CN102643785A - Method for targeted inhibition of infiltration and transfer of glioma cells and application of method - Google Patents
Method for targeted inhibition of infiltration and transfer of glioma cells and application of method Download PDFInfo
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Abstract
The invention discloses a method for targeted inhibition of infiltration and transfer of glioma cells, which includes targeted inhibition of JAM2 (junctional adhesion molecules 2) of the glioma cells. Experiments show that cell proliferative activity of the glioma cells can be inhibited in vivo by 30% by inhibition on the JAM2 of the glioma cells, cell migration activity can be inhibited by more than 80%. The method has superb clinical application prospect, and by the method, non-specificity of existing drug therapy on glioma cell cases, the defects in surgical therapy, radiotherapy, chemotherapy and endocrine therapy are overcome, and the range of targeted therapy is widened.
Description
Technical field
The present invention relates to a kind of target and suppress the method that glioma cell soaks into and shifts; The siRNA that can target suppresses glioma cell adhesion factor JAM2, shRNA, and neutrality antibody; Also comprise and use this siRNA, shRNA; And the neutrality antibody drug prepared, and this siRNA, shRNA, and neutrality antibody suppresses as the preparation target, and glioma cell soaks into and the application of the medicine that shifts.
Background technology
Neurospongioma is the malignant brain tumor that threatens China's people ' s health.Statistical figure show have 60% to be malignant brain tumor in the primary brain tumor approximately; The sickness rate of China is 8～100,000, and a little more than world's average attack rate, China malignant brain tumor patient estimates annual newly-increased more than 20 ten thousand people.Multiform glue blastoma patient's survival time (intermediate value) was generally 1 year, only survived by 5 years less than 3% patient.Glioblastoma clinical treatment means are mainly operation, chemotherapy and radiation, but all do not have fine clinical effectiveness at present, after therapeutic process finishes, and many patient's recurrences, transfer.Glioblastoma clinical treatment failure mainly due to tumour towards periphery cerebral tissue invasive growth, existing complex treatment measure target property poor, can not thoroughly kill due to the wellability tumour cell, and the invasive ability of glioma cell also increases along with the rising of grade malignancy.So the optimization of glioma treatment research strategy not only should be had in mind former position tumor treatment, also should put forth effort to control invasion and attack and the transfer of tumour cell to normal cerebral tissue and other histoorgans.Therefore, the glioma cell novel targets identifies that the targeted drug design has important clinical significance for development of new anticol matter tumor medicine.
Cell junction adhesion molecule (Junctional adhesion molecules is called for short JAM) is to be positioned at one type of gp that two immunoglobulin folding (VH-and C2 type) contained in the zone, extracellular.JAM albumen is expressed in the glutinous zone that connects between differentiation epithelial cell and epithelial cell usually.JAM analogue blood vessel epithelial cell connects adhesion molecule JAM2, and be accredited as and contain 298 amino acid, and specific expressed in areas of inflammation and tumor tissues arteries epithelial cell.Its function is brought into play in α 4 βs 1 and NK cell, the BMDC effect of JAM2 through its interact protein JAM3 and T cell surface.Javerzat etc. passed through the external embryonic blood vessel system cam of biochip technology high throughput analysis chicken (Chick chorio-allantoic membrane) in 2009; One type of height blood vessel tissue; Gene in ripe evolution changes; Discovery is compared with non-epithelial cell, and JAM2 expresses at blood vessel epithelial cell camber with the human similar gene order of other CAM gene.But it is not at present clear and definite as yet for the effect of JAM2 in tumor development.
