CN106074594A - The new application of Thallus Gracilariae polysaccharide and a kind of demethylation test kit and application - Google Patents
The new application of Thallus Gracilariae polysaccharide and a kind of demethylation test kit and application Download PDFInfo
- Publication number
- CN106074594A CN106074594A CN201610561746.9A CN201610561746A CN106074594A CN 106074594 A CN106074594 A CN 106074594A CN 201610561746 A CN201610561746 A CN 201610561746A CN 106074594 A CN106074594 A CN 106074594A
- Authority
- CN
- China
- Prior art keywords
- thallus gracilariae
- polysaccharide
- gracilariae polysaccharide
- application
- demethylation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/04—Rhodophycota or rhodophyta (red algae), e.g. Porphyra
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses Thallus Gracilariae polysaccharide as the new application of methylation of tumor suppressor inhibitor and the application in preparing antitumor drug thereof, also disclose a kind of demethylation test kit containing Thallus Gracilariae polysaccharide.The present invention discloses the epigenetic regulation mechanism of marine Chinese medicine Thallus Gracilariae polysaccharide effect of anti-lung cancer first from DNA methylation angle, find in pulmonary carcinoma, gastric cancer and cervical cancer cell that the antioncogenes such as SHP1, RAR β, MGMT, GADD45A, RASSF1A show as hyper-methylation state, its gene expression is closed or shows as low expression, and Thallus Gracilariae polysaccharide has demethylation and recovers the transcriptional regulation expressed these antioncogenes, this shows that Thallus Gracilariae polysaccharide is potential methylation inhibitor, has considerable prospect in the application in preparing antitumor drug.
Description
Technical field
The present invention relates to biomedicine field, be specifically related to the new application of Thallus Gracilariae polysaccharide and a kind of containing Thallus Gracilariae polysaccharide
Demethylation test kit and application.
Background technology
For methylation of tumor suppressor in tumor cell, carry out antineoplaston by demethylation and become the most swollen
One of study hotspot of tumor treatment.It is one of epigenetics phenomenon that DNA (deoxyribonucleic acid) (DNA) methylates, and refers in DNA first
Under the catalytic action of based transferase (DNMT), at cytosine and the cytosine 5 '-carbon of guanine dinucleotide (CpG) of genome
Position covalent bond combines 1 methyl group, forms the process of 5-methylcytosine (5mC).The change of methylation state be cause swollen
There is a key factor of development in tumor, shows as the offices such as the reduction of tumor cell gene group DNA entirety methylation level and CpG island
The exception of portion's methylation level raises, and abnormal DNA methylation particularly CpG island methylates by affecting genetic transcription, causes
Antioncogene transcriptional inactivation is also closed it and is expressed, increase chromosome structure many-sided generation promoting tumors such as unstability and
Development.
Owing to DNA methylation is a reversible epigenetics modification, the medicine with demethylation effect can
The reverse so that methylating, thus recover expression of tumor suppressor gene.In recent years, grind at the medicine acting as target spot with DNA demethylation
There has been bigger progress in originating party face.Such as, the ucleosides demethylation medicine having been enter into Clinical practice includes U-18496 and 5-
Nitrogen-2 '-deoxycytidine, all effective to treatment myelodysplastic syndrome.It is in the ucleosides demethyl of clinical experimental stage
Chemical medicine thing includes the fluoro-2 '-deoxycytidine of 5-and 1-(β-D-RIBOSE glycosides)-1,2-dihydropyrimidine-2-keto, respectively dialogue blood
Disease, skin carcinoma and myelodysplastic syndrome are effective；Newfound procaine, Pu Lu in non-nucleoside demethylation medicine
Caine amine all has potential using value to suppression pulmonary carcinoma development；Close according to deoxyribonucleic acid methyltransferase modelling
The novel demethylation medicine become includes RG108 and MG98, has certain suppression to make colon cancer, leukemia and renal carcinoma respectively
With.But these at present conventional demethylation medicines are mostly synthetics, have that toxic and side effects is big, active ingredient extracts pure
The defect such as change that preparation process is complicated or cost is bigger.
From developing original new drug angle, the research of marine drug is to develop the new type natural being source with Chinese medicine
One of the focus of active medicine and important directions.The most increasing about the report of Sargassum antitumor drug, China
Marine site is vast, and marine algae resource enriches, and utilizes algae to produce antitumorigenic substance the most potential.Resourceful Sargassum is utilized to develop low
Antitumor drug malicious, efficient is strengthening chemotherapeutical medicine curative effect, raising immunity of organisms, is extending life cycle, reduction relapse and metastasis
Rate and alleviate the aspects such as side effect and have positive meaning.If can be antitumor drug by the exploitation of resourceful Sargassum, must
New drug development and the cost of exploitation will be reduced.
