CN109908160A - Huang presss from both sides time application of the second glycosides as anti-tumor drug - Google Patents
Huang presss from both sides time application of the second glycosides as anti-tumor drug Download PDFInfo
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Abstract
The invention belongs to natural medicine technical fields, more particularly to yellow application of the folder time second glycosides as anti-tumor drug.The tumour includes osteosarcoma, breast cancer, cervical carcinoma, oophoroma, neuroblastoma and liver cancer.The present invention has found the yellow broad spectrum anticancer effect for pressing from both sides time second glycosides in research, has Inhibit proliferaton effect and a direct killing effect to kinds of tumor cells, but to normal endothelial cell and cardiac muscle cell and has no significant effect.Its antitumor mechanism mainly includes inhibiting tumor cell proliferation, promotes tumor death, and cell-cycle arrest inhibits neonate tumour blood vessel, tumour immunity etc..
Description
Technical field
The invention belongs to natural medicine technical fields, more particularly to yellow application of the folder time second glycosides as anti-tumor drug.
Background technique
Tumour is body under the effect of various carcinogenic factors, some cell of local organization loses pair at the genetic level
Its normal regulation grown leads to its clonal abnormality hyperplasia and the neoformation that is formed, is divided into benign tumour and malignant tumour two
Major class, wherein there is bigger threat to human life and health in malignant tumour.Currently, there are many treatment method of malignant tumour, early stage
Tumour is based on operative treatment, and Advanced cancers are based on Radiotherapy chemotherapy.Malignant tumour is easy transfer in early stage, gives surgical operation
Excision brings great difficulty；While tumors destroyed cell, patient is influenced by its toxic side effect and is shown Radiotherapy chemotherapy
Poor prognosis.And the front-line chemotherapeutic agents in currently available technology, which exist, is easy that drug resistance, easy to recur, side effect is big etc. asks
Topic.
In various malignant tumours, osteosarcoma (OS) is the most common Primary Malignant Bone Tumor in long bone, wherein children
With teenager's special hazard, child morbidity is 0.2-3/100,000 every year, and teenager is 0.8-11/10 ten thousand.Transfer is OS phase
Shut the main reason for dying.Although effective chemotherapeutics and new adjuvant chemotherapy make 5 years survival rates of patient increase to 66-
82%, only 30% metastatic OS patient reaches 5 years Event-free survival phases.What transfer can be detected in those survives for patient 5 years
Rate is down to 19%.In addition, even if 40% will continue to develop as secondary transfer in the patient of no primary transfer；In a Xiang Yan
In studying carefully, the survival rate for the patient that the high-level osteosarcoma of non-metastatic then shifts is respectively 5% and 23%, be respectively within 4 years lung and
Bone tumour.Meanwhile classic chemotherapy drug, simultaneously with biggish side effect, Patients with Osteosarcoma is poor to classic chemotherapy drug resistance
It is another major reason of Patients with Osteosarcoma survival rate stagnation.
Summary of the invention
There is the problems such as easy drug resistance, easy to recur, side effect is big, this hair for front-line chemotherapeutic agents in the prior art
It is bright to provide yellow application of the folder time second glycosides as anti-tumor drug.
And the present invention also provides a kind of methods for inhibiting external osteosarcoma proliferative cell proliferation.
And the present invention also provides a kind of broad-spectrum anti-cancer drug compositions.
To achieve the above object of the invention, the embodiment of the invention provides Huangs to press from both sides time application of the second glycosides as anti-tumor drug.
In terms of existing technologies, yellow to press from both sides the natural products that time second glycosides is a kind of cardiac glycosides, derive from middle cartridge bamboo
Peach section Luckynut Thevetia Seed (Thevetia peruviana (Pers.) K.Schum.) extract.The present invention has found Huang in research
The broad spectrum anticancer effect of time second glycosides is pressed from both sides, antitumor mechanism includes inhibiting tumor cell proliferation, promoting apoptosis, Cycle Arrest, suppression
System migration, angiogenesis inhibiting and tumour immunity etc. have Inhibit proliferaton effect and direct killing effect to kinds of tumor cells.
But Huang presss from both sides time second glycosides to normal endothelial cell and cardiac muscle cell and has no significant effect, as shown in Figure 24, Figure 15, the secondary second glycosides of Huang folder
To mouse weight, organ index, electrocardiogram simultaneously has no significant effect, it was demonstrated that the yellow time second glycosides that presss from both sides is to the selectivity and safety of tumour
Property.
Preferably, the tumour includes osteosarcoma, breast cancer, cervical carcinoma, oophoroma, neuroblastoma and liver cancer.It is yellow
Pressing from both sides time second glycosides has Inhibit proliferaton effect or direct killing effect to the tumour cell of above-mentioned tumour.
Preferably, the anti-tumor drug is anti-bone and flesh tumor medicine.
Preferably, the anti-tumor drug is anti-breast cancer medicines.This research finds that Huang presss from both sides time second by experiment in vivo
Glycosides can inhibit mouse breast cancer to grow.Currently, by having used targeting immunologic test point modulin (such as PD-1) in clinic
Having witnessed activating immune system being capable for the treatment of cancer.Huang presss from both sides time second glycosides and can significantly activate tumour immunity through a variety of ways and lower
The expression of PD-1, to inhibit the growth of 4T1 breast cancer.And inventor has found that although yellow press from both sides secondary second glycosides in body
Outer there is no the effects for inhibiting 4T1 Cells Proliferation of Human Breast Cancer, but can obviously inhibit the growth of 4T1 breast cancer in vivo.
Preferably, the anti-tumor drug is the drug for activating tumour immunity.Huang, which presss from both sides time second glycosides, can significantly activate tumour
It is immunized and lowers the expression of PD-1, to reach antitumor action.
