CN115105637B - Application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction - Google Patents
Application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction Download PDFInfo
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Abstract
The invention provides application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction. The invention discloses subconjunctival fibroblast acellular matrix for promoting the growth and proliferation of conjunctival epithelial cells; maintaining conjunctival epithelial stem cell characteristics; maintaining the activity of the notch pathway and the inhibition state of the wnt pathway in the in vitro differentiation process of conjunctival epithelial stem cells; promoting repair of large area conjunctival defects; promoting the expression of goblet cells and mucin after repair of conjunctival defects; reducing the fibrosis and scarring level of the matrix layer after conjunctival defect repair. The subconjunctival fibroblast acellular matrix prepared by the invention is a colorless transparent film, has certain mechanical strength and stretchability, can withstand surgical needle and line suturing, can construct the acellular matrix with the thickness obviously superior to that of adipose mesenchymal stem cells, has the Young modulus similar to that of normal conjunctival tissues, and is convenient for clinical popularization.
Description
Technical Field
The invention relates to the technical field of tissue functions and ophthalmic repair, in particular to application of conjunctival decellularized matrix in conjunctival reconstruction.
Background
Conjunctiva is an important component of the ocular surface, and its structural and functional integrity is critical to maintaining ocular surface homeostasis. Various clinically common refractory eye surface diseases such as chemical injury, thermal burn and the like can cause severe blepharoconjunctival adhesion, and large-area conjunctival defects can appear after surgical separation and adhesion, and conjunctival reconstruction is needed. The current common operation modes comprise autologous/allogenic conjunctival transplantation, orolabial mucosa transplantation, amniotic membrane transplantation and the like, but the current common operation modes are limited in clinical application due to the problems of limited tissue sources, graft immune rejection, poor repair effect, poor function replacement and the like, so that a brand-new conjunctival reconstruction mode needs to be developed.
Several polymer synthetic materials have been studied to find use in repairing conjunctival defects, but they have been deficient in biocompatibility and functional substitution, and often the stability of the tear film is seriously affected by the lack of conjunctival goblet cells (important single cell secretory glands in the conjunctiva, which secrete mucin, constitute the tear film mucus layer) after operation, and the ideal repairing effect cannot be achieved. With recent development of tissue engineering and stem cell technology, in vitro construction of functional conjunctiva has provided a possibility for solving the above problems. The acellular matrix is used as a natural biological material, has excellent biocompatibility, can provide adhesion surface and structural support for cell growth, is rich in growth factors, forms a tissue microenvironment favorable for cell growth, and is an excellent scaffold carrier for tissue regeneration and repair. The application of various acellular matrixes from different cell sources in the fields of bone tissue regeneration, wound repair and the like is carried out, and the inventor also constructs the acellular matrixes from adipose mesenchymal stem cells for conjunctival injury repair (published in the article) in the past, and has a certain effect of promoting conjunctival epithelial cell proliferation. However, the acellular matrix derived from the adipose-derived mesenchymal stem cells has poor mechanical strength, is difficult to be applied to clinical operation suturing, and has a good suitability for conjunctival stem cell growth microenvironment.
Disclosure of Invention
Aiming at the situation, the invention provides the application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction in order to make up the defects in the prior art.
The technical scheme for solving the problems is as follows:
the invention provides the use of subconjunctival fibroblast acellular matrix in the preparation of conjunctival structure and function repair products having at least one of the following functions:
1) Promote the growth and proliferation of conjunctival epithelial cells;
2) Maintaining conjunctival epithelial stem cell characteristics;
3) Maintaining the activity of the notch pathway and the inhibition state of the wnt pathway in the in vitro differentiation process of conjunctival epithelial stem cells;
4) Promoting repair of large area conjunctival defects;
5) Promoting the expression of goblet cells and mucin after repair of conjunctival defects;
6) Reducing the fibrosis and scarring level of the matrix layer after conjunctival defect repair.
In some embodiments of the invention, the conjunctival structure and function repair product comprises a functional conjunctival remodeling biomaterial that treats blepharoconjunctival adhesions.
The functional conjunctival reconstruction biological material for treating blepharocollosis adhesion is subconjunctival fibroblast acellular matrix.