Summary of the invention
We adopt the biochip technology analysis to compare 10 routine normal cerebral tissues and 50 routine multiform yeast cell knurl (GBM) gene differential expressions, and simultaneously, we have obtained the positive glioma stem cell of CD133 at separation from clinical high malignancy GBM tissue.Find through the biochip technology analysis, JAM2 not only in people GBM expression amount be higher than normal cerebral tissue, and expression amount also is significantly higher than at the negative glioma cell of CD133 and expresses in the positive stem cell of CD133.The present invention has identified the over-expresses of JAM2 in glioma cell and glioma stem cell; Through in the glioma cell culture system; The neutrality antibody that adds JAM2, perhaps through mediating the shRNA transfection through slow virus, the shRNA plasmid that said target is suppressed glioma cell adhesion factor JAM2 mixes with lentiviral vectors; Perhaps through mediating the shRNA transfection through transfection agents; The shRNA that said target is suppressed glioma cell adhesion factor JAM2 mixes with transfection agents, perhaps through the transfection of transfection reagent mediation siRNA, said target is suppressed the siRNA and methods such as transfection agents mixes of glioma cell adhesion factor JAM2; Target suppresses the genetic expression of JAM2, effectively suppresses glioma external and in-vivo tumour propagation, invasion and attack and migratory activity.
Can carry out the specific aim design according to the different target spots of JAM2 about siRNA, a plurality of different siRNA sequences can be arranged.For example: following 4 is four different siRNA action target spots of JAM2, and their complementary sequence is the siRNA sequence:
JAM2-homo-1001?GCTACGATGTCAAGACAAAGA
JAM2-homo-1191?GCCCGCAATTCTGTTGGATAT
JAM2-homo-1132?CTGGAACTCTGCAATTTAATA
JAM2-homo-1286?GGCCTTAGTGATTTCCGTTTG。
When the time spent of doing that the neutrality antibody of selecting to utilize JAM2 suppresses tumour cell adhesion factor JAM2, the neutrality antibody of JAM2 directly is used for glioma cell vitro culture system or body internal jugular vein drug administration by injection or topical, suppress the effect of JAM2.Used monoclonal mouse anti-human sequences are JAM2: FSAPKDQQVVTAVEYQEAILACKTPKKTVSSRLEWKKLGRSVSFVYYQQTLQGDFKNRAEMIDFNIRIKNVTRSDAGKYRCEVSAPSEQGQNLEEDTVTLEVLVAPAVPSCEVPSSALSGTVVELRCQDKEGNPAPEYTWFKDGIRLLENPRLGSQSTNSSYTMNTKTGTLQFNTVSKLDTGEYSCEARNSVGYRRCPGKRMQVDDLNISGIIAAVVVVALVISVCGLGVCYAQRKGYFSKETSFQKSNSSSKATTMSENDFKHTKSFII.
In addition, neutrality antibody can also comprise this antibody sequence is carried out the antibody sequence after humanization substitutes, promptly to the humanization monoclonal antibody sequence of this mouse source anti-people JAM2 monoclonal antibody.
The siRNA or the shRNA plasmid of said target JAM2 gene are mixed with transfection reagent, and transfection gets into glioma cell, the expression of reticent JAM2, thereby the effect that suppresses glioma cell propagation, soaks into, shifts.Transfection process both can have been used the transfection of the liposome-mediated siRNA that contains siRNA, also can use the transfection of the slow virus mediation shRNA that contains shRNA.Wherein transfection step is following: inoculation 106/ml glioma cell U87 cell to 6 well culture plate, and every hole adds RPMI-1640 and 10% foetal calf serum of 2ml, places the 5%CO2 incubator to cultivate; Spend the night, behind the cell attachment, treat that the cell attachment rate reaches 60% when above; The transfection mixture of preparation transfection reagent and siRNA, said transfection mixture adds the siRNA of 5 μ l, 2 μ M like 1 μ l liposome, perhaps adds JAM2 neutrality antibody 1mg/ml; Transfection mixture or neutrality antibody are added in the cell; Continue to cultivate 36-72 hour, and detected its transfection efficiency through WESTERN BLOT, shown in the result promptly to the proteic efficient that knocks out of JAM2.
