CN105395530A - New application of FTY720 - Google Patents

Abstract

The invention provides a new application of FTY720. By means of the new application, DM rat retina inflammatory cell infiltration, expression of cell factors and expression of adherence factors can be reduced; in addition, activation of NF-kappaB can be inhibited through the FTY720; the FTY720 has a protective effect on a blood retina barrier, vascular permeability is reduced, and expression of tight junction protein is kept; the FTY720 can be effectively applied to prepare medicine for diabetic retinopathy.

Description

The novelty teabag of FTY720

Technical field

The present invention relates to the novelty teabag of FTY720.

Background technology

One of Diabetic microvascular complication that diabetic renal papillary necrosis (DR) is, and for causing the one of the main reasons of work age cohort blinding.Inflammatory reaction plays an important role in the generation and development of DR.DR in early days with blood retina barrier destruction for principal character, there is retinal neovascularization in late period.At present, the treatment of DR is mainly and controls systemic conditions (comprising blood glucose, blood fat and blood pressure), the in vitro stereotomy treatment of anti-vegf treatment, full retinal laser and glass, but these methods are all the measures taked for symptom.There is no effective method and can treat DR from the cause of disease.

FTY720 (FTY720) is that the effective ingredient myriocin of extracting in Cordyceps removes chiral centre, changes side chain and the neotype immunosuppressant that synthesizes.FTY720, in vivo after the phosphorylation of E.C. 2.7.1.91 2, forms activity form FTY720-P.FTY720-P is the analog of sphingosine-1-phosphate (S1P), can act on other the 4 kinds of S1P receptors except S1P2 and play a role.

Present stage, FTY720 mainly had following application: FTY720 is the first sphingol 1-phosphate acceptor regulator class medicine preventing lymphocyte from leaving from lymph node, reduce the frequency of multiple sclerosis patients palindromia, delay multiple sclerosis patients sb.'s illness took a turn for the worse degree; It can also keep specific immunocyte in lymph node, prevents them from acting on central nervous system and causes damage, and being reversible to lymphocytic retentivity, thus makes the lymphocyte of patient's body-internal-circulation after treatment stops return to normal level.

Summary of the invention

Technical problem to be solved by this invention is the novelty teabag providing FTY720.

For solving the problems of the technologies described above, technical scheme of the present invention is:

FTY720 has the purposes reducing DM rat retina inflammatory cell infiltration and cytokine and adhesion factor and express.

FTY720 has the purposes suppressing NF-kB activation.

FTY720 has protective effect to blood retina barrier, reduces vascular permeability and maintains the expression of tight junction protein.

The purposes of FTY720 in the medicine become for the preparation for the treatment of of diabetic retinopathy.

The invention has the beneficial effects as follows:

The invention provides the novelty teabag of a kind of FTY720 (FTY720), DM rat retina inflammatory cell infiltration can be reduced, and cytokine and adhesion factor are expressed; In addition, FTY720 can also suppress the activation of NF-κ B; FTY720 has protective effect to blood retina barrier, reduces vascular permeability and maintains the expression of tight junction protein; FTY720 effectively can be applied to the medicine preparation of diabetic renal papillary necrosis.

Accompanying drawing explanation

Fig. 1 is rat retina HE staining examine retinal histopathology change figure in the embodiment of the present invention;

Fig. 2 is the microglia infiltration figure of the leukocyte of the rat retina CD45 positive and the macrophage/activation of the CD11b positive in the embodiment of the present invention;

Fig. 3 is rat retina inflammatory factor TNF-ɑ, IL-6 and IL-1 β expression figure in the embodiment of the present invention;

Fig. 4 is rat retina ICAM-1 and VCAM-1 expression in the embodiment of the present invention.Wherein, A is that immunohistochemical staining detects retina ICAM-1 and VCAM-1, and B is retina ICAM-1 and VCAM-1 positive cell statistics, and C is the mRNA relative expression spirogram of retina ICAM-1 and VCAM-1;

Fig. 5 is rat retina NF-κ B expression in the embodiment of the present invention.Wherein, A is that westernblot detects retina NF-kB protein expression, and B is the mRNA relative expression spirogram of retina NF-κ B;

