羊膜生物支架与组织工程化角膜上皮的构建
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摘要
由于多种原因，发生角膜损伤，角膜缘干细胞遭到不可逆性损害，导致角膜缘干细胞失去代偿能力，出现角膜上皮持续性缺损、角膜基质溶解溃疡、角膜严重的新生血管化和疤痕化以及角膜上皮结膜化等症状的眼表异常，其治疗问题非常棘手。羊膜移植是近年来临床治疗眼表异常的一种新方法。据报道羊膜移植对复发性翼状胬肉，急性或亚急性期眼热化学烧伤，伴有严重疤痕增殖的重度烧伤眼以及其它一些慢性炎症性眼病均有一定的疗效。但是，单纯羊膜不易操作，利用硝酸纤维素膜负载又不便于观察，报道的羊膜处理和保存方法比较复杂。另外，如果以羊膜为支架，构建组织工程化角膜上皮时，去掉上皮细胞保留基底膜的羊膜用于培养细胞载体时，细胞很难贴附。基于以上存在的问题，本研究制作出一种更便于操作的羊膜支架，得到一种处理和保存羊膜的简便方法，并且该羊膜利于细胞粘附，以此成功构建用于移植的羊膜角膜上皮植片。羊膜自体角膜上皮细胞植片移植可重建受损角膜。但自体角膜上皮细胞移植无法对Stevens-Johnson综合症和眼表瘢痕类天疱疮引起的眼角膜损伤进行修复。本研究首次用羊膜培养表皮干细胞构建羊膜植片，并成功地治疗了角膜干细胞缺损。
本研究，将人羊膜的上皮细胞和海绵层去除（同时去除基底膜结构），用纯甘油简化冻存法，－80℃保存5个月后，与新鲜羊膜相比较结构无明显差别，仍能使细胞生长良好。将此羊膜与自制的硝酸纤维素膜框架制成羊膜支架。该支架便于操作，羊膜在硝酸纤维素膜框架上贴合牢固，不会皱缩，不会漂浮，便于细胞的悬浮培养。在纤维素膜框架负载羊膜制成的羊膜支架上进行细胞培养，制成羊膜细胞植片。（申请专利，申请号：200410026031.0）
动物模型的制作过程：全身麻醉，保定，剪毛，清洁眼表，眼周消毒，眼球后部麻醉。对于家兔和山羊是通过碱烧伤的方法去除角膜缘组织，山羊则是在眼科手术显微镜下操作。对家兔模型分A、B、C和D 4组进行对照试验，4周后，A组所有试验家兔角膜表面血管化、结膜化，未发生睑球粘连、溃疡性穿孔，细胞印迹学检查为结膜表型，可作为实验性角膜缘干细胞移植的病理模型；B组5只模型中只有2只模型制作成功（2/5）；C组角膜表面透明，未见结膜和新生血管长入，细胞印迹学检查为角膜表型。不能用作模型；D组虽然4只为完全角膜缘干细胞缺失，但由于角膜溃疡穿孔，睑球粘连严重，不能用于移植实验。而另1只角膜表面透明，未见结膜和新生血管长入，细胞印迹学检查为角膜表型。山羊模型处理4周后，角膜表面血管化、结膜化，未发生睑球
粘连和溃疡性穿孔，并根据角膜混浊度和表面新生血管化、结膜化（细胞印迹学检查出现杯细胞）等指标，来判定属角膜缘干细胞完全缺失病理模型，可作为移植的实验动物模型。
模型建立后，将上述制备好的不同的羊膜－细胞植片分别移植给家兔和山羊。利用组织学检查及光镜、电镜进行观察。结果表明，所有实验兔，移植后10个月时，7例中，有2例视力恢复，效果明显，4例部分有效，1例无效；仅移植羊膜的4只家兔有3例无效，1例部分有效；未移植的3只家兔全部未见好转。本实验4只羊进行异体移植，除一只因手术不慎，损伤晶状体导致移植失败外，其余三只羊均实现角膜缘重建，眼表透明度增高。本实验共有9只羊进行了自体移植，其中5只至今六个月，有3只部分有效，眼表上皮化，实现角膜缘重建，2只未见好转；另外4只至今两个月，4只均部分有效，眼表上皮化，实现角膜缘重建。对照组共有6只羊，眼表均结膜化。（申请专利，申请号：200410026031.0） (申请专利，申请号：200410026154.4)
本研究说明，所制作的羊膜支架便于实验研究和临床操作；用冷冻保存方法可使羊膜长期保存，并且冷冻过的羊膜仍能使细胞在其上良好生长；羊膜去除基底膜后，细胞很容易贴附，并且能够生长良好，在其表面所培养的上皮细胞可以重构基底膜结构；该方法处理的羊膜可进行眼表重建，此种羊膜还可以联合皮肤表皮干细胞进行眼表重建。
Ocular surface disorders, due to irreversibly destroy of corneal limbal stem cells (CLSC) maintaining corneal epithelial renewal, result from malcompensation of limbal stem cells, which was characteriqed by persistent epithelial defects, corneal stromal melting, corneal neovascularization with scarring and conjunctival epithelial ingrowth. The treatment for ocular surface disorders was arduous. Recently, human amniotic membrane (HAM) transplantation was a new operation performed for ocular surface disorders. According to documents, it had partial curative effects on some ocular surface disorders including recurrent pterygium, acute thermal/chemical-burned eye, severe burned eye with proliferating scarring and some chronic inflammation. The pure HAM tissue can not be operated easily. Cells on HAM can not be observed conveniently if Nitrocellulose（NC） membrane which wasn’t been delt with is used to load HAM. The reported methods of disposing and conserving HAM are complex. And cells are difficult to adhere to HAM surface if it still contains basement membrane. For all these reasons, we try to make a new kind of HAM that can be easily and conveniently operated, prepared and preserved and is good for cells adhering. If the corneal epithelial cells successfully grow and form graft well in vitro on HAM carrier, the graft can be used into HAM-cornea. Autograft of corneal limbal epithelial cells carried by HAM can reconstruct injured cornea, but they can’t repair the cornea injured by Stevens-Johnson syndrome (SJS) and Ocular Cicatricial Pemphigoid (OCP). In this research we first used HAM to culture corneal limbal epithelial cells to make HAM grafts, and succeed in curing the lack of corneal limbal epithelial cells.
