CN101591655B - PKCalpha signal specifically-blocking siRNA and use thereof - Google Patents

Abstract

The invention discloses a PKCalpha signal specifically-blocking siRNA, which is one arbitrarily selected from the following double-stranded RNA molecules targeted at human PKCalpha: a sense strand of 5'-CGACGACUGUCUGUAGAAA-3' and an antisense strand of 5'-UUUCUACAGACAGUCGUCG-3', a sense strand of 5'-GGAUGGUACAAGUUGCUUA-3' and an antisense strand of 5'-GGCGUCCUGUUGUAUGAAA-3' or a sense strand of5'-GGCGUCCUGUUGUAUGAAA-3' and an antisense strand of 5'-UUUCAUACAACAGGACGCC-3'; or one arbitrarily selected from the following double-stranded RNA molecules targeted at mouse PKCalpha: a sense strand of 5'-GAAUGAGAGCAAACAGAAA-3' and an antisense strand of 5'-UUUCUGUUUGCUCUCAUUC-3', a sense strand of 5'-GCAAAGGACUUAUGACCAA-3' and an antisense strand of 5'-UUGGUCAUAAGUCCUUUGC-3', or a sense strand of 5'-GCCCAAAGUGUGUGGCAAA-3' and an antisense strand of 5'-UUUGCCACACACUUUGGGC-3'. The siRNA-PKCalpha is specifically targeted at hypotype PKCalpha and provides a novel pharmaceutical specific intervention targeting gene medicament for treating blinding eye diseases of the same type.

Description

SiRNA and application thereof that a kind of specific inhibition PKC signal transmits

Technical field

The present invention relates to a kind of biological targets medicine and application thereof, specifically, relate to siRNA and the application in preparation treatment illness in eye reason proliferative disease medicine thereof that a kind of specific inhibition PKC signal transmits.

Background technology

Illness in eye reason proliferative disease comprise proliferative vitreoretinopathy (proliferative vitreoretinopathy, PVR), filter behind traumatic proliferative vitreoretinopathy (T-PVR), diabetes proliferative vitreoretinopathy (PDR) and the glaucoma operation and steep scarring etc.Their common pathological characters are the scarrings that the hyperplasia of intraocular cell causes, cause retinal detachment and stop up and filter bubble, be one group of serious blinding illness in eye, be on cell and molecular level, form complicated cyberrelationship by various kinds of cell and humoral factor, and then (retinal pigment epithelium, RPE) cell is main, also comprises the hyperplasia reaction of spongiocyte, inoblast and vascular endothelial cell etc. with retinal pigment epithelium to regulate and control intraocular.

Present medicine, as using glucocorticosteroid inflammation-inhibiting process, suppress the cell proliferation process with antimetabolite, these medicines all are antitumoral cytotoxic medicines, comprise 5-FU (5-fluorouracil, 5-FU), daunomycin or title daunorubicin (daunorubicin or daunomycin), Colchicum autumnale are sealed (colchicine) and mitomycin etc.Yet all there are problems such as drug effect is weak, toxicity is bigger in these medicines, and lack the prospective study of contrast.So needs are sought, and some are safer, more efficient drug is prevented and treated this class disease.

Study a certain factor in this class disease complex network or the blocking-up of a certain cell and may compare difficulty, study perhaps more effective the signals-modulating at the merge downstream place of these complicated factor function cells.And protein kinase C (protein kinase C, PKC) be the desmo enzyme family of transducer cell external signal to the cell internal reaction, a series of processes relevant with biological phenomena such as the transmission of wide participation cell information, secretion, ionic channel adjusting, hyperplasia, differentiation and canceration are ideal signal blocker target spots.