Compared with prior art; The present invention uses the neutrality antibody of JAM2 or the method for siRNA, shRNA inhibition glioma cell infiltration and transfer makes the treatment of targeted cells adhesion factor JAM2 have widespread usage value; Make the clinical benefit of all glioma patients; The present invention is used to the neutralizing antibody of JAM2 or siRNA, shRNA to prepare and suppresses the medicine that glioma cell soaks into and shifts, and overcome chemotherapy non-selective to glioma, reaches the targeted therapy glioma, improves the purpose of prognosis of patients; Remedied the deficiency of topical therapeutic means such as operation, chemicotherapy, can the micrometastasis kitchen range have been played a role; Can overcome the toxic side effect of chemotherapy, enlarge the scope of targeted therapy body.
To achieve these goals, patent of the present invention adopts following technical scheme:
A kind of target suppresses the method that glioma cell soaks into and shifts, and said method is that target suppresses glioma cell adhesion factor JAM2.
As preferably, said glioma cell adhesion factor JAM2 also comprises the target spot that is positioned on the said glioma cell adhesion factor JAM2, and the sequence of said target spot includes:
JAM2-homo-1001?GCTACGATGTCAAGACAAAGA
JAM2-homo-1191?GCCCGCAATTCTGTTGGATAT
JAM2-homo-1132?CTGGAACTCTGCAATTTAATA
JAM2-homo-1286?GGCCTTAGTGATTTCCGTTTG。
As preferably, the method that said target suppresses glioma cell adhesion factor JAM2 does, utilizes the neutrality antibody of glioma cell adhesion factor JAM2 to suppress the effect of glioma cell adhesion factor JAM2.
As preferably; The sequence of said neutrality antibody is FSAPKDQQVVTAVEYQEAILACKTPKKTVSSRLEWKKLGRSVSFVYYQQTLQGDFK NRAEMIDFNIRIKNVTRSDAGKYRCEVSAPSEQGQNLEEDTVTLEVLVAPAVPSCE VPSSALSGTVVELRCQDKEGNPAPEYTWFKDGIRLLENPRLGSQSTNSSYTMNTKT GTLQFNTVSKLDTGEYSCEARNSVGYRRCPGKRMQVDDLNISGIIAAVVVVALVIS VCGLGVCYAQRKGYFSKETSFQKSNSSSKATTMSENDFKHTKSFII, perhaps the sequence of said neutrality antibody is carried out the antibody sequence that obtains after humanization substitutes.
As preferably; The method that the said neutrality antibody that utilizes glioma cell adhesion factor JAM2 suppresses the effect of glioma cell adhesion factor JAM2 does; Through said neutrality antibody directly being used for glioma cell vitro culture system; Body internal jugular vein drug administration by injection, perhaps the method for topical suppresses the effect of glioma cell adhesion factor JAM2.
As preferably; The method that said target suppresses glioma cell adhesion factor JAM2 does; Through slow virus or transfection agents mediation shRNA transfection; Perhaps, said shRNA or siRNA transfection are got into glioma cell, the expression of reticent glioma cell adhesion factor JAM2 through transfection agents mediation siRNA transfection.
As preferably, the antisense strand sequence of said siRNA is 5 '-UACUACGGCUGCUAUGAUG-3 '; The sequence of said shRNA is CCGGGCTCCTGAATACACATGGTTTCTCGAGAAACCATGTGTATTCAGGAGCTTTT TG; The sequence of said siRNA or shRNA also comprises the complementary sequence of the sequence that is positioned at the target spot on the glioma cell adhesion factor JAM2.
As preferably, the step of said transfection comprises: inoculation 10
6/ ml glioma cell to 6 well culture plate, every hole adds RPMI-1640 and 10% foetal calf serum of 2ml, places 5%CO
2Cultivate in the incubator, spend the night, behind the cell attachment, treat that the cell attachment rate reaches at 60% o'clock, preparation transfection reagent and siRNA, the perhaps transfection mixture of shRNA adds to said transfection mixture in the cell and to cultivate 36-72 hour.
