CN1915432B - Gene medication in use for treating tumor from epithelium, and preparation method - Google Patents
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Abstract
A genetic medicine for treating the epithelium originated tumor, such as breast cancer, stomach cancer, is a recombinant of gene14-3-3 alpha and adenovirus. Its preparing process includes such steps as obtaining the cDNA sequence of 14-3-3 alpha by RT-PCR method, testing the sequence, externally amplifying its whole length by RCR method, severing the amplified product and the shuttle plasmid of adenovirus by specific endonuclease, transinfecting HEK293 cell strain, and encapsulating the recombinant virus.
Description
Technical field
The present invention relates to a kind of genomic medicine, refer in particular to a kind of genomic medicine that is used for the treatment of epithelial origin tumors such as breast carcinoma, gastric cancer, oral squamous cell carcinomas, nasopharyngeal carcinoma, the invention still further relates to the preparation method of this genomic medicine.
Background technology
Traditional tumor therapeuticing method comprises chemotherapy, radiotherapy and operation etc., but from clinical therapeutic effect, all can not thoroughly effect a radical cure tumor.And because put, the non-specific lethal effect of chemotherapy, bigger to the toxicity of body, and life-time service can produce the treatment tolerance of tumor tissues.Operation is obvious to infantile tumour patient curative effect, but because a lot of tumors are found evening, can't implement, and post operative recurrence and branch problem are perplexing the surgeon always.If catch the different of gene expression between tumor cell and the normal cell, adopt the method for adenovirus mediated transfection, adjust the intragentic expression of tumor cell, by the mode of regulation and control physiological function, reach the purpose that suppresses tumor cell specifically.This so-called gene therapy methods has efficient height, high specificity and the advantage low to normal tissue toxicity.Since nineteen ninety America NI H carried out the first in the world gene therapy clinical trial, between the more than ten years after this, gene therapy brought the uncharted field of huge hope as one to the mankind, and its research is flourish, gets most of the attention.So far, 23 countries in 5 continents carry out in the whole world in the clinical trial of existing 1020 gene therapies.2004, the first in the world therapy of tumor medicine was born in China Shenzhen, makes gene therapy be applied to clinical becoming a reality as a kind of new therapy, made the development of world's gene therapy enter a new stage.
In order to open up the genomic medicine of oncotherapy, the present inventor adopts a species specific tumor suppressor gene 14-3-3 σ, and with it with thinking that at present the ideal carrier adenovirus of gene therapy packs (being Ad-14-3-3 σ), and be applied to clinical various epithelial origin tumor treatment.Select for use the reason of this gene to be that 14-3-3 σ expresses in normal person's epithelial tissue, but in the tumor of multiple epithelial origin such as breast carcinoma, gastric cancer, oral squamous cell carcinomas, nasopharyngeal carcinoma expression decreased, disappearance or functional defect.Our former studies shows that 14-3-3 σ crosses expression and can suppress propagation that Her2 crosses the breast cancer cell line mcf-7 of expression and Her18 and become tumor, yet can 14-3-3 σ suppress the nasopharyngeal carcinoma cell of epithelial origin and the cell that Akt crosses expression, and particularly breast carcinoma MDA-MB-361 cell does not appear in the newspapers both at home and abroad as yet.
Summary of the invention
A kind of genomic medicine that is used for the treatment of the epithelial origin tumor of providing a kind of therapeutic effect good, widely applicable is provided one of them purpose of the present invention.
Another object of the present invention provides a kind of can be mass-produced, the preparation method of the said gene medicine that cost is low.
Last purpose of the present invention is achieved in that a kind of genomic medicine that is used for the treatment of epithelial tissue source tumor, be primarily aimed at nasopharyngeal carcinoma and breast carcinoma, it is characterized in that by the people 14-3-3 σ gene with sequence shown in the SEQ ID NO:1 and the recombinant of adenovirus.
Back of the present invention one purpose is achieved in that the cDNA sequence of using the RT-PCR method to obtain 14-3-3 σ, through check order errorless after, its full length gene of external pcr amplification, with specific Cobra venom endonuclease cutting amplified production and adenovirus shuttle plasmid, transfection HEK293 cell strain, the packing of carrying out recombinant virus promptly.
The present invention selects for use 14-3-3 σ to carry out therapy of tumor, and 14-3-3 σ expression of gene product 14-3-3 σ albumen has advantages such as press down that the cancer approach is extensive, cancer suppressing action is definite and untoward reaction is little.As suppressing CDK2, CDK4 and CDK1 produce the retardation of cell cycle, make cell cycle arrest in the G2/M phase, directly suppress tumor proliferation, and help increasing the sensitivity of chemicotherapy; 14-3-3 σ albumen can also be regulated the expression of cancer suppressor protein p53, increases the active cancer suppressing action that produces of the latter; In addition, 14-3-3 σ has in the tumor cell of sudden change and can reach the effect that presses down tumor by regulating the Akt/PKB path that non-p53 relies at p53.The genetic fragment of 14-3-3 σ is shorter, helps obtaining effective virus recombinant.
The present invention selects the carrier of adenovirus as 14-3-3 σ gene transfection for use, adenovirus has relatively long history in the application of gene therapy, accumulated the lesson of a lot of successful experience and failure, its biological characteristics there is more comprehensive understanding, compare with some other viral vector, have safety (can not cause the sudden change of host chromosome), transfection efficiency height, carrying exogenous gene amount greatly, in vivo can the long term maintenance activity etc. advantage.Therefore, be the ideal carrier of present known treatment.
