CN106620678A - Novel application of PKA inhibitor H-89 - Google Patents

Abstract

The invention relates to an application of a PKA inhibitor H-89 as a PVR (proliferative vitroretinopathy) inhibitor. In comparison with the prior art, it is discovered for the first time that the PKA inhibitor H-89 has an inhibitory effect on PVR in in-vitro and in-vivo PVR models; the H-89 is capable of protecting change in an expression level of EMT-related genes, damage to tight junction and the like caused by EMT and the like in a PVR pathogenesis course; and meanwhile, a relation between TGF[beta] and a PKA signaling pathway in the PVR pathogenesis course is illustrated, and the TGF[beta] is capable of activating the PKA signaling pathway, while the inhibitory effect of the PKA inhibitor H-89 on the PVR can be achieved by inhibiting the EMT. The effect of the PKA signaling pathway in the PVR pathogenesis course is illustrated for the first time in the results, and the PKA inhibitor H-89 can serve as a medicine for treating the PVR.

Description

The new opplication of PKA inhibitor H-89

Technical field

The present invention relates to a kind of new application of PKA inhibitor H-89, belongs to biomedicine technical field.

Background technology

Proliferative vitreoretinopathy (PVR) is referred in rhegmatogenous detachment of retina rear vitreous body chamber and retina table The film hyperplasia in face and the pathology shunk, the film of these hyperplasia can pull and reopen repaired ceasma, cause new Ceasma and distort and block macula lutea, while PVR is also modal the reason for cause surgery of retinal detachment to fail, it was reported that Its incidence is 5.1%～11.7%, and in the surgery of retinal detachment for proving an abortion, 75% cause of disease is all attributed to PVR, because This its be also blinding main cause.Document report only have 11～25% PVR patient eyesight Jing after treatment can reach 0.2 with On.But the research so far to the pathogenesis of PVR is still indefinite, clinically without available effectively treatment medicine, still It is main to be treated by vitrectomy, but it is complex to perform the operation, and perform the operation can not thorough solve problem, it is postoperative easily again Send out, often over Repeated Operation, the visual function of patient is still gradually reduced even to be lost, and surgery cost is also costly, to trouble Person and family members bring heavy psychological burden and economic loss, thus the pathogenesis of further investigation PVR, seek safely and effectively Targeted drug treatment method aiding in the success of surgery of retinal detachment, the treatment for PVR have important clinical meaning and Social value.

In the pathogenesis of proliferative vitreoretinopathy, epithelial-mesenchymal (EMT) is considered as most important One of mechanism.EMT refers to that epithelial cell progressively transforms into the biological process with interstitial phenotype cells by specific program, Typical Epithelial form is such as lost, and is changed into fiber-like form, and lose the tight connection between cell, epithelial cell mark The expression such as note thing such as ZO-1, E-cad decline, and the expression such as mesenchymal cell markers thing such as a-SMA, FN, Vimentin increases, cell Hyperplasia, transfer ability strengthen, play the role of in multiple fiber disease important.RPE cells are in cytokine profiles Effect is lower to there is the important pathologic process that EMT is PVR.

The most study of the current EMT related mechanisms with regard to PVR is TGF signal beta paths, it is considered to be topmost The fibrillatable factor of PVR morbidities, all high expression in the vitreum, SRF and propagation film in PVR patient.TGF signal betas Path includes classical Smad signal paths and non-classical signal path.In classical TGF β-Smad signal paths, TGF β with I receptors are combined, then Direct Phosphorylation Smad2 and Smad3, are then combined again and gone in transporte to cells core to play with Smad4 Their effect.And non-classical TGF signal betas path mainly includes MAPK (MAPK), extracellular signal Adjust kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB or AKT)/mammal thunder Handkerchief mycin target protein (mTOR), the non-classical paths of wherein MAPK include p38MAPK, c-JNK and p42/44ERK.So far, exist In the Mechanism Study of PVR, also it is not involved with regard to PKA (PKA) signal path and TGF β and PKA signal path phases The research of mutual relation.

PKA is a silk-threonine kinase for depending on cAMP being widely present in cell, and PKA includes two regulations Subunit and two activation subunits, when intracellular c-AMP concentration is raised, cAMP is combined with subunit is adjusted, and is changed and is adjusted subunit structure As, regulation subunit and activation subunit dissociation are made, discharge activation subunit.The PKA activation subunits of activation can make intracellular some eggs White serine or threonine residues phosphorylation, then change the activity of these albumen, further have influence on the table of related gene Reach, such as part activation subunit can be transported to activating transcription factor c-AMP response element binding proteins (CREB) in core.PKA Various vital movements such as signal path and cell cycle, survival, propagation, differentiation and apoptosis are related.

And at present in the middle of the Mechanism Study of PVR, except TGF signal beta paths, also HGF (HGF), blood Platelet growth factor (PDGF), fibroblast growth factor (FGF), bone morphogenetic protein (BMPs) and connective tissue growth because Signal paths such as sub (CTGF), but also without the research about PKA signal paths, while also leading to without TGF β and PKA signal The research of road correlation.

