CN102784399A - Tumor suppressor gene NDRG2 based application for improving breast cancer chemotherapy sensibility - Google Patents
Tumor suppressor gene NDRG2 based application for improving breast cancer chemotherapy sensibility Download PDFInfo
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Abstract
The invention discloses a tumor suppressor gene NDRG2 based application for improving breast cancer chemotherapy sensibility. The tumor suppressor gene NDRG2 is transfected into a breast cancer cell through an expression vector or a virus vector, and the result shows that the tumor suppressor gene NDRG2 can improve the breast cancer chemotherapy sensibility and can be applied to preparation of relevant drugs. The NDRG2 prompts a deoxyribonucleic acid (DNA) damage ability of Adr induced p53 wild type breast cancer cells, improves morphology damage of Adr to p53 wild type breast cancer cells, improves a proliferation inhibiting ability of the Adr to the p53 wild type breast cancer cells, improves the breast cancer chemotherapy sensibility which is independent of specific chemotherapy drugs and further prompts apoptosis of the Adr induced p53 wild type breast cancer cells.
Description
Technical field
The invention belongs to the tumor treatment technical field, relate to the related gene that improves chemosensitivity, particularly improve the application of breast carcinoma chemosensitivity based on antioncogene NDRG2.
Background technology
Breast carcinoma is one of women's most common tumor, and sickness rate is the first place that the women suffers from tumor, accounts for 1/4 of women's whole body cancer, and the healthy of women in serious threat.It is main that the treatment of breast carcinoma combines chemotherapy, radiotherapy with operative treatment.At present, chemicals is still the common method of breast cancer treatment, in the therapeutic process of breast carcinoma, plays a significant role.But chemotherapy exists and fails to differentiate between the enemy and ourselves, side effect big, drug resistance more generally waits problem, is seriously restricting its clinical efficacy.
Amycin (Adr) is a kind of AGPM, can suppress the synthetic of RNA and DNA, and the tumor cell of various growth cycles is all had lethal effect, is a line medicine of breast cancer treatment.Its main toxic reaction has leukocyte and thrombocytopenia, alopecia, cardiac toxicity, feels sick, loss of appetite etc.These toxic and side effects are having a strong impact on the clinical application effect of amycin.Therefore, find that new chemosensitivity related gene can effectively improve the clinical efficacy of chemotherapeutic, and reduce its toxic and side effects, become the important means that improves chemosensitivity.
The NDRG2 gene is a kind of antioncogene of having reported, discovers that NDRG2 hangs down expression or do not have expression in the kinds of tumors tissue, and this low expression and patient's poor prognosis substantial connection.
Summary of the invention
The problem that the present invention solves is the application that improves the breast carcinoma chemosensitivity based on antioncogene NDRG2 is provided, and antioncogene NDRG2 can improve the breast carcinoma chemotherapeutic efficacy, selects for the clinical efficacy that effectively improves breast carcinoma provides new drug targets.
The present invention realizes through following technical scheme:
Antioncogene NDRG2 is in the application of the medicine of preparation anti-breast cancer.
Described medicine is the medicine that uses with chemotherapy drugs in combination.
Antioncogene NDRG2 depends on the expression of p53 albumen at breast cancer cell.
Described medicine is the medicine that promotes to express the proteic breast cancer cell damage of p53.
Described medicine is to promote inhibition to express the medicine of the propagation of the proteic breast cancer cell of p53.
Described medicine is the medicine that promotes the apoptosis of the proteic breast cancer cell of expression p53.
Expression that described p53 albumen is wild type or ectogenic expression.
Antioncogene NDRG2 improves the application of the medicine of breast carcinoma chemosensitivity in preparation.
Antioncogene NDRG2 through expression vector or viral vector transfection in breast cancer cell.
Described breast cancer cell is also expressed the transfection of the proteic expression vector of p53.
