CN104383521A - Use of alphaB-crystallin - Google Patents

Abstract

The invention discloses use of alphaB-crystallin in preparation of drug for inhibiting potassium ion channels in retinal ganglion cells. The alphaB-crystallin is injected into an acute high intraocular pressure rat model. Compared with a non-injected group, the expression quantities of kv1.3 and kv1.1 on a rat retina of an injected group are decreased, indicating that the alphaB-crystallin has a function of inhibiting potassium ion channels in retinal ganglion cells. The invention provides a novel candidate drug for treating related diseases by inhibiting potassium ion channels in retinal ganglion cells clinically.

Description

The purposes of α B-crystallin

Technical field

The invention belongs to biomedicine technical field, relate to the novelty teabag of α B-crystallin, be specifically related to the purposes of α B-crystallin in preparation suppression retinal ganglial cells in potassium-channel medicine.

Background technology

Retinal ganglial cells (RGCs) is the last leg that retina internal information transmits, and its aixs cylinder first forms amphiblestroid nerve fibre layer, then pools optic nerve, plays important conduction in pathways for vision.Optic nerve injury can cause optic neuropathy, thus causes the blindness, and this situation can be seen much ophthalmology is seriously ill, as optic neuritis, looks road occupying lesion, glaucoma, ischemic optic neuropathy and diabetic renal papillary necrosis etc.In recent years, the concern of lot of domestic and foreign scholar is received about the research of optic nerve protection.Research display, the key pathological basis of traumatic optic neuropathy optic nerve lesion is the excessive Apoptosis of RGCs and the aregeneratory of ganglionic cell.After optic nerve injury, the protection of RGCs mainly stopped or preventing it from apoptosis occurring.Block or reduce the apoptosis pathway of RGCs and increase the survival ability of RGCs, having become the research direction preventing optic neuropathy, preserve patient's visual function.

Research to show that on RGCs many kinds of potassium-channels exist, and these potassium-channels are at the growth of RGCs, neuraxon's regeneration, axon guidance, action potential and repeatedly serve very important effect in electric discharge conciliation.Kv1 passage family belongs to voltage gated k+ channel blocker.Research proves that the RGCs of rat expresses kv1.1, kv1.2, kv1.3, but do not express kv1.5, and pass through at rat intravitreal potassium channel blocker, find the apoptosis blocking kv1.1, kv1.3, RGCs can be reduced, only blocking kv1.2 only plays a part very little, blocks kv1.5 inoperative.Kv1.1 and kv1.3 promotes the spontaneous apoptosis of RGCs cell by the different paths in Apoptosis mechanism, and the exhaustion of kv1.1 adds the expression of anti-apoptosis factor Bcl-xL etc., and the exhaustion of kv1.3 decreases the expression of caspase-3, caspase-9 and Bad etc.Finally, the exhaustion of kv1.1 and kv1.3 all can increase the survival rate of the rear RGCs of optic nerve transection wound.

α B-crystallin is one of main protein ingredient of crystalline lens, along with going deep into of research, finds that it has certain existence at cornea, iris, retina, corpus ciliare, optic nerve and have important relation with the pathogenesis of ocular disease.α B-crystallin is the small heat shock protein of the common existence of a class, has powerful molecular chaperones effect.The seminar at inventor place has proved that α B-crystallin is to rat retina free of toxic effects, can promote the survival of RGCs after acute high intraocular pressure, and expresses by increasing GAP-43 in RGCs the axon regeneration promoting RGCs.

Although much research proves that α B-crystallin has protective effect to RGCs, its mechanism of action is still not clear.

Summary of the invention

The present invention finds to propose based on following experiment: give rat acute Ocular hypertensive model and inject with α B-crystallin, compared with not injecting, after injection α B-crystallin, in RGCs cell, kv1.1, kv1.3 express and reduce, Bcl-xL rising simultaneously, caspase-3 reduce, and show that α B-crystallin is the apoptosis regulating and controlling RGCs cell by controlling potassium-channel kv1.1 and kv1.3.

The invention provides the purposes of α B-crystallin in preparation suppression retinal ganglial cells in potassium-channel medicine.Described potassium-channel can be kv1.1, can be the combination of kv1.3 or kv1.1 and kv1.3.Kv1.1 and kv1.3 controls the apoptotic process of cell by different paths, suppresses separately the one in above-mentioned two kinds of potassium-channels just can reach and controls apoptotic object.In specific embodiment of the invention scheme, give rat acute Ocular hypertensive model with the injection of α B-crystallin, compared with not injecting, after injection α B-crystallin, the expression of kv1.1 and the kv1.3 two kinds of channel proteins in RGCs cell is all suppressed.