The content of the invention

The purpose of the present invention is exactly the especially relation between TGF β and PKA signal paths in order to inquire into the mechanism of PVR, And the mechanism of PVR is further elucidated, and a kind of active drug PKA inhibitor H-89 of PVR treatments are provided.

The purpose of the present invention can be achieved through the following technical solutions：

Present invention discover that in EMT cell models TGF beta induced in vitro and internal SD P of Rats VR model, PKA suppresses Agent H-89 can effectively suppress PVR.In vitro in model, we are lured using the TGF β of 10ng/ml in ARPE19 cells EMT is led, with the H-89 of 5-20uM the cell of EMT is processed, the table of EMT related gene a-SMA, Fibronectin etc. can be reduced Reach, and raise the expression of epithelium related gene ZO-1 etc., while close-connected destruction that EMT causes and tissue can also be protected thin The reconstruct of born of the same parents' skeleton.In testing in vivo, we utilize the ARPE19 cell (2.4*10 of intravitreal 8ul⁶) blood it is little The method of plate rich plasma (PRP) suspension successfully induces PVR models, then by intravitreal H-89 (final concentration 5-50uM) Treated, compared with saline control group, find H-89 can protect the SD rats caused due to PVR ERG decline with And the destruction of retinal structure.

A first aspect of the present invention, there is provided in cell model in vitro, PKA inhibitor H-89 can suppress ARPE19 There is the PVR processes of EMT process simulations in cell.Specifically, PKA inhibitor H-89 can in vitro from mRNA, albumen and The change of the EMT related genes that the aspects such as assignment of genes gene mapping protection TGF β cause.

A second aspect of the present invention, there is provided in SD P of Rats VR models in vivo, PKA inhibitor H-89 can suppress The symptom of PVR, and delay it to be in progress.

Specifically, described PKA inhibitor H-89 is used as the TGF beta induced increment of human retina epithelial cell and migration Inhibitor.

In the PVR models that ARPE19 cells combine the induction of platelet rich plasma intravitreal, PKA inhibitor H- 89 application can protect the decline of the electroretinogram b ripple wave amplitudes of the SD rats that PVR causes.

Described PKA inhibitor H-89 changes inhibitor as eyeball structure and cephacoria forms the application of accelerator. In the PVR models of ARPE19 cells joint platelet rich plasma intravitreal induction, the application energy of PKA inhibitor H-89 The formation of the change such as cephacoria of eyeball structure can also enough be protected.

Applications of the described PKA inhibitor H-89 as mesenchymal cell markers thing inhibitor.Combine blood in ARPE19 cells In the PVR models of platelet rich plasma intravitreal induction, the application of H-89 can reduce the table of EMT label a-SMA Reach.

A third aspect of the present invention, discloses in the pathogenic process of PVR, the relation of TGF β and PKA signal paths, finds During PVR, TGF β can activate PKA signal paths.In showing as cell model in vitro, TGF β 1 can dosage according to Rely the activity with time dependent increase PKA.

A fourth aspect of the present invention, there is provided PKA inhibitor H-89 as PVR medicines and PVR inhibitor foundation, There is provided a kind of means that PVR treatments are carried out by intravitreal H-89.

Specifically, PKA inhibitor H-89 is as prevention and/or treats answering for proliferative vitreoretinopathy medicine With.And PKA inhibitor H-89, used as the application of auxiliary surgery of retinal detachment medicine, PKA inhibitor H-89 are with vitreous chamber The form of injection is used.

A kind of a fifth aspect of the present invention, there is provided the medicine of prevention and/or treatment proliferative vitreoretinopathy, Main component is PKA inhibitor H-89.And there is provided a kind of medicine of auxiliary surgery of retinal detachment, main component is PKA suppressions Preparation H-89.

Present invention firstly discovers that PKA inhibitor H-89 makees in vitro and in internal PVR models to the suppression of PVR With H-89 can protect the gene expression dose of the EMT correlations caused due to such as EMT in PVR pathogenic processes to change and tight Destruction of close connection etc., and while illustrate in the pathogenic process of PVR, the relation of TGF β and PKA signal paths, TGF β can Activation PKA signal paths, and what PKA inhibitor H-89 may be realized to the inhibitory action of PVR by suppression EMT.These As a result show first in PVR pathogenic processes, the effect of PKA signal paths, and PKA inhibitor H-89 can be controlled as PVR The medicine for the treatment of.

Compared with prior art, the present invention has advantages below：

1st, the treatment of current PVR is still based on vitrectomy, but surgery cost is expensive, and needs hand repeatedly Art；And PKA inhibitor H-89 is micromolecular compound low price, it is readily available；

2nd, the drug therapy of PVR mainly has at present the medicine of glucocorticoid and non-steroidal anti-inflammatory drugs and inhibition of cell proliferation Thing etc., but at present the former application evidence be not also very fully, and the latter can wide spectrum suppress cell propagation, can bring serious Potential risks.And H-89 can effectively suppress PVR in vitro in vivo, while carrying out eye local by intravitreal Medication, the potential risk that systemic administration can be avoided to bring.

