CN102453715A

CN102453715A - Small molecular interfering RNA for glioma angiogenesis resistance with high efficiency, preparation method and application thereof

Info

Publication number: CN102453715A
Application number: CN2010105146001A
Authority: CN
Inventors: 郑大; 武佳; 叶思灵; 车丽花; 夏清梅; 潘讴东
Original assignee: SHANGHAI SHENGBO BIOMEDICAL TECHNOLOGY Co Ltd
Current assignee: SHANGHAI SHENGBO BIOMEDICAL TECHNOLOGY Co Ltd
Priority date: 2010-10-21
Filing date: 2010-10-21
Publication date: 2012-05-16

Abstract

The invention relates to a small molecular interfering RNA for inhibiting glioma angiogenesis, a preparation method and an application thereof. The gene is a base sequence as shown in SEQNO. 1. The invention design is a small molecular interfering RNA for inhibiting genes associated with glioma angiogenesis, and studies the promotion effect on the inhibition of glioma angiogenesis by using a glioma cell model. A new method is provided for the application of the small molecular interfering RNA in glioma treatment.

Description

Small molecules interference RNA, its preparation method and application thereof that a kind of efficient anti-glioma new vessel generates

Technical field

The present invention relates to small molecules interference RNA, its preparation method and application thereof that a kind of efficient anti-glioma new vessel generates.

Background technology

Last century the seventies; Folkman has proposed tumor growth first and has depended on the hypothesis in theory that neovascularity generates in " New England Journal of Medicine "; Along with the foundation of capillary endothelial cell culture technique after 8 years, the discovery of first angiogenesis inhibitor after 11 years, proteic purifying of first angiogenic activity etc. after 13 years; This viewpoint is supported by more and more evidences, and is made this field become the focus of tumor research gradually.Tumor-blood-vessel growth has been brought into play important effect in the formation of tumour and transfer process, suppressing tumor vascular generation is a Critical policies of modern treatment tumour.

RNA disturbs (RNA interference; RNAi), be meant that organism suppresses a kind of phenomenon that special endogenous mRNA expresses when importing with endogenous mRNA coding region homologous double-stranded RNA in the cell; And mRNA degrades, thereby causes the specific gene expression silencing.RNA interferes mechanism in cell, to occur with RNA molecule double chain form, when double-stranded RNA activates, can reduce the expression of endogenous mRNA molecular level.When target mRNA molecule disappeared, corresponding genetic expression was reticent.

Reduce the expression that the glioma new vessel generates crucial associated gene STAT3 through RNAi means target, for a new way is opened up in the treatment of cerebral glioma.Key in technical field herein and describe paragraph.

Summary of the invention

One of the object of the invention is to provide a kind of small molecules interference RNA of efficient anti-glioma new vessel generation.

Two of the object of the invention is to provide the preparation method of this small molecules interference RNA.

Three of the object of the invention is to provide the application of small molecules interference RNA.

For achieving the above object, the present invention adopts following technical scheme:

The small molecules interference RNA that a kind of efficient anti-glioma new vessel generates is characterized in that this gene is the base sequence shown in the SEQ NO.1.

A kind of small molecules interference RNA for preparing above-mentioned efficient anti-glioma new vessel generation; The concrete steps that it is characterized in that this method are: according to the specific requirement of carrier design among the SilenCircle TM RNAi Transcription Kit; Design is corresponding to STAT3 gene 2214-2232 position; That is: the oligonucleotide sequence of the shRNA of GAT CAT GGA TGC TAC CAA T is 53bp respectively, send Sangon Biotech (Shanghai) Co., Ltd. to synthesize; The oligonucleotide sequence of described shRNA is:

SEQ?NO.1:

Sense：?5’-aca?ccG?ATC?ATG?GAT?GCT?ACC?AAT?ttg?ctt?gaa?ATT?GGT?AGC?ATC?CAT?GAT?Ct-3’

Anti-sense:：5’-aaa?aaG?ATC?ATG?GAT?GCT?ACC?AAT?ttc?aag?caa?ATT?GGT?AGC?ATC?CAT?GAT?Cg-3’；

According to the specific requirement of carrier design in the SilenCircle TM RNAi Transcription Kit gene plasmid structure test kit, with oligonucleotide sequence and the P of the shRNA that designs ^SilenCirclePlasmid connects, and obtains the small molecules interference RNA that efficient anti-glioma new vessel generates.

The small molecules interference RNA that a kind of above-mentioned efficient anti-glioma new vessel generates generates the application in the medicine in the anti-glioma invasion and attack of preparation with anti-new vessel.

The present invention's design generates the RNAi of related gene to inhibition glioma new vessel, and uses the promoter action that its anti-glioma new vessel of glioma cell model research generates.For RNAi uses in the treatment of glioma a kind of novel method is provided.

[0012]

[0013]Embodiment:

One, the cultivation of glioma cell: glioma cell U251 clone is incubated at 37 ℃, 5%CO ₂Constant incubator.The cell of logarithmic phase takes off wall with trysinization, adds an amount of nutrient solution again to be easy to the mixing cell; It is moved in the 15ml centrifuge tube the centrifugal 3min of 1500rpm with pipettor; Remove supernatant; With the aseptic 1 * PBS of 5ml cell is hanged, and the piping and druming mixing; Add about 1ml cell suspension in the new culturing bottle that the 5ml fresh culture is housed, put into 37 ℃, 5%CO ₂Cultivate about about 2 days go down to posterity 1 time (checking that mainly cell has or not the confluent culture bottle) in the incubator.