Present invention also offers the purposes in the α B-crystallin medicine that Bcl-xL protein expression raises in preparation promotion retinal ganglial cells.In specific embodiment of the present invention, give rat acute Ocular hypertensive model with the injection of α B-crystallin, compared with not injecting, after injection α B-crystallin, in RGCs cell, Bcl-xL expresses and raises.

The present invention also provides happy α B-crystallin to suppress the purposes in the medicine of caspase-3 protein expression in retinal ganglial cells in preparation.In specific embodiment of the present invention, give rat acute Ocular hypertensive model with the injection of α B-crystallin, compared with not injecting, after injection α B-crystallin, in RGCs cell, caspase-3 expresses and reduces.

Present invention also offers the purposes of α B-crystallin in the medicine of preparation suppression retinal ganglion cells apoptosis.In specific embodiment of the present invention, give rat acute Ocular hypertensive model to inject with α B-crystallin, compared with not injecting, after injection α B-crystallin, in RGCs cell, Bcl-xL expresses and raises, caspase-3 expresses and reduces simultaneously, shows that the apoptotic process of RGCs is suppressed.

Present invention also offers the purposes of α B-crystallin in the medicine of preparation treatment optic nerve injury.The key pathological basis of optic nerve injury is the excessive Apoptosis of RGCs and the aregeneratory of ganglionic cell.After optic nerve injury, the protection of RGCs mainly stopped or preventing it from apoptosis occurring.

By setting up the animal model of optic nerve injury and carrying out experimentation, to seek effective Therapeutic Method, for studying optic nerve injury mechanism and treating significant.Optic nerve injury model is divided into animal model, the animal model of ischemic optic damage, the animal model of Bulbi hypertonia optic nerve injury of mechanicalness optic nerve injury both at home and abroad now.The animal model of Bulbi hypertonia optic nerve injury comprises acute temporary Bulbi hypertonia and Chronic persistent Bulbi hypertonia.Acute temporary Bulbi hypertonia can be realized by following method: (1) reverse headgear method; (2) vitreous chamber perfusion; (3) drug-induced method.The method of above-mentioned mentioned structure optic nerve injury model all can be used for the present invention.

In specific embodiment of the present invention, what select is the standby acute high intraocular pressure optic nerve injury model of reverse headgear normal saline legal system, and concrete building process is as follows: with 10% chloral hydrate (0.4ml/100g) row rat lower-left intraperitoneal injection of anesthesia.Rinse conjunctiva of right eye capsule with Levofloxacin Eye drop after general anesthesia success, then drip compound tropicamide eye drops and each one of Oxybuprocaine hydrochloride eye drops, two kinds of eye drop interval service time 5min.Under anatomic microscope, the 33G syringe needle being connected with normal saline transfusion device is done tunnel incision in limbus of corneae and thrust anterior chamber, note not hindering iris and crystalline lens, open infusion regulator to maximum.Keep liquid fluid level and rathole vertical dimension 156cm (produce 15.22kPa, be equivalent to the intraocular pressure of 120mmHg).When in Murphy's dropper no liquid instil and bulbar conjunctiva, iris, retinal pallor time illustrate acute high IOP model success.Be interrupted and drip Chibroxin and prevent bitot's patches with prevention infection.Continuous Perfusion 60min, extracts syringe needle, and visible bulbar conjunctiva and iris recover normal color, smear erythromycin eye ointment.The rat that ocular infection occurs after injuring crystalline lens, iris and puncture in paracentesis of anterior chamber process is rejected.

Advantage of the present invention and beneficial effect: the Late Cambrian dependency of α B-crystallin and kv1.1 and kv1.3 two kinds of channel proteins, namely α B-crystallin can suppress the expression of kv1.1 and kv1.3 two kinds of potassium-channel proteins, thus blocks the function of potassium-channel.According to above-mentioned discovery, the drug candidate of the disease that α B-crystallin is correlated with potassium-channel function as treatment can be developed.

Accompanying drawing explanation

Fig. 1 shows postoperative 7d QRT-PCR testing result;

Fig. 2 shows postoperative 14d QRT-PCR testing result;

Fig. 3 shows the Western blot result of postoperative 7d and 14d expressing quantity;

Fig. 4 shows the statistical result of postoperative 7d expressing quantity;

Fig. 5 shows the statistical result of postoperative 14d expressing quantity.