Summary of the invention
In view of prior art and the drawbacks described above of application, and in order to open up marine Chinese medicine anti-tumor active substance further
Medical value, the invention provides a kind of red algae plant Thallus Gracilariae polysaccharide new use as methylation of tumor suppressor inhibitor
Way and the application in preparing antitumor drug thereof.Thallus Gracilariae (Gracilariopsis lemaneiformis) be Rhodophyta,
True Rhodophyceae, Gigartinales, Gracilaria tenuistipitata section, Gracilaria tangleweed, be distributed mainly on China Shandong, Liaoning, Jiangsu, Fujian, Hainan
Deng coastal area.Thallus Gracilariae nature and flavor are sweet, cold, mainly enter Liver Channel, have aid digestion, solution long-pending greasy, bowel relieving harmonization of the stomach hemostasis blood pressure lowering, softening the hard mass
Reduce phlegm, effect of clearing away heat and promoting diuresis, antalgic.As marine pharmaceutical resource, Thallus Gracilariae polysaccharide has the biological action of uniqueness,
It is used to auxiliary and treats multiple disease.Thallus Gracilariae polysaccharide is mainly composed of D-galactose and 3,6-inner ether galactose, this length
Chain, high molecular natural polysaccharide compound due to its prominent biological activity, hypotoxicity and rich at nature, hence it is evident that
It is different from conventional cell toxicant series antineoplastic medicament.Due to Sargassum Thallus Gracilariae polysaccharide there is non-toxic efficient, easily prepared, medicine source is enriched
Feature, it is possible to increase tumour patient quality of life, therefore can be developed into demethylation test kit, as tumor antioncogene first
The favourable agents of base inhibitor, and will play a significant role in clinical antineoplastic diagnosis and treatment.The present invention is by following
Technical scheme realizes:
The invention provides the Thallus Gracilariae polysaccharide new application as methylation of tumor suppressor inhibitor.
Present invention also offers the application in preparing antitumor drug of the above-mentioned Thallus Gracilariae polysaccharide.
Further, described tumor includes pulmonary carcinoma, gastric cancer and cervical cancer.
Further, described tumor is pulmonary carcinoma.
Further, described Thallus Gracilariae polysaccharide is Sargassum Thallus Gracilariae polysaccharide.
Further, described Thallus Gracilariae polysaccharide acts on the antioncogene of tumor cell, and described antioncogene is included in institute
State the antioncogenes such as SHP1, RAR β, MGMT, GADD45A, RASSF1A of showing as hyper-methylation state in tumor cell.
Further, described Thallus Gracilariae polysaccharide acts on the concentration of tumor cell for not less than 50ug/mL.
Present invention also offers a kind of demethylation test kit, described demethylation test kit include Thallus Gracilariae polysaccharide or its
Pharmaceutically acceptable acid, alkali, salt, hydrate or ester, solvent and pharmaceutically acceptable adjuvant.
Further, described solvent is phosphate buffered saline(PBS), and described adjuvant is the distilled water of mass spectrum level.
Further, described Thallus Gracilariae polysaccharide or its pharmaceutically acceptable acid, alkali, salt, hydrate or the weight of ester
Number is 50 parts, and the parts by weight of described solvent are 900 parts, and the parts by weight of pharmaceutically acceptable adjuvant are 100 parts.
Thallus Gracilariae polysaccharide in the present invention can use trichloroacetic acid method (Trichloroacetic acid, TAC method) from red
Algae Thallus Gracilariae is extracted, then carries out stepwise elution with DEAE-Sephadex A-25 ion exchange column and purify, reclaim notable fraction
The holosaccharide obtained.Being mainly composed of D-galactose and 3,6-inner ether galactose, wherein D-galactose content is 27.36%, 3,
6-inner ether galactose content is 29.38%.Extracting method specifically can be found in: red algae Thallus Gracilariae polysaccharide and preparation, antitumor work
Property detection method and application [P], 201610006118.4.
Compared with prior art, the present invention has a following beneficial effect:
Present invention firstly discovers that Thallus Gracilariae polysaccharide can substantially suppress the effect of tumor cell methylation of tumor suppressor state.
Research finds, Thallus Gracilariae polysaccharide has the effect of demethylation for the tumor cell antioncogene of multiple different tissue sources,
Tumor cell includes pulmonary carcinoma, gastric cancer and cervical cancer etc., and antioncogene includes SHP1, RAR β, MGMT, GADD45A, RASSF1A etc..
The demethylation effect of Thallus Gracilariae polysaccharide is dependency action time, and in Thallus Gracilariae polysaccharide In vitro cell experiment working concentration is not
Seeing toxicity, have that medicine source is abundant, the feature of safety non-toxic, can be further developed as methylation of tumor suppressor inhibitor is anti-
Tumour medicine or demethylation test kit, have potential applicability in clinical practice.
Below with reference to accompanying drawing, the invention will be further described, with absolutely prove the purpose of the present invention, technical characteristic and
Technique effect.
