WO2015188664A1 - 一种角膜的脱细胞方法、脱细胞角膜基质及其制备方法 - Google Patents

一种角膜的脱细胞方法、脱细胞角膜基质及其制备方法 Download PDF

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WO2015188664A1
WO2015188664A1 PCT/CN2015/077422 CN2015077422W WO2015188664A1 WO 2015188664 A1 WO2015188664 A1 WO 2015188664A1 CN 2015077422 W CN2015077422 W CN 2015077422W WO 2015188664 A1 WO2015188664 A1 WO 2015188664A1
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cornea
decellularizing
water
reagent
corneal stroma
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PCT/CN2015/077422
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English (en)
French (fr)
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王维博
张斌
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深圳艾尼尔角膜工程有限公司
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Priority claimed from CN201410264542.XA external-priority patent/CN104001215B/zh
Priority claimed from CN201410265612.3A external-priority patent/CN104001217B/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials

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  • the invention relates to the field of medical instruments, in particular to a method for decellularizing a cornea, an acellular corneal stroma and a preparation method thereof.
  • Corneal disease is the second blind eye disease worldwide and is increasing at a rate of 1.5 to 2 million cases per year. Corneal transplantation is currently the only effective method for treating corneal blindness, but the extreme lack of donor corneal source restricts the development of corneal transplantation.
  • the corneal stroma of corneal stroma such as animals, has similar tissue structure, biophysical properties, and optical properties to human corneal stroma, but strong rejection after xenografts hinders the clinical application of heterogeneous corneas.
  • stromal cells in the corneal stroma are the main antigens causing matrix-type rejection, while collagen fibers as a corneal stroma structure are highly conserved among germ lines, have low antigenicity, and are not after cell-free xenogeneic corneal transplantation. A rejection reaction occurs. Therefore, the complete removal of the stromal cells of the animal to make it a cell-free corneal stroma may be an ideal substitute for human corneal stroma.
  • the main object of the present invention is to provide a corneal decellularization method, a decellularized corneal stroma and a preparation method thereof, which can completely preserve the corneal matrix collagen structure while removing the immunogenic cell components and soluble proteins in the cornea, thereby improving the acellular corneal stroma. Biocompatibility and resistance to degradation.
  • the present invention provides a method for decellularizing a cornea, comprising the steps of:
  • the picked cornea is placed in water and shaken to remove the epithelial layer;
  • steps B and C alternately to cause stromal cells and endothelial cells to fall off;
  • the decellularization reagent contains NaCl and EDTA.
  • the decellularization reagent is a mixture of a NaCl solution having a concentration of 2 to 5 mol/L and an EDTA solution having a concentration of 0.5 to 5 g/L.
  • the decellularization reagent has a pH of 7.0 to 7.4; and the decellularization reagent and/or water has a temperature of 2 to 37 °C.
  • the concentration of the NaCl solution is 3 mol/L, and the concentration of the EDTA solution is 3 g/L.
  • the oscillation time of the step A is 5 to 10 h, and the water is replaced every 1 to 2 hours.
  • the oscillation time of the step B is 1 to 2 h.
  • the oscillation time of the step C is 1 to 2 h.
  • the decellularization reagent or water is replaced every 1 to 2 hours during the duration of the step D.
  • the oscillating rotation speed of the step A, the step B, the step C or the step D is 100 to 200 rpm.
  • the invention provides a method for preparing a decellularized corneal stroma comprising the following steps:
  • the extracted cornea is placed in water and shaken to remove the epithelial layer;
  • the decellularizing reagent contains NaCl and EDTA, and the dehydrating agent contains glycerin.
  • the decellularization reagent is a mixture of a NaCl solution having a concentration of 2 to 5 mol/L and an EDTA solution having a concentration of 0.5 to 5 g/L.
  • the decellularization reagent has a pH of 7.0 to 7.4; and the decellularization reagent and/or water has a temperature of 2 to 37 °C.
  • the oscillation time of the step A is 5 to 10 hours, during which the water is replaced every 1 to 2 hours; the oscillation time of the step B is 40 to 70 hours, and the decellularization is alternately changed every 1 to 2 hours.
  • Reagent or water is used to determine whether the water is replaced every 1 to 2 hours.
  • the volume ratio of glycerin in the dehydrating agent is 50 to 90%.
  • the animal cornea was washed with a concentration of 2 to 5 g/L sodium bicarbonate solution for 2 to 10 min.
  • the water was replaced with a sodium hypochlorite solution having a concentration of 0.02 to 2 Torr and allowed to stand for 10 to 60 minutes.
  • step D the following steps are further included:
  • the decellularized corneal stroma was irradiated with cobalt-60.
  • embodiments of the present invention provide a decellularized corneal stroma prepared by the above method for preparing a decellularized corneal stroma.
  • the decellularized corneal stroma has a thickness of 150 to 550 ⁇ m and a diameter of 0.5 to 1.2 cm.
  • the method for decellularizing the cornea adopts a physical decellularization method, and repeatedly changes the tissue osmotic pressure by alternating use of the decellularizing reagent and water, thereby effectively removing corneal cell components and soluble proteins which are easy to cause an immune reaction, and at the same time,
  • the reagents used in the present invention are mild and are capable of completely retaining the corneal matrix collagen structure.