In addition, the present invention also comprises and a kind ofly suppresses according to target that glioma cell soaks into and the application of the method that shifts, is used to prepare target and suppresses the medicine that glioma cell soaks into and shifts.The effective constituent of this medicine is siRNA, shRNA, perhaps neutrality antibody.
In addition, the present invention comprises that also a kind of method that suppresses glioma cell infiltration and transfer according to said target is soaked into as preparation target inhibition glioma cell and the application of the medicine of transfer.Be above-mentioned siRNA, shRNA, the perhaps application of neutrality antibody in the medicine of the said target inhibition of preparation glioma cell infiltration and transfer.
The present invention has following beneficial effect with respect to prior art:
The infiltration and the transfer activity that can significantly suppress glioma cell; And target property with height; Can overcome existing pharmacological agent non-specific to the glioma case; Simultaneously can reach the targeted therapy glioma, improve the purpose of prognosis of patients, remedy the deficiency of topical therapeutic means such as operation, chemicotherapy.Can play a role to the micrometastasis kitchen range, overcome the toxic side effect of chemotherapy, enlarge the scope of targeted therapy, have splendid potential applicability in clinical practice body.
Description of drawings
Fig. 1 detects the proteic expression of brain tumor tissue of patient JAM2 for immunohistochemical methods.
JAM2 expresses Western Blot figure as a result to Fig. 2 among the glioma cell U251 for siRNA knocks out.
Fig. 3 is for being dyed hepatic cell in result that microscopically observed.
Fig. 4 is that transfection reagent transfection JAM2 siRNA after 48 hours, suppresses the map of glioma cell mobility to the U251 glioma cell.
Fig. 5 influences the U251 cell-proliferation activity for JAM2 shRNA target knocks out the JAM2 expression.
The influence that Fig. 6 migrates the U87 cells in vitro for the JAM2 neutrality antibody.
Fig. 7 knocks out back U251 cell animal body internal breeding activity influence for JAM2.
Embodiment
In order to make those skilled in the art person better understand patent of the present invention; Below in conjunction with the accompanying drawing in the patent working example of the present invention; Technical scheme in the patent working example of the present invention is carried out clear, complete description; Obviously, described embodiment only is a part of embodiment of patent of the present invention, rather than whole embodiment.Based on the embodiment in the patent of the present invention, those of ordinary skills are not making the every other embodiment that obtains under the creative work prerequisite, all should belong to the scope of patent protection of the present invention.
Embodiment 1: JAM2 expression in people's samples of human glioma
In order to study JAM2 gene expression in samples of human glioma, collected 60 routine glioma corrective surgery excision samples, done a series of related experiment.At first, with the situation that JAM2 in the methods analyst glioma of routine immunization groupization expresses, the result sees Fig. 1.
By Hematorylin with the nuclei dyeing au bleu, the showed cell distributed number.Represent the expression of JAM2 molecule by the yl moiety of DAB colour developing at cell peripheral.The result shows: the stained positive histocyte is arranged unordered; Closely porousness is inconsistent; The nucleus shape is not of uniform size, is the tumor tissue cell of excessive increase, and its pericellular pale brown look dyeing is also very abundant; Yellow hollow elliptical section is divided into blood vessel crosscut cross section, and closely link molecule JAM2 expresses more horn of plenty.The cell distribution of normal cerebral tissue, showed cell is less, is evenly distributed, and yellow staining cell does not have brain tumor tissue abundant.
Coloration result shows that brain tumor tissue JAM2 protein abnormal expression is abundant, is necessary further to do the relation that the gene knockout experiment discloses itself and tumor proliferation and invasion and attack, for being that the research that target carries out tumor therapeutic lays the first stone with the JAM2 molecule.Because the experiment condition restriction only takes the picked at random sample to experimentize, pathological tissue all is positive as a result, and healthy tissues is all negative, and brain tumor tissue JAM2 developed by molecule positive rate is 100%.
