CN101812122A - Protection effect of light-harvesting chromoprotein of algae on lens epithelial cells - Google Patents

Abstract

The invention belongs to the field of biological medicaments, and relates to a protection effect of a light-harvesting chromoprotein, namely, phycocyanin of algae on lens epithelial cells. The phycocyanin can obviously inhibit the reduction of the survival ratio of the lens epithelial cells and the increment of the content of reactive oxygen species (ROS) and malonaldehyde (MDA) caused by sodium selenite, and also can obviously inhibit the structure damage and the cell apoptosis of the lens epithelial cells caused by the sodium selenite; and therefore, the phycocyanin has a remarkable protection effect on the lens epithelial cells.

Description

A kind of provide protection of catching photopigment albumen of algae to lens epithelial cells

Technical field

The present invention relates to the bio-pharmaceutical field, be specifically related to a kind of photopigment albumen of catching of algae, promptly Phycocyanins, C-is to the provide protection of lens epithelial cells.

Background technology

Epidemiology and experimental study show that non-congenital cataract can be induced formation as uv-radiation, oxidation, undesired calcium metabolism, diabetes, selenium etc. by multiple stress factor.Although cataract is a result of various factor comprehensive action, oxidative stress is the common mechanism of its formation.Lens epithelial cells (lens epithelial cell, LEC) be that the lens intracellular metabolite enlivens part most, undertaking the function of lenticular growth, differentiation and injury repairing, this cell is monolayer alignment, it is unique cell that mitotic activity is arranged in the lens, can differentiate the lens fibers cell and produce crystallin, it is again lens and extraneous barrier.The specific position of LEC and function make its easy oxidated attack, cause oxidative damage.In case this confluent monolayer cells is impaired, lenticular growth will be influenced, even stop, and severe patient also can destroy lenticular self stability, cause that calcium infiltrates, the halfcystine enzyme activates, cytoskeleton is degraded, crystallin is assembled, power and water is separated matter and entered, finally cause phacoscotasmus.Therefore, the change of lens epithelium can not be ignored in cataractous morbidity.Search out compound, can be cataractous control new selection is provided with protection lens epithelial cells, also significant to the research of lens epithelial cells damage and protection mechanism.

Human lens epithelial clone (the Human lens epithelial cell line that Ibaraki etc. set up, hLEC) SRA01/04, the feature basically identical of itself and the former foster hLEC that is commissioned to train is widely used in studying lenticular differentiation, the cataractous cause of disease and screening anti-cataract medicine.

But certain density Sodium Selenite inducing cell generation oxidative damage is widely used in the research of cytoprotective medicine.

Phycobiliprotein be in the marine algae content rich catch the light action chromoprotein, to be that a class is red, the water soluble protein of indigo plant or purple, mainly be divided into phycoerythrin (Phycoerythrin, PE), Phycocyanins, C-(phycocyanin, PC), Allophyxoxyanin three major types such as (APC), wherein Phycocyanins, C-mainly is present in blue-green algae (Cyanophyta), red algae (Rhodophyta), latent algae (Cryptophyta) and the minority dinoflagellate (Pyrrophyta).The Phycocyanins, C-of separation and purification is sapphirine in solution, and sends purple fluorescence, has special absorption peak at wavelength 620nm, can A ₆₂₀/ A ₂₈₀Represent its purity.Phycocyanins, C-both can be used as natural pigment and had been widely used in industry such as food, makeup, dyestuff, simultaneously also have intense fluorescence and can be made into fluorescent reagent, fluorescent probe, fluorescent tracing material etc., be used for research fields such as clinical diagnose, immunochemistry and biotechnology.The activity of Phycocyanins, C-is very extensive, has anticancer, anti-oxidant, multiple biological activity such as treatment cerebral ischemia etc., but has not yet to see Phycocyanins, C-to the research work report of lens epithelial cells provide protection and use.

Summary of the invention

A kind of provide protection of catching the lens epithelial cells damage that photopigment albumen-Phycocyanins, C-causes Sodium Selenite that the present invention has studied algae.

Sodium Selenite can cause SRA01/04 cell generation oxidative damage, Phycocyanins, C-can obviously suppress the decline of the SRA01/04 cell survival rate that Sodium Selenite causes, significantly alleviate Sodium Selenite to the structural damage of SRA01/04 cellular form, also can reduce SRA01/04 intracellular reactive oxygen (reactive oxygen species, ROS) content and mda (malondialdehyde, MDA) level also significantly suppresses Sodium Selenite inductive SRA01/04 apoptosis.Damage has significant provide protection to Sodium Selenite inductive lens epithelial cells more than to show Phycocyanins, C-, is expected to become the damage of protection lens epithelial cells, especially prevents and treats cataractous medicine.

Phycocyanins, C-of the present invention can derive from blue-green algae (Cyanophyta), red algae (Rhodophyta) or latent algae (Cryptophyta), fresh frond or the dry algae powder of preferred blue-green algae (as spirulina etc.).