Description of the drawings

Figure 1A：H-89 mitigates the gene alteration that TGF β 1 and TGF β 2 cause in mRNA level in-site.

Figure 1B and 1C：H-89 mitigates the gene alteration that TGF β 1 cause in protein level.

Fig. 1 D：The positioning of the EMT GAP-associated protein GAPs that H-89 protection TGF β 1 cause changes and expression changes.

Fig. 2A and 2C：H-89 suppresses the cell that TGF β 1 cause to breed and migrate in scratch experiment.

Fig. 2 B and 2D：In Transwell migration experiments, H-89 suppresses the cell migration that TGF β 1 cause.

Fig. 3 A and 3B：H-89 treatments can retrieve the b ripple wave amplitudes of the SD rat retina electrographs that modeling causes in PVR bodies Decline.

Fig. 4：Eyeball frozen section immunofluorescence figure shows that H-89 can protect the cephacoria that PVR modelings cause to be formed, and drops The expression of low EMT labels a-SMA.

Fig. 5：Eyeball frozen section immunofluorescence figure shows that H-89 can protect the cephacoria that PVR modelings cause to be formed, and drops The expression of low EMT labels FN1.

Fig. 6 A and 6C：TGF β 1 can activate PKA paths, and increases of the TGF β 1 to PKA activity is dose-dependent.

Fig. 6 B and 6D：TGF β 1 can activate PKA paths, and increases of the TGF β 1 to PKA activity is time dependent.

Specific embodiment

Below in conjunction with the accompanying drawings the present invention is described in detail with specific embodiment.

In following examples, ARPE19 cells are purchased from ATCC, and culture medium is DMEM：F12 culture mediums contain 10% serum and 1% P/S.Culture environment is 37 DEG C, 5%CO₂With 95% air.TGF β 1, TGF β 2 are bought from R＆D companies.H-89 is purchased from Selleck Company, concentration is 5-50uM.Reverse transcription reagent box is PrimeScript^TMRT Master Mix are (public purchased from Takara Department), PCR kit is SuperReal PreMix Plus (SYBR Green) (purchased from Tiangeng company).The primer is by Suzhou Jin Weizhi companies synthesize.Transwell cells, 8uM apertures (are purchased from Corning Incorporated).PKA Activity Assay Kits are purchased from Promega companies.

Embodiment 1

PKA inhibitor H-89 suppresses EMT in cell model in vitro

ARPE19 cells EMT is induced：When ARPE19 cell confluency degree is about 40-50%, serum deprivation overnight starvation is carried out Process, then process the corresponding time with the TGF β 1 or TGF β 2 of 10ng/ml, have or intervene without H-89.EMT induces it Afterwards, cellular morphology is changed into fibre morphology by typical Epithelial, while closely connection is lost, and cell propagation is also accelerated.

Embodiment 2

Q-PCR detections mRNA change levels after EMT is induced and H-89 is processed：By normal control, the beta induced cells of TGF and The beta induced cells of TGF of H-89 process are collected in 1.5ml pipes with 1mlTrizol, are stripped total serum IgE.Through reverse transcription and reality When quantitative PCR analysis, using semiquantitative method calculate.

Key step is as follows：

1. in the sample collected to Trizol, the chloroform of 1/5th 200ul volumes is added, after acutely mixing, with 4 DEG C of the rotating speed of 12000rpm is centrifuged 10 minutes.

2. after being centrifuged, supernatant is transferred in new centrifuge tube, is careful not to get the albumin layer of centre, add equal-volume Isopropanol.

4 DEG C of the rotating speed of 3.12000rpm is centrifuged 10 minutes, and supernatant discarded washes precipitation 1-2 time with 75% ethanol.

4. will precipitate after drying at room temperature, with appropriate 20ul DEPC water dissolves.

5.Nanodrop is quantitative, and calculates the volume needed for reverse transcription.

6.RNA reverse transcriptions：

(1) system of 20ul, the RNA of 1000ng takes the reverse transcription reagents supermix of the Takara of 4ul, then adds ddH₂O Mend 20ul.

(2) reverse transcription program：37 DEG C 15 minutes, 85 DEG C 5 seconds, then can be standby in -20 DEG C of Refrigerator stores.

7. quantitative PCR

(1) cDNA10 times obtained after RNA reverse transcriptions is diluted as template, designs primer such as table 1.