Accompanying drawing explanation
Fig. 1 shows that the suppression of human normal cell line and three kinds of tumor cells is increased by the Thallus Gracilariae polysaccharide of the embodiment of the present invention 1
Grow action diagram；
Fig. 2 shows the Thallus Gracilariae polysaccharide of the embodiment of the present invention 2 demethyl to lung cancer A549 cell antioncogene SHP1
The research figure of change effect；
Fig. 3 shows the Thallus Gracilariae polysaccharide of the embodiment of the present invention 3 demethyl to lung cancer A549 cell antioncogene RAR β
The research figure of change effect；
Fig. 4 shows the Thallus Gracilariae polysaccharide of the embodiment of the present invention 4 demethyl to gastric cancer MKN45 cell antioncogene SHP1
The research figure of change effect；
Fig. 5 shows that cervical cancer mgmt gene promoter region sulphite is surveyed by the Thallus Gracilariae polysaccharide of the embodiment of the present invention 5
The action diagram in Tu JiCpG site, sequence peak；
Fig. 6 show the Thallus Gracilariae polysaccharide of the embodiment of the present invention 6 to pulmonary carcinoma SHP1 gene promoter zone methylation site and
The action diagram in CpG site；
Fig. 7 show the Thallus Gracilariae polysaccharide of the embodiment of the present invention 7 to pulmonary carcinoma SHP1, RAR β, MGMT, GADD45A,
The research figure of the expression of tumor suppressor gene effects such as RASSF1A；
Fig. 8 show the Thallus Gracilariae polysaccharide of the embodiment of the present invention 8 to gastric cancer SHP1, RAR β, MGMT, GADD45A,
The research figure of the expression of tumor suppressor gene effects such as RASSF1A；
Fig. 9 show the Thallus Gracilariae polysaccharide of the embodiment of the present invention 9 to cervical cancer SHP1, RAR β, MGMT, GADD45A,
The research figure of the expression of tumor suppressor gene effects such as RASSF1A.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.Following example will assist in the technology of this area
Personnel are further appreciated by the present invention, but limit the present invention the most in any form.It should be pointed out that, the ordinary skill to this area
For personnel, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement.These broadly fall into the present invention
Protection domain.
Embodiment 1
The Inhibit proliferaton effect to human normal cell line and three kinds of tumor cells of the Thallus Gracilariae polysaccharide
Experiment material: Thallus Gracilariae polysaccharide is the polysaccharide that extraction purification obtain from red algae plant Thallus Gracilariae.Thallus Gracilariae
It is 981 kinds, picks up from Wenzhou District of Zhejiang Province.People normal lung epithelial cell strain BEAS-2B and lung cancer cell types, stomach cancer cell line
MKN45 and cervical cancer cell lines Hela is purchased from Chinese Academy of Sciences's cell bank, and this room preserves cultivates.
Experimental technique: the Thallus Gracilariae polysaccharide powder being dried by purification is dissolved in lytic agent phosphate buffered saline(PBS) (phosphate
Buffer saline, PBS), it is made into 1mg/mL mother solution, filtration sterilization, subpackage ,-20 DEG C are frozen standby.Human lung carcinoma cell line
A549, stomach cancer cell line MKN45 and cervical cancer cell lines Hela are attached cell, respectively with 5 × 10 after cell recovery6/ L's
Inoculum density kind is in fresh RPMI-1640 (the RPMI1640 culture medium containing 89v/v%, the hyclone of 10v/v%
Green grass or young crops-the streptomycin of the 1U/mL of FBS, 1v/v%) in cultivate, be placed in 37 DEG C, saturated humidity, 5v/v%CO2Incubator is cultivated.
Within every 2 days, change liquid, pass on 1 time.Take the logarithm respectively BEAS-2B, A549, MKN45 and Hela cell of trophophase, with 0.25v/v%
Trypsinization, counting, with the RPMI-1640 culture fluid diluting cells containing 10v/v% hyclone, make 1 × 104/ mL cell
Suspension, is inoculated in 4 piece of 96 well culture plate, every hole 200 μ L, and making every porocyte density is 2 × 103Individual, often group set 4 parallel multiple
Hole, and set the most celliferous blank group, cultivates 24h, after cell attachment, every hole add containing variable concentrations Thallus Gracilariae polysaccharide (0,
25,50,100ug/mL) 200L culture medium change liquid, wherein 0ug/mL Thallus Gracilariae polysaccharide group is matched group.Cultivate the most continuously
After 48h, add culture medium 200L containing 10v/v%CCK-8 and change liquid, continue to cultivate 2.5h, use automatic elisa reading instrument to detect
Each hole light absorption value A of wavelength 450nm.Calculate growth of tumour cell suppression ratio by following equation, analyze drug on tumor to be measured thin
The impact of the proliferation activity of born of the same parents.