Embodiment 2: through the reticent glioma cell JAM2 of JAM2 siRNA gene transfection expression inhibiting cell migration experiment
The siRNA or the shRNA plasmid of said target JAM2 gene are mixed with transfection reagent, and transfection gets into glioma cell, the expression of reticent JAM2, thereby the effect that suppresses glioma cell propagation, soaks into, shifts.Wherein transfection step is following: inoculation 106/ml glioma cell U251 cell to 6 well culture plate, and every hole adds RPMI-1640 and 10% foetal calf serum of 2ml, places the 5%CO2 incubator to cultivate; Spend the night, behind the cell attachment, treat that the cell attachment rate reaches 60% when above; The transfection mixture of preparation transfection reagent and siRNA; Add the siRNA of 5 μ l, 2 μ M like 1 μ l liposome, to cell, continue to cultivate 36-72 hour; Detect its transfection efficiency through WESTERN BLOT, shown in the result promptly to the proteic efficient that knocks out of JAM2.
WESTERN BLOT detects step; The siRNA sequence of target JAM2 is mixed with transfection reagent; In-vitro transfection glioma cell line U251 cell, transfection extracted total protein, determination of protein concentration test kit (#23227 with the RIPA protein lysate after 72 hours; Thermo Pierce) measures protein concentration, 95 ℃ of hot 5min.Electrophoresis applied sample amount 20mg, behind electrophoresis, commentaries on classics film (pvdf membrane), protein blocking, an anti-JAM2 (H00058494-M01; Abnova; Taiwan) according to 4 ℃ of incubated overnight of 1:2000 extent of dilution, wash behind the film that HRP two is anti-hatched 2 hours according to 37 ℃ of 1:5000, add the ECL chemical luminescence for liquid after washing film; Sandwich magazine, exposure in the dark place.Through developing fixing, after the film oven dry, be scanned into electronic edition and preserve.The result is as shown in Figure 2, adopt different transfection reagent Lipofectamine (invitrogen, US) and PEI (25K da; Sigma), all can effectively knock out the JAM2 protein expression, GAPDH is the reference protein expression; As shown in Figure 2, in the present embodiment, transfection process both can have been used the transfection of the liposome-mediated siRNA that contains siRNA; Also can use the transfection of the slow virus mediation shRNA that contains shRNA; Each row of left, center, right are respectively and use Lipofectamine, PEI, and the result of control group among Fig. 2.
Cell migration suppresses experiment:
Albumen knocked out cells transfected after 48 hours; Trysinization is centrifugal; With serum-free DMEM dilution counting, (CORING is in cell USA) to be inoculated in transwell 24 orifice plates according to the density in 2000 in every hole; Cell below culture hole adds 20%FBSDMEM, puts into cell culture incubator and continues to cultivate 12 hours.Whether cell is arranged in the chamber under the microscopic examination, draw then and go up the chamber residual liquid, following chamber adds 600 μ lPBS, cleans twice.Add 500 μ l90% ethanol then in following chamber, room temperature is 30min fixedly, and is air-dry.Add 200 μ l0.1% Viola crystallinas, cell is immersed in wherein, 37 ℃ of dyeing 30min; Chamber residual liquid in the absorption after 75% alcohol swab is wiped ventricular cell, will be immersed in 500 μ l deionized water wash by cell; At last with the cell frame in the aperture of 24 orifice plates, take pictures, the counting; Being dyed hepatic cell is exactly the cell that passes film, result such as Fig. 3, shown in Figure 4.Wherein the left side is the result of siRNA group, and the right is the result of control group, and the result shows that transfection reagent transfection JAM2 siRNA after 48 hours, can suppress glioma cell 80% cell migration rate to the U251 glioma cell.
