CN103006706B - Amniotic membrane liposome for ocular surface reconstruction and preparation method and application thereof - Google Patents
Amniotic membrane liposome for ocular surface reconstruction and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an amniotic membrane liposome for ocular surface reconstruction and a preparation method and an application thereof. The preparation method comprises the following steps: aseptically separating an amniotic membrane, and homogenizing, performing ultrasonic extraction, centrifuging, taking supernatant and the like to prepare an amniotic membrane active liquid; adding phospholipid and cholesterol, dissolving with ethyl ether, performing reduced pressure evaporation in a rotary evaporator to remove the ethyl ether, and forming a film on the inner wall; adding the amniotic membrane active liquid and a plurality of glass beads, shaking and stirring, and completely desolventizing the lipid membrane adhered to the wall; and performing ultrasonic treatment, filtering and filling. The amniotic membrane liposome belongs to a bioadhesive eye drop in the field of biological medicines and can be applied to ocular surface reconstruction of multiple ocular diseases; the amniotic membrane liposome is reasonable in process, easy and convenient to prepare and significant in therapeutic effect; the amniotic membrane liposome has a structure similar to that of a cell membrane and has relatively strong affinity with an ocular surface tissue, so that the transmembrane transport efficiency is high; the amniotic membrane liposome is very high in biocompatibility, so that the amniotic membrane liposome can protect the amniotic membrane active substance from damage by an metabolic enzyme; and the amniotic membrane liposome is low in stimulation and low in toxicity and does not affect normal physiological functions of an eye, so that the amniotic membrane liposome is applicable to clinical popularization for use.
Description
Technical field
The present invention relates to a kind of bioadhesion eye drop, be specifically related to a kind of amniotic membrane liposome of rebuilding for eye table and its preparation method and application.Belong to biomedicine field.
Background technology
Amniotic membrane is the innermost layer of fetal membrane, by forming without blood vessel and vasculolymphatic substrate, basement membrane and simple cuboidal epithelium cell.Amniotic membrane contains large number of biological active component, mainly comprises various collagen and laminin，LN.In addition, also in amniotic membrane, find multiple somatomedin and a large amount of enzymes, as epidermal growth factor, basic fibroblast growth factor, neurotrophic factor, matrix metalloproteinase and other biological organized enzyme.Therefore, amniotic membrane has promotion Corneal epithelialization, and inflammation-inhibiting and new vessels, alleviate fibrosis, reduces synulotic effect.Transplantation of amniotic membrane is widely used in Acute Chemical wound and thermal burn in clinical, corneal ulcer, and pterygium, in the treatment that the eye table of the diseases such as the damaged and cicatrization of conjunctiva is rebuild.
Amnion transplantation operation need to invest eye table by the tight suture plaster of amniotic membrane with 9～10-0 suture, this operation is a kind of wound operation that has, due to the destruction of suture needle and the stimulation of suture, there is certain operation risk and complication: operation can cause the tissue injury at healthy position, suture stimulates and causes cicatrization, amniotic membrane dissolves biochemical action effect cannot be lasting, serious symptom chemical injury plant bed is limited cannot carry out transplant operation, post-operative infection, semipermeable membrane hides vision area and affects one's power of vision, it is not attractive in appearance that outward appearance is sent out crow, and suture stimulation causes sheds tears, and cannot open eyes etc.And commercialization amniotic membrane somewhat expensive, patient's operation compliance is poor.Therefore, amniotic membrane treatment need to be found the long-acting treatment means of a kind of noinvasive.
