CN101638635A - Method for obtaining corneal endothelium sample cells and construction of corneal metaplax layer in vitro - Google Patents
Method for obtaining corneal endothelium sample cells and construction of corneal metaplax layer in vitro Download PDFInfo
- Publication number
- CN101638635A CN101638635A CN200810041062A CN200810041062A CN101638635A CN 101638635 A CN101638635 A CN 101638635A CN 200810041062 A CN200810041062 A CN 200810041062A CN 200810041062 A CN200810041062 A CN 200810041062A CN 101638635 A CN101638635 A CN 101638635A
- Authority
- CN
- China
- Prior art keywords
- cell
- corneal
- bepcs
- endothelial
- altogether
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for induction formation of corneal endothelium sample cells, which comprises the step of co-culturing bone marrow derived endothelial progenitor cells (BEPCs) of whichthe concentration is 2-8*10<4> cells per milliliter and corneal endothelial cells (CECs) of which the concentration is 0.6-3*10<5> cells per milliliter so that the bone marrow derived endothelial progenitor cells are differentiated to form the corneal endothelium sample cells. The invention also discloses a corneal metaplax layer constructed by the corneal endothelium sample cells obtained by themethod, a preparation method and application thereof.
Description
Technical field
The present invention relates to organizational project and field of ophthalmology, relate in particular to the method and the external structure corneal posterior lamella that obtain the corneal endothelium like cell.
Background technology
Endothelial cell has been regulated and control the transparency of cornea by pumping function and barrier function.Become the human corneal endothelial cells number to wear with factor such as inflammation and descend along with age increase, ocular injury, operation, contact lense, but can't breed in vivo and compensate loss cell, can only increase the close apposition that keeps with flanking cell by cell volume, stop aqueous humor to infiltrate matrix too much.This is that the DNA that has blocked cell is synthetic, makes it be stuck in the G1 phase, can not breed regeneration by mitotic division because there is growth inhibiting factor again in the interior somatomedin deficiency of anterior chamber in the aqueous humor simultaneously.When corneal endothelial cell densities is lower than 400-700/mm
2The time, endothelium loses compensatory, corneal edema, visual deterioration or forfeiture.Endothelium loses compensatory disease such as bullous keratopathy, Fuch ' s corneal degeneration etc. all cause corneal endothelium pathology, the corneal opacity and visual deterioration clinically.It is penetrating keratoplasty that treatment corneal endothelium function is lost compensatory operation commonly used.Although conventional corneal transplantation has obtained bigger success, but still has weak point, comprise the postoperative astigmatism, ametropia, the suture infection that gets loose, donor source shortage and postoperative immunological rejection etc.Though cornea is in the position of a premunition absolution, still exists greater than 10% patient at the up keratoplasty of avascular cornea rejection takes place.Nineteen fifty-two Stocker has set up the endothelial cell tissue culture method, Maurice in 1972 has proposed the corneal endothelial transplantation of the tissue culture imagination to the unsound cornea of endotheliocyte has been opened up this brand-new research field of corneal endothelial transplantation (coneal endothelium transplantation) first on this basis.The entocornea of Xing Qiing divested transplanted endothelial cell art (Descemet stripping endothelialkeratoplasty in recent years, DSEK), utilize the little otch of scleral tunnel, tear descemet's membrane and endodermis off, transplant allosome metaplax layer matrix and descemet's membrane endodermis again, need not to sew up, compare traditional Penetrating Keratoplasty, wound is little, and immunological rejection further reduces, it is fast, astigmatic little that Postoperative visual acuity is recovered, but still have difficult problems such as donor shortage, the aging of cornea donor.Also the cell source is not enough to be difficult to use with no suitable carriers the vitro culture corneal endothelial transplantation of hot research because of being subjected at present.Therefore, seek seed cell and suitable biomaterial structure tissue engineering comea metaplax layer and to transplant be the task of top priority.
Organizational engineering (tissue engineering) is one and combines with cytobiology and materialogy, carries out new branch of science external or construct in vitro tissue or organ.Three big key elements of organizational project comprise microenvironment in seed cell, biomaterial and the body.Its basic skills be with cultured and amplified in vitro to be adsorbed in a kind of biocompatibility from the body normal tissue cell good and can be by the biological support of body degraded and absorbed, implanting to human body tissue defect position, at timbering material progressively in the degradation process, cell continues propagation justacrine matrix, the final respective organization that forms, it is damaged to reach repair tissue, rebuilds the purpose of function.
The seed cell that is used to make up the tissue engineering comea endothelium at present has endothelial cell, endothelial precursor cell and mesenchymal stem cells MSCs (MSC).1979, Gospodarowicz etc. were inoculated in the bovine corneal inner skin cell of cultivating on the corneal film of cat, are transplanted to after the vitro culture on the cat cornea of vessel endothelium and induced endothelial again, planted sheet and kept transparent and reach 7 days.The frontier that the endothelial cell carrier is cultivated and transplanted has been opened up in this experiment.But endotheliocyte source lacks, though externally can increase limited amount.2005, Mimura etc. found that the ability of cell proliferation of corneal endothelium periphery will obviously surpass the cell at center, and may there be the corneal endothelium precursor cell of similar stem cell in supposition at nearly corneal limbus and trabecular tissue structure interface place; There is research to obtain the corneal endothelium precursor cell of stem-like cell by peripheral corneal endothelial cell mass clone cultural method again, can clone agglomerate sample growing multiplication by external one-tenth, in the feature that still all has stem cell on the form on the biological characteristics such as proliferative ability, inject that to repair the rabbit corneal endothelium behind the anterior chamber damaged.But the separation and Culture of cornea precursor cell is difficulty very.Scholar's inducing and transplanting and carried out useful exploration at adult stem cell MSC arranged.Liu Xiaowei etc. are inoculated into from body BMSCs with rabbit and are transplanted to machinery on the gelatin film and remove in the rabbit corneal of endothelial cell, postoperative 8 all oedema alleviate gradually, it is transparent that cornea recovers, and 12 weeks of postoperative are divided into similar polygonal cell gradually, but intercellular gap is bigger.Can MSC be divided into endothelial cell or unknown number on earth, and the effect after the transplanting also needs long-term observation.
Another important factor that the tissue engineering comea metaplax layer makes up is a biomaterial scaffolds, and the growth of Chang Zuowei machinery mount sustenticular cell alleviates cell response, and As time goes on, changes their chemistry and mechanical characteristics gradually.Biocompatibility is its most important characteristic, and biomaterial is more near tissue microenvironment, and biocompatibility is good more.Ideal tissue engineering comea material should meet the following conditions: (1) good biocompatibility, and keep three-dimensional space and promote normal cell growth outside the pale of civilization with branch, do not disturb other physiological processs; (2) there is not bad tissue reaction; (3) material is made simply, and can reinvent by different shapes and size; (4) implant after, material can be degraded or absorb.The carrier that is used for tissue engineering comea metaplax layer structure at present has gelatin, collagen, amnion and PLGA, descemet's membrane etc.Jumblatt etc. cultivate endotheliocyte with gelatin film and transplant.Mimura etc. are inoculated into collagen sheet with human corneal endothelial cells the rabbit that tears descemet's membrane and endodermis off are transplanted, and cornea is bleach gradually.The bad mechanical strength of gelatin and collagen, operational difficulty is easy to degradation in vivo.Ishino etc. are cultivator endothelial cell on amnion first, and density surpasses 3000/mm
2, form is similar to the normal cornea endotheliocyte, behind the implantation rabbit corneal, does not find oedema, keeps the good transparency, but only keeps for 4 weeks, and ECD descends 27% after transplanting 7 days.It is poor that its weak point is that amnion and recipient cornea matrix attach property.Hadlock etc. plant the rabbit corneal endotheliocyte on PLGA, obtain individual layer rabbit corneal endothelial layer, prove that this material can be used for cell monolayer and transplants.Though PLGA is a degradable polymer, it is controlled to have degradation process, and remoldability is good, the physical strength height, advantages such as good biocompatibility, but lack the cell recognition signal, its hydrophobicity makes cell be difficult for plantation, and the activity that the acid degradation product also can pair cell has a negative impact.Mimura T etc. are inoculated into human corneal endothelial cells on the entocornea of removing endotheliocyte, and to the capable penetrating keratoplasty of nude mice cornea, cornea recovers transparent after 1 month, but this technology still is subjected to the difficult problem that donor lacks.
In sum, this area presses for to provide and enlarges the seed cell source that is used for corneal transplantation, and also pressing for provides Biodegradable material to be used to prepare corneal graft.
Summary of the invention
The present invention aims to provide a kind of method of inducing marrow endothelial progenitor cell to be differentiated to form the corneal endothelium like cell.
Another object of the present invention provides a kind of engineered corneal posterior lamella.
A further object of the present invention provides a kind of method for preparing engineered corneal posterior lamella.
In a first aspect of the present invention, a kind of method that forms the corneal endothelium like cell of inducing is provided, described method comprises step: with 2-8 * 10
4The marrow endothelial progenitor cell of individual/ml (bone marrow derived endothelialprogenitor cells, BEPCs) and 0.6-3 * 10
5(coneal endothlialcell CECs) cultivates the endothelial cell of individual/ml altogether, makes marrow endothelial progenitor cell be differentiated to form the corneal endothelium like cell; Preferably with 3-7 * 10
4The marrow endothelial progenitor cell of individual/ml and 0.8-1.2 * 10
5The endothelial cell of individual/ml is cultivated altogether.
In another preference, employed nutrient solution is DMEM/F12 or the DMEM that contains 1-5%v/v serum in cultivating altogether; Cultivated more preferably 8-10 days altogether 7-14 days.
In another preference, described marrow endothelial progenitor cell is consubstantiality of the same race, allogeneic or heterogenous allosome marrow endothelial progenitor cell; Described endothelial cell is consubstantiality of the same race, allogeneic or heterogenous allosome endothelial cell; Between marrow endothelial progenitor cell that described isolation is cultivated altogether and the endothelial cell consubstantiality of the same race, allogeneic or heterogenous allosome.
In another preference, described marrow endothelial progenitor cell is people's marrow endothelial progenitor cell; Described endothelial cell is a human corneal endothelial cells.
In another preference, described cultivate altogether to be selected to mix cultivate altogether or isolate altogether and cultivate; More preferably isolate altogether and cultivate.
In another preference, added endothelial cell culture supernatant and fresh medium again at the 3rd day that isolates cultivation altogether, change the liquid method by half amount more two days later at interval and add endothelial cell culture supernatant and fresh medium; Described nutrient solution is DMEM/F12 or the DMEM that contains 1-5%v/v serum; Described endothelial cell culture supernatant is the culture supernatant for the endothelial cell first-generation or s-generation cell.
