en) * 2007-11-30 2008-05-28 徐岩 Application of chip integration LED in treating photo-power tumor and treating equipment
CN106512230A (en) * 2016-12-08 2017-03-22 吉林大学 Blue light-all-transretinoic acid-nano-diamond synergistic treatment device for treating leukemia

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