CN107432873A - Application of glaucocalyxin A in preparation of anti-human osteosarcoma drugs - Google Patents
Application of glaucocalyxin A in preparation of anti-human osteosarcoma drugs Download PDFInfo
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- UCDVIBNDYLUWFP-MJTHGBBVSA-N glaucocalyxin a Chemical compound C([C@@H]1[C@@H](O)[C@]2(C(C1=C)=O)[C@H](O)C1)C[C@H]2[C@@]2(C)[C@H]1C(C)(C)C(=O)CC2 UCDVIBNDYLUWFP-MJTHGBBVSA-N 0.000 title claims abstract description 83
- UCDVIBNDYLUWFP-UHFFFAOYSA-N glaucocalyxin A Natural products C1C(O)C2(C(C3=C)=O)C(O)C3CCC2C2(C)C1C(C)(C)C(=O)CC2 UCDVIBNDYLUWFP-UHFFFAOYSA-N 0.000 title claims abstract description 82
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- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides application of glaucocalyxin A in preparing a medicament for resisting human osteosarcoma, and relates to the field of antitumor medicaments. A large number of experiments show that the glaucocalyxin A can inhibit the anti-apoptosis gene Bcl-2 and promote the protein expression of the apoptosis promoting gene Bax by up-regulating ROS, activate Caspase cascade reaction and induce MG-63 cell apoptosis. The effect of glaucocalyxin A in inducing apoptosis is also achieved by promoting phosphorylation of JNK and nuclear transcription of AP-1. The glaucocalyxin A with low concentration can inhibit the healing of scratches of the MG-63 cells of the osteosarcoma and can obviously inhibit the transmembrane capability of the Transwell of the MG-63 cells of the osteosarcoma, thereby inhibiting the migration of the MG-63 cells. Therefore, the glaucocalyxin A can be applied to preparing the anti-human osteosarcoma medicine.
Description
Technical field
The present invention relates to field of antineoplastic medicaments, and in particular to glaucocalyxin A answering in the medicine for preparing anti-human osteosarcoma
With.
Background technology
Human osteosarcoma is most common primary bone malignant tumour, and from interstitial cell, system develops, have grade of malignancy it is high,
The characteristics of growth is rapid, the early stage rate of transform is high.It is annual to there are about 200 ~ 3,000,000 people illness, early stage transfer easily occurs, it is most common
Metastasis site be lung, at present the 5 of Patients with Osteosarcoma year overall survival be 20%~30%, Most patients die from tumour turn
Move.Generally acknowledged clinical treatment of osteosarcoma pattern is both at home and abroad：" preoperative neoadjuvant chemotherapy+total surgical resection+adjuvant chemotherapy of patients ", bone
The multiple medicine joint of the single medicine application of the chemotherapy of sarcoma from the beginning gradually till now, the wherein selection of front-line chemotherapeutic agents are still
It is so the medicines such as adriamycin, methotrexate (MTX).But repeatedly there is resistance phenomenon with the use or the state of an illness of chemotherapeutics, and
And the tumour of transfer all brings certain difficulty to traditional chemoresistance, these situations to clinical treatment.Although chemotherapeutic
The application of thing significantly improves the therapeutic effect of osteosarcoma, but is still difficult to reach expected treatment results at present, in the past 30 years,
The survival rate of Patients with Osteosarcoma fails to be obviously improved always, early stage transfer be cause tumor prognosis difference one it is important because
Element.So finding, more efficiently chemotherapeutics is treated and more efficiently treatment method is to improve Patients with Osteosarcoma
Survival rate turn into current urgent requirement.
In recent years, the features such as Chinese medicine is because of its security and few side effects are as one of study hotspot of medical oncology.It is blue
Calyx A prime (glaucocalyxin A) is from Labiatae Rabdosia plant Rabdosia amethystoides HamRabdosia
The diterpene compound that separation and Extraction goes out in amethystoieds (Benth).Research has shown that:Glaucocalyxin A has antibacterial, resisted
The multiple biological activities such as oxidation.