In another preference, described endothelial cell culture supernatant is the culture supernatant for the endothelial cell first-generation or s-generation cell, and these cells every 1-2 days liquid feedings are once cultivated and promptly be can be used as supernatant liquor after the 3rd day as cultivating altogether.
In a second aspect of the present invention, a kind of purposes of corneal endothelium like cell of aforesaid induction method acquisition is provided, described purposes is to prepare corneal graft as seed cell.
In a third aspect of the present invention, a kind of engineered corneal posterior lamella is provided, it comprises:
(a) pharmaceutically acceptable Biodegradable material; With
(b) the corneal endothelium like cell of aforesaid induction method acquisition;
Described pharmaceutically acceptable Biodegradable material be the cornea acellular matrix (cornea acellularmatrix, CACM); Preferred porcine cornea acellular matrix (porcine cornea acellular matrix, PCACM).
In another preference, the content of the corneal endothelium like cell that aforesaid induction method obtains is 1000-4000 cell/mm
2Constructed corneal posterior lamella.
In a fourth aspect of the present invention, a kind of method for preparing engineered corneal posterior lamella is provided, described method comprises step:
(1) the corneal endothelium like cell that aforesaid induction method is obtained is inoculated in cornea acellular matrix (preferred porcine cornea acellular matrix), and inoculum density is 1000-4000 cell/mm
2The cornea acellular matrix; With
(2) cultivation obtains engineered corneal posterior lamella.
In another preference, vitro culture 4-10 days, more preferably 5-7 days.
In another preference, adding volume ratio in the step (2) is 1: the endothelial cell culture supernatant of 1-3 is cultivated with the DMEM/F12 or the DMEM that contain 1-5%v/v serum.
In view of the above, the present invention has enlarged the seed cell source that is used for corneal transplantation, and a kind of corneal graft also is provided.
Description of drawings
Fig. 1 has shown that density gradient centrifugation obtains myelomonocyte.
Fig. 2 has shown the form (inverted phase contrast microscope 100 *) of the people BEPCs of vitro culture; Wherein
A: former being commissioned to train supported 4 days, clone's form ovalization whirlpool shape, and B: cultivated the 6th day, cell is short fusiformis or Polygons.
Fig. 3 has shown people BEPCs picked-up Ac-LDL and in conjunction with UEA-1 situation (fluorescent microscope); Wherein
A: the 4th day former generation clone, can absorb Dil-Ac-LDL 200 *; B:BEPCs can in conjunction with UEA-1 200 *; C:Hoechst dye nuclear 200 *; D: with Dil+UEA+Hoechst stack, positive rate reaches 100% 200 *; E: the 2nd day cell of the first-generation, positive rate reach more than 90% 100 *.
Fig. 4 shown CD133, the CD34 of people BEPCs and Flk-1 expression 200 *; Wherein
A: people BEPCs expresses CD133; B: people BEPCs expresses CD34; C: people BEPCs expresses Flk-1.Three's positive rate is all more than 95%.
Fig. 5 has shown people BEPCs and people CECs VIII Collagen Type VI (496bp) expression, and both all express the VIII Collagen Type VI.
Fig. 6 has shown that Transwell isolates training mode figure altogether.
Fig. 7 has shown the form of former generation people CECs; Wherein
A: the 13rd day former generation the tissue block edge grow CECs cell 40 *; The B:CECs form be Polygons 200 *.
Fig. 8 has shown the form (inverted microscope 200 *) of people CECs inductive people BEPCs; Wherein
A: inductive BEPCs is not short fusiformis or spindle shape; B: people CECs inductive BEPCs is Polygons; C:CECs is Polygons; D: people LECs inductive BEPCs is short fusiformis or spindle shape.
Fig. 9 has shown the form of people CECs inductive people BEPCs, is Polygons (scanning electron microscope 1000 *).
Figure 10 shown people CECs induce people BEPCs AQP1 expression 200 *; Wherein
A: inductive people BEPCs does not express AQP1; B: people CECs inductive people BEPCs great majority are expressed AQP1; C: people CECs expresses AQP1; D: people LECs inductive BEPCs does not express AQP1.
Figure 11 has shown that RT-PCR detects AQP1 (218bp) expression, and people CECs expresses AQP1. and expresses AQP1 through people CECs inductive people BEPCs, does not express AQP1 without inductive people BEPCs.
Figure 12 has shown that people CECs inductive people BEPCs iuntercellular forms tight connection (transmission electron microscope 17500 *).
Figure 13 has shown the ZO-1 expression (immunofluorescence, 200 *) of people CECs inductive BEPCs; Wherein
A: inductive BEPCs does not express ZO-1; B: people CECs inductive BEPCs expresses ZO-1.
Figure 14 has shown the NSE expression (immunohistochemical methods 200 *) of people CECs inductive BEPCs; Wherein
A: inductive people BEPCs does not express NSE; B: people CECs inductive people BEPCs expresses NSE; C: people CECs expresses NSE.
Figure 15 has shown that PCACM sees substantially.
Figure 16 has shown that PCACM sees substantially in 24 orifice plates; Wherein PCACM (arrow indication) is close at the bottom of the ware, and is smooth smooth; The right side control wells does not have PCACM.
Figure 17 shown the histology form 200 of PCACM *; Wherein
A:HE dyeing, the collagen marshalling; B:Hoechst dyes nuclear, does not see that stroma cell is residual.
Figure 18 has shown the electron microscopic morphology of PCACM; Wherein
A:PCACM smooth surface (scanning electron microscope 1000 *); B:PCACM arrangement of collagen fibers neat fine and close (transmission electron microscope 9700 *).
Figure 19 has shown that the different densities inoculation induces BEPCs form (inverted phase contrast microscope, 100 *) on PCACM after 3 days; Wherein
A:1000 cell/mm2 inoculation, iuntercellular does not have effective connection; B:2000 cell/mm2 inoculation, cell is Polygons, connects closely; C:4000 cell/mm2 inoculation, cellular form is unclear, and overlapping growth is arranged.
Figure 20 has shown the form (transmission electron microscope 1000 *) of inductive BEPCs on PCACM of different vitro culture times; Wherein
A: vitro culture 4 days, cell becomes Polygons, visible microvillus, the intercellular substance is big; B: vitro culture 7 days, cell connects tight slabbing, does not see obvious intercellular substance.
Figure 21 has shown the histology form (HE dyeing) of cultivating inductive BEPCs with PCACM; Wherein
A: inductive BEPCs be monolayer growth BEPCs (black arrow indication) 100 *; B: inductive BEPCs closely be fitted in PCACM go up (black arrow indication) 400 *.
Figure 22 shown behind the BEPCs inoculation PCACM one week back form (transmission electron microscope 17500 *); Wherein TEM sees that cell and PCACM fit tightly (black arrow), and iuntercellular forms tight connection (white arrow).
Figure 23 has shown the situation in 4 weeks of post-transplantation; Wherein
A:BEPCs induces group, and cornea is more transparent, visible pupil; B:BEPCs does not induce group, and the obvious oedema of cornea can not be got a glimpse of pupil; C: simple material group, corneal edema muddiness; D: model group removes endothelium merely, the corneal edema muddiness.
Figure 24 has shown and induces group postoperative cornea in January to see substantially.
Figure 25 has shown post-transplantation 4 all histology (HE dyeing); Wherein
A, B: normal cornea, descemet's membrane is thinner, the complete A 100 * B 400 of epithelial lining *; C, D: induce group, PCACM and BEPCs and recipient cornea matrix face are fitted good, and epithelium layer is complete, do not see inflammatory cell invade C100 * D 400 *; E, F: model group, the normal cornea of cornea is thick, and epithelial lining has damaged, descemet's membrane and endodermis disappearance E 100 * F 200 *.
Figure 26 has shown that people BEPCs mixes cultivation back AQP1 expression (immunofluorescence) altogether with cat CECs; Wherein
A: people BEPCs do not express AQP1 100 *; B: cat CECs inductive people BEPCs, expression AQP1 150 *; C: cat CECs expression AQP1 200 *.
Embodiment
At the problem that lacks suitable seed cell in the corneal transplantation, the contriver is surprised to find through deep research, endothelial cell and marrow endothelial progenitor cell is cultivated altogether (the preferred isolation altogether cultivates), make marrow endothelial progenitor cell be induced differentiation, form the corneal endothelium like cell.Described corneal endothelium like cell all shows with the similar or identical characteristic of endothelial cell at the aspects such as expression of cellular form, pumping function, barrier function, surface antigen sign NES.
Further, the contriver is inoculated in the cornea acellular matrix with the above-mentioned corneal endothelium like cell that obtains, and has made up the engineered corneal posterior lamella that can be used for transplanting and repaired corneal endothelium damaged.
Induction method
The invention provides a kind of method that forms the corneal endothelium like cell of inducing, described method comprises step: with 2-8 * 10
4The marrow endothelial progenitor cell of individual/ml (bone marrow derived endothelial progenitorcells, BEPCs) and 0.6-3 * 10
5(coneal endothlial cell CECs) mixes cultivation altogether or isolation and cultivates altogether the endothelial cell of individual/ml, makes marrow endothelial progenitor cell be differentiated to form the corneal endothelium like cell; Preferably with 3-7 * 10
4The marrow endothelial progenitor cell of individual/ml and 0.8-1.2 * 10
5The endothelial cell of individual/ml is cultivated altogether, more preferably isolates altogether and cultivates.
In induction method provided by the invention, described isolation is cultivated altogether and is meant that between marrow endothelial progenitor cell and the endothelial cell, cell can not be in contact with one another, but the materials such as cytokine that produced in the culturing process can be contacted jointly by these two kinds of cells.Therefore, cultivating the materials such as cytokine that produced in the endothelial cell process can be contacted by marrow endothelial progenitor cell, thereby induces it to be differentiated to form the corneal endothelium like cell.
The utensil that can use various isolation well known in the art to cultivate is altogether cultivated in the isolation of being adopted in the induction method provided by the invention altogether, as long as can play the effect that described isolation is cultivated altogether.The wherein preferred isolation co-culture system of forming by 6 holes, 12 orifice plates or 24 orifice plates and suspension type cell pool; The base material of cell pool is that the aperture is the macromolecular material of 0.1-3.0 μ m, can allow macromole such as the various factors of emiocytosis and nutritive substance to pass through, but cell can't pass through, described macromolecular material is selected from polyester film (PolyethyleneTerephthalate, PET), poly-carbon ester film (poly carbonate, PC) or tetrafluoroethylene (polytetrafluoroethylene, PTFE).
