CN109444395A - A kind of living body on-spot study inducing tissue regeneration mechanism platform and its application method - Google Patents
A kind of living body on-spot study inducing tissue regeneration mechanism platform and its application method Download PDFInfo
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Abstract
A kind of living body on-spot study inducing tissue regeneration mechanism platform and its application method, the platform includes the cornea of rats of living body, the region of corneal epithelial cell is eliminated on the cornea of rats with one, cornea of rats hypothallus in the region has the surface for being sliced and being formed inwardly from the cutting mouth of setting separation but still the cornea of rats hypothallus to link together plants piece and cornea of rats hypothallus plant bed, tissue engineering material to be studied is placed in the cornea of rats hypothallus and plants in the chamber formed between piece and the separation surface of the cornea of rats hypothallus plant bed, the cornea of rats hypothallus plants piece and is connected at the cutting mouth by connecting material with the cornea of rats hypothallus plant bed.The platform can be observed by intravital microscope, and histotomy observation and molecular biological analysis can preferably realize the observation and assessment of repercussion effect between tissue engineering material and body.
Description
Technical field
The present invention relates to field of tissue engineering technology, more particularly to a kind of living body on-spot study inducing tissue regeneration mechanism platform
And its application method.
Background technique
Keratonosus is the second largest invertibity, the blinding eye disease of China or even the whole world after cataract, and corneal transplantation is
Clinically unique reliable, means of effectively recovering lost eyesight.Corneal donor serious plaque in China's is weary at present, although corneal graft is by full generation
Boundary accepts extensively, but wherein specific regeneration mechanism is not known also, exploitation and cornea regeneration for tissue engineering comea
Repair mechanisms research is today's society urgent need to solve the problem.
Rat is easy to raise since its growth cycle is fast, and genetic background understands, is good experimental animal selection.Rat
Cornea is since it is with Immune privilege characteristic, and corneal thickness is suitable for, and is suitble to make living body on-spot study inducing tissue regeneration machine
Platform.Cornea has transparent characteristic, it is directly observed with slit-lamp or ophthalmic operating microscope, observes
Its revascularization and cicatrization situation.Cutting for cornea is also very mature with cornea RNA extractive technique, can be used for analysis group
The case where knitting the variation and gene expression of morphosis in regenerative process.
Existing tissue engineering material mostly in skin, studied, rare using cornea as a kind of work by the positions such as bone
Body on-spot study inducing tissue regeneration mechanism platform.Since bone belongs to body interior structure, if wanting continuous observation inside it
Situation needs it is larger to injure experimental animal Test sites incision of skin.And skin does not have transparent characteristic, so that seeing
It is difficult to examine pathologic condition inside skin histology.Cornea belongs to body external structure, is a kind of relatively good with transparent characteristic
Living body on-spot study inducing tissue regeneration mechanism platform.
Summary of the invention
It is a primary object of the present invention to overcome the deficiencies of the prior art and provide a kind of living body on-spot study induced tissue again
Life platform and its application method.
To achieve the above object, the invention adopts the following technical scheme:
A kind of living body on-spot study inducing tissue regeneration mechanism platform, the cornea of rats including living body, the cornea of rats
The region of corneal epithelial cell is above eliminated with one, the cornea of rats hypothallus in the region, which has, passes through cutting from setting
Mouth is divided to be sliced inwardly and the surface separation formed but still the cornea of rats hypothallus plant piece to link together and rat
Corneal stroma plant bed, tissue engineering material to be studied are placed in the cornea of rats hypothallus and plant piece and the cornea of rats
In the chamber formed between the separation surface of hypothallus plant bed, the cornea of rats hypothallus plants piece and the cornea of rats matrix
Layer plant bed is connected at the cutting mouth by connecting material.
Further:
The connecting material is ophthalmologic operation suture or biogum.
The region for eliminating corneal epithelial cell is border circular areas.
Cornea of rats hypothallus in the region for eliminating corneal epithelial cell has one at the cutting mouth
Perpendicular to the notch that the surface of cornea of rats hypothallus extends downwardly, the notch, which is formed, enters cornea of rats base for cutting instrument
The channel of matter layer.