Fig. 6 is rat EB perfusion detection retinal blood vessels leak in the embodiment of the present invention.Wherein, A is that EB pours into inner nuclear layer retina, and B is that retinal EB seepage is quantitatively schemed;

Fig. 7 is rat retina tight junction protein ZO-1 in the embodiment of the present invention, Occludin and Claudin-5 expression.Wherein, A is that immunohistochemical staining detects retina ZO-1, Occludin and Claudin-5 Expression and distribution, B is that westernblot detects retina ZO-1, Occludin and Claudin-5 expression, and C is the mRNA relative expression spirogram of retina ZO-1, Occludin and Claudin-5.

Detailed description of the invention

In order to make those skilled in the art better understand technical scheme of the present invention, below in conjunction with the drawings and the specific embodiments, technical scheme of the present invention is described in further detail.

Embodiment 1

1. the foundation of rat diabetes (diabeticmellitus, DM) model

The choosing and raising of 1.1 laboratory animals

12-14 male Wistar rat in age in week 80, body weight 250-300g.Whole rat feeding is in standardization animal center specific pathogen free animal (SPF) level Animal House, and adopt standard particle forage feed, freely drink water and ingest, room temperature is (22 ± 2) DEG C, and relative humidity is 40%-60%.Adaptability is raised after 3-5 days for experiment.

1.2DM rat model producing principle

Antitumor drug streptozotocin (streptozotocin, STZ) can selective destruction beta cells of isolated rat islets, causes experimental type Ⅰ diabetes mellitus.

The preparation of 1.3DM model and standard

Weigh before modeling, disposable tail vein injection 2%STZ, dosage is detect fasting glucose after 50mg/kg, 48h, and the continuous 16.7mmol/L that is greater than for 3 times is modeling success.

2 FTY720s (FTY720) are to the therapeutical effect of laboratory animal and pharmacological research

Verify that using the DM model of above-mentioned preparation as experimental subject FTY720 is to its therapeutic effect, and investigate its pharmacological mechanism.

2.1 experimental technique

2.1.1DM the rearing conditions of rat

Raise in standardization animal center specific pathogen free animal (SPF) level Animal House, adopt standard particle forage feed, freely drink water and ingest, room temperature is (22 ± 2) DEG C, and relative humidity is 40%-60%.

2.1.2 administering mode and concrete ways

After modeling success, FTY720 treatment group rat gastric infusion every day, dosage is 0.3mg/kg, continuous 3 months.Due to FTY720 water soluble, therefore adopt distilled water to dissolve, and placebo group is not set.

2.1.3 retinal slice HE dyes

2.1.3.1 draw materials

Latter 3 months of modeling success, puts to death rat and complete taking-up eyeball.

2.1.3.2 paraffin section preparation

(1) 10% formalin+4% glacial acetic acid fixes eyeball, makes tissue, cell protein degeneration solidifies; Spend the night, normal saline flushing eyeball;

(2) dewater: eyeball is immersed successively 75% ethanol, 85% ethanol, 95% ethanol I, 95% ethanol II, 95% ethanol III, anhydrous alcohol I, anhydrous alcohol II, anhydrous alcohol III;

(3) transparent: eyeball immerses dimethylbenzene I, II, III successively;

(4) waxdip: immersed by eyeball (paraffin containing partial melting) in the embedded box of preheating, continues to add the paraffin of thawing to organizing whole submergence;

(5) cool: embedded box is inserted the fully rear taking-up of cooling in 4 DEG C of refrigerators;

(6) by embedded wax stone, use paraffin machine serial section, thickness is 5 μm, makes paraffin section.

2.1.3.3HE dyeing

(1) paraffin section de-waxing is to water: order is followed successively by, and dimethylbenzene embathes 2 5min altogether, and 100%, 95%, 90%, 80%, 70% ethanol embathes 3min × 5, tap water 10min;

(2) brazilwood extract dyeing 5min, washes lmin from the beginning;

(3) 1% hydrochloride alcohol differentiation 30s, wash lmin from the beginning, PBS returns blue 30s, washes lmin from the beginning;

(4) eosin stains 2min; Wash 3min from the beginning;

(5) gradient alcohol dehydration, dimethylbenzene is transparent;

(6) resinene rubber seal sheet, light Microscopic observation is also taken pictures.