Primer sequence table used in table 1q-PCR

Gapdh-Forward	AGGTCGGTGTGAACGGATTTG
		Gapdh-Reverse	TGTAGACCATGTAGTTGAGGTCA
ZO-1-Forward	ATTGTCGTCGCATGTAGATCC
		ZO-1-Revrse	GGGTTCATAGGTCAGATTAGGC
a-SMA-Forward	AATGCAGAAGGAGATCACGG
		a-SMA-Reverse	TCCTGTTTGCTGATCCACATC
N-Cad-Forward	CCCAAGACAAAGAGACCCAG
		N-Cad-Reverse	GCCACTGTGCTTACTGAATTG
FN1-Forward	GAGCTGCACATGTCTTGGGAAC
		FN1-Reverse	GGAGCAAATGGCACCGAGATA
Vimentin-Forward	CGTGAATACCAAGACCTGCTC
		Vimentin-Reverse	GGAAAAGTTTGGAAGAGGCAG

(2) using the SYBR Green real-time fluorescence quantitative PCR detection kits of Tiangeng company, the system of q-PCR is The cDNA templates of 20ul, 2ul, the primer of 1ul, the 2X PCR Mix of 10ul, the ddH of 7ul₂O, the expression of testing goal gene Amount.PCR amplification conditions are as follows：94 DEG C of denaturation 10 minutes, into circulation (95 DEG C of 5sec, 60 DEG C of 60sec), 40 circulations altogether, And collect solubility curve.

(3) data analysis, is processed, with Gapdh as internal reference using 2- △ △ Ct methods.

Experimental result as shown in Figure 1A, can be seen that Jing after TGF β 1 and TGF β 2 are processed, tight junction protein by figure ZO-1 expression declines, and the mRNA water of the activation subunit PRKACa of EMT related gene a-SMA, N-cad, Vimentin and PKA Flat expression substantially rises, and H-89 process can suppress the change of these gene mRNA expressions that TGF β cause.

Embodiment 3

Western Blot detections albumen change level after EMT is induced and H-89 is processed

1st, the extraction of albumen：Cell is seeded in the Tissue Culture Dish of 6cm, Jing after corresponding process, plus 150 μ l contain The RIPA lysates of Protease Inhibitor Cocktail, collection cell is scraped in new EP pipes with cell, is put and is incubated on ice Educate 30 minutes, 10000rpm, 4 DEG C are centrifuged 15 minutes, gently suction out supernatant into clean centrifuge tube, and -80 DEG C save backup.

2nd, the measure of protein concentration：Protein quantification adopts BCA sizing techniques, and concrete grammar is as follows：Prepare standard protein solution 2 μ g/ μ l, -20 DEG C save backup；Working solution is prepared according to the number of standard items and sample, reagent A and reagent B press 50:1 volume It is now with the current than fully mixing；Working solution cumulative volume=(standard items number+testing sample number) × (2 multiple holes numbers) × (200 μ l working solutions).Standard items are added in enzyme mark hole by the volume of 0 μ l, 1 μ l, 2 μ l, 4 μ l, 8 μ l, 16 μ l, are added per hole The BCA working solutions of 200 μ l, make calibration curve；Testing sample adds 2 μ l per hole, adds the BCA working solutions of 200 μ l, 37 DEG C Incubation 30 minutes；The absorbance at 560nm is determined with multi-function microplate reader.

3rd, the preparation of protein sample：According to the protein content and protein concentration that prepare loading, 5 × loading is added Buffer, mixes and is heated 10 minutes after 100 DEG C so as to sufficiently denaturation, 4 DEG C, and 12000rpm is centrifuged 2 minutes, and it is heavy to remove Form sediment, -80 DEG C of storages are standby.

4th, SDS-PAGE electrophoresis：The preparation of separation gel：10% separation gel (10ml) compound method is as follows：30% polypropylene Acid amides 3.3ml, ddH₂O 2.7ml, the 1M ammonium persulfate 0.1ml of Tris (PH 8.8) 3.8ml, 10%SDS 0.1ml, 10%, TEMED 0.004ml；Add APS and TEMED before encapsulating, fully mix after adding, rapid encapsulating.With liquid-transfering gun by prepare point During the glass plate for assembling is slowly added into from sol solution, separation gel solution height is about 6cm, reserves 1.5cm to concentration glue high Position, plus it is good after absolute ethyl alcohol be slowly added at the top of gel promote gelling, make gel surface be in straight line；Place 1 little When or so solidify completely until gel, illustrate that gel has solidified when there is a broken line between absolute ethyl alcohol and gel, outwell Absolute ethyl alcohol on glue, is blotted with filter paper.Concentration glue (4ml) method of preparation 5% is as follows：30% polyacrylamide 0.67ml, ddH₂The ammonium persulfate 0.04ml of O2.7ml, 1M Tris (PH 6.8) 0.5ml, 10%SDS 0.04ml, 10%, TEMED0.004ml；Add APS and TEMED before encapsulating, fully mix after adding, rapid encapsulating.It is dense by what is prepared with liquid-transfering gun Contracting sol solution is slowly added to the upper strata of separation gel, until the edge of glass plate, then carefully inserts comb, is careful not to produce gas Bubble, places about 1 hour or so gel, and comb is extracted after gel, prepares electrophoresis.By ready protein sample and protein labeling Molecule is added in well, prevents bubble from producing, and using Bio-Rad electrophoresis apparatuses, is started by filling it up with groove after electrophoresis liquid Electrophoresis, deposition condition is：80V voltages, 30 minutes, change 120V voltages, 30 minutes or so 1 hour.Electrophoresis to bromophenol blue is just run Go out the stopping of separation gel bottom.