Amount for nutritive substances such as abundant assurance cytokines in isolating cultivation altogether is unlikely to occur inducing the blank phase, can preferably adopt liquid feeding or half to measure the liquid method of changing, and interpolation endothelial cell culture supernatant and/or fresh medium reinforcement are induced.In a preference of the present invention, adopt per two days liquid feedings or half amount to change the liquid method and add endothelial cell culture supernatant and/or fresh medium; More preferably, it is the 3rd day that cultivates altogether isolating, directly add endothelial cell culture supernatant and fresh medium, the volume of the endothelial cell culture supernatant of adding is the 1/10-1/3 of liquid volume in the former isolation co-culture system, the volume of the fresh medium that adds is the 2/5-4/5 of liquid volume in the former isolation co-culture system, at interval two days later, half amount is changed the liquid method, remove liquid 1/2 in the former isolation co-culture system, add endothelial cell culture supernatant and fresh medium, the volume of the endothelial cell culture supernatant of adding is the 1/4-1/2 of liquid volume in the former isolation co-culture system, and the volume of the fresh medium of interpolation is the 1/4-1/2 of liquid volume in the former isolation co-culture system.Described nutrient solution can be the nutrient solution of cultivation endothelial cell well known in the art, and preferred DMEM/F12 or DMEM more preferably are DMEM/F12 or the DMEM that contains 1-5% serum.
Provided by the invention inducing in the method that marrow endothelial progenitor cell is differentiated to form the corneal endothelium like cell, employed marrow endothelial progenitor cell can be consubstantiality of the same race, allogeneic or heterogenous allosome marrow endothelial progenitor cell with respect to the acceptor of corneal transplantation; Employed endothelial cell can be consubstantiality of the same race, allogeneic or heterogenous allosome endothelial cell with respect to the acceptor of corneal transplantation; Between marrow endothelial progenitor cell that described isolation is cultivated altogether and the endothelial cell consubstantiality of the same race, allogeneic or heterogenous allosome.Marrow endothelial progenitor cell used in the present invention and/or endothelial cell can obtain by approach well known in the art, can cultivate by method well known in the art and obtain, for example can consult Asahara T, Murohara T, Sullivan A, et al.Isolation of putativeprogenitor endothelial cells for angiogenesis.Science, 1997,275 (5302): 964-967 obtains marrow endothelial progenitor cell Ishino Y, Sano Y, Nakamura T, et al.Amniotic membrane as a carrier for cultivated human corneal endothelial celltransplantation.Invest Ophthalmol Vis Sci, 2004,45 (3): 800-806 obtains endothelial cell.
The corneal endothelium like cell
Marrow endothelial progenitor cell is divided into the corneal endothelium like cell after inducing through method provided by the invention, and described corneal endothelium like cell has been expressed some characteristics of endothelial cell:
One, be Polygons, more approaching on the form with endothelial cell;
Two, great expression AQP1, RT-PCR has further proved this change from the mRNA level, shows the preliminary pumping function of having set up;
Three, can express ZO-1, set up iuntercellular and closely connected, the two is an important indicator of representing the endothelial cell barrier function, illustrate that marrow endothelial progenitor cell is induced by endothelial cell after, set up certain barrier function;
Four, (neurone-specific enolase NSE), shows that the conversion of phenotype has taken place cell to the neural Hydratase, phosphoenolpyruvate of great expression.
The cornea acellular matrix
The carrier that the present invention is used to make up corneal endothelial transplantation is thin (preferred 39.83 ± 3.85 μ m), transparent, smooth surface, and the collagen marshalling does not have hole; Preferred cornea acellular matrix more preferably is the porcine cornea acellular matrix.
In a preference of the present invention, get the fresh pig cornea, utilize 1%TritonX-100 to remove cell, and adopt and progressively cut thin cornea method, obtained thin and transparent cornea acellular matrix, the uniformity high resilience meets the demand of tissue engineering comea metaplax layer solid support material.This method can remove cell fully, and it is residual that HE dyeing and Hoechst dyeing there is no stroma cell.Scanning electron microscope sees that the PCACM smooth surface is smooth, and transmission electron microscope sees that PCACM collagen marshalling rule is fine and close.Contain the serum nutrient solution and soak, 4 ℃ of preservations can be preserved half a year at least, and acellular matrix is flat transparent still, can the oedema distortion.PBS washes several times and gets final product before taking.The cornea acellular matrix that takes out lies in the 24 new orifice plates, dries naturally 20 minutes, just can tightly be attached at the bottom of the ware, and open and flat opening possesses certain mechanical property, and adding nutrient solution can be not floating.Satisfied the mechanical characteristic of biomaterial so simultaneously.
The drying means of PCACM mainly contains lyophilize, vacuum-drying and seasoning.A preference of the present invention is to adopt natural seasoning.Though take off in the cell processes, corneal stroma expands, and collagen is loose, and through seasoning, it is thin and transparent that matrix is recovered again, collagen marshalling, imporosity.
Engineered corneal posterior lamella
Engineered corneal posterior lamella provided by the invention contains corneal endothelium like cell and the cornea acellular matrix through inducing the marrow endothelial progenitor cell differentiation to obtain provided by the invention.Wherein the inoculum density of corneal endothelium like cell is 1000-4000 cell/mm
2Corneal posterior lamella preferably is 1200-3000 cell/mm
2, more preferably be 1500-2500 cell/mm
2
In a preference of the present invention, PCACM prewets with nutrient solution earlier before inoculation, the corneal endothelium like cell is fully beaten to spare make single cell suspension, and the corneal endothelium like cell is inoculated on PCACM evenly.
The suitable vitro culture time is the basis of transplanting in the body.The keeping structure organized of the vigor of seed cell, matrix complex functionality and special phenotype successfully has decisive influence.Seed cell is in the extensive amplification procedure of cultured in monolayer in vitro, broken away from three-dimensional extracellular matrix environment in the body, " dedifferenting " that cell phenotype loses (dedifferentiation) phenomenon very easily takes place, cell proliferation simultaneously and specific cell epimatrix synthesis capability descend, and " wearing out " occur (aging).Carry out the corneal endothelium layer building and need between cell amplification and phenotypic differentiation are kept, seek a trim point.In a preference of the present invention, inductive BEPCs is inoculated into after PCACM goes up, go up liquid to the BEPCs successive induction with CECs, the vitro culture time was controlled at 4-10 days, and the time is too short, and inductive BEPCs density deficiency is so that cell connects tight, overlong time, cell can wear out.It is good that the vitro culture time in one week can make BEPCs and PCACM fit, and density and normal cornea ECD are similar, have guaranteed the function of inductive BEPCs and keeping of phenotype.
In another preference, the vitro culture time is a week (5-7 days).
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can arbitrary combination.All features that this case specification sheets is disclosed can with any composition forms and usefulness, each feature that is disclosed in the specification sheets can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore removing has special instruction, and the feature that is disclosed only is the general example of equalization or similar features.
Major advantage of the present invention is:
1, induce the corneal endothelium like cell that obtains as seed cell through endothelial cell marrow endothelial progenitor cell, wide material sources, safe;
2, with the Biodegradable material of cornea acellular matrix, satisfactory for result as the structure corneal posterior lamella;
3, the corneal posterior lamella that contains corneal endothelium like cell and cornea acellular matrix has the damaged ability of excellent repairing endothelial cell.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all per-cent and umber by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Related correlation parameter, experiment material and detection method among the embodiment
1. English breviary vocabulary
2. source of drawing material
Aborted fetus (14-18 week), Shanghai hospital for obstetrics and gynaecology provides, through Ethics Committee of Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University Sch examine by.
The fresh pig eyeball, pig slaughterhouse, Shanghai.
3. laboratory animal
Healthy cat, male and female are not limit, body weight 1.5～2.0kg, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University Sch's Animal House provides.
4. cell cultures is with liquid and reagent
(1) EGM-2, Clonetics company contains hydrocortisone 0.2ml, hfgf-β 2ml, VEGF 0.5ml, R3-IGF-1 0.5ml, ascorbic acid 0.5ml, GA-1000 0.5ml
(2) DMEM/F12, HYCLONE company
(3) DMEM, HYCLONE company
(4) Fn, Fibronectin, scleroproein, BioSource company
(5) Histopaque, 1.077g/ml, cellular segregation liquid, Sigma-Aldrich company
(6) 0.2% composite gum protoenzymes, Serva company
5. cell detection is with antibody and reagent
(1) Dil-AcLDL, the acetylize low-density lipoprotein of Dil mark (1,1 '-Dioctadecyl-3,3,3 ', 3 '-tetramethyl indocarbocyanine-acetylatedlow-density lipoprotein, Invitrogen company);
(2) FITC-UEA1, FITC mark Ulex europaeus lectin (FITC-conjugated lectin ulexeuropeaus agglutinin, Sigma company);
(3) sheep polyclone CD133 antibody (Santa Cruz, CA, dilution in 1: 200), the anti-sheep two of the rabbit of rhodamine mark anti-(Santa Cruz company, dilution in 1: 200);
(4) rabbit polyclonal CD34 antibody (Santa Cruz company, dilution in 1: 200), the anti-sheep two of Alexa Fluor 488 rabbits anti-(Invitrogen company, dilution in 1: 1000);
(5) Biotin is in conjunction with anti-mouse Flk-1 antibody (eBioscience company, 1: 200), biotinylation goat anti-mouse igg (Boster company, 1: 200), SABC (Boster company, 1: 100), DAB (Dako Real company, 1: 50).
(6) mouse mono-clonal AQP1 antibody (Santa Cruz, 1: 50), the sheep anti mouse two anti-(Santa Cruz, dilution in 1: 200) of corresponding two anti-FITC marks;
(7) rabbit polyclonal ZO-1 antibody (Zymed company, 1: 150), the goat-anti rabbit two anti-(BOSTER, dilution in 1: 200) of corresponding two anti-FITC marks;
(8) mouse mono-clonal γ Enolase (NSE-P1) antibody (Santa Cruz company, dilution in 1: 200), the anti-mouse two of corresponding two anti-HRP marks is anti-, DAB dilution in 1: 50.
6.RT-PCR detect with reagent, primer and reaction conditions
(1) RT-PCR detection reagent: Trizol (Gibco, the U.S.), TaKaRa RNA PCR Kit (Biotech), 1%Agarose (contains 0.5mg/L ethidium bromide, EB);
(2) VIII Collagen Type VI α 1 primer sequence (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd is synthetic)
Upstream 5 '-ACATGGGATACAATAAATATCCTGAAAAGG-3 '
Downstream 5 '-CCCCAGGGAGAGTATCTGCCAGATATGGGG-3 '
Product size 496bp.
β-actin is as internal reference, and the primer amplification fragment length is 318bp.
PCR reaction system: 1 μ lcDNA/20 μ l system.
PCR reaction conditions: 30 circulations, 94 ℃ of sex change, 1 minute; Anneal 55 ℃ 1 minute; Extend 72 ℃, 1 minute.
Gel electrophoresis: 1.2% sepharose.