The depth of the notch is 25-35 μm.
The depth of the notch is 30 μm.
The region for eliminating corneal epithelial cell is located at the central area of the cornea of rats.
A method of using the living body on-spot study inducing tissue regeneration mechanism platform, passing through slit-lamp or eye
Section's surgical operation microscope observe the cornea of rats of the living body on-spot study inducing tissue regeneration mechanism platform new vessels and
Cloudiness, and cornea of rats thickness is observed by OCT, the tissue engineering material studied with comprehensive assessment is big for inducing
The regenerated effect of mouse cornea tissue.
A method of using the living body on-spot study inducing tissue regeneration mechanism platform effect, passing through paraffin section
Or frozen section, by the cornea of rats of living body on-spot study inducing tissue regeneration mechanism platform slice carry out HE dyeing or
Person's immunofluorescence dyeing observes its cell distribution and institutional framework, and the tissue engineering material studied with comprehensive assessment is for luring
Lead the effect of cornea of rats regeneration.
A method of using the living body on-spot study inducing tissue regeneration mechanism platform effect, passing through the living body
The extraction of the cornea of rats tissue RNA of on-spot study inducing tissue regeneration mechanism platform carries out qPCR analysis, analyzing rat cornea
The expression conditions of tissue, the tissue engineering material studied with comprehensive assessment is to regenerated for induced rat cornea tissue
Effect.
The invention has the following beneficial effects:
The present invention provides a kind of living body on-spot study inducing tissue regeneration mechanism platform and its application methods, which can
To be observed by intravital microscope, histotomy observation and molecular biological analysis can preferably realize organizational project material
The observation and assessment of repercussion effect between material and body.
Compared with prior art, the invention has the following advantages that
The platform can preferably study the mutual of tissue engineering material and body due to its characteristic with Immune privilege
Effect, avoids the occurrence of immunological rejection phenomena and transplantation experiments is caused to fail.Simultaneously as cornea is a kind of transparent, external body
Body structure can easily observe it.Extraction to the slice and RNA of cornea, can analyze cornea tissue again
Raw repairing effect and expression conditions.
This platform realizes living body on-spot study inducing tissue regeneration mechanism environment construction, can be used for studying different materials use
Repelled the size influenced by immunity of organism in the effect and different materials of regeneration and restoration.
Detailed description of the invention
Fig. 1 is living body of embodiment of the present invention on-spot study inducing tissue regeneration mechanism platform sectional view.
Fig. 2 is living body of embodiment of the present invention on-spot study inducing tissue regeneration mechanism platform details display diagram.
Fig. 3 is living body of embodiment of the present invention on-spot study inducing tissue regeneration mechanism platform full three-dimensional plot.
Fig. 4 to fig. 6 is production living body of embodiment of the present invention on-spot study inducing tissue regeneration mechanism platform process demonstration graph.
Specific embodiment
It elaborates below to embodiments of the present invention.It is emphasized that following the description is only exemplary,
The range and its application being not intended to be limiting of the invention.
Refering to fig. 1 to Fig. 6, in one embodiment, a kind of living body on-spot study inducing tissue regeneration mechanism platform, including
The cornea of rats of living body eliminates the region of corneal epithelial cell 1 on the cornea of rats with one, big in the region
Mouse corneal stroma has the surface for being sliced and being formed inwardly from the cutting mouth of setting separation but still is connected to one
The cornea of rats hypothallus risen plants piece 5 and cornea of rats hypothallus plant bed 4, and tissue engineering material 6 to be studied is placed in described
Cornea of rats hypothallus is planted in the chamber formed between piece 5 and the separation surface 3 of the cornea of rats hypothallus plant bed 4, described
Cornea of rats hypothallus plants piece 5 and is connected at the cutting mouth by connecting material 7 with the cornea of rats hypothallus plant bed 4.
In a preferred embodiment, the connecting material 7 is ophthalmologic operation suture or biogum.
In a preferred embodiment, the region for eliminating corneal epithelial cell 1 is border circular areas.
