CN103006706A - 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 the 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 the large number of biological active component, mainly comprises various collagens and laminin，LN.In addition, also in amniotic membrane, find multiple somatomedin and a large amount of enzymes, such as epidermal growth factor, basic fibroblast growth factor, neurotrophic factor, matrix metalloproteinase and other biological organized enzyme.Therefore, amniotic membrane has the promotion Corneal epithelialization, and inflammation-inhibiting and new vessels alleviate fibrosis, reduces synulotic effect.Transplantation of amniotic membrane is widely used in clinical that Acute Chemical is hindered and thermal burn, corneal ulcer, and pterygium is in the treatment that the eye table of the diseases such as the damaged and cicatrization of conjunctiva is rebuild.
The amnion transplantation operation need to invest the eye table with the tight suture plaster of amniotic membrane with 9～10-0 suture, this operation is a kind of wound operation that has, because the destruction of suture needle and the stimulation of suture, have certain operation risk and complication: operation can cause the tissue injury at healthy position, suture stimulates and causes cicatrization, amniotic membrane dissolving biochemical action effect can't be lasting, serious symptom chemical injury plant bed is limited can't to 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 the suture stimulation causes sheds tears, and can't open eyes etc.And commercialization amniotic membrane somewhat expensive, patient's operation compliance is relatively poor.Therefore, the amniotic membrane treatment needs to seek 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 the cesarean Placenta Hominis, separate amniotic membrane after cleaning, after the liquid nitrogen grinding, volume ratio 1:5-10 adds medical saline by weight, and homogenate gets homogenate; Homogenate was placed 1-7 days at 0-4 ℃, 100-120 ℃ of heating 30-60 minute, filtered, and got filtrate; Regulate filtrate pH value to 3, leave standstill, filter, get filtrate; Regulate filtrate pH value to 7.2, the 0.20-0.22um membrane filtration is got filtrate under the aseptic condition, is the 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 or its salt 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. one cross property medicine peak concentration behind the topical, duration of efficacy is short.3. owing 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 the medicine transmission efficiency lower.5 amniotic membrane active component are by enzymatic degradation in the conjunctival sac.The higher phenomenon that then just descends rapidly of initial period tear Chinese medicine concentration not only easily causes potential risk of toxicity after the administration, 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 purpose of this invention is to provide a kind of amniotic membrane liposome of rebuilding for the eye table and its preparation method and application, preparation method of the present invention is easy rationally, and the amniotic membrane liposome for 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 purpose, the technical solution adopted in the present invention is:
At first, the present invention proposes a kind of preparation method of the amniotic membrane liposome of rebuilding for the 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 round-bottomed flask, use ether dissolution, reduction vaporization is removed ether on Rotary Evaporators, makes at inwall and forms a thin film, adds the amniotic membrane active liquid, add bead number piece, jolting is stirred, and makes the complete solution-off of adherent lipid film, after the supersound process, filter embedding, and get final product.
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 the trousers aseptic condition, with the normal saline solution flushing Placenta Hominis 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 for several times, until remove bloodstain, Placenta Hominis is dipped in the mentioned solution goes out amniotic membrane with medical calm blunt separation from chorion; After separating cuts into pieces amniotic membrane with doctor blade, and volume ratio 1:5-10 adds PBS liquid (concentration 0.01mol/L, PH7.4) by weight, under the aseptic condition, grinds amniotic membrane, and the refiner homogenized is with the unnecessary protein of Ultrasound Instrument extraction; Centrifugal, get supernatant, add vitamin C and ethyl hydroxybenzoate, mix and get final product.
Wherein, preferred, 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 in the described normal saline solution.
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 and 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 contain the phosphatidyl choline to be not less than 45% Ovum Gallus domesticus Flavus lecithin or soybean phospholipid, 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 are 3-5 times of ether volume.
The liposome prescription is in a specific embodiment of the present invention: 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 by weighing respectively recipe quantity, put in the round-bottomed flask, use the 2.5ml ether dissolution, 30 ℃ of reduction vaporizations are removed ether on Rotary Evaporators, make at inwall and form a thin film, add 10ml amniotic membrane active liquid, add diameter 3mm bead number piece, jolting was stirred 5 minutes, made the complete solution-off of adherent lipid film, ultrasonic (100w) 5 minutes.0.2 μ m Merlon microporous filter membrane press filtration is 2 times under the aseptic condition.Get the 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 the aseptic condition.