2.1.4 retina immunofluorescence dyeing

2.1.4.1 draw materials and frozen section preparation

(1) modeling success puts to death rat, complete taking-up eyeball in latter 3 months;

(2) immediately rat eye after freezing 10-15 second, is embedded with you OCT in liquid nitrogen;

(3) rat eye embedded by OCT is immediately freezing in ﹣ 80 DEG C of ultra cold storage freezers, takes out after 15 minutes;

(4) by freezing embedded tissue freezing microtome serial section, thickness is 5 μm, makes frozen section, and it is for subsequent use that section is temporarily frozen in ﹣ 20 DEG C.

2.1.4.2 immunofluorescence dyeing

(1) frozen section room temperature rewarming 30min;

(2), after ice acetone fixes 8min, ice PBS washes 3 times × 5min;

The BSA room temperature of (3) 3% closes 30min;

(4) drip primary antibodie CD45, CD11b, CD3 (equal 3%BSA dilution, dilution ratio is 1:200) respectively, 4 DEG C of overnight incubation, ice PBS washes 3 times × 5min;

(5) drip donkey Chinese People's Anti-Japanese Military and Political College Mus fluorescence two anti-(3%BSA dilutes, and dilution ratio is 1:400), room temperature lucifuge hatches 1h, and ice PBS washes 3 times × 5min;

(6) adopt containing DAPI mountant mounting.Fluorescence microscopy Microscopic observation, to take pictures.

2.1.5 retina immunohistochemical staining

2.1.5.1 draw materials and paraffin section preparation

With 2.1.3.1.1 and 2.1.3.1.2.

2.1.5.2 immunohistochemical staining

(1) paraffin section is placed in 65 DEG C of oven cooking cycle 2 hours, is cooled to room temperature after taking-up;

(2) dimethylbenzene takes off cured (dimethylbenzene I 10min → dimethylbenzene II 10min), graded ethanol aquation (100% ethanol I 5min → 100% ethanol II 5min → 95% ethanol 10min → 80% ethanol 10min), wash 1min from the beginning, distillation washing 1min;

(3) section immerses PH=6.4 citrate buffer, microwave heating, and after high fire heating 10min, low fire heating 10min, is cooled to room temperature after microwave oven takes out, and immerses distillation washing 5min;

(4) section is placed in 3%H ₂o ₂, level concussion 5min, distillation washing 5min

(5) add 5%BSA confining liquid, incubated at room 1h, get rid of confining liquid, do not wash;

(6) every block organization's dropping 30ul primary antibodie ZO-1 (thinner ratio 1:100), occludin (thinner ratio 1:100), claudin-5 (thinner ratio 1:100), ICAM-1 (thinner ratio 1:100), VCAM-1 (thinner ratio 1:200) are placed in wet box 4 DEG C of overnight incubation, the concussion of 0.1%PBS level washes 3 times, 5min/ time;

(7) drip 30ul biotin labeled two anti-(goat antirabbit or goat anti-mouse) (thinner ratio 1:200), the concussion of incubated at room temperature 1h, 0.1%PBS level washes 3 times, 5min/ time;

(8) drip 30ul horseradish peroxidase-biotin coupling three resist, and the concussion of incubated at room temperature 2h, 0.1%PBS level washes 3 times, 5min/ time;

(9) DAB colour developing: 1mlA liquid adds 50ulB liquid, matching while using; The DAB nitrite ion prepared is added drop-wise to upper covering of section all to organize, Microscopic observation does not occur being colored as standard with the painted good background of specificity, tap water color development stopping;

(10) tap water 10min after haematoxylin redyeing 1min;

(11) gradient alcohol dehydration (80% ethanol 1 minute → 95% ethanol 5 minutes → 100% ethanol I 5 minutes → 100% ethanol II 10 minutes), dimethylbenzene transparent (dimethylbenzene I 10 minutes → dimethylbenzene II 10 minutes);

(12) neutral gum mounting, light Microscopic observation is also taken pictures.