Compared with prior art, the present invention has following beneficial technical effects:
The application that improves the breast carcinoma chemosensitivity based on antioncogene NDRG2 provided by the invention; With antioncogene NDRG2 through expression vector or viral vector transfection in breast cancer cell; The result shows that antioncogene NDRG2 can improve the breast carcinoma chemosensitivity, thereby can use the preparation of related drugs:
NDRG2 has promoted Adr to induce the DNA damage ability of p53 wild type breast cancer cell, but in the MDA-MB-231 cell of p53 sudden change, expressing excessively of NDRG2 but can not promote the DNA damage that Adr causes; After in the MDA-MB-231 cell that the p53 transduction is got into the p53 sudden change, NDRG2 still can bring into play corresponding effect;
NDRG2 has improved the morphology damage of Adr to p53 wild type breast cancer cell; Not only microvillus disappears in the MCF-7 of NDRG2 high expressed cell line; And visible apoptotic body, significantly cavity shape lysosome appears, show as the significantly cell characteristics of apoptosis middle and advanced stage;
NDRG2 has improved the propagation inhibition ability of Adr to p53 wild type breast cancer cell, has detected NDRG2 simultaneously and has expressed the sensitivity to cisplatin, finds that NDRG2 also can improve the chemosensitivity of medicine cisplatin; This shows the concrete chemotherapy drug dependence of NDRG2 raising breast cancer cell chemosensitivity right and wrong.
In the breast cancer cell line MCF-7 of p53 wild type, NDRG2 has promoted the apoptosis of the inductive p53 wild type of Adr breast cancer cell.
Description of drawings
Fig. 1 packs successful fluorescence microscope efficiency of infection figure as a result for MCF-7 cell line;
Fig. 2 packs successful fluorescence microscope efficiency of infection figure as a result for MDA-MB-231 cell line;
Fig. 3-1～3-2 is the expression qualification result figure of NDRG2 stable transfection MCF-7 cell line; Wherein 3-1 is the variation of Real-time PCR detection rna level, and 3-2 is the variation that Western blot detects protein level;
Fig. 4-1～4-2 is that the MDA-MB-231 slow virus surely changes cell line NDRG2 expression qualification result figure; 4-1 is the variation that Western blot detects protein level for the variation of Real-time PCR detection rna level, 4-2;
The DNA damage degree detecting that Fig. 5 causes for different Adr concentration in the MCF-7 cell is figure as a result;
The DNA damage degree detecting that Fig. 6 causes for different Adr concentration in the MDA-MB-231 cell is figure as a result;
Fig. 7-1～7-2 is that variable concentrations Adr stress express relevance detection results figure by back NDRG2 in the MCF-7 cell line; 7-1 is the variation that Western blot detects protein level for the variation of Real-time PCR detection rna level, 7-2;
Fig. 8-1～8-2 for variable concentrations Adr handle the MDA-MB231 cell after NDRG2 express relevance detection results figure;
Fig. 9 is the NDRG2 detection of expression figure as a result of different time points behind the MDA-MB231 cellular stress;
Figure 10 remedies experiment detection NDRG2 gene expression testing result figure in the MDA-MB231 cell, carrying out the p53 gene;
Figure 11 is the cell injury testing result figure after the Adr of different time points handles the MCF-7 cell;
Figure 12 surely changes cell injury degree detecting (figure medium green color dot representes to damage the γ H2AX that express the back) after the cell line for Adr handles MCF-7NDRG2;
Figure 13 can obviously increase Adr to MCF-7 cells injury degree (expression of γ H2AX) for Western Blot detects NDRG2;
Figure 14 can not increase Adr to MDA-MB-231 cells injury degree for Western Blot detects NDRG2;
NDRG2 crosses the morphocytology change of expression back to Figure 15 in the MCF-7 cell for transmission electron microscope detects;
NDRG2 crosses the morphocytology change of expression back to Figure 16 in the MDA-MB-231 cell for transmission electron microscope detects;
Figure 17 analyzes the inductive MCF-7 cell inhibitory effect of Adr for MTT;
Figure 18 is the inductive MCF-7 cell inhibitory effect of Edu staining analysis Adr;
Figure 19 is the MCF-7 cell inhibitory effect of Edu staining analysis cisplatin induction;
Figure 20 analyzes the inductive MDA-MB-231 cell inhibitory effect of Adr for MTT;
Figure 21 analyzes the MDA-MB-231 cell inhibitory effect that Adr causes for Edu;
Figure 22-1～2 detects for the proliferative activity that MTT analyzes behind the NDRG2 adenovirus infection MDA-MB-231 cell;
Figure 23 detects the inductive MCF-7 apoptosis of Adr situation for AnnexinV dyeing;
Figure 24 detects the inductive MCF-7 apoptosis of Adr situation for TUNEL dyeing;
Figure 25 detects the inductive MDA-MB-231 apoptosis of Adr situation for AnnexinV dyeing;
Figure 26 detects the inductive MDA-MB-231 apoptosis of Adr situation for TUNEL dyeing.