The content of the invention
It is an object of the invention to provide application of the glaucocalyxin A in the medicine for preparing anti-human osteosarcoma.
It is a further object of the present invention to provide the pharmaceutical composition of anti-human osteosarcoma, its active component is glaucocalyxin A.
MTT experiment, DAPI dyeing and Annexin V-FITC/PI flow cytomery results show glaucocalyxin A
Can be with inducing cell MG-63 apoptosis in the concentration higher than 1.5 μm of ol/L, and dose dependent is presented.
DCF sonde methods, Western Blot and immunofluorescence results confirm that glaucocalyxin A is by raising ROS（Active oxygen
Cluster）Mitochondrial apoptosis path is inspired, and by activating JNK（c-Jun）Phosphorylation inspire AP-1 consideration convey record, effectively suppress
Human osteosarcoma cell MG-63 propagation.
Glaucocalyxin A can effectively suppress the cut of human osteosarcoma cell in the range of concentration is 0.75-3 μm of ol/L
Film ability is worn in wound healing, Transwell cells.
Found by many experiments, glaucocalyxin A can suppress anti-apoptotic genes expression Bcl-2 and promote rush to wither by raising ROS
Gene Bax protein expression is died, activates Caspase cascade reactions, induces MG-63 Apoptosis.Glaucocalyxin A is apoptosis-induced
Effect is also recorded by the phosphorylation and AP-1 consideration convey that promote JNK.It is thin that the glaucocalyxin A of low concentration can suppress osteosarcoma MG-63
Born of the same parents' cut heals, and can significantly inhibit osteosarcoma MG-63 cell Transwell and wear film ability, so as to suppress moving for MG-63 cells
Move.In summary, glaucocalyxin A can apply to prepare the medicine of anti-human osteosarcoma.
Brief description of the drawings
Fig. 1 shows influence of the various concentrations glaucocalyxin A to MG-63 cell survival rates, and C represents the concentration of glaucocalyxin A（μM）；Its
Middle Fig. 1（A）Middle glaucocalyxin A is 24h, Fig. 1 to the action time of MG-63 cells（B）Work of the middle glaucocalyxin A to MG-63 cells
It is 48h with the time.
Fig. 2 shows influence of the glaucocalyxin A to MG-63 apoptosis rates, and wherein Control represents Normal group,
1.5 μM, 3 μM, 6 μM of concentration for representing glaucocalyxin A in cell intervention.
The apoptosis rate of the double dye method measure cells of Fig. 3 Annexin V/PI, wherein Control represent negative control group, 1.5 μ
M, 3 μM, 6 μM of glaucocalyxin A interventions represented using 1.5 μM, 3 μM or 6 μM.
Fig. 4 glaucocalyxin As are to intracellular ROS（Reactive oxygen species）The influence of level change, control is negative control group,
1.5 μM, 3 μM, 6 μM of glaucocalyxin A interventions represented using 1.5 μM, 3 μM or 6 μM, 3 μM of+NAC represent the indigo plant using NAC and 3 μM
Calyx A prime is intervened simultaneously.
Fig. 5 glaucocalyxin As are to Bcl-2, Bax, caspase-3 albumen in MG-63 cells and Cytochrome c（Cell color
Plain C）The influence of expression, the title of albumen is shown per a line band left side, each row band cell processing is shown above figure
During glaucocalyxin A concentration.
Influence of Fig. 6 glaucocalyxin As to JNK pathway associated protein expressions, wherein each adhesive tape left side shows albumen
The title of band, top show the concentration of glaucocalyxin A in each cell processing procedure, and control represents negative control group.
The influence that Fig. 7 glaucocalyxin As and JNK inhibitor SP600125 record to AP-1 consideration conveys in MG-63 cells, the left side
Control represents negative control group, GLA（3μM）Represent the experimental group of 3 μM of glaucocalyxin A interventions, SP600125+GLA（3μM）Table
Show SP600125 and 3 μM of glaucocalyxin A while the experimental group intervened；AP-1, DAPI and Merge of top represent that AP-1 is respectively
Nuclear factor, DAPI are the fluorescent dye that can be combined with DNA, and Merge is the superposition of the fluorescence photo in two.