Liu Zuguo etc. have invented a kind of amniotic membrane extracting solution (200410027760.8): get cesarean Placenta Hominis, and separated amniotic membrane after cleaning, after liquid nitrogen grinding, volume ratio 1:5-10 adds medical saline by weight, and homogenate obtains homogenate; Homogenate is placed 1-7 days at 0-4 ℃, at 100-120 ℃ of heating 30-60 minute, filters, and gets filtrate; Regulate filtrate pH value to 3, standing, filter, get filtrate; Regulate filtrate pH value to 7.2, under aseptic condition, 0.20-0.22um membrane filtration, gets filtrate, is amniotic membrane extracting solution, 0-4 ℃ of preservation.Yu Yongbin etc. have invented a kind of amnion eye drops (200910178422.7) for the treatment of corneal alkali burn: write out a prescription as in every 100ml water for injection, comprise trehalose 1-2g, Hyaluronic Acid or its salt 0.5-1.5g, heparin Huo Qiyan 4000-6000 unit, sodium chloride 0.3-0.9g, benzalkonium bromide 0.001-0.003g, vitamin E 0.05-0.15g, amniotic homogenate supernatant 1-3ul.
These ophthalmic preparations have been avoided the drawback of amnion transplantation to a certain extent, but some deficiency of these pharmaceutical dosage forms: 1. lower eye bioavailability.2. after topical, one cross property medicine peak concentration, duration of efficacy is short.3. due to the body circulation Absorption of ocular movement and nasolacrimal duct system, can cause systemic adverse reactions and make a large amount of drug loss.4 topical ophthalmic organize medicine transmission efficiency lower.5 amniotic membrane active component are by enzymatic degradation in conjunctival sac.After administration, the higher phenomenon then just declining rapidly of initial period tear Chinese medicine concentration not only easily causes potential risk of toxicity, also must frequent drug administration.In order to overcome above-mentioned deficiency, development of new amniotic membrane ocular drug transmission system seems very necessary.
Summary of the invention
The object of this invention is to provide a kind of amniotic membrane liposome of rebuilding for eye table and its preparation method and application, preparation method of the present invention is easy rationally, and the amniotic membrane liposome preparing is evident in efficacy, slow release long-acting, the little clinical application that is suitable for of Ocular irritation.
In order to reach above object, the technical solution adopted in the present invention is:
First, the present invention proposes a kind of preparation method of the amniotic membrane liposome of rebuilding for eye table, it is characterized in that by phospholipid, cholesterol, ether and amniotic membrane active liquid prepare according to following steps:
The mixture of phospholipid and cholesterol is placed in to round-bottomed flask, with ether dissolution, on Rotary Evaporators, reduction vaporization is removed ether, makes on inwall, to form a thin film, adds amniotic membrane active liquid, add bead number piece, jolting is stirred, and makes the complete solution-off of adherent lipid film, after supersound process, filter embedding, obtain.
In the present invention, preferred, described amniotic membrane active liquid prepares according to following steps:
Get nosetiology and detect virus-free hepatitis, acquired immune deficiency syndrome (AIDS), the caesarean delivered fresh human placenta of the infectious diseases such as syphilis, under trousers aseptic condition, with the normal saline solution that contains 25-75 μ g/ml penicillin sodium, 25-75 μ g/ml streptomycin sulfate, 50-150 μ g/ml polygynax and 1.5-4.0 μ g/ml amphotericin B, rinse Placenta Hominis for several times, until remove bloodstain, Placenta Hominis is dipped in above-mentioned solution and goes out amniotic membrane with medical calm blunt separation from chorion; After separating, cuts into pieces amniotic membrane by doctor blade, and volume ratio 1:5-10 adds PBS liquid (concentration 0.01mol/L, PH7.4) by weight, under aseptic condition, grinds amniotic membrane, and refiner homogenized, extracts unnecessary protein by Ultrasound Instrument; Centrifugal, get supernatant, add vitamin C and ethyl hydroxybenzoate, mix and get final product.
Wherein, preferred, in described normal saline solution, contain 50 μ g/ml penicillin sodiums, 50 μ g/ml streptomycin sulfates, 100 μ g/ml polygynax and 2.5 μ g/ml amphotericin B.
Wherein, preferred, volume ratio 1:10 adds PBS liquid by weight.
Wherein, preferred, described homogenate is with 8000r/min homogenate 2 minutes; Described ultrasonic extraction is intensity 10w, ultrasonic 10 minutes; Described centrifugal be with 6500r/min centrifugal 10 minutes.
Wherein, preferred, add vitamin C and ethyl hydroxybenzoate to make its final concentration be respectively 0.5-2g/L and 0.15-0.3g/L.