Adopt 14-3-3 σ gene recombinant adenovirus to be applied to oncotherapy among the present invention, have following advantage:
(1) recombination adenovirus construction is simple, can large-scale production, reduce cost, and very broad clinical application prospect is arranged.
(2) adenovirus can carry genes of interest in the tumor cell efficiently, and longer a period of time of effective expression.
(3) have special and lethal effect efficiently to tumor cell, and untoward reaction is little.
(4) tumor (accounting for the overwhelming majority of whole tumors) for epithelial origin all has inhibitory action in various degree, wide application.
Description of drawings
Below in conjunction with drawings and Examples the present invention is described in further detail, but does not constitute any limitation of the invention.
Fig. 1 is the reorganization schematic flow sheet of 14-3-3 σ gene of the present invention and adenovirus;
Fig. 2 14-3-3 σ is to the influence (mtt assay) of nasopharyngeal carcinoma CNE2 cytoactive;
Fig. 3 14-3-3 σ is to the influence (BrdU experiment) of nasopharyngeal carcinoma CNE2 cell proliferation;
Fig. 4 14-3-3 σ is to the influence (soft-agar cloning forms experiment) of nasopharyngeal carcinoma CNE2 cell proliferation;
Fig. 5 14-3-3 σ becomes the influence of tumor nude mice to nasopharyngeal carcinoma CNE2 cell
Fig. 6 14-3-3 σ is to the influence (flow cytometer detection) of nasopharyngeal carcinoma CNE2 cell cycle;
Fig. 7 14-3-3 σ is to the influence (flow cytometer detection) of nasopharyngeal carcinoma CNE2 cell cycle;
Fig. 8 14-3-3 σ is to the apoptotic influence of nasopharyngeal carcinoma CNE2 (dna fragmentation detection kit);
Fig. 9 14-3-3 σ is to Akt and the active influence of CDK2 (vitro kinase analytic process);
14-3-3 σ expresses the relation of expressing with phosphorylation Akt (SABC method) that reaches in Figure 10 breast cancer tissue;
Protein expressions (Western Blot) such as Figure 11 breast carcinoma MDA-MB-361 cell 14-3-3 σ, p-Akt and p27;
Figure 12 14-3-3 σ is to the influence (mtt assay) of Rat1-Akt cytoactive;
Figure 13 14-3-3 σ is to the influence (BrdU experiment) of Rat1-Akt cell proliferation;
Figure 14 14-3-3 σ is to the influence (soft-agar cloning forms experiment) of Rat1-Akt cell proliferation;
Figure 15 14-3-3 σ becomes the influence (pressing down the tumor experiment in the halfbody) of tumor with the MDA-MB-361 cell to Rat1-Akt;
Figure 16 14-3-3 σ is to the tumor-inhibiting action (pressing down the tumor experiment in the body) of Rat1-Akt cell;
Figure 17 14-3-3 σ is to the influence (Western Bl σ t) of Akt albumen, Akt phosphorylation activity and phosphorylated substrate level;
Figure 18 14-3-3 σ is to Rat1-Akt cell p27 location influence (immunofluorescence).
The specific embodiment
The reorganization of embodiment 1 14-3-3 σ gene and adenovirus
Consult shown in Figure 1, use the RT-PCR method to obtain the cDNA sequence of 14-3-3 σ, through check order errorless after, external by PCR method its full length gene that increases, with specific Cobra venom endonuclease cutting amplified production and adenovirus shuttle plasmid, structure carries the shuttle plasmid of 14-3-3 σ gene, with the amplification 14-3-3 σ gene and signal peptide HA gene sub-clone to protokaryon plasmid PUC19, utilize Cla1 and BamH1 sub-clone to pQB1-AdCMV5 (gland-containing virus E1A) again, carry out cotransfection with QB1-viralDNA, the positive-virus clone obtains Ad-HA-14-3-3 σ genetic recombinants through the PCR screening, amplification, transfection HEK293 cell culture, the 14-3-3 σ gene recombinant adenovirus of crude separation is through super centrifugal purification, degerming packing, additive adds, cryopreservation.
Embodiment 214-3-3 σ is applied to the treatment of low differentiation nasopharyngeal carcinoma
1. tetramethyl azo azoles indigo plant (MTT) experiment detects cytoactive
RPIM1640 culture fluid with no calf serum is diluted to 3 * 10
7The cell suspension of cells/L is respectively 5,10,15 Ad-14-3-3 σ and Ad-β-gal or PBS (equivalent) transfectional cell again with MOI, and inoculates 96 well culture plates (100 μ l/well), establishes 5 multiple holes for every group.Put 37 ℃, 5%CO
2Cultivate 2h under the concentration conditions, every hole adds the RPIM1640 culture fluid 100 μ l that contain 20% calf serum, after continuing to cultivate 22h, 46h, 70h and 94h, add 3%MTT20 μ l/well, hatch 4h for 37 ℃, abandon supernatant, every hole adds DMSO100 μ l, behind the mixing 15min, detects the A value at 590nm place, and calculate and press down the tumor percentage rate, promptly press down tumor percentage rate (%)=(processed group A value-negative control group A value)/(control group A value-negative control group A value) * 100%.Repeat this experiment 3 times under identical conditions, getting its meansigma methods is experimental result.The MTT experimental result shows that adenovirus mediated 14-3-3 σ (MOI:5,10,15) transfection 24h, 48h, 72h and 96h all can suppress the CNE2 cell activity.Wherein, along with the prolongation of transfection time, inhibitory action is more obvious, has compared significant difference (P＜0.05) with Ad-β-gal or the PBS matched group of the identical MOI of transfection, and Ad-14-3-3 σ just can the pair cell activity produce obvious inhibitory action when low concentration, dose-dependence is not obvious.The result shows that 14-3-3 σ gene transfection can suppress the CNE2 cell activity, and inhibitory action is time-effect relationship, consults table 1 and shown in Figure 2.