The invention also provides a preparation method of the biological material, which comprises the steps of obtaining subconjunctival fibroblasts, in-vitro culture and amplification and preparation of acellular matrixes.
In some embodiments of the invention, the harvesting of subconjunctival fibroblasts comprises the specific steps of: washing conjunctival tissue, incubating with protease, scraping off epithelial layer, retaining subconjunctival matrix layer, digesting in digestive liquid until dispersed into single cell suspension, filtering to remove impurities, centrifuging, and retaining precipitate to obtain subconjunctival fibroblasts.
In some embodiments of the invention, the in vitro culture amplification comprises the following specific steps: resuspension of the obtained subconjunctival fibroblasts with a culture solution, inoculating the subconjunctival fibroblasts into a culture dish for culture, inoculating the cultured subconjunctival fibroblasts into a culture plate, culturing until the cells are fused, adding vitamin C into the culture solution, and continuously culturing in vitro.
In some embodiments of the invention, the preparation of the acellular matrix comprises the following specific steps: culturing until extracellular matrix forms membrane with proper thickness, performing decellularization treatment, removing genetic material DNA, and cleaning with PBS to obtain the biological material.
Compared with the prior art, the invention has the following beneficial effects:
1. the existing conjunctival reconstruction biological material has the problems of poor biocompatibility and functional substitution, incapability of effectively maintaining conjunctival stem cell characteristics and proliferation potential, less expression of goblet cells of conjunctival tissues after repair and poor ocular surface condition, and the existing adipose mesenchymal stem cell acellular matrix has the problems of low mechanical strength and intolerance to surgical suture.
2. The subconjunctival fibroblast acellular matrix prepared by the invention is a colorless transparent film, is easy to obtain, can be stably stored in vitro for a long time, has excellent biocompatibility, low immunogenicity and excellent mechanical strength, can bear surgical suture, can be used as a functional conjunctival reconstruction biological material for treating blepharocollosis, and has good treatment and repair effects on large-area conjunctival defects, ocular surface steady state serious damage and the like caused by blepharocollosis adhesion operation caused by acid-base chemical injury, thermal burn, steven-Johnson syndrome, ocular pemphigoid and the like.
Drawings
FIG. 1 shows preparation of subconjunctival fibroblast acellular matrix and mechanical strength test. Panel A is a general photograph, transparency and stretchability display of subconjunctival fibroblast decellularized matrix; panel B shows subconjunctival fibroblast decellularized matrix tolerating surgical needle and line suture; panel C is a comparison of HE staining and Masson staining of subconjunctival fibroblast decellularized matrix and adipose mesenchymal stem cell decellularized matrix; FIG. D is a statistical plot of tissue thickness of subconjunctival fibroblast decellularized matrix and adipose mesenchymal stem cell decellularized matrix; FIG. E is a graph of Young's modulus comparison statistics of subconjunctival fibroblast decellularized matrix and normal conjunctival tissue; panel F is a comparison of immunofluorescent staining of subconjunctival fibroblasts before and after extracellular matrix decellularization. Data are presented as mean ± standard deviation, using t-test; * Representing a statistical difference between the two groups, p < 0.05; * Representing a statistical difference between the two groups, p < 0.01; * Represents a statistical difference between the two groups, p < 0.001.
FIG. 2 is a graph of the biocompatibility assay of subconjunctival fibroblast decellularized matrix. FIG. A is a representative image of scanning electron microscope detection of subconjunctival fibroblasts before and after cell seeding by decellularized matrix; panel B is representative of the dead and alive staining of conjunctival epithelial cells seeded under conjunctival fibroblasts before and after decellularization of the matrix; c, a dead and alive dyeing result statistical graph; d is the result of CCK-8 detection statistics; e, a fluorescent staining representative graph of the EdU proliferation experiment; f, a statistical graph of the result of the EdU proliferation experiment; g is a representative graph of conjunctival epithelial cell stem index p63 fluorescent staining; the H diagram is a p63 fluorescent staining result statistical diagram; FIG. I is a representative graph of fluorescence staining of the conjunctival epithelial cell differentiation index CK 4; and J, a statistical graph of CK4 fluorescent staining results. Data are presented as mean ± standard deviation, using t-test; * Representing a statistical difference between the two groups, p < 0.05; * Representing a statistical difference between the two groups, p < 0.01; * Represents a statistical difference between the two groups, p < 0.001.