In the present invention, preferably, described phospholipid is to be not less than 45% Ovum Gallus domesticus Flavus lecithin or soybean phospholipid containing phosphatidylcholine, the weight ratio of C/PL is 1:2-2.5, the mixture of the C/PL of every 100mg 2-3ml ether dissolution, the consumption of amniotic membrane active liquid is 3-5 times of ether volume.
In a specific embodiment of the present invention, liposome formula is: soybean phospholipid 70mg; Cholesterol 30mg; Ether 2.5ml; Amniotic membrane active liquid 10ml.Its preparation method is: the soybean phospholipid 70mg and the cholesterol 30mg that take respectively recipe quantity, put in round-bottomed flask, use 2.5ml ether dissolution, on Rotary Evaporators, 30 ℃ of reduction vaporizations are removed ether, make on inwall, to form a thin film, add 10ml amniotic membrane active liquid, add diameter 3mm bead number piece, jolting is stirred 5 minutes, makes the complete solution-off of adherent lipid film, ultrasonic (100w) 5 minutes.Under aseptic condition, 0.2 μ m Merlon microporous filter membrane press filtration is 2 times.Get filtrate embedding, 0-4 ℃ of preservation.
In the present invention, preferred, described ultrasonic be intensity 100w, ultrasonic 5 minutes; Described filtration is 0.2 μ m Merlon microporous filter membrane press filtration 2 times under aseptic condition.
Secondly, the invention allows for a kind of amniotic membrane liposome of rebuilding for eye table being prepared by the method described in above any one.
Finally, the invention allows for the application of described amniotic membrane liposome in the medicine of rebuilding for the preparation of eye table.
Amniotic membrane liposome of the present invention can be applied in the treatment of eye table reconstruction, specifically comprise: the reparation of angle conjunctiva chemical injury, thermal burn, sclera wound repair after treatment of pterygium, the reparation of wound surface after conjunctiva large area Tumor resection, eyelid bulbar conjunctiva wound repair after symblepharon separation, the ulcer that the non-infective agent of cornea causes, conjunctiva is damaged, xerophthalmia, viral keratitis etc.
Amniotic membrane liposome of the present invention is compared with conventional art, and tool has the following advantages:
Compare with amnion transplantation operation, the complication that amniotic membrane liposome has effectively avoided operation to bring, as suture stimulation etc.; Because liposome viscosity is low, can use eye drop form administration, and as drug depot drug loosed time, effectively solve after amnion transplantation operation amniotic membrane and dissolve displacement and come off and draw the of short duration problem of role.
Compare liposome with eye drop and homogenate as a kind of novel eye medicinal carrier, there is multiple advantage.The sealing folliculus that liposome is comprised of one or more layers class lipid bilayer, in its bilayer or interlayer can comprise medicine, its similar cell membrane.Liposome particle diameter is to splash into the sense of eye foreign between 0.02-5 μ m time, does not affect the normal physiological function of eyes.Liposome Yu Yan table organization, conjunctiva cornea and sclera have stronger affinity, and bioavailability is high.After eye drip, disperse rapidly, strengthen the penetrance of medicine Dui Yan table organization.And easily merge with biomembrane, promotion medicine is to biomembranous permeability, therefore amniotic membrane active component higher across cornea transport efficacy in amniotic membrane liposome.Liposome has good biocompatibility, can protect amniotic membrane active substance to avoid the destruction of metabolic enzyme in tear and corneal epithelium.Amniotic membrane liposome can delay Slow release, thereby reduces the fluctuation of ocular drug concentration, and duration of efficacy is long.In addition, the phospholipid composition that forms liposome is nontoxic, and non-immunogenicity is safe and reliable, can reduce adverse effect.Amniotic membrane liposome has slow-releasing, targeting, cellular affinity and histocompatibility, and the protective effect to sealed amniotic membrane effective ingredient.Technique is reasonable, prepares easyly, evident in efficacy, and Ocular irritation is little, is suitable for clinical application.
Accompanying drawing explanation
Fig. 1 is the cornea rebirth blood vessel area statistics result of matched group and experimental group.