Domestic and international many studies show that, in PVR took place, RPE proliferation of cells (proliferation), divide a word with a hyphen at the end of a line (migration), the PKC that engulfs (phagocytosis) and collegen filament contraction behaviors such as (gel contraction) and RPE cell self had very important relation.These results suggest be after we will study paathogenic factor effect RPE cell, and the PKC signal of generation is a regulating cell and cause its behavioral disorder how.The present inventor uses the specific inhibitor of PKC and finds at home and abroad that first it is by suppressing the flow of calcium ions of RPE cell, suppress the generation (Ophthalmic Res.2005.37:128-35) of PVR, use the generation (Gao Qianying that PKC specific inhibitor hypericin can effectively suppress the experimental PVR of rabbit, favour is prolonged life, Wang Yusheng. hypericin suppresses the traumatic proliferative vitreoretinopathy of rabbit, eye Science, 2002,18 (4): 240-245).Determined 12 kinds of hypotypes of PKC, 10 kinds of PKC hypotypes to be arranged at the RPE cell expressing, and mainly be distributed in the cytoplasm from cell, albumen and gene level.

PKC also has wide expression in inoblast, spongiocyte and vascular endothelial cell, studies show that using pkc inhibitor also can block this class proliferation of cells, have preventive and therapeutic effect filtering illness in eye reason proliferative diseases such as bubble scarring behind traumatic proliferative vitreoretinopathy (T-PVR), diabetes proliferative vitreoretinopathy (PDR) and the glaucoma operation.

But if from the transmission of PKC level blocking-up bio signal, the signal transmission of all hypotypes of PKC all can be blocked, and influences the cells physiological function.

Aprinocarsen (ISIS 3521) is the antisense oligonucleotide of the mRNA of a kind of single-minded inhibition target PKC α, the paraplasm and the inducing apoptosis of tumour cell that can suppress tumour cell, be mainly used in malignant tumor patient, as patients such as non-small cell (type) lung cancer, ovarian cancers, be in clinical experimental stage at present.

Summary of the invention

The object of the present invention is to provide a kind of small molecules siRNA, this siRNA can block the signal transmission of PKC alpha hypotype specifically.

To achieve these goals, the present invention has taked following technical scheme:

The siRNA that a kind of specific inhibition PKC signal transmits, described siRNA are following in the double stranded rna molecule of people PKC α any one: positive-sense strand: 5 '-CGACGACUGUCUGUAGAAA-3 ', antisense strand 5 '-UUUCUACAGACAGUCGUCG-3 '; Positive-sense strand: 5 '-GGAUGGUACAAGUUGCUUA-3 ', antisense strand: 5 '-UAAGCAACUUGUACCAUCC-3 '; Or positive-sense strand: 5 '-GGCGUCCUGUUGUAUGAAA-3 ', antisense strand: 5 '-UUUCAUACAACAGGACGCC-3 '; Or described siRNA is following in the double stranded rna molecule of mouse PKC α any one: positive-sense strand: 5 '-GAAUGAGAGCAAACAGAAA-3 ', antisense strand: 5 '-UUUCUGUUUGCUCUCAUUC-3 '; Positive-sense strand: 5 '-GCAAAGGACUUAUGACCAA-3 ', antisense strand: 5 '-UUGGUCAUAAGUCCUUUGC-3 '; Or positive-sense strand: 5 '-GCCCAAAGUGUGUGGCAAA-3 ', antisense strand: 5 '-UUUGCCACACACUUUGGGC-3 '.

Preferably, described double stranded rna molecule 3 ' end also is provided with the suspension base (Over-hang) with two deoxynucleoside combinations, to increase the stability of siRNA.

Preferably, described double stranded rna molecule is a positive-sense strand: 5 '-GGCGUCCUGUUGUAUGAAA dAdT-3 ', antisense strand: 5 ' UUUCAUACAACAGGACG CC dAdT-3 '.

Another object of the present invention provides the application of this siRNA in preparation treatment illness in eye reason proliferative disease medicine, this siRNA can effectively block illness in eye reason proliferative disease, for the serious blinding illness in eye of this class provides a new specific biological treatment target spot and an interference method.