In a preferred embodiment, the cornea of rats hypothallus in the region for eliminating corneal epithelial cell 1 is in institute
Stating has a notch 2 extended downwardly perpendicular to the surface of cornea of rats hypothallus at cutting mouth, the notch 2 is formed for cutting
The channel for dividing instrument to enter cornea of rats hypothallus.
In a preferred embodiment, the depth of the notch is 25-35 μm.
In a more preferred embodiment, the depth of the notch is 30 μm.
In a preferred embodiment, the region for eliminating corneal epithelial cell is located at the center of the cornea of rats
Domain.
In another embodiment, a kind of to use living body on-spot study inducing tissue regeneration described in aforementioned any embodiment
The method of mechanism platform observes the living body on-spot study inducing tissue regeneration machine by slit-lamp or ophthalmic operating microscope
The new vessels and cloudiness of the cornea of rats of platform, and cornea of rats thickness is observed by OCT, with comprehensive assessment
The tissue engineering material studied effect regenerated for induced rat cornea tissue.
In another embodiment, a kind of use states living body on-spot study inducing tissue regeneration machine described in any embodiment
The method of platform effect, by paraffin section or frozen section, by the living body on-spot study inducing tissue regeneration mechanism
The cornea of rats slice of platform carries out HE dyeing or immunofluorescence dyeing, its cell distribution and institutional framework is observed, with synthesis
Assess studied tissue engineering material effect regenerated for induced rat cornea tissue.
In another embodiment, a kind of use states living body on-spot study inducing tissue regeneration machine described in any embodiment
The method of platform effect passes through the cornea of rats tissue RNA's of the living body on-spot study inducing tissue regeneration mechanism platform
It extracts, carries out qPCR analysis, the expression conditions of analyzing rat cornea tissue, the organizational project material studied with comprehensive assessment
Material is to effect regenerated for induced rat cornea tissue.
The present invention provides a kind of living body on-spot study inducing tissue regeneration mechanism platform and its application methods, which can
To be observed by intravital microscope, histotomy observation and molecular biological analysis can preferably realize organizational project material
The observation and assessment of repercussion effect between material and body.
Specific embodiments of the present invention are further described below in conjunction with attached drawing.
Refering to fig. 1 to Fig. 6, in one embodiment, a kind of living body on-spot study inducing tissue regeneration mechanism platform, including
Cornea of rats, the building of at least one chamber on the cornea of rats, cornea of rats is there are five structure, the corneal epithelium not struck off
The notch 2 of 1,30 μm of depth of cell, the cornea of rats hypothallus boundary line 3 of horizontal separation, cornea of rats hypothallus plant bed 4, rat
Corneal stroma plants piece 5.Cornea of rats hypothallus plant bed 4 and cornea of rats hypothallus are planted to be studied for carrying between piece 5
Tissue engineering material.The chamber is closed by 10-0 ophthalmologic operation suture or biogum, reuses 5-0 ophthalmologic operation
Suture sutures eyelid, and rat is avoided to scratch art eye, and eyedrops is added dropwise daily, smears eye ointment, avoids inflammatory reaction.
The intraperitoneal injection of 10% chloraldurate solution of 1.5ml can be used, rat is anaesthetized, and tetracaine hydrochloride eye drip is used
Liquid carries out ocular anesthesia, creates the notch 2 of 30 μm of depths on cornea of rats using 3.0 ㎜ jewel stab knives, uses 2.8 ㎜ jewels
Tunnel blade horizontal separation cornea of rats hypothallus is carried out the suture of hypothallus using Ai Erkang 10-0 ophthalmic sutures, uses Ling Qiao
5-0 ophthalmic sutures carry out the suture of eyelid, must different (tobramycin dexamethasone eye drops) and erythromycin ophthalmic ointment progress using allusion quotation
Postoperative nursing, to construct complete living body on-spot study inducing tissue regeneration mechanism platform.