  • the preparation method of the decellularized corneal stroma can repeatedly remove the immunogenic cell components and soluble proteins in the corneal stroma by the repeated use of the decellularizing reagent and water, and can obtain the intact decellularized cells.
  • Corneal stroma In addition, through the cutting process, a decellularized corneal stroma containing only the front elastic layer and the stromal layer can be prepared. Before the clinical use, the doctor only needs to select the corresponding specification model according to the size and depth of the patient's wound surface, and obtain a suitable diameter for the trephine. The acellular corneal stroma can be used. In this way, not only is the operation simple, but pollution can be avoided.
  • 1 is a comparative photograph of hematoxylin-eosin (HE) staining before and after decellularization of an embodiment of the corneal decellularization method of the present invention, wherein 1A is a hematoxylin before decellularization treatment. - E-red (HE) staining photo, 1B is a photo of hematoxylin-eosin (HE) staining after decellularization;
  • HE hematoxylin-eosin
  • 2 is a comparison diagram of transmission electron micrographs before and after decellularization treatment of an embodiment of the corneal decellularization method of the present invention, wherein 2A is a transmission electron micrograph before decellularization, and 2B is a decellularized treatment. Transmission electron micrograph photo.
  • Fig. 3 is a comparative photograph of hematoxylin-eosin (HE) staining after 60 days of transplantation into rabbits using the corneal decellularization method of the present invention, wherein 3A is before transplantation and without decellularization. Photograph of hematoxylin-eosin (HE) staining treated, and 3B is a photograph of hematoxylin-eosin (HE) staining after transplantation by decellularization;
  • 4 is a comparative photograph of hematoxylin-eosin (HE) staining before and after decellularization treatment of an acellular corneal stroma according to an embodiment of the present invention, wherein 4A is a hematoxylin before decellularization treatment.
  • HE hematoxylin-eosin
  • 4B is a photo of hematoxylin-eosin (HE) staining after decellularization
  • Figure 5 is a comparison of transmission electron micrographs before and after decellularization treatment of an acellular corneal stroma according to an embodiment of the present invention, wherein 5A is a transmission electron micrograph before decellularization, and 5B is a decellularized treatment. Transmission electron micrograph photo.
  • the invention provides a method for decellularizing a cornea, and in the first embodiment, the method for decellularizing the cornea comprises the following steps:
  • the extracted animal cornea was placed in water, and then shaken at a rotation speed of 200 rpm (revolutions per minute) for 5 hours (hours) in a constant temperature oscillator, during which water was changed every 1 hour to cause the epithelial layer to fall off.
  • the animal includes animals such as pigs, cows, and sheep, and the water used includes water for injection, pure water, physiological saline, and the like.
  • the collagen of the corneal stroma is swollen, the cornea gradually absorbs water and becomes thick, the corneal stromal cells and endothelial cells gradually rupture, and the corneal epithelial layer gradually falls off.
  • the cornea is in the shape of a flying saucer, and the corneal epithelial layer is completely detached.
  • the cornea was placed in a decellularization reagent containing HCl at a concentration of 5 mol/L NaCl and 5 g/L, and then oscillated for 2 h at 100 rpm in a thermostated shaker.
  • a decellularization reagent containing HCl at a concentration of 5 mol/L NaCl and 5 g/L
  • the hypertonic solution dehydrating agent Through the treatment of the hypertonic solution dehydrating agent, components such as corneal cell debris, heteroproteins, and polysaccharides which are susceptible to an immune reaction are precipitated.
  • the cornea was placed in water and shaken in a constant temperature oscillator at 200 rpm for 1 h.
  • the corneal cells were again in a hypotonic environment, and the unbroken cells were broken.
  • steps B and C Repeat steps B and C repeatedly for 40 hours to cause stromal cells and endothelial cells to fall off.
  • the physical osmotic pressure is repeatedly changed, thereby gently removing the immunogenic cell components and soluble proteins in the corneal stroma.
  • Decellularized corneal stroma Decellularized corneal stroma.
  • the NaCl solution is used to provide a hypertonic solution environment, and components apt to cause an immune reaction are precipitated;
  • the EDTA solution is used to protect the collagen structure of the corneal stroma.
  • step A the corneal epithelial layer is rapidly removed, and the method of repeatedly changing the osmotic pressure of the tissue by alternating use of the decellularizing reagent and water, the method is mild, and the immunogenic cell component and the soluble protein in the corneal stroma can be gradually removed, thereby facilitating the stepwise removal of the tissue osmotic pressure in the corneal stroma.
  • a well-derived corneal stroma with good biocompatibility and resistance to degradation is obtained.
  • the method of decellularizing the cornea comprises the following steps:
  • the extracted animal cornea was placed in water, and then shaken in a constant temperature oscillator at a rotation speed of 100 rpm (revolution per minute) for 10 hours, during which water was changed every 2 hours to cause the epithelial layer to fall off.
  • the cornea was placed in a decellularization reagent containing HCl at a concentration of 2 mol/L and 0.5 g/L, and then shaken in a constant temperature shaker at 200 rpm for 1 h.
  • the cornea was placed in water and then oscillated for 2 h at 100 rpm in a thermostated shaker.
  • steps B and C Repeat steps B and C repeatedly for 70 hours to cause stromal cells and endothelial cells to fall off.
  • the decellularization reagent has a pH of 7.0; the decellularization reagent and/or water has a temperature of 37 °C.