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

The application of the siRNA that specific inhibition PKC signal transmits in preparing treatment illness in eye reason proliferative disease medicine, described illness in eye reason proliferative disease is to filter reason such as the illness in eye of steeping scarring proliferative disease behind proliferative vitreoretinopathy, traumatic proliferative vitreoretinopathy, diabetes proliferative vitreoretinopathy and the glaucoma operation.

SiRNA of the present invention can make different dosage forms such as ophthalmic eye drop, injection liquid.

The multiple factor of the present inventor's applied molecular biology technical study vitro human RPE cell cycle and the relation of PKC hypotype find to have only PKC α by downward modulation p27 ^Kip1The formation of regulation and control RPE proliferation of cells and PVR plays keying action in PVR takes place.PKC α can regulate and control the G1/S weight break point of RPE cell cycle, can control cell and enter DNA synthesis phase (S phase) from stationary state (G1 phase), is also referred to as restriction point (restriction point), is that can cell cross the key point that enters hyperplasia stationary phase.That participate in regulation and control is cyclin (Cyclin) and cyclin dependent kinase (cyclin-dependentkinases, CDKs) the mixture Cdk2-CyclinE or the Cdk2-CyclinA of Xing Chenging, and cyclin-dependent kinase inhibitors (cyclin dependent kinase inhibitors CKIs) mainly is p27 ^Kip1

The present invention has following beneficial effect:

1) the siRNA-PKC alpha specific is at the PKC alpha hypotype, in blocking-up PKC α, can't cause the signal transmission blocking-up of other hypotype of PKC and influence the cells physiological function, thereby provide a kind of new drug specificity to intervene the genomic medicine of target spot for treating the serious blinding illness in eye of this class;

2) first Application PKC signal specific inhibitor siRNA-PKC αZu Duan illness in eye reason proliferative disease, effect is remarkable, and does not have present used Side effects of pharmaceutical drugs.

Description of drawings

Fig. 1 compares for eyeball of mouse HE dyeing;

A wherein: normal mouse eyeball; B: negative control group (Notarget siRNA); C:50nM concentration group; D:100nM concentration group; E:150nM concentration group; F:200nM concentration group; G:300nM concentration group;

Fig. 2 is the incidence of mouse PVR;

Contrast wherein: negative control group (Notarget siRNA); 1:50nM concentration group; 2:100nM concentration group; 3:150nM concentration group; 4:200nM concentration group; 5:300nM concentration group;

Fig. 3 accepts the RT-PCR of various processing after 24 hours figure as a result for the RPE cell;

Fig. 4 is P27 ^Kip1Accept PMA and Thy and handle 0-24 hour the mRNA (A) and the expression of albumen (B);

Fig. 5 accepts the various mRNA expressions of handling back PKC α for the RPE cell;

Fig. 6 is the influence figures of various processing to RPE cell proliferation;

Fig. 7 detects the proteic expression amount of PKC α in each treatment group RPE cell for Western blot method.

Embodiment

Below by specific embodiment the present invention is described.

Embodiment 1: utilize mouse siRNA treatment mouse PVR

1, trial model: mouse PVR model (the C57BL/6 mouse is available from Nanfang Medical Univ's Experimental Animal Center)

Totally 36 of the SPF level C57BL/6 mouse in this experimental selection 5-6 week, according to (Frenzel EM, Neely KA, Walsh AW such as Frenzel, et al.A new model of proliferative vitreoretinopathy, Invest Ophthalmol Vis Sci.1998Oct; 39 (11): method 2157-2164), at the dispase (neutral protease that need not to carry out injecting to vitreous space with microsyringe 3mm behind mouse right eye corneoscleral junction of 10 μ L under vitrectomy, retina incision or the refrigerated condition 2 μ L (0.2u/ μ L), GIBCO company), general 4～8 weeks the time have tangible PVR form, and have formed mouse PVR model.