Figure 3 shows subconjunctival fibroblast decellularized matrix maintaining conjunctival epithelial stem cell characteristics. FIG. A is a representative immunofluorescence staining chart of conjunctival epithelial stem cell marker ABCG2, which is obtained by respectively inoculating conjunctival epithelial stem cells into subconjunctival fibroblast decellularized matrix and a blank culture dish for in vitro culture; panel B is representative of immunofluorescent staining of conjunctival epithelial stem cell marker p 63; panel C is an immunofluorescent staining representation of the conjunctival epithelial cell differentiation marker keratin CK 4.
FIG. 4 shows the in vivo repair of large area conjunctival defects by subconjunctival fibroblasts decellularized matrix combined with conjunctival epithelial stem cells. FIG. A is a schematic diagram of the construction of a rabbit large-area conjunctival defect model and the operation of decellularized matrix transplantation; panel B shows the effect of reduzamine green staining on repairing conjunctival defects by subconjunctival fibroblast acellular matrix, NC is a blank control group, DM is a subconjunctival fibroblast acellular matrix group, DM-CjECs is a subconjunctival fibroblast acellular matrix composite conjunctival epithelial cell group, and DM-p75++ is a subconjunctival fibroblast acellular matrix composite conjunctival epithelial stem cell group; panel C is a representative graph of paraffin section HE, PAS, masson staining of each group of defect repair completion site tissue; panel D is a representative image of the results of immunofluorescent staining of frozen sections of tissue from each group of defect repair completion sites.
Detailed Description
The invention provides the use of subconjunctival fibroblast acellular matrix in the preparation of conjunctival structure and function repair products having at least one of the following functions:
1) Promote the growth and proliferation of conjunctival epithelial cells;
2) Maintaining conjunctival epithelial stem cell characteristics;
3) Maintaining the activity of the notch pathway and the inhibition state of the wnt pathway in the in vitro differentiation process of conjunctival epithelial stem cells;
4) Promoting repair of large area conjunctival defects;
5) Promoting the expression of goblet cells and mucin after repair of conjunctival defects;
6) Reducing the fibrosis and scarring level of the matrix layer after conjunctival defect repair.
In the present invention, the conjunctival structure and function repair product necessarily includes subconjunctival fibroblast acellular matrix, and takes subconjunctival fibroblast acellular matrix as an active ingredient for the aforementioned functions. In the conjunctival structure and function repair product of the invention, the active ingredient which plays the aforementioned role can be only subconjunctival fibroblast acellular matrix, and other products which can play similar roles can also be included.
In some embodiments of the invention, the conjunctival structure and function repair product comprises a functional conjunctival remodeling biomaterial that treats blepharoconjunctival adhesions. Further, the blepharocollosis includes blepharocollosis caused by acid-base chemical injury, thermal burn, steven-Johnson syndrome or ocular pemphigoid, etc.
The functional conjunctival reconstruction biological material for treating blepharocollosis adhesion is subconjunctival fibroblast acellular matrix.
The invention also provides a preparation method of the biological material, which comprises the steps of obtaining subconjunctival fibroblasts, in-vitro culture and amplification and preparation of acellular matrixes.
In some embodiments of the invention, the harvesting of subconjunctival fibroblasts comprises the specific steps of: washing conjunctival tissue, incubating with protease, scraping off epithelial layer, retaining subconjunctival matrix layer, digesting in digestive liquid until dispersed into single cell suspension, filtering to remove impurities, centrifuging, and retaining precipitate to obtain subconjunctival fibroblasts. In some embodiments of the invention, the wash medium is a penicillin-containing PBS buffer. In some embodiments of the invention, the incubation temperature is 4 ℃, and the incubation time is 10-18 hours, preferably 14 hours. In some embodiments of the invention, the protease is a Dispase II neutral protease at a concentration of 4-6 mg/mL, preferably 5mg/mL. In some embodiments of the invention, the digestive juice is 0.1% to 0.2% collagenase a, preferably 0.1%. In some embodiments of the invention, the digestion temperature is 37℃and the digestion time is 16 to 20 hours, preferably 18 hours.