  • the concentration of the NaCl solution was 3 mol/L, and the concentration of the EDTA solution was 3 g/L.
  • the pH of the decellularizing reagent is 7.4 as compared with the third embodiment; the temperature of the decellularizing reagent and/or water is 25 °C.
  • the temperature of the decellularizing reagent and/or water is 2 ° C as compared with the fourth embodiment.
  • the present invention provides a method for preparing a decellularized corneal stroma.
  • the method for preparing the decellularized corneal stroma comprises the following steps:
  • the extracted cornea is placed in a reagent bottle filled with water, and then shaken in a constant temperature oscillator at a rotation speed of 200 rpm (revolution per minute) for 5 hours (hours), during which water is changed every 1 hour.
  • the animal includes animals such as pigs, cows, and sheep, and the water used includes water for injection, pure water, physiological saline, and the like.
  • the collagen of the corneal stroma is swollen, the cornea gradually absorbs water and becomes thick, and the corneal stromal cells and endothelial cells gradually rupture.
  • the corneal epithelial layer gradually falls off.
  • the cornea is in the shape of a flying saucer, and the corneal epithelial layer is completely detached.
  • the water was replaced with a sodium hypochlorite solution having a concentration of 0.02 Torr for virus elimination treatment. After standing for 60 minutes, the sodium hypochlorite solution was poured out, and the residual sodium hypochlorite was washed away with water.
  • a dehydrating agent having a volume ratio of glycerin of 90% was added, and the mixture was shaken at a temperature of 25 ° C for 30 minutes (minutes) to obtain a decellularized cornea. Since the cornea after the decellularization treatment has a thickened thickness due to water absorption, the dehydration agent containing glycerin is used to remove the moisture in the cornea, and the thickness of the cornea can be made to the original thickness.
  • the obtained decellularized cornea was washed with a 2 g/L sodium bicarbonate solution for 10 min.
  • the glycerin content in the cornea can be reduced to a reasonable range, and the cornea can contain a certain amount of water.
  • the acellular cornea is cut to obtain an acellular corneal stroma, which contains only the front elastic layer and the matrix layer.
  • the resulting decellularized corneal stroma was sterilized by irradiation with cobalt-60 at a dose of 5 KGy.
  • the method of repeatedly changing the tissue osmotic pressure can gradually remove the immunogenic cell components and soluble proteins in the corneal stroma, thereby obtaining a complete decellularized corneal stroma.
  • a decellularized corneal stroma containing only the front elastic layer and the stromal layer can be prepared, so that the doctor only needs to select the corresponding specification model according to the size and depth of the patient's wound before clinical use, and obtain a suitable diameter by the trephine.
  • the acellular corneal stroma can be used. In this way, not only is the operation simple, but pollution can be avoided.
  • the extracted cornea is placed in a reagent bottle filled with water, and then shaken in a constant temperature oscillator at a speed of 150 rpm (revolution per minute) for 10 hours (hours), during which water is changed every 2 hours.
  • a constant temperature oscillator at a speed of 150 rpm (revolution per minute) for 10 hours (hours), during which water is changed every 2 hours.
  • the animal includes animals such as pigs, cows, and sheep, and the water used includes water for injection, pure water, physiological saline, and the like.
  • a decellularization reagent containing a concentration of 2 mol/L NaCl and 0.5 g/L of EDTA was added to the reagent bottle containing the animal cornea, and then oscillated at 100 rpm for 2 h in a thermostated shaker, The decellularization reagent was replaced with the water being oscillated in a constant temperature oscillator at 100 rpm for 2 h, so that the two solutions were used alternately and co-oscillated for 70 h.
  • a dehydrating agent having a volume ratio of glycerin of 50% was added, and the mixture was allowed to stand at a temperature of 2 ° C for 60 minutes (minutes) to obtain a decellularized cornea.
  • the obtained decellularized cornea was washed with a 5 g/L sodium bicarbonate solution for 2 min.
  • the acellular cornea is cut to obtain an acellular corneal stroma, which contains only the front elastic layer and the matrix layer.
  • the resulting decellularized corneal stroma was sterilized by irradiation with cobalt-60 at a dose of 25 KGy.
  • the pH of the decellularizing reagent was 7.0
  • the temperature of the decellularizing reagent and/or water was 37 °C.
  • the pH of the decellularizing reagent is 7.4 as compared with the third embodiment; the temperature of the decellularizing reagent and/or water is 2 °C.
  • the present invention also provides a decellularized corneal stroma.
  • the decellularized corneal stroma prepared by the aforementioned preparation method has a thickness of 150 ⁇ m and a diameter of 0.5 cm.
  • a cornea containing only the pre-corneal elastic layer and the matrix layer can be prepared.
  • the doctor does not need to treat the thickness of the material.
  • the doctor only needs to select the corresponding specification model according to the size and depth of the patient's wound surface, and the diarrhea can obtain the appropriate diameter of the acellular corneal stroma. In this way, not only is the operation simple, but pollution can be avoided.
  • the wound surface can be healed rapidly due to the smooth surface of the acellular corneal stroma prepared by the method.
  • the acellular corneal stroma prepared by the aforementioned preparation method has a thickness of 550 ⁇ m and a diameter of 1.2 cm.