The specific embodiment
Below in conjunction with concrete embodiment the present invention is done further detailed description, said is to explanation of the present invention rather than qualification.
1, the structure of NDRG2 expression carrier and virus packing
The expression vector establishment of NDRG2 is based on the pLenti6.3/V5-DEST of the Gateway system carrier of Invitrogen company.At first utilize molecule clone technology that NDRG2 is cloned into entry vector pENTR-3C (cloning through BamH I and EcoR I site); Utilize the LR recombinase that NDRG2 is recombinated to the pLenti6.3/V5-DEST carrier subsequently; Obtain the Plenti6.3-NDRG2 carrier, the NDRG2 gene order in the dna sequencing checking carrier is correct.
2, set up the steady commentaries on classics cell strain of NDRG2 high expressed
Select for use breast cancer cell MCF-7, MDA-MB-231 as target cell respectively, medicament Blasticidin S handles (blasticidin S) respectively in parental cell, screens suitable drug level.Blasticidin S be a kind of from streptomycin isolating ucleosides selective reagent, have very strong translation inhibitory action, most eukaryotic cells are had inhibited proliferation.And the cell of stable transfection Plenti6.3-NDRG2 carrier has resistance, can survive, and can about 2 weeks, obtain the stable transfected cells strain.
Press Lipofectamine2000 liposome transfection description with Plenti6.3-NDRG2 recombiant plasmid and viral packaging plasmid (PMD2G, pSPAX2) cotransfection 293T cell packing slow virus, change culture fluid after 6 hours, collect slow virus after about 48 hours.
The slow virus stock solution of collecting is filtered with 0.45 μ m filter and carried out packing.
Utilize slow virus to infect MCF-7 and MDA-MB-231 cell respectively, utilize the Blasticidin S screening cell of 8 μ g/mL after 48 hours, cross the cell of expressing NDRG2 and will continue survival.
Screening is approximately about January, the cytotostatic growth and in good condition after, a large amount of amplifying cells, frozen subsequent use.Utilize the Gateway slow virus Plenti6.3-NDRG2 of system carrier successfully to make up the NDRG2 high expressed stable cell line of breast carcinoma MDA-MB-231 (p53Mut) and MCF-7 (p53WT) cell line.
Stable cell line to obtaining utilizes Western blot and qRT-PCR that the expression status of genes of interest NDRG2 is analyzed respectively.Utilize red fluorescence Cherry labelling (negative control group), show that MCF-7 and MDA-MB-231 surely change cell line and make up successfully, its fluorescence microscope efficiency of infection result is respectively like Fig. 1, shown in Figure 2.
Simultaneously; Expression to NDRG2 is identified; Utilize qPCR and Western blot to prove conclusively the high expressed state of the NDRG2 of the cell that screens respectively; The testing result of MCF-7 cell line, MDA-MB-231 cell line shown in Fig. 3-1～3-2, Fig. 4-1～4-2, obviously will exceed contrast no matter can find out the expression of NDRG2 in the cell line after Real-time PCR detection or Western blot detection all show transfection respectively.The result shows that the steady commentaries on classics cell strain of NDRG2 high expressed makes up successfully.
For NDRG2 is expressed and breast cancer cell, and the difference of the effects that played down of two kinds of different p53 expressions of cell, adopted following detection means respectively, it is described respectively.
3, the explanation of detection method
3.1qRT-PCR detecting the mRNA of NDRG2 expresses
Design of primers: people NDRG2qRT-PCR primer:
UP:5’-GAGATATGCTCTTAACCACCCG-3’
DOWN:5’-GCTGCCCAATCCATCCAA-3’
People GAPDH qRT-PCR primer:
UP:5’-TTCGACAGTCAGCCGCATCTTCTT-3’
DOWN:5’-CAGGCGCCCAATACGACCAAATC-3’
3.2 extract total RNA and reverse transcription
Extract the MCF-7 of reorganization NDRG2 packaging virus, total RNA of MDA-MB-231, and will extract in 2 μ L RNA be dissolved among the 98 μ L TE buffer, measure its A260 and A280 value with ultraviolet spectrophotometer, the purity of analysis RNA.