Detailed description of the invention

Further illustrate the present invention below in conjunction with specific embodiment, embodiments of the invention only for explaining the present invention, and do not mean that and limit the scope of the invention.

The experimental technique used in following embodiment if no special instructions, is conventional method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Laboratory animal and grouping:

3 monthly age male cleaning grade SD rats 60, about body weight 220g, thered is provided by Test Animal Centre, Academy of Military Medical Sciences, P.L.A, credit number SCXK-(army) 2012-0004, raise in central laboratory of Armed hospital general, through slit lamp and ophthalmofundoscopy without Eye disease.Be divided into following three groups at random:

(1) acute high intraocular pressure group (A group): after acute high IOP model successfully builds, does not do other process.

(2) acute high intraocular pressure+intravitreal normal saline group (B group): after acute high IOP model is successfully set up, oozy glass body injecting normal saline 5 μ l.

(3) acute high intraocular pressure+intravitreal α B-crystallin group (C group): after acute high IOP model is set up, oozy glass body cavity injection α B-crystallin (concentration 1*10 ^-5g/L) 5 μ l.

Often organize 20, right eye is experimental eye.Respectively at postoperative 7d and 14d, often group gets 5 rat retinas, uses QRT-PCR method to detect the expression of kv1.1, kv1.3, Bcl-xL and caspase-3mRNA; During respectively at postoperative 7d and 14d, often group gets 5 rat retinas, uses western blot to detect the protein expression level of retina kv1.1, kv1.3, Bcl-xL and caspase-3.

The foundation of embodiment 1 rat acute Ocular hypertensive model

Method: with 10% chloral hydrate (0.4ml/100g) row rat lower-left intraperitoneal injection of anesthesia.Rinse conjunctiva of right eye capsule with Levofloxacin Eye drop after general anesthesia success, then drip compound tropicamide eye drops and each one of Oxybuprocaine hydrochloride eye drops, two kinds of eye drop interval service time 5min.Under anatomic microscope, the 33G syringe needle being connected with normal saline transfusion device is done tunnel incision in limbus of corneae and thrust anterior chamber, note not hindering iris and crystalline lens, open infusion regulator to maximum.Keep liquid fluid level and rathole vertical dimension 156cm (produce 15.22kPa, be equivalent to the intraocular pressure of 120mmHg).When in Murphy's dropper no liquid instil and bulbar conjunctiva, iris, retinal pallor time illustrate acute high IOP model success.Be interrupted and drip Chibroxin and prevent bitot's patches with prevention infection.Continuous Perfusion 60min, extracts syringe needle, and visible bulbar conjunctiva and iris recover normal color, smear erythromycin eye ointment.The rat that ocular infection occurs after injuring crystalline lens, iris and puncture in paracentesis of anterior chamber process is rejected.

Embodiment 2 rat acute Ocular hypertensive model vitreous chamber drug injection

The preparation of α B-crystallin solution: be dissolved in normal saline by commodity α B-crystallin dry powder (purchased from sigma), makes its final concentration be 1*10 ^-5g/L.

Method: after rat acute Ocular hypertensive model is set up, uses microsyringe, thrusts vitreous chamber under the microscope, from the syringe needle that Central corneal amplifies as seen, slowly inject, extract syringe needle after Rat Right cornea edge, is coated with erythromycin eye ointment in conjunctival sac.Wherein inject isotonic saline solution 5 μ l in B group rat vitreous chamber, in C group rat vitreous chamber, inject 1*10 ^-5the α B-crystallin solution 5 μ l of g/L.Gently press cornea with coverslip, sight glass body cavity injection site is with or without hemorrhage and seepage, and whether eye ground is reflective normal, optical fundus blood vessel traveling and full whether normal.Basis of microscopic observation confirms that vitreous chamber includes experiment in without hemorrhage, aphakia and injury of iris person.Chibroxin point art eye one, then smears art eye with erythromycin eye ointment.

Embodiment 3 QRT-PCR method detects the mRNA level in-site of kv1.1, kv1.3, Bcl-xL and caspase-3

Step:

1. retina Total RNAs extraction

During respectively at postoperative 7d and 14d, often group gets 5 rats, 10% chloral hydrate intraperitoneal anesthesia.Removal surgery eye to put in ice bath normal saline rinsing 3min*2 time.Under microscope, remove anterior ocular segment, take out retinal tissue.The total serum IgE in the explanation extraction retinal tissue of test kit (purchased from Beijing CoWin Bioscience Co., Ltd.) is extracted by ultrapure RNA.

