以纤维蛋白为基质网架的组织工程角膜上皮的构建及移植实验研究
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摘要
目前,对角膜缘干细胞缺损的难治性角膜上皮损伤的治疗大多数采用角膜移植术。但是,由于角膜组织来源于异体或自体,异体来源的角膜上皮会与患者角膜缘中的免疫细胞发生免疫排斥反应,导致手术失败;而自体来源的角膜上皮移植会受到数量限制,又造成健眼的损伤。组织工程学技术的发展和研究成果表明,组织工程人工角膜上皮将成为攻克这一难关的希望所在。本研究在以往研究工作的基础上,深入地进行了组织工程角膜上皮的构建与角膜上皮缺损移植修复方面的研究,为人角膜上皮缺损移植修复的治疗奠定了实验室基础。
本研究采用细胞生物学和组织工程学技术,成功地分离、培养了角膜缘干细胞和角膜上皮细胞,制备了纤维蛋白细胞外基质网架,这不仅为构建组织工程化角膜上皮提供细胞外基质网架,也为其它人工组织、器官的研究提供了新的支架材料。以纤维蛋白为基质网架,构建了组织工程化角膜上皮,经形态学检测结果显示,所构建的人工角膜上皮与天然角膜上皮极为相似,呈现出动态的增殖、分化过程。在此基础上,对全板层角膜上皮缺损动物模型进行了移植实验研究,取得了较好的修复效果。本研究为未来组织工程角膜上皮在临床上的应用提供了实验依据。
The study of stem cells of libus corneae make prominent progress in the therapy of the ocular surface disorder in recent years.Except normal amniotic membrane grafting and corneal transplanting, the development of tissue engineering provide a new therapy method:cultural stem cells of libus corneae transplantation.We used rabbit’s stem cells of libus corneae as semen cells and fibrin- based scaffold to build tissue engineering corneal epithelium. The tissue engineering corneal epithelium was used to repair the rabbit model of stem cells deficiency of libus corneae.The study estab- lished the work for using tissue engineering method to cure the disease caused by stem cells deficiency .
1.The construction of ranbbit stem cells deficiency model
The number of experimental rabbits is 40.According sterile microsurgical procedure step , sheared the right bulbar conjunctiva circle far from 3mm round the limbus cornea,and separated dornea opaca ,and separated 0.08mm superficial layers of limbus cornea,sheared corneal layers and 3mm bulbar conjunctiva.The cornea was dyed afer the operation has been done.The cornea was observed using lit lamp.After on week, the tiny blood vessel was appeared from edge to center,the conjunctival epithelium was also enter the cornea at the smame time . The papilla and the tunica coerulea can be seen at this time .Afer 2 weeks ,the conjunctiva was cover cornea.Several gross blood vessel branches was seen from conjunctiva to cornea,and its area was bigger than the half of the cornea.The round bigger cells was found using the cell blotting method,nucleus: cytoplasm is 1∶4～1∶5,PAS dying is positive .The chalice cells was dyed peach,it was means conjunctiva phenotype.After 4 weeks ,32 qulifified rabbits was choosed for transplantation,and they was separated into for 4 groups: the control group、the experimental groupⅠ( 2weeks after stem cell transplantation)、the experimental groupⅡ( 1 month after stem cell transplantation)、the experimental groupⅢ( 3months after stem cell transplantation).
2.The construction of fibrin scaffold
The fibrinogen powder was dissolved and duluted to 20mg/ml using PBS.The thrombin powder was dissolved and duluted to 10U/ml using PBS,and aprotinin was added into thrombin solution, the final concentration was 163μg/ml.Mixed the equal amount of fibrinogen and thrombin.The fibrin-based scaffold was transparent、semigel、plastic.The average thickness is about 1mm,and it was easily moved out of the culture plate.The diameter of the fibrin-based scaffold was between 70～108μm,the average diameter was 86μm,the average 3D vacant porosity was 70.4% mesured by medical image analysis instrument.All these means the fibrin-based scaffold provided enough space for cell growing.The fibrin-based scaffold was irregular multistrata network 3D structure observed under SEM.
The quality of fibrin-based scaffold was biocompatibility, absorbability,avirulent,fast-shaped ,longlasting,low degradation, and means it’s good bioframe material.
