CN102266561A - Application of Wnt/beta-catenin signal channel inhibitor to preparation of medicament for promoting apoptosis - Google Patents
Application of Wnt/beta-catenin signal channel inhibitor to preparation of medicament for promoting apoptosis Download PDFInfo
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Abstract
The invention belongs to the field of molecular genetics biology and discloses application of a Wnt/beta-catenin signal channel inhibitor to the preparation of a medicament for promoting apoptosis. The Wnt/beta-catenin signal channel inhibitor is preferably siRNA (small interfering Ribonucleic Acid) of beta-catenin. Chemical synthesis beta-catenin-siRNA is designed specific to beta-catenin genes, and granular cells are transferred with a lipoplast, so that the beta-catenin genes in pig ovary granular cells are decreased. A result shows that apoptotic cells in an experimental group are remarkably increased from 8.80 percent to 15.85 percent in comparison to a blank group after beta-catenin expression is inhibited, the difference is remarkable, and the beta-catenin-siRNA can promote the apoptosis of granular cells.
Description
Technical field
The invention belongs to the molecular genetic field of biology, relate to the application of Wnt/ β-catenin signal pathway inhibitor in the apoptotic medicine of preparation promotion.
Background technology
White (the β-of beta-catenin catenin) as the core transcription factor in the Wnt signal path, in development of ovary process, bringing into play important effect, its expression deletion or sudden change all can cause Wnt/ β-catenin signal transduction disorder, thereby body tissue or cell are produced material impact.Knock out Wnt4 and can cause mice, in knocking out the mice granular cell of β-catenin, form the follicle spline structure that includes disorderly and pleomorphism granulosa cell, can cause the generation of granular cell carcinoma in the sex reversal of part and the depletion of oocyte in utero.More than these find that explanation Wnt/ β-catenin has deep effect to propagation, differentiation and the survival of granular cell, and then the effect important to keeping of ovary normal function.Yet this conclusion also needs more experiment to support.
At present, this area is in experiment in vitro, and β-catenin promotes apoptosis or anti-apoptosis also exists dispute.Express the existing following report of apoptosis that β-catenin can promote cell but cross: discovery such as Kwonseop Kim cross express β-catenin will the fibroblastic apoptosis in various degree of inducing mouse embryonic, discoveries such as Marc S.Raab express the apoptosis that β-catenin will induce MM and HeLa cell.
About β-catenin is bigger by the arguement of which kind of machine-processed cell death inducing, the p53 signal path is thought in some researchs, p14ARF, cyclinD1, and some researchs think that LEF-1 and cyclin G1 do not participate in the inductive apoptosis of β-catenin, up-to-date studies show that, β-catenin also can regulate the apoptosis of some tumor cells by c-Jun/p73.And be apoptosis-induced not clear in gonad granulocyte by which kind of mechanism about β-catenin.
Summary of the invention
The objective of the invention is above-mentioned deficiency, the application of Wnt/ β-catenin signal pathway inhibitor in the apoptotic medicine of preparation promotion is provided at prior art.
Another object of the present invention provides a kind of method that promotes the granular cell apoptosis.
This present invention's purpose can be achieved through the following technical solutions:
The application of Wnt/ β-catenin signal pathway inhibitor in the apoptotic medicine of preparation promotion.
The application of preferred described Wnt/ β-catenin signal pathway inhibitor in the medicine of preparation promotion granular cell apoptosis.
Wherein, the siRNA of described Wnt/ β-preferred β-catenin of catenin signal pathway inhibitor.
Further preferred positive-sense strand sequence is SEQ ID No.1, and the antisense strand sequence is the siRNA of β-catenin of SEQ ID No.2, represents with β-catenin-siRNA.
A kind of method that promotes the granular cell apoptosis is to be SEQ ID No.1 with the positive-sense strand sequence, and the antisense strand sequence is the siRNA transfection particles cell of β-catenin of SEQ ID No.2.
The preferred 75nM of siRNA concentration wherein, preferred 36h of transfection time.