Computing formula: cell growth is with proliferation activity (%)=[A450 (dosing)-A450 (blank)]/[A450 is (right
According to)-A450 (blank)] × 100%；Inhibitory rate of cell growth=1-cell growth and proliferation activity (%)
Experimental result: from fig. 1, it can be seen that Thallus Gracilariae polysaccharide does not has Inhibit proliferaton effect to people's normal lung epithelial cell.And it is imperial
The growth of three kinds of inhibiting tumour cells and value-added effect must be increased dish polysaccharide along with the increase of activity, but at 25ug/
Within mL, act on the most inconspicuous (P > 0.05).And when activity is 50ug/mL, pulmonary carcinoma A549, adenocarcinoma of stomach MKN45 and palace
The growth of neck cancer Hela cell and multiplication capacity are 59.16%, 51.29% and 69.96% (P < 0.05) respectively.Activity is
Multiplication capacity 46.27%, 43.77% and 50.67% (P < 0.01) during 100ug/mL, to A549, MKN45 and Hela cell.
Visible, each experimental group is compared with matched group Inhibit proliferaton effect, and Thallus Gracilariae polysaccharide does not has toxicity to human normal cell line,
And the growth and multiplication capacity to tumor cell has inhibitory action, and along with the increase of concentration, cell proliferation vigor is the least, suppression
Effect increases the most obvious.Thallus Gracilariae polysaccharide presents obvious concentration to the action effect of growth of tumour cell and Proliferation Ability and depends on
Lai Xing is wherein the most notable to the tumor killing effect of pulmonary carcinoma.
Embodiment 2
Thallus Gracilariae polysaccharide is to the research to lung carcinoma cell antioncogene SHP1 demethylation effect
Experiment material: the Thallus Gracilariae polysaccharide that the extraction separation method that Thallus Gracilariae polysaccharide provides for using the present invention obtains becomes
Product, with embodiment 1.Lung carcinoma cell is cellar culture people in the RPMI1640 culture medium containing the hyclone (FBS) of 10%
Lung cancer cell types.
Experimental technique:
(1) tumor cell culture and drug treating: Thallus Gracilariae polysaccharide is acted on human lung carcinoma cell line A549 by the present embodiment.
Thallus Gracilariae polysaccharide activity selects 50ug/mL with reference to the concentration in embodiment 1, acts on 72 hours.The dragon palpus that purification is dried
Dish polysaccharide adds solvent PBS and is made into 1mg/mL mother solution, filtration sterilization, subpackage, and-20 DEG C frozen standby.By A549 cell with 5 ×
106The inoculum density kind of/L in fresh RMPI1640 culture fluid (the RPMI1640 culture medium containing 89v/v%, 10v/v%'s
Green grass or young crops-the streptomycin of the 1U/mL of hyclone FBS, 1v/v%) in cultivate, be placed in 37 DEG C, saturated humidity, 5v/v%CO2Incubator
Middle cultivation.Within every 2～3 days, change liquid or pass on 1 time.Experiment uses the cell of the exponential phase cultivated, and adds 50 μ g/ml PGL
After, and lung cell A549 co-cultivation 48,72h, with 0.25%Trypsin-EDTA trypsinization, PBS washing, cell counting
Instrument counts, and collects cell.
(2) extraction of genomic DNA: the cell collected is used PBS.The quantity of cell is adjusted to 2x103.Will
Cell suspension contains the lysis buffer of E.C. 3.4.21.64 (50mM Tris-HCl pH 8.5 at 500 μ l；25mM EDTA pH 8.0；
150mM NaCl；300mg/ml E.C. 3.4.21.64；In 0.5%SDS).Water-bath 65 DEG C, overnight.Phenol chloroform is used to be stripped
(phenol:chloroform:isoamyl alcohol=25:24:1, v/v/v), adds RNase (20U/ in supernatant
ml)(Ambion；Then carry out phenol extracting AM2286),.The NaAc of final concentration of 300mM and 2.5 times of volumes are added in extract
Dehydrated alcohol ,-80 DEG C preserve 30min.Being centrifuged after ethanol precipitation makes DNA be attached on tube wall, and the ethanol with 75% is clear
Air-dry again after washing one time, be finally suspended in 200ml TE (10mM Tris-HCl, 1mM EDTA, pH8.0).DNA concentration uses
ND-2000Nanodrop spectrophotometer detects.
(3) sulphite is modified: use EZ DNA Methylation-Gold Kit test kit (Zymo Research,
Los Angeles, USA) 400ng genomic DNA is carried out sulphite modification, modification step enters according to the description of manufacturer
OK.In the DNA extracting after final modification elution buffer (Elution Buffer) in 20 μ l test kits, it is placed in-20 DEG C
Refrigerator preserves.It is uracil that sulphite modification causes unmethylated Cytosines, converts in PCR cycle expands
For thymus pyrimidine, methylated cytosine then will not be converted, and becomes cytosine after PCR expands.