Get and extract the good total RNA of 0.5 μ g, carry out reverse transcription with the PrimeScript RT reverse transcription test kit of TaKaRa company and test, specifically carry out according to description operation.Reaction system is:
Reaction condition is: 37 ℃, and 15min; 85 ℃, 5sec
The final cDNA masterplate that obtains, the PCR that can be used for next step detects.
3.3Realtime PCR experiment
1) use SYBR Premix ExTaqII TaKaRa test kit to experimentize.
2) by reaction system application of sample to every hole.
3) adopt the two-step method performing PCR.
A. preparatory degeneration: 1cycle, 30s
The B.PCR reaction: 95 ℃, 5s; 60 ℃, 34s, 40cycles
4) carry out data analysis and tabulation with Graphpad Prism5 software
3.4Western-Blot detect the cell correlative protein expression
3.4.1 extraction cell protein:
1) culture dish is positioned on ice, inhales and abandon culture medium wherein, the PBS that slowly adds about 1mL pre-cooling along the ware wall carries out rinsing, discards residual liquid, repeats this process again with twice of cell rinsing.
2) add the cell pyrolysis liquid (amount of cell pyrolysis liquid decides according to the size of cell, density etc.) of about 150 μ L pre-coolings, scrape with cell and whole cells are scraped off as far as possible, after be transferred in the 1.5mL EP pipe.
3) place about 30min on ice, can intermittent concussion between resting period repeatedly, let the abundant cracking of cell (also can adopt ultrasonic direct cracking).4 ℃ centrifugal: 12000rpm, 20min.Draw supernatant, add Loading Buffer in proportion, boiling water boils 10min.
3.5BCA method protein quantification
1) gets 4 μ L and extract protein stock solution, add lysate 16 μ L and be diluted to 20 μ L.
2) carry out quantitatively with the BCA test kit: A liquid and B liquid with the mixed of 50:1, are added mixed working solution 200 μ L/ holes in 96 orifice plates.
3) add the good sample of 20 μ L protein standard substances and dilution in 96 orifice plates, carefully be positioned over 37 ℃ of incubators and hatch 30min.
4) with spectrophotometric determination sample absorbance.The production standard curve is finally confirmed protein sample concentration.
3.6 gel electrophoresis, commentaries on classics film and detection
1) separation gel of preparation 12% and 6% concentrated glue.
2) go up appearance, 80V carries out the upper strata gel electrophoresis.
When 3) observation Loading Buffer all gets into separation gel, transfer voltage, row lower floor gel electrophoresis to 120V.
4) etc. stop electrophoresis during bromophenol blue swimming to the separation degree that needs.Take out glue, get the NC film of suitable size, soak 20min at transfering buffering liquid.
5) in order filter paper, glue, NC film are put into transfer folder, constant voltage or constant current transferase 12 h or 12h
6) change during the film, the intermittent energising situation of observing guarantees to change smoothly film.
7) film is steeped into 5% the defatted milk powder for preparing in advance, sealed 2 hours.
8) prepare certain density one anti-solution according to tiring of antibody.TBST washs 3 5min.
9) resistive connection closes 1h or spends the night and fully combines.TBST washs 3 5min.
10) sealing fluorescence two is anti-, sweeps the film appearance at Odyssey and analyzes and obtain picture.
3.7, mtt assay detects cell inhibitory rate
Prepare fresh MTT solution: the MTT solution that 100mg Sigma MTT powder is dissolved in preparation 5mg/mL among the 20mL PBS.
Inoculating cell to 96 orifice plate, pair cell carries out synchronization process: adherent fully cell is carried out serum starvation processing 24 hours.
Cell adds chemotherapeutic amycin (Adr) to be handled 24 hours, and concentration is 0.2 μ g/ml.
Every hole adds 20 μ L0.5 μ g/ml MTT, cultivates 4 hours for 37 ℃ at constant incubator.
The careful suction abandoned supernatant in the hole.Every hole adds 150 μ L DMSO, and concussion 10min fully dissolves crystallization.
On the enzyme linked immunological appearance, select the 570nm wavelength to measure absorbance.
Carry out data analysis and tabulation drafting growth curve chart with Graphpad Prism5 software.