In some embodiments of the invention, the in vitro culture amplification comprises the following specific steps: resuspension of the obtained subconjunctival fibroblasts with a culture solution, inoculating the subconjunctival fibroblasts into a culture dish for culture, inoculating the cultured subconjunctival fibroblasts into a culture plate, culturing until the cells are fused, adding vitamin C into the culture solution, and continuously culturing in vitro. In some embodiments of the invention, the culture plate is a six well culture plate. In some embodiments of the invention, the vitamin C concentration is 10-20. Mu.g/mL, preferably 10. Mu.g/mL. The culture solution formula contains vitamin C, so that the fibroblast can be stimulated to secrete a large amount of extracellular matrix, and the acellular matrix after acellular has certain thickness and toughness and can be completely uncovered. In some embodiments of the invention, the in vitro continuous culture time is 2 weeks. In some embodiments of the invention, the culture medium is replaced every few days, preferably every 2 days, during the in vitro continuous culture.
In some embodiments of the invention, the preparation of the acellular matrix comprises the following specific steps: culturing to form a membrane with proper thickness on extracellular matrix, performing decellularization treatment, removing genetic material DNA, cleaning with PBS, and preserving at 4deg.C to obtain the biological material. In some embodiments of the invention, the decellularized solution used in the decellularization treatment is a solution containing 0.5-1% Triton-100 and 15-25 mM NH 4 PBS of OH, preferably 1% Triton-100 and 20mM NH 4 In some embodiments of the invention, the removal of genetic material DNA is performed by treating the extracellular matrix membrane with 100-200U/ml DNase; preferably, the extracellular matrix membrane is treated with 200U/ml DNase in PBS for 2h at 37 ℃.
The subconjunctival fibroblast acellular matrix prepared by the invention is a colorless transparent film (shown as A in figure 1), has certain mechanical strength and stretchability, and can resist surgical needle and line suturing (shown as B in figure 1). The subconjunctival fibroblast acellular matrix mainly comprises fibronectin and collagen, can provide a growth environment which is easy to adhere and beneficial to proliferation for conjunctival epithelial cells, and is beneficial to functional conjunctival reconstruction.
Early studies found that adipose mesenchymal stem cell-derived decellularized matrix can be used for ocular surface injury repair. In the present invention, the thickness of the constructable subconjunctival fibroblast acellular matrix is significantly better than that of adipose mesenchymal stem cell-derived acellular matrix (as shown in fig. 1C and D).
In the present invention, the Young's modulus of the subconjunctival fibroblast decellularized matrix is similar to that of normal conjunctival tissue (as shown by E in FIG. 1).
In the invention, the subconjunctival fibroblast acellular matrix can obviously promote the growth and proliferation of conjunctival epithelial cells.
In the present invention, the subconjunctival fibroblast decellularized matrix is effective in maintaining conjunctival epithelial stem cell characteristics, including but not limited to maintaining expression of conjunctival epithelial stem markers p75, p63 and ABCG2, inhibiting expression of conjunctival differentiated keratin CK 4.
In the invention, the subconjunctival fibroblast acellular matrix can maintain the activity of a notch pathway and the inhibition state of a wnt pathway in the in-vitro differentiation process of conjunctival epithelial stem cells, including but not limited to maintaining the expression of NICD and inhibiting the expression of beta-catenin/active-beta-catenin.
In the invention, the subconjunctival fibroblast acellular matrix can obviously promote the repair of large-area conjunctival defects.
In the invention, the subconjunctival fibroblast acellular matrix can obviously promote the expression of goblet cells and mucin after conjunctival defect repair. Goblet cells act as unicellular mucinous glands specifically expressed by the conjunctiva and secrete mucins important for maintaining ocular tear film stability.
In the invention, the subconjunctival fibroblast acellular matrix can obviously reduce the fibrosis and scarring level of the matrix layer after conjunctival defect repair.
Further, functional conjunctival remodeling actions of subconjunctival fibroblast acellular matrix include, but are not limited to, blepharoconjunctival adhesions caused by acid-base chemical injury, thermal burn, steven-Johnson syndrome, ocular pemphigoid, and the like. In the invention, the functional conjunctival reconstruction of the subconjunctival fibroblast acellular matrix is mainly realized by maintaining the stem cell characteristics of conjunctival epithelial stem cells, and the good proliferation activity of the conjunctival epithelial cells, the normal differentiation of goblet cells and the secretion of mucin are all dependent on the maintenance of the conjunctival epithelial stem cell characteristics.