2.RNA is quantitative

Draw 2 μ l RNA, make blank with air, detect RNA purity with ABI the quantitative analysis of nucleic acids instrument, the OD260/280 ratio of pure rna close to the ratio of the OD260/280 of 2.0, DNA close to 1.8.Adjust quantitative analysis instrument mensuration OD260 value with ABI and carry out accurate quantitative analysis.

3. reverse transcription

Carry out reverse transcription with HiFi-MMLV cDNA first chain synthetic agent box (purchased from Beijing CoWin Bioscience Co., Ltd.), experimental implementation is undertaken by product description, and step is as follows:

(1) by RNA template, primer, 5*RT mix and without RNase water dissolution and be placed in for subsequent use on ice.According to the form below adds the Part I of 20 μ l reaction systems in reaction tube, mixing.(see table 1)

The Part I of table 1 reaction system

Reagent name	Use amount	Final concentration
			Primer mixture	2μl	50μM
RNA template	2μl	10ng
			Without the water of RNase	To 15 μ l

Hatch 5min, ice bath 2-10min for (2) 65 DEG C, of short duration centrifugal.Add the Part II (see table 2) of reaction system.Inhale gently and play mixing, hatch 40min for 37 DEG C.Reaction terminates rear 70 DEG C of insulation 10min.

The Part I of table 2 reaction system

Reagent name	Use amount	Final concentration
			RT mix	4μl	1*
HiFi-MMLV enzymatic mixture	1μl	10ng

4.QPT-PCR reacts

(1) PCR primer design and synthesis: the online designed, designed in IDT primer Quest website, synthesizes (see table 3) by Shanghai biotechnology company limited.

Table 3 primer sequence

(2) QRT-PCR reaction system (see table 4).

Table 4 QRT-PCR reaction system

Reagent name	Use amount
		CDNA	2μl
2*SYBR Green Supermix	10μl
		Forward primer (10 μMs)	1μl
Reverse primer	1μl
		Without the water of RNase	6μl
Cumulative volume	20μl

(3) QRT-PCR reaction condition

Reaction condition: 95 DEG C of denaturation 10min; 95 DEG C of degeneration 15s; 58 DEG C of annealing extend 60s; 45 circulations.

5.QRT-PCR interpretation of result method

Result with the display of Ct value, the period that the experiences during threshold value that Ct value sets for the fluorescence signal arrival in each reaction tube.All pattern detection all repeat 3 times, adopt 2 ^{-△ △ ct}method carries out the relative quantitative assay of data.

Statistical analysis: data acquisition mean+SD represents, application spss17.0 statistical software, adopts one factor analysis of variance, and q checks, and P < 0.05 has statistical significance for difference.

6. result

Each group of kv1.1, kv1.3, Bcl-xL, caspase-3mRNA relative expression quantity result as shown in table 5 and table 6.One factor analysis of variance can obtain: postoperative 7d and 14d, C group rat retina kv1.1, kv1.3, Bcl-xL, caspase-3mRNA relative expression quantity compares with A group, B group respectively, and difference has statistical significance (P < 0.05); A group and B group compare, no significant difference.The relative expression quantity of C group rat retina kv1.1, kv1.3, caspase-3mRNA is lower, and the relative expression quantity of Bcl-xL mRNA is higher (see Fig. 1, Fig. 2).

The postoperative 7d of table 5, the relative expression quantity of each group rat retina kv1.1, kv1.3, Bcl-xL, caspase-3mRNA

Gene Name	A group	B group	C group
				kv1.1	1.104±0.238	1.008±0.243	0.507±0.136*
Kv1.3	1.132±0.301	1.168±0.215	0.659±0.078*
				Bcl-xL	1.094±0.579	0.980±0.072	2.030±0.551*
caspase-3	1.017±0.232	1.065±0.265	0.566±0.067*

Note: * represent component C not and A group, B group compare, difference has statistical significance (P < 0.05).

The postoperative 14d of table 6, the relative expression quantity of each group rat retina kv1.1, kv1.3, Bcl-xL, caspase-3mRNA

Gene Name

A group

B group

C group

kv1.1	1.027±0.192	0.961±0.185	0.503±0.110*
				kv1.3	1.021±0.245	1.102±0.237	0.511±0.201*
Bcl-xL	1.054±0.350	1.073±0.308	1.910±0.246*
				caspase-3	1.030±0.297	1.042±0.030	0.503±0.201*

Note: * represent component C not and A group, B group compare, difference has statistical significance (P < 0.05).