Influence of Fig. 8 glaucocalyxin As to MG-63 cell cut abilities, the left side represent that 0h, 24h represent patients before and after intervention respectively,
Following control represents negative control group, the cell of 0.5 μM of expression glaucocalyxin A intervention.
Influence of Fig. 9 glaucocalyxin As to MG-63 cell migration abilities, wherein Fig. 9（A）For negative control, Fig. 9（B）, Fig. 9
（C）, Fig. 9（D）The intervention concentration of middle glaucocalyxin A is respectively 0.25 μM, 0.5 μM and 1 μM.
Embodiment
Reagent and instrument：MEM dehydrated mediums (GIBCO, article No.：41500034)；Sodium acid carbonate（Shanghai Ling Feng chemistry examinations
Agent Co., Ltd）；Hyclone (Hyclone Corporation), trypsase (AMRESCO, USA), Thiazolyl blue (MTT,
), Sigma glaucocalyxin A（Shanghai Yuan Ye bio tech ltd）, DAPI dyestuffs（Green skies bio tech ltd）,
Annexin V-FITC/PI apoptosis kits（Chinese Kai base）；Multiskan MK3 type enzyme-linked immunosorbent assay instruments
(Thermo, USA), Nikon TS100 types inverted biologic microscopes (Nikon, Japan)；CO2Incubator (NUAIRE,
USA)；Biohazard Safety Equipment (NUAIRE, USA), flow cytometer (Becton, Dickinson and Company, USA).
Cell culture：Human osteosarcoma cell MG-63 cell lines are purchased from American type culture collection, i.e. ATCC cells
Storehouse.The conventional culture methods of osteosarcoma cell MG-63 cell lines：Using MEM nutrient solutions, it is placed in 37 DEG C, 5%CO2Cell culture incubator
Middle culture.
The mtt assay of embodiment 1 detects influence of the glaucocalyxin A to MG-63 cell survival rates
Influence of the glaucocalyxin A to MG-63 cell survival rates is investigated, the investigation scope of glaucocalyxin A administration concentration is 50 μM, 20 μ
M, 10 μM, 5 μM, 2 μM, 1 μM, 0.5 μM, 0.1 μM, 0.05 μM, specific method is as follows：Take and exponential phase is in culture dish
MG-63 cells, after digestion, MEM nutrient solution of the certain volume containing 10%FBS is added, cell concentration is adjusted to 5 × 104Individual/mL,
Then it is inoculated in per the μ L of hole 100 in 96 well culture plates, is placed in 5%CO2, cultivate 24h in 37 DEG C of incubators, remove nutrient solution, administration
Group, which is separately added into use, contains 2% FBS and 0.1%（Concentration expressed in percentage by volume, similarly hereinafter）DMSO MEM nutrient solutions configuration containing 50,20,
10th, the nutrient solution of 5,2,1,0.5,0.1,0.05 μM of glaucocalyxin As, add in blank control group and contain 2% FBS and 0.1% DMSO
MEM nutrient solutions, every group sets 6 multiple holes.After pharmaceutical intervention 24h, 48h, 15 μ L, 5mg/mL MTT are added per hole（Tetrazolium bromide）
Solution, continue to be put into CO2gas incubator and cultivate 4h, suck MTT solution, 150 μ L DMSO dissolving knots are respectively added per hole
Crystalline substance, absorbance of each hole under 492nm wavelength is detected using Multiskan MK3 types enzyme-linked immunosorbent assay instrument.Administration group is thin
Born of the same parents' survival rate is calculated with below equation：Survival rate=administration group absorbance/control group absorbance * 100%.