Preferably, the anti-tumor drug is the drug for inhibiting neonate tumour blood vessel.Neovascularization (angiogenesis)
It is the basis of tumour growth.Due to anoxic, tumor tissues generate and discharge angiogenesis growth factor, such as vascular endothelial growth
The factor (VEGF), acid and basic fibroblast growth factor (aFGF and bFGF) and platelet-derived endothelial cell are raw
The long factor (PD-ECGF), to form new blood vessel.Huang, which presss from both sides time second glycosides, can significantly inhibit endothelial cell vascularization, have and inhibit swollen
The activity of tumor angiogenesis.
And the embodiment of the invention also provides a kind of method for inhibiting external osteosarcoma proliferative cell proliferation, Huang is pressed from both sides
Secondary second glycosides is added in the culture solution of tumour cell, and Huang presss from both sides time final concentration of 10~100nM of the second glycosides in the culture solution.?
In 10~100nM concentration range, Huang, which presss from both sides time second glycosides, can inhibit endothelial cell vascularization, inhibit human osteosarcoma cell's migration.
And the embodiment of the invention also provides a kind of broad-spectrum anti-cancer drug compositions, which is characterized in that described is anti-
Time second glycosides is pressed from both sides containing yellow in tumors pharmaceutical combination.
Preferably, tablet, capsule, granule, oral solution, sustained release is made in the broad-spectrum anti-cancer drug composition
Preparation, controlled release preparation, nanometer formulation or injection, to facilitate clinical use.
Detailed description of the invention
Fig. 1 is that the different cells screened in embodiment 1 by high intension method press from both sides time IC of second glycosides to drug Huang50It is bent
Line；
Fig. 2 is that the different cells screened in embodiment 1 by CCK-8 method press from both sides time IC of second glycosides to drug Huang50It is bent
Line；
Fig. 3 is the effect picture of the U2OS cell cycle of high intension Fluorescence image analysis detection in embodiment 2；
Fig. 4 is the quantization figure of the U2OS cell cycle of high intension analysis in embodiment 2；
Fig. 5 is the effect picture of the U2OS cell cycle of flow cytomery in embodiment 2；
Fig. 6 is the quantization figure of the U2OS cell cycle of flow cytomery in embodiment 2；
Fig. 7 is the result figure of Flow cytometry U2OS Apoptosis in embodiment 3；
Fig. 8 is the quantization figure of Flow cytometry U2OS Apoptosis in embodiment 3；
Fig. 9 is that Western blot analyzes U2OS apoptosis-related protein Bax, Bcl-2 and Caspease-3 egg in embodiment 3
The effect picture of white expression；
Figure 10 is that U2OS cell carries out external cut experimental result in embodiment 4；
Figure 11 is endothelial cell mobility line chart in different time points in U2OS cell scratch experiment in embodiment 4；
Figure 12 is the picture of the 20th day three groups of mouse tumor after inoculated tumour cell in embodiment 5；
Figure 13 is the weight in wet base figure of each group tumour in embodiment 5；
Figure 14 is the size of tumour and the weight time history plot of mouse in embodiment 5；
Figure 15 is each organ index after three groups of mouse administrations in the 20th day after inoculated tumour cell in embodiment 5；
Figure 16 is electrocardiogram monitoring result after three groups of mouse administrations in the 19th day after inoculated tumour cell in embodiment 5；
Figure 17 is that solvent control group and high dose group tumor locus blood vessel CT develop in embodiment 6；
Figure 18 is the yellow blood for pressing from both sides time second glycosides group, blank control group, positive controls and being formed after being administered respectively in embodiment 6
Pipe number is quantitatively schemed；
Figure 19 is that EA.hy926 cell carries out external cut experimental result in embodiment 6；
Figure 20 is endothelial cell mobility broken line in different time points in EA.hy926 cell scratch experiment in embodiment 6
Figure；
Figure 21 is the tumour picture of the 4T1 tumor model mouse of micro-CT scanning in embodiment 6；
Figure 22 is the vascular distribution figure that ImageJ is extracted in embodiment 6；
Figure 23 is the histogram of the number of branch vessel in embodiment 6；
Figure 24 is the difference expression gene of tumor model blank control group (C) and tumor model administration group (H) in experimental example 7
(DEG) the volcano figure of the overall distribution (multiple variation 2 and p value 0.05) of difference expression gene；
Figure 25 is that hierarchical cluster indicates the significant and different expression in blank control group and administration group sample in experimental example 7
The general overview of transcript；
Figure 26 is preceding 10 signal paths of tumor model blank control group and tumor model administration group in experimental example 7；
Figure 27 is that the upstream of tumor model blank control group and tumor model administration group differential gene regulates and controls base in experimental example 7
Because of preceding 10 signal paths；
Figure 28 in tumour and is immunized for tumor model blank control group in experimental example 7 and tumor model administration group differential gene
Accounting in related target；
Figure 29 is point of differential gene relevant to tumour immunity in the mature access of Dendritic Cells (DCs) in embodiment 7
Analyse verification result.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, to this
Invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, not
For limiting the present invention.