Table 1:14-3-3 σ is to the influence of nasopharyngeal carcinoma CNE-2 cytoactive (X ± SD): %
2.BrdU experiment detects ability of cell proliferation
5-bromo-2 ' Brdurd (BrdU) labelling and detection kit be available from Roche company, experimental arrangement reference reagent box operation instruction., be respectively 5,10 Ad--14-3-3 σ and Ad-β-gal or PBS (equivalent) transfectional cell with MOI promptly, put 37 ℃ of 5%CO the cultivation slide of putting into lid through the exponential phase CNE2 of trypsinization cell
2Incubator is cultivated 30h, treats that it longly, inhales and abandon culture fluid when full to 50%, and every hole adds 200 μ l BrdU labelling culture fluid (1: 1000), 37 ℃, 5%CO
2Cultivate 15～30min under the condition, abandon supernatant, wash with washing liquid 1ml in every hole, and triplicate adds 70% ethanol and 50mM MGlycin mixed liquor (PH2.0) 200 μ l again, put fixed cell under-20 ℃ of conditions, behind the 20min, repeated washing three times adds working solution (1: 10) the 200 μ l of anti-BrdU, put 37 ℃, 5%CO
2Cultivate 30min under the condition, repeated washing three times, after blotting washing liquid, add an amount of counting fluid-tight sheet, observe down in fluorescence microscope, the positive cell number in 300 cells is observed in 5 visuals field of picked at random, calculate BrdU labelling positive percentage, i.e. BrdU positive percentage (%)=positive cell number/total cellular score * 100%.
It is that the CNE2 cell BrdU positive percentage of 5 or 10 Ad-HA-14-3-3 σ transfection is respectively 12% or 42% that the BrdU experimental result shows through MOI; And the CNE2 cell BrdU positive rate of handling with equivalent Ad-β-gal is respectively 86% or 75%, and the CNE2 cell BrdU positive rate that PBS handles is 99%, and the former with the latter comparing difference has statistical significance (P＜0.05), consults shown in Figure 3.
3. soft-agar cloning forms experiment and surveys ability of cell proliferation
Earlier 1% agar solution that preheating has been melted and equivalent contain the double RPIM1640 culture fluid of 30% calf serum mixing after, add 24 orifice plates, complete lower floor's agar, the RPIM1640 diluting cells with serum-free is 2 * 10 then
4/ ml, reuse MOI is 5,10,15 Ad--14-3-3 σ and Ad-β-gal or PBS (equivalent) transfectional cell, 37 ℃ hatch 2h after, respectively get 100 μ l and put into the double RPIM1640 culture fluid that contains 30% calf serum, simultaneously the 0.8% agar solution mixing that melts with the equivalent preheating, join and be covered with in the hole of solidifying lower floor's agar, the cell number that makes every hole is 2000, and 4 repeating holes are established in every hole, and culture plate is put into wet box, and place 37 ℃, 5%CO
2Cultivate 14d in the incubator, and every hole adding p-iodonitrotetrazolium violet (1mg/ml, PBS) solution 0.1ml dyes, and overnight incubation is taken out and is placed on 4 ℃ of refrigerators, treats that top-layer agar solidifies the back shooting fully and calculates the colony number.The soft agar colony forms experimental result and shows that also its colony number of Ad-HA-14-3-3 σ (MOI:5,10 and 15) cells transfected is less than matched group (with the cell after PBS or Ad-β-gal (MOI:5,10, the 15) processing) (P＜0.05), and MOI is a difference little (p＞0.05) between 5,10 or 15 the Ad-HA-14-3-3 σ transfection group, consult shown in Figure 4ly, these all point out cross expressing of 14-3-3 σ can suppress the multiplication capacity of nasopharyngeal carcinoma CNE2 cell.
4.Ad-14-3-3 σ is in vivo to the effect (zoopery) of nasopharyngeal carcinoma
The nude mice in age in 4-5 week is divided into five groups, and promptly MOI is that 5 and 10 Ad-HA-14-3-3 σ treatment group, MOI are 5 and 10 Ad-β-gal and PBS (equivalent) matched group, 6 every group.With MOI is 5,10 Ad-HA-14-3-3 σ, Ad-β-gal and PBS (equivalent) transfection CNE2 cell, collecting cell behind the 48h, and transferring cell number with the DMEM culture fluid is 1 * 10
7/ ml, the cell suspension of every subcutaneous injection 0.2ml of the right nape of nude mice portion duplicates lotus nasopharyngeal carcinoma nude mice model, measures the size of tumor in per then 3 days, to calculate tumor size and to press down tumor percentage rate, i.e. gross tumor volume (mm
3)=L * W
2/ 2; Tumour inhibiting rate (%)=(matched group tumor volume-experimental group tumor volume)/matched group tumor volume * 100%.Observe the time that mouse tumor forms appearance of respectively organizing in addition.
