培养的血管内皮细胞替代自体的角膜内皮细胞实验研究
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摘要
目的：利用血管内皮细胞和角膜内皮细胞之间某些生物学特性相似性，探讨和研究自体的血管内皮细胞替代角膜内皮细胞功能维持角膜脱水状态的可行性。
方法：取26只实验家兔分成三组：实验组(10只)、实验对照组(10只)、空白对照组(6只)。实验组家兔的角膜内表面移植培养的自体血管内皮细胞；实验对照组移植无角膜弹力层的自体植片；空白对照组直接移植带角膜内皮细胞的自体植片。术后15天内，应频繁注意各组角膜植片透明度的变化，并每隔7天A超检测各组植片术后厚度的变化情况。术后第16天，取下3组动物的角膜植片于倒置相差显微镜下观察各组角膜内皮面细胞分布的情况、形态学特征的变化以及对后弹力层代谢的影响。
结果：实验组角膜植片明显比实验对照组角膜植片透明，但比空白对照组植片稍混浊。实验组角膜植片与空白对照组植片之间的厚度差异性不明显(P＞0.05)，但以空白对照组角膜植片更接近正常角膜的厚度；实验组与实验对照组之间的植片厚度具有明显差异性(p＜0.05)，后者植片呈现高度水肿。同时，实验组角膜植片内表面移植的自体血管内皮细胞能较好地生长，且分布较均匀，形态呈铺路石样。病理切片：实验组角膜内皮面可见一层细胞生长，缺乏角膜后弹力层样物质；实验对照组角膜内皮面未见后弹力层样物质，无任何细胞生长；空白对照组角膜植片的内皮面可见明显的后弹力层样物质，有一层细胞生长。
结论：用自体的血管内皮细胞替代角膜内皮细胞维持角膜的脱水状态获得了一定的效果。
Purpose: Utilizing several similar characteristic role between vascular endotheliocyteand corneal endothelial cell to the feasibility of their succedaneous transplantationeach other for the therapy of bullous keratopathy.
Methods: 26 experimental rabbits were classified into the experimental group (10rabbits), the experimental control group (10 rabbits) and the blank control group (6rabbits). In experimental group, cultural autogeneic vascular endotheliocyte would begrafted on posterior corneal surface without corneal endothelium; in blank controlgroup, autogeneic corneal button with corneal endothelium would be transplanteddirectly to contralateral eyeball for contrasting observation of others two groups; inexperimental control group, all the autogeneic corneal button whose Descemet'smembrane were removed from corneal stroma layer would be sutured to the cornealbed of contralateral eyeball for the same purpose as in experimental control group.Corneal transparence of all animals was frequently observed within the postoperative15 days. A ultrasound apparatus was adopted to measure the postoperative thicknessvariability of corneal graft every 7 days in each groups. On the sixteenth day of thepostoperative, corneal grafts of all groups were taken out of their left eyeball and putunder the inverted phase contrast microscope for the observation of distribution stateof vascular endotheliocyte on posterior corneal surface without Descemet's membraneand their morphologic change.
Results: The clarity of corneal graft in the experimental group had an advantage overthat in the experimental control group, but next to that in the blank control group.Thickness value of corneal graft in each group was analyzed with T test as following: thickness difference of the graft was scarcely manifest between the experimentalgroup and blank control group (P＞0.05), but corneal thickness of the latter getespecially close to the normal cornea thickness. Thickness variability of the graft inthe experimental group was obviously different from that in the experimental group(P＜0.05), the serious corneal edema appeared in the latter group. Meanwhile,autogeneic vascular endotheliocyte could grow stably-uniformly in form of the pebbleon the posterior corneal surface of the experimental group. Pathological section hasshown that a layer of growing cell could be discovered on posterior corneal surface ofcorneal button in the experimental group, but lack of a membrane like Descemet'smembrane; no cell and material like membrane could be discovered on that of theexperimental control group; a layer of cell and a membrane like Descemet'smembrane could viewed on that of the blank control group.
Conclusions: it was significant that vascular endotheliocyte plays the part of cornealendothelial cell for the maintenance of corneal dehydration form.
方法：取26只实验家兔分成三组：实验组(10只)、实验对照组(10只)、空白对照组(6只)。实验组家兔的角膜内表面移植培养的自体血管内皮细胞；实验对照组移植无角膜弹力层的自体植片；空白对照组直接移植带角膜内皮细胞的自体植片。术后15天内，应频繁注意各组角膜植片透明度的变化，并每隔7天A超检测各组植片术后厚度的变化情况。术后第16天，取下3组动物的角膜植片于倒置相差显微镜下观察各组角膜内皮面细胞分布的情况、形态学特征的变化以及对后弹力层代谢的影响。
结果：实验组角膜植片明显比实验对照组角膜植片透明，但比空白对照组植片稍混浊。实验组角膜植片与空白对照组植片之间的厚度差异性不明显(P＞0.05)，但以空白对照组角膜植片更接近正常角膜的厚度；实验组与实验对照组之间的植片厚度具有明显差异性(p＜0.05)，后者植片呈现高度水肿。同时，实验组角膜植片内表面移植的自体血管内皮细胞能较好地生长，且分布较均匀，形态呈铺路石样。病理切片：实验组角膜内皮面可见一层细胞生长，缺乏角膜后弹力层样物质；实验对照组角膜内皮面未见后弹力层样物质，无任何细胞生长；空白对照组角膜植片的内皮面可见明显的后弹力层样物质，有一层细胞生长。
结论：用自体的血管内皮细胞替代角膜内皮细胞维持角膜的脱水状态获得了一定的效果。
Purpose: Utilizing several similar characteristic role between vascular endotheliocyteand corneal endothelial cell to the feasibility of their succedaneous transplantationeach other for the therapy of bullous keratopathy.