Beneficial effect: the present invention is directed to beta-catenin gene design chemosynthesis β-catenin-siRNA, by the liposome transfection granular cell to strike the beta-catenin gene that subtracts in the pig ovary granular cell.Grope through experiment repeatedly, determined the most effective interference chain, and to disturb concentration be 75nM that be 36 hours action time.Utilize when Annexin-V/PI is two to dye cells were tested by flow cytometry 75nM β-catenin-siRNA transfection particles cell, transfection efficiency is 64.49%.And detect transfection beta-catenin gene mRNA and expressing quantity after β-catenin-siRNA36 hour by quantitative fluorescent PCR and Westen blot, further confirm the jamming effectiveness of β-catenin-siRNA.The result shows, compares FAM-siRNA transfection group beta-catenin gene mRNA expression downward modulation 59.93% with the blank group, the protein level downward modulation, and significant difference (P＜0.05), and negative control group difference is not remarkable.It can be said that bright β-catenin-siRNA can suppress the expression of beta-catenin gene effectively, thereby suppress Wnt/ β-catenin signal path.Utilize the two apoptosis situations of dying the granular cell of flow cytometer detection transfection β-catenin-siRNA of Annexin-V/PI, the result shows, after β-catenin expression is suppressed, compare with the blank group, experimental group apoptosis cell obviously increase rises to 15.85%, significant difference (P＜0.05) by 8.80%.Illustrate that β-catenin-siRNA as Wnt/ β-catenin signal pathway inhibitor, can promote the apoptosis of granular cell.Bax and BCL-2 belong to the former apoptogene and the anti-apoptotic genes expression of BCL-2 family respectively, are the main moderators in the mitochondrion apoptosis pathway.The present invention utilizes real time RT-PCR to test and detects two with apoptosis relevant gene Bax and the variation of BCL-2 in the inductive granular cell apoptotic process of β-catenin, the result shows, striking in the pig ovary granular cell that subtracts β-catenin, the expression of BCL-2mRNA has descended 44.28%, and the mRNA expression of Bax has raised 32.76%.
Description of drawings
Fig. 1 FAM-siRNA transfection particles cell 48h fluorescence microscope (A) and light microscopic (B) observed result (200 *).
Fig. 2 flow cytometry FAM-siRNA transfection efficiency; A: blank group; The B:FAM transfection group.
Fig. 3 real time RT-PCR detects that the expression of β-catenin mRNA changes behind the transfection siRNA, and data are represented with means ± S.D, and test at every turn and establish 3 repetitions, and independent experiment 3 times at least.Compare with the blank group, the significant difference on the statistical significance is represented with * (P＜0.05).
Fig. 4 Western Blotting detects the proteic expression of β-catenin to be changed.
The diffusing point of a Fig. 5 apoptosis scattergram, A: blank group B:NCsiRNA negative control group C: β-catenin-siRNA experimental group.
The influence of the reticent β of Fig. 6-catenin pair cell expression of apoptosis-related genes, significant difference is represented p＜0.05 with *.
The specific embodiment
Embodiment 1
1.1 the collection of pig ovary and the selection of follicle
Gather the fresh pig ovary, place the normal saline that contains gentamycin, be transported to laboratory under 37 ℃ environment, selecting healthy ovary is experiment material.
1.2 granular cell separates and cultivates
Before the experiment, cell culture medium and PBS are placed 37 ℃ of preheatings of water-bath of aseptic Cytology Lab, and will test required equipment and put into the iuntercellular sterilization, centrifuge tube that culture bottle and autoclaving are crossed and 1mL rifle head are put into superclean bench; Ovary with containing two antibiosis reason normal saline washings 2 times, is cleaned 3-4 time with PBS, clean twice with alcohol swab, the selection diameter is that the follicle of 2-5mm carries out the granular cell collection; The syringe that use has the 20g syringe needle extracts the follicular fluid of 2-8mm diameter follicle and the granular cell on the ovarian follicle wall, and filters oocyte wherein, the centrifugal 3min of 800g with aseptic steel filter screen (aperture 100 μ m); Abandon supernatant, use the DMEM/F12 culture medium to clean once the granular cell of remainder, centrifugal again (800g, 3min); With DMEM/F12 culture medium re-suspended cell, expect blue dyeing mensuration cell viability for 0.2%.