MTT results are shown in Fig. 1, as a result show, compared with Normal group, after 24h is administered, and 50 μM of glaucocalyxin A（P ＜
0.05）Concentration can remarkably promote MG-63 apoptosis (IC50=23.42 μM)；48h after administration, 10 μM of glaucocalyxin A（P ＜
0.05）、20μM（P ＜ 0.05）、50μM（P ＜ 0.05）Concentration can remarkably promote MG-63 apoptosis (IC50=5.706 μM)；Its
Middle P ＜ 0.05 represent significant difference.The above results show：Glaucocalyxin A can effectively facilitate withering for human osteosarcoma MG-63 cells
Die, and show significant dose dependent and time dependence.
Influence of the DAPI of the embodiment 2 dyeing detection glaucocalyxin As to MG-63 apoptosis rates
MG-63 cells are inoculated in 6 orifice plates, after conventional method culture 24h, remove nutrient solution, experimental group, which is separately added into use, to be contained
The nutrient solution containing 1.5,3,6 μM of glaucocalyxin As of 2% FBS and 0.1% DMSO MEM nutrient solutions configuration, Normal group add
MEM nutrient solutions containing 2% FBS and 0.1%DMSO, supernatant is sucked after intervening 48h, washed three times with PBS, every time
5min, 30min is fixed with paraformaldehyde afterwards, PBS cleans 3 times, DAPI dyestuffs（4', 6- diamidino -2- phenyl Yin
Diindyl）8min is dyed, then is cleaned 3 times with PBS, finally in the intensity of fluorescence microscopy Microscopic observation blue-fluorescence.
DAPI coloration results, which are shown, sees Fig. 2, and compared with Normal group, after glaucocalyxin A administration 48h, its concentration is higher than 3 μ
M experimental group（6μM）Cell there is obvious apoptosis phenomenon.As a result show：Glaucocalyxin A can induce osteosarcoma cell
The apoptosis of MG-63 cells, and show dose dependent.
The apoptosis rate of the double dye method detection cells of the Annexin V-FITC/PI of embodiment 3
Using Annexin V-FITC/PI cell apoptosis detection kits（Chinese Kai base）The glaucocalyxin A of detection various dose is done
The pre- influence to MG-63 apoptosis rates in 48 hours.The kit include Binding buffer, AnnexinV-FITC and
Propidi μm of Iodide dyeing liquor.
MG-63 cells are inoculated in 6 orifice plates, after conventional method culture 24h, remove nutrient solution, experimental group, which is separately added into, adopts
The nutrient solution containing 1.5 μM, 3 μM or 6 μM glaucocalyxin As configured with the MEM nutrient solutions containing 2% FBS and 0.1%DMSO, it is negative right
The MEM nutrient solutions containing 2% FBS and 0.1%DMSO are added according to group, after intervening 48h, by pancreas egg of the MG-63 cells without EDTA
White enzymic digestion, 1200r/min centrifugation 10min, collects cell.Cell is resuspended in the Binding buffer for adding 500 μ L, then adds
5 μ l Annexin V-FITC are mixed, and are added 5 μ L Propidium Iodide dyeing liquors, are mixed at room temperature, and lucifuge is incubated
15min.Using flow cytomery apoptosis rate.
The double dye method results of Annexin V/PI such as Fig. 3.With the increase of glaucocalyxin A concentration, the apoptosis of MG-63 cells shows
It is incremented by as there is gradient.The summation of cell 48h apoptosis rate early apoptosis and late apoptic is 2.24% in negative control group,
When glaucocalyxin A concentration is 1.5,3,6 μM, MG-63 cells 48h apoptosis rate is respectively 34.71%, 46.10% and 61.90%.With
Negative control group is compared, and difference is statistically significant.As a result illustrate that glaucocalyxin A can effectively facilitate human osteosarcoma MG-63 cells
Apoptosis, and concentration dependent be present.