Experimental group and cellular control unit are after 5 and 10 Ad-HA-14-3-3 σ and Ad-β-gal or PBS handle in external elder generation through MOI, be inoculated in nude mice and duplicate tumor model, with observation 14-3-3 σ nasopharyngeal carcinoma CNE2 cell is become the influence of tumor, found that two Ad-HA-14-3-3 σ experimental group gross tumor volumes all are significantly less than matched group (Ad-β-gal or PBS handle) (P＜0.05).And Western blot found that 14-3-3 σ protein expression increases in the tumor tissues, and prompting 14-3-3 σ transfection can suppress the one-tenth tumor of CNE2 cell, consults table 2, shown in Figure 5.
Table 2 14-3-3 σ becomes the influence (X ± SD): % of tumor nude mice to nasopharyngeal carcinoma CNE2 cell
5.Ad-HA-14-3-3 σ suppresses the discussion of nasopharyngeal carcinoma CNE2 cell mechanism
5.1 the distribution of Flow cytometry cell cycle and apoptotic peak
With MOI respectively 5,10 and 15 Ad-HA-14-3-3 σ and Ad-β-gal or PBS (equivalent) transfection CNE-2 cell 36h, collecting cell after trypsinization, centrifugal, abandon supernatant and with behind the PBS washed cell 2 times, with 2 * 10
6Cell is resuspended among the 0.1ml PBS, under slight vibration, slowly drip 3ml 70% ethanol (4 ℃) fixed cell, 4 ℃ are spent the night, and abandon supernatant after centrifugal, with PBS washing 2 times, again cell is resuspended in (50 μ g/mL PI in the 0.5ml PI dye liquor, 20 μ g/mL RNaseA are dissolved in PBS, fresh preparation), 37 ℃ are detected with FACS after hatching 20min.
After FACS detection discovery Ad-14-3-3 σ transfection, the increase of G2 phase cell proportion appears in the CNE-2 cell, G2 phase cell proportion is respectively 72.6%, 72.6% and 42.5% when MOI5,10 and 15 concentration, compares obvious increase with blank group (17.7%); And use MOI is that Ad-β-gal of 5,10 and 15 contrasts as recombinant adenovirus, when with MOI being 5,10 and 15 processing CNE2 cells, its G2 phase cell proportion is respectively 19.7%, 20.3% and 16.4%, with blank group no significant difference (p〉0.05).Find simultaneously, 14-3-3 σ gene recombinant adenovirus transfection group is compared with blank group or Ad-β-gal matched group, the cell proportion of sub-G1 phase increases, consult shown in Figure 6, A is a blank among the figure, B is β-Gal recombinant adenovirus 5MOI, 10MOI and 15MOI group, and C is 14-3-3 σ recombinant adenovirus 5MOI, 10MOI and 15MOI.Wherein the increase of 15MOI Ad-14-3-3 σ group sub-G1 phase cell proportion is particularly evident, up to 19.8%.These promptings Ad-14-3-3 σ transfection CNE2 cell can cause retardance of CNE2 g2 phase and apoptosis, consults shown in Figure 7.
5.2DNA the apoptosis of crack fragment detection by quantitative cell
Be that 5,10 and 15 Ad-HA-14-3-3 σ and Ad-β-gal or PBS (equivalent) handle CNE-2 cell 16h with MOI respectively, collecting cell (comprises floating and attached cell, adding RIPA) liquid acts on 30min with cell lysis under the greenhouse, centrifugal then, get supernatant and diluting cells extract to suitable concentration, detect the dna cleavage fragment with cell death ELISA test kit, with the apoptosis degree of quantitative assessment cell.Specified operational procedure is with reference to dna cleavage fragment quantification kit description.
Fracture fragment with DNA in the dna cleavage segment quantification kit detection cell reflects the apoptosis level respectively.Found that transfection Ad-14-3-3 σ and transfection Ad-β-Gal matched group or blank group ratio, the fracture fragment showed increased (the P value is all less than 0.05) of DNA, wherein 5MOI and 10MOI group increase more obviously consult shown in Figure 8.Simultaneously cell cycle detects and shows, transfection 14-3-3 σ experimental group sub-G1 phase cell proportion increases, when MOI is 15, can consult up to 19.8% shown in Figure 7, A:G2 phase cell inhibitory rate among the figure; B: the ratio of inferior G1 phase cell.These promptings 14-3-3 σ transfection can be induced the CNE2 apoptosis.
5.3 the vitro kinase analytic process detects CDK2 and Akt activity
Earlier handle cell with Ad-HA-14-3-3 σ (MOI=5), matched group replaces with Ad-β-gal (MOI=5) or PBS (equivalent), and cell is after no calcium PBS washs, with about 400 μ l RIPI buffer cell lysis and add Ultrasonic Pulverization 30sec then.4 ℃ of centrifugal collection supernatants.Detect supernatant OD value with DC protein quantification test kit, calculate the sample protein concentration according to standard curve then, get the cell conditioned medium liquid of equal protein, do immunoprecipitation with anti-Akt antibody and anti-CDK2 monoclonal antibody, after washing adding 1 μ l contain 10 μ Ci[γ-
32P] Flag-p27 of ATP or GSK3 β and HH1 and sedimentary immune complex cultivate altogether, respectively organizes sample on the equivalent subsequently, and electrophoresis shows the level of phosphorylation p27 or GSK3 β and HH1 through autoradiography.
