人角膜缘干细胞增殖、分化及标记物的研究
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摘要
干细胞被认为是未分化的有增殖能力的，自我更新，产生大量分化后代细胞及重建组织的细胞。不仅胚胎有多能干细胞，在成年组织中还存在有组织特异性干细胞，包括血液，肝脏，脑组织，皮肤，角膜等。从组织获取的干细胞有着比我们认为的大得多的分化潜力，它们在组织创伤或发生疾病时能应激性增殖，并表现为非对称性分化。角膜缘干细胞不仅可以分化、增殖为角膜上皮细胞，更重要的是干细胞像一道屏障，阻止结膜上皮细胞移行至角膜表面，这对于保持角膜的透明性与正常生理功能有着重要的意义。角膜因其生理位置靠前，位于眼表，结构精细，所以非常容易受到病原体的感染及外界损害，又由于角膜在屈光系统中起着非常重要的作用，故一旦角膜受损，轻者视力降低，重者视力丧失，所以角膜病是我国第二位致盲眼病。随着现代眼科学的发展，许多角膜病所致的视力降低及盲目可以通过角膜移植复明，但对于某些角膜缘干细胞缺乏或功能障碍的疾病如无虹膜、化学和热烧伤、辐射损害、Stevens-Johnson综合征、眼瘢痕性天疱疮等，进行穿透性角膜移植和角膜成形术往往失败。若先施行角膜缘干细胞移植，改善或重建眼表结构，则可以提高移植成功率。因此对人角膜缘干细胞的体外培养、特异性标记物、增殖和分化的研究成为一大热点。本研究通过对人角膜各个部位上皮细胞的免疫组织化学染色；使用不同的方法培养角膜缘上皮干细胞并对比其培养成功率和生
长速度；用低钙培养基培养出角膜缘干细胞进行免疫细胞化学染色，以期筛选出较好的培养干细胞的方法和特异性较高的标记物，获得对人角膜缘干细胞增殖、分化的进一步了解。这对于研究因角膜缘干细胞缺乏或功能障碍导致的眼部疾病及基因转导治疗某些遗传疾病有着重要意义。
目的：通过对比得到较好的培养人角膜缘干细胞的方法；检测上皮性钙粘附蛋白（E-cadherin）在人角膜中央上皮，角膜边缘上皮及低钙环境下培养的人角膜缘干细胞上的表达来分析其与人角膜缘干细胞迁移,增殖,分化的关系及作为人角膜缘干细胞标记物的可能性；比较细胞角质蛋白K19（K19）、上皮性钙粘附蛋白（E-cadherin）及核p63蛋白(p63)三者作为人角膜缘干细胞标记物的可能性及特异性的高低。
通过以上的实验取得对人角膜缘干细胞特性的进一步了解,为分离,体外培养角膜缘干细胞提供更丰富的理论基础，以期将角膜缘干细胞移植及干细胞的基因转导治疗广泛的应用于以角膜缘干细胞缺乏或功能障碍为特征的眼表疾病患者。
方法
使用组织块培养法分别培养经与未经DispaseⅡ酶处理的人角膜缘组织，并对比两组培养成功率和从接种至见到细胞生长时间。
使用低钙（含Ca2+ 0.06mMol）培养基对人角膜缘组织进行组织块培养，并对培养出的细胞行免疫细胞化学染色，观察培养细胞E-cadherin的表达。
记录从人角膜缘组织块被培养至见到细胞生长的时间和培养成功率。
分别使用鼠抗人单克隆抗体K19、p63、E-cadherin对人角膜中央上皮，角膜边缘上皮进行免疫组织化学染色，分析三者作为人角膜缘干细胞特异性标记物的可行性及特异性的高低。
结果
未使用DispaseⅡ酶处理的角膜缘的培养成功率高于使用组。两组分别培养成功116块和108块；从接种至见到细胞生长的时间短于使用组，时间分别为5.48±0.23天和6.26±0.35天。经统计学分析差异有显著性。
免疫细胞化学证实，对人角膜缘组织使用低钙培养基培养出的细胞只有少量的E-cadherin表达。
在人角膜边缘上皮细胞上p63非常少量的表达，K19和E-cadherin大量表达；角膜中央上皮细胞上，p63和E-cadherin基本没有表达，K19仍有少量表达。
结论
在人角膜缘干细胞的组织块培养中，不用DispaseⅡ酶处理可以得到更快的细胞生长速度，更高的培养成功率。
E-cadherin在人角膜缘干细胞的分化及迁移过程中起着非常关键的作用。
p63作为人角膜缘干细胞标记物的特异性高于K19。
Stem cells have long been regarded as undifferentiated cells capable of proliferation, self-renewal, production of a large number of differentiated progeny, and regeneration of tissues. Generally, it has been thought that only embryonic stem cells are pluripotent. Indeed, extensive data support that stem cells exist in many adult tissues, including liver, brain, skin, cornea and so on. These stem cells have a high proliferative potential. They can be activated by wounding or disease. Limbal stem cells can maintain corneal epithelium long-term by proliferation and differentiation. It is more important that they like a barrier to prevent the transfer of conjunctival epithelium. These are very important to maintain normal physiology function and transparency of cornea. Cornea is located in the forepart of eyeball, and has elaborate structure. It’s very easy to be damaged or infected. If cornea is harmed, the vision will reduce or lose. So the corneal diseases are the second cause of the blindness. With the development of ophthalmology, many patients with cornea disease can be healed by corneal transplantation. This method, however, is effectless to treat ocular surface disease with limbal stem cell deficiency such as Stevens-Johnson syndrome, chemical and thermal burns,
radiation injury, extensive microbial infection, and aniridia. If using limbal stem cell transplantation first, The ocular surface structure can be improved, and the successful rate of corneal transplantation will be promoted. So many scholars pay their attention to limbal stem cell culture, special markers, proliferation and differentiation. This experiment used immunohistology and different methods of limbal stem cell culture to find a better way for stem cell culture and a special marker, make a deeper understand to human limbal stem cell.