3.8 immunofluorescence analysis
Cell climbing sheet: in super-clean bench, slide carefully is positioned over 24 orifice plates central authorities, will digests good cell seeding then on slide.
The chemotherapeutic amycin is handled cell, and process is with aforementioned.Fixed cell behind 24h or the 48h.
Supernatant is abandoned in suction, and with PBS washed cell 2 twice.4% paraformaldehyde incubated at room 15min fixed cell.
Penetratingization processing: after the cell that will fix washed 3 times with PBS, the 0.1%Triton-X100 room temperature that adds fresh configuration left standstill 15min.
Washing: the cell washing of Triton being handled with PBS 3 times, 5min at every turn.
Sealing: 5%BSA sealing 1 hour is put in section.
Sealing one is anti-: with an anti-solution of BSA preparation debita spissitudo, every hole guarantees the volume of at least 50 μ L, and sealing is spent the night.
Washing: wash each 5min 3 times.
Sealing two is anti-: with two suitable anti-solution of BSA preparation, guarantee every at least hole 50 μ L volumes.
Washing: wash each 5min 3 times.
DAPI dyeing:, guarantee every at least hole 50 μ L volumes with the suitable DAPI staining solution of BSA preparation.
Slide is taken out at the half-light place, carefully be placed on the slide.
Use the glycerol mounting, the confocal fluorescent microscopic examination is also taken a picture.
3.9 detecting organelle, transmission electron microscope changes
1) collect the cell that different pharmaceutical concentration is handled, centrifugal, careful operation guarantees that cell is in one.
2) with the glutaraldehyde fixative cell is fixed 2 hours, careful operation at 4 ℃.
3) sample that fixes being carried out Electronic Speculum detects.
4) interpretation of result and arrangement.
3.10Edu dyeing detects cell-proliferation activity
1) is seeded to 24 orifice plates, and gives the amycin processing, and collect the cell that different pharmaceutical concentration is handled respectively
2) Edu labeled cell: in the cultured cell of every hole, add the Edu dyeing liquor and dye, stop dyeing after 4 hours
5) cell fixation: abandon culture fluid, add 300 μ L4% paraformaldehyde fixative to 24 orifice plates, room temperature leaves standstill 30min
6) every hole glycine of adding freshly prepared 2mg/mL is hatched 10min
7) the PBS washing is 3 times, each 5min
8) abandon supernatant, every hole adds 300 μ L0.5%TritonX-100 and carries out penetratingization processing
9) the PBS washing is 3 times, each 5min.
10) according to the form below is prepared the 1X Apollo reactant liquor (existing with join at present) that every hole is not less than 50 μ L in proper order.
11) the PBS washing is 3 times, each 5min.
12) preparation 1X Hoechst33342 reactant liquor: Hoechst33342 (reagent F) dilutes with deionized water in the 1:100 ratio, keeps in Dark Place.
13) abandon supernatant, every hole adds 100 μ L33342 reactant liquors, and room temperature is hatched 30min at the half-light place.
14) the PBS washing is 3 times, each 5min.
15) with the DAPI 10min that dyes.
16) the PBS washing is 3 times, each 5min.
17) fragmentation is moved on the microscope slide mounting.
18) in confocal fluorescent microscopically observed result.
19) interpretation of result and summary.
3.11AnnexinV apoptosis is surveyed in dyeing
Belong to the cellular immunofluorescence technology, one anti-directly dyes with AnnexinV, notes detecting each component apoptosis difference observing cell and have on the basis of apoptosis, otherwise detects less than AnnexinV in intracellular variation.
3.12TUNEL dyeing detects apoptosis
1) PBS washs the cell of having handled, and the cell that has washed is dry.
2) add freshly prepared 4% paraformaldehyde of 300 μ L and under normal temperature condition, fix 1 hour.
3) the PBS washing is 3 times, each 5min.
4) under 4 ℃ of conditions, penetrate liquid 5min (0.1%Triton-X100) with what add sodium citrate.
5) add Label Solution and Enzyme Solution mixed liquor: will shift to an earlier date the Label Solution and the Enzyme Solution mixed liquor that prepare in proportion and add every hole.
6) with PBS washing 3 times, each 5min.
7) every hole adds TUNEL reaction mixture, and sample is passed on the microscope slide.
8) in the wet box of 37 ℃ of dark, hatch microscope slide.