Secondly, the invention allows for a kind of amniotic membrane liposome for the reconstruction of eye table that is prepared by above each described method.
At last, the invention allows for the application of described amniotic membrane liposome in the medicine of rebuilding for the preparation of the 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 behind the treatment of pterygium, the reparation of wound surface behind the conjunctiva large tracts of land Tumor resection, eyelid bulbar conjunctiva wound repair after symblepharon is separated, 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, has following advantage:
Compare with amnion transplantation operation, the complication that the amniotic membrane liposome has effectively avoided operation to bring is such as the suture stimulation etc.; Because liposome viscosity is low, can use the eye drop form administration, and as the drug depot drug loosed time, effectively solve the of short duration problem of effect that the rear amniotic membrane dissolving of amnion transplantation operation displacement comes off and causes.
Compare liposome with eye drop and homogenate as a kind of novel eye medicinal carrier, have 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.The liposome particle diameter is to splash into the sense of eye foreign between the 0.02-5 μ m time, does not affect the normal physiological function of eyes.Liposome and eye table organization, conjunctiva cornea and sclera have stronger affinity, and bioavailability is high.Disperse rapidly behind the eye drip, strengthen medicine to the penetrance of eye table organization.And easily merge with biomembrane, the promotion medicine is to biomembranous permeability, thus in the amniotic membrane liposome amniotic membrane active component to stride the cornea transport efficacy higher.Liposome has good biocompatibility, can protect the amniotic membrane active substance to avoid the destruction of metabolic enzyme in tear and the corneal epithelium.The amniotic membrane liposome can slowly discharge medicine, thereby reduces the fluctuation of ocular drug concentration, and duration of efficacy is long.In addition, the phospholipid composition that consists of liposome is nontoxic, and non-immunogenicity is safe and reliable, can reduce adverse effect.The amniotic membrane liposome has slow-releasing, targeting, cellular affinity and histocompatibility, and to the protective effect of the amniotic membrane effective ingredient sealed.Technique is reasonable, prepares easyly, evident in efficacy, and Ocular irritation is little, is suitable for clinical application.
Description of drawings
Fig. 1 is the cornea rebirth blood vessel area statistics result of matched group and experimental group.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of 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 make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
1 one kinds of preparations that are used for the amniotic membrane liposome of eye table reconstruction 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 the trousers aseptic condition, with normal saline (0.9%) the solution flushing Placenta Hominis 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 for several times, until remove bloodstain.Placenta Hominis is dipped in the mentioned solution goes out amniotic membrane with medical calm blunt separation from chorion.After separating cuts into pieces amniotic membrane with doctor blade, and weigh (10g), volume ratio 1:10 adds PBS liquid (100ml) by weight.Under the aseptic condition, grind amniotic membrane, refiner 8000r/min homogenate 2 minutes extracts unnecessary protein 10 minutes with Ultrasound Instrument (10w).Centrifugal 10 minutes of centrifuge 6500r/min gets supernatant, adds Vc and ethyl hydroxybenzoate and makes its final concentration reach respectively 0.5g/L Vc and 0.15g/L ethyl hydroxybenzoate, is mixed with into the amniotic membrane active liquid.
2, liposome prescription: 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 by weighing respectively recipe quantity, put in the round-bottomed flask, use the 2.5ml ether dissolution, 30 ℃ of reduction vaporizations are removed ether on Rotary Evaporators, make at inwall and form a thin film, add 10ml amniotic membrane active liquid, add diameter 3mm bead number piece, jolting was stirred 5 minutes, made the complete solution-off of adherent lipid film, ultrasonic (100w) 5 minutes.0.2 μ m Merlon microporous filter membrane press filtration is 2 times under the aseptic condition.Get the filtrate embedding, 0-4 ℃ of preservation.