Objective: To look for a better methods to culture human limbal stem cells, detective E-cadherin’s expression in human central, fringe epithelium and cultured limbal stem cells in low Ca2+, analyze it’s relationship with limbal stem cell’s transference, proliferation and differentiation and the probability to be a markers, and analyze the specialty to be a markers of E-cadherin, K19 and P63.
Through these experiments, grasp more basic theory to human limbal stem cell culture in vitro. So we can extensively use gene therapy and stem cells transplantation to treat the patients with diseases that have the character of limbal stem cells deficiency and function disorder.
Methods
Using or not using DispaseⅡ digest limbal tissues, analyze the time from culture to outgrow and their growing rate of two groups.
Low Ca2+ culture media is used to culture human limbal
stem cells. The cell expressions of E-cadherin are observed by immunocytochemistry.
Record the time from culture to outgrow and their growing rate of two groups.
The expression of K19, E-cadherin, P63 are observed by immunohistochemistry. The specialty is contrasted.
Results
The group digested by DispaseⅡ has slower speed of cell outgrow and lower growing rate than the other group. The numbers of tissues that can grow are 108 and 116. The times from culture to outgrow are 5.48±0.12days and 6.26± 0.18days.
Only few of cells cultured by low Ca2+ culture media express E-cadherin.
Few of limbal epithelium expresses P63, but a lot of cells express E-cadherin and K19.
Conclusion
It’s more beneficial to the human limbal stem cell culture if the tissue dose not digested by DispaseⅡ.
E-cadherin play a important role in the process of human limbal stem cell’s transference, proliferation and differentiation.
As a marker, P63’s specialty is higher than K19.
长速度；用低钙培养基培养出角膜缘干细胞进行免疫细胞化学染色，以期筛选出较好的培养干细胞的方法和特异性较高的标记物，获得对人角膜缘干细胞增殖、分化的进一步了解。这对于研究因角膜缘干细胞缺乏或功能障碍导致的眼部疾病及基因转导治疗某些遗传疾病有着重要意义。
目的：通过对比得到较好的培养人角膜缘干细胞的方法；检测上皮性钙粘附蛋白（E-cadherin）在人角膜中央上皮，角膜边缘上皮及低钙环境下培养的人角膜缘干细胞上的表达来分析其与人角膜缘干细胞迁移,增殖,分化的关系及作为人角膜缘干细胞标记物的可能性；比较细胞角质蛋白K19（K19）、上皮性钙粘附蛋白（E-cadherin）及核p63蛋白(p63)三者作为人角膜缘干细胞标记物的可能性及特异性的高低。
通过以上的实验取得对人角膜缘干细胞特性的进一步了解,为分离,体外培养角膜缘干细胞提供更丰富的理论基础，以期将角膜缘干细胞移植及干细胞的基因转导治疗广泛的应用于以角膜缘干细胞缺乏或功能障碍为特征的眼表疾病患者。
方法
使用组织块培养法分别培养经与未经DispaseⅡ酶处理的人角膜缘组织，并对比两组培养成功率和从接种至见到细胞生长时间。
使用低钙（含Ca2+ 0.06mMol）培养基对人角膜缘组织进行组织块培养，并对培养出的细胞行免疫细胞化学染色，观察培养细胞E-cadherin的表达。
记录从人角膜缘组织块被培养至见到细胞生长的时间和培养成功率。
分别使用鼠抗人单克隆抗体K19、p63、E-cadherin对人角膜中央上皮，角膜边缘上皮进行免疫组织化学染色，分析三者作为人角膜缘干细胞特异性标记物的可行性及特异性的高低。
结果
未使用DispaseⅡ酶处理的角膜缘的培养成功率高于使用组。两组分别培养成功116块和108块；从接种至见到细胞生长的时间短于使用组，时间分别为5.48±0.23天和6.26±0.35天。经统计学分析差异有显著性。
免疫细胞化学证实，对人角膜缘组织使用低钙培养基培养出的细胞只有少量的E-cadherin表达。
在人角膜边缘上皮细胞上p63非常少量的表达，K19和E-cadherin大量表达；角膜中央上皮细胞上，p63和E-cadherin基本没有表达，K19仍有少量表达。
结论
在人角膜缘干细胞的组织块培养中，不用DispaseⅡ酶处理可以得到更快的细胞生长速度，更高的培养成功率。
E-cadherin在人角膜缘干细胞的分化及迁移过程中起着非常关键的作用。
p63作为人角膜缘干细胞标记物的特异性高于K19。
Stem cells have long been regarded as undifferentiated cells capable of proliferation, self-renewal, production of a large number of differentiated progeny, and regeneration of tissues. Generally, it has been thought that only embryonic stem cells are pluripotent. Indeed, extensive data support that stem cells exist in many adult tissues, including liver, brain, skin, cornea and so on. These stem cells have a high proliferative potential. They can be activated by wounding or disease. Limbal stem cells can maintain corneal epithelium long-term by proliferation and differentiation. It is more important that they like a barrier to prevent the transfer of conjunctival epithelium. These are very important to maintain normal physiology function and transparency of cornea. Cornea is located in the forepart of eyeball, and has elaborate structure. It’s very easy to be damaged or infected. If cornea is harmed, the vision will reduce or lose. So the corneal diseases are the second cause of the blindness. With the development of ophthalmology, many patients with cornea disease can be healed by corneal transplantation. This method, however, is effectless to treat ocular surface disease with limbal stem cell deficiency such as Stevens-Johnson syndrome, chemical and thermal burns,