Adopt the vitro kinase analytic process to detect the active and active influence of CDK2 of 14-3-3 σ pair cell Akt, at first make immunoprecipitation, will have active nucleus then with anti-Akt antibody or anti-CDK2 antibody
32The p27 of the recombinant purification of P labelling and Gsk3 β are as the kinase whose substrate of Akt, and HH1 is as the kinase whose substrate of CDK2, by detecting the phosphorylation level of corresponding substrate, with reflection Akt kinases or the kinase whose activity of CDK2.The result shows that 14-3-3 σ can reduce Akt kinases and the kinase whose activity of CDK2, and the tumor-inhibiting action of prompting 14-3-3 σ may block cell cycle and apoptosis-induced the realization by reducing Akt and CDK2 kinase activity, consults shown in Figure 9.
Embodiment 3 14-3-3 σ are applied to Akt and cross in the treatment breast cancer tissue of the tumor of expression and human breast carcinoma adenocarcinoma (MDA-MB-361) or the cell 14-3-3 σ and proteic expression such as p-Akt or p27 and mutual relation 1.1 SABC methods and detect that 14-3-3 σ and the proteic expression of p-Akt collect 33 routine primary breast cancer patients' paraffin specimen in the patient breast cancer tissue, make section.Dewax to water with dimethylbenzene, citrate buffer is put in section, regulate pH to 6.0, heating promotes antigen retrieval.Monoclonal antibody 14-3-3 σ (c-18) with dilution in 1: 50, is splashed into microscope slide, put incubated at room 1h, phosphorylation Akt (ser473) also adds microscope slide and hatches by dilution in 1: 100.Dye with general immunohistochemical staining scheme then, use ABC zymolyte test kit labelled protein, redye microscope slide with haematoxylin, dehydration, transparent, mounting.Examine under a microscope the result, positive staining is that nucleus is pale brown color.When observed result, the painted depth degree of labelled protein on the microscope slide is divided into 1,2,3,4 four grade, and the painted quantity of labelled protein also is divided into 0,1 (positive cell＜10%=, 2 (positive cell 10%-50%), four grades of 3 (positive cell＞50%).As standard, judge comprehensively in conjunction with the painted depth and staining cell quantity whether separator protein 14-3-3 σ and p-Akt are high expressed or low the expression.Experimental result adopts the accurate probabilistic method of Fisher to carry out statistical procedures.
In 33 routine breast carcinoma specimen, respectively cut 2 white sheets, used anti-14-3-3 σ and anti-phosphorylation Akt (ser473) monoclonal antibody to do SABC respectively.The standard interpretation of just expressing according to labelled protein, and carry out statistical analysis.Find to have low express (42%) of 14 routine 14-3-3 σ albumen in the 33 routine breast carcinoma specimen.In the low tumor of expressing of these 14-3-3 σ, 75% phosphorylation Akt high expressed, P=0.0024.In the tumor tissues that has 14-3-3 σ to express, visible phosphorylation Akt (ser473) expresses and reduces in addition, consults among Figure 10 shown in the A figure.These results show that the expression of 14-3-3 σ in the breast carcinoma and the expression of phosphorylation Akt (ser473) are negative correlation, consult among Figure 10 shown in the B figure.
1.2 Western Blot method detects protein expressions such as breast carcinoma MDA-MB-361 cell 14-3-3 σ, p-Akt and p27
Tumor cell is after no calcium PBS flushing, and every dish is with 400 μ lRIPA buffer cell lysis and add Ultrasonic Pulverization 30sec.4 ℃ of centrifugal collection supernatants.Detect supernatant OD value with DC protein quantification test kit, calculate the sample protein concentration, adjust protein concentration, sample on the equal protein of every hole through the dilution of RIPA buffer according to standard curve.Adopt polyvinylidine fluoride film trace albumen, through ECL kit chemiluminescence detection box labelled protein, outcome record on Kodak X-omatic AR film (Eastman Kodak Co, Rochester, NY).
MDA-MB-361 is the MCF-7 of the neural overexpression of a HER2/, Western Blot method testing result shows to be compared with the Rat1 cell, MDA-MB-361 cell Akt crosses expression, simultaneously visible Akt phosphorylated substrate increases, the expression decreased of 14-3-3 σ and p27, its protein expression situation is similar to the Ratl-Akt cell, consults shown in Figure 11.
1.Ad-14-3-3 σ is at external effect to the Akt overexpressing cell (mtt assay, BrdU, soft agar colony form experiment)
2.1 cytoactive is surveyed in tetramethyl azo azoles indigo plant (MTT) experiment
The cell of trypsinization exponential phase is diluted to 3x10 with the DMEM culture fluid of no calf serum
7The cell suspension of cells/L is that 5 Ad-14-3-3 σ and Ad-β-gal or PBS (equivalent) transfection Akt cross the Rat1-Akt cell of expression with MOI respectively again, and inoculates 96 well culture plates (100 μ l/well), establishes 5 multiple holes for every group.Put 37 ℃, 5%CO
2Cultivate 2h under the concentration conditions, every hole adds the DMEM culture fluid 100 μ l that contain 20% calf serum, after continuing to cultivate 22h, 46h or 70h, add 3%MTT20 μ l/well, hatch 4h, abandon supernatant for 37 ℃, every hole adds DMSO100 μ l, leave standstill mixing behind the 15min, detect the A value at 590nm place, and calculating but tumor percentage rate (%).The gained experimental data is all within the range of linearity that detects.Press down the percentile computing formula of tumor: (processed group A value-negative control group A value)/(control group A value-negative control group A value) x100%.Repeat this experiment 3 times under identical conditions, the meansigma methods of getting 3 experiments is an experimental result.