2 one kinds of preparations that are used for the amniotic membrane liposome of eye table reconstruction 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 the trousers aseptic condition, with normal saline (0.9%) the solution flushing Placenta Hominis 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 for several times, until remove bloodstain.Placenta Hominis is dipped in the mentioned solution goes out amniotic membrane with medical calm blunt separation from chorion.After separating cuts into pieces amniotic membrane with doctor blade, and weigh (10g), volume ratio 1:5 adds PBS liquid (50ml) by weight.Under the aseptic condition, grind amniotic membrane, refiner 5000r/min homogenate 5 minutes extracts unnecessary protein 10 minutes with Ultrasound Instrument (10w).Centrifugal 10 minutes of centrifuge 7000r/min gets supernatant, adds Vc and ethyl hydroxybenzoate and makes its final concentration reach respectively 2.0g/L Vc and 0.3g/L ethyl hydroxybenzoate, is mixed with into the amniotic membrane active liquid.
2, liposome prescription: 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 by weighing respectively recipe quantity, put in the round-bottomed flask, use the 4.2ml ether dissolution, 30 ℃ of reduction vaporizations are removed ether on Rotary Evaporators, make at inwall and form a thin film, add 15ml amniotic membrane active liquid, add diameter 3mm bead number piece, jolting was stirred 5 minutes, made the complete solution-off of adherent lipid film, ultrasonic (100w) 5 minutes.0.2 μ m Merlon microporous filter membrane press filtration is 2 times under the aseptic condition.Get the filtrate embedding, 0-4 ℃ of preservation.
3 one kinds of preparations that are used for the amniotic membrane liposome of eye table reconstruction 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 the trousers aseptic condition, with normal saline solution (0.9%) the flushing Placenta Hominis 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 for several times, until remove bloodstain.Placenta Hominis is dipped in the mentioned solution goes out amniotic membrane with medical calm blunt separation from chorion.After separating cuts into pieces amniotic membrane with doctor blade, and weigh (10g), volume ratio 1:10 adds PBS liquid (100ml) by weight.Under the aseptic condition, grind amniotic membrane, refiner 8000r/min homogenate 2 minutes extracts unnecessary protein 10 minutes with Ultrasound Instrument (10w).Centrifugal 10 minutes of centrifuge 6500r/min gets supernatant, and it is 1.0g/L that adding Vc makes its final concentration, and it is 0.2g/L that ethyl hydroxybenzoate makes its final concentration, is prepared into the amniotic membrane active liquid.
2, liposome prescription: 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 by weighing respectively recipe quantity, put in the round-bottomed flask, use the 1.8ml ether dissolution, 30 ℃ of reduction vaporizations are removed ether on Rotary Evaporators, make at inwall and form a thin film, add 9ml amniotic membrane active liquid, add diameter 3mm bead number piece, jolting was stirred 5 minutes, made the complete solution-off of adherent lipid film, ultrasonic (100w) 5 minutes.0.2 μ m Merlon microporous filter membrane press filtration is 2 times under the aseptic condition.Get the filtrate embedding, 0-4 ℃ of preservation.
The application of test example 1 amniotic membrane liposome of the present invention in the 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 the 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.Concrete experimentation is as follows:
Experiment one
1 animal and grouping
Get 20 of standardization cleaning level healthy adult SD rats, male and female are not limit, and body weight 200-250g, examination with slitlamp microscope take right eye as object of study, are divided into 2 groups: matched group and experimental group without oculopathy at random.
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 behind the 20s, places that 1s dips in unnecessary alkali liquor on the dry filter paper, evenly be attached to the cornea of right eye central surface, keep 40s after, use rapidly the 10ml normal saline flushing.
3 methods
Control rats (n=10) uses the sodium hydroxide of 1mol/L to induce alkali burn, 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 the impaired cornea of slit lamp microscope assessment, observe after the 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 the limbus of corneae tangent line is as the criterion) and 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 use SPSS13.0 carries out statistical analysis, and P＜0.05 has statistical significance.
Experiment two
The Draize test: Ocular irritation is tested to examine or check by the Draize that slightly revises.8 white rabbits are divided into 2 groups, drip respectively amniotic membrane liposome or phosphate buffer 0.05mL in the conjunctiva of right eye capsule, and the eyelid 3 seconds of gently sleeping makes medicine evenly distribute at anterior corneal surface.After administration 1,2,4,12, with slit lamp microscope eye was checked in 24,48 and 72 hours, determine whether toxic reaction of cornea, conjunctiva and anterior chamber.At 24h and 72h with 2% fluorescein sodium chromoscopy epithelial damage.The each situations such as muddy congestion and edema record Ocular irritation integration of observing according to cornea, conjunctiva and iris, the stimulation degree of judgement tested material.0-3.9 nonirritant, the slight zest of 4-8.9,9-12.9 moderate zest, 13-16 severe zest.Eye irritant reaction standards of grading see Table 1.