With DMEM/F12 culture medium (containing 10% hyclone, penicillin and streptomycin) diluting cells, and count with blood cell counting plate; With cell with 10
6Individual/mL density is inoculated in the culture bottle, rocks culture bottle gently, and cell is uniformly dispersed, and places 37 ℃, 5%CO
2Incubator in cultivate; Cell changes a subculture every day.
1.3 passage
(1) converges when reaching 75%-95% when cell, can go down to posterity by pair cell.
(2) rock culture bottle gently, make cell detachment not adherent in the bottle, outwell culture medium.Add the 2mLPBS buffer, rock culture bottle gently, fully the residual culture medium of flush away.Repeat once.
(3) slowly in Tissue Culture Flask, add the 1mL0.25% trypsin and do not contain 0.02%EDTA) Digestive system, and rock gently, trypsin solution is covered bottle equably at the bottom of.
(4) take out after fast culture bottle being put into incubator 1-2min, microscopically is observed, treat that most cells is free after, the culture medium that contains 10% hyclone with 2-3ml stops digestion immediately.
(5) divide the bottle amplification culture: primary cell is by 1: 2-3 goes down to posterity.
1.4siRNA design synthetic
The pig beta-catenin gene sequence (serial number: NM_214367.1) that provides according to GenBank, respectively at the siRNA of 964,854,365 sites designs β-catenin (called after β-catenin-siRNA), do not have fluorescent labeling, be called β-catenin-siRNA-964, β-catenin-siRNA-854, β-catenin-siRNA-365, by the chemosynthesis of Shanghai Ji Ma company.Fluorescently-labeled nonsense sequence is FAM-NC-siRNA.
Table 1 siRNA sequence
1.5 cell transfecting (is example with 24 orifice plates)
With reference to FuGENE
HD Transfection Reagent description is used FuGENE
The amount that HD Transfection Reagent description is recommended is as starting point, and method is as follows:
When (1) the microscopically observation of cell reaches the degrees of fusion of 40-80%,, place 37 ℃, 5%CO to the culture medium that granular cell changes antibiotic-free and serum-free
2Adherent 24h in the incubator;
(2) to add a serum-free antibiotic-free culture medium cumulative volume be 51uL to drip shape with the FAM-NC-siRNA (20nM) of the 5.5uL of dilution, is solution A;
(3) with 2uLFuGENE
The HD transfection reagent adds solution A, notes the rifle head to be added to below the liquid level when adding, and gets solution B (V of transfection reagent and FAM-NC-siRNA: m is 3: 1);
(4) with solution B at room temperature mixing mixed liquor, static at least 20 minutes;
(5) take out culture plate, serum-free antibiotic-free culture medium flushing cell twice adds 10% antibiotic-free culture medium 450uL;
(6) mixed liquor is added in the hand-hole wave and culture plate, mixing gently;
(7) place incubator, change complete medium after 8 hours and continue to cultivate;
(8) experiment is divided into blank group (without any special handling), FAM-NC-siRNA organizes (transfection FAM-NC-siRNA contains the FAM label), utilizes fluorescence microscope whether green fluorescence is arranged behind the transfection 6h, judges that whether transfection is successful, the results are shown in Figure 1.As shown in Figure 1, granular cell green-emitting fluorescence, cellular morphology is fusiformis or polygon, shows the transfection success.
(9) as if granular cell green-emitting fluorescence, cell growth state is good, and then lucifuge 0.25% trypsin digestion cell stops digestion with the DMEM/F12 culture medium that contains 10%FBS, and centrifugal (2000rpm 5min) discards culture medium;
(10) lucifuge is cleaned cell 2 times with PBS, centrifugal (2000rpm, 5min);
(11) use the cells were tested by flow cytometry transfection efficiency, the result shows that transfection efficiency is 64.49% (Fig. 2).