9) washing is 3 times, each 5min.
10) examine with the DAPI transfect cell.
11) fluorescence co-focusing microscopically observed result.
12) interpretation of result and summary.
3.13 Flow Cytometry detects apoptosis
1) handle cell with amycin, method is the same.
2) collecting cell after 48 hours, peptic cell, PBS washing 2 times.
3) flow cytometer detects.Analysis result.
4, in the cell injury course of reaction that Adr causes, the dependency between antioncogene NDRG2 and the p53
For the dependency between clear and definite antioncogene NDRG2 and the amycin (Adr); Utilize the NDRG2 stable transfected cells strain of having set up, utilize qPCR and Western blot methods analyst NDRG2 to change for the expression in the MDA-MB-231 cell of saltant for the MCF-7 cell and the endogenous p53 of wild type respectively at endogenous p53.
γ H2AX with immune fluoroscopic examination phosphorylation estimates the cells injury degree, finds MCF-7 and MDA-MB231 all along with the concentration of it being handled Adr increases, and degree of injury increases the weight of (Fig. 5,6).
The expression of NDRG2 increases progressively with Adr concentration and increases in the MCF-7 cell, but when the Adr degree of injury surpasses certain concentration (0.6 μ g/ml), the expression of NDRG2 begins descend (Fig. 7-2) in the cell.The testing result of mRNA level has also measured consistent result (Fig. 7-1).
Yet, be among the MDA-MB-231 at the p53 mutant cell, along with the concentration of Adr increases progressively and the DNA damage degree increases, the mRNA level of NDRG2 and protein level all discovery can detectedly express (Fig. 8-1,8-2).Through detecting different time points DNA damage 0-96h afterwards, find yet detectedly to express (Fig. 9).Subsequently, it is MDA-MB-231 that wild type p53 is transfected into the p53 mutant cell through expression vector, finds NDRG2 gene expression rising (Figure 10).
Preliminary prompting, in the cell injury course of reaction that Adr causes, the expression of NDRG2 depends on the expression status of cell endogenous p53.
5, NDRG2 has promoted Adr to induce the DNA damage ability of p53 wild type breast cancer cell
Correlation detection shows that behind the DNA damage, NDRG2 performance function depends on the function of p53, in order further to explore the function of NDRG2 in the breast cancer cell chemosensitivity, has at first detected the cells injury degree.γ H2AX representes the histone H2AX tryptophan of phosphorylation, and it expresses to be generally believed it is relevant with the cells injury degree after damage, and is irrelevant with type of impairment.
Detect and find, the DNA damage degree that Adr processing tumor cell caused after 2 hours is (Figure 11) the most seriously.In the MCF-7 cell, with respect to matched group, the DNA damage effect that can obviously promote Adr is expressed in crossing of NDRG2.The γ H2AX that green point representative among Figure 12 stress be expressed expresses highly more, and the cells injury degree is obvious more.Simultaneously also confirmed result consistent (Figure 13) through Western Blot.
But in the MDA-MB-231 of p53 sudden change, expressing excessively of NDRG2 but can not promote the DNA damage (Figure 14) that Adr causes.
6, NDRG2 has improved the morphology damage of Adr to p53 wild type breast cancer cell
Further, through morphological observation the breast cancer cell degree of injury that causes of Adr.Utilize transmission electron microscope, find in the MCF-7 of p53 wild type cell, expressing excessively of NDRG2 can obviously promote cells injury.
Negative control group (Cherry) shows as after Adr handles that chromatin margination, after birth break, cellular edema, electron density reduce, a large amount of lysosome, the also visible phenomenon of more significantly breeding.And not only microvillus disappearance in the MCF-7 of NDRG2 high expressed cell line, and, show as cell characteristics (Figure 15) than tangible apoptosis middle and advanced stage it is thus clear that significantly cavity shape lysosome appears in apoptotic body,
It is thus clear that the expression of crossing of NDRG2 has improved the morphology damage of Adr to p53 wild type breast cancer cell.
And in the MDA-MB-231 of p53 sudden change, do not detect evident difference (Figure 16).
7, NDRG2 has improved the propagation inhibition ability of Adr to p53 wild type breast cancer cell
Utilize the propagation situation of mtt assay and Edu dyeing observation of cell respectively.