2.1.6 azovan blue (EB) vascular perfusion radiography inner nuclear layer retina

Latter 3 months of modeling success, each Stochastic choice of normal group, model group and FTY720 treatment group 5 rats, carry out EB blood vessel and pay close attention to radiography inner nuclear layer retina.

(1) anaesthetize: with the chloral hydrate 300mg/kg lumbar injection of 10% after rat weight;

(2) EB perfusion: tail vein injection 2%EB45mg/kg, circulation 2h;

(3) fixing: to extract eyeball after putting to death rat, be placed in 4% paraformaldehyde and fix 2h;

(4) peel off retina and spread sheet: cutting off along cornea edge, lens removal and vitreous body after removal cornea, iris; Retina is centered by optic disc, and radial incision 4 cutter, is placed on microscope slide, is carefully overturn by sclera, completely isolates retina, be laid in microscope slide with micro-toothed forceps;

(5) mounting: drip mounting after a little glycerol, be placed in confocal fluorescent basis of microscopic observation, photograph immediately.

2.1.7 retinal EB leakage measures

Latter 3 months of modeling success, each Stochastic choice of normal group, model group and FTY720 treatment group 6 rats, carry out retinal EB leakage mensuration.

(1) anaesthetize: with the chloral hydrate 300mg/kg lumbar injection of 10% after rat weight;

(2) EB perfusion: tail vein injection 2%EB45mg/kg, circulation 2h;

(3) fixing rat extremity and head, open thoracic cavity and expose heart, inserts pouring head until aorta by left ventricle, subduction left auricle, perfusion citrate buffer (0.05M, PH=3.5,37 DEG C of preheatings), perfusion pressure is 120mmHg, perfusion 2min;

(4) perfusion terminates rear rapid excision eyeball, cuts off along cornea edge, lens removal and vitreous body after removal cornea, iris, is separated retinal tissue and also claims its weight in wet base;

(5) retina is placed in Constant Temp. Oven inner drying (90 DEG C, 45min) (attention lucifuge), claims its dry weight.

(6) every retina and 120 μ l formamide solutions 70 DEG C hatch 18h, and extracting solution 4 DEG C of 15000rpm high speed centrifugation 30min, get the absorbance that supernatant surveys 620nm and 740nm respectively, ask its clean absorbance and A value.

(7) standard curve of EB dye strength in Methanamide is set up.According to the relation of EB and clean A value, obtain EB concentration in real solution, be multiplied by 120 μ l by concentration and draw retinal EB leakage (ng).Finally use retina dry weight (mg) markization EB leakage (ng), result is expressed as ng/mg.

2.1.8Westernblot rat retina tissue inflammatory correlation factor expressing quantity is detected

2.1.8.1 the extraction of total protein in tissue

(1) histiocyte lysate capable and 2ulPMSF that 100mg retinal tissue adds 1ml pre-cooling is got, inhibitors of phosphatases, homogenate on ice, ultrasonic degradation 90sec.

(2) leave standstill cracking 30min on ice, leave standstill every 5min pipettor in process and blow and beat 10 times.

(3) 4 DEG C of centrifugal 20min of 13000rpm, pipette supernatant and are sub-packed in clean EP pipe-80 DEG C and save backup.

2.1.8.2Western-blot method

(1) protein example pretreatment

Sample protein and 4 × SDS sample loading buffer press 3:1 (V/V)) mix, 100 DEG C of water-bath degeneration min, centrifugal 12,000rpm × 5min, remove impurity.