(4) detection methylation state: utilize methylation status of PTEN promoter (Methylation-specific PCR, MSP) side
The methylation state of method detection SHP1 gene promoter area uses.The mould that DNA after being modified by sulphite reacts as PCR
Plate, separately designs primer and methylates and non-methylated DNA to distinguish.Methylation-specific primer (MF:5 '-
GAACGTTATTATAGTATAGCGTTC-3’；MR:5 '-TCACGCATACGAACCCAAACG-3 ') be used for expanding methylated
DNA, and the non-specific primer that methylates (UF:5 '-GTGAATGTTATTATAGTATAGTGTTTGG-3 '；UR:5’-
TTCACACATACAAACCCAAACAAT-3 ') to be used for expanding non-methylated DNA, PCR reaction volume be 50 μ l, sub-including 5 μ l
The DNA profiling that sulfate is modified, 25 μ l Zymo TaqTM PreMix (Zymo Research, Los Angeles, USA), 1 μ l
Forward primer (10 μm ol/L), the 1 reverse primer of μ l (10 μm ol/L) and 18 μ l ddH2O.PCR reaction condition: 95 DEG C of denaturations
10min, 95 DEG C of degeneration 30s, 58 DEG C of renaturation 30s, 72 DEG C extend 30s, expand 39 circulations, and last 72 DEG C extend 7min.By 5 μ l
PCR primer carries out electrophoresis on addition 2% agarose gel, and gel imaging instrument is observed and taken pictures.
Result is as shown in Figure 2: only presents methylated band (M) in matched group electrophoretogram, and has no the non-bar that methylates
Band (U), shows in lung cancer A549 cell, and antioncogene SHP1 promoter region presents the state of high methylation.Thallus Gracilariae
After polysaccharide acts on lung carcinoma cell, the non-band that methylates occurs, and the band that methylates dies down, and shows that Thallus Gracilariae polysaccharide has suppression
The effect of antioncogene SHP1 hyper-methylation, along with the prolongation of Thallus Gracilariae polysaccharide action time, demethylation effect strengthens, presents
Time dependence.This explanation Thallus Gracilariae polysaccharide has the effect of suppression antioncogene hyper-methylation, and pulmonary carcinoma serves a kind of first
The effect of base inhibitor.
Embodiment 3
Thallus Gracilariae polysaccharide is to the research to lung carcinoma cell antioncogene RAR β demethylation effect
Experiment material: with embodiment 2.
Experimental technique: in addition to drug exposure times gradient, Gene Name and primer, remaining other with embodiment 2.Difference
After being that the implementation case adds 50 μ g/ml PGL, with lung cell A549 co-cultivation 24,48 and 72h.Selected antioncogene
For RAR β, and its Methylation-specific primer (MF:5 '-TCGAGAACGCGAGCGATTCG-3 '；MR:5’-
TCGTTTCGGTTGGATTGGTC-3 ') it is used for expanding methylated DNA, the non-specific primer that methylates (UF:5 '-
TTGAGAATGTGAGTGATTTGA-3’；UR:5 '-TTGTTTTGGTTGGATTGGTT-3 ') it is used for expanding non-methylated DNA.
Experimental result is as shown in Figure 3: only presents methylated band (M) in matched group electrophoretogram, and has no non-methyl
Changing band (U), show in lung cancer A549 cell, antioncogene RAR β promoter region presents the state of high methylation.Dragon
After palpus dish polysaccharide acts on lung carcinoma cell, the non-band that methylates occurs and gradually strengthens, and the band that methylates dies down, and shows dragon palpus
Dish polysaccharide has the effect of suppression antioncogene RAR β hyper-methylation, along with the prolongation of Thallus Gracilariae polysaccharide action time, demethyl
Change effect strengthens, presentative time dependency.This explanation Thallus Gracilariae polysaccharide has the effect of suppression antioncogene hyper-methylation, to lung
Cancer serves the effect of a kind of methylation inhibitor.
Embodiment 4
The research to the demethylation effect of stomach cancer cell antioncogene SHP1 of the Thallus Gracilariae polysaccharide
Experiment material: in addition to tumor cell replaces pulmonary carcinoma A549 by gastric cancer MKN45, other materials is with embodiment 2.
Experimental technique: except the tumor cell cultivated, collect is in addition to gastric cancer MKN45, and other are with embodiment 2.
Experimental result is as shown in Figure 4: only presents methylated band (M) in matched group electrophoretogram, and has no non-methyl
Changing band (U), show in gastric cancer MKN45 cell, antioncogene SHP1 promoter region presents the state of high methylation.Dragon
After palpus dish polysaccharide acts on lung carcinoma cell 48h, the non-band that methylates occurs, and the band that methylates dies down, after effect 72h, and methyl
Change band to disappear, rather than the band that methylates is brighter, show that Thallus Gracilariae polysaccharide has the work of suppression antioncogene SHP1 hyper-methylation
With, along with the prolongation of Thallus Gracilariae polysaccharide action time, demethylation effect strengthens, presentative time dependency.This illustrates Thallus Gracilariae
Polysaccharide has the effect of suppression antioncogene hyper-methylation, and gastric cancer serves the effect of a kind of methylation inhibitor.
Embodiment 5
The impact on cervical cancer mgmt gene promoter region sulphite order-checking Tu JiCpG site, peak of the Thallus Gracilariae polysaccharide
Experiment material: in addition to tumor cell replaces pulmonary carcinoma A549 with cervical cancer Hela cells, other materials is with embodiment 2.