In this study, we removed epithelial cells, sponge layer and with basement membrane from HAM. HAM was frozen and stored at －80℃ in pure glycerol for 5 months. As a result, it was indifferent from fresh HAM and can also make cells grow well. The HAM scaffold with self-made NC membrane frame can adhere?to the scaffold closely, without shrivelling and floating and is good for cells` floating culture. Then we cultured cells on the scaffold and made them into HAM-cells grafts.
Basiclly, the Animal models was made in similar methods of the general anaesthesia, settle, shear the eyelash, the ocular surface cleaning followed by eyeball anaesthesia. Rabbits were
used for alkili-burn eye and disinfection model, while goat cornea limbal was ablated micro-operation under ophthalmoscope. In general, it was the successful phothological model of total CLSC deficiency that those corneal surface were haze with neovasculavization and conjunctivalizaion, as well as the corneal stroma were integrate, symblepharon (adhesions of lid conjunctiva to globe conjunctiva) were not observed, which could be used for limbal stem cells transplants. To create the pathological model of CLSC deficiency, 20 nomal rabbits were randomly divided into A，B，C and D groups. In group A, the rabbits corneal limbal epithelium lamella were excised, and the central corneal epithelia were burned with 1N NaOH. In group B, corneal limbal epithelium lamella were all excised, in group C only the upper–half of limbal epithelium as removed, the central corneal epithelium of the both groups were erased with a swab soaked in physiological saline. In the group D, the corneal and limbal epitheliums were burned with a cotton swab socked in 1N NaOH. After 4 weeks the results showed that 100 percent of the rabbits in group A could be used. In group B only two fifths of animals could be used. All corneas of rabbits in group C were transparent, with intact corneal epithelium, and so could not be used as model. In group D, 4 rabbits with total CLSC deficiency so could not be used because of serious stroma perforation and symblepharon, another rabbit recovered as group C. That corneal limbal epithelium lamella excised and the center corneal epithelium burned with 1N NaOH is applicable method to create the pathological model of total CLSC deficiency. Aft
本研究，将人羊膜的上皮细胞和海绵层去除（同时去除基底膜结构），用纯甘油简化冻存法，－80℃保存5个月后，与新鲜羊膜相比较结构无明显差别，仍能使细胞生长良好。将此羊膜与自制的硝酸纤维素膜框架制成羊膜支架。该支架便于操作，羊膜在硝酸纤维素膜框架上贴合牢固，不会皱缩，不会漂浮，便于细胞的悬浮培养。在纤维素膜框架负载羊膜制成的羊膜支架上进行细胞培养，制成羊膜细胞植片。（申请专利，申请号：200410026031.0）
动物模型的制作过程：全身麻醉，保定，剪毛，清洁眼表，眼周消毒，眼球后部麻醉。对于家兔和山羊是通过碱烧伤的方法去除角膜缘组织，山羊则是在眼科手术显微镜下操作。对家兔模型分A、B、C和D 4组进行对照试验，4周后，A组所有试验家兔角膜表面血管化、结膜化，未发生睑球粘连、溃疡性穿孔，细胞印迹学检查为结膜表型，可作为实验性角膜缘干细胞移植的病理模型；B组5只模型中只有2只模型制作成功（2/5）；C组角膜表面透明，未见结膜和新生血管长入，细胞印迹学检查为角膜表型。不能用作模型；D组虽然4只为完全角膜缘干细胞缺失，但由于角膜溃疡穿孔，睑球粘连严重，不能用于移植实验。而另1只角膜表面透明，未见结膜和新生血管长入，细胞印迹学检查为角膜表型。山羊模型处理4周后，角膜表面血管化、结膜化，未发生睑球
粘连和溃疡性穿孔，并根据角膜混浊度和表面新生血管化、结膜化（细胞印迹学检查出现杯细胞）等指标，来判定属角膜缘干细胞完全缺失病理模型，可作为移植的实验动物模型。
模型建立后，将上述制备好的不同的羊膜－细胞植片分别移植给家兔和山羊。利用组织学检查及光镜、电镜进行观察。结果表明，所有实验兔，移植后10个月时，7例中，有2例视力恢复，效果明显，4例部分有效，1例无效；仅移植羊膜的4只家兔有3例无效，1例部分有效；未移植的3只家兔全部未见好转。本实验4只羊进行异体移植，除一只因手术不慎，损伤晶状体导致移植失败外，其余三只羊均实现角膜缘重建，眼表透明度增高。本实验共有9只羊进行了自体移植，其中5只至今六个月，有3只部分有效，眼表上皮化，实现角膜缘重建，2只未见好转；另外4只至今两个月，4只均部分有效，眼表上皮化，实现角膜缘重建。对照组共有6只羊，眼表均结膜化。（申请专利，申请号：200410026031.0） (申请专利，申请号：200410026154.4)
本研究说明，所制作的羊膜支架便于实验研究和临床操作；用冷冻保存方法可使羊膜长期保存，并且冷冻过的羊膜仍能使细胞在其上良好生长；羊膜去除基底膜后，细胞很容易贴附，并且能够生长良好，在其表面所培养的上皮细胞可以重构基底膜结构；该方法处理的羊膜可进行眼表重建，此种羊膜还可以联合皮肤表皮干细胞进行眼表重建。