Embodiment 1
The embodiment of the invention provides Huangs to press from both sides time second glycosides as broad-spectrum anti-cancer drug to kinds of tumor cells in-vitro multiplication
Active influence, specific as follows:
1, experimental method
1.1 experimental cell strains
EA.hy926 (human umbilical vein cell fusion cell strain), from Tianjin University Of Traditional Chinese Medicine's modern Chinese herbal medicine state key
Laboratory；
MYO (suckling mouse primary cardiomyocytes system), from modern Chinese herbal medicine National Key Laboratory, Tianjin University Of Traditional Chinese Medicine；
U2OS (cell line of human osteosarcoma), from modern Chinese herbal medicine National Key Laboratory, Tianjin University Of Traditional Chinese Medicine；
A2780 (human cervical carcinoma cell lines), from modern Chinese herbal medicine National Key Laboratory, Tianjin University Of Traditional Chinese Medicine；
Hela (human oophoroma cell line), from modern Chinese herbal medicine National Key Laboratory, Tianjin University Of Traditional Chinese Medicine；
MCF7 (three positive breast cancer cells strain of people) is tested from Tianjin University Of Traditional Chinese Medicine's modern Chinese herbal medicine state key
Room；
SK-N-SH (neuroblastoma cell), from modern Chinese herbal medicine National Key Laboratory, Tianjin University Of Traditional Chinese Medicine；
MDA-MB-231 (people's triple negative breast cancer cell strain), from Tianjin University Of Traditional Chinese Medicine's modern Chinese herbal medicine state key
Laboratory；
4T1-luc (the mouse mastopathy cell strain with luciferase), from Tianjin University Of Traditional Chinese Medicine's modern Chinese herbal medicine
National Key Laboratory.
1.2 cell culture processes
Using the DMEM high glucose medium containing 10%FBS, in 37 DEG C, 5%CO2Above-mentioned cell is carried out in incubator normal
Rule culture, selects 3-9 for cell, when being fused to 80%~90%, with 0.25% pancreatin digestion 1min or so, is used for subsequent reality
It tests.
1.3 sample preparation
Huang is pressed from both sides into time second glycosides and is configured to 10 with DMSO-1mol/L、10-2mol/L、10-3The solution of mol/L, and it is divided into 10 μ L/
Pipe, is placed in -20 DEG C of preservations.Take 10-2M mother liquor, with containing 1%CS-FBS without phenol red training base into being diluted to 10-3M、10-4M、10- 5M、10-6M、10-7M、10-8M、10-9The sample solution of M.1.4 high intension cell imaging system detection Huangs press from both sides time second glycosides to different thin
The influence of cytoactive
With 1% FBS by the cell inoculation in above-mentioned 1.2 in 96 porocyte culture plates, 8000, every hole cell is adherent
After culture for 24 hours, various concentration Huang in 1.3 is added and presss from both sides time second glycosides sample (10mmol/L, 1mmol/L, 100nmol/L, 10nmol/
L, 1nmol/L) be incubated for medicine is sucked out afterwards for 24 hours, nucleus is dyed with Hoechst33342 (1 μ g/L), 37 DEG C incubation
30min, 1 × PBS are cleaned 3 times.5 visuals field, 10 times of object lens are taken pictures, are counted among every hole selection under high intension analysis instrument
Calculate the number of each hole inner cell.One-way analysis of variance (n=3) is carried out to each group of data using SPSS16.0 statistical software, most
Small significant difference level set is * P < 0.05, P < 0.01 * *, and is taken statistics figure with Graph Pad Prism 6, as shown in Figure 1,
Respectively MCF-7, Hela, U2OS, A2780, SN-N-SH, hy926, MYO, 4T1 and MDA-MB-231 press from both sides time second glycosides to drug Huang
IC50(concentration of inhibitor when being suppressed half) curve.The IC of each cell50Value is as shown in table 1.
The yellow time second glycosides that presss from both sides of table 1 is to the IC of different cells50Value
| Cells | IC50(mol/L) |
| MCF7 | 5.452×10-8 |
| Hela | 1.708×10-8 |
| U2OS | 1.742×10-8 |
| A2780 | 2.687×10-8 |
| SK-N-SH | 5.076×10-8 |
| EA.hy926 | —— |
| MYO | —— |
| 4T1 | —— |
| MDA-MB-231 | —— |
By Fig. 1 and table 1 as it can be seen that it is yellow press from both sides time second glycosides have to A2780, Hela, MCF7, SK-N-SH, U2OS this five kinds of cells it is aobvious
The effect of work property Inhibit proliferaton, while to normal cell (EA.hy926, MYO) without remarkable effect, to 4T1 and MDA-MB-231 without
In-vitro Inhibitory Effect.IC50Value as shown in table 1, prompts the yellow time second glycosides that presss from both sides that cell Proliferation is inhibited to have selectivity, may be swollen to source of people
The lethal effect conspicuousness of oncocyte is better than normal cell, and it may be a comparatively safe anti-tumor drug that Huang, which presss from both sides time second glycosides,.
The detection of 1.5CCK-8 kit is yellow to press from both sides time influence of the second glycosides to different cell activity
With 1% FBS by the cell inoculation in above-mentioned 1.2 in 96 porocyte culture plates, 8000, every hole cell is adherent
After culture for 24 hours, various concentration Huang in 1.3 is added and presss from both sides time second glycosides sample (10mmol/L, 1mmol/L, 100nmol/L, 10nmol/
L, 1nmol/L) it is incubated for for 24 hours.Referring to CCK-8 kit specification, every hole adds 10%CCK-8 solution, and 37 DEG C of incubators are incubated for 2h,
Using3 multi-functional each hole absorbance values of read plate machine testing 450nm wavelength.As a result as shown in Fig. 2 and table 2, Fig. 2
Time EC of second glycosides is pressed from both sides to drug Huang for MCF7, MDA-MB-231, Hela, A2780, SK-N-SH, U2OS, hy926, MYO and 4T150
Curve.The result that CCK-8 kit detects and high intension cell imaging analysis result are consistent, Huang press from both sides time second glycosides to A2780,
Hela, MCF7, SK-K-SH, U2OS have significant inhibited proliferation, to 4T1 without In-vitro Inhibitory Effect.