The MTT experimental result shows adenovirus mediated 14-3-3 σ (MOI:5) transfection 24h, and 48h and 72h all can suppress the Rat1-Akt cell activity.Wherein, along with the prolongation of transfection time, inhibitory action is more obvious, and as when the transfection 72h, its suppression ratio is near 100%; Compared with transfection Ad-β-gal or PBS matched group significant difference (P＜0.05 〉, consult shown in Figure 12.The transfection that 14-3-3 σ gene is described can suppress the Rat1-Akt cell activity.
2.2BrdU experiment detects ability of cell proliferation (DNA's is synthetic)
5-bromo-2 ' Brdurd (BrdU) labelling and detection kit be available from Roche company, experimental arrangement reference reagent box operation instruction.The cultivation slide of putting into lid through the cell of the exponential phase of trypsinization, with MOI respectively 5 Ad--14-3-3 σ, Ad-β-gal and PBS (equivalent) transfection Rat1-Akt cell, put 37 ℃, 5%CO
2Incubator is cultivated 30h, treats that it longly, inhales and abandon culture fluid when full to 50%, and every hole adds 200 μ l BrdU labelling culture fluid (1: 1000), 37 ℃, 5%CO
2Cultivate 15～60min under the condition, abandon supernatant, it is inferior to give a baby a bath on the third day after its birth with washing liquid 1ml/well, add 70% ethanol and 50mMMGlycin mixed liquor (PH2.0) 200 μ l/well again, put fixed cell under-20 ℃ of conditions, behind the 20min, repeated washing three times, working solution (1: 10) the 200 μ l that every hole adds anti-BrdU put 37 ℃, 5%CO
2Cultivate 30min under the condition, repeated washing three times, after blotting washing liquid, add an amount of counting fluid-tight sheet, observe down in fluorescence microscope, the positive cell number in 300 cells is observed in 5 visuals field of picked at random, calculate BrdU labelling positive percentage, promptly equal positive cell number/total cellular score x100%.
The BrdU interpretation shows that Ad-HA-14-3-3 σ transfectional cell BrdU positive rate is 45%, and the cell BrdU positive rate of matched group after handling with PBS or Ad-β-gal is respectively 100% or 98%, both comparing differences have statistical significance (P＜0.05), consult shown in Figure 13.
2.3 soft-agar cloning forms experiment and surveys ability of cell proliferation
Earlier 1% agar solution that preheating has been melted and equivalent contain the double DMEM culture fluid of 30% calf serum mixing after, add 24 orifice plates, complete lower floor's agar, the DMEM diluting cells with serum-free is 2x10 then
4/ ml, reuse MOI is 5 Ad--14-3-3 σ and Ad-β-gal or PBS (equivalent) transfectional cell, 37 ℃ hatch 2h after, respectively get 100 μ l and put into the double DMEM culture fluid that contains 30% calf serum, simultaneously the 0.8% agar solution mixing that melts with the equivalent preheating, join and be covered with in the hole of solidifying lower floor's agar, the cell number that makes every hole is 2000, and 4 repeating holes are established in every hole, and culture plate is put into box, and place 37 ℃, 5%CO
2Cultivated 14 days in the incubator, take out every hole, back and add 0.1ml p-iodonitrotetrazolium violet (1mg/ml, PBS) solution-dyed, overnight incubation, taking-up is placed on 4 ℃ of refrigerators, treats that top-layer agar solidifies back shooting and the relative percent (the PBS processed group is made as 100%) that calculates colony number and colony fully.The soft agar colony forms experimental result and shows that also its colony number of Ad-HA-14-3-3 σ cells transfected is less than matched group (with the cell after PBS or the Ad-β-gal processing), consult shown in Figure 14ly, these all point out cross expressing of 14-3-3 σ can suppress the multiplication capacity of Rat1-Akt cell.
3. nude mice lotus tumor model observation 14-3-3 σ becomes the influence of tumor to the Akt overexpressing cell
3.1 press down tumor experiment (becoming the influence of tumor with reflection 14-3-3 σ pair cell) in the halfbody
The nude mice in age in 4-5 week is divided into 3 groups, i.e. Ad-HA-14-3-3 σ (MOI=5) treatment group and Ad-β-gal or PBS matched group, 6 every group.With MOI is that 5 Ad-HA-14-3-3 σ, Ad-β-gal and PBS (equivalent) transfection Akt cross the Rat1-Akt and the MDA-MB-361 cell of expression, collecting cell behind the 48h, and transferring cell number with the DMEM culture fluid is 1 * 10
7/ ml, the cell suspension that injects 0.2ml in the fat pad of and mammary gland subcutaneous respectively at the right nape of every nude mice portion duplicates lotus Rat1-Akt nude mice model, the size of per then 3 days measurement tumors 1 time, to calculate tumor size and tumour inhibiting rate, computing formula is: gross tumor volume (mm
3)=L * W
2/ 2; Tumour inhibiting rate (%)=(matched group tumor volume-experimental group tumor volume)/matched group tumor volume * 100%.Observe the time that the nude mice tumor forms appearance of respectively organizing in addition.