The Phycocyanins, C-preparation method is as follows:

Frond is suspended in the 50mmol/L phosphate buffered, multigelation, 4 ℃ of centrifugal (10000g * 10min), supernatant adds solid ammonium sulfate to 50% saturation ratio, 4 ℃ of standing over night, centrifugal (10000g * 10min), collecting precipitation, be dissolved in the small amounts of phosphoric acid salt buffer (10mmol/L, pH7.0) in, and in this damping fluid fully the dialysis, dialysate filter, be splined on above-mentioned phosphate buffered saline buffer equilibrated DEAE-Sepharose FF post, carry out gradient elution, collect blue elutriant with the phosphate buffered saline buffer that contains 0-1mol/L NaCl gradient, after the elutriant dialysis of collecting, be splined on above-mentioned same damping fluid equilibrated hydroxyapatite column, the phosphate buffered saline buffer of 10-300mmol/L gradient (pH7.0) gradient elution is collected A ₆₂₀/ A ₂₈₀Blue elutriant greater than 4, the dialysis back concentrates or freeze-drying is Phycocyanins, C-.

Description of drawings

Fig. 1 is the influence of Phycocyanins, C-to the SRA01/04 cellular form structure of Sodium Selenite damage.

A is the normal control group, and B is a model group, and C is a positive controls, and D is 100 μ g/ml Phycocyanins, C-groups, and E is 300 μ g/ml Phycocyanins, C-groups

Fig. 2 is that Phycocyanins, C-is to the apoptotic restraining effect of Sodium Selenite inductive SRA01/04.

M is dna molecular amount marker DL2000, the 1st, and the normal control group, the 2nd, model group, the 3rd, positive controls, 4 is 150 μ g/ml Phycocyanins, C-groups, and 5 is 300 μ g/ml Phycocyanins, C-groups, and 6 is 600 μ g/ml Phycocyanins, C-groups.

Embodiment

Embodiment 1

Phycocyanins, C-is to the provide protection of the SRA01/04 cell of Sodium Selenite damage

With SRA01/04 is target cell, and mtt assay is surveyed cell survival rate.The SRA01/04 cell of taking the logarithm vegetative period, 0.25% trypsin 0.02%EDTA) after the digestion, be prepared into cell suspension with the RPMI1640 that contains 10% new-born calf serum, be inoculated in 96 well culture plates, every hole 100 μ l, cultivate 12 hour cells adherent after, each test holes adds Sodium Selenite, and to make final concentration be 5 μ mol/L, the group of administration simultaneously adds different concns Phycocyanins, C-1000 μ g/ml respectively, 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, 62.5 μ g/ml, 31.25 μ g/ml cultivates 24h, adds 5%MTT 10 μ l/ holes, put 37 ℃, 5% CO ₂Incubator continues to cultivate 4h, abandons supernatant, adds DMSO 100 μ l/ holes, and vibrator vibration 10min surveys its light absorption value A at the 570nm place on the enzyme-linked immunosorbent assay instrument.The result shows that concentration all can significantly improve the survival rate (table 1) of cell greater than the Phycocyanins, C-of 62.5 μ g/ml.

Table 1 Phycocyanins, C-is to the influence of SRA01/04 cell survival rate

Compare with model group, ^*P＜0.01

Embodiment 2

Phycocyanins, C-is to the influence of the SRA01/04 cellular form structure of Sodium Selenite damage

The SRA01/04 cell of taking the logarithm vegetative period, 0.25% trypsin, 0.02% EDTA) after the digestion, is prepared into cell suspension, is inoculated in 96 well culture plates, every hole 100 μ l with the RPMI 1640 that contains 10% new-born calf serum.After treating cell attachment, it is 5 μ mol/L that each test holes adding Sodium Selenite makes final concentration, the group of administration simultaneously adds different concns Phycocyanins, C-300 μ g/ml respectively, 100 μ g/ml, and positive drug pirfenoxone group is set, administration concentration 5.3 μ g/ml, cultivate after the 24h, abandon the substratum supernatant, by auspicious-Ji Shi dye liquor test kit (building up bio-engineering research institute) available from Nanjing illustrate dyeing after, 96 orifice plates are positioned over observation of cell form (Fig. 1) under the inverted microscope.

The model group cellular form has been compared obvious change with normal group as can be seen from Figure, and cytolemma is obviously impaired, and entocyte overflows, part necrocytosis or distortion.Behind the Phycocyanins, C-effect 24h, 100 μ g/ml cellular fories all have certain recovery, cytolemma is complete gradually, nucleus is regularization gradually, and the cellular form cell of 300 μ g/ml group is tending towards normal, the film diffusion phenomena still less, test-results shows that Phycocyanins, C-can significantly alleviate Sodium Selenite to the structural damage of SRA01/04 cellular form.