The influence that embodiment 2 siRNA transfection particles cells are expressed β-catenin
2.1 cell transfecting (is example with 24 orifice plates)
With reference to FuGENE
HD Transfection Reagent description is used FuGENE
The amount that HD Transfection Reagent description is recommended is determined the best interference chain and its interference concentration of SiRNA as starting point according to following steps.
Transfection method:
When (1) the microscopically observation of cell reaches the degrees of fusion of 40-80%,, place 37 ℃, 5%CO to the culture medium that granular cell changes antibiotic-free and serum-free
2Adherent 24h in the incubator;
A certain amount of siRNA (20nM) that (2) will dilute is 51uL to drip shape adding serum-free antibiotic-free culture medium cumulative volume, is solution A (the single hole amount is calculated with final concentration 25,50,75 and 100nM concentration amounts respectively);
(3) with 2uLFuGENE
The HD transfection reagent adds solution A, notes the rifle head to be added to below the liquid level when adding, and gets solution B (V of transfection reagent and siRNA: m is 3: 1);
(4) with solution B mixing at room temperature, static at least 20 minutes;
(5) take out culture plate, serum-free antibiotic-free culture medium flushing cell twice adds 10% antibiotic-free culture medium 450uL;
(6) mixed liquor (4) is added in the hand-hole wave and culture plate, mixing gently;
(7) place incubator, change complete medium after 8 hours and continue to cultivate;
(8) experiment is divided into blank group (without any special handling), negative control group (transfection FAM-NC-siRNA, contain the FAM label), experimental group, experimental group is divided into 12 groups again, be respectively β-catenin-siRNA-964, β-catenin-siRNA-854, the β-catenin-siRNA-365 of final concentration 25,50,75 and 100nM concentration, amount to 12 groups; Check and analysis behind the transfection 36h.
2.2Real time RT-PCR analyzes β-catenin mRNA change of Expression situation
2.2.1 cell total rna extraction and cDNA's is synthetic
Extract the total RNA of the granular cell of respectively organizing transfection siRNA in 2.1 respectively, with random primer total RNA is carried out reverse transcription, the cDNA product of reverse transcription is placed-70 ℃ of preservations rapidly according to Ying Jun company product description.
2.2.2Real?time?RT-PCR
(1) 20 μ L reaction system
Real-time quantitative PCR 20 μ L reaction systems are as follows:
(2) use real time pcr amplification instrument to carry out the two-step method amplified reaction, response procedures is:
Use two-step method to carry out amplified reaction, response procedures is: 95 ℃ of degeneration 10min; 95 ℃ of 15sec, 58 ℃ of 1min repeat 40 circulations, each cyclic amplification and real-time analysis; 95 ℃ of 15sec60 ℃ of 1min, 95 ℃ of 15sec, 60 ℃ of 15sec.
(3) gene quantification.
As internal reference, each specimen repeats 3 times, is averaged the Ct value, presses 2-Δ Δ Ct method analyzing gene expression with GAPDH (glyceraldehyde 3-phosphate dehydro-genase).The relative concentration of mRNA is calculated by formula x=2-Δ Δ Ct, x represents the different amount of two initial substances between the group, Δ Δ Ct=Δ E-Δ C, Δ E=Ctexp-CtGAPDH exp, Δ C=Ctcontrol-CtGAPDH control, the amount of Ct value representative target gene amplification under the condition of a fixed threshold value.
Table 2 primer sequence and product thereof
Utilize real time RT-PCR to analyze the expression of β-catenin behind β-catenin-siRNA transfection 36h, the result shows, and is the highest at 365 site jamming effectivenesss among the siRNA of 964,854,365 3 sites design; When β-when catenin-siRNA-365 concentration was 75nM, expression decline 59.93% (p＜0.05) of the blank group β-catenin of experimental group was as Fig. 3.