Experimental group and cellular control unit in external elder generation after Ad-HA-14-3-3 σ and Ad-β-gal or PBS handle, be inoculated in nude mice and duplicate tumor model, to observe the tumor growth situation, found that transfection Ad-HA-14-3-3 σ experimental group gross tumor volume is significantly less than matched group (Ad-β-gal or PBS handle), difference has statistical significance (P＜0.05).Prompting 14-3-3 σ transfection can suppress the tumor that becomes of Rat1-Akt and MDA-MB-361 cell, consults shown in A among Figure 15, the B.
3.2 press down tumor experiment (with reflection 14-3-3 σ tumor-inhibiting action) in the body
With age in 4-5 week nude mice be divided into 5 groups, promptly 3 MOI are 5 Ad-HA-14-3-3 σ treatment group, Ad-β-gal and PBS matched group, 6 every group, every the right subcutaneous injection 0.2ml of nape portion cell suspension (1 * 10 of nude mice
7/ ml), duplicate nude mice lotus tumor model.Wherein treatment group is divided into 3 groups again, promptly every day drug administration by injection one-time continuous 15 times, per 3 days drug administration by injection are totally 5 times and first day drug administration by injection 1 time behind inoculated tumour only once, matched group injection Ad-β-gal (MOI=5) and PBS (equivalent), once a day.Measured the tumor size in same per three days 1 time, calculate gross tumor volume and tumour inhibiting rate, observation is simultaneously respectively organized the nude mice tumor and is formed the time that occurs.Put to death animal behind the 15d, get tumor tissues is measured expression such as 14-3-3 σ albumen with Western Blot method situation.
Press down the tumor experimental result in the body and show that also Ad-HA-14-3-3 σ treatment group gross tumor volume is significantly less than matched group (Ad-β-gal or PBS handle), the nude mice of wherein giving Ad-HA-14-3-3 σ treatment every day after 15 days its gross tumor volume at 50mm
3In, and gave the nude mice of an Ad-HA-14-3-3 σ treatment in per three days or per 15 days, its gross tumor volume is respectively 120mm
3Or 180mm
3, it all is 250mm that control group A d-β-gal or PBS handle its gross tumor volume
3, and three groups of different time limits give Ad-HA-14-3-3 σ treatment group tumor time of occurrence than matched group evening, wherein again to give Ad-HA-14-3-3 σ treatment group tumor time of occurrence the latest continuously every day, prompting 14-3-3 σ has tumor-inhibiting action in the body, consults shown in Figure 16.
4.Western Blot experiment
After no calcium PBS flushing, take by weighing about 100mg tumor tissues, be cut into behind the fragment by 1: 10 (w/v) adding RIPA buffer cell lysis and add Ultrasonic Pulverization 30sec.4 ℃ of centrifugal collection supernatants.Detect supernatant OD value with DC protein quantification test kit, calculate the sample protein concentration, adjust protein concentration, sample on the equal protein of every hole through the dilution of RIPA buffer according to standard curve.Adopt polyvinylidine fluoride film trace albumen, through ECL kit chemiluminescence detection box labelled protein, outcome record on Kodak X-omatic AR film (Eastman Kodak Co, Rochester, NY).
Measure 14-3-3 σ albumen, Akt albumen, Akt-473 or 308 site phosphorylations and phosphorylated substrate level in the tumor tissues with Western Blot method, found that, in continuous once a day 15 days treatment groups of intratumor injection administration nude mice tumor tissues, the proteic expression of 14-3-3 σ is arranged, and in matched group (Ad-β-gal or the PBS processed group) tumor tissues 14-3-3 σ protein expression lack as.This prompting Adenovirus Transfection 14-3-3 σ gene successful expression in the Rat1-Akt cell.The tumor tissues of finding simultaneously 14-3-3 σ protein expression is arranged is with the proteic minimizing of Akt, and Akt-308 site phosphorylation activity reduces and the minimizing of Akt phosphorylated substrate level, and Akt-473 site phosphorylation activity changes not quite, consults shown in Figure 17.These promptings 14-3-3 σ can suppress the level of the proteic expression of Akt, Akt phosphorylation activity and phosphorylated substrate.
5. immunofluorescence
The Rat1-Akt cell is respectively 5 Ad-HA-14-3-3 σ and Ad-β-gal or PBS (equivalent) (matched group) processing with MOI, use methanol then: acetone (1: 1v/v) behind the following fixedly 2min of room temperature, with the anti-p27 antibody incubation of rabbit 1h, the crosslinked anti-rabbit antibody (molecular probe) of reuse and Alexa568 is hatched 1h, with observation p27 location.For studying the Subcellular Localization of exogenous HA-14-3-3 σ, add earlier anti-HA monoclonal antibody (12CA5, Babco) hatch 1h after, add again with the crosslinked anti-Mus of Alexa488 two anti-(molecular probe) and hatch 1h.For making dyeing fully, above cell adds 1 μ l (0.1 μ g/ml), 4 ', 6 diamidinos-2-benzene indole hydrochloride (DAPI) simultaneously and (Sigma) hatches and do nuclear staining.Observe immunofluorescence in intracellular distribution with Axioplan2 type fluorescence microscope (Zeiss), to determine the Subcellular Localization of each protein molecular.