  • the porcine acellular corneal stroma prepared according to the above method and the subsequent method of the prior art was observed by morphological HE staining and ultrastructural transmission electron microscopy. The results show:
  • FIG. 1A is a photograph of hematoxylin-eosin (HE) staining before decellularization, in which the normal cornea has intact full-thickness corneal epithelial layer and monolayer corneal endothelial cells, and a large number of stromal cells exist in the corneal stroma;
  • 1B is a photograph of hematoxylin-eosin (HE) staining after decellularization, in which no intact cells, such as cortex and endothelial cells, were observed in the cornea after decellularization.
  • HE hematoxylin-eosin
  • 2A is a transmission electron micrograph of the natural porcine cornea before decellularization
  • 2B is a transmission electron micrograph after decellularization
  • 2B shows that the collagen fibers in the decellularized cornea are arranged neatly, compared with 2A, decellularized
  • the structure of the corneal matrix collagen is consistent with that of the native corneal collagen, indicating that the decellularization process does not cause damage to the corneal collagen structure.
  • 3A is a photograph of hematoxylin-eosin (HE) staining before transplantation and without decellularization, in which the normal cornea has intact full-thickness corneal epithelial layer and monolayer corneal endothelial cells, and there is a corneal stroma in the cornea. A large number of stromal cells; 3B is a photograph of hematoxylin-eosin (HE) staining after decellularization treatment, in which the cells were decellularized and then transplanted to rabbits for 60 days, and the corneal corneal matrix and rabbit cornea were healed.
  • HE hematoxylin-eosin
  • the porcine acellular corneal stroma prepared according to the above preparation method was observed by morphological HE staining and ultrastructural transmission electron microscopy.
  • FIG. 4A is a photograph of hematoxylin-eosin (HE) staining before decellularization, wherein the normal cornea has intact full-thickness corneal epithelial cells and monolayer corneal endothelial cells, and a large number of stromal cells exist in the corneal stroma; 4B is a photo of hematoxylin-eosin (HE) staining after decellularization, in which no intact cells, such as epithelial cells and endothelium, were observed in the cornea after decellularization.