3.The combinational cultivation of rabbit limus corneae tissue and fibrin-based scaffold
Take out of 110μm thickness limbus corneae of the left eye, about 2mm×2mm,and the tissue was digested for 10min using 0.1% trypsin.Then the tissue was shifted on the fibrin-baed scaffold and cultivated.
After24-36 hours,there’s cells migrated from the edge of limbus corneae tissue to the fibrin-based scaffold observed under the inverted microscope.The quality of cells were small,adherent and polygon .After 4 days, some cells growed as net-ballon style.After 6 days,there’s large area of cells ,the cells were ellipse and polygon.After 7 days, the cells were almost occupied the whole plate.After that the cells growed in layers .About 2 weeks it would be 3-4 layers.The cells and the fibrin has been the combination implant,it was slabstone shape.
The cells were different ,some were round and ellipse.Little kytoplam was in it,the bound of the cell membrane was clear,karyoplasmic ratio was above 1.There was tight junction between the cells,desmosome was clear.This means the cells culvated in vitro still had the ultramicrostructure feature of the epithelial cells.
The immunohistochemistry exam was done using anti-cytokeratin K3 McAb AE5. There had brown scattered and clustering granules in the cytoplasm .The positive results showed the cells were in the differentiating status,and there has normal corneal epithelial cells phenotype .
The immunohistochemistry exam was also done using anti-p63 McAb A4A. There had brown scattered and clustering granules in the nucleus.The positive result showed there’s stem cells which were low differentiated and highly hyperplasia .
4.The study of grafting tissue engineering corneal epithelium and repairing animal model of severe corneal epithelium deficiency
The grafting of tissue engineering corneal epithelium was after 6 weeks of animal models preparation.Separated conjunctiva and lower tissue along the sclerotica,cutted the vascularized epithelial tissue.The fibrin-based scaffold with the stem cells covered sclerotica,and it’s edge was 4mm larger than the limbus of cornea and the larger part was cutted.The 3mm edge of the fibrin-based scaffold was overlapped by the conjunctiva.The edge of the scaffold were fixed on the limbus of cornea using 10-0 nylon thread.
The thread was removed 3 days after the operation.The conjunctiva was congestion and dropsy,the blood vessel was appeared at the limbus of cornea.The status of conjunctiva were relieved 7 days after operation.There also had new blood vessel on the surface of cornea,but the vessel were degenerated from the center to the limbus of the cornea,the area of the blood vessel is smaller than 1/4 of the whole cornea.The dying area was reduced,the degree of corneal turbid was decreased, the translucency was increased.The conjunctiva congestion obvious released 14 days after operation,the blood vessel was only on the edge of the limbus of cornea,the area of dying relesed further.The blood vessel was almost vanished 1 month after the operation,the cornea was almost transparented.The cornea can’t be dyed.The cornea was ultimated transparented and there has no new blood vessel.The result of dying is negative.
The corneal epithelium is in the status of repairing 1 week after the operation,there’s 1-2 cell layers on the corneal surface.The cells layers were increased,but there has deficiency between corneal epithelium.The corneal epithelium recovered for normal epithelial appearance and order,the columnar stroma cells、upper ecderon pavement cells were likely as normal corneal epithelium.The cornea was intact normal corneal epithelium appearance .
There has few PAS (+) cells in the center of cornea using cell blotting examination 1 week after the grafting operation. There has no PAS (+) cells in the center of cornea 2 weeks after the grafting operation.The epithelium cell was large、fusiform or polygon.The number of the epithelium increased,tightly connected.
The cells were closer and poly-layer 1 month after grafting operationg ,there has no PAS (+) cells.There always have PAS(+) conjunctiva phenotype cells in the control group,there’s lots of PAS (+)cells.
The K3 protein was uniformed positive brown colored zone in the normal corneal epithelim using immunohistochemistry method.2weeks after grafting ,there’s thinner positive colored zone on the epithelial layer,but there’s breakpoint .1 month after grafting,the layer was positive continual colored zone.The layer is thinner than the normal one.3 months after grafting ,the intensity of the colored zone increase,and it’s successive and intact,just as the normal one.
p63 was all expressed 2 weeks、1month、3months after grafting, the most expressed location is in the limbus of cornea.
The express of MUC 5AC is negative 1month after grafting,there’s plenty MUC5AC positive cells in the control group.