5th, transferring film：After PAGE gel electrophoresis terminates, glass plate is pried open into rear careful separation and goes out gel, cut concentration The part of glue, retains separation gel, in being soaked in deionized water；The clip pvdf membrane similar to gel size is put into absolute methanol Immersion 2 minutes；The gel for cutting, filter paper and the pvdf membrane for being soaked with absolute methanol in advance are put into transferring film equilibrium liquid and are put down Weighing apparatus 10 minutes；Using the transferring film system of Bio-Rad, to press from both sides sandwich in the way of, the order from black flour to fine flour is followed successively by：Black flour (negative pole)-foam-rubber cushion-filter paper-gel-pvdf membrane-filter paper-sponge-fine flour (positive pole), filter paper, gel and pvdf membrane will align, Air entrapment is thoroughly removed, in being then placed in the transferring film instrument for filling transferring film liquid, transferring film condition is 300 milliamperes, 2 hours；Transferring film groove In being placed in ice bath.Transferring film may be referred to albumen Marker turn of upper pvdf membrane and successfully indicate as transferring film after terminating, also can be by film Dyeed with 1 × Ponceaux dye liquor, the dye liquor do not caught then is rinsed out with water it can be seen that the albumen on film.

6th, immuning hybridization reaction：Ponceaux is washed off with 1 × PBST, the BSA for configuring 5% closes liquid, and room temperature is closed 1 hour.Plus Enter diluted with PBST to resist, 4 spend night；1 × PBST washes film 3 times, and every time 10.The two of the HRP marks of addition PBST dilutions resist, Room temperature 1 hour.1 × PBST washes film 3 times, and every time 10.ECL chemiluminescence collection results, using Gapdh as control internal reference.

7th, data statistic analysis：With the gray analysis of Image J software western bands, then enter with corresponding Gapdh Row is compared, and then recycles Graphpad Prism softwares to carry out statistical analysis and mapping.

As shown in Figure 1B and Fig. 1 C, 1B is histogram to experimental result, and 1C is that the knot of the statistics after gray analysis is carried out to band Really, as seen from the figure Jing after the process of TGF β 1, tight junction protein ZO-1 expression declines, and EMT related gene a-SMA, N-cad, The expression of Vimentin, Fibronectin and PRKACa substantially rises, and H-89 process can save these that TGF β cause The change of genes protein level expression.

Embodiment 4

Immunofluorescence test albumen positioning and expression after EMT is induced and H-89 is processed

1st, cell is processed：Cell is inoculated on the slide being preset in culture dish, after Jing is processed accordingly, takes out glass Piece；

2nd, it is fixed：10 minutes are fixed with 4% paraformaldehyde, PBS washes 3 times, every time 5 minutes；

3rd, permeable membrane：0.1%tritonX-100 permeable membranes 10 minutes, PBS washes 3 times, every time 5 minutes；

4th, close：5% horse serum room temperature is closed 1 hour；

5th, one resists：Plus 5% horse serum room dilution one resist, being placed in wet box prevents drying, 4 DEG C of overnight incubations；

6th, two resist：PBS washes 3 times, every time 5 minutes；Plus 5% the dilution of horse serum room luciferase mark it is anti-with one of the same race The two of category source resist, and are placed in wet box, and 37 DEG C of 1, PBS of incubation of lucifuge wash 3 times, every time 5 minutes；

7th, core is contaminated：Nucleus is contaminated with 0.5 μ g/mlDAPI 3 minutes, PBS washes 3 times, every time 5 minutes；

8th, mounting：With fluorescence mountant mounting.

9th, take pictures：With laser confocal microscope, in oil mirror, 600 times of amplifications are taken pictures.

Experimental result as shown in the immunofluorescence figure in Fig. 1 D, Jing TGF β 1 process after, closely connecting between ARPE19 cells Connect destroyed, as shown in the dyeing of tight junction protein ZO-1；Cytoskeleton F-actin reconstruct becomes disorderly, and H-89 process Afterwards, the tight connection between cell is reappeared, and the disorder of the F-actin that the reconstruct of cytoskeleton causes also has mitigated.With EMT relevant albumen a-SMA, the Fibronectin and Vimentin and activation subunit PRKACa of PKA is Jing after the process of TGF β 1 After expression rises, and H-89 is processed, the expression rising of these albumen is all suppressed.

Embodiment 5

Scratch experiment detection cell migration ability after EMT is induced and H-89 is processed

1st, cell is processed：Cell is seeded in 96 orifice plates, is done after corresponding process (as described in example 1 above)；

2nd, cut：With 10uL liquid-transfering gun pipette tips on cell be in " one " stroke trace, then washed off for 3 times with PBS and floated Cell.The culture medium for adding alignment processing proceeds culture.