  • HE hematoxylin-eosin
  • 5A is a transmission electron micrograph of the natural porcine cornea before decellularization
  • 5B is a transmission electron micrograph after decellularization
  • 5B shows that the collagen fibers in the decellularized cornea are arranged neatly, compared with Fig. 5A, decellularized.
  • the corneal matrix collagen structure after treatment was consistent with the natural corneal collagen structure, indicating that the decellularization process did not cause damage to the corneal collagen structure.

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Abstract

一种角膜的脱细胞方法、一种脱细胞角膜基质及其制备方法，所述脱细胞方法包括以下步骤：A、将摘取的动物角膜置于水中，振荡处理，以使上皮层脱落；B、将所述角膜置于脱细胞试剂中，振荡处理；C、将所述角膜置于水中，振荡处理；D、交替重复步骤B和步骤C，以使基质细胞和内皮细胞脱落；其中所述脱细胞试剂含有NaCI和EDTA。所述角膜的脱细胞方法能够快速且温和地去除角膜上皮层和免疫原细胞成分，从而得到完好的脱细胞角膜基质，所述脱细胞角膜基质具有良好的生物相容性和抗降解能力。

Description

一种角膜的脱细胞方法、脱细胞角膜基质及其制备方法
本申请要求于2014年06月13日提交中国专利局、申请号为201410265612.3、发明名称为“一种角膜的脱细胞方法”以及于2014年06月13日提交中国专利局、申请号为201410264542.X、发明名称为“一种脱细胞角膜基质及其制备方法”的两项专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及医疗器械领域，尤其涉及一种角膜的脱细胞方法、脱细胞角膜基质及其制备方法。
背景技术
角膜病是全球范围内第二致盲眼病，并且以每年150～200万病例的速度递增。角膜移植是目前治疗角膜盲唯一有效的方法，但供体角膜来源的极端匮乏制约着角膜移植的开展。角膜基质如动物等动物的角膜基质具有与人角膜基质类似的组织结构、生物物理特性和光学特性，但异种移植后强烈的排斥反应阻碍了异种角膜在临床上的应用。近年来的研究发现，角膜基质内的基质细胞是引起基质型排斥反应的主要抗原，而作为角膜基质架构的胶原纤维在种系间高度保守，抗原性很低，无细胞的异种角膜移植后不产生排斥反应。因此，将动物的基质细胞完全脱去，使之成为无细胞的角膜基质可能是人角膜基质的理想替代物。
但现有技术生产的无细胞角膜基质在脱细胞过程中，采用的是胰酶、EDTA(乙二胺四乙酸)等溶液以及Triton-X100(聚乙二醇辛基苯基醚)、脱氧胆酸钠、SDS(十二烷基磺酸钠)等表面活性剂，此类方法虽然能去除细胞成分，但反应剧烈，对角膜基质破坏严重，其中使用胰酶处理易导致角膜基质降解，使用表面活性剂易导致表面活性剂残留，产生细胞毒性，在移植后容易引起毒性反应，不能达到良好的生物相容性。
发明内容
本发明的主要目的在于提供一种去除角膜中免疫原细胞成分和可溶性蛋白的同时能够完整保留角膜基质胶原结构的角膜脱细胞方法、脱细胞角膜基质及其制备方法，从而提高脱细胞角膜基质的生物相容性和抗降解能力。
为了实现上述目的，一方面，本发明提供一种角膜的脱细胞方法，包括以下步骤：
A、将摘取的动物角膜置于水中，振荡处理，以使上皮层脱落；
B、将所述角膜置于脱细胞试剂中，振荡处理；
C、将所述角膜置于水中，振荡处理；
D、交替重复步骤B和步骤C，以使基质细胞和内皮细胞脱落；
其中，所述脱细胞试剂含有NaCl和EDTA。
优选地，所述脱细胞试剂为：浓度为2～5mol/L的NaCl溶液与浓度为0.5～5g/L的EDTA溶液的混合液。
优选地，所述脱细胞试剂的PH值为7.0～7.4；所述脱细胞试剂和/或水的温度为2～37℃。
优选地，所述NaCl溶液的浓度为3mol/L，所述EDTA溶液的浓度为3g/L。
优选地，所述步骤A的振荡时间为5～10h，期间，每隔1～2h更换一次水。
优选地，所述步骤B的振荡时间为1～2h。
优选地，所述步骤C的振荡时间为1～2h。
优选地，在实施所述步骤D的持续时间内，每隔1～2h更换一次脱细胞试剂或水。
优选地，所述步骤A、步骤B、步骤C或步骤D的振荡转速为100～200rpm。
另一方面、本发明提供一种脱细胞角膜基质的制备方法，包括以下步骤：
A、将摘取的动物角膜放入水中，振荡处理，以使上皮层脱落；
B、将水更换为脱细胞试剂，振荡处理，期间，所述脱细胞试剂和水交替使用，以使基质细胞和内皮细胞脱落；
C、加入脱水剂，静置或振荡处理，得到脱细胞角膜；
D、将所述脱细胞角膜进行削切，得到脱细胞角膜基质，所述脱细胞角膜基质只含有前弹力层和基质层；