Experimental technique:
(1) tumor cell culture and drug treating: tumor cell selects Hela, and other are with embodiment 2 (1).
(2) extracting genome DNA and sulphite are modified: with embodiment 2 (2) and (3).
(3) use sulphite order-checking PCR (BSP) standard measure detection methylation state: forward primer: 5 '-
TTGTAGTTGATTTATTGATGGATTTA-3 ', downstream primer: 5 '-ACATATATACCTTACACACTCCAAAC-3 ', product
Length 329bp (includes 11 CpG, gi557889).PCR reaction volume is 50 μ l, the DNA mould modified including 5 μ l sulphite
Plate, 25 μ l Zymo TaqTM PreMix (Zymo Research, Los Angeles, USA), 1 μ l forward primer (10 μm ol/
L), the 1 reverse primer of μ l (10 μm ol/l) and 18 μ l ddH2O.PCR reaction condition: 95 DEG C of denaturations 10min, 95 DEG C of degeneration 30s,
58 DEG C of renaturation 60s, 72 DEG C extend 50s, expand 35 circulations, and last 72 DEG C extend 7min.5 μ l PCR primer are being added
GelRed. carry out electrophoresis on 2% agarose gel, use ZymocleanTMGel DNA Recovery Kit test kit
(Zymo Research, Los Angeles, USA) cuts glue and reclaims.The PCR primer of purification is cloned into pEASY-T1 carrier
(Invitrogen, CA, USA), often organizes and at least selects 9 clones and carry out Sanger order-checking.
(4) sulphite order-checking Tu JiCpG site, peak, promoter region is analyzed: utilize BLAST (Basic Local
Alignment Search Tool, http://blast.ncbi.nlm.nih.gov/) analyze through bisulfites
(Bisulfite) the cervical cancer mgmt gene promoter region Sanger sequencing data after modifying, in combination with Chromas software
Being analyzed order-checking peak figure, sequence alignment result is correct.MGMT promoter region sequence before and after being modified by Bisulfite is neat
Arrangement, and relatively the non-C → U that methylates → T converts and CpG methylation sites.
Experimental result is as shown in Figure 5: arrow: the CpG site signal of analysis.The DNA sequence of mgmt gene promoter region
In containing substantial amounts of CpG island, and the non-methylate DNA in CpG island C after Bisulfite modifies becomes U, becomes T through PCR amplification, says
Bright cervical cancer mgmt gene is implicitly present in methylation state.
Embodiment 6
Thallus Gracilariae polysaccharide is to lung carcinoma cell antioncogene promoter zone methylation site and the effect in CpG site
Experiment material: with embodiment 2.
Experimental technique: experimental operating conditions is with embodiment 5.Promoter zone methylation site and CpG Locus Analysis in Shoots method make
With QUMA software, the methylation state on each CpG island is carried out quantitative analysis.The overall methylated level of each process group is first
The quantity on base Hua CpG island and the ratio of total CpG number, be expressed as percent.
Experimental result is as shown in Figure 6: black circle represents methylated CpG site occurs；White circle represents non-methylated CpG position
Point.Along with the prolongation of Thallus Gracilariae polysaccharide action time, non-methylation sites (white point) accounting example gets more and more, demethylation effect
Strengthen.Show that Thallus Gracilariae polysaccharide has the suppression lung carcinoma cell methylated effect of antioncogene SHP1, and this demethyl is turned into
Use presentative time dependency.
Embodiment 7
The research to lung carcinoma cell expression of tumor suppressor gene effect of the Thallus Gracilariae polysaccharide
Experiment material: with embodiment 2.
Experimental technique:
(1) tumor cell culture and drug treating: tumor cell selects Hela, and other are with embodiment 2 (1).
(2) the detection Thallus Gracilariae polysaccharide effect impact on antioncogene mrna expression level: with RT-qPCR method detection dragon palpus
The mrna expression level of the antioncogene such as SHP1, RAR β, MGMT, GADD45A, RASSF1A before and after dish polysaccharide effect.TRIzol tries
A549 cell total rna before and after the process of agent extracting directly Thallus Gracilariae polysaccharide, detection RNA is dense for Nanodrop 2000 nucleic acid quantification instrument
Degree and purity.Take each experimental group 1 μ g total serum IgE, anti-according to Superscript IIIReverse Transcriptase description
It is transcribed into cDNA.Real-time qPCR detects RAR beta gene expression, and amplimer sequence is shown in Table 1.PCR reacts: SYBR
Green PCR Master Mix(2×)5μl；Forward primer (10 μMs) 0.4 μ l；Reverse primer (10 μMs) 0.4 μ l；cDNA 2μ
l；Moisturizing is to 10 μ l.PCR expands: 95 DEG C, 2min；95 DEG C, 10s；60 DEG C, 30s；72℃30s.Use ABI Step One instrument
And the situation of change of the expression of antioncogene before and after software analysis Thallus Gracilariae polysaccharide effect different time (24,48 and 72h).