The DCF methods of embodiment 4 detect glaucocalyxin A to intracellular ROS（Reactive oxygen species）The influence of level change
MG-63 cells are inoculated in 24 orifice plates, after conventional method culture 24h, are removed nutrient solution, are given various dose（1.5μM、3
μM、6μM）Glaucocalyxin A intervene 3h, separately take one group use 1mmol/L NAC（N-acetylcystein, ROS scavengers）With 3
μM glaucocalyxin A intervene 3h simultaneously, negative control group adds the MEM nutrient solutions containing 2% FBS and 0.1%DMSO.Glaucocalyxin A,
NAC intervenes cell after being configured to finite concentration as solvent using the MEM nutrient solutions containing 2% FBS and 0.1%DMSO.Medicine
After acting on 3h, every group of addition fluorescent dye DCFH-DA（Sigma companies of the U.S.）, lucifuge is incubated 30min in 37 DEG C of incubators.
24 orifice plates are taken out, suck fluorescence dye liquor, cell is cleaned 3 times using the PBS solution of 4 DEG C of precoolings, it is molten with 4% paraformaldehyde afterwards
Liquid fixes 30min, and PBS solution is cleaned 3 times, finally in the intensity of fluorescence microscopy Microscopic observation green fluorescence.
By as shown in Figure 4, after handling MG-63 cells 3h with glaucocalyxin A, the glaucocalyxin A of each concentration promotes MG-63
The increase of intracellular ROS contents, and the increase of dose dependent is presented in ROS contents levels, and 6 μM of glaucocalyxin A dramatically increases
Intracellular ROS is horizontal；And intervene simultaneously by NAC and glaucocalyxin A, MG-63 cells caused by glaucocalyxin A can be suppressed
Rise horizontal interior ROS.
The Western Blot methods of embodiment 5 detect glaucocalyxin A to mitochondrial apoptosis pathway associated protein expression
Influence
1. Protein Extraction and its assay
Take the logarithm the MG-63 cells in growth period, pancreatin digestion is made single cell suspension, cell density is adjusted, with every hole 2mL
（Cell number 2 × 105）Volume is inoculated in 6 orifice plates.After conventional method culture 24h, culture medium is discarded, experimental group is given different dense
Degree（1.5μM、3μM、6μM）Glaucocalyxin A（Using the MEM nutrient solutions containing 2% FBS and 0.1%DMSO as solvent）Intervene 48h,
Negative control group adds the MEM nutrient solutions containing 2% FBS and 0.1%DMSO, is washed twice, added with the PBS solution of 4 DEG C of precoolings after 24h
Enter 500 μ LRIPA lysates（Green skies biotech company of China）, gently blow and beat repeatedly, be placed in and crack 30 minutes on ice：4
DEG C, centrifuge 15 minutes under the conditions of 12000rmp, take supernatant as protein extract.Then BCA determination of protein concentration reagents are used
Protein content after box detection various dose glaucocalyxin A intervention 48h in cell.
Western blot are tested
The protein for taking SDS-PAGE electrophoretic separation said extracteds to arrive, specific method：10% separation gel and 4% concentration glue are configured,
Per the μ g albumen of hole loading 30,80V operations 25min, subsequent 120V run 65min, then by protein delivery to pvdf membrane, with containing
5% skimmed milk power PBS closing 2h after with 1: 1000 dilute rabbit Bax, Bcl-2, Cleaved caspase-3,
The primary antibody of Cytochrome C and β-actin albumen（CST companies of the U.S.）4 DEG C are incubated overnight, and TBST is washed 3 times, each 10min,
Then 2 h are incubated with the secondary antibody (the goat antirabbit lgG of horseradish peroxidase-labeled, CST companies of the U.S.) of 1: 1000 dilution,
TBST buffer solutions are washed 3 times, each 10min, are finally developed with chemoluminescence method.Clinx Science chemiluminescence imaging systems
Image is gathered, Clinx Chemi Image Analysis image softwares carry out gray value analysis.