其中，所述脱细胞试剂含有NaCl和EDTA，所述脱水剂含有甘油。
优选的，所述脱细胞试剂为：浓度为2～5mol/L的NaCl溶液与浓度为0.5～5g/L的EDTA溶液的混合液。
优选的，所述脱细胞试剂的pH值为7.0～7.4；所述脱细胞试剂和/或水的温度为2～37℃。
优选的，所述A步骤的振荡时间为5～10h，期间，每隔1～2h更换一次水；所述B步骤的振荡时间为40～70h，期间，每隔1～2h交替更换一次脱细胞试剂或水。
优选的，所述脱水剂中甘油的体积比为50～90％。
优选的，在所述C步骤之后、D步骤之前还包括以下步骤：
使用浓度为2～5g/L碳酸氢钠溶液清洗所述动物角膜2～10min。
优选的，在所述A步骤之后、B步骤之前还包括以下步骤：
将水更换成浓度为0.02～2‰的次氯酸钠溶液，静置10～60min。
优选的，在所述D步骤之后，还包括以下步骤：
将所述脱细胞角膜基质用钴-60辐照。
另一方面，本发明实施例提供一种脱细胞角膜基质，所述脱细胞角膜基质由上述脱细胞角膜基质制备方法制得。
优选的，所述脱细胞角膜基质的厚度为150～550μm，直径为 0.5～1.2cm。
本发明提供的角膜的脱细胞方法，采用物理脱细胞方法，通过脱细胞试剂和水的交替使用，反复改变组织渗透压，能够有效的去除容易引起免疫反应的角膜细胞成分和可溶性蛋白，同时，本发明所使用的试剂温和，能够完整的保留角膜基质胶原结构。
本发明提供的脱细胞角膜基质的制备方法，通过脱细胞试剂和水的交替使用，反复改变组织渗透压的方法，能逐步去除角膜基质中免疫原细胞成分和可溶性蛋白，从而得到完好的脱细胞角膜基质。此外，通过削切处理，可制备出只含有前弹力层和基质层的脱细胞角膜基质，临床使用前只需医生根据患者创面大小和深度选择对应的规格型号，环钻取得合适的直径大小的脱细胞角膜基质即可使用。这样，不仅操作简单，又能避免污染。
附图说明
图1为本发明的角膜脱细胞方法一实施例的脱细胞处理前和脱细胞处理后的苏木精-伊红(HE)染色照片对比图，其中，1A为脱细胞处理前的苏木精-伊红(HE)染色照片图，1B为脱细胞处理后的苏木精-伊红(HE)染色照片图；
图2为本发明的角膜脱细胞方法一实施例的脱细胞处理前和脱细胞处理后的透射电镜照片对比图，其中，2A为脱细胞处理前的透射电镜照片图，2B为脱细胞处理后的透射电镜照片图。
图3为采用本发明的角膜脱细胞方法进行脱细胞之后，移植给兔的动物实验60天后的苏木精-伊红(HE)染色照片对比图，其中，3A为移植前且未经脱细胞处理的苏木精-伊红(HE)染色照片图，3B为经脱细胞处理移植后的苏木精-伊红(HE)染色照片图；
图4为本发明的脱细胞角膜基质一实施例的脱细胞处理前和脱细胞处理后的苏木精-伊红(HE)染色照片对比图，其中，4A为脱细胞处理前的苏木精-伊红(HE)染色照片图，4B为脱细胞处理后的苏木精-伊红(HE)染色照片图；
图5为本发明的脱细胞角膜基质一实施例的脱细胞处理前和脱细胞处理后的透射电镜照片对比图，其中，5A为脱细胞处理前的透射电镜照片图，5B为脱细胞处理后的透射电镜照片图。
本发明目的的实现、功能特点及优点将结合实施例，参照附图做进一步说明。
具体实施方式
应当理解，此处所描述的具体实施方式为实现本发明目的的最佳实施方式，其仅仅用以解释本发明，并不用于限定本发明。
一方面，本发明提供一种角膜的脱细胞方法，在第一实施例中，所述角膜的脱细胞方法包括以下步骤：
A、将摘取的动物角膜置于水中，然后在恒温振荡器中以200rpm(转/每分钟)的转速振荡处理5h(小时)，期间，每隔1h换一次水，以使上皮层脱落。所述动物包括猪、牛、羊等动物，所用的水包括注射水、纯水、生理盐水等。在振荡处理过程中，角膜基质的胶原蛋白吸水蓬松，角膜逐渐吸水变厚，所述角膜基质细胞和内皮细胞逐渐破裂，角膜上皮层逐渐脱落。在振荡处理结束时，所述角膜呈飞碟状，所述角膜上皮层全部脱落。
B、将所述角膜置于含有浓度为5mol/L的NaCl和5g/L的EDTA的脱细胞试剂中，然后在恒温振荡器中以100rpm的转速振荡处理2h。通过高渗溶液脱水剂的处理，角膜细胞碎片、杂蛋白和多糖等易引起免疫反应的成分被析出。
C、将所述角膜置于水中，在恒温振荡器中以200rpm的转速振荡处理1h，所述角膜细胞再次处于低渗环境中，使未涨破的细胞破碎。
D、交替重复步骤B和步骤C累计40h，以使基质细胞和内皮细胞脱落。这样，通过所述含有NaCl与EDTA的混合液的脱细胞试剂与水的交替作用，以物理的方法反复改变组织渗透压，从而温和地去除角膜基质中免疫原细胞成分和可溶性蛋白的方法制备的脱细胞角膜基质。
其中，所述NaCl溶液用于提供高渗溶液环境，使易引起免疫反应的成分被析出；所述EDTA溶液用于保护所述角膜基质的胶原蛋白结构。
通过步骤A快速去除角膜上皮层，以及通过脱细胞试剂和水的交替使用，反复改变组织渗透压的方法，上述方法反应温和，能逐步去除角膜基质中免疫原细胞成分和可溶性蛋白，从而有利于得到完好的具有良好的生物相容性和抗降解能力的脱细胞角膜基质。
第二实施例中，所述角膜的脱细胞方法包括以下步骤：
A、将摘取的动物角膜置于水中，然后在恒温振荡器中以100rpm(转/每分钟)的转速振荡处理10h，期间，每隔2h换一次水，以使上皮层脱落。
B、将所述角膜置于含有浓度为2mol/L的NaCl和0.5g/L的EDTA的脱细胞试剂中，然后在恒温振荡器中以200rpm的转速振荡处理1h。
C、将所述角膜置于水中，然后在恒温振荡器中以100rpm的转速振荡处理2h。
D、交替重复步骤B和步骤C累计70h，以使基质细胞和内皮细胞脱落。