2, the design of PKC α siRNA is with synthetic

At first in the U.S. state-run biotechnology information center ncbi database, obtain the sequence total length of mouse PKC α mRNA, design three PKC α-siRNA and a negative control siRNA (Notarget siRNA).In addition, designed the suspension end of two bases of dTdT, to increase the stability of siRNA.The siRNA of design is synthetic by the sharp rich bio tech ltd in Guangzhou.With the ddH of synthetic siRNA powder with no RNA enzyme ₂The O dissolving, the siRNA mother liquor that is mixed with 20 μ M is standby in-20 ℃ of preservations.

Table 1 mouse PKC α-siRNA and negative control Notarget siRNA

3, test method

36 C57BL/6 mouse are divided into 6 groups at random, every group 6, A organizes negative control group (Notarget siRNA), and the 1-5 group is experimental group, and representing PKC α siRNA4 final concentration respectively is 50nM (1 group), 100nM (2 groups), 150nM (3 groups), 200nM (4 groups) and 300nM (5 groups).

Mouse vitreous space injection dispase uses chloral hydrate (4.3%) anesthetized mice in 1 week back, with the diffusing big mouse pupil of Tropicamide and Phenylephrine eye drop, under operating microscope, with the microsyringe of 10 μ L along mouse angle Gong Yuan inserting needle, arrive at the optic disk surface, at the medicine of vitreum intracavitary administration 2 μ L.Wherein to inject 2 μ L concentration be the NotargetsiRNA of 500nM to negative control group, the intraocular final concentration is 100nM, experimental group is injected the PKC α siRNA4 that 2 μ L concentration are 250nM, 500nM, 750nM, 1000nM, 1500nM respectively, and the intraocular final concentration is respectively 50nM (1 group), 100nM (2 groups), 150nM (3 groups), 200nM (4 groups), 300nM (5 groups).

Behind the vitreous space injectable drug, use CUY21EDIT multifunctional active electricity conversion instrument, carry out the live body electrotransfection.The preoperation inspection instrument is measured air resistance, and parameter (Resistance:0.8-1.5kohm is set; Volt:80-100V; Pon:50msec; Poff:950msec; Number:5; Ampere:0.08-0.15A).Two of mouse are coated hydroxypropyl methylcellulose ophthalmic solution, use electrode (CUY650P7) to be adjacent to cornea then, press Start then, begin to carry out transfection.

Observed for 4 weeks continuously behind the electrotransfection, the cervical vertebra dislocation method is put to death mouse, makes a collection of specimens.Bend tweezer with ophthalmology and win mouse bilateral eyeball, remove ball and organize outward,, make frozen section and carry out conventional H E dyeing and the immunohistochemical methods detection with OCT embedding eyeball.

4, test-results

A. eyeball structure changes

Drug effect will carry out HE dyeing after 4 weeks after the eyeball of mouse frozen section.Fig. 1 result shows, negative control group and experimental group 50nM concentration group: 6 eyeball total detachment of retina, and retina basic structure changes; Experimental group 100nM group, 150nM group, 200nM concentration group: have 4 retina shedding takes place, 2 retina shedding does not take place, and eyeball basic structure is intact; Experimental group 300nM concentration group: have 3 retina shedding takes place, 3 retina shedding does not take place, and eyeball structure is intact, and the PKC α siRNA restraining effect certain to having of PVR of 100nM-300nM is described.

The B.PVR incidence

As shown in Figure 2, negative control group and experimental group 50nM concentration group retina seriously break away from, and the PVR incidence is 100%, experimental group 100nM group, 150nM group, 200nM group, and the PVR incidence is 66.7%, and 300nM group PVR incidence is 50%.