Fig. 4 show the foundation structure schematic diagram of living body on-spot study inducing tissue regeneration mechanism platform, cornea of rats warp
Conventional transdermal disinfection, ANER DIAN type III thimerosal wipe 3 minutes, and tetracaine hydrochloride eye drip is added dropwise in 75% alcohol disinfecting 3 minutes
After liquid 1 minute, the use of diameter is that 2 ㎜ trepans press next clearly marking on cornea of rats, reuses No. 10 round end blades
By this region, corneal epithelial cell is struck off, and creates 30 μm of depths in cornea of rats hypothallus using 3.0 ㎜ jewel stab knives
Notch 2, reuse 2.8 ㎜ jewel tunnel blades and cornea of rats hypothallus separated from the position of 30 μm of depths, obtain
Cornea of rats hypothallus plant bed 4 and cornea of rats hypothallus vegetation 5.Fig. 5 show living body on-spot study inducing tissue regeneration machine
The tissue engineering material of platform transplants schematic diagram, drills through tissue engineering material using 1.5 ㎜ trepans, with tweezers by cornea of rats
Hypothallus vegetation 5 is opened, then is placed on diameter on plant bed for 1.5 ㎜ tissue engineering materials with iris repositor.Fig. 6 is shown
The tissue engineering material of living body on-spot study inducing tissue regeneration mechanism platform transplants rear enclosed figure, uses Ai Erkang 10-0 ophthalmology
Suture sutures cornea of rats hypothallus plant bed 4 and cornea of rats hypothallus vegetation 5, and air is discharged as far as possible in sewing process,
In order to avoid there is bubble in cornea of rats hypothallus.Clever bridge 5-0 ophthalmic sutures carry out the suture of eyelid, so as not to rat due to pain or
Itch scratches art eye, and eyelid suture is opened after 2 days.Postoperative dropwise addition be added dropwise allusion quotation must different (tobramycin dexamethasone eye drops) it is daily
3 times, until changing into after a week 2 times a day, erythromycin ophthalmic ointment or other anti-infective eye ointment each 1 time sooner or later are smeared, until after a week
Change daily 1 into.
Living body on-spot study inducing tissue regeneration mechanism platform can be according to arrangement, in slit-lamp or ophthalmic operating microscope
Lower observation vascularization of cornea degree and cloudiness observe cornea of rats thickness by OCT, judge oedema situation.
The cornea of rats is fixed in 4% poly methanol solution after can removing, and is cut by paraffin section or frost
Cornea of rats is sliced and carries out HE dyeing or immunofluorescence dyeing, observes its cell distribution and institutional framework by piece.
The cornea of rats carries out qPCR analysis, the gene table of analyzing rat cornea tissue by organizing the extraction of RNA
Up to situation, comprehensive assessment research material effect regenerated for induced rat cornea tissue.
The advantages that rat has growth cycle fast, is easy to raise, and genetic background understands, cornea of rats have Immune privilege special
Property, and corneal thickness is suitable for that can be good at carrying tissue engineering material.Cornea of rats has the transparency, so that external see
It is easy to examine.The application of Protocols in Molecular Biology has very the research of the effect and regeneration mechanism of research tissue engineering material
Big help.
The above content is combine it is specific/further detailed description of the invention for preferred embodiment, cannot recognize
Fixed specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs,
Without departing from the inventive concept of the premise, some replacements or modifications can also be made to the embodiment that these have been described,
And these substitutions or variant all shall be regarded as belonging to protection scope of the present invention.
Claims (10)
1. a kind of living body on-spot study inducing tissue regeneration mechanism platform, which is characterized in that the cornea of rats including living body, it is described
The region for eliminating corneal epithelial cell with one on cornea of rats, the cornea of rats hypothallus in the region have through from
The cutting mouth of setting is sliced inwardly and the surface separation formed but still the cornea of rats hypothallus plant to link together
Piece and cornea of rats hypothallus plant bed, tissue engineering material to be studied are placed in the cornea of rats hypothallus and plant piece and described
In the chamber formed between the separation surface of cornea of rats hypothallus plant bed, the cornea of rats hypothallus plants piece and the rat
Corneal stroma plant bed is connected at the cutting mouth by connecting material.
2. living body on-spot study inducing tissue regeneration mechanism platform as described in claim 1, which is characterized in that the connection material
Material is ophthalmologic operation suture or biogum.