Methods: 26 experimental rabbits were classified into the experimental group (10rabbits), the experimental control group (10 rabbits) and the blank control group (6rabbits). In experimental group, cultural autogeneic vascular endotheliocyte would begrafted on posterior corneal surface without corneal endothelium; in blank controlgroup, autogeneic corneal button with corneal endothelium would be transplanteddirectly to contralateral eyeball for contrasting observation of others two groups; inexperimental control group, all the autogeneic corneal button whose Descemet'smembrane were removed from corneal stroma layer would be sutured to the cornealbed of contralateral eyeball for the same purpose as in experimental control group.Corneal transparence of all animals was frequently observed within the postoperative15 days. A ultrasound apparatus was adopted to measure the postoperative thicknessvariability of corneal graft every 7 days in each groups. On the sixteenth day of thepostoperative, corneal grafts of all groups were taken out of their left eyeball and putunder the inverted phase contrast microscope for the observation of distribution stateof vascular endotheliocyte on posterior corneal surface without Descemet's membraneand their morphologic change.
Results: The clarity of corneal graft in the experimental group had an advantage overthat in the experimental control group, but next to that in the blank control group.Thickness value of corneal graft in each group was analyzed with T test as following: thickness difference of the graft was scarcely manifest between the experimentalgroup and blank control group (P＞0.05), but corneal thickness of the latter getespecially close to the normal cornea thickness. Thickness variability of the graft inthe experimental group was obviously different from that in the experimental group(P＜0.05), the serious corneal edema appeared in the latter group. Meanwhile,autogeneic vascular endotheliocyte could grow stably-uniformly in form of the pebbleon the posterior corneal surface of the experimental group. Pathological section hasshown that a layer of growing cell could be discovered on posterior corneal surface ofcorneal button in the experimental group, but lack of a membrane like Descemet'smembrane; no cell and material like membrane could be discovered on that of theexperimental control group; a layer of cell and a membrane like Descemet'smembrane could viewed on that of the blank control group.
Conclusions: it was significant that vascular endotheliocyte plays the part of cornealendothelial cell for the maintenance of corneal dehydration form.
引文
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1 Wolff RD, Volker H J, Alphen CC, at al. The histology of iatrogenic keratitis bullosa[J]. Ophthalmol, 1981, 50: 357-364.
2 Ruberti JW, Klyce SD. NaCL osmotic perturbation can modulate hydration control in rabbit cornea[J]. Exp Eye Res, 2003, 76: 349-359.
3 Alio JL, Shalaby AM, Attla WH, at al. Phacoemulsification in the anterior chamber: preliminary results[J]. Ophthalmol, 2002, 34: 59-73.
4 杨瑾，孙慧敏．大泡性角膜病变[J]．中国实用眼科杂志，2001，19(9)：646-649．
5 冯春茂，刘华，张秀兰，等．穿透性角膜移植治疗大泡性角膜病变[J]．眼科，学报，1994，10：244-247．
6 Pallone TL, Edwards A, Ma T, at al. Requirement of aquaporin-1 for NaCl-driven water transport across descending vasa recta[J]. J Clin Invest, 2000, 105:215-222.
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8 Nilius B, Droogmans G. Ion channels and their functional role in vascular endothelium[J]. Physiol Rev, 2001, 81: 1431.
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12 Gospodarowicz D, Greenburg G. The coating of bovine and rabbit corneas denuded of their endothelium with bovine corneal endothelial cells. Exp Eye Res, 1979, 28: 249-265.
13 刘小伟，赵家良．角膜内皮细胞移植．国外医学眼科学分册，2005，29(5)： 334-337．
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16 刘海伟，韩雅玲．血管平滑肌细胞异质性的研究进展．国外医学心血管疾病分册，2004，31(6)：323-325．
17 刘平，张运，胡维成．血管外膜及其成纤维细胞在血管增殖性病变中的作用．中华老年医学杂志，2005，24(12)：936-938．
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2 Ruberti JW, Klyce SD. NaCL osmotic perturbation can modulate hydration control in rabbit cornea[J]. Exp Eye Res, 2003, 76: 349-359.
3 Alio JL, Shalaby AM, Attla WH, at al. Phacoemulsification in the anterior chamber: preliminary results[J]. Ophthalmol, 2002, 34: 59-73.
4 杨瑾，孙慧敏．大泡性角膜病变[J]．中国实用眼科杂志，2001，19(9)：646-649．
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7 Benos D J, Stanton BA. Functional domains within the degenerin/epitheli.al sodium channel (DegIENaC) superfamily of ion channels[J]. J Physiol, 1999, 520:631-644.
8 Nilius B, Droogmans G. Ion channels and their functional role in vascular endothelium[J]. Physiol Rev, 2001, 81: 1431.
9 Fischbarg J. Mechanism of fluid transport across corneal endothelium and other epithelial layers: a possible explanation based on cyclic cell volume regulatory changes[J]. Br J Ophthalmol, 1997, 81: 85-89.
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12 Gospodarowicz D, Greenburg G. The coating of bovine and rabbit corneas denuded of their endothelium with bovine corneal endothelial cells. Exp Eye Res, 1979, 28: 249-265.
13 刘小伟，赵家良．角膜内皮细胞移植．国外医学眼科学分册，2005，29(5)： 334-337．
14 魏文利，关永源，孙家钧．血管平滑肌和内皮细胞Ca~(2+)内流机制及其与Cl~-通道的关系．中国药理学通报，1999，15(3)：212-215．
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17 刘平，张运，胡维成．血管外膜及其成纤维细胞在血管增殖性病变中的作用．中华老年医学杂志，2005，24(12)：936-938．
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