3rd, take pictures：Taken pictures in the cut time point of 0 hour, 24 hours and 48 hours respectively under the microscope.

4th, data analysis：The measurement of scratch width is carried out with Image J softwares, width data is obtained, is then recycled Graphpad Prism softwares carry out statistical analysis and mapping

As shown in Fig. 2A and Fig. 2 C, 2A is the image of different time points cut to data, and 2C is Jing Image J software statistics Result after analysis, we can see that Jing after TGF β 1 are processed, the proliferation apoptosis ability of ARPE19 cells is remarkably reinforced by figure, After cut is completely invisible after 48 hours, and H-89 is processed, the proliferation apoptosis ability of cell declines particularly evident, explanation H-89 can effectively suppress the proliferation apoptosis of ARPE19 cells.

Embodiment 6

Transwell experiments detection cell migration ability after EMT is induced and H-89 is processed

1. it is inoculated with：The Transwell cells of 8.0uM as 24 orifice plates in the middle of, by with the cell of different disposal in embodiment 1 Room on Transwell is inoculated in, upper room adds the culture medium containing the alignment processing factor of 1% serum of 100ul, lower room to add The culture medium of 10% serum of 600ul；

2. cell migration：Cell is placed in into 37 degree, is cultivated 24 hours in the incubator of 5% carbon dioxide, allow cell migration；

3. cell dyeing：The cell that upper room does not migrate gently is wiped away with cotton swab, then fix half an hour with methyl alcohol, cell It is appropriate to air-dry, then use eosin stains half an hour；

4. take pictures counting：Six visual field observation of cell immediately under 20 power microscopes, numeration.

As shown in Fig. 2 B and 2D, 2B is the migrating cell (part of light tone in figure) after eosin stains to data, and 2D is cell Statistics after counting, as seen from the figure, Jing after the process of TGF β 1, the cell of Transwell cell films is crossed in ARPE19 cell migrations Quantity substantially increases, and H-89 process can reduce about 1/3rd migrating cell number, illustrates that H-89 can effectively press down The migration of ARPE19 cells processed.

Embodiment 7

Intravitreal PVR modelings

1st, rat prepares：The SD rats of 6-8W are purchased from this Leco Corp., raise in Tongji University's animal center.160～ 180g weights, are randomly divided into three groups, PBS control group, model group (ARPE19+PRP), treatment group (ARPE19+PRP+H-89).

2nd, cell prepares：The ARPE19 cells of in vitro culture are taken, between P10-P15, cell concentration is adjusted and is caused per eye Cell quantity be 2.4*10⁶, assign to and be respectively in two EP pipes model combination treatment group, it is placed in standby on ice.

3rd, the preparation of platelet rich plasma (PRP)：Rat is fixed in mouse cage, rat-tail is exposed to outside cage.Before puncture Rat-tail is wiped with cotton ball soaked in alcohol, 5 number sword-shaped needles is connected with 1ml syringes, with liquaemin dilution Syringe needle Irrigator and injection Device；The right hand holds pin, left hand thumb it is upper, show that finger pinches rat-tail under, rat-tail is slightly curved bent so that blood vessel and syringe needle side at inserting needle To parallel, syringe needle punctures continuation inserting needle 3-5mm after epidermis parallel to rat-tail, and syringe needle is shown in blood back into tail vein, and left hand pinches mouse Tail, right hand push-and-pull 1ml syringe pistons, manufacture negative pressure carries out taking blood；Take after blood terminates, using rayon balls pressing pinprick position 30s, it is determined that decontroling rat after hemostasis.The blood of acquirement is transferred in the heparin tube containing 3.8% sodium citrate, low-temperature centrifugation, 1000g, 1min, its supernatant is PRP.

4th, prepared by ARPE19+PRP suspensions：It is resuspended with PRP respectively according to the cell concentration and injection volume that have calculated ARPE19 cells, model group is ARPE19+PRP, and treatment group is ARPE19+PRP+H-89；So that the final concentration of 5- of H-89 50uM。

5th, intravitreal：The yellow Jackets of rats by intraperitoneal injection 2% (1mL/400g body weight) is anaesthetized before injection, 1 × Su Mian Xin (0.1ml/200g) carries out of flaccid muscles, then gives Tropicamide mydriasis (the Wuxi Shanhe of a drop 0.5% Group, Jiangsu, China), 0.4% Oxybuprocaine surface anesthesia of a drop (Eisai Co Ltd, Tokyo, Japan). Under stereomicroscope, first with 1ml syringes, vertical inserting needle pricks an aperture from after corneal limbus, then again with injection needle from the hole to Injection respective liquid in vitreous chamber.The volume injected of per eye is 8ul.

Embodiment 8

SD rat retina electrographs ERG is gathered

1. instrument：APS full-automatic visions electrophysiologic study instrument (APS-2000) is purchased from Chongqing Kanghua Technology Co., Ltd..