All these results shows the repairing of corneal epithelium is progressive.The degree of corneal bedewing and the inflammatory reaction reduced,the number of new blood vessel is decreased.And the results of the control group is contrary.All these shows the tissue engineering corneal epithelium with the stem cells can repair and maintain a stable oscular surface.The normal protetive barrier function of the corneal epithelium is sustained by atutoallergic limbal stem cells transplantation.The proliferation and differentiation of the limbal stem cells can recover the integrated construction of corneal epithelium and prevent the growth of the new blood vessel and conjunctival epithelium ,and maintain the character of the corneal epithelium.
本研究采用细胞生物学和组织工程学技术,成功地分离、培养了角膜缘干细胞和角膜上皮细胞,制备了纤维蛋白细胞外基质网架,这不仅为构建组织工程化角膜上皮提供细胞外基质网架,也为其它人工组织、器官的研究提供了新的支架材料。以纤维蛋白为基质网架,构建了组织工程化角膜上皮,经形态学检测结果显示,所构建的人工角膜上皮与天然角膜上皮极为相似,呈现出动态的增殖、分化过程。在此基础上,对全板层角膜上皮缺损动物模型进行了移植实验研究,取得了较好的修复效果。本研究为未来组织工程角膜上皮在临床上的应用提供了实验依据。
The study of stem cells of libus corneae make prominent progress in the therapy of the ocular surface disorder in recent years.Except normal amniotic membrane grafting and corneal transplanting, the development of tissue engineering provide a new therapy method:cultural stem cells of libus corneae transplantation.We used rabbit’s stem cells of libus corneae as semen cells and fibrin- based scaffold to build tissue engineering corneal epithelium. The tissue engineering corneal epithelium was used to repair the rabbit model of stem cells deficiency of libus corneae.The study estab- lished the work for using tissue engineering method to cure the disease caused by stem cells deficiency .
1.The construction of ranbbit stem cells deficiency model
The number of experimental rabbits is 40.According sterile microsurgical procedure step , sheared the right bulbar conjunctiva circle far from 3mm round the limbus cornea,and separated dornea opaca ,and separated 0.08mm superficial layers of limbus cornea,sheared corneal layers and 3mm bulbar conjunctiva.The cornea was dyed afer the operation has been done.The cornea was observed using lit lamp.After on week, the tiny blood vessel was appeared from edge to center,the conjunctival epithelium was also enter the cornea at the smame time . The papilla and the tunica coerulea can be seen at this time .Afer 2 weeks ,the conjunctiva was cover cornea.Several gross blood vessel branches was seen from conjunctiva to cornea,and its area was bigger than the half of the cornea.The round bigger cells was found using the cell blotting method,nucleus: cytoplasm is 1∶4～1∶5,PAS dying is positive .The chalice cells was dyed peach,it was means conjunctiva phenotype.After 4 weeks ,32 qulifified rabbits was choosed for transplantation,and they was separated into for 4 groups: the control group、the experimental groupⅠ( 2weeks after stem cell transplantation)、the experimental groupⅡ( 1 month after stem cell transplantation)、the experimental groupⅢ( 3months after stem cell transplantation).
2.The construction of fibrin scaffold
The fibrinogen powder was dissolved and duluted to 20mg/ml using PBS.The thrombin powder was dissolved and duluted to 10U/ml using PBS,and aprotinin was added into thrombin solution, the final concentration was 163μg/ml.Mixed the equal amount of fibrinogen and thrombin.The fibrin-based scaffold was transparent、semigel、plastic.The average thickness is about 1mm,and it was easily moved out of the culture plate.The diameter of the fibrin-based scaffold was between 70～108μm,the average diameter was 86μm,the average 3D vacant porosity was 70.4% mesured by medical image analysis instrument.All these means the fibrin-based scaffold provided enough space for cell growing.The fibrin-based scaffold was irregular multistrata network 3D structure observed under SEM.
The quality of fibrin-based scaffold was biocompatibility, absorbability,avirulent,fast-shaped ,longlasting,low degradation, and means it’s good bioframe material.
3.The combinational cultivation of rabbit limus corneae tissue and fibrin-based scaffold
Take out of 110μm thickness limbus corneae of the left eye, about 2mm×2mm,and the tissue was digested for 10min using 0.1% trypsin.Then the tissue was shifted on the fibrin-baed scaffold and cultivated.