Adopt immunofluorescence to detect the influence of 14-3-3 σ to p27 Subcellular Localization in the Rat1-Akt cell, found that Rat1-Akt cell through Ad-β-gal (MOI=5) or PBS (equivalent) processing, its p27 is present in endochylema in a large number during 48h, 14-3-3 σ protein expression lack as; Find simultaneously to occur in the Rat1-Akt cell in a large amount of 14-3-3 σ protein expressions with behind the adenovirus mediated transfection 14-3-3 σ gene 48h, significantly reduce the location of p27 in the cytoplasm, and the p27 of karyon increases, and consults shown in Figure 180.Results suggest, the dislocation of p27 in endochylema of 14-3-3 σ Akt mediation capable of blocking.
Adnexa: SEQ ID NO:1 sequence
SEQUENCE?LISTING
＜110〉Zhongshan University: poplar, Hui Ling; In the summer, cloud flies; Lee, dream is grand; Huang, galaxy of literary talent; Soviet Union, brave; In the summer, flood is flat.
＜120〉a kind of epithelial origin oncogene medicine and preparation method thereof that is used for the treatment of
<140>200510102126.0；<141>2005-12-17；<160>1；<170>PatentIn?version
3.3；<210>1；<211>1336；<212>DNA；<213>Human?adenovirus?type?1；<220>；<221>
gene；<222>(1)..(1336)；<400>1。
gagagacaca?gagtccggca?ttggtcccag?gcagcagtta?gcccgccgcc?cgcctgtgtg 60
tccccagagc?catggagaga?gccagtctga?tccagaaggc?caagctggca?gagcaggccg 120
aacgctatga?ggacatggca?gccttcatga?aaggcgccgt?ggagaagggc?gaggagctct 180
cctgcgaaga?gcgaaacctg?ctctcagtag?cctataagaa?cgtggtgggc?ggccagaggg 240
ctgcctggag?ggtgctgtcc?agtattgagc?agaaaagcaa?cgaggagggc?tcggaggaga 300
aggggcccga?ggtgcgtgag?taccgggaga?aggtggagac?tgagctccag?ggcgtgtgcg 360
acaccgtgct?gggcctgctg?gacagccacc?tcatcaagga?ggccggggac?gccgagagcc 420
gggtcttcta?cctgaagatg?aagggtgact?actaccgcta?cctggccgag?gtggccaccg 480
gtgacgacaa?gaagcgcatc?attgactcag?cccggtcagc?ctaccaggag?gccatggaca 540
tcagcaagaa?ggagatgccg?cccaccaacc?ccatccgcct?gggcctggcc?ctgaactttt 600
ccgtcttcca?ctacgagatc?gccaacagcc?ccgaggaggc?catctctctg?gccaagacca 660
ctttcgacga?ggccatggct?gatctgcaca?ccctcagcga?ggactcctac?aaagacagca 720
ccctcatcat?gcagctgctg?cgagacaacc?tgacactgtg?gacggccgac?aacgccgggg 780
aagagggggg?cgaggctccc?caggagcccc?agagctgagt?gttgcccgcc?accgccccgc 840
cctgccccct?ccagtccccc?accctgccga?gaggactagt?atggggtggg?aggccccacc 900
cttctcccct?aggcgctgtt?cttgctccaa?agggctccgt?ggagagggac?tggcagagct 960
gaggccacct?ggggctgggg?atcccactct?tcttgcagct?gttgagcgca?cctaaccact 1020
ggtcatgccc?ccacccctgc?tctccgcacc?cgcttcctcc?cgaccccagg?accaggctac 1080
ttctcccctc?ctcttgcctc?cctcctgccc?ctgctgcctc?tgatcgtagg?aattgaggag 1140
tgtcccgcct?tgtggctgag?aactggacag?tggcaggggc?tggagatggg?tgtgtgtgtg 1200
tgtgtgtgtg?tgtgtgtgtg?tgtgcgcgcg?cgccagtgca?agaccgagat?tgagggaaag 1260
catgtctgct?gggtgtgacc?atgtttcctc?tcaataaagt?tcccctgtga?cactcaaaaa 1320
aaaaaaaaaa aaaaaa 1336
Claims (2)
1. the application of genomic medicine in the medicine of preparation treatment nasopharyngeal carcinoma, described treatment at be low differentiation nasopharyngeal carcinoma CNE2 cell strain, described genomic medicine is by the people 14-3-3 σ gene with sequence shown in the SEQ ID NO:1 and the recombinant of adenovirus.
2. the application of a kind of genomic medicine according to claim 1 in the medicine of preparation treatment nasopharyngeal carcinoma, the preparation of wherein said genomic medicine is to use the RT-PCR method to obtain the cDNA sequence of 14-3-3 σ, through check order errorless after, its full length gene of external pcr amplification, with specific Cobra venom endonuclease cutting amplified production and adenovirus shuttle plasmid, transfection HEK293 cell strain, the packing of carrying out recombinant virus promptly.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6740523B2 (en) * | 1997-12-18 | 2004-05-25 | The Johns Hopkins University | 14-3-3σ arrests the cell cycle |
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2005
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6740523B2 (en) * | 1997-12-18 | 2004-05-25 | The Johns Hopkins University | 14-3-3σ arrests the cell cycle |
Non-Patent Citations (1)
| Title |
|---|
| Heiko Hermeking et al.14-3-3sigma is a p53-regulated inhibitor of G2/M progression.Molecular Cell1.1997,13-11. * |
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| CN1915432A (en) | 2007-02-21 |
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