Western Blot experimental results Fig. 5 is shown, compared with negative control group, the intracellular Bcl-2 albumen of experimental group
Expression has reduced with the increase of glaucocalyxin A concentration, and corresponding is that Bax protein expressions significantly raise, and illustrates blue calyx
A prime can effectively induce MG-63 Apoptosis.With the gradual increase of glaucocalyxin A concentration, in MG-63 cell lines
Cleaved caspase-3 increase therewith, show that it causes the apoptotic response Cascaded amplification of Caspase series, final to promote carefully
Born of the same parents' apoptosis.The above results show glaucocalyxin A by inducing ROS further to activate mitochondrial apoptosis path, and induction human osteosarcoma is thin
Born of the same parents' MG-63 apoptosis.
The Western Blot methods of embodiment 6 detect influence of the glaucocalyxin A to JNK pathway associated protein expressions
MG-63 cells are inoculated in 6 orifice plates, after conventional method culture 24h, remove nutrient solution, experimental group gives various concentrations
（1.5μM、3μM、6μM）Glaucocalyxin A（Using the MEM nutrient solutions containing 2% FBS and 0.1%DMSO as solvent）Intervene 48h, it is negative right
The MEM nutrient solutions containing 2% FBS and 0.1%DMSO are added according to group.Then protein is extracted according to the method in embodiment 5, used
Western blot experiments detect the expression of JNK, p-JNK albumen in JNK paths using β-action as internal reference.Mtt assay is examined
Survey influence of glaucocalyxin A and JNK specific inhibitor the SP600125 addition to MG-63 cell survival rates.
Western Blot experimental results such as Fig. 6 is shown, compared with negative control group, p-JNK protein expressions are with blue calyx first
The increase of plain concentration and raise, other expressing quantities are constant.Illustrate that glaucocalyxin A can significantly promote JNK phosphorylation, it is right
Total protein does not influence.Meanwhile MTT results display addition JNK specific inhibitor SP600125 can effectively suppress blue calyx
The MG-63 Apoptosis that A prime inspires.Therefore, this experimental studies results shows that glaucocalyxin A can be by phosphorylation JNK albumen
Inspire Apoptosis.
Embodiment 7：The influence that immuno-fluorescence assay glaucocalyxin A and JNK inhibitor are recorded to AP-1 consideration conveys
MG-63 cells are inoculated in laser co-focusing capsule, after conventional method culture 24h, remove nutrient solution, experimental group adds respectively
Enter the nutrient solution containing 3 μM of glaucocalyxin As that is configured using the MEM nutrient solutions containing 2% FBS and 0.1% DMSO or containing 10 μM
SP600125（JNK specific inhibitor）Nutrient solution, negative control group add containing containing 2% FBS and 0.1%DMSO MEM
Nutrient solution, supernatant is sucked after intervening 48h, PBS is washed three times, each 3min, is consolidated afterwards with 4% paraformaldehyde solution
It is fixed, PBS cleaning, then with 0.5% Triton X-100 solution room temperature perforation 20min, PBS embathes, water suction
Paper blots PBS, and lowlenthal serum, room temperature closing 30min are added dropwise on slide, and blotting paper sops up confining liquid, do not washed, each
Enough AP-1 diluted primary antibody is added dropwise in ware（CST companies of the U.S.）And wet box is put into, 4 DEG C of overnight incubations；Second day
PBST buffer solutions embathe, and blotting paper blots the fluorescence secondary antibody for being added dropwise and having diluted after surplus liquid on creep plate（DyLight 488-
CST companies of the conjugated Affini-Pure goat anti-rabbit lgG secondary antibodies. U.S.）
（Step lucifuge afterwards）, 20-37 DEG C of incubation 1h, PBST buffer solutions embathe in wet box, and DAPI is added dropwise（4', 6- diamidino -2- phenyl
Indoles）Lucifuge is incubated 5min, and PBST buffer solutions embathe, wash away unnecessary DAPI, the liquid on creep plate is blotted with blotting paper, with containing
The mounting liquid of anti-fluorescence quenching（Green skies biotech company of China）Mounting, finally gather and scheme in fluorescence microscopy Microscopic observation
Picture.