After24-36 hours,there’s cells migrated from the edge of limbus corneae tissue to the fibrin-based scaffold observed under the inverted microscope.The quality of cells were small,adherent and polygon .After 4 days, some cells growed as net-ballon style.After 6 days,there’s large area of cells ,the cells were ellipse and polygon.After 7 days, the cells were almost occupied the whole plate.After that the cells growed in layers .About 2 weeks it would be 3-4 layers.The cells and the fibrin has been the combination implant,it was slabstone shape.
The cells were different ,some were round and ellipse.Little kytoplam was in it,the bound of the cell membrane was clear,karyoplasmic ratio was above 1.There was tight junction between the cells,desmosome was clear.This means the cells culvated in vitro still had the ultramicrostructure feature of the epithelial cells.
The immunohistochemistry exam was done using anti-cytokeratin K3 McAb AE5. There had brown scattered and clustering granules in the cytoplasm .The positive results showed the cells were in the differentiating status,and there has normal corneal epithelial cells phenotype .
The immunohistochemistry exam was also done using anti-p63 McAb A4A. There had brown scattered and clustering granules in the nucleus.The positive result showed there’s stem cells which were low differentiated and highly hyperplasia .
4.The study of grafting tissue engineering corneal epithelium and repairing animal model of severe corneal epithelium deficiency
The grafting of tissue engineering corneal epithelium was after 6 weeks of animal models preparation.Separated conjunctiva and lower tissue along the sclerotica,cutted the vascularized epithelial tissue.The fibrin-based scaffold with the stem cells covered sclerotica,and it’s edge was 4mm larger than the limbus of cornea and the larger part was cutted.The 3mm edge of the fibrin-based scaffold was overlapped by the conjunctiva.The edge of the scaffold were fixed on the limbus of cornea using 10-0 nylon thread.
The thread was removed 3 days after the operation.The conjunctiva was congestion and dropsy,the blood vessel was appeared at the limbus of cornea.The status of conjunctiva were relieved 7 days after operation.There also had new blood vessel on the surface of cornea,but the vessel were degenerated from the center to the limbus of the cornea,the area of the blood vessel is smaller than 1/4 of the whole cornea.The dying area was reduced,the degree of corneal turbid was decreased, the translucency was increased.The conjunctiva congestion obvious released 14 days after operation,the blood vessel was only on the edge of the limbus of cornea,the area of dying relesed further.The blood vessel was almost vanished 1 month after the operation,the cornea was almost transparented.The cornea can’t be dyed.The cornea was ultimated transparented and there has no new blood vessel.The result of dying is negative.
The corneal epithelium is in the status of repairing 1 week after the operation,there’s 1-2 cell layers on the corneal surface.The cells layers were increased,but there has deficiency between corneal epithelium.The corneal epithelium recovered for normal epithelial appearance and order,the columnar stroma cells、upper ecderon pavement cells were likely as normal corneal epithelium.The cornea was intact normal corneal epithelium appearance .
There has few PAS (+) cells in the center of cornea using cell blotting examination 1 week after the grafting operation. There has no PAS (+) cells in the center of cornea 2 weeks after the grafting operation.The epithelium cell was large、fusiform or polygon.The number of the epithelium increased,tightly connected.
The cells were closer and poly-layer 1 month after grafting operationg ,there has no PAS (+) cells.There always have PAS(+) conjunctiva phenotype cells in the control group,there’s lots of PAS (+)cells.
The K3 protein was uniformed positive brown colored zone in the normal corneal epithelim using immunohistochemistry method.2weeks after grafting ,there’s thinner positive colored zone on the epithelial layer,but there’s breakpoint .1 month after grafting,the layer was positive continual colored zone.The layer is thinner than the normal one.3 months after grafting ,the intensity of the colored zone increase,and it’s successive and intact,just as the normal one.
p63 was all expressed 2 weeks、1month、3months after grafting, the most expressed location is in the limbus of cornea.
The express of MUC 5AC is negative 1month after grafting,there’s plenty MUC5AC positive cells in the control group.
All these results shows the repairing of corneal epithelium is progressive.The degree of corneal bedewing and the inflammatory reaction reduced,the number of new blood vessel is decreased.And the results of the control group is contrary.All these shows the tissue engineering corneal epithelium with the stem cells can repair and maintain a stable oscular surface.The normal protetive barrier function of the corneal epithelium is sustained by atutoallergic limbal stem cells transplantation.The proliferation and differentiation of the limbal stem cells can recover the integrated construction of corneal epithelium and prevent the growth of the new blood vessel and conjunctival epithelium ,and maintain the character of the corneal epithelium.
引文
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