Dark adaptation：Visual electrophysiology functional check the previous day is done, SD rats are transferred to dark place, carry out dark adaptation.Second It starts to do.

2. the preparation of rat：Anaesthetized to the yellow Jackets of rats by intraperitoneal injection 2% (1mL/500g body weight), 1 × speed Sleep new (0.1ml/200g) carry out it is of flaccid muscles, then give the Tropicamide mydriasis of a drop 0.5% (Wuxi Shanhe Group, Jiangsu, China), 0.4% Oxybuprocaine surface anesthesia of a drop (Eisai Co Ltd, Tokyo, Japan), per eye Eyeball respectively applies some conductive pastes.Intercalative electrode：Ground wire is connect on rat tail, and negative pole is connect between the ear of rat two, and positive pole connects two eyes angles On film, it is careful not to contact on eyelid and sclera.

3. data acquisition：Software " visual electrophysiology figure ", point " FERG " are opened, then puts 1,2 passages, one logical for left eye Road, another is right eye channel, point " setting ", stimulates number of times to be 2 times, and frequency of stimulation is 0.05Hz；Stimulus intensity is clicked on successively (1) -0.0006325 (cd*s/m), (5) -0.006325 (cd*s/m), (9) -0.06325 (cd*s/m), each intensity is extremely 2min is spaced less.Point " oscillography ", when the baseline of ripple is steady, clicks on " collection ", waits and hears that collection is finished after a sound of " ticking ", Click on " preservation ".Click on again " setting ", change number of documents and stimulus intensity, the like.After finishing etc. all intensity, one is changed Rat.After all rats finish, open " file ", demarcated, double-click curve, curve is changed into white, clicks on " demarcation ".Mark After having determined a ripples, by space bar, b ripples are demarcated.All demarcation are finished, and are clicked on " printing ", save as .PDF forms.Click on the lower left corner Button, exits software, closes computer, closes amplifier.

Data as shown in figure 3, wherein 3A for ERG b waveform figures, 3B for b ripple wave amplitudes statistical chart, as seen from the figure, Jing Compared with PBS control group, b ripple wave amplitudes are decreased obviously model group, and treatment group is compared with model group, at Jing H-89 after injection induction After reason, b ripple wave amplitudes have gone up, and illustrate the decline of the ERG b ripple wave amplitudes that H-89 can be caused to PVR and have protective effect.

Embodiment 9

The preparation of eyeball frozen section

1. eyeball prepares：SD rats after PVR modelings collect eyeball sample in different time points, and the dislocation of SD rats is put to death Afterwards, eyeball is carefully extractd, notes retaining optic nerve；

2. fix：It is placed in 4% paraformaldehyde to fix overnight；

3. dissect：Eyeball is dissected in disecting microscope lower edge corneoscleral junction upper limb, cornea is carefully removed, iris and crystalline Body, forms complete optic cup, notices that the soft preventing and treating of operation causes detachment of retina；

4. it is dehydrated：Overnight it is dehydrated with 30% sucrose；

5. embed：4 DEG C of balances are embedded with organization embedding liquid OCT overnight；

6. liquid nitrogen flash freezer：By eyeball liquid nitrogen snap frozen, the position that eyeball is caused before freezing is tried one's best vertical center.- 80 DEG C of preservation frozen samples.

7. cut into slices：Machine is cut with ice carries out serial section by 8 μm of thickness, takes the section of optic papilla.Section is put In -80 DEG C of preservations, using front drying up.

Embodiment 10

Retina frozen section immunofluorescence

1. section prepares：The eyeball frozen section sample of gained carries out immunofluorescence dyeing detection in Example 9.

2. piece is baked：50 DEG C of roasting piece half an hour.

3. fix：4%PFA fixes 10 minutes, then with PBS three times, every time 5 minutes.

4. permeable membrane：With 0.25%Triton-X100 permeable membranes 15min, PBS is washed 3 times, each 5min.

5. close：5% horse serum room temperature is closed 1 hour；

6. one resists：Plus 5% horse serum dilution corresponding one resist, being placed in wet box prevents drying, 4 DEG C of overnight incubations.

7. two resist：PBS is washed 3 times, each 5min；Plus 5% horse serum dilution luciferase mark with one resist same kind The two of source resist, and are placed in wet box, and 37 DEG C of lucifuge is incubated 1 hour.

8.DAPI contaminates core：PBS is washed 3 times, each 5min；Nucleus 1min is contaminated with 0.5 μ g/mlDAPI, PBS washes 3 times, every time 5min。

9. mounting：With fluorescence mountant mounting.

10. take pictures observation：Take pictures under the microscope observation.

As shown in Figure 4 and Figure 5, it is to freeze eyeball section immunofluorescence picture, Fig. 4 and Fig. 5 is respectively EMT related gene a- The expression of SMA and FN1, we can see that in PBS control group completely without the expression of a-SMA and FN1, in mould by figure Type group, only cephacoria is not formed, and the expression of a-SMA and FN1 is significantly raised, and in H-89 treatment groups, a-SMA and FN1 Expression is decreased obviously, especially FN1, does not almost express.This further illustrates H-89 and ARPE19 joint PRP injections is drawn The cephacoria for rising is formed and EMT processes have inhibitory action, so as to demonstrate therapeutic actions of the H-89 to PVR.