3. living body on-spot study inducing tissue regeneration mechanism platform as claimed in claim 1 or 2, which is characterized in that described to go
In addition to the region of corneal epithelial cell is border circular areas.
4. living body on-spot study inducing tissue regeneration mechanism platform as described in any one of claims 1 to 3, which is characterized in that
Cornea of rats hypothallus in the region for eliminating corneal epithelial cell have at the cutting mouth one perpendicular to big
The notch that the surface of mouse corneal stroma extends downwardly, the notch, which is formed, enters the logical of cornea of rats hypothallus for cutting instrument
Road.
5. living body on-spot study inducing tissue regeneration mechanism platform as claimed in claim 4, which is characterized in that the notch
Depth is 25-35 μm.
6. living body on-spot study inducing tissue regeneration mechanism platform as claimed in claim 5, which is characterized in that the notch
Depth is 30 μm.
7. living body on-spot study inducing tissue regeneration mechanism platform as claimed in any one of claims 1 to 6, which is characterized in that institute
It states and eliminates the region of corneal epithelial cell and be located at the central area of the cornea of rats.
8. a kind of side using such as the described in any item living body on-spot study inducing tissue regeneration mechanism platforms of claim 1-7
Method characterized by comprising the living body on-spot study induced tissue is observed again by slit-lamp or ophthalmic operating microscope
The new vessels and cloudiness of the cornea of rats of life platform, and cornea of rats thickness is observed by OCT, with synthesis
Assess studied tissue engineering material effect regenerated for induced rat cornea tissue.
9. a kind of using such as the described in any item living body on-spot study inducing tissue regeneration mechanism platform effects of claim 1-7
Method characterized by comprising by paraffin section or frozen section, by the living body on-spot study inducing tissue regeneration
The cornea of rats slice of mechanism platform carries out HE dyeing or immunofluorescence dyeing, observes its cell distribution and institutional framework, with
The tissue engineering material that comprehensive assessment is studied effect regenerated for induced rat cornea tissue.
10. a kind of using such as the described in any item living body on-spot study inducing tissue regeneration mechanism platform effects of claim 1-7
Method characterized by comprising pass through the cornea of rats tissue of the living body on-spot study inducing tissue regeneration mechanism platform
The extraction of RNA carries out qPCR analysis, the expression conditions of analyzing rat cornea tissue, the tissue studied with comprehensive assessment
Engineering material is to effect regenerated for induced rat cornea tissue.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1579342A (en) * | 2004-04-28 | 2005-02-16 | 浙江大学医学院附属邵逸夫医院 | Exogenous cornea substrate without cells and its preparation method and use |
| CN101433478A (en) * | 2007-11-14 | 2009-05-20 | 上海交通大学医学院附属第九人民医院 | Whole layer biological cornea as well as construction method and use thereof |
| US20100080840A1 (en) * | 2008-07-31 | 2010-04-01 | Michael Cho | Hybrid superporous hydrogel scaffold for cornea regeneration |
| CN102294053A (en) * | 2011-07-24 | 2011-12-28 | 陕西省眼科研究所 | Acellular heterogeneous corneal stroma carrier and preparation method and application thereof |
| CN107929811A (en) * | 2017-12-15 | 2018-04-20 | 厦门大学 | A kind of tissue engineering comea |
-
2018
- 2018-10-31 CN CN201811291591.7A patent/CN109444395B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1579342A (en) * | 2004-04-28 | 2005-02-16 | 浙江大学医学院附属邵逸夫医院 | Exogenous cornea substrate without cells and its preparation method and use |
| CN101433478A (en) * | 2007-11-14 | 2009-05-20 | 上海交通大学医学院附属第九人民医院 | Whole layer biological cornea as well as construction method and use thereof |
| US20100080840A1 (en) * | 2008-07-31 | 2010-04-01 | Michael Cho | Hybrid superporous hydrogel scaffold for cornea regeneration |
| CN102294053A (en) * | 2011-07-24 | 2011-12-28 | 陕西省眼科研究所 | Acellular heterogeneous corneal stroma carrier and preparation method and application thereof |
| CN107929811A (en) * | 2017-12-15 | 2018-04-20 | 厦门大学 | A kind of tissue engineering comea |
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