The yellow time second glycosides that presss from both sides of table 2 is to the IC of different cells50Value
Embodiment 2
The embodiment of the invention provides Huangs to press from both sides time influence of the second glycosides as anti-bone and flesh tumor medicine to the osteosarcoma cell period.
1. high intension cell imaging system detection Huang presss from both sides time influence of the second glycosides to the osteosarcoma cell period
With 1% FBS by the cell inoculation in above-mentioned 1.2 in 96 porocyte culture plates, 8000, every hole cell is adherent
After culture for 24 hours, control group to DMEM, administration group give containing it is yellow press from both sides time second glycosides sample concentration be respectively 100nmol/L, 10nmol/L,
The DMEM of 1nmol/L.Every group of 3 multiple holes, cell is in 37 DEG C, 5%CO2, saturated humidity CMC model, effect up to after 24 hours,
It carries out immunofluorescence dyeing: marking EDU with fluorescent dye Alexa488 (green), identify S phase cell；Use phospho-histone
PHH3 and its fluorescent dye Alexa647 label (red), identifies M phase cell；With fluorescent dye Hochest33342 (blue)
All nucleus are identified, determine that (G1 phase cell chromosome is the diploid phase, and the G2 phase is to have silk for G1 phase and G2 phase cell threshold value
Division stage includes into the cell two sets of complete diploid chromosomes, so its G2 phase cell fluorescence expression quantity is G1 phase fluorescence table
Up to two times of amount, with this given threshold range, to distinguish G1 and G2 phase).Every hole in high intension analysis instrument experimental setup
Choose intermediate 13 visuals field, taken pictures with 10 times of object lens, choose three channels (Alexa647, Alexa488 and
Hoechst33324), the time for exposure is respectively 3000ms, 1000ms and 200ms, is calculated by the cell of different fluorochrome labels
The number of core is calculated by the cell nucleus number of different antibodies or dye marker with high intension analysis software and is marked by EdU thin
Karyon fluorescence intensity is indicated with mean ± standard deviation, carries out single factor test variance to each group of data using SPSS16.0 statistical software
Analysis, least significant difference level set are * P < 0.05, P < 0.001 * * P < 0.01, * * *.And with Graph Pad Prism 6
Take statistics figure, as a result as shown in figs. 34.
Cell number of the U2OS cell in the different cell cycles as shown in Figures 3 and 4 accounts for the ratio of entire cell cycle cell number:
At phase cell cycle G0/G1, with control group ratio, ratio shared by time administration group cell of second glycosides is pressed from both sides containing 100nM and 10nM Huang
Example is remarkably decreased (P < 0.01), presss from both sides ratio no difference of science of statistics shared by time administration group cell of second glycosides containing 10nM and 1nM Huang；
At phase cell cycle S, compared with the control group, containing 100nM Huang press from both sides ratio shared by the administration group cell of time second glycosides it is significant on
It rises (P < 0.01), 10nM and the yellow of 1nM press from both sides ratio no difference of science of statistics shared by time second glycosides cell；In phase cell cycle G2 and M
When the phase, compared with the control group, ratio no difference of science of statistics shared by each administration group cell.That is the yellow time second glycosides that presss from both sides of 100nM can be shown
It writes and the U2OS cell generation cell cycle S phase is induced to block (P < 0.01).The result is pressed from both sides time second glycosides with 100nM concentration Huang and is significantly inhibited
The result of cell Proliferation is mutually echoed.
2. Flow Cytometry methods detection is yellow to press from both sides time influence of the second glycosides to the cell cycle of osteosarcoma cell
The 3rd~9 generation U2OS cell is selected, with the DMEM high glucose medium containing 10%FBS, in 37 DEG C, 5%CO2Culture
Carry out routine culture in case, it is to be fused to 80% or so when, change 1% FBS it is synchronous for 24 hours, control group is to DMEM, and administration group is to being contained
Huang presss from both sides the DMEM that time second glycosides sample concentration is respectively 100nmol/L, 10nmol/L, 1nmol/L, is incubated for for 24 hours；Collect cell, number
Mesh is about 1 × 106~5 × 106A/mL, 1000r/min are centrifuged 3min, discard culture solution；It washing 1 time with PBS, PBS is removed in centrifugation,
The 70%v/v ethyl alcohol of ice pre-cooling is added, 4 DEG C are fixed 12 hours or more；Centrifugation discards fixer, adds 3mL PBS that 5min is resuspended,
500~1000r/min is centrifuged 5min, discards PBS；It is dyed with 1mL PI dye liquor, 4 DEG C are protected from light 30min.
Flow cytomery: PI argon ion excites fluorescence, and laser optical wavelength is 488nm, and transmitting optical wavelength is big
In 630nm, the histogram of red fluorescence analysis PI fluorescence intensity is generated；
When analyzing the histogram of PI fluorescence, in pairs or aggregation cell first is excluded with gate technique and sends out week fluorescent
Cell fragment, on the histogram of PI fluorescence, there is a hypodiploid peak before the G1/G0 phase in apoptotic cell.As with G1/G0 phase institute
Fluorescence intensity in position is 1.0, then the fluorescence intensity at typical its hypodiploid peak of apoptotic cell sample is 0.45, can
Do reference standard with the PI fluorescence intensity of the red blood cell of chicken and salmon, the two is respectively 0.35 and 0.7, it can be ensured that the two it
Between be not cell fragment but complete cell.As a result as shown in Figure 5, Figure 6.