Embodiment 3

Phycocyanins, C-is to the influence of ROS content in the SRA01/04 cell of Sodium Selenite damage

With the SRA01/04 cell inoculation of logarithmic phase in 24 orifice plates, every hole 500 μ l.After treating cell attachment, it is 5 μ mol/L that each test holes adding Sodium Selenite makes final concentration, the group of administration simultaneously adds different concns Phycocyanins, C-300 μ g/ml respectively, 100 μ g/ml, and positive drug pirfenoxone group is set, administration concentration 5.3 μ g/ml, cultivate after the 24h, abandon the substratum supernatant, PBS washs 24 orifice plates twice, it is 2 of 40 μ M ' that every hole adds final concentration, 7 ' dichloro fluorescin second, two fat (H ₂DCFDA) use liquid 200 μ L, place 30min in 37 ℃.Discard dye liquor, wash 24 orifice plates twice with PBS.Every hole adds 1M NaOH lysing cell 10min.NaOH partly is transferred in black 96 orifice plates every hole 50 μ L, each 3 multiple hole.Measure its fluorescence intensity in excitation wavelength/emission wavelength 485/530nm place.Remaining part is got 50 μ L measure each sample with micro-lowry method with protein determination kit (building up bio-engineering research institute available from Nanjing) protein content.Test-results shows that Phycocyanins, C-can significantly reduce the increase of ROS content in the Sodium Selenite inductive SRA01/04 cell, thereby alleviates oxidative damage (table 2).Table 2 Phycocyanins, C-is to the influence of ROS content in the SRA01/04 cell of Sodium Selenite damage

Compare with model group, ^*P＜0.01

Embodiment 4

Phycocyanins, C-is to the influence of MDA level in the SRA01/04 cell of Sodium Selenite damage

SRA01/04 cell with logarithmic phase is inoculated in 20cm ²In the culturing bottle, every hole 5ml.After treating cell attachment, it is 5 μ mol/L that each test holes adding Sodium Selenite makes final concentration, and the group of administration simultaneously adds different concns Phycocyanins, C-600 μ g/ml, 300 μ g/ml, 150 μ g/ml respectively.After cultivating 24h, discard nutrient solution, with 0.25% trypsin 0.02%EDTA) peptic cell, 1mL PBS blows down cell, is loaded on 6 1.5ml EP pipes respectively.Get and respectively organize cell pyrolysis liquid, measure protein content with protein determination kit (building up bio-engineering research institute) according to micro-lowry method, measure MDA content according to the specification sheets of MDA test kit (building up bio-engineering research institute) measuring method available from Nanjing available from Nanjing.Test-results explanation, Phycocyanins, C-can significantly reduce MDA level (table 3) in the SRA01/04 cell of Sodium Selenite damage.

Table 3 Phycocyanins, C-is to the influence of the hLEC cell MDA level of Sodium Selenite damage

Compare with model group, ^*P＜0.05, ^*P＜0.05

Embodiment 5

Phycocyanins, C-is to the apoptotic restraining effect of Sodium Selenite inductive SRA01/04

With the SRA01/04 cell inoculation of logarithmic phase in 6 orifice plates, every hole 2ml.After treating cell attachment, it is 5 μ mol/L that each test holes adding Sodium Selenite makes final concentration, and the group of administration simultaneously adds different concns Phycocyanins, C-600 μ g/ml respectively, 300 μ g/ml, 150 μ g/ml, and positive drug pirfenoxone group is set, administration concentration 5.3 μ g/ml, cultivate after the 24h, abandon the substratum supernatant, with 0.25% trypsin 0.02%EDTA) peptic cell, 2mL PBS blows down cell, and, be loaded on 6 1.5mLEP pipes respectively with behind the PBS washed cell 2 times.Specification sheets according to cytogene DNA extraction test kit (purchasing in Shanghai Jierui Biology Engineering Co., Ltd) extracts genomic dna.Get 20 μ l reaction product, 2% (w/v) agarose gel electrophoresis detects.Ultraviolet lamp is observed the DNA electrophoretic band down and is taken pictures.

Test-results (Fig. 2) as seen, DNA ladder appears in model group, 150 μ g/ml Phycocyanins, C-groups are only seen a small amount of conditions of streaking, 300 μ g/ml Phycocyanins, C-groups are suitable with positive group effect, 600 μ g/ml Phycocyanins, C-groups and normal group do not have significant difference.Therefore, Phycocyanins, C-can significantly suppress the apoptosis of Sodium Selenite inductive lens epithelial cells.

Claims (2)

1. a kind of photopigment albumen of catching of algae is meant Phycocyanins, C-(phycocyanin), derives from blue-green algae (Cyanophyta), red algae (Rhodophyta) or latent algae (Cryptophyta).

2. the described Phycocyanins, C-of claim 1 is characterized in that being used to prepare the medicine of protection lens epithelial cells damage.

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