As a result as shown in fig. 7, compared with negative control group, glaucocalyxin A can significantly promote AP-1（A kind of transcriptional activation
The factor）Consideration convey record.After adding JNK specific inhibitor SP600125, AP-1 consideration convey is recorded by a certain degree of suppression,
Illustrate that glaucocalyxin A rings MG-63 apoptosis by activating JNK phosphorylation and being made video recording and then promotion AP-1 consideration convey.
Influence of the glaucocalyxin A of embodiment 8 to MG-63 cell cut healing abilities
The MG-63 cells in exponential phase are taken to be inoculated in 12 orifice plates, conventional method culture 24h to cell is merged substantially.
With 200 μ L micropipette head in 12 orifice plates vertical cut, PBS solution rinse 2 times after, it is molten to give 0.5 μM of glaucocalyxin A
Liquid（Using the MEM nutrient solutions containing 2% FBS and 0.1%DMSO as solvent）Intervene 24h, add in negative control group containing 2% FBS and
0.1% DMSO MEM nutrient solutions.Observed respectively with inverted phase contrast microscope.
As a result it is as shown in Figure 8：Scratch experiment shows that glaucocalyxin A is after 24h is intervened, compared with negative control group, administration
The MG-63 cell migrations distance of group reduces, and concentration dependent is presented.After 0.5 μM of glaucocalyxin A solution processing, cell moves
Distance is moved significantly less than negative control group.
Influence of the glaucocalyxin A of embodiment 9 to MG-63 cell migration abilities
The MG-63 cells in exponential phase are taken, are resuspended after Trypsin Induced with the MEM nutrient solutions of serum-free, cytometer
Number；Transwell cells are placed in 24 orifice plates, cell upper strata adds the cell that 200 μ L contain various dose glaucocalyxin A and hanged
Liquid（Glaucocalyxin A concentration is respectively 0.25,0.5,1 μM, and solvent is the MEM nutrient solutions containing 0.1%DMSO）, every hole inoculation 5 ×
104Individual cell；The cell suspension for the MEM nutrient solutions that 200 μ L contain 0.1%DMSO is added in negative control group, is equally inoculated with per hole
5×104Individual cell；Transwell cells lower floor adds MEM nutrient solutions of the 600 μ L containing 10% hyclone and 0.1%DMSO.
Upper and lower layer nutrient solution is abandoned in 37 DEG C of culture 24h, suction, and PBS solution is cleaned 3 times；Add 600 μ L formaldehyde and fix 30min, PBS solution is clear
Wash 3 times；600 μ L Giemsa stains dyeing 30min is added, PBS solution is cleaned 3 times；It is with cotton swab that Transwell cells upper strata is thin
Born of the same parents wipe away；Transwell cells film is cut, neutral gum is closed on slide；Under microscope count through cell number
Mesh.
As a result as shown in figure 9, compared with negative control group, what glaucocalyxin A can substantially suppress osteosarcoma MG-63 wears film energy
Power, the inhibition highly significant of 0. 5 μM and 1 μM of glaucocalyxin A.
From embodiment 1-10 it will be seen that glaucocalyxin A inspires mitochondrial apoptosis by raising intracellular ROS leads to
Road, while JNK paths and AP-1 consideration convey record are activated, promote the apoptosis of cell.Found by lot of experiments, in mitochondria
The expression of related gene be changed, such as suppress anti-apoptotic genes expression Bcl-2 and promote apoptogene Bax expression, activation
Caspase cascade reactions, induce MG-63 Apoptosis.The glaucocalyxin A of low concentration can suppress osteosarcoma MG-63 cell cut
Healing, can significantly inhibit Transwell and wear film ability, so as to suppress the transfer of MG-63 cells.In summary, glaucocalyxin A
It can apply to prepare the medicine of anti-human osteosarcoma.
Claims (2)
1. application of the glaucocalyxin A in the medicine for preparing anti-human osteosarcoma.
2. the pharmaceutical composition of anti-human osteosarcoma, its active component is glaucocalyxin A.
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