As shown in Figure 5, Figure 6: cell number of the U2OS cell in the different cell cycles accounts for the ratio of entire cell cycle cell number
Value: it at phase cell cycle G0/G1, with control group ratio, is pressed from both sides shared by time administration group cell of second glycosides containing 10nM and 100nM Huang
Ratio is remarkably decreased (P < 0.01), presss from both sides ratio no difference of science of statistics shared by time administration group cell of second glycosides containing 1nM Huang；Thin
When born of the same parents' phase in period S, compared with the control group, containing 100nM Huang press from both sides ratio shared by the administration group cell of time second glycosides significantly rise (P <
0.01), the yellow of 10nM and 1nM presss from both sides ratio no difference of science of statistics shared by time second glycosides cell；In phase cell cycle G2 and M phase,
Compared with the control group, ratio no difference of science of statistics shared by each administration group cell.That is the yellow time second glycosides that presss from both sides of 100nM can be induced significantly
The U2OS cell generation cell cycle S phase blocks (P < 0.01).The result and 100nM concentration Huang press from both sides time second glycosides and significantly inhibit cell increasing
The result grown mutually is echoed.
Embodiment 3
The embodiment of the invention provides Huangs to press from both sides time influence of the second glycosides as anti-bone and flesh tumor medicine to apoptosis in osteosarcoma cells.
1, Flow cytometry Huang presss from both sides time influence of the second glycosides to U2OS Apoptosis
The 3rd~9 generation U2OS cell is selected, with the DMEM high glucose medium containing 10%FBS, in 37 DEG C, 5%CO2Culture
Carry out routine culture in case, it is to be fused to 80% or so when, change 1% FBS it is synchronous for 24 hours, control group is to DMEM, administration group difference
The DMEM that time second glycosides sample concentration is respectively 100nmol/L, 10nmol/L, 1nmol/L is pressed from both sides to containing yellow, is incubated for for 24 hours；Sop up culture
Base supernatant is added appropriate PBS rinse, jiggles cell culture vessel, allows PBS to cover entire vessel surface, later uses up PBS
It may blot net.
It is added appropriate pancreatin, 37 DEG C of incubations, every 1 minute or so when takes out primary, jiggles cell culture vessel or light
Cell culture vessel side wall is clapped, the whether smooth levitating of cell is observed.When cell energy smooth levitating, 3 times of bodies of added pancreatin are added
Long-pending culture medium (containing 10%FBS) terminates digestion reaction.1000rpm is centrifuged 5 minutes, supernatant is removed as far as possible clean.
Cell precipitation is resuspended with binding buffer, is placed in 50-55 DEG C of hot water, as osteosarcoma cell to be measured.
Blank control (Control): untreated, achromophil cell is taken to carry out just step voltage.
The mono- dye pipe of Annexin V: above-mentioned osteosarcoma cell to be measured is taken, with the mono- dye of Annexin V.
Nucleic acid list dye pipe: above-mentioned osteosarcoma cell to be measured is taken, is contaminated with PI dyestuff list.
Negative control (Negative Control): it prepares press from both sides time cell suspension conduct of second glycosides without yellow according to the above method
Negative control sample, the bis- dyes of Annexin V and PI are used to picture door.
Positive control (Positive Control): taking above-mentioned osteosarcoma cell to be measured, and Annexin V and nucleic acid dye are double
Dye.
Cell is resuspended in 100 μ L binding buffer that (cell number is generally 105It is a), it is added to specifications suitable
Measure reagent.
Room temperature is protected from light incubation 15 minutes.
It is put on ice for after 400 μ L binding buffer are added in stained specimens, and is used within 1 day
Attune NxT flow cytomery.
The result of Flow cytometry U2OS Apoptosis is as shown in Figure 7, Figure 8, and 10nM and the yellow of 100nM press from both sides time second glycosides
U2OS Apoptosis can be can induce.
2, by the yellow time second glycosides that presss from both sides of Western blot analysis and research to human osteosarcoma cell's U2OS related apoptosis albumen table
The influence reached
As a result as shown in figure 9, with actin (β-Actin) for internal reference, compared with blank control group, 10nM and 100nM
The yellow time second glycosides pro apoptotic protein Bax protein expression that presss from both sides there is conspicuousness to increase (P < 0.05, P < 0.05), the yellow of 1nM presss from both sides time second glycosides
No difference of science of statistics；Suppression apoptotic proteins Bcl-2 protein expression has conspicuousness to reduce (P < 0.05, P < 0.01), and the yellow of 1nM presss from both sides time second
Glycosides no difference of science of statistics；Caspase-3 activation has conspicuousness to increase (P < 0.01, P < 0.01) with non-activated ratio, 1nM's
Huang presss from both sides time second glycosides no difference of science of statistics.
The results show that with blank control group ratio, the yellow time second glycosides that presss from both sides of 10nM and 100nM can promote to wither Western blot
The expression of protein Bax is died, the expression of suppression apoptotic proteins Bcl-2 is inhibited, activation apoptosis executes PROTEIN C aspease-3.
Embodiment 4
The embodiment of the invention provides Huangs to press from both sides time second glycosides as anti-bone and flesh tumor medicine to the shadow of osteosarcoma cell transfer ability
It rings.
1, the yellow time second glycosides that presss from both sides is to inhibition osteosarcoma cell (U2OS) transfer ability research
U2OS cell carries out external cut experiment.As shown in Figure 10, the yellow time second glycosides that presss from both sides of 100nM and 10nM significantly inhibits
U2OS cell migration.
Shown in U2OS cell different time points mobility line chart 11, at 12h time point, and control group ratio, 100nM with
The yellow time second glycosides that presss from both sides of 10nM significantly inhibits U2OS cell migration (P < 0.05)；The yellow time second glycosides that presss from both sides of 1nM inhibits U2OS cell migration
No difference of science of statistics；At time point for 24 hours, compared with the control group, the yellow time second glycosides that presss from both sides of 100nM and 10nM significantly inhibit U2OS cell
It migrates (P < 0.01), the yellow time second glycosides that presss from both sides of 1nM inhibits U2OS cell migration no difference of science of statistics.