C. immunofluorescence detects

1) RPE cell, M ü ller cell expressing

Detect the significant albumen RPE65 of RPE cell by immunofluorescence, expression and distribution with the significant Protein G S of M ü ller cell, the result shows: visible RPE cell and M ü ller cell are weak positive expression in the normal mouse retina, retinal tissue RPE cell that negative control group, 50nM concentration group, 100nM concentration group, 150nM concentration group come off and Muller cell are strong positive expresses, and retinal tissue RPE cell and M ü ller cell that 200nM group and 300nM group come off are weak positive expression.Along with the rising of siRNA concentration, RPE cell and M ü ller cell expressing obviously reduce in the experimental group.

2) inoblast, star spongiocyte are expressed

Be detected as fibrocellular significant protein alpha-SMA by immunofluorescence, expression and distribution with the significant Protein G FAP of star spongiocyte, the result shows: visible inoblast, star spongiocyte are weak positive expression in the normal mouse retina, retinal tissue inoblast, the star spongiocyte that negative control group, 50nM concentration group, 100nM concentration group, 150nM concentration group come off is strong positive expresses, and 200nM group and 300nM form fibrocyte, star spongiocyte is weak positive expression.Along with the rising of siRNA concentration, inoblast and star spongiocyte are expressed obviously and are reduced in the experimental group.

Utilize mouse siRNA treatment mouse PVR, the result shows that PKC α siRNA can suppress the generation of PVR, the restraining effect that the siRNA of 100-300nM is certain to having of PVR, and concentration is high more, and restraining effect is obvious more; SiRNA suppresses the generation of PVR by the expression that suppresses the cell relevant with illness in eye reason proliferative disease, and along with the rising of siRNA concentration, RPE cell, M ü ller cell, inoblast and star spongiocyte are expressed obvious minimizing.

Embodiment 2: utilize people siRNA to suppress the propagation of RPE cell

1, experimental cell

RPE cell: in the ophthalmology center eye bank of middle mountain, get the eyeball in the donor after death 24 hours, under aseptic condition, cut eyeball from equator cloth annular, discard anterior ocular segment, remove vitreum, separate layer of retina,neuroepithelial, the deutomerite eyecup is placed in the eyeball holder, with mass percent is that 0.25% pancreatin adds eyecup, hatching digestion for 37 ℃ blows and beats gently with suction pipe after about 10 minutes, make the RPE cell detachment, the Digestive system that will contain the RPE cell moves into to be added with in advance and contains among the antibiotic DMEM that the quality percentage composition is 20% foetal calf serum and 1%, stops the pancreatin effect.Be that to make concentration be 2 * 10 for the antibiotic DMEM of 20% foetal calf serum and 1% with containing the quality percentage composition after centrifugal, the rinsing ⁴Cell/ml RPE cell suspension is used for inoculation culture.

2, the design of PKC α siRNA is with synthetic

At first in the U.S. state-run biotechnology information center ncbi database, obtain the sequence total length of people PKC α mRNA, design three PKC α siRNA and a negative control siRNA (Notarget siRNA).In addition, designed the suspension end of two bases, to increase the stability of siRNA.The siRNA of design is synthetic by the sharp rich bio tech ltd in Guangzhou.With the ddH of synthetic siRNA powder with no RNA enzyme ₂The O dissolving, the siRNA mother liquor that is mixed with 20 μ M is standby in-20 ℃ of preservations.

Table 2 people PKC α-siRNA and negative control Notarget siRNA

3, experimental technique

(1) cell cultures

The RPE cell suspension inoculation in 6 well culture plates, when cell confluency reaches 50%, is removed former substratum, and every hole adds 2ml and contains the DMEM substratum that the quality percentage composition is the antibiotic-free of 20% foetal calf serum.

(2) preparation transfection composite

Getting 60 μ l concentration is the liposome LipofectAMINE2000 of 2mg/ml, adds 300 μ l DMEM mixings, leaves standstill under the room temperature to get A liquid in 5 minutes; Get PKC α siRNA3 and Notarget siRNA that 180 μ l concentration are 100nM respectively, respectively add 300 μ l DMEM mixings and get B liquid, A liquid is mixed with B liquid, put upside down mixing lightly, not concussion, incubation is 20 minutes under the room temperature, forms the transfection composite of siRNA-LipofectAMINE2000 and Notarget siRNA-LipofectAMINE2000.