Embodiment 3: the shRNA of target JAM2 suppresses its cell proliferation experiment through slow-virus transfection glioma cell line U251 cell.
The shRNA of coding JAM2 is encapsulated through slow virus, stable transfection glioma cell U251 cell, wherein transfection step is following: inoculation 106/ml glioma cell U251 cell to 6 well culture plate; Every hole adds RPMI-1640 and 10% foetal calf serum of 2ml, places the 5%CO2 incubator to cultivate, and spends the night; Behind the cell attachment, treat that the cell attachment rate reaches 60% when above, the mixture of preparation slow virus and shRNA is to cell; Continue to cultivate 36-72 hour, and detected its transfection efficiency through WESTERN BLOT, shown in the result promptly to the proteic efficient that knocks out of JAM2; Detect influence through mtt assay, detect influence its invasion and attack through the cell strok method to its cell proliferation.The result shows, the JAM2 gene through slow-virus transfection is stable knock out after, but its cell-proliferation activity of vitro inhibition reaches 30%, suppresses it and migrates and actively surpass 80%.As shown in Figure 5.
Fig. 5 expresses the influence to the U251 cell-proliferation activity for JAM2 shRNA target knocks out JAM2.Wherein Control is a control group; U251-1 is that JAM2 knocks out group; U251-2 is the non-specific sequence control group of JAM2.The result shows that can suppress the glioma cell proliferation activity after the JAM2 target is pounded out surpasses 30%.
The experiment of embodiment 4:JAM2 neutrality antibody vitro inhibition glioma invasion and attack
Inoculation 106/ml glioma cell U87 cell to 6 well culture plate adds RPMI-1640 and 10% foetal calf serum, in vitro culture U87 glioma cell culture system; Add JAM2 neutrality antibody 1mg/ml, carry out scratch experiment, continued to cultivate 48 more as a child; Microscopically is observed, and takes pictures.The result is as shown in Figure 6, and JAM2 antibody can effectively suppress the external migratory activity of glioma cell, wherein, and A: cellular control unit line initial time microphotograph; B: cellular control unit line microphotograph after 48 hours; U87 cell line initial time microphotograph after the C:JAM2 antibody group; D:JAM2 antibody group U87 cell line 48 is microphotograph as a child.
Embodiment 5: gene knockout JAM2 expresses value-added influence in the glioma cell body
Adopt JAM2 siRNA or the external glioma cell U251 cell JAM2 expression of gene that knocks out of shRNA; Knock out efficient 50% and more than; Cell after the JAM2 stable gene knocked out and do not knock out cellular control unit according to the 106/ml subcutaneous vaccination in nude mice, raise 4 weeks of nude mice continuously.Measure tumor size weekly and change, raise finish after, dissect and win tumor tissues, measurement size and weighing calculates tumour inhibiting rate.The result is as shown in Figure 7, and after target knocked out JAM2 genetic expression, it was active significantly to suppress the internal breeding of glioma cell U251 cell paste, more than the inhibitory rate to 30%.
Fig. 7 knocks out the active influence of back U251 cell animal body internal breeding for JAM2.Wherein JAM-2 negative is the negative groups of cells of expressing of JAM2; Control is a control group JAM2 normal expression group.
The above results shows to have siRNA, the shRNA of ad hoc structure, and neutrality antibody has the exhibited strong inhibition effect to propagation, infiltration and the transfer of glioma cell.With above-mentioned siRNA, shRNA, perhaps neutrality antibody is that the medicine of effective constituent is a kind of new drug of very potential, effective anti-glioma, can be used to treat the neural system glioma by the mentioned component drug prepared, has broad application prospects.
Sequence table
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Claims (9)
1. a target suppresses the method that glioma cell soaks into and shifts, and it is characterized in that, said method is that target suppresses glioma cell adhesion factor JAM2.