The specific embodiment
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection of the present invention.
The preparation of 1 one kinds of amniotic membrane liposomees of rebuilding for eye table of embodiment
1, the preparation of amniotic membrane active liquid:
Get nosetiology and detect virus-free hepatitis, acquired immune deficiency syndrome (AIDS), the caesarean delivered fresh human placenta of the infectious diseases such as syphilis, under trousers aseptic condition, with normal saline (0.9%) solution that contains 50 μ g/ml penicillin sodiums, 50 μ g/ml streptomycin sulfates, 100 μ g/ml polygynax and 2.5 μ g/ml amphotericin B, rinse Placenta Hominis for several times, until remove bloodstain.Placenta Hominis is dipped in above-mentioned solution and goes out amniotic membrane with medical calm blunt separation from chorion.After separating, cuts into pieces amniotic membrane by doctor blade, and weigh (10g), volume ratio 1:10 adds PBS liquid (100ml) by weight.Under aseptic condition, grind amniotic membrane, refiner 8000r/min homogenate 2 minutes, extracts unnecessary protein 10 minutes by Ultrasound Instrument (10w).Centrifugal 10 minutes of centrifuge 6500r/min, gets supernatant, adds Vc and ethyl hydroxybenzoate to make its final concentration reach respectively 0.5g/L Vc and 0.15g/L ethyl hydroxybenzoate, is mixed with into amniotic membrane active liquid.
2, liposome formula: soybean phospholipid 70mg; Cholesterol 30mg; Ether 2.5ml; Amniotic membrane active liquid 10ml.
Preparation method: the soybean phospholipid 70mg and the cholesterol 30mg that take respectively recipe quantity, put in round-bottomed flask, use 2.5ml ether dissolution, on Rotary Evaporators, 30 ℃ of reduction vaporizations are removed ether, make on inwall, to form a thin film, add 10ml amniotic membrane active liquid, add diameter 3mm bead number piece, jolting is stirred 5 minutes, makes the complete solution-off of adherent lipid film, ultrasonic (100w) 5 minutes.Under aseptic condition, 0.2 μ m Merlon microporous filter membrane press filtration is 2 times.Get filtrate embedding, 0-4 ℃ of preservation.
The preparation of 2 one kinds of amniotic membrane liposomees of rebuilding for eye table of embodiment
1, the preparation of amniotic membrane active liquid
Get nosetiology and detect virus-free hepatitis, acquired immune deficiency syndrome (AIDS), the caesarean delivered fresh human placenta of the infectious diseases such as syphilis, under trousers aseptic condition, with normal saline (0.9%) solution that contains 25 μ g/ml penicillin sodiums, 75 μ g/ml streptomycin sulfates, 75 μ g/ml polygynax and 4 μ g/ml amphotericin B, rinse Placenta Hominis for several times, until remove bloodstain.Placenta Hominis is dipped in above-mentioned solution and goes out amniotic membrane with medical calm blunt separation from chorion.After separating, cuts into pieces amniotic membrane by doctor blade, and weigh (10g), volume ratio 1:5 adds PBS liquid (50ml) by weight.Under aseptic condition, grind amniotic membrane, refiner 5000r/min homogenate 5 minutes, extracts unnecessary protein 10 minutes by Ultrasound Instrument (10w).Centrifugal 10 minutes of centrifuge 7000r/min, gets supernatant, adds Vc and ethyl hydroxybenzoate to make its final concentration reach respectively 2.0g/L Vc and 0.3g/L ethyl hydroxybenzoate, is mixed with into amniotic membrane active liquid.
2, liposome formula: Ovum Gallus domesticus Flavus lecithin 100mg; Cholesterol 40mg; Ether 4.2ml; Amniotic membrane active liquid 15ml.