According to the invention, a rabbit conjunctival large-area defect model is prepared, conjunctival defects after blepharoconjunctival separation are simulated, and subconjunctival fibroblasts are adopted to remove cell matrixes and combine conjunctival epithelial stem cells to repair the defects. Through HE staining, PAS staining, masson staining and immunofluorescence staining detection on the defect repair completion area, the subconjunctival fibroblast decellularized matrix can obviously accelerate the repair speed of conjunctival defects, the repair of the subconjunctival tissue has the most complete structure of the composite epithelium, the expression of goblet cells and mucin is rich, the subconjunctival matrix fibrosis and scarring level is low, and the normal stem cell distribution of high expression of basal layer notch channels and low expression of wnt/beta-catenin channels in the composite epithelium is maintained.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts.
Example 1: preparation of subconjunctival fibroblast acellular matrix
1. Experimental method
Preparation of subconjunctival fibroblast acellular matrix comprises acquisition of subconjunctival fibroblasts, in vitro culture expansion and preparation of acellular matrix.
Subconjunctival fibroblasts were obtained: the human conjunctival tissue was taken from the eye tissue bank of the ninth human hospital affiliated to the university of Shanghai traffic medical college, all from voluntary donations under informed consent of the patients and passed ethical examination. Conjunctival tissue was rinsed several times in penicillin-containing PBS and incubated with dispese II neutral protease (5 mg/ml, roche Diagnostics GmbH, manheim, germany) for 14 hours at 4 ℃. The next day the epithelial layer was scraped off with sterile forceps, the subconjunctival matrix layer was retained, and digested with collagenase a (0.1%, roche Diagnostics GmbH) at 37 ℃ for 18 hours until dispersed as a single cell suspension which was filtered to remove impurities using a 70 μm cell filter.
In vitro culture and amplification: the cell suspension was centrifuged and inoculated into a 10cm dish, and the cell suspension was amplified by external culture with DMEM (Gibco) supplemented with 10% fetal bovine serum. The cultured subconjunctival fibroblasts were inoculated into a six-well culture plate, and after culturing until the cells were fused, vitamin C (Sigma-Aldrich) was added to the culture solution at a concentration of 50ug/ml to promote secretion of extracellular matrix. The culture was continued in vitro for about 2 weeks, during which time the culture medium was changed every 2 days.
Preparation of acellular matrix: after culturing to a suitable thickness of extracellular matrix to form a membrane, a membrane containing 1% Triton-100 (Sigma-Aldrich) and 20mM NH was used 4 PBS of OH (Sigma-Aldrich) was used as a decellularization solution, decellularized for 5 minutes to disrupt cell structure, and extracellular matrix membrane was treated with 200U/ml DNase-containing PBS at 37℃for 2 hours to remove genetic material DNA. Washing with PBS for 3 times, and storing at 4deg.C.
2. Analysis of results
The prepared subconjunctival fibroblast acellular matrix membrane has good transparency, certain mechanical strength and stretchability, and can withstand surgical needle and line suturing (figures 1A-B).
Example 2: mechanical property detection
1. Test method
The Young's modulus of the prepared subconjunctival fibroblast acellular matrix membrane sheet is detected by an atomic force microscope, and normal conjunctival tissues are used as a control group. 10 positions which are uniformly distributed are randomly selected for detection of each membrane, and 3 membranes cultured in different batches are detected for statistical analysis.
2. Test results
The young's modulus of subconjunctival fibroblast decellularized matrix was similar to normal conjunctival tissue with no statistical difference (fig. 1E).
Example 3: immunofluorescent staining
1. Dyeing method
Subconjunctival fibroblasts decellularized matrix or conjunctival epithelial stem cells seeded on subconjunctival fibroblasts decellularized matrix were fixed with 4% paraformaldehyde for 20 min at room temperature, membrane broken by permeation in PBS containing 0.3% Triton X-100 (Sigma-Aldrich) for 15 min, and then blocked with 5% donkey serum at room temperature for 1 hr. Primary antibodies against FN (Santa Cruz), COL1A1 (Abcam), COL3A (Abcam), p75 (Cell Signaling Technology), p63 (Abcam), ABCG2 (Abcam), CK4 (Abcam) were used for overnight incubation at 4 ℃. The next day the primary antibody was washed off, the samples incubated with Alexa fluorescent conjugated secondary antibody for 1 hour at room temperature, after extensive washing, stained with DAPI for 10 minutes at room temperature and photographed under a fluorescent microscope.