2. target according to claim 1 suppresses the method that glioma cell soaks into and shifts; It is characterized in that; Said glioma cell adhesion factor JAM2 also comprises the target spot that is positioned on the said glioma cell adhesion factor JAM2, and the sequence of said target spot comprises following arbitrary sequence:
Aminoacid sequence shown in the SEQ ID NO:1;
Aminoacid sequence shown in the SEQ ID NO:2;
Aminoacid sequence shown in the SEQ ID NO:3;
Aminoacid sequence shown in the SEQ ID NO:4.
3. target according to claim 1 suppresses the method that glioma cell soaks into and shifts; It is characterized in that; The method that said target suppresses glioma cell adhesion factor JAM2 is to utilize the effect of the neutrality antibody inhibition glioma cell adhesion factor JAM2 of glioma cell adhesion factor JAM2.
4. target according to claim 3 suppresses the method that glioma cell soaks into and shifts; It is characterized in that; The sequence of said neutrality antibody is the aminoacid sequence shown in the SEQ ID NO:5, perhaps the sequence of said neutrality antibody is carried out the antibody sequence that obtains after humanization substitutes.
5. target according to claim 3 suppresses the method that glioma cell soaks into and shifts; It is characterized in that; The method that the said neutrality antibody that utilizes glioma cell adhesion factor JAM2 suppresses the effect of glioma cell adhesion factor JAM2 does; Through said neutrality antibody directly being used for glioma cell vitro culture system, body internal jugular vein drug administration by injection, the perhaps effect of the method for topical inhibition glioma cell adhesion factor JAM2.
6. target according to claim 1 suppresses the method that glioma cell soaks into and shifts; It is characterized in that; The method that said target suppresses glioma cell adhesion factor JAM2 does, through slow virus or transfection agents mediation shRNA transfection, perhaps through transfection agents mediation siRNA transfection; Said shRNA or siRNA transfection are got into glioma cell, the expression of reticent glioma cell adhesion factor JAM2.
7. target according to claim 6 suppresses the method that glioma cell soaks into and shifts, it is characterized in that,
The antisense strand sequence of said siRNA is: the RNA sequence shown in the SEQ ID NO:6;
The sequence of said shRNA is: the RNA sequence shown in the SEQ ID NO:7;
The sequence of said siRNA or shRNA also comprises the complementary sequence of the sequence that is positioned at the target spot on the glioma cell adhesion factor JAM2, and the sequence of said target spot comprises following arbitrary sequence:
Aminoacid sequence shown in the SEQ ID NO:1;
Aminoacid sequence shown in the SEQ ID NO:2;
Aminoacid sequence shown in the SEQ ID NO:3;
Aminoacid sequence shown in the SEQ ID NO:4.
8. target according to claim 6 suppresses the method that glioma cell soaks into and shifts, and it is characterized in that the step of said transfection comprises: inoculation 10
6/ ml glioma cell to 6 well culture plate, every hole adds RPMI-1640 and 10% foetal calf serum of 2ml, places 5%CO
2Cultivate in the incubator, spend the night, behind the cell attachment, treat that the cell attachment rate reaches at 60% o'clock, preparation transfection reagent and siRNA, the perhaps transfection mixture of shRNA adds to said transfection mixture in the cell and to cultivate 36-72 hour.
9. the application of the method that suppresses the glioma cell infiltration according to the said target of claim 1-8 and shift is characterized in that, is used to prepare target and suppresses the medicine that glioma cell soaks into and shifts.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773767A (en) * | 2014-01-24 | 2014-05-07 | 华东理工大学 | Targeted ADGB inhibitory RNA and application thereof in preparing antitumor drugs |
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| CN103773767A (en) * | 2014-01-24 | 2014-05-07 | 华东理工大学 | Targeted ADGB inhibitory RNA and application thereof in preparing antitumor drugs |
| CN103773767B (en) * | 2014-01-24 | 2016-01-20 | 华东理工大学 | The inhibitory RNA of target ADGB and preparing the application in antitumor drug |
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