The measure of PKA activity in embodiment 11ARPE19 cell

1. cell carries out the respective handling of variable concentrations and different time sections with TGF β 1.

2. sample is received：Vitellophag, with the PKA Extraction buffer re-suspended cells of precooling, and is smashed carefully with ultrasound Born of the same parents.

3.PKA Activity determinations：

(1) solution is prepared：C-AMP- of the specification needed for PKA dilution buffer dilutions provided according to businessman Dependent Protein Kinase Catalytic Subunit to 2ug/ml.And each solution of system mixing is clicked, Operate on ice.

	Experimental group	Positive control	Negative control
				5X PKA Reaction buffer	5ul	5ul	5ul
A1Peptide	5ul	5ul	5ul
				5X PKA Activator solution	5ul	5ul	5ul
Peptide Protection solution	1ul	1ul	1ul
				Sample	1-10ul	●5ul PKA catalytic Subunit	0ul
ddH2O	To 25ul	To 25ul	To 25ul

Wherein sample and Catalytic Subunit individually add, by above-mentioned other solution it is mixed after after, take from ice Open, and 30 DEG C of waters 1 minute, sample and Catalytic Subunit are then added respectively, it is incubated at room temperature 30 minutes.

(2) thermal resistance is stopped：After being stopped with boiling water bath or 95 DEG C of 10 minutes thermal resistances, sample can be stored at 4-20 DEG C of dark, Before until running glue.

(3) glue is run：0.8% agarose gel is prepared with the Tristan-HCL (PH8.0) of 50mM.Before loading, in sample Plus the glycerine of the 80% of 1ul, it is ensured that sample is still in pipe.After loading, 100 volts, 15-20 minutes or until naked eyes bands visible Separate till understanding.

4.PKA active levels：

(1) tap rubber：Run and tapped rubber immediately after glue so that the volume after blob of viscose dissolving is about 250ul or so, is transferred to 1.5 In milliliter EP pipes, 95 DEG C of colloidal sols, not enough is supplemented to 250ul. with ddH2O

(2) 96 orifice plates are shifted：Prepare a 96 clean orifice plates, take the hot glue for having dissolved, the peptization of 75ul of 125ul The hole that 250ul is transferred to 96 orifice plates is taken in the EP pipes of the glacial acetic acid to 1 1.5 milliliters of solution liquid and 50ul after quick concussion In the middle of, all samples are added with this.

(3) ELIASA reading：Reading at 570nm wavelength, with negative control hole as blank.

As a result as shown in fig. 6, wherein 6A, 6C are to run the histogram after glue, 6B, 6D are statistical chart, as seen from the figure TGF β 1 can activate PKA paths, and it is active to increase PKA, and the process or dose-dependant and time dependent.This is further disclosed In vitro PKA paths are activated during PVR model Es MT, so as to suppress EMT for H-89, have therapeutic action to PVR There is provided foundation.

The above-mentioned description to embodiment is that invention is understood that and used for ease of those skilled in the art. Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general Principle is applied in other embodiment without through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability Field technique personnel announcement of the invention, the improvement made without departing from scope and modification all should be the present invention's Within protection domain.

Claims (10)

1. Applications of the 1.PKA inhibitor H-89 as PVR inhibitor.
2. 2. application according to claim 1, it is characterised in that PKA inhibitor H-89 is used as preventing and/or treat Hypertrophic The application of vitreoretinopathy medicine.
3. 3. application according to claim 2, it is characterised in that PKA inhibitor H-89 is used as auxiliary surgery of retinal detachment The application of medicine, PKA inhibitor H-89 is used in the form of intravitreal agent.
4. 4. application according to claim 1, it is characterised in that described PKA inhibitor H-89 is used as EMT in PVR mechanism The application of process inhibitor.
5. 5. application according to claim 1, it is characterised in that described PKA inhibitor H-89 is used as TGF beta induced people The inhibitor that retinal epithelial cells rise in value and migrate.
6. 6. application according to claim 1, it is characterised in that described PKA inhibitor H-89 changes as eyeball structure Inhibitor and cephacoria form the application of accelerator.
7. 7. application according to claim 1, it is characterised in that described PKA inhibitor H-89 are used as mesenchymal cell markers The application of thing inhibitor.
8. 8. application according to claim 1, it is characterised in that PKA paths are activated during PVR.
9. 9. application according to claim 8, it is characterised in that the work of TGF β dose-dependants and time dependent increase PKA Property.
10. It is 10. a kind of to prevent and/or treat proliferative vitreoretinopathy, and/or the medicine of auxiliary surgery of retinal detachment, Characterized in that, main component is PKA inhibitor H-89.