Preparation method: the Ovum Gallus domesticus Flavus lecithin 100mg and the cholesterol 40mg that take respectively recipe quantity, put in round-bottomed flask, use 4.2ml ether dissolution, on Rotary Evaporators, 30 ℃ of reduction vaporizations are removed ether, make on inwall, to form a thin film, add 15ml amniotic membrane active liquid, add diameter 3mm bead number piece, jolting is stirred 5 minutes, makes the complete solution-off of adherent lipid film, ultrasonic (100w) 5 minutes.Under aseptic condition, 0.2 μ m Merlon microporous filter membrane press filtration is 2 times.Get filtrate embedding, 0-4 ℃ of preservation.
The preparation of 3 one kinds of amniotic membrane liposomees of rebuilding for eye table of embodiment
1, the preparation of amniotic membrane active liquid:
Get nosetiology and detect virus-free hepatitis, acquired immune deficiency syndrome (AIDS), the caesarean delivered fresh human placenta of the infectious diseases such as syphilis, under trousers aseptic condition, with the normal saline solution (0.9%) that contains 50 μ g/ml penicillin sodiums, 50 μ g/ml streptomycin sulfates, 100 μ g/ml polygynax and 2.5 μ g/ml amphotericin B, rinse Placenta Hominis for several times, until remove bloodstain.Placenta Hominis is dipped in above-mentioned solution and goes out amniotic membrane with medical calm blunt separation from chorion.After separating, cuts into pieces amniotic membrane by doctor blade, and weigh (10g), volume ratio 1:10 adds PBS liquid (100ml) by weight.Under aseptic condition, grind amniotic membrane, refiner 8000r/min homogenate 2 minutes, extracts unnecessary protein 10 minutes by Ultrasound Instrument (10w).Centrifugal 10 minutes of centrifuge 6500r/min, gets supernatant, and adding Vc to make its final concentration is 1.0g/L, and it is 0.2g/L that ethyl hydroxybenzoate makes its final concentration, is prepared into amniotic membrane active liquid.
2, liposome formula: Ovum Gallus domesticus Flavus lecithin 60mg; Cholesterol 30mg; Ether 1.8ml; Amniotic membrane active liquid 9ml.
Preparation method: the Ovum Gallus domesticus Flavus lecithin 60mg and the cholesterol 30mg that take respectively recipe quantity, put in round-bottomed flask, use 1.8ml ether dissolution, on Rotary Evaporators, 30 ℃ of reduction vaporizations are removed ether, make on inwall, to form a thin film, add 9ml amniotic membrane active liquid, add diameter 3mm bead number piece, jolting is stirred 5 minutes, makes the complete solution-off of adherent lipid film, ultrasonic (100w) 5 minutes.Under aseptic condition, 0.2 μ m Merlon microporous filter membrane press filtration is 2 times.Get filtrate embedding, 0-4 ℃ of preservation.
The application of test example 1 amniotic membrane liposome of the present invention in eye table is rebuild
Amniotic membrane liposome (embodiment 1) is applied in the animal model treatment of cornea of rats alkali burn, finds that this liposome eye drop has the eye of promotion table and rebuilds, accelerate cornea associated with epithelial healing, suppress curative effect and the effect of neovascularization resulting.And this amniotic membrane liposome eye drop Ocular irritation is little.Specific experiment process is as follows:
Experiment one
1 animal and grouping
Get 20 of the clean level of standardization healthy adult SD rats, male and female are not limit, body weight 200-250g, and examination with slitlamp microscope, without oculopathy, be take right eye as object of study, is divided at random 2 groups: matched group and experimental group.
The foundation of 2 cornea of rats model of alkali burned
With chloral hydrate 0.3ml/100g intraperitoneal injection of anesthesia SD rat, times capable topical anesthesia of promise happiness eye drop point right eye.The circular filter paper sheet of diameter 3mm is soaked in the sodium hydroxide solution of concentration 1mol/L after 20s, is placed in 1s on dry filter paper and dips in unnecessary alkali liquor, be evenly attached to cornea of right eye central surface, keep, after 40s, using rapidly 10ml normal saline flushing.
3 methods
Control rats (n=10) is used the sodium hydroxide induction alkali burn of 1mol/L, then accepts local phosphate buffer (PBS) eye dripping, every day 4 times, continues 7d.Experimental group rat (n=10) is accepted local amniotic membrane liposome eye dripping after using the sodium hydroxide of 1mol/L to induce alkali burn, every day 4 times, continues 7d.