radiation injury, extensive microbial infection, and aniridia. If using limbal stem cell transplantation first, The ocular surface structure can be improved, and the successful rate of corneal transplantation will be promoted. So many scholars pay their attention to limbal stem cell culture, special markers, proliferation and differentiation. This experiment used immunohistology and different methods of limbal stem cell culture to find a better way for stem cell culture and a special marker, make a deeper understand to human limbal stem cell.
Objective: To look for a better methods to culture human limbal stem cells, detective E-cadherin’s expression in human central, fringe epithelium and cultured limbal stem cells in low Ca2+, analyze it’s relationship with limbal stem cell’s transference, proliferation and differentiation and the probability to be a markers, and analyze the specialty to be a markers of E-cadherin, K19 and P63.
Through these experiments, grasp more basic theory to human limbal stem cell culture in vitro. So we can extensively use gene therapy and stem cells transplantation to treat the patients with diseases that have the character of limbal stem cells deficiency and function disorder.
Methods
Using or not using DispaseⅡ digest limbal tissues, analyze the time from culture to outgrow and their growing rate of two groups.
Low Ca2+ culture media is used to culture human limbal
stem cells. The cell expressions of E-cadherin are observed by immunocytochemistry.
Record the time from culture to outgrow and their growing rate of two groups.
The expression of K19, E-cadherin, P63 are observed by immunohistochemistry. The specialty is contrasted.
Results
The group digested by DispaseⅡ has slower speed of cell outgrow and lower growing rate than the other group. The numbers of tissues that can grow are 108 and 116. The times from culture to outgrow are 5.48±0.12days and 6.26± 0.18days.
Only few of cells cultured by low Ca2+ culture media express E-cadherin.
Few of limbal epithelium expresses P63, but a lot of cells express E-cadherin and K19.
Conclusion
It’s more beneficial to the human limbal stem cell culture if the tissue dose not digested by DispaseⅡ.
E-cadherin play a important role in the process of human limbal stem cell’s transference, proliferation and differentiation.
As a marker, P63’s specialty is higher than K19.
引文
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Schwartz GS, Holland EG. Latrogenic limbal stem cell deficiency. Cornea, 1998, 7:31~37
Fujishima H, Shimazaki J, Tsubota K. Temporary corneal stem cell dysfunction after radiation therapy. Br J Ophthalmol, 1996, 120:368~375
Nishida K, Kinoshita S, Ohashi Y, Kuwayama Y, Yamamoto S. Ocular surface abnormalities in aniridia. Am J Ophthalmol, 1995, 120:368~375
Tseng SC, LI DQ. Comparison of protein kinase C subtype expression between normal and aniridic human ocular surface :implications for limbal stem cell dysfunction in aniridia. Cornea, 1996,15:168~178
Cotsarelis G, Cheng SZ, Dong G, et al. Exsistence of slow- cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell, 1989, 57:201~209
Kruse FE. Stem cells and corneal epithelial regeneration. Eye, 1994,8:170~183
S.Elizabeth James, Andrea Rowe, Luca Ilari, et al. The potential for eye bank limbal rings to generate cultured corneal epithelial allografts. Cornea, 2001, 20 : 488 ~ 494
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