The primer sequence of table 1 tumor cell antioncogene RT-qPCR
Experimental result is as shown in Figure 7: in matched group, the antioncogene such as SHP1, RAR β, MGMT, GADD45A, RASSF1A
Mrna expression level is low expression or closed mode.After Thallus Gracilariae polysaccharide acts on lung carcinoma cell, find SHP1, RAR β, MGMT,
The mrna expression of GADD45A, RASSF1A gene restarts, and along with the prolongation of Thallus Gracilariae polysaccharide action time, SHP1,
The mrna expression level of RAR β, MGMT, GADD45A and RASSF1A gene raises.This shows that lung carcinoma cell promoter region is abnormal high
Methylated antioncogene remains reticent, afunction, and with the expression of antioncogene after Thallus Gracilariae polysaccharide demethylation
Then can reactivate, thus reach to suppress the effect of tumor.
Embodiment 8
The research to stomach cancer cell expression of tumor suppressor gene effect of the Thallus Gracilariae polysaccharide
Experiment material: with embodiment 4.
Experimental technique: with embodiment 7.
Experimental result is as shown in Figure 8: in matched group, the antioncogene such as SHP1, RAR β, MGMT, GADD45A, RASSF1A
Mrna expression level is low expression or closed mode.After Thallus Gracilariae polysaccharide acts on stomach cancer cell, find SHP1, RAR β, MGMT,
The mrna expression of GADD45A, RASSF1A gene restarts, and along with the prolongation of Thallus Gracilariae polysaccharide action time, SHP1,
The mrna expression level of RAR β, MGMT, GADD45A and RASSF1A gene raises.This shows that stomach cancer cell promoter region is abnormal high
Methylated antioncogene remains reticent, afunction, and with the expression of antioncogene after Thallus Gracilariae polysaccharide demethylation
Then can reactivate, thus reach the effect suppressing gastric cancer that development occurs.
Embodiment 9
The research to cervical cancer cell expression of tumor suppressor gene effect of the Thallus Gracilariae polysaccharide
Experiment material: with embodiment 5.
Experimental technique: with embodiment 7.
Experimental result is as shown in Figure 9: in matched group, SHP1, RAR β, MGMT, GADD45A, RASSF1A in cervical cancer cell
It is low expression or closed mode Deng the mrna expression level of antioncogene.After Thallus Gracilariae polysaccharide acts on cervical cancer cell, find
The mrna expression of SHP1, RAR β, MGMT, GADD45A, RASSF1A gene restarts, and during along with Thallus Gracilariae polysaccharide effect
Between prolongation, the mrna expression level of SHP1, RAR β, MGMT, GADD45A and RASSF1A gene raises.This shows that cervical cancer is thin
The antioncogene of born of the same parents promoter region exception hyper-methylation remains reticent, afunction, and with Thallus Gracilariae polysaccharide demethylation
The expression of rear antioncogene then can reactivate, thus reaches the effect suppressing cervical cancer that development occurs.
In neoplastic process, the hyper-methylation of antioncogene is the key factor promoting growth of tumour cell.Start
The gene of sub-district CpG island hyper-methylation silence includes cell adhesion gene, mismatch repair gene, glutathione transferase etc., institute
The expression having these genes changes all can promote tumor growth, escape normal growth inhibited mechanism.The abnormal high first in promoter region
The antioncogene of base remains reticent, afunction, and after demethylation, antioncogene then can reactivate.Therefore,
DNA demethylation curable substance tumor.Sargassum Thallus Gracilariae polysaccharide has the effect of suppression antioncogene hyper-methylation, to tumor
Serve the effect of a kind of methylation inhibitor, the antineoplastic agent for methylation of tumor suppressor inhibitor can be further developed as
Thing or demethylation test kit, have clinical value.
The preferred embodiment of the present invention described in detail above.Should be appreciated that the ordinary skill of this area is without wound
The property made work just can make many modifications and variations according to the design of the present invention.Therefore, all technical staff in the art
The most on the basis of existing technology by the available technology of logical analysis, reasoning, or a limited experiment
Scheme, all should be in the protection domain being defined in the patent claims.
Claims (10)
1. Thallus Gracilariae polysaccharide is as the new application of methylation of tumor suppressor inhibitor.
The Thallus Gracilariae polysaccharide the most according to claim 1 application in preparing antitumor drug.
Application the most according to claim 2, it is characterised in that described tumor includes pulmonary carcinoma, gastric cancer and cervical cancer.
4. according to the application described in Claims 2 or 3, it is characterised in that described tumor is pulmonary carcinoma.
Application the most according to claim 2, it is characterised in that described Thallus Gracilariae polysaccharide is Sargassum Thallus Gracilariae polysaccharide.
Application the most according to claim 2, it is characterised in that what described Thallus Gracilariae polysaccharide acted on tumor cell presses down cancer base
Cause, described antioncogene be included in described tumor cell shows as SHP1, RAR β of hyper-methylation state, MGMT,
GADD45A, RASSF1A antioncogene.