Embodiment 5
The embodiment of the invention provides the yellow effects pressed from both sides time second glycosides and inhibit mouse breast cancer to grow as anti-breast cancer medicines.
Using ICR mouse, 18,4-5 week old, female is purchased from National Institute for Food and Drugs Control's Laboratory Animal Resource
Research institute, SPF grades.Room temperature raising, 12h are alternately illuminated, light application time 8:30a.m-8:30p.m.The use of animal is through Tianjin state
The approval of the border animal welfare committee, biological medicine joint study institute, meets Ethic review requirement.
Yellow folder time second glycosides solid powder is made into the mother liquor of 500mg/mL before experiment with DMSO, is dispensed into 4mLEP pipe,
5.5 μ L/ pipe, -20 DEG C of storages.
Mouse mastopathy cell in logarithmic phase growth is diluted to 5 × 10 with PBS7The cell suspension of/mL, by 5 ×
106It is a/only inoculum concentration, the oxter for being inoculated in nude mice is subcutaneous, grows into 300mm to it3(after inoculated tumour cell the 6th day),
Mouse is randomly divided into three groups by the size of gross tumor volume, i.e., yellow folder time second glycosides high dose group (1mg/kg), yellow folder time second glycosides are low
Dosage group (0.5mg/kg) and blank control group (physiological saline containing 0.2%DMSO), every group each 6, intraperitoneal injection is given daily
Medicine 1 time, and observe the state of mouse.Administration duration 15 days, an every 2 days mouse weights of measurement and tumour during entire treatment
Volume, and use following formula calculating gross tumor volume: the volume of tumour=long * wide squares/2.
It is cardiac glycoside compounds since Huang presss from both sides time second glycosides, the 19th day after inoculation, ethobrom was anaesthetized, electrocardiogram equipment
The heart function of mouse is detected, the yellow safety for pressing from both sides secondary second glycosides is detected.Pluck within 20th day eyeball and take blood, mouse cervical dislocation is put to death, take liver,
The heart, spleen, lung, kidney, thymus gland claim weight in wet base.Tumour is taken, claims weight in wet base, take pictures, be then placed in liquid nitrogen and be transferred to -80 DEG C of refrigerators after quick-frozen 2h
It saves, is used as subsequent experimental and investigates.
By Figure 12~14 as it can be seen that compared with blank control group, Huang, which presss from both sides time second glycosides high dose group, can significantly inhibit tumour growth
(P < 0.01), but the weight of three groups of 4T1 tumor model mouse changes over time no difference of science of statistics.The hearts of three groups of mouse, liver,
Spleen, lung, kidney, thymus gland and the weight in wet base of brain and the ratio of its own weight are as shown in figure 15, compared with control group ratio, high dose and low dosage
The heart of administration group mouse, liver, spleen, lung, kidney, the weight in wet base of thymus gland and brain and its own weight ratio all no difference of science of statistics, say
The bright yellow time second glycosides that presss from both sides is to the selectivity and safety of tumour.
The electrocardiogram of three groups of mouse of toy electrocardiogram equipment detection is as shown in figure 16, and heart function index is as shown in table 3, and compares
Group ratio, every heart function index no difference of science of statistics.
Embodiment 6
The embodiment of the invention provides Huangs to press from both sides time influence of the second glycosides as anti-breast cancer medicines to neonate tumour blood vessel ability.
1, the yellow time second glycosides that presss from both sides is to inhibition vascular endothelial cell tubule Forming ability research
Selecting EA.hy926, (human umbilical vein cell fusion cell is bought from Shanghai Inst. of Life Science, CAS
Cell resource center), it is incubated for the DMEM high glucose medium containing 10%FBS, is placed in 37 DEG C, 5%CO2Incubator carries out conventional
Culture.The 3rd~6 generation cell is selected, when being fused to 80%~90%, with 0.25% pancreatin digestion 1min or so, is used for subsequent reality
It tests.
Postdigestive EA.hy926 cell routine is inoculated in 75cm2In culture bottle, after adhere-wall culture 12h, no blood is changed into
Clear culture medium starvation is for 24 hours.BD matrigel shifts to an earlier date 4 DEG C and thaws overnight, 200 μ L sterilizing pipette tips, the pre-cooling of 96 orifice plates.In being carried out on ice chest
Operation, is rapidly added each hole bottom middle position (45 hole μ L/) for matrigel, and acutely concussion makes matrigel be paved with entire bottom hole,
It lies against 4 DEG C of refrigerators and stands 15min, room temperature is centrifuged 2500rpm, after 10min, in 37 DEG C, 5%CO2It is balanced in incubator
30min.Collected by trypsinisation cell, cell count.With every hole 1.5 × 104The density of a cell is inoculated in 96 orifice plates, blank
Control group is the culture medium containing 0.1%FBS, and the yellow folder time second glycosides of 100nmol/L is added in the cell culture medium of intervention group, positive
Control group adds 50ng/mL vascular endothelial growth factor (VEGF), respectively at 37 DEG C, 5%CO216h is cultivated in incubator.In height
Contain every hole under analysis instrument and choose intermediate 9 visuals field, is taken pictures with 10 times of object lens, as shown in figure 17.
The number for calculating vascular meshes structure in each hole carries out single factor test side to each group of data with SPSS16.0 statistical software
Difference analyses (n=3), and least significant difference level set is P < 0.05 * or P < 0.01 * *.As shown in figure 18, with blank control group
It compares, the yellow time second glycosides that presss from both sides of 100nM significantly inhibits endothelial cell vascularization (P < 0.01), and the VEGF of 50ng/mL can significantly promote
Into endothelial cell vascularization (P < 0.01).