2. Analysis of results
Subconjunctival fibroblasts were completely decellularized with the major components fibronectin and collagen (fig. 1F). Fluorescent staining of conjunctival epithelial cells seeded on subconjunctival fibroblast decellularized matrix expressed conjunctival epithelial stem cell marker p63 and specific keratin CK4 was observed and counted (fig. 2G-J). The subconjunctival fibroblast decellularized matrix was able to maintain the expression of stem cell markers p63 and ABCG2 during in vitro culture, inhibiting the expression of differentiated keratin CK4 (fig. 3).
Example 4: dead and alive dyeing
1. Dyeing method
Conjunctival epithelial cells were seeded at a density of 10 ten thousand per well in 24-well plates, blank and plated with subconjunctival fibroblast decellularization matrix, respectively, and two dead and alive staining reagents (Invitrogen) were added at concentrations of 1:10000 and 1:2000, respectively, incubated at 37℃for 15 minutes in the absence of light, and after washing off the staining reagents with PBS, photographed under a fluorescent microscope and counted.
2. Analysis of results
Conjunctival epithelial cells survived well on subconjunctival fibroblast decellularized matrix with no statistical differences in the proportion of dead cells compared to the blank (fig. 2B-C).
Example 5: CCK-8 detection
1. Detection method
Conjunctival epithelial cells were cultured in 6-well plates, blank and plated with subconjunctival fibroblast decellularized matrix, at a density of 1000 per well, with 6 replicates per subgroup. After cell attachment growth, 200. Mu. L Cell Counting Kit-8 solution (Dojindo, kumamoto, japan) was added to each well and incubated at 37℃for 4h in the absence of light. 100. Mu.L of the culture broth was aspirated every 24 hours and absorbance at 450nm was measured in 96-well plates.
2. Analysis of results
Conjunctival epithelial cells seeded under subconjunctival fibroblasts were good in proliferation, and proliferation rate was superior to that of the blank control group (fig. 2D).
Example 6: edU cell proliferation staining
1. Dyeing method
Conjunctival epithelial cells were inoculated into 24-well culture plates, which were blank and plated with subconjunctival fibroblast decellularized matrix, at a density of 5 ten thousand per well, and cultured, an EdU reagent (bi yun) was added at a ratio of 1:1000, and after further culturing at 37 ℃ for 2 hours, cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, washed with PBS, and incubated at room temperature for 30 minutes under light-shielding conditions, with an EdU mixed staining reagent prepared according to the specification. After washing with PBS, DAPI was added dropwise and stained at room temperature for 10 minutes, and photographs were taken under a fluorescence microscope and counted.
2. Analysis of results
Conjunctival epithelial cells seeded under conjunctival fibroblast decellularized matrix proliferated well, and the positive rate of EdU staining was superior to that of the blank control group (FIGS. 2E-F).
Example 7: construction of rabbit conjunctival defect model and subconjunctival fibroblast decellularized matrix transplantation
1. Experimental method
New Zealand white rabbits weighing about 2kg were anesthetized by intramuscular injection of sultai (approved by the ethical committee of experimental animals). The conjunctiva of the eyes was fully exposed, the defect area was marked with a sterile trephine of about 8mm diameter, and the conjunctiva was completely sheared with scissors to the scleral layer. The conjunctival defect site was marked by staining with lissamine green dye (Sigma-Aldrich) and photographing to record the size of the original defect. The animal experiment groups are as follows: blank control NC, transplanted subconjunctival fibroblast decellularized matrix DM, transplanted subconjunctival fibroblast decellularized matrix composite conjunctival epithelial cell DM-CjECs, transplanted subconjunctival fibroblast decellularized matrix composite conjunctival epithelial stem cell DM-p75++, and positive control PC. The subcombrane fibroblast acellular matrix is sutured to conjunctival defect by 10-0 sterile silk thread, and the levofloxacin eye drops are used for anti-infection and analgesic treatment after operation. Lissamine green staining is carried out on the defect part after one week and two weeks after operation, and the defect repair condition is recorded by photographing.