(3) AQP1 primer sequence (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd is synthetic):
Upstream 5 '-GTCCAGGACAACGTGAAGGT-3 '
Downstream 5 '-GAGGAGGTGATGCCTGAGAG-3 '
Product size 218bp.
PCR reaction: l μ lcDNA/20 μ l system.
Reaction conditions: 30 circulations, 94 ℃, 30 seconds; 65 ℃, 30 seconds; 72 ℃, 1 minute.
Gel electrophoresis: 1.8% sepharose.
7. cell cultures consumptive material
Cell pool (6 Well Millicell cellculture inserts, Millipore company, the U.S.) is fit to insert six orifice plates and uses.The film of cell pool is polyester (PET) film, and is transparent, film diameter 24mm, and film thickness 11 μ m, the useful area of film is 452mm
2, aperture 1.0 μ m, hole density 2 * 10
6
Microsurgery instruments set: diameter 7.5mm trepan, width 1.5mm diamond cutter, the improvement of 1ml syringe syringe needle, endothelium hook, corneal transplantation tweezer (Suzhou 66 Visual Science ﹠ Technology Co., Ltd.'s making), needle holder, microscissors, microforceps, double end iris repositor.
Viscoelastic agent (ProVisc 0.55ml, Alcon company); 10-0 nylon wire (health is treasured by Johnson ﹠ Johnson).
8. detection method
The AcLDL-UEA1 fluorescent dye
1) with 24 orifice plate cultivation of primary cells, get the cell in the 4th day former generation, PBS washes 1 time, renews bright nutrient solution 200ul/ hole, adds 10 μ g/mlDiI-AcLDL (dilution in 1: 100), and the rearmounted 37 ℃ of incubators of mixing were hatched 4 hours, and PBS washes 2 times.
2) 10% Paraformaldehyde 96 is fixed 20 minutes under the normal temperature, and PBS washes 2 times.
3) add 10 μ g/mlFITC-UEA1 (dilution in 1: 100), put 37 ℃ of incubators and hatched 2 hours, PBS washes 2 times.
4) Hoechst dyed nuclear 10 minutes, and PBS washes.
5) fluorescent microscope is observed down, and the positive cell that three fluorescence is all expressed is got 3 visuals field, and the counting positive cell is averaged.
The CD133/CD34/AQP1 immunofluorescence
1) cell is prepared: cell climbing sheet
2) fixing: 4% Paraformaldehyde 96 is fixed 10 minutes, PBS rinsing 3 times;
3) broken cell film: 0.25%Triton X-100 and 5%DMSO room temperature 10 minutes, PBS rinsing 3 times;
4) blocking-up endogenous peroxidase: 37 ℃ of baking ovens of hydrogen peroxide 15 minutes, PBS rinsing 3 times;
5) sealing non-specific antibody: CD133 group adds rabbit anteserum, and the CD34 group adds sheep blood serum, and AQP1 adds sheep blood serum, 37 ℃ of baking ovens 30 minutes;
6) one is anti-: after abandoning serum (not rinsing) add an anti-CD133, CD34, AQP1,4 ℃ of refrigerator overnight, PBS rinsing 3 times;
7) two is anti-: add corresponding two and resist, 37 ℃ of baking ovens were hatched 30 minutes;
8) observe: fluorescence microscope, take pictures.
Immunohistochemical methods detection of biological element is in conjunction with Flk-1 antibody
1) adds the two anti-preceding same immunofluorescences of step.
2) adding two resists: hatched 37 ℃ 30 minutes according to an anti-biotinylation sheep anti mouse of selecting
3) rinsing
4) adding three resists: ABC 1: 100,1 hour, 37 ℃
5) rinsing
6) colour developing: DAB colour developing 1-2 minute
7) lining dyes: bush uniformly dyeing 1min, and the hydrochloride alcohol differentiation, flowing water washes half an hour
8) dehydration of alcohol: 80%-90%-100%
9) transparence: dimethylbenzene I-II-III
10) resin mounting
Immunohistochemical methods detects NSE
1) cell is prepared: cell climbing sheet
2) fixing: 4% Paraformaldehyde 96 is fixed 10 minutes, PBS rinsing 3 times;
3) broken cell film: 0.25%Triton X-100 and 5%DMSO room temperature 10 minutes, PBS rinsing 3 times;
4) blocking-up endogenous peroxidase: 37 ℃ of baking ovens of hydrogen peroxide 15 minutes, PBS rinsing 3 times;
5) sealing non-specific antibody: CD133 group adds rabbit anteserum, and the CD34 group adds sheep blood serum, 37 ℃ of baking ovens 30 minutes;
6) one is anti-: after abandoning serum (not rinsing) add an anti-CD133, CD34,4 ℃ of refrigerator overnight, PBS rinsing 3 times;
7) two is anti-: add corresponding two dilutions in anti-1: 200,37 ℃ of baking ovens were hatched 30 minutes;
8) colour developing: DAB colour developing 1-2 minute
9) lining dyes: bush uniformly dyeing 1min, and the hydrochloride alcohol differentiation, flowing water washes half an hour
10) dehydration of alcohol: 80%-90%-100%
11) transparence: dimethylbenzene I-II-III
12) resin mounting
The scanning electron microscope example preparation process
1) preceding fixing: 2% glutaraldehyde PBS stationary liquid is fixed 2 hours 4 ℃.
2) rinsing: 4 ℃ of PBS damping fluid washing secondaries each 10 minutes.
3) back is fixing: 1% osmic acid PBS stationary liquid is fixed 2 hours 4 ℃.
4) rinsing: 4 ℃ of PBS damping fluid washing secondaries each 10 minutes.
5) dehydration: 30%-50%-70%-80%-95%-100%-100% ethanol dewaters step by step
The isoamyl acetate secondary was each 10 minutes in each 10 minutes.
6) drying: HITACHI HCP-2 critical point drying
7) conduction: BAL-TEC ion sputtering
8) observe: the Dutch Philips QUANTA-200 of company environmental scanning electronic microscope is observed
The sample for use in transmitted electron microscope preparation process
1) preceding fixing: 2% glutaraldehyde PBS stationary liquid is fixed 2 hours 4 ℃.
2) rinsing: 4 ℃ of PBS damping fluid washing secondaries each 10 minutes.
3) back is fixing: 1% osmic acid PBS stationary liquid is fixed 2 hours, 4 ℃
4) rinsing: each 10 minutes of PBS damping fluid washing secondary, 4 ℃.
5) dehydration: 30%-50%-70% ethanol dewatered each 10 minutes step by step
(70% ethanol contains 3% uranyl acetate), 4 ℃ of pieces dyed
80%-95%-100%-100% ethanol dewatered each 10 minutes step by step.
6) displacement: each 10 minutes of propylene oxide secondary.
7) soak into: Resins, epoxy 618 embedding liquid and propylene oxide 1: 12 hours
Resins, epoxy 618 embedding liquid and propylene oxide spend the night at 2: 1
Pure epoxy resin 618 embedding liquid soak into 37 ℃ 6 hours.
8) embedding: in 60 ℃ of baking ovens 48 hours.
9) section: LKB V-type ultramicrotome section.
10) dyeing: lead citrate electron stain.
11) observe: PHILIP (Dutch Philips company) CM-120 transmission electron microscope observing HE dyeing
1) roasting sheet: section is placed 70 ℃ of thermostat container bakings 30 minutes;
2) dewaxing and aquation: will cut into slices placed dimethylbenzene I, II each 10 minutes successively, dewaxing in 5 minutes among the dimethylbenzene III, 100%, 95%, 85%, 75% gradient alcohol aquation;
3) the flowing water flushing is 2 minutes;
4) haematoxylin dyeing is 10 minutes;
5) the tap water flushing is 1 minute;
6) 0.5% hydrochloride alcohol breaks up fast, flowing water flushing 30-60 minute;
7) 95% dehydration of alcohol is 1 minute;
8) Yihong dyeing is 2 minutes;
9) 95% alcohol, 100% alcohol, dimethylbenzene I, II, III;
10) dimethylbenzene is transparent, and the resinene mounting is waited to do the back mirror and observed down.
Frozen section Hoechst dyeing
1) OCT investing tissue piece;
2) conventional frozen section;
3) roasting sheet: section is placed 70 ℃ of thermostat container bakings 30 minutes;
4) under the 80% alcohol 2-3, the flowing water flushing;
5) Hoechst1:1000 dyeing;
6) fluorescent microscope is observed down.
People BEPCs mixes cultivation back AQP1 and the detection of mouse-anti people antinuclear antibodies altogether with cat CECs
1) the immunofluorescence step is the same
2) add anti-people's antinuclear antibodies (1: 50), 4 ℃ of refrigerator overnight after abandoning serum.
3) PBS shakes and washes, and adds corresponding two and resists, and 37 ℃ of baking ovens were hatched 30 minutes.
4) observer EPC expressing human antinuclear antibody under the fluorescent microscope
5) repeat the immunofluorescence step
6) add an anti-AQP1 again, spend the night
7) adding corresponding two again resists.
9. key instrument equipment
Inverted phase contrast microscope OLYMPUS company, Japan and NIKON company, Japan
Fluorescent microscope Nikon ECLIPSE E600/OLYMPUS
Dermatoscope OLYMPUS
Gel imaging system Tanon 2500
Three-flute PCR instrument Thermocycler 3000, Biometra, Germany
Nucleic acid-protein analyser DU 800 BECKMAN COULTER, the U.S.
QUANTA-200 environmental scanning electronic microscope PHILIPS Co., Holland
CM-120 transmission electron microscope PHILIPS Co., Holland
Freezing-microtome SHANDON CRYOTOME
Paraffin slicing machine Thermo ELECTRON CORPORATION
Operating microscope, optical instrument factory, 40 times of Zhenjiang
Slit lamp Xiamen Qiangben Science and Technology Co., Ltd
The ultrasonic corneal pachymeter Tomey of SP-3000 type company, Japan
Separation, cultivation and the evaluation of marrow endothelial progenitor cell (BEPCs)
1. experimental technique
1.1 the separation of people's marrow endothelial progenitor cell and cultivation
Get the aborted fetus bones of limbs in (14-18 week), put into and contain DMEM nutrient solution (low sugar, 10%FBS) in the culture dish, with vascular clamp bone is bitten into pieces, 150 mesh filter screens are filled in the centrifuge tube, remove bone chip, fat etc., 1500 rev/mins * 5 minutes are centrifugal, remove liquid, further remove fat, cell debris etc.Marrow is diluted to 6ml with nutrient solution to be beaten even, Histopaque density gradient centrifugation 400g * 30 minutes, as seen be divided into four layers (Fig. 1), the first layer is a nutrient solution, the second layer is cloud, be white mononuclear cell layer, the 3rd layer is transparent Histopaque liquid, and the 4th layer is sedimentary red corpuscle etc.Absorb the first layer, carefully draw the second layer and place new 50ml centrifuge tube, PBS 10ml dilutes centrifugal 250g * 10 minute, repeats 2 times, and thorough flush away Histopaque liquid is because this parting liquid pair cell is toxic.EGM-2 (10%FBS) nutrient solution mixing monocyte, 6 orifice plates of inoculation Fn shop layer, average 1 * 10
6Individual monocyte/hole places 37 ℃, 5%CO
2, 100% saturated humidity condition under cultivate.Add 40ng/ml Fn 2ml/ hole in 6 orifice plates, 4 ℃ of refrigerator overnight, second day sucking-off Fn (recyclable utilization again) is 6 orifice plates that Fn spreads layer after drying naturally on the super clean bench.Full dose was changed liquid in the 4th day, and PBS is the attached cell flush away not, adds EGM-2 (containing 5%FBS), and 37 ℃ of incubators are cultivated.Go down to posterity in the time of 7-10 days, earlier with PBS flush away nutrient solution, add 0.25% pancreatin again and digested 2-3 minute down for 37 ℃, when cell was round bright, the equivalent nutrient solution stopped digestion, with the cell sleaker cell is scraped off at the bottom of ware, gives and going down to posterity at 1: 4.