Ocular surface disorders, due to irreversibly destroy of corneal limbal stem cells (CLSC) maintaining corneal epithelial renewal, result from malcompensation of limbal stem cells, which was characteriqed by persistent epithelial defects, corneal stromal melting, corneal neovascularization with scarring and conjunctival epithelial ingrowth. The treatment for ocular surface disorders was arduous. Recently, human amniotic membrane (HAM) transplantation was a new operation performed for ocular surface disorders. According to documents, it had partial curative effects on some ocular surface disorders including recurrent pterygium, acute thermal/chemical-burned eye, severe burned eye with proliferating scarring and some chronic inflammation. The pure HAM tissue can not be operated easily. Cells on HAM can not be observed conveniently if Nitrocellulose（NC） membrane which wasn’t been delt with is used to load HAM. The reported methods of disposing and conserving HAM are complex. And cells are difficult to adhere to HAM surface if it still contains basement membrane. For all these reasons, we try to make a new kind of HAM that can be easily and conveniently operated, prepared and preserved and is good for cells adhering. If the corneal epithelial cells successfully grow and form graft well in vitro on HAM carrier, the graft can be used into HAM-cornea. Autograft of corneal limbal epithelial cells carried by HAM can reconstruct injured cornea, but they can’t repair the cornea injured by Stevens-Johnson syndrome (SJS) and Ocular Cicatricial Pemphigoid (OCP). In this research we first used HAM to culture corneal limbal epithelial cells to make HAM grafts, and succeed in curing the lack of corneal limbal epithelial cells.
In this study, we removed epithelial cells, sponge layer and with basement membrane from HAM. HAM was frozen and stored at －80℃ in pure glycerol for 5 months. As a result, it was indifferent from fresh HAM and can also make cells grow well. The HAM scaffold with self-made NC membrane frame can adhere?to the scaffold closely, without shrivelling and floating and is good for cells` floating culture. Then we cultured cells on the scaffold and made them into HAM-cells grafts.
Basiclly, the Animal models was made in similar methods of the general anaesthesia, settle, shear the eyelash, the ocular surface cleaning followed by eyeball anaesthesia. Rabbits were
used for alkili-burn eye and disinfection model, while goat cornea limbal was ablated micro-operation under ophthalmoscope. In general, it was the successful phothological model of total CLSC deficiency that those corneal surface were haze with neovasculavization and conjunctivalizaion, as well as the corneal stroma were integrate, symblepharon (adhesions of lid conjunctiva to globe conjunctiva) were not observed, which could be used for limbal stem cells transplants. To create the pathological model of CLSC deficiency, 20 nomal rabbits were randomly divided into A，B，C and D groups. In group A, the rabbits corneal limbal epithelium lamella were excised, and the central corneal epithelia were burned with 1N NaOH. In group B, corneal limbal epithelium lamella were all excised, in group C only the upper–half of limbal epithelium as removed, the central corneal epithelium of the both groups were erased with a swab soaked in physiological saline. In the group D, the corneal and limbal epitheliums were burned with a cotton swab socked in 1N NaOH. After 4 weeks the results showed that 100 percent of the rabbits in group A could be used. In group B only two fifths of animals could be used. All corneas of rabbits in group C were transparent, with intact corneal epithelium, and so could not be used as model. In group D, 4 rabbits with total CLSC deficiency so could not be used because of serious stroma perforation and symblepharon, another rabbit recovered as group C. That corneal limbal epithelium lamella excised and the center corneal epithelium burned with 1N NaOH is applicable method to create the pathological model of total CLSC deficiency. Aft
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