In the MCF-7 of p53 wild type cell, find that NDRG2 crosses the cell inhibitory effect (Figure 17,18) that expression can obviously improve Adr.
In order to detect the dependent/non-dependent that rises to chemotherapeutics of this sensitivity; Detected the sensitivity of the MCF-7 cell of the different expression status of NDRG2 simultaneously to the chemotherapeutic cisplatin; Find that the high expressed of NDRG2 also can improve the chemosensitivity (Figure 19) of medicine cisplatin, show that NDRG2 promotes that the chemosensitivity increase is not the chemotherapeutics dependency.
At the MDA-MB-231 of p53 sudden change, do not find detectedly to change (Figure 20,21) simultaneously.
In order to confirm the result of MDA-MB231 cell, cross with NDRG2 simultaneously and express the variation that adenovirus infection MDA-MB231 cell does not detect the propagation inhibition yet, with the result consistent (Figure 22-1,22-2) of stable transfected cells strain.
8, NDRG2 has promoted the apoptosis of the inductive p53 wild type of Adr breast cancer cell
Experiment through the front; Basic confirmation NDRG2 is to the propagation of breast cancer cell, the influence of damage; In order to confirm the influence of NDRG2, adopt TUNEL dyeing and AnnexinV colouring method to detect the influence of NDRG2 respectively to breast cancer cell apoptosis degree to the breast cancer cell chemosensitivity.
In the wild breast cancer cell line MCF-7 of p53, the high expressed of discovery NDRG2 can promote the apoptosis of cell.Compare with negative control group, (early apoptosis of having represented cell) taken place significantly to turn up (Figure 23) in AnnexinV.TUNEL dyeing finds that also apoptosis obviously increases (Figure 24).
And in the MDA-MB231 cell of p53 sudden change, compare the no significant difference as a result of AnnexinV and TUNEL (Figure 25,26) with cellular control unit.
Show that to sum up antioncogene NDRG2 can improve the breast carcinoma chemosensitivity fully, can pass through expression vector or viral vector transfection in breast cancer cell, thereby can be applicable to prepare the medicine that improves the breast carcinoma chemosensitivity; Thereby the preparation of the medicine of the anti-breast cancer that can be applicable to use with chemotherapy drugs in combination.In view of NDRG2 depends on the expression of wild type p53 albumen at the breast cancer tumour cell; And for the breast cancer cell of no wild type p53 protein expression; Can be through the exogenous p53 albumen of transduction; Thereby can realize that still NDRG2 acts on accordingly, perhaps with NDRG2, p53 gene integration in an expression vector, perhaps different expression vector is simultaneously or the front and back transfection.
Claims (10)
1. antioncogene NDRG2 is in the application of the medicine of preparation anti-breast cancer.
2. application as claimed in claim 1 is characterized in that, described medicine is the medicine that uses with chemotherapy drugs in combination.
3. application as claimed in claim 1 is characterized in that, antioncogene NDRG2 depends on the expression of p53 albumen at the breast cancer tumour cell.
4. application as claimed in claim 1 is characterized in that, described medicine is the medicine that promotes to express the proteic breast cancer cell damage of p53.
5. application as claimed in claim 1 is characterized in that, described medicine is to promote inhibition to express the medicine of the propagation of the proteic breast cancer cell of p53.
6. application as claimed in claim 1 is characterized in that, described medicine is the medicine that promotes the apoptosis of the proteic breast cancer cell of expression p53.
7. like any one described application of claim 3～6, it is characterized in that expression that described p53 albumen is wild type or ectogenic expression.
8. antioncogene NDRG2 improves the application of the medicine of breast carcinoma chemosensitivity in preparation.
9. application as claimed in claim 8 is characterized in that, antioncogene NDRG2 through expression vector or slow virus carrier transfection in breast cancer cell.
10. application as claimed in claim 9 is characterized in that described breast cancer cell is also expressed the transfection of the proteic expression vector of p53.
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| CN1641036A (en) * | 2004-12-20 | 2005-07-20 | 中国人民解放军第四军医大学 | Humandrg2 recombinant adenovirus expresion vector configuration and use of anti-tumour recombinant adenovirus |
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| CN105749283B (en) * | 2016-02-23 | 2019-04-02 | 南开大学 | Application based on transcription factor ZEB1 in the drug that preparation promotes chemosensitivity of breast cancer |
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