1.2 the routine of people BEPCs is identified
Get the cell and the 2nd day the cell of the first-generation in the 4th day former generation, add Dil-AcLDL and hatch, observation of cell is taken in the AcLDL situation, and hatches with FITC-UEA1, observes itself and the situation that combines of cytolemma.Fluorescent microscope is observed down, the positive cell that three fluorescence is all expressed, and positive cell is positive rate divided by total cell count.Get 3 visuals field, average.
Get on the cell climbing sheet that goes down to posterity after 7 days the BEPCs digestion, incubator adds the capacity nutrient solution again and spends the night after hatching and treating that most cells was attached on the creep plate in 4 hours, and the immunohistochemical methods of row CD133, CD34 and Flk-1 was identified with 4% Paraformaldehyde 96 fixed cell in second day
Set up blank group (not adding resists) and negative control group (human fibroblasts is available from Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University Sch organizational project laboratory).
1.3 people BEPCs VIII Collagen Type VI is identified
RT-PCR detects the VIII Collagen Type VI expression of the people BEPCs in the 7th day former generation, and with the positive control group of human corneal endothelial cells, embodiment 2 is seen in the cultivation of human corneal endothelial cells.
2 results
2.1BEPCs growth and propagation
In former generation, draws materials and generally can obtain 1.2 * 10
7Individual monocyte, the former pickup kind of BEPCs promptly has cell attachment after 24 hours, be Polygons, the growth of clone's sample, clone's form becomes little radial, does not see the wire arrangement.The rear clone number increased in 4 days, form ovalization whirlpool shape (Fig. 2 A), and cellular form is short fusiformis or Polygons.Cultivated the 6th day, the cell growth gradually loses clone's sample form, and cell is short fusiformis or Polygons (Fig. 2 B).Cultivated the 7th day, cell can cover with culture dish, during digestion cell in the form of sheets or lumps come off, need to be uniformly dispersed with the soft piping and druming of the adjustable micropipette rifle of 1ml.Can pass a generation, inoculum density 1 * 10 in 4-6 days later on
5Individual/ml, can pass for 7 generations at least, along with passage number increases, the height of cell inoculation density obviously influences the form of cell, and the low cell of inoculum density is into fiber-like, and the high cell of density is short fusiformis or Polygons, and back several generations cell inoculation density is with 2 * 10
5Individual/ml is advisable.
2.2BEPCs evaluation
2.2.1Ac-LDL and UEA-1 detects
The 4th day former generation of BEPCs, the growth of cell ovalization clone sample, can absorb Ac-LDL, cell is full, the fluorescence that takes on a red color (Fig. 3 A), and can be in conjunction with UEA-1, be green fluorescence (Fig. 3 B), be fluorescent orange after the stack, Hoechst dyes nuclear and is blue (Fig. 3 C), count the cell count that three looks all express and be positive rate (Fig. 3 D) divided by blue karyocyte number, positive rate reaches 100%.The people BEPCs first-generation, cell is short fusiformis, also can absorb Ac-LDL and in conjunction with UEA-1, positive rate reaches (Fig. 3 E) more than 90%.
2.2.2CD133 CD34 and Flk-1 detect
People BEPCs expresses CD133, and the fluorescence that takes on a red color (Fig. 4 A) is expressed CD34 simultaneously, is green fluorescence (Fig. 4 B), expresses Flk-1, is brown (Fig. 4 C).Positive rate all is higher than 95%.
2.3RT-PCR detect VIII Collagen Type VI expression
It is the same with people CECs that RT-PCR detects finder BEPCs, expresses VIII Collagen Type VI (Fig. 5).
The result shows that the people BEPCs form that the present invention tests separation and Culture becomes short fusiformis or Polygons, and is similar to endothelial cell on the form, can absorb Ac-LDL,, express CD133 in conjunction with UEA-1, CD34 and Flk-1, but also express the more special VIII Collagen Type VI of endothelial cell.
Marrow endothelial progenitor cell is mainly isolated myelomonocyte by density gradient centrifugation, cultivates by Fn shop layer and EGM-2 to obtain.
EPC lacks special surface marker, and the most frequently used authentication method is that EPC can absorb AcLDL and in conjunction with UEA-1, the surface antigen sign is expressed CD133, CD34 and Flk-1, and this cell is called functional endothelial progenitor cells.Early stage jejune EPC expresses hemopoietic stem cell sign CD133 and CD34, enter stage of more differentiation after the hemopoietic stem cell sign mistake that fades, begin to express the endotheliocyte sign, as VE cadherin, the vW factor, CD31 and nitricoxide synthase.The BEPCs that cultivates in the present invention's experiment can absorb Ac-LDL, in conjunction with UEA-1, and expresses CD133, CD34 and Flk-1 surface antigen.In order further to improve the purity of BEPCs, also can use CD133 magnetic bead sorting, perhaps airflow classification.
The VIII Collagen Type VI is secreted by endothelial cell, is one of moiety of forming descemet's membrane, also is the more specific sign of endothelial cell, through being usually used in the evaluation of endothelial cell.Contriver finder BEPCs also expresses the VIII Collagen Type VI.Certainly vascular endothelial cell also can be secreted the VIII Collagen Type VI, so be that BEPCs expresses the VIII Collagen Type VI or BEPCs has broken up the Collagen Type VI for vascular endothelial cell expression VI II, in order to get rid of false positive results, adopted the BEPCs in the 7th day former generation in the present invention's experiment, and done the evaluation of CD133, capable again VIII Collagen Type VI detects after proving BEPCs, has guaranteed the reliability of experimental result.
BEPCs induces differentiation to endothelial cell
1. experimental technique
1.1 the cultivation of people CECs amplification
Get the aborted fetus cornea, endothelium is faced down after striking off epithelium, be affixed on 50ml culturing bottle wall, treat after 5 minutes to add DMEM/F12 nutrient solution (10%FBS) 4ml, behind the wetting angle diaphragm, promptly culturing bottle was erected in the incubator after 2 hours, set level culturing bottle, make nutrient solution flood cornea.10-13 days can be with passage.First-generation CECs cultivates 24 orifice plates with Fn shop layer, and inoculum density is 5 * 10
4Individual/ml * 600 μ l/ holes, but went down to posterity available Fn shop layer 6 orifice plate later in 5-7 days.
1.2 human lens epithelial cell (Lens epithelial cells, cultivation amplification LECs)
Get the complete lens of aborted fetus, place the inserts (cell pool) of Transwell, cyst membrane is torn, be tiled on the film of cell pool, treat to add the wetting crystalline peplos of a small amount of DMEM (10%FBS) after 5 minutes, fill up nutrient solution after 2 hours.Can go down to posterity in 12-14 days, the back cell that goes down to posterity is cultivated with 6 orifice plates.
1.3 isolating altogether, people BEPCs and people CECs cultivate
Used BEPCs, CECs are the 1st generation and the 2nd generation.Experiment is divided into 3 groups:
The 1st group, people CECs induces experimental group, and cell pool was used a small amount of nutrient solution moistening 10 minutes in advance, inoculated 1 * 10
5Individual/ml CECs, the BEPCs that embodiment 1 obtains is then with 5 * 10
4Be inoculated in 6 orifice plates of shop layer, CECs is last, and BEPCs is following, isolate common cultivation (Fig. 6), isolate to cultivate altogether and adopt the Transwell co-culture system, be made up of 6 orifice plates and suspension type cell pool, the bottom of cell pool is polyester film (PolyethyleneTerephthalate, PET), aperture 1 μ m, transparent, can allow macromole such as the various factors of emiocytosis and nutritive substance to pass through, but cell can't pass through, and plays inducing action with this.General emiocytosis cytokine is to cultivate the 3rd day.Nutrient solution DMEM/F12 or the DMEM that contains 5%FBS, 1ml in the pond, 4ml outside the pond.
The 2nd group, do not induce control group, cultivate inoculum density 5 * 10 with the EGM-2 nutrient solution that contains 5%FBS
4Individual/ml, changed liquid once in per 2 days.
The 3rd group, the people CECs group that embodiment 2 obtains is cultivated inoculum density 5 * 10 with DMEM/F12 (10%FBS)
4Individual/ml, amount was changed liquid once in per 2 days half.
The 4th group, people LECs induces control group, and all methods only change people CECs into people LECs with first group.
Adopt per 2 days liquid feedings or partly measure the liquid method of changing, other adds the CECs culture supernatant and strengthens inducing, and gives when cell is intensive and going down to posterity.Cultivated altogether 7-14 days.
The 3rd day, add DMEM/F12 or DMEM (5%FBS) 1ml in the pond, the pond adds CECs supernatant liquor 1ml among the 3rd group of DMEM/F12 or DMEM (5%FBS) 1ml+
The 6th day, half amount was changed liquid in the pond, siphons away 1ml, adds fresh medium DMEM/F12 or DMEM (5%FBS) 1ml, and half measures and change liquid in the pond outside, siphons away 3ml, added CECs supernatant liquor 1.5ml among the 3rd group of DMEM/F12 or DMEM (5%FBS) 1.5ml+
The 9th day, with the 6th day.
Described endothelial cell culture supernatant is the culture supernatant for the endothelial cell first-generation or s-generation cell, and these cells every 1-2 days liquid feedings are once cultivated and promptly be can be used as supernatant liquor after the 3rd day as cultivating altogether.
1.4 cultivating the back altogether detects
Cultivate altogether after 7-14 days and the inductive cell detected from following four aspects:
Cellular form changes: the om observation cellular form;
Pumping function: immunofluorescence, RT-PCR detects the AQP1 expression;
Barrier function: the intercellular tight connection of electron microscopic observation, immunofluorescence detects Z0-1;
The surface antigen sign: immunohistochemical methods detects NSE;
Negative control all adopts human skin fibroblast (available from organizational project laboratory, attached the 9th the People's Hospital, the 9th the People's Hospital, Shanghai).