2.3Western blot analyzes the influence of β-catenin protein expression
2.3.1 total protein of cell extracts
(1) melts Western and IP cell pyrolysis liquid, mixing.Get the lysate of appropriate amount, add PMSF in several minutes before use, the ultimate density that makes PMSF is 1mM;
(2) remove culture fluid, cell is washed 2-3 time with PBS.The ratio that adds 100-200 μ l lysate according to the every hole of 6 orifice plates adds lysate.Several down with rifle piping and druming, lysate is fully contacted with cell.Usually lysate exposing cell 1-2 is after second, and cell will be cleaved.Scrape the cell in the culture dish scraped with cell and collect in the centrifuge tube;
(3) after the abundant cracking, the centrifugal 3-5 of 10000-14000g minute, get supernatant and insert in the new centrifuge tube in-70 ℃ of preservations.
2.3.2 the mensuration of protein concentration
(1) get 0.8ml protein standard preparation liquid and join in the tubulin standard (20mg BSA), fully be mixed with the protein standard solution of 25mg/ml after the dissolving, can use immediately after the preparation, also can-20 ℃ of long preservation.
(2) get an amount of 25mg/ml protein standard, being diluted to final concentration is 0.5mg/ml, for example gets 20 μ l 25mg/ml protein standards, adds 980 μ l diluents and can be mixed with the 0.5mg/ml protein standard.With PBS dilution standard product.0.5mg/ml protein standard after the dilution also can-20 ℃ of long preservation.
(3) quantity per sample adds an amount of BCA working solution of 1 volume BCA reagent B (50: 1) preparation, fully mixing by 50 volume BCA reagent A.
(4) standard substance are added in the standard substance hole of 96 orifice plates by 0,1,2,4,8,12,16,20 μ l, add the standard substance diluent and supply 20 μ l.
(5) add the proper volume sample in the sample well of 96 orifice plates, add the standard substance diluent to 20 μ l.
(6) each hole adds 200 μ l BCA working solutions, places 20-30 minute for 37 ℃.
(7) measure A562, the wavelength between the 540-595nm also can be accepted.Calculate the protein concentration of sample according to standard curve.
2.3.3Western?blot
The SDS-polyacrylamide gel of making 12.5%, get protein sample 15ng, last sample, with 80V voltage, separate about 2h after, each albumen sample that is extracted is transferred to pvdf membrane from gel respectively, it is as follows to change the film pre-treatment: adhesive tape, thick filter paper, pvdf membrane are put into changes film liquid I, a thick filter paper is in commentaries on classics film liquid II, and a thin filter paper is in changeing film liquid III.In 5% defatted milk powder solution room temperature sealing 2h, TBST washes film 3 times with pvdf membrane, each 10min; Adding one, anti-(β-catenin, GAPDH), 4 ℃ are spent the night.TBST adds two anti-(horseradish peroxidase goat anti-rabbit igg) (1: 5000) after washing film again, and incubated at room 1h, TBST wash film 3 times, each 10min; The ECL luminescent solution mixes back effect pvdf membrane 3min in the last darkroom, presses X-ray sheet 1min, X-ray film developing and photographic fixing.The result shows that 365 site jamming effectivenesss are the highest, and when siRNA concentration was 75nM, β-catenin-siRNA had inhibitory action (Fig. 4) at granular cell to the expression of β-catenin.
The influence of embodiment 3 siRNA transfection particles cell pair cell apoptosis and apoptosis correlation factor
3.1 cell transfecting: with embodiment 2, wherein experimental group siRNA only is β-catenin-siRNA-365 of 75nM.