By the result of Figure 17 and Figure 18 as it can be seen that the yellow time second glycosides that presss from both sides of 100nM can significantly inhibit endothelial cell vascularization, mention
Show that the yellow time second glycosides that presss from both sides there may be the activity for inhibiting neonate tumour blood vessel.
2, yellow to press from both sides time influence of the second glycosides to migration of vascular endothelial cells ability
EA.hy926 cell carries out external cut experiment.As shown in Figures 19 and 20, the yellow time second glycosides that presss from both sides of 100nM significantly inhibits
Endothelial cell migration.
Using the control group of the 1%FBS of not dosing as negative control, endothelial cell different time points mobility line chart is such as
Shown in Figure 20, at 12h time point, and control group ratio, 100nM it is yellow press from both sides time second glycosides significantly inhibit endothelial cell migration (P <
0.05)；The yellow time second glycosides that presss from both sides of 10nM and 1nM inhibits endothelial cell migration no difference of science of statistics, at time point for 24 hours, with control group
Compare, the yellow time second glycosides that presss from both sides of 100nM significantly inhibits endothelial cell migration (P < 0.01), and the yellow time second glycosides that presss from both sides of 10nM and 1nM inhibit
Endothelial cell migration no difference of science of statistics.
The experimental result of Figure 19 and Figure 20 is consistent with angiogenesis experimental result, prompts the yellow time second glycosides that presss from both sides that may have suppression
The potential of neonate tumour blood vessel processed.
3, the yellow time second glycosides that presss from both sides is to inhibition mouse interior tumor angiogenesis capability study
The 16th day (detailed modeling and administration process are shown in example 5), ethobrom is administered in grouping in tumor inoculation model mice
Anesthesia, radial artery detect the distribution situation of tumour peripheral vessels to contrast agent, micro-CT.
Figure 21 is the tumour picture of the 4T1 tumor model mouse of micro-CT scanning, and encircled is that mouse is subcutaneous swollen
Tumor, Figure 22 are the vascular distribution figure that ImageJ is extracted, and encircled is the vascular distribution situation on mouse subcutaneous tumor.Figure 23 is
The histogram of the number of branch vessel.It can intuitively see the vascular distribution situation around tumour, it is seen that it is high that Huang presss from both sides time second glycosides
Vascular distribution around dosage group tumour is obviously more obvious than solvent control group sparse, and the number of branch vessel is considerably less than solvent pair
According to group.
Experimental example 7
This experimental example provide it is yellow press from both sides time antitumor immune function Mechanism Study of the second glycosides as anti-breast cancer medicines, with normal
It advises RNA extraction method and RNA extraction is carried out to the tumour that refrigerator in embodiment 5 saves, gained RNA will be extracted and carry out oncogene
It investigates, detects the yellow time second glycosides that presss from both sides and inhibit mouse breast cancer mechanism of action.
1, experimental method
The acquisition of 1.1 samples and preparation
RNA degradation and pollution are monitored on 1% Ago-Gel: using NanoPhotometer spectrophotometer
(IMPLEN, CA, USA) checks RNA purity, usesIn 2.0FlurometerRNAAssay Kit is surveyed
It measures RNA concentration (Life Technologies, CA, USA), uses the RNA Nano of 2100 system of Bioanalyzer
6000Assay Kit (Agilent Technologies, CA, USA) assesses RNA integrality.
The library preparation of 1.2 transcript profiles sequencing
The input material that each sample uses 1 μ gRNA to prepare as RNA sample.It usesUltraTMRNA text
Library reagent preparation box (NEB, USA) generates sequencing library, and index codes are added in the sequence of attributes of each sample.Most
Afterwards, purified pcr product (AMPure XP system), and Library Quality is assessed in 2100 system of Agilent Bioanalyzer.
1.3 clusters and sequencing
Using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) in cBot Cluster
The cluster of coded samples is indexed on Generation System.In fasciation at rear, library prepared product is in Illumina
It is sequenced on Hiseq platform, and generates the pairing end reading of 125bp/150bp.
The analysis of 1.4 data
The control of a mass
The initial data (original reading) of fastq format is handled by internal perl script first.In this step, pass through
The reading comprising adapter is deleted, the reading comprising ploy-N and the low quality read from initial data read clean to obtain
Data (clean to read).Meanwhile Q20, Q30 and G/C content calculate clean data.All downstream analysis are based on high quality
Clean data.
B reading is mapped to reference to genome
The index of genome is referred to using Hisat2v2.0.4 building, and pairing tip cleaning is read and refers to genome
It compares.Select Hisat2 as mapping tool.
The quantization of c gene expression dose
It is mapped to the reading of each gene with HTSeq v0.9.1 meter record, is then based on each gene of length computation of gene
FPKM, and will reading count be mapped to the gene.
D Differential expression analysis
Using DESeq R packet (1.18.0) carry out two conditions/group Differential expression analysis (two biology of each condition
It repeats).Resulting P value is adjusted using the method for Benjamini and Hochberg to control false discovery rate.It is found by DESeq
The gene with whole value < 0.05 P be designated as differential expression.
E (DEGSeq duplicate for no biology) is before differential genes expression analysis, for each sequencing library,
It is read and is counted by a scaling normalization factor adjustment by edgeR program bag.It is carried out using DEGSeq R packet (1.20.0)
The Differential expression analysis of two kinds of conditions.P value is adjusted using Benjamini&Hochberg method.By 0.005 He of P value of correction
Log2 (multiple variation) is set as the threshold value of significant differential expression.