Embodiment 4 Western blot detects the protein expression level of kv1.1, kv1.3, Bcl-xL, caspase-3

1. retina total protein extraction

During respectively at postoperative 7d and 14d, often group gets 5 rats, 10% chloral hydrate intraperitoneal anesthesia.Removal surgery eye to put in ice bath normal saline rinsing 3min*2 time.Under microscope, remove anterior ocular segment, take out retinal tissue.Go flesh tissue to weigh 20mg, add pre-cooling RIPA Protein Extraction Reagent and protease inhibitor Proteaseinhibitor cocktail (purchased from Roche), homogenate, hatch 20min on ice, the centrifugal 20min of 13000rpm, gets supernatant, carries out protein quantification.

2.BCA protein quantification carries out (purchased from Pierce) according to the operating instruction in BCA protein quantification test kit

3. immunoblotting

(1) 10% separation gel is prepared, 5% concentrated glue.

(2) protein sample is by the amount loading in 32 μ g/ holes.

(3) deposition condition: concentrated glue constant voltage 70V, about 20min; Separation gel constant voltage 120V, determines electrophoresis dwell time by pre-dyed albumen marker.

(4) wet robin, transferring film condition: 300mA constant current, about 60min.

(5) close: 5% defatted milk-TBST, hatch 1h.

(6) primary antibodie is hatched: add primary antibodie (kv1.1lgG and kv1.3lgG (purchased from Abcam) in Mus source, Bcl-xL lgG and caspase-3lgG (purchased from CST) that 5% defatted milk-TBST dilutes, 4 DEG C of overnight incubation, next day, TBST washed 3 times, each 10min.

(7) two anti-hatch: add that 5% defatted milk-TBST dilutes two resist, and incubated at room 40min, TBST wash film 3 times, each 10min.

(8) expose: ECL is added drop-wise to memebrane protein face, reaction 3-5min, then exposes: 10s-30min, and development is fixing.

(9) carry out gray value analysis with labworks software to protein band, represent the expression of destination protein with the ratio of object band and internal reference β-actin band, every band all measures 4 times, averages, and represents with mean+SD.

4. result

Detection can obtain each group of associated protein band (as shown in Figure 3), carries out gray value analysis (see table 7 and table 8).One factor analysis of variance can obtain: postoperative 7d and 14d, C group rat retina kv1.1, kv1.3, Bcl-xL, caspase-3 protein expression level compares with A group, B group respectively, and difference has statistical significance (P < 0.05); A group and B group compare, no significant difference.C group rat retina kv1.1, kv1.3, caspase-3 protein expression level is lower, and the protein expression level of Bcl-xL is higher (see Fig. 4, Fig. 5).

The postoperative 7d of table 7, each group object band and β-actin band gray level ratio mean

Gene Name	A group	B group	C group
				kv1.1	0.485±0.021	0.470±0.061	0.388±0.030*
kv1.3	0.535±0.069	0.520±0.064	0.425±0.039*
				Bcl-xL	0.338±0.036	0.322±0.028	0.412±0.023*
caspase-3	0.475±0.022	0.467±0.044	0.413±0.042*

Note: * represent component C not and A group, B group compare, difference has statistical significance (P < 0.05).

The postoperative 14d of table 8, each group object band and β-actin band gray level ratio mean

Gene Name	A group	B group	C group
				kv1.1	0.468±0.047	0.452±0.069	0.378±0.028*
kv1.3	0.533±0.074	0.517±0.062	0.417±0.043*
				Bcl-xL	0.353±0.045	0.337±0.037	0.415±0.039*
caspase-3	0.362±0.055	0.345±0.036	0.275±0.029*

Note: * represent component C not and A group, B group compare, difference has statistical significance (P < 0.05).

Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.

Claims (8)

1. the purposes of α B-crystallin in preparation suppression retinal ganglial cells in potassium-channel medicine.

2. purposes according to claim 1, is characterized in that, in described suppression retinal ganglial cells, potassium-channel is the expression suppressing potassium-channel proteins in retinal ganglial cells.

3. purposes according to claim 2, is characterized in that, described potassium-channel proteins is kv1.1.

4. purposes according to claim 1, is characterized in that, described potassium-channel proteins is kv1.3.

5. purposes according to claim 1, is characterized in that, described potassium-channel proteins is kv1.1 and kv1.3.

6. α B-crystallin promotes the purposes in the medicine that in retinal ganglial cells, Bcl-xL protein expression raises in preparation.

7. α B-crystallin suppresses the purposes in the medicine of caspase-3 protein expression in retinal ganglial cells in preparation.

8. the purposes according to any one of claim 1-7, is characterized in that, described retinal ganglial cells comes from rat.