3.2Annexin the two cells were tested by flow cytometry granular cell apoptosis rates that dye of V/PI
(1) behind β-catenin-siRNA-365 transfection 36h, with the trypsin digestion cell that does not contain EDTA;
(2) the centrifugal collection suspension cell of 2000rpm 5min;
(3) the PBS washed cell twice, and 2000rpm 5min is centrifugal;
(4) add 500uLBinding Buffer suspension cell;
(6) behind the adding 5ul Annexin V-FITC mixing, add Propidium Iodide, mixing;
(7) room temperature lucifuge reaction 5-15min;
(8) carry out flow cytometer in the 1h and detect, excitation wavelength Ex=488nm, emission wavelength Em=530nm the results are shown in Table 3, Fig. 5.The result shows: the apoptosis rate of β-catenin-siRNA-365 transfection group cell is 15.85%, is higher than blank group 8.80%, significant difference (p＜0.05); The blank group is compared with negative control group, apoptosis rate no significant difference (p＞0.05).
Table 3 flow cytometer showed disturbs behind β-catenin the influence to the granular cell apoptosis
3.3Real time RT-PCR detects the expression of apafl
Method is with the 2.2.2 of embodiment 2, and use therein primer sequence sees Table 4.
Table 4 primer sequence and product thereof
Utilize real time RT-PCR to analyze the expression of interior Bcl-2 of transfection β-catenin-siRNA pig ovary granular cell and Bax, the result shows, after utilizing β-catenin-siRNA-365 to strike to subtract β-catenin, the expression of Bcl-2 is compared with the blank group, downward modulation 44.28% (p＜0.05), on the contrary, the expression of Bax has been risen 32.76% (p＜0.05) (Fig. 6).
The material that the embodiment of the invention relates to is as follows:
Laboratory animal and sample collecting
This research experiment animal is one commodity sow, is provided by ring pig slaughtering field, sky, Nanjing.
Main agents and test kit
FuGENE
HD transfection reagent: Roche company; Apoptosis test regent box: Kai Ji company; DMEM-F12 cultivates dry powder: GIBCO company; Newborn hyclone: people sea company; 0.25% pancreatin (containing EDTA): GIBCO company; Protein lysate, PMSF, BCA protein concentration test kit, ECL luminescent solution: green skies company; Film (Kodak): Kodak company; Developing powder, fixing powder: Wenzhou photographic equipment company; Pvdf membrane: millipore company; β-catenin antibody: Sigma company; GAPDH antibody: Santa Cruz company; Horseradish peroxidase goat anti-rabbit igg two is anti-: Santa Cruz company; The thick filter paper of Western: waterman company; Bromjophenol blue sample solution: give birth to the company that makes the science and technology prosperous; Albumen MARKER:Fermentas company; LiCl:Sigma company.
The solution preparation
The NaCl preparation: take by weighing the NaCl of 1g, be dissolved in the 20mL pure water, compound concentration is the storage liquid of 855mmol/L, and the time spent adds culture medium and is diluted to 25mmol/L.
Compositions such as serum-free DMEM/F-12 culture fluid, 10% serum antibiotic-free culture fluid, 10% serum, 0.25% antibiotic culture fluid, PBS buffer, 0.25% trypsin solution and compound method are seen appendix.
Tris-HCl (pH8.8): 1.5mol/L, 18.171g are dissolved in the 100mL pure water, deposit in 4 ℃ standby.
Tris-HCl (pH6.8): 1.0mol/L, 12.114g are dissolved in the 100mL pure water, deposit in 4 ℃ standby.
10%SDS:10g SDS powder is dissolved in the 100mL pure water, and room temperature is deposited.
Glue liquid A liquid: acrylamide 30g, two fork acrylamide 0.8g are dissolved in ultra-pure water, and final volume is 100mL, filters with qualitative filter paper and is stored in 4 ℃.
Glue liquid B liquid: 75mL1.5mol/L Tris-HCl, 4mL10%SDS, the 21mL ultra-pure water, final volume is 100mL, is stored in 4 ℃
Glue liquid C liquid: 50mL1.0mol/L Tris-HCl, 4mL10%SDS, the 46mL ultra-pure water, final volume is 100mL, is stored in 4 ℃
10% Ammonium Persulfate 98.5 (AP) is (W/V): get 100 μ g Ammonium Persulfate 98.5s, add the 1mL ultra-pure water, fresh preparation before using.