Two, the separation of human glioma cell: get the human glioma specimens from pri, immerse in 4 ℃ the KRBB solution. clean secondary, then sample is cut into small pieces; Be transferred in the triangular flask that fills 5mL digestive ferment liquid 37 ℃ of insulations in the constant temperature shaking table, shaking speed 100 r/min; Every 10min Glass tubing is blown and beaten once repeatedly; Place 30min, afterwards with the centrifugal 1000rpm of this suspension, centrifugal 5min; Remaining is glioma cell at last. under inverted microscope, glioma cell is rounded.

Three, the design of STAT3-RNAi plasmid and transfection: according to the specific requirement of carrier design among the SilenCircle TM RNAi Transcription Kit; Design is corresponding to STAT3 gene 2214-2232 position; That is: the oligonucleotide sequence of the shRNA of GAT CAT GGA TGC TAC CAA T; Be 53bp respectively, and synthesize.

The sequence that STAT3-RNAi is corresponding is:

SEQ?NO.1:

According to the specific requirement of carrier design among the SilenCircle TM RNAi Transcription Kit, with interference fragment and the P of the STAT3-RNAi that designs ^SilenCirclePlasmid connects, and referring to Fig. 1, glioma cell is advanced in transfection then.

The transfection of STAT3-RNAi plasmid: transfection previous day, planting 2 * 10 in the 60-mm petridish of the DMEM substratum that contains the 5ml antibiotic-free ⁶Individual glioma cell.Use the STAT3-RNAi expression vector of substratum (DMEM) the dissolving 8.0 μ g of 500 μ l serum-frees then, with 500 μ l substratum (DMEM) dissolving Lipofectamine ^TM2000 (20 μ l), mixing lightly, incubated at room is 5 minutes again.After hatching 5 minutes, with shRNA expression vector and Lipofectamine ^TM2000 abundant mixings, incubated at room 20 minutes is to form DNA-Lipofectamine ^TM2000 mixture.With DNA-Lipofectamine ^TM2000 mixtures join in the 60mm petridish, and cell is at 37 ℃ afterwards, 5% CO ₂Cultivated 6 hours in the incubator, change the normal cell nutrient solution.

Four, experiment is divided into groups and detected index normal control group: glioma cell, nutrient solution are the glioma cell nutrient solution; The RNAi group: the glioma cell of RNAi is crossed in transfection, and nutrient solution is the glioma cell nutrient solution.

The detection of glioma cell vasculogenesis: the cell suspension of respectively organizing after the transfection is placed 37 ℃, 5% CO ₂Incubator in cultivate 24h, get and respectively organize cell culture supernatant and add in the vascular endothelial cell, through the method and the HE dyeing of immunocytochemistry, detect the situation of new vessel generation, like Fig. 2.

Fig. 1 is oligonucleotide sequence primer and the P of shRNA ^SilenCircleThe interface chart of plasmid.

Fig. 2 Stat3-RNAi is to the influence of brain glioblastoma cell propagation, and figure is statistical analysis figure.Can know that by figure the cell new vessel number of transfection Stat3 RNA interference plasmid group and vessel branch digital display work are lower than the new vessel number and the vessel branch number of untransfected group and transfection empty carrier group.Mock-untransfected plasmid group among the figure; Empty vector-transfection empty carrier group; Stat3 RNAi-transfection Stat3 RNA interference plasmid group.

Five, result: explain that the tumor neogenetic blood vessels generation in the transfection Stat3 RNA interference plasmid group is suppressed, new vessel number and branches reduce significantly.This experimental result shows, expresses through the Stat3 in the RNAi technology downward modulation glioma, can suppress tumor neovasculature generation effectively.

SEQUENCE?LISTING

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Claims

1. the small molecules interference RNA that efficient anti-glioma new vessel generates is characterized in that this RNA sequence is the base sequence shown in the SEQ NO.1.

2. method for preparing the small molecules interference RNA that inhibition glioma new vessel according to claim 1 generates; The concrete steps that it is characterized in that this method are: according to the specific requirement of carrier design among the SilenCircle TM RNAi Transcription Kit; Design is corresponding to STAT3 gene 2214-2232 position; That is: the oligonucleotide sequence of the shRNA of GAT CAT GGA TGC TAC CAA T; Be 53bp respectively, serve sea living worker's biotechnology Services Co., Ltd and synthesize;

The oligonucleotide sequence sequence of described shRNA is:

SEQ?NO.1:

Sense：?5’-aca?ccG?ATC?ATG?GAT?GCT?ACC?AAT?ttg?ctt?gaa?ATT?GGT?AGC?ATC?CAT?GAT?Ct

-3’

According to the requirement of carrier design among the SilenCircle TM RNAi Transcription Kit, the shRNA sequence that designs is connected with the SilenCircle plasmid, obtain being used for the small molecules interference RNA that efficient anti-glioma new vessel generates.

3. the small molecules interference RNA that generates based on the described efficient anti-glioma new vessels of claim 1 is being produced the application that suppresses in glioma invasion and attack and the anti-glioma new vessels generation medicine.