4 morphological observations
Use slit lamp microscope to assess impaired cornea, observe after alkali burn 7 days experimental grouies and control rats eye expression condition and cornea rebirth blood vessel (CNV) growing state.Record CNV length (with continuous and flexibility is little and the new vessels length vertical with limbus of corneae tangent line is as the criterion) reference area.
CNV area computing formula: A=C/12 * 3.1416[r
2-(r-l)
2]
Wherein, C is the circumference hour number that CNV involves cornea, and l is the length that limbus of corneae stretches into cornea, and r is corneal radii 3mm.
5 application SPSS13.0 carry out statistical analysis, and P<0.05 has statistical significance.
Experiment two
Draize test: Ocular irritation is tested to examine or check by the Draize slightly revising.8 white rabbits are divided into 2 groups, drip respectively amniotic membrane liposome or phosphate buffer 0.05mL in conjunctiva of right eye capsule, and the eyelid 3 seconds of gently sleeping, makes medicine be uniformly distributed at anterior corneal surface.After administration 1,2,4,12, within 24,48 and 72 hours, with slit lamp microscope, eye is checked, determine the whether toxic reaction of cornea, conjunctiva and anterior chamber.At 24h and 2% fluorescein sodium chromoscopy epithelial damage for 72h.Each situations such as muddy congestion and edema of observing according to cornea, conjunctiva and iris record Ocular irritation integration, the stimulation degree of judgement tested material.0-3.9 nonirritant, zest that 4-8.9 is slight, 9-12.9 moderate zest, 13-16 severe zest.Eye irritant reaction standards of grading are in Table 1.
Table 1Draize Ocular irritation test scores table
Result
Experiment one
1 apparent examining: visible matched group eye ciliary congestion under slit lamp microscope, the obvious dilatation and congestion of limbus of corneae blood vessel, corneal opacity edema, epithelium limitation is damaged, alkali burn region is obvious, the vigorous transparency cornea of growing into of limbus of corneae place neovascularization growth, form even compact, part is invaded burn area, top bifurcated.Experimental group corneal epithelium is without obviously damaged, and corneal opacity degree is light compared with matched group, and limbus of corneae new vessels is relatively static, and hypertrophy is not obvious, without invading cornea central authorities trend.
As shown in Figure 1, experimental group cornea rebirth blood vessel area is less than matched group (P<0.05) to 2 cornea rebirth blood vessel areas
Experiment two
Adopt the white rabbit of amniotic membrane liposome or phosphate buffer eye drip, Draize test integration is respectively 0.75 ± 0.50,0.The integration of amniotic membrane liposome group is a little more than phosphate buffer group (p>0.05), and two groups of integrations are all less than 4 minutes.Therefore, amniotic membrane liposome is to eye nonirritant.
Corneal epithelial defect is not observed in 2% fluorescein sodium dyeing.Examination with slitlamp microscope shows that eye does not have the sign of toxicity or inflammatory reaction.Corneal transparency, conjunctiva does not have edema, and eyelid does not have ulcer.
Conclusion
Research shows, amniotic membrane liposome can maintain favourable biological effect in the healing of Corneal inflammation sexual trauma in vivo, is conducive to eye table and rebuilds, and can promote cornea associated with epithelial healing after alkali burn, reduces the corneal opacity and new vessels, and Dui Yan table organization zest is little.