(2) mensuration (Coomassie Brilliant Blue) of protein concentration

1) production standard curve;

2) quantity per sample, by reagent A: B is the proportions BCA reagent working solution of 50:1, fully mixes;

3) each hole adds 200 μ lBCA working solutions, places 30min for 37 DEG C;

4) consoluet protein standard substance, gets 10ul and adds PBS and be diluted to 100 μ l, make final concentration be 0.5mg/ml;

5) standard substance are pressed 0 μ l, 1 μ l, 2 μ l, 4 μ l, 8 μ l, 12 μ l, 16 μ l, 20 μ l are added in the standard sample wells of 96 orifice plates successively, add PBS and supply 20 μ l;

6) add 2ul sample in the sample well of 96 orifice plates, add PBS to 20 μ l;

7) microplate reader measures A562;

8) protein concentration is calculated according to standard curve;

(3) preparation of SDS-PAGE running gel:

1) glue glass plate is installed;

2) join 10% separation gel, after mixing, glue is poured between two glass plates to separation gel liquid level apart from short glass plate along about 1.5cm place;

3) on separation gel, inject one deck distilled water, ambient temperatare puts 30min;

4), when there is an obvious boundary between separation gel and deionized water, remove distilled water, blot residual moisture with filter paper;

5) the concentrated glue of preparation, be directly poured on separation gel by glue after mixing, insert clean comb immediately, ambient temperatare puts 30min.

(4) SDS-PAGE electrophoresis

1), when etc. concentrated glue is fully polymerized, support and glue is put into electrophoresis tank, then add electrophoretic buffer, take out comb;

2) take out albumen Marker and protein sample, in hole, add albumen Marker3ul and protein sample 20ul;

3) protein sample making alive 80V on concentrated glue, continues about 30min, makes protein sample slow transit through concentrated glue, then voltage is adjusted to 100V, continues 120min, when bromophenol blue arrives bottom separation gel, stops electrophoresis.

(5) transferring film

1) taken off by gel and be immersed in transferring film buffer, the labelling according to pre-dyed albumen Marker retains the gel section needed, and cuts the upper right corner and marks;

2) nitrocellulose filter 1, the Whatman filter paper 6 of cutting and gel formed objects, balance 10-15min in transferring film buffer;

3) according to the installation transferring film system of power-pole-face, 3 metafiltration paper, gel, nitrocellulose filter, 3 metafiltration paper, positive source face order.The bubble between filter paper, gel, nitrocellulose filter is carefully driven away in installation process;

4) cover transferring film instrument, red to red, black to black connection power supply;

5) transferring film in ice bath: voltage is adjusted to 100V, continues 1h;

6) close electrophresis apparatus, take out nitrocellulose filter, deduct one jiao and make positive and negative labelling, then use TBST rinsing.

(6) close

Be soaked in by cellulose membrane (5g milk powder+100mlTBST) in 5% defatted milk powder, under room temperature, shaking table hatches 1h.Then discard milk powder, TBST rinses 5min × 3 time.

(7) primary antibodie is hatched

Rabbit anti-TNF-ɑ, IL-6, IL-1 β and NF-κ B (p65) antibody (the equal 1:1000 of thinner ratio) 4 DEG C of overnight incubation.TBST rinses 5min × 3 time.

(8) two anti-hatch

Horseradish peroxidase-labeled goat anti-rabbit igg (1:5000 dilution) 37 DEG C hatches 1h.TBST rinses 5min × 3 time.

(9) develop the color

1) A in ECL luminescence reagent box and B solution equal-volume mixing for standby use is got.

2) ECL mixed liquor is dripped at the protein powder of film.

3) video picture of Bio-Rad company ChemiDocMP gel imaging system is applied

(10) quantitative analysis of object band

Application ImageJ analysis software measures the gray value of each the object band i.e. internal reference β-actin of its correspondence.Calculate the gray value of each sample corresponding index according to the gray value of relative gray values=object band gray value/same sample β-actin, represent the expression of destination protein.Statistical software is utilized to analyze each group of relative gray values, to remove the error that the uneven degraded of protein causes.

2.1.9qRT-PCR the mrna expression amount that rat retina organizes ICAM-1, VCAM-1 and NF-κ B (p65) is detected

2.1.9.1 retinal tissue sample collection

(1) Preoperative Method

1) operating room sterilization: ultraviolet lamps irradiates 30min, ventilation 15min;

2) surgical instrument disinfection: operating theater instruments high pressure steam sterilization half an hour, puts into baking box and dries for subsequent use.

