羊膜上培养的人角膜缘细胞对兔角膜上皮的功能重建
Functional reconstruction of rabbit corneal epithelium by human limbal cells cultured on amniotic membrane.
作者信息
Du Yiqin, Chen Jing, Funderburgh James L, Zhu Xiuan, Li Lingsong
机构信息
Peking University Stem Cell Research Center, Beijing China.
出版信息
Mol Vis. 2003 Dec 8;9:635-43.
PURPOSE
To investigate the phenotype of fetal and adult human limbal cells cultured on human amniotic membrane and the ability of cultured adult human limbal cells to repair limbal stem cell deficiency in a rabbit model.
METHODS
Human adult and fetal limbal cells were isolated and cultured either on plastic plates or on human amniotic membrane. Connexin43, p63, and keratins 3 and 12 (K3 and K12) were detected by immunofluorescence and RT-PCR. Limbal stem cell deficiency was established in rabbits using chemical ablation and mechanical debridement. Cultured adult human limbal cells were transplanted onto rabbit corneas one month after injury, then fixed and imbedded in paraffin forty days later. Immunofluorescent staining of human-nuclear antigen, p63, K3, and connexin43 identified human-specific cells, progenitor cells, and differentiated corneal epithelial cells, respectively.
RESULTS
Adult and fetal cultured limbal cells appeared similar in morphology. RT-PCR results showed that cells cultured from the human adult and fetal limbal area expressed both p63 and K12, whereas cells from central adult epithelium expressed K12 only. Immunofluorescent staining showed that more cells were p63 positive when cultured on human amniotic membrane than on plastic. Double staining for p63 and connexin43 showed some p63-positive cells co-expressing connexin43. After transplantation of adult human limbal cells cultured on human amniotic membrane, injured rabbit corneas were completely reconstructed exhibiting epithelial integrity, improved corneal clarity, and little or no neovascularization. The majority of repopulated epithelial cells expressed anti-human nuclear antibody. Cells expressing p63 occurred throughout the new epithelium.
CONCLUSIONS
During healing, expression of p63 is not limited to epithelial stem cells but may also mark transient amplifying progenitor cells. Culture on human amniotic membrane suppresses differentiation of limbal epithelial cells and promotes the proliferation of p63 expressing cells. Amniotic membrane-cultured human limbal cells fully reconstructed rabbit corneas having limbal stem cell deficiency, with human cells providing most of the cells of the new epithelium. Expression p63 is distributed throughout the reconstructed tissue.
目的
研究在人羊膜上培养的胎儿及成人角膜缘细胞的表型，以及培养的成人角膜缘细胞修复兔模型中角膜缘干细胞缺陷的能力。
方法
分离成人及胎儿角膜缘细胞，分别培养于塑料平板或人羊膜上。采用免疫荧光法和逆转录-聚合酶链反应（RT-PCR）检测连接蛋白43、p63以及角蛋白3和12（K3和K12）。通过化学消融和机械清创在兔眼建立角膜缘干细胞缺陷模型。损伤1个月后，将培养的成人角膜缘细胞移植到兔角膜上，40天后固定并石蜡包埋。通过人核抗原、p63、K3和连接蛋白43的免疫荧光染色分别鉴定人特异性细胞、祖细胞和分化的角膜上皮细胞。
结果
成人及胎儿培养的角膜缘细胞形态相似。RT-PCR结果显示，成人及胎儿角膜缘区域培养的细胞均表达p63和K12，而来自成人中央上皮的细胞仅表达K12。免疫荧光染色显示，在人羊膜上培养时p63阳性细胞比在塑料平板上更多。p63和连接蛋白43双重染色显示部分p63阳性细胞共表达连接蛋白43。将在人羊膜上培养的成人角膜缘细胞移植后，损伤的兔角膜完全重建，表现为上皮完整、角膜透明度提高，新生血管很少或无新生血管。大多数重新形成的上皮细胞表达抗人核抗体。表达p63的细胞分布于整个新生上皮。
结论
在愈合过程中，p63的表达不仅限于上皮干细胞，也可能标记短暂扩增的祖细胞。在人羊膜上培养可抑制角膜缘上皮细胞分化，促进表达p63细胞的增殖。羊膜培养的人角膜缘细胞可完全重建存在角膜缘干细胞缺陷的兔角膜，新上皮的大部分细胞由人细胞提供。p63表达分布于整个重建组织。
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