2. Analysis of results
Subconjunctival fibroblast decellularized matrix can significantly promote conjunctival injury repair, with the fastest repair rate in the DM-p75++ group (fig. 4A-B).
Example 8: HE/PAS/Masson staining
1. Experimental method
At the end of the second week animals were euthanized and each group of conjunctival tissue from the defect repair completed area was fixed in 4% paraformaldehyde and paraffin embedded tissue sections were performed. The paraffin sections of each group were subjected to hematoxylin-eosin staining, glycogen staining and masson staining, respectively, and tissue photographs were taken under an optical microscope.
2. Analysis of results
The subconjunctival fibroblast acellular matrix has the best effect on conjunctival injury repair, and the repair of the DM-p75++ group is obviously superior to other groups in terms of the morphology of the part of the multi-layered epithelial cells, the tissue integrity, the expression of goblet cells and mucin and the repair of the matrix layer (figure 4C).
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (8)
1. Use of subconjunctival fibroblast acellular matrix in the preparation of a conjunctival structure and function repair product which is a functional conjunctival remodeling biomaterial for the treatment of blepharoconjunctival adhesions; the preparation method of the biological material comprises the steps of obtaining subconjunctival fibroblasts, in-vitro culture and amplification and preparation of acellular matrixes; the conjunctival structure and function repair product has at least one of the following functions:
1) Promote the growth and proliferation of conjunctival epithelial cells;
2) Maintaining conjunctival epithelial stem cell characteristics;
3) Maintaining the activity of the notch pathway and the inhibition state of the wnt pathway in the in vitro differentiation process of conjunctival epithelial stem cells;
4) Promoting repair of large area conjunctival defects;
5) Promoting the expression of goblet cells and mucin after repair of conjunctival defects;
6) Reducing the fibrosis and scarring level of the matrix layer after conjunctival defect repair.
2. The use according to claim 1, further comprising one or more of the following features:
(2a) Maintaining conjunctival epithelial stem cell characteristics including maintaining expression of the dry markers p75, p63 and ABCG2, inhibiting expression of conjunctival differentiated keratin CK 4;
(3a) Maintains the expression of the notch pathway NICD and inhibits the expression of the wnt pathway beta-catenin/active-beta-catenin.
3. The use according to claim 1, wherein the obtaining subconjunctival fibroblasts comprises the specific steps of: washing conjunctival tissue, incubating with protease, scraping off epithelial layer, retaining subconjunctival matrix layer, digesting in digestive liquid until dispersed into single cell suspension, filtering to remove impurities, centrifuging, and retaining precipitate to obtain subconjunctival fibroblasts.
4. The use according to claim 3, further comprising one or more of the following features:
a) The washing medium is PBS buffer solution containing penicillin;
b) The incubation temperature is 4 ℃, and the incubation time is 10-18 h;
c) The protease is a Dispase II neutral protease with the concentration of 4-6 mg/mL;
d) The digestive juice is collagenase A accounting for 0.1 to 0.2 percent;
e) The digestion temperature is 37 ℃ and the digestion time is 16-20 h.
5. The use according to claim 1, wherein the in vitro culture amplification comprises the following specific steps: resuspension of the obtained subconjunctival fibroblasts with a culture solution, inoculating the subconjunctival fibroblasts into a culture dish for culture, inoculating the cultured subconjunctival fibroblasts into a culture plate, culturing until the cells are fused, adding vitamin C into the culture solution, and continuously culturing in vitro.
6. The use according to claim 5, further comprising one or more of the following features:
the concentration of vitamin C is 10-20 mug/mL;
ii) the culture medium is replaced every several days during the in vitro continuous culture process.
7. The use according to claim 1, wherein the preparation of the acellular matrix comprises the following specific steps: culturing until extracellular matrix forms membrane with proper thickness, performing decellularization treatment, removing genetic material DNA, and cleaning with PBS to obtain the biological material.
8. The use of claim 7, further comprising one or more of the following features:
(1) The decellularized solution used in the decellularization treatment contains 0.5-1% Triton-100 and 15-25 mM NH 4 PBS of OH;
(2) The specific operation of removing the genetic material DNA is to treat extracellular matrix membranes by using 100-200U/ml DNase.
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