2 results
2.1 the cultivation of people CECs amplification
Utilize the culturing bottle tissue mass cell culture, can make CECs adherent, the 4th day is that visible cell climbs out of from the tissue block edge, be short fusiformis, in the 13rd day former generation, visible CECs is comparatively dense, form is Polygons, and the extracellular matrix that justacrine is a large amount of attaches closely (Fig. 7) with culturing bottle.Can give and go down to posterity this moment.Going down to posterity of CECs cultivated and need be spread layer by Fn, can keep the Polygons form of cell preferably.CECs can pass for 6 generations at least, and along with generation increases, cellular form gradually is fusiformis.
2.2 the cultivation of people LECs amplification
Crystalline peplos is adherent very difficult, easily floating, utilizes the adhesive capacity of inserts film, can promote that cyst membrane is adherent.Can turn out people LECs by the inserts tissue mass cell culture, form is typical cube.
2.3BEPCs isolate cultivation altogether with CECs
Observe the 1st group of CECs inductive BEPCs form after 10 days under the inverted microscope and be Polygons, be paving stone sample (Fig. 8 B), with the 3rd group of CECs plesiomorphism (Fig. 8 C), the 2nd group not inductive BEPCs become short fusiformis or spindle shape (Fig. 8 A), the 4th group of LECs inductive BEPCs form is short fusiformis or spindle shape (Fig. 8 B).0.6-3 * 10
5Individual/ml CECs all can play inducing action, 2-8 * 10
4The marrow endothelial progenitor cell of individual/ml all can well be induced by CECs, and 3-7 * 10
4The marrow endothelial progenitor cell of individual/ml and 0.8-1.2 * 10
5The endothelial cell of individual/ml is isolated culture effect the best altogether, and derivative BEPCs form is obvious paving stone sample.
2.4 cultivating the back altogether detects
2.4.1 cellular form and ultrastructure
Scanning electron microscopic observation people CECs inductive people BEPCs is Polygons, and cell connects closely no gap (Fig. 9).
2.4.2 pumping function
Immunofluorescence detection finder CECs inductive BEPCs is most of to express aquaporin 1 (AQP1), is green fluorescence.Do not induce BEPCs and people LECs inductive BEPCs all not to express AQP1 (Figure 10)
Detect people CECs inductive people BEPCs great expression AQP1 from the mRNA level, inductive people BEPCs does not have only the AQP1 of trace to express (Figure 11) yet.
2.4.3 barrier function
Transmission electron microscope observing people CECs inductive people BEPCs is monolayer growth, and iuntercellular can form tight connection (Figure 12).
Immunofluorescence detects the tight join dependency albumen-1 of CECs inductive BEPCs express cell, and (ZonulaOccludens-1 ZO-1), is green fluorescence; Inductive BEPCs does not express ZO-1 (Figure 13).
2.4.4 the neural Hydratase, phosphoenolpyruvate of surface antigen sign (neurone-specific enolase, NSE)
Begin to express NSE through CECs inductive BEPCs major part, immunohistochemical methods detects sees brown particle, and color is than CECs shallow (Figure 14).
The result shows, adopting BEPCs and CECs to isolate altogether cultivates, add the CECs culture supernatant simultaneously, but combined induction BEPCs expresses some characteristics of endothelial cell, more approaching on the inductive BEPCs form with endothelial cell, tentatively set up pumping function and barrier function, and the conversion of phenotype has taken place, express NSE, for further the structure and the transplanting of corneal posterior lamella provide experiment basis; That is to say that BEPCs and CECs cultivate by isolating altogether, differentiation has obtained a kind of corneal endothelium like cell.Under the same isolation co-culture method, people LECs can not induce BEPCs to break up to the lens epithelial cells direction.
CECs excretory cytokine plays an important role in inducing.
It is the good in vitro study model that stem cell directional is induced differentiation that isolation co-culture system joint objective cell culture supernatant is induced.
Adopt liquid feeding or half amount to change the liquid method in the present invention's experiment, fully guaranteed the amount of cytokine, be unlikely to occur inducing the blank phase.Adopted the low density inoculation by inducing cell, guarantee fully to be induced, do not suppress and can not occur cell density too early.Contain various somatomedins in the serum,, provide enough nutritive ingredients to cell again simultaneously, adopt the nutrient solution that contains lower concentration serum to carry out common cultivation in the experiment in order to reduce the influence of these factors in inducing.
The detection index that the differentiation of stem cells state is changed mainly contains three at present: metamorphosis, physiological function analysis and tissue-specific genetic expression.The change of differentiation state is accompanied by tangible metamorphosis, and this is the most preliminary index the most intuitively.And whether the most essential variation of cytodifferentiation has promptly expressed tissue-specific albumen.From the angle of clinical treatment, the realization of physiological function is the index that the bit heterologous gene expression has more practice significance.Endothelial cell lacks special surface marker antigen, but relatively unique on form.Present hexagon or polygon form.Endothelial cell also has special pumping function and barrier function, is the important factor of keeping cornea half dewatering state and corneal transparency.
On the form, after CECs induced, BEPCs was Polygons more near endothelial cell.
The detection of pumping function mainly uses aquaporin 1 (AQP1) to weigh.Aquaporin (Aquaporins, AQPs) be a class has high selectivity to water molecules transmembrane transporter, mainly be present in cytolemma and cytoplasm, the quick transmembrane transport process of mediation water has vital role to keeping normal water metabolism and water balance.Endothelial cell and lens epithelial cells all can be expressed AQP1, and AQP1 plays an important role at the drainage procedure of corneal stroma to corneal endothelium to the anterior chamber.AQP1 is one of important indicator of weighing the endothelial cell pumping function.Immunofluorescence is found BEPCs great expression AQP1 after CECs induces from protein level, RT-PCR has further proved this change from the mRNA level, illustrate and induce back BEPCs to possess certain pumping function, and lens epithelial cells inductive BEPCs does not express AQP1, illustrates that BEPCs does not break up to the lens epithelial cells direction.
Transmission electron microscope understands that from morphology Shanghai Stock Exchange inducing back BEPCs iuntercellular can form closely connects.Immunofluorescence detects ZO-1 (Zonula Occludens-1, the tight join dependency albumen of cell) from protein level, finds to induce back BEPCs can express ZO-1.Iuntercellular closely connects and ZO-1 is an important indicator of representing the endothelial cell barrier function.After illustrating that BEPCs induces by CECs, set up certain barrier function.
Because vascular endothelial cell is not only similar on form to endothelial cell, also possesses pumping function, express VIII Collagen Type VI and AQP1, so through such inducing, can or can not make BEPCs become vascular endothelial cell, and be not endothelial cell? this must differentiate from the special sign of these two kinds of cells: (neurone-specific enolase NSE) is the specific proteins that high specific is present in neurone and neuroendocrine cell to neural Hydratase, phosphoenolpyruvate.Vascular endothelial cell is the mesoderm source, does not express NSE.BEPCs is the mesoderm source, does not also express NSE.Endothelial cell is the neural crest source, expresses NSE.Detect discovery by immunohistochemical methods, BEPCs does not express NSE, by inducing back great expression NSE, illustrates that the conversion of phenotype has taken place BEPCs, promptly to the differentiation of endothelial cell direction, rather than vascular endothelial cell.
The preparation of PCACM and detection
1. experimental technique
1.1PCACM preparation
Get the fresh pig cornea, soak 30min in 75% alcohol, mechanical process scrapes off corneal epithelium, immersion contains in the centrifuge tube of 1%TritonX-100, the 24h that vibrates on the shaking table changes the 1%TritonX-100 24h that vibrates again after with blade corneal film being divided into 4, corneal film is divided into two again, the TritonX-100 24h that vibrates changes PBS and shakes repeatedly and be washed till non-foam.Abandon epithelial lining and endodermis.Put into 24 orifice plates after corneal film blotted with filter paper, shakeout and place under the super clean bench, disinfection by ultraviolet light spends the night, and corneal film dries naturally.Exsiccant PCACM is added DMEM/F12 nutrient solution (containing 10% foetal calf serum) soak, 4 ℃ of refrigerators are preserved.
1.2PCACM histology
PCACM is with the fixing 24h of 4% Paraformaldehyde 96, routine paraffin wax embedded section, Hematorylin two Yihong (HE) dyeing; Conventional frozen section, Hoechst dyeing; The situation that removes of observation of cell.
1.3PCACM Electronic Speculum detect
PCACM is liquid-solid fixed with Electronic Speculum, the planeness on scanning electron microscopic observation PCACM surface, and transmission electron microscope observing collagen is arranged and cell removes the situation situation.
2 results
2.1PCACM general form
After the PCACM rehydration, thin and soft, flexible, smooth surface, the transparency height, still can be close to 24 orifice plate wares at the bottom of (Figure 15, Figure 16).
2.2PCACM histology
The CACM polishing is seen in HE dyeing, and arrangement of collagen fibers is neat, does not see that stroma cell is residual.Blue substrate nucleus (Figure 17) is not seen in Hoechst dyeing.
2.3PCACM Electronic Speculum detect
Scanning electron microscope is seen the PCACM surface finishing.Transmission electron microscope observing PCACM collagen queueing discipline, fine and close, be the rips shape, do not see stroma cell.(Figure 18).
The result shows, utilizes TritonX-100 to take off cell, and associating is progressively cut thin method and dried naturally, can remove corneal cell fully, obtains thin and transparent PCACM, has certain toughness.PCACM is that favorable tissue engineering cornea metaplax layer makes up material.
Embodiment 4
Inductive BEPCs and PCACM external structure corneal posterior lamella
1 material and method
1.1 material
The PCACM that inductive BEPCs that embodiment 2 obtains and embodiment 3 obtain
1.2 experimental technique
1.2.1 the cell of different vaccination density growing state on PCACM
With embodiment 2 obtain through CECs inductive BEPCs with 1000 cell/mm of no density
2(promptly 1 * 10
5Individual cells/well), 2000 cell/mm
2(promptly 4 * 10
5Individual cells/well), 4000 cell/mm
2(promptly 8 * 10
5Individual cells/well), be inoculated on the PCACM respectively, and corresponding inoculation does not have in the hole of PCACM observation of cell growing state in contrast.Nutrient solution personnel selection CECs supernatant liquor: DMEM/F12 (5%FBS) (1ml: 1ml) continue inducing culture.Inoculate back 3 days inverted phase contrast microscope observation of cell forms.