第三实施例中，在第二实施例的基础上，采用以下参数进行实施：
所述脱细胞试剂的pH值为7.0；所述脱细胞试剂和/或水的温度为37℃。
所述NaCl溶液的浓度为3mol/L，所述EDTA溶液的浓度为3g/L。
第四实施例中，与第三实施例相比，所述脱细胞试剂的pH值为7.4；所述脱细胞试剂和/或水的温度为25℃。
第五实施例中，与第四实施例相比，所述脱细胞试剂和/或水的温度为2℃。
另一方面，本发明提供了一种脱细胞角膜基质的制备方法，在第一实施例中，所述脱细胞角膜基质的制备方法包括以下步骤：
A、将摘取的动物角膜，放入装有水的试剂瓶中，然后在恒温振荡器中以200rpm(转/每分钟)的转速振荡处理5h(小时)，期间，每隔1h换一次水，以使上皮层脱落。所述动物包括猪、牛、羊等动物，所用的水包括注射水、纯水、生理盐水等。在振荡处理过程中，角膜基质的胶原蛋白吸水蓬松，角膜逐渐吸水变厚，所述角膜基质细胞和内皮细胞逐渐破裂， 角膜上皮层逐渐脱落。在振荡处理结束时，所述角膜呈飞碟状，所述角膜上皮层全部脱落。
将水更换成浓度为0.02‰的次氯酸钠溶液进行灭病毒处理，静置60min后倒去次氯酸钠溶液，再用水洗去残留的次氯酸钠。
B、在装有所述动物角膜的试剂瓶内加入含有浓度为5mol/L的NaCl和5g/L的EDTA的脱细胞试剂，然后在恒温振荡器中以200rpm的转速振荡处理1h后，将所述脱细胞试剂替换为所述水在恒温振荡器中以200rpm的转速振荡处理1h，如此这两种溶液交替使用，共振荡处理40h。
C、加入甘油所占体积比为90％的脱水剂，在25℃温度下振荡处理30min(分钟)，得到脱细胞角膜。脱细胞处理后的角膜由于吸水，厚度变厚，故使用含有甘油的脱水剂脱去角膜中的水分，能使角膜厚度达到原始厚度。
使用浓度为2g/L碳酸氢钠溶液清洗得到的脱细胞角膜10min。这样，既可以降低角膜中甘油含量至合理范围，又可以使角膜中含有一定水分。
D、将所述脱细胞角膜进行削切，得到脱细胞角膜基质，所述脱细胞角膜基质只含有前弹力层和基质层。
将得到的所述脱细胞角膜基质用用剂量为5KGy的钴-60辐照灭菌。
通过脱细胞试剂和水的交替使用，反复改变组织渗透压的方法，能逐步去除角膜基质中免疫原细胞成分和可溶性蛋白，从而得到完好的脱细胞角膜基质。此外，通过削切处理，可制备出只含有前弹力层和基质层的脱细胞角膜基质，使得临床使用前只需医生根据患者创面大小和深度选择对应的规格型号，环钻取得合适的直径大小的脱细胞角膜基质即可使用。这样，不仅操作简单，又能避免污染。
第二实施例中，包括以下步骤：
A、将摘取的动物角膜，放入装有水的试剂瓶中，然后在恒温振荡器中以150rpm(转/每分钟)的转速振荡处理10h(小时)，期间，每隔2h换一次水，以使上皮层脱落。所述动物包括猪、牛、羊等动物，所用的水包括注射水、纯水、生理盐水等。
将水更换成浓度为2‰的次氯酸钠溶液进行灭病毒处理，静置10min 后倒去次氯酸钠溶液，再用水洗去残留的次氯酸钠。
B、在装有所述动物角膜的试剂瓶内加入含有浓度为2mol/L的NaCl和0.5g/L的EDTA的脱细胞试剂，然后在恒温振荡器中以100rpm的转速振荡处理2h后，将所述脱细胞试剂替换为所述水在恒温振荡器中以100rpm的转速振荡处理2h，如此这两种溶液交替使用，共振荡处理70h。
C、加入甘油所占体积比为50％的脱水剂，在2℃温度下静置处理60min(分钟)，得到脱细胞角膜。
使用浓度为5g/L碳酸氢钠溶液清洗得到的脱细胞角膜2min。
D、将所述脱细胞角膜进行削切，得到脱细胞角膜基质，所述脱细胞角膜基质只含有前弹力层和基质层。
将得到的所述脱细胞角膜基质用用剂量为25KGy的钴-60辐照灭菌。
第三实施例中，在第二实施例的基础上，采用以下参数进行实施：所述脱细胞试剂的pH值为7.0；所述脱细胞试剂和/或水的温度为37℃。
第四实施例中，与第三实施例相比，所述脱细胞试剂的pH值为7.4；所述脱细胞试剂和/或水的温度为2℃。
本发明还提供一种脱细胞角膜基质，在第一实施例中，由前述制备方法制备得到的脱细胞角膜基质厚度为150μm，直径为0.5cm。通过削切处理，可制备出只含有角膜前弹力层与基质层的角膜。临床使用前，无需医生对材料进行厚度的处理，医生只需根据患者创面大小和深度选择对应的规格型号，环钻取得合适的直径大小的脱细胞角膜基质即可使用。这样，不仅操作简单，又能避免污染。此外，由于该方法制备的所述脱细胞角膜基质表面平整，创面能够快速愈合。
在第二实施例中，由前述制备方法制备得到的脱细胞角膜基质厚度为550μm，直径为1.2cm。
图1至图2，以猪角膜为实验对象，采用本发明提供的角膜的脱细胞 方法，得到以下实验结果：
取依据上述方法以及现有技术的后续方法所制得的猪脱细胞角膜基质进行形态学HE染色观察、超微结构透射电镜观察。结果显示：
图1中，1A为脱细胞处理前的苏木精-伊红(HE)染色照片图，其中正常角膜具有完整的全层角膜上皮层和单层角膜内皮细胞，角膜基质内存在大量基质细胞；1B为脱细胞处理后的苏木精-伊红(HE)染色照片图，其中脱细胞处理后，角膜中未观察到任何完整的细胞，如上皮层和内皮细胞。
图2中，2A为脱细胞处理前天然猪角膜的透射电镜照片图，2B为脱细胞处理后的透射电镜照片图，2B显示脱细胞角膜中胶原纤维排列整齐，与2A对比观察，脱细胞处理后的角膜基质胶原结构与天然角膜胶原结构一致，说明脱细胞过程未对角膜胶原结构造成破坏。