(2) sampling method

1) (general anesthesia) is anaesthetized: 10% chloral hydrate lumbar injection (300mg/kg) anesthesia;

2) 10min after anesthesia, lain on one's side by rat on operating-table, microforceps fixes rat eye, wipes out peribulbar group and is placed in normal saline flushing 3 times.Remove cornea, crystal and vitreous body, be separated retinal tissue with microforceps, the cryopreservation tube putting into RNase-free is at once for subsequent use.

2.1.9.2Trizol method extracts retinal tissue total serum IgE

(1), after taking-up retinal tissue is weighed, the PBS of pre-cooling washs 2 times, each 5min.

(2) retinal tissue ultrasonic disintegrator is pulverized, and every 100mg tissue adds 1mlTrizol.Room temperature places 5min, is transferred to by lysate in the 1.5mlEP pipe without enzyme;

(3) 0.2ml chloroform (chloroform: Trizol=1:5) is added, thermal agitation 15s (forbidding vortex), room temperature places 10min, 12000rpm4 DEG C of centrifugal 15min, sample is divided into three layers: bottom is peach organic facies, and upper strata is colourless aqueous phase and an intermediate layer;

(4) aqueous phase is transferred in new EP pipe, add the isopropyl alcohol (isopropyl alcohol: Trizol=1:2) of 0.5ml, room temperature places 10min, 12000rpm4 DEG C of centrifugal 10min, abandons supernatant;

(5) add 1ml75 ℅ ethanol (with the configuration of DEPC water) (ethanol: Trizol=1:1), vortex mixed, 7500rpm4 DEG C of centrifugal 5min, abandons supernatant;

(6) allow the RNA of precipitation at room temperature air-dry in fume hood;

(7) appropriate DEPC water dissolution precipitation is added according to precipitation capacity in EP pipe;

(8)-80 DEG C of Refrigerator stores are for subsequent use.

2.1.9.3 measure purity and the concentration of sample total serum IgE

(1) return to zero: get 100ulDEPC process water and be added in the color comparison tube of ultraviolet spectrophotometer, return to zero;

(2) sample rna content is measured: get 2ul sample rna+98ulDEPC process water and add in color comparison tube, read RNA concentration;

(3) sample rna concentration calculates: RNA concentration of specimens (ug/ml)=OD260 × 40ug/ml × 50;

(4) sample rna purity calculates: OD260/OD280 ratio detects RNA purity, and ratio illustrates that purity is better in 1.8 ~ 2.0 scopes;

(5) electrophoresis: sample this RNA5ul and be added in agarose gel loading hole, 130v constant voltage electrophoresis 10min.Observed result on gel imaging instrument, visible three bands and 28s, 18s, 5s, RNA is complete in band clear expression.

2.1.9.4 reverse transcription synthesis cDNA

(1) reaction system (see table 1) is prepared:

Table 1 reverse transcription reaction system

(2) reaction condition: hatch 30min for 42 DEG C, 85 DEG C of heating 5min, 4 DEG C of coolings.

(3) reverse transcription: by each component by adding shown in reaction system in 0.2mlRNase-Free micro-centrifuge tube, put into PCR instrument, according to reaction condition setting program, brings into operation.It is for subsequent use in-20 DEG C that reverse transcription terminates rear taking-up sample storage.

2.1.9.5qRT-PCR

(1) design of primers: according to design of primers principle, designs and synthesizes the Auele Specific Primer for rat ICAM-1, VCAM-1 and NF-κ B by Shanghai Sheng Gong biological engineering limited company.(see table 2)

Table 2qRT-PCR design of primers

(2) PCR reaction system (see table 3) is prepared

Table 3PCR reaction system

(3) qRT-PCR: reaction system is put into quantitative real time PCR Instrument, sets reaction as follows: denaturation 94 DEG C of 30s, degeneration 94 DEG C of 5s, anneal 57 DEG C of 10s, extends 72 DEG C of 10s, circulates 40 times.