1.2.2 the cell of different vitro culture times growing state on PCACM
With inductive BEPCs with 2000 cell/mm
2(promptly 4 * 10
5Individual cells/well) is inoculated on the PCACM nutrient solution personnel selection CECs supernatant liquor: DMEM/F12 (5%FBS) (1ml: 1ml) continue inducing culture, vitro culture 4 days, 7 days and 10 days, the growing state of scanning electric mirror observing cell on PCACM.
1.2.3 the compound back of cell and PCACM histology
With 2000 cell/mm
2(promptly 4 * 10
5Individual cells/well) inductive BEPCs is inoculated on the PCACM, nutrient solution personnel selection CECs supernatant liquor: DMEM/F12 (5%FBS) (1ml: 1ml) continue inducing culture, vitro culture one all backs Paraformaldehyde 96 is fixed, embedding becomes paraffin mass, do paraffin section, HE dyeing, observation of cell and material attach the collagen arranging situation of situation and PCACM.
1.2.4 the compound back of cell and PCACM Electronic Speculum detects
Cell and PCACM compound one all backs Electronic Speculum is liquid-solid fixed, line scanning electron microscopic observation cellular form, and transmission electron microscope observing cell and material attach situation.
2 results
2.1 the cell of different vaccination density growing state on PCACM
The cell inoculation of different densities is light microscopic observation down after 3 days, 1000 cell/mm
2The density of inoculation is low excessively, and iuntercellular can not form effective connection.2000 cell/mm
2The density of inoculation is more suitable, the cell outline of visible polygon, and cell connects closely, and cell can be cultivated a week, and form is kept well.4000 cell/mm
2The density of inoculation is higher, and cell reaches density very soon to be suppressed, and cellular form is unclear, and cell has overlapping growth, dead cell more (Figure 19).
2.2 the cell of different vitro culture times growing state on PCACM
With inductive BEPCs with 2000 cell/mm
2Be inoculated into PCACM and go up cultivation 4 days, scanning electron microscope sees that cell becomes Polygons, visible microvillus, and cell and lacuna are big, and cell can not form effective connection (Figure 20 A).Vitro culture the 7th day, scanning electron microscope see that cell connects closely in flakes, do not have obvious intercellular substance, cellular form good (Figure 20 B).Vitro culture 10 days, cell is very dense.
2.3 the compound back of inductive people BEPCs and PCACM histology form
After one week of inductive BEPCs inoculation material, HE dyeing sees that carrier is thin, smooth smooth, no corneal stroma cell residue; Inductive BEPCs is good in the PCACM surface growth, forms the successive individual layer, does not see that BEPCs invades PCACM; Inductive BEPCs and PCACM fit tightly, and do not have obvious gap, and cell is individual layer, similar to the normal cornea metaplax layer (Figure 21).
2.4 the compound back of inductive people BEPCs and PCACM ultrastructure
BEPCs inoculation PCACM one week back transmission electron microscope sees that cell is monolayer growth, and cell and material fit tightly, and iuntercellular forms tight connection (Figure 22).
The present invention is inoculated into inductive BEPCs after PCACM goes up, and we still go up liquid to the BEPCs successive induction with CECs, and the vitro culture time is controlled at a week, and the time is too short, inductive BEPCs density deficiency so that cell connect closely, overlong time, cell can wear out.It is good that the vitro culture time in one week can make BEPCs and PCACM fit, and density and normal cornea ECD are similar, have guaranteed the function of inductive BEPCs and keeping of phenotype.
By one week of vitro culture, visible inductive BEPCs well-grown on PCACM, cell becomes Polygons, and PCACM helps sticking of BEPCs, does not influence the growth and the hyperplasia of cell.Transmission electron microscope sees that iuntercellular forms tight connection, and cell and material attach closely.HE dyeing sees that cell becomes the successive monolayer growth, and is similar to the normal cornea metaplax layer.
The result shows, but people CECs inductive people BEPCs is inoculated into the thin metaplax layer of tissue engineering comea that PCACM goes up external structure, and the suitable vitro culture time is 4-10 days, especially 5-7 days better.
Embodiment 5
The corneal posterior lamella transplantation treatment corneal endothelium that makes up is damaged
1 material and method
1.1 material
Resulting corneal posterior lamella among the embodiment 4
1.2 method
1.2.1 operation method
The ketamine general anesthesia, the cat right eye is the operation eye, sterilization, drape, operating microscope operation down.Trepan is at anterior corneal surface central indented diameter 7.5mm, diamond cutter is in the capable scleral tunnel otch of corneoscleral junction 9 o ' clock positions, otch 2mm, inject viscoelastic agent and prop up the anterior chamber, 1ml syringe needle bending 30 degree enters the anterior chamber from otch after cutting off arrow slightly, along impression endodermis-descemet's membrane is torn, change and scrape interior dermatotome, the endodermis-descemet's membrane in the impression scope is scraped off together.Scrape clean back normal saline flushing.Enlarge otch to 5mm.Get cell material mixture in the culture dish (i.e. the corneal posterior lamella of Gou Jianing), cell is towards last, drill through the size of diameter 7.5mm with trepan, drip 1 viscoelastic agent, the corneal transplantation tweezer will be planted the sheet both sides and be turned up, and make the cell face interior, clamp from otch gently and send into the anterior chamber, to plant sheet gently with iris repositor and push aside, cell is towards the anterior chamber.Plant sheet and be close to the cornea inner face for making, can squeeze into air filled cavity, fixedly after 10-20 minute, inject physiological saline and make the anterior chamber, stitch 2 pins in incision with the 10-0 nylon wire.Art finishes, and erythromycin ophthalmic ointment is coated with eye, sends Animal House back to after benzylpenicillin sodium 400,000 units and dexamethasone sodium phosphate injection 0.5ml (2.5mg) intramuscular injection.Postoperative is observed weekly, and takes pictures, the angle measurement film thickness.
Experiment is divided into 4 groups, and first group for inducing group: the compound week back transplanting (corneal posterior lamella that embodiment 4 obtains) of people CECs inductive people BEPCs and material; Second group for not inducing group: the compound 1 week back transplanting of people BEPCs and material; The 3rd group is the material group, and simple PCACM transplants; The 4th group is model group, removes the descemet's membrane endodermis, not embedded material or cell.Every group of 4 right eyes that comprise 4 cats.
1.2.2 clinical follow
Postoperative is observed the art eye weekly, and in postoperative the 7th, 14, and 21 and 28 days slit lamp observation and taking pictures are measured corneal central thickness with ultrasonic thickness meter, and corneal thickness numerical value is all with mean ± standard deviation (X ± SD) expression.
1.2.3 histology
Postoperative 1 month gives the dark anesthesia back heart air tube that fans the air and puts to death, and takes off cornea, and 4% Paraformaldehyde 96 is fixed, row conventional organization section HE dyeing, and the position of observation of cell and material, and measure and plant sheet thickness.
2 results
2.1 post-transplantation is observed
Postoperative four groups of equal muddy oedema of cornea in 1 week can not be peeped into pupil; 2 week back BEPCs induce the group cornea to begin oedema to go down, and postoperative 4 all corneas are more transparent, visible pupil, and centre still has a little oedema muddiness, does not see that new vessel grows into.And other three groups after surgery 4 weeks corneal edema still, muddiness does not see pupil (Figure 23).
2.2 corneal thickness is measured
Do not induce in the cornea 1 month after surgery of group, simple material group and model group oedema serious, all the time can't measure thickness with ultrasonic thickness meter, BEPCs induces group to measure average 882 ± 31.8 μ m of corneal thickness after surgery 3 weeks, postoperative 4 all oedema further alleviate, corneal thickness is reduced to 612 ± 20.64 μ m, near thickness 503.3 ± 16.45 μ m of normal cornea.
2.3 form and histology
To induce group to draw materials, the gross examination of skeletal muscle cornea is more transparent, but it is obvious to plant sheet, and degraded is not planted the sheet edge and exceeded recipient cornea a little (Figure 24).
HE dyeing sees and induces the sheet of planting of group to be close to the recipient cornea hypothallus that inductive BEPCs becomes individual layer, tightly invests on the PCACM similar to normal cornea endodermis and descemet's membrane (Figure 25).Induce the corneal epithelium of group complete, hypothallus is not seen the inflammatory cell intrusion, and the PCACM density of planting sheet is higher, and collagen is arranged fine and close, is different from loose recipient cornea matrix.The model group cornea is thicker, and epithelial lining is destroyed, and the endothelium break is loose as, hypothallus, does not see that also inflammatory cell invades (Figure 25)
2.4 plant the sheet thickness measurement
The ultrasonic cornea thickness measuring of live body normal cornea thickness is 503.3 ± 16.45 μ m.Utilize dimensional measuring instrument in the Image-ProPlus image processing system, in conjunction with normal cornea thickness, can calculate that to plant sheet thickness be 39.83 ± 3.85 μ m, normal descemet's membrane-endodermis thickness is 13.22 ± 2.00 μ m, plants sheet thickness and is about 3 times of normal descemet's membrane-endodermis.
The present invention tests and adopts Descemet to tear to get interior dermepenthesis (Descemet stripping endothelialkeratoplasty, DSEK) operation plan, people CECs inductive BEPCs and PCACM back of compound one week of vitro culture are transplanted to the cat cornea inner face that tears descemet's membrane and endodermis off by the scleral tunnel otch of 5mm, cornea begins to recover transparent after 1 month, and does not induce group, simple material group and model group all still muddy.Explanation can be played the part physiological function of endothelial cell in vivo through inductive BEPCs.Utilize the tissue engineering comea metaplax layer energy repairing corneal endothelium of people CECs inductive BEPCs and the compound structure of PCACM damaged.
The present invention's experiment further specifies, and CECs inductive BEPCs has possessed certain pumping function and barrier function, and energy repairing corneal endotheliocyte is damaged, has a good application prospect.
Embodiment 6
Mixing to cultivate altogether makes BEPCs induce differentiation to endothelial cell
1. people BEPCs and cat CECs mixing co-culture experiments method
1.1 the cultivation of cat CECs amplification
Get the cat cornea, tear descemet's membrane and endodermis shred, 0.2% collagenase, 140 rev/mins of digestion of 37 ℃ of shaking tables 40 minutes added the 2ml0.25% trysinization 5 minutes again, stop digestion with volume DMEM/F12 (10%FBS), 1500 rev/mins centrifugal 5 minutes, the resuspended back of nutrient solution is with 1 * 10
5Individual/the ml inoculation.7-10 days visible cell confluent culture wares, 0.25% trysinization was gone down to posterity in 1: 2 to 1: 4, went down to posterity in later every 6-8 days 1: 16.