F passes through the analysis of IPA (Ingenuity Pathway Analysis) software implementation difference expression gene.2, it tests
As a result
Tumor tissues are subjected to transcriptome analysis, as shown in figure 24, wherein X-axis indicates gene expression in different samples
Change, the significant property of statistics of Y-axis expression gene expression difference, is the gene of up-regulation in the region of upper right side, in the region of upper left side
For the gene of downward.Blank control group and administration group difference expression gene totally 102 it can be seen from Figure 24, wherein raising
55, lower 47.Then software is analyzed with IPA to this 102 genes to analyze, as can see from Figure 25, control group
With the difference degree of administration group differential gene, two groups of gene color differences are bigger, and expression gene difference is bigger.
In ten accesses of Figure 26, first is Dendritic Cells (DCs) mature access, and Article 2 is adrenal medella
Signal path, Article 3 are natural killer cells signal path, and Article 4 is colorectal cancer transfer signal access, and Article 5 is
Ephrin a-signal access, Article 6 are eNOS signal path, Article 7 be γ-chain cell for being acted on by JAK1/JAK3 because
Subsignal access, Article 8 are macrophage, the effect path of fibroblast and endothelial cell in rheumatoid arthritis,
Article 9 is action pathway of the nuclear factor of activated T cells in immune response, and Article 10 is effect path of the leptin in obesity.
As can be seen from Figure 26 4 accesses are closely related with immune.It is first thin for auxiliary T in ten accesses in Figure 27
Born of the same parents break up access, Article 2 be kinase mediated immune response access, Article 3 be glucocorticoid receptor signal path, the 4th
Item is macrophage and t helper cell by IL-17A and IL-17F generation relevant cell factor signal path, and Article 5 is mould
Effect path of the formula identification receptor in bacterium and virus identification, Article 6 are logical for high mobility group protein B 1 (HMGB1) signal
Road, Article 7 are inflammation of asthma access, and Article 8 is that enterocyte passes through IL-17A and IL-17F generation relevant cell factor
Access, Article 9 are interleukin 15 (IL-15) signal path, and Article 10 is that activation t helper cell 1 and 2 signal of t helper cell are logical
Road.As can be seen from Figure 27,80% is closely related with immune pathway in preceding ten accesses.Blank control group and to yellow folder
The upstream regulating genes of second glycosides group difference expression gene carry out analysis and find that the highest access of P value is that T helper cell breaks up access,
T helper cell differentiation is pilot process in immune response, further proves that the yellow time second glycosides that presss from both sides is inhibited by activation immunization route
4T1 breast cancer.In addition, analyzing the differential gene that can be identified in Gene Card, as shown in figure 28, tumor model is empty
White control group and tumor model administration group differential gene ratio shared in tumour and immune related target are 73%.
This research, which to blank control group and to Huang presss from both sides time second glycosides group difference expression gene and carries out analysis, finds most crucial value most
High access is Dendritic Cells (DCs) mature access, and Dendritic Cells is the strongest professional antigen presenting cell of body function,
It can efficiently be absorbed, working process and present antigen, immature DC s have stronger transfer ability, and mature DCs can effectively swash
Primary tape T cell living, in starting, the key link for regulating and controlling and maintaining immune response.Generation, the development of DCs and tumour have
Substantial connection, the interior more then patient's good prognosis of DCs quantity infiltrated of most of solid tumor.This analysis result equally illustrates yellow folder time second
Glycosides inhibits 4T1 breast cancer by activation immunization route.Using RT-PCR method to related to tumour immunity in DCs maturation access
Differential gene carry out analysis verifying, as a result, it has been found that the yellow time second glycosides that presss from both sides can obviously raise FCGR3A and HLA-DRB1, lower
FGFR4, as shown in figure 29, the result are consistent with transcriptome analysis result.In addition, our significant genes to tumour immunity
The transcriptional level of PD-1 is analyzed, and is found the yellow expression pressed from both sides time second glycosides and obviously lowered PD-1, is further demonstrated Huang
Press from both sides the proliferation that time second glycosides inhibits 4T1 breast cancer cell in Mice Body by tumour immunity approach.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modification, equivalent replacement or improvement etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (9)
1. Huang presss from both sides time application of the second glycosides as anti-tumor drug.
2. yellow application of the folder time second glycosides as anti-tumor drug according to claim 1, which is characterized in that the tumour packet
Include osteosarcoma, breast cancer, cervical carcinoma, oophoroma, neuroblastoma and liver cancer.
3. yellow application of the folder time second glycosides as anti-tumor drug according to claim 1, which is characterized in that described is anti-swollen
Tumor medicine is anti-bone and flesh tumor medicine.
4. yellow application of the folder time second glycosides as anti-tumor drug according to claim 1, which is characterized in that described is anti-swollen
Tumor medicine is anti-breast cancer medicines.
5. yellow application of the folder time second glycosides as anti-tumor drug according to claim 1, which is characterized in that described is anti-swollen
Tumor medicine is the drug for activating tumour immunity.
6. yellow application of the folder time second glycosides as anti-tumor drug according to claim 1, which is characterized in that described is anti-swollen
Tumor medicine is the drug for inhibiting neonate tumour blood vessel.
7. a kind of method for inhibiting external osteosarcoma proliferative cell proliferation, which is characterized in that Huang is pressed from both sides time second glycosides and is added to tumour
In the culture solution of cell, Huang presss from both sides time final concentration of 10~100nM of the second glycosides in the culture solution.
8. a kind of broad-spectrum anti-cancer drug composition, which is characterized in that contain yellow folder in the antineoplastic pharmaceutical compositions
Second glycosides.
9. broad-spectrum anti-cancer drug composition as claimed in claim 8, which is characterized in that by the broad-spectrum anti-cancer drug
Tablet, capsule, granule, oral solution, sustained release preparation, controlled release preparation, nanometer formulation or injection is made in composition.
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