(4), after reaction terminates, instrument generates CT (thresholdcycles) value and melting curve figure automatically.With β-actin for internal reference.Each gene of every part of template arranges 3 repeating holes, and independently experiment repeats at least 3 times.Arrange and analytical data, draw block diagram.Analytical data is as follows:

Folds＝2 ^-ΔΔCt

-ΔΔCt＝(Ct1-Ct2)-(Ct3-Ct4)

Ct1: the critical cycle number of process sample testing gene

Ct2: the critical cycle number of process sample housekeeping gene (β-actin)

Ct3: the critical cycle number of check sample testing gene

Ct4: the critical cycle number of check sample housekeeping gene (β-actin)

2.1.10 statistical method

SPSS17.0 software is adopted to carry out statistical analysis.Between group, statistics is with mean ± standard deviation represent.One factor analysis of variance or independent sample T is adopted to check.Be that difference has statistical significance with P<0.05.

2.2 experimental result

2.2.1 respectively rat weight and blood glucose situation is organized

Each group of rat, in feeding process, increases and body weight steady-state growth with the monthly age.Control rats random blood sugar increases with the age and continues to maintain normal range, and DM group and DM+FTY720 group rat random blood sugar continue to be greater than 16.7mmol/l and increase without obviously changing with the age.(see table 4)

Table 44,8 and 12 weeks rat body weights and random blood sugar compare

Note: ^*p < 0.001vscontrol, ^#p < 0.01vsDM.

2.1.2 retinal histopathology checks HE coloration result

Light microscopy result shows, and each Rotating fields of matched group retina is clear, form is normal, cell arrangement neat (Fig. 1); The each layer of DM group rat retina is thinning, cell arrangement is disorderly, ganglion cell layer and inner nuclear layer cell quantity reduce, misaligned, nucleus swelling, volume increase, retinal tissue edema, part blood vessel is obviously expanded: each layer tissue edema of DM+FTY720 group rat retina alleviates, and cell arrangement is gradually regular.

2.1.3 retinitis sexual cell immunofluorescence dyeing

Adopt the method for immunofluorescence dyeing to detect retinitis sexual cell Infiltrating, comprise total leukocyte (CD45), the huge microglia (CD11b) biting/activate and T lymphocyte (CD3).Result display (Fig. 2), compared with Normal group, the section of DM group rat retina has a large amount of CD45 positive and CD11b positive cell to infiltrate, and is mainly distributed in ganglion cell layer; And FTY720 treatment group rat retina section CD45 is positive and CD11b positive cell is less.CD3 positive cell content is few, and result does not show.

2.1.4 retina expression of inflammatory cytokines

Westernblot method is adopted to detect the expression of cytokine in retinal tissue, result display (Fig. 3): compared with Normal group, TNF-ɑ (1.12 ± 0.13vs.0.42 ± 0.17 in DM group rat retina, P < 0.001), IL-6 (1.03 ± 0.23vs.0.24 ± 0.1, P < 0.001) and IL-1 β expression significantly increase (0.92 ± 0.08vs.0.31 ± 0.12, P < 0.001); And FTY720 treatment significantly can lower TNF-ɑ, IL-6 and IL-1 β expression (0.64 ± 0.18, P < 0.05; 0.73 ± 0.12, P < 0.05; 0.64 ± 0.11, P < 0.05).

2.1.5 retina adhesion factor is expressed

Adopt immunohistochemical staining and qRT-PCR method, detect albumen and the mrna expression amount of rat retina tissue adherence factor ICAM-1 and VCAM-1 respectively.Immunohistochemical staining result display (Fig. 4 A): compared with Normal group, DM rat retina ICAM-1 (18/HPF, P < 0.001), VCAM-1 (17/HPF, P < 0.001) expressing quantity significantly increases; And FTY720 treatment group ICAM-1 and VCAM-1 protein expression significantly reduce (7/HPF; 6/HPF).Consistent with immunohistochemical staining, qRT-PCR testing result display (Fig. 4 B, C): compared with Normal group, DM rat retina ICAM-1 (2.23 ± 0.17fold, P < 0.001), VCAM-1 (2.42 ± 0.33fold, P < 0.001) mrna expression amount significantly increases; And FTY720 treatment group ICAM-1 (1.01 ± 0.05fold, P < 0.001), VCAM-1 (1.24 ± 0.28fold, P < 0.001) mrna expression significantly reduces.