(3) transfection

When cell confluency reaches 60%, carry out transfection and handle.Each organizes the liquid that all adds the 1ml correspondence in each hole, and front and back softly push away shakes culture plate with mixing liquid, and culture plate is sent into the conventional cultivation of cell culture incubator.Each group is provided with 5 repetitions.

SiRNA group: siRNA-LipofectAMINE2000 transfection composite;

Negative control group: Notarget siRNA-LipofectAMINE2000 transfection composite;

The Thy group: contain the substratum of thymeleatoxin (specific specificity activates the chemical substance of PKC α), final concentration is 100nM;

Control group: blank group.

4, experimental result

RT-PCR result shows: cell accepts siRNA, Thy (100nM) or PMA (100nM) handled after 24 hours, in main cell cycle regulatory factors, has only p27 ^Kip1Variation has taken place in the expression amount of mRNA, prompting cell cycle repressible protein P27 ^Kip1It is the relevant cell cycle associated protein (Fig. 3) of the short people RPE propagation of PKC α.P27 ^Kip1Albumen is respectively in 1 hour (PMA) and 3 hours (Thy) obviously decline (Fig. 4).

The RPE cell is measured the expression of PKC α mRNA accepting various processing after 3 hours, and the result shows that behind Thy and the PMA effect RPE cell, the expression amount of PKC α mRNA increases, behind the siRNA effect RPE cell, and the expression amount of PKC α mRNA decline (Fig. 5).

After the transfection 2 days, each treatment group is all got 3 porocytes, adopts the cell counting count board method to count and calculate mean number respectively, and all the other 2 hole leach proteins are done the expression that Westernblot detects target protein PKC α.

Compare with the Control group, the RPE cell count of siRNA group obviously reduces, and shows that siRNA can significantly suppress the propagation of RPE cell, suppresses efficient and reaches 39.9% (Fig. 6).

β in the different treatment group-Actin band brightness is indifference almost, the total protein concentration basically identical is described, the PKC α protein expression of Control group has obvious band, and transfection the cell of PKC α siRNA3 almost detect proteic expression less than PKC α, experimental result has confirmed that further PKC α siRNA is in the specificity (Fig. 7) of protein level to the inhibition and the effect of expression of target gene.

Thereby siRNA of the present invention all can reduce the propagation that the mRNA level of PKC α suppresses the RPE cell in various degree, and is wherein with 50-300nM concentration effect the best, the most obvious with the restraining effect of PKC α siRNA3.At people RPE cell transfecting behind the siRNA3 of 100nM, the inhibition efficient of cell proliferation is 39.9%, and the siRNA of this concentration does not have influence to the form of RPE cell.

Sequence table

＜110〉Zhongshan Ophthalmic Center, Sun Yat-sen University

＜120〉a kind of siRNA and application thereof of specific inhibition PKC signal transmission

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Claims (4)

1. the siRNA that transmits of a specific inhibition PKC signal, it is characterized in that: described siRNA is following double stranded rna molecule at people PKC α: positive-sense strand: 5 '-GGCGUCCUGUUGUAUGAAA-3 ', antisense strand: 5 '-UUUCAUACAACAGGACGCC-3 '.

2. the siRNA that specific inhibition PKC signal according to claim 1 transmits, it is characterized in that: 3 ' end of described double stranded rna molecule also is hung with two deoxynucleotides.

3. the siRNA that specific inhibition PKC signal according to claim 2 transmits, it is characterized in that: described double stranded rna molecule is a positive-sense strand: 5 '-GGCGUCCUGUUGUAUGAAA dAdT-3 ', antisense strand: 5 ' UUUCAUACAACAGGACGCC dAdT-3 '.

4. the application of each described siRNA of claim 1-3 in preparation treatment proliferative vitreoretinopathy medicine.

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