Application the most according to claim 6, it is characterised in that described Thallus Gracilariae polysaccharide acts on the concentration of tumor cell and is
Not less than 50ug/mL.
8. a demethylation test kit, it is characterised in that described demethylation test kit includes Thallus Gracilariae polysaccharide or its pharmacy
Upper acceptable acid, alkali, salt, hydrate or ester, solvent and pharmaceutically acceptable adjuvant.
Demethylation test kit the most according to claim 8, it is characterised in that described solvent is phosphate buffered saline(PBS),
Described adjuvant is the distilled water of mass spectrum level.
Demethylation test kit the most according to claim 8 or claim 9, it is characterised in that described Thallus Gracilariae polysaccharide or its pharmacy
The parts by weight of upper acceptable acid, alkali, salt, hydrate or ester are 50 parts, and the parts by weight of described solvent are 900 parts, pharmacy
The parts by weight of upper acceptable adjuvant are 100 parts.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610561746.9A CN106074594A (en) | 2016-07-15 | 2016-07-15 | The new application of Thallus Gracilariae polysaccharide and a kind of demethylation test kit and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610561746.9A CN106074594A (en) | 2016-07-15 | 2016-07-15 | The new application of Thallus Gracilariae polysaccharide and a kind of demethylation test kit and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106074594A true CN106074594A (en) | 2016-11-09 |
Family
ID=57220393
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610561746.9A Pending CN106074594A (en) | 2016-07-15 | 2016-07-15 | The new application of Thallus Gracilariae polysaccharide and a kind of demethylation test kit and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106074594A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112375824A (en) * | 2020-11-13 | 2021-02-19 | 山东大学齐鲁医院 | Application of MSC as cervical cancer diagnosis, prognosis and/or treatment marker |
-
2016
- 2016-07-15 CN CN201610561746.9A patent/CN106074594A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 《ACS APPLIED MATERIALS & INTERFACES》 * |
| 《水产学报》 * |
| 《现代食品科技》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112375824A (en) * | 2020-11-13 | 2021-02-19 | 山东大学齐鲁医院 | Application of MSC as cervical cancer diagnosis, prognosis and/or treatment marker |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kreth et al. | Epigenetics in human gliomas | |
| Hauser et al. | Analysis of serum microRNAs (miR-26a-2*, miR-191, miR-337-3p and miR-378) as potential biomarkers in renal cell carcinoma | |
| ES2742310T3 (en) | Method and kit for non-invasive detection of EGFR gene mutations | |
| Bao et al. | Roles of microRNA-206 in osteosarcoma pathogenesis and progression | |
| Zhou et al. | miR-503 regulates metastatic function through Rho guanine nucleotide exchanger factor 19 in hepatocellular carcinoma | |
| CN101875972B (en) | Rapid detection of KRAS (Kirsten Rat Sarcoma) gene mutation | |
| CN108796086A (en) | A kind of circular rna circBCBM1 and its nondiagnostic fluorescence quantitative detecting method | |
| Chen et al. | Unraveling diverse roles of noncoding RNAs in various human papillomavirus negative cancers | |
| Su et al. | CircRNAs in lung adenocarcinoma: diagnosis and therapy | |
| CN109680064A (en) | Application of the YTHDF2 gene in bladder transitional cell carcinoma is diagnosed, prevented and treated | |
| CN108277279A (en) | A kind of application of lncRNA in diagnosing and/or treating breast cancer | |
| CN102399889A (en) | Method for detecting long-chain non-coding ribonucleic acid (RNA)-HOTAIRM1 | |
| CN106074594A (en) | The new application of Thallus Gracilariae polysaccharide and a kind of demethylation test kit and application | |
| CN108004322A (en) | A kind of applications of lncRNA in adenocarcinoma of lung is diagnosed and/or treated | |
| AU2011343954A1 (en) | Melanoma treatments | |
| Abolfathi et al. | The comparison and evaluation of the miR-16, miR-155 and miR-146a expression pattern in the blood of TB and NSCLC patients: A Research paper | |
| Han et al. | Structural maintenance of chromosomes 2 is identified as an oncogene in bladder cancer in vitro and in vivo. | |
| CN107177676A (en) | Long-chain non-coding RNA NONHSAT113026 is used for the purposes of Diagnosis of Renal Cell Carcinoma molecular marker | |
| CN112375824A (en) | Application of MSC as cervical cancer diagnosis, prognosis and/or treatment marker | |
| CN106167822A (en) | A kind of long-chain non-coding RNA and application thereof | |
| CN110387372A (en) | LncRNA is inhibited to express the application in curing gastric cancer | |
| CN102181520A (en) | Application of methylated SLC19A3 (solute carrier family 19 member 13) gene | |
| CN101270358A (en) | MiRNA sequence and uses thereof | |
| CN112494654A (en) | Pharmaceutical composition containing LncRNA HCG18 inhibitor and application thereof | |
| CN105169412A (en) | Application of NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) inhibition in treatment of tumor with abnormal RIZ1 (retinoblastoma protein-interacting zinc finger gene 1) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161109 |