2.1.6 retina NF-κ B expresses

The method of Westernblot and qRT-PCR is adopted to detect albumen and the mrna expression amount of rat retina NF-κ B (p65) respectively.Westernblot result display (Fig. 5 A): compared with Normal group, DM rat retina NF-kB protein expression significantly increases (1.43 ± 0.34vs0.93 ± 0.20, P < 0.05); And FTY720 treatment can suppress NF-kB protein to express (0.95 ± 0.31, P < 0.05).Consistent with Westernblot result, qRT-PCR testing result display (Fig. 5 B): compared with Normal group, DM rat retina NF-κ BmRNA expresses significantly increases by 1 (.92 ± 0.11fold, P < 0.001); And FTY720 treatment group NF-κ BmRNA expresses significantly minimizing (1.23 ± 0.09fold, P < 0.001).

2.1.7 retinal vascular permeability analysis

After EB vascular perfusion radiography inner nuclear layer retina, in confocal fluorescent basis of microscopic observation EB leakage scenarios (Fig. 6 A): rats in normal control group retinal vessel ne-leakage, the visible significantly EB seepage of DM group retinal vessels in rats and FTY720 treatment group has no obvious seepage.

In height linear correlation (r=0.9979, P=0.001) between statistical analysis EB concentration and clean A value, be Y=0.0702X+0.0125; Wherein, Y represents clean A value, and X represents EB concentration.Accordingly, the EB mass concentration corresponding to each group of clean A value of sample is calculated.The average leakage of normal rat retinal EB is the average leakage of 10.95 ± 1.95ng/mg, DM rat EB is 30.1 ± 2.3ng/mg; Both compare, and the average leakage of DM rat EB is significantly higher than normal group, and difference has statistical significance (P < 0.05).The average leakage of FTY720 treatment group EB is 17.28 ± 2.39ng/mg, and EB leakage reduces 42.57% compared with DM rat, and difference has statistical significance (P < 0.05).(Fig. 6 B).

2.1.8 retina tight junction protein is expressed

Immunohistochemical staining and Westernblot method is adopted to detect the expression of retina tight junction protein respectively.Immunohistochemical staining display (Fig. 7 A): normal rat retina tight junction protein ZO-1, Occludin and Claudin-5 great expression, and be mainly distributed in ganglion cell layer, nerve fibre layer, external plexiform layer and inner nuclear layer; DM rat retina tight junction protein is expressed and is obviously reduced, and FTY720 treatment group rat retina compact siro spinning technology is expressed then obviously to be increased.Westernblot detection display (Fig. 7 B): compared with normal rat, DM rat retina ZO-1, Occludin and Claudin-5 express all significantly to reduce and (are respectively 0.80 ± 0.16vs.1.35 ± 0.08, P < 0.05; 0.90 ± 0.18vs.1.26 ± 0.3, P < 0.05; 0.79 ± 0.1vs.1.38 ± 0.04, P < 0.05); FTY720 treatment significantly can reduce the loss of these three kinds of tight junction proteins, and difference has statistical significance (to be respectively 1.13 ± 0.12, P < 0.05; 1.19 ± 0.09, P < 0.05; 1.07 ± 0.15, P < 0.05).

2.3 conclusion

Above-mentioned experiment finds, FTY720 to DR treatment effectively.FTY720 can reduce DM rat retina inflammatory cell infiltration, and cytokine and adhesion factor are expressed.FTY720 can also suppress the activation of NF-κ B in addition.FTY720 has protective effect to blood retina barrier, reduces vascular permeability and maintains the expression of tight junction protein.

Above-mentioned detailed description of the novelty teabag of this FTY720 being carried out with reference to detailed description of the invention; illustrative instead of determinate; several embodiments can be listed according to institute's limited range; therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention.

Claims (4)

1. FTY720 has the purposes reducing DM rat retina inflammatory cell infiltration and cytokine and adhesion factor and express.

2. FTY720 has the purposes suppressing NF-kB activation.

3. FTY720 has protective effect to blood retina barrier, reduces vascular permeability and maintains the expression of tight junction protein.

4. the purposes of FTY720 in the medicine become for the preparation for the treatment of of diabetic retinopathy.