4% concentrates glue: 0.53mLA liquid, 1mLC liquid, 40uLAP liquid, 2.47mL distilled water, 7uLTMEMD
12.5% separation gel: 4.17mLA liquid, 2.5mLB liquid, 80uLAP liquid, 3.33mL distilled water, 7uLTMEMD
Tris-glycine electrophoretic buffer: Tris-Base 25mmol/L, Glycine 250mmol/L, SDS 0.1%, pH8.3.
Change film buffer I:7.571gTris, 7.505g glycine, 250mL10% methanol, 2250mL distilled water.
Change film buffer II:90.855gTris, 250mL10% methanol, 2250mL distilled water.
Change film buffer III:7.5725gTris, 250mL10% methanol, 2250mL distilled water.
10 * TBS:80g NaCl, 2gKCl, 30gTris is settled to 1L, transfers pH to 7.4
TBST (Tris salt contains the Tween-20 buffer): 1.65mL 20%Tween-20,700ml 1 * TBS, matching while using
Antibody confining liquid: the TBST buffer that contains 5%BSA or defatted milk powder.
Antibody lysate: one available antibodies confining liquid lytic antibody.
Claims (6)
1.Wnt/ the application of β-catenin signal pathway inhibitor in the apoptotic medicine of preparation promotion.
2. application according to claim 1 is characterized in that the application of described Wnt/ β-catenin signal pathway inhibitor in the medicine of preparation promotion granular cell apoptosis.
3. application according to claim 2 is characterized in that described Wnt/ β-catenin signal pathway inhibitor is the siRNA of β-catenin.
4. application according to claim 3, the siRNA positive-sense strand sequence that it is characterized in that described β-catenin is SEQ ID No.1, the antisense strand sequence is SEQ IDNo.2.
5. a method that promotes the granular cell apoptosis is characterized in that with the positive-sense strand sequence be SEQ ID No.1, and the antisense strand sequence is the siRNA transfection particles cell of β-catenin of SEQ ID No.2.
6. method according to claim 5, the siRNA concentration that it is characterized in that described β-catenin is 75nM, the transfection time is 36h.
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Cited By (4)
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| WO2013120394A1 (en) * | 2012-02-14 | 2013-08-22 | 昂科生物医学技术(苏州)有限公司 | Kit for detection or diagnosis of prostate cancer |
| CN106279390A (en) * | 2015-05-12 | 2017-01-04 | 复旦大学 | Protein B ach1 purposes in preparing cell Wnt signal path inhibitor |
| CN108559750A (en) * | 2018-01-16 | 2018-09-21 | 华南农业大学 | Applications of the STAT3 in pig ovary granular cell |
| CN111748559A (en) * | 2020-06-29 | 2020-10-09 | 华南农业大学 | Application of CTNNB1 gene in porcine ovarian granulosa cells |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013120394A1 (en) * | 2012-02-14 | 2013-08-22 | 昂科生物医学技术(苏州)有限公司 | Kit for detection or diagnosis of prostate cancer |
| CN106279390A (en) * | 2015-05-12 | 2017-01-04 | 复旦大学 | Protein B ach1 purposes in preparing cell Wnt signal path inhibitor |
| CN106279390B (en) * | 2015-05-12 | 2019-06-07 | 复旦大学 | Protein B ach1 is preparing the purposes in cell Wnt signal path inhibitor |
| CN108559750A (en) * | 2018-01-16 | 2018-09-21 | 华南农业大学 | Applications of the STAT3 in pig ovary granular cell |
| CN108559750B (en) * | 2018-01-16 | 2020-07-31 | 华南农业大学 | Application of STAT3 in porcine ovarian granulosa cells |
| CN111748559A (en) * | 2020-06-29 | 2020-10-09 | 华南农业大学 | Application of CTNNB1 gene in porcine ovarian granulosa cells |
| CN111748559B (en) * | 2020-06-29 | 2022-06-17 | 华南农业大学 | Application of CTNNB1 gene in porcine ovary granular cells |
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