图3中，3A为移植前且未经脱细胞处理的苏木精-伊红(HE)染色照片图，其中正常角膜具有完整的全层角膜上皮层和单层角膜内皮细胞，角膜基质内存在大量基质细胞；3B为经脱细胞处理移植后的苏木精-伊红(HE)染色照片图，其中经脱细胞处理然后移植给兔进行动物实验60天后，猪脱细胞角膜基质与兔角膜愈合完好，且猪脱细胞角膜基质完全整合且没有降解，兔基质细胞与上皮层细胞已经长入所述猪脱细胞角膜。由此，可进一步确认采用本发明提供的角膜脱细胞方法未破坏角膜基质胶原结构。
图4至图5，以猪角膜为实验对象，采用本发明提供的脱细胞角膜基质的制备方法，得到以下实验结果：
取依据上述制备方法所制得的猪脱细胞角膜基质进行形态学HE染色观察、超微结构透射电镜观察。
结果显示：
图4中，4A为脱细胞处理前的苏木精-伊红(HE)染色照片图，其中正常角膜具有完整的全层角膜上皮细胞和单层角膜内皮细胞，角膜基质内存在大量基质细胞；4B为脱细胞处理后的苏木精-伊红(HE)染色照片图，其中脱细胞处理后，角膜中未观察到任何完整的细胞，如上皮细胞和内皮 层。
图5中，5A为脱细胞处理前天然猪角膜的透射电镜照片图，5B为脱细胞处理后的透射电镜照片图，5B显示脱细胞角膜中胶原纤维排列整齐，与图5A对比观察，脱细胞处理后的角膜基质胶原结构与天然角膜胶原结构一致，说明脱细胞过程未对角膜胶原结构造成破坏。
上述实施方式仅仅为实现本发明目的的最佳实施方式，本发明并不局限于以上实施方式，在上述实施方式公开的技术内容下，还可以进行各种变化。凡是利用本发明说明书及附图内容所作的等效结构变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (20)

  1. 一种角膜的脱细胞方法，其特征在于，包括以下步骤：
    A、将摘取的动物角膜置于水中，振荡处理，以使上皮层脱落；
    B、将所述角膜置于脱细胞试剂中，振荡处理；
    C、将所述角膜置于水中，振荡处理；
    D、交替重复步骤B和步骤C，以使基质细胞和内皮细胞脱落；
    其中，所述脱细胞试剂含有NaCl和EDTA。
  2. 如权利要求1所述的角膜的脱细胞方法，其特征在于，所述脱细胞试剂为：浓度为2～5mol/L的NaCl溶液与浓度为0.5～5g/L的EDTA溶液的混合液。
  3. 如权利要求1所述的角膜的脱细胞方法，其特征在于，所述脱细胞试剂的pH值为7.0～7.4；所述脱细胞试剂和/或水的温度为2～37℃。
  4. 如权利要求2所述的角膜的脱细胞方法，其特征在于，所述NaCl溶液的浓度为3mol/L，所述EDTA溶液的浓度为3g/L。
  5. 如权利要求1所述的角膜的脱细胞方法，其特征在于，所述步骤A的振荡时间为5～10h，期间，每隔1～2h更换一次水。
  6. 如权利要求1所述的角膜的脱细胞方法，其特征在于，所述步骤B的振荡时间为1～2h。
  7. 如权利要求1所述的角膜的脱细胞方法，其特征在于，所述步骤C的振荡时间为1～2h。
  8. 如权利要求1所述的角膜的脱细胞方法，其特征在于，实施所述步骤D的持续时间为40～70h。
  9. 如权利要求8所述的角膜的脱细胞方法，其特征在于，在实施所述步骤D的持续时间内，每隔1～2h更换一次脱细胞试剂或水。
  10. 如权利要求1所述的角膜的脱细胞方法，其特征在于，所述步骤A、步骤B、步骤C或步骤D的振荡转速为100～200rpm。
  11. 一种脱细胞角膜基质的制备方法，其特征在于，包括以下步骤：
    A、将摘取的动物角膜放入水中，振荡处理，以使上皮层脱落；
    B、将水更换为脱细胞试剂，振荡处理，期间，所述脱细胞试剂和水交替使用，以使基质细胞和内皮细胞脱落；
    C、加入脱水剂，静置或振荡处理，得到脱细胞角膜；
    D、将所述脱细胞角膜进行削切，得到脱细胞角膜基质，所述脱细胞角膜基质只含有前弹力层和基质层；
    其中，所述脱细胞试剂含有NaCl和EDTA，所述脱水剂含有甘油。
  12. 如权利要求11所述的脱细胞角膜基质的制备方法，其特征在于，所述脱细胞试剂为：浓度为2～5mol/L的NaCl溶液与浓度为0.5～5g/L的EDTA溶液的混合液。
  13. 如权利要求12所述的脱细胞角膜基质的制备方法，其特征在于，所述脱细胞试剂的pH值为7.0～7.4；所述脱细胞试剂和/或水的温度为2～37℃。
  14. 如权利要求11所述的脱细胞角膜基质的制备方法，其特征在于，所述A步骤的振荡时间为5～10h，期间，每隔1～2h更换一次水；所述B步骤的振荡时间为40～70h，期间，每隔1～2h交替更换一次脱细胞试剂或水。
  15. 如权利要求11所述的脱细胞角膜基质的制备方法，其特征在于，所述脱水剂中甘油的体积比为50～90％。
  16. 如权利要求11所述的脱细胞角膜基质的制备方法，其特征在于，在所述C步骤之后、D步骤之前还包括以下步骤：
    使用浓度为2～5g/L碳酸氢钠溶液清洗所述动物角膜2～10min。
  17. 如权利要求11所述的脱细胞角膜基质的制备方法，其特征在于，在所述A步骤之后、B步骤之前还包括以下步骤：
    将水更换成浓度为0.02～2‰的次氯酸钠溶液，静置10～60min。
  18. 如权利要求11所述的脱细胞角膜基质的制备方法，其特征在于，在所述D步骤之后，还包括以下步骤：
    将所述脱细胞角膜基质用钴-60辐照。
  19. 一种脱细胞角膜基质，其特征在于，由权利要求11至18中任一项所述的制备方法制备得到。
  20. 如权利要求19所述的脱细胞角膜基质，其特征在于，所述脱细胞角膜基质的厚度为150～550μm，直径为0.5～1.2cm。
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