1.2 the separation of people BEPCs and cultivation are with embodiment 1
1.3 mixing with cat CECs altogether, cultivates people BEPCs
The BEPCs that chooses, cat CECs are the 1st generation and the 2nd generation.Experiment is divided into 3 groups: the 1st group, people BEPCs group is got 7-10 days cell, cultivates with the EGM-2 nutrient solution that contains 5%FBS, and inoculum density is 5 * 10
4Individual/ml; The 2nd group, mix cultivation group altogether, people's marrow endothelial progenitor cell: cat endothelial cell=1: 2-4, i.e. 3-7 * 10
4The marrow endothelial progenitor cell of individual/ml and 0.8-1.2 * 10
5The endothelial cell of individual/ml, nutrient solution DMEM/F12 or the DMEM that contains 5%FBS; The 3rd group, cat CECs group is cultivated with the DMEM/F12 that contains 10%FBS, and inoculum density is 5 * 10
4Individual/ml.Per 2 days liquid feedings or half amount are changed liquid, pass once generation in average 5-6 days.Cultivate the expression that detects AQP1 after 7-14 days altogether, people BEPCs mouse-anti people antinuclear antibodies mark.
2. people BEPCs mixes cultivation results altogether with cat CECs
2.1 the cultivation of cat CECs amplification
Can turn out a large amount of cat CECs with the cultivation of this kind cultural method, form is Polygons.
2.2 mixing with cat CECs altogether, cultivates people BEPCs
People BEPCs mix with cat CECs cultivate 7-14 days altogether after, immunofluorescence detect to find that people BEPCs does not express AQP1 (Figure 26 A), the people BEPCs expression AQP1 (Figure 26 B) after inducing with cat CECs, cat CECs expresses AQP1 (Figure 26 C).Utilize people's antinuclear antibodies can from cell mixing, detect people BEPCs easily.
With 3-7 * 10
4The people BEPCs of individual/ml and 0.8-1.2 * 10
5The cat CECs of individual/ml mixes cultivation altogether, can induce people BEPCs to break up to the corneal endothelium direction.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. induce the method that forms the corneal endothelium like cell for one kind, it is characterized in that described method comprises step: 2-8 * 10
4The marrow endothelial progenitor cell of individual/ml (bone marrow derived endothelial progenitorcells, BEPCs) and 0.6-3 * 10
5(coneal endothlial cell CECs) cultivates the endothelial cell of individual/ml altogether, makes marrow endothelial progenitor cell be differentiated to form the corneal endothelium like cell; Preferably with 3-7 * 10
4The marrow endothelial progenitor cell of individual/ml and 0.8-1.2 * 10
5The endothelial cell of individual/ml is cultivated altogether.
2. induction method as claimed in claim 1 is characterized in that, employed nutrient solution is DMEM/F12 or the DMEM that contains 1-5%v/v serum in cultivating altogether; Cultivated altogether 7-14 days; Preferred 8-10 days.
3. induction method as claimed in claim 1 is characterized in that, described marrow endothelial progenitor cell is consubstantiality of the same race, allogeneic or heterogenous allosome marrow endothelial progenitor cell; Described endothelial cell is consubstantiality of the same race, allogeneic or heterogenous allosome endothelial cell; Between marrow endothelial progenitor cell that described isolation is cultivated altogether and the endothelial cell consubstantiality of the same race, allogeneic or heterogenous allosome.
4. induction method as claimed in claim 1 is characterized in that, described cultivate altogether to be selected to mix cultivate altogether or isolate altogether and cultivate; The preferred isolation altogether cultivates.
5. induction method as claimed in claim 4, it is characterized in that, added endothelial cell culture supernatant and fresh medium again at the 3rd day that isolates cultivation altogether, change the liquid method by half amount more two days later at interval and add endothelial cell culture supernatant and fresh medium; Described nutrient solution is DMEM/F12 or the DMEM that contains 1-5%v/v serum; Described endothelial cell culture supernatant is the culture supernatant for the endothelial cell first-generation or s-generation cell.
6. the purposes as the corneal endothelium like cell of the arbitrary described induction method acquisition of claim 1-5 is characterized in that described purposes is to prepare corneal graft as seed cell.
7. engineered corneal posterior lamella is characterized in that it comprises:
(a) pharmaceutically acceptable Biodegradable material; With
(b) the corneal endothelium like cell that obtains as the arbitrary described induction method of claim 1-5;
Described pharmaceutically acceptable Biodegradable material be the cornea acellular matrix (cornea acellularmatrix, CACM); Preferred porcine cornea acellular matrix (porcine cornea acellular matrix, PCACM).
8. corneal posterior lamella as claimed in claim 7 is characterized in that, the content of the corneal endothelium like cell that obtains as the arbitrary described induction method of claim 1-5 is 1000-4000 cell/mm
2Constructed corneal posterior lamella.
9. a method for preparing engineered corneal posterior lamella is characterized in that, described method comprises step:
(1) will be inoculated in cornea acellular matrix (preferred porcine cornea acellular matrix) as the corneal endothelium like cell that the arbitrary described induction method of claim 1-5 obtains, inoculum density is 1000-4000 cell/mm
2The cornea acellular matrix; With
(2) cultivation obtains engineered corneal posterior lamella.
10. method as claimed in claim 9 is characterized in that, adding volume ratio in the step (2) is 1: the endothelial cell culture supernatant of 1-3 is cultivated with the DMEM/F12 or the DMEM that contain 1-5%v/v serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810041062A CN101638635A (en) | 2008-07-28 | 2008-07-28 | Method for obtaining corneal endothelium sample cells and construction of corneal metaplax layer in vitro |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810041062A CN101638635A (en) | 2008-07-28 | 2008-07-28 | Method for obtaining corneal endothelium sample cells and construction of corneal metaplax layer in vitro |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101638635A true CN101638635A (en) | 2010-02-03 |
Family
ID=41613815
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810041062A Pending CN101638635A (en) | 2008-07-28 | 2008-07-28 | Method for obtaining corneal endothelium sample cells and construction of corneal metaplax layer in vitro |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101638635A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103055348A (en) * | 2011-10-24 | 2013-04-24 | 北京清美联创干细胞科技有限公司 | Preparation method and application of autologous mesenchymal stem cell-loaded human amniotic membrane cornea paster |
| CN106282094A (en) * | 2016-10-13 | 2017-01-04 | 吴欣怡 | The method that the precursor in application on human skin source is induced to differentiate into corneal endothelium like cell |
| CN107427358A (en) * | 2015-03-26 | 2017-12-01 | 弗劳恩霍夫应用研究促进协会 | Limitans after cornea,artificial |
| CN108939161A (en) * | 2018-08-02 | 2018-12-07 | 陕西省眼科研究所 | A kind of humanization activity goes the preparation method of cell corneal stroma stent |
-
2008
- 2008-07-28 CN CN200810041062A patent/CN101638635A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103055348A (en) * | 2011-10-24 | 2013-04-24 | 北京清美联创干细胞科技有限公司 | Preparation method and application of autologous mesenchymal stem cell-loaded human amniotic membrane cornea paster |
| CN107427358A (en) * | 2015-03-26 | 2017-12-01 | 弗劳恩霍夫应用研究促进协会 | Limitans after cornea,artificial |
| CN107427358B (en) * | 2015-03-26 | 2020-06-12 | 弗劳恩霍夫应用研究促进协会 | Artificial cornea posterior limiting membrane |
| US11504225B2 (en) | 2015-03-26 | 2022-11-22 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Artificial Descemet construct |
| CN106282094A (en) * | 2016-10-13 | 2017-01-04 | 吴欣怡 | The method that the precursor in application on human skin source is induced to differentiate into corneal endothelium like cell |
| US20180105796A1 (en) * | 2016-10-13 | 2018-04-19 | Xinyi Wu | Method of Inducing and Differentiating Human Skin-Derived Precursors to Differentiate into Corneal Endothelial-like Cells |
| CN106282094B (en) * | 2016-10-13 | 2018-11-27 | 吴欣怡 | The method that the precursor in application on human skin source is induced to differentiate into corneal endothelium like cell |
| US10774307B2 (en) * | 2016-10-13 | 2020-09-15 | Xinyi Wu | Method of inducing and differentiating human skin-derived precursors to differentiate into corneal endothelial-like cells |
| CN108939161A (en) * | 2018-08-02 | 2018-12-07 | 陕西省眼科研究所 | A kind of humanization activity goes the preparation method of cell corneal stroma stent |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| San Choi et al. | Bioengineering endothelialized neo-corneas using donor-derived corneal endothelial cells and decellularized corneal stroma | |
| Mimura et al. | Transplantation of corneas reconstructed with cultured adult human corneal endothelial cells in nude rats | |
| CN100554411C (en) | Undifferentiated stem cell tissue system from corneal limbus | |
| US8628572B2 (en) | Corneal endothelial scaffolds and methods of use | |
| Nagata et al. | Evaluation of insulin secretion of isolated rat islets cultured in extracellular matrix | |
| JP5946046B2 (en) | Human corneal endothelial cell sheet | |
| ES2677945T3 (en) | Production of artificial tissues by tissue engineering using fibrin and agarose biomaterials | |
| CN1333818A (en) | Bioengineered tissue constructs and method for producing and using them | |
| CN104342402B (en) | A kind of bone marrow dedifferentes the cultural method of mescenchymal stem cell | |
| CN101432031A (en) | Conjunctival tissue system | |
| CN104755611A (en) | Method for producing retinal pigment epithelial cell sheet | |
| CN110051694B (en) | Urine-derived stem cell preparation, preparation thereof and application thereof in preparation of acute immune rejection medicament after organ transplantation | |
| CN101306207A (en) | Tissue engineered cornea epithelial transplantation membrane and preparation method and use thereof | |
| CN106661552A (en) | Cardiac cell culture material | |
| CN101638635A (en) | Method for obtaining corneal endothelium sample cells and construction of corneal metaplax layer in vitro | |
| Jurkunas et al. | Cultivated autologous limbal epithelial cell transplantation: new frontier in the treatment of limbal stem cell deficiency | |
| Amano et al. | Properties of corneas reconstructed with cultured human corneal endothelial cells and human corneal stroma | |
| Vemuganti et al. | Limbal stem cells: application in ocular biomedicine | |
| CN108774630A (en) | A kind of primary culture method of Microhyla ornata Skeletal Muscle Cell | |
| Castillo et al. | An improved method of isolating fetal human retinal pigment epithelium | |
| Gao et al. | Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue in vitro | |
| Cen et al. | Preliminary studies of constructing a tissue-engineered lamellar corneal graft by culturing mesenchymal stem cells onto decellularized corneal matrix | |
| Qiu et al. | Reconstruction of the ocular surface by autologous transplantation of rabbit adipose tissue-derived stem cells on amniotic membrane | |
| US20100203575A1 (en) | In Vitro Beating Heart Model | |
| Arpitha et al. | A method to isolate human limbal basal cells enriched for a subset of epithelial cells with a large nucleus/cytoplasm ratio expressing high levels of p63 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20100203 |