Table 1Draize Ocular irritation test scores table
The result
Experiment one
1 apparent examining: visible matched group eye ciliary congestion under the slit lamp microscope, the obvious dilatation and congestion of limbus of corneae blood vessel, corneal opacity edema, the epithelium limitation is damaged, the alkali burn zone 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.The experimental group corneal epithelium is without obviously damaged, and corneal opacity degree is light than matched group, and the limbus of corneae new vessels is relatively static, and hypertrophy is not obvious, without invading cornea central authorities trend.
2 cornea rebirth blood vessel areas as shown in Figure 1, experimental group cornea rebirth blood vessel area is less than matched group (P＜0.05)
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, the amniotic membrane liposome is to the eye nonirritant.
Corneal epithelial defect is not observed in the dyeing of 2% fluorescein sodium.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
Studies show that, the amniotic membrane liposome can be kept favourable biological effect in Corneal inflammation sexual trauma healing in vivo, is conducive to the eye table and rebuilds, and can promote cornea associated with epithelial healing after the alkali burn, reduces the corneal opacity and new vessels, and is little to eye table organization zest.
Claims (10)
1. a preparation method that is used for the amniotic membrane liposome of eye table reconstruction 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 round-bottomed flask, use ether dissolution, reduction vaporization is removed ether on Rotary Evaporators, makes at inwall and forms a thin film, adds the amniotic membrane active liquid, add bead number piece, jolting is stirred, and makes the complete solution-off of adherent lipid film, after the supersound process, filter embedding, and get final product.
2. preparation method as claimed in claim 1 is characterized in that 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 the trousers aseptic condition, with the normal saline solution flushing Placenta Hominis 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 for several times, until remove bloodstain, Placenta Hominis is dipped in the mentioned solution goes out amniotic membrane with medical calm blunt separation from chorion; After separating cuts into pieces amniotic membrane with doctor blade, and volume ratio 1:5-10 adds concentration 0.01mol/L by weight, and PH7.4PBS liquid under the aseptic condition, grinds amniotic membrane, and the refiner homogenized is with the unnecessary protein of Ultrasound Instrument extraction; Centrifugal, get supernatant, add vitamin C and ethyl hydroxybenzoate, mix and get final product.
3. preparation method as claimed in claim 2 is characterized in that containing in the described normal saline solution 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.
4. preparation method as claimed in claim 2 is characterized in that volume ratio 1:10 adds PBS liquid by weight.
5. preparation method as claimed in claim 2 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.
6. preparation method as claimed in claim 2 is characterized in that, adds vitamin C and ethyl hydroxybenzoate and makes its final concentration be respectively 0.5-2g/L and 0.15-0.3g/L.
7. preparation method as claimed in claim 1, it is characterized in that described phospholipid is to contain the phosphatidyl choline to be not less than 45% Ovum Gallus domesticus Flavus lecithin or soybean phospholipid, 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 are 3-5 times of ether volume.
8. 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 the aseptic condition.
9. a kind of amniotic membrane liposome for the reconstruction of eye table that is prepared by each described method of claim 1-8.
10. the application of amniotic membrane liposome claimed in claim 9 in the medicine of rebuilding for the preparation of the eye table.
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| CN106456675A (en) * | 2014-06-03 | 2017-02-22 | 组织技术公司 | Compositions and methods |
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| CN117530961A (en) * | 2023-12-21 | 2024-02-09 | 广州瑞泰生物科技有限公司 | Preparation method and application of amniotic membrane extract |
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| CN106456675A (en) * | 2014-06-03 | 2017-02-22 | 组织技术公司 | Compositions and methods |
| CN117070443A (en) * | 2023-10-18 | 2023-11-17 | 广州正源生物技术有限公司 | Separation method of human amniotic epithelial cells |
| 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 |
| CN117530961A (en) * | 2023-12-21 | 2024-02-09 | 广州瑞泰生物科技有限公司 | Preparation method and application of amniotic membrane extract |
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