Claims (9)
1. a preparation method for the amniotic membrane liposome of rebuilding for eye table, it is characterized in that by phospholipid, cholesterol, and ether and amniotic membrane active liquid prepare according to following steps:
The mixture of phospholipid and cholesterol is placed in to round-bottomed flask; with ether dissolution; on Rotary Evaporators, reduction vaporization is removed ether, makes on inwall, to form a thin film, adds amniotic membrane active liquid; add bead number piece; jolting is stirred, and makes the complete solution-off of adherent lipid film, after supersound process; filter embedding, obtain;
Wherein, described amniotic membrane active liquid prepares according to following steps:
Get nosetiology and detect virus-free hepatitis, acquired immune deficiency syndrome (AIDS), the caesarean delivered fresh human placenta of the infectious diseases such as syphilis, under trousers aseptic condition, with the normal saline solution that contains 25-75 μ g/ml penicillin sodium, 25-75 μ g/ml streptomycin sulfate, 50-150 μ g/ml polygynax and 1.5-4.0 μ g/ml amphotericin B, rinse Placenta Hominis for several times, until remove bloodstain, Placenta Hominis is dipped in above-mentioned solution and goes out amniotic membrane with medical calm blunt separation from chorion; After separating, cuts into pieces amniotic membrane by doctor blade, and volume ratio 1:5-10 adds concentration 0.01mol/L by weight, and PH7.4PBS liquid, under aseptic condition, grinds amniotic membrane, and refiner homogenized, extracts unnecessary protein by Ultrasound Instrument; Centrifugal, get supernatant, add vitamin C and ethyl hydroxybenzoate, mix and get final product.
2. preparation method as claimed in claim 1, is characterized in that containing 50 μ g/ml penicillin sodiums, 50 μ g/ml streptomycin sulfates, 100 μ g/ml polygynax and 2.5 μ g/ml amphotericin B in described normal saline solution.
3. preparation method as claimed in claim 1, is characterized in that volume ratio 1:10 adds PBS liquid by weight.
4. preparation method as claimed in claim 1, is characterized in that described homogenate is with 8000r/min homogenate 2 minutes; Described ultrasonic extraction is intensity 10w, ultrasonic 10 minutes; Described centrifugal be with 6500r/min centrifugal 10 minutes.
5. preparation method as claimed in claim 1, is characterized in that, adds vitamin C and ethyl hydroxybenzoate to make its final concentration be respectively 0.5-2g/L and 0.15-0.3g/L.
6. preparation method as claimed in claim 1, it is characterized in that described phospholipid is to be not less than 45% Ovum Gallus domesticus Flavus lecithin or soybean phospholipid containing phosphatidylcholine, the weight ratio of C/PL is 1:2-2.5, the mixture of the C/PL of every 100mg 2-3ml ether dissolution, the consumption of amniotic membrane active liquid is 3-5 times of ether volume.
7. preparation method as claimed in claim 1, it is characterized in that described ultrasonic be intensity 100w, ultrasonic 5 minutes; Described filtration is 0.2 μ m Merlon microporous filter membrane press filtration 2 times under aseptic condition.
8. a kind of amniotic membrane liposome of rebuilding for eye table being prepared by the method described in claim 1-7 any one.
9. the application of amniotic membrane liposome claimed in claim 8 in the medicine of rebuilding for the preparation of eye table.
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| CN117070443B (en) * | 2023-10-18 | 2024-02-06 | 广州正源生物技术有限公司 | Separation method of human amniotic epithelial cells |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712020A (en) * | 2004-06-23 | 2005-12-28 | 中山大学中山眼科中心 | Production of amniotic extractive liquid and use thereof |
| CN101095695A (en) * | 2007-08-10 | 2008-01-02 | 孙猛 | Liposome artificial tear eye drops |
| CN101658491A (en) * | 2009-09-24 | 2010-03-03 | 哈尔滨医科大学 | Amnion eye drops for curing cornea alkali burn |
| CN102631707A (en) * | 2012-05-04 | 2012-08-15 | 厦门大学 | Amnion-based biological material and preparation method and uses thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712020A (en) * | 2004-06-23 | 2005-12-28 | 中山大学中山眼科中心 | Production of amniotic extractive liquid and use thereof |
| CN101095695A (en) * | 2007-08-10 | 2008-01-02 | 孙猛 | Liposome artificial tear eye drops |
| CN101658491A (en) * | 2009-09-24 | 2010-03-03 | 哈尔滨医科大学 | Amnion eye drops for curing cornea alkali burn |
| CN102631707A (en) * | 2012-05-04 | 2012-08-15 | 厦门大学 | Amnion-based biological material and preparation method and uses thereof |
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